C3aR-activated monocytes induce increased Th17 responses. (A) Human CD4⁺ T cells respond to supernatants from LPS+C3aR-activated monocytes with increased IL-17 secretion. CD4⁺ T cells were activated with immobilized Abs to CD3 and CD28 in the presence of fresh media or supernatants derived from monocytes that had not been activated or activated with C3a agonist (C3aRag) alone, LPS, or LPS and C3aRag. Induction of Th1 (IFN-γ), Th2 (IL-4), and Th17 (IL-17) responses was assessed 3 days postactivation using the Cytokine Bead Array. (B) T-cell viability is unaffected by exposure to monocyte-derived supernatants. Experiments were performed as described in panel A but with inclusion of the Th17-skewing control condition (+IL-6, IL-1β, and IL-23) and cell viability was assessed 3 days post–T-cell activation. Data shown in panels A-B represent mean ± SD from 4 independently performed experiments (n = 4). (C) Functional inhibition of C3a-induced monocyte-derived IL-1β significantly decreases Th17 induction. Experiments were performed as in panel A but in the absence or presence of increasing amounts of soluble IL-1 receptor (sIL-1R), which functions as an IL-1R antagonist. Control cells (2 left bars) were activated in the presence of rIL-6 and rIL-23 but with or without rIL-1β. Bars represent the median values of each condition performed in duplicate with monocyte-derived supernatants from 3 distinct donors and with T cells isolated from 3 additional different donors (n = 3). (D) Neutralization of IL-1β, IL-6, and IL-23 does not abrogate Th17 induction. Experiments were performed as in panel A but in the absence or presence of 500 ng/mL sIL-1R and function-neutralizing mAbs to IL-6 and IL-23 (10 μg/mL each). Control cells (2 left bars) were activated either in the presence or absence of Th17-skewing cytokines. *P < .05; **P < .005 as determined by ANOVA. activ, activated; cytok, cytokine; MS, monocyte-derived supernatants; NA, nonactivated.