Sialidase facilitates Porphyromonas gingivalis immune evasion by reducing M1 polarization, antigen presentation, and phagocytosis of infected macrophages

Abstract

Background: Porphyromonas gingivalis (P. gingivalis), a major pathogen of periodontitis, can evade host immune defenses. Previously, we found that P. gingivalis W83 sialidase gene mutant strain (ΔPG0352) was more easily cleared by macrophages. The aims of this study were to investigate the effects of sialidase in P. gingivalis on the polarization, antigen presentation, and phagocytosis of infected macrophages and to clarify the mechanism of P. gingivalis immune evasion.

Methods: Human monocytes U937 were differentiated to macrophages and infected with P. gingivalis W83, ΔPG0352, comΔPG0352, and Escherichia coli (E. coli). The phagocytosis of macrophages was observed by transmission electron microscopy and flow cytometry. ELISA or Griess reaction were used to examine the levels of interleukin-12 (IL-12), inducible nitric oxide synthase (iNOS) and interleukin-10 (IL-10), and the expressions of CD68, CD80 and CD206 were determined by flow cytometry. The expression of major histocompatibility complex-II (MHC-II) was detected by immunofluorescence. A rat periodontitis model was established to determine the M1 and M2 polarization of macrophages.

Results: Compare with P. gingivalis W83, ΔPG0352 increased the levels of IL-12, iNOS, CD80, and MHC-II and inhibited the levels of IL-10 and CD206. Macrophages phagocytosed 75.4% of ΔPG0352 and 59.5% of P. gingivalis W83. In the rat periodontitis model, the levels of M1 and M2 macrophages in P. gingivalis W83 group were both higher than those in ΔPG0352 group, while the ratio of M1/M2 was higher in the ΔPG0352 group. Alveolar bone absorption was lower in ΔPG0352 group.

Conclusion: Sialidase facilitates P. gingivalis immune evasion by reducing M1 polarization, antigen presentation, and phagocytosis of infected macrophages.

Keywords: Porphyromonas gingivalis; macrophage; periodontitis; polarization; sialidase.

Figure 1

Morphological observation of macrophages infected with P. gingivalis W83, ΔPG0352, comΔPG0352, and E. coli. (A) Uninfected macrophages. Macrophages were infected with (B) P. gingivalis W83, (C) ΔPG0352, (D) comΔPG0352, and (E) E. coli. Black arrow: bacterial strains phagocytosed by macrophage; white arrows: pseudopods on the surface of macrophages; 25,000× magnification.

Figure 2

ΔPG0352 promoted M1 polarization of macrophage. (A) The levels of IL-12, iNOS, and IL-10. (B, C) The expression of M0 macrophages marker CD68. (D, E) The expression of M1 macrophage marker CD80. (F, G) The expression of M2 macrophage marker CD206. (D, F) Y-axis: number of cells; X-axis: relative intensity of fluorescence signal. Data are expressed as mean ± SD (n = 3), ^* P < 0.05, ^** P < 0.01, and ^*** P < 0.001.

Figure 3

ΔPG0352 enhanced the expression of MHC-II and phagocytosis ability of macrophages. (A) The expression of MHC-II was determined by immunofluorescence. Green: MHC-II; red: cell membrane; blue: cell nucleus (400×). (B) The mean fluorescence intensity of MHC-II was calculated. (C) The phagocytosis ability of macrophages to P. gingivalis W83, ΔPG0352, comΔPG0352, and E. coli was assessed by flow cytometry. (D) FITC⁺ percentage of macrophages following P. gingivalis W83, ΔPG0352, comΔPG0352, and E. coli. Data are expressed as mean ± SD (n = 3), ^* P < 0.05 and ^** P < 0.01.

Figure 4

Expressions of iNOS and CD163 in the rat periodontitis model. (A) The expression of iNOS in gingival tissues from maxillary was assessed by immunohistochemistry at 400 × magnification. (B) The expression of CD163 in gingival tissues from maxillary was assessed by immunohistochemistry at 400 × magnification. (C) The positive cells percentage of iNOS (%). (D) The positive cells percentage of CD163 (%). ^ns No statistical significance, ^* P < 0.05.

Figure 5

The ratio of M1 to M2 macrophages in the rat periodontitis model. (A) The expression of iNOS⁺F4/80⁺ in gingival tissues of rats. Red: macrophage; green: iNOS; blue: cell nucleus (400×). (B) The expression of CD206⁺F4/80⁺ in gingival tissues of rats. Green: macrophage; red: CD206; blue: cell nucleus (400×). (C, D) Percentage of iNOS⁺F4/80⁺ or CD206⁺F4/80⁺ macrophages. (E) The ratio of iNOS⁺F4/80⁺ to CD206⁺F4/80⁺ (M1/M2). Arrows indicate iNOS⁺F4/80⁺ or CD206⁺F4/80⁺ macrophages. Data are expressed as mean ± SD (n = 3), ^ns No statistical significance, ^* P < 0.05, ^** P < 0.01, and ^*** P < 0.001.

Figure 6

Alveolar bone resorption in the rat periodontitis model assessed by stereoscopic microscope. (A) Health control. (B) Ligation control. (C) P. gingivalis W83. (D) ΔPG0352. (E) comΔPG0352. (F) CEJ-ABC distance. Data are expressed as mean ± SD (n = 3), ^ns No statistical significance, ^*** P < 0.001 compared with the control and ligation control groups, ^* P < 0.05, and 100× magnification.