A dysbiotic microbiome triggers TH17 cells to mediate oral mucosal immunopathology in mice and humans

Abstract

Periodontitis is one of the most common human inflammatory diseases, yet the mechanisms that drive immunopathology and could be therapeutically targeted are not well defined. Here, we demonstrate an expansion of resident memory T helper 17 (T_H17) cells in human periodontitis. Phenocopying humans, T_H17 cells expanded in murine experimental periodontitis through local proliferation. Unlike homeostatic oral T_H17 cells, which accumulate in a commensal-independent and interleukin-6 (IL-6)-dependent manner, periodontitis-associated expansion of T_H17 cells was dependent on the local dysbiotic microbiome and required both IL-6 and IL-23. T_H17 cells and associated neutrophil accumulation were necessary for inflammatory tissue destruction in experimental periodontitis. Genetic or pharmacological inhibition of T_H17 cell differentiation conferred protection from immunopathology. Studies in a unique patient population with a genetic defect in T_H17 cell differentiation established human relevance for our murine experimental studies. In the oral cavity, human T_H17 cell defects were associated with diminished periodontal inflammation and bone loss, despite increased prevalence of recurrent oral fungal infections. Our study highlights distinct functions of T_H17 cells in oral immunity and inflammation and paves the way to a new targeted therapeutic approach for the treatment of periodontitis.

Fig. 1.. Th17 cells in human periodontitis

H&E of healthy (A) and periodontitis (B) gingiva. (C) CD3/T cell immunohistochemical staining in periodontitis (original magnification 15x). (D) mRNA expression for IFNg, IL4, IL17A and FOXP3 in health and periodontitis (n=3/6, health/periodontitis, unpaired t test, mean±SEM). (E-G) IL-17⁺CD45⁺ in health and periodontitis. FACS plot (E) and graph (F) showing numbers of CD45⁺IL-17⁺ cells per standardized biopsy (n=11/7, health/periodontitis, Mann-Whitney test, mean±SEM). (G) IL-17⁺ cellular sources in periodontitis (one-way ANOVA, Holm-Sidak’s multiple comparisons test, mean±SEM). (H-J) IL-17⁺CD4⁺ in health and periodontitis. FACS plot (H) and graph (I) showing numbers of CD4⁺IL-17⁺ cells per standardized biopsy (n=11/7, health/periodontitis, unpaired t test, mean±SEM). (J) Spearman correlation of CD4⁺IL-17⁺ cells with bone loss in mm (n=18 patients). (K-O) Th17 cells in periodontitis. FACS plot of CD4⁺IL-17⁺ (K), CD4⁺IL-17⁺ cells expressing CD45RO, CD45A, CCR7, CD69 (L). Frequencies of CD4⁺IL-17⁺ resident effector (rTem), resident central (rTcm), effector (Tem) and central (Tcm) memory T cells (M). CD4⁺IL-17⁺ cells co-producing IFNγ, GMCSF or IL-22, FACS Plot (N) and graph (O) (n=4–9, ANOVA and Holm-Sidak’s (M) or Tukey’s (O) multiple comparisons tests, mean±SEM). All p values are indicated in graphs.

Fig. 2.. Preferential expansion of Th17 cells in experimental periodontitis

(A) IL-17a mRNA expression in gingival tissues at baseline (CTL) and after ligature induced periodontitis (LIP) (n=10 mice per group, 2 separate experiments, Mann Whitney test, mean±SEM). (B-C) IL-17⁺CD45⁺ cells at baseline and LIP, in IL-17a^creR26R^eYFP mice. FACS plot (B) and graph (C) indicating numbers of CD45^eYFP+ cells per standardized tissue (n=12 per group, 3 separate experiments, unpaired t test, mean±SEM). (D-F) Proportions and numbers of IL-17⁺ cells at baseline and LIP. FACS plots (D) and graph (E) showing percentage of eYFP⁺ cells. (F) Graph showing numbers of eYFP⁺, per standardized gingival tissue (n=10, 3 separate experiments). (G) Ki67⁺ staining in IL-17⁺ cells in LIP (n=6, 2 separate experiments, one-way ANOVA and Tukey’s multiple comparisons test, mean±SEM for E-G). (H) Numbers of eYFP⁺ cells per gingival tissue in LIP with/without FTY720 (n=5, 2 separate experiments, Mann-Whitney test, mean±SEM). All p values are indicated in graphs.

Fig. 3.. Cytokine requirements for Th17 cell accumulation during experimental periodontitis

(A-F) IL-17 production by CD4⁺ cells at baseline (CTL) and LIP. Bar graphs and FACS plots show numbers of Th17 cells at CTL and after LIP in Il6^−/− and Il6^+/+ mice (A-B), Il1r1^−/− and Il1r1^+/+ mice (C, D), Il23a^−/− and Il23a^+/+ mice (E-F). All data are representative of 2–3 separate experiments, n=6–8 for each group, p values determined by Mann-Whitney test, mean±SEM.

