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Background & Aims: Defects in the colonic innate immune response have been associated with inflammatory bowel disease (IBD). Corticotropin-releasing hormone (CRH, or corticotropin-releasing factor [CRF]) is a neuropeptide that mediates the stress response in humans, is an immunomodulatory factor with proinflammatory effects, and regulates transcription of Toll-like receptors (TLR)-2 and TLR4. We investigated the role of CRF in an innate immunity–dependent mouse model of IBD. Methods: Crh −/− and wild-type (Crh +/+ ) mice, which are glucocorticoid insufficient, were given dextran sodium sulfate in their drinking water to induce colitis; in some experiments, mice were also given glucocorticoids. Phenotypes of mice were compared; tissues were analyzed by histology and for expression of immune mediators. Results: Crh −/− mice had more colonic inflammation than Crh +/+ mice, characterized by reduced numbers of crypts and severe epithelial damage and ulcerations. Colonic tissue levels of the proinflammatory factors interleukin-12 and prostaglandin E2 were increased in the Crh −/− mice. Colons of Crh −/− mice expressed lower levels of Tlr4 than wild-type mice before, but not after, colitis was induced. Administration of glucocorticoid at low levels did not prevent Crh −/− mice from developing severe colitis. Crh −/− mice were unable to recover from acute colitis, as indicated by their increased death rate. Conclusions: Mice deficient in CRF down-regulate TLR4 and are more susceptible to dextran sodium sulfate–induced colitis. CRF has anti-inflammatory effects in innate immunity–dependent colitis and its recovery phase; these are independent of glucocorticoid administration. CRF might therefore be developed as a therapeutic target for patients with IBD. [ABSTRACT FROM AUTHOR]

Background & Aims: Saccharomyces boulardii (Sb) is a probiotic yeast with anti-inflammatory and anti-microbial activities and has been used for decades in the prevention and treatment of a variety of human gastrointestinal disorders. We reported previously that Sb modulates host inflammatory responses through down-regulation of extracellular signal-regulated kinase (Erk)1/2 activities both in vitro and in vivo. The aim of this study was to identify upstream mediators responsible for extracellular signal-regulated kinase (Erk)1/2 inactivation and to examine the effects of Sb on tumor development in Apc Min mice. Methods: Signaling studies of colon cancer cells were done by western blot. Cell proliferation was measured by MTS and BrdU assay. Apoptosis was examined by flow cytometry, tunel assay and caspase assay. Apc Min mice were orally given Sb for 9 weeks before sacrifice for tumor analysis. Results: We found that the epidermal growth factor receptor (EGFR) was deactivated upon exposure to Sb, leading to inactivation of both the EGFR-Erk and EGFR-Akt pathways. In human colonic cancer cells, Sb prevented EGF-induced proliferation, reduced cell colony formation, and promoted apoptosis. HER-2, HER-3, and insulin-like growth factor-1 receptor were also found to be inactivated by Sb. Oral intake of Sb reduced intestinal tumor growth and dysplasia in C57BL/6J Min/+ (Apc Min ) mice. Conclusions: Thus, in addition to its anti-inflammatory effects, Sb inhibits EGFR and other receptor tyrosine kinase signaling and thereby may also serve a novel therapeutic or prophylactic role in intestinal neoplasia. [Copyright &y& Elsevier]

Background & Aims: Toll-like receptor (TLR)-dependent signaling was proposed as immunotherapeutic targets against invading pathogens and tumorigenesis. Here, we investigated whether TLR5-dependent signaling modulates colonic tumor development in mouse xenograft model of human colon cancer. Methods: The expression of myeloid differentiation factor 88 (MyD88) or TLR5 was stably knocked down in human colon cancer cells (DLD-1). Nude mice were subcutaneously implanted with MyD88-knocked down (KD), TLR5-KD, or control cells (n = 16) to examine the pathophysiology of tumor xenografts. Protein microarray assessed the differential expression of cytokines in these tumors. Leukocyte infiltration and tumor angiogenesis were assessed by immunohistochemistry with antibodies against neutrophil (Gr-1, 7/4) or macrophage-specific antigens (CD68, F4-80) and the vascular endothelial cell marker CD31, respectively. Tumor xenografts from DLD-1 cells were treated with flagellin (5.0 μg/kg, 1 injection/every 2 days for 3 weeks), and tumor regression and histopathology were examined. Results: Lack of MyD88 or TLR5 expression dramatically enhanced tumor growth and inhibited tumor necrosis in mouse xenografts of human colon cancer. In contrast, TLR5 activation by peritumoral flagellin treatment substantially increased tumor necrosis, leading to significant tumor regression. Tumors from MyD88-KD or TLR5-KD cells revealed the reduced production of neutrophil attracting chemokines (epithelial cell-derived neutrophil-activating peptide-78, macrophage-inflammatory protein α, and interleukin-8). Consequently, neutrophil infiltration was dramatically diminished in MyD88- or TLR5-KD xenografts, whereas tumor-associated macrophage infiltration or angiogenesis was not changed. Conclusions: TLR5 engagement by flagellin mediates innate immunity and elicits potent antitumor activity, indicating that TLR5-dependent signaling could be a potential immunotherapeutic target to modulate colonic tumors. [Copyright &y& Elsevier]

