Sex-Based Selectivity of PPARγ Regulation in Th1, Th2, and Th17 Differentiation

Abstract

Peroxisome proliferator-activated receptor gamma (PPARγ) has recently been recognized to regulate adaptive immunity through Th17 differentiation, Treg functions, and TFH responses. However, its role in adaptive immunity and autoimmune disease is still not clear, possibly due to sexual differences. Here, we investigated in vitro treatment study with the PPARγ agonist pioglitazone to compare Th1, Th2, and Th17 differentiation in male and female mouse splenic T cells. Pioglitazone treatment significantly inhibited various effector T cell differentiations including Th1, Th2, and Th17 cells from female naïve T cells, but it selectively reduced IL-17 production in male Th17 differentiation. Interestingly, pioglitazone and estradiol (E2) co-treatment of T cells in males inhibited differentiation of Th1, Th2, and Th17 cells, suggesting a mechanism for the greater sensitivity of PPARγ to ligand treatment in the regulation of effector T cell differentiation in females. Collectively, these results demonstrate that PPARγ selectively inhibits Th17 differentiation only in male T cells and modulates Th1, Th2, and Th17 differentiation in female T cells based on different level of estrogen exposure. Accordingly, PPARγ could be an important immune regulator of sexual differences in adaptive immunity.

Keywords: PPARγ; effector T cells; estrogen; pioglitazone; sex.

Figure 1

PPARγ activation by pioglitazone treatment inhibits Th1, Th2, and Th17 differentiation in female mouse T cells. MACS-purified CD62L^highCD44^low naïve T cells from the spleens of six- to eight-week-old female C57BL/6 mice were differentiated into Th1 and Th17 cells for three days and Th2 cells for five days in specific cytokine media for T cell–skewing conditions in the presence of DMSO or pioglitazone (20 μM). The proportions of IFN-γ–positive cells in Th1 differentiation (A,B); IL-4– and IL-13–producing cells in Th2 differentiation (C,D); and IL-17A–expressing cells in Th17 differentiation (E,F) were determined by flow cytometry and demonstrated as the dot plots. Values represent mean ± SEM (n = 5~6). * p < 0.05.

Figure 2

Cytokine production in female mouse Th1, Th2, and Th17 cells is reduced by pioglitazone treatment. The accumulated production of IFN-γ in Th1 cells (A); IL-4 and IL-13 in Th2 cells (B); and IL-17A in Th17 cells (C) in culture supernatants from female T cells treated with DMSO or pioglitazone was measured by ELISA assay. Values represent mean ± SEM (n = 5~6). * p < 0.05.

Figure 3

PPARγ activation by pioglitazone treatment selectively inhibits Th17 differentiation in male mouse T cells. MACS-purified CD62L^highCD44^low naïve T cells from the spleens of six- to eight-week-old male wild-type C57BL/6 mice were skewed into Th1 and Th17 cells for three days and Th2 cells for five days. The differentiated effector T cells were treated with DMSO or pioglitazone (20 μM), and the frequencies of IFN-γ–secreting cells in Th1 differentiation (A,B); IL-4– and IL-13–producing cells in Th2 differentiation (C,D); and IL-17A–positive cells in Th17 differentiation (E,F) were determined by flow cytometry and represented as the dot plots. Values represent mean ± SEM (n = 4~5). * p < 0.05; n.s: non-significant.

Figure 4

Selective inhibition of IL-17 production in Th17 cells from male mouse T cells by pioglitazone treatment. Accumulated cytokine expression of IFN-γ in Th1 cells (A); IL-4 and IL-13 in Th2 cells (B); and IL-17A in Th17 cells (C) in cultured supernatants from male T cells treated with DMSO or pioglitazone (20 μM) was measured by ELISA. Values represent mean ± SEM (n = 4~5). * p < 0.05; n.s: non-significant.

Figure 5

Pioglitazone and estradiol co-treatment inhibits Th1, Th2, and Th17 differentiation in male T cells. CD62L^highCD44^low naïve T cells were purified by magnetic-activated cell sorting (MACS) from the spleens of six- to eight-week-old male wild-type C57BL/6 mice and were differentiated into Th1 and Th17 cells for three days and Th2 cells for five days under lineage-specific skewing conditions with DMSO, pioglitazone (20 μM), E2 (5 nM), or pioglitazone (20 μM) + E2 (5 nM). The proportions of IFN-γ–producing cells in Th1 differentiation (A,B); IL-4– and IL-13–expressing cells in Th2 differentiation (C,D) and IL-17A–secreting cells in Th17 differentiation (E,F) were determined by flow cytometry and depicted as the dot plots. Values represent mean ± SEM (n = 4~5). * p < 0.05, ** p < 0.01, *** p < 0.001; n.s: non-significant.

Figure 6

Co-treatment with pioglitazone and estradiol suppresses lineage-specific cytokine production in male Th1, Th2, and Th17 cells. Cultured supernatants from male cells treated with DMSO, pioglitazone (20 μM), estradiol (5 nM), or pioglitazone (20 μM) + estradiol (5 nM) were analyzed by ELISA assay to determine the production of IFN-γ in Th1 cells (A); IL-4 and IL-13 in Th2 cells (B) and IL-17A in Th17 cells (C). Values represent mean ± SEM (n = 4~5). * p < 0.05, ** p < 0.01; n.s: non-significant.