**The Role of the Microbiota in Gastrointestinal Health and Disease**

Anne-Marie C. Overstreet, Amanda E. Ramer-Tait, Albert E. Jergens and Michael J. Wannemuehler

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/53920

## **1. Introduction**

Antony van Leeuwenhoek's observation of "animalcules" in the tarter of teeth in 1683 marked the beginning of humans trying to understand their relationship with microbes. Now com‐ monly referred to as "bacteria" or the "microbiota", we share our inner and outer space with these single-celled organisms. They are present all around us in the oceans, soil, leaves, and even air [1]. One of the microbial niches that has recently garnered much attention is the human body. In this niche, we share the table with 1014 microbial cells, which outnumber our eukary‐ otic cells ten to one [1, 2]. These organisms colonize most body sites, including the skin, oral cavity, gastrointestinal, urogenital and respiratory tracts. Additionally, there is 150 times more genetic material associated with our resident bacteria when compared to our own DNA and comprise what is known as our body's "second genome," a concept recently reviewed by Zhu et al. [3]. In an effort to understand the role that these organisms have on human development, health and disease, the National Institutes of Health launched the "Human Microbiome Project" in 2007 in an effort to elucidate this host-microbe interaction [4-6].

The role of bacteria is most obvious in the gastrointestinal tract (GIT). Bacteria in this niche have the ability to break down substances that otherwise could not be digested by GIT cells; they also produce metabolites needed by the body such as vitamin K (menaquinones), folate, B12, and riboflavin [7-12]. The GIT has a surface area the size of a tennis court and is home to 1011-1012 organisms per gram of colonic content [1, 13]. The predominant bacterial phyla present in the GIT include the Firmicutes and Bacteroidetes with low levels of Proteobacteria, Actino‐ bacteria, Fusobacteria, and Verrucomicrobia [14-16]. It has been noted that members of the predominant factions (the Firmicutes and Bacteroidetes) belong to only three groups, *Bacteroides, Clostridium coccoides* (cluster XIVa) and *Clostridium leptum* (cluster IV) [15, 17, 18].

© 2012 Overstreet et al.; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

There are also Archaea present in the GIT and have been found to belong to one phylotype, *Methanobrevibacter smithii* [14, 19].

*Clostridium difficile* infection, which was further associated with the development of wheeze, asthma, eczema, and food allergies throughout the first seven years of life [27]. Consistent with these findings, a Norwegian study documented an association between asthma and Cesarean delivered children [28]. Although the epidemiological connections between Cesarean delivery, the composition of the microbiota, and disease are still being studied, strong evidence exists that early colonization of the intestines with aerobic skin microbes is associated with health

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Another factor affecting early colonization of the human gut is determined by feeding method [29]. In one study of vaginally delivered infants, the fecal microbiota of breast-fed compared to formula-fed infants within the first month of life was analyzed using fluorescence *in situ* hybridization (FISH) to evaluate 11 separate groups of organisms [29]. *Bifidobacterium* spp. were the most prominent organisms found to colonize infants fed breast milk, comprising 69% of the bacterial population, with the next most prevalent group being the *Bacteroides* and *Prevotella* spp. making up 12%. With respect to the fecal microbiota of formula-fed infants, the microbiota was actually more diverse than that found in the breast-fed babies. Formula-fed infants had *Bifidobacterium* spp. as the most predominant group, however, they only comprised 32% of the bacterial population while *Bacteroides* and *Prevotella* spp. comprised 29% of the composition. Additionally, *Atopbium* spp. were found to account for 8% of the microbiota in formula-fed infants, but only comprised 1% of the community in the breast-fed infants.

The next major shift in the microbial population occurs during weaning. In a study consisting of infants from five European countries, the composition of the fecal microbiota at four weeks post-weaning was analyzed via FISH using 10 different probes [30]. When considering the results from all of the infants, it was noted that the microbial communities consisted primarily of *Bifidobacterium* (37%), *C. coccoides* (14%) and *Bacteroides* (14%). When samples were obtained from infants that were separated by geographic location, differences among the groups were noticeable. Infants from Spain had significantly greater *Lactobacillus* spp. and less *Bifidobacte‐ rium* spp. as compared to infants from the UK, Sweden, Germany, and Italy. The Spanish fecal samples also had significantly greater presence of *Bacteroides* spp. and *C. leptum* compared to those from the UK, Sweden, or Italy. The numbers of *C. leptum* was highest in samples obtained from German infants. The microbiota of infants from Spain also had significantly greater amounts of enterobacteria when compared to those from the UK, Sweden, or Germany, and higher numbers of *Streptococcus* spp. compared to those from Germany. The infants with the highest amount of *Bifidobacterium* spp. were from Sweden and the UK. Further separation of the data revealed that breast-fed infants had an increased percentage of *Bifidobacterium* spp. while *Bacteroides* spp. and *C. coccoides* were reduced post-weaning. Additionally, delivery method also influenced the microbiota composition, with vaginally delivered infants having

risks.

**1.3. Impact of feeding method on colonization**

**1.4. Microbial changes that occur after weaning**

higher numbers of *Bacteroides* present.

## **1.1. Initial colonization of the gastrointestinal tract**

Infants are born sterile and establishment of the GI microbiota begins shortly thereafter. Studies in mice have revealed that the first organisms to colonize include lactobacilli,fFlavo‐ bacteria and Group N Streptococci [20]. These three groups were found in the stomach and the small and large intestine. When the mice were 12 days of age, the flavobacteria disappeared from all three sites. Concurrently, there was a rapid increase in the presence of enterococci and slow lactose fermenting coliform bacilli reaching 109 bacteria per gram of tissue in the large intestine. This spike was transient however, and as the mice aged, a shift toward a more strict anaerobic population occurred in the large intestine. This ordered colonization occurs because aerotolerant organisms are able to colonize the GIT early in the presence of oxygen. Through their metabolism, they reduce the redox potential and allow for the later colonization and replication of strict anaerobes [21].

## **1.2. Impact of the method of delivery on colonization**

In humans, the method of delivery impacts the initial microbiota of the infant [22-24]. In a study of Venezuelan women, vaginal delivery was associated with initial colonization by *Lactobacillus*, *Prevotella*, *Atopobium*, and *Sneathia* spp.; in contrast Cesarean delivery was associated with colonization of *Staphylococcus, Propionibacterium*, and *Corynebacterium* spp. (organisms typically found on skin). It was also noted that the initial bacteria present in infants born vaginally were vertically transferred from the mother. This was not the case for those born via Cesarean. A Finnish study analyzed infant fecal samples collected between three days of age and six months of age to evaluate temporal colonization [23]. The results revealed that at three days of age, all but one infant, regardless of the delivery method, were colonized with aerobic bacteria. However, a higher percentage of vaginally delivered infants were colonized with *Bifidobacterium*-like (BLB) and *Lactobacillus*-like (LBB) bacteria as compared to Cesarean delivered infants. These differential colonization patterns were corrected by day ten for LBB and by day 30 for BLB. Additional characterization of the fecal microbiota revealed that at one month of age, infants born via Cesarean had a significant increase in *Clostridium perfringens* (25% to 56%); an increase not seen in vaginally delivered infants. This is an important obser‐ vation, as *C. perfringens* has been thought to play a role in sudden infant death syndrome (SIDS) [25]. Also of note, significant differences in the levels of *Bacteroides fragilis* were observed between the two methods of delivery [23]. Specifically, this bacterium was present at statisti‐ cally higher numbers in vaginally delivered infants as compared to Cesarean delivered infants at all time points throughout the six months of the study.

These differences in initial colonizers have been associated with adverse effects, as infants born via Cesarean are more susceptible to skin infections with methicillin resistant *Staphylococcus aureus* (MRSA). A 2004 report indicated that 82% of infant MRSA cases in Chicago and 64% in Los Angeles were delivered via Cesarean , all of which involved healthy, full-term infants [26]. An additional prospective cohort study indicated a link between Cesarean delivery and *Clostridium difficile* infection, which was further associated with the development of wheeze, asthma, eczema, and food allergies throughout the first seven years of life [27]. Consistent with these findings, a Norwegian study documented an association between asthma and Cesarean delivered children [28]. Although the epidemiological connections between Cesarean delivery, the composition of the microbiota, and disease are still being studied, strong evidence exists that early colonization of the intestines with aerobic skin microbes is associated with health risks.

## **1.3. Impact of feeding method on colonization**

There are also Archaea present in the GIT and have been found to belong to one phylotype,

Infants are born sterile and establishment of the GI microbiota begins shortly thereafter. Studies in mice have revealed that the first organisms to colonize include lactobacilli,fFlavo‐ bacteria and Group N Streptococci [20]. These three groups were found in the stomach and the small and large intestine. When the mice were 12 days of age, the flavobacteria disappeared from all three sites. Concurrently, there was a rapid increase in the presence of enterococci and

intestine. This spike was transient however, and as the mice aged, a shift toward a more strict anaerobic population occurred in the large intestine. This ordered colonization occurs because aerotolerant organisms are able to colonize the GIT early in the presence of oxygen. Through their metabolism, they reduce the redox potential and allow for the later colonization and

In humans, the method of delivery impacts the initial microbiota of the infant [22-24]. In a study of Venezuelan women, vaginal delivery was associated with initial colonization by *Lactobacillus*, *Prevotella*, *Atopobium*, and *Sneathia* spp.; in contrast Cesarean delivery was associated with colonization of *Staphylococcus, Propionibacterium*, and *Corynebacterium* spp. (organisms typically found on skin). It was also noted that the initial bacteria present in infants born vaginally were vertically transferred from the mother. This was not the case for those born via Cesarean. A Finnish study analyzed infant fecal samples collected between three days of age and six months of age to evaluate temporal colonization [23]. The results revealed that at three days of age, all but one infant, regardless of the delivery method, were colonized with aerobic bacteria. However, a higher percentage of vaginally delivered infants were colonized with *Bifidobacterium*-like (BLB) and *Lactobacillus*-like (LBB) bacteria as compared to Cesarean delivered infants. These differential colonization patterns were corrected by day ten for LBB and by day 30 for BLB. Additional characterization of the fecal microbiota revealed that at one month of age, infants born via Cesarean had a significant increase in *Clostridium perfringens* (25% to 56%); an increase not seen in vaginally delivered infants. This is an important obser‐ vation, as *C. perfringens* has been thought to play a role in sudden infant death syndrome (SIDS) [25]. Also of note, significant differences in the levels of *Bacteroides fragilis* were observed between the two methods of delivery [23]. Specifically, this bacterium was present at statisti‐ cally higher numbers in vaginally delivered infants as compared to Cesarean delivered infants

These differences in initial colonizers have been associated with adverse effects, as infants born via Cesarean are more susceptible to skin infections with methicillin resistant *Staphylococcus aureus* (MRSA). A 2004 report indicated that 82% of infant MRSA cases in Chicago and 64% in Los Angeles were delivered via Cesarean , all of which involved healthy, full-term infants [26]. An additional prospective cohort study indicated a link between Cesarean delivery and

bacteria per gram of tissue in the large

*Methanobrevibacter smithii* [14, 19].

74 Inflammatory Bowel Disease

replication of strict anaerobes [21].

**1.1. Initial colonization of the gastrointestinal tract**

slow lactose fermenting coliform bacilli reaching 109

**1.2. Impact of the method of delivery on colonization**

at all time points throughout the six months of the study.

Another factor affecting early colonization of the human gut is determined by feeding method [29]. In one study of vaginally delivered infants, the fecal microbiota of breast-fed compared to formula-fed infants within the first month of life was analyzed using fluorescence *in situ* hybridization (FISH) to evaluate 11 separate groups of organisms [29]. *Bifidobacterium* spp. were the most prominent organisms found to colonize infants fed breast milk, comprising 69% of the bacterial population, with the next most prevalent group being the *Bacteroides* and *Prevotella* spp. making up 12%. With respect to the fecal microbiota of formula-fed infants, the microbiota was actually more diverse than that found in the breast-fed babies. Formula-fed infants had *Bifidobacterium* spp. as the most predominant group, however, they only comprised 32% of the bacterial population while *Bacteroides* and *Prevotella* spp. comprised 29% of the composition. Additionally, *Atopbium* spp. were found to account for 8% of the microbiota in formula-fed infants, but only comprised 1% of the community in the breast-fed infants.

## **1.4. Microbial changes that occur after weaning**

The next major shift in the microbial population occurs during weaning. In a study consisting of infants from five European countries, the composition of the fecal microbiota at four weeks post-weaning was analyzed via FISH using 10 different probes [30]. When considering the results from all of the infants, it was noted that the microbial communities consisted primarily of *Bifidobacterium* (37%), *C. coccoides* (14%) and *Bacteroides* (14%). When samples were obtained from infants that were separated by geographic location, differences among the groups were noticeable. Infants from Spain had significantly greater *Lactobacillus* spp. and less *Bifidobacte‐ rium* spp. as compared to infants from the UK, Sweden, Germany, and Italy. The Spanish fecal samples also had significantly greater presence of *Bacteroides* spp. and *C. leptum* compared to those from the UK, Sweden, or Italy. The numbers of *C. leptum* was highest in samples obtained from German infants. The microbiota of infants from Spain also had significantly greater amounts of enterobacteria when compared to those from the UK, Sweden, or Germany, and higher numbers of *Streptococcus* spp. compared to those from Germany. The infants with the highest amount of *Bifidobacterium* spp. were from Sweden and the UK. Further separation of the data revealed that breast-fed infants had an increased percentage of *Bifidobacterium* spp. while *Bacteroides* spp. and *C. coccoides* were reduced post-weaning. Additionally, delivery method also influenced the microbiota composition, with vaginally delivered infants having higher numbers of *Bacteroides* present.

This study also reported results from infants who provided samples before (at six weeks of age) and four weeks after weaning [30]. Switching to solid food during this time caused a significant reduction in the presence of bifidobactera, enterobacteria, and *C. diffi‐ cile*/*C. perfringens* proportions. These reductions were accompanied by significant increas‐ es in both *C. leptum* and *C. coccoides* clusters. Again, location also played a significant role in the microbial differences at weaning. Spanish infants had the greatest increase in both *C. coccoides* and *C. leptum* post-weaning compared to the other four countries, while enter‐ obacteria was significantly decreased in the Spanish samples compared to those from the other countries. The samples from Spanish infants also had significant increases in *Ato‐ pobium* spp. compared to those from Sweden and Germany and *Streptococcus* spp. com‐ pared to Germany and Italy. The authors also report significant decreases post-weaning in the enterobacteria present in the Italian infant samples as compared to samples from Sweden and the UK. As mentioned previously, feeding and delivery method alter the composition of the microbiota at birth; this is also true after weaning. Formula-fed in‐ fants had higher proportions of *C. leptum* in their feces compared to breast-fed infants post-weaning. Infants born via Cesarean section possessed increased levels of *Bacteroides* spp. after weaning, whereas there was no change in the abundance of this group in vagi‐ nally delivered infants. Additionally, *Atopobium* spp. decreased in vaginally delivered chil‐ dren but increased in Cesarean delivered children post-weaning [30].

elicits the production of goblet cells and increases the heights of the mucosa, resembling what

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(a) (b)

**Figure 1.** Photomicrographs of cecal tissue from a) a germfree mouse and b) a mouse monoassociated with Brachy‐ *spira hyodysenteriae.* Note the changes in the cellularity, presence of goblet cells, and height of the mucosa in the ceca

In the small intestine, this change in epithelial cell transit time is doubled, going from 53 hours in a CONVR mouse to 115 hours in a GF mouse [33]. The most noticeable gross change in GF mice is the enlargement of the cecum, which can comprise up to 19% of the mouse's body weight [34]. The cecum of a GF mouse can assume a more normal size (4.5% body weight) following colonization with a mixture of *Lactobacillus* spp., Group N streptococci *, Bacteroides* spp., enterococci and coliform bacilli recovered from CONVR mice [34]. As shown in Figure 2, monoassociation with *B. hyodysenteriae* also returns the cecum to a more normal size.

Of interest, not all bacteria tested were able to return the cecum to a normal size. It was noted that colonizing mice with *Lactobacillus* spp. and Group N streptococci only was not sufficient to change the morphology of the cecum. However, when *Bacteroides* spp. colonized the mice, the morphology assumed a more normal size. Other differences between GF and CONVR mice include the consistency of the cecal contents, with the contents from a GF mouse being more liquid, hypotonic and alkaline than in a CONVR mouse [35]. Additionally, GI transit time is slower in GF mice, which are also prone to vitamin deficiency due to a lack of bacterial metabolism [36-38]. In GF rats, monoassociation with either a rat-derived *Escherichia coli* strain or a sarcina-like micrococcus was able to reverse the vitamin K deficiency. Research identifying the response to *Salmonella enteritis* serovar Typhimurium challenge indicated that there was a 50-fold greater translocation of bacteria to the mesenteric lymph node, and organisms were present in the blood of GF mice, something not seen in CONVR mice [37]. This phenomenon occurred due to the decreased transit time in the small intestine of GF mice, thereby allowing

the *Salmonella* to multiply in the host and translocate to other tissue sites.

from the monoassociated mouse 28 days after colonization with *B. hyodysenteriae*.

is seen in CONVR mice.

Studies describing development or maturation of the human microbiota reveal that as an individual ages, the microbiota shifts from predominantly facultative anaerobes to strict anaerobes, just as previously observed in rodents [20]. Notably, variations in initial coloniza‐ tion associated with different delivery and feeding methods seems to profoundly impact the microbial community post-weaning. Additionally, geographic location plays an important role in the initial colonization of the (GIT). In spite of all these variables, several themes for colonization can be described. Initially, *Bifidobacterium* spp. are the primary colonizers of the infant GIT along with other species such as *Bacteroides, Lactobacillus, Prevotella,* and *Atopobi‐ um.* As the infant matures, the oxygen presence in the gut is reduced and the infant begins eating solid food, which promotes a transition towards the growth and maintenance of the anaerobes found in the *C. coccoides* and *C. leptum* clusters.

#### **1.5. The microbiota and gastrointestinal homeostasis**

The commensal microbiota has a tremendous influence on the development and functional capabilities of the GIT of its host. Numerous studies have documented the effects that the microbiota has on GIT development in mice. Specifically, the morphology of the intestines differs significantly in animals devoid of microbes (i.e., germfree) compared to conventionallyreared (CONVR) mice [31]. The mucus layer is thinner and epithelial cells have a slower rate of turnover compared to those from conventional animals, primarily because of extended time in the S and G1 phases of the cell cycle [32]. This can be corrected by bacterial colonization. In Figure 1, panel A shows intestinal tissue from germfree (GF) mice and panel B shows tissue from a mouse colonized with *Brachyspira hyodysenteriae.* Colonization with *B. hyodysenteriae* elicits the production of goblet cells and increases the heights of the mucosa, resembling what is seen in CONVR mice.

This study also reported results from infants who provided samples before (at six weeks of age) and four weeks after weaning [30]. Switching to solid food during this time caused a significant reduction in the presence of bifidobactera, enterobacteria, and *C. diffi‐ cile*/*C. perfringens* proportions. These reductions were accompanied by significant increas‐ es in both *C. leptum* and *C. coccoides* clusters. Again, location also played a significant role in the microbial differences at weaning. Spanish infants had the greatest increase in both *C. coccoides* and *C. leptum* post-weaning compared to the other four countries, while enter‐ obacteria was significantly decreased in the Spanish samples compared to those from the other countries. The samples from Spanish infants also had significant increases in *Ato‐ pobium* spp. compared to those from Sweden and Germany and *Streptococcus* spp. com‐ pared to Germany and Italy. The authors also report significant decreases post-weaning in the enterobacteria present in the Italian infant samples as compared to samples from Sweden and the UK. As mentioned previously, feeding and delivery method alter the composition of the microbiota at birth; this is also true after weaning. Formula-fed in‐ fants had higher proportions of *C. leptum* in their feces compared to breast-fed infants post-weaning. Infants born via Cesarean section possessed increased levels of *Bacteroides* spp. after weaning, whereas there was no change in the abundance of this group in vagi‐ nally delivered infants. Additionally, *Atopobium* spp. decreased in vaginally delivered chil‐

76 Inflammatory Bowel Disease

dren but increased in Cesarean delivered children post-weaning [30].

anaerobes found in the *C. coccoides* and *C. leptum* clusters.

**1.5. The microbiota and gastrointestinal homeostasis**

Studies describing development or maturation of the human microbiota reveal that as an individual ages, the microbiota shifts from predominantly facultative anaerobes to strict anaerobes, just as previously observed in rodents [20]. Notably, variations in initial coloniza‐ tion associated with different delivery and feeding methods seems to profoundly impact the microbial community post-weaning. Additionally, geographic location plays an important role in the initial colonization of the (GIT). In spite of all these variables, several themes for colonization can be described. Initially, *Bifidobacterium* spp. are the primary colonizers of the infant GIT along with other species such as *Bacteroides, Lactobacillus, Prevotella,* and *Atopobi‐ um.* As the infant matures, the oxygen presence in the gut is reduced and the infant begins eating solid food, which promotes a transition towards the growth and maintenance of the

The commensal microbiota has a tremendous influence on the development and functional capabilities of the GIT of its host. Numerous studies have documented the effects that the microbiota has on GIT development in mice. Specifically, the morphology of the intestines differs significantly in animals devoid of microbes (i.e., germfree) compared to conventionallyreared (CONVR) mice [31]. The mucus layer is thinner and epithelial cells have a slower rate of turnover compared to those from conventional animals, primarily because of extended time in the S and G1 phases of the cell cycle [32]. This can be corrected by bacterial colonization. In Figure 1, panel A shows intestinal tissue from germfree (GF) mice and panel B shows tissue from a mouse colonized with *Brachyspira hyodysenteriae.* Colonization with *B. hyodysenteriae*

**Figure 1.** Photomicrographs of cecal tissue from a) a germfree mouse and b) a mouse monoassociated with Brachy‐ *spira hyodysenteriae.* Note the changes in the cellularity, presence of goblet cells, and height of the mucosa in the ceca from the monoassociated mouse 28 days after colonization with *B. hyodysenteriae*.

In the small intestine, this change in epithelial cell transit time is doubled, going from 53 hours in a CONVR mouse to 115 hours in a GF mouse [33]. The most noticeable gross change in GF mice is the enlargement of the cecum, which can comprise up to 19% of the mouse's body weight [34]. The cecum of a GF mouse can assume a more normal size (4.5% body weight) following colonization with a mixture of *Lactobacillus* spp., Group N streptococci *, Bacteroides* spp., enterococci and coliform bacilli recovered from CONVR mice [34]. As shown in Figure 2, monoassociation with *B. hyodysenteriae* also returns the cecum to a more normal size.

Of interest, not all bacteria tested were able to return the cecum to a normal size. It was noted that colonizing mice with *Lactobacillus* spp. and Group N streptococci only was not sufficient to change the morphology of the cecum. However, when *Bacteroides* spp. colonized the mice, the morphology assumed a more normal size. Other differences between GF and CONVR mice include the consistency of the cecal contents, with the contents from a GF mouse being more liquid, hypotonic and alkaline than in a CONVR mouse [35]. Additionally, GI transit time is slower in GF mice, which are also prone to vitamin deficiency due to a lack of bacterial metabolism [36-38]. In GF rats, monoassociation with either a rat-derived *Escherichia coli* strain or a sarcina-like micrococcus was able to reverse the vitamin K deficiency. Research identifying the response to *Salmonella enteritis* serovar Typhimurium challenge indicated that there was a 50-fold greater translocation of bacteria to the mesenteric lymph node, and organisms were present in the blood of GF mice, something not seen in CONVR mice [37]. This phenomenon occurred due to the decreased transit time in the small intestine of GF mice, thereby allowing the *Salmonella* to multiply in the host and translocate to other tissue sites.

protein known as Muc2 [42]. The importance of Muc2 has been highlighted by the development of Muc2-/- mice. These mice fail to gain weight, have diarrhea by seven weeks of age, occult blood present in their feces by eight weeks of age, and the majority of mice had gross bleeding and reversible rectal prolapse by nine weeks of age [45]. These mice also develop microscopic

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Underneath the mucus layers lay the intestinal epithelial cells, which are held together by tight junction proteins such as occludins, claudins, and junctional adhesion molecules [46]. These proteins seal the paracellular junction between the cells and regulate the entry of nutrients, ions and water. They also act as a barrier against bacterial entry. Loss of epithelial barrier function promotes a break in immunological tolerance and facilitates immunoreactivity towards the normal commensal microbiota (Figure 3). This phenomenon has been demon‐ strated in mouse studies employing 2,4,6 trinitrobenzensulfonic acid (TNBS), a chemical that disrupts the epithelial barrier to allow translocation of bacteria, resulting in the induction of both innate inflammatory responses and antigen-specific immune responses [47]. Subsequent to the loss of epithelial barrier function, these mice develop severe gastrointestinal inflamma‐ tion, which can be ameliorated by pretreating with antibiotics to reduce the microbial load [47].

The role of the commensal bacteria in regulating the immune response was elegantly demon‐ strated in a study by Wlodarska et al. [48]. Mice administered metronidazole were more susceptible to subsequent infection with the pathogen, *Citrobacter rodentium*. Metronidazoletreated mice had increased submucosal edema, ulcerations, mucosal hyperplasia and de‐ creased numbers of goblet cells. The author also noted enhanced expression of *IL-25* and *Reg3γ* mRNA and an increased presence of NK cells and macrophages in the lamina propria of these mice, indicating an increase in microbial stimulation. The inner mucous layer of metronida‐ zole-treated mice was significantly thinner and mRNA expression of genes encoding for the goblet-cell specific proteins Muc2, TFF3, and RelmB was also decreased in these mice [49, 50]. The thinning of the inner mucous layer following metronidazole treatment allowed for *C. rodentium* to more closely associate with the epithelium and promote production of the proinflammatory cytokines and chemokines TNF-α, IFN-γ, and MCP-1. This study highlights the contribution of the commensal microbiota in the production of the mucus barrier and subse‐

It has also been noted that specific members of the resident microbiota are able to illicit specific immune functions. Colonization with *Bacteroides fragilis* expressing polysaccharide A (PSA) suppressed development of T helper 17 (Th17) CD4+ T cells by promoting the development of

contact with the immune system but its production of PSA ensures that no inflammatory response is induced. Both prophylactic and therapeutic treatment of mice with PSA has been shown to ameliorate TNBS-induced colitis in mice [52]. More recently, work from Atarashi et al., eloquently demonstrated a role for indigenous *Clostridium* species in the induction of colonic Treg cells [53]. Colonization of GF mice with a cocktail of 46 *Clostridium* strains enriched for clusters IV and XIVa promoted TGF-β production from intestinal epithelial cells and

 T regulatory (Treg) CD4+ T cells and inducing IL-10 expression via TLR2 signaling [51]. Additional studies have demonstrated that purified PSA is sufficient to expand the numbers

Treg cells [52]. *B. fragilis*inhabits colonic crypts. This niche puts the organism in close

evidence of colitis as early as five weeks of age.

quently in maintaining mucosal homeostasis [48].

Foxp3+

of Foxp3+

**Figure 2.** Physical and morphological changes associated with microbial colonization. Shown here are ceca from a germfree mouse (left), monoassociated mouse (center), and conventional microbiota mouse (right). Note the in‐ creased size of the cecum from the GF mouse. The distal end of the cecum (appendix) of the monoassociated mouse has begun to undergo morphological changes, including the development of a lymphoid tissue known as a cecal ton‐ sil.

## **1.6. Effect of bacterial colonization on immune development**

In addition to its importance in maintaining gut homeostasis, the GI microbiota is also critical for normal priming and development of the immune system [39-41]. GF mice have reduced numbers of immune cells present in the lamina propria and have smaller Peyer's patches compared to CONVR mice. They also have a diminished capacity for antibody production, fewer plasma cells, smaller mesenteric lymph nodes, and reduced numbers of germinal centers compared to CONVR mice [39]. However, this underdeveloped immune system is fully capable of mounting a response comparable to that of a CONVR mouse when stimulated with bacterial antigen or protein [40, 41]. Macrophages from CONVR mice process antigen faster than those from GF mice, likely because the continued exposure of CONVR macrophages to bacterial antigen allows them to be "primed" for antigen degradation; a phenomenon that does not occur in GF mice [41]. The presence of bacteria in the GIT promotes decreased immunor‐ eactivity towards commensal organisms. The barrier between the gut microbiota and the underlying gastrointestinal associated lymphoid tissue consists of a single layer of epithelial cells covered by two layers of mucus [42, 43]. The inner layer of mucus is approximately 100 μm thick while the outer layer is approximately 700 μm in the rat colon [43]. The inner layer is firmly attached to the epithelial cells and devoid of bacteria, while the outer layer is "loose"and contains commensal organisms [44]. The major constituent of these layers is a protein known as Muc2 [42]. The importance of Muc2 has been highlighted by the development of Muc2-/- mice. These mice fail to gain weight, have diarrhea by seven weeks of age, occult blood present in their feces by eight weeks of age, and the majority of mice had gross bleeding and reversible rectal prolapse by nine weeks of age [45]. These mice also develop microscopic evidence of colitis as early as five weeks of age.

Underneath the mucus layers lay the intestinal epithelial cells, which are held together by tight junction proteins such as occludins, claudins, and junctional adhesion molecules [46]. These proteins seal the paracellular junction between the cells and regulate the entry of nutrients, ions and water. They also act as a barrier against bacterial entry. Loss of epithelial barrier function promotes a break in immunological tolerance and facilitates immunoreactivity towards the normal commensal microbiota (Figure 3). This phenomenon has been demon‐ strated in mouse studies employing 2,4,6 trinitrobenzensulfonic acid (TNBS), a chemical that disrupts the epithelial barrier to allow translocation of bacteria, resulting in the induction of both innate inflammatory responses and antigen-specific immune responses [47]. Subsequent to the loss of epithelial barrier function, these mice develop severe gastrointestinal inflamma‐ tion, which can be ameliorated by pretreating with antibiotics to reduce the microbial load [47].

The role of the commensal bacteria in regulating the immune response was elegantly demon‐ strated in a study by Wlodarska et al. [48]. Mice administered metronidazole were more susceptible to subsequent infection with the pathogen, *Citrobacter rodentium*. Metronidazoletreated mice had increased submucosal edema, ulcerations, mucosal hyperplasia and de‐ creased numbers of goblet cells. The author also noted enhanced expression of *IL-25* and *Reg3γ* mRNA and an increased presence of NK cells and macrophages in the lamina propria of these mice, indicating an increase in microbial stimulation. The inner mucous layer of metronida‐ zole-treated mice was significantly thinner and mRNA expression of genes encoding for the goblet-cell specific proteins Muc2, TFF3, and RelmB was also decreased in these mice [49, 50]. The thinning of the inner mucous layer following metronidazole treatment allowed for *C. rodentium* to more closely associate with the epithelium and promote production of the proinflammatory cytokines and chemokines TNF-α, IFN-γ, and MCP-1. This study highlights the contribution of the commensal microbiota in the production of the mucus barrier and subse‐ quently in maintaining mucosal homeostasis [48].

**Figure 2.** Physical and morphological changes associated with microbial colonization. Shown here are ceca from a germfree mouse (left), monoassociated mouse (center), and conventional microbiota mouse (right). Note the in‐ creased size of the cecum from the GF mouse. The distal end of the cecum (appendix) of the monoassociated mouse has begun to undergo morphological changes, including the development of a lymphoid tissue known as a cecal ton‐

In addition to its importance in maintaining gut homeostasis, the GI microbiota is also critical for normal priming and development of the immune system [39-41]. GF mice have reduced numbers of immune cells present in the lamina propria and have smaller Peyer's patches compared to CONVR mice. They also have a diminished capacity for antibody production, fewer plasma cells, smaller mesenteric lymph nodes, and reduced numbers of germinal centers compared to CONVR mice [39]. However, this underdeveloped immune system is fully capable of mounting a response comparable to that of a CONVR mouse when stimulated with bacterial antigen or protein [40, 41]. Macrophages from CONVR mice process antigen faster than those from GF mice, likely because the continued exposure of CONVR macrophages to bacterial antigen allows them to be "primed" for antigen degradation; a phenomenon that does not occur in GF mice [41]. The presence of bacteria in the GIT promotes decreased immunor‐ eactivity towards commensal organisms. The barrier between the gut microbiota and the underlying gastrointestinal associated lymphoid tissue consists of a single layer of epithelial cells covered by two layers of mucus [42, 43]. The inner layer of mucus is approximately 100 μm thick while the outer layer is approximately 700 μm in the rat colon [43]. The inner layer is firmly attached to the epithelial cells and devoid of bacteria, while the outer layer is "loose"and contains commensal organisms [44]. The major constituent of these layers is a

**1.6. Effect of bacterial colonization on immune development**

sil.

78 Inflammatory Bowel Disease

It has also been noted that specific members of the resident microbiota are able to illicit specific immune functions. Colonization with *Bacteroides fragilis* expressing polysaccharide A (PSA) suppressed development of T helper 17 (Th17) CD4+ T cells by promoting the development of Foxp3+ T regulatory (Treg) CD4+ T cells and inducing IL-10 expression via TLR2 signaling [51]. Additional studies have demonstrated that purified PSA is sufficient to expand the numbers of Foxp3+ Treg cells [52]. *B. fragilis*inhabits colonic crypts. This niche puts the organism in close contact with the immune system but its production of PSA ensures that no inflammatory response is induced. Both prophylactic and therapeutic treatment of mice with PSA has been shown to ameliorate TNBS-induced colitis in mice [52]. More recently, work from Atarashi et al., eloquently demonstrated a role for indigenous *Clostridium* species in the induction of colonic Treg cells [53]. Colonization of GF mice with a cocktail of 46 *Clostridium* strains enriched for clusters IV and XIVa promoted TGF-β production from intestinal epithelial cells and increased the number of Foxp3+ Treg cells in the colon. Furthermore, inoculation of young CONVR mice with *Clostridium* species facilitated resistance to both dextran sodium sulfate (DSS)- and oxazolone-induced colitis as well as increased systemic Immunoglobuin E (IgE) production as adults.

IL-22 likely stimulate intestinal epithelial cells to secrete antimicrobial peptides to limit

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In addition to promoting immune maturation, the commensal microbiota also helps to regulate fat storage. GF mice eat 29% more food than conventional mice yet have 42% less body fat and a decreased metabolic rate [55]. Colonization of GF mice with cecal con‐ tents from conventional mice for 14 days caused a 57% increase in total body fat with a concomitant 27% reduction in food intake. These "conventionalized" mice also had in‐ creases in leptin, fasting glucose, and insulin levels compared to GF mice; they also devel‐ oped insulin resistance. Levels of mRNA specific for the transcription factors, SREBP-1 and ChREBP, were also elevated leading to increased production of lipogenic enzymes. Fat formation is aided by the regulator lipoprotein lipase (LPL) and is inhibited by the *fiaf* gene product whose expression is suppressed in microbiota-bearing conventional mice. The authors suggest that the bacteria in the gut breakdown dietary polysaccharide into monosaccharaides that are then transported to the liver to activate lipogenic enzymes. This process promotes fat formation in the peripheral tissues due to the suppression of *fiaf*. In addition to the production of white adipose tissue, the microbiota may also play a role in eye health [56]. A recent study revealed differences in the lipid profiles in the lens and retinas of GF mice as compared to CONVR mice. The CONVR mice had reduced concentrations of multiple phosphatidylcholines and an overall reduced presence of phos‐ pholipids in the lens. The authors postulate these changes may be due to the increased exposure of the CONVR mice to more oxidative stress than their GF counterparts [56]. Together, these studies help us realize the effects that the gut microbiota may have on host systems that, at the surface, seem to have limited or no connection to the GIT.

Another health benefit that the microbiota provides its host revolves around the produc‐ tion of short-chain fatty acids (SCFA) such as acetate, propionate, and butyrate as end products of anaerobic fermentation [57]. These SCFA (predominantly butyrate) can be uti‐ lized as energy sources by eukaryotic cells. Members of the *Clostridium* clusters IV (e.g., *Faecalibacterium prausnitzii)* and XIVa (e.g., *Roseburia* spp. and *Eubacterium rectale*) are the primary butyrate producers in the GIT, and they comprise approximately 2-15% of the to‐ tal gut microbiota [14, 58-61]. Butyrate, a four-carbon fatty acid, is produced by bacteria via one of two metabolic pathways. The first pathway utilizes the enzymes phosphotrans‐ butyrylase and butyrate kinase to form butyrate from butyryl-CoA to yield one ATP per one molecule of butyrate produced [62, 63]. The second pathway, which is utilized by most organisms in the gut, uses butyryl-CoA acetate CoA transferase to form butyrate

Once produced, butyrate has multiple effects on gut health. A primary use is as a prefer‐ red energy source for colonocytes, and the mechanisms by which butyrate is utilized in the GIT is summarized in Figure 4 [66-69]. Two biomarkers of energy homeostasis, ATP

the growth of the pathogen and its infiltration into the colonic wall [54].

**1.7. Effect of bacterial colonization on other aspects of the body**

**1.8. Bacterial production of short chain fatty acids**

and acetyl-CoA from butyryl-CoA [64, 65].

between the commensal bacteria and the underlying immune cells. Panel B depicts acute inflammation. The epithelial cell barrier has been breached, allowing bacteria to be readily detected by the immune system and promote a pro-inflammatory immune response. **Figure 3.** Panel A shows normal mucosal homeostasis. The epithelial cells provide a barrier between the commensal bacteria and the underlying immune cells. Panel B depicts acute inflammation. The epithelial cell barrier has been breached, allowing bacteria to be readily detected by the immune system and promote a pro-inflammatory immune response.

