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. 2011 Sep;41(9):2741-52.
doi: 10.1002/eji.201041350. Epub 2011 Aug 4.

TLR activation enhances C5a-induced pro-inflammatory responses by negatively modulating the second C5a receptor, C5L2

Anne-Catherine Raby  1 , Benjamin Holst, James Davies, Chantal Colmont, Yves Laumonnier, Barbara Coles, Sanjoy Shah, Judith Hall, Nicholas Topley, Jörg Köhl, B Paul Morgan, Mario O Labéta
Affiliations
Free PMC article

TLR activation enhances C5a-induced pro-inflammatory responses by negatively modulating the second C5a receptor, C5L2

Anne-Catherine Raby et al. Eur J Immunol. 2011 Sep.
Free PMC article

Abstract

TLR and complement activation ensures efficient clearance of infection. Previous studies documented synergism between TLRs and the receptor for the pro-inflammatory complement peptide C5a (C5aR/CD88), and regulation of TLR-induced pro-inflammatory responses by C5aR, suggesting crosstalk between TLRs and C5aR. However, it is unclear whether and how TLRs modulate C5a-induced pro-inflammatory responses. We demonstrate a marked positive modulatory effect of TLR activation on cell sensitivity to C5a in vitro and ex vivo and identify an underlying mechanistic target. Pre-exposure of PBMCs and whole blood to diverse TLR ligands or bacteria enhanced C5a-induced pro-inflammatory responses. This effect was not observed in TLR4 signalling-deficient mice. TLR-induced hypersensitivity to C5a did not result from C5aR upregulation or modulation of C5a-induced Ca(2+) mobilization. Rather, TLRs targeted another C5a receptor, C5L2 (acting as a negative modulator of C5aR), by reducing C5L2 activity. TLR-induced hypersensitivity to C5a was mimicked by blocking C5L2 and was not observed in C5L2KO mice. Furthermore, TLR activation inhibited C5L2 expression upon C5a stimulation. These findings identify a novel pathway of crosstalk within the innate immune system that amplifies innate host defense at the TLR-complement interface. Unravelling the mutually regulated activities of TLRs and complement may reveal new therapeutic avenues to control inflammation.

