B cell subset distribution is altered in patients with severe periodontitis

Abstract

Several studies have recently highlighted the implication of B cells in physiopathogenesis of periodontal disease by showing that a B cell deficiency leads to improved periodontal parameters. However, the detailed profiles of circulating B cell subsets have not yet been investigated in patients with severe periodontitis (SP). We hypothesised that an abnormal distribution of B cell subsets could be detected in the blood of patients with severe periodontal lesions, as already reported for patients with chronic inflammatory diseases as systemic autoimmune diseases. Fifteen subjects with SP and 13 subjects without periodontitis, according to the definition proposed by the CDC periodontal disease surveillance work group, were enrolled in this pilot observational study. Two flow cytometry panels were designed to analyse the circulating B and B1 cell subset distribution in association with the RANKL expression. A significantly higher percentage of CD27+ memory B cells was observed in patients with SP. Among these CD27+ B cells, the proportion of the switched memory subset was significantly higher. At the same time, human B1 cells, which were previously associated with a regulatory function (CD20+CD69-CD43+CD27+CD11b+), decreased in SP patients. The RANKL expression increased in every B cell subset from the SP patients and was significantly greater in activated B cells than in the subjects without periodontitis. These preliminary results demonstrate the altered distribution of B cells in the context of severe periodontitis. Further investigations with a larger cohort of patients can elucidate if the analysis of the B cell compartment distribution can reflect the periodontal disease activity and be a reliable marker for its prognosis (clinical trial registration number: NCT02833285, B cell functions in periodontitis).

Fig 1. STROBE checklist.

Fig 2. Phenotype of B subsets in whole blood of patients with severe periodontitis (SP) and controls (No Periodontitis).

A) The gating strategy used after doublet exclusion allows the characterisation eight different and distinct B cell subsets. B) Circulating B cell distribution reveals an increase in the switched memory B cells and a decrease in the mature B cells in the SP patients in comparison with the controls. p value is indicated when significant. Plasmablasts: PB; Transitional B cells: TR; CD5⁺ B cells: CD5⁺; double negative IgD^-CD27^- B cells: DN; switched memory B cells: SwM; unswitched memory B cells: USwM; mature B cells: Mature and naïve B cells: Naïve.

Fig 3. Phenotype of B1 and CD5⁺ B cells in whole blood of patients with severe periodontitis (SP) and controls (No Periodontitis).

A) The gating strategy used after doublet exclusion allows the characterisation of B cells according to CD11b and CD5 expression but also the human B1 subset (CD20⁺CD69^-CD43⁺CD27⁺). B) Circulating B cell distribution according to CD11b and CD5 expression reveals a decrease in the CD11b⁺CD5⁺ B cells in the SP patients in comparison with the controls. C) B1 B cells are lower in the SP patients in comparison with the controls. D) Among B1 cells the CD11b⁺ B1 regulatory subset is markedly decrease in the SP patients. p value is indicated when significant.

Fig 4. Expression of RANKL in the B cell subsets from patients with severe periodontitis and controls (No Periodontitis).

A) Expression of RANKL in the eight distinct B cell subsets defined in Fig 1. B) Expression of RANKL in the circulating B cell distribution according to CD11b and CD5 expression. C) Expression of RANKL in B1 cells. Mean fluorescence intensity: MFI; Plasmablasts: PB; Transitional B cells: TR; CD5⁺ B cells: CD5⁺; double negative IgD^-CD27^- B cells: DN; switched memory B cells: SwM; unswitched memory B cells: USwM; mature B cells: Mature and naïve B cells: Naïve.

Fig 5. Expression of B cell activation markers in the B cell subsets from patients with severe periodontitis (SP) and controls (No Periodontitis).

A) The percentage of CD69⁺ B cells expressing RANKL was higher in the SP patients in comparison with the controls. B) The percentage of CD25⁺ B cells was higher in the SP patients in comparison with the controls. Mean fluorescence intensity: MFI, p value is indicated when significant.