Background
Chronic periodontitis, one of the most common and prevalent diseases in humans worldwide [
1], is defined as an infection-driven chronic inflammatory disease of the periodontium resulting in the destruction of gingival tissue, absorption of alveolar bone and eventually tooth loss [
2]. When infection occurs, the innate immune system will be the first line defense against pathogens and activates the adaptive immune system for sustained protection from such invasions [
3].
The intracellular multi-protein complexes, known as “NLR inflammasomes” play a central role in innate immunity. Among inflammasomes, the pyrin domain-containing-3 (NLRP3) inflammasome is the most studied [
4]. Gingival epithelial cells express a functional NLRP3 inflammasome that can be activated by pathogen- or danger-associated molecular patterns (PAMPs or DAMPs) [
4]. NLRP3 is assembled with the adaptor protein ASC (apoptosis-associated speck-like protein) into a multi-protein complex that governs caspase-1 activation and subsequent maturation the of pro-inflammatory cytokines interleukin (IL)-1β and/or IL-18 in the host response to periodontal infection [
4]. Gingival epithelial cells can sense and recognize PAMPs or DAMPs [
5] through stimulation of pathogen recognition receptors (PRRs) [
1,
3], with subsequent release of the pro-inflammatory cytokines IL-1β and IL-18 [
6]. The fundamental work by Bostanci et al. [
7] first indicated that NLR inflammasomes are involved in periodontal disease,which responses to
P. gingivalis infection both clinical and
in vitro studies.
Interleukin-1β (IL-1β), a proinflammatory cytokine belonging to the IL-1 family, is critical in the host defense against microbial infections [
5] and regulates innate immune and inflammatory responses. IL-18 is another proinflammatory cytokine belonging to the IL-1 family [
6] and has recently been described as an important element in the inflammasome system that activates caspase-1 and leads to the activation of the inflammation process. Recent evidence has demonstrated that the maturation and secretion of IL-1β and IL-18 are regulated by the NLRP3 inflammasome complex which contains the NLRP3 scaffold, caspase-1 and apoptotic speck protein containing a C-terminal caspase recruitment domain (ASC) [
8]. Excess IL-1β and IL-18 contribute to an increasing number of human inflammatory responses [
9]. Although IL-1β and IL-18 belong to the same cytokine family, their gene expression and secretion are differentially regulated in human monocytic cells in response to
P. gingivalis [
10]. Therefore, cytokines of the IL-1 family may participate
via different pathways in the complex pathogenesis of periodontitis [
10].
P. gingivalis, a gram-negative anaerobic bacterium [
11], has been confirmed to be a predominant periodontal pathogen [
12] and produces a number of potential virulence factors to perturb the host defense system [
13] and induce an inflammatory response in periodontal diseases [
14]. However, the effect of
P. gingivalis on activation of the NLRP3 inflammasome remains controversial. Lipopolysaccharide (LPS), as the major cell wall component of
P. gingivalis [
15], is considered an important virulence factor eliciting the inflammatory response in the periodontal disease [
16]. It seems to be unanimously agreed that LPS treatment of mononuclear phagocytes, [
17] macrophages [
18] and oral epithelial cells [
19] significantly induces the expression of NLRP3 and procaspase-1 at both the mRNA and protein levels. Studies have shown that any functional polymorphism in LPS-receptors affects the inflammatory process and the clinical outcomes of periodontal disease [
20]. The findings outlined above suggest that whole
P. gingivalis and
P. gingivalis LPS may have different effects on the activation of NLRP3 inflammasome system.
Extracellular ATP (adenosine triphosphate), one of the first activators described to induce NLRP3 inflammasome formation, is ascribed to the group of endogenous DAMPs released by dying or injured cells [
21,
22]. Its presence is negligible in healthy tissues, but may rise to high micromolar levels following tissue damage at sites of inflammation [
23]. Studies have shown ATP induced caspase-1 activation and subsequent IL-1β release [
24,
25]. Furthermore, Özlem et al. demonstrated that IL-1β was not secreted unless LPS-treated or infected gingival epithelial cells (GECs) were subsequently stimulated with ATP, and that ATP had no additional effect on NLRP3 or ASC expression in
P. gingivalis infected GECs [
26]. These results point out the need to further evaluate the role of ATP in NLRP3 inflammasome activation.
