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. 2018 Jun 18;9:1374.
doi: 10.3389/fimmu.2018.01374. eCollection 2018.

Multiple Regulatory Levels of Growth Arrest-Specific 6 in Mucosal Immunity Against an Oral Pathogen

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Free PMC article

Multiple Regulatory Levels of Growth Arrest-Specific 6 in Mucosal Immunity Against an Oral Pathogen

Maria Nassar et al. Front Immunol. .
Free PMC article

Abstract

Growth arrest-specific 6 (GAS6) expressed by oral epithelial cells and dendritic cells (DCs) was shown to play a critical role in the maintenance of oral mucosal homeostasis. In this study, we demonstrate that the induction of pathogen-specific oral adaptive immune responses is abrogated in Gas6-/- mice. Further analysis revealed that GAS6 induces simultaneously both pro- and anti-inflammatory regulatory pathways upon infection. On one hand, GAS6 upregulates expression of adhesion molecules on blood vessels, facilitating extravasation of innate inflammatory cells to the oral mucosa. GAS6 also elevates expression of CCL19 and CCL21 chemokines and enhances migration of oral DCs to the lymph nodes. On the other hand, expression of pro-inflammatory molecules in the oral mucosa are downregulated by GAS6. Moreover, GAS6 inhibits DC maturation and reduces antigen presentation to T cells by DCs. These data suggest that GAS6 facilitates bi-directional trans-endothelial migration of inflammatory cells and DCs, whereas inhibiting mucosal activation and T-cell stimulation. Thus, the orchestrated complex activity of GAS6 enables the development of a rapid and yet restrained mucosal immunity to oral pathogens.

Keywords: growth arrest-specific 6; immunoregulation; infection; mucosa; oral.

