Role and mechanism of the nod-like receptor family pyrin domain-containing 3 inflammasome in oral disease

Accepted Manuscript Title: Role and Mechanism of the Nod-Like Receptor Family Pyrin Domain-Containing 3 Inflammasome in Oral Disease Authors: Kejia Lv, Guohua Wang, Chenlu Shen, Xia Zhang, Hua Yao PII: DOI: Reference:

S0003-9969(18)30658-7 https://doi.org/10.1016/j.archoralbio.2018.10.003 AOB 4265

To appear in:

Archives of Oral Biology

Received date: Revised date: Accepted date:

14-5-2018 2-10-2018 3-10-2018

Please cite this article as: Lv K, Wang G, Shen C, Zhang X, Yao H, Role and Mechanism of the Nod-Like Receptor Family Pyrin DomainContaining 3 Inflammasome in Oral Disease, Archives of Oral Biology (2018), https://doi.org/10.1016/j.archoralbio.2018.10.003 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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Role and Mechanism of the Nod-Like Receptor Family Pyrin Domain-Containing 3 Inflammasome in Oral Disease

Kejia Lv1, Guohua Wang1, Chenlu Shen1, Xia Zhang2, Hua Yao1* 1: Department of Stomatology, First Affiliated Hospital, College of Medicine, Zhejiang University

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2: Department of Stomatology, Affiliated Yinzhou People Hospital, College of Medicine, Ningbo University

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*Corresponding author: Hua Yao Tel: (86)0571-87236338; E-mail: [email protected]

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Address: Department of Stomatology, First Affiliated Hospital, College of Medicine, Zhejiang

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University, No.79, Qing-chun road, Shang-cheng region, Hangzhou, Zhejiang, 310003, China.

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Highlights

Crucial molecular regulation of the NLRP3 inflammasome.

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Representative microbes and their ability to trigger and activate the

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inflammasome.

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The NLRP3 inflammasome participates in the process of several oral diseases.

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Objective: To summarize evidence and data from experimental studies regarding the role and mechanism of the Nod-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome in the pathogenesis of several representative oral diseases. Materials and methods: A literature search of PubMed and EBSCO was performed. The literature was searched using a combination of keywords, e.g., NLRP3 inflammasome, inflammation, microorganisms, oral inflammatory diseases, and oral immunological diseases. Results: The initiation and activation of the NLRP3 inflammasome are associated with the pathogenesis and progression of several representative oral diseases, including periodontitis, oral lichen planus, dental pulp disease, and oral cavity squamous cell carcinoma. Conclusions: The NLRP3 inflammasome plays a crucial role in the progression of inflammatory and adaptive immune responses. The possible role of the NLRP3 inflammasome in several oral diseases, including not only periodontitis and pulpitis but also mucosal diseases and oral cavity squamous cell carcinoma, may involve the aberrant regulation of inflammatory and immune responses. Understanding the cellular and molecular biology of the NLRP3 inflammasome is necessary because the NLRP3 inflammasome may be a potential therapeutic target for the treatment and prevention of oral inflammatory and immunological diseases.

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1Abbreviation：

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NLRP, Nod-like receptors-pyrin domain; IL, interleukin; Th17, T helper 17 cell; Treg, regulatory T cell; FOXP3, forkhead-box protein 3; RORγτ, retinoid‐acid receptor‐related orphan receptor gammaτ; LPS, Lipopolysaccharide; P.gingivalis, Porphyromonas gingivalis; A.actinomycetemcomitans, Aggregatibacter actinomycetemcomitans;

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NADPH, nicotinamide adenine dinucleotide phosphate.

Keywords: inflammation; microorganisms; NLRP3 inflammasome; oral

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inflammatory diseases; oral immunological diseases

Introduction:

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The innate immune system is the first line of defence against pathogen invasion and exogenous stimuli(Hato & Dagher, 2015; S. Lee, G. Y. Suh, S. W. Ryter, & A. M. K. Choi, 2016b). This system recognizes a large variety of pathogen-associated molecular patterns that are present during microbial infection as well as damageassociated molecular patterns that are produced during cell damage and tissue injury(Iwasaki & Medzhitov, 2015; Martin, 2014). Molecular sensing of pathogen/damage-associated molecular patterns is achieved by germline-encoded pattern recognition receptors, including the toll-like receptor family, the Nodlike receptor family, the C-type lectin receptor family and the absent in melanoma 2like receptor family(Kigerl, de Rivero Vaccari, Dietrich, Popovich, & Keane, 2014; Odendall & Kagan, 2017). Upon pattern recognition or cytokine receptor activation, 2

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these activated pattern recognition receptors initiate transcriptional signalling pathways, such as the transcriptional nuclear factor-kappa B pathway, which, in turn, mediate innate immune responses and produce pro-inflammatory cytokines. This process is also a prerequisite for adaptive immunity. Nod-like receptor contains a three-domain homologous region that includes an N-terminal effector domain, a central nucleotide-binding oligomerization domain and a C-terminal leucine-rich repeat domain, and this receptor can be further subdivided according to their variable N-termini(H. Guo, Callaway, & Ting, 2015; Sutterwala, Haasken, & Cassel, 2014; Ting et al., 2008). To date, five or more pattern recognition receptors have been reported to oligomerize and form inflammasomes, including Nod-like receptors-pyrin domain (NLRP) 1 and 3(H. Guo et al., 2015; S. Lee, G. Y. Suh, S. W. Ryter, & A. M. Choi, 2016a). Inflammasome formation is a response to cellular infection, cellular stress or tissue damage(Olsen & Yilmaz, 2016). The NLRP3 inflammasome is the most comprehensively characterized inflammasome with a wide array of stimuli. NLRP3 forms an inflammasome with apoptosis-associated speck-like protein containing a caspase-recruitment domain and pro-caspase-1(Zhou, Yazdi, Menu, & Tschopp, 2011). As a crucial intracellular multiprotein scaffold, the NLRP3 inflammasome contributes to the self-cleavage of dormant pro-caspase-1 into bio-active caspase-1, which in turn converts the precursors of pro-interleukin(IL)-1β and -18 into their final bio-active forms(He, Hara, & Nunez, 2016), (Liston & Masters, 2017; Stutz et al., 2017). Interleukin-1β and interleukin-18 are secreted and circulate systemically. Both cytokines are significant effectors that mediate inflammatory responses and adaptive immune reactions(Dinarello, 2009; D. Liu, Zeng, Li, Mehta, & Wang, 2017). In particular, IL-1β and IL-18 activity represent a focus of the host immune response in periodontitis(Delaleu & Bickel, 2004; Orozco, Gemmell, Bickel, & Seymour, 2006). Interleukin-1β, which was originally identified in the context of inducing fever and promoting T cell survival, plays a significant role in driving T helper 17 cells(Th17) and certain populations of innate lymphocytes(Mills, Dungan, Jones, & Harris, 2013). IL-18, which is a well-known interferon gamma-inducing factor(Garlanda, Dinarello, & Mantovani, 2013), has been reported to be accumulated in many chronic inflammatory disorders. Similar to IL-1β, IL-18 also regulates Th17 cell differentiation and forkhead-box protein 3(FOXP3) regulatory T cells(Treg) in intestinal epithelial cells(Harrison et al., 2015). Recent studies have shown that caspase-1-processed IL-1β and IL-18 promote IL-17 secretion and mediate autoimmunity by CD4 T cells(Harrison et al., 2015; Lalor et al., 2011). Two sequential steps, priming and triggering, are required for full activation of the NLRP3 inflammasome in macrophages and dendritic cells(Elliott & Sutterwala, 2015). First, toll-like receptors or other cytokine receptors discern pathogenassociated molecular patterns/damage-associated molecular patterns/environmental stress and regulate the transcription nuclear factor-kappa B, which induces the expression and stimulation of NLRP3, pro-IL-1β and pro-IL-18, as well as post3

