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. Author manuscript; available in PMC: 2016 May 1.
Published in final edited form as: Microbes Infect. 2015 Mar 27;17(5):369–377. doi: 10.1016/j.micinf.2015.03.010

Porphyromonas gingivalis attenuates ATP-mediated inflammasome activation and HMGB1 release through expression of a nucleoside-diphosphate kinase

Larry Johnson a,b,1, Kalina R Atanasova c,d,1, Phuong Q Bui a,b, Jungnam Lee c,d, Shu-Chen Hung a,b, Özlem Yilmaz c,d,*, David M Ojcius a,b,*
PMCID: PMC4426005  NIHMSID: NIHMS673985  PMID: 25828169

Abstract

Many intracellular pathogens evade the innate immune response in order to survive and proliferate within infected cells. We show that Porphyromonas gingivalis, an intracellular opportunistic pathogen, uses a nucleoside-diphosphate kinase (NDK) homolog to inhibit innate immune responses due to stimulation by extracellular ATP, which acts as a danger signal that binds to P2X7 receptors and induces activation of an inflammasome and caspase-1. Thus, infection of gingival epithelial cells (GECs) with wild-type P. gingivalis results in inhibition of ATP-induced caspase-1 activation. However, ndk-deficient P. gingivalis is less effective than wild-type P. gingivalis in reducing ATP-mediated caspase-1 activation and secretion of the proinflammatory cytokine, IL-1β, from infected GECs. Furthermore, P. gingivalis NDK modulates release of high-mobility group protein B1 (HMGB1), a pro-inflammatory danger signal, which remains associated with chromatin in healthy cells. Unexpectedly, infection with either wild-type or ndk-deficient P. gingivalis causes release of HMGB1 from the nucleus to the cytosol. But HMGB1 is released to the extracellular space when uninfected GECs are further stimulated with ATP, and there is more HMGB1 released from the cells when ATP-treated cells are infected with ndk-deficient mutant than wild-type P. gingivalis. Our results reveal that NDK plays a significant role in inhibiting P2X7-dependent inflammasome activation and HMGB1 release from infected GECs.

Keywords: innate immunity, purinergic receptor, Porphyromonas gingivalis, interleukins, inflammation

1. Introduction

Porphyromonas gingivalis is a Gram-negative opportunistic pathogen that colonizes the human oral epithelial tissues and plays a major role in the etiology of severe and chronic forms of periodontal disease [1]. The organism has been associated recently with a variety of other chronic and inflammatory conditions including orodigestive cancer, rheumatoid arthritis, liver disease, and diabetes [2, 3]. Gingival epithelial cells (GECs) are among the first cells in the oral cavity that are encountered and invaded by P. gingivalis, and they play an important barrier role while also sensing and responding to microbial colonizers [1, 2, 4].

Immune recognition of cellular damage and invading pathogens is carried out through pattern recognition receptors such as Toll-like receptors (TLRs) and purinergic P2X receptors situated on the plasma membrane, and nucleotide-binding oligomerization domain (NOD)-like receptors (NLRs) in the cytoplasm [5-7]. Ligation of the purinergic receptor, P2X7, typically leads to assembly of a complex of proteins, called the inflammasome, which results in activation of caspase-1 and subsequent processing and secretion of the pro-inflammatory cytokines, IL-1β and IL-18. The most widely studied inflammasome contains the NLR member, NLRP3, the adaptor protein, apoptosis-associated speck-like protein containing a CARD (ASC), and the protease, caspase-1 [7, 8]. The NLRP3 inflammasome can be activated by multiple stimuli, including the “danger signal,” extracellular ATP, which is released by damaged, stressed, or infected cells [7, 9]. GECs express functional P2X7, whose ligation by ATP leads to production of reactive oxygen species (ROS) and assembly and activation of the NLRP3 inflammasome [7, 10-13].

