Refractory neutrophil activation in type 2 diabetics with chronic periodontitis

Abstract

Background: Inflammation increases diabetes mellitus type 2 (T2DM) progression and severity. T2DM patients are at high risk of the rapid development of chronic periodontitis (CP). Topical presence, high numbers, and bactericidal effects of immune cells are challenged by augmented antigen-induced inflammation, which promotes both diseases.

Objectives: To investigate gingival cellular inflammatory responses in individuals with previously undiagnosed T2DM with CP or CP alone and in systemically and periodontally healthy controls (H) in vivo and to establish an ex vivo technique permitting quantitative and qualitative assessments of gingival crevicular immune cells.

Materials and methods: T2DM + CP, CP, and H individuals (n = 10, each) received a 2-week oral hygiene regimen (OHR). Afterwards, a noninvasive sampling technique was performed to evaluate gingival inflammation induced under standardized conditions in vivo, that is, in the absence of severe periodontal destruction and inflammation at clinically healthy sites. Stimuli (casein/test or phosphate-buffered saline w/o. Ca²⁺ or Mg²⁺ , PBS^(-/-) /control) were randomly applied contralaterally in the gingival sulci of participants' upper dentes canini. One day after completion of the OHR, gingival crevicular fluid (GCF) was kinetically assayed between the time of the baseline (BL) measurement and 55 minutes. Polymorphonuclear leukocyte (PMN) content (PMN_GCF ) was quantitated at an optimum time of 35 minutes. PMN_GCF counts reflect local inflammation. Ex vivo samples were fluorimetrically labeled, gated according to the donor's peripheral blood polymorphonuclear neutrophils (PMN_PB ), and then counted, employing flow cytometry.

Results: PMN_GCF counts in unstimulated gingival crevices (at BL) in the T2DM + CP group were higher than those in the CP and H groups. PMN_GCF counts were elevated in casein vs PBS^(-/-) -stimulated gingival crevices in all groups. Patients with T2DM + CP showed increased PMN_GCF counts compared to those with CP (P = .035) according to scatter plots. CD45⁺ counts in the stimulated sites in T2DM + CP patients were higher than those in CP and H patients (P = .041). Under stimulation conditions, the CD45⁺ counts differed from those under placebo conditions (P = .019), indicating augmented, inducible inflammatory leukocyte infiltrate in T2DM + CP patients.

Conclusions: This noninvasive technique permits quantitative assessment of (experimental) gingival inflammation in vivo, revealing an influence of T2DM + CP on the number of primary immune cells in the gingival crevice. Patients who are challenged with (local) leukocytosis are likely at risk of collateral damage to the gingival crevice neighboring tissues, favoring the severity and progression of CP and consequently T2DM (www.clinicaltrials.gov NCT01848379).

Keywords: chronic periodontitis; diabetes; flow cytometry; inflammation; neutrophils; periodontal immunology.