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.

Xenobiotic receptors and the regulation of intestinal homeostasis: harnessing the chemical output of the intestinal microbiota.

The commensal bacteria that reside in the gastrointestinal tract exist in a symbiotic relationship with the host, driving the development of the immune system and maintaining metabolic and tissue homeostasis in the local environment. The intestinal microbiota has the capacity to generate a wide array of chemical metabolites to which the cells of the intestinal mucosa are exposed. Host cells express xenobiotic receptors, such as the aryl hydrocarbon receptor (AhR) and the pregnane X receptor (PXR), that can sense and respond to chemicals that are generated by nonhost pathways. In this review, we outline the physiological and immunological processes within the intestinal environment that are regulated by microbial metabolites through the activation of the AhR and the PXR, with a focus on ligands generated by the stepwise catabolism of tryptophan.

Recruitment of α4ß7 monocytes and neutrophils to the brain in experimental colitis is associated with elevated cytokines and anxiety-like behavior.

BACKGROUND: Behavioral comorbidities, such as anxiety and depression, are a prominent feature of IBD. The signals from the inflamed gut that cause changes in the brain leading to these behavioral comorbidities remain to be fully elucidated. We tested the hypothesis that enhanced leukocyte-cerebral endothelial cell interactions occur in the brain in experimental colitis, mediated by α4ß7 integrin, to initiate neuroimmune activation and anxiety-like behavior. METHODS: Female mice treated with dextran sodium sulfate were studied at the peak of acute colitis. Circulating leukocyte populations were determined using flow cytometry. Leukocyte-cerebral endothelial cell interactions were examined using intravital microscopy in mice treated with anti-integrin antibodies. Brain cytokine and chemokines were assessed using a multiplex assay in animals treated with anti-α4ß7 integrin. Anxiety-like behavior was assessed using an elevated plus maze in animals after treatment with an intracerebroventricular injection of interleukin 1 receptor antagonist. RESULTS: The proportion of classical monocytes expressing α4ß7 integrin was increased in peripheral blood of mice with colitis. An increase in the number of rolling and adherent leukocytes on cerebral endothelial cells was observed, the majority of which were neutrophils. Treatment with anti-α4ß7 integrin significantly reduced the number of rolling leukocytes. After anti-Ly6C treatment to deplete monocytes, the number of rolling and adhering neutrophils was significantly reduced in mice with colitis. Interleukin-1ß and CCL2 levels were elevated in the brain and treatment with anti-α4ß7 significantly reduced them. Enhanced anxiety-like behavior in mice with colitis was reversed by treatment with interleukin 1 receptor antagonist. CONCLUSIONS: In experimental colitis, α4ß7 integrin-expressing monocytes direct the recruitment of neutrophils to the cerebral vasculature, leading to elevated cytokine levels. Increased interleukin-1ß mediates anxiety-like behavior.

Colitis-associated microbiota drives changes in behaviour in male mice in the absence of inflammation.

Inflammatory bowel diseases (IBD) are chronic inflammatory conditions of the gastrointestinal tract. IBD are associated with a high prevalence of cognitive, behavioural and emotional comorbidities, including anxiety and depression. The link between IBD and the development of behavioural comorbidities is poorly understood. As the intestinal microbiota profoundly influences host behaviour, we sought to determine whether the altered gut microbiota associated with intestinal inflammation contributes to the development of behavioural abnormalities. Using the dextran sulphate sodium (DSS) model of colitis, we characterized intestinal inflammation, behaviour (elevated plus maze and tail suspension test) and the composition of the microbiota in male mice. Cecal contents from colitic mice were transferred into germ-free (GF) or antibiotic (Abx)-treated mice, and behaviour was characterized in recipient mice. Gene expression was measured using qPCR. DSS colitis was characterized by a significant reduction in body weight and an increase in colonic inflammatory markers. These changes were accompanied by increased anxiety-like behaviour, an altered gut microbiota composition, and increased central Tnf expression. Transfer of the cecal matter from colitic mice induced similar behavioural changes in both GF and Abx-treated recipient mice, with no signs of colonic or neuroinflammation. Upon characterization of the microbiota in donor and recipient mice, specific taxa were found to be associated with behavioural changes, notably members of the Lachnospiraceae family. Behavioural abnormalities associated with intestinal inflammation are transmissible via transfer of cecal matter, suggesting that alterations in the composition of the gut microbiota play a key role in driving behavioural changes in colitis.

