The G protein-coupled estrogen receptor (GPER) agonist G-1 expands the regulatory T-cell population under TH17-polarizing conditions

Abstract

The transcription factor Foxp3 is critical to the suppressive phenotype of CD4+ regulatory T cells. Studies have clearly shown that numerous autoimmune diseases are marked by the presence of activated CD4+ T cells within the setting of chronic inflammation. Therefore, drugs capable of inducing Foxp3 expression in activated CD4+ T cells could be of great therapeutic interest. We have previously shown that the small molecule G-1, an agonist directed against the membrane-bound G protein-coupled estrogen receptor, can induce IL10 expression in naive CD4+ T cells. In addition, we and others have demonstrated that G-1 attenuates disease in an animal model of experimental autoimmune encephalomyelitis. Using ex vivo cultures of purified CD4+ T cells, we show that G-1 can elicit Foxp3 expression under TH17-polarizing conditions, which mimic the in situ inflammatory milieu of several autoimmune diseases. These findings build upon previous results demonstrating the immunosuppressive properties of the novel estrogenic small molecule G-1.

Figure 1. G-1 drives Foxp3 expression in CD4⁺ T cells

(A) CD4+GFP+ natural T_reg and CD4+CD62L^hiCD44^loGFP- naïve T cells (NTC) were isolated by FACS from Foxp3^egfp, GPERKO, and ERαKO mice. RNA was collected and reverse transcription used to make cDNA that was then used as the template for end-point PCR. PCR products confirmed the presence of GPER in nT_REG, NTC, and ERαKO but not GPERKO cells. GAPDH was used as a loading control. (B) Naïve T-cells (CD4⁺CD62L^hi ) were collected from spleen and inguinal lymph nodes of mice by FACS. Quantitative PCR was performed using GAPDH as an internal control and data were analyzed using the 2^ΔΔCT method. Average relative expression over 4 independent experiments is shown for the Transcription factors Foxp3, GATA3, RORγT, and T-bet. P values determined by Student’s t-test. Error bars = S.D. If no P value is indicated, P > 0.05.

Figure 2. G-1 induces Foxp3 under T(H)17-polarizing conditions

CD4⁺CD62L^hiCD44^lo naive T cells from Foxp3^egfp mice were collected by FACS and cultured for 4 days under the conditions indicated. (A) Representative histograms showing gating for GFP (Foxp3) expression analysis. (B) Summary of data from three to four independent experiments, with conditions for all panels indicated at the bottom of the figure. Individual wells were supplemented with either 100nM G-1 (Black bars) or DMSO (White bars). P values determined by Student’s t-test. *** = P < 0.0005, ** = P< 0.005, * = P < 0.05, N.S. = not significant. Error bars = S.D.

Figure 3. Foxp3+ cells induced under T(H)17-polarizing conditions do not express IL17

CD4⁺CD62L^HI naive T cells from Foxp3^egfp mice were collected by FACS and cultured for 4 days with IL6 + varying concentrations of TGFβ (0.5, 1.0, or 5.0 ng/mL). Cultures were supplemented with either DMSO or 100nM G-1, as indicated. Cells were subsequently stained for IL17A. Representative plots demonstrating IL17A and Foxp3 staining are shown. Data are from one of two independent experiments performed in triplicate.

Figure 4. G-1 has a modest effect on PD-1 surface expression

CD4⁺CD62L^hiCD44^lo naive T cells from Foxp3^egfp mice were collected by FACS and cultured for 4 days under the conditions indicated, supplemented with either DMSO or 100nM G-1, as indicated. Surface expression of PD-1 was determined by flow cytometry. (A, B) Representative histograms showing gating for analysis of PD-1 surface expression, quantified using geographic mean fluorescence intensity (GMFI), on DMSO- (grey line) or G-1- (black lines) treated cells, and isotype (rat IgG2b,κ) controls (shaded region). (C, D) Summary of data from three to four independent experiments showing relative GMFI for G-1 treated cells (black bars) relative to DMSO treated cells (white bars). Treatment conditions for all panels (AD) are indicated at the bottom of the figure. P values were determined by Student’s t-test. *** = P < 0.0005, ** = P< 0.005, * = P < 0.05, N.S. = not significant. Error bars = S.D.