Introduction

Periodontitis is a common oral disease which affectsglobally more than half of the adult population [1,2,3,4]. Periodontitis is initiated by subgingival plaque biofilms. According to Socransky's classification, subgingival plaque bacteria are classified into six major complexes : red, orange, green, yellow, purple, and Actinomyces [4]. It is known that these bacteria colonize in a certain order: yellow, green, and purple complexes, and Actinomyces are early colonizers; the red complex is a late colonizer; and the orange complex acts like a bridge between the early and late colonizers, called a bridging colonizer [4]. Therefore, the red complex bacteria are not detected in the absence of the orange complex bacteria or early colonizers. The red complex bacteria Porphyromonas gingivalis, Treponema denticola and Tannerella forsythia are highly associated with periodontitis and distinctly more pathogenic than the others [5].

In the gingival sulcus, two major defense mechanismsregulate plaque bacteria and protect from the invasion of pathogens into gingival tissues. One is antimicrobial peptides, such as human cathelicidin LL-37, human beta defensin-1, and human beta defensin-2, that directly kill bacteria. The other is phagocytosis by macrophages and neutrophils [6]. 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 [7,8,9,10].

Periodontal pathogens have diverse mechanisms to evadethe host defense machinery in the gingival sulcus. First, there is interleukin (IL)-8 degradation. IL-8 is upregulated in epithelial cells by invading bacteria and serve as a key chemokine for neutrophils [11]. It has been reported that P. gingivalis does not block the mRNA expression of IL-8 but degrades IL-8 protein with its proteases [12,13]. Another mechanism is resistance to LL-37. LL-37 is produced by epithelial cells and neutrophils as a part of the chemical barrier of the epithelia at the gingival sulcus. According to several studies, the late colonizers are more resistant to LL-37 than other bacteria [14,15,16,17]. A third mechanism is survival within macrophages. P. gingivalis can survive within macrophages through crosstalk among several immune receptors, such as TLR2, CXC-chemokine receptor 4 (CXCR4), complement receptor 3 (CR3) and complement 5a receptor (C5aR) [18,19]. A fourth mechanism is resistance to phagocytosis by neutrophils that also occurs by red complex bacteria [17,20]. Therefore, the enhancement of neutrophil function could be a good target to prevent periodontitis. It is known that various Toll-like receptor (TLR) ligands stimulate neutrophil function, including FcR-mediated phagocytosis [21]. 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.

Materials and Methods

Bacterial strains and growth conditions

All bacteria used in this study were obtained from theAmerican Type Culture Collection (Bethesda, MD, USA). P. gingivalis ATCC 49417 was cultured in brain heart infusion broth (BHI; BD Biosciences, Franklin Lakes, NJ, USA) with 5 μg/ml Hemin (Sigma, St Louis, MO, USA) and 10 μg/ml Vitamin K (Sigma) under anaerobic condition (5% H₂, 10% CO₂ and 85% N₂). Streptococcus sanguinis 804 (NCTC 10904) was cultured in BHI broth under aerobic condition. Bacteria in the log phase were harvested and washed twice with phosphate-buffered saline (PBS; Gibco, NY, USA). Bacterial concentration was determined by flow cytometry as previously described [21].

Preparation of fixed bacteria samples for the phagocytosis assay and measurement of reactive oxygen species (ROS) production

To prepare fixed bacterial samples, bacteria were collectedby centrifugation at 5,000 x g for 5 min at room temperature. The bacteria pellets were washed twice with PBS and then incubated with 3.7% paraformaldehyde for 20 min at room temperature. Finally, the samples were washed once and suspended in PBS. For phagocytosis assay, bacteria were stained with 5 μM 5[and-6]-carboxyfluorescein diacetate succinimidyl ester (CFSE; Molecular Probes, Eugene, OR, USA) at room temperature for 30 min. The bacteria were sonicated (5 cycles of 10 sec at the low power with sonicators, Fisher Scientific, Fair Lawn, USA) to dissociate aggregates and counted with a FACSCalibur (BD Bioscience, Franklin Lakes, NJ) to determine concentration.

