**3. Overview of animal IBD models**

generation and functional chambers). Both also enable exposure of large numbers of animals, for example, chambers of >700 L may each accommodate approximately 200 mice. The freedom of movement of animals during exposure results in minimal stress, although the animals require training to adjust to grid-caging systems and food is typically withdrawn to minimise oral uptake of aerosol constituents. One criticism of whole body exposures is that there is a high potential for compound uptake through non-inhalation routes because animals have surface contact with aerosol deposits on the cage surfaces and on their fur. In historical studies, up to 60% of aerosol constituents on the fur (pelt burden) were ingested following whole body exposures [28] and transdermal uptake may also be significant for some compounds. Because the skin is an effective barrier for drug transport, only potent drugs with appropriate physicochemical properties (low molecular weight and adequate solubility in aqueous and non-aqueous solvents) are suitable candidates for transdermal delivery [29–31]. Such mixed uptake mechanisms potentially occurring in whole body exposure systems complicate both dose estimations and require deconvolution of uptake amounts through oral/transdermal

184 Experimental Animal Models of Human Diseases - An Effective Therapeutic Strategy

Nose-only exposure chambers require restraint of the animals to permit only the head (nose) to be exposed to the test aerosol. This has the major advantage of decreasing deposition of aerosol constituents on the pelts, resulting in less oral uptake from grooming behaviour [32]. However, there are also disadvantages with this system, including technical asphyxiation (animal movements in the exposure tube may cut off their air supply); therefore, constant monitoring during the exposure period is required. In addition, because of stress associated with restraint in nose-only exposure systems, training is required to adapt animals to the technical procedures. Vehicle or fresh air exposures are also needed to help distinguish such stress-related effects from treatment effects [33]. The daily execution of nose-only exposures requires that animals be individually inserted into the exposure tubes, a technical aspect that

Measurement of dosages in an *in vivo* inhalation experiment is dependent upon many parameters, including deposition of the agent to the lungs (which itself is dependent upon aerosol droplet size), respiratory minute volume and body weight of the animal. This relationship is

DD <sup>=</sup> <sup>C</sup> <sup>×</sup> <sup>R</sup>M<sup>V</sup> <sup>×</sup> <sup>D</sup> <sup>×</sup> IF \_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_\_ Body weight (kg) (1)

where DD is the delivered dose (mg/kg); C is the concentration of substance (mg/L); RMV is

Among these parameters, the respiratory minute volume is important to determine the availability of compound for deposition and exchange in the lungs. This parameter may be calculated using allometric formulae relating body weights to minute volumes in laboratory animals [35, 36]. The alternative, direct measurement of the minute volume, as can be

may limit the numbers of animals that can be used in the experiments.

the respiratory minute volume (L/min) and IF is the inhalable fraction.

generally described by the following formula [34]:

and inhaled routes.

**2.2. Dose translatability**

The various types of animal models developed to study IBD may be divided into several categories depending on: the method of inducing the pathology (*chemically induced, bacteriainduced or genetically engineered*); the IBD subtype modelled in the animal (*UC or CD*); the site of inflammation (*colon, ileum, both sites or systemic*); and, in genetically engineered models, the gene modification strategy (*conventional transgenic* (*Tg*) *or knockout* (*KO*), *cell-specific conditional Tg or KO*, *inducible KO, knock-in, innate, mutagen-induced or spontaneous models*) [23, 38, 39]. The total number of IBD mouse models is growing, especially because of current genetic engineering approaches that accelerate development of new strains, so far, over 74 genetically engineered mouse models were reported to spontaneously develop intestinal inflammation [38]. The full description of all IBD models is beyond the scope of this chapter. However, **Table 1** summarises the most significant IBD murine models, highlighting their methods of pathology induction, IBD subtypes, sites of inflammation and mechanism of action (**Figure 1**). More detailed reviews of the different mouse models of IBD are available (e.g. see Refs. [23, 40, 41]).

There is a close agreement in many pathological findings among experimental IBD models and human disease. These include the molecular pathways and histological features of tissue injury, dysfunction of the immune system (including impact of the microbiome), genetic heterogeneity and primary defects in mucosal barrier function. All pathologies have been well established in several experimental models of colitis; therefore, these models closely resemble aspects of the human diseases. These common features enable exploration of specific pathological mechanisms, facilitating development of new therapeutic approaches. However, none of these models fully reflects human IBD, with each representing rather a small tile of a mosaic. This hinders a generalised view of the systemic consequences of IBD, often masking possible extra-intestinal implications [42].


The presence of such a multitude of mouse models indicates that IBD is mediated by complicated, multifactorial mechanisms. As expected, this complexity is greater in human beings, where environmental and clinical factors, such as smoking, diet, drugs, ethnicity, geographical area, social status, gender, stress and appendectomy, further modulate onset of IBD patholo-

Colon

**Table 1.** Classification of animal models of IBD. IBD subtype and site of inflammation predominantly addressed by the model, where applicable, are shown in bold font. DSS, dextran sulfate sodium; IBD, inflammatory bowel disease; DNBS, 2,4-dinitrobenzene sulfonic acid; TNBS, 2,4,6-trinitrobenzenesulfonic acid; UC, ulcerative colitis; CD, Crohn's disease;

**inflammation**

CD **Small intestine** Colon

CD Small intestine Colon

UC/CD Small intestine Colon

Adoptive transfer CD **Small intestine**

colon

Transgenic mouse UC Colon CD4+ T cell infiltration-

**Mechanism References**

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[167]

187

[168]

[169]

[173]

Loss of Paneth and goblet cells with impairment of mucosal defence

dependent inflammation

Th1-type inflammation [170]

