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.

Association of Circulating Fibrocytes With Fibrostenotic Small Bowel Crohn's Disease.

BACKGROUND: Fibrocytes are hematopoietic cells with features of mesenchymal cells found in the circulation and inflammatory sites implicated in promoting fibrosis in many fibroinflammatory diseases. However, their role(s) in the development of intestinal fibrosis is poorly understood. Here, we investigated a potential role of fibrocytes in the development of fibrosis in Crohn's disease (CD) and sought factors that may impact their development and function. METHODS: Plasma and mononuclear cells were collected from patients with and without fibrostenotic CD. Fibrocytes defined as CD11b+, CD34+, and Collagen 1+ were correlated with clinical assessments of fibrosis, including evaluation using intestinal ultrasound. We measured the levels of relevant circulating molecules via Luminex and studied the effect of patient plasma proteins on fibrocyte differentiation. RESULTS: Fibrocyte numbers were increased in CD patients with stricturing Crohn's disease compared with patients with an inflammatory phenotype (P = .0013), with strong correlation between fibrocyte numbers and acoustic radiation force impulse (ARFI), a measure of bowel elasticity on intestinal ultrasound (R = .8383, P = .0127). Fibrostenotic plasma was a more potent inducer of fibrocyte differentiation in both primary human monocytes and cell line and contained increased levels of cytokines implicated in fibrocyte differentiation compared with plasma from inflammatory patients. Interestingly, increased fibrocyte numbers at time of ultrasound were associated with escalation of medical therapy and endoscopic/surgical management of small bowel strictures at 30 months follow-up. CONCLUSIONS: Circulating fibrocytes strongly correlate with fibrostenotic disease in CD, and they may serve as predictors for escalation of medical +/- surgical therapy.

Intestinal strictures are thought to result from excessive deposition of extracellular matrix by activated mesenchymal cells. In this study, we provide evidence that supports a potential role of fibrocytes (bone marrow­derived mesenchymal precursors) in collagen deposition in Crohn's disease strictures. Inasmuch as fibrocyte numbers correlate with sonographic measures of bowel stiffness, fibrocyte numbers may predict the need for therapy escalation.

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.

Brain TNF drives post-inflammation depression-like behavior and persistent pain in experimental arthritis.

Patients with rheumatoid arthritis experience chronic pain, depression and fatigue, even when inflammation of the joints is well controlled. To study the relationship between arthritis, depression, and sustained pain when articular inflammation is no longer observed, we tested the hypothesis that brain TNF drives post-inflammation depression-like behavior and persistent pain in experimental arthritis. The murine model of antigen-induced arthritis (AIA) was used to evaluate the effects of knee inflammation on sustained pain and depression-like behavior. We measured joint pain using an automated dynamic plantar algesiometer and depression-like behavior with the tail suspension test. Cytokines were measured by Luminex assay and ELISA. TNF in the brain was blocked by intracerebroventricular injection of anti-TNF antibodies. Histological damage and elevated levels of cytokines were observed in the knee 24 h after antigen treatment, but not at 13 days. Reduced pain thresholds were seen 24 h and 13 days after treatment. Depression-like behavior was observed on day 13. Treatment with the antidepressant imipramine reduced both depression-like behavior and persistent pain. However, blocking joint pain with the analgesic dipyrone did not alter depression-like behavior. Elevated levels of TNF, CCL2, and CXCL-1 were observed in the hippocampus 24 h after treatment, with TNF remaining elevated at day 13. Intracerebroventricular infusion of an anti-TNF antibody blocked depression-like behavior and reduced persistent pain. We have demonstrated that depression-like behavior and pain is sustained in AIA mice after the resolution of inflammation. These changes are associated with elevated levels of TNF in the hippocampus and are dependent upon brain TNF. The findings reveal an important mechanistic link between the expression of chronic pain and depression in experimental arthritis. Furthermore, they suggest treating depression in rheumatoid arthritis may positively impact other debilitating features of this condition.

Proteomics and Imaging in Crohn's Disease: TAILS of Unlikely Allies.

