Regulation Of The Keratinocyte Inflammasome In Chronic Periodontitis

Back groundPeriodontal diseases are the main chronic infectious diseases of the oral cavity, which can affect the periodontal supporting tissues and/or result in the breakdown of the tooth-supporting tissues, even ultimately lead to resorption of the alveolar bone and tooth loss. Among these diseases, chronic periodontitis is the most common and prevalent chronic infections in humans worldwide.The gingival epithelium is a very important stratified squamous epithelium that interfaces between the external environment and prevents itself to be destroyed by the stimuli from outside, which can be classified into three types:the oral gingival epithelium; the sulcular epithelium and the junctional epithelium. The sulcular and the coronal margins of the junctional epithelium are in close contact with bacteria in the gingival sulcus and appear to be crucial sites with regard to the development of periodontal diseases. When the inflammation happened, amount of pathogenic microorganism including the periodontal pathogen, Porphyromonas gingivalis(Pg) begin to produce a broad array of potential virulence factors involve in tissue colonization and destroy the junction epithelium and the epithelial cells, which can sense and respond to the presence of bacteria, then make the epithelial barrier disappear, connective tissue attachment loss, bone resorption and finally convert to a pocket epithelium.The NLR family is an intracellular multiprotein complex containing different combinations of proteins, named’inflammasome’ and includes NODs, NLRPs, IPAP inflammasomes. Among these inflammasomes, NLRP3 inflammasome is the best-characterized multi-protein complex composed of the NLRP3, the ASC, and the caspase-1, which can recognize the pathogen- or danger-associated molecular patterns (PAMPs or DAMPs) and induces the maturation and secretion of IL-1β and the IL-18, which further lead to the recruitment of other immune cells to the affected cells or tissues and result in chronic inflammation and subsequent tissue damage.In this study, we will examine the inflammasome responses of the oral epithelial cell-culture model(H413 epithelial cell line) and try to elucidate this correlation with the affected periodontal tissues and tight junction proteins, which contributes to the further investigations into the immune mechanisms of NLRP3 inflammasome between bacteria and gingival epithelium and useful information for prevention and therapy of Periodontitis.Methods1. Tissue samplesGingival tissues were obtained with Ethics Committee approval and informed consent from 26 adult patients attending the periodontal clinic. Subjects had done the examination and X-ray strictly. Slides with sequential sections were stored in sealed boxes at -70℃ until required for immunostaining and confocal laser scanning microscopy.2. Oral epithelial cell cultureThe epithelial cell line (H413) derived from a human oralsquamous cell carcinoma displays stratified epithelialcell morphology and high CD24 expression in culture. The cloned cells were cultured in Eagle’s minimum essential medium (JMEM, Joklik modification, Sigma-Aldrich), penicillin/streptomycin(100 IU/ml, Sigma), and 10% fetal calf serum (FCS, CSL Limited, Victoria, Australia) at 378℃ in 5% CO2 Cultures were harvested with triple express (replacement for trypsin, Invitrogen, Australia) in PBS and sub-cultured every 3 days.3. Porphyromonas gingivalis culturePorphyromonas gingivalis (ATCC 33277) was cultured anaerobically for 24 h at 37℃ 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℃ for 15 min, washed twice and re-suspended in cold PBS, pH 7.3.4. Immunostaining and confocal laser scanning microscopyAll slides and cells were were treated with immunostaining. Confocal images were obtained with an Olympus Fluoview (FV) 1000 equipped with Olympus FV 10-MCPSU (405 nm,473 nm,633 nm) and NTT electronic Optil (559 nm) lasers. All fluorescence images prepared with confocal acquisition software (FV10-ASW 1.7) were stored and exported as TIF files.5. RNA isolation and quantitative real-time RT-PCRCells were harvested in 1ml of Trizol reagent (Invitrogen) and RNA extracted as per the Trizol protocol. For reverse transcription, the First-Strand cDNAs were synthesized with SuperScriptTM III Reverse Transcriptase (Invitrogen) according to the manufacturer’s (Invitrogen) protocol.6. Immunoassay-ELISA to quantify levels of IL-1β and IL-18A standard sandwich enzyme-linked immuno-sorbent assay (ELISA) was used to measure cytokine production of IL-1β and IL-18. 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℃.7. Immunoblots for NLRP3, ASC and caspase-1 proteins and tight junction proteinsProteins of the different conditions 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). Data were analysed after development of reactivity for proteins from control antibody.8. Statistical AnalysisAll data were analysed by Prism 3.03 software. paired t-test (mean ± S.D., two-tailed,95% CI range) was used for real-time RT-PCR and ELISA. P< 0.05 was considered statistically significant.Results1. Expression of NLRP3 and occludin in periodontal biopsiesComparison of minimally inflamed and periodontitis groups indicated statistically significantly different patterns to NLRP3 and occludin were observed between SE (Min/Perio) and JE/PE (Min/Perio).2. Inflammasome expression in response to P. gingivalis, LPS and ATP plus P. gingivalis / LPSThere was down-regulation of NLRP3 gene expression after P. gingivalis infection at 2 and 4 h compared with the control group. LPS elicits a striking immune response through up-regulation of the gene expression of NLRP3 and ASC, but not caspase-1. ATP activates the NLRP3 inflammasome, subsequently releasing cytokines IL-1β and IL-18. Meanwhile, gene expression of IL-1β up-regulated after P. gingivalis infection.3. Cytokines expression in response to P. gingivalis, LPS and ATP plus P. gingivalis / LPSThere was only up-regulated Claudin-4 gene expression after P. gingivalis infection compared with the control group. LPS elicits a striking immune response through up-regulation of the gene expression of Claudin-1、Claudin-15 and ZO-1. ATP activates the gene expression of Occludin、JAM-A、Claudin-1、Claudin-4、Claudin-15 and ZO-1.Conclusion1. P. gingivalis infection down-regulates NLRP3 inflammasome components and tight junction proteins except Claudin-4 in epithelial cells, which helps bacteria to infect cells more deeply.2. LPS elicits a striking immune response through up-regulation of the gene expression of NLRP3 and ASC, but down-regulation of the gene expression of Occludin、JAM-A and claudin-4, which indicates that LPS would be a key factor in eliciting the inflammatory response that leads to the diseased state and is considered an important virulence factor in destroying the first defense barrier of gingival epithelia cells.3. ATP activates the NLRP3 inflammasome, tight junction proteins, subsequently releasing cytokines IL-1β and IL-18, which support that extracellular ATP, as a danger signal, results in form of membrane pores that induce damage of membrane integrity or cause perturbation of the intracellular ion concentration.4. Caspase-1 is synthesised as an inactive zymogen. Its activation is inhibited after LPS stimulation until activated with ATP stimuli, which means caspase-1 is tightly regulated by inflammasomes.5. IL-1β is a critical cytokine in the host defence and its secretion 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.