Research ArticleMUCOSAL IMMUNITY

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

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Science Translational Medicine  17 Oct 2018:
Vol. 10, Issue 463, eaat0797
DOI: 10.1126/scitranslmed.aat0797
  • Fig. 1 TH17 cells in human periodontitis.

    (A) Hematoxylin and eosin staining of health and (B) diseased (periodontitis) gingiva. (C) CD3 immunohistochemical staining in periodontitis (original magnification, ×15). (D) mRNA expression for IFN-γ, IL4, IL17A, and FOXP3 in health (n = 3) and periodontitis (n = 6) (unpaired t test, mean ± SEM). (E to G) CD45+IL-17+ cells in health and periodontitis gingiva. Cell preparations from health and periodontitis were stimulated ex vivo (phorbol 12-myristate 13-acetate/ionomycin) before cytokine staining. (E) Representative FACS plot and (F) graph showing numbers of CD45+IL-17+ cells per standardized biopsy (n = 9 health, n = 6 periodontitis, Mann-Whitney test, mean ± SEM). (G) IL-17+ cellular sources in periodontitis [one-way analysis of variance (ANOVA), Holm-Sidak’s multiple comparisons test, mean ± SEM]. (H to J) CD4+ IL-17+ in health and periodontitis. (H) Representative FACS plot and (I) graph showing numbers of CD4+IL-17+ cells per standardized biopsy (n = 9 health, n = 7 periodontitis, unpaired t test, mean ± SEM). (J) Spearman correlation of CD4+IL-17+ cells with bone loss (in mm; n = 14 patients). (K to O) TH17 cells in periodontitis. (K) Representative FACS plot of CD4+IL-17+ and (L) CD4+IL-17+ cells expressing CD45RO, CD45RA, CCR7, and CD69. (M) Frequencies of CD4+IL-17+ rTEM, rTCM, TEM, and TCM memory T cells. (N) Representative FACS plot and (O) graph of CD4+IL-17+ cells coproducing IFN-γ (n = 9), GM-CSF (n = 4), or IL-22 (n = 4) after ex vivo stimulation. (M) ANOVA and Holm-Sidak’s or (O) Tukey’s multiple comparisons tests, mean ± SEM. All P values are indicated in graphs. ns, not significant.

  • Fig. 2 Expansion of TH17 cells in experimental periodontitis.

    (A) IL-17a mRNA expression in control gingival tissues (CTL, n = 10) and after ligature induced periodontitis (LIP, n = 11). Data combined from two experiments, Mann-Whitney test, mean ± SEM. (B and C) CD45+IL-17+ cells in CTL and LIP, in IL-17acreR26ReYFP mice. (B) Representative FACS plot and (C) graph indicating numbers of CD45eYFP+ cells per standardized tissue (n = 12 per group, data combined from three experiments, unpaired t test, mean ± SEM). (D to F) Proportions and numbers of IL-17+ cells in control and LIP. (D) Representative FACS plots and (E) graph showing percentage of eYFP+ cells. (F) Graph showing numbers of eYFP+ per standardized gingival tissue (n = 9, data combined from three experiments). (G) Ki67+ staining in IL-17+ cells in LIP [n = 6, data combined from two experiments, one-way ANOVA and Tukey’s multiple comparisons test, mean ± SEM for (E) to (G)]. (H) Numbers of CD4+eYFP+ cells per gingival tissue in LIP with or without FTY720 (n = 5, data combined from two experiments, Mann-Whitney test, mean ± SEM). All P values are indicated in graphs.

  • Fig. 3 Cytokine requirements for TH17 cell accumulation in periodontitis.

    IL-17 production by CD4+ cells in control and LIP. Representative FACS plots show numbers of TH17 cells after LIP and bar graphs show numbers of TH17 cells in control and LIP in (A and B) Il6−/− and Il6+/+ mice (n = 8, data combined from three experiments), (C and D) Il1r1−/− and Il1r1+/+ mice (n = 5, data combined from two experiments), and (E and F) Il23a−/− and Il23a+/+ mice (n = 6, data combined from three experiments). P values determined by Mann-Whitney test. Data were expressed as mean ± SEM. WT, wild-type.

  • Fig. 4 Disease-associated bacteria trigger TH17 cell expansion in periodontitis.

    (A) Microbiome composition at the operational taxonomic unit (OTU) level. The 10 most abundant OTUs are classified at the species level, and less dominant OTUs are shown combined at phylum level. Principal coordinates analysis (PCoA) plot of (B) global microbial community composition and (C) community structure in control and LIP. P values were determined using analysis of molecular variance (AMOVA), and 95% confidence ellipses were depicted. (D) Numbers of CD45+IL-17+ cells in control (n = 6) and after LIP without (n = 7) or with (n = 6) broad-spectrum ATB cocktail (ATB; doripenem-vancomycin-neomycin) (data combined from two experiments, one-way ANOVA and Tukey’s multiple comparisons test, mean ± SEM). (E) Numbers of IL-17+(CD4, TCRγδ, and ILC) in control (n = 6) and after LIP without (n = 7) or with (n = 6) ATBs (data combined from two experiments). (F) Numbers of CD4+IL-17+Ki67+ cells in control and LIP with or without antibiotics [n = 7; data combined from two experiments, Kruskal-Wallis test and Dunn’s multiple comparisons test, mean ± SEM for (E) and (F)]. (G) CD4+IL-17+ cell numbers (n = 6), (H) bone loss (in mm; n = 6 to 9), and (I) total oral microbial biomass (n = 5) in control and after LIP with or without ATB treatment (DOR, doripenem; VAN, vancomycin; NEO, neomycin; MET, metronidazole). Data combined from two experiments, one-way ANOVA and Tukey’s multiple comparisons test, mean ± SEM. All P values are indicated in graphs.

