Chronic smoke exposure is associated with autophagy in murine Peyer's patches

INTRODUCTION: Cigarette smoke causes oxidative stress, leading to smoke-induced autophagy in several organs. Autophagy is a homeostatic process regulating the turnover of proteins and cytoplasmatic organelles. However, recently it has also been associated with many autoimmune and inflammatory disorders, among which Crohn’s disease. The purpose of the present study was to investigate whether cigarette smoke exposure is associated with increased autophagy in Peyer’s patches and its epithelium. AIMS & METHODS: C57BL/6 mice were exposed to cigarette smoke or air. After 24 weeks, the animals were sacrificied and Peyer’s patches were collected. m RNA expression of autophagy-related genes was determined by RT-PCR. Transmission electron microscopy (TEM) was used to evaluate the presence of autophagic vesicles in the follicleassociated epithelium of Peyer’s patches. RESULTS: Expression of Beclin-1, a protein involved in the nucleation of autophagosomes, and of Atg5 and Atg7, which both play a role in the autophagosome vesicle elongation and completion, increased after chronic smoke exposure. Furthermore, electron microscopy of the follicle-associated epithelium demonstrated that the mean area of autophagic vesicles per epithelial cell increased considerably from 1.1 μm2 ± 0.4 μm2 in the air group to 2.4 μm2 ± 0.4 μm2 in the smoke group (p < 0.05). Epithelial cells had a significantly higher number of autophagic vesicles after smoke exposure (1.1 ± 0.1 after smoke exposure versus 0.5 ± 0.1 vesicles per cell after air exposure, p < 0.05), but the size of the vesicles did not differ between both groups. CONCLUSION: Here we provide the first evidence that chronic exposure to cigarette smoke is associated with autophagy in murine Peyer’s patches, and more in particular in the follicle-associated epithelium covering Peyer’s patches. Our findings can help to understand the role of smoking in the pathogenesis of inflammatory bowel disease, such as Crohn’s disease

Cigarette smoking alters intestinal barrier function and Peyer's Patch composition

Smokers have a two-fold increased risk to develop Crohn’s disease (CD). However, little is known about the mechanisms through which smoking affects CD pathogenesis. Interestingly, the Peyer’s patches in the terminal ileum are the sites where the first CD lesions develop. To investigate whether smoke exposure causes alterations in Peyer’s patches, we studied C57BL/6 mice after exposure to air or cigarette smoke for 24 weeks. First, barrier function of the follicle-associated epithelium overlying Peyer’s patches was evaluated. We demonstrate that chronic smoke exposure is associated with increased apoptosis in the follicle-associated epithelium. Furthermore, immune cell numbers and differentiation along with chemokine expression were determined in the ileal Peyer’s patches. We observed significant increases in total dendritic cells (DC), CD4+ T-cells (including regulatory T-cells) and CD8+ T-cells after smoke exposure compared with air-exposed animals. The CD11b+ DC subset almost doubled. Interestingly, these changes were accompanied by an up-regulated mRNA expression of the chemokines CCL9 and CCL20, which are known to attract CD11b+ DC towards the subepithelial dome of Peyer’s patches. Our results demonstrate that cigarette smoke exposure induces apoptosis in follicle-associated epithelium and is associated with immune cell accumulation in Peyer’s patches, changes which can predispose to the development of CD

Cigarette smoke induces apoptosis in the follicle-associated epithelium of murine Peyer's patches