Fig.4.. Expansion of Th17 cells in periodontitis disease-associated bacteria

(A) Microbiome composition at the OTU level. Most abundant OTUs are classified at the species-level (10) and less dominant OTUs are shown combined at phylum level. (B) Principal Coordinates Analysis (PCoA) plot of global microbial community composition and (C) community structure at baseline (CTL) and LIP, p values were determined using AMOVA and 95% confidence ellipse were depicted. (D) Numbers of CD45⁺IL-17⁺ cells at CTL and after LIP without/with broad-spectrum antibiotic cocktail (ATB = Doripenem-Vancomycin-Neomycin) (n=7 per group, 2 separate experiments, one-way ANOVA and Tukey’s multiple comparisons test, mean±SEM). (E) Numbers of IL-17⁺(CD4, TCRγδ and ILC) at CTL and after LIP without/with antibiotics (ATB) (n=7 per group, 2 separate experiments). (F) Numbers of CD4⁺IL-17⁺Ki67⁺ cells CTL and LIP with/without antibiotics (ATB) (n=7, 2 separate experiments, Kruskal-Wallis test and Dunn’s multiple comparisons test, mean±SEM for E-F). (G) CD4⁺IL-17⁺ cell numbers , (H) Bone loss in millimeters and (I) Total oral microbial biomass at CTL and after LIP with/without antibiotic treatment (DOR=Doripenem; VAN=Vancomycin; NEO=Neomycin; MET=Metronidazole) (n=6–9 per group, 2 separate experiments, one-way ANOVA and Tukey’s multiple comparisons test, mean± SEM). All p values are indicated in graphs.

Fig. 5.. Genetic inhibition of Th17 cells protects from periodontal bone loss

IL-17⁺ cells in Cd4^CreStat3 ^fl/fl mice and littermate controls before and after LIP. Representative FACS plots from LIP and graphs showing number of IL-17⁺ cells from CTL and LIP, for (A-B) TCRβ⁺CD4⁺IL-17⁺, (C-D) TCRγδ⁺IL-17⁺, (E-F) ILC(Lin^-CD90.2⁺),(lineage=TCRβ, TCRγδ, Ly6C, Ly6G, B220, CD11b, CD11c,), (G-H) CD45⁺IL-17⁺. (n=7 per group, 3 separate experiments). (I) Bone loss (in mm) after LIP in Cd4^CreStat3 ^fl/fl mice and littermate controls (n=9, 3 separate experiments). All p values were determined by unpaired t test and graphs showing mean±SEM. All p values are indicated in graphs.

Fig. 6.. RORγt targeting protects from inflammatory bone loss and reveals mechanisms of Th17 cell-driven periodontal inflammation

(A) Bone loss (in mm) after LIP in Lck^CreRorc^fl/fl mice and littermate controls (n=5 per group, 2 separate experiments). (B) Bone loss (in mm) after LIP in the presence/absence of RORγt inhibitor (GSK805) (n=6, 2 separate experiments). (C) Volcano plot of genes differentially expressed during LIP in the presence/absence of RORγt inhibitor (GSK805). Genes in red are p<0.05 and fold-change>1.5. (D) Heatmap depicts genes of interest from the top 30 downregulated genes with RORγt inhibitor (GSK805) in LIP, FDR ≤ 0.05. (E) Bone loss (in mm) with LIP in mice treated with anti-IL-17A or isotype control (n=8 per group) (F) Bone loss (in mm) with LIP in mice treated with anti-Ly6G or isotype control (n=5 per group, Mann-Whitney test). All other p values determined by unpaired t test and graphs depict mean±SEM unless otherwise stated.

Fig. 7.. Patients with genetic defects in Th17 cell differentiation present with reduced susceptibility to periodontal inflammation (A-B).

IL-17 production by CD4⁺ (CD45⁺CD3⁺TRCγδ^-CD56^-CD8^-) human gingival cells in health, AD-HIES and periodontitis patients. Representative FACS plots (A) and graph (B) indicating numbers of CD4⁺IL-17⁺ cells per standardized gingival biopsy (n=7–10). (C) Susceptibility to recurrent oral candidiasis in AD-HIES. Bar graph shows the number of patients with history of recurrent oral candidiasis (Thrush, n=31) or no history of candida infections (No-Thrush, n=5). (D-E). Periodontitis susceptibility in health, AD-HIES and periodontitis patients (Perio). (D) Periodontal inflammation in health, AD-HIES and periodontitis (n=29/25/27 patients). Bar graph shows frequency of bleeding sites (Inflammation score, per patient). (E) Periodontal bone loss in health, AD-HIES and Periodontitis (n=29/25/27 patients). Bar graph shows clinical attachment (level of bone loss). All p values in this figure were determined by one-way ANOVA and Holm-Sidak’s test, mean±SEM.