New Concepts of the Interplay Between the Gut Microbiota and the Enteric Nervous System in the Control of Motility.

Propulsive gastrointestinal (GI) motility is critical for digestive physiology and host defense. GI motility is finely regulated by the intramural reflex pathways of the enteric nervous system (ENS). The ENS is in turn regulated by luminal factors: diet and the gut microbiota. The gut microbiota is a vast ecosystem of commensal bacteria, fungi, viruses, and other microbes. The gut microbiota not only regulates the motor programs of the ENS but also is critical for the normal structure and function of the ENS. In this chapter, we highlight recent research that has shed light on the microbial mechanisms of interaction with the ENS involved in the control of motility. Toll-like receptor signaling mechanisms have been shown to maintain the structural integrity of the ENS and the neurochemical phenotypes of enteric neurons, in part through the production of trophic factors including glia-derived neurotrophic factor. Microbiota-derived short-chain fatty acids and/or single-stranded RNA regulates the synthesis of serotonin in enterochromaffin cells, which are involved in the initiation of enteric reflexes, among other functions. Further evidence suggests a crucial role for microbial modulation of serotonin in maintaining the integrity of the ENS through enteric neurogenesis. Understanding the microbial pathways of enteric neural control sheds new light on digestive health and provides novel treatment strategies for GI motility disorders.

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.

Metformin Prevents Hyperglycemia-Associated, Oxidative Stress-Induced Vascular Endothelial Dysfunction: Essential Role for the Orphan Nuclear Receptor Human Nuclear Receptor 4A1 (Nur77).

Vascular pathology is increased in diabetes because of reactive-oxygen-species (ROS)-induced endothelial cell damage. We found that in vitro and in a streptozotocin diabetes model in vivo, metformin at diabetes-therapeutic concentrations (1-50 µM) protects tissue-intact and cultured vascular endothelial cells from hyperglycemia/ROS-induced dysfunction typified by reduced agonist-stimulated endothelium-dependent, nitric oxide-mediated vasorelaxation in response to muscarinic or proteinase-activated-receptor 2 agonists. Metformin not only attenuated hyperglycemia-induced ROS production in aorta-derived endothelial cell cultures but also prevented hyperglycemia-induced endothelial mitochondrial dysfunction (reduced oxygen consumption rate). These endothelium-protective effects of metformin were absent in orphan-nuclear-receptor Nr4a1-null murine aorta tissues in accord with our observing a direct metformin-Nr4a1 interaction. Using in silico modeling of metformin-NR4A1 interactions, Nr4a1-mutagenesis, and a transfected human embryonic kidney 293T cell functional assay for metformin-activated Nr4a1, we identified two Nr4a1 prolines, P505/P549 (mouse sequences corresponding to human P501/P546), as key residues for enabling metformin to affect mitochondrial function. Our data indicate a critical role for Nr4a1 in metformin's endothelial-protective effects observed at micromolar concentrations, which activate AMPKinase but do not affect mitochondrial complex-I or complex-III oxygen consumption rates, as does 0.5 mM metformin. Thus, therapeutic metformin concentrations requiring the expression of Nr4a1 protect the vasculature from hyperglycemia-induced dysfunction in addition to metformin's action to enhance insulin action in patients with diabetes. SIGNIFICANCE STATEMENT: Metformin improves diabetic vasodilator function, having cardioprotective effects beyond glycemic control, but its mechanism to do so is unknown. We found that metformin at therapeutic concentrations (1-50µM) prevents hyperglycemia-induced endothelial dysfunction by attenuating reactive oxygen species-induced damage, whereas high metformin (>250 µM) impairs vascular function. However, metformin's action requires the expression of the orphan nuclear receptor NR4A1/Nur77. Our data reveal a novel mechanism whereby metformin preserves diabetic vascular endothelial function, with implications for developing new metformin-related therapeutic agents.

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.

Behavioural adaptations after antibiotic treatment in male mice are reversed by activation of the aryl hydrocarbon receptor.

