Activation of NLRP3 and AIM2 Inflammasomes by Porphyromonasgingivalis Infection

Eunjoo Park,a Hee Sam Na,a Yu-Ri Song,a Seong Yeol Shin,b You-Me Kim,c Jin Chunga

‹Department of Oral Microbiology, School of Dentistry, Pusan National University, Yangsan, Republic of Koreaa; Division of Integrative Biosciences & Biotechnologyb andDepartment of Life Sciences,c Pohang University of Science and Technology, Pohang, Republic of Korea

Porphyromonas gingivalis, a major periodontopathogen, is involved in the pathogenesis of periodontitis. Interleukin-1� (IL-1�), a proinflammatory cytokine, regulates innate immune responses and is critical for the host defense against bacterial infec-tion. However, excessive IL-1� is linked to periodontal destruction. IL-1� synthesis, maturation, and secretion are tightly regu-lated by Toll-like receptor (TLR) signaling and inflammasome activation. We found much higher levels of inflammasomecomponents in the gingival tissues from patients with chronic periodontitis than in those from healthy controls. To investigatethe molecular mechanisms by which P. gingivalis infection causes IL-1� secretion, we examined the characteristics of P. gingiva-lis-induced signaling in differentiated THP-1 cells. We found that P. gingivalis induces IL-1� secretion and inflammatory celldeath via caspase-1 activation. We also found that P. gingivalis-induced IL-1� secretion and pyroptic cell death required bothNLRP3 and AIM2 inflammasome activation. The activation of the NLRP3 inflammasome was mediated by ATP release, the P2X7

receptor, and lysosomal damage. In addition, we found that the priming signal via TLR2 and TLR4 activation precedes P. gingi-valis-induced IL-1� release. Our study provides novel insight into the innate immune response against P. gingivalis infectionwhich could potentially be used for the prevention and therapy of periodontitis.

Interleukin-1� (IL-1�), a proinflammatory cytokine, is critical inthe host defense against many pathogens and regulates innate

immune and inflammatory responses. IL-1� processing and se-cretion are tightly regulated by a two-step mechanism (1). Thefirst event is the transcription of the pro-IL-1� gene, dependenton the activation of nuclear factor-�B (NF-�B) by Toll-like recep-tors (TLRs). TLRs sense invading pathogens outside the cell and inintracellular endosomes and lysosomes (2). Each TLR detects dis-tinct pathogen-associated molecular patterns (PAMPs) derivedfrom bacteria, mycobacteria, fungi, and viruses (3). These includelipoproteins (recognized by TLR1, TLR2, and TLR6), double-stranded RNA (TLR3), lipopolysaccharide (LPS; TLR4), flagellin(TLR5), single-stranded RNA (TLR7 and TLR8), and DNA(TLR9). Upon recognition of PAMPs, TLRs recruit a specific set ofadaptor molecules that harbor a TIR domain, such as MyD88 andTRIF, and initiate downstream signaling events that lead to NF-�Bactivation, resulting in the upregulation of proinflammatory cy-tokines and chemokines. The second signal is the activation ofinflammasome that converts pro-IL-1� to IL-1�. The inflam-masome is composed of NLR or AIM2 family receptors and pro-caspase-1. An apoptosis-associated speck-like protein containinga caspase recruitment domain (ASC) is an adaptor protein with anN-terminal PYD and a C-terminal CARD. It links the PYD-con-taining NLR family member to procaspase-1, using its PYD tointeract with the PYD of the NLRs and its CARD to interact withthe CARD of procaspase-1. PYD-containing NLR family mem-bers assemble an inflammasome complex with ASC, which in turnrecruits and activates caspase-1 (4–6). Several members of theNLR family proteins participate in the formation of distinct in-flammasomes, including NLR family pyrin domain-containing 3(NLRP3; also known as cyropyrin or NALP3), NLR family CARDdomain-containing 4 (NLRC4; also known as IPAF), and NLRP1.Different inflammasomes are activated by various stimuli (7). Forexample, NLRP1 becomes activated by the lethal toxin producedby Bacillus anthracis, whereas NLRC4 responds to cytosolic flagel-

lin in cells infected with Salmonella, Legionella, and Pseudomonasspp. The NLRP3 inflammasome is activated by a large variety ofstimuli, including microbial products and endogenous signals,such as urate crystal, silica, amyloid fibrils, and ATP. BesidesNLRs, AIM2 family members can activate inflammasomes. AIM2is characterized by the presence of a pyrin domain and a DNA-binding HIN domain and activates caspase-1 by detecting cytoso-lic DNA (8). Upon activation, the NLR family members bind toadaptor protein ASC; in turn, the bound proteins recruit pro-caspase-1 for activation. Activated capsase-1 cleaves the proformof the cytokines IL-1� and IL-18 to their mature and secretedforms. Caspase-1 activation also induces a proinflammatory celldeath called pyroptosis and thereby removes the replicative nicheof intracellular pathogens (9). Assembly of the inflammasome re-quires a preceding priming signal via TLRs which is required toupregulate the expression of inflammasome receptors and thesubstrate pro-IL-1�, before the second signal can initiate inflam-masome complex formation (10). Although IL-1� is required forhost defense against pathogens, overreacted expression and secre-tion of this molecule can lead to tissue damage, and dysregulatedinflammasome activation is related to the pathogenesis of a varietyof inflammatory diseases (1, 11).

Periodontitis, one of the most common diseases, is an infec-

Received 22 July 2013 Returned for modification 4 August 2013Accepted 5 October 2013

Published ahead of print 14 October 2013

Editor: A. J. Bäumler

Address correspondence to Jin Chung, jchung@pusan.ac.kr.

Supplemental material for this article may be found at http://dx.doi.org/10.1128/IAI.00862-13.

Copyright © 2014, American Society for Microbiology. All Rights Reserved.

doi:10.1128/IAI.00862-13

112 iai.asm.org Infection and Immunity p. 112–123 January 2014 Volume 82 Number 1

on April 22, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

tion-driven chronic inflammatory disease of periodontium andthe major cause of tooth loss. Periodontitis is induced by peri-odontopathogens, such as Porphyromonas gingivalis, Tannerellaforsythia, and Treponema denticola. P. gingivalis, a Gram-negativeshort-rod anaerobe, is a predominant periodontal pathogen thatexpresses a number of virulence factors involved in the pathogen-esis of periodontitis (12–14). Virulence components of P. gingiva-lis, such as LPS, lipoproteins, gingipains, and fimbriae, are impor-tant in the induction of immune responses, including productionof cytokines and activation of inflammation-related signalingpathways (13, 15, 16). LPS, a strong inducer of IL-1�, inducescaspase-1 activation and a pyroptosis-like cell death in humanTHP-1 cells (17). The patterns of cytokine production from hu-man macrophages stimulated with purified bacterial components,LPS, or FimA differed from those observed when the cells werechallenged with P. gingivalis (18). Thus, to investigate the hostinflammatory responses evoked by P. gingivalis in vivo, we usedlive bacteria instead of bacterial components.

Since IL-1� has been linked with the destruction of periodon-tal tissue, examination of the interaction between live P. gingivalisand host cells leading to IL-1� release is necessary to understandthe process of periodontal diseases and identify valuable targetsfor periodontal treatment (19). Therefore, the aim of this studywas to elucidate the mechanism of P. gingivalis-induced IL-1�. Wefound that P. gingivalis-induced IL-1� secretion required bothNLRP3 and AIM2 inflammasome activation in THP-1 cells.Moreover, the activation of the NLRP3 inflammasome was medi-ated by ATP release, the P2X7 receptor (P2X7R), and lysosomaldamage. In addition, we also found that the priming signal pro-vided via the TLR2 and TLR4 activation signal is a prerequisite forP. gingivalis-induced IL-1� release.

