The Pregnane X Receptor and Indole-3-Propionic Acid Shape the Intestinal Mesenchyme to Restrain Inflammation and Fibrosis.

BACKGROUND & AIMS: Fibrosis is a common complication of inflammatory bowel diseases (IBDs). The pregnane X receptor (PXR) (encoded by NR1I2) suppresses intestinal inflammation and has been shown to influence liver fibrosis. In the intestine, PXR signaling is influenced by microbiota-derived indole-3-propionic acid (IPA). Here, we sought to assess the role of the PXR in regulating intestinal inflammation and fibrosis. METHODS: Intestinal inflammation was induced using dextran sulfate sodium (DSS). Fibrosis was assessed in wild-type (WT), Nr1i2-/-, epithelial-specific Nr1i2-/-, and fibroblast-specific Nr1i2-/- mice. Immune cell influx was quantified by flow cytometry and cytokines by Luminex. Myofibroblasts isolated from WT and Nr1i2-/- mice were stimulated with cytomix or lipopolysaccharide, and mediator production was assessed by quantitative polymerase chain reaction and Luminex. RESULTS: After recovery from DSS-induced colitis, WT mice exhibited fibrosis, a response that was exacerbated in Nr1i2-/- mice. This was correlated with greater neutrophil infiltration and innate cytokine production. Deletion of the PXR in fibroblasts, but not the epithelium, recapitulated this phenotype. Inflammation and fibrosis were reduced by IPA administration, whereas depletion of the microbiota exaggerated intestinal fibrosis. Nr1i2-deficient myofibroblasts were hyperresponsive to stimulation, producing increased levels of inflammatory mediators compared with WT cells. In biopsies from patients with active Crohn's disease (CD) and ulcerative colitis (UC), expression of NR1I2 was reduced, correlating with increased expression of fibrotic and innate immune genes. Finally, both CD and UC patients exhibited reduced levels of fecal IPA. CONCLUSIONS: These data highlight a role for IPA and its interactions with the PXR in regulating the mesenchyme and the development of inflammation and fibrosis, suggesting microbiota metabolites may be a vital determinant in the progression of fibrotic complications in IBD.

NR4A1 modulates intestinal smooth muscle cell phenotype and dampens inflammation-associated intestinal remodeling.

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+/+ and Nr4a1-/- littermates were subjected to shotgun proteomic analysis, proliferation, and bioenergetic assays. Proliferation was assessed in the presence and absence of NR4A1 agonists, cytosporone-B (Csn-B) and 6-mercaptopurine (6-MP). In vivo, we compared colonic smooth muscle thickening in Nr4a1+/+ and Nr4a1-/- mice using the chronic dextran sulfate sodium (DSS) model of colitis. Second, SAMP1/YitFc mice (a model of spontaneous ileitis) were treated with Csn-B and small intestinal smooth muscle thickening was assessed. SMCs isolated from Nr4a1-/- mice exhibited increased abundance of proteins related to cell proliferation, metabolism, and ECM production, whereas Nr4a1+/+ SMCs highly expressed proteins related to the regulation of the actin cytoskeleton and contractile processes. SMCs isolated from Nr4a1-/- mice exhibited increased proliferation and alterations in cellular metabolism, whereas activation of NR4A1 attenuated proliferation. In vivo, Nr4a1-/- mice exhibited increased colonic smooth muscle thickness following repeated cycles of DSS. Activating NR4A1 with Csn-B, in the context of established inflammation, reduced ileal smooth muscle thickening in SAMP1/YitFc mice. Targeting NR4A1 may provide a novel approach to regulate intestinal SMC phenotype, limiting excessive proliferation that contributes to stricture development in CD.

Nr4A1 modulates inflammation-associated intestinal fibrosis and dampens fibrogenic signaling in myofibroblasts.

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 (Nr4a1-/-) and their wild-type littermates (Nr4a1+/+). Lastly, intestinal myofibroblasts isolated from Nr4a1-/- and Nr4a1+/+ mice or primary human intestinal myofibroblasts were stimulated with transforming growth factor-ß1 (TGF-ß1), in the presence or absence of Csn-B or 6-MP, and proliferation and ECM gene expression assessed. Csn-B or 6-MP treatment significantly reduced ileal thickness, collagen, and overall ECM content in SAMP1/YitFc mice. This was associated with a reduction in proliferative markers within the mesenchymal compartment. Nr4a1-/- mice exposed to DSS exhibited increased colonic thickening and ECM content. Nr4a1-/- myofibroblasts displayed enhanced TGF-ß1-induced proliferation. Furthermore, Csn-B or 6-MP treatment was antiproliferative in Nr4a1+/+ but not Nr4a1-/- cells. Lastly, activating NR4A1 in human myofibroblasts reduced TGF-ß1-induced collagen deposition and fibrosis-related gene expression. Our data suggest that NR4A1 can attenuate fibrotic processes in intestinal myofibroblasts and could provide a valuable clinical target to treat inflammation-associated intestinal fibrosis.NEW & NOTEWORTHY Fibrosis and increased muscle thickening contribute to stricture formation and intestinal obstruction, a complication that occurs in 30%-50% of patients with CD within 10 yr of disease onset. More than 50% of those who undergo surgery to remove the obstructed bowel will experience stricture recurrence. To date, there are no drug-based approaches approved to treat intestinal strictures. In the current submission, we identify NR4A1 as a novel target to treat inflammation-associated intestinal fibrosis.

