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Carcinoembryogenic antigen cellular adhesion molecules (CEACAMs) are intercellular adhesion molecules highly expressed in intestinal epithelial cells. CEACAM1, -3, -5, -6, -7 are altered in patients suffering from colon cancer and inflammatory bowel diseases (IBD), but their role in the onset and pathogenesis of IBD is not well known. Herein, we aim to correlate CEACAM1, -3, -5, -6, -7 expression to the degree of inflammation in pediatric and adult IBD colon biopsies and to examine the regulation of CEACAMs on human intestinal epithelial cell lines (C2BBe1/HT29) by different IBD-associated triggers (cytokines, bacteria/metabolites, emulsifiers) and IBD-drugs (6-Mercaptopurine, Prednisolone, Tofacitinib). Biopsies from patients with pediatric Crohn's disease (CD) and adult ulcerative colitis (UC, active/inactive disease) showed a significant increase in CEACAM3, -5, -6 expression, while CEACAM5 expression was reduced in adult CD patients (active/inactive disease). Intestinal epithelial cells cultured with a pro-inflammatory cytokine cocktail and Adherent-invasive Escherichia coli (AIEC) showed a rapid induction of CEACAM1, -5, -7 followed by a reduced RNA and protein expression overtime and a constant expression of CEACAM3, correlating with IL-8 expression. Cells cultured with the emulsifier polysorbate-80 resulted in a significant induction of CEACAM3, -5, -6, -7 at a late time point, while SCFA treatment reduced CEACAM1, -5, -7 expression. No major alterations in expression of CEACAMs were noted on cells cultured with the commensal Escherichia coli K12 or the pathogen Salmonella typhimurium . IBD drugs, particularly Tofacitinib, significantly reduced cytokine-induced CEACAM1, -3, -5, -6, -7 expression associated with a reduced IL-8 secretion. In conclusion, we provide new evidence on the regulation of CEACAMs by different IBD-associated triggers, identifying a role of CEACAMs in IBD pathogenesis., Competing Interests: FS - Co-founder/shareholder of Alimentary Health Ltd, Tucana Health Ltd and Atlantia Food Clinical Trials Ltd. Scientific advisor to Kaleido Biosciences. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Saiz-Gonzalo, Hanrahan, Rossini, Singh, Ahern, Kelleher, Hill, O’Sullivan, Fanning, Walsh, Hussey, Shanahan, Nally, O’Driscoll and Melgar.)

Proteins of the BCL-2 family are evolutionarily conserved modulators of apoptosis that function as sensors of cellular integrity. Over the past three decades multiple BCL-2 family members have been identified, many of which are now fully incorporated into regulatory networks governing the mitochondrial apoptotic pathway. For some, however, an exact role in cell death signalling remains unclear. One such 'orphan' BCL-2 family member is BCL-G (or BCL2L14). In this study we analysed gastrointestinal expression of human BCL-G in health and disease states, and investigated its contribution to inflammation-induced tissue damage by exposing intestinal epithelial cells (IEC) to IFN-γ and TNF-α, two pro-inflammatory mediators associated with gut immunopathology. We found that both BCL-G splice variants - BCL-G S (short) and BCL-G L (long) - were highly expressed in healthy gut tissue, and that their mRNA levels decreased in active inflammatory bowel diseases (for BCL-G S ) and colorectal cancer (for BCL-G S/L ). In vitro studies revealed that IFN-γ and TNF-α synergised to upregulate BCL-G S/L and to trigger apoptosis in colonic epithelial cell lines and primary human colonic organoids. Using RNAi, we showed that synergistic induction of IEC death was STAT1-dependent while optimal expression of BCL-G S/L required STAT1, NF-κB/p65 and SWI/SNF-associated chromatin remodellers BRM and BRG1. To test the direct contribution of BCL-G to the effects of IFN-γ and TNF-α on epithelial cells, we used RNAi- and CRISPR/Cas9-based perturbations in parallel with isoform-specific overexpression of BCL-G, and found that BCL-G was dispensable for Th1 cytokine-induced apoptosis of human IEC. Instead, we discovered that depletion of BCL-G differentially affected secretion of inflammatory chemokines CCL5 and CCL20, thus uncovering a non-apoptotic immunoregulatory function of this BCL-2 family member. Taken together, our data indicate that BCL-G may be involved in shaping immune responses in the human gut in health and disease states through regulation of chemokine secretion rather than intestinal apoptosis.

Development of the human gut microbiota commences at birth, with certain bifidobacterial species representing dominant and early colonisers of the newborn gastrointestinal tract. The molecular basis of Bifidobacterium colonisation, persistence and presumed communication with the host has remained obscure. We previously identified tight adherence (Tad) pili from Bifidobacterium breve UCC2003 as an essential colonisation factor. Here, we demonstrate that bifidobacterial Tad pili also promote in vivo colonic epithelial proliferation. A significant increase in cell proliferation was detectable 5 days postadministration of B. breve UCC2003. Using advanced functional genomic approaches, bacterial strains either (a) producing the Tad 2003 pili or (b) lacking the TadE or TadF pseudopilins were created. Analysis of the ability of these mutant strains to promote epithelial cell proliferation in vivo demonstrated that the pilin subunit, TadE, is the bifidobacterial molecule responsible for this proliferation response. These findings were confirmed in vitro using purified TadE protein. Our data imply that bifidobacterial Tad pili may contribute to the maturation of the naïve gut in early life through the production of a specific scaffold of extracellular protein structures, which stimulate growth of the neonatal mucosa., (© 2018 John Wiley & Sons Ltd.)

