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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 (

Stricture formation is a common complication of Crohn's disease (CD), driven by enhanced deposition of extracellular matrix (ECM) and expansion of the intestinal smooth muscle layers. Nuclear receptor subfamily 4 group A member 1 (NR4A1) is an orphan nuclear receptor that exhibits anti-proliferative effects in smooth muscle cells (SMCs). We hypothesized that NR4A1 regulates intestinal SMC proliferation and muscle thickening in the context of inflammation. Intestinal SMCs isolated from Nr4a1

We proposed that circulating metabolites generated by the intestinal microbiota can affect vascular function. One such metabolite, indole 3-propionic acid (IPA), can activate the pregnane X receptor(PXR), a xenobiotic-activated nuclear receptor present in many tissues, including the vascular endothelium. We hypothesized that IPA could regulate vascular function by modulating PXR activity. To test this, Pxr+/+ mice were administered broad-spectrum antibiotics for 2 wk with IPA supplementation. Vascular function was evaluated by bioassay using aorta and pulmonary artery ring tissue from antibiotic-treated Pxr+/+ and Pxr−/−mice, supplemented with IPA, and using aorta tissue maintained in organ culture for 24 h in the presence of IPA. Endothelium-dependent, nitric oxide(NO)-mediated muscarinic and proteinase-activated receptor 2(PAR2)-stimulated vasodilation was assessed. Endothelial nitric oxide synthase (eNOS) abundance was evaluated in intact tissue or in aorta-derived endothelial cell cultures from Pxr+/+ and Pxr−/− mice, and vascular Pxr levels were assessed in tissues obtained from Pxr+/+ mice treated with antibiotics and supplemented with IPA. Antibiotic-treated Pxr+/+ mice exhibited enhanced agonist-induced endothelium-dependent vasodilation, which was phenocopied by tissues from either Pxr−/− or germ-free mice. IPA exposure reduced the vasodilatory responses in isolated and cultured vessels. No effects of IPA were observed for tissues obtained from Pxr−/− mice. Serum nitrate levels were increased in antibiotic-treated Pxr+/+and Pxr−/− mice. eNOS abundance was increased in aorta tissues and cultured endothelium from Pxr−/− mice. PXR stimulation reduced eNOS expression in cultured endothelial cells from Pxr+/+ but not Pxr−/− mice. The microbial metabolite IPA, via the PXR, plays a key role in regulating endothelial function. Furthermore, antibiotic treatment changes PXR-mediated vascular endothelial responsiveness by upregulating eNOS.

The pregnane X receptor (PXR) is a ligand-activated nuclear receptor that acts as a xenobiotic sensor, responding to compounds of foreign origin, including pharmaceutical compounds, environmental contaminants, and natural products, to induce transcriptional events that regulate drug detoxification and efflux pathways. As such, the PXR is thought to play a key role in protecting the host from xenobiotic exposure. More recently, the PXR has been reported to regulate the expression of innate immune receptors in the intestine and modulate inflammasome activation in the vasculature. In the current study, we report that activation of the PXR in primed macrophages triggers caspase-1 activation and interleukin-1β release. Mechanistically, we show that this response is nucleotide-binding oligomerization domain, leucine-rich repeat, and pyrin domain-containing 3-dependent and is driven by the rapid efflux of ATP and P2X purinoceptor 7 activation following PXR stimulation, an event that involves pannexin-1 gating, and is sensitive to inhibition of Src-family kinases. Our findings identify a mechanism whereby the PXR drives innate immune signaling, providing a potential link between xenobiotic exposure and the induction of innate inflammatory responses.

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)

Cancer cell lines have been the mainstay of intestinal epithelial experimentation for decades, due primarily to their immortality and ease of culture. However, because of the inherent biological abnormalities of cancer cell lines, many cellular biologists are currently transitioning away from these models and toward more representative primary cells. This has been particularly challenging, but recent advances in the generation of intestinal organoids have brought the routine use of primary cells within reach of most epithelial biologists. Nevertheless, even with the proliferation of publications that use primary intestinal epithelial cells, there is still a considerable amount of trial and error required for laboratories to establish a consistent and reliable method to culture three-dimensional (3D) intestinal organoids and primary epithelial monolayers. We aim to minimize the time other laboratories spend troubleshooting the technique and present a standard method for culturing primary epithelial cells. Therefore, we have described our optimized, high-yield, cost-effective protocol to grow 3D murine colonoids for more than 20 passages and our detailed methods to culture these cells as confluent monolayers for at least 14 days, enabling a wide variety of potential future experiments. By supporting and expanding on the current literature of primary epithelial culture optimization and detailed use in experiments, we hope to help enable the widespread adoption of these innovative methods and allow consistency of results obtained across laboratories and institutions. NEW & NOTEWORTHY Primary intestinal epithelial monolayers are notoriously difficult to maintain culture, even with the recent advances in the field. We describe, in detail, the protocols required to maintain three-dimensional cultures of murine colonoids and passage these primary epithelial cells to confluent monolayers in a standardized, high-yield and cost-effective manner.