MATERIALS AND METHODSReagents. We purchased chemicals and antibodies from the sources listedbelow. Phorbol 12-mystristate 13-acetate (PMA), hemin, vitamin K (3-phytyl-menadione), oxidized ATP (oxATP), KCl, and trichloroacetate(TCA) were from Sigma (St. Louis, MO). CA-074 methyl ester (CA-074Me) and pyrrolidine dithiocarbamate (PDTC; a NF-�B inhibitor) werepurchased from Calbiochem (San Diego, CA). Z-Trp-Glu(OMe)-His-As-p(OMe)-fluoromethylketone (Z-WEHD-FMK; caspase-1 inhibitor),benzyloxycarbonyl-V-A-D-O-methyl fluoromethyl ketone(Z-VAD-FMK; pancaspase inhibitor), anti-human IL-1� (catalog no. 2805), andanti-human NLRP3 (catalog no. AF6789) were from R&D Systems (Min-neapolis, MN). Anti-human ASC (catalog no. 4628), anti-human AIM2(catalog no. 8055), and anti-human caspase-1 (catalog no. 2225) antibod-ies and radioimmunoprecipitation assay buffer were from Cell SignalingTechnology (Beverly, MA). Anti-human caspase-1 (catalog no. sc-56036),anti-human NLRC4 (catalog no. sc-99056), anti-human TLR2 (catalogno. sc-21760), anti-human TLR4 (catalog no. sc-10741), anti-humanMyD88 (catalog no. sc-8196), anti-human TRIF (catalog no. sc-130902),anti-human TRAF6 (catalog no. sc-7221), and anti-�-actin (catalog no.sc-47778) antibodies were from Santa Cruz (Santa Cruz, CA).

GCF and gingival tissue sampling. We obtained gingival crevicularfluid (GCF) and gingival tissue from patients who were scheduled to un-dergo treatment at the Department of Periodontology of the Pusan Na-tional Dental School. All donors gave written informed consent, and theirrights were protected according to the protocol reviewed and approved bythe Institutional Review Board of Pusan National University. GCF sam-ples were collected from a periodontitis site (probing depth [PD] � 5 mm,calculus � 2 mm, and bleeding on probing [BOP]) as well as from ahealthy site (PD � 3 mm, no BOP). Sites for GCF collection were isolatedwith cotton rolls and gently air dried, and supragingival plaque was re-

moved. A collection strip was inserted 1 to 2 mm into the sulcus for 1 min.Samples were eluted from the strip in 300 �l of phosphate-buffered saline(PBS) by centrifugation in an Eppendorf tube. Eluates were stored at�80°C until assays were performed. Healthy gingival tissue was sampledfrom healthy participants’ tissue that showed no overt signs of gingivalinflammation at a probing depth of �3 mm. Periodontitis tissue wasobtained from sites with signs of gingival inflammation and a probingdepth of �5 mm. Gingival samples were collected from both healthy andperiodontitis sites after infiltration with an appropriate local anesthetic.Samples comprised the epithelial lining and a portion of the underlyingconnective tissue, and inflammation was confirmed by hematoxylin-eo-sin (H&E) staining. The gingival tissue specimens obtained were thor-oughly rinsed with sterile normal saline solution, transferred into Eppen-dorf tubes, and stored at �80°C until use.

Bacterial culture. P. gingivalis (strain 381) was grown in Gifu anaer-obic medium broth (Nissui, Japan), which contained 5 mg/ml hemin and0.5 mg/ml 3-phytyl-menadione (vitamin K) under anaerobic conditionsat 37°C. An optical density at 650 nm of 1.0 was determined to correlate to109 CFU/ml. The bacteria were washed and resuspended in RPMI me-dium to infect the THP-1 cells at a multiplicity of infection (MOI) of 1:10,1:50, or 1:100.

Cell treatment. Cells of the THP-1 cell line, a human acute monocyticleukemia cell line, were differentiated to macrophage-like cells by treat-ment with 50 nM PMA overnight. The differentiated cells were infectedwith live P. gingivalis for 6 or 24 h. In some experiments, the cells werepretreated with Z-WEHD-FMK, Z-VAD-FMK, oxATP, KCl, CA-074 Me,and dimethyl sulfoxide (DMSO) at the indicated concentrations for 30min before bacterial challenge.

Preparation of THP-1/ASC-GFP stable cell line. For generation ofretrovirus used for stable expression of green fluorescent protein-tagged ASC(ASC-GFP), HEK293T cells were transiently transfected with plasmids en-coding gag-pol, vesicular stomatitis virus G protein, and pMSCVneo-ASC-GFP. At 34 and 50 h posttransfection, the culture supernatant containingretroviral particles was collected and added to THP-1 cells. Retrovirus-trans-duced THP-1 cells were selected with 500 �g/ml G418 for 5 days.

ASC pyroptosome quantitation in live cells. THP-1/ASC-GFP cellswere seeded in 8-well chambers and then primed with PMA (50 nM).THP-1/ASC-GFP cells were pretreated with various agents and furtherchallenged with P. gingivalis. ASC pyroptosome formation was observedby use of a confocal laser scanning microscope (LSM 700; Carl Zeiss) atvarious time points. At least 400 cells were counted to enumerate thenumber of cells containing the ASC-GFP pyroptosome at the end of eachtime period. The percentage of cells with an ASC pyroptosome was calcu-lated by dividing the number of cells with an ASC pyroptosome by thetotal number of cells counted.

Real-time qRT-PCR. Total RNA was extracted with the TRIzol re-agent (Invitrogen) following the manufacturer’s instructions, and cDNAwas synthesized with a reverse transcription system (Bioneer Co., Dae-jeon, South Korea). For quantitative reverse transcriptase (qRT-PCR), thecDNA was amplified using TaqMan Universal PCR master mix (AppliedBiosystems) with gene-specific primers and fluorogenic TaqMan probeson an ABI 7500 real-time PCR system (Applied Biosystems) with thefollowing programs: a 10-min preincubation at 95°C and 40 cycles of 15 sat 95°C and 1 min at 60°C. The data were normalized relative to those forGAPDH (glyceraldehyde-3-phosphate dehydrogenase) as an endogenouscontrol. TaqMan assay primers and probes were purchased from Qiagen(Valencia, CA) (for GAPDH, catalog no. Hs99999905; for ASC, catalogno. Hs00203118; for NLRP3, catalog no. Hs00918082; for AIM2, catalogno. Hs00915710; for NLRC4, catalog no. Hs00892666).

RNA interference assay. Human small interfering RNAs (siRNAs) forASC, NLRP3, AIM2, NLRC4, TLR2, TLR4, MyD88, TRIF, TRAF6, andnontargeting control oligonucleotides were obtained from Qiagen (Va-lencia, CA). The sequences of each siRNA oligonucleotide in this pool areas follows: ASC siRNA, SI03086783; NLRP3 siRNA, SI03060323; NLRC4siRNA, SI04374685; AIM2 siRNA, SI04261432; TLR2 siRNA, SI00050029;

Inflammasome Activation by P. gingivalis Infection

January 2014 Volume 82 Number 1 iai.asm.org 113

on April 22, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

TLR4 siRNA, SI04951149; MyD88 siRNA, SI00300909; TRIF siRNA,RNF36; SI03027255; TRAF6 siRNA, SI03046043; and nontargeting con-trol siRNA oligonucleotides, 1027281. For siRNA experiments in THP-1cells, cells were seeded in 6-well plates at a density of 1 � 106 cells per wellin the presence of 50 nM PMA. The differentiated cells were then trans-fected with siRNA oligonucleotides (1,200 ng) for 48 h using the Attract-ene transfection reagent (Qiagen, Valencia, CA) in 1 ml of RPMI.