Therefore, the activation of the NLRP3 inflammasome complex in a cultured oral epithelial cell model by P. gingivalis infection, or P. gingivalis LPS, or ATP stimulation was tested. This provided further understanding of the mechanism behind periodontal inflammation.
Methods
Oral epithelial cell culture
The epithelial cell line (H413) derived from a human oral squamous cell carcinoma [
27], displays stratified epithelial cell morphology in culture. H413 cloned cell lines were established using a limit dilution method as described previously [
28]. The cloned cells were cultured in Eagle’s Minimum Essential Medium (JMEM, Joklik modification, Sigma-Aldrich, St Louis, MO, USA), penicillin/streptomycin (100 IU/ml, Sigma) and 10 % fetal calf serum (FCS, CSL Limited, Victoria, Australia) at 37 °C in 5 % CO
2 [
29]. Cultures were harvested with triple express (replacement for trypsin, Invitrogen, Life Technologies, Carlsbad, CA, USA) in PBS and sub-cultured every 3 days.
Bacterial cell culture
Porphyromonas gingivalis (ATCC 33277) was cultured anaerobically for 24 h at 37 °C in a trypticase soy broth supplemented with haemin (5 mg/ml, Sigma) and menadione (1 mg/ml, Sigma). On the day of cell treatment, bacteria were centrifuged at 5000 rpm, and 4 °C for 15 min, washed twice and re-suspended in cold PBS, pH 7.3.
Cell treatment
Confluent H413 cell cultures (5 × 10
6 cells in T-25 cm
2 flasks) were washed three times with PBS and infected with
P. gingivalis at a multiplicity of infection (MOI) of 100 bacterial cells per one epithelial cell [
30] for 2 and 4 h [
31,
32], or stimulated with 1-μg/mL ultrapure lipopolysaccharide (LPS) from
P. gingivalis (Invivogen, San Diego, CA, USA) in the cell growth media for 2 and 4 h. Uninfected and non-stimulated cells served as controls.
Experiments were also carried out after pre-incubating the cells with 5 mM adenosine triphosphate (ATP) (Invivogen) for 3 h prior to infection with P. gingivalis or stimulation with P. gingivalis LPS for 2 and 4 h. Cells pre-incubated with 5 mM ATP for 3 h were as controls for these groups.
RNA isolation and quantitative real-time RT-PCR
After treatment, cells were harvested in 1 ml of Trizol reagent (Invitrogen) and RNA extracted as per the Trizol protocol. For reverse transcription, the First-Strand cDNAs were synthesized with oligo (dT)12–18 (Invitrogen), 10 mM dNTP (Promega, Madison, WI, USA), 5× first stand buffer, RNaseOUT™ Recombinant RNase Inhibitor (Invitrogen) and SuperScript™ III Reverse Transcriptase (Invitrogen) according to the manufacturer’s protocol.
Primers for genes encoding inflammasome components (NLRP3, ASC and caspase-1) and the cytokines IL-1β and IL-18 (Table
1) were designed using Oligo Explorer software (1.1.0) and synthesised by Integrated DNA Technologies (IDT, Coralville, IA, USA). Real-time RT-PCR analyses were performed by SYBR Green based assays using the Stratagene MxPro-Mx3005P System (Agilent Technologies, Santa Clara, CA, USA). PCR reactions were conducted with 2 μl of diluted cDNA samples, 200 nM of each respective forward and reverse primer in a 20 μl final reaction mixture with Platinum SYBR Green qPCR SuperMix-UDG (Invitrogen). cDNA samples isolated from non-manipulated H413 clone-1 cells were quantified by PicoGreen kit (Invitrogen) and then used for constructing standard curves (2–2000 pg) by reference to the expression of the house keeping gene encoding β-actin. The PCR reactions for each gene were carried out in triplicate in 96-well plates, and initiated by activation at 95 °C for 2 min, followed by 40 PCR cycles of denaturation at 95 °C for 15 s, annealing and extension at 60 °C for 30 s. The results were analysed using MxPro 4.10 software.
