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Intestinal fibrosis is a common complication of the inflammatory bowel diseases (IBDs), contributing to tissue stiffening and luminal narrowing. Human nuclear receptor 4A 1 (NR4A1) was previously reported to regulate mesenchymal cell function and dampen fibrogenic signaling. NR4A1 gene variants are associated with IBD risk, and it has been shown to regulate intestinal inflammation. Here, we tested the hypothesis that NR4A1 acts as a negative regulator of intestinal fibrosis through regulating myofibroblast function. Using the SAMP1/YitFc mouse, we tested whether two pharmacological agents known to enhance NR4A1 signaling, cytosporone B (Csn-B) or 6-mercaptopurine (6-MP), could reduce fibrosis. We also used the dextran sulfate sodium (DSS) model of colitis and assessed the magnitude of colonic fibrosis in mouse nuclear receptor 4A 1 (

Here we characterized the murine dextran sulfate sodium (DSS) model of acute colitis. Specifically, we evaluated azithromycin and metronidazole treatment regimens to assess their effects on animal wellbeing, pathologic changes, barrier function, cytokine and chemokine profiles, and neutrophil migration in colon tissue. Azithromycin treatment significantly reduced the severity of colitis, as assessed through body weight change, water consumption, macroscopic lesions, and animal behaviors (activity level, climbing, and grooming), but did not alter food consumption or feeding behavior. Mucosal barrier function (evaluated by using FITC-labeled dextran) was decreased after DSS exposure; azithromycin did not significantly alter barrier function in mice with colitis, whereas metronidazole exacerbated the colitis-related deficit in barrier function. In addition, metronidazole appeared to exacerbate disease as assessed through water consumption and animal behaviors (overall activity, climbing, grooming, and drinking) but had no effect on weight loss, macroscopic lesions, or eating behavior. Pathologic changes were typical for DSS treatment. Antibiotic treatment resulted in reduced levels of proinflammatory cytokines and chemokines and decreased neutrophil adhesion and emigration in DSS-exposed mice. The results highlight the importance of clinical and behavioral assessments in addition to laboratory evaluation as tools to evaluate animal welfare and therapeutic efficacy in disease models. Data from this study suggest that azithromycin may convey some benefits in the mouse DSS colitis model through modulation of the immune response, including neutrophil migration into tissues, whereas metronidazole may exacerbate colitis.

Background Intestinal fibrosis and stricture formation are common complications of Crohn’s disease (CD). Recently, the stricturing phenotype has been recognized to be primarily attributable to hypertrophy/hyperplasia of smooth muscle, rather than an increase in fibrosis alone. Despite advances in treatment of CD, current therapies do little to prevent or reverse strictures. NR4A1 is an orphan nuclear receptor that has been reported as anti-fibrotic in non-intestinal systems and exhibits anti-proliferative effects in smooth muscle cells (SMCs). NR4A1 gene variants have been associated with increased risk of IBD, however, the mechanisms regulating NR4A1 expression and its role in intestinal SMC function has not been investigated. Aims To characterize and understand the role of NR4A1 activation in the regulation of mitogen-induced proliferation of intestinal SMCs. Methods Primary intestinal SMCs were isolated from Nr4a1+/+ and Nr4a1-/- mice. Furthermore, a commercially sourced human primary intestinal SMC line was used. To assess the response of SMCs to culture and identify growth differences, proliferation