Cytokine analysis. Tumor necrosis factor alpha (TNF-�) and IL-1�levels were measured by using enzyme-linked immunosorbent assay(ELISA) kits from eBioscience (San Diego, CA) following the manufac-turer’s instruction.

Immunoblot analysis. Procaspase-1 and pro-IL-1� in the gingivaltissue and cell extracts and their active forms precipitated by 10% TCAfrom the culture supernatant were analyzed by immunoblotting. The cellswere washed in ice-cold PBS and lysed. A total of 50 �g proteins wasseparated by 15% SDS-PAGE, transferred to a polyvinylidene difluoridemembrane, and probed with the indicated antibodies. Reactivity was de-termined by using horseradish peroxidase-conjugated anti-mouse, anti-rabbit, anti-sheep, or anti-goat IgG secondary antibody (diluted 1:5,000;Jackson ImmunoResearch Laboratories, Inc., PA), and the signals werevisualized using SuperSignal West Femto maximum-sensitivity substrate(Pierce, Rockford, IL) with an LAS-4000 Fujifilm luminescent image an-alyzer. The band intensities of the immunoblot were quantified with NIHImageJ software and are presented as a ratio relative to the intensity of�-actin.

Cell death assay. Cell death was measured with a lactate dehydroge-nase (LDH) cytotoxicity assay kit (CytoTox96 nonradioactive cytotoxicityassay; Promega). The percent cytotoxicity was calculated by the followingformula: 100 � [(experimental LDH release � spontaneous LDH re-lease)/(maximal LDH release � spontaneous LDH release)]. To deter-mine the maximal LDH release, cells were treated with 1% Triton X-100.

ATP determination. The extracellular ATP concentration was deter-mined with an ATP determination kit (Invitrogen, Carlsbad, CA) follow-ing the manufacturer’s instruction.

Statistics. Statistically significant differences between samples wereanalyzed with an unpaired, one-tailed Student’s t test. The data are shownas the mean � standard deviation (SD). A P value of less than 0.05 wasconsidered statistically significant.

RESULTSThe levels of IL-1� and inflammasome components are in-creased in periodontitis patients. Periodontitis is a chronic inflam-matory disease caused by major periodontopathogens. To describethe relationship between periodontitis and inflammasomes, we firstinvestigated if the level of IL-1� was higher in the GCF of periodon-titis patients. The level of IL-1� as well as that of TNF-� was about4-fold higher in the GCF of periodontitis patients (Fig. 1A). Next, weexamined the levels of inflammasome components in the gingivaltissues from healthy subjects and subjects with chronic periodontitis.As shown in Fig. 1B, caspase-1 was highly expressed in the gingivaltissue of periodontitis patients. The levels of expression of bothNLRP3 and AIM2 were also higher in the periodontitis gingival tis-sues than the healthy ones. These finding suggest that NLRP3 andAIM2 inflammasomes are activated in periodontitis tissues, leadingto caspase-1 activation and, in turn, IL-1� secretion.

P. gingivalis induces caspase-1 activation, IL-1� secretion,and inflammatory cell death. To determine whether P. gingivalisinduces inflammation, we examined proinflammatory cytokinesecretion. We differentiated cells of a human acute monocyticleukemia cell line (THP-1) to macrophage-like cells by treatmentwith PMA. The MOI- and time-dependent release of IL-1� andTNF-� after infection with P. gingivalis was detected in culturesupernatants by ELISA (Fig. 2A). P. gingivalis activated caspase-1,and the amount of processed caspase-1 subunit in the culturesupernatants was increased depending on time and the MOI of P.gingivalis (Fig. 2B and C). In addition to caspase-1 activation, P.gingivalis induced pro-IL-1� expression in the cells. These resultssuggest that P. gingivalis regulates caspase-1 activation, pro-IL-1�synthesis, and IL-1� secretion in THP-1 cells. Activation ofcaspase-1 not only leads to inflammation but also causes an in-flammatory form of cell death called pyroptosis. Formation of thepyroptosome might be a mechanism by which dying macrophagesrelease their cellular contents, such as lactate dehydrogenase(LDH), a marker of cell death. P. gingivalis dramatically induced

FIG 1 IL-1� and TNF-� secretion is increased in periodontitis patients. (A) GCF from healthy subjects and chronic periodontitis patients was analyzed fordetermining the concentrations of human IL-1� and TNF-� by ELISA. ***, P 0.001. (B) The expression of inflammasome components in the gingival tissuesof healthy subjects and chronic periodontitis patients was detected by immunoblotting. Each of nine gingival tissue specimens from healthy subjects and chronicperiodontitis patients was examined. Results represent those from one of three individual experiments. The numbers below the lanes indicate average ratiosnormalized with the level of �-actin expression.

Park et al.

114 iai.asm.org Infection and Immunity

on April 22, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

LDH release from THP-1 cells. Cytotoxicity was increased in anMOI- and time-dependent manner upon P. gingivalis infection(Fig. 2D). ASC plays a critical role in Salmonella enterica serovarTyphimurium-induced pyroptic cell death and is required for thesecretion of IL-1� induced by P. gingivalis (19, 20). To testwhether P. gingivalis induces ASC-dependent pyroptosome for-

mation, we generated a THP-1 cell line that stably expresses anASC-GFP fusion protein. The level of ASC-GFP expression wascomparable to that of the endogenous ASC protein (data notshown). ASC forms a single 1- to 2-mm supramolecular assemblyin each macrophage cell in response to stimulation with proin-flammatory stimuli (21). In unstimulated cells, ASC-GFP was

FIG 2 P. gingivalis enhances IL-1� secretion and caspase-1 activation, leading to inflammatory cell death. (A) PMA-primed THP-1 cells were infected with P. gingivalis(MOI, 100) for 6 or 24 h. Cell culture supernatants were assayed for human IL-1� and TNF-� by ELISA. The data represent mean values � SDs (n � 3). (B and C) TheIL-1� and caspase-1 secreted into the culture supernatants (sup.) and the pro-IL-1�, procaspase-1, and �-actin in the cell lysates (cell) were detected by immunoblotting.(B) PMA-primed THP-1 cells were infected with P. gingivalis (P.g) (MOI, 100) for 3, 6, or 24 h. (C) PMA-primed THP-1 cells were infected with P. gingivalis (MOI, 10,50, or 100) for 6 h. (D) The amount of the cytoplasmic enzyme LDH released into the culture supernatant was measured with an LDH cytotoxicity assay kit. The datarepresent mean values � SDs (n � 3). (E) Live PMA-primed ASC-GFP cells infected with P. gingivalis at each MOI for the indicated times were observed andphotographed by fluorescence confocal microscopy. The graph on the right shows the percentages of cells containing the ASC pyroptosome. The percentages of cellscontaining the ASC pyroptosome were calculated as described in Materials and Methods. *, P 0.05; **, P 0.01; ***, P 0.001.