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transcriptional protein modifications, such as NLRP3 deubiquitination and caspaserecruitment domain phosphorylation(D. Liu et al., 2017; Sutterwala et al., 2014). Emerging evidence has shown that lipopolysaccharide, lipoprotein, and flagellin from periodontal pathogens trigger several toll-like receptor signalling pathways to induce the NLRP3 inflammasome signalling cascade. Subsequently, microbial products or endogenous signals lead to potassium efflux, mitochondrial dysfunction, or lysosome rupture(Palova-Jelinkova et al., 2013). Within these stimuli, inactive NLRP3, caspase-recruitment domain and pro-caspase-1 were oligomerized, resulting in proteolytic cleavage of caspase-1 and the maturation of IL-1β and IL18(Frangogiannis, 2014). Extracellular ATP is a danger signalling molecule that activates the NLRP3 inflammasome in the second step(W. Guo, Wang, Liu, Yang, & Ye, 2015; O. Yilmaz et al., 2008). Emerging evidence has highlighted the role of extracellular ATP in regulation of the NLRP3 inflammasome by activating purinergic receptors 7, which are significant initiators of inflammatory responses and mediators of apoptosis responses(Almeida-da-Silva, Morandini, Ulrich, Ojcius, & Coutinho-Silva, 2016). Extracellular ATP is released during cellular stress or damage, serving as the most potent pro-inflammatory stimulus in host tissues. The role of extracellular ATP/purinergic receptors 7 signalling in inflammasome signalling has been widely studied in myeloid cells. Recently, the function of extracellular ATP/purinergic receptors 7 signalling was also explored in human primary gingival epithelial cells infected with P. gingivalis(Johnson et al., 2015). The NLRP3 inflammasome plays a role in mediating host metabolic responses, and the dysregulation of inflammasome components is associated with various inherited chronic inflammatory and immune disorders, such as arthropathy(Jin et al., 2011), lung disease(S. Lee et al., 2016b), vascular disease(D. Liu et al., 2017), diabetes and obesity(Jourdan et al., 2013) and Alzheimer’s disease(Heneka et al., 2013). With the acknowledgement of intricate microbial colonization in the oral cavity, developing therapies to combat the inflammatory response has proven to be more complex than previously thought. The complexity of the oral environment determines the diversity of oral diseases, ranging from different degrees of inflammatory diseases, autoimmune diseases and even tumourigenesis. Additionally, emerging evidence has shown that the NLRP3 inflammasome is involved in the onset of oral diseases, such as periodontitis, oral lichen planus, dental pulp disease, and oral cavity squamous cell carcinoma. The aim of this review is to provide a theoretical overview of the current knowledge regarding the role and mechanism of the NLRP3 inflammasome in regulating innate and adaptive immunity in several representative oral diseases. A better understanding of the cellular and molecular biology of the NLRP3 inflammasome is necessary because this inflammasome may be a potential therapeutic target for the treatment and prevention of oral inflammatory and immunological diseases.

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NLRP3 inflammasome and periodontal disease:

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Periodontitis is a common chronic oral inflammatory disease that is initiated by bacterial infection in the subgingival biofilm and subsequently progresses by an aberrant host response. It can lead to the collapse of tooth-supporting tissues and can influence systemic health(Anderson, 1979; Bergstrom, 2004; Moore & Moore, 1994; Socransky, 1977). In fact, numerous studies have shown a relationship between periodontitis and systemic disease, such as cardiovascular disease, rheumatoid arthritis, and type 2 diabetes(Ford, Yamazaki, & Seymour, 2007; Linden, Lyons, & Scannapieco, 2013; Seymour, Ford, Cullinan, Leishman, & Yamazaki, 2007). The bacterial infections of periodontitis are rather complex. Among over 500 bacterial species in the oral cavity, a bacterial complex termed the "red complex" comprising Porphyromonas gingivalis (P. gingivalis), Treponema denticola and Tannerella forsythia has been positively associated with worse periodontal lesions(Bodet, Chandad, & Grenier, 2007). A significant correlation between the red complex and the increasing expression of pro-inflammatory cytokines (IL-1β and -18) has been reported(Teles et al., 2010). These gram-negative anaerobic bacteria can express various virulence factors. The contributions of these bacterial species and their virulence factors to the aetiology and/or progression of periodontal disease are widely accepted. Certain toll-like receptors can recognize these periodontal pathogens as well as their virulence factors. For instance, toll-like receptor 5 responds to bacterial flagellin; toll-like receptor 2 is unique in that it forms heterodimers with toll-like receptor 1 and 6 in response to the detection of microbial cell wall components; tolllike receptor 4 is a vital receptor for bacterial Lipopolysaccharide (LPS) and endogenous ligands such as heat shock proteins-60 and 70(Hans & Hans, 2011; Parthiban & Mahendra, 2015). The toll-like receptor signalling pathways induce a cascade of molecules that involve activating the nuclear factor-kappa B, which subsequently promote the production of some cytokines(Ando-Suguimoto et al., 2014; S. R. Park et al., 2014; Ramos-Junior et al., 2015). The NLRP3 inflammasome is activated by periodontal pathogen-associated or damage-associated molecular patterns, which has been widely discussed(Belibasakis & Johansson, 2012; Bostanci, Meier, Guggenheim, & Belibasakis, 2011; W. Guo et al., 2015; J. J. Kim & Jo, 2013; W. L. Lu et al., 2017; E. Park et al., 2014; Taxman et al., 2012; Xue, Shu, & Xie, 2015; Yamaguchi, Kurita-Ochiai, Kobayashi, Suzuki, & Ando, 2015; Zhao, Liu, Pan, & Pan, 2014). In particular, IL-1β and IL-18 activity represent a focus of the host immune response in periodontitis(Delaleu & Bickel, 2004; Orozco et al., 2006). In this section, we mainly discuss representative periodontal pathogens and danger components that trigger and regulate the NLRP3 inflammasome-mediated inflammatory response in periodontal disease.

P. gingivalis: P. gingivalis is a keystone gram-negative opportunistic organism in chronic periodontitis that has been reported to interfere with innate immunity through multiple 5

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mechanisms(Hajishengallis & Lamont, 2014; Olsen, Lambris, & Hajishengallis, 2017). P. gingivalis is located almost exclusively in the subgingival crevice, inducing detrimental effects by perturbing the innate host response for its own benefit and offering an advantageous environment to co-habitants, such as Fusobacterium nucleatum and Treponema denticola(Andrian, Grenier, & Rouabhia, 2006; O. Yilmaz & Lee, 2015). Furthermore, the structural and functional integrity of the gingival epithelium can be destroyed by the multiple effects of P. gingivalis. We mainly discuss the two sequential steps by which P. gingivalis participates in NLRP3 inflammasome activation. Step 1 involves P. gingivalis-produced virulence factors such LPS, which promote toll-like receptor-dependent signalling and generate NLRP3, pro-IL-1β, and pro-IL-18. Step 2 involves P. gingivalis-induced danger signals, such as extracellular ATP and reactive oxygen species, which result in the secretion of bio-active cytokines. Several studies have also reported that NLRP3 is involved in the pathogenesis of P. gingivalis-induced infection. Bostanci et al.(Bostanci et al., 2009) were the first to reveal that the genes of NALP3 (widely known as NLRP3) and NLRP2, but not caspase-recruitment domain, are expressed at significantly higher levels in gingival tissues of patients with gingivitis, chronic periodontitis and generalized aggressive periodontitis than in tissues of healthy controls. Nevertheless, in vitro data demonstrated that P. gingivalis modulated the NALP3 inflammasome complex in a human monocytic cell line by enhancing NALP3 and downregulating NLRP2 and caspase-recruitment domain expression. Subsequently, Park et al.(E. Park et al., 2014) identified that P. gingivalis induces IL1β secretion and inflammatory cell death via NLRP3 and absent in melanoma 2 inflammasome activation. In their in vitro study, the priming signal was found to precede P. gingivalis-induced IL-1β release via toll-like receptor 2 and 4 activation. Yohei et al.(Yamaguchi, Kurita-Ochiai, Kobayashi, Suzuki, & Ando, 2017) demonstrated that P. gingivalis successfully induced gingival inflammation and bone loss via the NLRP3 inflammasome. In their in vivo mouse model, P. gingivalis was shown to significantly increase alveolar bone loss. In addition, the mRNA expression of pro-IL-1β, pro-IL-18, and receptor activator of nuclear factor kappa-B ligand and the protein expression of caspase-1, IL-1β and IL-18 were also increased in gingival tissue from wild-type mice with P. gingivalis injection. In NLRP3-knockout mice, the above changes were minimally affected regardless of P. gingivalis injection. The mRNA level of receptor activator of nuclear factor kappa-B ligand is also decreased in gingival tissue from NLRP3-knockout mice, while that of osteoprotegerin is increased. It should be noted that the receptor activator of nuclear factor kappa-B ligand/osteoprotegerin ratio served as a biomarker denoting bone resorption in periodontitis(Belibasakis & Bostanci, 2012). Thus, these results suggested that the NLRP3 inflammasome might exacerbate P. gingivalis-accelerated periodontal disease.

