Neutrophils drive sexual dimorphism in experimental periodontitis

The motivating premise of this study is to improve the treatment of periodontal disease by elucidating sex-specific mechanisms of periodontal disease progression. Men and women experience inflammation in fundamentally different ways and understanding the sex-specific biology leading to inflammation and bone loss in the periodontium will inevitably improve patient outcomes. We therefore examined clinical and immunological differences in the progression of periodontal disease using the ligature-induced periodontitis model. Periodontitis was induced in male and female C57BL/6j mice by tying a 5-0 silk suture around the left maxillary second molar. The ligature was left on for 7 or 21 days at which point maxillae were characterized for bone loss by μCT or immune infiltrate by flow cytometry. Neutrophil depletion was accomplished through systemic administration of a Ly6G antibody. Conditions were compared using two-way ANOVA with Tukey's multiple comparison correction from n≥5 animals. Ligature-induced periodontitis led to alveolar bone loss at both 7 and 21 days in both female and male mice. Males and females had approximately the same amount of linear bone loss 7 days post-ligature placement, while male mice had significantly more linear bone loss by day 21. Male mice had significantly more immune cells in their maxillae 7 days post ligature placement compared to female mice. Both male and female mice showed a shift in immune populations towards neutrophils, with no significant difference between males and females. Neutrophil counts were significantly elevated in male mice on day 7 but not day 21, while female mice did not have any statistically significant changes in neutrophil counts. Neutrophil depletion using a Ly6G antibody limited bone loss in male but not female mice relative to isotype antibody-treated controls. Analysis of single-cell sequencing data from human patients with periodontitis showed differences in neutrophil phenotypes that were also observed in a mouse model of periodontitis. Together, these data suggest a mechanistic role for neutrophil inflammation in sexual dimorphism in periodontitis.