Expression of NLRP3 and P2X7 transcripts in gingival tissues of chronic periodontitis patients and its correlation with P. gingivalis load and periodontal parameters
Introduction
The prolonged persistence of periodontal pathogens and imbalance in host immune response results in destruction of periodontal tissues leading to chronic periodontitis(Schenkein, 2006). The innate immune system is crucial for the initial defense against inflammation caused by these pathogenic microorganism (Medzhitov and Janeway, 2000).
As first line of defense, the innate immune system heavily relies on the presence of Pattern Recognition Receptors (PRR's) (Akira et al., 2006). Several pathogen recognition receptors such as TLR (Toll like receptors), RIG-I like receptors, and NLR (NOD like receptors) expressed in various cell types sense and respond to “Pathogen Associated Molecular Patterns” and “Danger Associated Molecular Patterns”. The stimulation of these receptors ultimately leads to recruitment of “inflammasomes”(Xue et al., 2015; Yilmaz and Lee, 2015).
“Inflammasomes” are an oligomeric assembly of multiprotein complex. Certain PRRs such as NLRP3 requires an adaptor protein ASC (apoptosis-associated speck-like protein containing a caspase-recruitment domain) and second signaling by P2X7 to recruit pro-caspase1 to inflammasome complex which ultimately leads to the activation of caspase-1, an enzyme, which is responsible for maturation and secretion of pro inflammatory cytokines like IL-1ß and IL-18 that are key cytokines in periodontal pathogenesis(Dagenais et al., 2012). There are 14 members of the NLRP family of proteins of which the NLRP3 is well studied and documented in conditions such as Alzheimer's disease, and arthritis and a handful of studies also document its role in periodontitis (García-Hernández et al., 2019; Kersse et al., 2011). Inflammasome regulating proteins are also reported to be upregulated resulting in active periodontal disease(Aral et al., 2020). Polymorphisms of NLRP3 are reported to increase susceptibility to periodontitis(de Alencar et al., 2020). Therefore inflammasomes, specifically the NLRP3 family, are found to be key regulators of the host immune response in periodontal inflammation.
NLRP3 has to be primed before being activated and this appears possible by at least three factors as understood currently: reactive oxygen species (ROS), signaling by P2X7 and Cathepsin B from lysosomes. The most important is the second signaling by P2X7 receptor, a family of cation permeable ligand-gated ion channel, especially in gingival epithelial cells(Kelley et al., 2019; Virgilio et al., 2017). In some cells such as dendritic cells, studies report NLRP3 activation independently of P2X7 (He et al., 2013).
Both NLRP3 inflammasome and the P2X7 receptor is reported to be modulated by Porphyromonas gingivalis pathogen for its survival in periodontal tissues. Authors have claimed that Porphyromonas gingivalis (P. gingivalis) is known to modulate NLRP3 inflammasome complex by means of its virulence factors such as lipopolysaccharide, capsule, fimbriae, and cysteine proteases. P. gingivalis fimbriae inhibited extra cellular ATP (eATP) induced IL-1β secretion via P2X7 receptor by secreting an enzyme nucleoside diphosphate kinase(Morandini et al., 2014). This enzyme is also reported to inhibit ATP-induced reactive oxygen species production via the P2X7/NADPH oxidase signaling in order to persist in gingival tissues(Choi et al., 2013). Also P. gingivalis is reported to suppress the inflammasome activation through other bacteria like Fusobacterium, due to its synergism with the bacteria. P. gingivalis is also found to suppress the endocytic pathways, leading to “pathogen mediated inflammasome inhibition”(Taxman et al., 2012). P2X7 is said to play a dual role in both eATP induced IL-1β secretion as well as for processing of pro- IL-1β (Ramos-Junior et al., 2015).
There are a handful of in vitro studies using subgingival film models reporting expression of NLRP3 in periodontitis. Few clinical studies analyzing the expression of NLRP3 in gingival tissues and/or saliva in periodontitis patients are also present in literature, where the authors have reported a significant increase in expression of NLRP3 (Belibasakis et al., 2013; Bostanci et al., 2009; Isaza-Guzmán et al., 2017; Xue et al., 2015). There are no in vivo studies on expression of P2X7 in human gingival tissues so far.
The expression of the NLRP3 inflammasome and P2X7 receptor has been reported in in-vitro setting and using animal models(Belibasakis et al., 2013; Bostanci et al., 2009; Xue et al., 2015; Yilmaz and Lee, 2015). The present study aims to investigate the same in the interproximal gingival tissues of periodontitis patients. To the best of our knowledge, expression of P2X7 in gingival tissue of chronic periodontitis patients has not been documented.
We also intend to assess the correlation of the mRNA expression of NLRP3 and P2X7 with P. gingivalis load in subgingival plaque samples and periodontal clinical parameters if any is present. Modulation of expression of NLRP3 and P2X7 genes by P. gingivalis has been reported and documented earlier in few studies (Choi et al., 2013; Olsen and Yilmaz, 2016). Though modulation of gene expression by specific pathogens is best assessed in in-vitro conditions, we have attempted to assess if any correlation is present between P. gingivalis load in subgingival plaque samples and the expression of genes in gingival tissues of subjects with chronic generalized periodontitis.
Section snippets
Materials and methods
The study participants, both male and female, with age groups ranging 20–60 years, were recruited from the outpatient pool of Department of Periodontology, Indira Gandhi Institute of Dental Sciences (IGIDS), Pondicherry. The study was in compliance with the Helsinki declaration and was approved by the Institutional review board and ethics committee, of the institute. Informed consent was obtained from each participant. After assessment of periodontal status, the study participants were divided
Clinical parameters and demographic data
The mean standard deviation for age, PPD and CAL for both the groups were recorded. (Table 1).
mRNA expression of NLRP3 and P2X7 in gingival tissues
There was no statistically significant difference in mRNA expression of NLRP3 in periodontitis patients (test group) when compared with healthy controls even though the average Δct of test group was marginally higher (Δct 2.01) than the controls (Δct 1.28) (Fig. 1). Whereas, a statistically significant difference in expression of P2X7 was observed between test and control groups. Interestingly P2X7
Discussion
The NLRP3 inflammasome is known to play an essential part in the innate immune response and is found contributing to a number of inflammatory diseases including periodontitis. In the present study, we have compared the mRNA expression of NLRP3 and P2X7 in gingival tissues of subjects with advanced chronic periodontitis (test group) and healthy controls. The results showed a statistically insignificant increase in expression of NLRP3 in chronic patients when compared to tissues from healthy
CRediT authorship contribution statement
Pratebha Balu (PB): Conceptualization, Visualisation, Methodology, Investigation, Writing, Original draft preparation
Ananya Sweta Venkatesan (ASV): Methodology, Writing, Original draft preparation
Jananni Muthu (JM): Writing- Reviewing and Editing
Vignesh Mariappan: (VM): Methodology, Statistics
Adithan Chandrasekaran (AC): Reviewing and Editing
Agieshkumar Balakrishna Pillai (ABP): Methodology, Supervision, Data curation, reviewing and editing
Saravanakumar Ravindran (SR): Writing- Reviewing and
Declaration of competing interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Acknowledgment
We thank Clinbiocare Technology LLP, Chennai and Pondicherry Centre for Biological Science and Educational trust for providing instrumentation facility.
Ethical approval
Informed Consent was obtained from patients and the study was performed in accordance with the Declaration of Helsinki.
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