Table 1
Primers used for real-time RT-PCR
NLRP3 | F-primer | GCTGGACCTGAGTGACAAC | 151 bp | Hs.159483 |
R-primer | GCTGAGTACCGAGGACAAAG |
ASC | F-primer | AGGCCTGCACTTTATAGACC | 174 bp | Hs.499094 |
R-primer | GCTGGTGTGAAACTGAAGAG |
caspase-1 | F-primer | GAAAAGCCATGGCCGACAAG | 205 bp | Hs.2490 |
R-primer | GCCCCTTTCGGAATAACGGA |
IL-1β | F-primer | GGCCCTAAACAGATGAAGTG | 90 bp | Hs.126256 |
R-primer | GTAGTGGTGGTCGGAGATTC |
IL-18 | F-primer | GCATCAACTTTGTGGCAAT | 161 bp | Hs.83077 |
R-primer | CCGATTTCCTTGGTCAAT |
β-actin | F-primer | ACTCTTCCAGCCTTCCTTC | 216 bp | Hs.520640 |
R-primer | GGAGCAATGATCTTGATCTTC |
Immunoassay-ELISA to quantify levels of IL-1β and IL-18
A standard sandwich enzyme-linked immuno-sorbent assay (ELISA) was used to measure cytokine production of IL-1β and IL-18. Briefly, supernatants of test (treated with P. gingivalis, P. gingivalis LPS, ATP+ P. gingivalis or ATP+ P. gingivalis LPS) and control cell cultures were collected at each hour (1, 2, 3, 4, 5, 6 h), then particles removed by centrifugation and analysed immediately or aliquoted and stored at −20 °C. Human IL-1β and IL-18 specific monoclonal and polyclonal antibodies (1 μg/ml) were pre-coated onto high binding 96-well plates (Corning Incorporated, USA) in carbonate buffer (pH 9.0) at 4 °C overnight. After removing the coating solution and washing the plate three times with 200-μl PBS/well, with the plate was blocked with 3 % bovine serum albumin (BSA, Sigma) at 4 °C overnight. The test samples were added to triplicate wells for 90 min at room temperature, and followed by washing with 0.05 % Tween20/PBS (TPBS) three times; then incubated with secondary antibody (goat-anti mouse/rabbit IgG, (DAKO, Glostrup, Denmark) conjugated with alkaline phosphatase (AP)) diluted 1:1500 in TPBS for 2 h at room temperature then washed with TPBS buffer 3 times. Bound conjugates were detected by pNPP (p-Nitrophenyl-phosphate) and the absorbance measured at 405 nm in a microplate reader (Bio-Rad, Hercules, CA, USA) after 15–30 min incubation at room temperature. Reactions were stopped by adding an equal volume of 1.00 M NaOH. The human IL-1β and IL-18 concentration of the samples were interpreted from a standard curve.
Immunoblots for NLRP3, ASC and caspase-1 proteins
To measure inflammasome protein expression, 2- and 4-h cultures of the different conditions (treated with P. gingivalis, P. gingivalis LPS, ATP 3 h + P. gingivalis or ATP 3 h + P. gingivalis LPS) were extracted in SDS sample buffer and separated by PAGE using gradient 5 to 12 % mini-gels, transferred to nitrocellulose membranes (Bio-Rad) and blocked overnight with 3 % BSA (Sigma) in 0.1 M Tris buffered salts solution pH 7.4 (TBS). Blotted antigens were incubated with rabbit polyclonal anti-human antibodies CIAS1/NALP3, TMS1/ASC, Caspase-1 (1 μg/ml, Abcam, Cambridge, UK), and β-actin (0.1 μg/ml, GenTex, Zeeland, MI, USA) as a loading control in 0.05 % Tween20/TBS for 4 h, washed three times and subsequently incubated with alkaline phosphatase (AP)-conjugated secondary antibody (goat-anti rabbit IgG, DAKO) diluted 1:1500 in Tween20/TBS for 2 h. Bound antibody was visualized with AP substrate (Bio-Rad) after development of reactivity for proteins from control antibody.
