Control of neutrophil inflammation at mucosal surfaces by secreted epithelial products

The human intestine is a large and delicately balanced organ, responsible for efficiently absorbing nutrients and selectively eliminating disease-causing pathogens. The gut architecture consists of a single layer of epithelial cells that forms a barrier against the food antigens and resident microbiota within the lumen. This barrier is augmented by a thick layer of mucus on the luminal side and an underlying lamina propria containing a resident population of immune cells. Attempted breaches of the intestinal barrier by pathogenic bacteria result in the rapid induction of a coordinated innate immune response that includes release of antimicrobial peptides, activation of pattern recognition receptors, and recruitment of various immune cells. In recent years, the role of epithelial cells in initiating this immune response has been increasingly appreciated. In particular, epithelial cells are responsible for the release of a variety of factors that attract neutrophils, the body\u27s trained bacterial killers. In this review we will highlight recent research that details a new understanding of how epithelial cells directionally secrete specific compounds at distinct stages of the inflammatory response in order to coordinate the immune response to intestinal microbes. In addition to their importance during the response to infection, evidence suggests that dysregulation of these pathways may contribute to pathologic inflammation during inflammatory bowel disease. Therefore, a continued understanding of the mechanisms by which epithelial cells control neutrophil migration into the intestine will have tremendous benefits in both the understanding of biological processes and the identification of potential therapeutic targets

Gain of Function Analysis Reveals Non-Redundant Roles for the Yersinia pestis Type III Secretion System Effectors YopJ, YopT, and YpkA [preprint]

Virulence of Yersinia pestis in mammals requires the type III secretion system, which delivers seven effector proteins into the cytoplasm of host cells to undermine immune responses. All seven of these effectors are conserved across Y. pestis strains, but three -- YopJ, YopT, and YpkA -- are apparently dispensable for virulence. Some degree of functional redundancy between effector proteins would explain both observations. Here, we use a combinatorial genetic approach to define the minimal subset of effectors required for full virulence in mice following subcutaneous infection. We found that a Y. pestis strain lacking YopJ, YopT, and YpkA is attenuated for virulence in mice, and that addition of any one of these effectors to this strain increases lethality significantly. YopJ, YopT, and YpkA likely contribute to virulence via distinct mechanisms. YopJ is uniquely able to cause macrophage cell death in vitro and to suppress accumulation of inflammatory cells to foci of bacterial growth in deep tissue, whereas YopT and YpkA cannot. The synthetic phenotypes that emerge when YopJ, YopT, and YpkA are removed in combination provide evidence that each enhances Y. pestis virulence, and that YopT and YpkA act through a mechanism distinct from that of YopJ

Enteroaggregative Escherichia coli Adherence Fimbriae Drive Inflammatory Cell Recruitment via Interactions with Epithelial MUC1

Enteroaggregative Escherichia coli (EAEC) causes diarrhea and intestinal inflammation worldwide. EAEC strains are characterized by the presence of aggregative adherence fimbriae (AAF), which play a key role in pathogenesis by mediating attachment to the intestinal mucosa and by triggering host inflammatory responses. Here, we identify the epithelial transmembrane mucin MUC1 as an intestinal host cell receptor for EAEC, demonstrating that AAF-mediated interactions between EAEC and MUC1 facilitate enhanced bacterial adhesion. We further demonstrate that EAEC infection also causes elevated expression of MUC1 in inflamed human intestinal tissues. Moreover, we find that MUC1 facilitates AAF-dependent migration of neutrophils across the epithelium in response to EAEC infection. Thus, we show for the first time a proinflammatory role for MUC1 in the host response to an intestinal pathogen. IMPORTANCE: EAEC is a clinically important intestinal pathogen that triggers intestinal inflammation and diarrheal illness via mechanisms that are not yet fully understood. Our findings provide new insight into how EAEC triggers host inflammation and underscores the pivotal role of AAFs-the principal adhesins of EAEC-in driving EAEC-associated disease. Most importantly, our findings add a new dimension to the signaling properties of the transmembrane mucin MUC1. Mostly studied for its role in various forms of cancer, MUC1 is widely regarded as playing an anti-inflammatory role in response to infection with bacterial pathogens in various tissues. However, the role of MUC1 during intestinal infections has not been previously explored, and our results describe the first report of MUC1 as a proinflammatory factor following intestinal infection

Gut microbiota regulation of P-glycoprotein in the intestinal epithelium in maintenance of homeostasis