**Figure 3** Panel A shows normal mucosal homeostasis. The epithelial cells provide a barrier

The segmented filamentous bacteria (SFB) is another microorganism of interest that has been implicated in the induction of Th17 responses [54]. Ivanov et al. found that C57BL/6 mice purchased from the Jackson Laboratory had significantly less IL-17-producing CD4+ T cells in the small intestine compared to those from Taconic Farms. This discrepancy could be corrected via intragastric gavage of the Jackson mice with contents from the small intestines of the Taconic mice. Using a 16S ribosomal RNA PhyloChip analysis, SFB were identified in the microbiota of mice from Taconic Farms but not in those from the Jackson Laboratory. Monoassociation of mice with SFB induced the production of Th17 cells and up-regulated genes encoding antimicrobial peptides and serum amyloid A (SAA). SAA co-cultured with naive CD4+ T cells and lamina propria derived dendritic cells induced Th17 cell differentiation *in vitro*. The authors concluded that SFB, which tightly adheres to and imbeds itself among the microvilli on the epithelial cell surface, in‐ duces the production of SAA by intestinal epithelial cells. In turn, the SAA acts on lami‐ na propria dendritic cells to stimulate the induction of Th17 cells. Colonization with SFB has also been shown to protect against *C. rodentium* infection. Th17 cytokines such as IL-22 likely stimulate intestinal epithelial cells to secrete antimicrobial peptides to limit the growth of the pathogen and its infiltration into the colonic wall [54].

## **1.7. Effect of bacterial colonization on other aspects of the body**

increased the number of Foxp3+

production as adults.

80 Inflammatory Bowel Disease

Treg cells in the colon. Furthermore, inoculation of young

**N**

**N**

**<sup>N</sup> <sup>N</sup>**

**B**

**T**

**<sup>T</sup> <sup>T</sup>**

**N**

**N**

**T**

CONVR mice with *Clostridium* species facilitated resistance to both dextran sodium sulfate (DSS)- and oxazolone-induced colitis as well as increased systemic Immunoglobuin E (IgE)

**B**

**Acute Inflammation A Normal Homeostasis B Acute Homeostasis**

**T M**

**T**

**Figure 3** Panel A shows normal mucosal homeostasis. The epithelial cells provide a barrier between the commensal bacteria and the underlying immune cells. Panel B depicts acute inflammation. The epithelial cell barrier has been breached, allowing bacteria to be readily detected by the immune system and promote a pro-inflammatory immune response.

**Figure 3.** Panel A shows normal mucosal homeostasis. The epithelial cells provide a barrier between the commensal bacteria and the underlying immune cells. Panel B depicts acute inflammation. The epithelial cell barrier has been breached, allowing bacteria to be readily detected by the immune system and promote a pro-inflammatory immune

The segmented filamentous bacteria (SFB) is another microorganism of interest that has been implicated in the induction of Th17 responses [54]. Ivanov et al. found that C57BL/6 mice purchased from the Jackson Laboratory had significantly less IL-17-producing CD4+ T cells in the small intestine compared to those from Taconic Farms. This discrepancy could be corrected via intragastric gavage of the Jackson mice with contents from the small intestines of the Taconic mice. Using a 16S ribosomal RNA PhyloChip analysis, SFB were identified in the microbiota of mice from Taconic Farms but not in those from the Jackson Laboratory. Monoassociation of mice with SFB induced the production of Th17 cells and up-regulated genes encoding antimicrobial peptides and serum amyloid A (SAA). SAA co-cultured with naive CD4+ T cells and lamina propria derived dendritic cells induced Th17 cell differentiation *in vitro*. The authors concluded that SFB, which tightly adheres to and imbeds itself among the microvilli on the epithelial cell surface, in‐ duces the production of SAA by intestinal epithelial cells. In turn, the SAA acts on lami‐ na propria dendritic cells to stimulate the induction of Th17 cells. Colonization with SFB has also been shown to protect against *C. rodentium* infection. Th17 cytokines such as

**T <sup>T</sup> <sup>M</sup> <sup>B</sup>**

**T T**

**Normal Homeostasis**

**T**

**M**

response.

In addition to promoting immune maturation, the commensal microbiota also helps to regulate fat storage. GF mice eat 29% more food than conventional mice yet have 42% less body fat and a decreased metabolic rate [55]. Colonization of GF mice with cecal con‐ tents from conventional mice for 14 days caused a 57% increase in total body fat with a concomitant 27% reduction in food intake. These "conventionalized" mice also had in‐ creases in leptin, fasting glucose, and insulin levels compared to GF mice; they also devel‐ oped insulin resistance. Levels of mRNA specific for the transcription factors, SREBP-1 and ChREBP, were also elevated leading to increased production of lipogenic enzymes. Fat formation is aided by the regulator lipoprotein lipase (LPL) and is inhibited by the *fiaf* gene product whose expression is suppressed in microbiota-bearing conventional mice. The authors suggest that the bacteria in the gut breakdown dietary polysaccharide into monosaccharaides that are then transported to the liver to activate lipogenic enzymes. This process promotes fat formation in the peripheral tissues due to the suppression of *fiaf*. In addition to the production of white adipose tissue, the microbiota may also play a role in eye health [56]. A recent study revealed differences in the lipid profiles in the lens and retinas of GF mice as compared to CONVR mice. The CONVR mice had reduced concentrations of multiple phosphatidylcholines and an overall reduced presence of phos‐ pholipids in the lens. The authors postulate these changes may be due to the increased exposure of the CONVR mice to more oxidative stress than their GF counterparts [56]. Together, these studies help us realize the effects that the gut microbiota may have on host systems that, at the surface, seem to have limited or no connection to the GIT.

#### **1.8. Bacterial production of short chain fatty acids**

Another health benefit that the microbiota provides its host revolves around the produc‐ tion of short-chain fatty acids (SCFA) such as acetate, propionate, and butyrate as end products of anaerobic fermentation [57]. These SCFA (predominantly butyrate) can be uti‐ lized as energy sources by eukaryotic cells. Members of the *Clostridium* clusters IV (e.g., *Faecalibacterium prausnitzii)* and XIVa (e.g., *Roseburia* spp. and *Eubacterium rectale*) are the primary butyrate producers in the GIT, and they comprise approximately 2-15% of the to‐ tal gut microbiota [14, 58-61]. Butyrate, a four-carbon fatty acid, is produced by bacteria via one of two metabolic pathways. The first pathway utilizes the enzymes phosphotrans‐ butyrylase and butyrate kinase to form butyrate from butyryl-CoA to yield one ATP per one molecule of butyrate produced [62, 63]. The second pathway, which is utilized by most organisms in the gut, uses butyryl-CoA acetate CoA transferase to form butyrate and acetyl-CoA from butyryl-CoA [64, 65].

Once produced, butyrate has multiple effects on gut health. A primary use is as a prefer‐ red energy source for colonocytes, and the mechanisms by which butyrate is utilized in the GIT is summarized in Figure 4 [66-69]. Two biomarkers of energy homeostasis, ATP and NADH/NAD+ levels, are both significantly reduced in only the colonic tissues of GF mice as compared to CONVR mice [70]. This observation indicates that GF mice have a reduction in TCA cycle activity, and subsequently less ATP is generated for cellular ener‐ gy. Moreover, this reduction in ATP was correlated with increased signs of energetic stress in colonocytes, including increased expression of 5'-adenosine monophosphate-acti‐ vated protein kinase (AMPK). Consistent with previous reports describing a role for AMPK in inducing autophagy [71], GF coloncytes also expressed elevated levels of the autophagosome marker LC3-11. Transmission election microscopic analysis revealed that significantly more GF colonocytes were undergoing autophagy than colonocytes from CONVR mice. Colonizing GF mice with a conventional microbiota reversed these effects, as did incubation of isolated colonocytes with butyrate. Additional experiments employ‐ ing the fatty-acid oxidation blocker, etomoxir, demonstrated that colonocytes consume butyrate as an energy source and not as a histone deacetylase (HDAC) inhibitor, another known function of butyrate [72, 73].

Butyrate functions as an HDAC inhibitor by blocking cellular deacetylase activity and al‐ lowing histone acetylation [72, 73]. Histone modification causes changes in cellular gene expression patterns; compounds and molecules that can elicit these modifications are be‐ ing studied as potential anti-cancer therapeutics [74]. A comparative study of gene ex‐ pression patterns in HT-29 cells (a colon carcinoma-derived cell line), treated with either butyrate or trichostain A (a known HDAC inhibitor) revealed that both substances had similar effects on gene expression [75]. Upregulated genes (21 total) were found to regu‐ late the cell cycle, signal transduction, DNA repair and genome transcription. Only two genes were down-regulated—lactoferrinδ and MAPKAP kinase. It is also important to note that both butyrate and trichostain A inhibited the growth of HT-29 cells by creating an arrest in the G1 phase of the cell cycle [76]. Butyrate may also down-regulate pro-in‐ flammatory responses via its HDAC activity, as incubation of butyrate with inflamed bi‐ opsy samples or LPS-induced peripheral blood mononuclear cells (PBMCs) reduced the mRNA expression of IL-6, TNF-α, TNF-β, and IL-1β [77]. Additionally, in murine studies of TNBS- and DSS-induced colitis, and in ulcerative colitis (UC) patients, administration of butyrate enemas ameliorates disease activity via NFκB inhibition [77-79]. Butyrate has been shown to decrease both COX-2 and PGE2 expression in HT-29 cells stimulated with TNF-α [80].

The information presented in this section clearly defines a central role for the gut micro‐ biota in many physiological processes; the microbiota even has a tremendous impact on the host's health status. Causal links likely exist between the methods by which infants are delivered, their gut microbial colonization patterns and subsequent health concerns, including asthma, eczema and, food allergies. Associations between gut bacterial com‐ munities and obesity, diabetes, and cardiovascular disease have also been documented [81-83]. A significant body of literature also links changes in the composition of the gut microbiota with inflammatory bowel disease (IBD), which is discussed in detail in the following section [84-89].

**Figure 4. Microbial regulation of colonocyte metabolism.** Schematic depicting how dietary fiber is fermented by microbes into butyrate in the lumen of the colon, which is then transported into the colonocyte. In the colonocyte, butyrate promotes oxidative metabolism and inhibits autophagy. Based on transcriptome and proteome experiments, enzymes regulated by microbes are shown in boxes. In all cases, boxed enzymes that function in β-oxidation and the TCA cycle are downregulated in GF colonocytes, revealing that microbes positively regulate their expression. Diminish‐ ed ATP results in phosphorylation of AMPK and p27, which culminates in autophagy. (Cell Metab. 2011 May 4;13(5):

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83

517-5) Reprinted with permission from the publisher license number 2977121495018

and NADH/NAD+ levels, are both significantly reduced in only the colonic tissues of GF mice as compared to CONVR mice [70]. This observation indicates that GF mice have a reduction in TCA cycle activity, and subsequently less ATP is generated for cellular ener‐ gy. Moreover, this reduction in ATP was correlated with increased signs of energetic stress in colonocytes, including increased expression of 5'-adenosine monophosphate-acti‐ vated protein kinase (AMPK). Consistent with previous reports describing a role for AMPK in inducing autophagy [71], GF coloncytes also expressed elevated levels of the autophagosome marker LC3-11. Transmission election microscopic analysis revealed that significantly more GF colonocytes were undergoing autophagy than colonocytes from CONVR mice. Colonizing GF mice with a conventional microbiota reversed these effects, as did incubation of isolated colonocytes with butyrate. Additional experiments employ‐ ing the fatty-acid oxidation blocker, etomoxir, demonstrated that colonocytes consume butyrate as an energy source and not as a histone deacetylase (HDAC) inhibitor, another

Butyrate functions as an HDAC inhibitor by blocking cellular deacetylase activity and al‐ lowing histone acetylation [72, 73]. Histone modification causes changes in cellular gene expression patterns; compounds and molecules that can elicit these modifications are be‐ ing studied as potential anti-cancer therapeutics [74]. A comparative study of gene ex‐ pression patterns in HT-29 cells (a colon carcinoma-derived cell line), treated with either butyrate or trichostain A (a known HDAC inhibitor) revealed that both substances had similar effects on gene expression [75]. Upregulated genes (21 total) were found to regu‐ late the cell cycle, signal transduction, DNA repair and genome transcription. Only two genes were down-regulated—lactoferrinδ and MAPKAP kinase. It is also important to note that both butyrate and trichostain A inhibited the growth of HT-29 cells by creating an arrest in the G1 phase of the cell cycle [76]. Butyrate may also down-regulate pro-in‐ flammatory responses via its HDAC activity, as incubation of butyrate with inflamed bi‐ opsy samples or LPS-induced peripheral blood mononuclear cells (PBMCs) reduced the mRNA expression of IL-6, TNF-α, TNF-β, and IL-1β [77]. Additionally, in murine studies of TNBS- and DSS-induced colitis, and in ulcerative colitis (UC) patients, administration of butyrate enemas ameliorates disease activity via NFκB inhibition [77-79]. Butyrate has been shown to decrease both COX-2 and PGE2 expression in HT-29 cells stimulated with

The information presented in this section clearly defines a central role for the gut micro‐ biota in many physiological processes; the microbiota even has a tremendous impact on the host's health status. Causal links likely exist between the methods by which infants are delivered, their gut microbial colonization patterns and subsequent health concerns, including asthma, eczema and, food allergies. Associations between gut bacterial com‐ munities and obesity, diabetes, and cardiovascular disease have also been documented [81-83]. A significant body of literature also links changes in the composition of the gut microbiota with inflammatory bowel disease (IBD), which is discussed in detail in the

known function of butyrate [72, 73].

82 Inflammatory Bowel Disease

TNF-α [80].

following section [84-89].

**Figure 4. Microbial regulation of colonocyte metabolism.** Schematic depicting how dietary fiber is fermented by microbes into butyrate in the lumen of the colon, which is then transported into the colonocyte. In the colonocyte, butyrate promotes oxidative metabolism and inhibits autophagy. Based on transcriptome and proteome experiments, enzymes regulated by microbes are shown in boxes. In all cases, boxed enzymes that function in β-oxidation and the TCA cycle are downregulated in GF colonocytes, revealing that microbes positively regulate their expression. Diminish‐ ed ATP results in phosphorylation of AMPK and p27, which culminates in autophagy. (Cell Metab. 2011 May 4;13(5): 517-5) Reprinted with permission from the publisher license number 2977121495018

## **2. Introduction to inflammatory bowel disease**

In 1932, three physicians, Burrill Crohn, Leon Ginzburg, and Gordon Oppenheimer described a disease of unknown etiology in the terminal ileum of young adults [90]. The disease was characterized as being similar to UC (fever, diarrhea and weight loss) and having ulcerations of the mucosa that would eventually lead to stenosis of the lumen and formation of multiple fistulas. Eventually, surgical intervention was used to resect the affected portion of the intestinal tract and patients recovered with little signs of the disease persisting. In their 1932 report, Crohn and colleagues discuss observations from other physicians regarding observa‐ tions of granulomas in the small and large intestine of unknown etiology being classified under the umbrella term "benign granulomas". Still today Crohn's disease (CD) and UC are both included under the umbrella term "inflammatory bowel disease." Crohn and colleagues described the disease as beginning at the ileocecal junction with lesions separated by normal mucosa [90]. They described inflammation of the submucosa and the muscularis resulting in a markedly thickened bowel wall. The presence of "giant cells" was also noted, which they attributed to vegetable matter becoming entrapped in the ulcers and become encapsulated during healing. The authors concluded that the presence of these giant cells in the granulom‐ atous lesion led others to believe that the inflammation was due to an unusual form of tuberculosis. However, the authors could not find any evidence of tuberculosis in their 14 patients.

Clinically, the patients with IBD were characterized as young adults with fever, diarrhea, dull abdominal pain, and vomiting; they were also weak, anemic and had poor appetite. Upon physical examination, the author noted five commonalties: 1) a mass in the right iliac region, 2) evidence of fistula formation, 3) emaciation and anemia, 4) approximately half of the subjects had undergone an appendectomy, and 5) evidence of intestinal obstruction. Treatment was supportive and surgical resection of the affected portion of the intestinal tract was recom‐ mended; 13 of their 14 patients had no symptoms post-operatively. For the one patient that developed recurrent symptoms, it was later determined that not all of the affected intestinal tissue had been resected during the first surgery.

**Figure 5. Inflammatory bowel disease susceptibility loci.** The loci (depicted by lead gene name) attaining genome wide significance (p < 5x10-8) are shown for Crohn's disease (CD, red), ulcerative colitis (UC, blue) and IBD (black where p < 5x10-8 in CD and UC; red were p < 5x10-8 in CD and p < 5x10-4 in UC; blue where p < 5x10-8 in UC and p < 5x10-4 in

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One of the first candidate genes linked to IBD susceptibility was *Nod2.* Located on chromosome 16, the defective allele affects the activation of the transcription factor NF-κB. NOD2 is expressed in antigen-presenting cells, macrophages, lymphocytes, ileal Paneth cells, and intestinal epithelial cells and functions as an intracellular microbial recognition molecule [96-100]. NOD2 senses muramyl dipeptide (MDP), a minimally bioactive motif of peptido‐ glycan from both Gram-negative and Gram-positive bacteria. Once activated by its ligand, NOD2 undergoes a conformational change to expose its CARD domains and recruits the kinase RIP2 to the complex which associates with NOD2 via homophillic CARD-CARD interactions [97]. This process leads to the activation of IKK and the subsequent degradation of IκB and

Petnicki-Ocwieja et al. demonstrated that MDP stimulation of ileal crypts induced bactericidal secretions capable of killing *E. coli* [102]. Moreover, the same authors showed that NOD2 was

CD. Reprinted with permission from the publisher license number: 2977121069437

ultimately results in the translocation of NF-κB to the nucleus [96, 101].

*2.1.1. Nod2*

Eighty years after the first published article of what would become known as "Crohn's disease," there is still no cure for this disease nor do we fully understand its etiology. Un‐ fortunately, the number of people being diagnosed with IBD is increasing yearly [91]. There is not one specific, causative agent of IBD; instead the etiology is thought to be multifactorial in nature, with host genetics, environmental factors, the induction of aberrant immune responses, and the gastrointestinal microbiota all contributing to disease pathogenesis.

## **2.1. Genetic factors affecting IBD**

As of 2011, genome-wide meta-analyses had identified 71 susceptibility loci associated with CD, 47 with UC and 28 associated with both, some of which are shown in Figure 5 [92-95]. Here, we focus on genetic variants within specific genes involved in mediating host responses to microbial components.

**Figure 5. Inflammatory bowel disease susceptibility loci.** The loci (depicted by lead gene name) attaining genome wide significance (p < 5x10-8) are shown for Crohn's disease (CD, red), ulcerative colitis (UC, blue) and IBD (black where p < 5x10-8 in CD and UC; red were p < 5x10-8 in CD and p < 5x10-4 in UC; blue where p < 5x10-8 in UC and p < 5x10-4 in CD. Reprinted with permission from the publisher license number: 2977121069437

#### *2.1.1. Nod2*

**2. Introduction to inflammatory bowel disease**

tissue had been resected during the first surgery.

**2.1. Genetic factors affecting IBD**

to microbial components.

patients.

84 Inflammatory Bowel Disease

In 1932, three physicians, Burrill Crohn, Leon Ginzburg, and Gordon Oppenheimer described a disease of unknown etiology in the terminal ileum of young adults [90]. The disease was characterized as being similar to UC (fever, diarrhea and weight loss) and having ulcerations of the mucosa that would eventually lead to stenosis of the lumen and formation of multiple fistulas. Eventually, surgical intervention was used to resect the affected portion of the intestinal tract and patients recovered with little signs of the disease persisting. In their 1932 report, Crohn and colleagues discuss observations from other physicians regarding observa‐ tions of granulomas in the small and large intestine of unknown etiology being classified under the umbrella term "benign granulomas". Still today Crohn's disease (CD) and UC are both included under the umbrella term "inflammatory bowel disease." Crohn and colleagues described the disease as beginning at the ileocecal junction with lesions separated by normal mucosa [90]. They described inflammation of the submucosa and the muscularis resulting in a markedly thickened bowel wall. The presence of "giant cells" was also noted, which they attributed to vegetable matter becoming entrapped in the ulcers and become encapsulated during healing. The authors concluded that the presence of these giant cells in the granulom‐ atous lesion led others to believe that the inflammation was due to an unusual form of tuberculosis. However, the authors could not find any evidence of tuberculosis in their 14

Clinically, the patients with IBD were characterized as young adults with fever, diarrhea, dull abdominal pain, and vomiting; they were also weak, anemic and had poor appetite. Upon physical examination, the author noted five commonalties: 1) a mass in the right iliac region, 2) evidence of fistula formation, 3) emaciation and anemia, 4) approximately half of the subjects had undergone an appendectomy, and 5) evidence of intestinal obstruction. Treatment was supportive and surgical resection of the affected portion of the intestinal tract was recom‐ mended; 13 of their 14 patients had no symptoms post-operatively. For the one patient that developed recurrent symptoms, it was later determined that not all of the affected intestinal

Eighty years after the first published article of what would become known as "Crohn's disease," there is still no cure for this disease nor do we fully understand its etiology. Un‐ fortunately, the number of people being diagnosed with IBD is increasing yearly [91]. There is not one specific, causative agent of IBD; instead the etiology is thought to be multifactorial in nature, with host genetics, environmental factors, the induction of aberrant immune responses, and the gastrointestinal microbiota all contributing to disease pathogenesis.

As of 2011, genome-wide meta-analyses had identified 71 susceptibility loci associated with CD, 47 with UC and 28 associated with both, some of which are shown in Figure 5 [92-95]. Here, we focus on genetic variants within specific genes involved in mediating host responses One of the first candidate genes linked to IBD susceptibility was *Nod2.* Located on chromosome 16, the defective allele affects the activation of the transcription factor NF-κB. NOD2 is expressed in antigen-presenting cells, macrophages, lymphocytes, ileal Paneth cells, and intestinal epithelial cells and functions as an intracellular microbial recognition molecule [96-100]. NOD2 senses muramyl dipeptide (MDP), a minimally bioactive motif of peptido‐ glycan from both Gram-negative and Gram-positive bacteria. Once activated by its ligand, NOD2 undergoes a conformational change to expose its CARD domains and recruits the kinase RIP2 to the complex which associates with NOD2 via homophillic CARD-CARD interactions [97]. This process leads to the activation of IKK and the subsequent degradation of IκB and ultimately results in the translocation of NF-κB to the nucleus [96, 101].

Petnicki-Ocwieja et al. demonstrated that MDP stimulation of ileal crypts induced bactericidal secretions capable of killing *E. coli* [102]. Moreover, the same authors showed that NOD2 was required for the bactericidal activity of crypt secretions of the terminal ileum. The composition of the ileal microbiota of the NOD2-/- mice was significantly altered in comparison to their WT counterparts. Specifically, NOD2-/- mice had increased numbers of Firmicutes, specifically *Bacillus* species and *Bacteroides* species. Wild-type mice were able to clear an infection of *Helicobacter hepaticus,* an opportunistic mouse pathogen, within seven days while NOD2-/- mice remained colonized with *H. hepaticus* for at least 14 days post-infection [102]. Also of note, the presence of a gut microbiota was required for the expression of NOD2. GF mice possessed decreased levels of NOD2—a phenotype that could be reversed following monoassociation with either Gram-negative or Gram-positive organisms. Together, these results outline a reciprocal regulatory relationship between NOD2 expression and the gut microbiota.

*ATG16L1.* When AIEC are present, this reduction in bacterial clearance likely leads to increased production of pro-inflammatory cytokines, which could tip the immune balance to an inflammatory state. Thus, it appears that the presence of bacterial provocateurs (e.g., AIEC) in the microbiota increases the risk of developing IBD in individuals possessing polymorphisms

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The mucosal immune system is charged with the monumental task of balancing respon‐ siveness and tolerance to a tremendous number of environmental antigens, including those from both food and bacteria. Although the exact cause of IBD remains elusive, sig‐ nificant evidence supports the hypothesis that GIT inflammation is initiated and perpetu‐ ated by a dysregulated immune response directed against the gut microbiota resulting in deleterious responses in genetically susceptible individuals following an environmental trigger. The host with a genetic predisposition for IBD may possess defects in epithelial permeability and/or altered regulation (i.e., NOD2 deficiency) of commensal bacteria. Po‐ tential environmental triggers may include smoking, certain medications, or a gastroin‐ testinal illness that induces a break in homeostasis. Regardless of the specific genetic or environmental trigger, what ensues is an exaggerated, inappropriate mucosal immune re‐ sponse characterized by chronic activation of T cells and the production of cytokines and

The success of cytokine-targeted immunotherapies for a subset of IBD patients supports the idea that these chemical messengers of the immune system play an important role in disease pathogenesis. However, the nature of the immune phenotypes observed in CD and UC patients do differ [108]. The immune response of CD is typically associated with a T helper 1 (Th1) phenotype while UC is characterized by a T helper 2 (Th2) phenotype [108]. Isolated lamina propria (LP) CD4+ T cells from CD patients produce IFN-γ when stimulated via the CD2/CD28 pathway; in contrast, LP CD4+ T cells from UC patients se‐ creted mostly IL-5 [109]. Another cytokine with a strong link to CD is IL-12 [110]. Messen‐ ger RNA for IL-12p40 in LP mononuclear cells (LPMC) was found in 85 % of CD patients, a percentage significantly higher than that found in healthy and UC GIT tissue samples. Similarly, IL-12p35 mRNA was detectable in the LPMC of 92 % of CD patients; expres‐ sion was again significantly higher than for healthy and UC patients. Elevated levels of IL-12 were also detectable in the serum of CD patients [110], and this potent Th1-promot‐ ing cytokine is able to induce IFN-γ production from LP lymphocytes (LPL) isolated from CD patients [111]. Additionally, studies employing a murine model of TNBS-induced col‐ itis have revealed that administration of anti-IL-12 antibodies ameliorated disease severi‐

ty, presumably by decreasing IFN-γ secretion from LP CD4+ T cells [112].

In UC, production of the cytokines IL-5 and IL-13 appear to mediate the disease process. Using LPMC from IBD patients, those with UC had increased production of IL-13 and IL-5 upon *in vitro* restimulation as compared to LPMCs recovered from either CD patients or healthy individuals [113]. Levels of IFN-γ produced by LPMC from UC patients were similar to those

CD161+

produced by cells from healthy controls. The authors specifically identified that CD4+

in genes such as *ATG16L* or *NOD2.*

**2.2. The immune response and IBD**

other inflammatory mediators.

#### *2.1.2. ATG16L1*

Another gene implicated in the pathogenesis of IBD that regulates cellular autophagy is *ATG16L1* [95, 103]. Autophagy is a process utilized by cells to recycle cellular compo‐ nents. This process may also be utilized by cells as a non-apoptotic method of program‐ med cell death [104]. *ATG16L1* is expressed by CD4+ and CD8+ T cells, CD19+ B cells, epithelial cells, and macrophages [105, 106]. Studies by Caldwell et al. utilizing mice en‐ gineered to express low or hypomorphic levels of *ATG16L1* revealed that this gene is in‐ deed an autophagy-related protein. Elevated levels of cytosolic proteins normally degraded by a rapamycin-induced degradation pathway were found in the cells of the *ATG16L1* hypomorphic (ATG16L1HM) mice [95]. Additionally, cells from ATG16L1HM mice possessed fewer autophagosomes following rapamycin treatment or nutrient deprivation. The small intestines and colons of ATG16L1HM mice had no morphological defects in crypt height or villus length but did possess abnormalities found in their Paneth cells. Specifically, lysozyme within Paneth cells was found to be either depleted/absent or dif‐ fuse, which is in contrast to the normal and orderly packaging of lysozyme within gran‐ ules. Decreased levels of lysozyme were also observed in the ileal mucus layer of ATG16L1HM mice. However, there were intact granules observed in the crypt lumen, in‐ dicating a potential role of *ATG16L1* in the maintenance of the Paneth cell granule exocy‐ tosis pathway. The authors observed no differences between WT and ATG16L1HM mice in terms of resistance to a challenge with *Listeria monocytogenes*. However, others have re‐ ported a reduction in intracellular bacteria targeted to autophagic vacuoles following in vitro infections of HeLa or Caco-2 cells with *S. typhimurium* in conjunction with siRNA to reduce the expression of *ATG16L1* [105, 107]. Additional work by Caldwell et al. re‐ vealed that 100 % of ileocolic resection specimens obtained from humans with the *ATG16L1* risk allele had abnormal Paneth cells that looked similar to those found in the ATG16L1HM mice [95].

Using siRNA to reduce *ATG16L1* expression in THP-1 macrophages revealed an increase in intracellular adherent and invasive *E. coli* (AIEC) as compared to normal THP-1 infected macrophages [106]. AIEC promote enhanced production of TNF-α and IL-6 from macrophag‐ es. Silencing of *ATG16L1* expression resulted in significant increases in both cytokines. The authors postulate that bacterial clearance is less efficient in individuals with the risk alleles for *ATG16L1.* When AIEC are present, this reduction in bacterial clearance likely leads to increased production of pro-inflammatory cytokines, which could tip the immune balance to an inflammatory state. Thus, it appears that the presence of bacterial provocateurs (e.g., AIEC) in the microbiota increases the risk of developing IBD in individuals possessing polymorphisms in genes such as *ATG16L* or *NOD2.*

## **2.2. The immune response and IBD**

required for the bactericidal activity of crypt secretions of the terminal ileum. The composition of the ileal microbiota of the NOD2-/- mice was significantly altered in comparison to their WT counterparts. Specifically, NOD2-/- mice had increased numbers of Firmicutes, specifically *Bacillus* species and *Bacteroides* species. Wild-type mice were able to clear an infection of *Helicobacter hepaticus,* an opportunistic mouse pathogen, within seven days while NOD2-/- mice remained colonized with *H. hepaticus* for at least 14 days post-infection [102]. Also of note, the presence of a gut microbiota was required for the expression of NOD2. GF mice possessed decreased levels of NOD2—a phenotype that could be reversed following monoassociation with either Gram-negative or Gram-positive organisms. Together, these results outline a

reciprocal regulatory relationship between NOD2 expression and the gut microbiota.

Another gene implicated in the pathogenesis of IBD that regulates cellular autophagy is *ATG16L1* [95, 103]. Autophagy is a process utilized by cells to recycle cellular compo‐ nents. This process may also be utilized by cells as a non-apoptotic method of program‐ med cell death [104]. *ATG16L1* is expressed by CD4+ and CD8+ T cells, CD19+ B cells, epithelial cells, and macrophages [105, 106]. Studies by Caldwell et al. utilizing mice en‐ gineered to express low or hypomorphic levels of *ATG16L1* revealed that this gene is in‐ deed an autophagy-related protein. Elevated levels of cytosolic proteins normally degraded by a rapamycin-induced degradation pathway were found in the cells of the *ATG16L1* hypomorphic (ATG16L1HM) mice [95]. Additionally, cells from ATG16L1HM mice possessed fewer autophagosomes following rapamycin treatment or nutrient deprivation. The small intestines and colons of ATG16L1HM mice had no morphological defects in crypt height or villus length but did possess abnormalities found in their Paneth cells. Specifically, lysozyme within Paneth cells was found to be either depleted/absent or dif‐ fuse, which is in contrast to the normal and orderly packaging of lysozyme within gran‐ ules. Decreased levels of lysozyme were also observed in the ileal mucus layer of ATG16L1HM mice. However, there were intact granules observed in the crypt lumen, in‐ dicating a potential role of *ATG16L1* in the maintenance of the Paneth cell granule exocy‐ tosis pathway. The authors observed no differences between WT and ATG16L1HM mice in terms of resistance to a challenge with *Listeria monocytogenes*. However, others have re‐ ported a reduction in intracellular bacteria targeted to autophagic vacuoles following in vitro infections of HeLa or Caco-2 cells with *S. typhimurium* in conjunction with siRNA to reduce the expression of *ATG16L1* [105, 107]. Additional work by Caldwell et al. re‐ vealed that 100 % of ileocolic resection specimens obtained from humans with the *ATG16L1* risk allele had abnormal Paneth cells that looked similar to those found in the

Using siRNA to reduce *ATG16L1* expression in THP-1 macrophages revealed an increase in intracellular adherent and invasive *E. coli* (AIEC) as compared to normal THP-1 infected macrophages [106]. AIEC promote enhanced production of TNF-α and IL-6 from macrophag‐ es. Silencing of *ATG16L1* expression resulted in significant increases in both cytokines. The authors postulate that bacterial clearance is less efficient in individuals with the risk alleles for

*2.1.2. ATG16L1*

86 Inflammatory Bowel Disease

ATG16L1HM mice [95].

The mucosal immune system is charged with the monumental task of balancing respon‐ siveness and tolerance to a tremendous number of environmental antigens, including those from both food and bacteria. Although the exact cause of IBD remains elusive, sig‐ nificant evidence supports the hypothesis that GIT inflammation is initiated and perpetu‐ ated by a dysregulated immune response directed against the gut microbiota resulting in deleterious responses in genetically susceptible individuals following an environmental trigger. The host with a genetic predisposition for IBD may possess defects in epithelial permeability and/or altered regulation (i.e., NOD2 deficiency) of commensal bacteria. Po‐ tential environmental triggers may include smoking, certain medications, or a gastroin‐ testinal illness that induces a break in homeostasis. Regardless of the specific genetic or environmental trigger, what ensues is an exaggerated, inappropriate mucosal immune re‐ sponse characterized by chronic activation of T cells and the production of cytokines and other inflammatory mediators.

The success of cytokine-targeted immunotherapies for a subset of IBD patients supports the idea that these chemical messengers of the immune system play an important role in disease pathogenesis. However, the nature of the immune phenotypes observed in CD and UC patients do differ [108]. The immune response of CD is typically associated with a T helper 1 (Th1) phenotype while UC is characterized by a T helper 2 (Th2) phenotype [108]. Isolated lamina propria (LP) CD4+ T cells from CD patients produce IFN-γ when stimulated via the CD2/CD28 pathway; in contrast, LP CD4+ T cells from UC patients se‐ creted mostly IL-5 [109]. Another cytokine with a strong link to CD is IL-12 [110]. Messen‐ ger RNA for IL-12p40 in LP mononuclear cells (LPMC) was found in 85 % of CD patients, a percentage significantly higher than that found in healthy and UC GIT tissue samples. Similarly, IL-12p35 mRNA was detectable in the LPMC of 92 % of CD patients; expres‐ sion was again significantly higher than for healthy and UC patients. Elevated levels of IL-12 were also detectable in the serum of CD patients [110], and this potent Th1-promot‐ ing cytokine is able to induce IFN-γ production from LP lymphocytes (LPL) isolated from CD patients [111]. Additionally, studies employing a murine model of TNBS-induced col‐ itis have revealed that administration of anti-IL-12 antibodies ameliorated disease severi‐ ty, presumably by decreasing IFN-γ secretion from LP CD4+ T cells [112].

In UC, production of the cytokines IL-5 and IL-13 appear to mediate the disease process. Using LPMC from IBD patients, those with UC had increased production of IL-13 and IL-5 upon *in vitro* restimulation as compared to LPMCs recovered from either CD patients or healthy individuals [113]. Levels of IFN-γ produced by LPMC from UC patients were similar to those produced by cells from healthy controls. The authors specifically identified that CD4+ CD161+ NK T cells were the primary source of IL-13 in UC patients, as these cells produced 30-fold more IL-13 than NK T cells from CD patients following *in vitro* restimulation.

antigens, while IgG1, IgG2, IgG3 were the predominant isotypes reacting with the bacterial antigens in serum samples from CD patients [126]. Conversely, other reports have described an increase in IgG1 antibodies in UC patients and an increased in IgG2 antibodies in CD patients [127-130]. Additional analyses have determined that IgG antibodies in CD patients are primarily directed towards bacterial cytoplasmic proteins rather than membrane associated proteins [126]. Despite the presence of many types of antibodies in both the mucosal secretions and serum of IBD patients, no direct evidence for their involvement in IBD immunopatho‐ genesis has been reported, indicating that they may simply be a marker for immune respon‐

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89

Although there are allelic differences in many people with IBD, genetics alone cannot completely account for the development of IBD nor the increase in the incidence of IBD worldwide. Studies of monozygotic twins best highlight this concept, as there are disease concordance rates of only 50 % for CD and only 20 % for UC [132, 133]. Of interest, a British study assessing discordant twins with CD found an association with mumps in‐ fection, smoking, and oral contraceptive usage with the development of CD [134]. Addi‐ tionally, the twin(s) with CD had suffered both a medical illness, more episodes of gastroenteritis, and spent more time with animals. Smoking is one confounding environ‐ mental factor that is of special interest, because it appears to have a protective effect on UC, but increases the risk of CD [132, 135-137]. Another factor that appears to be protec‐ tive for UC is an appendectomy; its effect on CD is not as evident [138-140]. The equiva‐ lent procedure performed in mice has been shown to ameliorate colitis in both chemically-induced and genetically-engineered murine colitis models [141, 142]. Modest associations between oral contraceptive use and IBD have been documented, while oth‐ ers have found breast-feeding to be protective against both UC and CD [143, 144]. A meta-analysis performed by investigators in New Zealand identified other potential envi‐ ronmental factors, including being an only child, using antibiotics prior to and during adolescence (four or more courses in year), and having a pet in the house during child‐ hood [145]. IBD also tends to occur in extended families as first and second degree rela‐ tives of IBD patients reported the occurrence of disease with a significantly higher frequency than the general population [146]. These finding strengthen the interconnec‐

tion between genetics, environmental factors, and the incidence of IBD.