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Figures

Figure 1
Figure 1
Sensitivity to C5a of PBMCs pre-exposed to TLR ligands. (A–C) Levels of IL-8 in culture supernatants of PBMCs (1.5×105/well) (A) stimulated for 14 h with the indicated concentrations of C5a and (B) after washing and re-culture following pre-exposure (14 h) to LPS (100 pg/mL or as indicated), Pam3Cys (100 ng/mL), Zymosan (1 μg/mL), Flagellin (5 μg/mL), Imiquimod (3 μg/mL) or mock-pre-exposure (no TLR ligand). IL-8 concentrations were estimated by subtracting the background levels of IL-8 present in cultures not activated with C5a and pre-exposed or not to TLR ligands from the corresponding C5a-activated samples (IL-8 background levels (ng/mL): No ligand/No C5a, 1.6±0.7; +LPS, 2.3±1.2; +Pam3Cys, 1.5±0.6; +Zymosan, 1.3±0.9; +Flagellin, 6.9±2.5; +Imiquimod, 5.3±1.1; n≥4). (C) IL-8 fold increases were determined by comparing IL-8 levels – after background subtraction – between C5a-stimulated (10 nM) cell samples pre-exposed and not pre-exposed to LPS. (D) Levels of IL-6 in culture supernatants of PBMCs stimulated for 14 h with the indicated concentrations of C5a, after washing and re-culture following pre-exposure to LPS. (E and F) Determination of (E) IL-6 and IL-8 mRNA levels in RNA samples and (F) NF-κB concentrations in the nuclear extracts of PBMCs pre-exposed or not to LPS and subsequently stimulated with C5a as described for A–D. (E) mRNA levels are relative to control (No LPS/No C5a). Results are from one experiment (+SD) representative of at least four for each ligand (A, B) or three (C–F). *p<0.05, **p<0.01, ***p<0.005 (TLR-pre-exposed versus TLR not pre-exposed, paired Student's t-test).
Figure 2
Figure 2
Sensitivity to C5a of human blood cells pre-exposed to LPS or E. coli. (A) C5a-induced levels of IL-8 and (B) fold increase in IL-8 concentration in blood cell culture supernatants following whole blood (100 μL/well) pre-exposure or not to LPS (500 pg/mL, A, left and B) or E. coli (1×108 CFU/mL). Pre-exposure to TLR ligands followed by C5a stimulation, and estimation of C5a-induced IL-8 concentrations and fold increases were as described for Fig. 1. (A) Results are from one experiment (+SD) representative of three. *p<0.05, ***p<0.005 (LPS- or E. coli-treated versus mock-treated, paired Student's t-test). (B) Response profile of 15 healthy blood donors.
Figure 3
Figure 3
Ex vivo blood cell sensitivity to C5a of TLR4 signalling-deficient and WT mice. KC and MCP-1 levels in blood cell culture supernatants (100 μL whole blood/condition) of C3H/HeJ and C3H/HeN (WT) mice stimulated (14 h) ex vivo with C5a following a challenge (1 h) i.p. with LPS (50 μg/mouse) or PBS (no LPS). The C5a-induced chemokine concentrations were estimated by background subtraction as described for Fig. 1. Values are expressed as the mean+SEM (n=5/condition).*p<0.05, **p<0.01, ***p<0.005 (LPS-treated versus no LPS, paired Student's t-test).
Figure 4
Figure 4
Effect of a pre-exposure to LPS on cell sensitivity to C5a and C5aR expression. (A) Fold increase in the levels of IL-8 – determined as described for Fig. 1– in culture supernatants of PBMCs (1.5×105/well) pre-exposed or not to LPS (100 pg/mL) for the indicated times (starting from 30 min), and subsequently activated (14 h) with C5a (10 nM). (B) C5aR cell-surface expression levels on gated monocytes or neutrophils (inset) at different times following 1×106 PBMC (monocytes) or 100 μL whole blood (neutrophils) pre-exposure (30 min) to 100 pg/mL (PBMCs) or 500 pg/mL (whole blood) LPS or a mock-pre-exposure (no LPS), and subsequent activation or not with C5a (10 nM) for the indicated times. (C) C5aR mRNA levels determined by RT-qPCR in RNA samples extracted from PBMC aliquots taken from the experiment described in (B) at the end of the culture (12 h). Results are from one experiment (A and C, ±SD) representative of three. *p<0.05, ***p<0.005 (LPS-pre-exposed versus not pre-exposed, paired Student's t-test).
Figure 5
Figure 5
C5a-induced Ca2+ mobilization following PBMCs pre-exposure to LPS. C5a (10 nM)-induced changes in monocyte cell fluorescence over 180 s, as a measure of intracellular Ca2+ mobilization, detected by flow cytometry following PBMC (1×106/condition) pre-exposure or not to LPS (100 pg/mL) for the indicated times and staining with the Ca2+-chelating fluorescent dye, Fluo3-AM. Background fluorescence was determined before addition of C5a (time 0) in cells pre-exposed or not to LPS. Results are expressed as normalized (Ca2+)i: the ratio between the mean fluorescence intensity at time t after C5a addition and that at time 0. A representative experiment out of four is shown.
Figure 6
Figure 6
Effect of TLR activation on C5L2 receptor activity. (A) Western blot analysis and densitometric scanning of HMGB1 levels in cytoplasmic cell extracts of PBMCs (0.5×106/condition) pre-exposed (14 h) or not to LPS (100 pg/mL) and subsequently activated (14 h) or not with C5a (10 nM). The IL-8 levels in the culture supernatants of this experiment are shown in the lower left inset. Results are of one experiment representative of five. *p<0.05; **p<0.01; ***p<0.005, paired Student's t-test. (B) Western blot analysis and densitometric scanning of C5a (60 nM)-induced levels of HMGB1 in culture supernatants of whole blood cells (100 μL whole blood/condition) from TLR4 signalling-deficient (C3H/HeJ) and WT mice (n=5/condition) challenged (1 h) i.p. with LPS (50 μg/mouse) or PBS (No LPS). Results shown for each condition are of the pooled supernatants of the five mice.
Figure 7
Figure 7
C5L2 receptor expression and cell sensitivity to C5a. (A) HMGB1 (left) and IL-8 (right) levels detected in cytoplasmic cell extracts and culture supernatants respectively, following PBMC (0.5×106/condition) stimulation (14 h) with C5a (2.5 nM) in the absence or presence of an anti-C5L2 blocking mAb (5 μg/mL) or the same mAb denatured by boiling (Ø). Results are from one experiment representative of four (HMGB1) and three (IL-8). ***p<0.005, paired Student's t-test. (B) Levels of KC in blood cell culture supernatants (100 μL whole blood/condition) of C5L2KO and WT mice challenged (1 h) i.p. with LPS (50 μg/mouse) or PBS (no LPS) and stimulated (14 h) ex vivo with C5a. C5a-induced KC concentrations were estimated by background subtraction as described for Fig. 1. Values are expressed as the mean+SEM (n=5/condition). *p<0.05, ***p<0.005, (LPS-treated versus no LPS, paired Student's t-test). (C) Western blots analysis of C5L2 levels in cell lysates of PBMCs (0.5×106/condition) pre-exposed or not to LPS and subsequently activated with C5a (10 nM). Results are representative of three experiments. Densitometry of Western blots are shown in A and C; *p<0.05, ***p<0.005, paired Student's t-test.
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