Bacterial sensitisation [171]

NF-κB signalling dysregulation

IL-12-driven Th1 hyper-response

CD Colon Epithelial cell dysfunction [172]

Animal Inhalation Models to Investigate Modulation of Inflammatory Bowel Diseases

CD Small intestine TNF-α overproduction [64]

Clinical and pre-clinical findings suggested divergent effects of smoking or smoke constituents on the pathophysiology of the gut depending mainly on two conditions, the IBD subtype and the route of administration of the active substance (such as nicotine or CS). Active human smoking is difficult to mimic under laboratory conditions, while classical *in vitro* approaches have translational limitations. Thus, several animal models have been used to assess the impact of CS, nicotine or non-nicotine CS constituents on intestinal pathophysiology [47]. Both genetic- and chemically induced IBD models have been used and effects of various treatment regimens on gut inflammation in these systems are summarised in **Table 2**. There is a general consensus that CS and nicotine administration do not cause macroscopic or histological damage or inflammation in the healthy gut. However, differences in immune cell recruitment [48], cytokine secretion [49–51], mucosal barrier [52, 53] and oxidative stress were

**3.1. Inhalation studies investigating the effect of CS in rodent models of IBD**

observed [54, 55], although without evident tissue damage.

**IBD model Model category IBD subtype Site of** 

NEMO−/− CD Small intestine/

engineered, conditional KO

XBP1−/− Genetically

IL-7 Tg mice (IL-7 overexpression)

STAT4 Tg mice (STAT4 overexpression)

HLA-B27 Tg mice

DNN-cadherin/ keratin8−/−

CD45RB high-transfer

TNFΔARE Mutation

DNCB, Dinitrochlorobenzene.

knock-in

gies [43–46].


**IBD model Model category IBD subtype Site of** 

186 Experimental Animal Models of Human Diseases - An Effective Therapeutic Strategy

Indomethacin CD **Small intestine**

C3H/HejBir Spontaneous CD **Small intestine**

IL-2−/− UC Colon/systemic

NOD2−/− CD Small intestine

IL-23−/− CD Small intestine

A20−/− UC/CD Colon

DSS Chemically

Salmonella induced

Adherent invasive *E*. *coli*

IL-10−/− Genetically

induced

Bacterially induced

engineered/ knockouts (KO) **inflammation**

TNBS **CD**/UC Colon Hapten-dependent

DNBS CD/UC Colon Hapten-dependent

Oxazolone UC Colon Hapten-dependent

Iodoacetamide UC Colon Sulphydryl (SH)

DNCB **UC**/CD Colon Hapten-dependent

UC **Colon**

TGF-β−/− UC/CD Systemic Macrophage

Acetic acid UC Colon Epithelial cell damage [152] Carrageenan UC Colon Epithelial cell damage [153]

Colon

UC Colon Bacterial colonisation-

Small intestine

Colon

(no small intestine)

Colon

MDR1A−/− UC Colon Accumulation of bacterial

Gαi2−/− UC Colon Impaired T/B cell function

TCRα−/− UC Colon Th2-type inflammation [75]

Colon

Small intestine

SAMP1/4it CD Small intestine Epithelial cell dysfunction [40]

CD **Small intestine** Colon

UC Colon Epithelial cell damage [147, 148]

**Mechanism References**

[149]

[150]

[151]

[155]

[156]

[157]

[158]

[159]

[160]

[161]

[162]

[163]

[164]

[165, 166]

immunogenic response

immunogenic response

immunogenic response

compound (e.g. glutathione) blocker

immunogenic response

induced inflammation

Bacterial-dependent epithelial cell damage

hyperactivation and impaired Treg function

Impaired T cell/Treg

TNF-induced NF-κB signalling dysregulation

products and increased T

and epithelial cell damage

Impaired Th17 cell

function

NF-κB and TLR2 signalling

function

dysregulation

cell activation

Epithelial cell dysfunction [39]

Impaired Treg function [74]

Epithelial cell damage [154]

**Table 1.** Classification of animal models of IBD. IBD subtype and site of inflammation predominantly addressed by the model, where applicable, are shown in bold font. DSS, dextran sulfate sodium; IBD, inflammatory bowel disease; DNBS, 2,4-dinitrobenzene sulfonic acid; TNBS, 2,4,6-trinitrobenzenesulfonic acid; UC, ulcerative colitis; CD, Crohn's disease; DNCB, Dinitrochlorobenzene.

The presence of such a multitude of mouse models indicates that IBD is mediated by complicated, multifactorial mechanisms. As expected, this complexity is greater in human beings, where environmental and clinical factors, such as smoking, diet, drugs, ethnicity, geographical area, social status, gender, stress and appendectomy, further modulate onset of IBD pathologies [43–46].

#### **3.1. Inhalation studies investigating the effect of CS in rodent models of IBD**

Clinical and pre-clinical findings suggested divergent effects of smoking or smoke constituents on the pathophysiology of the gut depending mainly on two conditions, the IBD subtype and the route of administration of the active substance (such as nicotine or CS). Active human smoking is difficult to mimic under laboratory conditions, while classical *in vitro* approaches have translational limitations. Thus, several animal models have been used to assess the impact of CS, nicotine or non-nicotine CS constituents on intestinal pathophysiology [47]. Both genetic- and chemically induced IBD models have been used and effects of various treatment regimens on gut inflammation in these systems are summarised in **Table 2**. There is a general consensus that CS and nicotine administration do not cause macroscopic or histological damage or inflammation in the healthy gut. However, differences in immune cell recruitment [48], cytokine secretion [49–51], mucosal barrier [52, 53] and oxidative stress were observed [54, 55], although without evident tissue damage.