Antibiotics are used in the treatment of IBD to reduce microbial load and dampen in‐ flammatory immune responses. However, their use prior to disease diagnosis is now be‐ ing identified as a risk factor for developing IBD. A Danish study of IBD patients revealed that antibiotic users were 1.84 times more likely to develop IBD, which correlat‐ ed to a 12 % increase in disease risk for each course of antibiotics taken [147]. Further analysis revealed that antibiotic users were 3.41 times more likely to develop CD than UC, which correlated to an increased risk of 18 % per course of antibiotics used. Specifi‐

siveness and/or dysregulation [131].

*2.3.1. Antibiotic usage*

**2.3. Environmental factors affecting IBD**

More recent work has implicated the T helper 17 (Th17) lineage of CD4+ T cells in the chronic inflammation observed in IBD [114, 115]. The development of Th17 T cells is dependent upon the presence of both IL-6 and TGF-β [115]. Colonic biopsies from UC and CD patients expressed higher levels of IL-17A mRNA as compared to those from healthy controls [115-117]. Immu‐ nohistochemical analysis of these tissues identified increased numbers of IL-17A+ cells in both the LP and the epithelium of UC and CD patients as compared to controls [116]. Additional work also noted the presence of IL-17+ cells in patients with active IBD [114]. However, the IL-17+ cells were found predominantly in the LP of UC patients but in the submucosa and muscularis propria of CD patients [114]. These different locations of IL-17+ cells complements prior observations that CD lesions often present as transmural while those in UC are superfi‐ cial. Also of note, a comparison of biopsies from patients with active versus inactive disease revealed that IL-17+ cells were only increased in numbers during active disease. The authors also determined that both T cells and monocytes/macrophages are a source of IL-17, and that IBD patients have elevated levels of IL-17 in their sera as compared to undetectable levels of this cytokine in the sera of healthy individuals.

The maintenance of Th17 T cells requires the presence of IL-23 [115]. Of interest, a gene variant significantly associated with CD encodes for the IL-23R, which, along with IL-12Rβ1, com‐ prises the IL-23 receptor complex [118, 119]. This receptor complex interacts with IL-23 (a heterodimer of IL-12p40 and IL-23p19) to direct the production of a TH17 immune response [120-122]. Anti-IL-23 antibodies have been shown to both prevent and ameliorate established disease in a T-cell transfer model of murine colitis [123]. Studies of human tissues demonstrate that IL-23R expression is upregulated not only on IL-17 producing CD4+ T cells, but also on IFN-γ<sup>+</sup> T cells in both UC and CD patients [115]. Stimulation of LP CD4+T cells from UC patients with IL-23 significantly increased IL-17 production; in contrast, IL-23 stimulation of LP CD4+ T cells from CD patients resulted in enhanced IFN-γ secretion [115]. In addition to promoting the production of TH17 cells, IL-23 also inhibits the production of Foxp3+ Treg cells [124] and suppresses production of IL-10, a key regulatory cytokine [125].

Another important hallmark of the immune response observed in IBD is the production of IgG antibodies against the normal commensal microbiota [126]. Increased intestinal permeability, be it through genetic predisposition or environmental trigger, promotes enhanced immunor‐ eactivity to bacterial antigens. The mucosal immunoglobulin profile of healthy adults consists predominantly of IgA with only a small amount of IgG. In contrast, patients with active IBD presented with significantly higher amounts of IgG in their mucosal secretions with no difference in amounts of IgA as compared to controls. The IgG antibodies present in IBD patients were identified as binding to non-pathogenic bacterial commensals, including *E. coli, B. fragilis, C. perfringens, Klebsiella aerogenes, and Enterobacter faecalis.* Patients with CD had significantly higher titers than UC patients, while antibody titers to these commensal bacteria were at or below the limit of detection in serum samples from healthy controls. Notable differences were also observed in the isotypes of IgG present in UC versus CD patients. Specifically, UC patients produced predominantly IgG1 and IgG3 antibodies to bacterial antigens, while IgG1, IgG2, IgG3 were the predominant isotypes reacting with the bacterial antigens in serum samples from CD patients [126]. Conversely, other reports have described an increase in IgG1 antibodies in UC patients and an increased in IgG2 antibodies in CD patients [127-130]. Additional analyses have determined that IgG antibodies in CD patients are primarily directed towards bacterial cytoplasmic proteins rather than membrane associated proteins [126]. Despite the presence of many types of antibodies in both the mucosal secretions and serum of IBD patients, no direct evidence for their involvement in IBD immunopatho‐ genesis has been reported, indicating that they may simply be a marker for immune respon‐ siveness and/or dysregulation [131].

## **2.3. Environmental factors affecting IBD**

NK T cells were the primary source of IL-13 in UC patients, as these cells produced 30-fold

inflammation observed in IBD [114, 115]. The development of Th17 T cells is dependent upon the presence of both IL-6 and TGF-β [115]. Colonic biopsies from UC and CD patients expressed higher levels of IL-17A mRNA as compared to those from healthy controls [115-117]. Immu‐ nohistochemical analysis of these tissues identified increased numbers of IL-17A+ cells in both the LP and the epithelium of UC and CD patients as compared to controls [116]. Additional

cells were found predominantly in the LP of UC patients but in the submucosa and

prior observations that CD lesions often present as transmural while those in UC are superfi‐ cial. Also of note, a comparison of biopsies from patients with active versus inactive disease revealed that IL-17+ cells were only increased in numbers during active disease. The authors also determined that both T cells and monocytes/macrophages are a source of IL-17, and that IBD patients have elevated levels of IL-17 in their sera as compared to undetectable levels of

The maintenance of Th17 T cells requires the presence of IL-23 [115]. Of interest, a gene variant significantly associated with CD encodes for the IL-23R, which, along with IL-12Rβ1, com‐ prises the IL-23 receptor complex [118, 119]. This receptor complex interacts with IL-23 (a heterodimer of IL-12p40 and IL-23p19) to direct the production of a TH17 immune response [120-122]. Anti-IL-23 antibodies have been shown to both prevent and ameliorate established disease in a T-cell transfer model of murine colitis [123]. Studies of human tissues demonstrate

 T cells in both UC and CD patients [115]. Stimulation of LP CD4+T cells from UC patients with IL-23 significantly increased IL-17 production; in contrast, IL-23 stimulation of LP CD4+ T cells from CD patients resulted in enhanced IFN-γ secretion [115]. In addition to promoting the production of TH17 cells, IL-23 also inhibits the production of Foxp3+ Treg cells [124] and

Another important hallmark of the immune response observed in IBD is the production of IgG antibodies against the normal commensal microbiota [126]. Increased intestinal permeability, be it through genetic predisposition or environmental trigger, promotes enhanced immunor‐ eactivity to bacterial antigens. The mucosal immunoglobulin profile of healthy adults consists predominantly of IgA with only a small amount of IgG. In contrast, patients with active IBD presented with significantly higher amounts of IgG in their mucosal secretions with no difference in amounts of IgA as compared to controls. The IgG antibodies present in IBD patients were identified as binding to non-pathogenic bacterial commensals, including *E. coli, B. fragilis, C. perfringens, Klebsiella aerogenes, and Enterobacter faecalis.* Patients with CD had significantly higher titers than UC patients, while antibody titers to these commensal bacteria were at or below the limit of detection in serum samples from healthy controls. Notable differences were also observed in the isotypes of IgG present in UC versus CD patients. Specifically, UC patients produced predominantly IgG1 and IgG3 antibodies to bacterial

T cells in the chronic

cells complements

T cells, but also on

cells in patients with active IBD [114]. However, the

more IL-13 than NK T cells from CD patients following *in vitro* restimulation.

More recent work has implicated the T helper 17 (Th17) lineage of CD4+

muscularis propria of CD patients [114]. These different locations of IL-17+

that IL-23R expression is upregulated not only on IL-17 producing CD4+

suppresses production of IL-10, a key regulatory cytokine [125].

work also noted the presence of IL-17+

this cytokine in the sera of healthy individuals.

IL-17+

88 Inflammatory Bowel Disease

IFN-γ<sup>+</sup>

Although there are allelic differences in many people with IBD, genetics alone cannot completely account for the development of IBD nor the increase in the incidence of IBD worldwide. Studies of monozygotic twins best highlight this concept, as there are disease concordance rates of only 50 % for CD and only 20 % for UC [132, 133]. Of interest, a British study assessing discordant twins with CD found an association with mumps in‐ fection, smoking, and oral contraceptive usage with the development of CD [134]. Addi‐ tionally, the twin(s) with CD had suffered both a medical illness, more episodes of gastroenteritis, and spent more time with animals. Smoking is one confounding environ‐ mental factor that is of special interest, because it appears to have a protective effect on UC, but increases the risk of CD [132, 135-137]. Another factor that appears to be protec‐ tive for UC is an appendectomy; its effect on CD is not as evident [138-140]. The equiva‐ lent procedure performed in mice has been shown to ameliorate colitis in both chemically-induced and genetically-engineered murine colitis models [141, 142]. Modest associations between oral contraceptive use and IBD have been documented, while oth‐ ers have found breast-feeding to be protective against both UC and CD [143, 144]. A meta-analysis performed by investigators in New Zealand identified other potential envi‐ ronmental factors, including being an only child, using antibiotics prior to and during adolescence (four or more courses in year), and having a pet in the house during child‐ hood [145]. IBD also tends to occur in extended families as first and second degree rela‐ tives of IBD patients reported the occurrence of disease with a significantly higher frequency than the general population [146]. These finding strengthen the interconnec‐ tion between genetics, environmental factors, and the incidence of IBD.

#### *2.3.1. Antibiotic usage*

Antibiotics are used in the treatment of IBD to reduce microbial load and dampen in‐ flammatory immune responses. However, their use prior to disease diagnosis is now be‐ ing identified as a risk factor for developing IBD. A Danish study of IBD patients revealed that antibiotic users were 1.84 times more likely to develop IBD, which correlat‐ ed to a 12 % increase in disease risk for each course of antibiotics taken [147]. Further analysis revealed that antibiotic users were 3.41 times more likely to develop CD than UC, which correlated to an increased risk of 18 % per course of antibiotics used. Specifi‐ cally, usage of penicillin V and extended spectrum penicillins were associated with the greatest disease risks. A Swedish study focused on the use of antibiotics from birth to age 5, a time when the microbiota and immune response are still developing and/or ma‐ turing, as a risk factor for IBD. They reported an association between a diagnosis of pneumonia, subsequent antibiotic treatment and the onset of both pediatric and adult CD [148]. Another study by Card et al. revealed a statistically significant association be‐ tween antibiotic usage 2 to 5 years prior to diagnosis of CD in patients from the United Kingdom [149]. This finding was confirmed in a Canadian study, which reported that the more antibiotics taken within 2 to 5 years of diagnosis, the greater the risk of devel‐ oping IBD [150]. That same study found that disease risk was weakly associated with penicillin use and greatly associated with metronidazole use, which was prescribed pri‐ marily for "non-infectious gastroenteritis." This association with antibiotic usage may be explained by the failure of the gut microbiota to reestablish its normal community struc‐ ture and function following a course of antibiotics [151]. This alteration in the microbio‐ ta or dysbiosis may be a predisposing factor to the onset of IBD in a susceptible individual.

gene and the presence of MAP [183]. In this study, 68 % of CD patients were positive for MAP DNA based on PCR analysis as compared to 21 % of the healthy individuals. Fifty-one percent of the CD patients were carriers of NOD2 polymorphisms as compared to 21 % of healthy individuals, and 74 % of the CD patients possessing one of the mutat‐ ed NOD2 alleles were positive for MAP DNA. Even with of all of the data collected, the debate continues as to whether or not MAP plays an important role in the pathogenesis

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Another organism implicated in IBD pathogenesis is adherent-invasive *Escherichia coli* (AIEC). First described in 1998, AIEC isolates were recovered from the ileal mucosa of CD patients [186]. These organisms are found in higher numbers in CD patients (22 %) as compared to healthy controls (6 %) [187]. These isolates were able to adhere to Caco-2 cells and did not posses any of the virulence genes associated with known *E. coli* pathotypes (e.g., ETEC, EHEC) [186]. A characterization of expressed adhesions in AIEC strains revealed the *Pap* and *Sfa* adhesion or colonization factors, both of which are found in uropathogenic *E. coli* strains; however many of the AIEC strains did not possess any of the known adhesions associated with pathogenic *E. coli* (e.g., intimin). Some of the cytotoxic strains were found to express the *hly* operon, and it was subsequently demonstrated that these organisms express a type 1 pili similar in sequence to an *E. coli* strain associated with avian colisepticemia and meningitis [188]. Type 1 pili are involved in both the adherence and internalization of *E. coli* into host cells. They specifically bind to the CEACAM6 receptor on ileal enterocytes, which is expressed at significantly higher levels in the inflamed small intestinal epithelium of CD patients as

Further research involving the prototypic AIEC strain, LF82, has shown that this pathogenic group of *E. coli* invades epithelial cells via engagement of actin microtubules and microfila‐ ments and replicates intracellularly even without possessing any of the known invasive determinants found in other invasive *E. coli* strains [190]. AIEC strains are also able to survive and replicate within macrophages without causing apoptosis; they also induce production of the pro-inflammatory cytokine TNF-α [191]. LF82 is also able to induce *in vitro* aggregation of peripheral blood mononuclear cells (PBMCs), an observation reminiscent of the granulomas

In addition to being detected with increased frequency in human IBD patients, AIEC strains have also been isolated from Boxer dogs with granulomatous colitis. These *E. coli* isolates were found to have the same adherent-invasive phenotype as human AIEC strains. The Boxer dog isolates were also of the same phylotype (B2 and D) and possessed similar virulence gene profiles as LF82. In contrast to the human AIEC strains, however, the canine AIEC strains were only isolated from diseased dogs and not healthy controls. Simpson and colleagues also demonstrated that remission of colitis in Boxer dogs could be achieved by treatment with

enrofloxacin, the use of which resulted in the eradication of AIEC *E. coli* [193].

of CD [184, 185].

*2.3.3. Adherent-invasive Escherichia coli*

compared to healthy controls [189].

observed in the colonic tissue of CD patients [192].

#### *2.3.2. MAP*

Another potential environmental risk factor for the development of IBD is a chronic pathogenic infection in the GIT. To date, no one particular organism has been found to be the causative agent of IBD. Although many microbial pathogens have been implicated as causative, only two have been significantly investigated. The first organism hypothe‐ sized to be associated with IBD was *Mycobacterium avium* subsp. *paratuberculosis,* which causes Johne's disease in cattle [152, 153]. The granulomatous inflammation observed in CD patients led many early researchers to investigate various types of *Mycobacterial* spp. in CD pathogenesis but confirmation was never achieved (reviewed in [154]). The first report of *M. paratuberculosis*-like organisms isolated from patients with CD in was pub‐ lished 1984. Now known as *M. avium* subsp. *paratuberculosis* (MAP), there is still no con‐ sensus as to whether or not it plays a role in the onset of IBD in a subset of genetically susceptible patients [155]. Using PCR to amplify the DNA insertion element IS*900*, which is specific to MAP, in biopsy sections, Sanderson et al. reported that 65 % of adult CD patients examined were PCR positive [156]. Dell'Isola et al. reported that 72 % of pedia‐ tric CD patients examined were PCR positive for the presence of MAP [156, 157]. While many studies have reported the presence of MAP DNA in CD patients, just as many have found MAP DNA in samples from healthy individuals and UC patients [156-162]. Moreover, multiple studies have failed to detect MAP-specific DNA in any CD patient sample examined [163-169]. The ability to detect MAP in some tissue samples and not others may be due, in part, to the organism's fastidiousness, slow growth, and/or the presence of PCR inhibitors in fecal/tissue samples that inhibit the detection of IS900. Se‐ rological evidence also fails to provide definitive answers for the involvement of MAP in the pathogenesis of IBD. Some studies assessing the presence of serum antibody titers against MAP-derived antigens find an association with IBD [170-176] while others do not [177-182]. More recent work has identified a possible link between variants in the *Nod2* gene and the presence of MAP [183]. In this study, 68 % of CD patients were positive for MAP DNA based on PCR analysis as compared to 21 % of the healthy individuals. Fifty-one percent of the CD patients were carriers of NOD2 polymorphisms as compared to 21 % of healthy individuals, and 74 % of the CD patients possessing one of the mutat‐ ed NOD2 alleles were positive for MAP DNA. Even with of all of the data collected, the debate continues as to whether or not MAP plays an important role in the pathogenesis of CD [184, 185].

## *2.3.3. Adherent-invasive Escherichia coli*

cally, usage of penicillin V and extended spectrum penicillins were associated with the greatest disease risks. A Swedish study focused on the use of antibiotics from birth to age 5, a time when the microbiota and immune response are still developing and/or ma‐ turing, as a risk factor for IBD. They reported an association between a diagnosis of pneumonia, subsequent antibiotic treatment and the onset of both pediatric and adult CD [148]. Another study by Card et al. revealed a statistically significant association be‐ tween antibiotic usage 2 to 5 years prior to diagnosis of CD in patients from the United Kingdom [149]. This finding was confirmed in a Canadian study, which reported that the more antibiotics taken within 2 to 5 years of diagnosis, the greater the risk of devel‐ oping IBD [150]. That same study found that disease risk was weakly associated with penicillin use and greatly associated with metronidazole use, which was prescribed pri‐ marily for "non-infectious gastroenteritis." This association with antibiotic usage may be explained by the failure of the gut microbiota to reestablish its normal community struc‐ ture and function following a course of antibiotics [151]. This alteration in the microbio‐ ta or dysbiosis may be a predisposing factor to the onset of IBD in a susceptible

Another potential environmental risk factor for the development of IBD is a chronic pathogenic infection in the GIT. To date, no one particular organism has been found to be the causative agent of IBD. Although many microbial pathogens have been implicated as causative, only two have been significantly investigated. The first organism hypothe‐ sized to be associated with IBD was *Mycobacterium avium* subsp. *paratuberculosis,* which causes Johne's disease in cattle [152, 153]. The granulomatous inflammation observed in CD patients led many early researchers to investigate various types of *Mycobacterial* spp. in CD pathogenesis but confirmation was never achieved (reviewed in [154]). The first report of *M. paratuberculosis*-like organisms isolated from patients with CD in was pub‐ lished 1984. Now known as *M. avium* subsp. *paratuberculosis* (MAP), there is still no con‐ sensus as to whether or not it plays a role in the onset of IBD in a subset of genetically susceptible patients [155]. Using PCR to amplify the DNA insertion element IS*900*, which is specific to MAP, in biopsy sections, Sanderson et al. reported that 65 % of adult CD patients examined were PCR positive [156]. Dell'Isola et al. reported that 72 % of pedia‐ tric CD patients examined were PCR positive for the presence of MAP [156, 157]. While many studies have reported the presence of MAP DNA in CD patients, just as many have found MAP DNA in samples from healthy individuals and UC patients [156-162]. Moreover, multiple studies have failed to detect MAP-specific DNA in any CD patient sample examined [163-169]. The ability to detect MAP in some tissue samples and not others may be due, in part, to the organism's fastidiousness, slow growth, and/or the presence of PCR inhibitors in fecal/tissue samples that inhibit the detection of IS900. Se‐ rological evidence also fails to provide definitive answers for the involvement of MAP in the pathogenesis of IBD. Some studies assessing the presence of serum antibody titers against MAP-derived antigens find an association with IBD [170-176] while others do not [177-182]. More recent work has identified a possible link between variants in the *Nod2*

individual.

90 Inflammatory Bowel Disease

*2.3.2. MAP*

Another organism implicated in IBD pathogenesis is adherent-invasive *Escherichia coli* (AIEC). First described in 1998, AIEC isolates were recovered from the ileal mucosa of CD patients [186]. These organisms are found in higher numbers in CD patients (22 %) as compared to healthy controls (6 %) [187]. These isolates were able to adhere to Caco-2 cells and did not posses any of the virulence genes associated with known *E. coli* pathotypes (e.g., ETEC, EHEC) [186]. A characterization of expressed adhesions in AIEC strains revealed the *Pap* and *Sfa* adhesion or colonization factors, both of which are found in uropathogenic *E. coli* strains; however many of the AIEC strains did not possess any of the known adhesions associated with pathogenic *E. coli* (e.g., intimin). Some of the cytotoxic strains were found to express the *hly* operon, and it was subsequently demonstrated that these organisms express a type 1 pili similar in sequence to an *E. coli* strain associated with avian colisepticemia and meningitis [188]. Type 1 pili are involved in both the adherence and internalization of *E. coli* into host cells. They specifically bind to the CEACAM6 receptor on ileal enterocytes, which is expressed at significantly higher levels in the inflamed small intestinal epithelium of CD patients as compared to healthy controls [189].

Further research involving the prototypic AIEC strain, LF82, has shown that this pathogenic group of *E. coli* invades epithelial cells via engagement of actin microtubules and microfila‐ ments and replicates intracellularly even without possessing any of the known invasive determinants found in other invasive *E. coli* strains [190]. AIEC strains are also able to survive and replicate within macrophages without causing apoptosis; they also induce production of the pro-inflammatory cytokine TNF-α [191]. LF82 is also able to induce *in vitro* aggregation of peripheral blood mononuclear cells (PBMCs), an observation reminiscent of the granulomas observed in the colonic tissue of CD patients [192].

In addition to being detected with increased frequency in human IBD patients, AIEC strains have also been isolated from Boxer dogs with granulomatous colitis. These *E. coli* isolates were found to have the same adherent-invasive phenotype as human AIEC strains. The Boxer dog isolates were also of the same phylotype (B2 and D) and possessed similar virulence gene profiles as LF82. In contrast to the human AIEC strains, however, the canine AIEC strains were only isolated from diseased dogs and not healthy controls. Simpson and colleagues also demonstrated that remission of colitis in Boxer dogs could be achieved by treatment with enrofloxacin, the use of which resulted in the eradication of AIEC *E. coli* [193].

## **2.4. The microbiota and IBD**

Although no single organism has been implicated in the induction of IBD, a preponderance of studies indicates that the GI microbial community of IBD patients is different from that of healthy individuals. This imbalance is known as "dysbiosis". Characteristics of a dysbiotic community in IBD include a reduction in members of the *C. leptum* and *C. cocciodes* clusters (members of the Firmicutes phyla and major butyrate producers) and an increase in *Entero‐ bacteriaceae* and members of the Bacteroidetes, thereby leading to a reduction in the microbial diversity of the gut. Another notable difference is the greater concentration of mucosallyassociated bacteria in IBD patients as compared to healthy individuals [194-197]. Using fluorescence *in situ* hybridization (FISH), patients with CD were found to have predominantly *Enterobacteriaceae, γ-Proteobacteria,* or *Bacteroides/Prevotella* adherent to their mucosa and present in their submucosa [195]. Another FISH-based analysis revealed that *Bacteroides* spp. were the dominant gut bacteria, representing up to 80 % of the total mucosally-adherent bacterial population in some samples. Of interest was the authors' additional finding that treatment of IBD patients with mesalamine (an anti-inflammatory therapeutic drug) signifi‐ cantly reduced the numbers of mucosally-adherent bacteria [198].

involvement of the microbiota in the development of IBD in a genetically susceptible host, as

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One specific member of the *C. leptum* cluster, *Faecalibacterium prausnitzii*, has recently been the subject of many published studies. This organism is a major butyrate producer that also possesses some anti-inflammatory properties [59, 207]. A study by Sokol et al. described a reduction of both *C. leptum* and *C. coccoides* clusters in the stool of patients with active IBD along with a skewed Firmicutes/Bacteroidetes ratio [203]. The authors specifically identified a reduction in *F. prausnitzii* in IBD patients, confirming results ob‐ tained using biopsy samples from twins analyzed via qPCR [205]. In other work, dena‐ turing gradient gel electrophoresis (DGGE) analysis of biopsy samples also demonstrated a significant decrease in *Faecalibacterium* spp. along with increased levels of *E. coli* and *Clostridium* spp. in CD patients as compared to healthy individuals [208]. Using fecal cyl‐ inders and 11 different FISH probes to analyze sections GIT tissue specimens, Swidsinski and colleagues found the presence of *F. prausnitzii* to be significantly reduced in CD pa‐ tients [209]. Of special note, analysis of samples from IBD patients given high-dose corti‐

sol or infliximab to reduce inflammation revealed a dramatic increase (>14 x109

flammation plays a major role in shaping the intestinal microbiota.

[212], induced no detectable changes in microbial load.

levels of *F. prausnitzii* within days of initiating treatment. This increase was short-lived, however, as levels of *F. prausnitzii* decreased when the cortisol dose was reduced or the time between infliximab infusions was increased. Together, these data indicate that in‐

Work in murine models of colitis also demonstrates that inflammation in the gut, be it chemically or bacterially induced, causes an increase in *Enterobacteriaceae* [210]. Colonization of mice with *Citrobacter rodentium* resulted in a reduction of the total number of bacteria in the colon at 7 and 14 days post-infection; this decrease coincided with the highest levels of both *C. rodentium* and intestinal inflammation [211]. Further analysis specifically determined that members of the Cytophaga-Flavobacter-Bacteroides phylum were decreased. Infection with *C. rodentium* infection is self-limiting, and the total bacterial levels returned to normal following pathogen clearance. These changes in the bacterial load were not simply associated with colonization of a new organism to the microbial community, such as *Campylobacter jejuni*, an organism that does not cause intestinal inflammation in immunocompetent CONVR mice

The induction of GIT inflammation in mice via administration of dextran sulfate sodium (DSS) in the drinking water for seven days was associated with increased numbers of aerobic bacteria, specifically *Entercococcus faecalis*[210]. Bacteroidetes were eliminated from the community and the total number of bacteria was also decreased. A dramatic increase in the numbers of a nonpathogenic *E. coli* following DSS treatment was also observed. The authors subsequently colonized IL-10-/- mice, which spontaneously develop colitis [213], with this non-pathogenic *E. coli*. Upon the development of GIT inflammation, the *E. coli* was able to proliferate to the same level observed in DSS treated mice, but displacing the Firmicutes phylum instead of the Bacteroidetes. These results indicate that regardless of the source, GIT inflammation creates a niche that favors the colonization and expansion of *Enterobacteriaceae.* Collectively, these data suggest that the induction of inflammation following the colonization of the GIT by a bacterial

) in the

only one of the twins developed disease.

Sample origin is an important factor to consider when interpreting data for microbial analyses. Although stool samples are easy to obtain, it may not accurately reflect the microbial com‐ munity in the cecum and proximal colon of patients [14]. In a comparative study of the microbial populations detected in cecal versus fecal samples, more anaerobes and *Bifidobacte‐ rium* spp. were detected in fecal samples using culture-based methods [199]. Molecular probe hybridization revealed that facultative anaerobes represented by *Lactobacillus, Enterococcus,* and *E. coli* were higher in numbers in cecal contents, yet the number of strict anaerobes represented by the *Bacteroides, C. leptum,* and *C. coccoides* groups were significantly lower in the cecal contents. Differences in microbial composition have also been observed when comparing colonic biopsies versus feces [200]. Analysis of feces from Japanese IBD patients showed that *Faecalibacterium* spp. were significantly decreased in CD patients, while *Bacter‐ oides* spp. were significantly increased only in patients with active IBD [201]. Based on the Shannon diversity index, this study demonstrated that microbial diversity was significantly reduced in CD patients both during active disease and remission as compared to healthy individuals.

Several studies have noted a specific decrease in the numbers of *C. leptum* and *C. coccoides* clusters present in colonic contents or feces in IBD patients [18, 197, 202-206]. A reduction in the *C. coccoides* group in UC patients and *C. leptum* in CD patients has been shown using FISH probes on patient feces [202]. Another study employing a combination of PCR and FISH analysis of fecal samples found a reduced presence of both *C. leptum* and *C. coccoides* clusters in IBD patients [197]. Additional analyses revealed significant decreases in the concentrations of the SCFAs (e.g., butyric and propionic acid) in the feces of IBD patients [197]. In other work, high throughput sequencing demonstrated reduced numbers of *Faecalibacterium, Ruminococ‐ caceae*, *Alistipes, Collinsella,* and *Roseburia* and increased numbers of *Enterobacteriaceae* in a twin with ileal CD as compared to the healthy twin [204]. This latter study again emphasizes the involvement of the microbiota in the development of IBD in a genetically susceptible host, as only one of the twins developed disease.

**2.4. The microbiota and IBD**

92 Inflammatory Bowel Disease

individuals.

Although no single organism has been implicated in the induction of IBD, a preponderance of studies indicates that the GI microbial community of IBD patients is different from that of healthy individuals. This imbalance is known as "dysbiosis". Characteristics of a dysbiotic community in IBD include a reduction in members of the *C. leptum* and *C. cocciodes* clusters (members of the Firmicutes phyla and major butyrate producers) and an increase in *Entero‐ bacteriaceae* and members of the Bacteroidetes, thereby leading to a reduction in the microbial diversity of the gut. Another notable difference is the greater concentration of mucosallyassociated bacteria in IBD patients as compared to healthy individuals [194-197]. Using fluorescence *in situ* hybridization (FISH), patients with CD were found to have predominantly *Enterobacteriaceae, γ-Proteobacteria,* or *Bacteroides/Prevotella* adherent to their mucosa and present in their submucosa [195]. Another FISH-based analysis revealed that *Bacteroides* spp. were the dominant gut bacteria, representing up to 80 % of the total mucosally-adherent bacterial population in some samples. Of interest was the authors' additional finding that treatment of IBD patients with mesalamine (an anti-inflammatory therapeutic drug) signifi‐

Sample origin is an important factor to consider when interpreting data for microbial analyses. Although stool samples are easy to obtain, it may not accurately reflect the microbial com‐ munity in the cecum and proximal colon of patients [14]. In a comparative study of the microbial populations detected in cecal versus fecal samples, more anaerobes and *Bifidobacte‐ rium* spp. were detected in fecal samples using culture-based methods [199]. Molecular probe hybridization revealed that facultative anaerobes represented by *Lactobacillus, Enterococcus,* and *E. coli* were higher in numbers in cecal contents, yet the number of strict anaerobes represented by the *Bacteroides, C. leptum,* and *C. coccoides* groups were significantly lower in the cecal contents. Differences in microbial composition have also been observed when comparing colonic biopsies versus feces [200]. Analysis of feces from Japanese IBD patients showed that *Faecalibacterium* spp. were significantly decreased in CD patients, while *Bacter‐ oides* spp. were significantly increased only in patients with active IBD [201]. Based on the Shannon diversity index, this study demonstrated that microbial diversity was significantly reduced in CD patients both during active disease and remission as compared to healthy

Several studies have noted a specific decrease in the numbers of *C. leptum* and *C. coccoides* clusters present in colonic contents or feces in IBD patients [18, 197, 202-206]. A reduction in the *C. coccoides* group in UC patients and *C. leptum* in CD patients has been shown using FISH probes on patient feces [202]. Another study employing a combination of PCR and FISH analysis of fecal samples found a reduced presence of both *C. leptum* and *C. coccoides* clusters in IBD patients [197]. Additional analyses revealed significant decreases in the concentrations of the SCFAs (e.g., butyric and propionic acid) in the feces of IBD patients [197]. In other work, high throughput sequencing demonstrated reduced numbers of *Faecalibacterium, Ruminococ‐ caceae*, *Alistipes, Collinsella,* and *Roseburia* and increased numbers of *Enterobacteriaceae* in a twin with ileal CD as compared to the healthy twin [204]. This latter study again emphasizes the

cantly reduced the numbers of mucosally-adherent bacteria [198].

One specific member of the *C. leptum* cluster, *Faecalibacterium prausnitzii*, has recently been the subject of many published studies. This organism is a major butyrate producer that also possesses some anti-inflammatory properties [59, 207]. A study by Sokol et al. described a reduction of both *C. leptum* and *C. coccoides* clusters in the stool of patients with active IBD along with a skewed Firmicutes/Bacteroidetes ratio [203]. The authors specifically identified a reduction in *F. prausnitzii* in IBD patients, confirming results ob‐ tained using biopsy samples from twins analyzed via qPCR [205]. In other work, dena‐ turing gradient gel electrophoresis (DGGE) analysis of biopsy samples also demonstrated a significant decrease in *Faecalibacterium* spp. along with increased levels of *E. coli* and *Clostridium* spp. in CD patients as compared to healthy individuals [208]. Using fecal cyl‐ inders and 11 different FISH probes to analyze sections GIT tissue specimens, Swidsinski and colleagues found the presence of *F. prausnitzii* to be significantly reduced in CD pa‐ tients [209]. Of special note, analysis of samples from IBD patients given high-dose corti‐ sol or infliximab to reduce inflammation revealed a dramatic increase (>14 x109 ) in the levels of *F. prausnitzii* within days of initiating treatment. This increase was short-lived, however, as levels of *F. prausnitzii* decreased when the cortisol dose was reduced or the time between infliximab infusions was increased. Together, these data indicate that in‐ flammation plays a major role in shaping the intestinal microbiota.

Work in murine models of colitis also demonstrates that inflammation in the gut, be it chemically or bacterially induced, causes an increase in *Enterobacteriaceae* [210]. Colonization of mice with *Citrobacter rodentium* resulted in a reduction of the total number of bacteria in the colon at 7 and 14 days post-infection; this decrease coincided with the highest levels of both *C. rodentium* and intestinal inflammation [211]. Further analysis specifically determined that members of the Cytophaga-Flavobacter-Bacteroides phylum were decreased. Infection with *C. rodentium* infection is self-limiting, and the total bacterial levels returned to normal following pathogen clearance. These changes in the bacterial load were not simply associated with colonization of a new organism to the microbial community, such as *Campylobacter jejuni*, an organism that does not cause intestinal inflammation in immunocompetent CONVR mice [212], induced no detectable changes in microbial load.

The induction of GIT inflammation in mice via administration of dextran sulfate sodium (DSS) in the drinking water for seven days was associated with increased numbers of aerobic bacteria, specifically *Entercococcus faecalis*[210]. Bacteroidetes were eliminated from the community and the total number of bacteria was also decreased. A dramatic increase in the numbers of a nonpathogenic *E. coli* following DSS treatment was also observed. The authors subsequently colonized IL-10-/- mice, which spontaneously develop colitis [213], with this non-pathogenic *E. coli*. Upon the development of GIT inflammation, the *E. coli* was able to proliferate to the same level observed in DSS treated mice, but displacing the Firmicutes phylum instead of the Bacteroidetes. These results indicate that regardless of the source, GIT inflammation creates a niche that favors the colonization and expansion of *Enterobacteriaceae.* Collectively, these data suggest that the induction of inflammation following the colonization of the GIT by a bacterial provocateur along with perturbations of the microbiota together have a deleterious impact on the microbiota and mucosal homeostasis.

Mutations in innate immune receptors in German shepherd dogs (TLR5, NOD2) have been linked to IBD susceptibility which in the presence of an inappropriate enteric microbiota may lead to upregulated pro-inflammatory cytokine production (e.g., IL-17, IL-22, TNF-α) and reduced bacterial clearance, thereby promoting chronic intestinal inflammation [218, 219]. Commensal bacterial antigens are likely to be important in disease pathogenesis because it has been observed that boxer dogs with granulomatous colitis (GC) show clinical remission following the eradication of mucosally associated AIEC that share a novel adherent and invasive pathotype which bears phylogenetic similarity with AIEC strains recovered from patients with ileal Crohn's disease [193, 220, 221]. Moreover, genome-wide analysis in affected boxer dogs has identified disease-associated single nucleotide polymorphisms (SNPs) in a gene (*NCF2*) involved with killing intracellular bacteria [222]. Still others have shown that

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cells are significantly decreased in the intestines of dogs with IBD suggesting that

chronic mucosal inflammation may involve an imbalance in the intestinal dendritic cell population leading to aberrant immune activation [223]. Molecular analysis of the intestinal microbiome in different breeds of dogs with IBD have consistently shown that diseased tissues are enriched with members of the families *Enterobacteriaceae* and *Clostridiaceae*[224, 225]. These bacteria are believed to contribute to the pathogenesis of GIT inflammatory disease in dogs as in humans [187, 226]. A recent trial using high throughput 16S rDNA sequencing methods (ie, 454 pyrosequencing) on intestinal biopsies of IBD dogs revealed a dysbiosis in the mucosallyadherent microbiota with an increase in sequences belonging to Proteobacteria and a decrease in Bacteroidetes, Fusobacteria, and the Clostridiales [227]. Taken together, these studies suggest that chronic intestinal inflammation of canine IBD may be due to overly aggressive adaptive immune responses to enteric bacteria (or fungi) [225] in hosts with genetic defects that fail to properly regulate microbial killing, mucosal barrier function, or immune responses. As in human IBD, environmental factors (diet, microbiota imbalances) likely govern the onset

of inflammation or reactivation and modulate genetic susceptibility to disease.

