Disruption of Monocyte and Macrophage Homeostasis in Periodontitis

Abstract

Monocytes and macrophages are major cellular components of the innate immunity that play essential roles in tissue homeostasis. The contribution of different subsets of monocytes/macrophages to periodontal health and disease has not been fully elucidated. Type 2 diabetes mellitus (T2DM) is a risk factor for periodontitis. We hypothesized that the monocyte/macrophage signaling is perturbed in periodontitis-affected sites versus periodontally healthy sites and that this perturbation plays a critical role in the pathogenesis of periodontitis. Pairs of gingival tissue samples (each from a periodontally healthy and a periodontitis-affected site of the same patient) were harvested from 27 periodontitis patients, with and without T2DM. Each sample was processed to form a single-cell suspension, and a flow-cytometry panel was designed and validated to study monocyte and macrophage phenotypes. In separate experiments, the transcriptional changes associated with a pro-inflammatory phenotype were also examined in monocyte/macrophage subsets obtained from peripheral blood of patients with T2DM versus diabetes-free controls. A significantly higher proportion of intermediate (CD14+CD16+) monocytes was observed in periodontitis-affected tissues compared to healthy tissues. These monocytes overexpressed HLA-DR and PDL1 molecules, suggesting their activated inflammatory status. PDL1 increase was specific to intermediate monocytes. The ratio of M1/M2 macrophages was also significantly higher in periodontally affected sites, signifying an imbalance between inflammatory and repair mechanisms. We found a significantly higher expression of PDL1 in overall monocytes and M1 macrophages in periodontitis-affected sites compared to controls. Importantly, we identified a subpopulation of M1 macrophages present in periodontally affected tissues which expressed high levels of CD47, a glycoprotein of the immunoglobulin family that plays a critical role in self-recognition and impairment of phagocytosis. Analysis of the transcriptional landscape of monocytes/macrophages in gingival tissue of T2DM patients with periodontitis revealed a significant disruption in homeostasis toward a proinflammatory phenotype, elevation of pro-inflammatory transcription factors STAT1 and IRF1, and repression of anti-inflammatory JMJD3 in circulating monocytes. Taken together, our results demonstrate disruption of myeloid-derived cell homeostasis in periodontitis, with or without T2DM, and highlight a potentially significant role of these cell types in its pathogenesis. The impact of macrophage and monocyte signaling pathways on the pathobiology of periodontitis should be further evaluated.

Keywords: CD47; diabetes; macrophages; monocytes; periodontitis.

Copyright © 2020 Almubarak, Tanagala, Papapanou, Lalla and Momen-Heravi.

Figures

FIGURE 1

Single cell suspensions from matched periodontitis-affected and healthy gingival tissues were prepared and stained for UV Zombie, CD45, Lin (B220, CD19, Gr1, and Ter119), HLA-DR, CD16, CD14. (A,B) Representative counter plots of intermediate (CD14+CD16+) and non-classical monocytes (CD14–CD16+). Frequency of both cell types was higher in periodontitis-affected tissue compared to matched controls (n = 21; p < 0.05). (C,D) Representative counter plots and frequency of HLA-DR cells which are gated from live, CD45+Lin–CD16+ cells in periodontitis-affected gingival tissue and healthy gingival tissue (n = 21). Frequency of CD45+Lin–CD16 + HLA-DR+ cells was significantly higher in periodontitis-affected tissue compared to health (p < 0.05). (E,F) Representative counter plots (values indicate percentage of parent population) and frequency of HLA-DR cells which are gated from live, CD45+Lin–CD14+ cells in the periodontitis-affected gingival tissue and healthy gingival tissue (n = 21). Frequency of CD45+Lin–CD14+HLA-DR+ cells was significantly higher in periodontitis-affected tissue compared to health (p < 0.05). Data presented as mean ± SEM and p less than 0.05 was defined as significant (the symbol “*” indicates p < 0.05 compared to the healthy control).

FIGURE 2

Single cell suspensions from matched periodontitis-affected and healthy gingival tissues were prepared and stained for UV Zombie, CD45, Lin, HLA-DR, CD16, CD14, and PDL1. (A–C): Representative counter plots of CD14+PDL1+ and CD16+PDL1+ cells. Frequency of both cell types was higher in periodontitis-affected tissue compared to matched controls (n = 14; p < 0.05). (D,E): Representative counter plots of intermediate and non-classical monocytes based on HLA-DR and PDL1 expression. HLA-DR+ PDL1+ population was only detectable in intermediate and not in non-classical monocytes. (F) Flow cytometry histogram of FITC-labeled CD47 expression in monocytes in periodontitis-affected tissue (black line) compared to healthy tissue (red line). The histogram was normalized to the number of events and depicts increase in CD47 in periodontitis-affected sites. Data presented as mean ± SEM and p less than 0.05 was defined as significant (the symbol “*” indicates p < 0.05).