**Figure 1.** Schematic view of major inflammatory and anti-inflammatory mechanisms implicated in inflammatory bowel diseases and the potential role of a nicotinic anti-inflammatory pathway. Top: altered microbiota in the colonic lumen and/or epithelial-damaging factors (e.g., DSS in experimentally induced colitis) lead to the disruption of the epithelial barrier function and the consequent infiltration of bacteria and other antigens. Middle: various inflammatory processes can be triggered in the lamina propria by the infiltrating bacteria (DSS-induced epithelial barrier; "Barrier dysfunction and epithelial permeability" and "Nicotinic anti-inflammatory pathway" sectors), haptens (oxazolone- and TNBS-induced inflammation, "Differential nicotine effects in UC-like (oxazolone) and CD-like (TNBS) colitis" sector) or by endogenous dysregulation of the balance between Th1/Th17-driven and Th2-driven immune activities, (genetically engineered mouse models; "Immune regulation" section). A hypothetical role of nicotinic receptor-mediated anti-inflammatory response is depicted in the "Nicotinic anti-inflammatory pathway" sector. Bottom: the colonic vasculature is symbolized as a tube running perpendicular to the cross section of the colon. The blood stream delivers leukocytes recruited by cytokine shedding from the local inflammatory sites and enables the perpetuation of the inflammation, e.g., via circulating T-cells. Systemically provided nicotine could increase the anti-inflammatory nicotinic signaling that is naturally transmitted by acetylcholine shed from the efferents of the vagus nerve that innervate the colonic wall. For details of these mechanisms, see Chapter 4.1 to 4.4. Modified from: De Jonge & Ulloah (2007), Ordas et al. (2012), Xu et al. (2014).

Consistent with results of human epidemiological studies, CS had opposing effects on development of CD (negatively) and UC (positively) in several, but not all, of their respective IBD models. Only a few of these studies used inhalation exposure (**Table 2**) and most of their findings mimicked the effects of smoking in humans with IBD.

Thus, the dichotomous effects of CS inhalation, on development of CD versus UC, were perfectly reproduced using two different rat IBD models [54–60]. 2,4,6-trinitrobenzenesulphonic acid (TNBS) and 2,4-dinitrobenzene sulphonic acid (DNBS) were instilled into the rat colon to induce, respectively, CD- and UC-like symptoms. Indeed, pre-exposure of rats to CS increased acute (2–24 h post-induction) intestinal inflammation in the TNBS-induced colitis (CD-like) model [54–57]. The authors used a ventilated smoking chamber filled with a fixed concentration of smoke, delivered by burning commercial cigarettes at a constant rate (2 or 4%, vol/ vol, smoke/air) [61]. These results showed that promotion of neutrophil infiltration, as well as free radical production with the accumulation of reactive oxygen metabolites in the intestinal

**IBD model** TNBS colitis

CD—rat

Cigarette smoke (inhalation)

Mucosal damage: ↑

MPO activity: ↑

LTB4 level: ↑

GSH level: ↓

ROM generation: ↑

TNF-α protein: ↑

SOD activity: ↓

iNOS activity: ↑

COX2 protein: ↑

LTB4 level: ↓

Low dose: ↓

[78, 79]

High dose: ↑ or no effect

PGE2 level: =

MPO activity: ↓

Histology score: ↓

iNOS protein: ↓

Serum IL-1: =

CD—mouse

Subcutaneous nicotine

Histology score: ↑

↑

[77]

DAI scoring: ↑

Treg/Th17 cell ratio: ↓

α7nAChR expression in T cells: no

Carbon monoxide (inhalation)

Oral TCDD

Iodoacetamide

CD—mouse

Oral nicotine

Histology score: ↓

↓

[73]

MPO activity: ↓

TNF-α protein and RNA: ↓

Histology score: ↓

↓

[174]

Animal Inhalation Models to Investigate Modulation of Inflammatory Bowel Diseases

Colon cytokine proteins: ↓

Gene expression

Immune cells in MLN and colon

Mucosal damage: J↑; C↓

Jejunitis: ↑

[175]

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189

Colitis: ↓

iNOS activity: J NA; C=

MPO activity: J=; C NA

PGE2 level: J↓; C↓

Histology score: J↑; C↓

Oral nicotine

**IBD subtype—species**

**Treatment**

**Endpoint observed**

**Effects on intestinal** 

**References**

**inflammation**

↑

[54–57]


Consistent with results of human epidemiological studies, CS had opposing effects on development of CD (negatively) and UC (positively) in several, but not all, of their respective IBD models. Only a few of these studies used inhalation exposure (**Table 2**) and most of their find-

see Chapter 4.1 to 4.4. Modified from: De Jonge & Ulloah (2007), Ordas et al. (2012), Xu et al. (2014).

**Figure 1.** Schematic view of major inflammatory and anti-inflammatory mechanisms implicated in inflammatory bowel diseases and the potential role of a nicotinic anti-inflammatory pathway. Top: altered microbiota in the colonic lumen and/or epithelial-damaging factors (e.g., DSS in experimentally induced colitis) lead to the disruption of the epithelial barrier function and the consequent infiltration of bacteria and other antigens. Middle: various inflammatory processes can be triggered in the lamina propria by the infiltrating bacteria (DSS-induced epithelial barrier; "Barrier dysfunction and epithelial permeability" and "Nicotinic anti-inflammatory pathway" sectors), haptens (oxazolone- and TNBS-induced inflammation, "Differential nicotine effects in UC-like (oxazolone) and CD-like (TNBS) colitis" sector) or by endogenous dysregulation of the balance between Th1/Th17-driven and Th2-driven immune activities, (genetically engineered mouse models; "Immune regulation" section). A hypothetical role of nicotinic receptor-mediated anti-inflammatory response is depicted in the "Nicotinic anti-inflammatory pathway" sector. Bottom: the colonic vasculature is symbolized as a tube running perpendicular to the cross section of the colon. The blood stream delivers leukocytes recruited by cytokine shedding from the local inflammatory sites and enables the perpetuation of the inflammation, e.g., via circulating T-cells. Systemically provided nicotine could increase the anti-inflammatory nicotinic signaling that is naturally transmitted by acetylcholine shed from the efferents of the vagus nerve that innervate the colonic wall. For details of these mechanisms,