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.

The inflammatory bowel diseases (IBDs) are chronic inflammatory disorders with a complex etiology. IBD is thought to arise in genetically susceptible individuals in the context of aberrant interactions with the intestinal microbiota and other environmental risk factors. Recently, the pregnane X receptor (PXR) was identified as a sensor for microbial metabolites, whose activation can regulate the intestinal epithelial barrier. Mutations in NR1I2, the gene that encodes the PXR, have been linked to IBD, and in animal models, PXR deletion leads to barrier dysfunction. In the current study, we sought to assess the mechanism(s) through which the PXR regulates barrier function during inflammation. In Caco-2 intestinal epithelial cell monolayers, tumor necrosis factor-α/interferon-γ exposure disrupted the barrier and triggered zonula occludens-1 relocalization, increased expression of myosin light-chain kinase (MLCK), and activation of c-Jun N-terminal kinase 1/2 (JNK1/2). Activation of the PXR [rifaximin and [[3,5-Bis(1,1-dimethylethyl)-4-hydroxyphenyl]ethenylidene]bis-phosphonic acid tetraethyl ester (SR12813); 10 μM] protected the barrier, an effect that was associated with attenuated MLCK expression and JNK1/2 activation. In vivo, activation of the PXR [pregnenolone 16α-carbonitrile (PCN)] attenuated barrier disruption induced by toll-like receptor 4 activation in wild-type, but not Pxr-/-, mice. Furthermore, PCN treatment protected the barrier in the dextran-sulfate sodium model of experimental colitis, an effect that was associated with reduced expression of mucosal MLCK and phosphorylated JNK1/2. Together, our data suggest that the PXR regulates the intestinal epithelial barrier during inflammation by modulating cytokine-induced MLCK expression and JNK1/2 activation. Thus, targeting the PXR may prove beneficial for the treatment of inflammation-associated barrier disruption in the context of IBD.

BACKGROUND Mycophenolate mofetil (MMF) is commonly prescribed after transplantation and has major advantages over other immunosuppressive drugs, but frequent gastrointestinal (GI) side-effects limit its use. The mechanism(s) underlying MMF-related GI toxicity have yet to be elucidated. METHODS To investigate MMF-related GI toxicity, experimental mice were fed chow containing MMF (0.563%) and multiple indices of toxicity, including weight loss and colonic inflammation, were measured. Changes in intestinal microbial composition were detected using 16S rRNA Illumina sequencing, and downstream PICRUSt analysis was used to predict metagenomic pathways involved. Germ-free (GF) mice and mice treated with orally administered broad-spectrum antibiotics (ABX) were utilized to interrogate the importance of the microbiota in MMF-induced GI toxicity. RESULTS Mice treated with MMF exhibited significant weight loss, related to loss of body fat and muscle, and marked colonic inflammation. MMF exposure was associated with changes in gut microbial composition, as demonstrated by a loss of overall diversity, expansion of Proteobacteria (specifically Escherichia/Shigella), and enrichment of genes involved in lipopolysaccharide (LPS) biosynthesis, which paralleled increased levels of LPS in the feces and serum. MMF-related GI toxicity was dependent on the intestinal microbiota, as MMF did not induce weight loss or colonic inflammation in GF mice. Furthermore, ABX prevented and reversed MMF-induced weight loss and colonic inflammation. CONCLUSIONS An intact intestinal microbiota is required to initiate and sustain the GI toxicity of MMF. MMF treatment causes dynamic changes in the composition of the intestinal microbiota that may be a targetable driver of the GI side-effects of MMF.

