Figure 2.

Th9 cells enhance osteoclastogenesis in an IL-9 dependent manner: Spleen was harvested and processed for negative selection of CD4⁺CD25^- naïve T cells. After estimation of CD4⁺ T cells purity (>95 %), naïve T cells were cultured in Th9 differentiation conditions for 72 h. (A) Zebra plot showing the differentiation of Th9 cells in the presence of 17-β Estradiol. (B) Bar graphs representing the percentage of CD4⁺IL-9⁺ Th9 cells. (C) MFI of IL-9. (D) Bar graph representing the level of IL-9 in the culture supernatant of Th9 cells in the absence or presence of estrogen. (E) BMCs and Th0/Th9 were co-cultured in cell culture plate in the presence of M-CSF (30 ng/mL) and RANKL (60 ng/mL) for 4 d. Th9 cells were differentiated from naïve T cells for 72 h prior to co-cultures. Photomicrographs represent TRAP positive cells. (F) Number of TRAP positive cells with more than 3 nuclei. (G) Osteoclast differentiation was induced in BMCs with M-CSF (30 ng/mL) and RANKL (60 ng/mL) with or without IL-9 at different concentrations for 4 d. Giant multinucleated cells were stained with TRAP and cells with ≥ 3 nuclei were considered as mature osteoclasts. Photomicrographs at 20× magnification were taken. (H) Number of TRAP positive cells with more than 3 nuclei. (I) Photomicrographs represent TRAP positive multinucleated cells in absence and presence of RANKL. (J) Graph representing number of TRAP positive cells with more than 3 nuclei. Data are expressed as mean ± SEM. Data were analyzed by unpaired Student t test and analyzed by one-way ANOVA. ^*p ≤ 0.05, ^**p ≤ 0.01, ^***p ≤ 0.001, ^****p ≤ 0.0001) compared with the indicated group. Abbreviation: BMC, bone marrow cells.