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

#### **4.1. Nicotinic anti-inflammatory pathway**

**IBD model, induction**

(OECD TG 412 recommendation)

Rat (Sprague Dawley), TNBS enema

Rat (Sprague Dawley), TNBS enema

Rat (Sprague Dawley), TNBS enema

Rat (Sprague Dawley), TNBS enema

Mouse (C57BL/6), DSS in drinking

Rat (Sprague Dawley), DNBS enema

water

**Study design Exposure duration Inhalation** 

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

6 h/day; 5 (7) days/ week; 28 days

1 h/day; 4 days pre-induction

1 h/day; 4 days pre-induction

1 h/day; 4 days pre-induction

1 h/day; 8 days pre-induction

2 week (5 days/ week) pre-induction and 1 week post-induction

5–40 min/day, 15 days preinduction and 2 day post-induction

At least 5 males and 5 females per group, 3 dose levels of test article, filtered air and/or vehicle

8–10 rats/group (males only), 1 dose level, fresh air control

10–12 rats/group (males only), 2 dose levels, fresh air control

6–8 rats/group (males only), 1 dose level, fresh air control

10 rats/group (males only), 2 dose levels, fresh air control

6–10 mice/group (males only), 1 dose level, fresh air control

6–8 rats/group, 3 dose levels, fresh air control (10 rats/group)

control

**technology**

Nose-only preferred, whole body acceptable, detailed description of exposure chamber to be given

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

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

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

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

Whole body, InExpose chamber (Scireq) and rotary smoking machine

Nose-only, puffwise smoke injection into

chamber

**CS/inhalant characterisation**

Analytical characterisation; respirable particle size (1–3 μm MMAD), nominal and actual test article concentration (mass per volume) to be indicated, constant concentration during exposure period

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

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

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

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

3R4F reference cigarettes, mainstream smoke from 5 cigarettes (8 puffs per cigarette), no concentration/ characterisation

2R1 reference cigarette, 5, 20 or 40 puffs/day (undiluted), no concentration/ characterisation **References**

[92]

[56]

[55]

[54]

[57]

[22]

[58]

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 cytokines [98, 99]. This pathway was reported to be one of the most likely explanations for CS-associated anti-inflammatory responses in the gut. Mapping the relevant neuronal circuits revealed that efferent vagus nerve fibres innervated the small intestine and proximal colon [100]. Vagotomised mice were more susceptible than normal mice to developing colitis after exposure to DSS and had increased levels of NF-κB and cytokines, such as IL-1β, IL-6 and TNF-α [101–103]. Pretreatment with nicotine reversed these effects through activation of α7nAChR, identified as the major receptor involved in nicotinic anti-inflammatory pathways [99, 104]. Potential therapeutic applications of selective α7nAChR agonists, such as the partial α7 agonists 3-(2,4-dimethoxybenzylidene)-anabaseine (GTS-21) and anatabine citrate, and of α7nAChR-positive allosteric modulators, was explored in pre-clinical and clinical studies [105–109]. Moreover, additional nAChR subtypes, such as α4β2, α3β4, α3β2 and α6, were also proposed as targets for nicotine treatment [110–112], increasing the complexity, but also the therapeutic potential, of this approach. Although research on the mechanisms involved in nicotinic anti-inflammatory pathways has highlighted the pharmacological potential of nAChR agonists, studies showing contradictory results obtained with specific α7nAChR ligands [82] suggested that these compounds should be used with caution in patients with IBD.

proteins [128]. However, no intestinal barrier changes were identified in the colons of control or CS-exposed mice, suggesting that there was CS-related organ specificity and, thus, possibly

Animal Inhalation Models to Investigate Modulation of Inflammatory Bowel Diseases

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

199

Much evidence supports the strong impact of environmental factors on gut microbiota, and smoking has recently been investigated as a potential factor shaping the microbiota. This potential connection implied new possibilities regarding the role of smoking in IBD development. Thus, studies targeting selected bacterial groups reported that patients with active CD, who also smoked, had microbial profiles different from those of non-smoking patients with CD. Similar results were found in healthy smoking controls, suggesting that the association related not to intestinal inflammation but, instead, to a direct impacts of smoking on the microbiota [129, 130]. Differences between mice and humans at the level of the gut microbiota limit the usefulness of mouse models, relevant to CS, gut microbiota and IBD. However, a few studies using rats and mice were consistent with observations in humans, indicating CS-dependent shifts in gut microbiota compositions [131–133]. These observations supported a possible role for CS in shaping the gut microbiome, with potential, though still unknown,