Background: Recently, cigarette smoking has been associated with the development of several auto-immune diseases, including rheumatoid arthritis and inflammatory bowel disease (IBD). The cellular and molecular mechanisms through which cigarette smoking predisposes to IBD are unknown. Cigarette smoke-induced apoptosis is described in several in vivo and in vitro experiments, and might play a role in the pathogenesis of several smoke-associated diseases. The aim of this study was to quantify apoptosis in normal murine Follicle-Associated Epithelium (FAE) and compare this to apoptosis in FAE of smoking mice. Methods: 8 C57BL/6 male mice were exposed to cigarette smoke for 24 weeks (chronic exposure); a control group of 8 mice was exposed to air during the same period. After 24 weeks the mice were sacrificed and Peyer’s patches of each mouse were dissected for histology. Immunohistochemistry for caspase-3 was performed on paraffin-embedded tissue sections of 11 Peyer’s patches of smoking animals and 11 Peyer’s patches of controls. To compare apoptotic activities between smokers and controls, the apoptotic index (percentage of apoptotic cells per 100 cells) in the FAE was calculated. An unpaired student T-test was applied. Results: A statistically significant increase in apoptosis of FAE cells was observed in smoking mice compared to air-exposed mice (P=0.002). In the FAE of smoking animals, the mean apoptotic index was 1.82 with a range of 1.11 to 3.00, whereas the mean apoptotic index in non-smoking animals was 0.92 (range 0.24 -2.06). Most apoptotic cells in both groups were seen at the apex of the FAE. Conclusion: We quantified rates of apoptosis in the FAE of murine Peyer’s patches. Furthermore we compared apoptosis in the FAE of smoking mice versus non-smoking siblings and observed an increased apoptotic index in the FAE of smoking animals. Our results demonstrate that cigarette smoke induces a significant increase of apoptosis in the FAE of murine Peyer’s patches and may point to a role for smoking in the pathogenesis of intestinal inflammation. Further investigation needs to clarify whether this increase in apoptosis influences normal function of the FAE

Translational research into the effects of cigarette smoke on inflammatory mediators and epithelial TRPV1 in Crohn’s disease

Crohn's disease is a pathological condition of the gastro-intestinal tract, causing severe transmural inflammation in the ileum and/or colon. Cigarette smoking is one of the best known environmental risk factors for the development of Crohn's disease. Nevertheless, very little is known about the effect of prolonged cigarette smoke exposure on inflammatory modulators in the gut. We examined the effect of cigarette smoke on cytokine profiles in the healthy and inflamed gut of human subjects and in the trinitrobenzene sulphonic acid mouse model, which mimics distal Crohn-like colitis. In addition, the effect of cigarette smoke on epithelial expression of transient receptor potential channels and their concurrent increase with cigarette smoke-augmented cytokine production was investigated. Active smoking was associated with increasedIL-8transcription in ileum of controls (p < 0,001; n = 18-20/group). In the ileum, TRPV1 mRNA levels were decreased in never smoking Crohn's disease patients compared to healthy subjects (p <0,001; n = 20/group). In the colon, TRPV1 mRNA levels were decreased (p = 0,046) in smoking healthy controls (n = 20/group). Likewise, healthy mice chronically exposed to cigarette smoke (n = 10/group) showed elevated ilealCxcl2(p = 0,0075) and colonicKcmRNA levels (p = 0,0186), whereas TRPV1 mRNA and protein levels were elevated in the ileum (p = 0,0315). Although cigarette smoke exposure prior to trinitrobenzene sulphonic acid administration did not alter disease activity, increased pro-inflammatory cytokine production was observed in the distal colon (Kc: p = 0,0273; Cxcl2: p = 0,104; Il1-beta: p = 0,0796), in parallel with the increase ofTrpv1mRNA (p < 0,001). We infer that CS affects pro-inflammatory cytokine expression in healthy and inflamed gut, and that the simultaneous modulation of TRPV1 may point to a potential involvement of TRPV1 in cigarette smoke-induced production of inflammatory mediators

The effect of cigarette smoke exposure on the development of inflammation in lungs, gut and joints of TNFΔARE mice