Another danger signal molecule, high mobility group box 1 protein (HMGB1), can be secreted after inflammasome activation [14, 15]. In healthy cells, HMGB1 is associated with chromatin and enhances the binding of regulatory proteins to a variety of genes [14]. Outside the cell, HMGB1 has potent and diverse immunomodulatory functions after binding to its receptor, RAGE, TLR2 or TLR4 [16]. Extracellular HMGB1 has been linked with different inflammatory conditions, from endotoxemia, ischemia and liver injury to squamous cell carcinoma [17-19]. Increased HMGB1 release and RAGE expression is also described to possibly play a role in diabetes-associated periodontitis [20]. Recently, HMGB1 release has been shown to be regulated by inflammasomes [21-23]. However, there is still little known on how different microorganisms or their components regulate the expression and secretion of HMGB1.

As a successful facultative intracellular organism, P. gingivalis has evolved an array of virulence factors and associated mechanisms that allow the organism's survival and spread in the gingival epithelium, and evasion of recognition by the host immune system [1]. Prominent virulence factors include gingipains and a secreted homologue of nucleoside-diphosphate kinases (NDKs) [24-27].

NDKs catalyze the transfer of orthophosphates from nucleoside triphosphates, such as ATP, to nucleoside diphosphates, thus enabling it to control nucleotide pools inside the cells [24]. Additionally, secreted NDKs can be used by intracellular persistent bacteria, such as Mycobacterium tuberculosis and P. gingivalis, to mask their presence from the host immune system by scavenging extracellular ATP [9, 28-30]. Unlike many other infectious bacteria, P. gingivalis can stimulate the production of biologically inactive pro-IL-1β in infected GECs, but does not induce significant release of the secreted active form of IL-1β [12]. Active IL-1β secretion in P. gingivalis-infected GECs only occurs after stimulation with extracellular ATP [12]. _ENREF _10 Our previous studies have shown that P. gingivalis NDK secretion is important for maximal inhibition of ATP-triggered P2X7-mediated apoptosis in infected GECs as well as ATP-induced generation of cellular ROS [13, 31, 32]. These studies imply a potentially critical role for P. gingivalis NDK in the modulation of the inflammasome-mediated host response and IL-1β secretion. The current knowledge on the mechanisms of inflammasome activation has been predominantly derived from cells of the myeloid lineage [33]. Epithelial cells in general, including GECs, have attracted less attention. Additionally the mechanisms of regulation of other inflammasome-dependent danger-signals, such as HMGB1, are widely unexplored. Therefore, we aimed to examine the potential role of the effector molecule NDK on ATP-mediated inflammasome activation and pro-inflammatory cytokine release. We hypothesize that the release of NDK inhibits caspase-1 activation and secretion of pro-inflammatory cytokines and HMGB1 in P. gingivalis-infected GECs.

2. Materials and Methods

2.1. Primary and Immortalized GECs

Healthy gingival tissue was obtained by oral surgery to produce primary GECs cultures as previously described [34]. No subject recruitment per se was done. Adult patients were selected at random and anonymously from those presenting at the University of Florida Dental Clinics for tooth crown lengthening or impacted third molar extraction. Gingival tissue that would otherwise be discarded was collected after informed written consent by the patient. This study is approved by the Institutional Review Board under the University of Florida. Cells were cultured as monolayers in serum-free keratinocyte growth medium (Lonza) at 37°C in a 5% CO2 incubator. The human immortalized GEC (HIGK) cell line was obtained as previously described [35]. Cells were cultured in defined keratinocytes serum-free media (K-SFM, Life Technologies) at 37 °C in a 5% CO2 incubator.

2.2. Porphyromonas gingivalis Culture

Porphyromonas gingivalis ATCC 33277 and its isogenic ndk-deficient mutant were cultured anaerobically for 24 h at 37 °C in trypticase soy broth supplemented with yeast extract (1 mg/ml), hemin (5 mg/ml), and menadione (1 mg/ml). Erythromycin (10 mg/ml) was added to the media as a selective agent for the growth of the ndk-deficient mutant strain, which was previously described (29). Bacteria were grown for 24 h and collected by centrifugation at 6000 × g for 10 min at 4 °C. The bacteria were then washed twice and resuspended with Dulbecco's phosphate-buffered saline (PBS) (Life Technologies). Quantification of bacteria was determined using a Klett-Summerson photoelectric colorimeter.

