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Background & Aims Cathelicidin (encoded by Camp) is an anti-microbial peptide in the innate immune system. We examined whether macrophages express cathelicidin in colons of mice with experimental colitis and patients with inflammatory bowel disease; we investigated its signaling mechanisms. Methods Quantitative, real-time, reverse transcription PCR, bacterial 16S PCR, immunofluorescence, and small interfering (si)RNA analyses were performed. Colitis was induced in mice using sodium dextran sulfate (DSS); levels of cathelicidin were measured in human primary monocytes. Results Expression of cathelicidin increased in the inflamed colonic mucosa of mice with DSS-induced colitis, compared with controls. Cathelicidin expression localized to mucosal macrophages in inflamed colon tissues of patients and mice. Exposure of human primary monocytes to E coli DNA induced expression of Camp mRNA, which required signaling by ERK; expression was reduced by siRNAs against toll-like receptor (TLR)9 and MyD88. Intracolonic administration of bacterial DNA to wild-type mice induced expression of cathelicidin in colons of control mice and mice with DSS-induced colitis. Colon expression of cathelicidin was significantly reduced in TLR9 −/− mice with DSS-induced colitis. Compared with wild-type mice, Camp −/− mice developed a more severe form of DSS-induced colitis, particularly after intracolonic administration of E coli DNA. Expression of cathelicidin from bone marrow-derived immune cells regulated DSS induction of colitis in transplantation studies in mice. Conclusions Cathelicidin protects against colitis induction in mice. Increased expression of cathelicidin in monocytes and experimental models of colitis involves activation of TLR9–ERK signaling by bacterial DNA. This pathway might be involved in pathogenesis of ulcerative colitis.

Background and Aims Neurotensin (NT) promotes colon cancer and inflammation via NT receptor-1 (NTR1). MicroRNAs regulate protein synthesis by targeting mRNAs. We determined the microRNA signature of NTR1 stimulation on human colonic (NCM460) epithelial cells. Methods RNA from NT-stimulated NCM460 cells overexpressing NTR1 was used for microarray expression analysis. NF-κB binding sites were identified by sequence homology, ChIP-assay and qPCR. Tumorigenesis was assessed by the soft agar assay and HCT-116 tumor xenografts in SCID mice. Down-stream targets of NT-regulated microRNAs were identified via bioinformatics, real time PCR and Western blot. Results NT stimulated differential expression of 38 microRNAs. We identified NF-κB binding sites on miR-21 and miR-155, previously implicated in tumor growth. NT increased the number of colonies of HCT-116 cells and antisense-microRNAs against miR-21 and/or miR-155 inhibited this response (p<0.001). NT administration (i.p.) increased the rate of tumor growth in xenograft tumors while miR-21 and/or miR-155 antisense attenuated this response. Since potential downstream targets of miR-21 and miR-155 are PTEN and SOCS1, respectively, and both are upstream of Akt, we investigated the effect of NT on Akt activation. NT activated Akt in HCT-116 cells, an effect inhibited by miR-21 and/or miR-155 antisense (p<0.001). We report for the first time PPP2CA phosphatase as a miR-155 target exerting the effects of NT on Akt. Conclusions NT stimulates miR-21 and miR-155 expression in colonocytes via Akt and NF-κB, and both microRNAs mediate colon tumor growth in response to NT. Importantly, this NT-microRNA circuit is found perturbated in human colon cancers and correlates with tumor stage, suggesting its relevance to human colon carcinogenesis.

Creeping fat has long been recognized as an indicator of Crohn’s disease activity. Although most patients with Crohn’s Disease (CD) have normal or low BMI, the ratio of intra-abdominal fat to total abdominal fat is far greater than that of controls. The obesity epidemic has instructed us on the inflammatory nature of hypertrophic adipose tissue and similarities between mesenteric depots in obese and CD patients can be drawn. However, several important physiological differences exist between these two depots as well. While the molecular basis of the cross-talk between mesenteric adipose and the inflamed intestine in CD is largely unknown, novel evidence implicate neuropeptides along with adipocyte-derived paracrine mediators (adipokines) as potential targets for future investigations and highlight adipose tissue physiology as a potential important determinant in the course of IBD.

