Introduction

Periodontitis is the most prevalent chronic inflammatory human disease and is considered as a major problem in the global burden of oral diseases [1]. It is characterized by the breakdown of tooth-supporting tissues producing teeth loss. Porphyromonas gingivalis (P. gingivalis) is asaccharolytic gram-negative anaerobic rod that is a major etiological agent in the initiation and progression of chronic periodontal disease [2]. P. gingivalis is also implicated in certain systemic conditions such as atherosclerosis, aspiration pneumonia, and rheumatoid arthritis [3,4].

Before initiating an infection, bacterial pathogens come into contact with the human skin, respiratory or gastro-intestinal system and interact with different host cells such as epithelial cell, PMN cell, and macrophage. As sentinels of infection, macrophages are one of the first cell types to encounter pathogens and the frontline of defense when combating bacterial infection [5]. During the host cell-pathogen interaction, macrophages can die in many ways such as apoptosis, necrosis, pyroptosis and autophagy, and sometimes they are intertwined involving with different and complex underlying mechanisms [6]. Cell death appears to play a significant role in macrophage and triggers an inflammatory response. Programmed cell death (PCD) different from necrosis is an active cell death that is mediated by a series of gene expression events [7,8]. PCD can be further classified into apoptosis, autophagy, programmed necrosis (necroptosis), and pyroptosis [9]. Although PCD is involved in many inflammatory diseases, its correlation with periodontitis is unclear. The purpose of the present study aimed to evaluate macrophage cell death in response to P. gingivalis infection.

Materials and Methods

Cell treatment

The human monocyte cell line, THP-1 cells were maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum (FBS) and were cultured at 37°C in 5% CO₂ incubator. To induce cell death, THP-1 cells were infected with P. gingivalis for 24 h. To inhibit autophagic cell death, THP-1 cells were pretreated with 5 mM of 3-methyladenine (3-MA; sigma, MO, USA) for 30 min and were infected with P. gingivalis for 24 hr. For confocal microscopy, THP-1 cells were differentiated into macrophage-like cells with 50 ng/ml of Phorbol 12-mystristate 13-acetate (PMA; sigma, MO, USA).

Bacterial culture

P. gingivalis (strain 381) were grown in gifu anaerobic medium (GAM; Nissui, Japan) broth, which contained hemin (5mg/mL) and 3-phytyl-menadione (vitamin K, 0.5mg/ml) at 37°C in an anaerobic chamber under an atmosphere containing 90% N₂, 5% H₂, and 5% CO₂. An optical density (OD) of 1.0 (650 nm) was determined to correlate to 1x10^⁹ colony forming units/mL. To prepare the bacteria for infection, an overnight culture was diluted to an OD 650 nm of 1.0 in GAM broth. The bacteria were washed, resuspended in RPMI media, and were used to infect THP-1 cells at a multiplicity of infection (MOI) of 50 and 100.

Autophagy determined by flow cytometry

Cells were harvested and autophagy flux was determined using a Cyto-ID autophagy detection kit (Enzo Life Sciences, NY, USA) according to the manufacturer’s instructions. Briefly, the cells were collected by centrifuge and stained in a solution with Cyto-ID Detection Reagent covered from light for 30 min at 37°C. Cells were then analyzed by FACS verse (BD bioscience, NJ, USA). Data were presented as the mean fluorescence intensity of Cyto-ID.

Quantifying autophagy with AO staining

THP-1 cells were seeded in 8-well-chamber slides. Cells were pre-treated with or without 3-MA for 30 min followed by infection with P. gingivalis (MOI 50, 100) for 24 hr and stained with 100 μg/ml of AO in serum-free medium at 37 °C for 15 min. Then, cells were washed with PBS and fluorescent micrographs were obtained using confocal laser-scanning microscope (LSM 700, Carl Zeiss, Germany).

Apoptosis analyses with flow cytometry

Apoptosis was analyzed by flow cytometry using annexin-V and propium iodine (PI) double staining according to the manufacturer’s protocol (Invitrogen, CA, USA). Briefly, THP-1 cells were infected with P. gingivalis for 24 h. THP-1 cells were collected and were washed with cold PBS and annexinbinding buffer. Then, annexin V and PI working solution were added to cells. After incubated at room temperature for 15 min, the stained cells were analyzed with flow cytometry (FACS verse, BD, NJ, USA).