2.3. Infection of Primary and Immortalized GECs with P. gingivalis and Treatment with ATP and Inhibitors

Primary GECs and HIGK cells were seeded onto 6-well plates and upon reaching 75-80% confluence the cells were infected with either wild-type or ndk-deficient P. gingivalis at a multiplicity of infection (MOI) of 100 which has previously been shown to be biologically relevant [27, 36, 37]. Cells used for HMGB1 Western blot assays were collected after 9 hours of infection. For cells treated with the caspase-1 inhibitor, YVAD (z-YVAD-fmk, R&D Systems) [10, 38, 39], YVAD was added to the media at a final concentration of 50μM and incubated at 37°C in a 5% CO2 incubator. The cells were treated with 3 mM ATP (Sigma) 30 minutes after addition of YVAD, or 6 hour after infection and incubated for additional 3 h. Cell-culture fluids were collected and centrifuged at 100 × g to remove cell debris. Protease inhibitors (PhosStop, Roche) were added to all samples to prevent protein degradation.

2.4. Measurement of IL-1β Secretion by ELISA

Samples from at least three separate experiments were collected for each infection and treatment condition. Samples were cleared of cellular debris by centrifugation at 800 × g for 5 min and cell-free supernatants were used for IL-1β ELISA assay. IL-1β secretion levels were measured using Human IL-1β ELISA kit II (BD Biosciences). ELISAs were performed as per manufacturer's instructions. Briefly, samples, controls, and standards were diluted with assay diluent, loaded onto a 96-well plate, and incubated for 2 h at room temperature. Wells were washed and incubated with the detection antibody for 1 h at room temperature. After washing, substrate was added to all wells. The reactions were stopped with stop solution and optical densities were read at an absorbance of 450 nm using a Synergy MX plate reader (BioTek Instruments). P-values were calculated using two-tailed Student's t-test.

2.5. Western Blot

Proteins from cell-free culture fluids were concentrated using trichloroacetic acid (TCA) [40]. TCA at 100% was added to supernatants for a final concentration of 20% TCA. Samples were vortexed and incubated on ice. After incubation, samples were centrifuged and supernatants were discarded. The pellets were washed twice with cold acetone and allowed to air-dry. Pellets were resuspended with sample buffer (Thermo Scientific) and boiled. Protein concentration in samples was measured using the BioRad protein assay. Samples were loaded at equal final protein concentrations and run on SDS-PAGE gels, followed by transfer onto PVDF membranes. Membranes were blocked with 5% BSA for one hour at RT, and then incubated with primary antibodies against caspase-1 (Cell Signaling) or HMGB1 (Cell Signaling). After one hour, blots were washed and incubated with secondary anti-rabbit HRP (Millipore) for another hour. Finally, membranes were washed and exposed to Luminata Forte (Millipore) substrate. Images were acquired using ChemiDoc XRS+ system (Bio-Rad) and analyzed using NIH-ImageJ.

2.6. Immunofluorescence Examination of Intracellular IL-1β and HMGB1

Primary GECs were cultured in 4-well culture plates (Nunc) containing round glass inserts. Cells were infected at a confluence of 75-80% with either wild-type P. gingivalis or the ndk-deficient mutant strain at an MOI of 100. Six hours later, cells were treated with ATP at a final concentration of 3 mM for an additional 3 h. Cells were washed three times with PBS and fixed with 4% paraformaldehyde for 15 min. Cells were permeabilized using 0.01% Triton X-100 solution in PBS and incubated with the primary antibodies. IL-1β was detected using an FITC-conjugated mouse monoclonal antibody (R&D Systems). HMGB1 was detected using a rabbit polyclonal anti-human HMGB1 antibody (Cell Signaling). P. gingivalis infection was detected using anti-P. gingivalis rabbit polyclonal antibody, coupled with a secondary goat-anti-rabbit polyclonal antibody (Invitrogen), and all inserts were mounted onto glass slides using VectaShield mounting medium (Fisher) containing DAPI stain. The intensity of IL-1β and HMGB1 staining was measured using NIH-ImageJ, and measurements of at least 25 cells, originating from at least three separate replicate experiments, were averaged.

2.7. Statistical Analysis

Data are reported as mean ±standard deviation (SD). Statistical significance was calculated by two-tailed Student's t-test and was considered significant at P < 0.05.