Isolation of neutrophils

The use of human materials was approved by theInstitutional Review Board at the SNU School of Dentistry (S-D2009005). Leftover human peripheral blood samples were provided by the Department of Diagnostic Medicine, Seoul National University Hospital. To minimize inter-subject variation, a mixture of 10 blood samples (1 ml each) was diluted in 1 x Dulbecco's phosphate-buffered saline (DPBS; Gibco, NY, USA) at 1:1, layered on a 10 ml FICOLLPAQUE ™ plus (Amersham BioSchiencem, Uppsala, Sweden), and centrifuged at 720 x g for 40 min. After centrifugation, the pellet that contains the red blood cells (RBCs) and polymorphonuclear cells (PMNs) was obtained, and the PMNs were purified by lysing RBCs using hypotonic NaCl solutions. The purified cells were suspended in RPMI 1640 medium (HyClone, Logan, UT, USA) supplemented with 10% heat inactivated fetal bovine serum (FBS; HyClone), 100 U/ml penicillin, and 100 μg/ml streptomycin (Gibco) and incubated on ice.

Phagocytosis assay

To examine the phagocytosing ability of neutrophils,purified neutrophils (1 x 10⁵/assay) were incubated with CFSE-labeled bacteria at a cell to bacteria ratio of 1:25 in the presence of various concentrations of Pam₃CSK₄ (Invivogen, San Diego, CA, USA) for 60 min at 37℃. Incubated neutrophils were washed with DPBS. After quenching the fluorescence of the extracellular bacteria with 500 μl trypan blue (Gibco), the cells were analyzed with FACSCalibur. A negative control was prepared by adding the same amount of CFSE-labeled bacteria to neutrophils fixed with 3.7% formaldehyde. The phagocytosing ability of cells was solely analyzed for live cells that were gated based on the forward scatter and FL-3 fluorescence of trypan blue.

Enhanced chemiluminescence assay for ROS measurement

In 96-well plates, purified neutrophils (1x10⁵/assay/100 μl)were incubated with unlabeled fixed bacteria at a cell to bacteria ratio of 1:25 in the presence of 0.5 mM luminol and various concentrations of Pam₃CSK₄ at 37℃ for 100 min. All the cells and reagents were resuspended in RPMI medium supplemented with 10% heat inactivated FBS and 2 mM L-glutamine. As a negative and positive control, neutrophils were stimulated with medium alone and 0.1 μM phorbol 12-myristate 13-acetate (Sigma), respectively. After plating, the plate was immediately placed in a microplate luminometer (BMGLabtech, Ortenberg, Germany), and light emission in relative light units was recorded every 10 min during the 100 min incubation at 37℃.

Antibiotic protection assay

To examine the intracellular killing of live S. sanguinis andP. gingivalis within neutrophils, purified neutrophils (1 x 10⁵ cells/ 500 μl per FACS tube) were incubated with those bacteria at a cell to bacteria ratio of 1:25 in the presence of various concentrations of Pam₃CSK₄ at 37℃ for 30 min. Then, the extracellular bacteria were killed by incubating the neutrophils in antibiotic (gentamicin 30 μg/ml, amphotericin 15 μg/ml, and amoxicillin 5 μg/ml)-containing fresh medium for 30 min. The antibiotic-treated neutrophils were washed three times with cold DPBS and further cultured in fresh medium without antibiotics at 37℃ for 1, 2, or 4 h. The neutrophils were lysed with sterile distilled water for 10 min to liberate intracellular bacteria. The lysates were plated onto agar plates and incubated under the appropriate condition until colonies were observed by the naked eye.

Statistical analysis

All experiments were performed at least three times intriplicate. Statistical difference between the untreated control and the treated sample was analyzed with t-test. P values of < 0.05 were considered statistically significant.

Result

Pre-stimulation of neutrophils with Pam₃CSK₄ enhances the phagocytosis of S. sanguinis and concomitant ROS production.

To investigate the effect of TLR2 activation on thephagocytic function of neutrophils, neutrophils were pre-stimulated with Pam₃CSK₄, a synthetic TLR2 ligand, and incubated with CFSE-labeled S. sanguinis. Pre-stimulation with Pam₃CSK₄ increased not only the percentage of neutrophils that phagocytosed S. sanguinis but also the amount of S. sanguinis phagocytosed by each neutrophil (Fig. 1A Left and Right panels). Upon phagocytosis of the microbes, neutrophils produce ROS, one of the major weapons to kill phagocytosed microbes [23]. Therefore, the effect of pre-stimulation with Pam₃CSK₄ on ROS production was also evaluated. Non-stimulated neutrophils produced ROS upon exposure to S. sanguinis as expected. Pre-stimulation with Pam₃CSK₄ increased the ROS production by neutrophils more than twofold (Fig. 1B). These results suggest that Pam₃CSK₄ increases the phagocytosing ability and ROS production.

