The effect of Toll-like receptor 2 activation on the non-opsonic phagocytosis of oral bacteria and concomitant ROS production by human neutrophils

Abstract

Background

Periodontitis is a chronic inflammatory disease that results in major damage to periodontal tissues, especially the gingiva and underlying alveolar bones, leading to eventual tooth loss. Chronic/cyclic neutropenia, leukocyte adhesion deficiency syndrome, Papillon-Lefèvre syndrome and Chédiak-Higashi syndrome are associated with severe periodontitis, suggesting the importance of neutrophils in the maintenance of periodontal health. It is known that various Toll-like receptor (TLR) ligands stimulate neutrophil function, including FcR-mediated phagocytosis. The aim of this study was to investigate whether the stimulation of TLR2 can enhance the non-opsonic phagocytosis of oral bacteria and subsequent killing in neutrophils.

Methods

As the source of human neutrophils, peripheral blood polymorphonuclear cells were isolated by the ficoll-hypaque method and subsequent lysis of red blood cells using hypotonic NaCl solution. Streptococcus sanguinis 804 (NCTC 10904) and Porphyromonas gingivalis ATCC 49417 were used after fixation with 3.7 % paraformaldehyde, except for the intracellular survival assay where live bacteria were used. Bacteria were stained with 5[and-6]-carboxyfluorescein diacetate succinimidyl ester (CFSE) for fluorescence application. Neutrophils were incubated with S. sanguinis or P. gingivalis in the presence of various concentrations of Pam3CSK4 and subjected to the measurement of phagocytosis and reactive oxygen species (ROS) by flow cytometry and enhanced chemiluminescence, respectively. To examine the intracellular killing of live S. sanguinis and P. gingivalis within neutrophils, antibiotic protection assay was performed.

Results

Pam3CSK4 significantly increased the phagocytosis of both bacterial species in a dose-dependent manner. Pam3CSK4 alone induced ROS production from neutrophils and also increased concomitant ROS production induced by bacteria. Interestingly, incubation with P. gingivalis and Pam3CSK4 decreased the amounts of ROS compared with those produced by Pam3CSK4 alone, indicating a possibility that P. gingivalis survives within neutrophils. However, neutrophils efficiently both phagocytosed species even in the absence of Pam3CSK4. Stimulation with Pam3CSK4 increased the adhesion of P. gingivalis, but not that of S. sanguinis, to neutrophils, suggesting that TLR2-assisted phagocytosis in neutrophils works through different mechanisms depending on the target bacterial species.