Clostridium difficile mediates intestinal inflammation by releasing toxin A (TxA), a potent enterotoxin. Cathelicidins (Camp as gene name, LL-37 peptide in humans and mCRAMP peptide in mice) are antibacterial peptides that also posses anti-inflammatory properties.
To determine the role of cathelicidins in models of Clostridium difficile infection and TxA-mediated ileal inflammation and cultured human primary monocytes.
Wild-type (WT) and mCRAMP-deficient (Camp−/−) mice were treated with an antibiotic mixture and infected orally with C difficile. Some mice were intracolonically given mCRAMP daily for 3 days. Ileal loops were also prepared in WT mice and treated with either saline or TxA and incubated for 4 h, while some TxA-treated loops were injected with mCRAMP.
Intracolonic mCRAMP administration to C difficile-infected WT mice showed significantly reduced colonic histology damage, apoptosis, tissue myeloperoxidase (MPO) and tumour necrosis factor (TNF)α levels. Ileal mCRAMP treatment also significantly reduced histology damage, tissue apoptosis, MPO and TNFα levels in TxA-exposed ileal loops. WT and Camp−/− mice exhibited similar intestinal responses in both models, implying that C difficile/TxA-induced endogenous cathelicidin may be insufficient to modulate C difficile/TxA-mediated intestinal inflammation. Both LL-37 and mCRAMP also significantly reduced TxA-induced TNFα secretion via inhibition of NF-κB phosphorylation. Endogenous cathelicidin failed to control C difficile and/or toxin A-mediated inflammation and even intestinal cathelicidin expression was increased in humans and mice.
Exogenous cathelicidin modulates C difficile colitis by inhibiting TxA-associated intestinal inflammation. Cathelicidin administration may be a new anti-inflammatory treatment for C difficile toxin-associated disease.
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.
Neurotensin (NT) is a highly expressed gastrointestinal (GI) neuropeptide, which modulates GI motility, secretion and cell growth as well as intestinal inflammation. Since EGF receptor is highly expressed in human colon cancer cells, we sought to examine whether NT stimulation contributes to the EGFR overexpression using nontransformed colonocyte NCM460 cells. The results show that NT treatment caused a significant increase in EGFR protein expression and gene transcription. Pretreatment with MAP kinase pathway inhibitor PD98059 blocked NT-induced EGFR expression. As the EGFR promoter has a functional Egr-1 site, previously shown to mediate its transcription in response to hypoxia, we examined the role of Egr-1 in the NT response. We first show that NT stimulated Egr-1 expression, which can be inhibited by PD98059. We also determined whether NT increases Egr-1 binding to its site within the EGFR promoter. The data indicate that NT enhanced the amount of Egr-1 binding to the EGFR Egr-1 site and that this binding was significantly decreased by PD98059. To verify that Egr-1 mediated NT-induced EGFR transcription, Egr-1 siRNA was used to knock down its expression. The data show that transfection of Egr-1 siRNA significantly inhibited NT-stimulated EGFR transcription. Together, our results suggest that NT can stimulate MAP kinase-mediated Egr-1 and EGFR gene expression in human colonocytes. Our results may be relevant to the mechanisms by which NT participates in the development of colon cancer.
neurotensin; early growth response gene-1; EGF receptor
Antimicrobial peptides (AMPs) are important components of innate immunity. They are often expressed in response to colonic inflammation and infection. Over the last several years, the roles of several antimicrobial peptides have been explored. Gene expression of many AMPs (beta defensin HBD2-4 and cathelicidin) is induced in response to invasion of gut microbes into the mucosal barrier. Some AMPs are expressed in a constitutive manner (alpha defensin HD 5-6 and beta defensin HBD1), while others (defensin and bactericidal/permeability increasing protein BPI) are particularly associated with Inflammatory Bowel Disease (IBD) due to altered defensin expression or development of autoantibodies against Bactericidal/permeability increasing protein (BPI). Various AMPs have different spectrum and strength of antimicrobial effects. Some may play important roles in modulating the colitis (cathelicidin) while others (lactoferrin, hepcidin) may represent biomarkers of disease activity. The use of AMPs for therapeutic purposes is still at an early stage of development. A few natural AMPs were shown to be able to modulate colitis when delivered intravenously or intracolonically (cathelicidin, elafin and SLPI) in mouse colitis models. New AMPs (synthetic or artificial non-human peptides) are being developed and may represent new therapeutic approaches against colitis. This review discusses the latest research developments in the AMP field with emphasis in innate immunity and pathophysiology of colitis.
