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. 2022 Feb 3;3:787360. doi: 10.3389/ftox.2021.787360

FIGURE 2.

FIGURE 2

Effect of AhR ligands on IL-22 mRNA expression. BMM derived from (A) WT, (B) AhR−/−, and (C) RelB−/− mice were treated with I3C (50 μM), TCDD (1 nM), and FICZ (100 nM), in non-activated and LPS- (1.0 μg/ml) activated BMM for 24 h. The expression was corrected against the housekeeping gene ß-actin. Results are presented as mean ± SEM and the y-axis represents mRNA expression level as fold increase above control. (D) Effect of gene silencing of RelA on the expression of IL-22. BMM derived from WT mice were transfected with scrambled siRNA as control or siRNA specific for RelA for 24 h and then treated with I3C (50 μM), TCDD (1 nM), and FICZ (100 nM), in non-activated and LPS- (1.0 μg/ml) activated BMM for 24 h. (E) BMM derived from WT, AhR−/−, and RelB−/− mice were stimulated with TCDD in presence or absence of LPS. After 24 h, IL-22 production was determined in cell supernatant by ELISA. (F) Effect of TCDD on IL-22 expression in BMM is independent of RORγt and JAK/STAT. BMM were pre-treated with antagonists of AhR (CH223191, 10 μM), antagonists of RORγt (SR-2211, 5 μM) and the JAK/STAT inhibitor JSI-124 (5 μM) for 10 min and then treated with 1 nM TCDD for 24 h. (G) Effect of TCDD on IL-22 mRNA expression in CD4+ T cells depends on RORγt and JAK/STAT. CD4+ T cells were stimulated with IL-21 (30 ng/ml) for 24 h in presence or absence of TCDD (1 nM). To inhibit AhR, RORγt and JAK/STAT, CD4+ T cells were pre-treated with antagonists of AhR (CH223191, 10 μM), antagonists of RORγt (SR-2211, 5 μM) and the JAK/STAT inhibitor JSI-124 (5 μM) for 10 min and then treated with 1 nM TCDD for 24 h. Cells were harvested for RNA extraction and IL-22 expression analysis via qPCR. Results are presented as mean ± SEM of triplicates from three independent experiments. Lowercase letters indicate significant differences between control and treatment groups. a significantly higher than control, p < 0.05; b significantly higher than AhR ligands or LPS alone, p < 0.05, c significantly lower than BMM derived from wt mice, p < 0.05.