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

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

Cigarette smoking and inflammatory bowel disease : insights into microbial and immune homeostasis

For decades, cigarette smoking has been known to modulate the development and course of inflammatory bowel diseases (IBD). IBD include two major types of disease: Crohn’s disease (CD; affecting the ileum) and ulcerative colitis (UC; affecting the colon). Intriguingly, cigarette smoking exerts a dual effect on IBD: it has a detrimental effect on Crohn’s disease, while it has been shown to be protective for ulcerative colitis. Despite the epidemiological evidence, many of the cellular and molecular mechanisms underlying this differential effect remain to be revealed. Moreover, little is known of the effect cigarette smoking on microbial and immune homeostasis in the gut. Previous studies within our research group have shed light on the effect of cigarette smoke on the Peyer’s patches (main lymphoid organs in the gut) in the ileum of mice. It was demonstrated that chronic cigarette smoke exposure induces the recruitment of immune cells (CD4+ and CD8+ T cells, CD11b+ dendritic cells) to the subepithelial dome of the murine Peyer’s patches. In addition, chronic cigarette smoke exposure induced autophagy and apoptosis in the follicle-associated epithelium overlying these Peyer’s patches. The current thesis wants to elaborate further on these initial findings and aims to provide additional insights on the effect of cigarette smoke on the gut immune system and even the gut microbiome. In the first part of this thesis, we study the effect of chronic smoke exposure on the composition and activity of the gut microbiome and the production of immune factors in mice. A first finding is that the composition and activity of the microbiome shifts in the colon in response to chronic smoke exposure. Moreover, the activity of the Lachnospiraceae sp. is increased. Secondly, we find an increase in the expression of mucins, the building blocks of the mucus layer in response to smoking: MUC2 and MUC3 were increased in the ileum, while MUC4 increases in the colon. Thirdly, we find that although no microbial changes are induced by smoking in the ileum, the expression of immune factors is altered. The expression of ileal CXCL2 is increased and ileal IFN-γ is decreased after chronic smoke exposure. In the proximal colon, an increase in IL-6 and a decrease in TGF-β is denoted due to chronic smoke exposure. Interestingly, these findings highlight the dual effect of smoking on the gut: smoke exposure affects the immune system in the ileum, whereas it affects the microbiome in the colon. The local effect of smoking therefore strongly depends on the gut compartment. The second part of this thesis elaborates on the effect of smoking in the context of intestinal inflammation. We make use of two different Crohn-like inflammation mouse models: TNBS-induced colitis as a model for Crohn-like colitis and the TNFΔARE model for Crohn-like ileitis. In the TNBS colitis model, we show that smoke exposure further increased inflammation-induced cytokine/chemokine production, however does not exacerbate clinical disease parameters and histology. In the TNFΔARE model, cigarette smoke exposure induces no changes in cytokine/chemokine production, clinical disease parameters or histology. It seems that the effect of cigarette smoke is too subtle to further worsen full-blown intestinal inflammation, however, cigarette smoke does affect cytokine and chemokine production during ongoing inflammation. The third and final part of this thesis focuses on the effect of cigarette smoking on the production of cytokines and chemokines, especially IL-8, and the expression of the transient receptor potential channel TRPV1. A first finding is that cigarette smoking boosts IL-8 expression in healthy and inflamed gut of both human and mice. Using a human database of currently and never smoking Crohn’s disease patients, we demonstrate that IL-8 increases in currently smoking human subjects and this is often paralleled with an increase of TRPV1. TRP channels have been shown to modulate cigarette smoke-induced IL-8 expression in the airways. In mice experiments, we find a simultaneous increase of the mouse IL-8 homologue CXCL2 and TRPV1 in the ileum. In an IBD mouse model (TNBS-induced colitis), we also observe a parallel smoke-induced increase of CXCL2 and TRPV1 in the inflamed distal colon. The simultaneous increase of IL-8 and TRPV1 in the gut due to cigarette smoking suggests a link between both, however many more research is needed into this matter. In conclusion, this thesis provides important insights into the effect of cigarette smoke on the gut microbiome and immune system. In the colon, cigarette smoking mainly affects the microbiome, while in the ileum, the production of immune factors is induced. Furthermore, cigarette smoking increases cytokine and chemokine expression in healthy and inflamed gut of both human and mice, and this parallels smoke-induced expression of TRPV1