Inflammasome Activation by P. gingivalis Infection

January 2014 Volume 82 Number 1 iai.asm.org 115

on April 22, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

evenly distributed in the cytoplasm and nucleus. After stimulationwith P. gingivalis, the ASC-GFP fluorescence accumulated inbright large spots in the cytoplasm, indicating the formation of theASC pyroptosome. Quantitative analysis revealed that the num-ber of pyroptosome-containing cells increased in an MOI- andtime-dependent manner after stimulation with P. gingivalis (Fig.2E). These data suggest that P. gingivalis induces the formation ofthe pyroptosome, leading to pyroptosis (inflammatory cell death)in THP-1 cells.

P. gingivalis-induced IL-1� secretion and proinflammatorycell death are dependent on caspase-1. Caspase-1 is required forthe processing and subsequent release of IL-1�. To confirm that P.gingivalis-induced IL-1� secretion was caused by caspase-1 acti-vation, the cells were pretreated with the caspase inhibitorZ-WEHD-FMK or Z-VAD-FMK prior to P. gingivalis challenge.As shown in Fig. 3A and B, Z-WEHD-FMK or Z-VAD-FMK in-hibited caspase-1 activation and IL-1� secretion, indicating thatP. gingivalis-induced IL-1� secretion is dependent on caspase-1activation. To determine whether P. gingivalis-induced pyropto-some formation and cell death are dependent on caspase-1 acti-vation, cells were pretreated with the caspase inhibitor and thenstimulated with P. gingivalis. Pretreatment with a caspase inhibi-tor blocked LDH release and the formation of an ASC pyropto-some in response to P. gingivalis infection (Fig. 3C and D). Thesedata indicate that P. gingivalis-induced pyroptosis is dependent oncaspase-1 activation.

P. gingivalis-induced IL-1� secretion requires both NLRP3and AIM2 inflammasomes. Next, we investigated whether the

expression of inflammasome components is regulated by P. gingi-valis. P. gingivalis infection increased the expression of NLRP3 andAIM2 in a time-dependent manner but did not affect the expres-sion of ASC and NLRC4 (Fig. 4A and B). To determine whichinflammasome components are involved in P. gingivalis-inducedIL-1� secretion, we individually knocked down ASC, NLRP3,AIM2, and NLRC4 with specific siRNAs. Treatment with siRNAsalmost completely blocked the expression of each protein (see Fig.S1A in the supplemental material). Using the knockdown cells, wefound that ASC, NLRP3, or AIM2 knockdown significantly inhib-ited P. gingivalis-induced caspase-1 activation and subsequentIL-1� secretion without affecting the expression of procaspase-1(Fig. 4C and D). In contrast, the silencing of NLRC4 did not in-hibit caspase-1 activation or IL-1� secretion (Fig. 4C). The LDHrelease induced by P. gingivalis infection was also strongly inhib-ited in THP-1 cells transfected with siRNA for ASC, NLRP3, orAIM2 (Fig. 4E). To find out which inflammasome components P.gingivalis-induced pyroptosome formation is dependent on, ASC-GFP cells were transfected with siRNAs and then stimulated withP. gingivalis. Silencing of NLRP3 or AIM2 blocked the formationof an ASC pyroptosome in response to P. gingivalis infection,whereas the silencing of NLRC4 had a marginal effect (Fig. 4F).These results indicate that P. gingivalis-induced caspase-1 activa-tion and subsequent IL-1� secretion are mediated by both NLRP3and AIM2 inflammasomes.

P. gingivalis-induced caspase-1 activation is mediated byATP, potassium depletion, and cathepsin B. The NLRP3 inflam-masomes are activated in response to danger signals, such as ATP,

FIG 3 P. gingivalis-induced IL-1� secretion and proinflammatory cell death are dependent on caspase-1. (A to C) THP-1 cells were pretreated with Z-WEHD-FMK (30 �M) or Z-VAD-FMK (30 �M) for 30 min before P. gingivalis infection for 6 h. (A) The IL-1� and caspase-1 secreted into the culture supernatant (sup.)and the pro-IL-1�, procaspase-1, and �-actin in the cell lysates (cell) were detected by immunoblotting. (B and C) Cell culture supernatant was collected andassayed for IL-1� secretion by ELISA (B) and the cytoplasmic enzyme LDH (C). The data represent mean values � SDs (n � 3). (D) Live PMA-primed ASC-GFPcells were pretreated with Z-WEHD-FMK or Z-VAD-FMK for 30 min before P. gingivalis infection for 6 h or 24 h. The graph on the right shows the percentagesof cells containing the ASC pyroptosome. **, P 0.01; ***, P 0.001.

Park et al.

116 iai.asm.org Infection and Immunity

on April 22, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

released from stressed cells (11, 22). Extracellular ATP binds to theP2X7 receptor (P2X7R), and the subsequent potassium efflux isthought to account for NLRP3 inflammasome activation (23). Toexamine whether P. gingivalis induces ATP release, THP-1 cellswere infected with P. gingivalis and the ATP concentration in theculture supernatant was measured. P. gingivalis significantly in-duced ATP release (Fig. 5A; see Fig. S2A in the supplemental ma-terial). To evaluate whether the effects of P. gingivalis on caspase-1activation and IL-1� secretion are mediated through P2X7R, wepretreated the cells with oxATP, a P2X7R antagonist that blocksthe interaction of ATP with P2X7R, for 30 min and then infectedthe cells with P. gingivalis for 6 or 24 h. The oxATP stronglyblocked the caspase-1 activation and IL-1� secretion induced byP. gingivalis (Fig. 5B and C; see Fig. S2B and C in the supplementalmaterial). Furthermore, oxATP treatment inhibited the inductionof cytotoxicity and ASC-dependent pyroptosome formation by P.gingivalis infection (Fig. 5D and K; see Fig. S2D and K in thesupplemental material). These results indicate that ATP has a crit-ical role in P. gingivalis-induced NLRP3 inflammasome activa-tion.

Potassium (K) efflux is a common event induced by a broadrange of inflammasome stimuli and is important in caspase-1 ac-tivation, IL-1� processing, and cell death. Consequently, activa-tion can be specifically blocked by the addition of extracellular K

(23, 24). To examine whether K efflux affects P. gingivalis-in-duced IL-1� secretion and caspase-1 activation, THP-1 cells werepretreated with extracellular KCl at a 30 mM concentration for 30min and then infected with P. gingivalis for 6 or 24 h. The in-creased extracellular KCl concentration significantly reduced P.gingivalis-induced caspase-1 activation and IL-1� secretion (Fig.5E and F; see Fig. S2E and F in the supplemental material). Giventhat depletion of intracellular potassium by bacterial toxins hasbeen shown to induce pyroptic cell death of THP-1 cells, we askedwhether intracellular K depletion also plays a role in P. gingivalis-induced LDH release and the formation of an ASC pyroptosome.To answer this question, we examined the effect of inhibiting K

efflux by increasing the extracellular K concentration to 30 mM.Inhibition of the K efflux strongly reduced the cytotoxicity andASC pyroptosome formation (Fig. 5G and K; see Fig. S2G and K inthe supplemental material). Our results suggest that K efflux is

FIG 4 Caspase-1 activation, IL-1� secretion, cell cytotoxicity, and pyroptic cell death induced by P. gingivalis infection are mediated by NLRP3 and AIM2inflammasome activation. (A) Real-time PCR measurement of ASC, NLRP3, AIM2, NLRPC4, and IL-1� mRNA expression in PMA-primed THP-1 cells atdifferent times after P. gingivalis infection (MOI, 100). (B) Immunoblot analysis of ASC, NLRP3, AIM2, NLRPC4, and �-actin in P. gingivalis-infected cells. Thenumbers below the lanes indicate average ratios relative to controls normalized to the level of �-actin expression. (C to E) THP-1 cells were transfected with ASC,NLRP3, AIM2, or NLRC4 siRNA for 48 h. siRNA-transfected cells were infected with P. gingivalis (MOI, 100) for 6 h or 24 h. The IL-1� and caspase-1 secretedinto the culture supernatants (sup.) and the procaspase-1 and �-actin in the cell lysates (cell) were detected by immunoblotting (C). Cell culture supernatant wascollected and assayed for IL-1� secretion by ELISA (Con., control) (D) and the cytoplasmic enzyme LDH (E). The data represent mean values � SDs (n � 3). (F)Live PMA-primed ASC-GFP cells were transfected with ASC, NLRP3, AIM2, or NLRC4 siRNA for 48 h and infected with P. gingivalis (MOI, 100) for 6 h or 24h and observed by fluorescence confocal microscopy. The graph on the right shows the percentages of cells containing ASC pyroptosome. *, P 0.05; **, P 0.01;***, P 0.001.

