Complement activation product C5a is a selective suppressor of TLR4-induced, but not TLR3-induced, production of IL-27(p28) from macrophages

Abstract

There is accumulating evidence that the complement activation product, C5a, can orchestrate cellular immune functions. IL-27(p28/EBI3) is an emerging key player essential for regulating inflammatory responses and T cells. In this article, we report that C5a robustly suppressed IL-27(p28) gene expression and release in peritoneal macrophages. These cells from C57BL/6J mice abundantly produced IL-27(p28) after engagement of either the TLR3 (polyinosinic-polycytidylic acid) or TLR4 (LPS) receptor. Genetic deficiency of either TLR4 or LBP completely incapacitated the ability of macrophages to secrete IL-27(p28) in response to LPS. IL-27(p28)-producing macrophages also expressed the C5aR receptor, thus displaying an IL-27(p28)(+)F4/80(+)C5aR(+) phenotype. C5a suppressed IL-27(p28) in LPS-stimulated macrophages via interactions with the C5aR receptor rather than the C5L2 receptor. After endotoxemia, C5aR(-/-) mice displayed higher plasma levels of IL-27(p28) compared with C57BL/6J mice. C5a did not affect the release of IL-27(p28) or the frequency of IL-27(p28)(+)F4/80(+) macrophages after engagement of TLR3. Mechanistically, LPS activated both the NF-κB and the PI3K/Akt pathways, whereas C5a activated only the PI3K/Akt pathway. Engagement of PI3K/Akt was inhibitory for IL-27(p28) production, because PI3K/Akt pharmacologic blockade resulted in increased amounts of IL-27(p28) and reversed the suppressive effects of C5a. Blockade of PI3K/Akt in endotoxemic C57BL/6J mice resulted in higher generation of IL-27(p28). In contrast, the PI3K/Akt pathway was not involved in TLR3-mediated release of IL-27(p28). These data provide new evidence about how complement activation may selectively interfere with production of T cell regulatory cytokines by APCs in the varying contexts of either bacterial (TLR4 pathway) or viral (TLR3 pathway) infection.

Figure 1

Characterization of IL-27(p28) release from macrophages after activation of TLR3 or TLR4. (A) Peritoneal elicited macrophages (PEM) from C57BL/6J (Wt) mice were incubated with agonists for various TLR-agonists (all 1 µg/ml) and secreted IL-27(p28) detected by ELISA after 10h. (B) Macrophages from Wt mice, LBP^−/− mice and TLR4^−/− mice were incubated with LPS (50ng/ml) for 10 h before detection of IL-27(p28). (C) Dose response studies of IL-27(p28) release by PEM after TLR4-activation by LPS (10 µg/ml – 10 ng/ml), 10 h. (D) Dose response curve of IL-27(p28) production after TLR3-activation by Poly (I:C) (10 µg/ml – 500 ng/ml), 10 h. (E) Time course of IL-27(p28) release after LPS (1 µg/ml). (F) Time course of IL-27(p28) production after Poly (I:C) (10 µg/ml). All experiments shown were done with thioglycollate elicited PEM from C57BL/6J (Wt) mice. Data are representative of at least 3 independent experiments. *p < 0.05, error bars represent s.e.m.

Figure 2

C5a mediates inhibition of IL-27(p28) from TLR4-activated macrophages. (A) ELISA of IL-27(p28) levels in supernatants from macrophages after incubation with LPS alone, the combination of LPS and different concentrations of recombinant mouse C5a (100-10 nM) or C5a alone (100 nM), 10 h.(B) Suppression of IL-27(p28) production from LPS-activated macrophages by co-incubation with C5a (100 nM) at different time points. (C) RT-PCR of mRNA levels for IL-27(p28) in macrophages after LPS alone or in combination with C5a (100 nM). (D) Effects of recombinant mouse C5a_desArg (100 nM) on IL-27(p28) secretion by LPS-activated macrophages, 10h. (E) IL-27(p28) release from macrophages after co-stimulation with LPS and formyl-methionyl-leucyl-phenylalanine (FMLP, 4 µM) compared to LPS alone, n.s. denotes not significant. All experiments shown were done with PEM from C57BL/6J (Wt) mice and LPS was used as 1 µg/ml. Data are representative of at least 3 independent experiments. *p < 0.05, error bars represent s.e.m.

Figure 3

C5a suppresses IL-27(p28) via the C5aR but not the C5L2 receptor. (A) Flow cytometry analysis of the C5aR receptor together with the macrophage surface marker F4/80 on PEM from Wt mice or C5aR^−/− mice. (B) PEM were left as resting controls (Ctrl) or stimulated with LPS (1 µg/ml) for 12 h, both in the presence of Monensin (2 µM). Cells were then analyzed for F4/80, C5aR and intracellular IL-27(p28) by flow cytometry. Dot-plots were gated to show only F4/80⁺ events. (C) PEM of the strains C57BL/6J, C5aR^−/− and C5L2^−/− were incubated with LPS and increasing concentrations of C5a (0, 10, 100 nM) for 10 h. (D) Relative inhibition of LPS-induced IL-27(p28) release in macrophages from C57BL6/J, C5aR^−/− or C5L2^−/− mice by C5a (100 nM). Levels of IL-27(p28) with LPS alone were used as 100% for each individual strain. All experiments were done with thioglycollate elicited macrophages (PEM) and LPS was used as 1 µg/ml. Data are representative of at least 3 independent experiments. *p < 0.05, error bars represent s.e.m.

