Activation of α2B/2C adrenergic receptor ameliorates ocular surface inflammation through enhancing regulatory T cell function

Abstract

There is an unmet need for effectively treating dry eye disease (DED), a T cell-mediated chronic, inflammatory ocular surface disorder. Given the potential of nonneuronal adrenergic system in modulating T cell response, we herein investigated the therapeutic efficacy and the underlying mechanisms of a specific alpha 2 adrenergic receptor agonist (AGN-762, selective for α2B/2C receptor subtypes) in a mouse model of DED. Experimental DED was treated with the AGN-762 by oral gavage, either at disease induction or after disease establishment, and showed sustained amelioration, along with reduced expression of DED-pathogenic cytokines in ocular surface tissues, decreased corneal MHC-II⁺CD11b⁺ cells and lymphoid Th17 cells, and higher function of regulatory T cells (Treg). In vitro culture of DED-derived effector T helper cells (Teff) with AGN-762 failed to suppress Th17 response, while culture of DED-Treg with AGN-762 led to enhanced suppressive function of Treg and their IL-10 production. Adoptive transfer of AGN-762-pretreated DED-Treg in syngeneic B6.Rag1^-/- mice effectively suppressed DED Teff-mediated disease and Th17 response, and the effect was abolished by the neutralization of IL-10. In conclusion, our findings demonstrate that α2B/2C adrenergic receptor agonism effectively ameliorates persistent corneal epitheliopathy in DED by enhancing IL-10 production from Treg and thus restoring their immunoregulatory function.

Fig. 1.. α2B/2C agonism suppresses induction and progression of acute corneal epitheliopathy in dry eye disease (DED).

(A) Treatment with the α2B/2C agonist (AGN-762) at the time of DED induction effectively prevents the development of severe corneal epitheliopathy, and decreases the ocular surface inflammation. (B) Treatment with the α2B/2C agonist after DED induction effectively suppresses the acute progression of corneal epitheliopathy, and decreases the ocular surface inflammation. Corneal fluorescein staining (CFS) score was used to assess disease severity. ***, p < 0.001; ****, p < 0.0001 vs untreated or vehicle group; n = 10 eyes (5 mice) /group; data were analyzed by one-way ANOVA followed by Bonferroni’s multiple comparisons post hoc test. Cytokine mRNA levels were quantified relative to the naive (non-DED) group (relative level = 1) at the end of treatment; both eyes of each mouse were pooled together for analysis; *, p < 0.05; **, p < 0.01; data were analyzed by unpaired t test.

Fig. 2.. α2B/2C agonism suppresses chronic corneal epitheliopathy in dry eye disease (DED) and restores immunohomeostasis.

(A) Treatment with the α2B/2C agonist (AGN-762) after the development of chronic DED effectively ameliorates the disease, assessed by corneal fluorescein staining (CFS) score. ****, p < 0.0001 vs untreated or vehicle group; n = 30 eyes (15 mice) /group from two independent experiments. (B) Corneal inflammation was assessed at the end of treatment by quantifying mature MHC-II⁺CD11b⁺ cells using flow cytometry and protein levels of IL-1β and IL-6 using ELISA. The upper panel shows the gating strategy for flow cytometry analysis. (C) Conjunctival inflammation was assessed by quantifying infiltrating CD4⁺ T cells using flow cytometry and protein levels of IL-17A, IL-17F, and IFN-γ using ELISA. The upper panel of the flow plots shows the gating strategy for flow cytometry analysis. (D) Draining lymph node (DLN) Th17 response was determined by flow cytometry. The upper panel shows the gating strategy for flow cytometry analysis. (E) The suppressive function of Treg cells on cell proliferation was quantified using the BrdU incorporation assay and compared with the proliferative responses in the absence of Treg (0 % suppression). For flow cytometry of corneal and conjunctival tissues, samples were pooled from 5 eyes in each group. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; data shown were from one representative experiment out of two performed and were analyzed by one-way ANOVA followed by Bonferroni’s multiple comparisons post hoc test.

Fig. 3.. α2B/2C agonism provides sustained amelioration of corneal epitheliopathy and suppresses disease exacerbation.

(A) A 7-day treatment with the α2B/2C agonist (AGN-762) in chronic DED (day 21 – 28) effectively maintains therapeutic efficacy for months (day 28 – 56), and suppresses disease exacerbation in treated animals upon re-challenged with desiccating stress (day 56 – 63). Disease severity was assessed by corneal fluorescein staining (CFS) score. ***, p < 0.001; ****, p < 0.0001 vs untreated or vehicle group; n = 20 eyes (10 mice) /group from two independent experiments. (B) Corneal inflammation was assessed at day 63 by quantifying mature MHC-II⁺CD11b⁺ cells using flow cytometry and protein levels of IL-1β and IL-6 using ELISA. The same gating strategy for flow cytometry analysis was used as shown in Fig. 2B. (C) Conjunctival inflammation was assessed at day 63 by quantifying protein levels of IL-17A, IL-17F, and IFN-γ using ELISA. (D) Draining lymph node Th17 response was determined by flow cytometry at day 63. The same gating strategy for flow cytometry analysis was used as shown in Fig. 2D. (E) The suppressive function of Treg cells on cell proliferation was quantified using the BrdU incorporation assay and compared with the proliferative responses in the absence of Treg (0 % suppression). For flow cytometry of corneal and conjunctival tissues, samples were pooled from 5 eyes in each group. *, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001; n.s., not significant; data shown were from one representative experiment out of two performed and were analyzed by one-way ANOVA followed by Bonferroni’s multiple comparisons post hoc test.

