Local complement-targeted intervention in periodontitis: proof-of-concept using a C5a receptor (CD88) antagonist

Abstract

When excessively activated or deregulated, complement becomes a major link between infection and inflammatory pathology including periodontitis. This oral inflammatory disease is associated with a dysbiotic microbiota, leads to the destruction of bone and other tooth-supporting structures, and exerts an adverse impact on systemic health. We have previously shown that mice deficient either in complement C5a receptor (C5aR; CD88) or TLR2 are highly and similarly resistant to periodontitis, suggesting that a cross-talk between the two receptors may be involved in the disease process. In this paper, we show that C5aR and TLR2 indeed synergize for maximal inflammatory responses in the periodontal tissue and uncover a novel pharmacological target to abrogate periodontitis. Using two different mouse models of periodontitis, we show that local treatments with a C5aR antagonist inhibited periodontal inflammation through downregulation of TNF, IL-1β, IL-6, and IL-17 and further protected against bone loss, regardless of the presence of TLR2. These findings not only reveal a crucial cooperation between C5aR and TLR2 in periodontal inflammation but also provide proof-of-concept for local targeting of C5aR as a powerful candidate for the treatment of human periodontitis.

FIGURE 1. C5aR and TLR2 synergize in periodontal inflammation

(A, B) C5a and/or Pam3CSK4 (Pam), or PBS control, were microinjected into the gingiva of wild-type mice as outlined in Materials and Methods. (C, D) C5a and Pam in combination were microinjected into the gingiva of wild-type, Tlr2^-/-, and C5ar^-/- mice. The wild-type mice were additionally pretreated with C5aRA or iC5aRA control (ctrl) microinjections (each compound at 5 μg per site) 1h prior to the combined Pam and C5a treatment. All mice were euthanized 24h later and gingiva were dissected to assess the indicated cytokine responses at the mRNA (A and C) or protein (B and D) level. The cytokine mRNA expression levels were normalized against GAPDH mRNA and expressed as fold induction relative to the transcript levels of PBS-microinjected mice, which were assigned an average value of 1 (in C, the value for the PBS controls is indicated by a dashed line in lieu of bars, for clarity). Data are means ± SD (n = 6 mice per group) from one of two independent experiments with similar results. *p < 0.01 between the indicated groups.

FIGURE 2. C5aRA prevents periodontal inflammation

Groups of mice were microinjected in the gingiva with the indicated amount (μg) of C5aRA (or PBS control) five times at two-day intervals prior to oral inoculation with P. gingivalis (Pg), as indicated on the left panel (numbers indicate days). A group of mice was inoculated with vehicle alone (Sham) to serve as the baseline for the host response. One week after the last inoculation, the gingiva were dissected and analyzed by qPCR for mRNA expression of the indicated cytokines (normalized against GAPDH mRNA levels and presented as fold change relative to the transcript levels of sham-infected mice, which were assigned an average value of 1). Data are means ± SD (n = 3 mice per group) from one of two independent experiments yielding similar results. *, significant (p < 0.01) induction of cytokine expression in Pg-infected (without C5aRA) compared to sham-infected mice; ^•, significant (p < 0.01) inhibition of cytokine expression by C5aRA.

FIGURE 3. C5aRA reverses periodontal inflammation

Groups of mice were first inoculated with P. gingivalis (Pg), as indicated on the left panel, and two weeks later were locally administered 1 μg of C5aRA or iC5aRA control (or PBS), every three days for a total of four times. Three days later, the mice were euthanized. Gingiva were dissected and analyzed by qPCR for mRNA expression of the indicated cytokines (normalized against GAPDH mRNA levels and presented as fold change relative to the transcript levels of sham-infected mice, which were assigned an average value of 1). Data are means ± SD (n = 3 mice per group) from one of two independent experiments with similar results. *, significant (p < 0.01) induction of cytokine expression in Pg-infected (without C5aRA) vs. sham-infected mice; ^•, significant (p < 0.01) inhibition of cytokine expression by C5aRA.

FIGURE 4. C5aRA prevents induction of P. gingivalis-instigated bone loss

(A) Groups of mice were microinjected in the gingiva with 1 μg of C5aRA or iC5aRA control followed by oral inoculation with P. gingivalis (Pg) or vehicle only (Sham), as indicated on the left panel (numbers indicate days), and were euthanized six weeks later. Bone loss measurements were performed in defleshed maxillae. Data are means ± SD (n = 6 mice per group) from one of two independent experiments with similar results; negative values indicate bone loss in Pg-inoculated mice relative to sham controls. (B) Quantitative detection of Pg by qPCR of the ISPg1 gene, in the periodontal tissue of Pg-inoculated or sham-inoculated wild-type (WT) or C5ar^-/- mice, four or fourteen days after the last Pg inoculation. The Pg numbers are total per tissue (maxilla). Each symbol represents an individual mouse and small horizontal lines indicate the mean. The experiment was performed twice yielding similar results. *, p < 0.01 compared to corresponding sham control.

FIGURE 5. C5aRA therapeutically inhibits induction of P. gingivalis-instigated bone loss

Groups of mice were orally inoculated with P. gingivalis (or vehicle only; Sham) followed by microinjection in the gingiva with 1 μg of C5aRA or iC5aRA control, as indicated on the left panel (numbers indicate days). Mice were euthanized at the specified time point and bone loss measurements were performed in defleshed maxillae. Data are means ± SD (n = 6 mice per group) from one of two independent experiments with similar results; negative values indicate bone loss in P. gingivalis (Pg)-inoculated mice relative to sham controls. *, p < 0.01 compared to corresponding sham control.

FIGURE 6. Pharmacologic or genetic ablation of C5aR inhibits ligature-induced periodontitis

(A and B) Periodontitis was induced by placing a silk ligature around the second maxillary molar of mice. C5aRA (or PBS control) was microinjected at 1 μg in the palatal gingiva of the ligated second maxillary molar, one day before placement of the ligature and every day thereafter until the day before sacrifice (day 5). (A) Bone loss was measured in defleshed maxillae. (B) Dissected gingiva were processed for qPCR to determine mRNA expression of the indicated molecules (normalized against GAPDH mRNA and expressed as fold change in the transcript levels in the ligated site relative to those of the contralateral unligated site, which were assigned an average value of 1). (C) Determination of ligature-induced periodontal bone loss in wild-type or C5ar^-/- mice at day 5, performed as above. Data are means ± SD (n = 5 to 6 mice per group) from one of two independent experiments with similar results; negative values in A and C indicate bone loss relative to the unligated contralateral tooth. *, p < 0.05; **, p < 0.01 between the indicated groups (A, C) or as compared to PBS control (B).