Luminal Antigens Research Articles

When the levee breaks: The etiologic role of epithelial barrier function defects in ileitis

When the levee breaks: The etiologic role of epithelial barrier function defects in ileitis

Methotrexate‐induced mucositis in mucin 2‐deficient mice

The mucin Muc2 or Mycin2 (Muc2), which is the main structural component of the protective mucus layer, has shown to be upregulated during chemotherapy-induced mucositis. As Muc2 has shown to have protective capacities, upregulation of Muc2 may be a counter reaction of the intestine protecting against mucositis. Therefore, increasing Muc2 protein levels could be a therapeutic target in mucositis prevention or reduction. Our aim was to determine the role of Muc2 in chemotherapy-induced mucositis. Mucositis was induced in Muc2 knockout (Muc2(-/-)) and wild type (Muc2(+/+)) mice by injecting methotrexate (MTX). Animals were weighed and sacrificed on Days 2-6 after MTX treatment and jejunal segments were analyzed. Before MTX treatment, the small intestine of Muc2(+/+) and Muc2(-/-) mice were similar with respect to epithelial morphology and proliferation. Moreover, sucrase-isomaltase and trefoil factor-3 protein expression levels were comparable between Muc2(+/+) and Muc2(-/-) mice. Up to Day 3 after MTX treatment, percentages of weight-loss did not differ. Thereafter, Muc2(+/+) mice showed a trend towards regaining weight, whereas Muc2(-/-) mice continued to lose weight. Surprisingly, MTX-induced intestinal damage of Muc2(-/-) and Muc2(+/+) mice was comparable. Prior to MTX-injection, tumor necrosis factor-alpha and interleukin-10 mRNAs were upregulated in Muc2(-/-) mice, probably due to continuous exposure of the intestine to luminal antigens. Muc2 deficiency does not lead to an increase in chemotherapy-induced mucositis. A possible explanation is the mechanism by which Muc2 deficiency may trigger the immune system to release interleukin-10, an anti-inflammatory cytokine before MTX-treatment.

Endocytic pathways of luminal antigens intersect MHC class I and class II pathways in multivesicular late endosomes – Antigen trafficking in intestinal epithelial cells studied in vivo in Crohn's disease patients

Endocytic pathways of luminal antigens intersect MHC class I and class II pathways in multivesicular late endosomes – Antigen trafficking in intestinal epithelial cells studied in vivo in Crohn's disease patients

Immune privilege in the gut: the establishment and maintenance of non‐responsiveness to dietary antigens and commensal flora

Immune privilege in the gut is the result of a complex interplay between the gut microbiome, gut luminal antigens, and the intestinal epithelial barrier. Composed of both physical and immunochemical components, the intestinal barrier secretes immunoregulatory mediators that promote the generation of tolerogenic antigen-presenting cells, phagocytic innate immune cells characterized by 'inflammatory anergy', and regulatory cells of the adaptive immune system. Innate immune cells mediate controlled transepithelial transport of luminal antigens as far as the mesenteric lymph nodes, where the intestinal and peripheral immune systems intersect. This promotes the generation of adaptive regulatory lymphocytes that actively suppress effector cell responses against gut luminal antigens and flora. The net result is the generation of tolerance to dietary antigens and the maintenance of gut homeostasis. Dysregulation of this complex immunoregulatory network leads to diseases such as food allergy and inflammatory bowel disease. Future therapies for these diseases will likely involve the functional restoration of the barrier and regulatory cell functions at the epithelial/luminal interface.

The pathogenetical role of intestinal permeability in type 1 diabetes

Objectives. The pathogenesis of type 1 diabetes mellitus (DM1), involving genetical and environmental factors, is still unclear; recent studies suggest that an increased intestinal permeability due to disregulation of enterocytic tight-junction system, could determine the paracellular passage of luminal antigens in the blood with consequent development of autoimmune reactions. The aim of our study is to evaluate the role of gluten in DM1.

Molecular characterisation of the gut microflora of healthy and inflammatory bowel disease cats using fluorescence in situ hybridisation with special reference to Desulfovibrio spp.