Statistical analysis
All data were analysed by paired
t-test (mean ± S.D., two-tailed, 95 % CI range) from at least three consecutive experiments for real-time RT-PCR and ELISA. For western blot quantification, the densitometric analysis was performed on the grey level intensity of target bands relative to control β-actin bands derived from scanned films, processed by using Gene Tool image analysis software (GeneToos, version 4.02; Syngene, Cambridge, UK) [
33].
P < 0.05 was considered statistically significant.
Discussion
In this study,
P. gingivalis infection down-regulates NLRP3 inflammasome components, including NLRP3, ASC and caspase-1 in epithelial cells at all time points, which indicates that
P. gingivalis may either up-regulate or down-regulate NLRP3 expression, depending on the cell type [
35]. In this model,
P. gingivalis may dampen the endpoint innate immune responses by inhibiting the activation of NLRP3 inflammasome and evading host surveillance, offering a survival advantage to all co-habiting organisms of the biofilm [
35]. Meanwhile, up-regulated gene expression of IL-1β after
P. gingivalis infection, demonstrates that IL-1β is a critical cytokine in the host defence against
P. gingivalis infection [
5]. Moreover, IL-1β is not NLRP3 or caspase-1 dependent [
5,
36] and other factors may also influence the IL-1β protein secretion. Therefore, we speculate that the secretion of IL-1β in the early stages of
P. gingivalis infection would play a very important role in combating the invading pathogen as part of the innate immune response [
37]. This is consistent with Dinarello’s conclusion that IL-1β is one of the earliest cytokines to be secreted during the initial phases of inflammation and participates in almost all events involved in the activation and regulation of inflammation [
36]. This kind of inflammasome-independent IL-1β activation can substantially contribute to tissue inflammation [
38].
In the present study, LPS elicits a striking immune response through up-regulation of the gene expression of NLRP3 and ASC, but not caspase-1, which indicates that
P. gingivalis LPS would be a key factor in eliciting the inflammatory response that leads to the diseased state [
16] and is considered an important virulence factor in the pathogenesis of periodontal disease. However, Jain and Darveau’s research has elucidated that activation of NLRP3 and ASC after proinflammatory stimuli such as LPS may be involved in the apoptosis of host cells, which supports the idea of
P. gingivalis-induced cell death [
39], which would then facilitate periodonto-pathogens to invade and destroy epithelial tissues. In addition, caspase-1 is synthesised as an inactive zymogen. Its activation is tightly regulated by inflammasomes and associated with a rapid and lytic form of cell death known as pyroptosis [
40]. This could explain the mechanism that caspase-1 activation is inhibited after
P. gingivalis LPS stimulation until activated with ATP stimuli in the present study.
ATP is a very efficient extracellular distress signal [
41]. As one of the first activators described to induce NLRP3 inflammasome formation, it is ascribed to the group of endogenous DAMPs, which come from dying cells [
22]. In this study, we demonstrate that ATP activates the NLRP3 inflammasome, subsequently releasing cytokines IL-1 β and IL-18, even though the effect is very transient and the concentration may not be high enough to maintain the level of activated NLRP3 inflammasome. These findings support that extracellular ATP, as a danger signal, results in assembly of NLRP3 inflammasome and secretion of mature cytokines in
P. gingivalis-infected cells [
26]. In addition, a common denominator of extracellular ATP activating NLRP3 has the ability to form membrane pores that induce damage of membrane integrity or cause perturbation of the intracellular ion concentration [
22].
Moreover, after stimulation with ATP and
P. gingivalis LPS, the reduction of ASC gene expression may serve as a mechanism for shutting down inflammation, thus avoiding overzealous immune responses [
32]. Similarly, the epithelial cell line (H413) used in this study has the function to restrain the activity and secretion of IL-1β [
42] to protect the cell against the tissue destruction. This can explain our finding showing a postponed increase of IL-1β in response to ATP plus
P. gingivalis LPS stimulation.
Competing interests
All authors declare that they have no competing interests.
Authors’ contributions
WG performed the experimental studies and data analysis, and contributed to the draft of manuscript. PW contributed to data analysis of western blot. ZHL supported WG. and provided oversight for this study. PSY contributed to the revision of manuscript. PY provided oversight for this study and the revision of manuscript. All authors read and approved the final manuscript.