High basal and activated P-p65 by IL-17 in autoimmune B cells of BXD2 mice. MACS-purified CD19⁺ B cells from B6, BXD2 or BXD2-Il17ra^−/− mice were treated with or without IL-17 (30 ng/ml) for 5 min. A, Whole cell lysates were prepared and analyzed by Western blot analysis with P-p65 and p65 Abs to detect their respective proteins (30 μg in each lane). GAPDH was used as a loading control. The results are representative of three different experiments. B, Western blot analysis of the levels of P-p65 and GAPDH in purified B cells from B6 and BXD2 mice after stimulation with IL-17 by the indicated doses and time points. C and D, Flow cytometry analysis of the levels of P-p65 in GC (Fas⁺PNA⁺) and non-GC (Fas⁻PNA⁻) B cells from the indicated mouse strains. Cells were either unstimulated or stimulated with IL-17 (30 ng/ml) for 5 min. C, Representative staining for P-p65 in cells gated on GC or non-GC B cells. D, the percentages of P-p65⁺ GC and non-GC B cells are graphed as the mean ± SEM. The data in C and D are representative of three independent experiments (*p < 0.05; **p < 0.01; between B6 and BXD2 B cells). E, Real-time quantitative PCR analysis of the levels of Il17ra in purified B cells from B6 mice, non-GC (Fas⁻PNA⁻) and GC (Fas⁺PNA⁺) B cells from BXD2 mice. The results represent the mean ± SEM. N≥5 from each group. ***p < 0.005, between B6 and BXD2 B cells. F, Flow cytometry analysis of the expression of IL17RA in B cells from the indicated mouse strains. The percentages of IL17RA in PNA⁺ and PNA⁻ B cells are graphed as the mean ± SEM. The data in F are representative of three independent experiments. *p < 0.05, between B6 and BXD2 B cells.

IL-17 rapidly activates the canonical NF-κB signaling pathway in BXD2 B cells. Purified splenic B cells from BXD2 mice were treated with IL-17 (30 ng/ml). Cells were harvested at the indicated time points. A–F, The levels of the indicated protein in total cell lysates (A), cytoplasmic extract (C), and nuclear extract (E) were analyzed by Western blot analysis with the respective Abs. Quantitation of the indicated protein after normalization to the levels of GAPDH or SP1 transcription factor is graphed as the mean ± SEM for total cell lysates (B), cytoplasmic extract (D), and nuclear extract (F). The results are representative of three independent experiments. *p < 0.05; **p < 0.01 compared with unstimulated control.

NF-κB peptide inhibitor released the migration arrest of BXD2 B cells by IL-17. A, BXD2 B cells were preincubated with peptide inhibitor for 1 h and followed by stimulation with IL-17 (30 ng/ml) for 6 h before being harvested for Western blot analysis of RGS16. The peptide inhibitor is NF-κB p65-Ser²⁷⁶ inhibitory peptide, Peptide control is a nonfunctional peptide that corresponds to the PTD sequence of the inhibitor peptide. B, Quantitation of the expression of RGS16 after normalization to the levels of GAPDH is graphed as the mean ± SEM. *p < 0.05; ***p < 0.005 between the indicated comparisons. C, BXD2 B cells, after treatment with peptide inhibitor (1 h) and IL-17 (4 h) sequentially, were subjected to a transwell chamber to determine the CXCL12-mediated (100 ng/ml) chemotactic response. The chemotaxis index was calculated by dividing the number of cells that migrated in response to chemokine by the number of cells that migrated in the absence of CXCL12. *p < 0.05; **p < 0.01 between the indicated comparisons. The results are mean ± SEM from three different experiments.

Traf6 and Traf3ip2 are involved in IL-17-mediated upregulation of Rgs16 in 70Z/3 pre-B cells. 70Z/3 pre-B cells were transfected with siRNA for Traf6, Traf3ip2, or scrambled siRNA. After 24 h of siRNA transfection, the cells were treated with IL-17 (30 ng/ml). A, The expression of Traf6 (upper panel) and Traf3ip2 (lower panel) at the mRNA levels was measured by quantitative real-time PCR. B and C, Western blot analysis of the levels of P-p65 in the indicated siRNA-transfected 70Z/3 cells (B). Quantitation of P-p65 after normalization with the levels of GAPDH is graphed as the mean ± SEM (C). D and E, siRNA-transfected 70Z/3 cells were stimulated with IL-17 (30 ng/ml) for 4 h, the expression of Rgs16 was analyzed by real-time PCR (D), and the migration response of these cells to CXCL12 was analyzed by a transwell migration chamber (E). *p < 0.05 between the indicated comparisons. n = 3; mean ± SEM for all panels.

IL-17 rapidly activates nuclear translocation of P-p65 in BXD2 B cells. A and B, MACS-purified CD19⁺ B cells from BXD2 (A) or B6 (B) mice were treated with or without IL-17 (30 ng/ml) for the indicated time points. The cells were fixed and permeablized, and stained with primary P-p65 Ab followed by a FITC-conjugated secondary Ab (Green). The nuclei were stained with DAPI (Blue). The magnification of the objective lens is shown on the top.

IL-17 upregulated the levels of Rgs16 and Rgs13 in B cells. A–C, Total RNA was isolated from the purified spleen B cells of BXD2, B6 or BXD2-Il17ra^−/− mice at different time points after treatment with IL-17 (30 ng/ml). The mRNA levels of Rgs16 (A), Rgs13 (B), and Rgs1 (C) were measured by quantitative real-time PCR and were normalized to Gapdh. Significant increases relative to the untreated control are shown. *p < 0.05; **p < 0.01 compared with unstimulated control. The results are mean ± SEM from three different experiments.

IL-17 upregulated RGS16 via the P-p65 pathway in 70Z/3 pre-B cells. A and B, 70Z/3 pre-B cells were treated with IL-17 (30 ng/ml) at the indicated time points. Whole-cell lysates were prepared and analyzed by Western blot analysis with Abs against P-p65. *p < 0.05; **p < 0.01 denotes significant increase relative to the untreated control and normalized to GAPDH. C and D, 70Z/3 pre-B cells were treated with IL-17 (30 ng/ml) for the indicated time points. Whole-cell lysates were prepared and analyzed by Western blot analysis with Abs against RGS16. *p < 0.05; **p < 0.01 denotes significant increase relative to the untreated control. E and F, 70Z/3 cells were preincubated with peptide inhibitor for 1 h, and followed by stimulation with IL-17 (30 ng/ml) for 6 h before being harvested for Western blot analysis of RGS16. **p < 0.01 denotes significant decrease relative to the inhibitor control peptide. The results are mean ± SEM from three different experiments.