Simultaneous stimulation with Pam₃CSK₄ also enhances phagocytosing activity and concomitant ROS production in neutrophils.

Next, the effect of simultaneous stimulation with Pam₃CSK₄ on the phagocytosis of S. sanguinis was examined.Simultaneous stimulation presented a similar result with that of pre-stimulation, presenting the maximal effect by 1 μg/ml Pam₃CSK₄ (Fig. 2A Left panel). Simultaneous stimulation with Pam₃CSK₄ on the phagocytosis of P. gingivalis was also examined. The phagocytosis of P. gingivalis was increased by Pam₃CSK₄, also presenting the maximal effect by 1 μ g/ml. However, the enhancing effect was greater for S. sanguinis than for P. gingivalis (Fig. 2A Right panel). Stimulation with Pam₃CSK₄ induced ROS production by itself and increased the bacteria-induced ROS production for both S. sanguinis and P. gingivalis in a dose-dependent manner (Fig. 2B Left panel). Different from the effect on phagocytosis, the maximal effect on ROS production was observed by 10 μg/ml. Interestingly, P. gingivalis attenuated the Pam₃CSK₄ (1 and 10 μg/ml)- induced ROS production (Fig. 2B Right panel).

Neutrophils efficiently kill phagocytosed bacteria even in the absence of a TLR2 stimulator

The increased ROS production induced by Pam₃CSK₄ mayassist intracellular killing of phagocytosed bacteria in neutrophils. To determine the effect of Pam₃CSK₄ stimulation on the intracellular killing of bacteria, antibiotic protection assay was performed. Right after phagocytosis and killing of extracellular bacteria (0 h), the enhancement of phagocytosis by Pam₃CSK₄ was evident for both bacterial species. After h of further culture, all P. gingivalis and 80% of S. sanguinis were killed even in the absence of Pam₃CSK₄ stimulation. The presence of 1 μg/ml Pam₃CSK₄ induced a threefold increase in the phagocytosis of S. sanguinis, 89% of which was eliminated in h (Fig. 3).

Discussion

Phagocytosis is the primary defense mechanism againstbacteria. It involves several events including the recognition of pathogens through various cell surface receptors, receptor-mediated uptake, fusion of the phagosomes with lysosomes, generation of ROS, and killing of pathogens in the phagolysosomes [24]. A number of studies have shown that TLRs on the cell surface of neutrophils recognize various microbial structures such as lipopolysaccharides (TLR4), lipoproteins (TLR2), and bacterial DNA (TLR9), resulting in the production of cytokines and chemokines or the inhibition of neutrophil apoptosis [25,26,27]. The present study showed that TLR2 activation enhances the phagocytosing ability and ROS production in neutrophils.

Stimulation of neutrophils with a TLR2 ligand increasedphagocytosis of both S. sanguinis and P. gingivalis. Compared to S. sanguinis, P. gingivalis was resistant to phagocytosis by neutrophils even in the presence of Pam₃CSK₄. It is similar to the fact that T. denticola was resistant to phagocytosis by neutrophils compared to S. sanguinis, even after opsonization with specific antibodies [28]. Although the effect of Pam₃CSK₄ stimulation was not compared with the effect of opsonizing antibodies in parallel, 1 μg/ml Pam₃CSK₄ increased the phagocytosis of S. sanguinis about fourfold, which was comparable to the effect of specific opsonizing antibodies shown in the previous study [28].

Induction of ROS production by TLR2 activation inneutrophils has been already reported [25,29]. Therefore, it is not surprising that neutrophils exposed to bacteria produced increased amounts of ROS in the presence of Pam₃CSK₄. The increase in ROS production may be necessary to handle the increased amounts of phagocytosed bacteria. One interesting finding is attenuation of the Pam₃CSK₄-induced ROS production by P. gingivalis, which suggests the potential survival of P. gingivalis within neutrophils. However, the small number of phagocytosed P. gingivalis did not survive within neutrophils even in the absence of Pam₃CSK₄.

In summary, TLR2 activation increases phagocytosis ofbacteria and ROS production by neutrophils. Although P. gingivalis is poorly phagocytosed even by the TLR2-activated neutrophils, TLR2 activation of neutrophils may help to reduce the colonization of P. gingivalis by efficiently eliminating S. sanguinis, an early colonizer, in subgingival biofilm.