Antimicrobial peptides; colitis; infection; microflora; protein; Crohn’s disease; ulcerative colitis
Neurotensin receptor-1 (NTR-1) is overexpressed in colon cancers and colon cancer cell lines, Signaling through this receptor stimulates proliferation of colonocyte-derived cell lines and promotes inflammation and mucosal healing in animal models of colitis. Given the causal role of this signaling pathway in mediating colitis and the importance of inflammation in cancer development we tested the effects of NTR-1 in mouse models of inflammation-associated and sporadic colon cancer using NTR-1 deficient (Ntsr1−/−) and wild type (Ntsr1+/+) mice. In mice treated with azoxymethane (AOM) to model sporadic cancer, NTR-1 had a significant effect on tumor development with Ntsr1+/+ mice developing over 2-fold more tumors than Ntsr1−/− mice (p = 0.04). There was no effect of NTR-1 on the number of aberrant crypt foci or tumor size, suggesting that NT/NTR-1 signaling promotes the conversion of precancerous cells to adenomas. Interestingly, NTR-1 status did not affect tumor development in an inflammation-associated cancer model where mice were treated with AOM followed by two cycles of 5% dextran sulphate sodium (DSS). In addition, colonic molecular and histopathologic analyses were performed shortly after a single cycle of DSS. NTR-1 status did not affect colonic myeloperoxidase activity or histopathologic scores for damage and inflammation. However, Ntsr1−/− mice were more resistant to DSS-induced mortality (p = 0.01) and had over 2-fold higher colonic expression levels of Il6 and Cxcl2 (p < 0.04), cytokines known to promote tumor development. These results represent the first direct demonstration that targeted disruption of the Ntsr1 gene reduces susceptibility to colon tumorigenesis.
Neurotensin receptor-1; azoxymethane; dextran sulphate sodium; aberrant crypt foci; colon cancer
Obesity is an important risk factor for colon cancer in humans, and numerous studies have shown that a high fat diet enhances colon cancer development. As both increased adiposity and high fat diet can promote tumorigenesis, we examined the effect of diet-induced obesity, without ongoing high fat diet, on colon tumor development. C57BL/6J male mice were fed regular chow or high fat diet for 8 weeks. Diets were either maintained or switched resulting in four experimental groups: regular chow (R), high fat diet (H), regular chow switched to high fat diet (RH), and high fat diet switched to regular chow (HR). Mice were then administered azoxymethane to induce colon tumors. Tumor incidence and multiplicity were dramatically smaller in the R group relative to all groups that received high fat diet at any point. The effect of obesity on colon tumors could not be explained by differences in aberrant crypt foci number. Moreover, diet did not alter colonic expression of pro-inflammatory cytokines tumor necrosis factor-α, interleukin-6, interleukin-1β, and interferon-γ, which were measured immediately after azoxymethane treatment. Crypt apoptosis and proliferation, which were measured at the same time, were increased in the HR relative to all other groups. Our results suggest that factors associated with obesity – independently of ongoing high fat diet and obesity – promote tumor development because HR group animals had significantly more tumors than R group, and these mice were fed the same regular chow throughout the entire carcinogenic period. Moreover, there was no difference in the number of aberrant crypt foci between these groups, and thus the effect of obesity appears to be on subsequent stages of tumor development when early preneoplastic lesions transition into adenomas.
The current global outbreak of Clostridium difficile infection exemplifies the major public health threat posed by clostridial glucosylating toxins. In the western world, C. difficile infection is one of the most prolific causes of bacterial-induced diarrhea and potentially fatal colitis. Two pathogenic enterotoxins, TcdA and TcdB, cause the disease. Vancomycin and metronidazole remain readily available treatment options for C. difficile infection, but neither is fully effective as is evident by high clinical relapse and fatality rates. Thus, there is an urgent need to find an alternative therapy that preferentially targets the toxins and not the drug-resistant pathogen. Recently, we addressed these critical issues in a Nature Medicine letter, describing a novel host defense mechanism for subverting toxin virulence that we translated into prototypic allosteric therapy for C. difficile infection. In this addendum article, we provide a continued perspective of this antitoxin mechanism and consider the broader implications of therapeutic allostery in combating gut microbial pathogenesis.