Colonic inflammation is associated with decreased tissue oxygenation, significantly affecting gut homeostasis. However, the crosstalk between O 2 consumption and supply in the inflamed tissue are not fully understood. Using a murine model of colitis, we analysed O 2 in freshly prepared samples of healthy and inflamed colon tissue. We developed protocols for efficient ex vivo staining of mouse distal colon mucosa with a cell-penetrating O 2 sensitive probe Pt-Glc and high-resolution imaging of O 2 concentration in live tissue by confocal phosphorescence lifetime-imaging microscopy (PLIM). Microscopy analysis revealed that Pt-Glc stained mostly the top 50-60 μm layer of the mucosa, with high phosphorescence intensity in epithelial cells. Measured O 2 values in normal mouse tissue ranged between 5 and 35 μM (4-28 Torr), tending to decrease in the deeper tissue areas. Four-day treatment with dextran sulphate sodium (DSS) triggered colon inflammation, as evidenced by an increase in local IL6 and mKC mRNA levels, but did not affect the gross architecture of colonic epithelium. We further observed an increase in oxygenation, partial activation of hypoxia inducible factor (HIF) 1 signalling, and negative trends in pyruvate dehydrogenase activity and O 2 consumption rate in the colitis mucosa, suggesting a decrease in mitochondrial respiration, which is known to be regulated via HIF-1 signalling and pyruvate oxidation rate. These results along with efficient staining with Pt-Glc of rat and human colonic mucosa reveal high potential of PLIM platform as a powerful tool for the high-resolution analysis of the intestinal tissue oxygenation in patients with inflammatory bowel disease and other pathologies, affecting tissue respiration.

A role for the IL-36 family of cytokines has been identified in the pathogenesis of psoriasis. Although significant mechanistic overlap can exist between psoriasis and inflammatory bowel disease (IBD), to date there have been no reports investigating the IL-36 family in gastrointestinal inflammation. Here we demonstrate that expression levels of IL-36α are specifically elevated in the colonic mucosa of ulcerative colitis patients. This elevated expression is mirrored in the inflamed colonic mucosa of mice, wherein IL-36 receptor deficiency confirmed this pathway as a mediator of mucosal inflammation. Il36r-/- mice exhibited reduced disease severity in an acute DSS-induced model of colitis in association with decreased innate inflammatory cell infiltration to the colon lamina propria. Consistent with these data, infection with the enteropathogenic bacteria Citrobacter rodentium, resulted in reduced innate inflammatory cell recruitment and increased bacterial colonization in the colons of il36r-/- mice. Il36r-/- mice also exhibited altered T helper cell responses in this model, with enhanced Th17 and reduced Th1 responses, demonstrating that IL-36R signaling also regulates intestinal mucosal T-cell responses. These data identify a novel role for IL-36 signaling in colonic inflammation and indicate that the IL-36R pathway may represent a novel target for therapeutic intervention in IBD.

The main aim of the present study was to investigate the effects of dietary trans-10, cis-12-conjugated linoleic acid (t10c12-CLA) on intestinal microbiota composition and SCFA production. C57BL/6 mice (n 8 per group) were fed a standard diet either supplemented with t10c12-CLA (0·5 %, w/w) (intervention) or with no supplementation (control), daily for 8 weeks. Metabolic markers (serum glucose, leptin, insulin and TAG, and liver TAG) were assessed by ELISA commercial kits, tissue long-chain fatty acids and caecal SCFA by GC, and microbial composition by 16S rRNA pyrosequencing. Dietary t10c12-CLA significantly decreased visceral fat mass (P< 0·001), but did not affect body weight (intervention), when compared with no supplementation (control). Additionally, lipid mass and composition were affected by t10c12-CLA intake. Caecal acetate, propionate and isobutyrate concentrations were higher (P< 0·05) in the t10c12-CLA-supplemented group than in the control group. The analysis of the microbiota composition following 8 weeks of t10c12-CLA supplementation revealed lower proportions of Firmicutes (P= 0·003) and higher proportions of Bacteroidetes (P= 0·027) compared with no supplementation. Furthermore, t10c12-CLA supplementation for 8 weeks significantly altered the gut microbiota composition, harbouring higher proportions of Bacteroidetes, including Porphyromonadaceae bacteria previously linked with negative effects on lipid metabolism and induction of hepatic steatosis. These results indicate that the mechanism of dietary t10c12-CLA on lipid metabolism in mice may be, at least, partially mediated by alterations in gut microbiota composition and functionality.

More microbes are resident in the distal colon than any other part of the body, and this microbiota has the capacity to influence enteric nerve development, excitability, and gastrointestinal function. Germ-free (GF) mice are a valuable tool in interrogating the communication between microbiota and host. Despite the intimate relationship which exists between the microbiota and the colonic mucosa-submucosa, there is a paucity of studies examining the influence of the microbiota on secretogogue-evoked responses. To this end, we investigated both epithelial and neural-evoked ion transport, and the response elicited by two commensal organisms, in colonic mucosa-submucosa preparations from GF mice in Ussing chambers. Baseline electrical parameters, short-circuit current and transepithelial resistance, were comparable between tissues from GF and conventional animals. Noteworthy, however, was a hyper-responsiveness of GF colon to forskolin stimulation. In contrast, the absence of the microbiota did not influence the tissue response to bethanechol. Moreover, responses to the sodium-channel activator, veratridine, and the TRPV1 receptor agonist, capsaicin were preserved in GF mice relative to conventional tissues. Similarly, the short-circuit current response to two well-characterized commensal organisms occurred independent of an interaction with the host microbiota. This is the first comprehensive characterization of secretomotor responses in GF colon., (© 2014 John Wiley & Sons Ltd.)