BACKGROUND: P-glycoprotein (P-gp) plays a critical role in protection of the intestinal epithelia by mediating efflux of drugs/xenobiotics from the intestinal mucosa into the gut lumen. Recent studies bring to light that P-gp also confers a critical link in communication between intestinal mucosal barrier function and the innate immune system. Yet, despite knowledge for over 10 years that P-gp plays a central role in gastrointestinal homeostasis, the precise molecular mechanism that controls its functional expression and regulation remains unclear. Here, we assessed how the intestinal microbiome drives P-gp expression and function. RESULTS: We have identified a functional core microbiome of the intestinal gut community, specifically genera within the Clostridia and Bacilli classes, that is necessary and sufficient for P-gp induction in the intestinal epithelium in mouse models. Metagenomic analysis of this core microbial community revealed that short-chain fatty acid and secondary bile acid production positively associate with P-gp expression. We have further shown these two classes of microbiota-derived metabolites synergistically upregulate P-gp expression and function in vitro and in vivo. Moreover, in patients suffering from ulcerative colitis (UC), we find diminished P-gp expression coupled to the reduction of epithelial-derived anti-inflammatory endocannabinoids and luminal content (e.g., microbes or their metabolites) with a reduced capability to induce P-gp expression. CONCLUSION: Overall, by means of both in vitro and in vivo studies as well as human subject sample analysis, we identify a mechanistic link between cooperative functional outputs of the complex microbial community and modulation of P-gp, an epithelial component, that functions to suppress overactive inflammation to maintain intestinal homeostasis. Hence, our data support a new cross-talk paradigm in microbiome regulation of mucosal inflammation. Video abstract

Intestinal P-glycoprotein exports endocannabinoids to prevent inflammation and maintain homeostasis

Neutrophil influx into the intestinal lumen is a critical response to infectious agents, but is also associated with severe intestinal damage observed in idiopathic inflammatory bowel disease. The chemoattractant hepoxilin A3, an eicosanoid secreted from intestinal epithelial cells by the apically restricted efflux pump multidrug resistance protein 2 (MRP2), mediates this neutrophil influx. Information about a possible counterbalance pathway that could signal the lack of or resolution of an apical inflammatory signal, however, has yet to be described. We now report a system with such hallmarks. Specifically, we identify endocannabinoids as the first known endogenous substrates of the apically restricted multidrug resistance transporter P-glycoprotein (P-gp) and reveal a mechanism, which we believe is novel, for endocannabinoid secretion into the intestinal lumen. Knockdown or inhibition of P-gp reduced luminal secretion levels of N-acyl ethanolamine-type endocannabinoids, which correlated with increased neutrophil transmigration in vitro and in vivo. Additionally, loss of CB2, the peripheral cannabinoid receptor, led to increased pathology and neutrophil influx in models of acute intestinal inflammation. These results define a key role for epithelial cells in balancing the constitutive secretion of antiinflammatory lipids with the stimulated secretion of proinflammatory lipids via surface efflux pumps in order to control neutrophil infiltration into the intestinal lumen and maintain homeostasis in the healthy intestine

Modulation of Neutrophil Function by a Secreted Mucinase of Escherichia coli O157∶H7

Escherichia coli O157∶H7 is a human enteric pathogen that causes hemorrhagic colitis which can progress to hemolytic uremic syndrome, a severe kidney disease with immune involvement. During infection, E. coli O157∶H7 secretes StcE, a metalloprotease that promotes the formation of attaching and effacing lesions and inhibits the complement cascade via cleavage of mucin-type glycoproteins. We found that StcE cleaved the mucin-like, immune cell-restricted glycoproteins CD43 and CD45 on the neutrophil surface and altered neutrophil function. Treatment of human neutrophils with StcE led to increased respiratory burst production and increased cell adhesion. StcE-treated neutrophils exhibited an elongated morphology with defective rear detachment and impaired migration, suggesting that removal of the anti-adhesive capability of CD43 by StcE impairs rear release. Use of zebrafish embryos to model neutrophil migration revealed that StcE induced neutrophil retention in the fin after tissue wounding, suggesting that StcE modulates neutrophil-mediated inflammation in vivo. Neutrophils are crucial innate effectors of the antibacterial immune response and can contribute to severe complications caused by infection with E. coli O157∶H7. Our data suggest that the StcE mucinase can play an immunomodulatory role by directly altering neutrophil function during infection. StcE may contribute to inflammation and tissue destruction by mediating inappropriate neutrophil adhesion and activation

Computer-guided design of optimal microbial consortia for immune system modulation

Manipulation of the gut microbiota holds great promise for the treatment of diseases. However, a major challenge is the identification of therapeutically potent microbial consortia that colonize the host effectively while maximizing immunologic outcome. Here, we propose a novel workflow to select optimal immune-inducing consortia from microbiome compositicon and immune effectors measurements. Using published and newly generated microbial and regulatory T-cell (Treg) data from germ-free mice, we estimate the contributions of twelve Clostridia strains with known immune-modulating effect to Treg induction. Combining this with a longitudinal data-constrained ecological model, we predict the ability of every attainable and ecologically stable subconsortium in promoting Treg activation and rank them by the Treg Induction Score (TrIS). Experimental validation of selected consortia indicates a strong and statistically significant correlation between predicted TrIS and measured Treg. We argue that computational indexes, such as the TrIS, are valuable tools for the systematic selection of immune-modulating bacteriotherapeutics