The inflammatory cytokine TNF-alpha is a central mediator in many immune-mediated diseases, such as Crohn's disease (CD), spondyloarthritis (SpA) and chronic obstructive pulmonary disease (COPD). Epidemiologic studies have shown that cigarette smoking (CS) is a prominent common risk factor in these TNF-dependent diseases. We exposed TNF Delta ARE mice; in which a systemic TNF-alpha overexpression leads to the development of inflammation; to 2 or 4 weeks of air or CS. We investigated the effect of deregulated TNF expression on CS-induced pulmonary inflammation and the effect of CS exposure on the initiation and progression of gut and joint inflammation. Upon 2 weeks of CS exposure, inflammation in lungs of TNF Delta ARE mice was significantly aggravated. However, upon 4 weeks of CS-exposure, this aggravation was no longer observed. TNF Delta ARE mice have no increases in CD4+ and CD8+ T cells and a diminished neutrophil response in the lungs after 4 weeks of CS exposure. In the gut and joints of TNF Delta ARE mice, 2 or 4 weeks of CS exposure did not modulate the development of inflammation. In conclusion, CS exposure does not modulate gut and joint inflammation in TNF Delta ARE mice. The lung responses towards CS in TNF Delta ARE mice however depend on the duration of CS exposure

The effect of smoking on intestinal inflammation: what can be learned from animal models?

AbstractEpidemiological evidence demonstrates that smoking is the most important environmental risk factor in Crohn's disease while it positively interferes with the disease course of ulcerative colitis. However, the underlying mechanisms through which smoking exerts this divergent effect and affects pathogenesis of inflammatory bowel disease are largely unknown. Animal smoke models are good models to investigate the impact of cigarette smoke on intestinal physiology and inflammation. They enable one to explore the interaction of smoke components and the gut on cellular and molecular level, clarifying how smoking interferes with normal gut function and with disease course in inflammatory conditions. This review describes the currently used animal models for studying the impact of cigarette smoke on the intestinal tract. We first discuss the different methods for simulation of smoking. Furthermore, we focus on the effect of smoke exposure on normal gut physiology and immunology, on experimental (entero)colitis, and on inflammation-induced neoplasia. Based on this current knowledge, a hypothesis is formulated about the mechanisms through which cigarette smoke interferes with the gut in normal and pathological conditions

β-glucan microparticles as mucosal delivery system in oral vaccine development

Several enteric pathogens infect the body following oral uptake and cause life-threatening diseases such as cholera, dysentery and typhoid fever. Oral vaccination is essential to generate protective local immunity against intestinal pathogens. However, antigen delivery to the inductive sites for mucosal immunity (the small intestine Peyer’s patches, PP) has proven to be particularly challenging. Recently, there has been a lot of interest in the use of microparticles as antigen delivery systems in the development of more efficient mucosal vaccines. We evaluated the potential of β-glucan microcapsules to deliver antigen transmucosally in the PP regions of the murine small intestine. β-glucan microparticles (2-4 µm) were prepared from Saccharomyces cerevisiae and loaded with FITC or Alexa Fluor 488-conjugated bovine serum albumin. Firstly, the appropriateness of β-glucan particles as antigen delivery systems for oral and intestinal applications was assessed in stability tests. Fluorimetric analysis demonstrated that the BSA concentration within particles is stable during the first 12 hours of simulated gastric or simulated intestinal fluid treatment. Secondly, β-glucan particles were administered to male C57BL/6 mice (8-10 weeks old) via intestinal loops at a particle concentration of 100*106/ml. After one hour of incubation, transmucosal particle transport and uptake in the PP was evaluated by flow cytometry, confocal microscopy and transmission electron microscopy. Using flow cytometry, we could not observe any particle uptake in the main antigen presenting cell population, the dendritic cells, however, a modest particle uptake was repeatedly detected in the B-cell population. Confocal microscopic images showed localization of β-glucan particles in the follicle associated epithelium and B-cell internalization. Moreover, transmission electron microscopy demonstrated transcellular transport of yeast particles in M-cells. In conclusion, stability tests show that antigen concentration of β-glucan particles remains stable in gastric and intestinal environment. This means that the β-glucan particles can be administered orally without prior enteric coating and are suitable for mucosal delivery of antigen in the murine gastro-intestinal tract. Our data suggest that M-cells, but not subepithelial dendritic cells, are crucial for the transmucosal transport of β-glucan particles from the intestinal lumen to the PP and transcytosis of antigen to underlying antigen presenting cells and immune cells