Platelet and neutrophil responses to Porphyromonas gingivalis in human whole blood

Abstract

Porphyromonas gingivalis is a causative agent for periodontal disease. Binding of platelets to this gram-negative anaerobe can regulate host hemostatic (thrombus forming) and immune (neutrophil interacting) responses during bacterial infection. Additionally, in response to bacterial pathogens neutrophils can release their DNA, forming highly prothrombotic neutrophil extracellular traps (NETs), which then further enhance platelet responses. This study evaluates the role of P. gingivalis on platelet expression of CD62P, platelet-neutrophil interactions, and labeled neutrophil-associated DNA. Human whole blood was preincubated with varying P. gingivalis concentrations, with or without subsequent addition of adenosine diphosphate (ADP). Flow cytometry was employed to measure platelet expression of CD62P using PerCP-anti-CD61 and PE-anti-CD62P, platelet-neutrophil interactions using PerCP-anti-CD61 and FITC-anti-CD16, and the release of neutrophil DNA using FITC-anti-CD16 and Sytox Blue labeling. Preincubation with a high (6.25 × 10⁶ CFU/mL) level of P. gingivalis significantly increased platelet expression of CD62P in ADP treated and untreated whole blood. In addition, platelet-neutrophil interactions were significantly increased after ADP stimulation, following 5-22 min preincubation of blood with high P. gingivalis CFU. However, in the absence of added ADP, platelet-neutrophil interactions increased in a manner dependent on the preincubation time with P. gingivalis. Moreover, after ADP addition, 16 min preincubation of whole blood with P. gingivalis led to increased labeling of neutrophil-associated DNA. Taken together, the results suggest that the presence of P. gingivalis alters platelet and neutrophil responses to increase platelet activation, platelet interactions with neutrophils, and the level of neutrophil antimicrobial NETs.

Keywords: Porphyromonas gingivalis; flow cytometry; platelet activation; platelet-neutrophil interaction; thrombosis.

FIGURE 1

The impact of Porphyromonasgingivalis preincubation on CD62P expression in the absence and presence of ADP. Whole blood was preincubated with P. gingivalisW83, a wild type strain, at 37°C. Three preincubation times were used: 15, 30, and 60 min. Platelet activation was assessed via whole blood flow cytometry, with ADP (a) or without ADP (b) stimulation. The investigated whole blood preincubations include no P. gingivalis (■, ◻), heat-killed (HK) P. gingivalis (▲, △), low P. gingivalis CFU (◆, ◇), and high P. gingivalis CFU (●, ◯). Calculated CFU for P. gingivalis was 0 (No), 6.25 × 10⁶ (HK), 1.25 × 10⁶ (Low), and 6.25 × 10⁶ (High) CFU/ml. CD62P expression is presented as means ± SEM based on experiments performed in triplicate (n = 6). In the presence of added ADP, the P. gingivalis CFU significantly increased and preincubation time decreased the expression of CD62P in whole blood (Kruskal–Wallis, p < .05 and p < .001, respectively). In the absence of added ADP, the P. gingivalis CFU and preincubation time significantly increased CD62P expression in whole blood (Kruskal–Wallis, p < .001, for both)

FIGURE 2

Time-dependent effect of Porphyromonas gingivalis on whole blood platelet–neutrophil interactions. P. gingivalis W83 was preincubated in whole blood for four-time durations: 5, 10, 16, and 22 min. The calculated P. gingivalis CFU was 6.25 × 10⁶ CFU/ml at 37°C. Four whole blood treatments were assessed: no ADP treatment and no P. gingivalis preincubation (◻), no ADP treatment with P. gingivalis preincubation (■), with ADP treatment and no P. gingivalis preincubation (◻), and with ADP treatment with P. gingivalis preincubation (●). Levels of platelet–neutrophil interactions are presented as means ± SEM from experiments performed in triplicate (n = 12). With (a) or without (b) added ADP, significant P. gingivalis effects were measured on platelet–neutrophil interactions (linear mixed model (LMM) p <.0001). (b) In the absence of added ADP, there was a significant interaction effect between P. gingivalis and preincubation duration for heterotypic interactions between platelets and neutrophils (LMM, p < .0001)

FIGURE 3

Impact of Porphyromonas gingivalis on labeled neutrophil-associated DNA. P. gingivalisW83 was preincubated in whole blood for 16 or 35 min at 37°C. Following agonist treatment (PBS or ADP), the percentage of cells with accessible DNA associated with CD16⁺ neutrophils was evaluated via whole blood flow cytometry. Concentration of bacterial culture was 6.25 × 10⁶ CFU/ml. The percentage of CD16⁺Sytox Blue⁺ neutrophils for whole blood preincubated with P. gingivalis in the absence and presence of ADP are presented as means ± SEM from experiments performed in triplicate (n = 12). In the presence of added ADP, significant increases in labeled neutrophil-associated DNA were measured in whole blood preincubated with P. gingivalis (Dwass, Steel, Critchlow-Fligner, p < .05). *p < .05; n.s., not significant