Inflammasome Activation by P. gingivalis Infection

January 2014 Volume 82 Number 1 iai.asm.org 117

on April 22, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

needed for P. gingivalis-mediated NLRP3 inflammasome activa-tion.

Phagosomal uptake of monosodium urate (MSU), silica crys-tals, aluminum salts, or fibrillar amyloid-� induces lysosomaldamage and the leakage of lysosomal proteases, specifically, ca-thepsin B, into the cytosol, leading to the activation of the NLRP3inflammasome and IL-1� secretion (25, 26). We examinedwhether P. gingivalis-induced caspase-1 activation and IL-1� se-

cretion are dependent on cathepsin B. THP-1 cells were eitheruntreated (control) or pretreated with a cathepsin B inhibitor(CA-074 Me, 10 �M) or DMSO for 30 min and subsequentlyinfected with P. gingivalis for 6 or 24 h. The inhibition of cathepsinB with CA-074 Me almost completely inhibited P. gingivalis-in-duced caspase-1 activation and IL-1� secretion (Fig. 5H and I; seeFig. S2H and I in the supplemental material). To further examinethe role of cathepsin B, we analyzed the LDH release and the for-

FIG 5 ATP and potassium (K) efflux are involved in P. gingivalis-induced NLRP3 activation. (A) THP-1 cells were infected with P. gingivalis for 6 h. Theextracellular ATP concentration was determined using an ATP determination kit. The data are shown as mean values � SDs (n � 3). (B to J) THP-1 cells werepretreated with oxATP (300 �M), KCl (50 mM), CA-074 Me (10 �M), or DMSO for 30 min before P. gingivalis infection for 6 h. Secreted IL-1� and caspase-1were detected by immunoblotting (B, E, and H). Cell culture supernatant was collected and assayed for IL-1� secretion by ELISA (C, F, and I) and the release ofthe cytoplasmic enzyme LDH (D, G, and J). (K) Live PMA-primed ASC-GFP cells were pretreated with oxATP (300 �M), KCl (50 mM), or CA-074 Me (10 �M)for 30 min prior to infection with P. gingivalis for 6 h and observed and photographed by fluorescence confocal microscopy. The graph on the right shows thepercentages of cells containing ASC pyroptosome. DMSO was used as a negative control. *, P 0.05; **, P 0.01; ***, P 0.001.

Park et al.

118 iai.asm.org Infection and Immunity

on April 22, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

mation of the ASC pyroptosome in the presence of CA-074 Me.The cathepsin B inhibitor significantly decreased the extent of P.gingivalis-induced cytotoxicity and ASC pyroptosome formation(Fig. 5J and K; see Fig. S2J and K in the supplemental material).These results indicate that the effect of P. gingivalis on NLRP3inflammasome activation is also mediated by lysosomal disrup-tion. Our data suggest that ATP, K efflux, and cathepsin B arecritical in P. gingivalis-induced NLRP3 inflammasome activationand subsequent IL-1� secretion.

P. gingivalis-induced inflammasome activation requires thepreceding TLR signaling. The production of mature IL-1� re-quires the activation of two pathways: (i) pattern recognition re-ceptors such as TLRs increase pro-IL-1� expression throughNF-�B activation, and (ii) inflammasomes convert pro-IL-1� toIL-1�. The surface components of P. gingivalis, such as LPS, lipo-proteins, and fimbriae, interact with host TLRs and activate theinnate immune responses (13). We found that P. gingivalis infec-tion increases pro-IL-1� gene expression and protein synthesis(Fig. 2A and B). Because TLR2 and TLR4 are the key moleculesrequired for the cytokine production induced by Gram-negativeperiodontal bacteria (27, 28), we tested the roles of TLR2 andTLR4 in P. gingivalis-induced IL-1� secretion. Knockdown ofTLR2 and/or TLR4 with specific siRNAs inhibited P. gingivalis-mediated pro-IL-1� induction as well as caspase-1 activation andIL-1� secretion (Fig. 6A and B; see Fig. S3A to C in the supple-mental material). The silencing of TLR2 and/or TLR4 also de-creased the extent of cytotoxicity and ASC pyroptosome forma-tion induced by P. gingivalis (Fig. 6C and D; see Fig. S3D and E inthe supplemental material).

Next, we examined the roles of downstream signaling mole-cules of the TLR2 and TLR4 pathways. By silencing of MyD88and/or TRAF6 expression, we observed a significant downregula-tion of P. gingivalis-induced caspase-1 activation and IL-1� secre-tion (Fig. 6E and F; see Fig. S3F to H in the supplemental material).Similarly, the silencing of TRIF and/or TRAF6 downregulated thecaspase-1 activation and the IL-1� release stimulated by P. gingi-valis (Fig. 6H and I; see Fig. S3J and K in the supplemental mate-rial). These results show that MyD88 and TRIF, TLR adaptor mol-ecules, are required for the activation of P. gingivalis-inducedcaspase-1 and IL-1� secretion. Knockdown of MyD88, TRIF,and/or TRAF6 strongly blocked LDH release and the formation ofthe ASC pyroptosome (Fig. 6G and J and K; see Fig. S3I, L, and Min the supplemental material). These results indicate that TLRsignaling is the prerequisite for P. gingivalis-induced inflam-masome activation leading to IL-1� secretion. We also usedPDTC, an NF-�B inhibitor, to determine whether NF-�B activa-tion is involved in pro-IL-1� induction. The induction of pro-IL-1� expression by P. gingivalis was inhibited by pretreatment ofthe cells with PDTC (Fig. 6L; see Fig. S3N in the supplementalmaterial). PDTC pretreatment also inhibited P. gingivalis-inducedcaspase-1 activation, IL-1� secretion, LDH release, and the for-mation of the ASC pyroptosome (Fig. 6L to O; see Fig. S3N to Q inthe supplemental material), suggesting a role of NF-�B for inflam-masome activation.