Cell death assay

Cell death was measured with a lactate dehydrogenase (LDH) Cytotoxicity assay kit (CytoTox 96 non radio active cytotoxicity assay; Promega, WI, USA). The percent cytotoxicity was calculated by the following formula: 100 X [(experimental LDH release - spontaneous LDH release)/(maximal LDH release - spontaneous LDH release)]. To determine the maximal LDH release, cells were treated with 1% Triton X-100

Cytokine analysis

The amounts of TNF-α and IL-1β released into the culture media after P. gingivalis infection were analyzed by using an ELISA kit purchased from Biolegend (San Diego, CA, USA). Cytokine level was measured by the manufacturer’s instruction. The plates were read in an ELISA reader (Tecan, Männedorf, Switzerland) at 450/570nm.

Statistics

Statistically significant differences between samples were analyzed with the SPSS 13.0 statistical software program (SPSS Inc., IL, USA). The data were shown as the mean ± SD. p value of < 0.05 was considered statically significant. * p < 0.05 vs control, # p<0.05 vs P. g MOI 100. *p < 0.05, **p < 0.001, and ***p < 0.005.

Results

1.P. gingivalis induced autophagosome formation in THP-1 cells.

To determine if P. gingivalis induce autophagy in THP-1 cells, autophagosome formation was examined by cyto-ID staining and was quantitated by FACS. THP-1 cells infected with P. gingivalis showed significantly increased mean fluorescence intensity suggesting autophagosome formation was induced by P. gingivalis infection. Also, 3-MA, which inhibit autophagy, decreased the autophagosome formation induced by P. gingivalis infection (Fig. 1).

To confirm autophagosome formation by P. gingivalis, P. gingivalis-infected THP-1 cells were stained with acridine orange. As shown in Fig. 2A, control cells primarily displayed green fluorescence with minimal red fluorescence, indicating a lack of acidic vesicular organelles (AVOs). In P. gingivalisinfected THP-1 cells, Cells showed an increased red fluorescent AVOs in both MOI 50 and 100 (Fig 2). The numbers of P. gingivalis-induced AVOs forming cells were decreased by 3-MA treatment (Fig 2). Moreover, in bright field apoptotic blebs were significantly increased in P. gingivalis-infected THP-1 cells treated with 3-MA. Taken together, P. gingivalis infection induced autophagy in THP-1 cells and inhibiting autophagy seemed to drive cell death into apoptosis.

2.Inhibition of autophagy by 3-MA increased P. gingivalis-induced apoptosis in THP-1 cells.

To test the effect of inhibition of autophagy by 3-MA, cell apoptosis was detected by annexin-V and PI staining. As shown in Fig. 3, the infection of THP-1 cells by P. gingivalis showed similar apoptosis rate compared to the control. In THP-1 cells treated with 3-MA and infected with P. gingivalis, significantly higher proportion of cells were positive for early and late apoptosis (Fig 3). These results indicate that P. gingivalis does not directly induce apoptosis, but apoptosis is significantly induced by inhibiting autophagy in THP-1 cells infected with P. gingivalis.

3.Effect of inhibiting autophagy by 3-MA on P. gingivalis-induced cell death and cytokine production.

To determine total cell death, we analyzed LDH release in the cell supernatant. Compared with control, P. gingivalis infection significantly increased cell cytotoxicity in a MOI-dependent manner. When 3-MA was pretreated, LDH release was significantly increased compared to P. gingivalis infection (Fig. 4A).

Finally, we tested the relation between cell death and inflammatory response in P. gingivalis-infected THP-1 cells. P. gingivalis infection significantly induced IL-1β and TNF-α production in THP-1 cells and inhibiting autophagy by 3-MA slightly increased IL-1β production while TNF-α production was significantly increased compared to P. gingivalis infection (Fig 4B). Taken together, P. gingivalis can induce inflammatory cytokine production and cell death in THP-1 cells and autophagy inhibition significantly increased inflammatory cytokine production and cell death.

Discussion

The maintenance of a balance between the host response and microorganisms is important to preserve health. Bacteriatriggered cell death is an intrinsic immune defense mechanism in response to microbial infection. Understanding the cell death by bacterial pathogens will provide insight into new therapeutic approaches for the control of infection [12]. Programmed cell death (PCD) different from necrosis is an active cell death [7,8]. and is classified into apoptosis, autophagy, necroptosis, and pyroptosis [9]. Until now, the relationship between PCD and inflammatory diseases, especially periodontitis is not specified. Thus, in this stidy, we examine macrophage cell death in response to P. gingivalis infection, because macrophage plays key roles in inflammation and immune responses.

