Characterization of oral polymorphonuclear neutrophils in periodontitis patients: a case-control study

Abstract

Background: Maintaining oral health is a continuous and dynamic process that also involves the immune system. Polymorphonuclear neutrophils (PMNs) migrate from blood circulation and become apparent in the oral fluid. Controversies exist regarding the specific role of the oral PMNs (oPMNs) in the presence of chronic oral inflammation, such as periodontitis. In this study we characterized cell counts, activation status, apoptosis, and reactive oxygen species (ROS) generation by oPMNs and circulatory (cPMNs), and the salivary protease activity, in subjects with and without periodontitis.

Methods: Venous blood and oral rinse samples were obtained from 19 patients with untreated periodontitis and 16 control subjects for PMN isolation. Apoptosis and expression of cell activation markers CD11b, CD63, and CD66b were analyzed using flow cytometry. Constitutive ROS generation was detected using dihydrorhodamine123. Additionally, ROS production in response to stimulation was evaluated in samples incubated with 10 μM phorbol myristate acetate (PMA) or Fusobacterium nucleatum. Total protease activity was measured using substrate PEK-054.

Results: Periodontitis patients presented with over 4 times higher oPMN counts compared to controls (p = 0.007), which was a predictor for the total protease activity (r² = 0.399, P = 0.007). More oPMNs were apoptotic in periodontitis patients compared to the controls (P = 0.004). All three activation markers were more expressed on the oPMNs compared to the cPMNs (p < 0.05), and a higher expression of CD11b on the oPMNs from periodontitis patients was observed compared to the control subjects (P = 0.024). Constitutive ROS production per oPMN was higher compared to the cPMN (P < 0.001). Additional analysis showed that the oPMNs retained their ability to respond to stimulation, with no apparent differences between the periodontitis and control subjects.

Conclusions: Higher numbers of oral PMNs, being more apoptotic and having increased levels of degranulation markers were found in periodontitis compared to periodontal health. However, since the oPMNs in periodontitis were responsive to ex vivo stimulation, we conclude that the oPMNs are active in the oral ecosystem. It is currently unknown whether the oPMN counts, which correlated with the detected protease levels, are detrimental in the long term for the oral mucosa integrity.

Trial registration: This study was retrospectively registered at the ISRCTN registry (trial ID ISRCTN15252886 ). Registration date August 11, 2017.

Keywords: Apoptosis; Degranulation; Periodontitis; Polymorphonuclear neutrophilic granulocytes; Reactive oxygen species.

Fig. 1

Apoptosis analysis of PMNs from controls (n = 16) and periodontitis subjects (n = 19) quantified by flow cytometry. The percentages of propidium iodide positive (PI⁺) PMNs were calculated, representing apoptotic cells. The different cell populations are expressed as a percentage of the total PMN cell population and are given as mean ± SD. *Comparisons periodontitis group versus control group, Student’s T-test, *P < 0.05. ^#Comparisons oPMN versus cPMN within subjects, Paired T-test, ^#P < 0.001

Fig. 2

Mean fluorescence intensity (MFI) of cellular expression of (a) CD11b, (b) CD63 and (c) CD66b, on PMNs isolated from controls (n = 16) and periodontitis subjects (n = 19). PMNs were gated according to CD16 expression and the sideward scatter profile. Expressions of surface markers of interest were corrected for the non-specific binding of the isotype control antibodies. Data are mean ± SD. *Comparisons periodontitis group versus control group, Student’s T-test, *P = 0.024. ^#Comparisons oPMN versus cPMN within subjects, paired T-test, all ^#P < 0.001

Fig. 3

Mean fluorescence intensity (MFI) of reactive oxygen species (ROS) produced by PMNs isolated from controls (n = 16) and periodontitis subjects (n = 19). a Non-stimulated ROS production was calculated and expressed as mean ± SD. b Stimulated ROS production produced by cPMNs when incubated with phorbol myristate acetate (+PMA) or F. nucleatum (+F.nuc). c Stimulated ROS production produced by oPMNs when incubated with phorbol myristate acetate (+PMA) or F. nucleatum (+F.nuc). Ratios were labeled as fold increase and were calculated for the stimulated ROS samples (MFI of the PMA-stimulated or F.n.-stimulated samples, divided by the MFI of the non-stimulated ROS samples). ^#Comparisons oPMN versus cPMN within subjects, Paired T-test, ^#P < 0.001. ^γComparisons to corresponding non-stimulated samples (panel a), Student’s T-test, all ^γP < 0.05

Fig. 4

Total protease activity measured from oral rinses originating from controls (n = 16) and periodontitis subjects (n = 16). a Total protease activity was measured as relative fluorescence per unit (RF U) and expressed as mean ± SD. *Comparisons periodontitis group versus control group, Student’s T-test, *P < 0.001. b Total protease activity was only predicted by oPMN counts in periodontitis patients (dashed line, r² = 0.3989, P = 0.0065) and not in the control group (straight line, r² = 0.0629, P = 0.3486)