Background/Purpose: Microbiome studies in autoimmune diseases including rheumatoid arthritis and inflammatory bowel disease demonstrate alterations in relative abundance of specific bacterial species (dysbiosis). The immunologic influence of dysbiosis is only now being appreciated as important in disease pathogenesis. As intraepithelial lymphocytes (IELs) are anatomically positioned in intimate association with epithelial cells, it is intriguing to consider how the commensal flora might impact IEL function. In contrast to IELs of the small intestine, which have been better studied, the role of IELs in the colon during health and disease remains unclear.

Methods: Epithelial cells were harvested from wild type C57Bl/6 mice, lymphocytes stained for T cell surface markers, and cells evaluated by flow cytometry. IELs were also magnetically sorted from the epithelial cell harvest and stimulated ex vivo for five hours with PMA and ionamycin. Then IEL RNA was purified and analyzed by qRT-PCR. To model dysbiosis, mice were treated with 1 mg/ml ampicillin, metronidazole, and neomycin and 0.5 mg/ml vancomycin in the drinking water for one week. Recolonization of mice occurred by cohousing antibiotic treated mice with unmanipulated littermates. Trafficking of IELs was studied using the KiKGR transgenic mouse in which violet light exposure via colonoscopy photoconverts green fluorescent cells to red.

Results: Our studies demonstrate that the major subpopulation of IELs in the colon (52%) is CD3+ CD4- CD8- TCRβ+ (versus the small intestine where 72% are CD3+ CD8αα+ TCRγδ+). These colonic double negative IELs express cell surface markers consistent with activated lymphocytes (CD44+ CD69+ CD62L-) and produce a balance between inflammatory and regulatory cytokines. We next aimed to understand the role of colonic microbiota in shaping this population of cells. Treatment of mice with broad-spectrum antibiotics significantly decreased the number of IELs and shifted cytokine expression to a more pathogenic phenotype. Not only did the number of mature, activated T cells significantly decrease by about 75%, but also the remaining IELs expressed significantly more IL-17 (nearly 70-fold higher) in the antibiotic treated mice compared to untreated controls. The impact of antibiotics was reversible, as recolonization of mice resulted in normalization of the IEL numbers, phenotype, and cytokine expression. Finally, using a transgenic mouse in which colonoscopy-guided green-to-red photoconverstion labels IELs in the distal colon, we demonstrate that IELs traffic to extraintestinal sites, including the spleen, lung, and liver.

Conclusion: These data strongly support our hypothesis that colonic microbiota dynamically influence IEL function which, in turn, influences downstream systemic immune responses. By understanding the development and function of colonic IELs in health and disease, we hope to identify a potential pathway for the development of autoimmunity.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/microbiota-modulate-intraepithelial-lymphocyte-presence-and-function/