Background & Aims: Gastrin induces the expression of cyclooxygenase (COX)-2 and interleukin (IL)-8; however, the mechanism(s), especially in gastric epithelial cells, is not well understood. Here, we have determined the intracellular mechanisms mediating gastrin–dependent gene expression. Methods: AGS-E human gastric cancer cell line stably expressing cholecystokinin-2 receptor was treated with amidated gastrin-17. Real-time polymerase chain reaction, Western blot, and enzyme-linked immunosorbent assay were performed to determine COX-2 and IL-8 expression and Akt, Erk, and p38 phosphorylation. Gene promoter activity was determined by luciferase assay. Electrophoretic mobility shift assay analysis was performed for nuclear factor κB (NF-κB) and activator protein-1 activity. RNA stability was determined after actinomycin D treatment. HuR localization was determined by immunocytochemistry. Results: Gastrin induced COX-2 and IL-8 expression in AGS-E cells, which was inhibited by phosphatidylinositol 3′ kinase (PI3K) and p38 inhibitors. Gastrin-mediated Akt activation was observed to be downstream of p38. IL-8 expression was dependent on COX-2–mediated prostaglandin E2 synthesis. In the presence of an NF-κB inhibitor MG132, IL-8 transcription was inhibited, but not that of COX-2. This was confirmed after knockdown of the p65 RelA subunit of NF-κB. Further studies showed that COX-2 gene transcription is regulated by activator protein-1. Gastrin increased the stability of both COX-2 and IL-8 messenger RNA (mRNA) in a p38-dependent manner, the half-life increasing from 31 minutes to 8 hours and approximately 4 hours, respectively. Gastrin, through p38 activity, also enhanced HuR expression, nucleocytoplasmic translocation, and enhanced COX-2 mRNA binding. Conclusions: Gastrin differentially induces COX-2 and IL-8 expression at the transcriptional and posttranscriptional levels by PI3K and p38 mitogen-activated protein kinase pathways, respectively. [Copyright &y& Elsevier]

Background & Aims: Substance P (SP) is an 11-amino acid peptide that belongs to the tachykinin family of peptides. SP acts in the brain and in the periphery as a neuropeptide, neurotransmitter, and hormone affecting diverse physiologic pathways, mainly via its high-affinity neurokinin-1 receptor (NK-1R). Its presence in the hypothalamus and other areas of the brain that regulate feeding as well as in the stomach and small intestine prompted us to investigate its role on appetite control and energy balance. Methods: CJ 012,255 (CJ), a SP antagonist that binds to NK-1R, was injected into lean, diet-induced obese (DIO), and genetically obese (ob/ob) mice, and its effects on body weight, adiposity, and insulin sensitivity were investigated. Results: CJ administration prevented weight gain and accumulation of fat after 2 weeks of high-fat feeding, whereas similar CJ treatment in obese mice (following 3 months of high-fat diet) resulted in weight loss, reduction in adiposity, and improvement of insulin sensitivity, in part because of inhibition of food intake. The effects of SP in the control of energy balance are, at least in part, leptin independent because CJ treatment was also effective in leptin-deficient mice. Peripheral SP administration resulted in a mild, dose-dependent increase in food intake, evident 3 hours post-SP injection. Conclusions: CJ reduces appetite and promotes weight loss in mice. We speculate that NK-1R antagonists, already tested in clinical trials for various diseases, may represent a potential target against obesity. [Copyright &y& Elsevier]