Currently, the processes described in Sections 4.1–4.4 have been those most explored as potential links between CS and IBD development. However, there are several other possible mechanisms, indicative of how environmental factors might exponentially increase complexity of

In UC, fasting colonic motility increased, whereas motor responses to food significantly decreased [134]. Observations in experimental animals and humans showed that nicotine promoted smooth muscle relaxation, reducing symptoms, such as diarrhoea and urgency

Smoking and nicotine may also affect UC by reducing eicosanoid-mediated inflammatory responses. Two studies independently demonstrated this specific effect in humans and rab-

Patients with UC have significantly higher rectal blood flow than normal controls, but smoking decreased rectal blood flow to within normal ranges [139–141]. However, changes in blood flow can affect intestinal inflammation in opposing ways. Decreasing blood flow can reduce levels of inflammatory mediators that reach the mucosal surface, while long-term impairment

consequences for evolution of inflammation-related disorders, such as IBD.

without significantly influencing inflammation [135–137].

explaining the opposing effects of smoking on CD and UC.

**4.4. Gut microbiota**

**4.5. Other potential mechanisms**

*4.5.2. Eicosanoid-mediated inflammation*

IBD pathology.

bits [53, 138].

*4.5.3. Rectal blood flow*

*4.5.1. Colon motility*

#### **4.2. Immune regulation**

The immunosuppressive effects of cigarette smoking, on both cellular and humoral immunity, have long been recognised [113–115]. Studies exploring how nicotine or CS can suppress the immune system indicated that, in nicotine-treated animals, T cells did not enter the cell cycle and proliferate as expected. Similar effects were observed in smokers and in animals exposed to CS [116–118]. Several studies described the implications of CS for different immune cell types, as well as the diverse actions of nicotine or CS, depending on the pathological environment, for example, UC or CD, in which the immune cells originated [77, 99, 112, 119–122]. For instance, when stimulated by lipopolysaccharide, peripheral blood mononuclear cells derived from smokers showed decreased IL-8 release only if subjects were also CD patients [122]. Similarly, the same investigators demonstrated that smokers with CD had significantly lower IL-10 (anti-inflammatory)/IL-12 (pro-inflammatory) ratios than non-smokers or smokers with UC. As suggested in some reports, the differential signalling of dendritic cells from CD (Th1-like) and UC patients exposed to cigarette smoke extract (CSE) *in vitro* could play a role in the opposing responses of cigarette smoke exposure, that is, a Th1-like response in CD, with increased Foxp3-positive CD4 T cells [121].

#### **4.3. Barrier dysfunction and intestinal permeability**

The intestinal mucosa is one of the most important physical barriers against external threats. Changes in intestinal permeability are crucial for the development of IBD [123] and several studies implicated CS in regulating barrier integrity. However, the effects of smoking on intestinal permeability are controversial. Several *in vitro* and *in vivo* observations, in studies using humans or rodents, suggested that decreased intestinal permeability in smokers might explain the protective effects of smoking in UC [53, 124–127]. In contrast, a recent article reported that mice exposed to CS exhibited increased intestinal permeability and bacterial translocation, intestinal villi atrophy, damaged tight junctions and abnormal tight junction proteins [128]. However, no intestinal barrier changes were identified in the colons of control or CS-exposed mice, suggesting that there was CS-related organ specificity and, thus, possibly explaining the opposing effects of smoking on CD and UC.