The xenobiotic sensing pregnane X receptor regulates tissue damage and inflammation triggered by C difficile toxins.

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 pregnane X receptor and its microbiota-derived ligand indole 3-propionic acid regulate endothelium-dependent vasodilation.

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.

Pregnane X Receptor Activation Triggers Rapid ATP Release in Primed Macrophages That Mediates NLRP3 Inflammasome Activation.

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-1ß 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.

Effects of Azithromycin on Behavior, Pathologic Signs, and Changes in Cytokines, Chemokines, and Neutrophil Migration in C57BL/6 Mice Exposed to Dextran Sulfate Sodium.

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.

An intact microbiota is required for the gastrointestinal toxicity of the immunosuppressant mycophenolate mofetil.

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.

Hyperglycaemic impairment of PAR2-mediated vasodilation: Prevention by inhibition of aortic endothelial sodium-glucose-co-Transporter-2 and minimizing oxidative stress.

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.

A simple, cost-effective method for generating murine colonic 3D enteroids and 2D monolayers for studies of primary epithelial cell function.

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.

Constitutive androstane receptor regulates the intestinal mucosal response to injury.

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.

Pregnane X Receptor Activation Attenuates Inflammation-Associated Intestinal Epithelial Barrier Dysfunction by Inhibiting Cytokine-Induced Myosin Light-Chain Kinase Expression and c-Jun N-Terminal Kinase 1/2 Activation.

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.

Pregnane X receptor agonists enhance intestinal epithelial wound healing and repair of the intestinal barrier following the induction of experimental colitis.

The intestinal epithelial barrier plays a key role in the maintenance of homeostasis within the gastrointestinal tract. Barrier dysfunction leading to increased epithelial permeability is associated with a number of gastrointestinal disorders including the inflammatory bowel diseases (IBD) - Crohn's disease and ulcerative colitis. It is thought that the increased permeability in patients with IBD may be driven by alterations in the epithelial wound healing response. To this end considerable study has been undertaken to identify signaling pathways that may accelerate intestinal epithelial wound healing and normalize the barrier dysfunction observed in IBD. In the current study we examined the role of the pregnane X receptor (PXR) in modulating the intestinal epithelial wound healing response. Mutations and reduced mucosal expression of the PXR are associated with IBD, and others have reported that PXR agonists can dampen intestinal inflammation. Furthermore, stimulation of the PXR has been associated with increased cell migration and proliferation, two of the key processes involved in wound healing. We hypothesized that PXR agonists would enhance intestinal epithelial repair. Stimulation of Caco-2 intestinal epithelial cells with rifaximin, rifampicin and SR12813, all potent agonists of the PXR, significantly increased wound closure. This effect was driven by p38 MAP kinase-dependent cell migration, and occurred in the absence of cell proliferation. Treating mice with a rodent specific PXR agonist, pregnenolone 16α-carbonitrile (PCN), attenuated the intestinal barrier dysfunction observed in the dextran sulphate sodium (DSS) model of experimental colitis, an effect that occurred independent of the known anti-inflammatory effects of PCN. Taken together our data indicate that the activation of the PXR can enhance intestinal epithelial repair and suggest that targeting the PXR may help to normalize intestinal barrier dysfunction observed in patients with IBD. Furthermore, our data provide additional insight into the potential mechanisms through which rifaximin elicits its clinical efficacy in the treatment of IBD.

The P2Y6 receptor mediates Clostridium difficile toxin-induced CXCL8/IL-8 production and intestinal epithelial barrier dysfunction.

C. difficile is a Gram-positive spore-forming anaerobic bacterium that is the leading cause of nosocomial diarrhea in the developed world. The pathogenesis of C. difficile infections (CDI) is driven by toxin A (TcdA) and toxin B (TcdB), secreted factors that trigger the release of inflammatory mediators and contribute to disruption of the intestinal epithelial barrier. Neutrophils play a key role in the inflammatory response and the induction of pseudomembranous colitis in CDI. TcdA and TcdB alter cytoskeletal signaling and trigger the release of CXCL8/IL-8, a potent neutrophil chemoattractant, from intestinal epithelial cells; however, little is known about the surface receptor(s) that mediate these events. In the current study, we sought to assess whether toxin-induced CXCL8/IL-8 release and barrier dysfunction are driven by the activation of the P2Y6 receptor following the release of UDP, a danger signal, from intoxicated Caco-2 cells. Caco-2 cells express a functional P2Y6 receptor and release measurable amounts of UDP upon exposure to TcdA/B. Toxin-induced CXCL8/IL-8 production and release were attenuated in the presence of a selective P2Y6 inhibitor (MRS2578). This was associated with inhibition of TcdA/B-induced activation of NFκB. Blockade of the P2Y6 receptor also attenuated toxin-induced barrier dysfunction in polarized Caco-2 cells. Lastly, pretreating mice with the P2Y6 receptor antagonists (MSR2578) attenuated TcdA/B-induced inflammation and intestinal permeability in an intrarectal toxin exposure model. Taken together these data outline a novel role for the P2Y6 receptor in the induction of CXCL8/IL-8 production and barrier dysfunction in response to C. difficile toxin exposure and may provide a new therapeutic target for the treatment of CDI.