Amphiregulin-producing γδ T cells are vital for safeguarding oral barrier immune homeostasis

Abstract

γδ T cells are enriched at barrier sites such as the gut, skin, and lung, where their roles in maintaining barrier integrity are well established. However, how these cells contribute to homeostasis at the gingiva, a key oral barrier and site of the common chronic inflammatory disease periodontitis, has not been explored. Here we demonstrate that the gingiva is policed by γδ T cells with a T cell receptor (TCR) repertoire that diversifies during development. Gingival γδ T cells accumulated rapidly after birth in response to barrier damage, and strikingly, their absence resulted in enhanced pathology in murine models of the oral inflammatory disease periodontitis. Alterations in bacterial communities could not account for the increased disease severity seen in γδ T cell-deficient mice. Instead, gingival γδ T cells produced the wound healing associated cytokine amphiregulin, administration of which rescued the elevated oral pathology of tcrδ^-/- mice. Collectively, our results identify γδ T cells as critical constituents of the immuno-surveillance network that safeguard gingival tissue homeostasis.

Keywords: amphiregulin; mucosal immunology; γδ T cells.

Fig. 1.

Heterogeneous γδ T cells are resident in the gingiva and can be generated from circulating precursors. (A) Representative FACS plot showing TCRβ and TCRγδ staining on live, CD45⁺, CD3⁺ gingiva cells. (B and C) Representative FACS plot and bar graphs showing percentage γδ T cells positive for (B) CD44 and CD27 and (C) IL-17 and IFNγ. Data, from five separate experiments. (D) Representative FACS plots showing staining of Vγ1, Vγ4, Vγ5, and Vγ6 on adult gingiva γδ T cells. Data representative of four experiments. (E) CD45.2⁺ host mice were sublethally irradiated before receipt of CD45.1⁺ donor bone marrow. Bar graph shows degree of chimerism of γδ T cells in gingiva of control and chimeric mice. Data representative of two separate experiments with three to four mice per group. Results are expressed as means ± SEM.

Fig. 2.

The gingival γδ T cell network is rapidly remodeled after birth. (A and B) Representative FACS plot and pie chart showing frequencies of Vγ⁺ subsets in the gingiva of day 0 pups (n = 6). (C and D) Graphs showing (C) frequencies of specific Vγ⁺ subsets and (D) total number of gingival γδ T cells (A = adult; n = 6–13 mice per time). (E and F) Graphs show (E) total number of gingival γδ T cells and (F) frequencies of specific Vγ⁺ subsets in the gingiva of GF mice at day 1 (n = 3) and day 7 (n = 8) after birth. (G and H) Graphs show (G) total number of gingival γδ T cells and (H) frequencies of Vγ⁺ subsets in gingiva of control mice or mice experiencing acute gingival damage. Data representative of two experiments with two to three mice per group. (I and J) Graphs show (I) total number of gingival γδ T cells and (J) frequencies of Vγ⁺ subsets in gingiva of mice aged on normal control diet (control) or on a hard pellet diet (hard) since weaning. Data representative of two experiments with two to three mice per group. *P < 0.05 as determined by unpaired Student’s t test. **P < 0.05 as determined by one-way ANOVA. Results are expressed as means ± SEM.

Fig. 3.

γδ T cells promote gingival homeostasis and protect against periodontal bone loss in mouse models of periodontitis. (A) CEJ–ABC distances in maxilla of 24-wk-old wild-type (open circles) and tcrδ^−/− (closed squares) mice (n = 7–12 mice per group). (Left) CEJ–ABC distance measured at six defined points across the molars. (Right) Graph shows total CEJ–ABC distance. (B) CEJ–ABC distances were measured in maxilla of separately housed control and tcrδ^−/− mice in which experimental periodontitis had been introduced. Change in bone heights was determined by subtracting the CEJ–ABC for periodontitis/ligated molars from naive molars of mice of the same genotype. (Left) CEJ–ABC distance measured at six defined points across the molars. (Right) Graph shows total change in bone heights in periodontitis mice compared with unligated controls. Data representative of three experiments with four to five mice per group. *P < 0.05 as determined by unpaired Student’s t test. Results are expressed as means ± SEM.

Fig. 4.

Altered oral microbial communities in the absence of γδ T cells are not responsible for elevated periodontal pathology. (A) Graph showing oral microbiome composition depicting most abundant operational taxonomic unit (n = 7–10). (B) Levels of Aa 16S were determined by qPCR assay. Graph shows levels relative to those in control mice. Data representative of two experiments, with four to six mice per group. (C) CEJ–ABC distances were measured in maxilla of cross-fostered/cohoused control and tcrδ^−/− mice in which experimental periodontitis had been introduced. Graph shows total change in bone heights in periodontitis mice compared with unligated control. Data representative of two experiments with three to four mice per group. (D and E) Experimental periodontitis was induced in control and tcrδ^−/− mice that had been treated with the antibiotic Sulfamethoxazole-Trimethoprim for 4–5 d before induction and throughout the course of disease. (D) Graph shows levels of Aa 16S in mice treated with antibiotics, relative to those in control mice, as determined by qPCR. (E) CEJ–ABC distances were measured in maxilla of antibiotic-treated control and tcrδ^−/− mice in which experimental periodontitis had been introduced. Graph shows total change in bone heights in periodontitis mice compared with unligated controls. Data representative of two experiments with four to six mice per group. *P < 0.05, **P < 0.005 as determined by unpaired Students t test. Results are expressed as means ± SEM.

Fig. 5.

Gingival γδ T cells produce Areg to limit oral pathology. (A and B) Volcano plots comparing gene expression of gingiva versus (A) spleen and (B) intestinal epithelium (intestinal intraepithelial lymphocytes; IEL) γδ T cells. (C) Gene expression signatures of gingival γδ T cells were examined using PANTHER to identify enriched gene ontology terms describing biological processes. Graph outlines terms enriched in gingiva γδ T cells. (D) Relative expression of Areg in gingival tissues of wild-type and tcrδ^−/− mice. Expression in tcrδ^-/− gingiva presented relative to that in wild-types, data from six to seven separate mice. (E) Representative FACS plots gated on gingival γδ T cells stained for Areg. Cells were restimulated with PMA, ionomycin, IL-6, IL-1β, and IL-23 with Brefeldin A. (Left) FACS plot is unstained for Areg. (Right) FACS plots shows representative staining for Areg. Graph shows percentage Areg⁺TCRγδ⁺ cells in unstained (FMO), spleen (SPL), and gingiva (GING) samples from four experiments. (F) CEJ–ABC distances in maxilla of aged wild-type (open circles) and Areg^−/− mice (closed squares; n = 7–8 mice per group). (Left) CEJ–ABC distance was measured at six defined points across the molars. (Right) Graph shows total CEJ–ABC distance. (G and H) Experimental periodontitis was induced in control and tcrδ^−/− mice, and mice received PBS or Areg i.v. every other day three times. (G) CEJ–ABC distance was measured at six defined points across the molars, and changes in bone heights determined in (Left) control mice and (Right) tcrδ^−/− mice. (H) Graph shows total change in bone heights in ligated/periodontitis molars compared with unligated molars. Data representative of three experiments with two to five mice per group. *P < 0.05 as determined by unpaired Student’s t test. **P < 0.05; ***P < 0.0001, as determined by one-way ANOVA. Results are expressed as means ± SEM.