Thus, the dichotomous effects of CS inhalation, on development of CD versus UC, were perfectly reproduced using two different rat IBD models [54–60]. 2,4,6-trinitrobenzenesulphonic acid (TNBS) and 2,4-dinitrobenzene sulphonic acid (DNBS) were instilled into the rat colon to induce, respectively, CD- and UC-like symptoms. Indeed, pre-exposure of rats to CS increased acute (2–24 h post-induction) intestinal inflammation in the TNBS-induced colitis (CD-like) model [54–57]. The authors used a ventilated smoking chamber filled with a fixed concentration of smoke, delivered by burning commercial cigarettes at a constant rate (2 or 4%, vol/ vol, smoke/air) [61]. These results showed that promotion of neutrophil infiltration, as well as free radical production with the accumulation of reactive oxygen metabolites in the intestinal

ings mimicked the effects of smoking in humans with IBD.

188 Experimental Animal Models of Human Diseases - An Effective Therapeutic Strategy


**IBD model** DSS Colitis

UC—mouse

Oral nicotine

**IBD subtype—species**

**Treatment**

**Endpoint observed**

Histology score: ↓

DAI scoring: =

MPO activity: =

PGE2 level: ↓

DAI scoring: ↓

↓

[50]

Histology score: ↓

miRNA expression

Colon cytokine RNA: ↓

↓

[22]

MPO activity: ↓

Infiltrating immune cells

DAI scoring: ↓

Cigarette smoke (inhalation)

Mucosal damage: =

No effect

[66]

Colon cell proliferation: =

Colon cell apoptosis: =

Colon angiogenesis: ↑

Bcl2/VEGF protein: ↑

DAI scoring: ↓

↓

[176]

Histology score: ↓

MPO activity: ↓

TNF-α and IL-6 mRNA: ↓

DAI scoring: ↓

↓

[81, 102]

Animal Inhalation Models to Investigate Modulation of Inflammatory Bowel Diseases

Histology score: ↓

Colon TNF-α protein: ↓

MPO activity: ↓

Colon cytokines mRNA: ↓

DAI scoring: =

DAI scoring: =

Histology score: =

Colon TNF-α protein: =

No effect No effect

[81]

[81]

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Oral cotinine Subcutaneous nicotine

Intraperitoneal nicotine

Oral TCDD

DAI scoring: =

No effect

[81, 82]

Histology score: ↓

Colon TNF-α protein: ↑

Histology score: ↓

↓

[177]

191

Colon TNF-α RNA/protein: ↓

MPO mRNA: ↓

Oral nicotine

Subcutaneous nicotine

Cigarette smoke (inhalation)

Subcutaneous nicotine

**Effects on intestinal** 

**References**

**inflammation**

↓

[80]


**IBD model** IL-10−/− mice DNBS colitis

UC—rat

Cigarette smoke (inhalation)

Histology score: ↑

↑

[58]

Mucosal damage: ↑

MPO activity: ↑

Mucosal damage: ↓

↓

[59]

190 Experimental Animal Models of Human Diseases - An Effective Therapeutic Strategy

MPO activity: ↓

LTB4 level: ↓

ROM generation: ↓

Colon cytokine proteins: ↓

Cigarette smoke (inhalation)

Mucosal damage: ↓

↓

[59]

MPO activity: ↓

LTB4 level: ↓

ROM generation: ↓

Colon cytokine proteins: ↓

Cigarette smoke (inhalation)

Histology score: ↓

↓

[60]

Mucosal damage: ↓

MPO activity: ↓

iNOS activity: ↓

LTB4 level: ↓

Colon cytokine proteins: ↓

Oxazolone colitis

UC—mouse

Subcutaneous nicotine

Histology score: ↓

↓

[77]

DAI scoring: ↓

Treg/Th17 cell ratio: ↑

α7nAChR expression in T cells

Subcutaneous nicotine

CD—mouse

Oral nicotine

**IBD subtype—species**

**Treatment**

**Endpoint observed**

Mucosal damage: J↑; C↓

Histology score: J↑; C↓

Gene expression

Carbon monoxide (inhalation)

Histology score: ↓

↓

[71]

Colon cytokine proteins: ↓

Gene expression

**Effects on intestinal** 

**References**

**inflammation**

Jejunitis: ↑

[52]

Colitis: ↓


**Table 2.** Effects of cigarette smoke or related compounds in experimental models of IBD. tissues, contributed significantly to the potentiating effects of CS on intestinal inflammation. In contrast, in DNBS-treated rats (UC-like model), CS inhalation improved macroscopic signs of colitis at the mucosal level and decreased the levels of colonic pro-inflammatory cytokines [59, 60]. In these latter papers, Ko et al. used a similar inhalation method to the aforementioned study [61], but with a different time of exposure and a few "homemade" modifications to the smoking chamber. One study, conducted in DNBS-treated rats exposed to CS for 15 days before and 2 days after DNBS instillation, showed increased macroscopic and histological damage in the CS-exposed rat colon [58]. Noteworthy, this study used a different inhalation method than did the others. Rats were exposed to a rhythmic inhalation of smoke, with only the nose exposed to the specialized chamber [62], and this chamber was filled with

