It is well known that inflammatory conditions of the intestinal mucosa result in compromised barrier function. Inflammation is characterized by an influx into the mucosa of immune cells that ...influence epithelial function by releasing proinflammatory cytokines such as IFN-gamma and TNF-alpha. Mucosal barrier function is regulated by the epithelial apical junctional complex (AJC) consisting of the tight junction and the adherens junction. Since the AJC regulates barrier function, we analyzed the influence of IFN-gamma and TNF-alpha on its structure/function and determined the contribution of apoptosis to this process using a model intestinal epithelial cell line, T84, and IFN-gamma and TNF-alpha. AJC structure/function was analyzed by confocal microscopy, biochemical analysis, and physiologic measurement of epithelial gate/fence function. Apoptosis was monitored by determining cytokeratin 18 cleavage and caspase-3 activation. IFN-gamma induced time-dependent disruptions in epithelial gate function that were potentiated by coincubation with TNF-alpha. Tight junction fence function was somewhat disrupted. Cytokine treatment was associated with internalization of AJC transmembrane proteins, junction adhesion molecule 1, occludin, and claudin-1/4 with minimal effects on the cytoplasmic plaque protein zonula occludens 1. Detergent solubility profiles of junction adhesion molecule 1 and E-cadherin and their affiliation with "raft-like" membrane microdomains were modified by these cytokines. Inhibition of cytokine-induced apoptosis did not block induced permeability defects; further emphasizing their primary influence on the epithelial AJC structure and barrier function. Our findings for the first time clearly separate the proapoptotic effects of IFN-gamma and TNF-alpha from their abilities to disrupt barrier function.
Flagellin, the structural component of bacterial flagella, is secreted by pathogenic and commensal bacteria. Flagellin activates proinflammatory gene expression in intestinal epithelia. However, only ...flagellin that contacts basolateral epithelial surfaces is proinflammatory; apical flagellin has no effect. Pathogenic Salmonella, but not commensal Escherichia coli, translocate flagellin across epithelia, thus activating epithelial proinflammatory gene expression. Investigating how epithelia detect flagellin revealed that cell surface expression of Toll-like receptor 5 (TLR5) conferred NF-kappaB gene expression in response to flagellin. The response depended on both extracellular leucine-rich repeats and intracellular Toll/IL-1R homology region of TLR5 as well as the adaptor protein MyD88. Furthermore, immunolocalization and cell surface-selective biotinylation revealed that TLR5 is expressed exclusively on the basolateral surface of intestinal epithelia, thus providing a molecular basis for the polarity of this innate immune response. Thus, detection of flagellin by basolateral TLR5 mediates epithelial-driven inflammatory responses to Salmonella.
The intercellular tight junction is the rate-limiting barrier in the paracellular pathway for permeation by ions and larger solutes. A variety of widely used electrical and flux approaches are used ...in the analyses of solute permeation through this pathway; however, each has limitations in practice. It is now clear that solute permeation across tight junctions is dynamically regulated by intracellular events with a common effector mechanism apparently tied to the cytoskeleton. These pathways, which regulate tight junction solute permeability, are targets that produce epithelial barrier dysfunction in a variety of disease states. However, regulation of solute permeation across the junctional barrier may also represent a potential means to improve bioavailability of orally administered bioactive solutes.
Epithelial intercellular junctions regulate cell-cell contact and mucosal barrier function. Both tight junctions (TJs) and adherens junctions (AJs) are regulated in part by their affiliation with the ...F-actin cytoskeleton. The cytoskeleton in turn is influenced by Rho family small GTPases such as RhoA, Rac1, and Cdc42, all of which constitute eukaryotic targets for several pathogenic organisms. With a tetracycline-repressible system to achieve regulated expression in Madin-Darby canine kidney (MDCK) epithelial cells, we used dominant-negative (DN) and constitutively active (CA) forms of RhoA, Rac1, and Cdc42 as tools to evaluate the precise contribution of each GTPase to epithelial structure and barrier function. All mutant GTPases induced time-dependent disruptions in epithelial gate function and distinct morphological alterations in apical and basal F-actin pools. TJ proteins occludin, ZO-1, claudin-1, claudin-2, and junctional adhesion molecule (JAM)-1 were dramatically redistributed in the presence of CA RhoA or CA Cdc42, whereas only claudins-1 and -2 were redistributed in response to CA Rac1. DN Rac1 expression also induced selective redistribution of claudins-1 and -2 in addition to JAM-1, whereas DN Cdc42 influenced only claudin-2 and DN RhoA had no effect. AJ protein localization was unaffected by any mutant GTPase, but DN Rac1 induced a reduction in E-cadherin detergent solubility. All CA GTPases increased the detergent solubility of claudins-1 and -2, but CA RhoA alone reduced claudin-2 and ZO-1 partitioning to detergent-insoluble membrane rafts. We conclude that Rho family GTPases regulate epithelial intercellular junctions via distinct morphological and biochemical mechanisms and that perturbations in barrier function reflect any imbalance in active/resting GTPase levels rather than simply loss or gain of GTPase activity.
