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Yukako Taketani, Man Yu, Yihe Chen, Reza Dana; Interleukin-6 is Responsible for Regulatory T cell Dysfunction in Dry Eye Disease. Invest. Ophthalmol. Vis. Sci. 2018;59(9):3298.
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Increased frequencies of IL-17+CD4+ T helper (Th17) cells and their resistance to regulatory T cell (Treg)-mediated suppression have been established in dry eye disease (DED). However, it is still unclear how Th17 immunity affects the function of Tregs in the course of DED. The aim of this study was to assess phenotypic and functional changes of Tregs in response to major Th17-associated cytokines, IL-6, IL-17, and IL-23 in a mouse model of DED.
DED was induced in female C57BL/6 mice using a controlled environment chamber for 14 days. CD4+CD25+Foxp3+ Tregs were isolated from draining lymph nodes (DLN) of normal and DED mice (n=5). The mRNA level of cytokine receptors, IL-6R, IL-17RA, and IL-23R, were examined using RT-PCR. To determine the optimal dose of IL-6 for in vitro experiments, Tregs isolated from DLN of normal mice were co-cultured with different doses of IL-6 (0.01, 0.1, 1, 10, and 100ng/ml) for 48 hours, and frequencies of Tregs and their expression of Foxp3 were evaluated using flow cytometry. Isolated Tregs were then co-cultured with 10ng/ml IL-6 for 48 hours, and the expression of surface inhibitory molecules CTLA-4, GITR, LAG-3, and PD-1 were examined using flow cytometry.
mRNA expression of IL-6R, IL-17RA, and IL-23R in Tregs were significantly increased in DED mice compared to normal controls (p=0.005, 0.006, and 0.003, respectively), and IL-6 receptor showed the highest expression levels compared to IL-17RA and IL-23R in Tregs from DED mice. Culturing Tregs with 0.1, 1, 10, and 100ng/ml of IL-6 resulted in a significant decrease in Treg recovery rate (p=0.002, 0.002, 0.002, and 0.008, respectively) and Foxp3 expression levels (p=0.006, 0.004, 0.005, and 0.005, respectively), with the highest decrease observed with 10ng/ml dose. In addition, there was a significant decrease in expression of LAG-3 and PD-1 by Tregs cultured with 10 ng/ml IL-6 compared to Tregs cultured in media alone (p=0.005 and 0.02, respectively).
Our results show that Tregs increase their mRNA expression of IL-6 receptor in response to desiccating stress and express lower levels of Foxp3, PD-1, and LAG-3 upon exposure to IL-6. These findings suggest a role for IL-6 in inducing Treg dysfunction in DED.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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