The intestinal microbiota plays an important role in regulating brain functions and behaviour. Microbiota-dependent changes in host physiology have been suggested to be key contributors to psychiatric conditions. However, specific host pathways modulated by the microbiota involved in behavioural control are lacking. Here, we assessed the role of the aryl hydrocarbon receptor (Ahr) in modulating microbiota-related alterations in behaviour in male and female mice after antibiotic (Abx) treatment. Mice of both sexes were treated with Abx to induce bacterial depletion. Mice were then tested in a battery of behavioural tests, including the elevated plus maze and open field tests (anxiety-like behaviour), 3 chamber test (social preference), and the tail suspension and forced swim tests (despair behaviour). Behavioural measurements in the tail suspension test were also performed after microbiota reconstitution and after administration of an Ahr agonist, ß-naphthoflavone. Gene expression analyses were performed in the brain, liver, and colon by qPCR. Abx-induced bacterial depletion did not alter anxiety-like behaviour, locomotion, or social preference in either sex. A sex-dependent effect was observed in despair behaviour. Male mice had a reduction in despair behaviour after Abx treatment in both the tail suspension and forced swim tests. A similar alteration in despair behaviour was observed in Ahr knockout mice. Despair behaviour was normalized by either microbiota recolonization or Ahr activation in Abx-treated mice. Ahr activation by ß-naphthoflavone was confirmed by increased expression of the Ahr-target genes Cyp1a1, Cyp1b1, and Ahrr. Our results demonstrate a role for Ahr in mediating the behaviours that are regulated by the crosstalk between the intestinal microbiota and the host. Ahr represents a novel potential modulator of behavioural conditions influenced by the intestinal microbiota.

Gut microbiota profiles and fecal beta-glucuronidase activity in kidney transplant recipients with and without post-transplant diarrhea.

Post-transplant diarrhea is a common complication after solid organ transplantation and is frequently attributed to the widely prescribed immunosuppressant mycophenolate mofetil (MMF). Given recent work identifying the relationship between MMF toxicity and gut bacterial ß-glucuronidase activity, we evaluated the relationship between gut microbiota composition, fecal ß-glucuronidase activity, and post-transplant diarrhea. We recruited 97 kidney transplant recipients and profiled the gut microbiota in 273 fecal specimens using 16S rRNA gene sequencing. We further characterized fecal ß-glucuronidase activity in a subset of this cohort. Kidney transplant recipients with post-transplant diarrhea had decreased gut microbial diversity and decreased relative gut abundances of 12 genera when compared to those without post-transplant diarrhea (adjusted p value < .15, Wilcoxon rank sum test). Among the kidney transplant recipients with post-transplant diarrhea, those with higher fecal ß-glucuronidase activity had a more prolonged course of diarrhea (≥7 days) compared to patients with lower fecal ß-glucuronidase activity (91% vs 40%, p = .02, Fisher's exact test). Our data reveal post-transplant diarrhea as a complex phenomenon with decreased gut microbial diversity and commensal gut organisms. This study further links commensal bacterial metabolism with an important clinical outcome measure, suggesting fecal ß-glucuronidase activity could be a novel biomarker for gastrointestinal-related MMF toxicity.

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.

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.

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.

Exaggerated IL-15 and Altered Expression of foxp3+ Cell-Derived Cytokines Contribute to Enhanced Colitis in Nlrp3-/- Mice.

The pathogenesis of Crohn's disease (CD) involves defects in the innate immune system, impairing responses to microbes. Studies have revealed that mutations NLRP3 are associated with CD. We reported previously that Nlrp3-/- mice were more susceptible to colitis and exhibited reduced colonic IL-10 expression. In the current study, we sought to determine how the loss of NLRP3 might be altering the function of regulatory T cells, a major source of IL-10. Colitis was induced in wild-type (WT) and Nlrp3-/- mice by treatment with dextran sulphate sodium (DSS). Lamina propria (LP) cells were assessed by flow cytometry and cytokine expression was assessed. DSS-treated Nlrp3-/- mice exhibited increased numbers of colonic foxp3+ T cells that expressed significantly lower levels of IL-10 but increased IL-17. This was associated with increased expression of colonic IL-15 and increased surface expression of IL-15 on LP dendritic cells. Neutralizing IL-15 in Nlrp3-/- mice attenuated the severity of colitis, decreased the number of colonic foxp3+ cells, and reduced the colonic expression of IL-12p40 and IL-17. These data suggest that the NLRP3 inflammasome can regulate intestinal inflammation through noncanonical mechanisms, providing additional insight as to how NLRP3 variants may contribute to the pathogenesis of CD.

Challenges in IBD Research 2024: Preclinical Human IBD Mechanisms.