C. difficile; allostery; dietary supplement; inositol phosphate; S-nitrosylation; toxin
Lin28A and Lin28B selectively block the expression of let-7 microRNAs and function as oncogenes in a variety of human cancers. Lin28A recruits a TUTase (Zcchc11/TUTase4) to let-7 precursors to block processing by Dicer in the cell cytoplasm. Here we find that unlike Lin28A, Lin28B represses let-7 processing through a TUTase-independent mechanism. Lin28B functions in the nucleus by sequestering primary let-7 transcripts and inhibiting their processing by the Microprocessor. The inhibitory effects of Zcchc11 depletion on the tumorigenic capacity and metastatic potential of human cancer cells and xenografts is restricted to Lin28A-expressing tumors. Furthermore, the majority of human colon and breast tumors analyzed exclusively express either Lin28A or Lin28B. Lin28A is expressed in HER2-overexpressing breast tumors while Lin28B expression characterizes triple-negative breast tumors. Overall our results illuminate the distinct mechanisms by which Lin28A and Lin28B function, and have implications for the development of new strategies for cancer therapy.
Lin-28; Lin28A; Lin28B; let-7; microRNA (miRNA); TUTase; Zcchc11; TUTase4; TUT4; Cancer
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.
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.
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.
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.
Cramp; LL-37; IBD; mouse models; endogenous inhibitors; immune regulation
Psychological stress plays a role in the exacerbation of functional lower urinary tract disorders such as painful bladder syndrome and overactive bladder. To better understand the mechanism underlying this relationship, we characterized changes in micturition, anxiety-related behavior, and bladder pathology in rats exposed to repeated water avoidance (WA) stress.
Twenty-four Wistar rats were subjected to WA stress or sham. Immediately following acute (day 1) and chronic (day 10) stress or sham, rats were placed in a metabolic cage for a 2-hour voiding behavior assessment. Voiding parameters were compared to baseline values obtained prior to stress. Four animals from each group were sacrificed on day 10 and bladders harvested for histologic and gene expression studies. The remaining 8 animals per group underwent repeated voiding assessment every 3 days for 1 month followed by 10 days of repeat WA stress or sham. Bladder histology and gene expression were studied.
Rats exposed to WA stress developed a significant increase in micturition frequency and decrease in latency to void, voiding interval and volume of first void compared to sham and baseline. Alterations in micturition persisted for approximately 1 month. Stressed rats showed increased fecal pellet excretion and anxiety-like behavior. Additionally, bladder specimens from stressed animals revealed increased angiogenesis, and increased total and activated mast cells.
In rats, repeated psychological stress results in lasting alterations in micturition frequency, interval, and volume. This rodent model may represent a valid tool for studying syndromes characterized by increased urinary frequency.
Water avoidance stress; overactive bladder; painful bladder syndrome/interstitial cystitis; animal model; urinary frequency
Background & Aims
Corticotropin-releasing factor receptor-1 (CRF1) mediates the stress-induced colonic motor activity. Less is known about the role of CRF2 in the colonic response to stress.
We studied colonic contractile activity (CCA) in rats and CRF2-/-, CRF-overexpressing, and wild-type mice using still manometry; we analyzed defecation induced by acute, partial-restraint stress (PRS), and/or intraperitoneal (IP) injection of CRF ligands. In rats, we monitored activation of the colonic longitudinal muscle myenteric plexus (LMMP) neurons and localization of CRF1 and CRF2 using immunohistochemical and immunoblot analyses. We measured phosphorylation of ERK1/2 by CRF ligands in primary cultures of LMMP-neurons (PC-LMMPn) and cAMP production in HEK-293 cells transfected with CRF1 and/or CRF2.
