Comparative analysis of immune cell subsets in peripheral blood from patients with periodontal disease and healthy controls

Abstract

Periodontitis is a chronic inflammatory disease caused by the colonization of teeth by the bacterial plaque biofilm and the resultant host immune responses in adjacent periodontal tissues. Disease severity can vary dramatically between patients with periodontitis, with some subjects displaying inflammation without bony destruction (gingivitis), while others experience chronic progressive or rapidly aggressive gingival connective tissue damage and bone loss. To determine whether peripheral immune dysregulation is associated with periodontitis, we performed extensive analysis of immune cell subsets in peripheral blood from patients with chronic or aggressive periodontitis versus periodontally healthy control subjects. Peripheral blood mononuclear cells (PBMC) from patients with chronic periodontitis or aggressive periodontitis and from periodontally healthy controls were analysed by 8-10-colour flow cytometry for the frequencies of various lymphocyte subsets, including interleukin (IL)-17-, interferon (IFN)-γ-, tumour necrosis factor (TNF)-α- and IL-10-producing cells, and the frequencies and phenotype of monocytes. Cytokine levels in serum from the different groups were determined by Luminex assay. We found no significant differences in the frequencies of major immune cell populations [CD4⁺ T cells, CD8⁺ T cells, γδ T cells, CD4⁺ CD45RO⁺ CD25⁺ CD127^low regulatory T cells (T_regs ), CD19⁺ B cells, CD14⁺ monocytes] or of cytokine-producing T cells, or in the phenotype of CD14⁺ monocytes in peripheral blood from these patient cohorts. Additionally, no significant differences were observed in serum levels of prototypical inflammatory cytokines. These results suggest that the local gingival inflammatory response is not reflected by obvious changes in major blood immune cell subset frequencies.

Keywords: T helper 17 cells; Tregs; periodontal disease; peripheral blood mononuclear cells.

Figure 1

Frequencies of major immune cell populations in peripheral blood mononuclear cells (PBMC) from healthy controls and patients with periodontal disease. PBMC were isolated from peripheral blood from patients with chronic periodontitis (CP, n = 15) or aggressive periodontitis (AP, n = 15) as well as periodontally healthy controls (HC, n = 13). (a) Representative gating strategy and (b) cumulative data showing the frequencies of cell populations within live‐gated single cells. Each symbol represents an individual donor. Lines show the mean with standard error of the mean (s.e.m.). Log‐transformed data were analysed by one‐way analysis of variance followed by Bonferroni’s multiple comparison test.

Figure 2

Frequencies of CD4⁺CD45RO⁺CD25⁺CD127^low regulatory T cells in peripheral blood mononuclear cells (PBMC) from healthy controls and patients with periodontal disease. PBMC were isolated from peripheral blood from patients with chronic periodontitis (CP, n = 15) or aggressive periodontitis (AP, n = 15) as well as periodontally healthy controls (HC, n = 13). Cells were surface‐stained and then permeabilized and stained intranuclearly for forkhead box protein 3 (FoxP3). Representative dot‐plots and cumulative data showing the frequencies of CD4⁺CD45RO⁺ T cells (a) and CD25⁺CD127^low cells within CD4⁺CD45RO⁺ T cells (b). (c) Representative histogram plots showing the expression levels of FoxP3 in CD4⁺CD25⁺CD127^low T cells. Each symbol represents an individual donor. Lines show the mean with standard error of the mean (s.e.m.). Log‐transformed data were analysed by one‐way analysis of variance with Bonferroni’s multiple comparison test.

Figure 3

Expression of activation markers on CD14⁺ monocytes from healthy controls and patients with periodontal disease. Peripheral blood mononuclear cells (PBMC) were isolated from peripheral blood from patients with chronic periodontitis (CP, n = 12) or aggressive periodontitis (AP, n = 11), as well as periodontally healthy controls (HC, n = 8). (a) Representative gating strategy to identify CD14⁺ monocytes. (b) Representative histogram plots and (c) cumulative data showing the geometric mean fluorescence intensity (MFI) for the surface expression of indicated markers determined by flow cytometry on CD14⁺ monocytes. Each symbol represents an individual donor. Lines show the mean with standard error of the mean (s.e.m.). Log‐transformed data were analysed by one‐way analysis of variance with Bonferroni’s multiple comparison test.

Figure 4

Frequencies of cytokine producing CD4⁺ T cells in PBMC from healthy controls and patients with periodontal disease. Peripheral blood mononuclear cells (PBMC) were isolated from peripheral blood from patients with chronic periodontitis (CP, n = 15) or aggressive periodontitis (AP, n = 15), as well as periodontally healthy controls (HC, n = 13). Cells were stimulated ex vivo with phorbol myristate acetate (PMA) and ionomycin for 3 h prior to intracellular cytokine staining. (a) Representative gating strategy and (b) cumulative data showing the frequencies for the interleukin (IL)‐17‐, interferon (IFN)‐γ‐, tumour necrosis factor (TNF)‐α‐ and IL‐10‐expressing cells within CD4⁺ T cells as determined based on fluorescence‐minus‐one (FMO) control. Each symbol represents an individual donor. Lines show the mean with standard error of the mean (s.e.m.). Log‐transformed data were analysed by one‐way analysis of variance with Bonferroni’s multiple comparison test.

Figure 5

Frequencies of cytokine‐producing cells CD8⁺ and γδ T cells in peripheral blood mononuclear cells (PBMC) from healthy controls and patients with periodontal disease. PBMC were isolated from peripheral blood from patients with chronic periodontitis (CP, n = 15) or aggressive periodontitis (AP, n = 15) as well as periodontally healthy controls (HC, n = 13). (a,b) Representative dot‐plots and cumulative data for the frequencies of IL‐17⁺, IFN‐γ⁺ and TNF‐α⁺ cells within CD8⁺ T cells (a) or γδ T cells (b). Each symbol represents an individual donor. Lines show the mean with standard error of the mean (s.e.m.). Log‐transformed data were analysed by one‐way analysis of variance with Bonferroni’s multiple comparison test.

Figure 6

Cytokine levels in serum from patients with periodontitis or gingivitis, or from healthy controls. Serum from patients with chronic periodontitis (n = 15) or aggressive periodontitis (n = 15), as well as periodontally healthy controls (n = 13) was analysed. Cytokine levels in 25 µl of serum were determined by Luminex. Each symbol represents an individual subject; the median and interquartile range is shown, dotted lines indicate the minimum detectable concentration of that particular mediator. Log‐transformed data were analysed by one‐way analysis of variance with Bonferroni’s multiple comparison test. *P<0.05.