Preclinical human inflammatory bowel disease (IBD) mechanisms is one of 5 focus areas of the Challenges in IBD Research 2024 document, which also includes environmental triggers, novel technologies, precision medicine, and pragmatic clinical research. Herein, we provide a comprehensive overview of current gaps in inflammatory bowel diseases research that relate to preclinical research and deliver actionable approaches to address them with a focus on how these gaps can lead to advancements in IBD interception, remission, and restoration. The document is the result of multidisciplinary input from scientists, clinicians, patients, and funders and represents a valuable resource for patient-centric research prioritization. This preclinical human IBD mechanisms section identifies major research gaps whose investigation will elucidate pathways and mechanisms that can be targeted to address unmet medical needs in IBD. Research gaps were identified in the following areas: genetics, risk alleles, and epigenetics; the microbiome; cell states and interactions; barrier function; IBD complications (specifically fibrosis and stricturing); and extraintestinal manifestations. To address these gaps, we share specific opportunities for investigation for basic and translational scientists and identify priority actions.

To address the unmet medical needs of patients with inflammatory bowel diseases (IBD) and move toward cures, preclinical human-relevant research must center on mechanistic questions pertinent to patients with IBD in the 3 areas of disease interception, remission, and restoration.

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.

The Nlrp3 inflammasome promotes myocardial dysfunction in structural cardiomyopathy through interleukin-1ß.

Heart failure is associated with a low-grade and chronic cardiac inflammation that impairs function; however, the mechanisms by which this sterile inflammation occurs in structural heart disease remain poorly defined. Cardiac-specific heterozygous overexpression of the calcineurin transgene (CNTg) in mice results in cardiac hypertrophy, inflammation, apoptosis and ventricular dilatation. We hypothesized that activation of the Nlrp3 inflammasome, an intracellular danger-sensing pathway required for processing the pro-inflammatory cytokine interleukin-1ß (IL-1ß), may contribute to myocardial dysfunction and disease progression. Here we report that Nlrp3 mRNA was increased in CNTg mice compared with wild-type. Consistent with inflammasome activation, CNTg animals had increased conversion of pro-caspase-1 to cleaved and activated forms, as well as markedly increased serum IL-1ß. Blockade of IL-1ß signalling via chronic IL-1 receptor antagonist therapy reduced cardiac inflammation and myocyte pathology in CNTg mice, resulting in improved systolic performance. Furthermore, genetic ablation of Nlrp3 in CNTg mice reduced pro-inflammatory cytokine maturation and cardiac inflammation, as well as improving systolic performance. These findings indicate that activation of the Nlrp3 inflammasome in CNTg mice promotes myocardial inflammation and systolic dysfunction through the production of pro-inflammatory IL-1ß. Blockade of IL-1ß signalling with the IL-1 receptor antagonist reverses these phenotypes and offers a possible therapeutic approach in the management of heart failure.

The airway epithelium nucleotide-binding domain and leucine-rich repeat protein 3 inflammasome is activated by urban particulate matter.

BACKGROUND: The airway epithelium is the first line of defense against inhaled insults and therefore must be capable of coordinating appropriate inflammatory and immune responses. OBJECTIVE: We sought to test the hypothesis that the nucleotide-binding domain and leucine-rich repeat protein 3 (NLRP3) inflammasome, an intracellular danger-sensing complex, plays a critical role in airway epithelium-mediated immune responses to urban particulate matter (PM) exposure. METHODS: In this study we (1) identified NLRP3 and caspase-1 expression in human airway epithelium bronchus and primary cells, (2) characterized NLRP3 inflammasome-mediated IL-1ß production from human airway epithelium in response to PM, and (3) performed in vivo PM exposure experiments with wild-type and Nlrp3(-/-) mice. RESULTS: Our results demonstrate that human airway epithelium contains a functional NLRP3 inflammasome that responds to PM exposure with caspase-1 cleavage and production of IL-1ß. Exposure of Nlrp3(-/-) and wild-type mice to PM in vivo demonstrates NLRP3-dependent production of IL-1ß in the lung, airway neutrophilia, and increases in CD11c(+hi)/MHC class II(+hi) cell numbers in intrathoracic lymph nodes. CONCLUSION: Our study is the first to characterize airway epithelial NLRP3 inflammasome-mediated immune responses to PM exposure, which might have implications in patients with asthma and other lung diseases.