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Double-stranded RNA-dependent kinase was reported to be a danger-sensing molecule at the crossroad of several pathways implicated in bone metabolism processes, including periodontitis(Haneji, 2017; B. Lu et al., 2012). Double-stranded RNA-dependent kinase was also reported to promote inflammation in response to microbial infection by regulating the NLRP3 inflammasome(Yoshida et al., 2017). The activation of double-stranded RNA-dependent kinase is mediated by toll-like receptor 2/4-dependent signalling after recognizing the extracellular milieu and pathogen-associated molecular patterns, such as flagellin and LPS(Cabanski et al., 2008; Hsu et al., 2004). Upon pattern activation, the activated toll-like receptors initiate transcriptional nuclear factor-kappa B signalling pathways. The mechanisms by which double-stranded RNA-dependent kinase mediates the activation of nuclear factor-kappa B have been well studied(Zamanian-Daryoush, Mogensen, DiDonato, & Williams, 2000). Yoshida et al.(Yoshida et al., 2017) reported that double-stranded RNA-dependent kinase modulates the expression of NLRP3 by regulating the nuclear factor-kappa B pathway in P. gingivalis-infected osteoblasts. Toll-like receptor 2 is likely sensed by LPS from P. gingivalis, which, in turn, mediates the translocation of nuclear factor-kappa B to the nucleus, resulting in an increase in NLRP3 expression, and this process is involved in the activation of double-stranded RNAdependent kinase. The abovementioned studies suggest that in certain aspects, P. gingivalis with its specific pathogenic capabilities might modulate the initiation and activation of the NLRP3 inflammasome. However, other scholars believe that activation of the NLRP3 inflammasome in oral epithelial cell model not only depends on P. gingivalis infection but also on stimulation by the danger signalling molecule, extracellular ATP(W. Guo et al., 2015; O. Yilmaz et al., 2008). Extracellular ATP, which is considered the most potent pro-inflammatory stimulus involved in the second step of NLRP3 inflammasome formation, is released by cellular stress or damage(Gombault, Baron, & Couillin, 2012; Ö. Yilmaz, 2015). Extracellular ATP activates purinergic receptors 7 to transmit pathogen invasion signals(Almeida-da-Silva et al., 2016). purinergic receptors 7 is a cation-permeable ligand-gated ion channel that permits K+ efflux and gradually results in a pore on the membrane by recruiting hemichannel pannexin-1(Kahlenberg & Dubyak, 2004). The ligation of purinergic receptors 7 by extracellular ATP can result in phospholipase D activation and/or reactive oxygen species production, both of which can lead to elimination of intracellular pathogens(Coutinho-Silva, Corrêa, Sater, & Ojcius, 2009). However, continuous stimulation by the ATP-purinergic receptors 7 signal can induce either cell death or apoptosis(Lenertz, Gavala, Zhu, & Bertics, 2011). The significance of purinergic receptors 7 in inflammasome signalling has recently been explored in human gingival epithelial cells, which serve as the first line of protection against invading pathogens(Hung et al., 2013). A recent study reported that nucleoside diphosphate kinase, an evolved virulence factor of P. gingivalis, affects ATP hydrolysis and then blocks the ATP-purinergic 7

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receptors 7 interaction(Choi et al., 2013). Another in vitro study also examined the role of nucleoside diphosphate kinase in the modulation of the inflammasomemediated host response and IL-1β secretion in both human immortalized and primary gingival epithelial cells(Johnson et al., 2015). Human immortalized gingival epithelial cell lines infected with wild-type P. gingivalis showed attenuated ATP-induced caspase-1 activation, whereas these cell lines infected with nucleoside diphosphate kinase-deficient P. gingivalis exhibited higher protein levels of activated caspase-1. Similarly, primary gingival epithelial cells infected with wild-type P. gingivalis showed lower ATP-induced IL-1β secretion than cells infected with nucleoside diphosphate kinase-deficient P. gingivalis(Johnson et al., 2015). Taken together, these findings suggest that P. gingivalis-nucleoside diphosphate kinase can interfere with extracellular ATP-purinergic receptors 7 signalling, inhibit ATP-mediated inflammasome activation, and evade the innate immune response. This sequence of events ultimately leads to P. gingivalis destroying the periodontal tissue and contributing to disease along with other host and bacterial factors during this process. Indeed, the anaerobic environment in periodontal pockets also leads to reduced oxygen levels, and hypoxia plays some role in accelerating the process of periodontitis(Motohira et al., 2007) by inducing the activation of hypoxia-inducible factor α and nuclear factor-kappa B in periodontal ligament cells in cooperation with P. gingivalis(Golz et al., 2015; X. J. Yu et al., 2015). Increasing evidence suggests that the paradoxical increase in reactive oxygen species generation plays a significant role in the pathologic mechanisms associated with the hypoxic feature(Giussani et al., 2014; Jefferson, Escudero, Johnson, Swenson, & Hurtado, 2014; Pandey, Patnaik, Muresanu, Sharma, & Sharma, 2012). The pathogenesis of periodontitis is also associated with an imbalance between reactive oxygen species and the antioxidant defence system(C. Liu et al., 2017). reactive oxygen species are integral components of multiple cellular pathways and function as antimicrobial effector molecules and signalling molecules that regulate processes such as NF-kB transcriptional activity(Dan Dunn, Alvarez, Zhang, & Soldati, 2015). Mitochondria and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase are the main sources of cellular reactive oxygen species. Excessive accumulation of reactive oxygen species leads to intracellular oxidative stress, which causes mitochondrial DNA damage, the oxidation of mitochondrial proteins and, subsequently, the progression of chronic inflammatory diseases, such as metabolic syndrome, type 2 diabetes, hypertension, and Alzheimer's disease(J. W. Yu & Lee, 2016). If the overproduced reactive oxygen species, mostly by hyperactive neutrophils, cannot be neutralized by the antioxidant defence system in tooth-supporting tissue, the periodontal tissue can be destroyed, and periodontitis occurs(Y. Wang, Andrukhov, & Rausch-Fan, 2017). Several periodontopathic bacterial pathogens can also induce the generation of reactive oxygen species and even oxidative stress(Golz et al., 2014; Kurita-Ochiai, Jia, Cai, Yamaguchi, & Yamamoto, 2015; M. C. Lee, 2014). For example, P. gingivalis can effectively

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accommodate reactive oxygen species levels in infected primary gingival epithelial cells through the stimulation with extracellular ATP(Johnson et al., 2015). An in vitro study showed that LPS from P. gingivalis combined with hypoxia induces the accumulation of reactive oxygen species and catalase in periodontal ligament cells(Golz et al., 2014). The levels of NADPH oxidase 4 significantly increase after hypoxic or P. gingivalis LPS stimulation in periodontal ligament cells, and this increase is more pronounced after exposure to a combination of these two stimuli. NADPH oxidase 4 is a major source of reactive oxygen species generation and can be detected in kidney cells, endothelial cells, fibroblasts, keratinocytes, and osteoclasts. Compared with the other family members, NADPH oxidase 4 is highly and ubiquitously expressed in these cells; thus, it serves as the main indicator for detecting reactive oxygen species(Gray et al., 2016; Krause, 2004; Lozhkin et al., 2017). A previous research group also found that prolonged exposure to both stimuli leads to a reduction in catalase(Golz et al., 2014). It was demonstrated that the elevation in reactive oxygen species production and the reduction in catalase, which were induced by a combination of hypoxia and P. gingivalis LPS under the same condition, increased oxidative stress in the periodontium and contributed to periodontitis. Reactive oxygen species/oxidative stress serves as a trigger in the second step to activate NLRP3 inflammasomes(Abais, Xia, Zhang, Boini, & Li, 2015). An in vivo experiment showed that hypoxia-inducible factor α and the NLRP3 inflammasomes (NLRP3, cleaved-caspase-1, IL-1β, and caspase-1-induced cell death) were highly expressed in human periodontitis tissue(Cheng et al., 2017). In addition, a previous group also reported that P. gingivalis LPS slightly downregulated the protein expression of NLRP3, pro-IL-1β and IL-1β in normoxic mouse gingival fibroblasts. However, hypoxia reversed these results. These above indicators were significantly promoted in response to the synergistic effects of P. gingivalis LPS and hypoxia. Furthermore, the protein level of pro-IL-1β was increased, indicating the involvement of NF-kB transcriptional activity(Cheng et al., 2017). These findings suggest that P. gingivalis combined with hypoxia can induce obvious NLRP3/caspase-1 activation in periodontitis. Therapies directed against oxidative stress are likely efficient in downregulating the activation of NLRP3 inflammasomes in periodontal tissue. Co-enzyme Q10, an antioxidant with anti-aging abilities, can suppress inflammatory reactions in periodontal tissue induced by oxidative stress(Hanioka, Tanaka, Ojima, Shizukuishi, & Folkers, 1994; Prakash, Sunitha, & Hans, 2010). The NLRP3 inflammasomes (NLRP3, caspase-recruitment domain, and caspase-1) in periodontal tissues of experimental rats treated with co-enzyme Q10 are less active than those in tissues of control rats(Yoneda et al., 2013). According, we suggest that P. gingivalis is a representative periodontal pathogen that, combined with hypoxia, increases oxidative stress in periodontal ligament fibroblasts. Oxidative stress may be a link between NLRP3 inflammasome activation and the development of periodontitis. Future

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research should enhance the understanding of oxidative stress in periodontal tissue, which may improve diagnosis and treatment strategies for oral diseases. Currently, periodontitis is well known to influence the pathogenesis of diabetes. A bidirectional relationship exists between diabetes mellitus and inflammatory periodontal disease. Scientific evidence has confirmed that periodontitis is a chronic localized infection of the oral cavity and can be a source of bacteria. In addition, poor glycaemic control contributes to a worse periodontal condition(Bascones-Martinez, Munoz-Corcuera, & Bascones-Ilundain, 2015),(Llambes, Arias-Herrera, & Caffesse, 2015). Kuo et al.(Kuo et al., 2016) studied human gingival fibroblasts treated under a condition of high glucose and P. gingivalis invasion. The mRNA expression of NLRP3, IL-1β and sterol regulatory element binding protein 1C increased in HG-Pgtreated human gingival fibroblasts, and it should be noted that sterol regulatory element binding protein 1C is a vital transcription factor in the cholesterol and fatty acid metabolic response(Y. Wang, Viscarra, Kim, & Sul, 2015). In addition, in vitro data also demonstrated that after blocking the expression of sterol regulatory element binding protein 1C, the mRNA levels of NLRP3 inflammasomes (NLRP3, caspase-1 and IL-1β) in the high glucose plus P. gingivalistreated human gingival fibroblasts were significantly lower than those in the control group(Kuo et al., 2016). Furthermore, thee data also demonstrated that high glucose plus P. gingivalis invasion induces activation of the Janus kinase 2 pathway, ultimately enhancing NLRP3 expression in human gingival fibroblasts. Thus, these experimental data suggest that mechanisms of P. gingivalis and its action in modulating NLRP3 inflammasome signalling are likely important factors in the development of the dysbiotic stage of periodontal disease. Understanding the roles and regulatory mechanisms of the NLRP3 inflammasome is essential for developing potential treatment approaches against pathogenic infections.