3. Results

3.1. NDK Inhibits Caspase-1 Activation

We previously showed that ATP stimulation of P2X7 resulted in assembly with P2X4 and pannexin-1 and generation of reactive oxygen species (ROS) [10]. Stimulation with ATP alone could induce caspase-1 activation in GECs, but P. gingivalis infection diminished the effect of ATP [12], likely through hydrolysis of extracellular ATP by secretion of the NDK homologue [31]. Therefore, we aimed here to evaluate the effects of P. gingivalis NDK on ATP-mediated inflammasome activation during infection of GECs. As shown in Figure 1A and B, HIGKs infected with wild-type P. gingivalis showed reduced ATP-induced caspase-1 activation, whereas cells infected with ndk-deficient P. gingivalis resembled uninfected cells, with higher levels of secreted (activated) caspase-1. This suggests that the NDK homologue of P. gingivalis regulates caspase-1 activation and may therefore influence secretion of pro-inflammatory cytokines.

Figure 1. NDK modulates ATP-induced caspase-1 activation in GECs.

Figure 1

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Cells were infected or uninfected with wild-type P. gingivalis or ndk-deficient P. gingivalis for 6 h, and then treated with 3 mM ATP for 3 h. (A) Caspase-1 (20 kDa) activation in HIGK cell supernatants was detected by SDS-PAGE Western blot assay. (B) Average of three Western blot experiments, quantified by densitometry. Error bars represent the standard deviations (± SD) of at least two independent experiments ran in at least duplicates (*P<0.05 Student t-test).

3.2. NDK Inhibits IL-1β secretion

Our previous studies have shown that P. gingivalis-infected or LPS-primed GECs require stimulation with a second danger signal, such as ATP, in order to release active IL-1β [12]. In order to evaluate the role of NDK in inhibiting signaling by ATP, we infected primary GECs with P. gingivalis followed by stimulation with ATP, and examined the supernatant for IL-1β by ELISA. The results demonstrated significantly lower ATP-induced IL-1β secretion in wild-type P. gingivalis-infected GECs, compared to ndk-deficient P. gingivalis-infected GECs or uninfected cells (Fig. 2).

Figure 2. NDK hydrolysis of ATP inhibits secretion of IL-1β in ATP-induced GECs.

Figure 2

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Cells were infected or uninfected with wild-type P. gingivalis or ndk-deficient P. gingivalis for 6 h, and then treated with 3 mM ATP for 3 h. ELISA of IL-1β secretion was measured in supernatants of P. gingivalis-infected primary GECs treated with ATP. Error bars represent the standard deviations (± SD) of at least two independent experiments ran in at least duplicates (** P<0.001 and *** P<0.0001 Student t-test).

Immunofluorescence staining for IL-1β showed that ndk-deficient P. gingivalis induces higher levels of IL-1β accumulation within primary GECs (Fig. 3A) and there is a significant decrease in fluorescence intensity upon stimulation with ATP. In contrast, GECs infected with wild-type P. gingivalis showed reduced IL-1β release and relatively higher intracellular staining after ATP treatment, compared with cells infected with ndk-deficient P. gingivalis (Fig. 3B), suggesting that NDK produced by wild-type P. gingivalis inhibits ATP-mediated IL-1β secretion.

Figure 3. NDK affects intracellular IL-1β accumulation and ATP-dependent release in primary GECs.

Figure 3

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GECs were infected or uninfected with wild-type P. gingivalis or ndk-deficient P. gingivalis for 6 h, and then treated with 3 mM ATP for 3 h. (A) Immunofluorescence staining of DAPI-labeled nuclei (blue), Alexa-Fluor-594-labeled P. gingivalis (red), and fluorescein-isothiocyanate (FITC)-labeled IL-1β (green). Images were taken at 40X magnification, and scale bars represent 10 μm. (B) Quantification analysis for mean fluorescence intensity was performed using NIH-ImageJ. Error bars represent the standard deviations (± SD) of at least 25 independent cells (*P<0.05 and *** P<0.0001 Student t-test).