Figures

Figure 1
Figure 1
Diminished pathogen-specific adaptive immunity in Gas6−/− mice. (A) Schematic presentation of the murine periodontitis model used in this study. Antibiotic pre-treated mice were infected via oral gavage, three times at 2-day intervals, with 1 × 1010 CFU of Porphyromonas gingivalis strain 53977 (Pg). Six weeks after the last inoculation, the mice were analyzed. (B) IFN-γ production by restimulated splenocytes quantified by ELISA representing the mean value ± SEM (n = 8). (C) Pg-specific IgG titers measured by ELISA in the plasma of the mice, representing the mean of OD450 values ± SEM (n = 8). (D–F) Flow cytometry analysis of the percentages of CD45+ cells (D), CD4+ T cells (E), and FOXP3-expressing CD4+ T regulatory cells (F) in the gingival tissues of infected mice (n = 5). Representative data of one out of three independent experiments is shown.
Figure 2
Figure 2
Lack of inflammation-induced bone loss in Gas6−/− mice. (A) Representative μCT sections of the second upper molar demonstrating the residual bone volume measured from the cemento-enamel junction (CEJ) and alveolar bone crest (ABC). White arrows indicate lesions in the alveolar bone. (B) Three-dimensional quantification of the residual alveolar bone. Data are presented as the volume of alveolar bone in the buccal plate and represent the mean of eight mice per group ± SEM. (C) Quantification by RT-qPCR of the expression levels of RNAKL in the gingiva of infected mice, graphs represent the mean of five mice per group ± SEM. Representative data of one out of two independent experiments is shown.
Figure 3
Figure 3
Lack of innate infiltrate in the gingiva of infected Gas6−/− mice. (A) Schematic presentation of an acute oral infection. Mice were infected once with Pg by oral gavage and analyzed either 1, 3, or 7 days after infection. (B) H&E and immunofluorescence stained histological section of the lower jaw and RT-qPCR analyses of GAS6 and PROS1 expression in the gingiva of WT mice prior to and 3 days after Pg infection. Bar graphs represent the mean of four mice per group ± SEM. Immunohistochemical staining is shown for GAS6 (green), PROS1 (red) and nuclei are stained with hoechst (blue). Enlarged images of the framed area are shown to the right. (C) Percentages of total CD45+ cells, neutrophils, monocytes, and APCs in the gingiva 24 h after infection. Bar graphs represent the mean of five mice per group ± SEM. (D) Representative FACS plot showing the frequencies of MHCII+CD11c+ cells (APCs) in the gingiva of WT and Gas6−/− mice 24 h after infection. Representative data of one out of three independent experiments is shown. Abbreviations: BE, buccal epithelium; GE, gingival epithelium; SE, sulcular epithelium; JE, junctional epithelium; LP, lamina propria; GAS6, growth arrest-specific 6.
Figure 4
Figure 4
Elevated expression of pro-inflammatory cytokines in Gas6−/− mice following infection with Pg. (A) Expression levels of TNF-α, IL-1β, and CCL2 in the gingiva 24 h after a single infection with Pg. Data are presented as relative mRNA expression analyzed by RT-qPCR and represent the mean of five mice ± SEM. (B) Serum levels of TNF-α, on days 0, 1, and 3 days post-infection, measured by ELISA. Graphs represent the mean of five mice ± SEM. Representative data of one out of three independent experiments is shown. (C) Peripheral blood samples were collected from naive and infected mice 24 h after infection for flow cytometry analysis. Representative FCAS plots and bar graphs demonstrate the percentages of neutrophils in the blood. Bar graphs represent the mean of five mice per group ± SEM. Representative data of one out of three independent experiments is shown.
Figure 5
Figure 5
Infiltration of innate leukocytes to the gingiva is blocked in infected Gas6−/− mice. (A) Immunofluorescence staining of AXL or growth arrest-specific 6 (GAS6) (green), CD31 (red), and hoechst (blue) in gingival lamina propria cross sections of Pg-infected and naive WT and Gas6−/− mice. Representative images of at least three independent experiments. Scale bars represent 20 µm. (B) Expression of P-selectin, ICAM-1, and VCAM-1 in the gingiva of WT and Gas6−/− mice 24 h following infection with Pg. Relative mRNA expression levels are presented using RT-qPCR analysis representing the mean of five mice per group ± SEM. Representative data of one out of two independent experiments is shown. (C,D) Quantification of inflammatory cells in the blood and gingiva following a single infection of WT and Gas6−/− mice with Pg. (C) Gating strategy to identify neutrophils, APCs, Ly6Clow, and Ly6Chigh monocytes in the blood using flow cytometry analysis. (D) Time course analysis of the frequencies of CD45+ cells, neutrophils, and Ly6Chigh monocytes in the blood versus gingiva in WT and Gas6−/− mice, representing the mean of five mice per group ± SEM. Representative data of one out of two independent experiments is shown.
Figure 6
Figure 6
Growth arrest-specific 6 (GAS6) enhances migration of oral dendritic cells (DCs) to the draining lymph nodes (LNs). (A) Mice were infected once with Pg and the expression levels of CCL19 and CCL21 chemokines was analyzed in the gingiva using RT-qPCR 1 or 3 days later. Data represent the mean of five mice per group ± SEM. (B) Immunofluorescence staining of AXL or GAS6 (green) and LYVE1 (red), in gingival lamina propria cross sections of Pg-infected and naive WT and Gas6−/− mice. Representative images of two independent experiments. (C) The oral mucosa of WT and Gas6−/− mice were painted with FITC/DBP solution, 2 days later the draining cervical LNs were collected and processed for analysis by flow cytometry. Left plot—representative image demonstrating the segregation of CD45+CD11c+ cells into distinct DC subsets based on the expression of MHC class II and CD11c (RLN-DC—LN resident DCs; pDC—plasmacytoid DCs). Upper panel—representative FACS plots and bar graphs illustrating the percentages of FITC-positive cells among migratory DCs of Gas6−/− and WT mice, representing the mean of five mice per group ± SEM. Lower panel—representative FCAS histograms showing MHCII expression levels on total migratory DCs and RLN-DCs. Gray histograms represent MHC staining on T cells which do not express MHCII. Bar graphs present the mean florescence intensity (MFI) of MHCII expression on the noted DC populations and represent the mean of five mice per group ± SEM.
Figure 7
Figure 7
Growth arrest-specific 6 (GAS6) inhibits dendritic cell (DC) maturation and limits T-cell activation by DCs. (A) BMDCs generated from WT and Gas6−/− mice were stimulated with LPS for 24 h. Representative FACS histograms and graphs demonstrating the expression and mean florescence intensity (MFI) of MHCII (top) and CD86 (bottom) on BMDCs, respectively. Gray histograms represent staining on MHC-negative cells in the culture. (B) DCs purified from cervical lymph nodes (LNs) of Gas6−/− and WT mice were stimulated with LPS for 24 h. Representative FACS plots demonstrating the expression of CD86, MHCII, and CCR7 are presented. (C,D) DCs enriched from cervical LNs of Gas6−/− and WT mice were stimulated with LPS and OVA peptides and then co-cultured with naive CFSE-labeled OT-II CD4+ T cells or OT-I CD8+ T cells. (C) Three days post-stimulation the dilution in CFSE levels on the T cells was analyzed by flow cytometry, representative FACS histograms are presented, numbers indicate the mean of three repeats per group ± SEM. (D) Bar graphs show the concentrations of IFN-γ secreted to the supernatant by activated CD8+ T cells and represent the mean of four repeats per group ± SEM. Representative data of one out of two independent experiments is shown.
Figure 8
Figure 8
Pro- and anti-inflammatory roles for growth arrest-specific 6 (GAS6) in the oral mucosa. Upon infection, GAS6 facilitates extravasation of neutrophils and Gr1high monocytes to the oral mucosa by upregulating expression of adhesion molecules on blood endothelial cells. GAS6 also enhances migration of mucosal dendritic cells (DCs) to the lymph node by upregulating CCL19/CCL21 expression, chemokines mediating this process via interaction with CCR7 on DCs. Both processes involve the GAS6-mediated trans-endothelial migration, indicating its pro-inflammatory function. On the other hand, GAS6 downregulates the production of pro-inflammatory cytokines and chemokines by epithelial cells. In the LN, GAS6 inhibits antigen presentation and T cell activation by DCs, via reducing expression of the maturation-associated molecules MHC and CD86. Interestingly, CCR7, an additional molecule associated with DC maturation, is not downregulated by GAS6, thus complementing the upregulation of CCR7 ligands by GAS6 in the mucosa to promote migration. Such apparently opposing roles of GAS6 during infection enable the induction of swift innate inflammatory responses, while facilitating the development of a restrained adaptive immune response.

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