Summary The human host has co-evolved with the collective of bacteria species, termed microbiota, in a complex fashion that affects both innate and adaptive immunity. Differential regulation of regulatory T-cell and effector T-cell responses are a direct result of specific microbial species present within the gut, and this relationship is subject is dysregulation during inflammation and disease. The microbiota varies widely between individuals and has a profound effect on how one reacts to various environmental stimuli, particularly if a person is genetically predisposed to an immune-mediated inflammatory disorder such as inflammatory bowel disease (IBD), including Crohn’s disease (CD) and ulcerative colitis (UC). Approximately half of all CD patients have elevated antibodies to CBir1, a microbiota flagellin common to mice and humans, demonstrating flagellins as immunodominant antigens in the intestines. This review focuses on the use of flagellins as probes to study microbiota specific responses in the context of health and disease as well as probes of innate and adaptive responses employed by the host to deal with the overwhelming bacterial presence of the microbiota.

The comprehensive collection of bacterial species, termed microbiota, within human and other mammalian hosts has profound effects on both innate and adaptive immunity. Multiple host innate mechanisms contribute to intestinal homeostasis, including epithelial production of protective mucin layers maintaining spatial segregation in the intestine as well as epithelial cell secretion of a broad range of antimicrobial peptides. Additionally, epithelial cells employ autophagy to contain and eliminate invading bacteria; interestingly, genetic variants in specific autophagy genes are linked to susceptibility to Crohn's disease. Innate lymphoid cells, which rapidly respond to cytokine and microbial signals, have emerged as important regulators of the intestinal immune response to the microbiota. With regard to adaptive immunity, specific microbial species stimulate induction of regulatory T cells while others induce effector T cells within the gut. Such stimulation is subject to dysregulation during inflammation and disease, contributing to ‘dysbiosis' or an abnormal microbiota composition that has been associated with a variety of immune-mediated inflammatory disorders, including celiac disease. The microbiota communicates with the immune system and vice versa; thus, an abnormal microbiota composition likely translates into an altered host immune response, though the exact mechanisms of such are not yet clear. Immunoglobulin A plays a critical role in limiting bacterial access to the host and in maintaining mutualism with the microbiota. Perturbation of the mucosal barrier via infection or other means can induce effector T cells reactive to the intestinal microbiota, and these cells can persist as memory cells for extended periods of time and potentially serve as pathogenic effector cells upon re-encounter with antigen. Health is associated with a diverse microbiota that functions to maintain the balance between T effector and T regulatory cells in the intestine. Whether dysbiosis can be reversed in immune-mediated disease, thus restoring health, is a question of intense interest for this active area of research.

BACKGROUND AND AIMS: Crohn's disease and ulcerative colitis are characterized by dysregulated adaptive immune responses to the microbiota in genetically susceptible individuals, but the specificity of these responses remains largely undefined. Therefore, we developed a microbiota antigen microarray to characterize microbial antibody reactivity, particularly to human-derived microbiota flagellins, in inflammatory bowel disease. METHODS: Sera from healthy volunteers (n ¼ 87) at the University of Alabama at Birmingham and from patients recruited from the Kirklin Clinic of University of Alabama at Birmingham Hospital, including patients with Crohn's disease (n ¼ 152) and ulcerative colitis (n ¼ 170), were

Toll-like receptor (TLR) 5 has been shown to maintain intestinal homeostasis and regulate host defense against enterobacterial infection. However, how TLR5 expression is regulated and its function in the intestine have not been fully elucidated. Here we demonstrate that mucosal dendritic cells (DCs), but not splenic DCs, express high levels of TLR5 protein. Alternatively spliced Tlr5 transcripts were identified but it did not explain the selective expression of TLR5 on mucosal DCs. Treatment with various bacterial ligands downregulated BMDC TLR5 expression, while retinoic acid and host stromal cell-derived signals promoted TLR5 expression in a TGF-β-independent mechanism. Signaling through TLR5 restrained regulatory T (Treg) cell generation, and accordingly, TLR5−/− mice displayed increased frequencies of Foxp3+ Treg cells in the intestinal lamina propria. Our data indicate that bacterial and host factors differentially regulate DC TLR5 expression. TLR5 signaling regulates immune responses towards the microbiota via modulation of the Treg/effector T cell balance.

Programed cell death promotes homeostatic cell turnover in the epithelium but is dysregulated in cancer.  The glycosyltransferase ST6Gal-I is known to block homeostatic apoptosis through α2,6-linked sialylation of the death receptor TNFR1 in many cell types. However, its role has not been investigated in gastric epithelial cells or  gastric tumorigenesis. We determined that human gastric antral epithelium rarely expressed ST6Gal-I, but the number of ST6Gal-I-expressing epithelial cells increased significantly with advancing premalignancy leading to cancer. The mRNA expression level of ST6GAL-I and SOX9 in human gastric epithelial cells correlated positively with one another through the premalignancy cascade, indicating that increased epithelial cell expression of ST6Gal-I is associated with premalignant progression. To determine the functional impact of increased ST6Gal-I, we generated human gastric antral organoids from epithelial stem cells and differentiated epithelial monolayers from gastric organoids. Gastric epithelial stem cells strongly expressed ST6Gal-I, suggesting a novel biomarker of stemness. In contrast, organoid-derived epithelial monolayers expressed markedly reduced ST6Gal-I and underwent TNF-induced, caspase-mediated apoptosis, consistent with homeostasis. Conversely, epithelial monolayers generated from gastric cancer stem cells retained high levels of ST6Gal-I and resisted TNF-induced apoptosis, supporting prolonged survival.  Protection from TNF-induced apoptosis was dependent on ST6Gal-I overexpression, since forced ST6Gal-I overexpression in normal gastric stem cell-differentiated monolayers inhibited TNF-induced apoptosis, and cleavage of α2,6-linked sialic acids from gastric cancer organoid-derived monolayers restored susceptibility to TNF-induced apoptosis. These findings implicate upregulated ST6Gal-I expression in blocking homeostatic epithelial cell apoptosis in gastric cancer pathogenesis, suggesting a mechanism for prolonged epithelioid tumor cell survival.