3.3. NDK Modulates the Release of HMGB1

We next evaluated whether P. gingivalis NDK affects release of another danger signaling molecule, HMGB1. Under normal conditions, HMGB1 is found within the nucleus of eukaryotic cells and is associated with DNA, where it functions as a regulator of gene expression [17]. However, HMGB1 can act as a danger signaling molecule when released by stressed cells or cells undergoing necrosis [17]. In a previous study using Salmonella-infected macrophages, it was shown that HMGB1 release is caspase-1 activation-dependent [41]. We therefore hypothesized that NDK may inhibit HMGB1 release due to its ability to inhibit ATP-mediated caspase-1 activation. To study this possibility, we examined the release of HMGB1 by Western blot analysis. Our results showed that ATP treatment induces HMGB1 release from uninfected GECs, and that wild-type P. gingivalis infection is more effective than infection with ndk-deficient P. gingivalis in inhibiting ATP-induced HMGB1 release (Fig. 4A and B).

Figure 4. NDK affects release of HMGB1 in GECs.

Figure 4

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Cells were infected or uninfected with wild-type P. gingivalis or ndk-deficient P. gingivalis for 6 h, and then treated with 3 mM ATP for 3 h. (A) HMGB1 secretion in GEC cell supernatants was imaged by SDS-PAGE Western blot assay. (B) Average of two Western blot experiments, quantified by densitometry. Error bars represent the standard deviations (± SD) (*P<0.05 Student t-test).

Due to the localization of HMGB1 in the nucleus under quiescent conditions, we hypothesized that HMGB1-release may require relocalization from the nucleus through the cytosol and to the extracellular space. We therefore performed immunofluorescence staining against HMGB1 and confirmed the expected nuclear localization of HMGB1 in uninfected untreated primary GECs (Figure 5A). However, when uninfected cells were treated with ATP, we observed a relocation of HMGB1 from the nucleus to the extracellular space, which was attenuated in cells treated with the caspase-1 inhibitor. Moreover, there was relocation of HMGB1 from the nucleus to the cytosol in GECs infected with either the wild-type or ndk-deficient P. gingivalis strain. Upon ATP stimulation, the intensity of HMGB1 in the cytosol was significantly decreased in the cells infected with ndk-deficient P. gingivalis, consistent with the release of HMGB1 from the cell demonstrated in the Western blot assay (Figure 5A and B). The cells infected with the wild-type P. gingivalis showed a substantially higher intensity of HMGB1 in the cytosol after ATP stimulation, compared to cells infected with the ndk-deficient P. gingivalis, supporting the proposed role of P. gingivalis NDK in inhibiting ATP-mediated HMGB1 release.

Figure 5. NDK or P. gingivalis infection affects intracellular HMGB1 localization in primary GECs or ATP–mediated HMGB1 secretion.

Figure 5

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Cells were infected or uninfected with wild-type P. gingivalis or ndk-deficient P. gingivalis for 6 h, and then treated with 3 mM ATP for 3 h. YVAD inhibitor was administered at a final concentration of 50 μM prior to ATP stimulation. (A) Immunofluorescence staining representing DAPI-labeled nuclei (blue), Alexa-Fluor-594-labeled P. gingivalis (red), and Alexa-Fluor-488-labeled HMGB1 (green). Images were taken at 40X magnification, and scale bars represent 10 urn. (B) Quantification analysis for mean fluorescence intensity was performed using NIH-ImageJ. Error bars represent the standard deviations (± SD) of at least 25 independent cells (**P<0.001 Student t-test).