  • Fig. 5 Genetic ablation of TH17 cells prevents periodontal bone loss.

    Gingival IL-17+ cells in Cd4CreStat3 fl/fl mice and littermates in control and LIP. Representative FACS plots from LIP and graphs showing the number of IL-17+ cells from control and LIP for (A and B) TCRβ+CD4+IL-17+, (C and D) TCRγδ+IL-17+, (E and F) ILC (LinCD90.2+) (lineage: TCRβ, TCRγδ, Ly6C, Ly6G, B220, CD11b, and CD11c), and (G and H) CD45+IL-17+ (n = 6 per group, data combined from three experiments). (I) Bone loss (in mm) after LIP in Cd4CreStat3 fl/fl mice (n = 10) and littermates (n = 9). Data combined from three separate experiments. All P values were determined by unpaired t test and graphs depict mean ± SEM. All P values are indicated in graphs.

  • Fig. 6 RORγt inhibition in experimental periodontitis.

    (A) Bone loss (in mm) after LIP in LckCreRorcfl/fl mice (n = 5) and littermates (n = 6). Data combined from two experiments. (B) Bone loss (in mm) after LIP in the presence of GSK805 (n = 7) or vehicle (n = 6). Data combined from two experiments. (C) Volcano plot of genes differentially expressed during LIP in the presence of GSK805 or vehicle. Genes in red are P < 0.05 and fold change > 1.5 (GSK805 versus vehicle treatment). (D) Heatmap depicts genes of interest from the top 30 down-regulated genes with GSK805 in LIP [false discovery rate (FDR) < 0.05]. Each column represents a single sample. (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 Reduced periodontitis susceptibility in patients with genetic defects in TH17 cell differentiation.

    (A and B) IL-17 production by CD4+ (CD45+CD3+TCRγδCD56CD8) human gingival cells in healthy volunteers, AD-HIES patients, and periodontitis patients (periodontitis). (A) Representative FACS plots and (B) graph indicating numbers of CD4+IL-17+ cells per standardized gingival biopsy (healthy volunteers, n = 9; AD-HIES patients, n = 4; and periodontitis patients, n = 7). (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 and E) Periodontitis susceptibility in healthy volunteers, AD-HIES patients, and periodontitis patients. (D) Bar graph shows percentage of bleeding sites in healthy volunteers (n = 29), AD-HIES patients (n = 25), and periodontitis (n = 27) patients. (E) Bar graph shows (in mm) clinical attachment loss in healthy volunteers (n = 29), AD-HIES patients (n = 25), and periodontitis (n = 27) patients. All P values in this figure were determined by one-way ANOVA and Holm-Sidak’s test. Data were expressed as mean ± SEM.

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/10/463/eaat0797/DC1

    Materials and Methods

    Fig. S1. Frequencies of IL-17+ cells in human gingiva (related to Fig. 1).

    Fig. S2. Ligature-induced periodontitis model (related to Figs. 2 to 6).

    Fig. S3. TCR activation in gingiva (related to Fig. 3).

    Fig. S4. Effects of ATB treatments (related to Fig. 4).

    Fig. S5. Foxp3 and RORγt in Cd4CreStat3fl/fl mice (related to Fig. 5).

    Fig. S6. IL-17–secreting cells in LckCreRorcfl/fl mice (related to Fig. 6).

    Fig. S7. IL-17–secreting cells during RORγt treatment (related to Fig. 6).

    Fig. S8. Neutrophils in LIP and periodontitis (related to Fig. 6).

    Fig. S9. IL-17 cell sources in AD-HIES (related to Fig. 7).

    Table S1. Primary data.

    Reference (58)

  • The PDF file includes:

    • Materials and Methods
    • Fig. S1. Frequencies of IL-17+ cells in human gingiva (related to Fig. 1).
    • Fig. S2. Ligature-induced periodontitis model (related to Figs. 2 to 6).
    • Fig. S3. TCR activation in gingiva (related to Fig. 3).
    • Fig. S4. Effects of ATB treatments (related to Fig. 4).
    • Fig. S5. Foxp3 and RORγt in Cd4CreStat3fl/fl mice (related to Fig. 5).
    • Fig. S6. IL-17–secreting cells in LckCreRorcfl/fl mice (related to Fig. 6).
    • Fig. S7. IL-17–secreting cells during RORγt treatment (related to Fig. 6).
    • Fig. S8. Neutrophils in LIP and periodontitis (related to Fig. 6).
    • Fig. S9. IL-17 cell sources in AD-HIES (related to Fig. 7).
    • Reference (58)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S1 (Microsoft Excel format). Primary data.

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