In rats, a selective agonist of CRF2 (urocortin 2) reduced CRF-induced defecation (>50%), CCA, and Fos expression in the colonic LMMP. A selective antagonist of CRF2 (astressin2-B) increased these responses. Urocortin 2 reduced PRS-induced CCA in wild-type and CRF-overexpressing mice, whereas disruption of CRF2 increased PRS-induced CCA and CRF-induced defecation. CRF2 co-localized with CRF1 and neuronal nitric oxide synthase in the rat colon, LMMP, and PC-LMMPn. CRF-induced phosphorylation of ERK in PC-LMMPn; this was inhibited or increased by a selective antagonist of CRF1 (NBI35965) or astressin2-B, respectively. The EC50 for the CRF-induced cAMP response was 8.6 nM in HEK-293 cells that express only CRF1; this response was suppressed 10-fold in cells that express CRF1 and CRF2.
In colon tissues of rodents, CRF2 activation inhibits CRF1 signaling in myenteric neurons and the stress-induced colonic motor responses. Disruption of CRF2 function impairs colonic coping responses to stress.
colonic contraction; myenteric neurons; nNOS; stress response
Clostridium difficile-associated diarrhea and pseudomembranous colitis are typically treated with vancomycin or metronidazole, but recent increases in relapse incidence and the emergence of drug-resistant strains of C. difficile indicate the need for new antibiotics. We previously isolated coprisin, an antibacterial peptide from Copris tripartitus, a Korean dung beetle, and identified a nine-amino-acid peptide in the α-helical region of it (LLCIALRKK) that had antimicrobial activity (J.-S. Hwang et al., Int. J. Pept., 2009, doi:10.1155/2009/136284). Here, we examined whether treatment with a coprisin analogue (a disulfide dimer of the nine peptides) prevented inflammation and mucosal damage in a mouse model of acute gut inflammation established by administration of antibiotics followed by C. difficile infection. In this model, coprisin treatment significantly ameliorated body weight decreases, improved the survival rate, and decreased mucosal damage and proinflammatory cytokine production. In contrast, the coprisin analogue had no apparent antibiotic activity against commensal bacteria, including Lactobacillus and Bifidobacterium, which are known to inhibit the colonization of C. difficile. The exposure of C. difficile to the coprisin analogue caused a marked increase in nuclear propidium iodide (PI) staining, indicating membrane damage; the staining levels were similar to those seen with bacteria treated with a positive control for membrane disruption (EDTA). In contrast, coprisin analogue treatment did not trigger increases in the nuclear PI staining of Bifidobacterium thermophilum. This observation suggests that the antibiotic activity of the coprisin analogue may occur through specific membrane disruption of C. difficile. Thus, these results indicate that the coprisin analogue may prove useful as a therapeutic agent for C. difficile infection-associated inflammatory diarrhea and pseudomembranous colitis.
Low-grade colonic mucosal inflammation has been postulated to have an important role in the pathophysiology of irritable bowel syndrome (IBS). The objectives of this study were (i) to identify serum and tissue-based immunological and neuroendocrine markers associated with mucosal inflammation in male (M) and female (F) patients with non-post-infectious IBS (non-PI-IBS) compared with healthy controls and (ii) to assess possible correlations of such markers with IBS symptoms.
Sigmoid mucosal biopsies were obtained from 45 Rome II positive IBS patients without a history of PI-IBS (26 F, 35.5% IBS-C, 33.3% IBS-D, 31.1% IBS-A/M) and 41 healthy controls (22 F) in order to measure immunological markers (serum cytokine levels, colonic mucosal mRNA levels of cytokines, mucosal immune cell counts) and neuroendocrine markers associated with mucosal inflammation (corticotropin releasing factor- and neurokinin (NK)-related ligands and receptors, enterochromaffin cells). Symptoms were measured using validated questionnaires.
Of all the serum and mucosal cytokines measured, only interleukin-10 (IL-10) mRNA expression showed a group difference, with female, but not male, patients showing lower levels compared with female controls (18.0 ± 2.9 vs. 29.5 ± 4.0, P = 0.006). Mucosal mRNA expression of NK-1 receptor was significantly lower (1.15 ± 0.19 vs. 2.66 ± 0.56, P = 0.008) in female, but not male, patients compared with healthy controls. No other significant differences were observed.