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Furthermore, the effect of CS on the development of small intestinal inflammation (CD-like pathophysiology) was studied in a TNFΔARE mouse model [63]. In this mouse model, a knockin mutation determines the deletion of the AU-region of the TNF-α mRNA, resulting in a systemic TNF-α overproduction and the consequent development of chronic Crohn's-like ileitis and inflammatory arthritis [64]. The authors exposed the mice to CS 4 times a day with 30 min smoke-free intervals, 5 days per week for 2 or 4 weeks [65]. Contrarily to what obtain in human and rat CD, in this model CS did not modulate gut inflammation. Both molecular (e.g. inflammatory and autophagy gene expression) and histopathological endpoints were

In contrast to its effects in CD rodent models, CS exposure for 2 weeks decreased UC-like inflammation in an acute DSS-induced colitis model in mice [22]. Montbarbon et al. showed a significant decrease in macroscopic and histological colon damage, as well as in colonic proinflammatory cytokine expression, in DSS-exposed mice after CS inhalation. Interestingly, this study highlighted a pivotal role for a specific intestinal lymphocyte type, iNKT, in the CS-dependent protection of the colon. The authors used a ventilated smoking chamber of the InExpose® System and exposed the mice to the mainstream smoke of research cigarettes 5 days per week (5 cigarettes/day). However, a previous study, in a long-term mouse model of DSS-mediated chronic colitis, showed a CS-dependent increase in inflammation-associated colon adenoma/adenocarcinoma formation. Although specific inflammatory endpoints were not reported, the number of colon adenomas/adenocarcinomas was significantly increased in the CS-exposed mice [66]. This tumour formation was associated with inhibition of cellular apoptosis and supported by increased angiogenesis. As a possible explanation for this discrepancy, this study used Balb/c mice while the protective effects of CS [22] were observed in C57BL/6 mice. Opposite responses in Balb/c mice, compared with C57BL/6 and other mouse strains, were also reported for other chemical inducers of IBD [67]. Moreover, a different inhalation method was applied in the Balb/c mouse study. These mice were exposed to 2 or 4% CS

In the context of inhalation studies aimed to understand the major CS component responsible for the observed anti-inflammatory effects in the intestine, three studies on the anti-inflammatory properties of carbon monoxide (CO) in IBD models are notable. Indeed, CO, a prominent component of CS long considered as just being a toxic gas [68], was recently shown to exert

mainstream smoke from a high tar, unfiltered cigarette.

not affected by CS smoke compared to fresh air exposed mice.

in a ventilated smoking chamber for 1 h per day.

tissues, contributed significantly to the potentiating effects of CS on intestinal inflammation. In contrast, in DNBS-treated rats (UC-like model), CS inhalation improved macroscopic signs of colitis at the mucosal level and decreased the levels of colonic pro-inflammatory cytokines [59, 60]. In these latter papers, Ko et al. used a similar inhalation method to the aforementioned study [61], but with a different time of exposure and a few "homemade" modifications to the smoking chamber. One study, conducted in DNBS-treated rats exposed to CS for 15 days before and 2 days after DNBS instillation, showed increased macroscopic and histological damage in the CS-exposed rat colon [58]. Noteworthy, this study used a different inhalation method than did the others. Rats were exposed to a rhythmic inhalation of smoke, with only the nose exposed to the specialized chamber [62], and this chamber was filled with mainstream smoke from a high tar, unfiltered cigarette.

Furthermore, the effect of CS on the development of small intestinal inflammation (CD-like pathophysiology) was studied in a TNFΔARE mouse model [63]. In this mouse model, a knockin mutation determines the deletion of the AU-region of the TNF-α mRNA, resulting in a systemic TNF-α overproduction and the consequent development of chronic Crohn's-like ileitis and inflammatory arthritis [64]. The authors exposed the mice to CS 4 times a day with 30 min smoke-free intervals, 5 days per week for 2 or 4 weeks [65]. Contrarily to what obtain in human and rat CD, in this model CS did not modulate gut inflammation. Both molecular (e.g. inflammatory and autophagy gene expression) and histopathological endpoints were not affected by CS smoke compared to fresh air exposed mice.

In contrast to its effects in CD rodent models, CS exposure for 2 weeks decreased UC-like inflammation in an acute DSS-induced colitis model in mice [22]. Montbarbon et al. showed a significant decrease in macroscopic and histological colon damage, as well as in colonic proinflammatory cytokine expression, in DSS-exposed mice after CS inhalation. Interestingly, this study highlighted a pivotal role for a specific intestinal lymphocyte type, iNKT, in the CS-dependent protection of the colon. The authors used a ventilated smoking chamber of the InExpose® System and exposed the mice to the mainstream smoke of research cigarettes 5 days per week (5 cigarettes/day). However, a previous study, in a long-term mouse model of DSS-mediated chronic colitis, showed a CS-dependent increase in inflammation-associated colon adenoma/adenocarcinoma formation. Although specific inflammatory endpoints were not reported, the number of colon adenomas/adenocarcinomas was significantly increased in the CS-exposed mice [66]. This tumour formation was associated with inhibition of cellular apoptosis and supported by increased angiogenesis. As a possible explanation for this discrepancy, this study used Balb/c mice while the protective effects of CS [22] were observed in C57BL/6 mice. Opposite responses in Balb/c mice, compared with C57BL/6 and other mouse strains, were also reported for other chemical inducers of IBD [67]. Moreover, a different inhalation method was applied in the Balb/c mouse study. These mice were exposed to 2 or 4% CS in a ventilated smoking chamber for 1 h per day.