Listeria monocytogenes is a Gram-positive bacterium that can cause septicemia and meningitis. TLRs are central receptors of the innate immune system that drive inflammatory responses to invading microbes such as L. monocytogenes. Although intestinal epithelial cells (IECs) represent the initial point of entry used by L. monocytogenes for infection, the innate immune response to L. monocytogenes in these cells has been poorly characterized to date. The aim of this study was to determine which TLRs are involved in mediating the immune response to L. monocytogenes in IECs. We performed an RNA interference screen of TLRs 1-10 in the HT-29 IEC cell line and observed the most significant reduction in chemokine output following silencing of TLR10. This effect was also observed in the macrophage cell line THP-1. The chemokines CCL20, CCL1, and IL-8 were reduced following knockdown of TLR10. Silencing of TLR10 resulted in increased viability of L. monocytogenes in both HT-29 and THP-1 cells. TLR10 was found to be predominantly expressed intracellularly in epithelia, and activation required viable L. monocytogenes. NF-κB activation was seen to require TLR2 in addition to TLR10. Taken together, these data indicate novel roles for TLR10 in sensing pathogenic infection in both the epithelium and macrophages and have identified L. monocytogenes as a source of ligand for the orphan receptor TLR10.

Enhanced barrier function is one mechanism whereby commensals and probiotic bacteria limit translocation of foreign antigens or pathogens in the gut. However, barrier protection is not exhibited by all probiotic or commensals and the strain-specific molecules involved remain to be clarified. We evaluated the effects of 33 individual Lactobacillus salivarius strains on the hydrogen peroxide (H(2)O(2))-induced barrier impairment in human epithelial Caco-2 cells. These strains showed markedly different effects on H(2)O(2)-induced reduction in transepithelial resistance (TER). The effective strains such as UCC118 and CCUG38008 attenuated H(2)O(2)-induced disassembly and relocalization of tight junction proteins, but the ineffective strain AH43324 did not. Strains UCC118 and CCUG38008 induced phosphorylation of extracellular signal-regulated kinase (ERK) in Caco-2 cells, and the ERK inhibitor U0126 attenuated the barrier-protecting effect of these strains. In contrast, the AH43324 strain induced phosphorylation of Akt and p38, which was associated with an absence of a protective effect. Global transcriptome analysis of UCC118 and AH43324 revealed that some genes in a bacteriocin gene cluster were upregulated in AH43324 under TER assay conditions. A bacteriocin-negative UCC118 mutant displayed significantly greater suppressive effect on H(2)O(2)-induced reduction in TER compared with wild-type UCC118. The wild-type strain augmented H(2)O(2)-induced phosphorylation of Akt and p38, whereas a bacteriocin-negative UCC118 mutant did not. These observations indicate that L. salivarius strains are widely divergent in their capacity for barrier protection, and this is underpinned by differences in the activation of intracellular signaling pathways. Furthermore, bacteriocin production appears to have an attenuating influence on lactobacillus-mediated barrier protection.

Different rates of bacterial translocation across the gut mucosa have been reported but few studies have examined translocation of commensals at the level of the gut epithelial microfold (M) cell. We used an in vitro M-cell model to quantify translocation and determine the transcriptional response of M cells to various commensal bacteria. The transport kinetics and gene expression profile of M cells in response to different bacterial strains, namely Lactobacillus salivarius, Escherichia coli and Bacteroides fragilis, was assessed. Bacterial strains translocated across M cells with different efficiencies; E. coli and B. fragilis translocated with equal efficiency whereas L. salivarius translocated with less efficiency. Microarray analysis of the M cell response showed both common and differential gene expression changes between the bacterial strains. In the presence of bacteria, but not control beads, up-regulated genes were mainly involved in transcription regulation whereas pro-inflammatory and stress response genes were primarily up-regulated by E. coli and B. fragilis, but not L. salivarius nor beads. Translocation of bacteria and M-cell gene expression responses were confirmed in murine M cells following bacterial challenge in vivo. These results demonstrate that M cells have the ability to discriminate between different commensal bacteria and modify subsequent immune responses., (Published 2012. This article is a U.S. Government work and is in the public domain in the USA.)

