Figure 3.

IL-1β induces NF-κB factors that regulate Il17a/f transcription and Th17 differentiation. A, C57BL/6 CD4⁺ T cells were Th17-polarized for 5 days before live cell isolation was performed using a Ficoll gradient, and cells were rested overnight. On day 6, Th17 cells were restimulated for the indicated time frames with IL-23 (5 ng/ml) and/or IL-1β (20 ng/ml), and nuclear extracts were prepared. Nuclear translocation of STAT3 and the NF-κB factors RelA, c-Rel, and p50 were evaluated by immunoblotting. Blots were stripped and reprobed with β-actin as a loading control. The splice site at which two separate blots were merged is indicated with a black vertical line. B, FACS-sorted naïve CD4⁺ T cells from Rela^fl/fl.Cd4.cre⁺, Rel^−/−, or WT B6 littermate mice were labeled with CFSE and cultured under Th17-polarizing conditions in the absence or presence of IL-21 (10 ng/ml) for 3 days. Cells were stimulated with PMA plus ionomycin stimulation for 5 h in the presence of monensin before intracellular cytokine staining for IL-17A. Proliferation was assessed by quantifying CFSE uptake. Flow cytometry plots are gated on live CD4⁺ cells, and numbers represent the percentage of cells present in the designated quadrant. Data are representative of at least three independent experiments. C, CD4⁺ T cells isolated from WT, Rela^fl/fl.Cd4.cre⁺, or Rel^−/− mice were differentiated under Th1- or Th17-polarizing conditions (± addition of CFSE). On day 3 of polarization, proliferation was assessed by CFSE analysis. Plots are gated on live CD4⁺ T cells. D, naïve CD4⁺ T cells from the indicated mice were cultured under Th17 conditions as in Fig. 1 (for 3 days as in A and assayed directly (top panel) or for 5 days and assayed following restimulation (48 h) as indicated (bottom panel). The amount of IL-17 in culture supernatants was quantified by ELISA. Results represent mean ± S.E. of three independent experiments. *, p < 0.05. and #, p < 0.01 versus WT IL-17A production. E, FACS-sorted naïve CD4⁺ from Rela^fl/fl.Cd4.cre⁺, Rel^−/−, or WT B6 mice were cultured under Th17 conditions for 72 h and then processed for mRNA quantification by real-time PCR for the indicated genes. Data were normalized to β2m and are expressed as relative difference (n-fold) compared with WT; values for Rel^−/− are set at 1. Results represent mean ± S.E. of three independent experiments. *, p < 0.05, and #, p < 0.01 versus WT mRNA expression.