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A. actinomycetemcomitans Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans) has been recognized as a gram-negative capnophilic coccobacillus oral anaerobe that is closely related to localized aggressive periodontitis(Gholizadeh et al., 2017; Kawamoto et al., 2009). It is a much more powerful bacterium than P. gingivalis with respect to its role in inducing the differentiation and activation of dendritic cells and promoting macrophage cell death through IL-1β expression(Lv, Zhu, Liu, & Xue, 2015). An in vitro study showed that apoptosis of human osteoblastic MG63 cells infected with A. actinomycetemcomitans was due to activation of the NLRP3 inflammasome(Zhao et al., 2014). The induction of apoptosis can subsequently exacerbate bone resorption during the pathogenic processes of periodontal disease. A. actinomycetemcomitans can produce many virulence factors to evade host innate defence, such as LPS, leukotoxin and cytolethal distending toxin(Herbert, Novince, & Kirkwood, 2016). A. actinomycetemcomitans leukotoxin can selectively kill human 10

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leukocytes by triggering apoptosis or lysis depending on these virulence factors(Kato, Kowashi, & Demuth, 2002). This process is mediated by the activation of caspase-1, which further leads to the secretion of bio-active IL-1β(Kelk, Johansson, Claesson, Hanstrom, & Kalfas, 2003). In addition, cytokines belonging to the IL-1 family, including IL-1β and IL-18, are critical for the inflammatory host response, and their levels are elevated in gingival crevicular fluid, gingival tissues and periodontitis(Bostanci et al., 2009; Kelk et al., 2003). Kelk P et al.(Kelk et al., 2011) proposed that leukotoxin-induced IL-1β and IL-18 secretion involved pore formation on the cell membrane and activation of intracellular signalling pathways. In their study, the leukotoxin-induced IL-1β secretion in macrophages was modified by blockage of Ca2+ influx or K+ efflux. In addition, after the addition of oxidized ATP, purinergic receptors 7 antagonist, the cell death induced by leukotoxin was inhibited, indicating that purinergic receptors 7 is also involved in leukotoxin-induced proinflammatory monocyte/macrophage death. Thus, it is tempting to speculate that the involvement of K+ efflux and ATP/purinergic receptors 7 signalling by leukotoxin may participate in inflammasome-dependent IL-1β secretion in periodontitis. Cytolethal distending toxin, a family of heat-labile protein cytotoxins, regulates A. actinomycetemcomitans infection site function in macrophages by reducing phagocytosis and interfering with the secretion of pro-inflammatory/antiinflammatory cytokines(Ando-Suguimoto et al., 2014). A study reported that cytolethal distending toxin activates the NLRP3 inflammasome in human monocytic cell lines, leading to the release of pro-inflammatory cytokines, including IL-1β, IL-6 and tumour necrosis factor-α, within 5 h and IL-18 within 48 h. In addition, caspase-1 levels were also elevated(Shenker et al., 2015). Two signals are proposed to be linked to activation of the NLRP3 inflammasome. The first signal blocks phosphatidylinositol 3-kinase signalling and activates glycogen synthase kinase 3β, which subsequently activates the nuclear factor-kappa B signalling pathway. The second signal induces the generation of extracellular ATP, the efflux of K+ and the utilization of endogenous reactive oxygen species. In contrast, a separate in vitro study demonstrated the regulation of A. actinomycetemcomitans for enhancing the expression of NLRP3 in human mononuclear leukocytes, irrespective of leukotoxin and cytolethal distending toxin. However, expression of the adaptor molecule caspase-recruitment domain and caspase-1 was not affected(Belibasakis & Johansson, 2012). Based on this observation, it is speculated that other unknown molecules are involved in regulation of the NLRP3 inflammasome other than these two well-documented virulence factors from A. actinomycetemcomitans. In conclusion, A. actinomycetemcomitans regulates NLRP3 inflammasome expression and increases the levels of IL-1β and IL-18 in periodontal disease. However, the underlying mechanisms of the NLRP3 inflammasome in response to A. actinomycetemcomitans infection have not been fully elucidated. In addition to major virulence factors, other unknown molecules are involved in the host immune cascades 11

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responding to A. actinomycetemcomitans infection, confirming the high pathogenic profile of this species. Further studies identifying the NLRP3 inflammasome components related to A. actinomycetemcomitans invasion are needed and would significantly contribute to the recognition of the pathogen and its correlation with aggressive periodontitis.

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Other periodontal pathogens Treponema denticola is a highly pathogenic spirochete that can cause periodontal tissue damage(Teles et al., 2010). It mainly contributes to synergy and enhances the ability of P. gingivalis to regulate inflammasome signalling and increase the colonization of other periodontal pathogenic bacteria. Jun et al.(Jun, Lee, Lee, & Choi, 2012) reported that the direct interaction between Treponema denticola surface protein and cell membrane integrin α5β1 resulted in ATP release and K+ efflux, which are the main events in NLRP3 inflammasome activation. Fusobacterium nucleatum is another periodontal pathogen associated with a wide range of human diseases(Han, 2015). It also co-aggregates with diverse bacterium and ultimately exacerbates periodontitis by inducing immune cell death(Mima et al., 2015). Fusobacterium nucleatum infection has also been reported to activate nuclear factor-kappa B transcriptional signalling and can serve as an endogenous damage-associated molecular pattern to initiate the NLRP3 inflammasome in gingival epithelial cells(Bui et al., 2016). Outer membrane vesicles from gram-negative bacteria, such as P. gingivalis, Treponema denticola, and Tannerella forsythia, are powerful proteoliposomes that regulate microbial interactions(Schwechheimer & Kuehn, 2015). Cecil et al.(Cecil et al., 2017) demonstrated that pathogen outer membrane vesicles can deliver damage/pathogen-associated molecular patterns to the cell surface or cytosolic receptors and subsequently induce nuclear factor-kappa B activation and inflammasome complexes, such as NLRP3 and absent in melanoma 2.

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It is undeniable that the NLRP3 inflammasome plays a crucial role in the pathogenesis of periodontitis. Inflammasomes may be regulated by other molecules in addition to the aforementioned virulence factors of the infecting microorganism. Further studies investigating the mechanisms by which periopathogens modulate NLRP3 inflammasome signalling and their association with other emerging infectious cytokine stimuli, such as viruses, may be important for the prevention and control of periodontal disease in the future.

NLRP3 inflammasome and dental pulp disease: Microorganisms have long been considered the primary aetiological agents of dental pulp lesions. Following exogenous stimulation or damage, the pulp-dentin complex triggers immunoprotective mechanisms(Jontell, Okiji, Dahlgren, & Bergenholtz, 1998). Once bacterial infection invades the dentin-pulp interface, the microflora 12