In the context of inhalation studies aimed to understand the major CS component responsible for the observed anti-inflammatory effects in the intestine, three studies on the anti-inflammatory properties of carbon monoxide (CO) in IBD models are notable. Indeed, CO, a prominent component of CS long considered as just being a toxic gas [68], was recently shown to exert

**IBD model** TCRα−/− mice

*Clostridium* 

UC—mouse

Intraluminal nicotine

MPO activity: ↓

↓ Colon;

[178]

No effect in ileum

LTB4 level: ↓

Luminal fluid: ↓

Substance P release: ↓

↑, potentiating effect; ↓, attenuating effect; =, no changes; TCDD, 2,3,7,8-tetrachlorodibenzo-p-dioxin; TCR, T cell receptor; NA, not applicable; ROM, reactive oxygen metabolites;

DAI, disease activity index (for further details please see the reference), MPO, myeloperoxidase; LTB4, leukotriene B4; PGE2, prostaglandin E2; SOD, superoxide dismutase 2; COX,

192 Experimental Animal Models of Human Diseases - An Effective Therapeutic Strategy

cyclooxygenase; iNOS, nitric oxide synthase.

**Table 2.**

Effects of cigarette smoke or related compounds in experimental models of IBD.

*difficile* ToxA

UC—mouse

Carbon monoxide (inhalation)

Histology score: ↓

Colon cytokines RNA/protein: ↓

**IBD subtype—species**

**Treatment**

**Endpoint observed**

**Effects on intestinal** 

**References**

**inflammation**

↓

[72]

potent cell protective effects because of its anti-inflammatory, anti-apoptotic and anti-oxidant capabilities [69, 70]. In three different studies, inhaled CO consistently decreased inflammation in chemically induced and genetic mouse models of UC and CD, respectively [71–73]. In particular, the same group of researchers [71, 72] exposed two different knockout mouse models, IL-10−/− [74] and TCRα−/− [75, 76], to CO at a concentration of 250 ppm (part per million) or compressed air (control), attempting to recapitulate, at least in part, CS effects on the development of CD and UC, respectively. IL-10−/− mice were generated by gene targeting in 1993 by Kuhn et al. [74], introducing two stop codons in exon 1 and 3 of the IL-10 gene in murine ES cells. These mice are characterised by extensive Th1-mediated enterocolitis originated by an antigen-driven uncontrolled immune response mainly resembling human CD condition. T cell receptor (TCR) α knockout mice were generated with a similar gene targeting approach [76], thus integrating a neomycin cassette in the first exon of the TCRα locus. In these mutant mice, the intestinal mucosal immunoregulatory mechanisms are negatively affected, triggering the development of UC-like symptoms [75]. Surprisingly, CO inhalation suppressed inflammation in both models, regardless of their IBD subtype, through a heme oxygenase (HO)-1 dependent pathway. The anti-inflammatory capabilities of CO were also confirmed in a TNBS-induced mouse model of CD. Mice were exposed to CO at 200 ppm, beginning after TNBS administration and throughout the remaining study period (3 days) [73]. Thus, the increased colonic damage induced by TNBS was significantly inhibited by the CO treatment, with a consistent suppression of inflammatory markers, such as TNF-α levels and myeloperoxidase (MPO) activity.

**3.2. Limits and pitfalls of studies using inhalation mouse models**

ers, CS exposure schedules, endpoints and observation periods.

timeframe as the COPD and CVD models.

Among the aforementioned studies, only a few used inhalation exposure (**Table 2**) models were observed, although many of the findings mimicked human smoking effects in IBD, the results were still variable. Such heterogeneity in observed CS effects on experimentally induced colitis is not unexpected, given variability in animal species and strains, IBD induc-

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When comparing such quality-relevant exposure conditions, group sizes were usually sufficient, but most of the studies used only male mice or rats, instead of both genders as recommended by the Organisation for Economic Co-operation and Development (OECD) test guidelines. Only one rat study employed the preferable nose-only inhalation mode [58]. Many of the papers did not describe the exposure chambers sufficiently and explanations of exposure concentration parameters (such as number of puffs, flow rate and chamber volume) often did not enable derivation of the standard Total Particulate Matter (TPM) or smoke constituent concentration values, in a weight per volume unit (e.g. mg/L). The most evident heterogeneity among studies, however, was in exposure schedules and durations. The CS inhalation studies in IBD models typically used daily exposure durations no longer than one hour, with none using the recommended 6 h/day duration. Some studies pre-exposed the animals a few days before IBD induction and discontinued CS exposure after the induction treatment, while others continued exposure until the end of the study or began CS inhalation after IBD induction [59]. To explore more systematically the effects of inhaled CS or CS constituents on IBD in various models, there is a clear need to harmonise exposure conditions to be closer to minimal standards for inhalation toxicity studies. This is particularly true for exposure schedules and durations, as well as for documentation of meaningful concentration measurements in the exposure atmospheres (**Table 3**). Finally, to elucidate the molecular mechanisms of IBD-CS interactions, beyond the current knowledge, it will be necessary to combine robust IBD models (UC and CD), well-controlled, state-of-the-art inhalation exposure design and technology and disease-specific endpoints with systems-wide molecular profiling. We conducted systems toxicology-oriented inhalation studies using mouse models to investigate effects of CS and candidate modified risk tobacco products in chronic obstructive and cardiovascular diseases [33, 88–91]. These studies demonstrated the feasibility and suitability of this approach for identifying the molecular basis of disease mechanisms and the biological impacts of CS. The study design and inhalation exposure technology were based on the OECD guidelines TG412 and TG413 for 28 and 90 days inhalation toxicity studies, respectively [92, 93]. Satellite groups were included to provide material for the additional molecular investigations and a similar study was conducted on rats exposed to nicotine aerosols [33]. A very detailed description of the study design and methodology was provided [94] and this might serve as a template for new IBD inhalation studies. Of course, adaptations will be necessary, based on specifications of the IBD models. For example, most chemically induced IBD models require acute, rather than subchronic or chronic, observation periods, while the genetically engineered IBD models develop the disease in a similar

As highlighted in the aforementioned reports, although CS or CS component inhalation studies in mouse models seem to recapitulate most epidemiological observations in humans, differences in the inhalation methodologies are many and frequent, making impossible a clear and solid comparison between the studies.