DISCUSSION

Periodontal disease, the major cause of adult tooth loss, is charac-terized by a chronic inflammation caused by oral bacterial infec-tion. Overgrowth of periodontal pathogens can result from a de-ficiency in the host defense system or modification of the

subgingival environment. P. gingivalis, an oral black-pigmentedGram-negative bacterium, is primarily found in deep periodontalpockets, especially in sites with active disease (29). Upon P. gingi-valis infection, a dense infiltration of inflammatory cells, includ-ing monocytes and macrophages, occurs in the periodontitis tis-sues. The inflammatory cells activate cytokine secretion as adefense against bacterial infection. Although cytokines are ini-tially protective in the elimination of infected bacteria, overpro-duction of proinflammatory cytokines is related to periodontaldestruction, including periodontal attachment loss, destruction ofcollagen, and alveolar bone resorption. The cytokine concentra-tion in GCF and saliva is higher in patients with aggressive perio-dontitis than in healthy subjects, and these effects are associatedwith autoimmune diseases (30, 31). Thus, understanding themechanisms of P. gingivalis-induced immune cell signaling couldprovide useful information for prevention and therapy of perio-dontitis. Little is known about the molecular mechanisms bywhich P. gingivalis infection causes IL-1� secretion in human im-mune cells. Therefore, we investigated the effect of P. gingivalisinfection on inflammasome activation, which is important in thesecretion of IL-1� during host innate immune responses.

First, we compared the levels of proinflammatory cytokines inGCF between healthy and periodontitis subjects. As expected,IL-1� and TNF-� production was significantly higher in GCFfrom periodontitis patients. Next, we measured the protein levelsof inflammasome components in healthy and periodontitis gingi-val tissues and found that the NLRP3 and AIM2 proteins wereupregulated in the gingival tissues from periodontitis patients.Moreover, the expression of caspase-1, which converts pro-IL-1�into mature IL-1�, was greatly enhanced in periodontitis gingivaltissues. This is the first study reporting the upregulation ofNLRP3, AIM2, and caspase-1 in periodontitis gingival tissues.Based on these results, we directly measured the TNF-� and IL-1�secretion from differentiated THP-1 cells after infection with P.gingivalis. P. gingivalis stimulated the secretion of IL-1� in anMOI- and time-dependent manner as well as enhanced the ex-pression of pro-IL-1�. The amount of secreted caspase-1 in-creased depending on the time and MOI of P. gingivalis infection,without affecting the procaspase-1 levels in the cells. The inhibi-tion of caspase-1 with Z-WEHD-FMK or Z-VAD-FMK blocked P.gingivalis-induced IL-1� secretion in THP-1 cells, suggesting thatIL-1� maturation is mediated by active caspase-1.

Pyroptosis is an important event in inflammatory processes bywhich activated macrophages rapidly release large amounts of ac-tivated cytokines, such as IL-1�, into the extracellular space (21).Additionally, pyroptosis can restrict the replication of intracellu-lar pathogens by removing the replicative niche. In SalmonellaTyphimurium infection, the activation of the NLRC4 inflam-masome and pyroptosis is temporally correlated with bacterialclearance (32). We found that P. gingivalis also induced pyropticcell death via caspase-1 activation. It is tempting to speculate thatpyroptic cell death plays a role in the clearance of P. gingivalisduring inflammatory responses, but further studies would beneeded to confirm its role.

Our study also showed that the expression of the componentsof NLRP3 and AIM2 inflammasomes, namely, ASC, NLRP3,AIM2, and caspase-1, was increased in response to P. gingivalisinfection and each was required for IL-1� secretion, suggestingthat P. gingivalis-induced caspase-1 activation and IL-1� secretionare dependent on the NLRP3 and AIM2 inflammasomes in THP-1

Inflammasome Activation by P. gingivalis Infection

January 2014 Volume 82 Number 1 iai.asm.org 119

on April 22, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

cells. In contrast to our findings, it was recently reported thatNLRP3 expression is downregulated by subgingival biofilm in pri-mary gingival fibroblast tissue, partly due to P. gingivalis (33). Thediscrepancy between studies may be attributed to the different celltypes studied or to the difference between a live single-type bacte-rial infection and a biofilm infection.

The NLRP3 inflammasome is a cytosolic protein complex thatis essential for processing and secretion of IL-1� via activation of

caspase-1 (1). The diverse types of NLRP3 inflammasome activa-tors, such as uric acid, asbestos, silica, and extracellular ATP, couldinduce reactive oxygen species synthesis and K efflux, leading tocaspase-1 activation and IL-1� secretion (34). We found that P.gingivalis activates ATP release. Furthermore, the inhibition of K

efflux by increasing the extracellular K concentration blockedthe caspase-1 activation, IL-1� secretion, and pyroptic cell deathinduced by P. gingivalis. Inhibition of cathepsin B also reduced

FIG 6 P. gingivalis-induced inflammasome activation requires the preceding TLR signaling. (A to C) THP-1 cells were transfected with TLR2 and/or TLR4siRNA for 48 h. siRNA-transfected cells were infected with P. gingivalis (MOI, 100) for 6 h. The IL-1� and caspase-1 secreted into the culture supernatants (sup.)and the pro-IL-1� and �-actin in the cell lysates (cell) were detected by immunoblotting (A). Cell culture supernatant was assayed for IL-1� secretion by ELISA(B) and the cytoplasmic enzyme LDH (C). The data represent mean values � SDs (n � 3). (D) Live PMA-primed ASC-GFP cells were transfected with TLR2and/or TLR4 siRNA for 48 h and then infected with P. gingivalis (MOI, 100) for 6 h. (E to J) THP-1 cells were transfected with MyD88, TRIF, and/or TRAF6 siRNAfor 48 h. siRNA-transfected cells were infected with P. gingivalis (MOI, 100) for 6 h. The IL-1� and caspase-1 secreted into the culture supernatants and thepro-IL-1�, procaspase-1, and �-actin in the cell lysates were detected by immunoblotting (E and H). Cell culture supernatant was collected and evaluated forIL-1� secretion by ELISA (F and I) and release of the cytoplasmic enzyme LDH (G and J). The data represent mean values � SDs (n � 3). (K) Live PMA-primedASC-GFP cells were transfected with MyD88, TRIF, and/or TRAF6 siRNA for 48 h and then infected with P. gingivalis (MOI 100) for 6 h. (L to N) THP-1 cellswere pretreated with PDTC (300 �M) for 1 h prior to infection with P. gingivalis for 6 h. (L) The secreted IL-1� and caspase-1 were detected by immunoblotting.Cell culture supernatant was assayed for IL-1� secretion by ELISA (M) and the release of the cytoplasmic enzyme LDH (N). (O) Live PMA-primed ASC-GFP cellswere pretreated with PDTC (300 �M) for 1 h prior to infection with P. gingivalis for 6 h and observed by fluorescence confocal microscopy. *, P 0.05; **, P 0.01; ***, P 0.001.

Park et al.

120 iai.asm.org Infection and Immunity

on April 22, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

the caspase-1 activation and IL-1� production induced by P.gingivalis, suggesting a potential role of ATP, K efflux, andcathepsin B in P. gingivalis-mediated activation of the NLRP3inflammasome.

AIM2 binding to cytosolic double-stranded DNA (dsDNA) viathe HIN200 domain recruits the adaptor ASC through a PYDdomain and activates caspase-1 (35). AIM2 controls inflam-masome activation, IL-1� secretion, and cell death in response tobacterial dsDNA. Recent research found that AIM2 detects cyto-solic DNA released from lysed Listeria monocytogenes or Franci-sella tularensis (36, 37). In this study, we showed for the first timethat P. gingivalis infection induces AIM2 expression and promotescaspase-1 activation, IL-1� secretion, and pyroptic cell deaththrough the AIM2 inflammasome. Moreover, we detected P. gin-givalis and its DNA in the cytoplasm of THP-1 cells (data notshown), suggesting that AIM2 can detect P. gingivalis DNA.Therefore, P. gingivalis induces caspase-1 activation and IL-1�secretion via the AIM2 inflammasome as well as the NLRP3 in-flammasome. Similarly, NLRP3, AIM2, and NLRC4 inflam-masomes all contribute to caspase-1 activation in macrophageswith Listeria monocytogenes (38).