Background & Aims: Clostridium difficile toxin A causes acute inflammation and fluid secretion in experimental animals and patients with C difficile infection. We previously reported that toxin A increased cyclooxygenase-2/prostaglandin E2 (PGE2) expression and apoptosis in human colonocytes. Here, we assessed the role of secreted PGE2 in inflammation and enterocyte apoptosis in toxin A enteritis. Methods: Effects of PGE2 and PGE2 blockade on toxin A-induced apoptosis of human colonocytes (NCM460) and of PGE2 or toxin A on the Fas ligand (FasL) induction were analyzed by flow cytometry and Western blot. Functional activity of elevated FasL on colonocytes was assessed by coculture of colonocytes with Fas bearing Jurkat T cells. The involvement of PGE2-dependent Fas/FasL activation in toxin A enteritis was further assessed in either scid or FasL and Fas deficient mice. Results: Inhibition of cyclooxygenase-2 by NS-398 and of PGE2 using a blocking antibody markedly attenuated apoptosis in colonocytes exposed to toxin A. Enhanced expression and release of FasL followed PGE2 or toxin A exposure in vivo and in vitro and also was significantly attenuated by treatment with NS-398 and PGE2 blocking antibody. PGE2 acting through an EP1 receptor activated nuclear factor-κB, which induced transcription of FasL. Toxin A enteritis was accompanied by increased cellular infiltration, fluid secretion, and mucosal damage in control mice, but this response was markedly reduced in both Fas−/− and FasL−/− mice. Conclusions: Toxin A enteritis involves release of PGE2, which activates the Fas/FasL system, causing enterocyte apoptosis and inflammation. [Copyright &y& Elsevier]

Background & Aims: We studied the role of protease-activated receptor 2 (PAR2) and its activating enzymes, trypsins and tryptase, in Clostridium difficile toxin A (TxA)-induced enteritis. Methods: We injected TxA into ileal loops in PAR2 or dipeptidyl peptidase I (DPPI) knockout mice or in wild-type mice pretreated with tryptase inhibitors (FUT-175 or MPI-0442352) or soybean trypsin inhibitor. We examined the effect of TxA on expression and activity of PAR2 and trypsin IV messenger RNA in the ileum and cultured colonocytes. We injected activating peptide (AP), trypsins, tryptase, and p23 in wild-type mice, some pretreated with the neurokinin 1 receptor antagonist SR140333. Results: TxA increased fluid secretion, myeloperoxidase activity in fluid and tissue, and histologic damage. PAR2 deletion decreased TxA-induced ileitis, reduced luminal fluid secretion by 20%, decreased tissue and fluid myeloperoxidase by 50%, and diminished epithelial damage, edema, and neutrophil infiltration. DPPI deletion reduced secretion by 20% and fluid myeloperoxidase by 55%. In wild-type mice, FUT-175 or MPI-0442352 inhibited secretion by 24%–28% and tissue and fluid myeloperoxidase by 31%–71%. Soybean trypsin inhibitor reduced secretion to background levels and tissue myeloperoxidase by up to 50%. TxA increased expression of PAR2 and trypsin IV in enterocytes and colonocytes and caused a 2-fold increase in Ca2+ responses to PAR2 AP. AP, tryptase, and trypsin isozymes (trypsin I/II, trypsin IV, p23) caused ileitis. SR140333 prevented AP-induced ileitis. Conclusions: PAR2 and its activators are proinflammatory in TxA-induced enteritis. TxA stimulates existing PAR2 and up-regulates PAR2 and activating proteases, and PAR2 causes inflammation by neurogenic mechanisms. [Copyright &y& Elsevier]

Background & Aims: Barrier functions across epithelia and endothelia are essential for homeostatic tissue regulation. Astroglia interact with cerebral endothelia to maintain the blood-brain barrier. Whether similar interactions between astrocyte-like enteric glia and epithelia regulate intestinal barrier function is not known. Methods: Fluorescent permeability markers were used to measure intestinal barrier function in vivo after conditional ablation of enteric glia in transgenic mice. Enteric glial cell regulation of epithelial barrier integrity then was modeled in vitro using coculture. Glial-derived barrier-inducing factors were characterized using size-exclusion chromatography and mass spectrometry. Epithelial barrier integrity was assessed by transepithelial resistance readings and by quantitative measurement of tight-junction–associated protein expression by quantitative polymerase chain reaction and Western blot. Results: We show that ablation of enteric glial cells in transgenic mice causes intestinal mucosal barrier dysfunction, resulting in inflammation. Glial-derived s-nitrosoglutathione (GSNO) was identified as a potent inducer of mucosal barrier function in vitro and in vivo and of attenuated tissue inflammation after ablation of enteric glia in transgenic mice. GSNO regulation of mucosal barrier function was associated directly with an increased expression of perijunctional F-actin and tight-junction–associated proteins zonula occludens-1 and occludin. GSNO also significantly restored mucosal barrier function in colonic biopsy specimens from patients with Crohn’s disease, a well-described inflammatory permeability disorder associated with enteric glial-cell disruption. Conclusions: Enteric glia therefore share the ability of astrocytes to regulate tight-junction integrity, and cellular interactions comparable with those maintaining blood-brain barrier function also regulate epithelial permeability at mucosal surfaces. [Copyright &y& Elsevier]

