Porphyromonas gingivalis-induced production of reactive oxygen species, tumor necrosis factor-α, interleukin-6, CXCL8 and CCL2 by neutrophils from localized aggressive periodontitis and healthy donors: modulating actions of red blood cells and resolvin E1

Abstract

Background and objectives: Porphyromonas gingivalis is regarded as a significant contributor in the pathogenesis of periodontitis and certain systemic diseases, including atherosclerosis. P. gingivalis occasionally translocates from periodontal pockets into the circulation, where it adheres to red blood cells (RBCs). This may protect the bacterium from contact with circulating phagocytes without affecting its viability.

Material and methods: In this in vitro study, we investigated whether human peripheral blood neutrophils from 10 subjects with localized aggressive periodontitis (LAgP) and 10 healthy controls release the proinflammatory cytokines interleukin (IL)-6, tumor necrosis factor α (TNF-α), the chemokine (C-X-C motif) ligand 8 (CXCL8; also known as IL-8) and chemokine (C-C motif) ligand 2 (CCL2; also known as monocyte chemotactic protein-1) and intracellular reactive oxygen species (ROS) in response to challenge with P. gingivalis. In addition, the impact of RBC interaction with P. gingivalis was investigated. The actions of resolvin E1 (RvE1), a known regulator of P. gingivalis induced neutrophil responses, on the cytokine and ROS responses elicited by P. gingivalis in cultures of neutrophils were investigated.

Results: Upon stimulation with P. gingivalis, neutrophils from subjects with LAgP and healthy controls released similar quantities of IL-6, TNF-α, CXCL8, CCL2 and intracellular ROS. The presence of RBCs amplified the release of IL-6, TNF-α and CCL2 statistically significant in both groups, but reduced the generation of ROS in the group of healthy controls, and showed a similar tendency in the group of subjects with LAgP. RvE1 had no impact on the production of intracellular ROS, TNF-α, IL-6, CXCL8 and CCL2 by neutrophils from either group, but tended to reduce the generation of ROS in subjects with LAgP in the absence of RBCs.

Conclusions: Our data support that binding to RBCs protects P. gingivalis from ROS and concomitantly enhances neutrophil release of proinflammatory cytokines providing a selective advantage for P. gingivalis growth.

Keywords: Porphyromonas gingivalis; aggressive periodontitis; cytokines; neutrophils; red blood cells; resolvin.

Fig. 1. A–D: P. gingivalis–induced neutrophil cytokine and chemokine responses

5×10⁵ neutrophils, from either healthy controls (n=10) or subjects with localized aggressive periodontitis (LAgP) (n=10), suspended in RPMI-medium containing 20% (v/v) AB serum, were stimulated with 1×10⁷ P. gingivalis for 1 hour at 37°C in presence (■) or absence (○) of 3.6×10⁷ autologous red blood cells (RBCs). Culture supernatants were thereafter assessed for content of A) TNF-α, B) IL-6, C) CCL2 and D) CXCL8. The presence of RBCs bound to neutrophils significantly increased release of TNFα, IL-6 and CCL2. There were no differences in the response of LAgP and healthy control neutrophils under any conditions. The lower limits of detection provided by the manufacturer were A) 0.16, B) 0.11, C) 1.9 and D) 0.13 as indicated with a dotted line. ***p<0.0001.

Fig. 2. P. gingivalis–induced generation of reactive oxygen species (ROS) in neutrophils

Dihydroethidium (DHE)-labeled 5×10⁵ neutrophils (PMN), from either healthy controls (n=6) (○) or subjects with localized aggressive periodontitis (LAgP) (n=6) (■) were suspended in media containing 20% (v/v) AB serum and stimulated with 1×10⁷ Porphyromonas gingivalis (P.g.) for 1 hour at 37°C. ROS is expressed as median fluorescence intensity of individual donors, in i) unstimulated, non-labelled neutrophils, ii) unstimulated DHE-labeled neutrophils and iii) DHE-labeled neutrophils stimulated with P.g. for 1 hour. Mean and 95% confidence intervals are shown (*p-values <0.05).

Fig. 3. Red blood cells (RBC) and resolvin E1 (RvE1) reduce P. gingivalis–induced generation of reactive oxygen species (ROS) in neutrophils

Dihydroethidium (DHE)-labeled 5×10⁵ neutrophils (PMN), from either healthy controls (n=6) (□) or subjects with localized aggressive periodontitis (LAgP) (n=6) (■) were suspended media containing 20% (v/v) AB serum and stimulated with 1×10⁷ Porphyromonas gingivalis (P.g.) for 1 hour at 37°C in absence or presence of 3.6×10⁷ autologous red blood cells (RBCs). All experiments were performed in triplicate. Half of the cultures received RvE1 (final concentration: 10nM). Each box represents the intracellular content of reactive oxygen species (ROS) of either healthy controls or patients with LAgP. Boxes indicate minimum and maximum with line at the mean. ROS are expressed as median fluorescence intensity (*p-values <0.05). RvE1 reduces intracellular ROS as do RBC; however, the additive effect is statistically significant only in subjects with LAgP (p<0.05), although there is a clear trend in controls as well (p=0.0652).

Fig. 4. A–D: Relative effect of resolvin E1 (RvE1) on P. gingivalis–induced neutrophil cytokine and chemokine responses

5×10⁵ neutrophils from either healthy controls (n=10) (○) or subjects with localized aggressive periodontitis (LAgP) (n=10) (■), suspended in RPMI-medium containing 20% (v/v) AB serum were stimulated with 1×10⁷ P. gingivalis for 1 hour at 37°C in presence of 3.6×10⁷ autologous red blood cells (RBCs). Half of the cultures received RvE1 (final concentration: 10nM). Culture supernatants were assessed for content of A) TNF-α, B) IL-6, C) CCL2 and D) CXCL8. The data points represents mean ± 95% confidence intervals of the cytokine levels observed in presence of RvE1 normalized to those observed in absence of RvE1 (=100%). No statistically significant differences (p-values >0.05) from 100% were observed.