Figure 4.

NETs potentiate IL-17/Th17 inflammation in periodontitis. (A–C) Flow cytometry analysis of IL-17a^creR26R^eYFP mice oral gingival tissues with or without LIP (4 d) treated with DNase-I (n = 4) or vehicle (n = 4) control. (A) FACS plot of CD45^eYFP+ and (B and C) quantification of CD45^eYFP+ and CD4^eYFP+ cells (LIP, 4 d, numbers of cells/per standardized block). (D–F) Flow cytometry analysis in WT mice with or without LIP (n = 4, 4 d). (D) FACS plot and quantification of (E) CD45⁺TLR2⁺ or (F) CD4⁺TLR2⁺ cells. (G) Immunofluorescence for citH3 and CD3 in mice without/with LIP (n = 3, 18 h). Scale bars, 50 µm; representative images shown. (H) Total oral microbial biomass in mice treated with DNase-I (n = 8) or vehicle (n = 7) treated mice at 18 h and 6 d after LIP. (I and J) 16S rRNA sequencing of LIP microbial communities from DNase-I and vehicle-treated mice at 18 h and 6 d (n = 7–8). (I) Plots show alpha-diversity based on the Shannon index. (J) Principal Coordinate analysis (PCoA) of beta-diversity based on Bray-Curtis dissimilarity. Vehicle and DNase-I are not significantly different regarding beta-diversity at either 18 h or 6 d. Data are representative of three (B, C, E, F, and H) independent experiments. Graphs show the mean ± SEM. *P < 0.05, **P < 0.01. Unpaired t test (B, C, E, F, H, and I) or permutational multivariate ANOVA (J).