IL-10 expression by Th17 cells correlates with their response to glucocorticoids

Abstract

Purpose: We have previously shown that CD4⁺ T cells from steroid refractory (SR) uveitis patients demonstrate both increased IL-17 expression and a failure to up-regulate IL-10 in response to glucocorticoids; leading to a significantly reduced ratio of IL-10 to IL-17 in CD4⁺ cells from these patients. Hence, due to the relationship between these two cytokines and the clinical SR uveitis phenotype, we sought to determine the capacity of the synthetic glucocorticoid dexamethasone (Dex) to up-regulate IL-10 in Th17 cells and how production of IL-10 from these cells affects their response to Dex.

Methods: All experiments used blood from healthy donors (N=4). CD4⁺ T cells were isolated and cultured with anti CD3/CD28 microbeads and 1x10^-6M Dex for 4 days. Post culture, Dex induced IL-10 cells were isolated using capture antibodies (Miltenyi Biotech) and sorted (BD influx). Their suppression of CD4⁺CD25^- effector cells was functionally assessed in a standard proliferation assay. Similarly, Th1 and Th17 CD4⁺ T cells were isolated on the basis of differential expression of CXCR3, CCR4, CCR6 and CD161 and cultured as described. On day 4, intracellular IL-17, IFNγ and IL-10 expression was determined by flow cytometry (BD, LSRII). To assess the effects of IL-10 on suppression by Dex, sorted Th1 and Th17 cells were cultured with IL-2 and autologous irradiated APCs for 14 days, after which intracellular cytokine expression was determined by flow cytometry. Cells were then cultured with 1x10^-6M Dex for 24hours and proliferation was quantified using tritiated thymidine incorporation.

Results: Dex induced IL-10⁺ CD4⁺ T cells exhibited functional suppression of CD4⁺CD25^- cells (1 Treg:15 effector cell ratio). Both Th17 and Th1 cells were able to up-regulate IL-10 expression after culture with Dex. However, the suppression of proliferation by Dex in Th17 cells correlated positively with the level of IL-10 in these cells.

Conclusions: IL-10 and IL-17 are key cytokines involved in the clinical SR phenotype. In normal volunteers Dex up regulates IL-10 in CD4⁺ T cells and these cells are functionally suppressive. Further, in Th17 cells from normal volunteers, Dex induces increased IL-10 expression and expression of IL-10 in this cell subset correlates with Dex mediated suppression of proliferation suggesting this may function as a negative feedback mechanism to curb immune responses.<br />