was measured via trypan blue exclusion and EdU incorporation. In mouse and human SMCs, proliferation was induced by platelet-derived growth factor-BB (PDGF-BB) and NR4A1 activation was assessed by pretreating with selective agonists, cytosporone B (Csn-B) and 6-mercaptopurine (6-MP), at various concentrations. Mass spectrometry was used to characterize proteomic differences between Nr4a1+/+ and Nr4a1-/- SMCs. Expression levels of NR4A1 were assessed by qPCR and western blot after mitogen exposure and Csn-B treatment. Results Nr4a1-/- cells exhibited a significantly higher rate of proliferation compared to Nr4a1+/+ cells, under both basal and mitogen-exposed conditions. Proteomic analysis showed that Nr4a1-/- SMCs exhibited increased expression of proteins related to the cell cycle and metabolism, compared to Nr4a1+/+ SMCs. Pretreating human intestinal SMCs with Csn-B and 6-MP significantly attenuated proliferation induced by PDGF-BB. Similar effects were observed in Nr4a1+/+ SMCs, however, the anti-proliferative effect of Csn-B was absent in Nr4a1-/- cells. Furthermore, NR4A1 expression was rapidly induced by Csn-B and PDGF-BB, the latter response suggesting the existence of a potential negative feedback mechanism to control mitogen-induced SMC proliferation. Conclusions Our results suggest that NR4A1 is a critical regulator of intestinal SMC proliferation and its induction by mitogens may contribute to a negative feedback loop to control smooth muscle growth. These data support targeting NR4A1 to treat excessive smooth muscle hypertrophy/hyperplasia that contributes to tissue remodelling observed in fibrostenotic CD. Funding Agencies CAG, CCC, CIHR

Hyperglycaemia is a major contributor to diabetic cardiovascular disease with hyperglycaemia-induced endothelial dysfunction recognized as the initiating cause. Coagulation pathway-regulated proteinase-activated receptors (PARs) that can regulate vascular tone in vivo cause eNOS-mediated endothelium-dependent vasodilation; but, the impact of hyperglycaemia on this vasodilatory action of PAR stimulation and the signalling pathways involved are unknown. We hypothesized that vascular sodium-glucose co-transporter 2 activity and hyperglycaemia-induced oxidative stress involving Src-kinase, EGF receptor-kinase, Rho-kinase and protein-kinase-C biochemical signalling pathways would compromise PAR2-mediated endothelium-dependent vasodilation. Using an organ culture approach, wherein murine aorta rings were maintained for 24 h at hyperglycaemic 25 mM versus euglycaemic 10 mM glucose, we observed severely blunted acetylcholine/muscarinic and PAR2-mediated endothelial eNOS/NO-dependent vasodilation. PEG-catalase, superoxide-dismutase, and NADPH-oxidase inhibition (VAS2870) and either SGLT2-inhibition (canagliflozin/dapagliflozin/empagliflozin) or antioxidant gene induction (sulforaphane), prevented the hyperglycaemia-induced impairment of PAR2-mediated vasodilation. Similarly, inhibition of Src-kinase, EGF receptor-kinase, protein kinase-C and Rho-kinase also preserved PAR2-mediated vasodilation in tissues cultured under hyperglycaemic conditions. Thus, intracellular hyperglycaemia, that can be prevented with an inhibitor of the SGLT2 cotransporter that was identified in the vascular tissue and tissue-derived cultured endothelial cells by qPCR, western blot and immunohistochemistry, leads to oxidative stress that compromises PAR2-mediated NOS-dependent vasodilation by an NAPDH oxidase/reactive-oxygen-species-triggered signalling pathway involving EGFR/Src/Rho-kinase and PKC. The data point to novel antioxidant therapeutic strategies including use of an SGLT2 inhibitor and sulforaphane to mitigate hyperglycaemia-induced endothelial dysfunction.