Background & Aims: Matrix metalloproteases (MMPs) mediate pathogenesis of chronic intestinal inflammation. We characterized the role of the gelatinase (GelE), a metalloprotease from Enterococcus faecalis, in the development of colitis in mice., Methods: Germ-free, interleukin-10-deficient (IL-10(-/-)) mice were monoassociated with the colitogenic E faecalis strain OG1RF and isogenic, GelE-mutant strains. Barrier function was determined by measuring E-cadherin expression, transepithelial electrical resistance (TER), and translocation of permeability markers in colonic epithelial cells and colon segments from IL-10(-/-) and TNF(ΔARE/Wt) mice. GelE specificity was shown with the MMP inhibitor marimastat., Results: Histologic analysis (score 0-4) of E faecalis monoassociated IL-10(-/-) mice revealed a significant reduction in colonic tissue inflammation in the absence of bacteria-derived GelE. We identified cleavage sites for GelE in the sequence of recombinant mouse E-cadherin, indicating that it might be degraded by GelE. Experiments with Ussing chambers and purified GelE revealed the loss of barrier function and extracellular E-cadherin in mice susceptible to intestinal inflammation (IL-10(-/-) and TNF(ΔARE/Wt) mice) before inflammation developed. Colonic epithelial cells had reduced TER and increased translocation of permeability markers after stimulation with GelE from OG1RF or strains of E faecalis isolated from patients with Crohn's disease and ulcerative colitis., Conclusions: The metalloprotease GelE, produced by commensal strains of E faecalis, contributes to development of chronic intestinal inflammation in mice that are susceptible to intestinal inflammation (IL-10(-/-) and TNF(ΔARE/Wt) mice) by impairing epithelial barrier integrity., (Copyright © 2011 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Crohn's disease and ulcerative colitis are inflammatory bowel diseases (IBD) characterized by chronic relapsing mucosal inflammation. Tumour necrosis factor (TNF)-α, a known agonist of the mitogen-activated protein kinase (MAPK) pathway, is a key cytokine in this process. We aimed first to determine whether p38 MAPK is activated in IBD inflamed mucosa, and then studied the effect of four different p38α inhibitory compounds on MAPK phosphorylation and secretion of proinflammatory cytokines by IBD lamina propria mononuclear cells (LPMCs) and organ culture biopsies. In vivo phospho-p38α and p38α expression was evaluated by immunoblotting on intestinal biopsies from inflamed areas of patients affected by Crohn's disease and ulcerative colitis, and from normal mucosa of sex- and age-matched control subjects. Both mucosal biopsies and isolated LPMCs were incubated with four different p38α selective inhibitory drugs. TNF-α, interleukin (IL)-1β and IL-6 were measured in the organ and cell culture supernatants by enzyme-linked immunosorbent assay. We found higher levels of phospho-p38α in the inflamed mucosa of IBD patients in comparison to controls. All the p38α inhibitory drugs inhibited p38α phosphorylation and secretion of TNF-α, IL-1β and IL-6 from IBD LPMCs and biopsies. Activated p38α MAPK is up-regulated in the inflamed mucosa of patients with IBD. Additionally, all the p38α selective inhibitory drugs significantly down-regulated the activation of the MAPK pathway and the secretion of proinflammatory cytokines., (© 2010 The Authors. Clinical and Experimental Immunology © 2010 British Society for Immunology.)

Purpose: The objectives of this study were, firstly, to determine the diversity of the host's gut microbiota in irritable bowel syndrome (IBS) using a culture-independent method (DGGE of the 16S rRNA gene) and, secondly, to examine mucosal biopsies of IBS patients and compare them to their own fecal microbiota., Methods: The diversity of the dominant microbiota in the fecal material of IBS patients was compared to a healthy control group. In addition, we compared the mucosal and fecal microbiota of IBS patients., Results: Statistical analysis of the mean similarity data for these groups indicated a significant difference (P < 0.001) between IBS (n = 47) and healthy controls (n = 33) with significantly more variation in the gut microbiota of healthy volunteers than that of IBS patients. The average intra-individual similarity between the mucosa and luminal microbiota was 84%, which indicates that different communities were present at the two sites. This difference, however, is similar to that previously described between these two niches in control subjects. The average inter-individual similarity of the bacterial communities on the mucosa and in the lumen of IBS was not significantly different (P > 0.05)., Conclusions: IBS impacts equally on both bacterial communities in the IBS host and a significant difference in the gut microbiota exists between fecal samples from IBS patients and healthy controls. The reason for this difference is unclear and various possible explanations are available, but much more work is required to determine the underlying reason for this observation.

Background: Human intestinal epithelial cells (IECs) secrete the chemokine CCL20 in response to infection by various enteropathogenic bacteria or exposure to bacterial flagellin. CCL20 recruits immature dendritic cells and lymphocytes to target sites. Here we investigated IEC responses to various pathogenic and commensal bacteria as well as the modulatory effects of commensal bacteria on pathogen-induced CCL20 secretion. HT-29 human IECs were incubated with commensal bacteria (Bifidobacterium infantis or Lactobacillus salivarius), or with Salmonella typhimurium, its flagellin, Clostridium difficile, Mycobacterium paratuberculosis, or Mycobacterium smegmatis for varying times. In some studies, HT-29 cells were pre-treated with a commensal strain for 2 hr prior to infection or flagellin stimulation. CCL20 and interleukin (IL)-8 secretion and nuclear factor (NF)-kappaB activation were measured using enzyme-linked immunosorbent assays., Results: Compared to untreated cells, S. typhimurium, C. difficile, M. paratuberculosis, and flagellin activated NF-kappaB and stimulated significant secretion of CCL20 and IL-8 by HT-29 cells. Conversely, B. infantis, L. salivarius or M. smegmatis did not activate NF-kappaB or augment CCL20 or IL-8 production. Treatment with B. infantis, but not L. salivarius, dose-dependently inhibited the baseline secretion of CCL20. In cells pre-treated with B. infantis, C. difficile-, S. typhimurium-, and flagellin-induced CCL20 were significantly attenuated. B. infantis did not limit M. Paratuberculosis-induced CCL20 secretion., Conclusion: This study is the first to demonstrate that a commensal strain can attenuate CCL20 secretion in HT-29 IECs. Collectively, the data indicate that M. paratuberculosis may mediate mucosal damage and that B. infantis can exert immunomodulatory effects on IECs that mediate host responses to flagellin and flagellated enteric pathogens.