FIGURE 3

Single cell suspensions from matched periodontitis-affected and healthy gingival tissues were prepared and stained for UV Zombie, CD45, Lin, CD64, MHC II, CD80, CD163, CD206, PDL1, CD47. (A,B) Representative counter plots of M1 macrophages (CD80+MHC II+). Frequency of M1 macrophages was higher in periodontitis-affected tissue compared to controls (n = 21 pairs; p < 0.05). (C,D) Representative counter plots of M2 macrophages (CD206+CD163+). Frequency of M2 macrophages was lower in periodontitis-affected tissue compared to controls (n = 6 pairs; p < 0.05). (E,F) Flow cytometry histogram of CD47 (FITC) and PDL1 (Qdot 655-A) expression in M1 macrophages in periodontitis-affected tissue (red line) compared to the healthy tissue (black line) shows higher expression of both markers in periodontitis-affected sites. The histogram was normalized to the number of events. (G,H) Flow cytometry histogram of CD47 (FITC) and PDL1 (Qdot655-A) expression in M2 macrophages in periodontitis-affected tissue (black line) compared to the healthy tissue (red line) shows change in both markers in periodontitis affected sites in T2DM (the symbol “*” indicates p < 0.05 compared to the healthy control).

FIGURE 4

Single cell suspensions from matched periodontitis-affected and healthy gingival tissues harvested from patients with T2DM were prepared and stained for UV Zombie, CD45, Lin (B220, CD19, Gr1 and Ter119), HLA-DR, CD16, CD14. (A–C) Representative counter plots of intermediate (CD14+CD16+) and non-classical monocytes (CD14–CD16+). Frequency of intermediate monocytes was significantly higher in periodontitis-affected tissue compared to matched controls (n = 6; p < 0.05). (D) Flow cytometry histogram of CD47 (FITC) and PDL1 (Qdot655-A) expression in M2 macrophages in periodontitis-affected tissue (red line) compared to the healthy tissue (black line) shows higher PDL1 expression in periodontitis affected sites in T2DM (the symbol “*” indicates p < 0.05 compared to the healthy control).

FIGURE 5

Single cell suspensions from matched periodontitis-affected and healthy gingival tissues from T2DM patients were prepared and stained for UV Zombie, CD45, Lin, CD64, MHC II, CD80, CD163, CD206, PDL1, CD47. (A,B) Representative counter plots of M1 macrophages (CD80+MHC II+). Frequency of M1 macrophages was higher in periodontitis-affected tissue compared to control tissue in T2DM (n = 6 pairs; p < 0.05). (C,D) Representative counter plots of M2 macrophages (CD206+CD163+). Frequency of M2 macrophages was lower in periodontitis-affected tissue compared to control tissue in T2DM (n = 6 pairs; p < 0.05). (E,F) Flow cytometry histogram of CD47 (FITC) and PDL1 (Qdot 655-A) expression in M1 macrophages in periodontitis-affected tissue (red line) compared to the healthy tissue (black line) shows higher expression of both markers in periodontitis-affected sites in T2DM. The histogram was normalized to the number of events. (G,H) Flow cytometry histogram of CD47 (FITC) and PDL1 (Qdot655-A) expression in M2 macrophages in periodontitis-affected tissue (red line) compared to the healthy tissue (black line) shows change in both markers in periodontitis affected sites in T2DM (the symbol “*” indicates p < 0.05 compared to the healthy control).

FIGURE 6

Primary human monocytes were isolated from the circulation of individuals with and without T2DM (at least 5 donors in each group). mRNA was isolated and qPCR was performed to analyze gene expression. (A–E) IRF1, STAT1, STAT2, HIF1α, JMJD3 levels were quantified by qPCR. 18 s was used as the endogenous normalizer. Data presented as mean ± SEM and p less than 0.05 was defined as significant (the symbol “*” indicates p < 0.05).

FIGURE 7

(A) THP1-derived macrophages were challenged with P. gingivalis LPS (10 ng/ml) and IFNγ (20 ng/ml) (M1 phenotype), or IL4 (40 g/ml) + IL13 (20 ng/ml) (M2 phenotype). Level of CD47 mRNA was quantified by qPCR after 24 h (at least 5 donors in each group). Data were normalized to 18 s. (B) Luciferase reporter assay was used to asses NF-κB and CD47 promoter activity in THP1-derived macrophages after challenge with P. gingivalis LPS or SN50 (20 μM). (C) THP1-derived macrophages were challenged with P. gingivalis LPS (20 ng/ml) and/or SN50 (20 μM) and level of CD47 mRNA was quantified by qPCR. Data were normalized to 18 s. (D,E) Primary human macrophages were challenged with P. gingivalis LPS (10 ng/ml) and IFNγ (20 ng/ml) (M1 phenotype), or IL4 (40 g/ml) + IL13 (20 ng/ml) (M2 phenotype). Levels of CD47 and SIPRα mRNA were quantified by qPCR after 24 h (at least 5 donors in each group). Data presented as mean ± SEM and p less than 0.05 was defined as significant (the symbol “*” depicts p < 0.05 compared to the unstimulated control, unless otherwise noted in the figure).