Inflammatory bowel disease (IBD) is a common cause of chronic large bowel diarrhoea in cats. Although the aetiology of IBD is unknown, an immune-mediated response to a luminal antigen is thought to be involved. As knowledge concerning the colonic microflora of cats is limited and requires further investigation, the purpose of this study was to determine the presence of specific bacterial groups in normal and IBD cats, and the potential role they play in the health of the host. Total bacterial populations, Bacteroides spp., Bifidobacterium spp., Clostridium histolyticum subgp., Lactobacillus-Enterococcus subgp. and Desulfovibrio spp. were enumerated in 34 healthy cats and 11 IBD cats using fluorescence in situ hybridisation. The study is one of the first to show the presence of Desulfovibrio in cats. Total bacteria, Bifidobacterium spp. and Bacteroides spp. counts were all significantly higher in healthy cats when compared with IBD cats, whereas Desulfovibrio spp. (producers of toxic sulphides) numbers were found to be significantly higher in colitic cats. The information obtained from this study suggests that modulation of bacterial flora by increasing bifidobacteria and decreasing Desulfovibrio spp. may be beneficial to cats with IBD. Dietary intervention may be an important aspect of their treatment.

Correlation between cyclical epithelial barrier dysfunction and bacterial translocation in the relapses of intestinal inflammation

Although several factors have been implicated in the pathogenesis of inflammatory bowel disease (IBD), the mechanisms underlying the recurrent relapses have not yet been clarified. We hypothesized that epithelial barrier dysfunction, associated with intestinal motor disturbances, could play a key role in exacerbation of inflammatory illness due to an increased uptake of luminal antigen and bacterial translocation. Indomethacin administration to rats induced a long-lasting oscillation of active and quiescent phases of inflammation associated with phases of hypo and hypermotility. Rats selected at either active or quiescent phase and from 2 to 60 days after indomethacin treatment were used. Short-circuit current; conductance and HRP flux were evaluated in small intestinal segments mounted in Ussing Chambers. Enterocyte endosomes containing HRP and ultrastructural damage were assessed by electron microscopy. Bacterial translocation was determined by cultures from mesenteric lymph nodes. Rats with induced enteritis in both phases demonstrated a long-lasting increase of epithelial paracellular permeability. In contrast, transcellular permeability was only disturbed during the active phases, coinciding with bacterial translocation and the increase in inflammatory parameters. Furthermore, although mithochondrial damage was observed throughout the inflammatory state, alterations were worse during the active phase. The sustained enhancement of paracellular permeability could facilitate the constant passage of luminal antigens through the mucosa, and hence, be the basis for chronicity. By contrast, transcellular permeability only increases during the active phases, when hypomotility and bacterial translocation are also present, suggesting this factor may play a critical role in the course of acute relapses in IBD.

The Vagus Nerve: A Tonic Inhibitory Influence Associated With Inflammatory Bowel Disease in a Murine Model

The recently proposed Inflammatory Reflex describes an interaction between the vagus nerve and peripheral macrophages, resulting in attenuation of proinflammatory cytokine release in response to systemic exposure to bacterial endotoxin. The purpose of this study was to determine whether a similar vagus/macrophage axis modulates the inflammatory responses in the colon in mice. We assessed the Disease Activity Index (DAI), macroscopic and histologic damage, serum amyloid-P level, and myeloperoxidase activity in colitis induced by administration of dextran sodium sulfate (DSS) in healthy and vagotomized C57BL/6 and in mice deficient in macrophage-colony stimulating factor (M-CSF)-induced and in hapten-induced colitis. A pyloroplasty was performed in vagotomized mice. DAI, macroscopic and histologic scores, myeloperoxidase activity, levels of serum amyloid-P, and colonic tissue levels of interleukin (IL)-1beta, IL-6, and tumor necrosis factor-alpha were increased significantly in vagotomized mice 5 days post-DSS and 3 days after hapten-induced colitis compared with sham-operated mice that received DSS or the hapten. Pretreatment with nicotine significantly decreased each of these markers in vagotomized mice with DSS colitis, and all markers except DAI and IL-6 in sham-operated DSS-treated mice. Conversely, hexamethonium treatment significantly increased each of these markers in the sham-operated DSS-treated mice. Vagotomy had no effect on the colitis in M-CSF-deficient mice. The vagus nerve plays a counterinflammatory role in acute colitis via a macrophage-dependent mechanism, involving hexamethonium-sensitive nicotinic receptors. The identification of a counterinflammatory neural pathway would open new therapeutic avenues for treating acute exacerbations of inflammatory bowel disease.