Immune cell counts and levels of cytokines and neuropeptides that are associated with inflammation were not significantly elevated in the colonic mucosa of non-PI-IBS patients, and did not correlate with symptoms. Thus, these findings do not support that colonic mucosal inflammation consistently has a primary role in these patients. However, the finding of decreased IL-10 mRNA expression may be a possible biomarker of IBS and warrants further investigation.
Several clinical trials and experimental studies strongly suggest a place for Saccharomyces boulardii as a biotherapeutic agent for the prevention and treatment of several gastrointestinal diseases. S. boulardii mediates responses resembling the protective effects of the normal healthy gut flora. The multiple mechanisms of action of S. boulardii and its properties may explain its efficacy and beneficial effects in acute and chronic gastrointestinal diseases that have been confirmed by clinical trials. Caution should be taken in patients with risk factors for adverse events. This review discusses the evidence for efficacy and safety of S. boulardii as a probiotic for the prevention and therapy of gastrointestinal disorders in humans.
efficacy; gastrointestinal disorders; probiotic; Saccharomyces boulardii; safety
The global prevalence of severe Clostridium difficile infection highlights the profound clinical significance of clostridial glucosylating toxins1–4. Virulence is dependent on the autoactivation of a toxin cysteine protease5–9, which is promoted by the allosteric cofactor inositol hexakisphosphate (InsP6)10–17. Host mechanisms that protect against such exotoxins are poorly understood. It is increasingly appreciated that the pleiotropic functions attributed to nitric oxide (NO), including host immunity, are in large part mediated by S-nitrosylation of proteins18,19. Here we show that C. difficile toxins are S-nitrosylated by the infected host and that S-nitrosylation attenuates virulence by inhibiting toxin self-cleavage and cell entry. Notably, InsP6- and inositol pyrophosphate (InsP7)-induced conformational changes in the toxin enabled host S-nitrosothiols to transnitrosylate the toxin catalytic cysteine, which forms part of a structurally conserved nitrosylation motif. Moreover, treatment with exogenous InsP6 enhanced the therapeutic actions of oral S-nitrosothiols in mouse models of C. difficile infection. Allostery in bacterial proteins has thus been successfully exploited in the evolutionary development of nitrosothiol-based innate immunity and may provide an avenue to new therapeutic approaches.
BACKGROUND & AIMS
The corticotrophin-releasing hormone (CRH) family of peptides modulates intestinal inflammation and the CRH receptor 2 (CRHR2) suppresses postnatal angiogenesis in mice. We investigated the functions of CRHR1 and CRHR2 signaling during intestinal inflammation and angiogenesis.
The activities of CRHR1 and CRHR2 were disrupted by genetic deletion in mice or with selective antagonists. A combination of in vivo, ex vivo, and in vitro measures of angiogenesis were used to determine their activity. CRHR1−/− mice and CRHR2−/− mice with dextran sodium sulfate-induced colitis were analyzed in comparison with wild-type littermates (controls).
Colitis was significantly reduced in mice in which CRHR1 activity was disrupted by genetic deletion or with an antagonist, determined by analyses of survival rate, weight loss, histological scores, and cytokine production. Inflammation was exacerbated in mice in which CRHR2 activity was inhibited by genetic deletion or with an antagonist, compared with controls. The inflamed intestines of CRHR1−/− mice had reduced microvascular density and expression of vascular endothelial growth factor (VEGF)-A, whereas the intestines of CRHR2−/− mice had increased angiogenesis and VEGF-A levels. An antagonist of VEGFR2 activity alleviated colitis in CRHR2−/− mice. Ex vivo aortic vessel outgrowth was reduced when CRHR1 was deficient but increased when CRHR2 was deficient. The CRHR1 preferred agonist CRH stimulated tube formation, proliferation, and migration of cultured intestinal microvascular endothelial cells by phosphorylating Akt whereas the specific CRHR2 agonist Urocortin III had opposite effects.