Fig. 4.. α2B/2C agonism by AGN-762 promotes IL-10 production by DED-Treg and restores the function of DED-Treg in vitro.

(A) Flow cytometry plots show the purity of freshly sorted Treg cells defined as CD4⁺CD25⁺Foxp3⁺ cells that were subjected to cell cultures in vitro. (B) Flow cytometric analysis of DED-Treg after 24 h culturing with AGN-762, with representative dot plots shown (the upper panel shows the gating strategy), and frequencies and mean fluorescence intensity (MFI) of Foxp3 summarized in the bar graphs. The frequencies were summarized from three independent experiments and MFI was summarized from one representative experiment. (C) ELISA assay on the culture supernatants for the levels of IL-10 and TGF-β. Data were summarized from two independent experiments. (D) The suppressive function of Treg cells on cell proliferation was quantified using the BrdU incorporation assay and compared with the proliferative responses in the absence of Treg (0 % suppression). *, p < 0.05; data were analyzed by one-way ANOVA followed by Bonferroni’s multiple comparisons post hoc test.

Fig. 5.. α2B/2C agonism (by AGN-762 at the higher concentration) enhances DED-derived effector T cells (Teff) in vitro.

(A) Flow cytometry plots show the purity of freshly sorted Teff cells defined as CD4⁺CD25⁻Foxp3⁻ cells that were subjected to cell cultures in vitro. (B) Flow cytometric analysis of CD4⁺CD25⁻ Teff after 24 h culturing with AGN-762, with representative dot plots shown (the upper panel shows the gating strategy), and frequencies of IL-17A⁺IFN-γ⁻ Th17, IL-17A⁺IFN-γ⁺ Th17/1, and IL-17A⁻IFN-γ⁺ Th1 summarized in the bar graphs. (C) ELISA assay on the culture supernatants for the levels of IL-17A and IFN-γ. Data shown were from one representative experiment out of two performed. *, p < 0.05; data were analyzed by one-way ANOVA followed by Bonferroni’s multiple comparisons post hoc test.

Fig. 6.. Adoptive transfer of ex vivo AGN-762-treated DED-Treg effectively reduces corneal epitheliopathy severity and Th17 immunity.

(A) Treg cells were isolated from DED mice and in vitro cultured with 1 μM AGN-762 or vehicle for 24 h. The freshly sorted effector CD4⁺ T cells from DED (Teff) along with normal Treg, DED-Treg pre-treated with the vehicle, or DED-Treg pre-treated with AGN-762 were adoptively transferred to Rag1^−/− mice, which were subsequently exposed to desiccating stress for 6 days. Disease severity was assessed by corneal fluorescein staining (CFS) score, and summarized from two independent experiments. n = 10–14 eyes (5–7 mice) /group. (B) Flow cytometric analysis of T cell response in the draining lymph nodes of Rag1^−/− recipients at day 6, with representative dot plots shown on the left (the upper panel shows the gating strategy) and frequencies of IL-17-producing CD4 + T cells (including both IL-17A⁺IFN-γ⁻ Th17 and IL-17A⁺IFN-γ⁺ Th17/1) summarized from two independent experiments on the right. *, p < 0.05; **, p < 0.01; data were analyzed by one-way ANOVA followed by Bonferroni’s multiple comparisons post hoc test.

Fig. 7.. Neutralization of IL-10 abrogates AGN-762-mediated restoration of Treg immunosuppressive function.

(A) DED-Treg cells were pre-treated with 1 μM AGN-762 or vehicle for 24 h and then subjected to suppressive function assay in the presence of anti-IL-10 neutralizing antibody or isotype IgG control. (B) Treg cells were isolated from DED mice and in vitro cultured with 1 μM AGN-762 for 24 h. The freshly sorted effector CD4⁺ T cells from DED (Teff) along with DED-Treg pre-treated with AGN-762 were adoptively transferred to Rag1^−/− mice, which were subsequently exposed to desiccating stress for 6 days. The recipients received treatment with anti-IL-10 antibody or control IgG immediately after adoptive transfer (day 0) as well as at day 3. Disease severity was assessed by corneal fluorescein staining (CFS) score, and summarized from two independent experiments. n = 16 eyes (8 mice) /group (C) Flow cytometric analysis of T cell response in the draining lymph nodes of Rag1^−/− recipients at day 6, with representative dot plots shown on the left and frequencies of total T cells and IL-17-producing CD4 + T cells (including both IL-17A⁺IFN-γ⁻ Th17 and IL-17A⁺IFN-γ⁺ Th17/1) summarized from two independent experiments on the right. *, p < 0.05; **, p < 0.01; data were analyzed by one-way ANOVA followed by Bonferroni’s multiple comparisons post hoc test (A) or unpaired t test (B and C).