Contribution of effector T helper 17/1 (eTh17/1) versus effector Th17 cells (eTh17) to memory pool in dry eye disease

Abstract

Purpose : Our previous work has demonstrated that memory Th17 cells (mTh17; CD4⁺CD44^hiIL-17⁺) mediate the chronicity of dry eye disease (DED). However, the differential contribution of effector T cell subsets to mTh17 pool as well as the factors that mediate this conversion are not clear. This study was conducted to determine the effector precursors which give rise to mTh17 cells, and to investigate factors that promote their conversion into mTh17 cells in DED.

Methods : Six- to eight-week-old female C57BL/6 mice were exposed to desiccating stress in a controlled environment chamber for 14 days (acute DED), and were subsequently housed in a standard vivarium for additional 14 days (chronic DED). At day 14 and 28, draining lymph nodes (DLNs) were collected and expression of T-bet and RORγt by effector Th17/1 (eTh17/1; CD4⁺IL-17⁺IFNγ⁺), effector Th17 (eTh17; CD4⁺IL-17⁺) and mTh17 cells were analyzed using flow cytometry. The levels of IL-23 and substance P (SP) in DLN were analyzed at day 14 (acute DED) and day 21 (during memory induction) using real time RT-PCR, and the expression of their respective receptors (IL-23R and NK1R) by eTh17/1 and eTh17 cells were analyzed using flow cytometry. Furthermore, CD4⁺CD44^lo T effectors were isolated from DLNs at day 14 and were cultured in medium supplemented with IL-23 or SP for 48 hours, and frequencies of mTh17 cells were assessed.

Results : eTh17 and eTh17/1 cells were analyzed at day 14 and mTh17 cells were analyzed at day 28. RORγt was highly expressed by all the eTh17, eTh17/1 and mTh17 cells. In contrast, T-bet was only expressed at high levels by eTh17/1 and mTh17 cells (p<0.05), but not by eTh17 cells. During the memory induction phase, there was a significant increase in expression of IL-23 and SP (~ 3-fold increase for both compared to naïve mice, p<0.05) in DLN. Moreover, eTh17/1 expressed significantly higher levels of IL-23R and NK1R compared to eTh17 cells (p<0.05). Finally, in vitro culture of CD4⁺CD44^lo T effectors in the presence of IL-23 or SP led to increased generation of mTh17 pool by 2.73 folds and 3.05 folds, respectively, as compared to the control (p<0.05).

Conclusions : Our data suggest that T-bet⁺ eTh17/1 cells are the principal effector subsets giving rise to mTh17 cells in DED. Conversion of eTh17/1 to mTh17 cells is correlated with IL-23 and SP signaling.

This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.