Effect of TLR4 and complement synergy on Th17-cell development at low C5a and LPS dosages and in an antigen immunization model. (A) WT and DAF^−/− mice were treated with LPS (20 mg/kg intraperitoneally) and the indicated dose of C5a (0-5 μg/mouse intraperitoneally). Serum C5a and IL-6 levels were measured at 1 hour and 3 hours, respectively. *P < .01, Student t test; all comparisons are with WT mice treated with LPS alone. (B) Th17 cell–promoting activity of mouse sera from panel A (collected at 3 hours after treatment). (C) Serum IL-6 levels in WT mice treated with 2 mg/kg LPS alone or in combination with 0.2 μg per mouse C5a. *P < .01, Student t test. (D) Th17-promoting activity of sera from mice in panel C. Experiments panels in B and D were performed with naive CD4⁺ T cells activated with plate-bound anti-CD3/CD28 and are representative of 2 independent experiments. Values in panels A and C are the mean ± SEM, n = 3 for each group. (E) ELISA of IL-17 levels in the cell culture medium of restimulated splenocytes from mice immunized with MOG_38-50 in IFA containing LPS (100 μg per mouse) or LPS plus C5a (1 μg per mouse). Values are mean ± SEM, n = 4 for each group. *P < .01, Student t test, comparison with WT mice immunized with LPS alone; **P < .01, Student t test, comparison with DAF^−/− mice.