CRHR1 promotes intestinal inflammation, as well as endogenous and inflammatory angiogenesis whereas CRHR2 inhibits these activities.
neuropeptide; inflammatory bowel disease; PI3K; HIMECs
TL1A is a member of the TNF superfamily and its expression is increased in the mucosa of inflammatory bowel disease patients. Moreover, a subset of Crohn's disease (CD) patients with the risk TL1A haplotype is associated with elevated TL1A expression and a more severe disease course. To investigate the in vivo role of elevated TL1A expression, we generated two transgenic (Tg) murine models with constitutive Tl1a expression in either lymphoid or myeloid cells. Compared to wildtype (WT) mice, constitutive expression of Tl1a in either lymphoid or myeloid cells showed mild patchy inflammation in the small intestine, which was more prominent in the ileum. In addition, mice with constitutive Tl1a expression exhibited enhanced intestinal and colonic fibrosis compared to WT littermates. The percentage of T cells expressing the gut homing chemokine receptors CCR9 and CCR10 was higher in the Tl1a Tg mice compared to WT littermates. Sustained expression of Tl1A in T cells also lead to increased Foxp3+ Treg cells. T cells or antigen presenting cells (APC) with constitutive expression of Tl1a were found to have a more activated phenotype and mucosal mononuclear cells exhibit enhanced Th1 cytokine activity. These results indicated an important role of TL1A in mucosal T cells and APC function and showed that up-regulation of TL1A expression can promote mucosal inflammation and gut fibrosis.
C. difficile toxin A impairs tight junction function of colonocytes by glucosylation of Rho family proteins causing actin filament disaggregation and cell rounding. We investigated the effect of toxin A on focal contact formation by assessing its action on focal adhesion kinase (FAK) and the adapter protein paxillin. Exposure of NCM460 human colonocytes to toxin A for 1 hour resulted in complete dephosphorylation of FAK and paxillin, while protein tyrosine phosphatase activity was reduced. Blockage of toxin A-associated glucosyltransferase activity by co-incubation with UDP 2′3′dialdehyde did not reduce toxin A-induced FAK and paxillin dephosphorylation. GST-pull down and in vitro kinase activity experiments demonstrated toxin A binding directly to the catalytic domain of Src with suppression of its kinase activity. Direct binding of toxin A to Src, independent of any effect on protein tyrosine phosphatase or Rho glucosylation, inhibits Src kinase activity followed by FAK/paxillin inactivation. These mechanisms may contribute to toxin A-inhibition of colonocyte focal adhesion that occurs in human colonic epithelium exposed to toxin A.
Urocortin II (UcnII) is a neuropeptide that binds with high affinity to the corticotropin‐releasing hormone receptor 2 (CRHR2) in peripheral tissues. UcnII is synthesised in the intestine, but its role in human intestinal inflammation is largely unknown.
Responses of human colonic epithelial cells expressing CRHR2 to stimulation by UcnII were measured using ELISA, western blot analysis, real‐time reverse transcription‐PCR (RT‐PCR) and interleukin (IL)8 promoter activity. Expression levels of CRHR2 and UcnII in human colitis were determined by immunofluorescence and real‐time RT‐PCR in mucosal biopsies from patients with Crohn's and ulcerative colitis, and in human intestinal xenografts after exposure to Clostridium difficile toxin A.
It is reported here that expression of CRHR2 mRNA and protein in human colonic epithelial cells (HT‐29) are increased by exposure to C difficile toxin A or tumour necrosis factor (TNF)α. Stimulation of non‐transformed NCM460 colonocytes overexpressing CRHR2α receptor with UcnII resulted in a time‐ and concentration‐dependent increase in IL8 production. UcnII stimulation also led to activation of nuclear factor‐κB (NF‐κB) and mitogen‐acivated protein (MAP) kinase in these cells, as evidenced by degradation of IκBα and phosphorylation of the p65 subunit of NF‐κB and extracellularly regulated kinase (ERK) 1/2. Furthermore, expression of UcnII and CRHR2 mRNA was increased in mucosal samples of patients with inflammatory bowel disease, and after exposure of human intestinal xenografts to C difficile toxin A.
These results suggest that UcnII has pro‐inflammatory effects in human intestinal cells via the CRHR2α receptor and may play an important role in the pathophysiology of colitis in humans.
