RvE1 Impacts the Gingival Inflammatory Infiltrate by Inhibiting the T Cell Response in Experimental Periodontitis

Abstract

Periodontitis is a chronic inflammatory disease associated with the formation of dysbiotic plaque biofilms and characterized by the progressive destruction of the alveolar bone. The transition from health to disease is characterized by a shift in periodontal immune cell composition, from mostly innate (neutrophils) to adaptive (T lymphocytes) immune responses. Resolvin E1 (RvE1) is a specialized pro-resolution mediator (SPMs), produced in response to inflammation, to enhance its resolution. Previous studies have indicated the therapeutic potential of RvE1 in periodontal disease; however, the impact of RvE1 in the microbial-elicited osteoclastogenic immune response remains uncharacterized in vivo. In the present study, we studied the impact of RvE1 on the gingival inflammatory infiltrate formation during periodontitis in a mouse model. First, we characterized the temporal-dependent changes of the main immune cells infiltrating the gingiva by flow cytometry. Then, we evaluated the impact of early or delayed RvE1 administration on the gingival immune infiltration and cervical lymph nodes composition. We observed a consistent inhibitory outcome on T cells -particularly effector T cells- and a protective effect on regulatory T cells (Tregs). Our data further demonstrated the wide range of actions of RvE1, its preventive role in the establishment of the adaptive immune response during inflammation, and bone protective capacity.

Keywords: Periodontal disease; RvE1; SPMs (specialized pro-resolving mediators); T cells; inflammation.

Figure 1

Gingival immune-infiltrate shift during experimental periodontitis progression. (A) Experimental design. (B) Representative palatal views of maxillary molars from animals at baseline (D0) or sacrificed at days 1, 3, 5, or 10 after ligature placement. Bone loss area is highlighted in orange. (C) Bone loss measurements from the cementoenamel junction to alveolar bone crest. (D) Identification of gingival neutrophils (PMN, CD45⁺LY6G^high LY6C^midCD11b⁺), macrophages (Mac, CD45⁺CD64⁺CD11b⁺MHCII⁺), and T cells (CD45⁺CD3⁺) as clustered by FlowSOM. tSNE graphs of concatenated data (4 animals per group) showing CD45+ gingival cells. (E) Comparison of the peak frequencies of PMN, T cells, and macrophages within CD45⁺ gingival cells over time. Dotted line represents day 3 (highlighting transition from innate to adaptive immune response) (F) Percentages and (G) cell number of neutrophils, T cells, and macrophages in gingiva. ^#P<0.05 PMN vs T cell and Mac; ^&P < 0.05 Mac vs T cell and PMN; *P < 0.05; **P 4< 0.01; ***P < 0.001.

Figure 2

Temporal-dependent changes of the gingival immune-infiltrate in experimental periodontitis due to early RvE1 administration. (A) Experimental design. (B) Representative palatal views of maxillary molars from animals sacrificed at days 1, 3, 5, 10 after ligature application with or without RvE1 treatment (D1-D10 in yellow), baseline (D0) and ligature without treatment at day 10 (D10 white) as references. Bone loss area is highlighted in orange. (C) Bone loss in RvE1-treated and non-treated animals (n=5). (D) Identification of neutrophils (PMN, CD45⁺Ly6G⁺), macrophages (Mac, CD45⁺CD11b⁺CD64⁺), and T cell (CD45⁺CD3⁺) subsets in tSNE graphs of concatenated data (n = 4 animals per group) within CD45⁺ gingival cells. (E) Comparison of the frequencies of PMN, T cells, and macrophages within CD45⁺ gingival cells (F–H) Comparison of the frequency of each cell subtype within CD45⁺ gingival cells over time in animals with or without RvE1 treatment. ^#P < 0.05 T cells vs PMN and Mac; ^##P < 0.01 T cells vs PMN and Mac; *P < 0.05.

Figure 3

Impact of early RvE1 administration on CD4⁺ cells in cervical lymph nodes. (A, B) Representative dot plots indicating the frequency of CD4⁺ and CD4⁺Foxp3⁺ cells among all cells from cervical lymph nodes of animals with ligature-induced periodontitis (10 days; ligature), pre-treated with RvE1 (RvE1 + Ligature) or baseline (no ligature). (C, D) Representative histograms indicating the expression of IL-17 or IFNγ in CD4⁺ cells. (E–H) Frequency of total CD4+ cells, CD4⁺Foxp3⁺ cells, and CD4⁺ IL-17 or IFNγ⁺ cells in cervical lymph nodes (n = 4). FMO, Fluorescence minus one. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 4

Impact of delayed RvE1 administration on the gingival immune-infiltrate composition. (A) Experimental design. (B) Representative palatal views of maxillary molars from animals at baseline (BAS), untreated ligature-induced periodontitis (D10), or treated with RvE1 after day 1 (RvE1). Bone loss area highlighted in orange. (C) Bone loss measurements from the cement-enamel junction to alveolar bone crest. (D) tSNE plots of concatenated data (n = 4 animals per group), and (E) pie charts indicating the frequency of neutrophils (PMN, CD45⁺Ly6G⁺), macrophages (Mac, CD45⁺CD11b⁺CD64⁺) and T cells (CD45⁺CD3⁺), Dendritic cells (DCs, CD45⁺CD11c⁺MHCII⁺), B cells (CD45⁺CD19⁺MHCII⁺), and other cells within gingival CD45⁺ cells. (F–I) Percentages of neutrophils, macrophages, T cells, and DCs in CD45⁺ gate. (J) Heat map representing the log2 fold-change gingival expression of mRNA of cytokines. (K, L) Gingival mRNA expression of IL-17 and IL-6. *P < 0.05, **P < 0.01.

Figure 5

RvE1-induced changes in leukocyte composition in cervical lymph nodes. (A) tSNE plots of concatenated data (n = 4 animals per group), and (B) pie charts indicating the frequency neutrophils (PMN), macrophages (Mac), T cells, dendritic cells (DCs), B cells, and other cells within the CD45⁺ cell population in cervical lymph nodes from animals at baseline, untreated ligature-induced periodontitis (Ligature), or treated with RvE1 from day 1 (RvE1). (C, D) Percentages of T and B cells within CD45⁺ cells in cervical lymph nodes. *P < 0.05.

Figure 6

Impact of delayed RvE1 administration on Treg cells from cervical lymph nodes. (A) Representative plots indicating the gating strategy and percentage of CD4⁺CD25⁺Foxp3⁺IL17⁺ cells in cervical lymph nodes of animals at baseline, with ligature-induced periodontitis (Ligature), or treated with RvE1 from day 1 (RvE1). (B) Quantification of the percentage and (C) total number of Treg cells. (D) Quantification of the percentage and (E) cell number of IL-17⁺ Treg cells. *P < 0.05, ***P < 0.001.