Figure 5.

PAK1 promotes Th17 differentiation by inducing IL‐6 expression in macrophages. Peritoneal macrophages (5 × 10⁵) were separated from WT or PAK1‐deficient mice and stimulated with SEA (25 μg/mL) for 24 h in a 6‐well plate, the mRNA of IL‐6 was analysed via real‐time quantitative PCR (A), and cellular supernatant was collected and measured the level of IL‐6 via ELISA (B), n = 5. (C‐D, E‐F) SEA‐activated WT or PAK1‐deficient macrophages (5 × 10⁵) co‐cultured with WT CD4⁺ T cells (1 × 10⁶) in the presence or absence of mouse IL‐6‐neutralizing antibody (5 μg/mL) or IgG controls for 24 h, flow cytometry analysed the proportion of CD3⁺CD4⁺IL‐17⁺ (Th17) and CD4⁺CD25⁺ Foxp3⁺ (Treg) cells. (G, H) The mRNA and protein levels of IRF1 was identified by real‐time quantitative PCR and Western blot, n = 5. (I) We stimulated RAW264.7 with IPA3, which was an inhibitor of PAK1. Western blot analysis of IRF1 protein from cytoplasm and nucleus in RAW264.7 after SEA (25μg/mL) stimulation for 24 h. (J‐K) Immunofluorescence of IRF1 (Red) in primary peritoneal macrophages was observed in the presence of PBS or SEA (25 μg/mL) (Scale bars, 50 μm), n = 8. Data were representative of one experiment from two replicate experiments. Data were presented as mean ± SEM. *P < .05; **P < .005; ***P < .001. (L) The peritoneal macrophages (5 × 10⁵) were treated with PBS or SEA (25 μg/mL) for 24 h, phosphorylated‐p65 and total p65 detected via Western blotting. (M) RAW264.7 cells were transfected with the plasmid overexpressing PAK1, and the protein of PAK1 was analysed by Western blotting. (N) We separated the protein from cytoplasm and nucleus in WT and PAK1‐overexpressed RAW264.7 after SEA (25 μg/mL) stimulation for 24 h, and IRF‐1 expression was analysed by Western blotting. (O) Western blot analysed the expression of total p65 and phosphorylated‐p65 protein in WT and PAK1‐overexpressed RAW264.7 incubated with SEA (25 μg/mL) for 24 h. (P) Macrophages were transfected with siRNA targeting IRF1 (si‐IRF1), and the protein of IRF1 was analysed by Western blot. (Q) Western blot analysed the protein levels of IL‐6 and phosphorylated p65 in WT and IRF1 knockdown macrophages in the presence or absence of SEA. Data were one representative experiment from two experiments with similar results (I, L‐Q)