The route of administration was relevant on the final effect also when single CS components, such as nicotine, were administered to IBD mouse models or patients [47]. Thus, in a TNBS mouse model of CD, the detrimental effects of subcutaneous nicotine administration [77] contrasted with the dose-dependent bivalent effect of nicotine administered in the drinking water, that is, positive at low and negative at high concentrations [78, 79]. Furthermore, subcutaneous or oral nicotine administration to rats treated with DNBS led to, respectively, decreased or increased colon inflammation [58, 59]. Finally, while oral or subcutaneous nicotine administration attenuated inflammation caused by DSS treatment in mice [50, 80], intraperitoneal nicotine injection had no effects [81, 82]. Inconsistencies related to different routes of administration of CS components were also observed in human studies [83–86]. Overall, these observations suggested that the route of administration of a CS-related compound, such as nicotine, is important to consider in treating colitis. In animal models, it is clear that mimicking the nicotine intake profiles in smokers (inhalation) could result in increased treatment efficacy. This idea was supported in humans by the conflicting results obtained by local nicotine application (enemas) [87]. Therefore, although the colon may be an important site of action for CS components, the responsible molecule for the observed effects might act on many peripheral and central inflammatory pathways, such as vagus-related anti-inflammatory nicotinic signalling, or might require intermediate metabolic transformations.

#### **3.2. Limits and pitfalls of studies using inhalation mouse models**

potent cell protective effects because of its anti-inflammatory, anti-apoptotic and anti-oxidant capabilities [69, 70]. In three different studies, inhaled CO consistently decreased inflammation in chemically induced and genetic mouse models of UC and CD, respectively [71–73]. In particular, the same group of researchers [71, 72] exposed two different knockout mouse models, IL-10−/− [74] and TCRα−/− [75, 76], to CO at a concentration of 250 ppm (part per million) or compressed air (control), attempting to recapitulate, at least in part, CS effects on the development of CD and UC, respectively. IL-10−/− mice were generated by gene targeting in 1993 by Kuhn et al. [74], introducing two stop codons in exon 1 and 3 of the IL-10 gene in murine ES cells. These mice are characterised by extensive Th1-mediated enterocolitis originated by an antigen-driven uncontrolled immune response mainly resembling human CD condition. T cell receptor (TCR) α knockout mice were generated with a similar gene targeting approach [76], thus integrating a neomycin cassette in the first exon of the TCRα locus. In these mutant mice, the intestinal mucosal immunoregulatory mechanisms are negatively affected, triggering the development of UC-like symptoms [75]. Surprisingly, CO inhalation suppressed inflammation in both models, regardless of their IBD subtype, through a heme oxygenase (HO)-1 dependent pathway. The anti-inflammatory capabilities of CO were also confirmed in a TNBS-induced mouse model of CD. Mice were exposed to CO at 200 ppm, beginning after TNBS administration and throughout the remaining study period (3 days) [73]. Thus, the increased colonic damage induced by TNBS was significantly inhibited by the CO treatment, with a consistent suppression of inflam-

194 Experimental Animal Models of Human Diseases - An Effective Therapeutic Strategy

matory markers, such as TNF-α levels and myeloperoxidase (MPO) activity.

and solid comparison between the studies.

As highlighted in the aforementioned reports, although CS or CS component inhalation studies in mouse models seem to recapitulate most epidemiological observations in humans, differences in the inhalation methodologies are many and frequent, making impossible a clear

The route of administration was relevant on the final effect also when single CS components, such as nicotine, were administered to IBD mouse models or patients [47]. Thus, in a TNBS mouse model of CD, the detrimental effects of subcutaneous nicotine administration [77] contrasted with the dose-dependent bivalent effect of nicotine administered in the drinking water, that is, positive at low and negative at high concentrations [78, 79]. Furthermore, subcutaneous or oral nicotine administration to rats treated with DNBS led to, respectively, decreased or increased colon inflammation [58, 59]. Finally, while oral or subcutaneous nicotine administration attenuated inflammation caused by DSS treatment in mice [50, 80], intraperitoneal nicotine injection had no effects [81, 82]. Inconsistencies related to different routes of administration of CS components were also observed in human studies [83–86]. Overall, these observations suggested that the route of administration of a CS-related compound, such as nicotine, is important to consider in treating colitis. In animal models, it is clear that mimicking the nicotine intake profiles in smokers (inhalation) could result in increased treatment efficacy. This idea was supported in humans by the conflicting results obtained by local nicotine application (enemas) [87]. Therefore, although the colon may be an important site of action for CS components, the responsible molecule for the observed effects might act on many peripheral and central inflammatory pathways, such as vagus-related anti-inflamma-

tory nicotinic signalling, or might require intermediate metabolic transformations.

Among the aforementioned studies, only a few used inhalation exposure (**Table 2**) models were observed, although many of the findings mimicked human smoking effects in IBD, the results were still variable. Such heterogeneity in observed CS effects on experimentally induced colitis is not unexpected, given variability in animal species and strains, IBD inducers, CS exposure schedules, endpoints and observation periods.