The clinical manifestations of IBD are diverse and are influenced by the organ(s) involved, presence of active versus inactive disease, and physiologic complications seen with enteric plasma protein loss and/or micronutrient (cobalamin) deficiency [215, 216, 228, 229]. Canine IBD is a disease that predominantly affects middle-aged animals. Vomiting and diarrhea are most commonly observed and are often accompanied by decreased appetite and weight loss. Gastric and duodenal inflammation is associated with vomiting and small bowel diarrhea while colonic involvement causes large bowel diarrhea with blood, mucus, and straining. The clinical course of IBD is generally cyclical and is characterized by spontaneous exacerbations and remissions. Importantly, the clinical signs of IBD are not disease specific and share numerous over-lapping features with other canine disorders. A diagnosis of IBD is one of exclusion and requires careful elimination of IBD mimics [230]. The possible causes for chronic intestinal inflammation may be excluded through the integration of history, physical findings, clinicopathological testing, diagnostic imaging, and histopathology of intestinal biopsies. A baseline CBC, biochemistry profile, urinalysis, and diagnostic imaging are useful in eliminat‐ ing the most common systemic and metabolic disorders (e.g., renal disease, hepatopathy,

*2.5.1. Clinical and diagnostic features*

CD11c+

## **2.5. Canine IBD**

In addition to affecting humans, IBD can also occur in dogs as well [214]. As in human IBD, the interactions between genetics, the mucosal immune system, inflammation, and environ‐ mental factors (ie, diet and imbalances in the intestinal microbiome) all contribute to the pathogenesis of canine IBD (Figure 6) [215-217].

**Figure 6.** The etiology for canine IBD involves complex interactions between host genetics, mucosal immunity, and the enteric microbiota. Therapeutic intervention with diet, antibiotics and immunosuppressive drugs is aimed at re‐ ducing inflammation and dysbiosis.

Mutations in innate immune receptors in German shepherd dogs (TLR5, NOD2) have been linked to IBD susceptibility which in the presence of an inappropriate enteric microbiota may lead to upregulated pro-inflammatory cytokine production (e.g., IL-17, IL-22, TNF-α) and reduced bacterial clearance, thereby promoting chronic intestinal inflammation [218, 219]. Commensal bacterial antigens are likely to be important in disease pathogenesis because it has been observed that boxer dogs with granulomatous colitis (GC) show clinical remission following the eradication of mucosally associated AIEC that share a novel adherent and invasive pathotype which bears phylogenetic similarity with AIEC strains recovered from patients with ileal Crohn's disease [193, 220, 221]. Moreover, genome-wide analysis in affected boxer dogs has identified disease-associated single nucleotide polymorphisms (SNPs) in a gene (*NCF2*) involved with killing intracellular bacteria [222]. Still others have shown that CD11c+ cells are significantly decreased in the intestines of dogs with IBD suggesting that chronic mucosal inflammation may involve an imbalance in the intestinal dendritic cell population leading to aberrant immune activation [223]. Molecular analysis of the intestinal microbiome in different breeds of dogs with IBD have consistently shown that diseased tissues are enriched with members of the families *Enterobacteriaceae* and *Clostridiaceae*[224, 225]. These bacteria are believed to contribute to the pathogenesis of GIT inflammatory disease in dogs as in humans [187, 226]. A recent trial using high throughput 16S rDNA sequencing methods (ie, 454 pyrosequencing) on intestinal biopsies of IBD dogs revealed a dysbiosis in the mucosallyadherent microbiota with an increase in sequences belonging to Proteobacteria and a decrease in Bacteroidetes, Fusobacteria, and the Clostridiales [227]. Taken together, these studies suggest that chronic intestinal inflammation of canine IBD may be due to overly aggressive adaptive immune responses to enteric bacteria (or fungi) [225] in hosts with genetic defects that fail to properly regulate microbial killing, mucosal barrier function, or immune responses. As in human IBD, environmental factors (diet, microbiota imbalances) likely govern the onset of inflammation or reactivation and modulate genetic susceptibility to disease.

#### *2.5.1. Clinical and diagnostic features*

provocateur along with perturbations of the microbiota together have a deleterious impact on

In addition to affecting humans, IBD can also occur in dogs as well [214]. As in human IBD, the interactions between genetics, the mucosal immune system, inflammation, and environ‐ mental factors (ie, diet and imbalances in the intestinal microbiome) all contribute to the

**Figure 6.** The etiology for canine IBD involves complex interactions between host genetics, mucosal immunity, and the enteric microbiota. Therapeutic intervention with diet, antibiotics and immunosuppressive drugs is aimed at re‐

the microbiota and mucosal homeostasis.

pathogenesis of canine IBD (Figure 6) [215-217].

**2.5. Canine IBD**

94 Inflammatory Bowel Disease

ducing inflammation and dysbiosis.

The clinical manifestations of IBD are diverse and are influenced by the organ(s) involved, presence of active versus inactive disease, and physiologic complications seen with enteric plasma protein loss and/or micronutrient (cobalamin) deficiency [215, 216, 228, 229]. Canine IBD is a disease that predominantly affects middle-aged animals. Vomiting and diarrhea are most commonly observed and are often accompanied by decreased appetite and weight loss. Gastric and duodenal inflammation is associated with vomiting and small bowel diarrhea while colonic involvement causes large bowel diarrhea with blood, mucus, and straining. The clinical course of IBD is generally cyclical and is characterized by spontaneous exacerbations and remissions. Importantly, the clinical signs of IBD are not disease specific and share numerous over-lapping features with other canine disorders. A diagnosis of IBD is one of exclusion and requires careful elimination of IBD mimics [230]. The possible causes for chronic intestinal inflammation may be excluded through the integration of history, physical findings, clinicopathological testing, diagnostic imaging, and histopathology of intestinal biopsies. A baseline CBC, biochemistry profile, urinalysis, and diagnostic imaging are useful in eliminat‐ ing the most common systemic and metabolic disorders (e.g., renal disease, hepatopathy, hypoadrenocorticism) causing chronic GI signs in dogs. The measure of clinical disease activity by means of quantifiable indices is well established in human IBD [231-233].

tion and maintenance of remission of clinical signs have not been determined for most protocols [216, 230]. In general, the administration of corticosteroids (i.e., prednisone, prednisolone or budesonide), antimicrobials (i.e., metronidazole or tylosin), and immuno‐ suppressive drugs (i.e., cyclosporine, chlorambucil, azathioprine) used alone or in some combination are effective in inducing clinical remission in most animals. Some dogs will

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The rationale for nutritional therapy of IBD is that restricting exposure to antigens (i.e., dietary proteins) known to evoke sensitivity will reduce exaggerated host responses and attenuate intestinal inflammation. Other indications for specialized nutrition include the presence of decreased appetite, impaired nutrient absorption, or enteric plasma protein loss seen with moderate-to-severe mucosal inflammation. While evidence-based observations indicate that most dogs respond favorably to dietary intervention, the superiority of one novel protein source versus another or the advantage in feeding an intact protein elimination diet versus a hydrolyzed protein elimination diet has not been shown to date. Modifying the dietary n3:n6 fatty-acid ratio may also modulate inflammatory responses by reducing production of proinflammatory metabolites [237]. There is relatively sparse clinical data investigating prebiotic

Canine IBD represents a common and frustrating GI disorder in veterinary medicine. More research is needed to unravel the mechanisms responsible for disease development and to translate these findings directly to human IBD. The primary features of IBD in humans and

> **Feature Human IBD Canine IBD** Genetic basis Yes Likely

> > pANCA, CRP, calprotectin)

Involves the microbiota Yes Yes Hematochezia Yes Yes Diarrhea Yes Yes Definitive Diagnosis GI biopsy GI biopsy

Responsive to anti-inflammatory drugs Yes Yes

Responsive to antibiotics Yes Yes Spontaneous GI flares Yes Yes

Disease activity assessment Clinical indices, biomarkers (ASCA,

**Table 1.** Comparative features of IBD in humans and dogs.

Etiology Unknown but multifactorial Unknown but multifactorial

Clinical indices, biomarkers (pANCA, CRP, calprotectin ?)

or probiotic therapy for canine IBD (see subsequent section on probiotics).

require intermittent or life-long drug therapy.

*2.5.3. Future directions in canine IBD*

animals are remarkably similar (Table 1).

A canine IBD activity index (CIBDAI) used for assessment of inflammatory activity in dogs has been recently designed [234]. Similar to other indices, the magnitude of the numerical score is proportional to the degree of inflammatory activity. This index serves as the principal measure of response to a therapeutic regimen and may be used to tailor medical therapy for an individual patient's needs [235]. Intestinal biopsies are required to confirm histopatholog‐ ical inflammation and to determine the extent of mucosal disease. Diagnostic endoscopy is preferred since this technique allows for direct assessment of mucosal abnormalities and the acquisition of targeted biopsy specimens. The microscopic findings in canine IBD consist of minimal to pronounced inflammatory cell (lymphoplasmacytic) infiltration of the intestinal lamina propria accompanied by varying degrees of mucosal architectural disruption similar to that observed in tissue from human IBD patients (Figure 7).

**Figure 7.** Histopathological lesions of (A) crypt distortion with abscessation, (B) diffuse villous atrophy, and (C) mucos‐ al ulceration seen in duodenal biopsies of dogs with IBD.

Unfortunately, biopsy interpretation is notoriously subjective and suffers from extensive intraobserver variability and the technical constraints of procurement/processing artifacts inherent in evaluation of endoscopic specimens [236]. Although several histopathological scoring schemes have been proposed there are no uniform grading criteria that pathologists can universally agree on. One small study has resulted in development of a 'simplified model system' for defining intestinal inflammation of IBD that is presently being tested in a separate clinical trial.

#### *2.5.2. Therapeutic approach*

Treatment principles for canine IBD are empirical and consist of combination therapy us‐ ing both dietary and pharmacologic interventions. As compared to clinical trials evaluat‐ ing the efficacy of therapy for CD and UC, only one randomized, controlled drug trial for canine IBD has been reported [235]. There are, however, abundant evidence-based ob‐ servations that feeding elimination diets and administering corticosteroids, immunosup‐ pressive drugs, and/or select antibiotics are useful in the clinical management of canine IBD. Some clinicians prefer a sequential approach to nutritional and drug therapy for IBD. The optimal drug or drug combinations as well as duration of therapy for induc‐ tion and maintenance of remission of clinical signs have not been determined for most protocols [216, 230]. In general, the administration of corticosteroids (i.e., prednisone, prednisolone or budesonide), antimicrobials (i.e., metronidazole or tylosin), and immuno‐ suppressive drugs (i.e., cyclosporine, chlorambucil, azathioprine) used alone or in some combination are effective in inducing clinical remission in most animals. Some dogs will require intermittent or life-long drug therapy.

The rationale for nutritional therapy of IBD is that restricting exposure to antigens (i.e., dietary proteins) known to evoke sensitivity will reduce exaggerated host responses and attenuate intestinal inflammation. Other indications for specialized nutrition include the presence of decreased appetite, impaired nutrient absorption, or enteric plasma protein loss seen with moderate-to-severe mucosal inflammation. While evidence-based observations indicate that most dogs respond favorably to dietary intervention, the superiority of one novel protein source versus another or the advantage in feeding an intact protein elimination diet versus a hydrolyzed protein elimination diet has not been shown to date. Modifying the dietary n3:n6 fatty-acid ratio may also modulate inflammatory responses by reducing production of proinflammatory metabolites [237]. There is relatively sparse clinical data investigating prebiotic or probiotic therapy for canine IBD (see subsequent section on probiotics).

## *2.5.3. Future directions in canine IBD*

hypoadrenocorticism) causing chronic GI signs in dogs. The measure of clinical disease activity

A canine IBD activity index (CIBDAI) used for assessment of inflammatory activity in dogs has been recently designed [234]. Similar to other indices, the magnitude of the numerical score is proportional to the degree of inflammatory activity. This index serves as the principal measure of response to a therapeutic regimen and may be used to tailor medical therapy for an individual patient's needs [235]. Intestinal biopsies are required to confirm histopatholog‐ ical inflammation and to determine the extent of mucosal disease. Diagnostic endoscopy is preferred since this technique allows for direct assessment of mucosal abnormalities and the acquisition of targeted biopsy specimens. The microscopic findings in canine IBD consist of minimal to pronounced inflammatory cell (lymphoplasmacytic) infiltration of the intestinal lamina propria accompanied by varying degrees of mucosal architectural disruption similar

**Figure 7.** Histopathological lesions of (A) crypt distortion with abscessation, (B) diffuse villous atrophy, and (C) mucos‐

Unfortunately, biopsy interpretation is notoriously subjective and suffers from extensive intraobserver variability and the technical constraints of procurement/processing artifacts inherent in evaluation of endoscopic specimens [236]. Although several histopathological scoring schemes have been proposed there are no uniform grading criteria that pathologists can universally agree on. One small study has resulted in development of a 'simplified model system' for defining intestinal inflammation of IBD that is presently being tested in a separate

Treatment principles for canine IBD are empirical and consist of combination therapy us‐ ing both dietary and pharmacologic interventions. As compared to clinical trials evaluat‐ ing the efficacy of therapy for CD and UC, only one randomized, controlled drug trial for canine IBD has been reported [235]. There are, however, abundant evidence-based ob‐ servations that feeding elimination diets and administering corticosteroids, immunosup‐ pressive drugs, and/or select antibiotics are useful in the clinical management of canine IBD. Some clinicians prefer a sequential approach to nutritional and drug therapy for IBD. The optimal drug or drug combinations as well as duration of therapy for induc‐

by means of quantifiable indices is well established in human IBD [231-233].

to that observed in tissue from human IBD patients (Figure 7).

al ulceration seen in duodenal biopsies of dogs with IBD.

clinical trial.

96 Inflammatory Bowel Disease

*2.5.2. Therapeutic approach*

Canine IBD represents a common and frustrating GI disorder in veterinary medicine. More research is needed to unravel the mechanisms responsible for disease development and to translate these findings directly to human IBD. The primary features of IBD in humans and animals are remarkably similar (Table 1).


**Table 1.** Comparative features of IBD in humans and dogs.

Recent advances in clinical indices, histopathological standards, and the development of species-specific immunologic reagents and innovative molecular tools have made the dog an excellent 'spontaneous' animal model to study chronic immunologically-mediated intestinal inflammation. In addition, the dog has higher genomic sequence similarity to that of humans than do mice, a species traditionally used for comparative disease genetics [238]. However, clinical manifestations of complex disease in the mouse do not compare to the human form as closely as they do in the dog. Furthermore, the lifespan of the dog is much shorter than that of a human; thus, clinical trials aimed at treatment of IBD can be carried out much quicker and yield results that should have relevant application to human trials [230, 235, 239].

Clofazamine has also been documented to significantly affect the rate of remission in CD patients [241]. This drug, used to treat leprosy, is similar to ciprofloxacin, in that it has both anti-bacterial and anti-inflammatory properties [247-249]. It is only effective against Grampositive organisms, and its effectiveness increases in anaerobic environments [250]. Its effects on the immune system include increasing the presence of lysosomal enzymes in macrophages [251] and increasing phagocytosis by macrophages resulting in enhanced uptake and digestion of immune complexes [252]. Clofazamine has also been shown to inhibit TCR-mediated IL-2 production by T cells, thereby limiting T cell activation, a component of the pathogenesis of

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The most commonly used corticosteroids used are prednisolone, methylprednisolone, and budesonide [254]. These drugs are very effective at inducing remission, however, they are not without side effects. In a study comparing prednisolone and budesonide, both were found to be effective at inducing remission [255]. However, patients on budesonide had reduced evidence of adrenal axis suppression and peripheral leucocyte counts compared to those treated with prednisolone. These results indicate that budesonide is a safer choice yet it is still as effective as prednisolone. Corticosteroids are able to prevent NFκB activation [256] as well as block infiltration of neutrophils, prevent vasodilation and enhanced vascular permeability and downregulate the production of pro-inflammatory cytokines [254]. Although it is well established that these corticosteroids have an anti-inflammatory effect, little research has been conducted on the effects they have on the gut microbiota. A study by Swidsinski et al., mentioned previously, indicated that administration of cortisol increased the population of *F.*

Drugs such as methotrexate, 6-mercaptopurine (6-MP) and azathioprine (the prodrug of 6-MP) work by inhibiting the proliferation and activation of lymphocytes and decreasing the production of pro-inflammatory cytokines [254]. 6-mercaptopurine (6-MP) and azathioprine are purine antagonists and inhibit cellular metabolism by interfering with DNA replication [257, 258]. Methotrexate is a folic acid analog that inhibits DNA synthesis and, therefore, has an anti-proliferative effect [259]. Antibacterial effects, including growth inhibition of MAP,

Sulfasalazine was the first 5-ASA-type drug developed and is a combination of sulfapyridine (an antimicrobial) and salicyclic acid (an anti-inflammatory) to form a pro-drug. Upon entering the colon, this pro-drug is cleaved by colonic bacteria into the two separate molecules [260, 261]. Unfortunately, the frequency of gastrointestinal side effects was quite high due to the sulfapyridine moiety [262, 263]. It was later determined that the active moiety is 5-aminosali‐ cyclic acid (5-ASA, mesalazine) [264]. More recently formulations of this pro-drug have eliminated the sulfapyridine moiety and replaced it with either a second salicyclic acid moiety

IBD [253].

*2.6.2. Corticosteroids*

*prausnitzii* in a dose dependent manner [209].

have also been demonstrated for methotrexate and 6-MP [258].

*2.6.3. Immunosuppressive therapy*

*2.6.4. 5-aminosalicyclic acid (5-ASA)*

#### **2.6. Treatment for human IBD**

Treatments for human IBD patients typically involve the use of anti-inflammatory drugs, antibiotics, and there is a growing trend of probiotics and prebiotics being studied to determine their effects on disease activity. When patients fail to respond to treatment, the last and most drastic treatment option is surgery to resect sections of the inflamed bowel [240]. Most treatment options are primarily focused on reducing the inflammation associated with IBD. Some of these drugs also impact the microbiota and those will be discussed below.

#### *2.6.1. Antibiotics*

Given the importance of the microbiota in the pathogenesis of IBD, antibiotic therapy may seem an obvious treatment option. A meta-analysis by Khan et al. in 2011 reviewed random‐ ized controlled trials utilizing antibiotics in the treatment of IBD [241]. Rifamycin derivatives, ciprofloxacin, and clofazamine all induced remission in CD patients. Rifaximin (a rifamycin derivative) is effective against both Gram-negative and Gram-positive anaerobes and aerobes; it is also poorly absorbed after oral administration, resulting in little to no systemic side effects [242, 243]. Analysis of the effect of the drug using an *in vitro* continuous culture method with fecal samples from CD patients revealed increases in beneficial bacteria post-rifaximin administration [244]. Significant increases in *Bifidobacterium* spp., the *Atopobium* cluster, and *F. prausnitzii* were noted, as were increases in levels of lactate, acetate, and propionate as determined by 1 H-NMR spectroscopy [244].

Ciprofloxacin is a fluoroquinolone with broad-spectrum antibiotic activity [245]. In addition to having anti-bacterial properties, this drug has also been shown to have immunomodulatory properties as well [246]. In a TNBS mouse model of colitis, administration of ciprofloxacin ameliorated disease as compared to mice given ceftazidime (an antibacterial with a similar spectrum of activity compared to ciprofloxacin) [245]. Clinically, mice treated with ciproflox‐ acin did not lose weight and had reduced histopathological inflammatory scores associated with their colons. Ciprofloxacin treated mice also had reduced expression of IL-1β, IL-8, and TNF-α as measured from colonic homogenates as well as reduced expression of NF-κB. Since ceftazidime was less effective in ameliorating GIT inflammation, there appears to be additional benefits (i.e., anti-inflammatory) to the use of ciprofloxacin in addition to its spectrum of antimicrobial activity.

Clofazamine has also been documented to significantly affect the rate of remission in CD patients [241]. This drug, used to treat leprosy, is similar to ciprofloxacin, in that it has both anti-bacterial and anti-inflammatory properties [247-249]. It is only effective against Grampositive organisms, and its effectiveness increases in anaerobic environments [250]. Its effects on the immune system include increasing the presence of lysosomal enzymes in macrophages [251] and increasing phagocytosis by macrophages resulting in enhanced uptake and digestion of immune complexes [252]. Clofazamine has also been shown to inhibit TCR-mediated IL-2 production by T cells, thereby limiting T cell activation, a component of the pathogenesis of IBD [253].

## *2.6.2. Corticosteroids*

Recent advances in clinical indices, histopathological standards, and the development of species-specific immunologic reagents and innovative molecular tools have made the dog an excellent 'spontaneous' animal model to study chronic immunologically-mediated intestinal inflammation. In addition, the dog has higher genomic sequence similarity to that of humans than do mice, a species traditionally used for comparative disease genetics [238]. However, clinical manifestations of complex disease in the mouse do not compare to the human form as closely as they do in the dog. Furthermore, the lifespan of the dog is much shorter than that of a human; thus, clinical trials aimed at treatment of IBD can be carried out much quicker and

Treatments for human IBD patients typically involve the use of anti-inflammatory drugs, antibiotics, and there is a growing trend of probiotics and prebiotics being studied to determine their effects on disease activity. When patients fail to respond to treatment, the last and most drastic treatment option is surgery to resect sections of the inflamed bowel [240]. Most treatment options are primarily focused on reducing the inflammation associated with IBD.

Given the importance of the microbiota in the pathogenesis of IBD, antibiotic therapy may seem an obvious treatment option. A meta-analysis by Khan et al. in 2011 reviewed random‐ ized controlled trials utilizing antibiotics in the treatment of IBD [241]. Rifamycin derivatives, ciprofloxacin, and clofazamine all induced remission in CD patients. Rifaximin (a rifamycin derivative) is effective against both Gram-negative and Gram-positive anaerobes and aerobes; it is also poorly absorbed after oral administration, resulting in little to no systemic side effects [242, 243]. Analysis of the effect of the drug using an *in vitro* continuous culture method with fecal samples from CD patients revealed increases in beneficial bacteria post-rifaximin administration [244]. Significant increases in *Bifidobacterium* spp., the *Atopobium* cluster, and *F. prausnitzii* were noted, as were increases in levels of lactate, acetate, and propionate as

Ciprofloxacin is a fluoroquinolone with broad-spectrum antibiotic activity [245]. In addition to having anti-bacterial properties, this drug has also been shown to have immunomodulatory properties as well [246]. In a TNBS mouse model of colitis, administration of ciprofloxacin ameliorated disease as compared to mice given ceftazidime (an antibacterial with a similar spectrum of activity compared to ciprofloxacin) [245]. Clinically, mice treated with ciproflox‐ acin did not lose weight and had reduced histopathological inflammatory scores associated with their colons. Ciprofloxacin treated mice also had reduced expression of IL-1β, IL-8, and TNF-α as measured from colonic homogenates as well as reduced expression of NF-κB. Since ceftazidime was less effective in ameliorating GIT inflammation, there appears to be additional benefits (i.e., anti-inflammatory) to the use of ciprofloxacin in addition to its spectrum of

yield results that should have relevant application to human trials [230, 235, 239].

Some of these drugs also impact the microbiota and those will be discussed below.

H-NMR spectroscopy [244].

**2.6. Treatment for human IBD**

98 Inflammatory Bowel Disease

*2.6.1. Antibiotics*

determined by 1

antimicrobial activity.

The most commonly used corticosteroids used are prednisolone, methylprednisolone, and budesonide [254]. These drugs are very effective at inducing remission, however, they are not without side effects. In a study comparing prednisolone and budesonide, both were found to be effective at inducing remission [255]. However, patients on budesonide had reduced evidence of adrenal axis suppression and peripheral leucocyte counts compared to those treated with prednisolone. These results indicate that budesonide is a safer choice yet it is still as effective as prednisolone. Corticosteroids are able to prevent NFκB activation [256] as well as block infiltration of neutrophils, prevent vasodilation and enhanced vascular permeability and downregulate the production of pro-inflammatory cytokines [254]. Although it is well established that these corticosteroids have an anti-inflammatory effect, little research has been conducted on the effects they have on the gut microbiota. A study by Swidsinski et al., mentioned previously, indicated that administration of cortisol increased the population of *F. prausnitzii* in a dose dependent manner [209].

## *2.6.3. Immunosuppressive therapy*

Drugs such as methotrexate, 6-mercaptopurine (6-MP) and azathioprine (the prodrug of 6-MP) work by inhibiting the proliferation and activation of lymphocytes and decreasing the production of pro-inflammatory cytokines [254]. 6-mercaptopurine (6-MP) and azathioprine are purine antagonists and inhibit cellular metabolism by interfering with DNA replication [257, 258]. Methotrexate is a folic acid analog that inhibits DNA synthesis and, therefore, has an anti-proliferative effect [259]. Antibacterial effects, including growth inhibition of MAP, have also been demonstrated for methotrexate and 6-MP [258].

## *2.6.4. 5-aminosalicyclic acid (5-ASA)*

Sulfasalazine was the first 5-ASA-type drug developed and is a combination of sulfapyridine (an antimicrobial) and salicyclic acid (an anti-inflammatory) to form a pro-drug. Upon entering the colon, this pro-drug is cleaved by colonic bacteria into the two separate molecules [260, 261]. Unfortunately, the frequency of gastrointestinal side effects was quite high due to the sulfapyridine moiety [262, 263]. It was later determined that the active moiety is 5-aminosali‐ cyclic acid (5-ASA, mesalazine) [264]. More recently formulations of this pro-drug have eliminated the sulfapyridine moiety and replaced it with either a second salicyclic acid moiety (disodium azodisalicylate [265]) or an inert carrier (balsalazide [266]) thereby reducing the side effects.

Anti-TNF-α therapy works via multi-factorial mechanisms. First, it neutralizes TNF-α by blocking its ability to bind to TNF receptors, thus, inhibiting the pro-inflammatory response. Second, Anti-TNF-α binds to cell surface bound TNF-α on CD4+ T cells and macrophages, resulting in both complement- and antibody-dependent cell-mediated cytotoxicity [282]. All three monoclonal antibodies bind to and neutralize both soluble and membrane forms of TNFα [281]. Infliximab and Adalimumab both mediate complement- and antibody-dependent cellmediated cytotoxicity; however, Certolizumab only mediates complement-dependent cellular cytotoxicity as it lacks of an Fc region. Furthermore, Infliximab and Adalimumab induce apoptosis in peripheral blood lymphocytes and monocytes, as well as cause degranulation and loss of membrane integrity of PMNs. These activities were not induced with Certolizumab. Lastly, all three monoclonal antibodies inhibit the production of IL-1β after LPS stimulation in vitro, suggesting that there is a sequential production of pro-inflammatory cytokines induced by microbial components. Infliximab and Adalimumab both inhibit T cell prolifera‐ tion in mixed lymphocyte reactions in vitro, again suggesting that anti-TNF-α monoclonal antibodies ameliorate the inflammation associated with IBD via more than one mode of action [283]. The impact of these therapies on the microbiota, however, is not well studied. As previously mentioned, treatment with Infliximab resulted in increased levels of *F. prausnitzii* after administration [209]. As mentioned above, it is clear that the host inflammatory response often negatively impacts (i.e., shapes) the composition of the GIT microbiota, and that controlling mucosal inflammation benefits the health of the microbiota as well. Otherwise, there have been no specific studies performed to directly evaluate the role of anti-TNF-α

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An estimated 70 % of IBD patients have reported using complementary and alternative medicine (CAM) products at some time to treat their symptoms In a Canadian study of IBD patients, some of the most commonly used CAM treatments included massage therapy, chiropractic visits, probiotics, herbs, and fish oils [284]. A systematic review of the literature on the use of herbal medicines reveals some anti-inflammatory benefits associated with the administration of these herbs to both animals and humans [285]. It should be stressed that prior to utilizing any herbal remedy, which are potentially biologically active, patients need to consult their physician. This is especially important because the number two reason patients using CAM gave as to why they sought these products was that "natural therapy is safe"[286]. Some of the biological properties associated with CAM products include the reduction of proinflammatory cytokines, increased antioxidant production, inhibition of leukotriene B4,

Increasing evidence supports a potential therapeutic role for prebiotic and probiotic therapy in human IBD [287, 288]. If IBD in dogs is indeed driven by loss of tolerance to components of the intestinal microbiota as it is in humans, then prebiotics and probiotics may also prove beneficial as primary or adjunct therapies with diet and drugs. *Probiotics* are living microor‐ ganisms that, upon ingestion in sufficient numbers, impart health benefits beyond those of inherent basic nutrition [289]. Lactobacilli and Bifidobacterium have been the most commonly

decreased NF-κB activation, and inhibition of platelet activation [285].

therapies on the gut microbiota.

*2.6.6. Complementary and alternative therapies*

In addition to being an anti-inflammatory agent [267-270], 5-ASA also affects the gut micro‐ biota. As mentioned previously, patients taking mesalamine had a reduction in mucosaadherent bacteria [198, 271]. 5-ASA has also been shown to inhibit the growth of MAP [272] and *Bacteroides* spp. [273] and moderately inhibit the growth of *C. difficile* and *C. perfringens* in culture [273]. This drug also effects bacterial gene expression, as *Salmonella enterica* serovar Typhimurium incubated with 5-ASA had no change in growth but differentially expressed 110 genes [274]. Those genes characterized were found to be involved in invasion, metabolism, and antibiotic and stress resistance. *In vitro* assays revealed attenuation in the invasiveness of *S. enterica* serovar Typhimurium towards HeLa cells when pretreated with 5-ASA. These results indicate that in addition to inhibiting host-mediated inflammation, 5-ASA also has the potential to affect the intestinal microbiota.

#### *2.6.5. Anti-TNF monoclonal antibodies*

The induction of TNF-α is most often a downstream event following the interaction of phlogistic microbial components with toll-like receptors (TLRs) on host cells. The interest in TNF-α as a therapeutic target for IBD treatment began when the expression and secretion of this cytokine was found to be increased in IBD patients [275, 276]. For example, pediatric patients with either active UC or CD present with elevated levels of TNF-α in their stool [275]. Additionally, the incubation of GIT tissue sections in culture medium has demonstrated that significantly elevated levels of TNF-α are spontaneously secreted from inflamed tissue of both UC and CD patients when compared to the amounts produced by non-inflamed tissue and tissue from otherwise healthy individuals [276]. The predominant cell type producing the TNFα has been shown to be the macrophage [276]. Based on the central role TNF-α appears to play in the pathogenesis of IBD, there was interest in developing a therapeutic approach to control the harmful effects of this cytokine.

Clinically, the use of monoclonal antibodies to treat IBD patients began with Infliximab, an IgG1 murine-human chimeric monoclonal antibody specific for TNF-α, which was approved for human use in 1998 for CD [277, 278]. This monoclonal antibody consists of human constant regions and murine antigen binding regions [277]. These chimeric antibodies reduce the risk of immunoreactivity that occurs when murine antibodies are used. In addition to being less immunoreactive, this chimeric antibody had improved binding and neutralization character‐ istics for TNF-α than that of the original murine antibody [277]. Another monoclonal anti-TNF antibody, Adalimumab, is a fully humanized IgG1 antibody that avoids the induction of antispecies IgG that neutralize the effectiveness of the anti-TNF-α reagent [278, 279]. Lastly, Certolizumab is a monoclonal antibody fragment with a polyethylene glycol moiety (PEGy‐ lated) [278]. Certolizumab lacks the crystallizable fragment (Fc) portion of the immunoglobulin molecule and is an IgG4 isotype unlike Infliximab and Adalimumab, which are IgG1 antibodies [278, 280, 281]. In addition, the PEGylation increases the half-life of the antibody thereby reducing the frequency of administration.

Anti-TNF-α therapy works via multi-factorial mechanisms. First, it neutralizes TNF-α by blocking its ability to bind to TNF receptors, thus, inhibiting the pro-inflammatory response. Second, Anti-TNF-α binds to cell surface bound TNF-α on CD4+ T cells and macrophages, resulting in both complement- and antibody-dependent cell-mediated cytotoxicity [282]. All three monoclonal antibodies bind to and neutralize both soluble and membrane forms of TNFα [281]. Infliximab and Adalimumab both mediate complement- and antibody-dependent cellmediated cytotoxicity; however, Certolizumab only mediates complement-dependent cellular cytotoxicity as it lacks of an Fc region. Furthermore, Infliximab and Adalimumab induce apoptosis in peripheral blood lymphocytes and monocytes, as well as cause degranulation and loss of membrane integrity of PMNs. These activities were not induced with Certolizumab. Lastly, all three monoclonal antibodies inhibit the production of IL-1β after LPS stimulation in vitro, suggesting that there is a sequential production of pro-inflammatory cytokines induced by microbial components. Infliximab and Adalimumab both inhibit T cell prolifera‐ tion in mixed lymphocyte reactions in vitro, again suggesting that anti-TNF-α monoclonal antibodies ameliorate the inflammation associated with IBD via more than one mode of action [283]. The impact of these therapies on the microbiota, however, is not well studied. As previously mentioned, treatment with Infliximab resulted in increased levels of *F. prausnitzii* after administration [209]. As mentioned above, it is clear that the host inflammatory response often negatively impacts (i.e., shapes) the composition of the GIT microbiota, and that controlling mucosal inflammation benefits the health of the microbiota as well. Otherwise, there have been no specific studies performed to directly evaluate the role of anti-TNF-α therapies on the gut microbiota.

## *2.6.6. Complementary and alternative therapies*

(disodium azodisalicylate [265]) or an inert carrier (balsalazide [266]) thereby reducing the side

In addition to being an anti-inflammatory agent [267-270], 5-ASA also affects the gut micro‐ biota. As mentioned previously, patients taking mesalamine had a reduction in mucosaadherent bacteria [198, 271]. 5-ASA has also been shown to inhibit the growth of MAP [272] and *Bacteroides* spp. [273] and moderately inhibit the growth of *C. difficile* and *C. perfringens* in culture [273]. This drug also effects bacterial gene expression, as *Salmonella enterica* serovar Typhimurium incubated with 5-ASA had no change in growth but differentially expressed 110 genes [274]. Those genes characterized were found to be involved in invasion, metabolism, and antibiotic and stress resistance. *In vitro* assays revealed attenuation in the invasiveness of *S. enterica* serovar Typhimurium towards HeLa cells when pretreated with 5-ASA. These results indicate that in addition to inhibiting host-mediated inflammation, 5-ASA also has the

The induction of TNF-α is most often a downstream event following the interaction of phlogistic microbial components with toll-like receptors (TLRs) on host cells. The interest in TNF-α as a therapeutic target for IBD treatment began when the expression and secretion of this cytokine was found to be increased in IBD patients [275, 276]. For example, pediatric patients with either active UC or CD present with elevated levels of TNF-α in their stool [275]. Additionally, the incubation of GIT tissue sections in culture medium has demonstrated that significantly elevated levels of TNF-α are spontaneously secreted from inflamed tissue of both UC and CD patients when compared to the amounts produced by non-inflamed tissue and tissue from otherwise healthy individuals [276]. The predominant cell type producing the TNFα has been shown to be the macrophage [276]. Based on the central role TNF-α appears to play in the pathogenesis of IBD, there was interest in developing a therapeutic approach to control

Clinically, the use of monoclonal antibodies to treat IBD patients began with Infliximab, an IgG1 murine-human chimeric monoclonal antibody specific for TNF-α, which was approved for human use in 1998 for CD [277, 278]. This monoclonal antibody consists of human constant regions and murine antigen binding regions [277]. These chimeric antibodies reduce the risk of immunoreactivity that occurs when murine antibodies are used. In addition to being less immunoreactive, this chimeric antibody had improved binding and neutralization character‐ istics for TNF-α than that of the original murine antibody [277]. Another monoclonal anti-TNF antibody, Adalimumab, is a fully humanized IgG1 antibody that avoids the induction of antispecies IgG that neutralize the effectiveness of the anti-TNF-α reagent [278, 279]. Lastly, Certolizumab is a monoclonal antibody fragment with a polyethylene glycol moiety (PEGy‐ lated) [278]. Certolizumab lacks the crystallizable fragment (Fc) portion of the immunoglobulin molecule and is an IgG4 isotype unlike Infliximab and Adalimumab, which are IgG1 antibodies [278, 280, 281]. In addition, the PEGylation increases the half-life of the antibody thereby

effects.

100 Inflammatory Bowel Disease

potential to affect the intestinal microbiota.

*2.6.5. Anti-TNF monoclonal antibodies*

the harmful effects of this cytokine.

reducing the frequency of administration.

An estimated 70 % of IBD patients have reported using complementary and alternative medicine (CAM) products at some time to treat their symptoms In a Canadian study of IBD patients, some of the most commonly used CAM treatments included massage therapy, chiropractic visits, probiotics, herbs, and fish oils [284]. A systematic review of the literature on the use of herbal medicines reveals some anti-inflammatory benefits associated with the administration of these herbs to both animals and humans [285]. It should be stressed that prior to utilizing any herbal remedy, which are potentially biologically active, patients need to consult their physician. This is especially important because the number two reason patients using CAM gave as to why they sought these products was that "natural therapy is safe"[286]. Some of the biological properties associated with CAM products include the reduction of proinflammatory cytokines, increased antioxidant production, inhibition of leukotriene B4, decreased NF-κB activation, and inhibition of platelet activation [285].