Background Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of Johne’s disease (JD) in ruminants. Following infection, JD may present as enteritis, leading to wasting, often causing premature culling of livestock. Beyond veterinary medicine, several mycobacterium species, including MAP, have been implicated in human gastrointestinal disease. While MAP has been incriminated in causing Crohn’s disease (a claim that has yet to be substantiated), there are confirmed cases of MAP infection in immunocompromised individuals, causing profuse diarrhea, fever, and drastic weight loss. Given the economic burden associated with MAP infection, considerable efforts have sought to understand its dynamics. However, these processes have not been completely characterized, hindering our ability to generate novel anti-infective agents. While the current paradigm suggests that MAP travels to the small intestine, gaining entry through the epithelium, the exact cellular tropism and the mechanism(s) of entry are not well defined. Therefore, we have developed an ex vivo enteroid-based system to visualize invasion of MAP in distinct cells of the intestinal epithelium using a GFP-expressing MAP strain. With this, we sought to test the hypothesis that MAP invasion occurs via M cells through receptor-mediated transcytosis. Aims 1) Characterize experimental system and visualize MAP invasion 2) Determine cellular tropism 3) Uncover mechanisms underlying MAP invasion Methods Enteroids (2D and 3D) were generated and M cell differentiation induced via addition of RANKL. Confluent ileal monolayers were exposed to GFP-expressing MAP strain (K10 pWES4). Confocal microscopy was performed, and barrier function was measured via transepithelial electrical resistance (TEER). Results We generated 3D enteroids and confluent enteroid-derived monolayers with functional M cells capable of transcytosis. MAP was detected mainly within M cells. We further confirmed this finding using a human in vitro M cell model, the Caco-2/Raji-B co-culture system. Furthermore, alterations in TEER following MAP exposure in monolayers cultured with RANKL, triggering M cell differentiation, suggest the existence of a novel mechanism by which MAP disrupts the barrier to invade the mucosa. Conclusions Our results suggest that MAP translocates across the epithelium predominantly via M cells, as shown both in a human and murine model. This newly optimized approach provides an experimental system that will enable us to better characterize M cell-MAP interactions, with the hopes of identifying new therapeutic targets to prevent the spread of MAP and reduce economic impact of JD. Beyond MAP infection, this novel ex vivo system has potential to elucidate other host-pathogen interactions. Funding Agencies Natural Sciences and Engineering Research Council of Canada (NSERC)

Background and Purpose The pathogenesis of the inflammatory bowel diseases (IBD), comprising Crohn's disease (CD) and ulcerative colitis (UC), involves aberrant interactions between a genetically susceptible individual, their microbiota and environmental factors. Alterations in xenobiotic receptor expression and function are associated with increased risk for IBD. Here, we have assessed the role of the constitutive androstane receptor (CAR), a xenobiotic receptor closely related to the pregnane X receptor, in the regulation of intestinal mucosal homeostasis. Experimental Approach CAR expression was assessed in intestinal mucosal biopsies obtained from CD and UC patients, and in C57/Bl6 mice exposed to dextran sulphate sodium (DSS; 3.5% w/v in drinking water) to evoke intestinal inflammation and tissue damage. CAR-deficient mice were exposed to DSS and mucosal healing assessed. Modulation of wound healing by CAR was assessed in vitro. The therapeutic potential of CAR activation was evaluated, using 3,3′,5,5′-tetrachloro-1,4-bis(pyridyloxy)benzene (TCPOBOP), a selective rodent CAR agonist. Key Results CAR expression was reduced in CD and UC samples, compared with expression in healthy controls. This was reproduced in our DSS studies, where CAR expression was reduced in colitic mice. CAR-deficient mice exhibited reduced healing following DSS exposure. In vitro, CAR activation accelerated intestinal epithelial wound healing by enhancing cell migration. Lastly, treating mice with TCPOBOP, following induction of colitis, enhanced mucosal healing. Conclusion and Implications Our results support the notion that xenobiotic sensing is altered during intestinal inflammation, and suggest that CAR activation may prove effective in enhancing mucosal healing in patients with IBD.

