Abstract
Purpose :
We have demonstrated that T regulatory cells (Tregs) become dysfunctional during dry eye disease (DED) being less able to control pathogenic Th17 cells. Th17-associated cytokines (IL-6, IL-17, IL-23) are implicated in altering Treg function; however, the precise functions of these cytokines on Treg in DED have remained unclear.
Methods :
Using a controlled environmental chamber, female C57BL/6 mice were exposed to desiccating stress (relative humidity 10% vs. ~40% normal environment) to induce DED. Western blot of IL-6R, IL-17RA, IL-23R was performed on isolated Tregs (using magnetic activated sorting) from naïve and DED animals. Mean Fluorescent Intensity (MFI) of FoxP3, CD25, CTLA-4 and GITR of naïve and DED Tregs was measured using flow cytometry. Isolated naïve Tregs were in vitro stimulated with 20 ng/ml of IL-6, IL-17A and 30 ng/ml of IL-23 separately for 24 and 72hs. MFIs of IL-6R, IL-17RA, IL-23R, FoxP3, CD25, CTLA-4 and GITR were analysed. The cytokine-stimulated Tregs were then co-cultured with in vitro differentiated Th17 cells for 72hs. Treg suppression assay was calculated as % of inhibition of Th17 cells by flow cytometry. IL-10 and TGFβ1 supernatant levels (in Treg-Th17 co-culture) were quantified by ELISA.
Results :
DED Tregs expressed higher levels of cytokine receptors IL-6R (p=0.004), IL-17RA (p<0.001), IL-23R (p=0.002) and lower levels of FoxP3 (p<0.001), CD25 (p=0.013) and CTLA-4 (p=0.022) as compared to naïve Tregs. Culturing Tregs with each of the three Th17-associated cytokines all led to a significant decrease in FoxP3 (p<0.01), CD25 (p<0.05) and CTLA-4 (p<0.05) expression. Conversely, IL-6 and IL-17 enhanced expression of IL-6R (p<0.001 for both), IL-17RA (p<0.05 for both), IL-23R (p<0.001 for IL-6; p<0.05 for IL-17). The suppressive function on Th17 by cytokine-stimulated Tregs was reduced with IL-6 being the most potent at rendering Tregs dysfunctional (31.2 ± 4.2% vs. 63.4 ± 2.3% naïve; p=0.043). IL-6-stimulated Tregs produced significantly less IL-10 (79.6 ± 6.5 pg/ml) as compared to unstimulated Tregs (198.6 ± 19.4; p<0.001).
Conclusions :
These results suggest that IL-6 is the most potent cytokine that drives Treg dysfunction in DED, and thus can be a promising therapeutic target for restoring Treg function and reducing disease severity.
This abstract was presented at the 2022 ARVO Annual Meeting, held in Denver, CO, May 1-4, 2022, and virtually.