Objective: To systematically examine mucosal biopsies for differences in cytokine gene expression and protein secretion., Material and Methods: The study included 59 females with irritable bowel syndrome (IBS) and 39, otherwise healthy, female volunteers presenting for colonoscopy. Colonic biopsies from subsets were studied by microarray analysis (IBS, n=9; controls, n=8), quantitative reverse transcription-polymerase chain reaction (qRT-PCR) (IBS, n=22; controls, n=21), and ex vivo biopsy culture (IBS, n=28, controls, n=10). Biopsies from patients with active colitis were used as inflammatory disease controls., Results: While gene array analysis revealed extensive overlapping between controls and IBS patients, reduced expression of genes linked to chemokine function was evident among the IBS patients alone. Differential expression was confirmed by qRT-PCR or ex vivo biopsy culture for 5 out of 6 selected genes. Reduced secretion of chemokines (IL-8, CXCL-9 and MCP-1) but not pro-inflammatory cytokines (TNF-alpha, IL-6 and IL-1beta) was established on the basis of the ex vivo biopsy cultures. These findings were in marked contrast to the IBD patients who demonstrated increased production of both chemokines and pro-inflammatory cytokines., Conclusions: Despite the expected heterogeneity of the disorder, differences in mucosal chemokine signalling were evident in this cross-sectional study of IBS patients at the level of both gene expression and protein secretion, with IBS patients demonstrating a consistent deficit in the expression and secretion of chemokines known to play a critical role in mucosal defence.

Intestinal microbiota is a positive health asset that exerts a conditioning effect on intestinal homeostasis. Resident bacteria deliver regulatory signals to the epithelium and instruct mucosal immune responses. Recent research has revealed a potential therapeutic role for the manipulation of the microbiota and exploitation of host-microbial signalling pathways in the maintenance of human health and treatment of various mucosal disorders. A variety of pharmabiotic strategies, such as the use of specific members of the microbiota, their surface components, or metabolites, as well as genetically modified commensal bacteria, are being investigated for their ability to enhance the beneficial components of the microbiota. It is clear that engagement with host cells is central to pharmabiotic action, and several strain-specific mechanisms of action have been elucidated. However, the molecular details underpinning these mechanisms remain almost entirely unknown. Understanding how pharmabiotics exert their beneficial effects is critical for the establishment of definitive selection criteria for certain pharmabiotic strategies for specific clinical conditions. Scientifically accredited evidence of efficacy and studies to elucidate the molecular mechanisms of host-microbiota interactions are needed to lend credence to the use of pharmabiotic strategies in clinical medicine.

Intestinal epithelial cells (IECs) and dendritic cells (DCs) play a pivotal role in antigen sampling and the maintenance of gut homeostasis. However, the interaction of commensal bacteria with the intestinal surface remains incompletely understood. Here we investigated immune cell responses to commensal and pathogenic bacteria. HT-29 human IECs were incubated with Bifidobacterium infantis 35624, Lactobacillus salivarius UCC118 or Salmonella typhimurium UK1 for varying times, or were pretreated with a probiotic for 2 hr prior to stimulation with S. typhimurium or flagellin. Gene arrays were used to examine inflammatory gene expression. Nuclear factor (NF)-kappaB activation, interleukin (IL)-8 secretion, pathogen adherence to IECs, and mucin-3 (MUC3) and E-cadherin gene expression were assayed by TransAM assay, enzyme-linked immunosorbent assay (ELISA), fluorescence, and real-time reverse transcriptase-polymerase chain reaction (RT-PCR), respectively. IL-10 and tumour necrosis factor (TNF)-alpha secretion by bacteria-treated peripheral blood-derived DCs were measured using ELISA. S. typhimurium increased expression of 36 of the 847 immune-related genes assayed, including NF-kappaB and IL-8. The commensal bacteria did not alter expression levels of any of the 847 genes. However, B. infantis and L. salivarius attenuated both IL-8 secretion at baseline and S. typhimurium-induced pro-inflammatory responses. B. infantis also limited flagellin-induced IL-8 protein secretion. The commensal bacteria did not increase MUC3or E-cadherin expression, or interfere with pathogen binding to HT-29 cells, but they did stimulate IL-10 and TNF-alpha secretion by DCs. The data demonstrate that, although the intestinal epithelium is immunologically quiescent when it encounters B. infantis or L. salivarius, these commensal bacteria exert immunomodulatory effects on intestinal immune cells that mediate host responses to flagellin and enteric pathogens.

This paper aims to examine the contribution of all three of the MAP kinase signaling pathways to Fas-induced IL-8 up-regulation in HT29 colon epithelial cells.