Background Failure to resolve inflammation is often associate with the complications of Crohn’s Disease (CD). The pregnane X receptor (PXR), a xenobiotic receptor, is recognized for its role in suppressing inflammation and has recently been shown to influence fibrogenesis in the liver. In the intestine, PXR-signaling can be influenced by the microbial tryptophan metabolite indole-3- propionic acid (IPA), which can modulate intestinal inflammation, in turn influencing fibrogenesis, resolution and healing. This suggests that the gut microbiota could modulate mucosal homeostasis and resolution of inflammation via microbial metabolites Aims To understand and characterize the interplay between microbial complexity and the regulation of host inflammatory and healing responses, specifically focusing on the PXR and its microbial metabolite ligand IPA. Methods Intestinal inflammation was induced using DSS (1%, 1.5%, 2% and 3.5%) for 5 days followed by healing for 25 days in C57Bl/6 stably derived moderately diverse mouse microbiota 2 (sDMDMm2) colonized gnotobiotic and C57Bl/6 specific pathogen free (SPF) mice. Inflammation, architectural changes and fibrosis were assessed using Haemotoxylin and Eosin and Masson-Trichrome histological stains. Weight was recorded daily for the first 10 days and every other day after for 25 days, for a total of 30 days. Fecal lipocalin was quantified in samples collected throughout the study to assess inflammation. Innate immune cell influx was measured by flow cytometry, and the microbiota assessed via 16S rRNA sequencing. Results The gnotobiotic sDMDMm2 mice were exquisitely sensitive to DSS-induced colitis, exhibiting significantly increased mortality and morbidity at 2% and 3.5% w/v DSS compared to the SPF group. To elicit the same degree of disease to assess recovery, sDMDMm2 mice were exposed to 1.5% DSS and SPF mice to 3.5% DSS. Following 25 days recovery, sDMDMm2 colonized mice showed increased levels of fecal lipocalin 2, as compared to the SPF mice. DSS-treated sDMDMm2 mice supplemented with IPA during their recovery presented lower levels of fecal lipocalin, similar to colitic SPF mice. IPA supplemented sDMDMm2 mice also exhibited greater overall survival, with no significant differences in neutrophil count compared to mice given H20 during recovery. Conclusions A model system with a less complex microbiota (sDMDMm2) has a higher susceptibility to acute inflammation and a diminished capacity to resolve said inflammation. Addition of the microbial metabolite IPA normalized the recovery of the sDMDMm2 colonized mice, to a response indistinguishable from SPF mice, while also increasing survival. These data highlight the importance of microbial complexity in the regulation of intestinal mucosal homeostasis. Funding Agencies CAG, CCC, CIHR

Clostridioides difficile (formerly Clostridium difficile; C difficile), the leading cause of nosocomial antibiotic‐associated colitis and diarrhea in the industrialized world, triggers colonic disease through the release two toxins, toxin A (TcdA) and toxin B (TcdB), glucosyltransferases that modulate monomeric G‐protein function and alter cytoskeletal function. The initial degree of the host immune response to C difficile and its pathogenic toxins is a common indicator of disease severity and infection recurrence. Thus, targeting the intestinal inflammatory response during infection could significantly decrease disease morbidity and mortality. In the current study, we sought to interrogate the influence of the pregnane X receptor (PXR), a modulator of xenobiotic and detoxification responses, which can sense and respond to microbial metabolites and modulates inflammatory activity, during exposure to TcdA and TcdB. Following intrarectal exposure to TcdA/B, PXR‐deficient mice (Nr1i2−/−) exhibited reduced survival, an effect that was associated with increased levels of innate immune cell influx. This exacerbated response was associated with a twofold increase in the expression of Tlr4. Furthermore, while broad‐spectrum antibiotic treatment (to deplete the intestinal microbiota) did not alter the responses in Nr1i2−/− mice, blocking TLR4 signaling significantly reduced TcdA/B‐induced disease severity and immune responses in these mice. Lastly, to assess the therapeutic potential of targeting the PXR, we activated the PXR with pregnenolone 16α‐carbonitrile (PCN) in wild‐type mice, which greatly reduced the severity of TcdA/B‐induced damage and intestinal inflammation. Taken together, these data suggest that the PXR plays a role in the host's response to TcdA/B and may provide a novel target to dampen the inflammatory tissue damage in C difficile infections.

