Activation of α2B receptor ameliorates dry eye disease through enhancing regulatory T cell functions

Abstract

Purpose : The adrenergic receptor (AR) family is widely expressed by immune cells and involved in modulating their functions. This study investigated the therapeutic effect of a α2B agonist on chronic dry eye disease (DED) and its underlying immunoregulatory mechanisms.

Methods : Chronic DED was induced in 6-8 week-old female C57BL/6 mice by placing them in an environmental desiccating stress for 14 days followed by a standard vivarium for additional 14 days. 100 μl of the α2B agonist (AGN-762, 1mg/kg) was administrated 3 times a day by oral gavage from day 21 to 28. Clinical disease severity was evaluated by corneal fluorescein staining (CFS). In addition, the expression levels of IL-17A and IL-17F in conjunctiva were analyzed by qPCR and ELISA, and the frequencies of Foxp3⁺ regulatory T cells (Tregs) in draining lymph nodes were analyzed using flow cytometry. Furthermore, Tregs isolated from DED mice were cultured in the presence of AGN-762 (1 μM) for 24 hours. After culture, the frequencies of recovered Tregs and the IL-10 levels in the supernatants were analyzed by flow cytometry and ELISA, respectively. The function of cultured Tregs was further assessed by the suppression assay.

Results : DED treated with AGN-762 showed significantly decreased CFS scores (3.8±0.2 vs. 5.7±0.3, P<0.0001), and reduced expressions of IL-17A and IL-17F in conjunctiva in both mRNA (~75% and 50% decrease, P=0.04 and 0.003, respectively) and protein levels (both ~95% decrease, P=0.001 and <0.0001, respectively). In addition, AGN-762 treatment led to significant increase of Treg frequencies (13.1±0.3% vs. 9.8±0.3%, P<0.0001) and Foxp3 expressions by Tregs (measured by mean fluorescein intensity, MFI: 392±5 vs. 321±5, P<0.0001). In vitro culture of the Tregs isolated from DED in the presence of the AGN-762 effectively enhanced Foxp3 levels (MFI: 108±2 vs. 100±2, P=0.02) and promoted IL-10 secretion by Tregs (1.5-fold increase, P=0.04). Furthermore, the regulatory function of DED Tregs, which was impaired in suppressing effector T cells (suppression capability: 26.48±3.99%), was restored to a level (40.96±1.30%, P=0.006) even higher than that in normal Tregs (35.41±5.62%) after co-culture with AGN-762.

Conclusions : Our data suggest that the α2B agonist ameliorates DED through restoring Treg suppressive functions on effector Th17 cells.

This is a 2020 ARVO Annual Meeting abstract.