Saccharomyces boulardii (Sb) is a probiotic yeast with anti-inflammatory and antimicrobial 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 Erk1/2 MAP kinase activities both in vitro and in vivo. The aim of this study was to identify upstream mediators responsible for Erk1/2 inactivation and to examine the effects of Sb on tumor development in ApcMin mice. We found that the EGF receptor 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 IGF-1R were also found to be inactivated by Sb. Oral intake of Sb reduced intestinal tumor growth and dysplasia in C57BL/6J Min/+ (ApcMin) mice. Thus, in addition to its anti-inflammatory effects, S. boulardii inhibits EGFR and other receptor tyrosine kinase signaling and thereby may also serve a novel therapeutic or prophylactic role in intestinal neoplasia.
Background & Aims
Toll-like receptor (TLR)-dependent signaling pathways have been proposed as immunotherapeutic targets against invading pathogens and tumorigenesis. Here we investigated whether TLR5-dependent signaling modulates colonic tumor development in a mouse xenograft model of human colon cancer.
The expression of MyD88 or TLR5 was stably knocked down in human colon cancer cells (DLD-1). Nude mice were subcutaneously implanted with MyD88-KD, TLR5-KD, or control cells (n=16) to examine the pathophysiology of tumor xenografts. Protein micro-array 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 PECAM-1/CD31, respectively. Tumor xenografts from DLD-1 cells were treated with flagellin (5.0 µg/kg, one injection/every 2 days for 3 weeks) and tumor regression and histopathology of these tumors were examined.
Lack of MyD88 or TLR5 expression dramatically enhanced tumor growth and inhibited tumor necrosis in mouse xenograft of human colon cancer. In contrast, TLR5 activation by peritumoral flagellin treatment substantially increased tumor necrosis, leading to significant tumor regression. Tumors from MyD88- or TLR5-KD cells revealed the reduced production of neutrophil attracting chemokines (ENA-78, MIP3α, and IL-8). Consequently, neutrophil infiltration was dramatically diminished in MyD88 or TLR5 deficient tumor xenografts, while tumor-associated macrophage infiltration or angiogenesis was not changed.
TLR5 engagement by flagellin mediates innate immunity and elicits potent anti-tumor activity, indicating that TLR5-dependent signaling could be a potential immunotherapeutic target to modulate colonic tumors.
Toll-like receptor 5; innate immunity; flagellin; colon cancer; anti-tumor activity
Substance P (SP) participates in acute intestinal inflammation via binding to the G-protein-coupled neurokinin-1 receptor (NK-1R) and release of nuclear factor κ B (NF-κB)-driven proin-flammatory cytokines from colonic epithelial cells. However, the signal transduction pathways by which SP-NK-1R interaction induces NF-κB activation and interleukin-8 (IL-8) production are not clear. Here, we examined participation of protein kinase C (PKC) in SP-induced IL-8 production in human nontransformed NCM460 colonocytes stably transfected with the human NK-1R (NCM460-NK-1R cells). SP (10−7 M) induced an early (1 min) phosphorylation of the PKC isoforms PKCδ, PKCθ, and PKCϵ, followed by I-κB kinase, IκBα, and p65 phosphorylation. Depletion of PKC by phorbol-12-myristate-13-acetate (10 µM) blocked SP-induced IκBα and p65 phosphorylation and IL-8 production. The PKCδ inhibitor rottlerin at a low concentration (1 µM), but not pseudosubstrate PKCθ and PKCϵ inhibitors (10 µM), significantly reduced IL-8 secretion. PKCδ silencing by RNA interference reduced PKCδ protein expression and SP-induced PKCδ phosphorylation that was associated with diminished IL-8 promoter and NF-κB luciferase activities in response to SP. Moreover, overexpression of wild-type PKCδ increased SP-induced IL-8 promoter- and NF-κB-driven luciferase activities that were rottlerin-sensitive. We conclude that PKCδ plays an important role in SP-induced proinflammatory signaling in human colonocytes.
