Regulation of Toll-like receptor-mediated inflammatory response by complement in vivo

Abstract

Toll-like receptors (TLRs) and complement are 2 components of innate immunity that are critical for first-line host defense and elicitation of adaptive immune responses. Many pathogen-associated molecular patterns activate both TLR and complement, but whether and how these 2 systems, when coactivated in vivo, interact with each other has not been well studied. We demonstrate here a widespread regulation of TLR signaling by complement in vivo. The TLR ligands lipopolysacharride (TLR4), zymosan (TLR2/6), and CpG oligonucleotide (TLR9) caused, in a complement-dependent manner, strikingly elevated plasma interleukin-6 (IL-6), tumor necrosis factor alpha (TNF-alpha), and IL-1beta, and/or decreased plasma IL-12 levels in mice deficient in the membrane complement inhibitor decay-accelerating factor (DAF). A similar outcome was observed in wild-type mice cotreated with the TLR ligands and cobra venom factor, a potent complement activator. The regulatory effect of complement on TLR-induced cytokine production in vivo was mediated by the anaphylatoxin receptors C5aR and C3aR. Additionally, changes in lipopolysaccharide (LPS)-induced cytokine production in DAF-deficient mice correlated with increased mitogen-activated protein kinase and nuclear factor-kappaB activation in the spleen. These results reveal a strong interaction between complement and TLR signaling in vivo and suggest a novel mechanism by which complement promotes inflammation and modulates adaptive immunity.

Figure 1

LPS sensitivity of wild-type (WT) and DAF^−/− mice. (A-C) ELISA assays of plasma levels of IL-6 (A), TNF-α (B), and IL-1β (C) in C57BL/6 WT and DAF^−/− mice at various time points after LPS challenge. (D) Northern blot analysis of IL-6 mRNA levels in the spleen, lung, and fat of C57BL/6 WT and DAF^−/− mice. Each lane represents an individual animal. (E) ELISA assays of plasma IL-12p40 levels in C57BL/6 WT and DAF^−/− mice at various time points after LPS challenge. (F) ELISA assays of plasma IL-6, TNF-α, and IL-1β levels in BALB/c WT and DAF^−/− mice 3 hours after LPS challenge. (G-H) Comparison of plasma IL-6, TNF-α (G), and IL-12p40, IL-12p70 (H) levels in C57BL/6 WT, DAF^−/−, and CD59^−/− mice 3 hours after LPS challenge. (I) Correlation plot of plasma IL-6 and LPS levels in C57BL/6 WT and DAF^−/− mice 3 hours after LPS challenge. N = 4 for each group in panels A-C and E. N = 2 for each group in panel D. N = 4-12 for each group in panels F-I. Values shown are mean ± SEM. *P < .05, **P < .001, Student t test.

Figure 2

Effect of complement on LPS-induced cytokine production in vivo. (A) ELISA assays of activated C3 products in plasmas of wild-type (WT) and DAF^−/− mice at various time points after LPS treatment. Percentage of C3 activation was relative to that of a mouse plasma sample activated in vitro by CVF. (B) ELISA assays of plasma IL-6 and IL-12p40 levels in WT, DAF^−/−, C3^−/−, and DAF^−/−/C3^−/− mice 3 hours after LPS challenge. (C) ELISA assays of plasma IL-6 and IL-12p40 levels in WT mice 3 hours after CVF, LPS, or CVF/LPS treatment. (D) Effect of a C3a receptor antagonist (C3aRa) and a C5a receptor antagonist (C5aRa) on LPS-induced plasma IL-6 levels in DAF^−/− mice. Polyethylene glycol 400 (PEG) was used as a vehicle control. Antagonists were administered 30 minutes before LPS injection. (E) ELISA assays of plasma IL-6 and IL-12p40 levels in WT, C3aR^−/−, and C5aR^−/− mice 3 hours after LPS or LPS/CVF treatment. N = 4-6 mice per group for panels A-E. Values shown are mean (± SEM); *P < .05, **P < .01, Student t test.

