Interactions between the microbiota and distal gut are important for the maintenance of a healthy intestinal barrier; dysbiosis of intestinal microbial communities has emerged as a likely contributor to diseases that arise at the level of the mucosa. Intraepithelial lymphocytes (IELs) are positioned within the epithelial barrier, and in the small intestine, function to maintain epithelial homeostasis. We hypothesized that colonic IELs promote epithelial barrier function through the expression of cytokines in response to interactions with commensal bacteria. 16S rRNA profiling revealed that candidate bacteria in the order Bacteroidales are sufficient to promote IEL presence in the colon, which in turn, produce IL-6 in a MyD88-dependent fashion. IEL-derived IL-6 is functionally important in the maintenance of the epithelial barrier as IL-6−/− mice were noted to have increased paracellular permeability, decreased claudin-1 expression, and a thinner mucus-gel layer, all of which were reversed by transfer of IL-6+/+ IELs, leading to protection of mice in response to Citrobacter rodentium infection. Therefore, we conclude that microbiota provide a homeostatic role for epithelial barrier function through regulation of IEL-derived IL-6.

BackgroundDysbiosis occurs in spondyloarthritis (SpA) and inflammatory bowel disease (IBD), which is subdivided into Crohn’s disease (CD) and ulcerative colitis (UC). The immunologic consequences of alterations in microbiota, however, have not been defined. Intraepithelial lymphocytes (IELs) are T cells within the intestinal epithelium that are in close contact with bacteria and are likely to be modulated by changes in microbiota. We examined differences in human gut-associated bacteria and tested correlation with functional changes in IELs in patients with axial SpA (axSpA), CD, or UC, and in controls.MethodsWe conducted a case-control study to evaluate IELs from pinch biopsies of grossly normal colonic tissue from subjects with biopsy-proven CD or UC, axSpA fulfilling Assessment of SpondyloArthritis International Society (ASAS) criteria and from controls during endoscopy. IELs were harvested and characterized by flow cytometry for cell surface markers. Secreted cytokines were measured by ELISA. Microbiome analysis was by 16S rRNA gene sequencing from rectal swabs. Statistical analyses were performed with the Kruskal-Wallis and Spearman’s rank tests.ResultsThe total number of IELs was significantly decreased in subjects with axSpA compared to those with IBD and controls, likely due to a decrease in TCRβ+ IELs. We found strong, significant negative correlation between peripheral lymphocyte count and IEL number. IELs secreted significantly increased IL-1β in patients with UC, significantly increased IL-17A and IFN-γ in patients with CD, and significantly increased TNF-α in patients with CD and axSpA as compared to other cohorts. For each disease subtype, IELs and IEL-produced cytokines were positively and negatively correlated with the relative abundance of multiple bacterial taxa.ConclusionsOur data indicate differences in IEL function among subjects with axSpA, CD, and UC compared to healthy controls. We propose that the observed correlation between altered microbiota and IEL function in these populations are relevant to the pathogenesis of axSpA and IBD, and discuss possible mechanisms.Trial registrationClinicalTrials.gov, NCT02389075. Registered on 17 March 2015.Electronic supplementary materialThe online version of this article (10.1186/s13075-018-1639-3) contains supplementary material, which is available to authorized users.