Introduction: Ileal pouch-anal anastomosis (IPAA) remains the gold standard for patients with refractory ulcerative colitis. Pouchitis causes considerable morbidity in 40% of patients with IPAA. This study examined the role of increased epithelial apoptosis in the etiology of pouchitis., Methods: Following ethical approval pouch biopsies taken from patients with a history of pouchitis were compared with age-matched controls from patients who were pouchitis free. Apoptosis was detected immunohistochemically using a monoclonal antibody (M30) and terminal deoxyribonucleotidyl transferase (TDT)-mediated dUTP-digoxigenin end labeling (TUNEL). Villous atrophy was assessed histologically and correlated with levels of apoptosis. Epithelial Fas-ligand (L) was also assessed immunohistochemically., Results: A significant increase in TUNEL staining was seen at the epithelial but not at the lamina propria level for known pouchitis patients versus controls (0.091 vs 0.035; P < 0.01). Similarly, epithelial M30 immunoreactivity (0.225 vs 0.082; P < 0.05) and villous atrophy (0.035 vs 0.10; P < 0.05) were significantly increased in pouches with previous pouchitis when compared with normal pouches. Upregulation of Fas-L expression was characteristic of this epithelium. Mononuclear cells were strongly positive for Fas-L. Increased epithelial levels of apoptosis correlated with increased levels of villous atrophy., Conclusions: Our data suggest a role for elevated Fas-Fas-L (CD95-CD95L)-mediated epithelial apoptosis in the etiology of pouchitis. Increased levels of villous atrophy may result from increased apoptosis and thereby predispose to infection by otherwise apathogenic organisms., (Copyright 2001 Academic Press.)

Homeostasis in the colonic epithelium is achieved by a continuous cycle of proliferation and apoptosis, in which imbalances are associated with disease. Inflammatory bowel disease (IBD) and colon cancer are associated with either excessive or insufficient apoptosis of colonic epithelial cells, respectively. By using two colonic epithelial cell lines, HT29 and SW620, we investigated how the epithelial cell's sensitivity to apoptosis was regulated by the proinflammatory cytokine interferon-gamma (IFN-gamma). We found that IFN-gamma sensitized HT29 cells, and to a lesser extent SW620, to diverse inducers of apoptosis of physiologic or therapeutic relevance to the colon. These apoptosis inducers included Fas (CD95/APO-1) ligand (FasL), short-chain fatty acids, and chemotherapeutic drugs. The extent of IFN-gamma-mediated apoptosis sensitization in these two cell lines correlated well with the degree of IFN-gamma-mediated upregulation of the proapoptotic protease caspase-1. Although IFN-gamma alone effectively sensitized HT29 cells to apoptosis, inclusion of the protein synthesis inhibitor cyclohexamide (CHX) during apoptotic challenge was necessary for maximal sensitization of SW620. The requirement of CHX to sensitize SW620 cells to apoptosis implies a need to inhibit translation of antiapoptotic proteins absent from HT29. In particular, the antiapoptotic protein Bcl-2 was strongly expressed in SW620 cells but absent from HT29. Our results indicate that IFN-gamma increases the sensitivity of colonic epithelial cells to diverse apoptotic stimuli in concert, via upregulation of caspase-1. Our findings implicate caspase-1 and Bcl-2 as important central points of control determining the general sensitivity of colonic epithelial cells to apoptosis., (Copyright 2000 Wiley-Liss, Inc.)

The close anatomic juxtaposition of epithelial cells with lymphocytes lining the intestinal tract facilitates communication between the two cell types. This intercellular dialogue is important for mucosal development and has a conditioning effect on mucosal structure, function, and response to tissue injury. Lymphoepithelial communication is bi-directional, and mediated in large part, by shared ligands and receptors. The chemical messengers involved include cytokines, growth factors, local hormones, and products of arachidonate metabolism. The interdependency between the epithelium and adjacent lymphoid cells is such that the epithelium is considered to have a central role in the mucosal immune system and is an active participant in both the afferent and efferent limbs of the mucosal immune response. The molecular crosstalk between the epithelium and adjacent lymphocytes is just one aspect of a more complex network of intercellular signalling within the intestinal mucosa and upon which the integrity of the mucosa is dependent. Thus, there are extensive interactions between nerve and immune cells and between the enteric flora and the epithelium and amongst intestinal mesenchymal cells including fibroblasts and vascular endothelial cells. Disruption of any aspect of the mucosal microenvironment, as has been achieved with selective genetically engineered murine models, is associated with impaired mucosal defence and inflammation.

Reciprocal communication between the immune system and the neuroendocrine system is mediated via a common chemical language of shared ligands and receptors. The neuropeptide substance P (SP) has been implicated as a mediator of immunomodulation. The evidence for substance P receptors on human lymphocytes is, however, controversial. The aims of the present study are to investigate substance P receptor (SPR) expression in human peripheral and mucosal mononuclear cells and to identify cellular sites of expression in human colonic mucosa. Using reverse-transcriptase PCR, we demonstrate that PBMC isolations are negative for SPR mRNA expression, whereas lamina propria mononuclear cell (LPMC) isolations express on average eight SPR mRNA transcripts per cell. In situ hybridization performed on surgically resected colonic tissue confirms the expression of SPR mRNA in LPMC in vivo. SPR mRNA signal was detected in LPMC, lymphoid follicles, and epithelium. The complementary technique of immunohistochemistry gave a similar distribution of SPR expression that colocalized with CD45 immunoreactivity. Dual-fluorochrome flow cytometry revealed SPR expression by CD4, CD45RO, CD45RA, CD8, CD19, and CD14 LPMC subsets, but not PBMC. Our findings suggest that SPR expression is distinctive of human colonic mucosal mononuclear cells and support a direct role for SP in mucosal immunomodulation.