BACKGROUND: Fibrosis is a serious and irreversible complication of Crohn’s disease (CD) that can lead to intestinal obstruction and has no effective treatment beyond surgical resection. The pathogenesis of fibrosis is incompletely understood however, it is thought to involve an aberrant repair response following inflammation-associated tissue damage. The pregnane X receptor (PXR), a xenobiotic receptor, is recognized for its role in suppressing inflammation and has been show to influence fibrogenesis in the liver. In the intestine, PXR-signaling is influenced by the tryptophan metabolite indole-3-propionic acid (IPA), which can affect intestinal inflammation, in turn influencing fibrogenesis. How microbial metabolite sensing through the PXR influences intestinal fibrotic responses has not been explored. AIMS: To understand the impact PXR-signaling has on intestinal fibrosis and determine whether it can be modulated by IPA. METHODS: Intestinal inflammation was induced using DSS (3.5%) for 5 days followed by healing for 25 days. Fibrosis in the colon was assessed using Masson’s trichrome and Sirius Red histological stains in wild type (WT), PXR(-/-), epithelial-specific PXR(-/-) and fibroblast-specific PXR(-/-) mice. Immune cell influx was measured by flow cytometry and cytokine concentrations by Luminex. Fibroblasts isolated from WT and PXR(-/-) mice were stimulated with cytomix (TNF-α, IL-1β, and IFN-γ) or LPS in the presence or absence of IPA for 24 hours and assessed for gene expression. To examine the role of microbial metabolites in fibrosis, the microbiota was depleted using a cocktail of broad-spectrum antibiotics and some mice treated with IPA. RESULTS: Following recovery from DSS, WT mice showed clear evidence of colonic fibrosis, a response that was exacerbated in PXR(-/-) mice and correlated with greater neutrophil infiltration and levels of innate cytokines (e.g. CXCL2, GMCSF, GCSF,) in the colon. This phenotype was not observed when PXR deficiency was limited to the epithelial cells, but was reproducible in mice with fibroblast-specific PXR-deficiency. Mechanistically, PXR(-/-) fibroblasts were hyper-responsive to proinflammatory cytokines and LPS, producing increased levels of CXCL2, GM-CSF and G-CSF compared to WT cells. Importantly, the microbial metabolite IPA was able to block the expression of these cytokines. Depletion of the microbiota completely suppressed systemic levels of IPA and exacerbated intestinal fibrosis, an effect that was reversed by IPA treatment. CONCLUSIONS: PXR signaling in intestinal fibroblasts restrains their inflammatory responses, reducing fibrogenesis. Luminal sensing of the bacterial derived indole, IPA, via PXR may be involved in this process, highlighting a microbial metabolite-sensing pathway in fibroblasts that could be targeted to prevent intestinal fibrosis observed in CD. FUNDING AGENCIES: CAG, CCC, CIHRAbbvie/CAG/CIHR, Alberta Innovates, Canadian Foundation for Innovation, US Department of Defense

BACKGROUND: Intestinal fibrosis is a common complication of IBD, comprises of excessive accumulation of scar tissue in the intestinal wall. Fibrosis is thought to result from an aberrant response to injury resulting in excessive extracellular matrix (ECM) deposition, which can lead to tissue stiffening and intestinal stricture formation. NR4A1 is an orphan nuclear receptor previously reported to regulate mesenchymal cell function and dampen fibrogenic signaling. More recently, NR4A1 gene variants were found to be associated with IBD risk, and its signaling shown to play a role in regulating intestinal inflammation. AIMS: To test the hypothesis that NR4A1 acts as a negative regulator of intestinal fibrosis through regulating, myofibroblast function. METHODS: Using the SAMP1-YitFc mouse, a spontaneous model of ileitis that exhibits extensive intestinal tissue remodelling, we administered NR4A1 agonists cytosporone B (CSN-B) or 6-mercaptopurine (6MP), beginning at 10 weeks of age (the age reported to be associated with established ileal inflammation in this model). Bodyweight and fecal lipocalin were assessed throughout the treatment period. After 12 weeks of NR4A1 agonist exposure, mice were sacrificed and intestinal