In vivo TAK1 gene silencing in arthritic mice interferes with downstream JNK and NF-κB signaling cascades and pro-inflammatory cytokines. DBA/1 mice were immunized with bovine type II collagen (CII) on day 0 and boosted on day 21. Mice were injected intravenously from arthritis onset (days 25 and 28) with 0.5 mg/kg of TAK-1 siRNA lipoplexes (siTAK1). Control groups were injected with either PBS or the nontargeting siRNA lipoplexes (siCT). Twenty-four hours after the last injection, spleen cells were isolated from siTAK1-, siCT-, or PBS-treated mice for subsequent experiments. (A-B) Cultured splenic cells were activated overnight with anti-CD3/CD28 Abs, and cells were stained with anti-CD4 fluorescein isothiocyanate together with anti–IL-17A phycoerythrin and anti–IFN-γ APC mAbs. Gating was performed on the basis of CD4⁺ cells. (A) Representative dot plots for the detection of IFN-γ- and IL-17A-expressing CD4⁺ T cells in the spleen by intracellular cytokine staining assay and FACS analysis. (B) Histograms indicate mean percentages of IL-17A (Th17)– and IFN-γ (Th1)–expressing CD4⁺ T cells in the spleen. (C) ELISA analyses of TNF-α, IFN-γ, and IL-17A secretion in cultured splenic cells activated overnight with anti-CD3/CD28 Abs. (D-E) Protein extracts were prepared from freshly isolated splenocytes. (D) Western blot analyses of TAK1 inhibition and downstream signaling cascades. (E) Relative expression levels of signaling mediators in arthritic mice were quantified as described in the Methods. The highest expression level of the indicated signaling mediator in each group (PBS, siCT, and siTAK1) was arbitrarily considered as 100%. Results in panels A-E are the mean ± SEM of 5-6 mice per group. * P < .05; ** P < .01; *** P < .001 versus controls.