Increasing evidence supports a potential therapeutic role for prebiotic and probiotic therapy in human IBD [287, 288]. If IBD in dogs is indeed driven by loss of tolerance to components of the intestinal microbiota as it is in humans, then prebiotics and probiotics may also prove beneficial as primary or adjunct therapies with diet and drugs. *Probiotics* are living microor‐ ganisms that, upon ingestion in sufficient numbers, impart health benefits beyond those of inherent basic nutrition [289]. Lactobacilli and Bifidobacterium have been the most commonly used human probiotics, but multi-strain cocktails (e.g., VSL#3), *E. coli* Nissle 1917, and nonbacterial *Saccharomyces boulardii* have also been used as probiotics [226]. Probiotic bacteria have measurable host benefits, including the ability to improve epithelial barrier function, modulate the mucosal immune system, and alter the intestinal flora [290]. *Prebiotics* are nondigestible dietary carbohydrates, such as lactosucrose, fructo-oligosaccharides (FOS), psylli‐ um, bran, which beneficially stimulate the growth and metabolism of endogenous enteric bacteria upon consumption [291]. Beneficial effects of prebiotics are also associated with the production of short chain fatty acids (SCFA) due to fermentation by colonic bacteria. *Synbiot‐ ics* are combinations of probiotics and prebiotics that are an emerging therapeutic modality. Increasing evidence supports a therapeutic role for probiotics, prebiotics, and synbiotics in treating gastrointestinal diseases of humans, including infectious diarrhea, *H. pylori* infection, irritable bowel syndrome, lactase deficiency, and IBD [84]. A comparison of prebiotic and probiotic preparations is outlined in Table 2.

pro-inflammatory chemokine IP-10 [300]. The most recent studies with VSL#3 treatment of UC show increased fecal concentrations of beneficial bacterial species, improved clinical, endo‐ scopic and histopathological scores in most patients, and higher rates of remission compared to placebo [298]. The most recent studies with VSL#3 treatment of UC show increased fecal concentrations of beneficial bacterial species, improved clinical, endoscopic and histopatho‐ logical scores in most patients, and higher rates of remission compared to placebo [298]. VSL#3 is available without prescription and can be ordered via the internet or obtained locally at the

**Probiotics Prebiotics**

Non-digestible carbohydrate which stimulate replication of protective enteric bacteria when consumed

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Fructo-oligosaccharide (FOS) Galacto-oligosaccharide (GOS) Inulin Lactulose Psyllium Bran Beet pulp, pumpkin Resistant starch

Stimulates replication of beneficial bacteria (*Bifidobacterium*) Enhances production of SCFA (butyrate) Improved intestinal barrier function Decreases proinflammatory cytokines

pharmacy in the U.S. [301].

Definition Live microorganisms which, given in

Examples *E. coli* Nissle 1917

Protective Mechanisms Alters microbiota to suppress pathogens

**Table 2.** Basic Features of Probiotics and Prebiotics

adequate amounts confer health benefits to the host

> VSL#3 *Lactobacillus* species *Bifidobacterium* species Saccharomyces boulardii Prostora Max® Forti Flora® Proviable-DC®

Improved intestinal barrier function Increased production of antimicrobial peptides Decreased expression of proinflammatory cytokines

Recent studies have also shown that dogs with IBD have distinctly different duodenal microbial communities compared to healthy dogs. Current treatments for IBD include the administration of nonspecific anti-inflammatory drugs which may confer serious side effects and do not address the underlying basis for disease, namely, altered microbial composition. The use of probiotics offers an attractive, physiologic, and non-toxic alternative to shift the balance to protective species and treat canine IBD. The authors (AEJ) have initiated a clinical trial to investigate the clinical, microbiologic, and anti-inflammatory effects of probiotic VSL#3 in the treatment of canine IBD. We hypothesize that VSL#3 used as an adjunct to standard therapy (i.e., elimination diet and prednisone) will induce a beneficial alteration of enteric

#### *2.6.7. Probiotics*

VSL#3 is one of the most commonly used probiotic cocktails and contains a very high bacterial concentration per gram of product characterized by greater number of different bacterial species as compared to traditional probiotic preparations [292]. This commercially prepared formulation consists of 450 billion bacteria/g of viable lyophilized bacteria comprised of eight bacterial strains (*Lactobacillus casei, L plantarum, L. bulgaricus, L. acidolphilus, Bifidobacterium longum, B. breve, B. infantis* and *Streptococcus thermophiles*). While the exact mechanism of action of VSL#3 is unknown, several studies have demonstrated the effects of VSL#3 on epithelial barrier function and down regulation of cytokine secretion from immune cells. For example, Madsen [293] has shown in *in vitro* studies that epithelial barrier function could be enhanced by exposure to a soluble factor secreted by VSL#3 bacteria. Moreover, this same study demonstrated that VSL#3 did not alter the ability of the epithelial cell to activate a mucosal inflammatory response to a bacterial invasion. Studies with VSL#3 formulations have also been conducted in several animal models of colitis, inflammatory liver disease, sepsis, and irritable bowel syndrome (IBS). In models of experimentally-induced colitis, these studies demonstrat‐ ed that VSL#3 normalized gut permeability and barrier function, and that VSL#3 modulated inflammatory and immune responses [226, 294]. Animal models of sepsis have also demon‐ strated that VSL#3 administration reduced bacterial translocation and significantly attenuated damage to the liver and intestinal mucosa [295]. The use of VSL#3 as an innovative probiotic preparation, developed specifically to balance the intestinal microbiota, is supported by studies in humans with IBD (ulcerative colitis [296], pouchitis) and in other patients with diverse gastrointestinal disorders, such as IBS [297, 298].

Studies have shown VSL#3 to induce remission of inflammation in 77 % of adult UC patients with no adverse effects [298] and 56 % of pediatric UC patients [299]. These same pediatric UC patients had a reduction in disease activity index and sigmoidoscopy scores following VSL#3 treatment. They also had reduced levels of the pro-inflammatory cytokines TNF-α and IFNγ following therapy [299]. *In vitro* analysis of the effects of VSL#3 on Mode-K epithelial cells revealed that only one of the eight organisms, *L. casei*, inhibited TNF-induced secretion of the pro-inflammatory chemokine IP-10 [300]. The most recent studies with VSL#3 treatment of UC show increased fecal concentrations of beneficial bacterial species, improved clinical, endo‐ scopic and histopathological scores in most patients, and higher rates of remission compared to placebo [298]. The most recent studies with VSL#3 treatment of UC show increased fecal concentrations of beneficial bacterial species, improved clinical, endoscopic and histopatho‐ logical scores in most patients, and higher rates of remission compared to placebo [298]. VSL#3 is available without prescription and can be ordered via the internet or obtained locally at the pharmacy in the U.S. [301].


#### **Table 2.** Basic Features of Probiotics and Prebiotics

used human probiotics, but multi-strain cocktails (e.g., VSL#3), *E. coli* Nissle 1917, and nonbacterial *Saccharomyces boulardii* have also been used as probiotics [226]. Probiotic bacteria have measurable host benefits, including the ability to improve epithelial barrier function, modulate the mucosal immune system, and alter the intestinal flora [290]. *Prebiotics* are nondigestible dietary carbohydrates, such as lactosucrose, fructo-oligosaccharides (FOS), psylli‐ um, bran, which beneficially stimulate the growth and metabolism of endogenous enteric bacteria upon consumption [291]. Beneficial effects of prebiotics are also associated with the production of short chain fatty acids (SCFA) due to fermentation by colonic bacteria. *Synbiot‐ ics* are combinations of probiotics and prebiotics that are an emerging therapeutic modality. Increasing evidence supports a therapeutic role for probiotics, prebiotics, and synbiotics in treating gastrointestinal diseases of humans, including infectious diarrhea, *H. pylori* infection, irritable bowel syndrome, lactase deficiency, and IBD [84]. A comparison of prebiotic and

VSL#3 is one of the most commonly used probiotic cocktails and contains a very high bacterial concentration per gram of product characterized by greater number of different bacterial species as compared to traditional probiotic preparations [292]. This commercially prepared formulation consists of 450 billion bacteria/g of viable lyophilized bacteria comprised of eight bacterial strains (*Lactobacillus casei, L plantarum, L. bulgaricus, L. acidolphilus, Bifidobacterium longum, B. breve, B. infantis* and *Streptococcus thermophiles*). While the exact mechanism of action of VSL#3 is unknown, several studies have demonstrated the effects of VSL#3 on epithelial barrier function and down regulation of cytokine secretion from immune cells. For example, Madsen [293] has shown in *in vitro* studies that epithelial barrier function could be enhanced by exposure to a soluble factor secreted by VSL#3 bacteria. Moreover, this same study demonstrated that VSL#3 did not alter the ability of the epithelial cell to activate a mucosal inflammatory response to a bacterial invasion. Studies with VSL#3 formulations have also been conducted in several animal models of colitis, inflammatory liver disease, sepsis, and irritable bowel syndrome (IBS). In models of experimentally-induced colitis, these studies demonstrat‐ ed that VSL#3 normalized gut permeability and barrier function, and that VSL#3 modulated inflammatory and immune responses [226, 294]. Animal models of sepsis have also demon‐ strated that VSL#3 administration reduced bacterial translocation and significantly attenuated damage to the liver and intestinal mucosa [295]. The use of VSL#3 as an innovative probiotic preparation, developed specifically to balance the intestinal microbiota, is supported by studies in humans with IBD (ulcerative colitis [296], pouchitis) and in other patients with

Studies have shown VSL#3 to induce remission of inflammation in 77 % of adult UC patients with no adverse effects [298] and 56 % of pediatric UC patients [299]. These same pediatric UC patients had a reduction in disease activity index and sigmoidoscopy scores following VSL#3 treatment. They also had reduced levels of the pro-inflammatory cytokines TNF-α and IFNγ following therapy [299]. *In vitro* analysis of the effects of VSL#3 on Mode-K epithelial cells revealed that only one of the eight organisms, *L. casei*, inhibited TNF-induced secretion of the

probiotic preparations is outlined in Table 2.

diverse gastrointestinal disorders, such as IBS [297, 298].

*2.6.7. Probiotics*

102 Inflammatory Bowel Disease

Recent studies have also shown that dogs with IBD have distinctly different duodenal microbial communities compared to healthy dogs. Current treatments for IBD include the administration of nonspecific anti-inflammatory drugs which may confer serious side effects and do not address the underlying basis for disease, namely, altered microbial composition. The use of probiotics offers an attractive, physiologic, and non-toxic alternative to shift the balance to protective species and treat canine IBD. The authors (AEJ) have initiated a clinical trial to investigate the clinical, microbiologic, and anti-inflammatory effects of probiotic VSL#3 in the treatment of canine IBD. We hypothesize that VSL#3 used as an adjunct to standard therapy (i.e., elimination diet and prednisone) will induce a beneficial alteration of enteric bacteria leading to induction and maintenance of remission in dogs with IBD. A randomized, controlled clinical trial of eight weeks duration will assess the efficacy of standard therapy in conjunction with VSL#3 versus standard therapy alone in the management of canine IBD. There is a need for additional data to be generated to provide proof of efficacy in probiotic therapy before these agents can be applied to widespread clinical use. These studies will also provide highly relevant insight into the anti-inflammatory effects of probiotics for treatment of human and canine IBD.

and *F. prausnitzii* also has other anti-inflammatory properties. In animal models, prebiot‐ ics have been shown to be effective as well. The severity of DSS-induced colitis in rats was attenuated by administering inulin orally as evidenced by a reduction in histopatho‐ logical scores and myeloperioxidase accumulation in the colons [315]. In addition, the acidity of the colonic contents increased in rats fed inulin as well as an increase in *Lacto‐ bacillus* spp. in the feces. In a multicenter trial feeding UC patients germinated barley foodstuff (GBF), patients consuming GBF had improved clinical activity index scores [316]. GBF has also been shown to be effective in maintaining remission [317] and reduc‐ ing pro-inflammatory cytokine levels in UC patients [318]. In CD patients, dietary sup‐ plementation with fructo-oligosaccharide (FOS) reduced their disease activity index, increased fecal *Bifidobacterium* spp. levels as well as increased production of IL-10 and ex‐

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Scientific studies have also investigated the effects of dietary supplementation with prebiotics on the intestinal microbiota of healthy dogs and cats. In one study, FOS supplemented at 0.75 % dry matter produced qualitative and quantitative changes in the fecal flora of healthy cats [320]. Compared with samples from cats fed a basal diet, increased numbers of lactobacilli and *Bacteroides* spp. and decreased numbers of *E. coli* were associated with cats fed the FOS supplemented diet. However, bacteriologic examination of the duodenal juice in these same cats showed wide variation in the composition of the duodenal microbiota, across sampling periods, which was not affected by FOS supplementation [321]. Moreover, healthy Beagle dogs fed a 1 % FOS diet over a three-month trial showed inconsistent fecal excretion of *Lactobacil‐ lus* spp. and *Bifidobacterium* spp. [322]. While FOS supplementation has been shown to have health benefits, these studies demonstrate that FOS does not have an adverse affect on the microbiota and suggest that it may have positive physiological benefits as seen in humans. This observation and the lack of significant side effects associated with FOS supplementation provide evidence that FOS should be considered as an attractive alternative or adjunct therapy

Many different mouse models have been utilized in IBD research to elucidate the roles that bacteria, genetics, the immune response, and environment play in the induction and mainte‐ nance of IBD [323]. They fall into two main categories: chemically-induced and geneticallyengineered models. The composition of the microbiota in the various mouse models is also discussed. Regardless of the strain of mouse employed in chemically-induced or geneticallyengineered models, there are only five options for its microbiota—conventional, specific pathogen free, restricted, gnotobiotic, or germ-free. Note that the word gnotobiotic (gnostos– "known" bios –"life") indicates that all the organisms present, regardless of the numbers of species, are known and does not apply only to mice that are completely devoid of microbes

pression of TLR-2 and TLR-4 from lamina propria dendritic cells [319].

for IBD in dogs and cats.

(i.e., germfree).

**3. Mouse models of IBD**

Another popular probiotic is *E. coli* Nissle 1917 also known as Mutaflor®. This probiotic has been shown to be just as effective as mesalazine in achieving and maintaining remission of inflammation in UC patients [302-304]. It has recently been shown that when *E. coli* Nissle 1917 is genetically modified to produce the quorum-sensing molecule AI-2, it affected the beneficial probiotic properties of this organism [305]. Colonization of healthy newborn infants with *E. coli* Nissle 1917 has shown that the presence of pathogenic bacteria in the gut was significantly reduced compared to non-colonized infants [306]. *In vitro* studies have shown *E. coli* Nissle 1917 is able to reduce the invasive ability of multiple pathogenic organisms [307], and also to reduce the adherent and invasive ability of AIEC [308]. *E. coli* Nissle 1917 was also shown to either inhibit the growth (49 %) or overgrowth (30 %) of 79 % of uropathogenic organisms recovered from children with urinary tract infections [309]. Unlike VSL#3, Mutaflor® cannot be purchased in the U.S. due to its reclassification by the F.D.A. from a "medical food" (which is what VSL#3 is classified as) to a "biologic" [301, 310].

There are few reports on the use of probiotic bacteria in dogs and cats. Recent *in vitro* studies have confirmed the capacity of a lyophilized probiotic cocktail (e.g., three different *Lactobacil‐ lus* spp strains) to modulate the expression of regulatory versus pro-inflammatory cytokines in dogs with chronic enteropathies [311]. However, a clinical trial using this same probiotic cocktail fed to dogs with food-responsive diarrhea failed to induce consistent patterns of regulatory (e.g., beneficial) cytokine expression in spite of obvious clinical improvement [312]. One commercially-manufactured probiotic (FortiFlora™–*Enterococcus faecium* SF68, Nestle Purina) is reported to potentially control diarrhea and enhance immune responses in dogs and cats. Several recent trials attest to the short-term efficacy of probiotics in treating acute diarrhea in dogs and cats [313]. The link between the intestinal microbiota and gastrointestinal health in companion animals is now obvious. Future developments in the pharmabiotic field must include performance of randomized clinical trials to determine the role of probiotics and prebiotics in the management of canine chronic enteropathy. One large multicenter trial investigating the efficacy of VSL#3 in reducing inflammatory activity of canine IBD is presently underway

#### *2.6.8. Prebiotics*

Prebiotics are substances that can be used to promote specific changes in the microbiota. Administration has been shown to shift the microbiota in healthy adults; for example, in‐ dividuals who consume either soluble corn fiber or polydextrose had increases in *F. prausnitzii* and those who consumed the soluble corn fiber had increases in *Roseburia* spp. [314]. As mentioned previously, both of these organisms metabolically produce butyrate and *F. prausnitzii* also has other anti-inflammatory properties. In animal models, prebiot‐ ics have been shown to be effective as well. The severity of DSS-induced colitis in rats was attenuated by administering inulin orally as evidenced by a reduction in histopatho‐ logical scores and myeloperioxidase accumulation in the colons [315]. In addition, the acidity of the colonic contents increased in rats fed inulin as well as an increase in *Lacto‐ bacillus* spp. in the feces. In a multicenter trial feeding UC patients germinated barley foodstuff (GBF), patients consuming GBF had improved clinical activity index scores [316]. GBF has also been shown to be effective in maintaining remission [317] and reduc‐ ing pro-inflammatory cytokine levels in UC patients [318]. In CD patients, dietary sup‐ plementation with fructo-oligosaccharide (FOS) reduced their disease activity index, increased fecal *Bifidobacterium* spp. levels as well as increased production of IL-10 and ex‐ pression of TLR-2 and TLR-4 from lamina propria dendritic cells [319].

Scientific studies have also investigated the effects of dietary supplementation with prebiotics on the intestinal microbiota of healthy dogs and cats. In one study, FOS supplemented at 0.75 % dry matter produced qualitative and quantitative changes in the fecal flora of healthy cats [320]. Compared with samples from cats fed a basal diet, increased numbers of lactobacilli and *Bacteroides* spp. and decreased numbers of *E. coli* were associated with cats fed the FOS supplemented diet. However, bacteriologic examination of the duodenal juice in these same cats showed wide variation in the composition of the duodenal microbiota, across sampling periods, which was not affected by FOS supplementation [321]. Moreover, healthy Beagle dogs fed a 1 % FOS diet over a three-month trial showed inconsistent fecal excretion of *Lactobacil‐ lus* spp. and *Bifidobacterium* spp. [322]. While FOS supplementation has been shown to have health benefits, these studies demonstrate that FOS does not have an adverse affect on the microbiota and suggest that it may have positive physiological benefits as seen in humans. This observation and the lack of significant side effects associated with FOS supplementation provide evidence that FOS should be considered as an attractive alternative or adjunct therapy for IBD in dogs and cats.

## **3. Mouse models of IBD**

bacteria leading to induction and maintenance of remission in dogs with IBD. A randomized, controlled clinical trial of eight weeks duration will assess the efficacy of standard therapy in conjunction with VSL#3 versus standard therapy alone in the management of canine IBD. There is a need for additional data to be generated to provide proof of efficacy in probiotic therapy before these agents can be applied to widespread clinical use. These studies will also provide highly relevant insight into the anti-inflammatory effects of probiotics for treatment

Another popular probiotic is *E. coli* Nissle 1917 also known as Mutaflor®. This probiotic has been shown to be just as effective as mesalazine in achieving and maintaining remission of inflammation in UC patients [302-304]. It has recently been shown that when *E. coli* Nissle 1917 is genetically modified to produce the quorum-sensing molecule AI-2, it affected the beneficial probiotic properties of this organism [305]. Colonization of healthy newborn infants with *E. coli* Nissle 1917 has shown that the presence of pathogenic bacteria in the gut was significantly reduced compared to non-colonized infants [306]. *In vitro* studies have shown *E. coli* Nissle 1917 is able to reduce the invasive ability of multiple pathogenic organisms [307], and also to reduce the adherent and invasive ability of AIEC [308]. *E. coli* Nissle 1917 was also shown to either inhibit the growth (49 %) or overgrowth (30 %) of 79 % of uropathogenic organisms recovered from children with urinary tract infections [309]. Unlike VSL#3, Mutaflor® cannot be purchased in the U.S. due to its reclassification by the F.D.A. from a "medical food" (which

There are few reports on the use of probiotic bacteria in dogs and cats. Recent *in vitro* studies have confirmed the capacity of a lyophilized probiotic cocktail (e.g., three different *Lactobacil‐ lus* spp strains) to modulate the expression of regulatory versus pro-inflammatory cytokines in dogs with chronic enteropathies [311]. However, a clinical trial using this same probiotic cocktail fed to dogs with food-responsive diarrhea failed to induce consistent patterns of regulatory (e.g., beneficial) cytokine expression in spite of obvious clinical improvement [312]. One commercially-manufactured probiotic (FortiFlora™–*Enterococcus faecium* SF68, Nestle Purina) is reported to potentially control diarrhea and enhance immune responses in dogs and cats. Several recent trials attest to the short-term efficacy of probiotics in treating acute diarrhea in dogs and cats [313]. The link between the intestinal microbiota and gastrointestinal health in companion animals is now obvious. Future developments in the pharmabiotic field must include performance of randomized clinical trials to determine the role of probiotics and prebiotics in the management of canine chronic enteropathy. One large multicenter trial investigating the efficacy of VSL#3 in reducing inflammatory activity of canine IBD is presently

Prebiotics are substances that can be used to promote specific changes in the microbiota. Administration has been shown to shift the microbiota in healthy adults; for example, in‐ dividuals who consume either soluble corn fiber or polydextrose had increases in *F. prausnitzii* and those who consumed the soluble corn fiber had increases in *Roseburia* spp. [314]. As mentioned previously, both of these organisms metabolically produce butyrate

of human and canine IBD.

104 Inflammatory Bowel Disease

underway

*2.6.8. Prebiotics*

is what VSL#3 is classified as) to a "biologic" [301, 310].

Many different mouse models have been utilized in IBD research to elucidate the roles that bacteria, genetics, the immune response, and environment play in the induction and mainte‐ nance of IBD [323]. They fall into two main categories: chemically-induced and geneticallyengineered models. The composition of the microbiota in the various mouse models is also discussed. Regardless of the strain of mouse employed in chemically-induced or geneticallyengineered models, there are only five options for its microbiota—conventional, specific pathogen free, restricted, gnotobiotic, or germ-free. Note that the word gnotobiotic (gnostos– "known" bios –"life") indicates that all the organisms present, regardless of the numbers of species, are known and does not apply only to mice that are completely devoid of microbes (i.e., germfree).

## **3.1. Chemically-induced models**

#### *3.1.1. DSS*

Dextran sulfate sodium (DSS) is formed by the esterification of dextran with chlorosulphonic acid [324]. Administered *ad libtum* in the drinking water, this compound causes enterocolitis in mice with disease severity being dependent on mouse strain, DSS molecular mass, and sulfur content [325-328]. Within three to seven days after the addition of DSS (1 to 10 % w/v) to the drinking water, mice exhibit loose stools, weight loss, and occult blood. Upon necropsy of mice treated with DSS, cecal atrophy and colonic shortening are noted, and histopathological examination reveals mucosal ulceration, inflammatory infiltrate, and hyperplastic epithelium in the colonic mucosa. It has also been reported that glandular dropout occurs prior to signs of inflammation. Although the exact mechanism of action for DSS-induced colitis is currently not known, it is thought to manifest epithelial toxicity [329, 330]. Other reports indicate that DSS increases mucosal permeability within three days of administration (before the appear‐ ance of inflammatory infiltrate) [331]. Studies have also shown that DSS is taken up by macrophages in the colon and mesenteric lymph node and by Kupffer cells in the liver [326, 330]. When macrophages become laden with DSS, they have a reduced ability to perform normal homeostatic functions such as tissue repair and phagocytosis of bacteria [332]. DSS was demonstrated to be cytotoxic to Caco-2 cells, binding to their nucleus, causing cell cycle arrest and reduced production of reactive oxygen species [333].

inflammatory cell infiltrate that produced IFN-γ [336]. In that same study, researchers found that feeding mice TNBS-haptenized colonic protein caused the mice to develop or‐ al tolerance. These mice subsequently failed to develop colitis after TNBS administration or the transfer of CD4<sup>+</sup> T cells from mice with TNBS-induced colitis. T cells from these

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107

Oxazolone is a haptenating agent that causes a disease in mice similar to UC when mixed with ethanol and administered rectally [337, 338]. SJL/J mice rapidly develop diarrhea and weight loss that peaks at day two post-administration with a 50 % mortality rate by day four. At day two, the distal half of the colon becomes hemorrhagic and edematous and histologically shows signs of superficial inflammation. There is epithelial cell erosion, gob‐ let cell depletion, edema, and inflammatory cell infiltrate composed of neutrophils and eo‐ sinophils. This is similar to what is observed microscopically in the colonic tissues of human UC patients. These mice also develop elevated levels of IL-4 and IL-5, but no IFNγ, indicating that oxazolone induces a Th2 response. Elevated levels of TGF-β are also noted and may play in a role in the induction of disease in only part of the colon. The model has also been examined for its role in determining efficacy of IBD treatments. BALB/c mice given either 5-aminosalicylic acid (5-ASA) or sodium prednisolone phos‐ phate intra-rectally prior to and during induction of oxazolone colitis had decreased se‐ verity of disease [337]. The disease is self-limiting, and the mice that survive beyond day four show increased weight gain and are healthy by days 10-12 post-administration.

In 1993, Kuhn et al. discovered that mice deficient in the anti-inflammatory cytokine IL-10 spontaneously develop enterocolitis [213]. This model has been popular in IBD research and this genetic deficiency has been crossed onto many different genetic backgrounds of mouse [339-345]. The availability of different strains has highlighted the role that genetics plays in enterocolitis, as the severity of disease is strain dependent. The order of severity from most severe to least severe is as follows: C3Bir > 129 > BALB/c or NOD/Lt > C57BL/ 6 or C57BL/10. In addition to strain differences, development of entercolitis is also de‐ pendent on the microbiota, as GF IL-10-/- do not develop enterocolitis and disease is atte‐ nuated after administration of antibiotics to IL-10-/- mice harboring a conventional microbiota [346, 347]. The lack of IL-10 does not affect the development of B or T cells, but its absence does result in a lack of regulatory T cells [213, 348]. Studies to under‐ stand the development of disease in these mice have shown that B cells (while present in high numbers in the lamina propria) are not needed for the initiation of disease, but that disease is mediated by CD4<sup>+</sup> T cells [342, 349]. Transfer studies using RAG2-/- mice as re‐ cipients have specifically shown that naive CD4<sup>+</sup> T cells are capable of inducing colitis, and CD45RBlow CD4+ T cells from IL-10-/- mice can induce disease in these RAG2-/- mice

tolerant mice secreted elevated amounts of TGF-β, IL-4, and IL-10 [336].

*3.1.3. Oxazolone*

**3.2. Genetically engineered models**

*3.2.1. IL-10-/-*

Microbial populations of the GIT are altered after DSS administration, with the microbiota of the treated mice having increased numbers of *Bacteroidaceae* and *Clostridium* spp. [326]. This result indicates that the changes to the microbiota may play a contributory role (e.g., reduction of butyrate production) in the induction of DSS-induced disease. However, DSS induces more severe colitis and increased mortality in GF mice as compared to their conventional counter‐ parts [334, 335]. GF mice given either 5% or 1% DSS died at days three or 14, respectively, after the start of DSS administration [335]. Collectively, these studies indicate that the composition of the microbiota may influence the sensitivity of mice to DSS-induced colitis as well as support the idea that the resident microbiota affords cytoprotective benefits for the host.

#### *3.1.2. TNBS (2,4,6,-trinitrobenzne sulfonic acid)*

TNBS is a haptenating agent that causes a disease similar to CD when mixed with etha‐ nol and administered rectally as an enema. Mice treated with TNBS develop a pan-colitis with the peak of clinical signs, such as diarrhea and rectal prolapse, occurring two to four weeks post-administration [112]. Microscopically, transmural inflammation is noted along with neutrophil infiltration, loss of goblet cells, edema, and granulomas. T cells isolated from the lamina propria secrete elevated levels of IFN-γ and IL-2 following stimulation with anti-CD3 and anti-CD28. However, administration of anti-IL-12 antibod‐ ies after induction of TNBS-induced colitis reduced the disease severity, and treated mice also showed reduced production of IFN-γ [112]. Further studies have shown that CD4+ T cells recovered from mice with TNBS-induced colitis could induce mild colitis when adoptively transferred into naive control mice; the colitic lesions were characterized by inflammatory cell infiltrate that produced IFN-γ [336]. In that same study, researchers found that feeding mice TNBS-haptenized colonic protein caused the mice to develop or‐ al tolerance. These mice subsequently failed to develop colitis after TNBS administration or the transfer of CD4<sup>+</sup> T cells from mice with TNBS-induced colitis. T cells from these tolerant mice secreted elevated amounts of TGF-β, IL-4, and IL-10 [336].

### *3.1.3. Oxazolone*

**3.1. Chemically-induced models**

Dextran sulfate sodium (DSS) is formed by the esterification of dextran with chlorosulphonic acid [324]. Administered *ad libtum* in the drinking water, this compound causes enterocolitis in mice with disease severity being dependent on mouse strain, DSS molecular mass, and sulfur content [325-328]. Within three to seven days after the addition of DSS (1 to 10 % w/v) to the drinking water, mice exhibit loose stools, weight loss, and occult blood. Upon necropsy of mice treated with DSS, cecal atrophy and colonic shortening are noted, and histopathological examination reveals mucosal ulceration, inflammatory infiltrate, and hyperplastic epithelium in the colonic mucosa. It has also been reported that glandular dropout occurs prior to signs of inflammation. Although the exact mechanism of action for DSS-induced colitis is currently not known, it is thought to manifest epithelial toxicity [329, 330]. Other reports indicate that DSS increases mucosal permeability within three days of administration (before the appear‐ ance of inflammatory infiltrate) [331]. Studies have also shown that DSS is taken up by macrophages in the colon and mesenteric lymph node and by Kupffer cells in the liver [326, 330]. When macrophages become laden with DSS, they have a reduced ability to perform normal homeostatic functions such as tissue repair and phagocytosis of bacteria [332]. DSS was demonstrated to be cytotoxic to Caco-2 cells, binding to their nucleus, causing cell cycle

Microbial populations of the GIT are altered after DSS administration, with the microbiota of the treated mice having increased numbers of *Bacteroidaceae* and *Clostridium* spp. [326]. This result indicates that the changes to the microbiota may play a contributory role (e.g., reduction of butyrate production) in the induction of DSS-induced disease. However, DSS induces more severe colitis and increased mortality in GF mice as compared to their conventional counter‐ parts [334, 335]. GF mice given either 5% or 1% DSS died at days three or 14, respectively, after the start of DSS administration [335]. Collectively, these studies indicate that the composition of the microbiota may influence the sensitivity of mice to DSS-induced colitis as well as support

TNBS is a haptenating agent that causes a disease similar to CD when mixed with etha‐ nol and administered rectally as an enema. Mice treated with TNBS develop a pan-colitis with the peak of clinical signs, such as diarrhea and rectal prolapse, occurring two to four weeks post-administration [112]. Microscopically, transmural inflammation is noted along with neutrophil infiltration, loss of goblet cells, edema, and granulomas. T cells isolated from the lamina propria secrete elevated levels of IFN-γ and IL-2 following stimulation with anti-CD3 and anti-CD28. However, administration of anti-IL-12 antibod‐ ies after induction of TNBS-induced colitis reduced the disease severity, and treated mice also showed reduced production of IFN-γ [112]. Further studies have shown that CD4+ T cells recovered from mice with TNBS-induced colitis could induce mild colitis when adoptively transferred into naive control mice; the colitic lesions were characterized by

the idea that the resident microbiota affords cytoprotective benefits for the host.

arrest and reduced production of reactive oxygen species [333].

*3.1.2. TNBS (2,4,6,-trinitrobenzne sulfonic acid)*

*3.1.1. DSS*

106 Inflammatory Bowel Disease

Oxazolone is a haptenating agent that causes a disease in mice similar to UC when mixed with ethanol and administered rectally [337, 338]. SJL/J mice rapidly develop diarrhea and weight loss that peaks at day two post-administration with a 50 % mortality rate by day four. At day two, the distal half of the colon becomes hemorrhagic and edematous and histologically shows signs of superficial inflammation. There is epithelial cell erosion, gob‐ let cell depletion, edema, and inflammatory cell infiltrate composed of neutrophils and eo‐ sinophils. This is similar to what is observed microscopically in the colonic tissues of human UC patients. These mice also develop elevated levels of IL-4 and IL-5, but no IFNγ, indicating that oxazolone induces a Th2 response. Elevated levels of TGF-β are also noted and may play in a role in the induction of disease in only part of the colon. The model has also been examined for its role in determining efficacy of IBD treatments. BALB/c mice given either 5-aminosalicylic acid (5-ASA) or sodium prednisolone phos‐ phate intra-rectally prior to and during induction of oxazolone colitis had decreased se‐ verity of disease [337]. The disease is self-limiting, and the mice that survive beyond day four show increased weight gain and are healthy by days 10-12 post-administration.

## **3.2. Genetically engineered models**

## *3.2.1. IL-10-/-*

In 1993, Kuhn et al. discovered that mice deficient in the anti-inflammatory cytokine IL-10 spontaneously develop enterocolitis [213]. This model has been popular in IBD research and this genetic deficiency has been crossed onto many different genetic backgrounds of mouse [339-345]. The availability of different strains has highlighted the role that genetics plays in enterocolitis, as the severity of disease is strain dependent. The order of severity from most severe to least severe is as follows: C3Bir > 129 > BALB/c or NOD/Lt > C57BL/ 6 or C57BL/10. In addition to strain differences, development of entercolitis is also de‐ pendent on the microbiota, as GF IL-10-/- do not develop enterocolitis and disease is atte‐ nuated after administration of antibiotics to IL-10-/- mice harboring a conventional microbiota [346, 347]. The lack of IL-10 does not affect the development of B or T cells, but its absence does result in a lack of regulatory T cells [213, 348]. Studies to under‐ stand the development of disease in these mice have shown that B cells (while present in high numbers in the lamina propria) are not needed for the initiation of disease, but that disease is mediated by CD4<sup>+</sup> T cells [342, 349]. Transfer studies using RAG2-/- mice as re‐ cipients have specifically shown that naive CD4<sup>+</sup> T cells are capable of inducing colitis, and CD45RBlow CD4+ T cells from IL-10-/- mice can induce disease in these RAG2-/- mice [348, 349]. This latter observation implicates IL-10 as a central mediator of regulatory T cells (CD25+ Foxp3+ CD4+ T cells). It has also been shown that IL-12 and IFN-γ are need‐ ed for initiation but not the continuation of colitis [342, 350]. The increase in the produc‐ tion of IL-12 and IFN-γ along with undetectable levels of IL-4 indicates that the enterocolitis in these mice is mediated by a Th1 immune response, similar to that ob‐ served in humans with IBD [342, 346].

large intestine. The mucosa, submucosa, and muscularis all presented with inflammatory cell infiltrates (macrophages and CD4+ T cells predominately), and there was also a loss of goblet cells, epithelial cell hyperplasia, and deep fissure ulcers. Elevated levels of IFNγ were present in these mice, and treatment with anti-IFN-γ or anti-TNF (α and β) anti‐ bodies ameliorated disease in these mice. However, the protection garnered by using the antibodies was not as great as when CD45RBlo CD4+ T cells were transferred along with CD45RBhi CD4+ T cells [362]. This disease type mimics CD with respect to the type of in‐ flammatory response generated (Th1) and the type of intestinal inflammation present (transmural infiltrate of CD4+ T cells) [365]. There is also a definite microbial component in this model of disease, as restricted microbiota mice have less severe disease as com‐

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Although not considered a microbial cause of IBD, the presence of *Helicobacter* spp. does adjuvant or predispose mice to the onset of colitis in some models. In our lab, we utilize a dual hit model of colitis consisting of both *Helicobacter bilis* colonization and low-dose (1.5%) DSS

**Figure 8.** To increase sensitivity to a colitic insult, ASF-bearing C3H/HeN:Tac mice were colonized with a bacterial pro‐

vocateur, *Helicobater bilis*. An otherwise non-colitic low dose of DSS is then administered resulting in colitis.

pared to their SPF counterparts [364].

to elicit colitis, as shown in Figure 8 [366].

**3.4. Bacterial-induced models**

*3.4.1. Helicobacter spp.*

## *3.2.2. Mdr1a-/-*

In 1994, mice lacking the gene *mdr1a* were generated. This gene encodes a P-glycoprotein, which is a drug efflux pump, thus protecting host cells from the build-up of toxic compounds. It was noted later that these mice spontaneously develop a colitis similar to human IBD and that disease can be exacerbated by colonization with *Helicobacter bilis* [351, 352]. These *mdr1a-/* mice have reduced growth rates compared to their WT counterparts and their histological lesions begin in the proximal colon and proceed distally as disease severity progresses. *Mdr1a-/* males are more susceptible to the onset of severe disease than females [353, 354]. *Mdr1a-/-* mice also have increased epithelial permeability and reduced phosphorylation of both occludin and ZO-1, tight junction proteins, compared to WT counterparts [354]. They exhibit increased bacterial translocation with bacteria detected in both the spleen and lymph nodes that correlates with disease severity. Therefore, the induction of disease is associated with a defect in the epithelial barrier function of the GI tract. In addition to the similar disease progression found in IBD patients, studying these mice are of interest because the human MDR1 gene has been mapped on a loci that that is associated with susceptibility of IBD, although this rela‐ tionship is under debate [355-358].

## *3.2.3. TRUC*

TRUC mice are both T-bet-/- and RAG-/- and spontaneously develop colitis by four weeks of age [359, 360]. T-bet (T-box expressed in T cells) is a transcription factor that aids in the development of a Th1 response [359]. These mice have increased permeability of their colonic epithelium that increases with age and increased rate of epithelial cell death. Microscopically, there is inflammatory cell infiltrate, goblet cell dropout, crypt loss, and the presences of ulcers. The only cytokine elevated in these mice is TNF-α and disease can be ameliorated using an anti-TNF-α antibody. The microbiota also contributes to disease in this model, as treatment with antibiotics was able to "cure" the mice of their colitis. Additionally, the TRUC colitic microbiota can be horizontally transferred to both WT and RAG-/- mice, and 16S rRNA analysis of feces from TRUC mice revealed that the presence of *Klebsiella pneumoniae* and *Proteus mirabilis* correlate with colitis. Interestingly, GF TRUC mice colonized with these two organ‐ isms alone do not develop colitis unless a more complete microbiota is present [361].