Substance P (SP) via its neurokinin-1 receptor (NK-1R) regulates several gastrointestinal functions. We previously reported that NK-1R-mediated chloride secretion in the colon involves formation of PG. PGE2 biosynthesis is controlled by cyclooxygenase-1 (COX-1) and COX-2, whose induction involves the STATs. In this study, we examined whether SP stimulates PGE2 production and COX-2 expression in human nontransformed NCM460 colonocytes stably transfected with the human NK-1R (NCM460-NK-1R cells) and identified the pathways involved in this response. SP exposure time and dose dependently induced an early (1-min) phosphorylation of JAK2, STAT3, and STAT5, followed by COX-2 expression and PGE2 production by 2 h. Pharmacologic experiments showed that PGE2 production is dependent on newly synthesized COX-2, but COX-1 protein. Inhibition of protein kinase Cθ (PKCθ), but not PKCε and PKCδ, significantly reduced SP-induced COX-2 up-regulation, and JAK2, STAT3, and STAT5 phosphorylation. Pharmacological blockade of JAK inhibited SP-induced JAK2, STAT3, and STAT5 phosphorylation; COX-2 expression; and PGE2 production. Transient transfection with JAK2 short-interferring RNA reduced COX-2 promoter activity and JAK2 phosphorylation, while RNA interference of STAT isoforms showed that STAT5 predominantly mediates SP-induced COX-2 promoter activity. Site-directed mutation of STAT binding sites on the COX-2 promoter completely abolished COX-2 promoter activity. Lastly, COX-2 expression was elevated in colon of mice during experimental colitis, and this effect was normalized by administration of the NK-1R antagonist CJ-12,255. Our results demonstrate that SP stimulates COX-2 expression and PGE2 production in human colonocytes via activation of the JAK2-STAT3/5 pathway.
Clostridium difficile toxin A (TxA), a key mediator of antibiotic-associated colitis, requires binding to a cell surface receptor prior to internalization. Our aim was to identify novel plasma membrane TxA binding proteins on human colonocytes. TxA was coupled with biotin and cross-linked to the surface of HT29 human colonic epithelial cells. The main colonocyte binding protein for TxA was identified as glycoprotein 96 (gp96) by coimmunoprecipitation and mass spectrum analysis. gp96 is a member of the heat shock protein family, which is expressed on human colonocyte apical membranes as well as in the cytoplasm. TxA binding to gp96 was confirmed by fluorescence immunostaining and in vitro coimmunoprecipitation. Following TxA binding, the TxA-gp96 complex was translocated from the cell membrane to the cytoplasm. Pretreatment with gp96 antibody decreased TxA binding to colonocytes and inhibited TxA-induced cell rounding. Small interfering RNA directed against gp96 reduced gp96 expression and cytotoxicity in colonocytes. TxA-induced inflammatory signaling via p38 and apoptosis as measured by activation of BAK (Bcl-2 homologous antagonist/killer) and DNA fragmentation were decreased in gp96-deficient B cells. We conclude that human colonocyte gp96 serves as a plasma membrane binding protein that enhances cellular entry of TxA, participates in cellular signaling events in the inflammatory cascade, and facilitates cytotoxicity.
Clostridium difficile-associated colitis is an increasing cause of morbidity and mortality in hospitalized patients, with high relapse rates following conventional therapy. We sought to determine the efficacy of rifaximin, a novel nonabsorbed antibiotic, in the hamster model of C. difficile-associated diarrhea (CDAD). Hamsters received clindamycin subcutaneously and 24 h later were infected by gavage with one of two C. difficile strains: a reference strain (VPI 10463) and a current epidemic strain (BI17). Vancomycin (50 mg/kg of body weight) or rifaximin (100, 50, and 25 mg/kg) were then administered orally for 5 days beginning either on the same day as infection (prevention) or 24 h later (treatment). Therapeutic effects were assessed by weight gain, histology, and survival. We found that rifaximin was as effective as vancomycin in the prevention and treatment of colitis associated with the two C. difficile strains that we examined. There was no relapse after treatment with vancomycin or rifaximin in hamsters infected with the BI17 strain. Hamsters infected with the VPI 10463 strain and treated with rifaximin did not develop relapsing infection within a month of follow-up, whereas the majority of vancomycin-treated animals relapsed (0% versus 75%, respectively; P < 0.01). In conclusion, rifaximin was found to be an effective prophylactic and therapeutic agent for CDAD in hamsters and was not associated with disease recurrence. These findings, in conjunction with the pharmacokinetic and safety profiles of rifaximin, suggest that it is an attractive candidate for clinical use for CDAD.