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drastically changes. Subsequently, gram-negative anaerobic bacteria dominate and activate the initial pulpal immune response. Lipoteichoic acid, LPS, noxious metabolic by-products and other bacterial toxins induce pulpal and periapical inflammatory reactions, ranging from irreversible pulpitis and pulp necrosis to periapical disease(Jang et al., 2015; Love & Jenkinson, 2002). Human dental pulp fibroblasts, also known as human dental pulp cells, are the core constituent of dental pulp that defend against bacterial invasion in dental pulp tissues and maintain the structural integrity of the connective tissue(Zhai et al., 2013). Several studies have investigated the pulpal innate immune response through the NLRP3 inflammasome pathway and its function as a versatile platform to detect bacterial pathogens(W. Jiang et al., 2015; S. I. Lee et al., 2015; Song et al., 2012; A. Zhang et al., 2015). Upon activation, the NLRP3 inflammasome regulates the secretion and bioactivity of IL-1β. Song et al.(Song et al., 2012) were the first to show the mRNA and protein expression of NLRP3 in normal human dental pulp tissues and human dental pulp cells. However, the role and function of the NLRP3 inflammasome in human dental pulp cells were not described in the previous study. Muramyl dipeptide is the minimal structural subunit of peptidoglycans, responsible for some of their immunogenicity(Baschang, 1989). Muramyl dipeptide is widely distributed in the cell walls of both gram-negative and gram-positive bacteria and acts synergistically with LPS to stimulate the release of both pro- and anti-inflammatory cytokines by myeloid cells(Wolfert, Murray, Boons, & Moore, 2002). To investigate the involvement of the NLRP3 inflammasome in dental pulp inflammation, Sang et al.(S. I. Lee et al., 2015) cultured muramyl dipeptide-induced human dental pulp cells in vitro. The mRNA expression levels of NLRP3, caspase-recruitment domain, caspase-1 and IL-β were all enhanced in a dose- and time-dependent manner. Moreover, silencing the NLRP3 gene induced decreases in the muramyl dipeptideinduced expression of human beta defensin 2 and cytokines such as nitric oxide synthase-derived nitric oxide, cyclooxygenase 2 and prostaglandin E2, whose levels are well known to increase during the progression of pulpitis(Miyauchi et al., 1996). These results indicate that local inhibition of NLRP3 may reduce the impact of cytokine-mediated host destructive processes in pulpitis. Jiang et al.(W. Jiang et al., 2015) demonstrated that NLRP3 inflammasome expression varies in different degrees of pulpitis. The gene and protein expression levels of NLRP3 inflammasomes (NLRP3, caspase-1, and IL-1β) were much higher in irreversible pulpitis tissue than in reversible pulpitis tissue and normal pulp tissue(W. Jiang et al., 2015). The group also found that purinergic receptors 7/ATPgated ion channels and K+ efflux are involved in ATP-plus-LPS-induced NLRP3 inflammasome activation in human dental pulp fibroblasts. The expression of purinergic receptors 7 and NLRP3 inflammasomes (NLRP3, caspase-1 and IL-1β) were increased in human dental pulp fibroblasts treated with ATP-plus-LPS over time. However, after treatment with a high extracellular K+ concentration or a selective K+ channel inhibitor, the mRNA and protein levels of IL-1β decreased 13

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significantly. The levels of IL-1β mRNA and protein were used as indicators of inflammasome activation. In addition, IL-1β secretion was also reduced in ATP-plusLPS-induced human dental pulp fibroblasts after using N-acetylcysteine, which serves as an antioxidant to neutralize reactive oxygen species. N-acetylcysteine can not only protect against oxidative damage but also work anti-inflammatory by inhibiting nuclear factor-kappa B pathways(X. Liu, Nie, Huang, & Xie, 2015; Pan et al., 2018). Taken together, it is reasonable to speculate that extracellular ATP-plus-LPS activates purogenic purinergic receptors 7 ATP-gated ion channels, which, in turn, contribute to K+ efflux and the formation of membrane pores. Therefore, extracellular LPS gains access to the cytosol and directly activates the NLRP3/caspase-1 inflammasome. Furthermore, a low K+ concentration in the cytoplasm leads to reactive oxygen species generation, which subsequently activates the NLRP3/caspase-1 pathway and the release of IL-1β in human dental pulp fibroblasts. K+ efflux has also been shown to be associated with reactive oxygen species generation in other studies(Hoeberichts et al., 2010; Y. Zhou et al., 2011). Research investigating the mechanisms of NLRP3 inflammasome activation in human dental pulp fibroblasts is ongoing. Zhang et al.(A. Zhang et al., 2015) demonstrated that the toll-like receptor 4/myeloid differentiation primary response gene 88/nuclear factor-kappa B transcriptional pathway is likely engaged in the initiation of the NLRP3 inflammasome in LPS-stimulated human dental pulp fibroblasts. When LPS-stimulated human dental pulp fibroblasts were pre-incubated with these transcriptional inhibitors, NLRP3 inflammasome expression was markedly reduced. The previous group also found that the reactive oxygen species/superoxide level was much higher in ATP-treated human dental pulp fibroblasts than in control human dental pulp fibroblasts. In human dental pulp fibroblasts pre-treated with Nacetylcysteine, the production of reactive oxygen species was inhibited despite ATP stimulation. However, N-acetylcysteine did not affect the expression of NLRP3, caspase-1 or IL-1β in ATP-treated human dental pulp fibroblasts. Thus, ATP likely induces reactive oxygen species generation, which subsequently promotes NLRP3 inflammasome activation. However, reactive oxygen species are not sufficient in the process of inflammasome activation. Collectively, these experimental results indicate that the NLRP3 inflammasome is expressed in inflamed dental pulp tissue and functions as a certain defence component during pulpitis. The toll-like receptor 4-dependent transcriptional pathway is engaged in initiation of the NLRP3 inflammasome in LPS-stimulated human dental pulp fibroblasts. ATP and K+ efflux both promote reactive oxygen species generation in human dental pulp fibroblasts, serving as a second signal that activates the NLRP3 inflammasome and IL-1β release. A good understanding of the role of the NLRP3 inflammasome in pulpal innate immunity may provide new insight for the development of future novel therapies.

NLRP3 inflammasome and oral lichen planus: 14

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Oral lichen planus belongs to a group of common, chronic, immunoinflammatory diseases that occur in the oral mucosa(Sarode, Sarode, & Patil, 2014). The prevalence of oral lichen planus is higher in women than in men. It is estimated that approximately 1–2% of the adult population worldwide will develop oral lichen planus, and 15% will develop cutaneous lichen planus(Axell & Rundquist, 1987). There is no clear consensus regarding the etiopathogenesis of oral lichen planus. Both antigen-specific immune mechanisms and nonspecific immune mechanisms are believed to be involved in the pathogenesis of oral lichen planus(Roopashree et al., 2010). Thi et al.(Thi Do, Phoomak, Champattanachai, Silsirivanit, & Chaiyarit, 2018) recently provided evidence suggesting that O-linked β-N-acetylglucosamine promotes nuclear factor-kappa B signalling molecules and ultimately induces the constitutive expression of the NLRP3 inflammasome in oral lichen planus patients. O-linked β-Nacetylglucosamine, which is considered a dynamic regulatory mechanism of keratinocyte differentiation and cell adhesion, has already been detected in oral lichen planus lesional mucosae(Sohn et al., 2014). Nuclear factor-kappa B, NLRP3, caspaserecruitment domain, caspase-1, and IL-1β expression is present in the oral epithelia and connective tissues of oral lichen planus patients at higher levels than in those of control subjects(Thi Do et al., 2018). The increased level of NLRP3 inflammasomes in oral lichen planus is also in agreement with previous studies in other chronic mucosal diseases(E. H. Kim, Park, Park, & Lee, 2015; Lazaridis et al., 2017). In addition, previous studies have demonstrated increased expression levels of nuclear factor-kappa B and IL-18 in oral lichen planus patients, respectively(Rusanen, 2017; Y. Zhang et al., 2012). Thus, it is tempting to postulate that nuclear factor-kappa B signalling and the NLRP3 inflammasome are related to the molecular mechanism of oral lichen planus. The increased expression of the NLRP3 inflammasome in oral epithelial cells and the basal layer of oral lichen planus lesions may also reflect cellular responses to pathological stimuli. Previous studies have demonstrated the altered expression of heat shock proteins 60 and 70 in oral lichen planus lesions(Chaiyarit et al., 1999; Di & Gao, 2003). Heat shock proteins are molecular chaperones that regulate conformational changes, translocation, assembly and degradation of cellular proteins(Dubaniewicz, 2013). Numerous studies have highlighted the role of heat shock proteins as damage-associated molecular patterns in activating the NLRP3 inflammasome pathway(Lamkanfi & Dixit, 2014; Maslanik et al., 2013). Considering these observations, one could speculate that aberrant expression of damage-associated molecular patterns molecules, such as heat shock proteins 60 and 70, may induce activation of the NLRP3 inflammasome in oral lichen planus due to pathological stimuli. As mentioned previously, the NLRP3 inflammasome is not only an innate responder to pathogenic and danger signals but can also induce adaptive immunity(Brydges et al., 2009; M. Chen, Wang, Chen, & Meng, 2011; Gris et al., 2010; Meng, Zhang, Fuss, Kitani, & Strober, 2009). The excessive production of IL-1β and IL-18 resulting 15