#### **3.3. CD45RBhi CD4+ T-cell transfer model**

C.B.-17 *scid* mice administered CD45RBhi CD4+ T cells develop a wasting disease which is not seen if CD45RBlo CD4+ T cells or CD45RBhi CD8+ T cells are adoptively transferred [362-364]. Disease occurs three to five weeks post-administration and is limited to the large intestine. The mucosa, submucosa, and muscularis all presented with inflammatory cell infiltrates (macrophages and CD4+ T cells predominately), and there was also a loss of goblet cells, epithelial cell hyperplasia, and deep fissure ulcers. Elevated levels of IFNγ were present in these mice, and treatment with anti-IFN-γ or anti-TNF (α and β) anti‐ bodies ameliorated disease in these mice. However, the protection garnered by using the antibodies was not as great as when CD45RBlo CD4+ T cells were transferred along with CD45RBhi CD4+ T cells [362]. This disease type mimics CD with respect to the type of in‐ flammatory response generated (Th1) and the type of intestinal inflammation present (transmural infiltrate of CD4+ T cells) [365]. There is also a definite microbial component in this model of disease, as restricted microbiota mice have less severe disease as com‐ pared to their SPF counterparts [364].

#### **3.4. Bacterial-induced models**

## *3.4.1. Helicobacter spp.*

[348, 349]. This latter observation implicates IL-10 as a central mediator of regulatory T cells (CD25+ Foxp3+ CD4+ T cells). It has also been shown that IL-12 and IFN-γ are need‐ ed for initiation but not the continuation of colitis [342, 350]. The increase in the produc‐ tion of IL-12 and IFN-γ along with undetectable levels of IL-4 indicates that the enterocolitis in these mice is mediated by a Th1 immune response, similar to that ob‐

In 1994, mice lacking the gene *mdr1a* were generated. This gene encodes a P-glycoprotein, which is a drug efflux pump, thus protecting host cells from the build-up of toxic compounds. It was noted later that these mice spontaneously develop a colitis similar to human IBD and that disease can be exacerbated by colonization with *Helicobacter bilis* [351, 352]. These *mdr1a-/* mice have reduced growth rates compared to their WT counterparts and their histological lesions begin in the proximal colon and proceed distally as disease severity progresses. *Mdr1a-/* males are more susceptible to the onset of severe disease than females [353, 354]. *Mdr1a-/-* mice also have increased epithelial permeability and reduced phosphorylation of both occludin and ZO-1, tight junction proteins, compared to WT counterparts [354]. They exhibit increased bacterial translocation with bacteria detected in both the spleen and lymph nodes that correlates with disease severity. Therefore, the induction of disease is associated with a defect in the epithelial barrier function of the GI tract. In addition to the similar disease progression found in IBD patients, studying these mice are of interest because the human MDR1 gene has been mapped on a loci that that is associated with susceptibility of IBD, although this rela‐

TRUC mice are both T-bet-/- and RAG-/- and spontaneously develop colitis by four weeks of age [359, 360]. T-bet (T-box expressed in T cells) is a transcription factor that aids in the development of a Th1 response [359]. These mice have increased permeability of their colonic epithelium that increases with age and increased rate of epithelial cell death. Microscopically, there is inflammatory cell infiltrate, goblet cell dropout, crypt loss, and the presences of ulcers. The only cytokine elevated in these mice is TNF-α and disease can be ameliorated using an anti-TNF-α antibody. The microbiota also contributes to disease in this model, as treatment with antibiotics was able to "cure" the mice of their colitis. Additionally, the TRUC colitic microbiota can be horizontally transferred to both WT and RAG-/- mice, and 16S rRNA analysis of feces from TRUC mice revealed that the presence of *Klebsiella pneumoniae* and *Proteus mirabilis* correlate with colitis. Interestingly, GF TRUC mice colonized with these two organ‐

isms alone do not develop colitis unless a more complete microbiota is present [361].

C.B.-17 *scid* mice administered CD45RBhi CD4+ T cells develop a wasting disease which is not seen if CD45RBlo CD4+ T cells or CD45RBhi CD8+ T cells are adoptively transferred [362-364]. Disease occurs three to five weeks post-administration and is limited to the

 **T-cell transfer model**

served in humans with IBD [342, 346].

tionship is under debate [355-358].

*3.2.2. Mdr1a-/-*

108 Inflammatory Bowel Disease

*3.2.3. TRUC*

**3.3. CD45RBhi CD4+**

Although not considered a microbial cause of IBD, the presence of *Helicobacter* spp. does adjuvant or predispose mice to the onset of colitis in some models. In our lab, we utilize a dual hit model of colitis consisting of both *Helicobacter bilis* colonization and low-dose (1.5%) DSS to elicit colitis, as shown in Figure 8 [366].

**Figure 8.** To increase sensitivity to a colitic insult, ASF-bearing C3H/HeN:Tac mice were colonized with a bacterial pro‐ vocateur, *Helicobater bilis*. An otherwise non-colitic low dose of DSS is then administered resulting in colitis.

mice compared to the uninfected control *mdr1a-/-* mice [352]. The disease phenotypes between spontaneous versus *Helicobacter*-induced colitis in *mdr1a-/-* mice were different, with epithelial ulceration not present in the *Helicobacter-*induced colitis. When *mdr1a-/-* mice were co-colonized with both *Helicobacter* species, the morbidity and mortality rate was between that of mice colonized by either *H. bilis* or *H. hepaticus* and the colitis that developed was characterized by dysplasia [372]. *H. bilis* was also able to out compete *H. hepaticus in vivo* as evidenced by recovery of higher numbers of *H. bilis*, suggesting that these two species may compete for

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Additionally, colonization of IL-10-/- C57BL/6 mice with *Helicobacter* species results in the induction of colitis in otherwise disease-free mice [373]. It was also shown that the onset and severity of colitis was species specific in relation to colonization by *H. bilis* or *H. hepaticus* [374]. With respect to C3Bir.129 IL-10-/- mice, the presence of *Helicobacter* spp. is required for the spontaneous onset of disease [339]. Further studies show that colonization of RAG1-/- mice with *Helicobacter* species fails to elicit clinical signs of disease after nine months of colonization and only very mild colonic inflammation was detected at necropsy [374]. Taken together, these observations indicate that disease induction can be mediated by an aberrant adaptive immune response initiated by bacterial provocateurs entering an otherwise stable host-microbe

Similar to the need for the a resident microbiota in the TRUC model of colitis, mice colonized with *Brachyspira hyodysenteriae* also require the presence of a microbiota for the induction of typhlocolitis. *B. hyodysenteriae* is an anaerobic spirochete that is the causative agent of swine dysentery [375]. While the pathogenesis of *B. hyodysenteriae* is associated with the production of a ß-hemolysin [376], disease does not develop in the absence of a resident microbiota as was demonstrated by the inoculation of germfree pigs [377-379]. The importance of the microbiota was further demonstrated when the microbiota of C3H/HeSnJ or BALB/c mice was depleted by adding a cocktail of antibiotics (rifampicin, colistin, spectinomycin, spiramycin, and vancomycin) to their drinking water. As can be seen in Figure 10, the antibiotic cocktail depleted the numbers of Gram-positive, Gram-negative, and strict anaerobes by as much as 5 to 7 log10 (Nibbelink and Wannemuehler, unpublished observations). On day seven, antibiotic treated and sham treated mice were inoculated with 1 x 108 *B. hyodysenteirae* strain B204 and severity of disease was evaluated at 5, 10, and 15 days post-infection (DPI). As can be seen in Figure 11, the sham-treated mice developed severe typhlocolitis while the antibiotic treated mice had no lesions. At 15 DPI, the antibiotics were withdrawn from the drinking water of the remaining *B. hyodysenteriae-*infected mice to allow the microbiota to recover. On day 30 PI, the mice that had been treated with antibiotics through 15 DPI now presented with severe typhlocolitis. The presence and severity of typhlocolitis in these mice correlated with the presence of TNF-specific mRNA in the cecal tissue of the *B. hyodysenteriae-*infected mice (data not shown). Lastly, *B. hyodysenteriae-*induced typhlocolitis could also be prevented when the host's inflammatory response was inhibited [380, 381]. Collectively, these observations indicate that certain bacterial provocateurs (*K. pneumoniae, P. mirabilis,* and *B. hyodysenteriae)*

similar niches in the GIT.

environment.

*3.4.2. Brachyspira hyodysenteriae*

**Figure 9.** Mice colonized with *Helicobacter bilis* and subsequently administered a low dose of DSS exhibit colitis that is not seen with *H. bilis* or DSS alone. Disease is characterized grossly by colonic shortening, edema, cecal atrophy, en‐ larged lymphoid aggregates, and blood present in the contents. Microscopically, the disease presents with inflamma‐ tory cell infiltrate, crypt hyperplasia, glandular dropout, and cellular erosion

It has been noted that colonization of mice with *H. bilis* alone can change mucosal gene expression and alter the immune response in C3H/HeN mice bearing the altered Schaedler's flora (ASF) [366, 369]. Genes involved in T cell receptor signaling, the survival and activation of peripheral B cells, and chemotaxis are a few examples of the host genes that were upregu‐ lated by *H. bilis* colonization. Genes that were down regulated were involved in fatty acid metabolism and detoxification. After *H. bilis* colonization, serum antibodies directed at antigens derived from members of the ASF are induced [366, 369]. *H. hepaticus* colonization of A/JCr mice also induced changes in gene expression in the cecum, with female mice being more susceptible to the onset of disease [370, 371].

It appears that an over zealous host response to the introduction of the novel organism (i.e., provocateur) predisposes certain strains of mice for the onset of typholocoltiis following a secondary colitic insult. In a study comparing A/JCr mice (that develop mild inflammation) to C57BL/6 (who do not develop inflammation) mice, cecal gene expression profiles revealed that A/JCr mice had more genes differentially regulated (176) compared to C57BL/6 (80). Differ‐ entially expressed genes were predominantly those associated with immune response, chemotaxis, signal transduction, and antigen processing in the A/JCr mice while the genes upregulated in the C57BL/6 mice were predominately associated with immunoglobulin production.

In the *mdr1a-/-* mouse, it is not simply the presence of *Helicobacter* that results in the induction of colitis but the specific species of *Helicobacter* influences the induction of a differential immune response. In *mdr1a-/-* mice, *H. bilis* colonization causes colitis as early as three weeks post-infection, whereas *H. hepaticus* colonization ameliorated the severity of colitis in these mice compared to the uninfected control *mdr1a-/-* mice [352]. The disease phenotypes between spontaneous versus *Helicobacter*-induced colitis in *mdr1a-/-* mice were different, with epithelial ulceration not present in the *Helicobacter-*induced colitis. When *mdr1a-/-* mice were co-colonized with both *Helicobacter* species, the morbidity and mortality rate was between that of mice colonized by either *H. bilis* or *H. hepaticus* and the colitis that developed was characterized by dysplasia [372]. *H. bilis* was also able to out compete *H. hepaticus in vivo* as evidenced by recovery of higher numbers of *H. bilis*, suggesting that these two species may compete for similar niches in the GIT.

Additionally, colonization of IL-10-/- C57BL/6 mice with *Helicobacter* species results in the induction of colitis in otherwise disease-free mice [373]. It was also shown that the onset and severity of colitis was species specific in relation to colonization by *H. bilis* or *H. hepaticus* [374]. With respect to C3Bir.129 IL-10-/- mice, the presence of *Helicobacter* spp. is required for the spontaneous onset of disease [339]. Further studies show that colonization of RAG1-/- mice with *Helicobacter* species fails to elicit clinical signs of disease after nine months of colonization and only very mild colonic inflammation was detected at necropsy [374]. Taken together, these observations indicate that disease induction can be mediated by an aberrant adaptive immune response initiated by bacterial provocateurs entering an otherwise stable host-microbe environment.

## *3.4.2. Brachyspira hyodysenteriae*

**Figure 9.** Mice colonized with *Helicobacter bilis* and subsequently administered a low dose of DSS exhibit colitis that is not seen with *H. bilis* or DSS alone. Disease is characterized grossly by colonic shortening, edema, cecal atrophy, en‐ larged lymphoid aggregates, and blood present in the contents. Microscopically, the disease presents with inflamma‐

It has been noted that colonization of mice with *H. bilis* alone can change mucosal gene expression and alter the immune response in C3H/HeN mice bearing the altered Schaedler's flora (ASF) [366, 369]. Genes involved in T cell receptor signaling, the survival and activation of peripheral B cells, and chemotaxis are a few examples of the host genes that were upregu‐ lated by *H. bilis* colonization. Genes that were down regulated were involved in fatty acid metabolism and detoxification. After *H. bilis* colonization, serum antibodies directed at antigens derived from members of the ASF are induced [366, 369]. *H. hepaticus* colonization of A/JCr mice also induced changes in gene expression in the cecum, with female mice being

It appears that an over zealous host response to the introduction of the novel organism (i.e., provocateur) predisposes certain strains of mice for the onset of typholocoltiis following a secondary colitic insult. In a study comparing A/JCr mice (that develop mild inflammation) to C57BL/6 (who do not develop inflammation) mice, cecal gene expression profiles revealed that A/JCr mice had more genes differentially regulated (176) compared to C57BL/6 (80). Differ‐ entially expressed genes were predominantly those associated with immune response, chemotaxis, signal transduction, and antigen processing in the A/JCr mice while the genes upregulated in the C57BL/6 mice were predominately associated with immunoglobulin

In the *mdr1a-/-* mouse, it is not simply the presence of *Helicobacter* that results in the induction of colitis but the specific species of *Helicobacter* influences the induction of a differential immune response. In *mdr1a-/-* mice, *H. bilis* colonization causes colitis as early as three weeks post-infection, whereas *H. hepaticus* colonization ameliorated the severity of colitis in these

tory cell infiltrate, crypt hyperplasia, glandular dropout, and cellular erosion

more susceptible to the onset of disease [370, 371].

production.

110 Inflammatory Bowel Disease

Similar to the need for the a resident microbiota in the TRUC model of colitis, mice colonized with *Brachyspira hyodysenteriae* also require the presence of a microbiota for the induction of typhlocolitis. *B. hyodysenteriae* is an anaerobic spirochete that is the causative agent of swine dysentery [375]. While the pathogenesis of *B. hyodysenteriae* is associated with the production of a ß-hemolysin [376], disease does not develop in the absence of a resident microbiota as was demonstrated by the inoculation of germfree pigs [377-379]. The importance of the microbiota was further demonstrated when the microbiota of C3H/HeSnJ or BALB/c mice was depleted by adding a cocktail of antibiotics (rifampicin, colistin, spectinomycin, spiramycin, and vancomycin) to their drinking water. As can be seen in Figure 10, the antibiotic cocktail depleted the numbers of Gram-positive, Gram-negative, and strict anaerobes by as much as 5 to 7 log10 (Nibbelink and Wannemuehler, unpublished observations). On day seven, antibiotic treated and sham treated mice were inoculated with 1 x 108 *B. hyodysenteirae* strain B204 and severity of disease was evaluated at 5, 10, and 15 days post-infection (DPI). As can be seen in Figure 11, the sham-treated mice developed severe typhlocolitis while the antibiotic treated mice had no lesions. At 15 DPI, the antibiotics were withdrawn from the drinking water of the remaining *B. hyodysenteriae-*infected mice to allow the microbiota to recover. On day 30 PI, the mice that had been treated with antibiotics through 15 DPI now presented with severe typhlocolitis. The presence and severity of typhlocolitis in these mice correlated with the presence of TNF-specific mRNA in the cecal tissue of the *B. hyodysenteriae-*infected mice (data not shown). Lastly, *B. hyodysenteriae-*induced typhlocolitis could also be prevented when the host's inflammatory response was inhibited [380, 381]. Collectively, these observations indicate that certain bacterial provocateurs (*K. pneumoniae, P. mirabilis,* and *B. hyodysenteriae)*

may fail to induce disease in the absence of an appropriate resident microbiota. As depicted in Figure 12, the etiology of colitis is complex and may require the presence of a microbial provocateur, the resident microbiota, and a host inflammatory response.

Laboratories [54]. This lack of consistency in microbiota from mice of the same strain has even been identified at different facilities from the same vendor (Overstreet and Wannemuehler unpublished observation). Therefore, comparison of studies evaluating the microbiota of mice are difficult because there are hundreds of unknown bacterial species present in the murine microbiota and there is no standardized microbiota used by investigators. The advent of nextgen sequencing has helped to alleviate some of this challenge as all of the organisms in the gut can be identified based on 16S rRNA sequences. However, it still does not resolve the issue related to the use of disparate strains of mice with varying microbiota from different suppliers.

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**Figure 11.** Assessment of *Brachyspira hyodysenteriae-*induced typhlocolitis in C3H/HeSnJ mice treated with an antibi‐ otic cocktail. While *B. hyodysenteriae* colonized the antibiotic-treated mice to the same extent as it did the sham-treat‐ ed mice, there was no evidence of typhlocolitis in the antibiotic treated mice through 15 days post-infection (DPI). However, 15 days after the antibiotics were withdrawn (30 DPI), the mice that had been treated with the antibiotics

had developed severe disease once the microbiota recovered.

**Figure 10.** To assess the role of the microbiota in *Brachyspira hyodysenteriae-*induced typhlocolitis, C3H/HeSnJ mice were treated with an antibiotic cocktail to deplete the resident microbiota. Six days later, mice were infected with *B. hyodysenteriae.* The data indicate that there was a five to seven log10 reduction in the resident microbiota.

## **3.5. Conventional mice**

The majority of commercially available mice harbor a "conventional" microbiota. This simply means that the composition of the community is unknown. There are different types of conventionally-reared mice. For example, Taconic Farms maintain two types of conventional microbiota mice, restricted flora™ (RF) and murine pathogen free™ (PF). RF mice are not colonized by β-hemolytic *Streptococcus* species, *K. pneumoniae, K. oxytoca, Pseudomonas aerugi‐ nosa,* or *Staphylococcus aureus.* Mice that are PF are *Helicobacter* free but can contain organisms not found in RF mice [382]. Additionally, mice on the same background purchased from different vendors can harbor different microbiota, as highlighted in work by Ivanov and colleagues demonstrating the presence of SFB in C57BL/6 mice from Taconic but not Jackson Laboratories [54]. This lack of consistency in microbiota from mice of the same strain has even been identified at different facilities from the same vendor (Overstreet and Wannemuehler unpublished observation). Therefore, comparison of studies evaluating the microbiota of mice are difficult because there are hundreds of unknown bacterial species present in the murine microbiota and there is no standardized microbiota used by investigators. The advent of nextgen sequencing has helped to alleviate some of this challenge as all of the organisms in the gut can be identified based on 16S rRNA sequences. However, it still does not resolve the issue related to the use of disparate strains of mice with varying microbiota from different suppliers.

may fail to induce disease in the absence of an appropriate resident microbiota. As depicted in Figure 12, the etiology of colitis is complex and may require the presence of a microbial

**Figure 10.** To assess the role of the microbiota in *Brachyspira hyodysenteriae-*induced typhlocolitis, C3H/HeSnJ mice were treated with an antibiotic cocktail to deplete the resident microbiota. Six days later, mice were infected with *B.*

The majority of commercially available mice harbor a "conventional" microbiota. This simply means that the composition of the community is unknown. There are different types of conventionally-reared mice. For example, Taconic Farms maintain two types of conventional microbiota mice, restricted flora™ (RF) and murine pathogen free™ (PF). RF mice are not colonized by β-hemolytic *Streptococcus* species, *K. pneumoniae, K. oxytoca, Pseudomonas aerugi‐ nosa,* or *Staphylococcus aureus.* Mice that are PF are *Helicobacter* free but can contain organisms not found in RF mice [382]. Additionally, mice on the same background purchased from different vendors can harbor different microbiota, as highlighted in work by Ivanov and colleagues demonstrating the presence of SFB in C57BL/6 mice from Taconic but not Jackson

*hyodysenteriae.* The data indicate that there was a five to seven log10 reduction in the resident microbiota.

**3.5. Conventional mice**

112 Inflammatory Bowel Disease

provocateur, the resident microbiota, and a host inflammatory response.

**Figure 11.** Assessment of *Brachyspira hyodysenteriae-*induced typhlocolitis in C3H/HeSnJ mice treated with an antibi‐ otic cocktail. While *B. hyodysenteriae* colonized the antibiotic-treated mice to the same extent as it did the sham-treat‐ ed mice, there was no evidence of typhlocolitis in the antibiotic treated mice through 15 days post-infection (DPI). However, 15 days after the antibiotics were withdrawn (30 DPI), the mice that had been treated with the antibiotics had developed severe disease once the microbiota recovered.

Although the information gathered from these studies has been useful in analyzing the immune response to specific organisms, trying to relate the resultant disease to that char‐ acteristic of IBD is marginal at best because of the differences in the complexities of the microbiota. IBD itself is a multi-factorial disease and it has been fairly well-established that the role of bacteria in IBD is associated with a shift in community dynamics (i.e., dysbiosis) and not the presence/absence of one particular species. A perfect example of this complexity is the fact that *K. pneumonia* and *P. mirabilis* fail to induce colitis in GF

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Also referred to as gnotobiotic, defined microbiota (DM) mice have a microbiota in which all members are known and are housed in flexible film isolators to maintain this status [388]. One of the most established DM mouse models harbors the "Altered Schaedler Flora" (ASF). Developed by Dr. Rodger Orcutt and colleagues as a request from the National Cancer Institute, these eight microbial species were originally used to standardized the microbiota of the rodents used as founders in their breeding colonies [389]. He chose to modify his mentor's (Dr. Russ Schaedler) cocktail of organisms by eliminating facultative anaerobes (*Escherichia coli* var. mutabilis and *Streptococcus fecalis*) and the anaerobic *Streptococcus* and *Clostridia* spp. that formed the original "Schaedler Flora". Dr. Orcutt added four additional species (see Table 4 below). This resulted in an anaerobic community devoid of any cocci or spore-forming blunt ended rods, which comprise the main isolator contaminants, making it easier to monitor for contamination of the gnotobiotic isolator. This new community was dubbed the "Altered Schaedler Flora" and was utilized in the breeding stock by major commercial mouse vendors in the US in that era. The ASF consists of members with different morphologies so that identification using fecal smears for microscopic evaluation was possible. The members, whose full genomes have yet to be sequenced, have had 16S rRNA sequencing performed to assist

Multiple studies have demonstrated the stability of this community both when maintained under gnotobiotic housing conditions or when part of a conventionalized microbiota [391-394]. In our own lab, all eight ASF members have been stably maintained in our breeding colony for over 12 years, indicating the remarkable stability of this model microbial community over time. PCR primers have been developed for each of the eight ASF members as well as groupspecific FISH probes [391, 394]. Therefore, the effects of any perturbation of the ASF, such as with antibiotics, inflammation, or CAM treatments can be monitored by bacterial abundance as well as spatial redistribution using qPCR and FISH, respectively. All of the organisms can be cultured and whole cell sonicates produced to measure the immune response to each organism individually (something that is impossible to do with a conventional microbiota). Additionally, this community, although limited in scope, is able to synthesize all the metabo‐ lites needed by the mouse and maintains near normal cecal shape and size, something not possible in GF mice. It is important to note, however, that some of the characteristics of ASF mice are more similar to GF than conventional mice. Both ASF and GF mice have high fecal tryptic activity and possess the ability to degrade mucin and β-aspartylglycine. They also

TRUC mice (Figure 12) [361].

**3.7. Defined microbiota mice**

with the identification of the organisms [390].

**Figure 12.** The pathogenesis of inflammatory bowel disease is complex. Studies from animal models indicate that the etiology of disease involves the presence of a bacterial provocateur, the resident microbiota, and the host response.

#### **3.6. Germfree mice**

GF mice are completely devoid of microbial life. As mentioned previously, this does grossly affect the anatomy of these mice, which is most evident by the enlarged cecum being the most prominent feature [34]. The discovery that many GF mouse strains that carry genetic deficiencies associated with IBD (notably the IL-10-/-) do not develop colitis has led to the popularity of GF models to study the role of bacteria in the pathogenesis of IBD [346]. To determine if an organism is capable of initiating colitis, GF mice are monoassociated with a single bacterial species and then monitored for clinical signs of disease. Some of the bacterial strains used to date to evaluate their ability to induce dis‐ ease in IL-10-/- mice are shown in Table 3.


**Table 3.** Bacterial strains used to monoassociate GF mice to examine the ability of the strain to induce colitis

Although the information gathered from these studies has been useful in analyzing the immune response to specific organisms, trying to relate the resultant disease to that char‐ acteristic of IBD is marginal at best because of the differences in the complexities of the microbiota. IBD itself is a multi-factorial disease and it has been fairly well-established that the role of bacteria in IBD is associated with a shift in community dynamics (i.e., dysbiosis) and not the presence/absence of one particular species. A perfect example of this complexity is the fact that *K. pneumonia* and *P. mirabilis* fail to induce colitis in GF TRUC mice (Figure 12) [361].

## **3.7. Defined microbiota mice**

**Figure 12.** The pathogenesis of inflammatory bowel disease is complex. Studies from animal models indicate that the etiology of disease involves the presence of a bacterial provocateur, the resident microbiota, and the host response.

GF mice are completely devoid of microbial life. As mentioned previously, this does grossly affect the anatomy of these mice, which is most evident by the enlarged cecum being the most prominent feature [34]. The discovery that many GF mouse strains that carry genetic deficiencies associated with IBD (notably the IL-10-/-) do not develop colitis has led to the popularity of GF models to study the role of bacteria in the pathogenesis of IBD [346]. To determine if an organism is capable of initiating colitis, GF mice are monoassociated with a single bacterial species and then monitored for clinical signs of disease. Some of the bacterial strains used to date to evaluate their ability to induce dis‐

> **Organism Colitis Severity Time of Disease Onset Reference** *Enterococcus faecalis* Severe 10-12 weeks p.i. [383, 384]

*Escherichia coli (*from WT SPF mouse) Moderate 3 weeks p.i. [383] *Pseudomonas flourescens* No disease - [383] *Helicobacter hepaticus* No disease - [385] *Lactococcus lactis* No disease - [384] *Viridans* group *Streptococcus* No disease - [386] *Clostridium sordellii* No disease - [386] *Bacteroides vulgatus* No disease - [386] *Bifidobacterium animalis* Mild 23 weeks p.i. [387] *Bifidobacterium infantis* No disease - [387]

**Table 3.** Bacterial strains used to monoassociate GF mice to examine the ability of the strain to induce colitis

**3.6. Germfree mice**

114 Inflammatory Bowel Disease

ease in IL-10-/- mice are shown in Table 3.

Also referred to as gnotobiotic, defined microbiota (DM) mice have a microbiota in which all members are known and are housed in flexible film isolators to maintain this status [388]. One of the most established DM mouse models harbors the "Altered Schaedler Flora" (ASF). Developed by Dr. Rodger Orcutt and colleagues as a request from the National Cancer Institute, these eight microbial species were originally used to standardized the microbiota of the rodents used as founders in their breeding colonies [389]. He chose to modify his mentor's (Dr. Russ Schaedler) cocktail of organisms by eliminating facultative anaerobes (*Escherichia coli* var. mutabilis and *Streptococcus fecalis*) and the anaerobic *Streptococcus* and *Clostridia* spp. that formed the original "Schaedler Flora". Dr. Orcutt added four additional species (see Table 4 below). This resulted in an anaerobic community devoid of any cocci or spore-forming blunt ended rods, which comprise the main isolator contaminants, making it easier to monitor for contamination of the gnotobiotic isolator. This new community was dubbed the "Altered Schaedler Flora" and was utilized in the breeding stock by major commercial mouse vendors in the US in that era. The ASF consists of members with different morphologies so that identification using fecal smears for microscopic evaluation was possible. The members, whose full genomes have yet to be sequenced, have had 16S rRNA sequencing performed to assist with the identification of the organisms [390].

Multiple studies have demonstrated the stability of this community both when maintained under gnotobiotic housing conditions or when part of a conventionalized microbiota [391-394]. In our own lab, all eight ASF members have been stably maintained in our breeding colony for over 12 years, indicating the remarkable stability of this model microbial community over time. PCR primers have been developed for each of the eight ASF members as well as groupspecific FISH probes [391, 394]. Therefore, the effects of any perturbation of the ASF, such as with antibiotics, inflammation, or CAM treatments can be monitored by bacterial abundance as well as spatial redistribution using qPCR and FISH, respectively. All of the organisms can be cultured and whole cell sonicates produced to measure the immune response to each organism individually (something that is impossible to do with a conventional microbiota). Additionally, this community, although limited in scope, is able to synthesize all the metabo‐ lites needed by the mouse and maintains near normal cecal shape and size, something not possible in GF mice. It is important to note, however, that some of the characteristics of ASF mice are more similar to GF than conventional mice. Both ASF and GF mice have high fecal tryptic activity and possess the ability to degrade mucin and β-aspartylglycine. They also cannot convert bilirubin to urobilinogens or cholesterol to coprostanol [395]. Interestingly, this same research team compared these parameters in CD patients versus healthy subjects and the characteristics of the microbial metabolism associated with the microbiota of CD patients were very similar to those of the ASF [396]. This study suggests that there is benefit to the use of the ASF in mouse models of IBD.

munity. This addition caused the reduction of the original *E. coli* to drop to undetectable levels at day two post-addition. Later *L. johnsonii* also reached undetectable levels. *B. longum* decreased initially but them returned to normal titer levels. Lastly, when the original triassociated mice were exposed to conventional mouse feces, the numbers of the original three

In this chapter, we have discussed the three most common types of microbiota available for use in IBD research. Because it is clear that IBD is associated with an imbalance in the microbial community, the use of GF mice (which cannot mimic a "community" dynamic) may be less useful in unraveling the complexities of a multifactorial disease in place of defined microbiota and conventional microbiota mice. Ideally, one would want to use a simplified community (such as the ASF) where the actions of all organisms can be assessed. To understand the dynamic interactions that occur between microbes and between the microbes and the host, it will be important to start with what is known in order to begin the unraveling the enigmatic

, Amanda E. Ramer-Tait3

1 Department of Veterinary Microbiology and Preventive Medicine, Iowa State University,

3 Department of Food Science and Technology, University of Nebraska-Lincoln, Lincoln,

[1] Whitman, W.B., Coleman, D.C., and Wiebe, W.J., Prokaryotes: the unseen majority. Proceedings of the National Academy of Sciences of the United States of America,

[2] Savage, D.C., Microbial ecology of the gastrointestinal tract. Annual Review of Mi‐

[3] Zhu, B., Wang, X., and Li, L., Human gut microbiome: the second genome of human

[4] Structure, function and diversity of the healthy human microbiome. Nature, 2012.

2 Department of Veterinary Clinical Sciences, Iowa State University, Ames, IA, USA

, Albert E. Jergens2

and

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117

organisms decreased.

**Author details**

Ames, IA, USA

NE, USA

**References**

1998. 95(12): p. 6578-83.

486(7402): p. 207-14.

crobiology, 1977. 31: p. 107-33.

body. Protein & Cell, 2010. 1(8): p. 718-25.

Anne-Marie C. Overstreet1

Michael J. Wannemuehler1

nature of gut health and disease.

Other defined microbiota mouse studies have been published [397-400]. A study using ten bacterial species specifically chosen for their metabolic function were used to colonize GF mice [397]. Using microbial RNA-seq, the authors were able to build a model relating perturbation of the microbial community to changes in diet. By modeling the functional capacity of a gnotobiotic community under different conditions, this model and similar approaches can be used to unravel the operational dynamics of the gut microbiome with respect to nutrient utilization such that the microbiota might be manipulated to improve human and animal health [397]. Another study colonized mice with *E. rectale* and *B. thetaiotamicron*, and the authors then assessed changes in bacterial gene expression using Affymetrix GeneChips to show that the cross-talk between these two organisms affected up- and down-regulated genes in response to one another.


**Table 4.** Members of the altered Schaedler flora

Three human commensals, *E. coli, B. longum,* and *L. johnsonii,* have also been used to colonize GF mice to create a gnotobiotic community [398]. This community was used to identify the effects that the introduction of novel organisms has on the community dynamic. When colonized with a second *Lactobacillus, L. paracasei,* it was noted that both *Lactobacillus* spp. were able to co-habitate reaching similar fecal titers. However, that was not the same when a fifth organism was added, a second *B. longum* strain. This new strain was only maintained in the mouse at detectable levels for three days. Lastly, a second *E. coli* strain was added to the original community of three. This new *E. coli* reached the highest titer of any organism in the com‐ munity. This addition caused the reduction of the original *E. coli* to drop to undetectable levels at day two post-addition. Later *L. johnsonii* also reached undetectable levels. *B. longum* decreased initially but them returned to normal titer levels. Lastly, when the original triassociated mice were exposed to conventional mouse feces, the numbers of the original three organisms decreased.

In this chapter, we have discussed the three most common types of microbiota available for use in IBD research. Because it is clear that IBD is associated with an imbalance in the microbial community, the use of GF mice (which cannot mimic a "community" dynamic) may be less useful in unraveling the complexities of a multifactorial disease in place of defined microbiota and conventional microbiota mice. Ideally, one would want to use a simplified community (such as the ASF) where the actions of all organisms can be assessed. To understand the dynamic interactions that occur between microbes and between the microbes and the host, it will be important to start with what is known in order to begin the unraveling the enigmatic nature of gut health and disease.

## **Author details**

cannot convert bilirubin to urobilinogens or cholesterol to coprostanol [395]. Interestingly, this same research team compared these parameters in CD patients versus healthy subjects and the characteristics of the microbial metabolism associated with the microbiota of CD patients were very similar to those of the ASF [396]. This study suggests that there is benefit to the use

Other defined microbiota mouse studies have been published [397-400]. A study using ten bacterial species specifically chosen for their metabolic function were used to colonize GF mice [397]. Using microbial RNA-seq, the authors were able to build a model relating perturbation of the microbial community to changes in diet. By modeling the functional capacity of a gnotobiotic community under different conditions, this model and similar approaches can be used to unravel the operational dynamics of the gut microbiome with respect to nutrient utilization such that the microbiota might be manipulated to improve human and animal health [397]. Another study colonized mice with *E. rectale* and *B. thetaiotamicron*, and the authors then assessed changes in bacterial gene expression using Affymetrix GeneChips to show that the cross-talk between these two organisms affected up- and down-regulated genes

ASF356 Most closely related to *Clostridium propionicum* (92% max identity) [390]

ASF500 *Clostridium* sp. with no known related organism in GenBank Database [390] ASF502 Most closely related to *Ruminococcus gnavus* (92% max identity) [390]

Three human commensals, *E. coli, B. longum,* and *L. johnsonii,* have also been used to colonize GF mice to create a gnotobiotic community [398]. This community was used to identify the effects that the introduction of novel organisms has on the community dynamic. When colonized with a second *Lactobacillus, L. paracasei,* it was noted that both *Lactobacillus* spp. were able to co-habitate reaching similar fecal titers. However, that was not the same when a fifth organism was added, a second *B. longum* strain. This new strain was only maintained in the mouse at detectable levels for three days. Lastly, a second *E. coli* strain was added to the original community of three. This new *E. coli* reached the highest titer of any organism in the com‐

ASF519 *Parabacteroides goldsteinii* (99% max identity) [404, 405]

ASF360 *Lactobacillus intestinalis* (99% max identity) [401]

ASF361 *Lactobacillus murinus [390]* ASF457 *Mucispirillum schaedleri [402]* ASF492 *Eubacterium plexicaudatum* [403]

Member of *Clostridium* cluster XIV

Member of *Clostridium* cluster XIV

Member of *Clostridium* cluster XIV

**Strain Identity**

of the ASF in mouse models of IBD.

116 Inflammatory Bowel Disease

in response to one another.

**Table 4.** Members of the altered Schaedler flora

Anne-Marie C. Overstreet1 , Amanda E. Ramer-Tait3 , Albert E. Jergens2 and Michael J. Wannemuehler1

1 Department of Veterinary Microbiology and Preventive Medicine, Iowa State University, Ames, IA, USA

2 Department of Veterinary Clinical Sciences, Iowa State University, Ames, IA, USA

3 Department of Food Science and Technology, University of Nebraska-Lincoln, Lincoln, NE, USA

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[391] Sarma-Rupavtarm, R.B., Ge, Z.M., Schauer, D.B., Fox, J.G., and Polz, M.F., Spatial distribution and stability of the eight microbial species of the altered Schaedler flora in the mouse gastrointestinal tract. Applied and Environmental Microbiology, 2004.

[392] Stehr, M., Greweling, M.C., Tischer, S., Singh, M., Blocker, H., et al., Charles River al‐ tered Schaedler flora (CRASF) remained stable for four years in a mouse colony housed in individually ventilated cages. Laboratoy Animals, 2009. 43(4): p. 362-70.

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[394] Alexander, A.D., Orcutt, R.P., Henry, J.C., Baker, J., Bissahoyo, A.C., et al., Quantita‐ tive PCR assays for mouse enteric flora reveal strain-dependent differences in com‐ position that are influenced by the microenvironment. Mammalian Genome, 2006.

