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Comparative Study
. 2011 Jan 15;186(2):869-77.
doi: 10.4049/jimmunol.1003252. Epub 2010 Dec 13.

The C5a receptor impairs IL-12-dependent clearance of Porphyromonas gingivalis and is required for induction of periodontal bone loss

Shuang Liang  1 , Jennifer L Krauss, Hisanori Domon, Megan L McIntosh, Kavita B Hosur, Hongchang Qu, Fenge Li, Apostolia Tzekou, John D Lambris, George Hajishengallis
Affiliations
Comparative Study

The C5a receptor impairs IL-12-dependent clearance of Porphyromonas gingivalis and is required for induction of periodontal bone loss

Shuang Liang et al. J Immunol. .

Abstract

The C5a anaphylatoxin receptor (C5aR; CD88) is activated as part of the complement cascade and exerts important inflammatory, antimicrobial, and regulatory functions, at least in part, via crosstalk with TLRs. However, the periodontal pathogen Porphyromonas gingivalis can control C5aR activation by generating C5a through its own C5 convertase-like enzymatic activity. In this paper, we show that P. gingivalis uses this mechanism to proactively and selectively inhibit TLR2-induced IL-12p70, whereas the same pathogen-instigated C5aR-TLR2 crosstalk upregulates other inflammatory and bone-resorptive cytokines (IL-1β, IL-6, and TNF-α). In vivo, the ability of P. gingivalis to manipulate TLR2 activation via the C5a-C5aR axis allowed it to escape IL-12p70-dependent immune clearance and to cause inflammatory bone loss in a murine model of experimental periodontitis. In the latter regard, C5aR-deficient or TLR2-deficient mice were both resistant to periodontal bone loss, in stark contrast with wild-type control mice, which is consistent with the interdependent interactions of C5aR and TLR2 in P. gingivalis immune evasion and induction of bone-resorptive cytokines. In conclusion, P. gingivalis targets C5aR to promote its adaptive fitness and cause periodontal disease. Given the current availability of safe and effective C5aR antagonists, pharmacological blockade of C5aR could act therapeutically in human periodontitis and reduce associated systemic risks.