A defect in epithelial barrier integrity is not required for a systemic response to bacterial antigens or intestinal injury in T cell receptor-α gene-deficient mice

Genetically induced disruption of the intestinal epithelial barrier leads to development of intestinal inflammation. In the interleukin-10 gene-deficient inflammatory bowel disease (IBD) mouse model, for instance, a primary defect in intestinal epithelial integrity occurs before the development of enterocolitis. In humans, a causal role for epithelial barrier disruption is still controversial. Although studies with first-degree relatives of IBD patients suggests an underlying role of impaired barrier function, a primary epithelial barrier defect in IBD patients has not been confirmed. The purpose of this article is to examine whether a primary epithelial barrier disruption is a prerequisite for the development of intestinal inflammation or whether intestinal inflammation can develop in the absence of epithelial disruption. We examined the intestinal epithelial integrity of the T cell receptor (TCR)-alpha gene-deficient mouse model of IBD. In vivo colonic permeability, determined by mannitol transmural flux, was assessed in 6-week-, 12-week-, and 25-week-old TCR-alpha gene-deficient and wild-type control mice using a single-pass perfusion technique. Mice were scored for intestinal histological injury and intestinal cytokine levels measured in organ cultures. Systemic responses to bacterial antigens were determined through 48-h spleen cell cultures stimulated with sonicate derived from endogenous bacterial strains. In contrast with previous findings in the interleukin-10 gene-deficient IBD model, TCR-alpha gene-deficient mice did not demonstrate evidence of primary intestinal epithelial barrier disruption at any age, despite developing a moderate to severe colitis within 12 weeks. A rise in intestinal interferon (IFN)-gamma levels preceded the onset of mucosal inflammation and then correlated closely with the degree of intestinal inflammation and injury. Spleen cells from TCR-alpha gene-deficient mice released IFN-gamma in response to stimulation with endogenous luminal bacterial antigens, a finding that suggests that the systemic response to bacterial antigens occurred independently of epithelial barrier disruption. Intestinal inflammation and a systemic response to bacterial antigens can develop in the absence of a measurable disruption of intestinal permeability.

Neonatal Fc receptor for IgG regulates mucosal immune responses to luminal bacteria

The neonatal Fc receptor for IgG (FcRn) plays a major role in regulating host IgG levels and transporting IgG and associated antigens across polarized epithelial barriers. Selective expression of FcRn in the epithelium is shown here to be associated with secretion of IgG into the lumen that allows for defense against an epithelium-associated pathogen (Citrobacter rodentium). This pathway of host resistance to a bacterial pathogen as mediated by FcRn involves retrieval of bacterial antigens from the lumen and initiation of adaptive immune responses in regional lymphoid structures. Epithelial-associated FcRn, through its ability to secrete and absorb IgG, may thus integrate luminal antigen encounters with systemic immune compartments and as such provide essential host defense and immunoregulatory functions at the mucosal surfaces.

This Month in Gastroenterology

This Month in Gastroenterology

New mediators of immunity and inflammation in inflammatory bowel disease

In both Crohn's disease and ulcerative colitis, the tissue damage results from an inappropriate or exaggerated immune response to antigens of the gut microflora. This review summarizes current knowledge regarding the role of immune-inflammatory mediators in the pathogenesis of inflammatory bowel disease. Despite having a common basis in overresponsiveness to luminal antigens, Crohn's disease and ulcerative colitis are immunologically distinct entities. Crohn's disease is associated with a Th1 T cell-mediated response, characterized by enhanced production of interferon-gamma and tumor necrosis factor-alpha. Interleukin (IL)-12 and, possibly, IL-23 govern the Th1 cell differentiation, but optimal induction and stabilization of polarized Th1 cells would require additional cytokines, such as IL-15, IL-18 and IL-21. In ulcerative colitis, the local immune response is less polarized, but it is characterized by CD1-reactive natural killer T cell production of IL-13. Beyond these differences, Crohn's disease and ulcerative colitis share important end-stage effector pathways of intestinal injury, which are mediated by an active cross-talk between immune and non-immune mucosal cells. The clarification of the complex network of immune-inflammatory mediators operating in the gut of patients with inflammatory bowel disease has led to the identification of new targets that could, in turn, drive the development of effective biological therapies.