We previously reported that P. gingivalis induces autophagy in PMA-differentiated THP-1-derived macrophages and in turn, macrophages eliminate P. gingivalis through an autophagic response, which can lead to the restriction of an excessive inflammatory response by downregulating interleukin-1β production [1]. Autophagy is a physiologically essential cellular process for the degradation of long-lived proteins and damaged organelles in lysosomes. Autophagy also modulates immunity through the direct elimination of microorganisms, the control of inflammation and adaptive immunity, and the secretion of immune mediators [13]. Many studies demonstrated a function of autophagy in vitro in defense against invading pathogens including Streptococcus, Shigella flexneri, Mycobacterium tuberculosis, Salmonella typhimurium, and Toxoplasma gondii [14,15]. Induction of autophagy inhibited M. tuberculosis survival in infected macrophages [16]. In the study, we used CytoID to quantify autophagosome formation in THP-1 cells infected with P. gingivalis. Cyto-ID reagent which specifically fluorescense in autophagic vesicles, detects autophagic flux in lysosomally inhibited live cells using a novel dye that selectively labels accumulated autophagic vacuoles [17]. P. gingivalis induced autophagy in a dose-dependent manner (Fig 1). To visualize autophagosome formation, THP-1 cells were stained with acridine orange. Acridine orange is a lysotropic dye that accumulates in acidic organelles in a pH-dependent manner. At neutral pH, acridine orange is a hydrophobic green fluorescent molecule. However, within acidic vesicles, acridine orange becomes protonated and trapped within the organelle and forms aggregates that emit bright red fluorescence [18]. Autophagosome formation was significantly increased in P. gingivalis infected THP-1 cells. Treatment of 3-MA, an autophagy inhibitor, inhibited autophagy formation when detected both by CytoID and by acridine orange staining (Fig. 1 and Fig. 2). Taken together, THP-1 cells induced autophagy to resist against P. gingivalis infection.

When autophagosome formation was observed under confocal microscopy, THP-1 cells treated with 3-MA and P. gingivalis showed bleb formation (Fig. 2). To examine the effect of autophagy inhibition on apoptosis, cells were stained with annexin-V and propium iodine. The inhibition of autophagy by 3-MA significantly enhanced P. gingivalis-induced cell apoptosis, although P. gingivalis infection did not induce any apoptosis, P. gingivalis has been reported to be anti-apoptotic for epithelial [19] while heat killed P. gingivalis is proapoptotic. P. gingivalis cell wall components such as gingipain proteases can also directly induce apoptosis [20]. Taken together, although P. gingivalis has the ability to induce apoptosis in THP-1 cells, autophagic host response may be the first line defense mechanism to resist apoptotic cell death. When autophagy is inhibited, P. gingivalis may induce host cell apoptosis. Apoptosis of fibroblasts has been suggest to be associated with inflammation in human gingiva [21]. In vitro studies show that P. gingivalis can modulate apoptosis in the following cell types: fibroblasts, endothelial cells and lymphocytes and apoptosis has been proposed as a mechanism to explain the extensive tissue destruction in chronic periodontitis lesions [22].

To determine total cell death, LDH assay was carried out in THP-1 cells infected with P. gingivalis. P. gingivalis-infected THP-1 cells with 3MA pretreatment showed significantly increased LDH release compared to P. gingivalis-infected THP-1 cells. Inhibition of autophagy by 3-MA should have increased total cell death through the increase of cellular apoptosis (Fig 4A). Cell death plays an important role in the regulation of inflammation. The maintenance of tissue homeostasis necessitates both the recognition and removal of invading microbial pathogens as well as the clearance of dying cells [23].

To determine inflammatory response, cytokines were determined in THP-1 cells infected with P. gingivalis. P. gingivalis infection significantly induced both IL-1β and TNF- α production. When 3-MA was pretreated to THP-1 cells, P. gingivalis infection increased IL-1β and TNF-α production. Excessive IL-1β and TNF-α production can lead to tissue damage and inflammatory disease including periodontitis [24]. Thus, autophagic host response may be the first line of defense mechanism for macrophage cells to resist bacterial infection and minimize host inflammatory response.

In summary, THP-1 cells infected with P. gingivalis induced autophagy. Inhibition of autophagy induced apoptotic cell death and increased IL-1β and TNF-α production in THP-1 cells infected with P. gingivalis. Cellular analysis for autophagy and apoptosis may provide a new understanding on the interaction between host macrophages and P. gingivalis and may provide a new strategy for prophylactic and therapeutic treatment of P. gingivalis infection.