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 × 10 6 CFU/mL) level of P. gingivalis significantly increased platelet expression of CD62P in ADP treated and untreated whole blood. In addition, plateletneutrophil 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.

Neutrophil Extracellular DNA Traps in Response to Infection or Inflammation, and the Roles of Platelet Interactions

“…Preincubating whole blood with live Porphyromonas gingivalis results in increased platelet–neutrophil interactions in the presence or absence of added ADP [ 172 ]. However, the highest interaction levels are observed in the presence of ADP stimulation, even for the shortest P. gingivalis preincubation time of 5 min.…”

Local and Systemic Effects of Porphyromonas gingivalis Infection

“…However, platelet aggregation depends on the ratio of platelet/bacteria, consistent with the possibility of either a threshold phenomenon or multiple competing platelet interactions. In whole blood, platelet expression of CD62P, an adhesion molecule expressed on surfaces of activated platelets, increases following preincubation of high P. gingivalis colony-forming units (CFU) with or without subsequent ADP stimulation [ 263 ]. Conversely, there is a trend towards higher CD62P expression even in response to low P. gingivalis CFU, particularly as preincubation time is extended.…”

“…Platelet-neutrophil interactions are believed to be mediated by an interaction between platelet P-selectin and neutrophil P-selectin glycoprotein ligand-1 (PSGL-1) [ 269 ]. Such interaction is enhanced following ADP-mediated platelet activation [ 263 ]. Platelet-neutrophil interactions are also enhanced in the presence of P. gingivalis in a preincubation time-dependent manner.…”

“…Moreover, bacterial exposure to whole blood can trigger the neutrophil release of nuclear DNA, also known as neutrophil extracellular traps (NETs). Such release of NETs in response to P. gingivalis is known to be at least in part dependent on an interaction between activated platelets and neutrophils [ 263 ]. This implies that the interaction of P. gingivalis with the various blood cells does not only potentially alter their cell-specific functions in response to this pathogen but can also impact their physiologic cell-cell interactions.…”

Porphyromonas gingivalis regulates atherosclerosis through an immune pathway

“…Platelets activated by P. gingivalis express P-selectin on their surface, which binds to the receptor P-selectin glycoprotein-1 on leukocytes, forming platelet-leukocyte aggregates that not only converge a variety of leukocytes (neutrophils, monocytes) to the site of inflammation and clear bacteria but also induce a coagulation-inflammatory series of responses ( 224 ). Further studies have indicated that P. gingivalis induces increased platelet-neutrophil aggregate formation, enhances platelet-neutrophil interactions, and induces the release of NETs, which in turn promotes late thrombosis in AS ( 225 , 226 ). However, no studies on the effects of P. gingivalis on other types of leukocyte-platelet interactions have been seen.…”