Microscopic colitis demonstrates a T helper cell type 1 mucosal cytokine profile

Background: Microscopic colitis (MC) is an inflammatory disorder of unknown aetiology. Aim: To characterise the mucosal cytokine profile of MC, with a view to understanding its potential pathogenic mechanisms. Methods:...

Phenotype and distribution of dendritic cells in the porcine small intestinal and tracheal mucosa and their spatial relationship to epithelial cells

Dendritic cells (DC) as key mediators of tolerance and immunity perform crucial immunosurveillance functions at epithelial surfaces. In order to induce an immune response, the DC have to gain access to antigens present at the luminal surface of mucosal epithelia. The mechanisms of this process are still largely unclear. We have therefore analysed the distribution of DC in the porcine intestinal and respiratory mucosa and their spatial relationship to epithelial cells by immunohistology. Immunofluorescence analysis of cryosections taken from jejunal Peyer's patches and double-stained for DC and M cells (specialised for antigen uptake) have revealed that 35.2+/-3.9% of M cells are located directly adjacent to DC in the subepithelial domes, representing possible antigen transfer sites. In normal jejunal villi, a rare population of lamina propria DC extending cytoplasmic processes between enterocytes has been identified as a possible correlate for direct luminal antigen uptake. Like small intestinal DC, DC in the porcine trachea mostly co-express CD16 with MHC-II. Tracheal DC have been found at high densities both above and below the basement membrane (BM) of the tracheal epithelium, with 32.4 DC/mm BM and 23.0 DC/mm BM, respectively. The intraepithealial DC population forms a dense network, with many of the cytoplasmic processes being directed towards the tracheal lumen. Our morphological analyses indicate that DC at mucosal epithelial sites are ideally positioned for the uptake of luminal antigens.

Measuring the Quality of Endoscopy

There has been recent interest into the potential role of cellular and molecular mechanisms in the pathophysiology of irritable bowel syndrome (IBS). Although the intestinal mucosa of IBS patients is endoscopically and histologically "normal," it contains an increased number of activated T lymphocytes and mast cells, along with evidence of an increased release of mediators known to signal to epithelial, neuronal, and muscle cells leading to intestinal dysfunction. In this issue, Dunlop et al. provide evidence of increased intestinal permeability in patients with diarrhea predominant IBS. There is now consistent evidence indicating that mucosal barrier defects allow the passage of an increased load of luminal antigens of dietary and bacterial origin which, in turn, elicit the activation of mucosal immune responses involved in the generation of diarrhea. Further work has now to be done to better understand the interplay among luminal factors, epithelial cells, and mucosal immunocytes in the pathogenesis of IBS.

A Functional Role of Flip in Conferring Resistance of Crohn’s Disease Lamina Propria Lymphocytes to FAS-Mediated Apoptosis

There is evidence that, in Crohn's disease (CD), lamina propria T lymphocytes (LPLs) are resistant to FAS-mediated apoptosis and that this defect contributes to the mucosal T-cell accumulation. In this study we examined the functional role of Flip, a Flice inhibitor protein, in the resistance of CD LPL to FAS-mediated apoptosis. Biopsy specimens and LPLs were taken from CD and ulcerative colitis (UC) patients and normal controls and analyzed for Flip by Western blotting. We also examined whether inhibition of Flip by antisense oligonucleotide restored the susceptibility of CD LPLs to FAS-induced apoptosis. LPL apoptosis was assessed by flow cytometry. After FAS stimulation, the rate of apoptosis of CD3+ LPLs was higher in normal controls and patients with UC than in patients with CD. Enhanced expression of both long and short Flip isoforms was seen in biopsy specimens and purified CD3+ and CD45RO+ LPLs of CD patients in comparison with UC patients and normal controls. No increase in Flip was documented in untreated celiac disease mucosa, thus suggesting the possibility that induction of Flip in the gut does not simply rely on the ongoing inflammation. Finally, we showed that inhibition of Flip by antisense oligonucleotide reverted the resistance of CD LPLs to FAS-induced apoptosis. Data suggest a role for Flip in the resistance of CD LPLs to FAS-mediated apoptosis.