Background & Aims: The neurokinin 1 receptors (NK1Rs) and substance P (SP) have been implicated in the stress and/or pain pathways involved in chronic pain conditions. Here we examined the participation of NK1Rs in sustained visceral hyperalgesia observed in rats exposed to chronic psychological stress. Methods: Male Wistar rats were exposed to daily 1-hour water avoidance stress (WA) or sham WA for 10 consecutive days. We tested intraperitoneal or intrathecal injection of the NK1R antagonist SR140333 on the visceromotor reflex to colorectal distention in both groups at day 11. Real-time reverse-transcription polymerase chain reaction, Western blot, and immunohistochemistry were used to assess the expression of NK1Rs and/or SP in samples of colon, spinal cord, and dorsal root ganglia. Results: Both intraperitoneal and intrathecal SR140333 injection diminished the enhanced visceromotor reflex to colorectal distention at day 11 in stressed rats but did not affect the response in control animals. Real-time polymerase chain reaction and Western blotting demonstrated stress-induced up-regulation of spinal NK1Rs. Immunohistochemistry showed an increased number of NK1R-expressing neurons in the laminae I of the dorsal horn in stressed rats. The expression of NK1Rs was decreased in colon from stressed rats compared with control. The expression of SP gene precursor in dorsal root ganglia was unchanged in stressed rats compared with controls. Conclusions: Stress-induced increased NK1R expression on spinal neurons and the inhibitory effect of intrathecal NK1R antagonist on visceral hyperalgesia support the key contribution of spinal NK1Rs in the molecular pathways involved in the maintenance of visceral hyperalgesia observed after chronic WA. [Copyright &y& Elsevier]

Background & Aims: Clostridium difficile toxin A causes marked apoptosis of colonocytes in vivo and in vitro, which contributes to the formation of ulcers and pseudomembranes. We investigated the role of p53-dependent pathways and p38 mitogen-activated protein kinase (p38) in toxin A–induced colonocyte apoptosis. Methods: The effects of the activation of p53 and p53-dependent pathways including p21(WAF1/CIP1) were assessed in nontransformed human colonic NCM460 epithelial cells exposed to toxin A. Phosphorylation of p53 protein by p38 was measured by in vitro kinase assay, whereas p21 induction by activated p53 was determined by gel shift assays and RNA silencing (small interfering RNA). The relationship between colonocyte apoptosis and p38/p53-dependent pathways was studied in intact mice. Results: Toxin A stimulated p38 and p53 activation and induced cell cycle arrest (G2–M) with persistent expression of p21(WAF1/CIP1). Blockage of p38 by SB203580 inhibited p53 phosphorylation and induction of p21(WAF1/CIP1). In intact mice, p38 blockade suppressed toxin A–mediated destruction of intestinal villi, p21(WAF1/CIP1) expression, and enterocyte apoptosis. In addition, toxin A–mediated p21(WAF1/CIP1) and Bak induction, cytochrome c release, and caspase-3 activation were markedly attenuated in p53-silenced colonocytes, despite active p38. Overexpression of p21(WAF1/CIP1) triggered apoptosis and increased toxin A–associated colonocyte apoptosis. Conclusions:: The signaling pathway for colonocyte apoptosis following toxin A exposure involves p38-dependent activation of p53 and subsequent induction of p21(WAF1/CIP1), resulting in cytochrome c release and caspase-3 activation through Bak induction. [Copyright &y& Elsevier]

Background & Aims: Clostridium difficile toxin B (TxB) mediates acute inflammatory diarrhea characterized by neutrophil infiltration and intestinal mucosal injury. In a xenograft animal model, TxB was shown to induce interleukin (IL)-8 gene expression in human colonic epithelium. However, the precise mechanisms of this TxB response are unknown. The aim of this study was to investigate the TxB-mediated proinflammatory pathway in colonocytes. Methods: The effect of TxB on epidermal growth factor receptor (EGFR), extracellular signal-regulated kinase (ERK) 1/2 signaling pathway and IL-8 gene expression was assessed in nontransformed human colonic epithelial NCM460 cells. TxB regulation of EGFR-ERK1/2 signaling pathways was determined using immunoblot analysis, confocal microscopy, and enzyme-linked immunosorbent assay, whereas IL-8 gene expression was measured by luciferase promoter assay. Results: TxB activates EGFR and ERK1/2 phosphorylation with subsequent release of IL-8 from human colonocytes. Pretreatment with either the EGFR tyrosine kinase inhibitor, AG1478, or an EGFR-neutralizing antibody blocked both TxB-induced EGFR and ERK activation. By using neutralizing antibodies against known ligands of EGFR, we found that the activation of EGFR and ERK1/2 phosphorylation was mediated by transforming growth factor-α (TGF-α). Inhibition of matrix metalloproteinase (MMP) decreased TGF-α secretion and TxB-induced EGFR and ERK activation. Inhibition of MMP, EGFR, and ERK activation significantly decreased TxB-induced IL-8 expression. Conclusions: TxB signals acute proinflammatory responses in colonocytes by transactivation of the EGFR and activation of the ERK/MAP kinase pathway. [Copyright &y& Elsevier]