Crohn's disease (CD) is a chronic relapsing inflammatory bowel disease (IBD) that may be marked by debilitating symptoms of abdominal pain and obstruction. The etiology and pathogenesis of the disease are not fully understood, and treatment with corticosteroids, biologics, and surgical intervention are the usual therapeutic options. Diagnosis, disease activity, and therapeutic response are currently assessed by endoscopy, cross-sectional imaging, and biomarkers. However, challenges remain regarding the efficacy of the drugs and safety of these imaging techniques. There are also limitations with current clinical and laboratory tools for diagnosis, disease progression, and treatment response. Here, we discuss how the integration of proteomics and activity-based probes, along with intestinal ultrasound, an easily repeatable and well-tolerated diagnostic imaging modality, can address these challenges and may provide a novel precision medicine-based approach for the treatment of CD.

Targeting anti-fibrotic pathways in Crohn's disease - The final frontier?

Intestinal fibrosis with stricture formation affects up to half of patients with Crohn's disease (CD), resulting in impaired quality of life, increased risk of surgical intervention, and associated patient morbidity. The underlying pathophysiologic mechansisms responsible for initiating and perpetuating intestinal fibrosis are complex, dynamic, and implicate both inflammation-dependent and independent pathways. Previously thought to be an irreversible complication of long-standing inflammation unresponsive to medical therapy, fibrostenotic CD has been traditionally managed with endoscopic or surgical approaches. However, recent advances in our understanding of the humoral, cellular, and environmental pathways driving intestinal fibrosis has the potential to fundamentally change these management paradigms for CD-related strictures. Furthermore, the promise of fibrosis treatments in other organ systems has encouraged hope that anti-fibrotic treatment approaches for CD may be within reach. Here, we summarize the key breakthroughs in our molecular understanding of intestinal fibrosis, review current medical, endoscopic, and surgical treatment approaches to CD-related strictures, propose future directions for anti-fibrotic therapy in CD, and identify crucial research questions in this field that require additional investigation.

Minimizing Hyperglycemia-Induced Vascular Endothelial Dysfunction by Inhibiting Endothelial Sodium-Glucose Cotransporter 2 and Attenuating Oxidative Stress: Implications for Treating Individuals With Type 2 Diabetes.

This overview deals with mechanisms whereby hyperglycemia-induced oxidative stress compromises vascular endothelial function and provides a background for a recently published study illustrating the beneficial impact of endothelial sodium-glucose cotransporter 2 (SGLT2) inhibitors in attenuating hyperglycemia-induced vascular dysfunction in vitro. The data provide new insight that can possibly lead to improved drug therapy for people with type 2 diabetes. The working hypotheses that underpinned the experiments performed are provided, along with the findings of the study. For the causes of hyperglycemia-induced vascular endothelial dysfunction, the findings point to the key roles of: 1) functional endothelial SGLT2; 2) oxidative stress-induced signalling pathways including mammalian sarcoma virus kinase, the EGF receptor-kinase and protein kinase C; and 3) mitochondrial dysfunction triggered by hyperglycemia was mitigated by an SGLT2 inhibitor in the hyperglycemic mouse aorta vascular organ cultures. The overview sums up the approaches implicated by the study that can potentially counteract the detrimental impact of hyperglycemia on vascular function in people with diabetes, including the clinical use of SGLT2 inhibitors for those with type 2 diabetes already being treated, for example, with metformin, along with dietary supplementation with broccoli-derived sulforaphane and tetrahydrobiopterin. The caveats associated with the study for extending the findings from mice to humans are summarized, pointing to the need to validate the work using vascular tissues from humans. Suggestions for future clinical studies are made, including the assessment of the impact of the therapeutic strategies proposed on measurements of blood flow in subjects with diabetes.

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.

Antibiotic treatment affects the expression levels of copper transporters and the isotopic composition of copper in the colon of mice.

Copper is a critical enzyme cofactor in the body but also a potent cellular toxin when intracellularly unbound. Thus, there is a delicate balance of intracellular copper, maintained by a series of complex interactions between the metal and specific copper transport and binding proteins. The gastrointestinal (GI) tract is the primary site of copper entry into the body and there has been considerable progress in understanding the intricacies of copper metabolism in this region. The GI tract is also host to diverse bacterial populations, and their role in copper metabolism is not well understood. In this study, we compared the isotopic fractionation of copper in the GI tract of mice with intestinal microbiota significantly depleted by antibiotic treatment to that in mice not receiving such treatment. We demonstrated variability in copper isotopic composition along the length of the gut. A significant difference, ∼1.0‰, in copper isotope abundances was measured in the proximal colon of antibiotic-treated mice. The changes in copper isotopic composition in the colon are accompanied by changes in copper transporters. Both CTR1, a copper importer, and ATP7A, a copper transporter across membranes, were significantly down-regulated in the colon of antibiotic-treated mice. This study demonstrated that isotope abundance measurements of metals can be used as an indicator of changes in metabolic processes in vivo. These measurements revealed a host-microbial interaction in the GI tract involved in the regulation of copper transport.