Background: Relationships between specific microbes and proper immune system development, composition, and function have been reported in a number of studies. However, researchers have discovered only a fraction of the likely relationships. "Omic" methodologies such as 16S ribosomal RNA (rRNA) sequencing and time-of-flight mass cytometry (CyTOF) immunophenotyping generate data that support generation of hypotheses, with the potential to identify additional relationships at a level of granularity ripe for further experimentation. Pairwise linear regressions between microbial and host immune features provide one approach for quantifying relationships between "omes", and the differences in these relationships across study cohorts or arms. This approach yields a top table of candidate results. However, the top table alone lacks the detail that domain experts such as microbiologists and immunologists need to vet candidate results for follow-up experiments. Results: To support this vetting, we developed VOLARE (Visualization Of LineAr Regression Elements), a web application that integrates a searchable top table, small in-line graphs illustrating the fitted models, a network summarizing the top table, and on-demand detailed regression plots showing full sample-level detail. We applied VOLARE to three case studies-microbiome:cytokine data from fecal samples in human immunodeficiency virus (HIV), microbiome:cytokine data in inflammatory bowel disease and spondyloarthritis, and microbiome:immune cell data from gut biopsies in HIV. We present both patient-specific phenomena and relationships that differ by disease state. We also analyzed interaction data from system logs to characterize usage scenarios. This log analysis revealed that users frequently generated detailed regression plots, suggesting that this detail aids the vetting of results. Conclusions: Systematically integrating microbe:immune cell readouts through pairwise linear regressions and presenting the top table in an interactive environment supports the vetting of results for scientific relevance. VOLARE allows domain experts to control the analysis of their results, screening dozens of candidate relationships with ease. This interactive environment transcends the limitations of a static top table.

Interactions between the microbiota and distal gut are important for the maintenance of a healthy immune system. Dysbiosis of colon bacteria has emerged as a likely contributor to diseases that arise at the level of the mucosa. Intraepithelial lymphocytes (IELs) are positioned within the epithelial barrier, and in the small intestine, function to maintain epithelial homeostasis. We hypothesized that IELs of the colon modulate epithelial barrier function through the liberation of cytokines stimulated by interactions with resident bacteria. Our data demonstrate that IL-6 is a major cytokine secreted by colonic IELs in a microbe-dependent fashion. We identify Alistipes species of the phylum Bacteroidetes as candidates to recruit IELs and stimulate their IL-6 secretion. IEL-derived IL-6 is functionally important in the maintenance of the epithelial barrier as IL-6−/− and antibiotic-treated mice were noted to have increased paracellular permeability and closer interaction with luminal bacteria. IL-6 was found to signal in colonic epithelial cells and resulted in increased epithelial barrier integrity and claudin1 expression in model epithelia. Therefore, we conclude that the host microbiota provides a homeostatic role for epithelial barrier function through regulation of IEL derived IL-6.

Dysbiosis occurs in spondyloarthritis (SpA) and inflammatory bowel disease (IBD), subdivided into Crohn’s Disease (CD) and Ulcerative Colitis (UC). The immunologic consequences of dysbiosis have not been defined. Intraepithelial lymphocytes (IELs) are T cells within the intestinal epithelium that are in close contact with bacteria, and as such, are likely to be modulated by dysbiosis. We correlated IELs with resident bacteria in SpA, IBD, and controls. Subjects with biopsy-proven IBD (N=10 with CD and 7 with UC), SpA fulfilling ASAS criteria (N=5), and healthy controls (N=15) were evaluated for fecal microbiome by 16S rRNA sequencing and IELs from colon biopsies analyzed by flow cytometry and ELSIA. Subjects with SpA had significantly lower numbers of IELs compared to controls (p=0.03). Subjects with CD had significantly increased IL-17A, (p=0.03) and IFN-γ (p<0.01) whereas those with UC had higher IL-1β (p=0.01) compared to controls. Both subjects with CD and SpA had significantly increased secretion of TNF-α (p=0.04). Correlating cytokines to bacteria revealed an association between TNF-α and the Simpson Diversity Index in subjects with UC (Spearman’s r=0.943, p<0.01); in subjects with CD, Fusobacterium had a negative correlation with TNFα+IFNγ+IL-1β (Spearman’s r=−0.786, p=0.02). Sequencing results from subjects with SpA are pending. Our data indicate differences in IEL function among subjects with SpA, CD, and UC compared to healthy controls. We hypothesize that the correlations between dysbiosis and IEL function are relevant to the pathogenesis of SpA and IBD. Future studies will be aimed at further understanding mucosal T cell and microbial interactions and may offer new therapeutic targets in these diseases.