BACKGROUND: The current treatments available for C. difficile infections (CDI) target the bacteria, however they do nothing to treat the damaging inflammation triggered by the virulence factors, TcdA and TcdB. Indeed, enhanced chemokine/cytokine expression during CDI is associated with poor clinical outcomes. Interestingly, a number of rifamycin antibiotics have been shown to have potent activity against C. difficile, while also exhibiting anti-inflammatory effects through their ability to activate the pregnane X receptor (PXR) and inhibit NFκB signaling. In the current study, we sought to test the hypothesis that targeting the PXR could prove to be an effective strategy to reduce C. difficile toxin-induced inflammation and tissue damage. AIMS: 1) To assess the ability of PXR agonists to attenuate TcdB-induced CXCL8/IL-8 production in vitro 2) To test whether PXR activation can attenuate TcdA/B-induced damage and inflammation in an in vivo model of C. difficile toxin exposure 3) Determine the mechanism by which activation of the PXR attenuates TcdA/B-induced damage and inflammation METHODS: We first assessed the ability of the PXR to modulate the production of the neutrophil chemokine CXCL8/IL-8 from human colonic intestinal epithelial cells (IECs;Caco-2), a mediator highly expressed during CDI. Caco-2 IECs were treated for 16 hours with purified C. difficile TcdB (2.5μg/mL) in the presence of PXR agonists at various concentrations (rifampicin, SR12813). In addition to culture supernatants, cell lysates were used to evaluate cell death and toxin function by immunoblot for caspase-3 and glucosylated RAC1, respectively. We then assessed whether PXR activation could attenuate toxin-induced inflammation and tissue damage in vivo. Mice were administered TcdA/B (25μg;intrarectal administration) in the presence of PCN(25mg/kg), a rodent specific PXR agonist. After 4 hours of TcdA/B exposure, colonic tissues were harvested and the expression of IL-17A, CXCL1, CXCL2 and CXCL10 were assessed by qPCR and neutrophil infiltration assessed by flow cytometry. RESULTS: Activation of the PXR with known agonists attenuated C. difficile TcdB-induced CXCL8/IL-8 release in Caco-2 IECs. This effect was not due to alterations in IEC survival, nor due to a direct inhibition of TcdB function by PXR agonists. In our in vivo studies, we found that PXR activation with PCN attenuated TcdA/B induced expression of the pro-inflammatory cytokines IL-17A, CXCL1, CXCL2, CXCL10 and significantly reduced neutrophil infiltration in the colon. CONCLUSIONS: Although there are many emerging treatments for CDI, drug tolerability, disease recurrence, resistance and dosing remain problematic. Taken together, these data will help us expand our knowledge of the PXR’s role in host-pathogen interactions, and its potential to be modulated for the treatment of inflammation and tissue damage in CDI. FUNDING AGENCIES: CCCDr. Keith Sharkey’s CCFC Chair in IBD Research, The Dr. Lloyd Sutherland Investigator in IBD/GI Research, Canadian Foundation for Innovation, Canada Research Chairs Program

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: The ability of the intestinal microbiota to influence drug responses has been recognized, however the mechanisms through which this occurs remain unexplored. Work in germ free mice has demonstrated that colonization with different microbiota influences the expression of cytochrome P450 (CYP) enzymes in the liver. Like the liver, the small intestine (SI) expresses CYP enzymes including CYP3A11 which can metabolize over 60% of commercially available drugs. The activity of CYP enzymes in the SI has been shown to influence circulating levels of orally administered drugs, but how the microbiota affects this process is unknown. AIMS: To investigate if distinct microbiota differentially modulate host drug-metabolism in the SI to influence drug activity and efficacy. METHODS: SFB-free (SFB-) mice were obtained from Jackson (Jax) and SFB+ mice were obtained from Taconic (Tac). Feces from SFB+ Tac mice were mixed in PBS and orally gavaged to mice. 14 days later sections of ileum were used for PCR array or digested in collagenase to isolate lamina propria cells for flow cytometry. A monoclonal antibody for Thy1.2 was used to deplete innate lymphoid cells (ILCs) in RAG1(-/-) mice (lacking T- and B-cells). CYP3A11 activity was determined through the colourmetric breakdown of the substrate 7-benzyloxyresorufin. RESULTS: PCR array analysis of ileal sections revealed CYP3A11 as one of the most downregulated genes in both SFB+ Tac mice and Jax mice colonized with a SFB+ microbiota when compared to SFB- Jax mice. Further analysis showed that colonization of Jax mice with a SFB+ microbiota induced IL-22 production by type 3 innate lymphoid cells (ILC3). Increase IL-22 production positively correlated with fecal levels of SFB and reduced ileal CYP3A11 expression. Colonization of IL-22 KO mice with a SFB+ microbiota had no effect on the ileal expression of CYP3A11. Furthermore, depletion of ILCs in RAG1(-/-) mice colonized with a SFB+ microbiota prevented a decrease in the expression of CYP3A11. In mouse SI enteroid cultures, recombinant IL-22 dose-dependently reduced the expression of CYP3A11, an effect that was blocked by the STAT3 inhibitor Stattic. IL-22 treatment also significantly decreased the ability of SI enteroids to metabolize CYP3A11 specific substrates. CONCLUSIONS: Our data suggest that colonization with a specific microbiota can influence the expression and activity of the drug metabolising enzyme CYP3A11. This occurs through the production of IL-22 by SI ILC3, which down regulated CYP3A11 in a STAT3-specific manor and altered the ability of the small intestine to metabolise CYP3A11 specific substrates. These findings provide an understanding of how the intestinal microbiota modulates host drug metabolism, and how the microbiota can be manipulated to render various drug therapies, such as those for IBD, more effective. FUNDING AGENCIES: CAG, CCC, CIHRAlberta Innovates

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