The gastroenteritis-causing pathogen Salmonella typhimurium induces profound transcriptional changes in intestinal epithelia resulting in the recruitment of neutrophils whose presence is the ...histopathologic hallmark of salmonellosis. Here we used cDNA microarray expression profiling to define the molecular determinants that mediate such changes in model intestinal epithelia. Enteropathogenic Salmonella induced a classical proinflammatory gene expression program similar to that activated by the canonical proinflammatory agonist TNF-alpha. Nonproinflammatory bacteria, both commensals (Escherichia coli) and systemic pathogens (S. typhi), did not activate this expression profile. While S. typhimurium strains lacking the SPI-1-encoded type III system were fully proinflammatory, strains lacking the genes for the flagellar structural component flagellin were nearly devoid of proinflammatory signaling. Lastly, the epithelial proinflammatory response could be largely recapitulated by basolateral addition of purified flagellin. Thus, S. typhimurium flagellin is the major molecular trigger by which this pathogen activates gut epithelial proinflammatory gene expression.
Epithelia of the vertebrate intestinal tract characteristically maintain an inflammatory hyporesponsiveness toward the lumenal prokaryotic microflora. We report the identification of enteric ...organisms (nonvirulent Salmonella strains) whose direct interaction with model human epithelia attenuate synthesis of inflammatory effector molecules elicited by diverse proinflammatory stimuli. This immunosuppressive effect involves inhibition of the inhibitor κB/nuclear factor κB (IκB/NF-κB) pathway by blockade of IκB-α degradation, which prevents subsequent nuclear translocation of active NF-κB dimer. Although phosphorylation of IκB-α occurs, subsequent polyubiquitination necessary for regulated IκB-α degradation is completely abrogated. These data suggest that prokaryotic determinants could be responsible for the unique tolerance of the gastrointestinal mucosa to proinflammatory stimuli.
Physician membership organizations vary in the extent of their engagement in activities to address health disparities. Increasing engagement of those organizations not already highly active in this ...critical area is, thus, an opportunity. Studies that provide definitional contours of key issues, like disparities, are necessary and must be iteratively refined. However, parallel activities of intervention with measured outcomes to assess the effects of these interventions are necessary to truly address major problems in the health care system. To date, work in the problem definition category exceeds work toward intervention in and mitigation of these problems with measured outcomes. Many problems in health care, including disparities, are now sufficiently understood that it is time to shift focus toward bold intervention with measured outcomes. Optimal approaches that yield superior outcomes generally require collaboration across the provider-payer spectrum and the private sectors, including physicians, hospitals, insurers, etc. Stakeholders are now free to act in such coordinated fashion; it only requires social capital that permits cooperation and compromise. Interventions for problems such as health care disparities can be developed in the private sector and mirrored by government payers if physicians and organizations can get real about collaborating to implement outcomes-based initiatives to improve the health of all patients.
The mechanism by which neutrophils polymorphonuclear leukocyte (PMNs) are stimulated to move across epithelial barriers at mucosal surfaces has been basically unknown in biology. IL-8 has been shown ...to stimulate PMNs to leave the bloodstream at a local site of mucosal inflammation, but the chemical gradient used by PMNs to move between adjacent epithelial cells and traverse the tight junction at the apical neck of these mucosal barriers has eluded identification. Our studies not only identify this factor, previously termed pathogen-elicited epithelial chemoattractant, as the eicosanoid hepoxilin A3 ( hepA3) but also demonstrate that it is a key factor promoting the final step in PMN recruitment to sites of mucosal inflammation. We show that hepA3 is synthesized by epithelial cells and secreted from their apical surface in response to conditions that stimulate inflammatory events. Our data further establish that hepA3 acts to draw PMNs, via the establishment of a gradient across the epithelial tight junction complex. The functional significance of hepA3 to target PMNs to the lumen of the gut at sites of inflammation was demonstrated by the finding that disruption of the 12-lipoxygenase pathway (required for hepA3 production) could dramatically reduce PMN-mediated tissue trauma, demonstrating that hepA3 is a key regulator of mucosal inflammation.
The anti-inflammatory eicosanoid lipoxin A(4) (LXA(4)), aspirin-triggered 15-epi-LXA(4), and their stable analogs down-regulate IL-8 secretion and subsequent recruitment of neutrophils by intestinal ...epithelia. In an effort to elucidate the mechanism by which these lipid mediators modulate cellular proinflammatory programs, we surveyed global epithelial gene expression using cDNA microarrays. LXA(4) analog alone did not significantly affect expression of any of the >7000 genes analyzed. However, LXA(4) analog pretreatment attenuated induction of approximately 50% of the 125 genes up-regulated in response to the gastroenteritis-causing pathogen Salmonella typhimurium. A major subset of genes whose induction was reduced by LXA(4) analog pretreatment is regulated by NF-kappaB, suggesting that LXA(4) analog was influencing the activity of this transcription factor. Nanomolar concentrations of LXA(4) analog reduced NF-kappaB-mediated transcriptional activation in a LXA(4) receptor-dependent manner and inhibited induced degradation of IkappaBalpha. LXA(4) analog did not affect earlier stimulus-induced signaling events that lead to IkappaBalpha degradation, such as S. typhimurium-induced epithelial Ca(2+) mobilization or TNF-alpha-induced phosphorylation of IkappaBalpha. To establish the in vivo relevance of these findings, we examined whether LXA(4) analogs could affect intestinal inflammation in vivo using the mouse model of DSS-induced inflammatory colitis. Oral administration of LXA(4) analog (15-epi-16-para-fluoro-phenoxy-LXA(4), 10 microg/day) significantly reduced the weight loss, hematochezia, and mortality that characterize DSS colitis. Thus, LXA(4) analog-mediated down-regulation of proinflammatory gene expression via inhibition of the NF-kappaB pathway can be therapeutic for diseases characterized by mucosal inflammation.
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