Background: The bacterial chemotactic peptide N-formyl-methionine-leucine-phenylalanine (FMLP) is produced by enteric flora and is one of the factors implicated in contributing to inflammatory bowel disease. Expression of receptors for FMLP on human phagocytes (polymorphs and monocytes) is well established, but there is conflicting evidence regarding the potential expression of FMLP receptors on other cells within the mucosa, particularly the epithelial cells., Aims: To map FMLP receptors within intestinal mucosa using several different experimental approaches., Methods and Results: Radioligand binding assays with 'H-FMLP' revealed no specific binding to primary cultured colonic enterocytes or to the cell line HT29, whereas neutrophils, as expected, exhibited specific binding with a Kd of 19 nM and approximately 2 x 10(4) receptors per cell. FITC labelled FMLP exhibited specific, displaceable binding on flow cytometry to neutrophils and monocytes but not to 10 gastrointestinal epithelial cell lines. Isolated lamina propria lymphocytes and peripheral blood lymphocytes exhibited no binding. To confirm the absence of receptors on epithelia, reverse transcription polymerase chain reaction for mRNA for the classic FMLP receptor was performed. While the presence of message was detected in activated peripheral blood phagocytes, it was not detected in epithelial cell lines. To exclude the possibility of FMLP binding to other receptors such as tachykinin receptors on epithelia, FITC labelled FMLP binding in tissue sections confirmed that the binding is subepithelial--that is, in the lamina propria., Conclusion: Receptors for FMLP are subepithelial and map to the lamina propria of the gastrointestinal mucosa.

Conventional wisdom dictates that ulcerative colitis affects contiguous areas of the colon and is most severe in the rectum, and that the finding of rectal sparing or patchy involvement should raise suspicions of Crohn's disease. We and others have noted occasional rectal sparing and patchy involvement in patients with ulcerative colitis. Therefore, we prospectively studied the prevalence of patchiness, including rectal sparing, in treated cases of ulcerative colitis. Consecutive patients with longstanding ulcerative colitis were studied. The left colon was divided into three zones for scoring degree of activity, and biopsy specimens from each zone were graded for histologic activity by a blinded observer. Patchiness by endoscopy or histology was defined as (1) frank rectal sparing (normal appearance endoscopically; absence of inflammation of the lamina propria and crypts histologically); (2) areas of greater inflammation proximally than distally; or (3) discrete areas of patchiness endoscopically within any one zone. Of 39 patients evaluated, 17 (44%) had endoscopic evidence of patchiness, including 5 (13%) with rectal sparing. Thirteen (33%) had histologic evidence of patchiness, including 6 (15%) with rectal sparing. Both endoscopic and histologic patchiness were seen in 9 patients (23%). The patchy and nonpatchy groups did not differ in regard to the use of rectal therapy. In patients with treated ulcerative colitis, the finding of rectal sparing or patchiness should not necessarily indicate a change in the diagnosis to Crohn's disease.

T cell activation can lead to local tissue injury in organ culture studies of human fetal jejunum, either directly through cytotoxicity or indirectly by the release of cytotoxic cytokines. The goal of this study was to establish in vitro whether cytotoxic cytokines can be released by isolated colonic T cells and what cytokine interactions are required for killing of human colonic epithelial cells. Cytokine-containing supernatants were induced by incubating unseparated lamina propria lymphocytes (LPL) or mucosal T cell subpopulations (separated by indirect panning) with anti-CD3 and/or K562 target cells for 18 h at 37 degrees C. Cytokines were measured by cytotoxicity assays using L929 (murine fibroblast) and HT-29 (human colonic tumour) lines as target cells in combination with blocking anti-cytokine antibodies. Supernatants derived from unseparated, CD4+ (greater than 95% pure) and CD8+ (greater than 90% pure) LPL were cytotoxic to L929 targets (350 U/ml, 230 U/ml and 100 U/ml tumour necrosis factor-alpha, respectively). All or nearly all of the cytotoxicity was due to the presence of tumour necrosis factor-alpha (little or no tumour necrosis factor-beta was detected). These same supernatants were cytotoxic (up to 32% lysis at 1/4 dilution) to HT-29 targets in a 48-h 111In release assay. Recombinant tumour necrosis factor-alpha and interferon-gamma alone produced minimal killing of HT-29, but together killed the HT-29 target cells. Anti-tumour necrosis factor-alpha or anti-interferon-gamma alone blocked killing of HT-29 target cells by LPL-derived supernatants, although anti-tumour necrosis factor-beta had no effect upon killing of HT-29. These results demonstrate that human LPL T cells, triggered by addition of anti-CD3 and target cells, produce tumour necrosis factor-alpha and interferon-gamma, both of which are required for optimal killing of HT-29. Simultaneous release of these cytokines in the vicinity of epithelial cells during immune responses could play an important role in the mucosal damage in chronic inflammatory states such as inflammatory bowel disease.