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**Chapter 4**

**Fibrosis in Crohn's Disease**

Additional information is available at the end of the chapter

The objectives of this chapter are to review clinical and pathophysiologic aspects of fibrosis in inflammatory bowel disease, particularly in Crohn's disease. Potential therapeutic strat‐ egies and the current status of preclinical animal models to evaluate these therapeutic strat‐

Crohn's disease (CD) and ulcerative colitis (UC) are chronic, relapsing inflammatory gastro‐ intestinal diseases which often onset in young adulthood. Unlike in UC, where inflamma‐ tion is limited to the mucosa, CD patients frequently develop transmural disease which can extend to involve the muscularis and serosa. While inflammatory disease accounts for much of the symptomatology associated with CD, significant morbidity results from fibrotic le‐

CD can be broadly categorized as having three major clinical subtypes: stricturing, penetrat‐ ing or inflammatory (nonstricturing nonpenetrating) disease [1]. This categorization is re‐ flected by the inclusion of disease behavior ( stricturing, penetrating or inflammatory) as one of three variables elected for inclusion in the Vienna classification in 1998 [2]. Review of natural history data from shows that the majority of patients undergo progression from in‐ flammatory disease to development of complications including stricture [3]. Even though most patients present with inflammatory disease only and later progress to a more compli‐ cated disease phenotype, a subset of patients will present with stricturing or penetrating dis‐ ease. In population-based studies, 19-36% of patients newly diagnosed with CD present

> © 2012 Diehl; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,

© 2012 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution,

distribution, and reproduction in any medium, provided the original work is properly cited.

and reproduction in any medium, provided the original work is properly cited.

with disease complications such as strictures, fistulas or abscesses [4-6].

Lauri Diehl

**1. Introduction**

http://dx.doi.org/10.5772/46222

egies will also be discussed.

**2. Clinical considerations**

sions and their resulting complications.


**Chapter 4**

## **Fibrosis in Crohn's Disease**

Lauri Diehl

[398] Denou, E., Rezzonico, E., Panoff, J.M., Arigoni, F., and Brussow, H., A Mesocosm of *Lactobacillus johnsonii, Bifidobacterium longum, and Escherichia coli* in the mouse gut.

[399] Mahowald, M.A., Rey, F.E., Seedorf, H., Turnbaugh, P.J., Fulton, R.S., et al., Charac‐ terizing a model human gut microbiota composed of members of its two dominant bacterial phyla. Proceedings of the National Academy of Sciences of the United

[400] Faith, J.J., Rey, F.E., O'Donnell, D., Karlsson, M., McNulty, N.P., et al., Creating and characterizing communities of human gut microbes in gnotobiotic mice. The ISME

[401] Pena, J.A., Li, S.Y., Wilson, P.H., Thibodeau, S.A., Szary, A.J., et al., Genotypic and phenotypic studies of murine intestinal lactobacilli: species differences in mice with and without colitis. Applied and Environmental Microbiology, 2004. 70(1): p. 558-68.

[402] Robertson, B.R., O'Rourke, J.L., Neilan, B.A., Vandamme, P., On, S.L., et al., *Mucispir‐ illum schaedleri* gen. nov., sp. nov., a spiral-shaped bacterium colonizing the mucus layer of the gastrointestinal tract of laboratory rodents. International Journal of Sys‐

[403] Wilkins, T.D., Fulghum, R.S., and Wilkins, J.H., *Eubacterium plexicaudatum* sp. nov., an anaerobic bacterium with a subpolar tuft of flagella, isolated from a mouse cecum.

[404] Sakamoto, M. and Benno, Y., Reclassification of *Bacteroides distasonis, Bacteroides gold‐ steinii* and *Bacteroides merdae* as *Parabacteroides distasonis* gen. nov., comb. nov., *Para‐ bacteroides goldsteinii* comb. nov. and *Parabacteroides merdae* comb. nov. International Journal of Systematic and Evolutionary Microbiology, 2006. 56(Pt 7): p. 1599-605. [405] Song, Y., Liu, C., Lee, J., Bolanos, M., Vaisanen, M.L., et al., "*Bacteroides goldsteinii* sp. nov." isolated from clinical specimens of human intestinal origin. Journal of Clinical

tematic and Evolutionary Microbiology, 2005. 55(Pt 3): p. 1199-204.

International Journal of Systemic Bacteriology, 1974. 24(4): p. 408-411.

DNA and Cell Biology, 2009. 28(8): p. 413-22.

States of America, 2009. 106(14): p. 5859-64.

journal, 2010. 4(9): p. 1094-8.

150 Inflammatory Bowel Disease

Microbiology, 2005. 43(9): p. 4522-7.

Additional information is available at the end of the chapter

http://dx.doi.org/10.5772/46222

## **1. Introduction**

The objectives of this chapter are to review clinical and pathophysiologic aspects of fibrosis in inflammatory bowel disease, particularly in Crohn's disease. Potential therapeutic strat‐ egies and the current status of preclinical animal models to evaluate these therapeutic strat‐ egies will also be discussed.

## **2. Clinical considerations**

Crohn's disease (CD) and ulcerative colitis (UC) are chronic, relapsing inflammatory gastro‐ intestinal diseases which often onset in young adulthood. Unlike in UC, where inflamma‐ tion is limited to the mucosa, CD patients frequently develop transmural disease which can extend to involve the muscularis and serosa. While inflammatory disease accounts for much of the symptomatology associated with CD, significant morbidity results from fibrotic le‐ sions and their resulting complications.

CD can be broadly categorized as having three major clinical subtypes: stricturing, penetrat‐ ing or inflammatory (nonstricturing nonpenetrating) disease [1]. This categorization is re‐ flected by the inclusion of disease behavior ( stricturing, penetrating or inflammatory) as one of three variables elected for inclusion in the Vienna classification in 1998 [2]. Review of natural history data from shows that the majority of patients undergo progression from in‐ flammatory disease to development of complications including stricture [3]. Even though most patients present with inflammatory disease only and later progress to a more compli‐ cated disease phenotype, a subset of patients will present with stricturing or penetrating dis‐ ease. In population-based studies, 19-36% of patients newly diagnosed with CD present with disease complications such as strictures, fistulas or abscesses [4-6].

© 2012 Diehl; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2012 The Author(s). Licensee InTech. This chapter is distributed under the terms of the Creative Commons Attribution License http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

Intestinal stricture is a common and serious complication of long term CD. Critical intestinal stricture formation will occur in at least one-third of CD patients within 10 years of onset [7-9]. By contrast, fibrosis associated with UC is generally limited to the mucosa and stric‐ ture formation is rare although rectal strictures, when they occur, can be problematic to manage [10]. Advances in CD treatment have yet to make significant impact on the inci‐ dence of strictures and the associated morbidity [11].

and fibrostenotic strictures [25]. A recent study confirms that, while the combination of MRenterography and ultrasound as well as the combination of 18FDG-PET/CT and ultrasound are highly efficient in detecting CD strictures, no current imaging techniques shows suffi‐ cient sensitivity or specificity to reliably differentiate inflamed from fibrotic strictures [26]. The clinical need for this for such differentiation remains high and may require both techno‐ logic advancement and establishment of criteria for grading or distinguishing strictures which contain both fibrotic and inflammatory components [27]. [25]. In clinical practice, a variety of diagnostic tools including imaging techniques and inflammatory biomarkers are often applied in an effort to obtain sufficient evidence to inform treatment decisions [28].

Fibrosis in Crohn's Disease http://dx.doi.org/10.5772/46222 153

Therapeutic strategies have evolved over the past decade and having the ability to predict disease outcomes could guide the clinician's choice of therapy. The goals of CD treatment should include 1.) steroid-free sustained clinical remission; 2.) mucosal healing; 3.) potential induction and maintenance of radiological healing; 4.) prevention of surgery; 5.) mainte‐ nance of normal gastrointestinal function; and 6.) prevention of disability [6]. There are a number of therapeutic options available for the treatment of inflammation in CD patients, however none of these have been demonstrated to be effective in preventing preventing or treating fibrostenosis thereby failing to achieve at least 2 of the 6 major treatment goals in patients with fibrostenotic disease. There have been some reports of regression of strictures and of overall benefit following infliximab treatment in a subset of patients with small bow‐ el stricturing disease [18, 29, 30]. This data is largely anecdotal and remains controversial as

In CD, disease location (ileal vs. colonic) remains relatively stable but clinical behavior can alter significantly over time [8, 31, 32]. During the first few years of disease, inflammatory forms predominate, whereas, after 40 years, most patients have experienced complications and are classified as having penetrating or stricturing disease[33]. However, the rate at which disease behavior evolves can vary widely between CD patients and those differences would determine therapeutic strategies if rapidly progressing patients could be identified. For example, initiation of more aggressive treatment early in the course of disease has the potential to result in better outcomes, however, these therapies can also lead to greater risk of toxicity and adverse effects [34, 35]. Decisions on whether to use a conservative or an ag‐ gressive treatment strategy in newly diagnosed patients could be informed by the ability to

A number of retrospective studies have identified specific disease characteristics that may be of use in predicting risk for individual CD patients. These include an initial CD diagnosis under age 40, need for steroid therapy at diagnosis, and perianal fistulizing disease [31, 36]. Localization of inflammation to the small bowel has also been identified as predictive of progression to more complicated disease and higher rate of surgery [37]. However, all of these clinical features tend to correlate with the presence of small intestinal disease and do

identify patients at higher risk for developing disabling or complicated disease.

large controlled trials have not yet been performed.

**3. Fibrosis risk factors**

While surgical resection is a highly effective short term treatment, remission after surgical resection in CD is only temporary. The disease course in CD patients postsurgery is relative‐ ly consistent and has allowed development of a postoperative recurrence model [12, 13]. In this model, a focal inflammatory infiltrate forms in the ileum above the anastomosis fol‐ lowed by aphthous ulcers which are endoscopically visible in two-thirds of patients within 3 months after surgery. These patients go on to develop extensive superficial and deep ulcers that precede the development of a new stricture. Postoperative reoccurrence is very com‐ mon with fewer than 5% of patients having normal endoscopy results 10 years after surgery. Symptoms occur on average 2-3 years after lesions are observed.

In patients with a fibrostenotic disease behavior, symptomatic strictures tend to return de‐ spite medical therapy and this leads to repeated bowel resections and eventually to short bowel syndrome [14-16]. Endoscopic balloon dilation can provide a treatment option for pa‐ tients with fibrostenotic strictures 7 cm or less in length although there is some risk of perfo‐ ration [17, 18]. Strictureplasty can be a useful bowel-preserving surgical option for stenosing small bowel CD in patients with multiple obstructions and in those vulnerable to short bow‐ el syndrome [19]. The incidence of postoperative recurrence is very similar between strictur‐ eplasty and resection [11].

Strictures can arise due to either inflammatory or fibrotic processes. There is evidence that direct steroid injection can provide symptom relief in some CD patients with anastomotic strictures, presumably in patients with active inflammation and relatively little fibromuscu‐ lar proliferation at the anastomotic site [18]. Given the risk of multiple bowel resection sur‐ geries and the possibility of short bowel syndrome, the ability to differentiate between inflammatory or fibrotic strictures could be used to drive treatment decisions. Endoscopybased techniques such as colonoscopy, small intestinal endoscopy, and capsule endoscopy have high clinical utility but only visualize the mucosal surface. Cross-sectional imaging techniques can be used to visualize deep layers of the intestinal wall and assess for stric‐ tures. Computed tomography enterography (CTE) has become the most widely used crosssectional imaging technology for CD although concern about cumulative radiation exposure, particularly in young patients, has led to interest in alternative imaging modali‐ ties [20, 21].

Imaging methods such as magnetic resonance (MR) enterography or ultrasonography are ef‐ fective in determining the anatomic location and length of affected intestinal segments and these techniques, as well as CTE, have shown a good correlation with endoscopy [22-24]. However, distinguishing stricture composition remains a challenge. In 2006, the European Crohn's and Colitis Organization (ECCO) stated in their consensus report on treatment of CD that current techniques are insufficiently accurate to differentiate between inflammatory and fibrostenotic strictures [25]. A recent study confirms that, while the combination of MRenterography and ultrasound as well as the combination of 18FDG-PET/CT and ultrasound are highly efficient in detecting CD strictures, no current imaging techniques shows suffi‐ cient sensitivity or specificity to reliably differentiate inflamed from fibrotic strictures [26]. The clinical need for this for such differentiation remains high and may require both techno‐ logic advancement and establishment of criteria for grading or distinguishing strictures which contain both fibrotic and inflammatory components [27]. [25]. In clinical practice, a variety of diagnostic tools including imaging techniques and inflammatory biomarkers are often applied in an effort to obtain sufficient evidence to inform treatment decisions [28].

Therapeutic strategies have evolved over the past decade and having the ability to predict disease outcomes could guide the clinician's choice of therapy. The goals of CD treatment should include 1.) steroid-free sustained clinical remission; 2.) mucosal healing; 3.) potential induction and maintenance of radiological healing; 4.) prevention of surgery; 5.) mainte‐ nance of normal gastrointestinal function; and 6.) prevention of disability [6]. There are a number of therapeutic options available for the treatment of inflammation in CD patients, however none of these have been demonstrated to be effective in preventing preventing or treating fibrostenosis thereby failing to achieve at least 2 of the 6 major treatment goals in patients with fibrostenotic disease. There have been some reports of regression of strictures and of overall benefit following infliximab treatment in a subset of patients with small bow‐ el stricturing disease [18, 29, 30]. This data is largely anecdotal and remains controversial as large controlled trials have not yet been performed.

In CD, disease location (ileal vs. colonic) remains relatively stable but clinical behavior can alter significantly over time [8, 31, 32]. During the first few years of disease, inflammatory forms predominate, whereas, after 40 years, most patients have experienced complications and are classified as having penetrating or stricturing disease[33]. However, the rate at which disease behavior evolves can vary widely between CD patients and those differences would determine therapeutic strategies if rapidly progressing patients could be identified. For example, initiation of more aggressive treatment early in the course of disease has the potential to result in better outcomes, however, these therapies can also lead to greater risk of toxicity and adverse effects [34, 35]. Decisions on whether to use a conservative or an ag‐ gressive treatment strategy in newly diagnosed patients could be informed by the ability to identify patients at higher risk for developing disabling or complicated disease.

## **3. Fibrosis risk factors**

Intestinal stricture is a common and serious complication of long term CD. Critical intestinal stricture formation will occur in at least one-third of CD patients within 10 years of onset [7-9]. By contrast, fibrosis associated with UC is generally limited to the mucosa and stric‐ ture formation is rare although rectal strictures, when they occur, can be problematic to manage [10]. Advances in CD treatment have yet to make significant impact on the inci‐

While surgical resection is a highly effective short term treatment, remission after surgical resection in CD is only temporary. The disease course in CD patients postsurgery is relative‐ ly consistent and has allowed development of a postoperative recurrence model [12, 13]. In this model, a focal inflammatory infiltrate forms in the ileum above the anastomosis fol‐ lowed by aphthous ulcers which are endoscopically visible in two-thirds of patients within 3 months after surgery. These patients go on to develop extensive superficial and deep ulcers that precede the development of a new stricture. Postoperative reoccurrence is very com‐ mon with fewer than 5% of patients having normal endoscopy results 10 years after surgery.

In patients with a fibrostenotic disease behavior, symptomatic strictures tend to return de‐ spite medical therapy and this leads to repeated bowel resections and eventually to short bowel syndrome [14-16]. Endoscopic balloon dilation can provide a treatment option for pa‐ tients with fibrostenotic strictures 7 cm or less in length although there is some risk of perfo‐ ration [17, 18]. Strictureplasty can be a useful bowel-preserving surgical option for stenosing small bowel CD in patients with multiple obstructions and in those vulnerable to short bow‐ el syndrome [19]. The incidence of postoperative recurrence is very similar between strictur‐

Strictures can arise due to either inflammatory or fibrotic processes. There is evidence that direct steroid injection can provide symptom relief in some CD patients with anastomotic strictures, presumably in patients with active inflammation and relatively little fibromuscu‐ lar proliferation at the anastomotic site [18]. Given the risk of multiple bowel resection sur‐ geries and the possibility of short bowel syndrome, the ability to differentiate between inflammatory or fibrotic strictures could be used to drive treatment decisions. Endoscopybased techniques such as colonoscopy, small intestinal endoscopy, and capsule endoscopy have high clinical utility but only visualize the mucosal surface. Cross-sectional imaging techniques can be used to visualize deep layers of the intestinal wall and assess for stric‐ tures. Computed tomography enterography (CTE) has become the most widely used crosssectional imaging technology for CD although concern about cumulative radiation exposure, particularly in young patients, has led to interest in alternative imaging modali‐

Imaging methods such as magnetic resonance (MR) enterography or ultrasonography are ef‐ fective in determining the anatomic location and length of affected intestinal segments and these techniques, as well as CTE, have shown a good correlation with endoscopy [22-24]. However, distinguishing stricture composition remains a challenge. In 2006, the European Crohn's and Colitis Organization (ECCO) stated in their consensus report on treatment of CD that current techniques are insufficiently accurate to differentiate between inflammatory

dence of strictures and the associated morbidity [11].

Symptoms occur on average 2-3 years after lesions are observed.

eplasty and resection [11].

152 Inflammatory Bowel Disease

ties [20, 21].

A number of retrospective studies have identified specific disease characteristics that may be of use in predicting risk for individual CD patients. These include an initial CD diagnosis under age 40, need for steroid therapy at diagnosis, and perianal fistulizing disease [31, 36]. Localization of inflammation to the small bowel has also been identified as predictive of progression to more complicated disease and higher rate of surgery [37]. However, all of these clinical features tend to correlate with the presence of small intestinal disease and do not necessarily identify which patients with small bowel disease are at greatest risk of devel‐ oping fibrostenosing disease.

**4. Serologic biomarkers**

or treatment of bowel stricture.

developing fibrostenotic disease [62].

fibrostenotic disease.

Serologic markers may identify CD patients at higher risk of developing disease-related complications. Some of the best characterized serologic markers associated with CD are di‐ rected against microbial peptides. Most of the available data is from cross sectional studies in which the patient samples analyzed have been collected at various times in the disease course allowing for comparison of serum from before, concomitant with, and after diagnosis

Fibrosis in Crohn's Disease http://dx.doi.org/10.5772/46222 155

Disease progression from non-complicated CD to stricturing and/or penetrating phenotypes has been significantly associated with the presence and magnitude of serologic response to microbial antigens. This concept was initially triggered by the observation that high anti-Saccharomyces cerevisiae antibody (ASCA) levels were found to be associated with fibroste‐ nosing and penetrating disease and with the need for surgery [52]. This observation was repeated in another cross sectional study where ASCA positive patients were more likely to undergo surgery within 3 years of diagnosis than ASCA negative patients [53]. Time to first complication was shown to be shorter in ASCA positive pediatric CD patients than in ASCA negative patients [54]. In addition, anti-I2 (an antibody directed against *Pseudomonas* fluores‐ cens), anti-OmpC (the outer membrane porin protein of *Escherichia* coli) and anti-CBir1 (an‐ ti-flagellin) levels have also been shown to associated with fibrostenotic disease [55-60]. A multi-center study evaluated the association of ASCA, anti-I2, anti-OmpC, and anti-CBir1 reactivity with disease course in a large cohort of pediatric CD patients and found that the frequency of fibrostenotic or penetrating disease increased in parallel with the number of antigens recognized [61]. Combining anti-microbial antibody titers and evaluation of NOD2 variants or other gene polymorphisms may improve detection of patients at higher risk of

The predictive value of other serologic markers for fibrostenotic disease has been evaluated on a much more limited basis than the anti-microbial antibodies. C-reactive protein (CRP) is widely used to monitor inflammatory disease activity and one prospective study found a significant association between CRP levels and subsequent risk of intestinal resection in pa‐ tients with ileal disease [63]. Despite the prominent place of extracellular matrix proteins in composition of fibrostenotic lesions, little association has been found with levels of these molecules in the circulation [64]. However, one study did find that higher levels of plasma fibronectin were associated with stricture formation in CD patients [65]. Growth factors have also been evaluated in a limited fashion. Serum levels of YKL-40, a mammalian glyco‐ protein member of the chitinase family, has been reported as increased in CD patients with stricturing disease compared to those without strictures [66]. Another study found serum levels of basic fibroblast growth factor, a cytokine promoting fibroblast activation and prolif‐ eration, were higher in CD patients with intestinal strictures compared to patients with fis‐ tulizing or inflammatory phenotypes [67]. Prospective studies will be required to determine which, if any, of these serologic tests may have potential as a clinically useful biomarker of

Genetic polymorphisms play an important role in susceptibility to CD so the use of genetic markers to provide risk stratification and drive clinical decisions is very attractive. The ob‐ servation that some CD patients are susceptible to stricture development early and often in their clinical course while others never develop a stenosing phenotype argues for the exis‐ tence of a genetic background which predisposes to stricturing behavior in CD.

One of the genes linked with susceptibility to CD is the CARD15/NOD2 gene which encodes a protein involved in bacterial recognition and activation of nuclear factor kB. Carriers of two mutant alleles have 17 to 42 times the risk while carriers of one mutation have 1.5 to 3 times the risk of developing CD [38, 39]. There is a significant body of evidence suggesting that the main NOD2/CARD15 variants (Arg702Trp, Gly908Arg, and Leu1007insC) are asso‐ ciated with risk for developing stenotic disease and increased need for surgery [40-44]. However, this finding is not reproducible in every CD cohort evaluated [45, 46]. One large meta-analysis of the NOD2/CARD15 literature indicated that carrying at least one NOD2/ CARD15 variant increased the risk of both small intestinal disease and of the stenosing phe‐ notype [47]. The discrepancy between studies may be due to differences in definitions of dis‐ ease behavior between studies and, perhaps, to the particular genetic epidemiological analyses used. At present, understanding whether the relationship between the NOD2/ CARD15 variants and a stenosing phenotype is a true association or whether it instead re‐ flects aspects of disease duration and ileal localization remains a matter of controversy.

Other gene polymorphisms have been described as associated with a fibrostenotic pheno‐ type although the existing data is much less extensive than that available for NOD2/ CARD15. The ATG16L1 gene encodes for a protein involved in autophagy and mutations in this gene have been associated with stricturing disease as well as perianal involvement in CD [48]. CX3CR1, the receptor of CX3CL1 (fractalkine), is involved in regulation of inflam‐ matory response and the V249I polymorphism has been reported to be associated with in‐ testinal strictures [49]. A recent study linked the receptor for advanced glycation endproducts (RAGE) -374T/A polymorphism to protection from stricturing phenotype in CD. The polymorphism increases RAGE gene transcription which may provide protection by increasing levels of soluble RAGE leading to neutralization of proinflammatory media‐ tors [50]. Some fibrostenosis-related polymorphisms have been observed in combination with NOD2/CARD15. CXCL16 is a chemokine involved in bacterial defense mechanisms. CD patients with at least one CXCL16 p.Ala181Val allele and one CARD15/NOD2 variant had a higher incidence of stricturing and penetrating phenotype as well as stenosis as pa‐ tients with the NOD2 variant alone [51].

Despite their potential, genetic markers may never fully be able to predict the clinical course of CD. The low frequency, incomplete penetrance, and interplay with other genetic poly‐ morphisms greatly complicate interpretation of genetic markers. In addition, environmental factors can modulate disease history and impact phenotypic features. It is probable that ge‐ netic markers will need to be integrated with other clinical and serologic information in or‐ der to be useful predictors of disease course and to inform treatment decisions.

## **4. Serologic biomarkers**

not necessarily identify which patients with small bowel disease are at greatest risk of devel‐

Genetic polymorphisms play an important role in susceptibility to CD so the use of genetic markers to provide risk stratification and drive clinical decisions is very attractive. The ob‐ servation that some CD patients are susceptible to stricture development early and often in their clinical course while others never develop a stenosing phenotype argues for the exis‐

One of the genes linked with susceptibility to CD is the CARD15/NOD2 gene which encodes a protein involved in bacterial recognition and activation of nuclear factor kB. Carriers of two mutant alleles have 17 to 42 times the risk while carriers of one mutation have 1.5 to 3 times the risk of developing CD [38, 39]. There is a significant body of evidence suggesting that the main NOD2/CARD15 variants (Arg702Trp, Gly908Arg, and Leu1007insC) are asso‐ ciated with risk for developing stenotic disease and increased need for surgery [40-44]. However, this finding is not reproducible in every CD cohort evaluated [45, 46]. One large meta-analysis of the NOD2/CARD15 literature indicated that carrying at least one NOD2/ CARD15 variant increased the risk of both small intestinal disease and of the stenosing phe‐ notype [47]. The discrepancy between studies may be due to differences in definitions of dis‐ ease behavior between studies and, perhaps, to the particular genetic epidemiological analyses used. At present, understanding whether the relationship between the NOD2/ CARD15 variants and a stenosing phenotype is a true association or whether it instead re‐ flects aspects of disease duration and ileal localization remains a matter of controversy.

Other gene polymorphisms have been described as associated with a fibrostenotic pheno‐ type although the existing data is much less extensive than that available for NOD2/ CARD15. The ATG16L1 gene encodes for a protein involved in autophagy and mutations in this gene have been associated with stricturing disease as well as perianal involvement in CD [48]. CX3CR1, the receptor of CX3CL1 (fractalkine), is involved in regulation of inflam‐ matory response and the V249I polymorphism has been reported to be associated with in‐ testinal strictures [49]. A recent study linked the receptor for advanced glycation endproducts (RAGE) -374T/A polymorphism to protection from stricturing phenotype in CD. The polymorphism increases RAGE gene transcription which may provide protection by increasing levels of soluble RAGE leading to neutralization of proinflammatory media‐ tors [50]. Some fibrostenosis-related polymorphisms have been observed in combination with NOD2/CARD15. CXCL16 is a chemokine involved in bacterial defense mechanisms. CD patients with at least one CXCL16 p.Ala181Val allele and one CARD15/NOD2 variant had a higher incidence of stricturing and penetrating phenotype as well as stenosis as pa‐

Despite their potential, genetic markers may never fully be able to predict the clinical course of CD. The low frequency, incomplete penetrance, and interplay with other genetic poly‐ morphisms greatly complicate interpretation of genetic markers. In addition, environmental factors can modulate disease history and impact phenotypic features. It is probable that ge‐ netic markers will need to be integrated with other clinical and serologic information in or‐

der to be useful predictors of disease course and to inform treatment decisions.

tence of a genetic background which predisposes to stricturing behavior in CD.

oping fibrostenosing disease.

154 Inflammatory Bowel Disease

tients with the NOD2 variant alone [51].

Serologic markers may identify CD patients at higher risk of developing disease-related complications. Some of the best characterized serologic markers associated with CD are di‐ rected against microbial peptides. Most of the available data is from cross sectional studies in which the patient samples analyzed have been collected at various times in the disease course allowing for comparison of serum from before, concomitant with, and after diagnosis or treatment of bowel stricture.

Disease progression from non-complicated CD to stricturing and/or penetrating phenotypes has been significantly associated with the presence and magnitude of serologic response to microbial antigens. This concept was initially triggered by the observation that high anti-Saccharomyces cerevisiae antibody (ASCA) levels were found to be associated with fibroste‐ nosing and penetrating disease and with the need for surgery [52]. This observation was repeated in another cross sectional study where ASCA positive patients were more likely to undergo surgery within 3 years of diagnosis than ASCA negative patients [53]. Time to first complication was shown to be shorter in ASCA positive pediatric CD patients than in ASCA negative patients [54]. In addition, anti-I2 (an antibody directed against *Pseudomonas* fluores‐ cens), anti-OmpC (the outer membrane porin protein of *Escherichia* coli) and anti-CBir1 (an‐ ti-flagellin) levels have also been shown to associated with fibrostenotic disease [55-60]. A multi-center study evaluated the association of ASCA, anti-I2, anti-OmpC, and anti-CBir1 reactivity with disease course in a large cohort of pediatric CD patients and found that the frequency of fibrostenotic or penetrating disease increased in parallel with the number of antigens recognized [61]. Combining anti-microbial antibody titers and evaluation of NOD2 variants or other gene polymorphisms may improve detection of patients at higher risk of developing fibrostenotic disease [62].

The predictive value of other serologic markers for fibrostenotic disease has been evaluated on a much more limited basis than the anti-microbial antibodies. C-reactive protein (CRP) is widely used to monitor inflammatory disease activity and one prospective study found a significant association between CRP levels and subsequent risk of intestinal resection in pa‐ tients with ileal disease [63]. Despite the prominent place of extracellular matrix proteins in composition of fibrostenotic lesions, little association has been found with levels of these molecules in the circulation [64]. However, one study did find that higher levels of plasma fibronectin were associated with stricture formation in CD patients [65]. Growth factors have also been evaluated in a limited fashion. Serum levels of YKL-40, a mammalian glyco‐ protein member of the chitinase family, has been reported as increased in CD patients with stricturing disease compared to those without strictures [66]. Another study found serum levels of basic fibroblast growth factor, a cytokine promoting fibroblast activation and prolif‐ eration, were higher in CD patients with intestinal strictures compared to patients with fis‐ tulizing or inflammatory phenotypes [67]. Prospective studies will be required to determine which, if any, of these serologic tests may have potential as a clinically useful biomarker of fibrostenotic disease.

## **5. Histologic features**

Stricture due to fibrostenotic change resulting in chronic obstruction is a major pathologic event in chronic CD. Histologically, CD strictures are characterized by hyperplasia of the in‐ testinal muscle layers which is typically manifest as islands of smooth muscle cells in the submucosa surrounded by dense collagen deposits. These regions of smooth muscle prolif‐ eration may become so extensive that they obliterate the submucosa [68]. Transmission elec‐ tron microscopy studies show alteration of muscle cells of the muscularis propria, especially the inner muscle layer, including hypertrophy, synthesis and deposition of collagen, and fo‐ cal cellular necrosis [69].

this lesion. Several studies have shown an association between the occurrence of epitheliod

Fibrosis in Crohn's Disease http://dx.doi.org/10.5772/46222 157

Tissue injury or inflammation triggers a cascade of wound healing activities in the sur‐ rounding cell populations. Normal wound healing is a tightly regulated and coordinated series of events triggered by secretion of mediators from activated immune and mesenchy‐ mal cells which induce cell proliferation, migration, and extracelluar matrix (ECM) produc‐ tion. Wound healing activity is followed by resolution of inflammation and tissue remodeling. A balance must be achieved between processes involved in ECM production and degradation and those involved in cellular hyperplasia (proliferation and cell death). In the intestinal tract, tissue repair and regeneration are of great importance in mucosal ho‐ meostasis and intestinal barrier function. Rapid wound healing and restitution of an intact mucosal barrier is crucial for controlling mucosal inflammation. However, excessive wound healing response can result in fibrosis and stricture formation while insufficient tissue repair

The classic model of wound healing has 4 phases: hemostasis, inflammation, proliferation, and remodeling [76, 77]. In the hemostasis phase, platelet degranulation and fibrin forma‐ tion provide both hemostasis and a provisional matrix for subsequent healing events to take place. Cytokine and chemokine expression, initially by the innate immune system and later including the adaptive immune system, drives the inflammatory phase. During the prolifer‐ ative phase, activated fibroblasts and myofibroblasts secrete collagen and other matrix mole‐ cules which provide a granulation tissue scaffold on which tissue structure repair can commence. During the proliferative phase, cytokines and growth factors regulate reconstitu‐ tion of the mucosal epithelium allowing closing of the epithelial defect. Angiogenesis and lymphangiogenesis also take place during this phase and there is expansion of the fibro‐ blast/myofibroblast population with concomitant ECM production. Finally, in the remodel‐ ing phase, myofibroblasts produce matrix-modifying molecules which assist in the restoring anatomic structural integrity and completing the transition from wound to normal or near

When severe mucosal tissue damage occurs, myofibroblasts migrate to the edges of the tis‐ sue defect. The ability of myofibroblasts to migrate to the wound area and synthesize ECM proteins is critical in proliferative phase of intestinal wound healing [78]. Migration of sube‐ pithelial myofibroblasts can be mediated by a variety of soluble factors such as transforming growth factor –β (TGFβ), insulin-like growth factor (IGF-1), platelet-derived growth factor-AB (PDGF-AB), and epidermal growth factor (EGF) [79]. Fibronectin, synthesized by myofi‐ broblasts, is essential and is largely responsible for autocrine induction of intestinal

Wound healing and myofibroblast migration can be affected by chronic inflammation [79]. Subepithelial myofibroblasts isolated from CD patients show a significant reduction in mi‐

granulomas, especially at presentation, and a more aggressive disease course [75].

**6. Pathophysiology of fibrosis**

can result in fistula formation.

normal intestine architecture.

myfibroblast migration [80].

Despite the general categorization of strictures as inflammatory, fibrostenotic or both, fibro‐ sis is often well correlated with inflammation and the majority of strictures contain some de‐ gree of both processes [70]. When histologic tissue inflammation and fibrosis were compared in a relatively small cohort of patients undergoing surgical resection, the authors found that all specimens which were significantly fibrotic were also significantly inflamed [71]. This may accurately reflect the reality of stricturing disease in many patients, however, this has not yet been confirmed in studies of larger patient cohorts and the results may be skewed because histologic evaluation is only possible in patients undergoing surgical resec‐ tion. This excludes stricture patients who either respond to aggressive medical therapy or who undergo bowel-sparing procedures which may inadvertently exclude many patients who fall on either end of the inflammation/fibrosis spectrum.

Further consideration of the relationship between histologic categorization and disease behavior is needed and new histologic scoring systems may be required which consider cellular composition or other features in order to more effectively categorize strictures. One retrospective study has been published where biopsies were evaluated to determine if certain histologic characteristics correspond to eventual development of complicated CD [72]. The authors report that severe lymphoid infiltration of the lamina propria with crypt atrophy and absence of intraepithelial lymphocytes correlates with non-stricturing/ non-penetrating disease while these features were absent in 80% of CD patients with stricturing disease. Once again, these findings were based on a small cohort size and need further evaluation but do accord with the larger concept of inflammatory vs. fibros‐ tenotic stricture processes.

Other studies comparing histologic features with biologic behavior are limited. At least two studies have noted an association of mast cells in the submucosa and especially in the mus‐ cularis propria with stricture formation in CD patients [73, 74]. When compared to normal bowel or non-strictured CD bowel, mast cell numbers were significantly higher in the thick‐ ened muscularis propria of CD strictures. No increase in mast cells was associated with ul‐ cerative colitis or other intestinal inflammatory conditions. Also, epitheliold granulomas have been implicated as a risk factor for progression to complicated disease behavior. Epi‐ theliod granulomas are one of the most characteristic histologic features in biopsies or re‐ sected tissue from patients with CD although only about 15-25% of patients present with this lesion. Several studies have shown an association between the occurrence of epitheliod granulomas, especially at presentation, and a more aggressive disease course [75].

## **6. Pathophysiology of fibrosis**

**5. Histologic features**

156 Inflammatory Bowel Disease

cal cellular necrosis [69].

tenotic stricture processes.

Stricture due to fibrostenotic change resulting in chronic obstruction is a major pathologic event in chronic CD. Histologically, CD strictures are characterized by hyperplasia of the in‐ testinal muscle layers which is typically manifest as islands of smooth muscle cells in the submucosa surrounded by dense collagen deposits. These regions of smooth muscle prolif‐ eration may become so extensive that they obliterate the submucosa [68]. Transmission elec‐ tron microscopy studies show alteration of muscle cells of the muscularis propria, especially the inner muscle layer, including hypertrophy, synthesis and deposition of collagen, and fo‐

Despite the general categorization of strictures as inflammatory, fibrostenotic or both, fibro‐ sis is often well correlated with inflammation and the majority of strictures contain some de‐ gree of both processes [70]. When histologic tissue inflammation and fibrosis were compared in a relatively small cohort of patients undergoing surgical resection, the authors found that all specimens which were significantly fibrotic were also significantly inflamed [71]. This may accurately reflect the reality of stricturing disease in many patients, however, this has not yet been confirmed in studies of larger patient cohorts and the results may be skewed because histologic evaluation is only possible in patients undergoing surgical resec‐ tion. This excludes stricture patients who either respond to aggressive medical therapy or who undergo bowel-sparing procedures which may inadvertently exclude many patients

Further consideration of the relationship between histologic categorization and disease behavior is needed and new histologic scoring systems may be required which consider cellular composition or other features in order to more effectively categorize strictures. One retrospective study has been published where biopsies were evaluated to determine if certain histologic characteristics correspond to eventual development of complicated CD [72]. The authors report that severe lymphoid infiltration of the lamina propria with crypt atrophy and absence of intraepithelial lymphocytes correlates with non-stricturing/ non-penetrating disease while these features were absent in 80% of CD patients with stricturing disease. Once again, these findings were based on a small cohort size and need further evaluation but do accord with the larger concept of inflammatory vs. fibros‐

Other studies comparing histologic features with biologic behavior are limited. At least two studies have noted an association of mast cells in the submucosa and especially in the mus‐ cularis propria with stricture formation in CD patients [73, 74]. When compared to normal bowel or non-strictured CD bowel, mast cell numbers were significantly higher in the thick‐ ened muscularis propria of CD strictures. No increase in mast cells was associated with ul‐ cerative colitis or other intestinal inflammatory conditions. Also, epitheliold granulomas have been implicated as a risk factor for progression to complicated disease behavior. Epi‐ theliod granulomas are one of the most characteristic histologic features in biopsies or re‐ sected tissue from patients with CD although only about 15-25% of patients present with

who fall on either end of the inflammation/fibrosis spectrum.

