Calprotectin (S100A8/A9) Is an Innate Immune Effector in Experimental Periodontitis

Abstract

Upregulated in inflammation, calprotectin (complexed S100A8 and S100A9; S100A8/A9) functions as an innate immune effector molecule, promoting inflammation, and also as an antimicrobial protein. We hypothesized that antimicrobial S100A8/A9 would mitigate change to the local microbial community and promote resistance to experimental periodontitis in vivo. To test this hypothesis, S100A9^-/- and wild-type (WT; S100A9^+/+) C57BL/6 mice were compared using a model of ligature-induced periodontitis. On day 2, WT mice showed fewer infiltrating innate immune cells than S100A9^-/- mice; by day 5, the immune cell numbers were similar. At 5 days post ligature placement, oral microbial communities sampled with swabs differed significantly in beta diversity between the mouse genotypes. Ligatures recovered from molar teeth of S100A9^-/- and WT mice contained significantly dissimilar microbial genera from each other and the overall oral communities from swabs. Concomitantly, the S100A9^-/- mice had significantly greater alveolar bone loss than WT mice around molar teeth in ligated sites. When the oral microflora was ablated by antibiotic pretreatment, differences disappeared between WT and S100A9^-/- mice in their immune cell infiltrates and alveolar bone loss. Calprotectin, therefore, suppresses emergence of a dysbiotic, proinflammatory oral microbial community, which reduces innate immune effector activity, including early recruitment of innate immune cells, mitigating subsequent alveolar bone loss and protecting against experimental periodontitis.

Keywords: calprotectin; experimental periodontitis; inflammation; innate immunity; microbiome.

FIG 1

Microbial alpha diversity (i.e., within samples) during inflammation (5 days postligation) is greater in S100A9^−/− than WT mice. Alpha diversity is represented by the Shannon index for taxonomic richness and evenness. Greater diversity within the sample constitutes a higher Shannon index value. Significant differences between group means were determined using the Mann-Whitney U test. Swab/ligature comparisons using the Mann-Whitney U test were made pairwise within-mouse. Between-group comparisons are indicated by bars with P values. P values of <0.05 were considered significant (n = 7 to 8 for each treatment group).

FIG 2

WT and S100A9^−/− mice have distinct oral microbes in response to ligation. The relative overall abundances of the most prevalent genera are represented by beta diversity. Genotype differences were evaluated using a Mann-Whitney U test. P values of <0.05 were considered significant (n = 7 to 8 for each treatment group).

FIG 3

Interstitial immune cell infiltrates differ in S100A9^−/− and WT mice following ligature placement. At 2 or 5 days post ligature placement, anesthetized mice were given 1.25 μg of FITC-conjugated anti-CD45 (CD45:FITC) MAb in a retro-orbital injection and euthanized 3 min later. Gingival cells were processed, stained, and counted by flow cytometry as reported in the Materials and Methods. Live immune cell numbers were normalized to 10,000 live nonimmune cells to account for variations in tissue size and sample lost during processing. (A) Flow cytometry gating strategy used to identify specific immune cell types of interest. Live interstitial immune cells were identified as Zombie Aqua^lo, CD45:FITC⁻, CD45:PE⁺; live neutrophils as Zombie Aqua^lo, CD45:FITC⁻, CD45:PE⁺, CD11b^hi, Ly-6G⁺, MHCII⁻, CD3⁻; live nonneutrophil granulocytes as Zombie Aqua^lo, CD45:FITC⁻, CD45:PE⁺, CD11b^hi, Ly-6G⁻, MHCII-, CD3⁻; and live CD11b^hi dendritic cells and tissue macrophages as Zombie Aqua^lo, CD45:FITC⁻, CD45:PE⁺, CD11b^hi, Ly-6G⁻, MHCII⁺, CD3⁻. (B) Effects of ligation and antibiotic suppression of the oral microbiota on the inflammatory cell infiltrate in WT and S100A9^−/− mice at day 2. Summary data of at least three independent experiments are shown. Gingival tissues were harvested from sites in mice that were nonligated (open triangles), ligated (filled circles), or ligated after prophylactic antibiotic treatment (open circles). Cells were quantified using flow cytometry. Individual data points and means ± SEM are plotted. Data were analyzed by two-way ANOVA; *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001; ns, not significant. (C) At day 5 post ligature placement, S100A9^−/− and WT mice show similar levels of infiltrating gingival immune cells. Individual data points from at least two independent experiments are plotted as means ± SEM. Data are for nonligated (open triangles) and ligated (filled circles) mice. Mean cell numbers were compared using two-tailed Student’s t test; **, P < 0.01; ****, P < 0.0001; ns, not significant.

FIG 4

S100A8/A9 expression protects against alveolar bone loss in ligature-induced periodontitis. Harvested maxillae were imaged using micro-CT and reconstructed in 3D as described in the Materials and Methods. A volume of interest encompassing ligated and proximal molars and their roots was set using fixed anatomical landmarks. Bone volume was converted into cubic millimeters by reconstruction from the voxel size. Bone volume differences were analyzed by an unpaired independent Student’s t test. P values are indicated (n = 7 to 8 for each treatment group).