#### **4.4. Gut microbiota**

cytokines [98, 99]. This pathway was reported to be one of the most likely explanations for CS-associated anti-inflammatory responses in the gut. Mapping the relevant neuronal circuits revealed that efferent vagus nerve fibres innervated the small intestine and proximal colon [100]. Vagotomised mice were more susceptible than normal mice to developing colitis after exposure to DSS and had increased levels of NF-κB and cytokines, such as IL-1β, IL-6 and TNF-α [101–103]. Pretreatment with nicotine reversed these effects through activation of α7nAChR, identified as the major receptor involved in nicotinic anti-inflammatory pathways [99, 104]. Potential therapeutic applications of selective α7nAChR agonists, such as the partial α7 agonists 3-(2,4-dimethoxybenzylidene)-anabaseine (GTS-21) and anatabine citrate, and of α7nAChR-positive allosteric modulators, was explored in pre-clinical and clinical studies [105–109]. Moreover, additional nAChR subtypes, such as α4β2, α3β4, α3β2 and α6, were also proposed as targets for nicotine treatment [110–112], increasing the complexity, but also the therapeutic potential, of this approach. Although research on the mechanisms involved in nicotinic anti-inflammatory pathways has highlighted the pharmacological potential of nAChR agonists, studies showing contradictory results obtained with specific α7nAChR ligands [82]

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

suggested that these compounds should be used with caution in patients with IBD.

The immunosuppressive effects of cigarette smoking, on both cellular and humoral immunity, have long been recognised [113–115]. Studies exploring how nicotine or CS can suppress the immune system indicated that, in nicotine-treated animals, T cells did not enter the cell cycle and proliferate as expected. Similar effects were observed in smokers and in animals exposed to CS [116–118]. Several studies described the implications of CS for different immune cell types, as well as the diverse actions of nicotine or CS, depending on the pathological environment, for example, UC or CD, in which the immune cells originated [77, 99, 112, 119–122]. For instance, when stimulated by lipopolysaccharide, peripheral blood mononuclear cells derived from smokers showed decreased IL-8 release only if subjects were also CD patients [122]. Similarly, the same investigators demonstrated that smokers with CD had significantly lower IL-10 (anti-inflammatory)/IL-12 (pro-inflammatory) ratios than non-smokers or smokers with UC. As suggested in some reports, the differential signalling of dendritic cells from CD (Th1-like) and UC patients exposed to cigarette smoke extract (CSE) *in vitro* could play a role in the opposing responses of cigarette smoke exposure, that is, a Th1-like response in CD,

The intestinal mucosa is one of the most important physical barriers against external threats. Changes in intestinal permeability are crucial for the development of IBD [123] and several studies implicated CS in regulating barrier integrity. However, the effects of smoking on intestinal permeability are controversial. Several *in vitro* and *in vivo* observations, in studies using humans or rodents, suggested that decreased intestinal permeability in smokers might explain the protective effects of smoking in UC [53, 124–127]. In contrast, a recent article reported that mice exposed to CS exhibited increased intestinal permeability and bacterial translocation, intestinal villi atrophy, damaged tight junctions and abnormal tight junction

**4.2. Immune regulation**

with increased Foxp3-positive CD4 T cells [121].

**4.3. Barrier dysfunction and intestinal permeability**

Much evidence supports the strong impact of environmental factors on gut microbiota, and smoking has recently been investigated as a potential factor shaping the microbiota. This potential connection implied new possibilities regarding the role of smoking in IBD development. Thus, studies targeting selected bacterial groups reported that patients with active CD, who also smoked, had microbial profiles different from those of non-smoking patients with CD. Similar results were found in healthy smoking controls, suggesting that the association related not to intestinal inflammation but, instead, to a direct impacts of smoking on the microbiota [129, 130]. Differences between mice and humans at the level of the gut microbiota limit the usefulness of mouse models, relevant to CS, gut microbiota and IBD. However, a few studies using rats and mice were consistent with observations in humans, indicating CS-dependent shifts in gut microbiota compositions [131–133]. These observations supported a possible role for CS in shaping the gut microbiome, with potential, though still unknown, consequences for evolution of inflammation-related disorders, such as IBD.

#### **4.5. Other potential mechanisms**

Currently, the processes described in Sections 4.1–4.4 have been those most explored as potential links between CS and IBD development. However, there are several other possible mechanisms, indicative of how environmental factors might exponentially increase complexity of IBD pathology.