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from NLRP3 activation can lead to many autoinflammatory diseases(Dinarello, 2009; Hoffman, Mueller, Broide, Wanderer, & Kolodner, 2001; D. Liu et al., 2017; Shalev et al., 2007; Ting, Kastner, & Hoffman, 2006). In addition, IL-1β has been demonstrated to orchestrate the differentiation of Th17 cells(Chung et al., 2009), and IL-18 was shown to play roles in limiting homeostatic Th17 cell differentiation and promoting FOXP3+ Treg cell function to minimize tissue pathology during experimental colitis(Harrison et al., 2015). Canonical IL-18/IL-18R1 signalling plays an essential role in the epithelial/T cell immune-regulatory axis in the intestine(Hedl, Zheng, & Abraham, 2014). Furthermore, the Th17/Treg cell response has been shown to be closely related to immune diseases, such as systemic lupus erythaematosus and allergic rhinitis(Handono, Pratama, Endharti, & Kalim, 2015; Ni et al., 2015). The Th17/Treg imbalance is positively correlated with the clinical course and severity of these diseases and has been used as a diagnostic marker and therapeutic target. Oral lichen planus is a T cell-mediated autoimmunity in the oral mucosa. Many studies have shown that Th17 and Treg cells are also involved in the immune response in oral lichen planus. Tao et al.(Tao et al., 2010) were the first to show that the number of FOXP3+ Treg cells was much higher in oral lichen planus lesions than in normal mucosal tissue. Interestingly, the frequency of FOXP3+ Treg cells in erythaematous/erosive oral lichen planus was higher than that in reticular oral lichen planus, and the density of FOXP3+ T cells in the lesions was negatively correlated with the severity of the disease. Another group also detected and compared the expression of the retinoid‐acid receptor‐related orphan receptor gamma τ (RORγτ) and FOXP3 in oral lichen planus lesions(Xie, Feng, Zhu, & Ding, 2014), as RORγτ and FOXP3 are the most reliable transcription factors of pro-inflammatory Th17 cells and Treg cells, respectively(Harrison et al., 2015; Ivanov et al., 2006). The expression levels of both RORγτ and FOXP3 in oral lichen planus lesions were higher than those in the normal mucosal group. Furthermore, the RORγτ/FOXP3 ratio was significantly higher in an atrophic-erosive oral lichen planus group than in a reticular oral lichen planus group and a normal mucosal group. Thus, there is an imbalance in RORγτ/FOXP3 in the lesion tissue of oral lichen planus, further suggesting that the Th17/Treg imbalance is a possible pathogenic mechanism of atrophic-erosive oral lichen planus(Xie et al., 2014). Patel D et al.(Patel, Gaikwad, Challagundla, Nivsarkar, & Agrawal-Rajput, 2018) recently showed that spleen tyrosine kinase regulates the NLRP3 inflammasome pathway to disrupt the Th17/Treg balance and finally leads to allergic asthma. Spleen tyrosine kinase is an immune receptor that activates nuclear factor-kappa B signalling and NLRP3 inflammasomes, which, in turn, promote the release of IL-1β and IL18(Gross et al., 2006). The release of IL-1β promotes the differentiation Th17 cells and results in Th17/Treg imbalance, which contributes to inflammation and tissue damage.

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Taking these findings into account, it is possible that damage-associated molecular patterns induce activation of the NLRP3 inflammasome in oral lichen planus, and the NLRP3 inflammasome/IL-1β/IL-18 axis is associated with activation Th17/Treg imbalance in the pathological development of oral lichen planus. Considering these findings, the NLRP3 inflammasome may interplay between oral lichen planusspecific immunity and nonspecific immunity. However, whether NLRP3 participates in the pathological development of oral lichen planus by mediating the Th17/Treg axis is unclear. Thus, further innovative studies must be performed.

NLRP3 inflammasome and oral cavity squamous cell carcinoma:

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oral cavity squamous cell carcinoma represents a group of serious, malignant diseases of the head and neck region(Reid, Winn, Morse, & Pendrys, 2000). Despite great improvements in medical treatment, including surgery, radiotherapy and chemotherapy, the 5-year overall survival rate of oral cavity squamous cell carcinoma patients is only 50% because of recurrence and distant metastasis(L. Chen et al., 2017; Warnakulasuriya, 2009). Aberrant NLRP3 inflammasome activation has been recently reported to promote tumour growth in multiple cancer types, including lung cancer, bladder cancer, breast cancer and gastrointestinal cancer(Kolb, Liu, Janowski, Sutterwala, & Zhang, 2014; Kong et al., 2015; Mearini et al., 2017; Paugh et al., 2015). It has been widely reported that the NLRP3 inflammasome is an essential part of the innate immune system and plays an important role in the pathogenesis of oral cavity squamous cell carcinoma(Bae et al., 2017; L. Chen et al., 2017; Feng, Luo, Wang, Zhang, & Chen, 2018; Feng et al., 2017b; Huang et al., 2017; Wu et al., 2016b). Wu et al.(Wu et al., 2016a) were the first to report that NLRP3 and its inflammasomeassociated proteins (caspase-recruitment domain, IL-1β, and caspase-1) are highly expressed in oral cavity squamous cell carcinoma tumour tissues. Furthermore, the group also demonstrated that high expression of caspase-recruitment domain was closely correlated with various clinicopathological characteristics and worse patient survival. Their studies using oral cavity squamous cell carcinoma cell lines (SAS cells) and an in vivo lymph node metastasis model of oral cavity squamous cell carcinoma confirmed that caspase-recruitment domain contributes to oral cavity squamous cell carcinoma cell metastasis. Accumulating evidence suggests that caspase-recruitment domain is a bipartite protein that plays different roles in tumours(Guan et al., 2003). For instance, in primary melanoma, a high level of caspase-recruitment domain expression was shown to resist tumourigenesis, while in metastatic melanoma, caspase-recruitment domain enhances tumourigenic pathways by mediating nuclear factor-kappa B signalling and inflammasome-initiated IL-1β secretion. Furthermore, caspase-recruitment domain can also function as a tumour suppressor and is downregulated in cancers such as breast cancer and hepatocellular carcinoma(Conway et al., 2000; Guan et al., 2003; C. Zhang et al., 2007). Such tumourigenic suppression is partially mediated by aberrant methylation. Conversely, 17

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the expression of caspase-recruitment domain is highly elevated in nasopharyngeal carcinoma cells(L. C. Chen et al., 2012). Bae et al.(Bae et al., 2017) reported that NLRP3 inflammasome activation also plays crucial roles in the survival and invasiveness of head and neck squamous cell carcinoma. Moreover, the expression of purinergic receptors 7 was upregulated in A253 cells (derived from human squamous carcinoma in submandibular glands). Inhibition of purinergic receptors 7 or/and the NLRP3 inflammasome decreased/blocked the invasiveness of A253 cells. Chen et al.(L. Chen et al., 2017) also found that the NLRP3 inflammasome/IL-1β pathway promotes tumourigenesis in head and neck squamous cell carcinoma. After treatment with the NLRP3 inhibitor MCC950 (PZ0280), anti-tumour immune responses were observed in head and neck squamous cell carcinoma mouse models. Thus, blocking the NLRP3 inflammasome may improve anti-tumour immune responses in head and neck squamous cell carcinoma. Some experts believe that the role of the NLRP3 inflammasome in tumourigenesis might be correlated with cancer stem cells(Basiorka et al., 2016). Cong et al. found that the NLRP3 inflammasome was upregulated and associated with carcinogenesis and cancer stem cell self-renewal activation in head and neck squamous cell carcinoma(Huang et al., 2017). These cancer stem cells are involved in tumour recurrence and have already been identified in several solid tumours, including breast cancer, brain tumours, lung cancer, colon cancer, and melanoma(Dawood, Austin, & Cristofanilli, 2014; Sandoval & Harris, 2014). Feng et al.(Feng et al., 2017a) showed that NLRP3 inflammasome activation induced 5-fluorouracil resistance of oral cavity squamous cell carcinoma both in vitro and in vivo. 5-fluorouracil is an effective commercially available drug for the treatment of oral cavity squamous cell carcinoma, but its clinical effectiveness is often limited due to the chemotherapy tolerance of tumour cells. In vitro study, Feng et al.(Feng et al., 2017a) showed that 5-fluorouracil promoted intracellular reactive oxygen species generation in oral cavity squamous cell carcinoma cells, which in turn activated NLRP3 inflammasome and IL-1β release, both of which could mediate chemoresistance. In addition, microRNA-22 has been implicated as an effective anti-tumour regulator in the context of carcinogenesis(X. Jiang et al., 2016). Surprisingly, it was reported that microRNA-22 suppresses oral cavity squamous cell carcinoma cell proliferation, migration, and invasion by targeting the expression of NLRP3 in oral cavity squamous cell carcinoma tissues and oral cavity squamous cell carcinoma cell lines(Feng et al., 2018). The expression of microRNA-22 in oral cavity squamous cell carcinoma was significantly lower than in adjacent non-tumour mucosa tissues. However, NLRP3 expression was significantly higher in oral cavity squamous cell carcinoma tissues than in non-tumour tissues. When microRNA-22 was overexpressed in oral cavity squamous cell carcinoma cell lines in vitro, NLRP3 mRNA and protein expression is significantly reduced. These results indicate that 18

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Outlook to future NLRP3 inflammasome studies:

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miR-22 may play a suppressive role in oral cavity squamous cell carcinoma by targeting NLRP3. Taken together, these findings suggest that the NLRP3 inflammasome is highly important for the pathogenesis of oral cavity squamous cell carcinoma. Inhibition of the tumour microenvironment through the NLRP3 inflammasome/IL-1β pathway and understanding the suppressive role of microRNA-22 in targeting NLRP3 are likely to provide new insights into the effective prevention, control and treatment of oral cavity squamous cell carcinoma.