Tissue injury or inflammation triggers a cascade of wound healing activities in the sur‐ rounding cell populations. Normal wound healing is a tightly regulated and coordinated series of events triggered by secretion of mediators from activated immune and mesenchy‐ mal cells which induce cell proliferation, migration, and extracelluar matrix (ECM) produc‐ tion. Wound healing activity is followed by resolution of inflammation and tissue remodeling. A balance must be achieved between processes involved in ECM production and degradation and those involved in cellular hyperplasia (proliferation and cell death). In the intestinal tract, tissue repair and regeneration are of great importance in mucosal ho‐ meostasis and intestinal barrier function. Rapid wound healing and restitution of an intact mucosal barrier is crucial for controlling mucosal inflammation. However, excessive wound healing response can result in fibrosis and stricture formation while insufficient tissue repair can result in fistula formation.

The classic model of wound healing has 4 phases: hemostasis, inflammation, proliferation, and remodeling [76, 77]. In the hemostasis phase, platelet degranulation and fibrin forma‐ tion provide both hemostasis and a provisional matrix for subsequent healing events to take place. Cytokine and chemokine expression, initially by the innate immune system and later including the adaptive immune system, drives the inflammatory phase. During the prolifer‐ ative phase, activated fibroblasts and myofibroblasts secrete collagen and other matrix mole‐ cules which provide a granulation tissue scaffold on which tissue structure repair can commence. During the proliferative phase, cytokines and growth factors regulate reconstitu‐ tion of the mucosal epithelium allowing closing of the epithelial defect. Angiogenesis and lymphangiogenesis also take place during this phase and there is expansion of the fibro‐ blast/myofibroblast population with concomitant ECM production. Finally, in the remodel‐ ing phase, myofibroblasts produce matrix-modifying molecules which assist in the restoring anatomic structural integrity and completing the transition from wound to normal or near normal intestine architecture.

When severe mucosal tissue damage occurs, myofibroblasts migrate to the edges of the tis‐ sue defect. The ability of myofibroblasts to migrate to the wound area and synthesize ECM proteins is critical in proliferative phase of intestinal wound healing [78]. Migration of sube‐ pithelial myofibroblasts can be mediated by a variety of soluble factors such as transforming growth factor –β (TGFβ), insulin-like growth factor (IGF-1), platelet-derived growth factor-AB (PDGF-AB), and epidermal growth factor (EGF) [79]. Fibronectin, synthesized by myofi‐ broblasts, is essential and is largely responsible for autocrine induction of intestinal myfibroblast migration [80].

Wound healing and myofibroblast migration can be affected by chronic inflammation [79]. Subepithelial myofibroblasts isolated from CD patients show a significant reduction in mi‐ gration response when compared to cells from control patients [81]. Similar reduction in fi‐ broblast migration can be induced by treatment with tumor necrosis factor (TNF) or gamma interferon (IFN-g) suggesting that an inflammatory environment can induce changes in my‐ ofibroblast function [82]. However, environmental impact on fibroblast migration is com‐ plex. For example, fibroblasts from lung tissue with dense fibrosis show higher PDGFdriven migratory potential than do fibroblasts from tissues at an early stage of fibrosis [83]. A recent paper compared migratory potential in colonic fibroblasts isolated from CD pa‐ tients with either fistulizing (penetrating) or fibrotic (stricturing) disease [81]. These authors showed that, while migratory potential is reduced in CD patients with fistulizing disease, there is in an increase in fibroblast migratory potential in patients with fibrotic disease.

cularis mucosa is populated largely by V+/A- and V+/A+ cells rather than the V-/A+ smooth muscle cells seen in normal muscularis mucosa from non-CD patients. This suggests a tran‐ sition from an enteric smooth muscle cell phenotype toward a fibroblast or myofibroblast

Fibrosis in Crohn's Disease http://dx.doi.org/10.5772/46222 159

Mesenchymal cells including myofibroblasts as well as smooth muscle cells of the muscula‐ ris mucosa and muscularis propria are the main producers of ECM proteins in the intestine. These ECM proteins include structural proteins such as collagen, matricellular proteins such as osteopontin and thrombospondin, and other specialized proteins such as vitronectin and fibronectin. Collagen is the major ECM component associated with intestinal fibrosis. The most common collagen subtypes in normal intestine are type I, type III, and type V in order of abundance. In intestinal fibrosis, there is an increase in total collagen as well as specific

Fibronectin and vitronectin are ligands for the αVβ3 integrin and, in the presence of fibro‐ nectin, smooth muscle IGF-1-stimulated IGF-1 receptor activation is augmented [98]. This suggests that increased production of these proteins by smooth muscle cells at sites of intes‐ tinal stricture could activate αVβ3 integrin and further increase secretion of collagen as well as promote cellular proliferation creating a positive feedback loop which could further sub‐ vert the normal healing process. Fibronectin is also an important mediator in focal adhesion kinase (FAK) signaling pathways involved in cell migration [99]. Myofibroblasts synthesize abundant fibronectin which is largely responsible for the autocrine induction of intestinal

The balance between formation and breakdown of ECM proteins determines the net dep‐ osition in tissues. In intestinal fibrosis, mechanisms to degrade ECM fail to keep pace with deposition. Matrix metalloproteinases (MMPs), a large family of proteolytic en‐ zymes, are responsible for the breakdown of ECM components. The proteolytic activity of MMPs is controlled by tissue inhibitors of metalloproteinases (TIMPs) and an imbal‐ ance between MMP and TIMP activity can result in excessive deposition of ECM pro‐ teins with subsequent fibrosis [101]. Higher levels of constitutive TIMP-1 expression have been shown in intestinal myofibroblast culture derived from fibrotic CD patients than

Transforming growth factor –β (TGFβ) is a pleotrophic cytokine and one of the most influ‐ ential factors in fibrotic processes. It is a component of Th17 as well as regulatory T cell type immune responses as well as a profibrotic mediator. TGFβ exerts profibrotic effects through its ability to regulate collagen expression and extracellular matrix dynamics. There are three isoforms of TGFβ: TGFβ1, TGFβ2, and TGFβ3. TGFβ1 activates the canonical Smad signal‐ ing cascade leading to translocation of the Smad receptor complex into the nucleus and reg‐ ulation of gene transcription including ECM genes such as collagen I, collagen III, and fibronectin [103]. TGFβ also induces EMT in organ-fibrosis inducing diseases [104] and can to induce EndoMT in vitro via a "noncanonical" signaling pathway [105]. In CD patients, TGFβ1 and TGFβ3 are increased in in smooth muscle, fibroblasts and myofibroblasts from

the strictured region when compared to normal intestine [102, 106].

and relative increases in collagen types III and V [95-97].

myofibroblast migration [100].

those from normal individuals [102].

phenotype.

Fibrosis in CD is thought to result from an excessive wound healing response. For reasons that are not wholly understood, the wound repair process in a subset of CD patients contin‐ ues to progress rather than reaching a termination and allowing for tissue remodeling. Ulti‐ mately, the fibrotic process leads to thickening of the intestinal wall and luminal narrowing which can result in bowel obstruction. There are three hallmark pathological features which characterize intestinal stricutres in CD: proliferation of mesenchymal cells including myofi‐ broblasts, smooth muscle cells and fibroblasts; hypertrophy of smooth muscle cells and my‐ ofibroblasts ; and accumulation of excess extracellular matrix proteins [84].

Mesenchymal cells in the intestine can be broadly classed as fibroblasts, smooth muscle cells or myofibroblasts on the basis of immunostaining properties with antibodies to vimentin (V) and smooth muscle actin (A) [85, 86]. Fibroblasts are typically V+/A- and are present in the intestinal submucosal and serosa. Subepithelial myofibroblasts (SEMF) are found adjacent to intestinal epithelial cells and are V+/A+. Intestinal smooth muscle cells of the muscularis mucosa and muscularis propria are normally V-/A+. All of these mesenchymal cell types have been implicated in collagen production in CD patients [69, 87, 88].

Activated fibroblasts or myofibroblasts in tissues undergoing a fibrotic process may be de‐ rived from a variety of sources. There are three general mechanisms which allow for tissue accumulation of these cells: proliferation of existing tissue fibroblasts, recruitment of fibro‐ blast precursor cells from bone marrow, and transformation either of epithelial cells via epi‐ thelial to mesenchymal transistion (EMT), or of endothelial cells by endothelial to mesenchymal transition (EndoMT) [89, 90]. Proliferation and activation of tissue fibroblasts occurs in response to profibrotic signals from infiltrating inflammatory cells or from colonic epithelial cells exposed to proinflammatory cytokines [91]. Soluble inflammatory mediators also drive recruitment of fibroblast precursor cells (fibrocytes) from bone marrow. These fi‐ brocytes migrate from the bloodstream into tissues undergoing pathologic fibrosis in re‐ sponse to specific chemokine gradients [89]. EMT and EndoMT are induced by TGFβ [92]. The relative significance of each of mechanisms discussed above to activated fibroblast/ myofibroblast accumulation at the site of injury in CD is not yet fully understood

Smooth muscle hyperplasia surrounded by collagen deposits is the major histologic feature of fibrostenotic CD. This smooth muscle proliferation expands and disrupts the muscularis mucosa. Thickening of the muscular layer is associated with an increase in the number of vimentin-positive cells [93, 94]. In severely affected tissue, even histologically normal mus‐ cularis mucosa is populated largely by V+/A- and V+/A+ cells rather than the V-/A+ smooth muscle cells seen in normal muscularis mucosa from non-CD patients. This suggests a tran‐ sition from an enteric smooth muscle cell phenotype toward a fibroblast or myofibroblast phenotype.

gration response when compared to cells from control patients [81]. Similar reduction in fi‐ broblast migration can be induced by treatment with tumor necrosis factor (TNF) or gamma interferon (IFN-g) suggesting that an inflammatory environment can induce changes in my‐ ofibroblast function [82]. However, environmental impact on fibroblast migration is com‐ plex. For example, fibroblasts from lung tissue with dense fibrosis show higher PDGFdriven migratory potential than do fibroblasts from tissues at an early stage of fibrosis [83]. A recent paper compared migratory potential in colonic fibroblasts isolated from CD pa‐ tients with either fistulizing (penetrating) or fibrotic (stricturing) disease [81]. These authors showed that, while migratory potential is reduced in CD patients with fistulizing disease, there is in an increase in fibroblast migratory potential in patients with fibrotic disease.

158 Inflammatory Bowel Disease

Fibrosis in CD is thought to result from an excessive wound healing response. For reasons that are not wholly understood, the wound repair process in a subset of CD patients contin‐ ues to progress rather than reaching a termination and allowing for tissue remodeling. Ulti‐ mately, the fibrotic process leads to thickening of the intestinal wall and luminal narrowing which can result in bowel obstruction. There are three hallmark pathological features which characterize intestinal stricutres in CD: proliferation of mesenchymal cells including myofi‐ broblasts, smooth muscle cells and fibroblasts; hypertrophy of smooth muscle cells and my‐

Mesenchymal cells in the intestine can be broadly classed as fibroblasts, smooth muscle cells or myofibroblasts on the basis of immunostaining properties with antibodies to vimentin (V) and smooth muscle actin (A) [85, 86]. Fibroblasts are typically V+/A- and are present in the intestinal submucosal and serosa. Subepithelial myofibroblasts (SEMF) are found adjacent to intestinal epithelial cells and are V+/A+. Intestinal smooth muscle cells of the muscularis mucosa and muscularis propria are normally V-/A+. All of these mesenchymal cell types

Activated fibroblasts or myofibroblasts in tissues undergoing a fibrotic process may be de‐ rived from a variety of sources. There are three general mechanisms which allow for tissue accumulation of these cells: proliferation of existing tissue fibroblasts, recruitment of fibro‐ blast precursor cells from bone marrow, and transformation either of epithelial cells via epi‐ thelial to mesenchymal transistion (EMT), or of endothelial cells by endothelial to mesenchymal transition (EndoMT) [89, 90]. Proliferation and activation of tissue fibroblasts occurs in response to profibrotic signals from infiltrating inflammatory cells or from colonic epithelial cells exposed to proinflammatory cytokines [91]. Soluble inflammatory mediators also drive recruitment of fibroblast precursor cells (fibrocytes) from bone marrow. These fi‐ brocytes migrate from the bloodstream into tissues undergoing pathologic fibrosis in re‐ sponse to specific chemokine gradients [89]. EMT and EndoMT are induced by TGFβ [92]. The relative significance of each of mechanisms discussed above to activated fibroblast/

ofibroblasts ; and accumulation of excess extracellular matrix proteins [84].

have been implicated in collagen production in CD patients [69, 87, 88].

myofibroblast accumulation at the site of injury in CD is not yet fully understood

Smooth muscle hyperplasia surrounded by collagen deposits is the major histologic feature of fibrostenotic CD. This smooth muscle proliferation expands and disrupts the muscularis mucosa. Thickening of the muscular layer is associated with an increase in the number of vimentin-positive cells [93, 94]. In severely affected tissue, even histologically normal mus‐

Mesenchymal cells including myofibroblasts as well as smooth muscle cells of the muscula‐ ris mucosa and muscularis propria are the main producers of ECM proteins in the intestine. These ECM proteins include structural proteins such as collagen, matricellular proteins such as osteopontin and thrombospondin, and other specialized proteins such as vitronectin and fibronectin. Collagen is the major ECM component associated with intestinal fibrosis. The most common collagen subtypes in normal intestine are type I, type III, and type V in order of abundance. In intestinal fibrosis, there is an increase in total collagen as well as specific and relative increases in collagen types III and V [95-97].

Fibronectin and vitronectin are ligands for the αVβ3 integrin and, in the presence of fibro‐ nectin, smooth muscle IGF-1-stimulated IGF-1 receptor activation is augmented [98]. This suggests that increased production of these proteins by smooth muscle cells at sites of intes‐ tinal stricture could activate αVβ3 integrin and further increase secretion of collagen as well as promote cellular proliferation creating a positive feedback loop which could further sub‐ vert the normal healing process. Fibronectin is also an important mediator in focal adhesion kinase (FAK) signaling pathways involved in cell migration [99]. Myofibroblasts synthesize abundant fibronectin which is largely responsible for the autocrine induction of intestinal myofibroblast migration [100].

The balance between formation and breakdown of ECM proteins determines the net dep‐ osition in tissues. In intestinal fibrosis, mechanisms to degrade ECM fail to keep pace with deposition. Matrix metalloproteinases (MMPs), a large family of proteolytic en‐ zymes, are responsible for the breakdown of ECM components. The proteolytic activity of MMPs is controlled by tissue inhibitors of metalloproteinases (TIMPs) and an imbal‐ ance between MMP and TIMP activity can result in excessive deposition of ECM pro‐ teins with subsequent fibrosis [101]. Higher levels of constitutive TIMP-1 expression have been shown in intestinal myofibroblast culture derived from fibrotic CD patients than those from normal individuals [102].

Transforming growth factor –β (TGFβ) is a pleotrophic cytokine and one of the most influ‐ ential factors in fibrotic processes. It is a component of Th17 as well as regulatory T cell type immune responses as well as a profibrotic mediator. TGFβ exerts profibrotic effects through its ability to regulate collagen expression and extracellular matrix dynamics. There are three isoforms of TGFβ: TGFβ1, TGFβ2, and TGFβ3. TGFβ1 activates the canonical Smad signal‐ ing cascade leading to translocation of the Smad receptor complex into the nucleus and reg‐ ulation of gene transcription including ECM genes such as collagen I, collagen III, and fibronectin [103]. TGFβ also induces EMT in organ-fibrosis inducing diseases [104] and can to induce EndoMT in vitro via a "noncanonical" signaling pathway [105]. In CD patients, TGFβ1 and TGFβ3 are increased in in smooth muscle, fibroblasts and myofibroblasts from the strictured region when compared to normal intestine [102, 106].

TGFβ family proteins are important in regulating the synthesis and breakdown of ECM pro‐ teins [107]. TGFβ1 downregulates MMP expression and enhances the expression of TIMP-1 [101]. Characterization of the role of TGFβ expression in disease has been done using myofi‐ broblast cultures. Myofibroblasts from normal intestine predominantly express TGFβ3 while those patients with fibrotic CD had significantly lower expression of TGFβ3 and higher lev‐ els of TGFβ1 and TGFβ2 [102, 108].

While many murine models of inflammatory colitis or enteritis exist, these models are gen‐ erally not suitable for study the pathogenesis of stricture formation or for testing interven‐ tion strategies because they generate very little intestinal fibrosis. Fibrotic models have been challenging to develop given the inherent resistance of mice when compared to other spe‐ cies in development of fibrotic disease [94]. However, progress is being made in this area.

Fibrosis in Crohn's Disease http://dx.doi.org/10.5772/46222 161

Ileocecal resection is a common surgical intervention in CD and is associated with high rates of disease recurrence[120]. After surgery, recurrence of inflammation and/or fibrosis typical‐ ly occurs at the anastomosis and in the small intestine immediately upstream of the anasto‐ mosis. A model of ileocecal resection in IL-10 gene knockout mice has been described which develops inflammation and fibrosis both at the anastomosis site and in other regions of the small intestine [121]. This approach is attractive because it models a major clinical feature of CD fibrostenotic disease and is highly relevant to future clinical trials where therapeutics targeting CD fibrosis will likely be evaluated for prevention of postsurgical recurrence. However, IL-10 null mice do not spontaneously develop small intestinal inflammation and this surgical approach may need to be combined with one of the existing murine ileitis mod‐

Chronic TNBS treatment has been used to induce colitis in mice as well as rats. In the mouse, TNBS with concomitant administration of ethanol as an epithelial barrier disrupt‐ er induces intestinal ulceration and inflammation. This model is widely used to investi‐ gate acute inflammation in the gut. Chronic TNBS treatment has been tested in an effort to develop a more robust intestinal fibrosis model in mouse [122]. This model has been reported to have some common features with CD including transmural inflammation and stricturing with proximal dilation and fibrosis. Affected animals have increased ex‐ pression of MMP-1 and collagen type 1. Fibrosis in this model can be enhanced by treat‐ ment with indomethacin, a cyclooxygenase (COX) inhibitor which can block the anti-

Dextran sulfate sodium added to drinking water is frequently used to induce epithelial in‐ jury and acute colitis in mice. Fibrosis with associated increase in collagen, TGFβ, and ma‐ trix metalloproteinase expression has been described in C57BL6 mice following a single 5 day cycle of DSS exposure [124]. The authors were also able to show an increase in fibro‐ blasts (V+/A-) and myofibroblasts (V+/A+ ) cells in the mucosa and submucosa. While this likely reflects a primary intestinal wound healing response rather than the chronic fibrotic process suggested by the authors, it is worth considering if this could be a useful pathway model which might allow rapid testing the effect of therapeutic candidates on specific ele‐ ments of the wound healing/fibrotic response. Other groups have investigated the effect of multiple cycles of DSS exposure on fibrotic response in FVB-N and C57BL6 mice [125]. A single cycle of DSS exposure in C57BL6 mice does result in ECM deposition followed by mucosal repair and normalized mucosal architecture. Multiple cycles of DSS exposure did not result in enhanced fibrosis in FVB-N mice, however, it did result in prolongation of a fibrotic response in C57BL6 mice as measured by procollagen α1(I) promoter-GFP reporter transgene reporter activity. Further characterization will be needed to determine the utility

els to achieve the most relevant preclinical model.

fibrotic effects of COX-2 [123].

of this model.

Insulin-like growth factor has also been implicated in the pathogenesis of stricture forma‐ tion [109]. Intestinal smooth muscle cells express IGF-1 which activates the IGF-1 recep‐ tor thereby regulating smooth muscle cell hyperplasia by simultaneously stimulating proliferation and inhibiting apoptosis [110, 111]. Studies using CD tissue from patients undergoing intestinal resection show increased expression of both IGF-1 as well as syner‐ gistic IGF binding protein 5 (IGFBP-5) in lesional tissue [112]. Localization studies show IGF-1 is upregulated in smooth muscle cells in regions of stricture when compared to tis‐ sue from surgical margins.

## **7. Preclinical models**

A major challenge facing scientists interested in developing treatments for CD-associated fi‐ brotic disease is the need for a robust animal model which develops morphologic features and utilizes pathogenic processes similar to those characterized for the human disease. Pre‐ clinical models should provide a consistent environment for testing intervention strategies and quantifying outcomes. At present, no animal model exists which reproduces the unique histologic features associated with CD intestinal strictures. There are several models which address some aspects of CD stricture pathogenesis and those are reviewed below.

Intestinal inflammation and fibrosis can be induced in rats by injection of peptidoglycanpolysaccharide (PG-PS) into the intestinal wall or by repeated rounds of trinitrobenzene sul‐ fonic acid (TNBS) treatment [113, 114]. PG-PS injection causes acute inflammation which peaks by 2 days followed by remission. Spontaneous reactivation of inflammation occurs in genetically susceptible rat strains by 12-17 days and is characterized by progressive trans‐ mural granulomatous enterocolitis [115]. Multiple cycles of intrarectal injection of TNBS in ethanol also induces a granulomatous transmural inflammatory response which becomes dominated by chronic inflammation and fibrosis after cycle 4[113, 116]. This model features transmural collagen deposition which is most prominent in the submucosa. Smooth muscle proliferation and expansion into the submucosal space is not a feature of these models.

Given that no existing preclinical model completely mimics changes found in human CD fi‐ brostenosis, assessment of the value of models should be based on the presence of pathways of interest as well as tractability in testing potential therapeutic entities. These rat models show transmural inflammation associated with transmural fibrosis as well as overexpres‐ sion of TGFβ and/or IGF-1 in a manner consistent with human disease [116-119]. However, given the availability of reagents and other research tools, mouse models of intestinal fibro‐ sis are more desirable to the research community.

While many murine models of inflammatory colitis or enteritis exist, these models are gen‐ erally not suitable for study the pathogenesis of stricture formation or for testing interven‐ tion strategies because they generate very little intestinal fibrosis. Fibrotic models have been challenging to develop given the inherent resistance of mice when compared to other spe‐ cies in development of fibrotic disease [94]. However, progress is being made in this area.

TGFβ family proteins are important in regulating the synthesis and breakdown of ECM pro‐ teins [107]. TGFβ1 downregulates MMP expression and enhances the expression of TIMP-1 [101]. Characterization of the role of TGFβ expression in disease has been done using myofi‐ broblast cultures. Myofibroblasts from normal intestine predominantly express TGFβ3 while those patients with fibrotic CD had significantly lower expression of TGFβ3 and higher lev‐

Insulin-like growth factor has also been implicated in the pathogenesis of stricture forma‐ tion [109]. Intestinal smooth muscle cells express IGF-1 which activates the IGF-1 recep‐ tor thereby regulating smooth muscle cell hyperplasia by simultaneously stimulating proliferation and inhibiting apoptosis [110, 111]. Studies using CD tissue from patients undergoing intestinal resection show increased expression of both IGF-1 as well as syner‐ gistic IGF binding protein 5 (IGFBP-5) in lesional tissue [112]. Localization studies show IGF-1 is upregulated in smooth muscle cells in regions of stricture when compared to tis‐

A major challenge facing scientists interested in developing treatments for CD-associated fi‐ brotic disease is the need for a robust animal model which develops morphologic features and utilizes pathogenic processes similar to those characterized for the human disease. Pre‐ clinical models should provide a consistent environment for testing intervention strategies and quantifying outcomes. At present, no animal model exists which reproduces the unique histologic features associated with CD intestinal strictures. There are several models which

Intestinal inflammation and fibrosis can be induced in rats by injection of peptidoglycanpolysaccharide (PG-PS) into the intestinal wall or by repeated rounds of trinitrobenzene sul‐ fonic acid (TNBS) treatment [113, 114]. PG-PS injection causes acute inflammation which peaks by 2 days followed by remission. Spontaneous reactivation of inflammation occurs in genetically susceptible rat strains by 12-17 days and is characterized by progressive trans‐ mural granulomatous enterocolitis [115]. Multiple cycles of intrarectal injection of TNBS in ethanol also induces a granulomatous transmural inflammatory response which becomes dominated by chronic inflammation and fibrosis after cycle 4[113, 116]. This model features transmural collagen deposition which is most prominent in the submucosa. Smooth muscle proliferation and expansion into the submucosal space is not a feature of these models.

Given that no existing preclinical model completely mimics changes found in human CD fi‐ brostenosis, assessment of the value of models should be based on the presence of pathways of interest as well as tractability in testing potential therapeutic entities. These rat models show transmural inflammation associated with transmural fibrosis as well as overexpres‐ sion of TGFβ and/or IGF-1 in a manner consistent with human disease [116-119]. However, given the availability of reagents and other research tools, mouse models of intestinal fibro‐

sis are more desirable to the research community.

address some aspects of CD stricture pathogenesis and those are reviewed below.

els of TGFβ1 and TGFβ2 [102, 108].

160 Inflammatory Bowel Disease

sue from surgical margins.

**7. Preclinical models**

Ileocecal resection is a common surgical intervention in CD and is associated with high rates of disease recurrence[120]. After surgery, recurrence of inflammation and/or fibrosis typical‐ ly occurs at the anastomosis and in the small intestine immediately upstream of the anasto‐ mosis. A model of ileocecal resection in IL-10 gene knockout mice has been described which develops inflammation and fibrosis both at the anastomosis site and in other regions of the small intestine [121]. This approach is attractive because it models a major clinical feature of CD fibrostenotic disease and is highly relevant to future clinical trials where therapeutics targeting CD fibrosis will likely be evaluated for prevention of postsurgical recurrence. However, IL-10 null mice do not spontaneously develop small intestinal inflammation and this surgical approach may need to be combined with one of the existing murine ileitis mod‐ els to achieve the most relevant preclinical model.

Chronic TNBS treatment has been used to induce colitis in mice as well as rats. In the mouse, TNBS with concomitant administration of ethanol as an epithelial barrier disrupt‐ er induces intestinal ulceration and inflammation. This model is widely used to investi‐ gate acute inflammation in the gut. Chronic TNBS treatment has been tested in an effort to develop a more robust intestinal fibrosis model in mouse [122]. This model has been reported to have some common features with CD including transmural inflammation and stricturing with proximal dilation and fibrosis. Affected animals have increased ex‐ pression of MMP-1 and collagen type 1. Fibrosis in this model can be enhanced by treat‐ ment with indomethacin, a cyclooxygenase (COX) inhibitor which can block the antifibrotic effects of COX-2 [123].

Dextran sulfate sodium added to drinking water is frequently used to induce epithelial in‐ jury and acute colitis in mice. Fibrosis with associated increase in collagen, TGFβ, and ma‐ trix metalloproteinase expression has been described in C57BL6 mice following a single 5 day cycle of DSS exposure [124]. The authors were also able to show an increase in fibro‐ blasts (V+/A-) and myofibroblasts (V+/A+ ) cells in the mucosa and submucosa. While this likely reflects a primary intestinal wound healing response rather than the chronic fibrotic process suggested by the authors, it is worth considering if this could be a useful pathway model which might allow rapid testing the effect of therapeutic candidates on specific ele‐ ments of the wound healing/fibrotic response. Other groups have investigated the effect of multiple cycles of DSS exposure on fibrotic response in FVB-N and C57BL6 mice [125]. A single cycle of DSS exposure in C57BL6 mice does result in ECM deposition followed by mucosal repair and normalized mucosal architecture. Multiple cycles of DSS exposure did not result in enhanced fibrosis in FVB-N mice, however, it did result in prolongation of a fibrotic response in C57BL6 mice as measured by procollagen α1(I) promoter-GFP reporter transgene reporter activity. Further characterization will be needed to determine the utility of this model.

*Salmonella* species are facultative intracellular gram negative bacteria which cause a range of illnesses including, but not limited to, enterocolitis [126]. *Salmonella enterica* serovar Typhi‐ murium is an enteric bacterial pathogen which normally causes little intestinal pathology in mice but instead mimics human typhoid. However, a model which utilizes oral streptomy‐ cin pretreatment has been developed which allows study of *S*. Typhimurium-induced cecal inflammation [127]. This work has been extended by utilizing attenuated *S*. Typhimurium strains or by infecting resistant mouse strains which carry a functional nramp1 gene to in‐ duce chronic infection which results in intestinal fibrosis characterized by transmural colla‐ gen deposition and accumulation of fibroblasts in the intestinal submucosa [128]. While increase in collagen deposition is observed throughout the colon, the most intense lesions are present in the cecum. The *Salmonella* model of intestinal fibrosis is unique in that it is induced by bacterial colitis. It results in a relatively long term fibrotic process where fibrosis can be observed in the cecal submucosa at least to day 40 post infection. Similar to human CD, increased TGFβ and IGF-1 are associated with fibrosis in this model.

mouse model of intestinal fibrosis [123]. Much more research will be needed to determine if

Fibrosis in Crohn's Disease http://dx.doi.org/10.5772/46222 163

Resveratrol (trans-3,5,4'-trihydroxystilbene) is a phytoalexin found in a variety of plant products including berries, peanuts, grapes and red wine. It has been shown to reduce in‐ flammation in rat colitis [136]. Resveratrol has also been shown to reduce activation of NFkB in TNBS colitis [137]. A recent paper reports that resveratrol exposure results in decreased collagen synthesis as well as apoptosis in rat intestinal smooth muscle cells [138]. While the data on resveratrol is quite preliminary, the data is of interest because it targets

Anti-inflammatory and anti-fibrotic effects of the cholesterol lowering 3-hydroxy-3-methyl‐ glutaryl-CoA reductase inhibitors (statins) have been reported. Statins may play an anti-fi‐ brotic role through inhibition of the activation and proliferation of fibroblasts and by inducing apoptosis of activated fibroblasts [139]. Angiotensin type 1 receptor blockers [140] and the angiotensin-converting enzyme inhibitor captopril [141] have also been proposed as

Fibrosis has traditionally been considered an irreversible process. Further testing of these and other agents which have potential to block initiation or inhibit progression of fibrosis may also reveal if medical treatment has the potential to reverse existing fibrotic lesions. Re‐ search to further characterize the underlying pathophysiologic processes involved in fibros‐ tenotic disease and to test potential therapeutic approaches remains important to the goal of

[1] Greenstein, A.J., et al., *Perforating and non-perforating indications for repeated operations*

[2] Gasche, C., et al., *A simple classification of Crohn's disease: Report of the working party for the World Congresses of Gastroenterology, Vienna 1998.* Inflammatory Bowel Diseases,

[3] Peyrin-Biroulet, L., et al., *The Natural History of Adult Crohn ' s Disease in Population-Based Cohorts.* American Journal of Gastroenterology, 2010. 105: p. 289–297.

*in Crohn's disease: evidence for two clinical forms.* Gut, 1988. 29: p. 588-592.

RA has promise as a fibrosis modifying agent in CD.

smooth muscle rather than the fibroblasts or myofibroblasts.

fibrosis inhibitors.

**Author details**

Lauri Diehl

**References**

fully meeting CD therapeutic needs.

Department of Pathology, Genentech, USA

2000. 6(1): p. 8-15.

## **8. Prevention or treatment of CD fibrosis**

While considerable progress has been made, the pathophysiology of fibrostenotic disease in CD patients is incompletely understood. The drug development challenges this creates are greatly compounded by the absence of a well defined and widely accepted preclinical ani‐ mal model of intestinal fibrosis. Recognition of the unmet need for medical interventions which can effectively prevent or treat CD fibrostenotic disease drives ongoing research in both areas. Despite the challenges, a number of potential therapeutic agents or pathways have undergone preliminary testing. A few of these results are summarized below. The data available for all of these agents is quite limited.

Prostaglandins (PGE1 and 2) are known to inhibit smooth muscle proliferation as well as fi‐ broblast proliferation induced by proinflammatory cytokines [129, 130]. Reduced PGE2 lev‐ els are associated with development of fibrosis in idiopathic pulmonary fibrosis (IPF) [131] and indomethacin treatment, which inhibits PGE2, increases fibrosis in the chronic murine TNBS model of colon fibrosis. However, mice deficient in prostaglandin endoperoxide syn‐ thase (Ptgs) 2, an enzyme involved in prostaglandin production, showed deficient wound healing following full-thickness colonic biopsy so the effects of prostaglandins may be com‐ plex and, perhaps, dependent on the stage of wound healing [132]. Phosphatidyl choline, a polyunsaturated fatty acid which is a precursor to PGE2, has been shown to decrease stric‐ ture formation in the rat TNBS intestinal fibrosis model [133]. These data suggest a role for PGE2 in intestinal wound healing and fibrosis but the potential for a therapeutic role re‐ quires further investigation.

The steroid hormone retinoic acid (RA) is another potential agent for modification of fibrosis in CD. RA has been shown to have effects on human fibroblast proliferation in cells isolated from IPF lungs [134] and to protect against bleomycin-induced pulmonary fibrosis in mice [135]. More recently, RA has been shown to reduce intestinal fibrosis in the chronic TNBS mouse model of intestinal fibrosis [123]. Much more research will be needed to determine if RA has promise as a fibrosis modifying agent in CD.

Resveratrol (trans-3,5,4'-trihydroxystilbene) is a phytoalexin found in a variety of plant products including berries, peanuts, grapes and red wine. It has been shown to reduce in‐ flammation in rat colitis [136]. Resveratrol has also been shown to reduce activation of NFkB in TNBS colitis [137]. A recent paper reports that resveratrol exposure results in decreased collagen synthesis as well as apoptosis in rat intestinal smooth muscle cells [138]. While the data on resveratrol is quite preliminary, the data is of interest because it targets smooth muscle rather than the fibroblasts or myofibroblasts.

Anti-inflammatory and anti-fibrotic effects of the cholesterol lowering 3-hydroxy-3-methyl‐ glutaryl-CoA reductase inhibitors (statins) have been reported. Statins may play an anti-fi‐ brotic role through inhibition of the activation and proliferation of fibroblasts and by inducing apoptosis of activated fibroblasts [139]. Angiotensin type 1 receptor blockers [140] and the angiotensin-converting enzyme inhibitor captopril [141] have also been proposed as fibrosis inhibitors.

Fibrosis has traditionally been considered an irreversible process. Further testing of these and other agents which have potential to block initiation or inhibit progression of fibrosis may also reveal if medical treatment has the potential to reverse existing fibrotic lesions. Re‐ search to further characterize the underlying pathophysiologic processes involved in fibros‐ tenotic disease and to test potential therapeutic approaches remains important to the goal of fully meeting CD therapeutic needs.

## **Author details**

Lauri Diehl

*Salmonella* species are facultative intracellular gram negative bacteria which cause a range of illnesses including, but not limited to, enterocolitis [126]. *Salmonella enterica* serovar Typhi‐ murium is an enteric bacterial pathogen which normally causes little intestinal pathology in mice but instead mimics human typhoid. However, a model which utilizes oral streptomy‐ cin pretreatment has been developed which allows study of *S*. Typhimurium-induced cecal inflammation [127]. This work has been extended by utilizing attenuated *S*. Typhimurium strains or by infecting resistant mouse strains which carry a functional nramp1 gene to in‐ duce chronic infection which results in intestinal fibrosis characterized by transmural colla‐ gen deposition and accumulation of fibroblasts in the intestinal submucosa [128]. While increase in collagen deposition is observed throughout the colon, the most intense lesions are present in the cecum. The *Salmonella* model of intestinal fibrosis is unique in that it is induced by bacterial colitis. It results in a relatively long term fibrotic process where fibrosis can be observed in the cecal submucosa at least to day 40 post infection. Similar to human

While considerable progress has been made, the pathophysiology of fibrostenotic disease in CD patients is incompletely understood. The drug development challenges this creates are greatly compounded by the absence of a well defined and widely accepted preclinical ani‐ mal model of intestinal fibrosis. Recognition of the unmet need for medical interventions which can effectively prevent or treat CD fibrostenotic disease drives ongoing research in both areas. Despite the challenges, a number of potential therapeutic agents or pathways have undergone preliminary testing. A few of these results are summarized below. The data

Prostaglandins (PGE1 and 2) are known to inhibit smooth muscle proliferation as well as fi‐ broblast proliferation induced by proinflammatory cytokines [129, 130]. Reduced PGE2 lev‐ els are associated with development of fibrosis in idiopathic pulmonary fibrosis (IPF) [131] and indomethacin treatment, which inhibits PGE2, increases fibrosis in the chronic murine TNBS model of colon fibrosis. However, mice deficient in prostaglandin endoperoxide syn‐ thase (Ptgs) 2, an enzyme involved in prostaglandin production, showed deficient wound healing following full-thickness colonic biopsy so the effects of prostaglandins may be com‐ plex and, perhaps, dependent on the stage of wound healing [132]. Phosphatidyl choline, a polyunsaturated fatty acid which is a precursor to PGE2, has been shown to decrease stric‐ ture formation in the rat TNBS intestinal fibrosis model [133]. These data suggest a role for PGE2 in intestinal wound healing and fibrosis but the potential for a therapeutic role re‐

The steroid hormone retinoic acid (RA) is another potential agent for modification of fibrosis in CD. RA has been shown to have effects on human fibroblast proliferation in cells isolated from IPF lungs [134] and to protect against bleomycin-induced pulmonary fibrosis in mice [135]. More recently, RA has been shown to reduce intestinal fibrosis in the chronic TNBS

CD, increased TGFβ and IGF-1 are associated with fibrosis in this model.

**8. Prevention or treatment of CD fibrosis**

162 Inflammatory Bowel Disease

available for all of these agents is quite limited.

quires further investigation.

Department of Pathology, Genentech, USA

## **References**


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**Section 2**

**Management of Disease**