Figure 4

C5a inhibition of IL-27(p28) is specific for the TLR4- but not the TLR3-pathway. (A) Relative effects of C5a on IL-27(p28) release by macrophages (PEM) after activation by either LPS or Poly (I:C), 20 h, ELISA. Levels of IL-27(p28) were set to 100% for LPS and Poly (I:C) alone, respectively, n.s. indicates not significant. (B) Flow cytometry analysis of intracellular IL-27(p28) and surface F4/80 in PEM after 12 h incubation with either LPS and Poly (I:C) alone or in combination with C5a. All experiments shown were done with thioglycollate elicited macrophages (PEM) from C57BL/6J mice. LPS and Poly (I:C) were both used as 1 µg/ml and C5a as 100 nM. Data are representative of at least 3 independent experiments. *p < 0.05, error bars represent s.e.m.

Figure 5

LPS and Poly (I:C) mediate release of IL-27(p28) in vivo. (A) Survival curves of C57BL/6J (Wt) mice (n=8–12 per group) after i.p. injection with different doses of LPS from E. coli (0111:B4). (B) RT-PCR of IL-27(p28) gene expression in spleen homogenates from Wt mice 6 h after administration of either LPS (10 mg/kg BW i.p., n=6) or Poly (I:C) (10 mg/kg BW i.p., n=5). Sham mice (n=3) received an i.p. injection with PBS. (C) Levels of circulating plasma IL-27(p28) as detected by ELISA after 6 h from the same experiment as shown in (B). *p < 0.05, error bars represent s.e.m.

Figure 6

Absence of the C5aR receptor correlates with increased IL-27(p28) levels in vivo. C57BL/6J (n=8), C5aR^−/− (n=6) and C5L2^−/− (n=6) mice were injected with LPS (10 mg/kg BW i.p.). Plasma was collected after 12 h and IL-27(p28) detected by ELISA.*p < 0.05, error bars represent s.e.m. and n.s. denotes not significant compared to C57BL/6J.

Figure 7

C5a suppression of LPS-induced IL-27(p28) is related to differential roles of the NFκB and PI3K-Akt signaling pathways. (A) Effects of different doses of the IκB-inhibitor Bay11-7082 on IL-27(p28) production from Wt macrophages after LPS, 6h, ELISA.(B) Wt macrophages were stimulated for 2 h with LPS alone, the combination of LPS and C5a (100 nM) or left as untreated controls (Ctrl). Nuclear lysates were prepared an analyzed for NFκB activity for binding of its DNA consensus sequence (5’-GGGACTTTCC-3’) by immunodetection. (C) Bead-based assay for phospho-Akt (threonine 308). Wt macrophages were stimulated for 30 min with LPS and C5a (100 nM) alone or in combination, or left as un stimulated controls. (D) Macrophages were stimulated with either LPS, Poly (I:C) or left as un stimulated controls. Cells were fixated after 60 min and phosphorylation of Akt (threonine 308) was analyzed by flow cytometry. Histograms were gated to only show F4/80⁺ cells. (E) RT-PCR of mRNA levels for IL-27(p28) from macrophages treated with LPS alone or in combination with wortmannin (1 µM) or Bay11-7082 (10 µM), 6h.(F) Effects of specific inhibition of the PI3K-Akt pathway by wortmannin (1–0.1 µM) for the release of IL-27(p28) from LPS-activated Wt macrophages, ELISA, 6 h. (G) Differential effects of wortmannin (1 µM) on production of IL-27(p28) when induced by either LPS or by Poly (I:C), 20 h. (H) PI3K-Akt blockade by wortmannin reverses C5a-mediated suppression of IL-27(p28) release from macrophages after LPS, 8 h, ELISA.(I) Plasma levels of IL-27(p28) in C57BL/6J Wt mice 8 h after endotoxemia. Mice had been injected with wortmannin (1 mg/kg BW i.p., n=4) or vehicle (n=5) 1 h before LPS (10 mg/kg BW i.p.). All data shown except (I) were done with thioglycollate elicited macrophages from C57BL/6J (Wt) mice. Wortmannin and Bay11-7082 were added 1 h before LPS (1 µg/ml), Poly (I:C) (1 µg/ml) or C5a (100 nM). Data are representative of 3 independent experiments or were performed with the numbers of mice as indicated. *p < 0.05, error bars represent s.e.m.

Figure 8

IL-27(p28) is suppressed by C5a. The proposed simplified scheme summarizes the interactions of C5a and IL-27(p28) in macrophages, highlighting the differential roles of the intracellular signaling pathways involved.