Complement promotes the development of inflammatory T-helper 17 cells through synergistic interaction with Toll-like receptor signaling and interleukin-6 production

Abstract

Toll-like receptors (TLRs) and complement are 2 major components of innate immunity that provide a first-line host defense and shape the adaptive immune responses. We show here that coincidental activation of complement and several TLRs in mice led to the synergistic production of serum factors that promoted T-helper cell 17 (Th17) differentiation from anti-CD3/CD28 or antigen-stimulated T cells. Although multiple TLR-triggered cytokines were regulated by complement, Th17 cell-promoting activity in the serum was correlated with interleukin (IL)-6 induction, and antibody neutralization of IL-6 abrogated the complement effect. By using both in vitro and in vivo approaches, we examined in more detail the mechanism and physiologic implication of complement/TLR4 interaction on Th17-cell differentiation. We found that the complement effect required C5a receptor, was evident at physiologically relevant levels of C5a, and could be demonstrated in cultured peritoneal macrophages as well as in the setting of antigen immunization. Importantly, despite an inhibitory effect of complement on IL-23 production, complement-promoted Th17 cells were functionally competent in causing autoimmunity in an adoptive transfer model of experimental autoimmune encephalomyelitis. Collectively, these data establish a link between complement/TLR interaction and Th17-cell differentiation and provide new insight into the mechanism of action of complement in autoimmunity.

Figure 1

Complement C5a synergizes with TLR4 to produce serum factors that drive Th17-cell differentiation. (A) Mouse CD4⁺ T cells were activated by plate-bound anti-CD3/CD28 in the presence of 5% serum from WT mice treated with PBS, LPS, CVF, or LPS+CVF. Culture medium and recombinant IL-6 + TGF-β were used as negative and positive controls, respectively, for Th17-cell differentiation. (B) CD4⁺ T cells were activated as in panel A in the presence of 5% serum from C3^−/−, C3aR^−/−, and C5aR^−/− mice treated with LPS + CVF. (C) CD4⁺ T cells were activated as in panel A in the presence of 5% serum from WT mice treated with LPS, C5a, and LPS + C5a or from C5aR^−/− mice treated with LPS + C5a. Cells were cultured for 3 days after activation, and IFN-γ and IL-17–producing cells were detected by flow cytometry after intracellular staining. Data in panels A and B were from the same experiment, whereas data in panel C were from a separate experiment.

Figure 2

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.

Figure 3

TLR4/C5aR cosignaling on macrophages enhances their capacity to differentiate Th17 cells. (A) Thioglycollate-elicited peritoneal macrophages from WT mice were stimulated with C5a, LPS, LPS + C5a, or vehicle control (medium) for 20 hours, and IL-6 levels in the supernatant were measured by ELISA. (B) Macrophages were stimulated for 20 hours as in panel A, and naive CD4⁺ T cells from WT mice were then added to the culture and activated by anti-CD3 for 3 days in the presence of recombinant TGF-β1. Some wells were treated with a C5aR antagonist at the time of macrophage stimulation by LPS and C5a (Ant-0 hour) or at the time of T-cell addition and activation (Ant-20 hour). Th17-cell frequencies were determined by FACS after intracellular staining. (C) Macrophage and CD4⁺ T-cell cocultures were set up and analyzed as in panel B except that CD4⁺ T cells were from C5aR^−/− mice. Data are representative of 2 independent experiments. Wells were set up in triplicates, and values shown are mean ± SEM. *P < .01 by Student t test.

Figure 4

Complement-TLR4 interaction promotes the development of pathogenic Th17 cells from antigen-experienced autoreactive CD4⁺ T cells. WT mice were immunized with MOG_38-50 in CFA. After 10 days, cells from draining lymph nodes were isolated and restimulated with MOG_38-50 peptide for 4 days in the presence of 5% serum from WT mice treated with LPS or LPS + C5a or in the presence of recombinant cytokines or vehicle control (medium). (A) Cells were analyzed by FACS for IFN-γ and IL-17 production after intracellular staining. (B) Cells were analyzed by FACS for IL-10 and IL-17 production after intracellular staining. (C) Twenty million CD4⁺ T cells propagated by in vitro restimulation in the presence of LPS- or LPS/C5a-treated mouse serum were adoptively transferred into naive mice (n = 6 for each group). Clinical EAE scores were determined daily. Data are representatives of 2 independent experiments.

Figure 5

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.

Figure 6

C5a enhances TLR2- and TLR9-dependent serum IL-6 production and Th17 cell–promoting activity. (A) WT mice were treated intraperitoneally with zymosan (1 g/kg), CPG1826 (20 mg/kg), and polyI:C (15 mg/kg) alone or in combination with C5a (0.2 μg per mouse). Sera were collected after 3 hours (6 hours for polyI:C), and IL-6 levels were determined by ELISA. Values shown are the mean ± SEM, n = 3 for each group, *P < .01 by Student t test; ns indicates not statistically significant. (B) Naive mouse CD4⁺ T cells were activated by plate-bound anti-CD3/CD28 for 3 days in the presence of 5% serum from mice in panel A, and production of IFN-γ and IL-17 by the activated T cells was assessed by intracellular staining and flow cytometry. Plots are representative of 2 independent experiments.