IL-6 is critical for the increased Th17 cell–promoting activity in mouse serum after coincidental TLR4 and complement activation. (A) Serum levels of IL-6, TNF-α, IL-1β, IL-10, IL-12, and IL-23 in WT, C3^−/−, C3aR^−/−, and C5aR^−/− mice, as measured by ELISA at 3 hours after intraperitoneal injection with PBS, CVF, LPS, or LPS + CVF. (B) Serum TGF-β levels in WT mice 3 hours after challenge with LPS or LPS + CVF. *P < .01, Student t test. (C) Serum IL-6 levels in WT or C5aR^−/− mice 3 hours after challenge with LPS, recombinant mouse C5a, or LPS + C5a. n = 4 for each group of mice in panels A through C. Values shown are mean ± SEM. (D) Mouse CD4⁺ T cells were activated by plate-bound anti-CD3/CD28 in the presence of 5% serum from WT or IL-6^−/− mice treated with LPS or LPS + C5a. Some sera from WT mice treated with LPS + C5a were depleted of 1 or more cytokines with neutralizing antibodies or isotype IgG controls, as indicated. Cells were cultured for 3 days after activation, and IFN-γ and IL-17–producing cells were detected by flow cytometry after intracellular staining. Plots are representative of 2 independent experiments.