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Figures

Figure 1
Figure 1. C5aR signaling inhibits TLR2-dependent IL-12p70 induction in P. gingivalis-activated macrophages
Mouse peritoneal macrophages were primed with IFN-γ (0.1 µg/ml) and stimulated with medium only (Med), P. gingivalis (MOI 10:1), or E. coli LPS (Ec-LPS; 0.1 µg/ml), as indicated. IFN-γ priming was performed in those experiments (A–D) investigating IL-12p70 regulation. Wild-type P. gingivalis (Pg) was used in all experiments but panel B additionally includes the use of an isogenic mutant (KDP128) which is deficient in all three gingipain genes. In A and B, the macrophages were additionally treated or not with C5a (50 nM), in the absence or presence of C5aRA (1 µM). In C, the macrophages were from wild-type or TLR2-deficient (Tlr2−/−) mice. In D, the macrophages were pretreated with U0126 (10 µM) or wortmannin (WTM; 100 nM) for 1h prior to treatments with C5a, P. gingivalis, or Ec-LPS. In E, the macrophages were stimulated with P. gingivalis as in panel A, but without IFN-γ priming, to measure levels of cytokines other than IL-12p70. Culture supernatants were assayed for induction of the indicated cytokines after 24h of incubation. Data are means ± SD (n = 3 sets of macrophages) from typical experiments performed three (A) or two (B–E) times yielding consistent results. Asterisks show statistically significant (p < 0.01) inhibition (A–D; IL-12p70) or enhancement (E; IL-6 and TNF-α) of cytokine production, whereas black circles indicate statistically significant (p < 0.01) reversal of these modulatory effects. In B, the upward arrow shows a significant difference (p < 0.05) between KDP128 and Pg under no-treatment conditions. In D, inverse triangles show significant (p < 0.01) U0126 or WTM effects on P. gingivalis- or LPS-induced IL-12p70.
Figure 2
Figure 2. C5aR signaling regulates P. gingivalis-induced and TLR2-dependent cytokine production in vivo
10–12 week-old wild-type (WT) mice, which were pretreated or not with C5aRA (i.p.; 25 µg/mouse), as well as mice deficient in C5aR (C5ar−/−) or TLR2 (Tlr2−/−), were i.p. infected with P. gingivalis (5×107 CFU). Peritoneal lavage was performed 5h postinfection and the peritoneal fluid was used to measure the levels of the indicated cytokines. Mice not infected with P. gingivalis had undetectable levels of the cytokines investigated. Data are means ± SD (n = 5 mice). *, p < 0.01 and **, p < 0.01 vs. WT+PBS control.
Figure 3
Figure 3. Inhibition of C5aR signaling promotes the in vivo clearance of P. gingivalis by augmenting IL-12
(A) Wild-type (WT) mice were pretreated or not with C5aRA (i.p.; 25 µg/mouse), in the presence or absence of goat polyclonal anti-mouse IL-12 IgG, anti-mouse IL-23p19 IgG, or equal amount of non-immune IgG (i.p.; 0.1 mg/mouse). The mice were then infected i.p. with P. gingivalis (5×107 CFU). (B) Similar experiment in which C5aRA-treated mice were replaced by C5aR-deficient (C5ar−/−) mice. (C) WT and C5ar−/− mice were infected i.p. with wild-type P. gingivalis or the isogenic KDP128 mutant (both at 5×107 CFU). Peritoneal lavage was performed 24h postinfection and the peritoneal fluid was used to determine viable P. gingivalis CFU counts. Data are shown for each individual mouse with horizontal lines indicating mean values. *, p < 0.01 vs. controls. The inverted triangles indicate significant (p < 0.01) reversal of the effects of C5aRA or C5aR deficiency by anti-IL-12. In C, the downward arrow shows significant (p < 0.01) difference between KDP128 and the wild-type organism.
Figure 4
Figure 4. Comparative modulatory effects of C5a and C5adesArg on IL-12p70 production and antimicrobial activities in P. gingivalis-challenged macrophages
Groups of mouse peritoneal macrophages were incubated with P. gingivalis (Pg; MOI = 10:1) in the absence or presence of C5a or C5adesArg (at 10 or 50 nM) and assayed for (A) induction of IL-12p70 (after 24h), (C) generation of cAMP (1h), (D) NO2− (24h), and (E) viable counts (CFU) of internalized bacteria (24h). In panel B, the macrophages were pretreated with C5aRA (1 µM), the dual C5aR/C5a-like receptor-2 antagonist A8Δ71–73 (1 µM), or the C3aR antagonist SB290157 (5 µM) to determine the receptor by which C5adesArg (50 nM) inhibits IL-12p70 production. Data are means ± SD (n = 3 sets of macrophages) from one of two independent sets of experiments yielding consistent results. *, p < 0.05 and **, p < 0.01 compared to no C5a or C5adesArg (0 nM). In B, black circles indicate statistically significant (p < 0.01) reversal of the inhibitory effect of C5adesArg. In panels C–E, no significant differences were found between C5a and C5adesArg when tested at 50 nM.
Figure 5
Figure 5. Comparison of C5a and C5adesArg in intracellular Ca2+ mobilization
Mouse peritoneal macrophages (A) or neutrophils (B) were loaded with the ratiometric calcium indicator Indo-1 AM and stimulated with C5a or C5adesArg at the indicated concentrations (lower concentrations were used for neutrophils since they are more sensitive to C5a than macrophages (50)). Ca2+ mobilization was measured in a spectrofluorometer and the traces are representative of three experiments.
Figure 6
Figure 6. C5aR and TLR2 deficiencies protect against periodontal bone loss
Mice deficient in C5aR [C5ar−/−] (A, BALB/c; B, C57BL/6) or TLR2 [Tlr2−/−] (C; BALB/c) and appropriate wild-type controls were orally infected or not with P. gingivalis and assessed for induction of periodontal bone loss six weeks later. Mice used in these experiments were 10–12 week-old. (D) Induction of naturally occurring periodontal bone loss in 16-month-old wild-type or C5ar−/− BALB/c mice relative to their young counterparts (≤ 12 weeks of age). (E) Representative images of P. gingivalis-induced bone loss under wild-type or C5aR- or TLR2-deficient conditions: P. gingivalis-infected C5ar−/− or Tlr2−/− mice display considerably smaller CEJ-ABC distances (yellow arrows) compared to infected wild-type mice, but quite comparable to those of sham-infected wild-type mice. Data are means ± SD (n = 5 mice). *, p < 0.01 compared to corresponding sham-infected controls (A and B) or young counterparts (C).
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