ObjectiveNeurotensin (NT) mediates colonic inflammation through its receptor neurotensin receptor 1 (NTR1). NT stimulates miR-133α expression in colonic epithelial cells. We investigated the role of miR-133α in NT-associated colonic inflammation in vitro and in vivo.DesignmiR-133α and aftiphilin (AFTPH) levels were measured by quantitative PCR. Antisense (as)-miR-133α was administrated intracolonicaly prior to induction of 2, 4, 6-trinitrobenzene sulfonic acid (TNBS)-induced colitis and dextran sodium sulfate (DSS)-induced colitis. The effect of AFTPH was examined by gene silencing in vitro.ResultsNT increased miR-133α levels in NCM-460 overexpressing NTR1 (NCM460-NTR1) and HCT-116 cells. NT-induced p38, ERK1/2, c-Jun, and NF-κB activation, as well as IL-6, IL-8 and IL-1β messenger RNA (mRNA) expression in NCM-460-NTR1 cells were reduced in miR-133α-silenced cells, while overexpression of miR-133α reversed these effects. MiR-133α levels were increased in TNBS (2 day) and DSS (5 day) colitis, while NTR1 deficient DSS-exposed mice had reduced miR-133α levels, compared to wild-type colitic mice. Intracolonic as-miR-133α attenuated several parameters of colitis as well expression of proinflammatory mediators in the colonic mucosa. In silico search coupled with qPCR identified AFTPH as a downstream target of miR-133α, while NT decreased AFTPH expression in NCM-460-NTR1 colonocytes. Gene silencing of AFTPH enhanced NT-induced proinflammatory responses and AFTPH levels were downregulated in experimental colitis. Levels of miR-133α were significantly upregulated, while AFTPH levels were downregulated in colonic biopsies of patients with ulcerative colitis compared to controls.ConclusionsNT-associated colitis and inflammatory signalling are regulated by miR-133α-AFTPH interactions. Targeting of miR-133α or AFTPH may represent a novel therapeutic approach in inflammatory bowel disease.

Clostridium difficile infection (CDI) is mediated by two major exotoxins, toxin A (TcdA) and toxin B (TcdB), which damage the colonic epithelial barrier and induce inflammatory responses. The function of the colonic vascular barrier during CDI has not been studied. Here we report increased colonic vascular permeability in CDI mice and elevated vascular endothelial growth factor A (VEGF-A) which was induced by infection with a TcdA and/or TcdB-producing strain in vivo but not with a TcdA−TcdB− isogenic mutant. TcdA or TcdB also induced VEGF-A in human colonic mucosal biopsies. Hypoxia-inducible factor (HIF) signaling appeared to mediate toxin-induced VEGF production in colonocytes, which can further stimulate human intestinal microvascular endothelial cells. Neutralization of VEGF-A and inhibition of its signaling pathway each attenuated CDI in vivo. Compared to healthy controls, CDI patients had significantly higher serum VEGF-A, which subsequently decreased after treatment. Our findings indicate critical roles for toxin-induced VEGF-A and colonic vascular permeability in CDI pathogenesis. It may also implicate the pathophysiological significance of gut vascular barrier in response to virulence factors of enteric pathogens. As an alternative to pathogen-targeted therapy, this study may enable new host-directed therapeutic approach for severe, refractory CDI.

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Peripheral administration of a specific neurokinin-1 receptor (NK-1R) antagonist to mice leads to reduced weight gain and circulating levels of insulin and leptin after high-fat diet (HFD). Here, we assessed the contribution of substance P (SP) and NK-1R in diet-induced obesity using NK-1R deficient [knockout (KO)] mice and extended our previous findings to show the effects of SP-NK-1R interactions on adipose tissue-associated insulin signaling and glucose metabolic responses. NK-1R KO and wild-type (WT) littermates were fed a HFD for 3 wk, and obesity-associated responses were determined. Compared with WT, NK-1 KO mice show reduced weight gain and circulating levels of leptin and insulin in response to HFD. Adiponectin receptor mRNA levels are higher in mesenteric fat and liver in NK-1 KO animals compared with WT, after HFD. Mesenteric fat from NK-1R KO mice fed with HFD has reduced stress-activated protein kinase/c-Jun N-terminal kinase and protein kinase C activation compared with WT mice. After glucose challenge, NK-1R KO mice remove glucose from the circulation more efficiently than WT and pair-fed controls, suggesting an additional peripheral effect of NK-1R-mediated signaling on glucose metabolism. Glucose uptake experiments in isolated rat adipocytes showed that SP directly inhibits insulin-mediated glucose uptake. Our results further establish a role for SP-NK-1R interactions in adipose tissue responses, specifically as they relate to obesity-associated pathologies such as glucose intolerance and insulin resistance. Our results highlight this pathway as an important therapeutic approach for type 2 diabetes.