The release of IL-1� is strictly controlled by a two-signal system(1), whereas a single signal is generally sufficient for the secretionof other cytokines. First, a transcriptional response must be acti-

vated via NF-�B activation, leading to synthesis of pro-IL-1�.Subsequently, the separate second signal pathway initiates thecaspase-1-dependent maturation and secretion of IL-1� via in-flammasome activation. We found that P. gingivalis infection ac-tivates both signals and results in IL-1� secretion. NF-�B-depen-dent TLR signaling was required for the synthesis of pro-IL-1� byP. gingivalis infection. It was reported that NF-�B-dependent TLRsignaling upregulates the expression of NLRP3 (10). Consistentwith this, our data showed that P. gingivalis infection stimulatedthe expression of NLRP3 as well as AIM2 at the mRNA and pro-tein levels.

TLRs are expressed in multiple cell types at the site of peri-odontal infection, and it was previously shown that P. gingivalissignals inflammatory cytokine production from mouse peritonealmacrophages through both TLR2 and TLR4 (27, 28). Here, wefound that P. gingivalis utilizes both TLR2 and TLR4 for stimula-tion of IL-1� secretion in THP-1 cells. The specific response ini-tiated by individual TLRs depends on the recruitment of a singleadaptor protein or combinations of adaptor proteins, such asMyD88, TIRAP, and TRIF. TLR2 and/or TLR4 recruits MyD88and TRIF to activate NF-�B and mitogen-activated protein ki-nases for induction of inflammatory cytokines. We found thatthe adaptor proteins MyD88 and TRIF are required for theeffects of P. gingivalis infection on caspase-1 activation and

FIG 7 Infection with P. gingivalis triggers the activation of NLRP3 and AIM2 inflammasomes via TLR2 and TLR4 signaling, leading to IL-1� secretion andpyroptic cell death.

Inflammasome Activation by P. gingivalis Infection

January 2014 Volume 82 Number 1 iai.asm.org 121

on April 22, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

IL-1� secretion. These results imply that the transcription ofNLRP3, AIM2, and pro-IL-1� via TLR signaling is a prerequi-site for P. gingivalis-induced inflammasome activation andIL-1� secretion.

In summary, infection with P. gingivalis triggered the activa-tion of NLRP3 and AIM2 inflammasomes via TLR2 and TLR4signaling, leading to IL-1� secretion and pyroptic cell death. Inaddition, P. gingivalis-induced NLRP3 inflammasome activationwas dependent on ATP release, K efflux, and cathepsin B (Fig. 7).Our study provides novel insight into the innate immune re-sponse against P. gingivalis infection which could potentially beused for the potential prevention and therapy of periodontitis.

ACKNOWLEDGMENTS

We thank Jeomil Choi and Juyeon Lee of Pusan National University Den-tal Hospital for providing GCF and the gingival tissues used in this study.

A National Research Foundation of Korea (NRF) grant funded by theSouth Korea government (MEST; no. 2012R1A2A2A01015470) sup-ported this research.

REFERENCES1. Dinarello CA. 2009. Immunological and inflammatory functions of the

interleukin-1 family. Annu. Rev. Immunol. 27:519 –550. http://dx.doi.org/10.1146/annurev.immunol.021908.132612.

2. Medzhitov R. 2001. Toll-like receptors and innate immunity. Nat. Rev.Immunol. 1:135–145. http://dx.doi.org/10.1038/35100529.

3. Akira S, Takeda K. 2004. Toll-like receptor signalling. Nat. Rev. Immu-nol. 4:499 –511. http://dx.doi.org/10.1038/nri1391.

4. Martinon F, Mayor A, Tschopp J. 2009. The inflammasomes: guardiansof the body. Annu. Rev. Immunol. 27:229 –265. http://dx.doi.org/10.1146/annurev.immunol.021908.132715.

5. Pedra JH, Cassel SL, Sutterwala FS. 2009. Sensing pathogens and dangersignals by the inflammasome. Curr. Opin. Immunol. 21:10 –16. http://dx.doi.org/10.1016/j.coi.2009.01.006.

6. Schroder K, Tschopp J. 2010. The inflammasomes. Cell 140:821– 832.http://dx.doi.org/10.1016/j.cell.2010.01.040.

7. Broz P, Monack DM. 2011. Molecular mechanisms of inflammasomeactivation during microbial infections. Immunol. Rev. 243:174 –190. http://dx.doi.org/10.1111/j.1600-065X.2011.01041.x.

8. Fernandes-Alnemri T, Yu JW, Datta P, Wu J, Alnemri ES. 2009. AIM2activates the inflammasome and cell death in response to cytoplasmicDNA. Nature 458:509 –513. http://dx.doi.org/10.1038/nature07710.

9. Miao EA, Rajan JV, Aderem A. 2011. Caspase-1-induced pyroptotic celldeath. Immunol. Rev. 243:206 –214. http://dx.doi.org/10.1111/j.1600-065X.2011.01044.x.

10. Bauernfeind FG, Horvath G, Stutz A, Alnemri ES, MacDonald K, SpeertD, Fernandes-Alnemri T, Wu J, Monks BG, Fitzgerald KA, Hornung V,Latz E. 2009. Cutting edge: NF-kappaB activating pattern recognition andcytokine receptors license NLRP3 inflammasome activation by regulatingNLRP3 expression. J. Immunol. 183:787–791. http://dx.doi.org/10.4049/jimmunol.0901363.

11. Franchi L, Eigenbrod T, Munoz-Planillo R, Nunez G. 2009. The inflam-masome: a caspase-1-activation platform that regulates immune re-sponses and disease pathogenesis. Nat. Immunol. 10:241–247. http://dx.doi.org/10.1038/ni.1703.

12. Lamont RJ, Jenkinson HF. 1998. Life below the gum line: pathogenicmechanisms of Porphyromonas gingivalis. Microbiol. Mol. Biol. Rev. 62:1244 –1263.

13. Pathirana RD, O’Brien-Simpson NM, Reynolds EC. 2010. Host immuneresponses to Porphyromonas gingivalis antigens. Periodontology 2000 52:218 –237. http://dx.doi.org/10.1111/j.1600-0757.2009.00330.x.

14. Haffajee AD, Socransky SS. 1994. Microbial etiological agents of destruc-tive periodontal diseases. Periodontology 2000 5:78 –111. http://dx.doi.org/10.1111/j.1600-0757.1994.tb00020.x.

15. Grenier D, Tanabe S. 2010. Porphyromonas gingivalis gingipains trigger aproinflammatory response in human monocyte-derived macrophagesthrough the p38alpha mitogen-activated protein kinase signal transductionpathway. Toxins 2:341–352. http://dx.doi.org/10.3390/toxins2030341.

16. Wilensky A, Polak D, Houri-Haddad Y, Shapira L. 2013. The role of

RgpA in the pathogenicity of Porphyromonas gingivalis in the murineperiodontitis model. J. Clin. Periodontol. 40:924 –932. http://dx.doi.org/10.1111/jcpe.12139.

17. Zhang D, Chen L, Li S, Gu Z, Yan J. 2008. Lipopolysaccharide (LPS) ofPorphyromonas gingivalis induces IL-1beta, TNF-alpha and IL-6 productionby THP-1 cells in a way different from that of Escherichia coli LPS. InnateImmun. 14:99–107. http://dx.doi.org/10.1177/1753425907088244.