Figure 3

Effect of complement on LPS-induced cytokine production by splenocytes and peritoneal macrophages in vitro. (A) ELISA assays of IL-6 production by wild-type (WT) and DAF^−/− mouse splenocytes in culture. Splenocytes from LPS-challenged (30 minutes before harvest) mice were cultured for 3 hours in the presence or absence of C5a (50 nM) and C3a (200 nM). (B,C) ELISA assays of IL-6 (B) and TNF-α (C) production by WT and DAF^−/− mouse peritoneal macrophages in culture. Cells were stimulated by various concentrations of LPS for 5 hours. (D) ELISA assays of IL-6 production by WT and DAF^−/−/C3^−/− mouse peritoneal macrophages in culture. Cells were stimulated by 1000 ng/mL LPS for 5 hours. (E) ELISA assays of IL-6 production by WT mouse peritoneal macrophages stimulated for 5 hours with LPS (100 or 1000 ng/mL) in the presence or absence of C5a (50 nM) and C3a (200 nM). Cells from 4-5 mice were pooled and assayed in triplicate wells. Values shown are the mean ± SEM. *P < .05, **P < .01, Student t test.

Figure 4

Role of NF-κB activation and MAP kinase phosphorylation in the LPS sensitivity phenotype of DAF^−/− mice. (A) Western blot analysis showing the time course of IκBα phosphorylation in wild-type (WT) and DAF^−/− mouse spleens after LPS challenge. Each time point represents an individual mouse. (B) Western blot analysis of IκBα phosphorylation in the spleens of 4 WT and 4 DAF^−/− mice at 30 minutes after LPS challenge. (C) Western blot analysis of IκBα levels in the spleens of 4 WT and 4 DAF^−/− mice at 60 minutes after LPS challenge. (D) Effect of C5a (50 nM) on LPS (100 ng/mL)–induced activation of an NF-κB luciferase reporter gene and TNF-α production in RAW264.7 cells. Cells were transiently transfected with the reporter gene plasmid together with a human C5aR cDNA construct. NT, No treatment. (E) Western blot analysis showing the time course of ERK1/2 phosphorylation in WT and DAF^−/− mouse spleens after LPS challenge. Each time point represents an individual mouse. (F) Western blot analysis of JNK phosphorylation in the spleens of 4 WT and 4 DAF^−/− mice at 60 minutes after LPS challenge. Relative amount of each protein was expressed as the ratio between the protein and β-actin signals on Western blots.

Figure 5

Complement regulates TLR2/6 and TLR9 activation. (A) ELISA assays of plasma IL-6 levels in wild-type (WT) and DAF^−/− mice after zymosan treatment. (B) ELISA assays of plasma IL-6 and IL-12p40 levels in WT and MyD88^−/− mice 3 hours after zymosan or zymosan/CVF treatment. (C) ELISA assays of plasma IL-6 and IL-12p40 levels in WT, DAF^−/−, DAF^−/−/C3^−/−, and DAF^−/−/C5aR^−/− mice 3 hours after CpG treatment. (D) ELISA assays of plasma IL-6 and IL-12p40 levels in WT mice 3 hours after CpG, CVF, or CpG/CVF treatment. (E) ELISA assays of plasma IL-12p40 levels in WT, C5aR^−/−, and C3aR^−/− mice 3 hours after CpG or CpG/CVF treatment. Two mice were in the MyD88^−/− groups in panel B; 4 to 7 mice were in all other groups. Values shown are the mean (± SEM). *P < .05, **P < .001, Student t test.

Figure 6

Role of IL-10 in complement-mediated IL-12 inhibition. (A) ELISA assays of plasma IL-10 levels in wild-type (WT) and DAF^−/− mice 3 hours after LPS, CVF, or LPS/CVF treatment. (B) ELISA assays of plasma IL-12p40 levels in WT and IL-10^−/− mice 3 hours after LPS or LPS/CVF treatment. (C) ELISA assays of IL-10 production by cultured WT mouse peritoneal macrophages 5 hours after LPS and/or C5a (50 nM) and C3a (200 nM) stimulation. (D) ELISA assays of IL-12p40 production by cultured WT mouse peritoneal macrophages 5 hours after LPS and/or C5a (50 nM) and C3a (200 nM) stimulation in the presence or absence of anti–IL-10 mAb (5 ng/mL). Four to 6 mice were used per group for panels A and B. Macrophages from 4 to 5 mice were pooled and assayed in triplicate in panels C and D. Values shown are the mean (± SEM). *P < .05, **P < .001, Student's t test.

Figure 7

Diagram showing proposed interaction between complement and the TLR pathways. PAMPs such as LPS and zymosan can activate both pathways. Activated complement regulates TLR signaling through the G-protein-coupled anaphylatoxin receptors C5aR and C3aR, MAPKs, NF-κB, and likely other transcription factors. In the absence of the complement regulatory protein DAF, complement activation and its effect on TLR signaling is amplified. The absence of DAF may be mimicked by strong complement activators such as CVF or pathological conditions such as sepsis.