Effect of in vivo TAK1 gene silencing on systemic CIA hallmarks. DBA/1 mice were immunized with bovine type II collagen (CII) on day 0 and boosted on day 21. From arthritis onset, mice were weekly injected intravenously with 0.5 mg/kg of either siTAK1- (black bars) or siCT- (white bars) lipoplexes. Serum samples from the experiment shown in Figure 4B were collected overtime. (A) Serum levels of IFN-γ, TNF-α, IL-1β, and IL-6 were analyzed on day 44 by ELISA. (B-C) Anti-CII IgG1 (B) or IgG2a (C) isotype antibody levels were measured by ELISA in control- and TAK1- siRNA-treated mice. Results are expressed as arbitrary units, as described in “Anti–type II collagen assays.” (D-G) At euthanasia, splenocytes from the different groups were collected, and isolated cells were either not stimulated (NS) or stimulated with bCII antigen (20 μg/mL). Levels of TNF-α (D), IL-6 (E), IFN-γ (F), and IL-17A (G) were determined by ELISA after 24 hours of culture. (H) Splenic T cells were stimulated with increasing concentrations of bCII. As a positive control, splenocytes were stimulated with concanavalin A (5 μg/mL). Antigen-specific T-cell proliferation was quantified by the CellTiter-Glo luminescent cell viability assay 3 days later. Results are the mean ± SEM of 7-8 mice per group and are representative of 3 separate experiments. * P < .05, ** P < .01.