A Trypsin-Sensitive Proteoglycan from the Tapeworm Hymenolepis diminuta Inhibits Murine Neutrophil Chemotaxis in vitro by Suppressing p38 MAP Kinase Activation.

It has emerged that neutrophils can play important roles in the host response following infection with helminth parasites. Mice infected with the tapeworm, Hymenolepis diminuta, are protected from some inflammatory conditions, accompanied by reduced neutrophil tissue infiltration. Thus, the ability of a phosphate-buffered saline-soluble extract of the worm (H. diminuta extract [HdE]) was tested for (1) its ability to activate murine neutrophils (Ca2+ mobilization, reactive oxygen species (ROS) and cytokine production); and (2) affect neutrophil chemotaxis in vitro to the penta-peptide, WKYMVm, the chemokine, KC, and leukotriene B4. HdE was not cytotoxic to neutrophils, elicited a Ca2+ response and ROS, but not, cytokine (KC, interleukin-10, tumour necrosis factor-α) generation. HdE is not a chemotactic stimulus for murine neutrophils. However, a heat- and trypsin-sensitive, acid-insensitive proteoglycan (sensitive to sodium metaperiodate) in the HdE significantly reduced neutrophil chemotaxis towards WKYMVm or KC, but not LTB4. The latter suggested that the HdE interfered with p38 mitogen-activated protein kinase signalling, which is important in WKYMVm chemotaxis. Corroborating this, immunoblotting revealed reduced phosphorylated p38, and the downstream signal heat-shock protein-27, in protein extracts from HdE + WkYMVm treated cells compared to those exposed to the penta-peptide only. We speculate that HdE can be used to modify the outcome of neutrophilic disease and that purification of the bioactive proteoglycan(s) from the extract could be used as a template to design immunomodulatory drugs targeting neutrophils.

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.

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.

Giardia duodenalis infection reduces granulocyte infiltration in an in vivo model of bacterial toxin-induced colitis and attenuates inflammation in human intestinal tissue.

Giardia duodenalis (syn. G. intestinalis, G. lamblia) is a predominant cause of waterborne diarrheal disease that may lead to post-infectious functional gastrointestinal disorders. Although Giardia-infected individuals could carry as much as 106 trophozoites per centimetre of gut, their intestinal mucosa is devoid of overt signs of inflammation. Recent studies have shown that in endemic countries where bacterial infectious diseases are common, Giardia infections can protect against the development of diarrheal disease and fever. Conversely, separate observations have indicated Giardia infections may enhance the severity of diarrheal disease from a co-infecting pathogen. Polymorphonuclear leukocytes or neutrophils (PMNs) are granulocytic, innate immune cells characteristic of acute intestinal inflammatory responses against bacterial pathogens that contribute to the development of diarrheal disease following recruitment into intestinal tissues. Giardia cathepsin B cysteine proteases have been shown to attenuate PMN chemotaxis towards IL-8/CXCL8, suggesting Giardia targets PMN accumulation. However, the ability of Giardia infections to attenuate PMN accumulation in vivo and how in turn this effect may alter the host inflammatory response in the intestine has yet to be demonstrated. Herein, we report that Giardia infection attenuates granulocyte tissue infiltration induced by intra-rectal instillation of Clostridium difficile toxin A and B in an isolate-dependent manner. This attenuation of granulocyte infiltration into colonic tissues paralled decreased expression of several cytokines associated with the recruitment of PMNs. Giardia trophozoite isolates that attenuated granulocyte infiltration in vivo also decreased protein expression of cytokines released from inflamed mucosal biopsy tissues collected from patients with active Crohn's disease, including several cytokines associated with PMN recruitment. These results demonstrate for the first time that certain Giardia infections may attenuate PMN accumulation by decreasing the expression of the mediators responsible for their recruitment.