When comparing such quality-relevant exposure conditions, group sizes were usually sufficient, but most of the studies used only male mice or rats, instead of both genders as recommended by the Organisation for Economic Co-operation and Development (OECD) test guidelines. Only one rat study employed the preferable nose-only inhalation mode [58]. Many of the papers did not describe the exposure chambers sufficiently and explanations of exposure concentration parameters (such as number of puffs, flow rate and chamber volume) often did not enable derivation of the standard Total Particulate Matter (TPM) or smoke constituent concentration values, in a weight per volume unit (e.g. mg/L). The most evident heterogeneity among studies, however, was in exposure schedules and durations. The CS inhalation studies in IBD models typically used daily exposure durations no longer than one hour, with none using the recommended 6 h/day duration. Some studies pre-exposed the animals a few days before IBD induction and discontinued CS exposure after the induction treatment, while others continued exposure until the end of the study or began CS inhalation after IBD induction [59]. To explore more systematically the effects of inhaled CS or CS constituents on IBD in various models, there is a clear need to harmonise exposure conditions to be closer to minimal standards for inhalation toxicity studies. This is particularly true for exposure schedules and durations, as well as for documentation of meaningful concentration measurements in the exposure atmospheres (**Table 3**). Finally, to elucidate the molecular mechanisms of IBD-CS interactions, beyond the current knowledge, it will be necessary to combine robust IBD models (UC and CD), well-controlled, state-of-the-art inhalation exposure design and technology and disease-specific endpoints with systems-wide molecular profiling. We conducted systems toxicology-oriented inhalation studies using mouse models to investigate effects of CS and candidate modified risk tobacco products in chronic obstructive and cardiovascular diseases [33, 88–91]. These studies demonstrated the feasibility and suitability of this approach for identifying the molecular basis of disease mechanisms and the biological impacts of CS. The study design and inhalation exposure technology were based on the OECD guidelines TG412 and TG413 for 28 and 90 days inhalation toxicity studies, respectively [92, 93]. Satellite groups were included to provide material for the additional molecular investigations and a similar study was conducted on rats exposed to nicotine aerosols [33]. A very detailed description of the study design and methodology was provided [94] and this might serve as a template for new IBD inhalation studies. Of course, adaptations will be necessary, based on specifications of the IBD models. For example, most chemically induced IBD models require acute, rather than subchronic or chronic, observation periods, while the genetically engineered IBD models develop the disease in a similar timeframe as the COPD and CVD models.


**4. Mechanisms of IBD pathogenesis with possible relationship to CS** 

**Study design Exposure duration Inhalation** 

1 h/day; 3 days post-induction

1 h/day; 3 days preinduction, 4 day post-induction

3 cycles of: 7 days DSS + CS (1 h/day) followed by 14 days

recovery

4 week (daily duration not indicated)

4 week (daily duration not indicated)

3 day (permanent) post-induction

**Table 3.** Comparison of exposure conditions in published inhalation studies using rodent IBD models.

7 rats/group, 2 dose levels, fresh air control

6–8 rats/group, 1 dose level, fresh air control

5–12 mice/group (males only), 2 dose levels, fresh air control

10 mice/group (5 males and 5 females), 1 dose level, fresh air control

12 mice/group (males only), 1 dose level, fresh air control

12 mice/group, 1 dose level, fresh air control

**technology**

Whole body, ventilated smoking chamber (20 L) with 5–6 rats, smoke generated with peristaltic pump smoke, no characterisation

Animal Inhalation Models to Investigate Modulation of Inflammatory Bowel Diseases

Whole body, ventilated smoking chamber (20 L) with 5–6 rats, smoke generated with peristaltic pump

Whole body, ventilated smoking chamber (20 L), smoke generated with peristaltic pump

Whole body, 3.70 ft<sup>2</sup> plexiglass animal chamber, 12 L/min flow rate

Whole body, 3.70 ft<sup>2</sup> plexiglass animal chamber, 12 L/min flow rate

Whole body, acrylic

chamber

**CS/inhalant characterisation**

"Camel" cigarettes, 2 and 4% v/v smoke, no characterisation

"Camel" cigarettes, 2 and 4% v/v smoke, no characterisation

CO gas, 250 ppm in air, continuous measurement

CO gas, 250 ppm in air, continuous measurement

CO gas, 200 ppm in air, continuous measurement

"Kings" cigarettes, 4% v/v; no concentration/ characterisation

http://dx.doi.org/10.5772/intechopen.69569

**References**

197

[59]

[60]

[66]

[72]

[71]

[73]

The vagus nerve transmits signals by releasing acetylcholine that, in turn, stimulates neuronal and immune cells via their nicotinic acetylcholine receptors (nAChR) [95, 96]. These are ligand-gated ion channels expressed not only in neuronal cells, but also in most mammalian non-neuronal cell types, though different cell type-specific downstream signalling functions [97]. In the nicotinic anti-inflammatory pathway, nAChR activation by acetylcholine or other ligands inhibits the downstream NF-κB pathway, attenuating production of TNF-α and other

**constituents**

**IBD model, induction**

Rat (Sprague Dawley), DNBS enema

Rat (Sprague Dawley), DNBS enema

Mouse (Balb/c), DSS in drinking

TCRα−/− mouse (C57BL/6)

IL-10−/− mouse (C57BL/6)

Mouse (C57BL/6), TNBS enema

water

**4.1. Nicotinic anti-inflammatory pathway**


**Table 3.** Comparison of exposure conditions in published inhalation studies using rodent IBD models.