Immunogenetic phenotypes in inflammatory bowel disease

The currently accepted etiopathogenic hypothesis suggests that the chronic intestinal inflammation and related systemic manifestations characteristic of inflammatory bowel disease (IBD) are due to an overly aggressive or pathologic immune response to resident luminal bacterial constituents. Predisposing factors are genetic dysregulation of mucosal immune responses and/or barrier function, with onset triggered by environmental stimuli. These factors and their interactions may also be important determinants of disease phenotype and disease progression. The emergence of immunogenetic phenotypes lends support to the proposed hypothesis that susceptibility genes regulate distinct immune processes, driven by luminal antigens, expressed as specific immune phenotypes which in turn influence clinical phenotypes in IBD patient.

Persorption of Luminal Antigenic Molecule and Its Specific Antibody via Apoptotic Epithelial Cells of Intestinal Villi and Peyer's Patches into Peripheral Blood in Rats

The possibility of persorption of bovine serum albumin (BSA) molecules from mucous epithelial cells and its mechanism were investigated in rats orally pre-immunized by BSA for 14 consecutive days. In the small and large intestines, both the BSA antigen (BSA-Ag) and its specific antibody (SpAb) were absorbed by the epithelial cells at the late apoptotic stage (ApoEp), and were subsequently transcytosed by membranes of the small vesicles. The basal cytoplasms containing highly-concentrated BSA-Ag and SpAb were occasionally fragmented into small cytoplasmic droplets that were secreted into the lamina propria. In Peyer's patches, both BSA-Ag and SpAb were more actively absorbed and transcytosed toward the dome area by the ApoEp of the dome apex than by the M cells. BSA-Ag and SpAb were finally persorbed into the portal blood and lymph, but were never secreted into the bile. They were also engulfed by macrophage-like cells in the villous lamina propria, mesenteric lymph node and spleen, and by hepatocytes in the liver. These findings suggest that sensitized soluble luminal antigens are taken up by ApoEp in the small intestine and are finally persorbed into the peripheral blood. The uptake of luminal antigen might be mediated by its luminal SpAb.

Epithelial cells as sentinels in mucosal immune barrier

The mucosal surface of the body is exposed to a vast array of exogenous antigens and microorganisms. Epithelial cells evoke minimal immune response to food ingredients and commensal bacteria, while they release an array of antimicrobial peptides and CXC chemokines in response to bacterial invasion or inflammatory stimuli. The mucosal antigens are transported from the gut lumen to organized lymphoid follicles by specialized epithelial M cells residing in follicle-associated epithelium (FAE). An alternative pathway of antigen uptake with neonatal Fc receptor (FcRn) is also reported. Furthermore, intestinal dendritic cells underneath epithelium directly take up luminal antigens, where epithelial fractalkine expression plays a critical role in the guidance of dendrite extrusion. Epithelial cells express polymeric Ig receptor (pIgR) that is essential for the luminal secretion of dimeric IgA produced in the lamina propria. Furthermore, soluble factors released by mucosal epithelial cells condition dendritic cells, which in turn promote Th2 response. These multiple lines of evidence clearly suggest the significant role of epithelial cells at the front line of mucosal immune defense.

Chronic Psychological Stress in Rats Induces Intestinal Sensitization to Luminal Antigens

There is increasing evidence that stress plays a role in the pathophysiology of chronic intestinal disorders, but the mechanisms remain unclear. Previous studies in rats have revealed that stress decreases gut barrier function and allows excessive uptake of luminal material. Here, we investigated whether chronic psychological stress acts to induce sensitization of intestinal tissues to oral antigens. Rats were subjected to 1 hour per day of water avoidance stress or sham stress daily for 10 days, and horseradish peroxidase (HRP) was delivered by gavage on day 5. Studies to determine sensitization were conducted on day 20. All stressed rats developed HRP-specific IgE antibodies, antigen-induced intestinal secretion, and increased numbers of inflammatory cells in gut mucosa. Luminal HRP was absorbed more readily by enterocytes of stressed animals. In addition, stressed rats had increased expression of interleukin-4 and decreased expression of interferon-gamma in gut mucosa, a cytokine profile that is typical of allergic conditions. Treatment of stressed rats with an antagonist to corticotropin-releasing hormone (previously shown to inhibit stress-enhanced gut permeability) eliminated the manifestations of intestinal hypersensitivity. Our results indicate that the presence of oral antigen during chronic psychological stress alters the immune response (to sensitization rather than oral tolerance) and causes subsequent antigen-induced gut pathophysiology.