Evaluation of a zinc chelate on clinical swine dysentery under field conditions

2020 The Author(s). Background: Brachyspira hyodysenteriae is the primary cause of swine dysentery, characterized by bloody to mucoid diarrhea due to mucohaemorhagic colitis in pigs and primarily affects pigs during the grow/finishing stage. Control and prevention of B. hyodysenteriae consists of administration of antimicrobial drugs, besides management and adapted feeding strategies. A worldwide re-emergence of the disease has recently been reported with an increasing number of isolates demonstrating decreased susceptibility to several crucially important antimicrobials in the control of swine dysentery. A novel non-antibiotic zinc chelate has been reported to demonstrate positive effects on fecal quality and consistency, general clinical signs, average daily weight gain and B. hyodysenteriae excretion during and after a 6-day oral treatment. The objective of the present study was to evaluate the zinc chelate (Intra Dysovinol® 499 mg/ml (ID); Elanco) on naturally occurring swine dysentery due to B. hyodysenteriae under field conditions in the Netherlands. Results: Oral administration of zinc chelate resulted in improvement of general clinical signs from 3 days onwards in the ID-treated group combined with a significantly better total fecal score at 14 days post-treatment. Overall, average daily weight gain was better in the ID-treated group over the entire study period (0-14 days) and during the 8 days following the end of ID-treatment. A significant reduction (4.48 vs. 0.63 log10 cfu/g feces; ID-treated vs. control) in B. hyodysenteriae excretion was observed during the 6-day treatment period with a high percentage of animals (58.3 vs. 12.3%; ID-treated vs. control) with no excretion of B. hyodysenteriae from their feces. No additional antimicrobial treatment was needed in the ID-treated group, whereas 35% of the pigs in the control group were treated with an antibiotic at least once. No mortality occurred in both groups. No adverse events were reported during and following the ID-treatment. Conclusions: Zinc chelate - administered as a Zn-Na2-EDTA complex - is a non-antibiotic treatment for swine dysentery that reduces B. hyodysenteriae shedding with 4.48 log10 cfu/g feces within its 6-day treatment while improving general clinical signs (90.0 vs. 73.6% animals with normal score) and total fecal score within 2-4 days following administration in naturally infected pigs. The positive effects of ID treatment remain for at least 8 days after cessation of oral ID therapy. Pigs remaining in a highly contaminated environment may be re-infected following the end of ID treatment, however, this is not different to standard antimicrobial therapy. Therefore, control of swine dysentery should combine an efficacious treatment with additional management practices to reduce the environmental infection pressure in order to limit re-infection as much as possible. The ID treatment resulted in a higher growth rate and improved general health, whereas no mortality was observed and no additional therapeutic treatments were necessary in contrast to the control pigs

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

Oocyst shedding patterns of Eimeria species and their association with management and performance at ten rose veal starter farms in the Netherlands.

Coccidiosis at rose veal starter farms is often diagnosed however, this was the first study performed considering this topic on this type of rearing unit. The objective of this study was to identify Eimeria species faecal shedding patterns at ten Dutch rose veal starter farms during rearing. Further objectives were to investigate associations with (gut) health, production and management decisions. Faecal samples from twelve randomly selected calves per farm were collected weekly during 9 consecutive weeks. Thereafter samples were pooled in a predetermined composition of six. These calves were clinically evaluated every sample visit and weighed at the first and last visit. Laboratory tests included a flotation test of the samples. If this yielded Eimeria oocysts, both oocysts per gram feces (OPG) and species differentiation were established using a modified McMaster method. Management parameters and technical herd results were identified after finalizing the study period using a questionnaire. Studied patterns in oocyst shedding included the pool's cumulated OPG, maximum OPG level and the number of sample days (SD) that OPG exceeded a confirmed level. Statistical analysis included univariate and multivariate analysis. Associated (p 1000 OPG for E. alabamensis 141 g lower ADG. From the questionnaire we identified lower cumulated OPG of all Eimeria species except for E. bovis at farms where the units were cleaned before arrival of the calves. Conclusion: As a rule, on Dutch rose veal starter farms, mixed Eimeria infections occur, but shedding patterns differ between farms. Clinical and growth performance is related to OPG patterns found. Cleaning units before arrival of calves lowers oocyst shedding during the rearing period

Beta-glucan particles as novel antigen delivery systems: towards oral vaccination

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