4. Discussion

The healthy human oral mucosa is colonized by a diversity of organisms, including high numbers of different bacterial species in addition to fungi and protozoa [42]. These microbes can reside within pockets between the teeth and epithelium to form biofilms and carry on as commensals or potential pathogenic colonizers [43]. Some of these microorganisms have evolved elaborate virulence factors to ensure their survival within the oral mucosa without eliciting an immediate destructive host immune response [1]. Among these organisms, P. gingivalis uses its major fimbriae to adhere to the host-cell membrane and quickly localize to the endoplasmic reticulum rich perinuclear region [13, 27, 44]. P. gingivalis infection can also inhibit the host apoptotic response in GECs by activating the PI3K/Akt pathway and modulating expression of Bcl-2 family proteins [1, 45]. Finally, P. gingivalis secretes an enzyme homologue of the evolutionarily conserved NDKs [13,31], which is critical for suppression of cellular ROS production and ATP-induced host cell death [13, 31]. In the present study, we show that the lack of NDK significantly reduces the inhibition of ATP-dependent inflammasome activation and subsequent release of pro-inflammatory cytokines by GECs. Our previous studies have elucidated the role of this effector of P. gingivalis as an important modulator of cellular ROS generation, as well as demonstrated that the ndk-deficient strain has significantly less intracellular survival ability compared to its isogenic wild strain upon ATP-stimulation in primary GECs [12, 13, 31]. Further, there was a significant intracellular recovery of the ndk-deficient strain after treatment with the potent ROS-inhibitor N-acetyl-cysteine (NAC) and complementation of the ndk gene restored the observed phenotype [13]. Since ROS generation contributes to inflammasome activation [46], our results showing a suppression of caspase-1 activation by wild-type P. gingivalis infection was expected. Our previous studies have extensively demonstrated that P. gingivalis is a facultative intracellular bacterium and in the GECs, the majority of bacteria rapidly enters and resides in the GECs [1, 36, 47]. Further, our previous studies displayed that GECs that are infected with P. gingivalis had intact membranes while their supernatant showed a time-dependent release of the small NDK molecule [13]. Altogether, this evidence points that the majority of the NDK in supernatants are derived from the intracellular bacteria, rather than incidental extracellular P. gingivalis.

Previous studies have shown that IL-1β secretion requires two signals. Recognition of a pathogen-associated molecular pattern by a pattern recognition receptor such as TLR provides the first signal and stimulates pro-IL-1β production [48]. A second signal, from a danger signal such as ATP, activates the inflammasome and caspase-1, resulting in processing of pro-IL-1β and secretion of the mature cytokine, IL-1β [48]. We hypothesized that ATP-dependent IL-1β secretion would be reduced by infection with wild-type P. gingivalis due to its ability to produce NDK, which should scavenge extracellular ATP. The findings in this study confirm the anti-inflammatory role of NDK in infected GECs.

We also investigated whether P. gingivalis infection could affect the release by GECs of another danger signal, HMGB1 [49, 50]. Unexpectedly, our studies also revealed that P. gingivalis induces translocation of HMGB1 from the nucleus to the cytosol. However, similarly to IL-1β secretion, ATP was required as a secondary stimulus to release HMGB1 from the cell (Fig. 6). Furthermore, ndk-deficient P. gingivalis infection was less effective than infection with wild-type P. gingivalis in inhibiting HMGB1 release from infected GECs, which also required caspase-1 activation.

Figure 6. Proposed model for the role of NDK in HMGB1 localization during P. gingivalis infection.

Figure 6

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(A) P. gingivalis inhibits ATP-mediated caspase-1 activation and maturation of IL-1β by NDK hydrolysis of ATP. (B) P. gingivalis modulates caspase-1 dependent HMGB1 release from the host cell [12, 13].

NDKs have recently attracted attention due to their involvement in a variety of chronic inflammatory diseases, and increased expression of human NDK was recently described in affected tissues of oral squamous cell carcinoma patients [51]. In addition, P. gingivalis is associated with oral squamous cell carcinomas and other chronic diseases [3, 52]. Our findings also support studies showing that HMGB1 is another possible candidate for an early inflammatory disease marker [53, 54]. Collectively, these results suggest that P. gingivalis NDK dampens the host-immune response by decreasing release of pro-inflammatory cytokine, IL-1β, and the pro-inflammatory danger signal, HMGB1, from infected GECs. Identification of NDK as a key mediator used by P. gingivalis to modulate inflammation suggests that therapeutic strategies against periodontitis could also target this effector.

Acknowledgments

This study was supported by the NIDCR grants R01DE016593 and R01DE019444, and a University of California Presidential Chair. We would like to thank the Stem Cell Instrumentation Foundry (SCIF), UC Merced, for instrument support.

Footnotes

Conflict of interest: The authors declare no conflict of interest.

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