Alterations in microRNA (miRNA) and in the intestinal barrier are putative risk factors for irritable bowel syndrome (IBS). We aimed to identify differentially expressed colonic mucosal miRNAs, their targets in IBS compared to healthy controls (HCs), and putative downstream pathways. Twenty-nine IBS patients (15 IBS with constipation [IBS-C], 14 IBS with diarrhea [IBS-D]), and 15 age-matched HCs underwent sigmoidoscopy with biopsies. A nCounter array was used to assess biopsy specimen-associated miRNA levels. A false discovery rate (FDR) < 10% was considered significant. Real-time polymerase chain reaction (PCR) was used to validate differentially expressed genes. To assess barrier function, trans-epithelial electrical resistance (TEER) and dextran flux assays were performed on Caco-2 intestinal epithelial cells that were transfected with miRNA-inhibitors or control inhibitors. Protein expression of barrier function associated genes was confirmed using western blots. Four out of 247 miRNAs tested were differentially expressed in IBS compared to HCs (FDR < 10%). Real-time PCR validation suggested decreased levels of miR-219a-5p and miR-338-3p in IBS (P =.026 and P =.004), and IBS-C (P =.02 and P =.06) vs. HCs as the strongest associations. Inhibition of miR-219a-5p resulted in altered expression of proteasome/barrier function genes. Functionally, miR-219a-5p inhibition enhanced the permeability of intestinal epithelial cells as TEER was reduced (25-50%, P <.05) and dextran flux was increased (P <.01). Additionally, inhibition of miR-338-3p in cells caused alterations in the mitogen-activated protein kinase (MAPK) signaling pathway genes. Two microRNAs that potentially affect permeability and visceral nociception were identified to be altered in IBS patients. MiR-219a-5p and miR-338-3p potentially alter barrier function and visceral hypersensitivity via neuronal and MAPK signaling and could be therapeutic targets in IBS. [Display omitted] [ABSTRACT FROM AUTHOR]

Background & Aims Persistent activation of the inflammatory response contributes to the development of inflammatory bowel diseases, which increase the risk of colorectal cancer. We aimed to identify microRNAs that regulate inflammation during the development of ulcerative colitis (UC) and progression to colitis-associated colon cancer (CAC). Methods We performed a quantitative polymerase chain reaction analysis to measure microRNAs in 401 colon specimens from patients with UC, Crohn’s disease, irritable bowel syndrome, sporadic colorectal cancer, or CAC, as well as subjects without these disorders (controls); levels were correlated with clinical features and disease activity of patients. Colitis was induced in mice by administration of dextran sodium sulfate (DSS), and carcinogenesis was induced by addition of azoxymethane; some mice also were given an inhibitor of microRNA214 (miR214). Results A high-throughput functional screen of the human microRNAome found that miR214 regulated the activity of nuclear factor-κB. Higher levels of miR214 were detected in colon tissues from patients with active UC or CAC than from patients with other disorders or controls and correlated with disease progression. Bioinformatic and genome-wide profile analyses showed that miR214 activates an inflammatory response and is amplified through a feedback loop circuit mediated by phosphatase and tensin homolog (PTEN) and PDZ and LIM domain 2 (PDLIM2). Interleukin-6 induced signal transducer and activator of transcription 3 (STAT3)-mediated transcription of miR214. A miR214 chemical inhibitor blocked this circuit and reduced the severity of DSS-induced colitis in mice, as well as the number and size of tumors that formed in mice given azoxymethane and DSS. In fresh colonic biopsy specimens from patients with active UC, the miR214 inhibitor reduced inflammation by increasing levels of PDLIM2 and PTEN. Conclusions Interleukin-6 up-regulates STAT3-mediated transcription of miR214 in colon tissues, which reduces levels of PDLIM2 and PTEN, increases phosphorylation of AKT, and activates nuclear factor-κB. The activity of this circuit correlates with disease activity in patients with UC and progression to colorectal cancer. [ABSTRACT FROM AUTHOR]