Mucosal mast cell (MMC) suspensions obtained from the rat intestinal lamina propria by collagenase digestion (35.2 +/- 3.2% MMC) were enriched to 65.5 +/- 5.2% MMC by the use of a discontinuous gradient (30%/80%) of Percoll. Further purification to 95.7 +/- 1.3% MMC was achieved using velocity sedimentation at unit gravity (Sta-Put). Analysis of the cells throughout the purification procedure confirms that the purified MMC are representative of the MMC in the initial isolated cell suspension. No differences were seen in terms of size, histamine content, protease content and responsiveness to secretagogues among the initial isolated population, the Percoll-enriched population and the Sta-Put-purified population. This study represents a major advance in mast cell research in that, for the first time, mast cells isolated from a homogeneous in vivo mucosal source have been obtained at levels of purity sufficient for specific biochemical characterization. Such characterization will aid in the interpretation of the role of MMC in disease and will provide a firm basis of knowledge of the form and function of intestinal MMC for comparison with mast cells derived from other mucosal sites or cultured in vitro from various organs.

Recent reports suggesting that the actions of certain neuroenteric peptides may be mediated in part by the secretion of histamine and other mast cell contents could have important implications for gastrointestinal motility and secretion. However, evidence for a mast cell-hormonal interaction is based on studies using peritoneal or cutaneous mast cells. Because intestinal mucosal mast cells (MMC) differ functionally from peritoneal mast cells (PMC), we compared the effects of several neurotransmitters and intestinal hormones on histamine secretion from two mast cell types in the rat. MMC hyperplasia was induced in rats by infection with the nematode Nippostrongylus brasiliensis, and MMC were isolated from the small intestine by collagenase digestion. Substance P, somatostatin, vasoactive intestinal polypeptide (VIP), neurotensin, and bradykinin had a potent secretagogue effect on (10(-7) to 10(-4)M) PMC which was temperature-, energy-, and calcium-dependent. In contrast to PMC, MMC released significant amounts of histamine only when challenged with substance P. Acetylcholine, bombesin, motilin, and pentagastrin had no secretory effect on either PMC or MMC. The differences between PMC and MMC in responsiveness to peptides could not be attributed to the MMC isolation procedure because PMC treated similarly or mixed with MMC suspensions retained their responsiveness to these stimuli. Our results extend the concept of neurocrine control of mast cell function, but indicate that mast cells from different sites have distinct profiles of responsiveness to regulatory peptides.

Non-major histocompatibility complex-restricted cytotoxicity triggered by antibodies to the CD3 component of the human T-cell receptor complex is thought to be an indirect measure of in vivo primed cytotoxic T-cell activity. We have used this technique to examine the lytic activity of freshly isolated T cells from noninflamed human colonic mucosa. Anti-CD3-triggered T-cell (anti-CD3-T) cytotoxicity was found in all mucosal specimens studied. The mucosal anti-CD3-T effectors do not have Fc receptors for immunoglobulin G, and are therefore distinct from T gamma cells, which mediate antibody-dependent cellular cytotoxicity. The surface antigen phenotype of mucosal anti-CD3-Ts is CD2+, CD3+, CD8+, CD4-, CD16-, and Leu7-. In contrast, peripheral blood anti-CD3-T effectors are Leu7+. Although non-major histocompatibility complex-restricted, mucosal anti-CD3-T cytotoxicity has considerable target specificity, which differs from that of natural killer and lymphokine-activated killer cells. The profile of target cell susceptibility and the inhibitory effects of anti-CD45 antibody suggest that the CD45 molecule on the effector cell may be an important determinant of anti-CD3-T sensitivity. As anti-CD3-triggered lysis may be a marker of in vivo primed mucosal T cells of undetermined antigen specificity, this technique might have important implications in inflammatory bowel disease, where the antigen(s) inciting the mucosal immune reactivity is not certain.

Human intestinal lamina propria mononuclear cells have been shown to mediate mitogen-induced cellular cytotoxicity, antibody-dependent cellular cytotoxicity, and lymphokine activated killer cell function. However, although natural killer cells have been demonstrated in the gut mucosa of rodents, recent reports found little or no spontaneous cytotoxic activity in the lamina propria of the human gut. Using the natural killer cell-related monoclonal antibody NKH-1, which has not previously been applied to studies of mucosal killer cell function, we have shown by immunofluorescence that 2%-3% of enzymatically dispersed lamina propria lymphocytes are NKH-1+. A "panning" technique was then used to enrich for the NKH-1+ cells. Panned cells were consistently greater than or equal to 80% NKH-1+ by indirect immunofluorescence. Unlike their counterparts in the peripheral blood, the mucosal NKH-1+ cells were Leu-11-. Although unseparated lamina propria lymphocytes failed to exhibit natural killer activity against K562 targets in 4-h chromium release assays at effector to target ratios of up to 100:1, the NKH-1+ cells were cytolytically active at ratios of less than 5:1. Mucosal lymphocytes depleted of natural killer cells (NKH-1-) exhibited cytotoxic activity when cultured for 72 h with interleukin-2. The precursors of the lymphokine culture activated phenomenon were NKH-1-, Leu-11-, T4-, T3-, T11+, and T8+. Although lamina propria T3+ cells did not exhibit spontaneous or culture activated cytotoxicity, they were shown to exhibit nonspecific anti-CD3 (anti-T3)-induced T-cell cytotoxicity. In conclusion, functional natural killer and lymphokine activated killer cells are both present in the human gut mucosa and represent distinct populations of cytotoxic cells. In addition, anti-CD3-induced cytotoxicity is a feature of mucosal T cells. These mucosal killer cell subsets differ phenotypically from those previously described in the peripheral blood.