#### *4.5.1. Colon motility*

In UC, fasting colonic motility increased, whereas motor responses to food significantly decreased [134]. Observations in experimental animals and humans showed that nicotine promoted smooth muscle relaxation, reducing symptoms, such as diarrhoea and urgency without significantly influencing inflammation [135–137].

#### *4.5.2. Eicosanoid-mediated inflammation*

Smoking and nicotine may also affect UC by reducing eicosanoid-mediated inflammatory responses. Two studies independently demonstrated this specific effect in humans and rabbits [53, 138].

#### *4.5.3. Rectal blood flow*

Patients with UC have significantly higher rectal blood flow than normal controls, but smoking decreased rectal blood flow to within normal ranges [139–141]. However, changes in blood flow can affect intestinal inflammation in opposing ways. Decreasing blood flow can reduce levels of inflammatory mediators that reach the mucosal surface, while long-term impairment of rectal mucosal microvascular blood flow can result in a higher incidence of anastomotic breakdown in chronic smokers [140].

**Conflict of interest**

Morris International.

**Author details**

Giuseppe Lo Sasso<sup>1</sup>

**References**

1490-1497

2009;**361**(21):2066-2078

, Walter K. Schlage<sup>2</sup>

2 WK Schlage Biology Consulting, Bergisch Gladbach, Germany

3 Philip Morris International Research Laboratories Pte Ltd, Singapore

bowel disease. Journal of Crohn's & Colitis. 2013;**7**(12):982-1018

lished and evolving therapies. Lancet. 2007;**369**(9573):1641-1657

\*Address all correspondence to: julia.hoeng@pmi.com

Gastroenterology. 1989;**24**(suppl 170):2-6

Gastroenterology. 2004;**126**(6):1561-1573

Authors are employees of Philip Morris International. Philip Morris International is the sole source of funding and sponsor of this project. W.K. Schlage is contracted and paid by Philip

, Blaine Phillips<sup>3</sup>

[1] Abraham C, Cho JH. Inflammatory bowel disease. New England Journal of Medicine.

[2] Tontini GE, et al. Differential diagnosis in inflammatory bowel disease colitis: State of the art and future perspectives. World Journal of Gastroenterology. 2015;**21**(1):21-46 [3] Annese V, et al. European evidence based consensus for endoscopy in inflammatory

[4] Lennard-Jones J. Classification of inflammatory bowel disease. Scandinavian Journal of

[5] Baumgart DC, Sandborn WJ, Inflammatory bowel disease: Clinical aspects and estab-

[6] Hommes DW, van Deventer SJ. Endoscopy in inflammatory bowel diseases.

[7] Sartor RB. Mechanisms of disease: Pathogenesis of Crohn's disease and ulcerative colitis.

[8] Fuss IJ, et al. Nonclassical CD1d-restricted NK T cells that produce IL-13 characterize an atypical Th2 response in ulcerative colitis. Journal of Clinical Investigation. 2004;**113**(10):

[9] Fuss IJ, et al. Disparate CD4+ lamina propria (LP) lymphokine secretion profiles in inflammatory bowel disease. Crohn's disease LP cells manifest increased secretion of

Nature Clinical Practice. Gastroenterology & Hepatology. 2006;**3**(7):390-407

1 Philip Morris International R&D, Philip Morris Products S.A, Neuchatel, Switzerland

, Manuel C. Peitsch<sup>1</sup>

Animal Inhalation Models to Investigate Modulation of Inflammatory Bowel Diseases

and Julia Hoeng<sup>1</sup>

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

\*

201

#### *4.5.4. Non-nicotine-mediated effects*

Although nicotine is considered to be the major mediator of CS effects on intestinal inflammation, there is a clear evidence for involvement of other smoke constituents in CS-dependent responses. Both UC and CD mouse models were affected by carbon monoxide (CO) inhalation [71–73, 142]. These studies suggested that the mechanism through which CO protected against intestinal inflammation involved promoting bactericidal activities of macrophages [142]. Nitric oxide (NO) was also suggested as contributing to beneficial CS effects, based on its relaxant effects on colonic smooth muscle from UC patients [143]. Moreover, physiological NO, derived from nicotine-stimulated intestinal neuronal cells, functioned as a mediator in smooth muscle relaxation in the colons of DSS-treated mice [137].