18. Zhou Q, Amar S. 2006. Identification of proteins differentially expressedin human monocytes exposed to Porphyromonas gingivalis and its puri-fied components by high-throughput immunoblotting. Infect. Immun.74:1204 –1214. http://dx.doi.org/10.1128/IAI.74.2.1204-1214.2006.

19. Taxman DJ, Zhang J, Champagne C, Bergstralh DT, Iocca HA, Lich JD,Ting JP. 2006. Cutting edge: ASC mediates the induction of multiplecytokines by Porphyromonas gingivalis via caspase-1-dependent and -in-dependent pathways. J. Immunol. 177:4252– 4256.

20. Hwang I, Park S, Hong S, Kim EH, Yu JW. 2012. Salmonella promotesASC oligomerization-dependent caspase-1 activation. Immune Netw. 12:284 –290. http://dx.doi.org/10.4110/in.2012.12.6.284.

21. Fernandes-Alnemri T, Wu J, Yu JW, Datta P, Miller B, Jankowski W,Rosenberg S, Zhang J, Alnemri ES. 2007. The pyroptosome: a supramo-lecular assembly of ASC dimers mediating inflammatory cell death viacaspase-1 activation. Cell Death Differ. 14:1590 –1604. http://dx.doi.org/10.1038/sj.cdd.4402194.

22. Mariathasan S, Weiss DS, Newton K, McBride J, O’Rourke K, Roose-Girma M, Lee WP, Weinrauch Y, Monack DM, Dixit VM. 2006.Cryopyrin activates the inflammasome in response to toxins and ATP.Nature 440:228 –232. http://dx.doi.org/10.1038/nature04515.

23. Petrilli V, Papin S, Dostert C, Mayor A, Martinon F, Tschopp J. 2007.Activation of the NALP3 inflammasome is triggered by low intracellularpotassium concentration. Cell Death Differ. 14:1583–1589. http://dx.doi.org/10.1038/sj.cdd.4402195.

24. Walev I, Reske K, Palmer M, Valeva A, Bhakdi S. 1995. Potassium-inhibited processing of IL-1 beta in human monocytes. EMBO J. 14:1607–1614.

25. Hornung V, Bauernfeind F, Halle A, Samstad EO, Kono H, Rock KL,Fitzgerald KA, Latz E. 2008. Silica crystals and aluminum salts activatethe NALP3 inflammasome through phagosomal destabilization. Nat. Im-munol. 9:847– 856. http://dx.doi.org/10.1038/ni.1631.

26. Hentze H, Lin XY, Choi MS, Porter AG. 2003. Critical role for cathepsinB in mediating caspase-1-dependent interleukin-18 maturation andcaspase-1-independent necrosis triggered by the microbial toxin nigeri-cin. Cell Death Differ. 10:956 –968. http://dx.doi.org/10.1038/sj.cdd.4401264.

27. Hajishengallis G, Martin M, Sojar HT, Sharma A, Schifferle RE, De-Nardin E, Russell MW, Genco RJ. 2002. Dependence of bacterial proteinadhesins on Toll-like receptors for proinflammatory cytokine induction.Clin. Diagn. Lab. Immunol. 9:403– 411. http://dx.doi.org/10.1128/CDLI.9.2.403-411.2002.

28. Zhou Q, Desta T, Fenton M, Graves DT, Amar S. 2005. Cytokineprofiling of macrophages exposed to Porphyromonas gingivalis, its lipo-polysaccharide, or its FimA protein. Infect. Immun. 73:935–943. http://dx.doi.org/10.1128/IAI.73.2.935-943.2005.

29. Kuula H, Salo T, Pirila E, Tuomainen AM, Jauhiainen M, Uitto VJ,Tjaderhane L, Pussinen PJ, Sorsa T. 2009. Local and systemic responsesin matrix metalloproteinase 8-deficient mice during Porphyromonas gin-givalis-induced periodontitis. Infect. Immun. 77:850 – 859. http://dx.doi.org/10.1128/IAI.00873-08.

30. Hernandez M, Dutzan N, Garcia-Sesnich J, Abusleme L, Dezerega A,Silva N, Gonzalez FE, Vernal R, Sorsa T, Gamonal J. 2011. Host-pathogen interactions in progressive chronic periodontitis. J. Dent. Res.90:1164 –1170. http://dx.doi.org/10.1177/0022034511401405.

31. Yue Y, Liu Q, Xu C, Loo WT, Wang M, Wen G, Cheung MN, Bai LJ,Dou YD, Chow LW, Hao L, Tian Y, Li JL, Yip AY, Ng EL. 2013.Comparative evaluation of cytokines in gingival crevicular fluid and salivaof patients with aggressive periodontitis. Int. J. Biol. Markers 28:e108 –e112. http://dx.doi.org/10.5301/jbm.5000014..

32. Miao EA, Leaf IA, Treuting PM, Mao DP, Dors M, Sarkar A, WarrenSE, Wewers MD, Aderem A. 2010. Caspase-1-induced pyroptosis is aninnate immune effector mechanism against intracellular bacteria. Nat.Immunol. 11:1136 –1142. http://dx.doi.org/10.1038/ni.1960.

33. Belibasakis GN, Guggenheim B, Bostanci N. 2013. Down-regulation ofNLRP3 inflammasome in gingival fibroblasts by subgingival biofilms: in-

Park et al.

122 iai.asm.org Infection and Immunity

on April 22, 2020 by guest

http://iai.asm.org/

Dow

nloaded from

volvement of Porphyromonas gingivalis. Innate Immun. 19:3–9. http://dx.doi.org/10.1177/1753425912444767.

34. Leemans JC, Cassel SL, Sutterwala FS. 2011. Sensing damage by theNLRP3 inflammasome. Immunol. Rev. 243:152–162. http://dx.doi.org/10.1111/j.1600-065X.2011.01043.x.

35. Hornung V, Ablasser A, Charrel-Dennis M, Bauernfeind F, Horvath G,Caffrey DR, Latz E, Fitzgerald KA. 2009. AIM2 recognizes cytosolicdsDNA and forms a caspase-1-activating inflammasome with ASC. Na-ture 458:514 –518. http://dx.doi.org/10.1038/nature07725.

36. Fernandes-Alnemri T, Yu JW, Juliana C, Solorzano L, Kang S, Wu J,Datta P, McCormick M, Huang L, McDermott E, Eisenlohr L, Landel

CP, Alnemri ES. 2010. The AIM2 inflammasome is critical for innateimmunity to Francisella tularensis. Nat. Immunol. 11:385–393. http://dx.doi.org/10.1038/ni.1859.

37. Kim S, Bauernfeind F, Ablasser A, Hartmann G, Fitzgerald KA, Latz E,Hornung V. 2010. Listeria monocytogenes is sensed by the NLRP3 andAIM2 inflammasome. Eur. J. Immunol. 40:1545–1551. http://dx.doi.org/10.1002/eji.201040425.

38. Wu J, Fernandes-Alnemri T, Alnemri ES. 2010. Involvement of theAIM2, NLRC4, and NLRP3 inflammasomes in caspase-1 activation byListeria monocytogenes. J. Clin. Immunol. 30:693–702. http://dx.doi.org/10.1007/s10875-010-9425-2.

Inflammasome Activation by P. gingivalis Infection

January 2014 Volume 82 Number 1 iai.asm.org 123

on April 22, 2020 by guest

http://iai.asm.org/

Dow

nloaded from