Inflammatory bowel disease cause-specific mortality: a primer for clinicians.

BACKGROUND: Inflammatory bowel disease (IBD), including Crohn's disease (CD) and ulcerative colitis (UC) is perceived to harbor significant morbidity but limited excess mortality, thought to be driven by colon cancer, compared with the general population. Recent studies suggest mortality rates seem higher than previously understood, and there are emerging threats to mortality. Clinicians must be up to date and able to clearly convey the causes of mortality to arm individual patients with information to meaningfully participate in decisions regarding IBD treatment and maintenance of health. METHODS: A MEDLINE search was conducted to capture all relevant articles. Keyword search included: "inflammatory bowel disease," "Crohn's disease," "ulcerative colitis," and "mortality." RESULTS: CD and UC have slightly different causes of mortality; however, malignancy and colorectal cancer-associated mortality remains controversial in IBD. CD mortality seems to be driven by gastrointestinal disease, infection, and respiratory diseases. UC mortality was primarily attributable to gastrointestinal disease and infection. Clostridium difficile infection is an emerging cause of mortality in IBD. UC and CD patients have a marked increase in risk of thromboembolic disease. With advances in medical and surgical interventions, the exploration of treatment-associated mortality must continue to be evaluated. CONCLUSIONS: Clinicians should be aware that conventional causes of death such as malignancy do not seem to be as significant a burden as originally perceived. However, emerging threats such as infection including C. difficile are noteworthy. Although CD and UC share similar causes of death, there seems to be some differences in cause-specific mortality.

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.

Effects of nitric oxide and reactive oxygen species on HIF-1α stabilization following clostridium difficile toxin exposure of the Caco-2 epithelial cell line.

BACKGROUND/AIMS: Stabilization of the hypoxia-inducible factor (HIF-1α) is proposed to provide a protective host-response to C. difficile intoxication. Here, we aimed to elucidate whether nitric oxide and/or reactive oxygen species produced during C. difficile toxin exposure could influence HIF-1α stability and initiate protection against epithelial cell damage. METHODS/RESULTS: HIF-1α and inducible nitric oxide synthase (iNOS) proteins were up-regulated whereas factor-inhibiting HIF-1 (FIH-1) protein was down-regulated in Caco-2 epithelial cell monolayers with in vitro toxin exposure. We demonstrate using the biotin-switch assay that the stabilization of HIF-1α protein occurred via iNOS-dependent nitrosylation. Inhibition of iNOS activity by selective inhibitor (1400W) attenuated HIF-1α stabilization and exacerbated toxin-dependent disruptions in Caco-2 monolayer morphology and tight junctional integrity in vitro. Treatment of Caco-2 cell monolayers with N-actylcysteine (NAC), a scavenger of reactive oxygen species (ROS), attenuated toxin-dependent increases in iNOS and HIF-1α protein levels but had no effect on FIH-1 responses. In addition, mice that were exposed to C. difficile toxin in vivo also demonstrated a significant increase in HIF-1α protein and nitrosylation levels. CONCLUSION: Taken together, these data suggest that important synergistic actions exist between nitric oxide and ROS to stabilize HIF-1α and its innate, protective actions in the context of C. difficile toxin-mediated epithelial injury.

Activation of neuronal P2X7 receptor-pannexin-1 mediates death of enteric neurons during colitis.

Inflammatory bowel diseases (IBDs) are chronic relapsing and remitting conditions associated with long-term gut dysfunction resulting from alterations to the enteric nervous system and a loss of enteric neurons. The mechanisms underlying inflammation-induced enteric neuron death are unknown. Here using in vivo models of experimental colitis we report that inflammation causes enteric neuron death by activating a neuronal signaling complex composed of P2X7 receptors (P2X7Rs), pannexin-1 (Panx1) channels, the Asc adaptor protein and caspases. Inhibition of P2X7R, Panx1, Asc or caspase activity prevented inflammation-induced neuron cell death. Preservation of enteric neurons by inhibiting Panx1 in vivo prevented the onset of inflammation-induced colonic motor dysfunction. Panx1 expression was reduced in Crohn's disease but not ulcerative colitis. We conclude that activation of neuronal Panx1 underlies neuron death and the subsequent development of abnormal gut motility in IBD. Targeting Panx1 represents a new neuroprotective strategy to ameliorate the progression of IBD-associated dysmotility.