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. 2010 Feb 16;3(109):ra11.
doi: 10.1126/scisignal.2000697.

Microbial Hijacking of Complement-Toll-Like Receptor Crosstalk

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Free PMC article

Microbial Hijacking of Complement-Toll-Like Receptor Crosstalk

Min Wang et al. Sci Signal. .
Free PMC article

Abstract

Crosstalk between complement and Toll-like receptors (TLRs) coordinates innate immunity. We report a previously unknown immune subversion mechanism involving microbial exploitation of communication between complement and TLRs. Porphyromonas gingivalis, a major oral and systemic pathogen with complement C5 convertase-like activity, synergizes with C5a (fragment of complement protein C5) to increase cyclic adenosine monophosphate (cAMP) concentrations, resulting in suppression of macrophage immune function and enhanced pathogen survival in vitro and in vivo. This synergy required TLR2 signaling, a pertussis toxin- and thapsigargin-sensitive C5a receptor pathway, with protein kinase A and glycogen synthase kinase-3beta as downstream effectors. Antagonistic blockade of the C5a receptor abrogated this evasive strategy and may thus have important therapeutic implications for periodontitis and atherosclerosis, diseases in which P. gingivalis is implicated. This first demonstration of complement-TLR crosstalk for immunosuppressive cAMP signaling indicates that pathogens may not simply undermine complement or TLRs (or both) as separate entities, but may also exploit their crosstalk pathways.

Figures

Figure 1
Figure 1
Immunosubversive effects of C5a on macrophages. (A–D) Peritoneal mouse macrophages were left untreated (A,B) or primed with 100 ng/ml IFN-γ (C,D) overnight, washed, and incubated with P. gingivalis (Pg; MOI=10:1) in the presence or absence of C3a (200 nM) or C5a (50 nM). Viable counts of internalized bacteria at 24 hours (A and C) or 48 hours (B and D) post-infection were determined by CFU enumeration. (E) Macrophages were incubated with medium only or with Pg in the presence or absence of C5a for the indicated times and assayed for induction of intracellular cAMP. (F) Similar experiment as in E, involving 1-hour incubation and the use of a specific C5a receptor antagonist (C5aRA; 1 μM), as indicated. (G) Unprimed or IFN-γ–primed macrophages were assayed for NO2 after 24-hour incubation with or without Pg and/or C5a, which acted in the absence or presence of C5aRA. (H–I) Similar experiments for induction of cAMP (H) and NO2 (I) using macrophages from both wild-type and C5aR-deficient (C5ar−/−) mice. Data are means ± SD (n = 3) from typical experiments performed three (A–D, F, G) or two (E, H–I) times yielding consistent results. *, P < 0.05 and **, P < 0.01 vs. medium (med) control treatments. •, P < 0.01 in C5a+Pg vs. Pg alone. Inverted triangles indicate significant (P < 0.01) reversal of C5a effects by C5aRA or C5aR deficiency.
Figure 2
Figure 2
C5a-mediated inhibition of nitric oxide and promotion of P. gingivalis survival is cAMP- and PKA-dependent. (A and B) Mouse macrophages were pretreated or not with SQ22536 (cAMP synthesis inhibitor; 200 μM), H89 (PKA inhibitor; 5 μM), chelerythrin (protein kinase C inhibitor; 5 μM), PKI 6-22 (peptide inhibitor of PKA; 1 μM), or KT5823 (peptide inhibitor of protein kinase G; 1 μM), and then infected with P. gingivalis (Pg; MOI=10:1) with or without C5a (50 nM), as indicated. (C) Macrophages were pretreated with 1 mM L-NAME (or D-NAME) and/or 1 μM C5aRA and then infected with Pg with or without C5a. (D) Macrophages were incubated with Pg and C5a in the absence or presence of SQ22536 or PKI 6-22, added prior to Pg and C5a (“0 time delay”) or with increasing delay times, as indicated. NO2 production (A) and viable counts of internalized bacteria (B–D) were determined at 24 hours postinfection. In D, the dashed line indicates Pg CFU in the absence of inhibitors (13.7±2.7[×104] CFU). Results are means ± SD (n = 3) from typical experiments performed at least twice with consistent results. *, P < 0.05 and **, P < 0.01 vs. corresponding controls. •, P < 0.01 in C5a+Pg plus inhibitor or antagonist vs. C5a+Pg only. In C, the inverted triangle shows significant (P < 0.01) reversal of the C5aRA effect.
Figure 3
Figure 3
P. gingivalis exploits C5aR signaling to inhibit nitric oxide production and promote its survival in vivo. (A) Wild-type (WT) mice were i.p. pretreated with C5aRA (1 mg/Kg body weight) or PBS control, followed by i.p. infection of these mice, as well as mice deficient in C5aR (C5ar−/−), with 5×107 CFU P. gingivalis. (B and C) Wild-type mice were i.p. pretreated or not with C5aRA with or without L-NAME or D-NAME (0.1 ml of 12.5 mM solution, corresponding to 0.34 mg per mouse) followed by P. gingivalis i.p. infection. Peritoneal fluid was collected 24 hours postinfection and used to determine viable P. gingivalis CFU (A and C) and NO2 production (B). Data are from typical experiments performed twice yielding consistent findings and represent means ± SD (n = 5) or are shown for each individual mouse with horizontal lines denoting mean values. *, P < 0.01 vs. controls. The inverted triangles show significant (P < 0.01) reversal of the C5aRA effects.
Figure 4
Figure 4
Synergistic activation of the cAMP-PKA pathway requires C5aR-TLR2 crosstalk. Macrophages pretreated with 1 μM thapsigargin (TG), 5 mM EGTA, 100 ng/ml pertussis toxin (PTX) (A) or 1μg/ml AMD3100 (B–D) were stimulated with P. gingivalis (Pg; MOI=10:1; 1 hour) with or without 50 nM C5a and assayed for cAMP (A–C) or PKA activity (D). PKA assay specificity was confirmed using PKI-6-22 and an irrelevant kinase inhibitor (KT5823). Forskolin (20 μM; 10-min) served as positive control in experiments with Tlr2−/− macrophages (C and D). (E) PKA activities in freshly explanted peritoneal macrophages from Pg-infected mice (activities of indicated receptor-deficient cells expressed as % wild-type activity). (F) Macrophages pretreated with 1 μM PKI-6-22 or 25 μM PD98059 (PD; control) were stimulated with Pg, with or without C5a, and assayed for GSK3β Ser9-phosphorylation and total GSK3β. (G) Macrophages stimulated with Pg with or without C5a (50 nM), SB216763 (10 μM), or 8-Br-cAMP (100 μM) were assayed for iNOS expression (4 hours) or NO2 (24 hours). (H) Confocal colocalization of P. gingivalis (green), C5aR (red), and TLR2 (blue), as better shown in the bottom right merge image. (I) FRET between the indicated donors and acceptors measured from the increase in donor (Cy3 or FITC) fluorescence after acceptor (Cy5 or TRITC) photobleaching. Data are means ± SD (n=3 except for E, n=5) from typical experiments performed at least twice with consistent results. *, P<0.05; **, P <0.01 between the indicated groups or vs. controls (E and I). (K) Pg induces weak TLR2-dependent cAMP induction (left), whereas CXCR4 or C5aR signaling alone fails to induce cAMP (middle). However, Pg-induced TLR2 signaling with concomitant activation of C5aR and, to a lesser extent, CXCR4 synergistically enhances the immunosuppressive cAMP-PKA pathway that inactivates GSK3β and impairs iNOS-dependent killing.

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