SAP deficiency aggravates periodontitis possibly via C5a-C5aR signaling-mediated defective macrophage phagocytosis of Porphyromonas gingivalis

Abstract

Introduction: Serum amyloid P component (SAP) regulates the innate immune system and microbial diseases. Periodontitis is an inflammatory oral disease developed by the host immune system's interaction with the dysbiotic oral microbiome, thereby SAP could play a role in periodontitis pathogenicity.

Objectives: To investigate the role of SAP in oral microbiome modulation and peridontitis pathogenicity.

Methods: In this study, wildtype and SAP-knockout (KO) mice were used. Ligature-based periodontitis was developed in mice. Oral microbiome diversity was analyzed by 16 s rRNA sequencing. Macrophages and Porphyromonas gingivalis (P. gingivalis) co-culture system analyzed the effect of SAP in macrophage phagocytosis of P. gingivalis.

Results: The level of SAP was upregulated in the periodontitis-affected periodontium of humans and mice but not in the liver and blood circulation. Periodontal macrophages were the key source of upregulated SAP in periodontitis. SAP-KO aggravated periodontal inflammation, periodontitis, and a higher number of M1-type inflammatory macrophage infiltration in the periodontium. The oral microbiome of SAP-KO periodontitis mice was altered with a higher abundance of Porphyromonas at the genus level. SAP-KO macrophages showed compromised phagocytosis of P. gingivalis in the co-culture system. Co-culture of SAP-KO macrophages and P. gingivalis induced the C5a expression and exogenous SAP treatment nullified this effect. Exogenous recombinant SAP treatment did not affect P. gingivalis growth and opsonization. PMX205, an antagonist of C5a, treatment robustly enhanced P. gingivalis phagocytosis by SAP-KO macrophages, indicating the involvement of the C5a-C5aR signaling in the compromised P. gingivalis phagocytosis by SAP-KO macrophages.

Conclusion: SAP deficiency aggravates periodontitis possibly via C5a-C5aR signaling-mediated defective macrophage phagocytosis of P. gingivalis. A higher abundance of P. gingivalis during SAP deficiency could promote M1 macrophage polarization and periodontitis. This finding suggests the possible protecting role of elevated levels of periodontal SAP against periodontitis progression.

Keywords: C5a-C5aR signaling; Macrophages; Periodontitis; Porphyromonas gingivalis; Serum amyloid P component.

Graphical abstract

Fig. 1

The expression of SAP is upregulated in periodontitis periodontium. (a) Expression of SAP protein in gingiva tissue of healthy and periodontitis individuals (n = 18). (b) Relative SAP mRNA expression in the periodontium of control and periodontitis wildtype mice (n = 4). Expression of SAP protein in the gingiva (c), serum (d), and liver (e) of control and periodontitis wildtype mice (n = 6). (f) SAP immunohistochemistry images of control and periodontitis wildtype mice. (g) Quantification of immunohistochemistry results (n = 3). (h) Relative mRNA expression of SAP in BMDM with or without LPS treatment (n = 4). (i-k) Western blots of SAP protein expression in the periodontium of control and periodontitis wildtype mice (n = 3), as well as in BMDM with or without LPS treatment (n = 8). (l) Immunofluorescence image of BMDB with or without LPS treatment. (m) Quantification of SAP expression (n = 6). Data are presented as mean ± SD. The significant difference among the groups, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, not significant. WT: wildtype.

Fig. 2

SAP deficiency aggravates periodontitis and inflammation. (a) Micro-CT 3D reconstruction images of the maxilla of wildtype and SAP-KO mice. (b) CEJ-ABC distance was analyzed from Micro-CT images (n = 5). (c) Histological images of H&E and TRAP staining, and representative CD34 and CD45 immunohistochemistry images of the periodontium in wildtype and SAP-KO periodontitis mice. Quantitative analysis of CEJ-ABC distance (d) and osteoclasts (e) from histological images (n = 5). Quantitative analysis of CD34 (f) and CD45 (g) positive cells from immunohistochemistry images (n = 3). (h-j) Inflammatory cytokine mRNA expression in the periodontium of wildtype and SAP-KO periodontitis mice (n = 3). (k-m) Protein level expression of inflammatory cytokines detected by ELISA (n = 5). Red dot line, CEJ level; Black dot line, ABC level; Blue double arrow line, CEJ-ABC distance. Yellow “R”, tooth root; Yellow “B”, alveolar bone. Black arrow, osteoclasts. Data are presented as mean ± SD. The significant difference among the groups, *p < 0.05, **p < 0.01, ***p < 0.001, ns, not significant. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Fig. 3

SAP deficiency enhances the infiltration of macrophages and M1 macrophage polarization in periodontitis. (a) Immunohistochemistry (F4/80) images of periodontium of wildtype mice and SAP-KO periodontitis mice. (b) Quantitative analysis of F4/80 positive cells from immunohistochemistry images (n = 3). FACS images of macrophages sorting using FITC-CD11b and PE-F4/80 (c), CD16/32, and CD206 specific antibody (d). Quantification of the ratio of CD11b⁺F4/80⁺ (M0 macrophages) (e), CD16/32⁺ (M1 macrophages) cells (f), and CD206⁺ (M2 macrophages) cells (g) from FACS data (n = 7). CD86 (h) and CD206 (i) mRNA expression of LPS-treated BMDM of wildtype and SAP-KO mice (n = 4). Data are presented as mean ± SD. The significant difference among the groups, *p < 0.05, **p < 0.01, ***p < 0.001, ns, not significant.

Fig. 4

SAP deficiency alters the oral microbiome in periodontitis. (a) The taxonomic composition of the microbial communities at the phylum level (relative abundance greater than 0.01 % at least one sample (n = 3). (b) The composition of the microbial communities at the genus level There was a statistical difference in the relative abundance of P. gingivalis at the genus level. (n = 3). (c) The hierarchical heatmap at the genus level (n = 3). The beta diversity was determined based on the PCoA (d), and LEfSe (e) (n = 3). Data are presented as mean ± SD. The significant difference among the groups, *p < 0.05.

Fig. 5

SAP deficiency inhibits macrophage phagocytosis of P. gingivalis. (a) FACS images of phagocytosis of P. gingivalis by BMDM. (b-d) Quantitative analysis of phagocytosis rate from FACS analysis (n = 3). (e) Immunofluorescence image of phagocytosis of P.gingivalis by BMDM at 1 h. (f) Quantitative analysis of phagocytosis rate from immunofluorescence staining (n = 4). Data are presented as mean ± SD. The significant difference among the groups, **p < 0.01, ****p < 0.0001.

Fig. 6

SAP deficiency inhibits macrophage phagocytosis of P. gingivalis by activating the C5a-C5aR pathway. Expression of C5a in P.gingivalis and BMDM co-culture for 30 min (a), 1 h (b), and 2 h (c), respectively (n = 4). Quantitative PCR analysis of C5aR (d), TLR2 (e), and TLR1 (f) mRNA expression in BMDM (n = 6). (g) FACS images of phagocytosis of P. gingivalis by BMDM of SAP-KO mice with or without PMX205. (h-j) Quantitative analysis of phagocytosis rate (n = 5). Data are presented as mean ± SD. The significant difference among the groups, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, not significant.