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The NLRP3 inflammasome is a major regulator of the innate immune system and plays a crucial role in the progression of inflammatory and adaptive immune responses. Many extensive and in-depth studies have elucidated the possible roles of the NLRP3 inflammasome in several oral diseases, including not only periodontitis and pulpitis but also mucosal diseases and oral cavity squamous cell carcinoma, which may involve the aberrant regulation of other inflammatory and immune responses. Via IL-1β and IL-18, the NLRP3 inflammasome is also linked to Th17 cell and Treg cell differentiation, which are crucial for immune and bone homeostasis(Deng et al., 2010). In addition, recent studies have reported that Th17/Treg imbalance may cause the pathogenesis of oral lichen planus(Patel et al., 2018), periodontitis(Karthikeyan, Talwar, Arun, & Kalaivani, 2015; Okui, Aoki, Ito, Honda, & Yamazaki, 2012; L. Wang, Wang, Jin, Gao, & Lin, 2014), and periapical lesions(Wei et al., 2013; Yang et al., 2014). However, there is currently no direct evidence clarifying the relationship between the NLRP3 inflammasome/IL-1β/IL-18 axis and Th17/Treg imbalance in these diseases. Although further research is needed in these areas, we envision many promising future investigations of the NLRP3 inflammasome signalling cascade for the identification of therapeutic targets to treat oral inflammatory and immune diseases.

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Conflict of Interest Disclosures: Authors declare no conflict of interest. Ethical approval: Ethical Approval was not necessary for the review paper

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Financial support: This work was supported by the Ministry of Health in Zhejiang Province, China (Grant No. LY14H140001)

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References:

Abais, J. M., Xia, M., Zhang, Y., Boini, K. M., & Li, P. L. (2015). Redox regulation

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of NLRP3 inflammasomes: ROS as trigger or effector? Antioxid Redox

N

Signal, 22(13), 1111-1129.

A

Almeida-da-Silva, C. L. C., Morandini, A. C., Ulrich, H., Ojcius, D. M., &

M

Coutinho-Silva, R. (2016). Purinergic signaling during Porphyromonas

TE D

gingivalis infection. Biomed J, 39(4), 251-260. Anderson, D. L. (1979). Etiology of periodontal disease. J Can Dent Assoc,

EP

45(12), 669-672.

Ando-Suguimoto, E. S., da Silva, M. P., Kawamoto, D., Chen, C., DiRienzo, J.

CC

M., & Mayer, M. P. (2014). The cytolethal distending toxin of

A

Aggregatibacter

actinomycetemcomitans

inhibits

macrophage

phagocytosis and subverts cytokine production. Cytokine, 66(1), 46-53.

Andrian, E., Grenier, D., & Rouabhia, M. (2006). Porphyromonas gingivalisepithelial cell interactions in periodontitis. J Dent Res, 85(5), 392-403. Axell, T., & Rundquist, L. (1987). Oral lichen planus--a demographic study. 20

21

Community Dent Oral Epidemiol, 15(1), 52-56. Bae, J. Y., Lee, S. W., Shin, Y. H., Lee, J. H., Jahng, J. W., & Park, K. (2017). P2X7 receptor and NLRP3 inflammasome activation in head and neck cancer. Oncotarget, 8(30), 48972-48982.

immunostimulants. Tetrahedron, 45(20), 6331-6360.

IP T

Baschang, G. (1989). Muramylpeptides and lipopeptides: Studies towards

SC R

Bascones-Martinez, A., Munoz-Corcuera, M., & Bascones-Ilundain, J. (2015).

[Diabetes and periodontitis: A bidirectional relationship]. Med Clin (Barc),

U

145(1), 31-35.

A

N

Basiorka, A. A., McGraw, K. L., Eksioglu, E. A., Chen, X., Johnson, J., Zhang,

M

L., . . . List, A. F. (2016). The NLRP3 inflammasome functions as a driver of the myelodysplastic syndrome phenotype. Blood, 128(25), 2960-2975.

TE D

Belibasakis, G. N., & Bostanci, N. (2012). The RANKL-OPG system in clinical periodontology. J Clin Periodontol, 39(3), 239-248. G.

EP

Belibasakis,

N.,

&

Johansson,

A.

(2012).

Aggregatibacter

CC

actinomycetemcomitans targets NLRP3 and NLRP6 inflammasome expression in human mononuclear leukocytes. Cytokine, 59(1), 124-130.

A

Bergstrom, J. (2004). Tobacco smoking and chronic destructive periodontal disease. Odontology, 92(1), 1-8.

Bodet, C., Chandad, F., & Grenier, D. (2007). [Pathogenic potential of Porphyromonas gingivalis, Treponema denticola and Tannerella

21

22

forsythia, the red bacterial complex associated with periodontitis]. Pathol

Biol (Paris), 55(3-4), 154-162. Bostanci, N., Emingil, G., Saygan, B., Turkoglu, O., Atilla, G., Curtis, M. A., & Belibasakis, G. N. (2009). Expression and regulation of the NALP3

IP T

inflammasome complex in periodontal diseases. Clin Exp Immunol, 157(3), 415-422.

SC R

Bostanci, N., Meier, A., Guggenheim, B., & Belibasakis, G. N. (2011).

Regulation of NLRP3 and AIM2 inflammasome gene expression levels

U

in gingival fibroblasts by oral biofilms. Cell Immunol, 270(1), 88-93.

A

N

Brydges, S. D., Mueller, J. L., McGeough, M. D., Pena, C. A., Misaghi, A.,

M

Gandhi, C., . . . Hoffman, H. M. (2009). Inflammasome-mediated disease animal models reveal roles for innate but not adaptive immunity.

TE D

Immunity, 30(6), 875-887.

Bui, F. Q., Johnson, L., Roberts, J., Hung, S. C., Lee, J., Atanasova, K. R., . . .

EP

Ojcius, D. M. (2016). Fusobacterium nucleatum infection of gingival

CC

epithelial cells leads to NLRP3 inflammasome-dependent secretion of

A

IL-1beta and the danger signals ASC and HMGB1. Cell Microbiol, 18(7), 970-981.

Cabanski, M., Steinmuller, M., Marsh, L. M., Surdziel, E., Seeger, W., & Lohmeyer, J. (2008). PKR regulates TLR2/TLR4-dependent signaling in murine alveolar macrophages. Am J Respir Cell Mol Biol, 38(1), 26-31.

22

23

Cecil, J. D., O'Brien-Simpson, N. M., Lenzo, J. C., Holden, J. A., Singleton, W., Perez-Gonzalez, A., . . . Reynolds, E. C. (2017). Outer Membrane Vesicles Prime and Activate Macrophage Inflammasomes and Cytokine Secretion In Vitro and In Vivo. Front Immunol, 8, 1017.

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Chaiyarit, P., Kafrawy, A. H., Miles, D. A., Zunt, S. L., Van Dis, M. L., & Gregory, R. L. (1999). Oral lichen planus: an immunohistochemical study of heat

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shock proteins (HSPs) and cytokeratins (CKs) and a unifying hypothesis of pathogenesis. J Oral Pathol Med, 28(5), 210-215.

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Chen, L., Huang, C. F., Li, Y. C., Deng, W. W., Mao, L., Wu, L., . . . Sun, Z. J.

A

N

(2017). Blockage of the NLRP3 inflammasome by MCC950 improves

M

anti-tumor immune responses in head and neck squamous cell carcinoma. Cell Mol Life Sci.

TE D

Chen, L. C., Wang, L. J., Tsang, N. M., Ojcius, D. M., Chen, C. C., Ouyang, C. N., . . . Chang, Y. S. (2012). Tumour inflammasome-derived IL-1beta

EP

recruits neutrophils and improves local recurrence-free survival in EBV-

CC

induced nasopharyngeal carcinoma. EMBO Mol Med, 4(12), 1276-1293.

A

Chen, M., Wang, H., Chen, W., & Meng, G. (2011). Regulation of adaptive immunity by the NLRP3 inflammasome. Int Immunopharmacol, 11(5), 549-554.

Cheng, R., Liu, W., Zhang, R., Feng, Y., Bhowmick, N. A., & Hu, T. (2017). Porphyromonas

gingivalis-Derived

23

Lipopolysaccharide

Combines

24

Hypoxia to Induce Caspase-1 Activation in Periodontitis. Front Cell Infect

Microbiol, 7. Choi, C. H., Spooner, R., DeGuzman, J., Koutouzis, T., Ojcius, D. M., & Yilmaz, Ö. (2013). P. gingivalis-Nucleoside-diphosphate-kinase Inhibits ATP-

IP T

Induced Reactive-Oxygen-Species via P2X(7) Receptor/NADPHOxidase Signaling and Contributes to Persistence. Cell Microbiol, 15(6),

SC R

961-976.

Chung, Y., Chang, S. H., Martinez, G. J., Yang, X. O., Nurieva, R., Kang, H.