tissues isolated for assessment of inflammatory markers and tissue ECM content. Histological analyses were performed to quantify tissue damage, inflammation, and remodelling. To determine the anti-proliferative effects of NR4A1, intestinal myofibroblast isolated from Nr4a1(+/+) and Nr4a1(-/-) mice were exposed to TGF-β, treated with CSN-B or 6MP, and proliferation and ECM gene expression assessed. RESULTS: SAMP1-YitFc treated with CSN-B or 6MP showed reduced body weight loss and a moderate reduction in inflammatory markers compared to the untreated mice. Ileal thickness and tissue collagen content was reduced in mice treated with CSN-B or 6MP. In vitro, Nr4a1(-/-) intestinal myofibroblasts exhibited enhanced TGF-β-induced proliferation compared to Nr4a1(+/+) cells. Lastly, CSN-B and 6-MP each reduced TGF-β-induced proliferation in Nr4a1(+/+), but not Nr4a1(-/-), myofibroblasts. CONCLUSIONS: These data point towards targeting NR4A1 for the treatment of inflammation-associated fibrosis and suggest that 6MP, a commonly used agent for the treatment of IBD, may exhibit anti-fibrotic effects that should be reassessed in clinical studies. FUNDING AGENCIES: CIHR

BACKGROUND: Fibrosis contributes to intestinal stricture and obstruction in 30–50% of Crohn’s disease patients. The pathogenesis of fibrosis is incompletely understood and has virtually no effective treatments. The pregnane X receptor (PXR), a xenobiotic receptor involved in detoxification responses, has been identified as a modulator of fibrosis in hepatic stellate cells. Of the number of foreign ligands for the PXR, the tryptophan metabolite indole-3-propionic acid (IPA) produced by the intestinal commensal Clostridium sporogenes can bind to the PXR to mediate signalling events that protect intestinal barrier function. The role of the PXR in intestinal fibrosis and if microbial metabolite sensing can affect intestinal fibrotic responses is unknown. AIMS: To examine the role of the PXR and its ability to sense microbial metabolites in the modulation of intestinal fibrosis. METHODS: Intestinal inflammation was induced using DSS (3.5%) for 5 days followed by healing for 25 days. Fibrosis was assessed using Masson’s trichrome and Sirius Red staining of colonic sections. Mouse primary colonic fibroblasts were grown from wild type (WT) and PXR-/- mice and stimulated with cytomix (TNFα, IL1β, and IFNγ) for 24 hours to assess cytokine production by Luminex. Fibroblasts were also stimulated with cytomix in the presence of the PXR agonists PCN or IPA and assessed for gene expression via qPCR. NFkB activity was assessed by Western blot for phos-p65. To examine the microbiota’s role in fibrosis, after the 5-day course of DSS the microbiota was depleted for 25 days with an antibiotic cocktail (vancomycin, neomycin, metronidazole, ampicillin). C. sporogenes DNA was detected in feces using qPCR. RESULTS: Following a 25-day recovery after DSS, WT mice demonstrated clear intestinal fibrosis. When compared to WT mice, PXR-/- mice demonstrated significantly greater levels of fibrosis. Mouse primary fibroblasts were found to express the PXR. Following stimulation with cytomix, PXR-/- fibroblasts produced dramatically higher levels of inflammatory cytokines including eotaxin, CXCL2, G-CSF, GM-CSF, IL-9, and IL-15. Importantly, PCN and the microbial metabolite IPA suppressed the expression of these cytokines. These effects in PXR-/- fibroblasts may be linked to the increased activity of NFkB that was observed both basally and after stimulation with cytomix compared to WT fibroblasts. Depletion of the microbiota supressed levels of C. sporogenes DNA in feces and exacerbated intestinal fibrosis. CONCLUSIONS: PXR signaling in intestinal fibroblast appears to be required to restrain inflammation and fibrosis. Luminal sensing of bacterial derived indoles (i.e. IPA) via PXR may be involved in this process, highlighting a xenobiotic-microbiota axis that could be targeted to prevent the intestinal fibrosis observed in Crohn’s disease. FUNDING AGENCIES: CCCBeverley Phillips Rising Stars Program, The Dr. Lloyd Sutherland Investigator in IBD/GI Research, Canadian Foundation for Innovation, Canada Research Chairs Program, U.S. Department of Defense