U

S., . . . Dong, C. (2009). Critical regulation of early Th17 cell

A

N

differentiation by interleukin-1 signaling. Immunity, 30(4), 576-587.

M

Conway, K. E., McConnell, B. B., Bowring, C. E., Donald, C. D., Warren, S. T., & Vertino, P. M. (2000). TMS1, a novel proapoptotic caspase recruitment

TE D

domain protein, is a target of methylation-induced gene silencing in human breast cancers. Cancer Res, 60(22), 6236-6242.

EP

Coutinho-Silva, R., Corrêa, G., Sater, A. A., & Ojcius, D. M. (2009). The P2X(7)

CC

receptor and intracellular pathogens: a continuing struggle. Purinergic

Signal, 5(2), 197-204.

A

Dan Dunn, J., Alvarez, L. A., Zhang, X., & Soldati, T. (2015). Reactive oxygen species and mitochondria: A nexus of cellular homeostasis. Redox Biol, 6, 472-485. Dawood, S., Austin, L., & Cristofanilli, M. (2014). Cancer stem cells:

24

25

implications for cancer therapy. Oncology (Williston Park), 28(12), 11011107, 1110. Delaleu, N., & Bickel, M. (2004). Interleukin-1 beta and interleukin-18: regulation and activity in local inflammation. Periodontol 2000, 35, 42-

IP T

52.

Deng, S., Xi, Y., Wang, H., Hao, J., Niu, X., Li, W., . . . Chen, G. (2010).

SC R

Regulatory effect of vasoactive intestinal peptide on the balance of Treg and Th17 in collagen-induced arthritis. Cell Immunol, 265(2), 105-110.

U

Di, P., & Gao, Y. (2003). [Studies of heat shock protein 60 and heat shock

A

N

protein 70 in oral lichen planus]. Zhonghua Kou Qiang Yi Xue Za Zhi,

M

38(4), 275-278.

Dinarello, C. A. (2009). Immunological and inflammatory functions of the

TE D

interleukin-1 family. Annu Rev Immunol, 27, 519-550. Dubaniewicz, A. (2013). Microbial and human heat shock proteins as 'danger

EP

signals' in sarcoidosis. Hum Immunol, 74(12), 1550-1558.

CC

Elliott, E. I., & Sutterwala, F. S. (2015). Initiation and perpetuation of NLRP3 inflammasome activation and assembly. Immunol Rev, 265(1), 35-52.

A

Feng, X., Luo, Q., Wang, H., Zhang, H., & Chen, F. (2018). MicroRNA-22 suppresses cell proliferation, migration and invasion in oral squamous cell carcinoma by targeting NLRP3. J Cell Physiol. Feng, X., Luo, Q., Zhang, H., Wang, H., Chen, W., Meng, G., & Chen, F.

25

26

(2017a). The role of NLRP3 inflammasome in 5-fluorouracil resistance of oral squamous cell carcinoma. J Exp Clin Cancer Res, 36. Feng, X., Luo, Q., Zhang, H., Wang, H., Chen, W., Meng, G., & Chen, F. (2017b). The role of NLRP3 inflammasome in 5-fluorouracil resistance

IP T

of oral squamous cell carcinoma. J Exp Clin Cancer Res, 36(1), 81.

Ford, P. J., Yamazaki, K., & Seymour, G. J. (2007). Cardiovascular and oral

SC R

disease interactions: what is the evidence? Prim Dent Care, 14(2), 5966.

U

Frangogiannis, N. G. (2014). The inflammatory response in myocardial injury,

A

N

repair, and remodelling. Nat Rev Cardiol, 11(5), 255-265.

M

Garlanda, C., Dinarello, C. A., & Mantovani, A. (2013). The interleukin-1 family: back to the future. Immunity, 39(6), 1003-1018.

TE D

Gholizadeh, P., Pormohammad, A., Eslami, H., Shokouhi, B., Fakhrzadeh, V., & Kafil, H. S. (2017). Oral pathogenesis of Aggregatibacter

EP

actinomycetemcomitans. Microb Pathog, 113, 303-311.

CC

Giussani, D. A., Niu, Y., Herrera, E. A., Richter, H. G., Camm, E. J., Thakor, A.

A

S., . . . Allison, B. J. (2014). Heart disease link to fetal hypoxia and oxidative stress. Adv Exp Med Biol, 814, 77-87.

Golz, L., Memmert, S., Rath-Deschner, B., Jager, A., Appel, T., Baumgarten, G., . . . Frede, S. (2014). LPS from P. gingivalis and hypoxia increases oxidative stress in periodontal ligament fibroblasts and contributes to

26

27

periodontitis. Mediators Inflamm, 2014, 986264. Golz, L., Memmert, S., Rath-Deschner, B., Jager, A., Appel, T., Baumgarten, G., . . . Frede, S. (2015). Hypoxia and P. gingivalis synergistically induce HIF-1 and NF-kappaB activation in PDL cells and periodontal diseases.

IP T

Mediators Inflamm, 2015, 438085.

Gombault, A., Baron, L., & Couillin, I. (2012). ATP release and purinergic

SC R

signaling in NLRP3 inflammasome activation. Front Immunol, 3, 414.

Gray, S. P., Di Marco, E., Kennedy, K., Chew, P., Okabe, J., El-Osta, A., . . .

U

Jandeleit-Dahm, K. A. (2016). Reactive Oxygen Species Can Provide

A

N

Atheroprotection via NOX4-Dependent Inhibition of Inflammation and

M

Vascular Remodeling. Arterioscler Thromb Vasc Biol, 36(2), 295-307. Gris, D., Ye, Z., Iocca, H. A., Wen, H., Craven, R. R., Gris, P., . . . Ting, J. P.

TE D

(2010). NLRP3 plays a critical role in the development of experimental autoimmune encephalomyelitis by mediating Th1 and Th17 responses.

EP

J Immunol, 185(2), 974-981.

CC

Gross, O., Gewies, A., Finger, K., Schafer, M., Sparwasser, T., Peschel, C., . . .

A

Ruland, J. (2006). Card9 controls a non-TLR signalling pathway for innate anti-fungal immunity. Nature, 442(7103), 651-656.

Guan, X., Sagara, J., Yokoyama, T., Koganehira, Y., Oguchi, M., Saida, T., & Taniguchi, S. (2003). ASC/TMS1, a caspase-1 activating adaptor, is downregulated by aberrant methylation in human melanoma. Int J

27

28

Cancer, 107(2), 202-208. Guo, H., Callaway, J. B., & Ting, J. P. (2015). Inflammasomes: mechanism of action, role in disease, and therapeutics. Nat Med, 21(7), 677-687. Guo, W., Wang, P., Liu, Z., Yang, P., & Ye, P. (2015). The activation of pyrin

from

Porphyromonas

gingivalis

and

extracellular

IP T

domain-containing-3 inflammasome depends on lipopolysaccharide adenosine

SC R

triphosphate in cultured oral epithelial cells. BMC Oral Health, 15(1), 133. Hajishengallis, G., & Lamont, R. J. (2014). Breaking bad: manipulation of the

U

host response by Porphyromonas gingivalis. Eur J Immunol, 44(2), 328-

A

N

338.

M

Han, Y. W. (2015). Fusobacterium nucleatum: a commensal-turned pathogen.

Curr Opin Microbiol, 23, 141-147.

TE D

Handono, K., Pratama, M. Z., Endharti, A. T., & Kalim, H. (2015). Treatment of low doses curcumin could modulate Th17/Treg balance specifically on

EP

CD4+ T cell cultures of systemic lupus erythematosus patients. Cent Eur

CC

J Immunol, 40(4), 461-469.

A

Haneji, T. (2017). Roles of PKR in differentiation and apoptosis of bone-related cells. Anat Sci Int, 92(3), 313-319.

Hanioka, T., Tanaka, M., Ojima, M., Shizukuishi, S., & Folkers, K. (1994). Effect of topical application of coenzyme Q10 on adult periodontitis. Mol

Aspects Med, 15 Suppl, s241-248.

28

29

Hans, M., & Hans, V. M. (2011). Toll-like receptors and their dual role in periodontitis: a review. J Oral Sci, 53(3), 263-271. Harrison, O. J., Srinivasan, N., Pott, J., Schiering, C., Krausgruber, T., Ilott, N. E., & Maloy, K. J. (2015). Epithelial-derived IL-18 regulates Th17 cell

IP T

differentiation and Foxp3(+) Treg cell function in the intestine. Mucosal

Immunol, 8(6), 1226-1236.

SC R

Hato, T., & Dagher, P. C. (2015). How the Innate Immune System Senses

Trouble and Causes Trouble. Clin J Am Soc Nephrol, 10(8), 1459-1469.

U

He, Y., Hara, H., & Nunez, G. (2016). Mechanism and Regulation of NLRP3

A

N

Inflammasome Activation. Trends Biochem Sci, 41(12), 1012-1021.

M

Hedl, M., Zheng, S., & Abraham, C. (2014). The IL18RAP region disease polymorphism decreases IL-18RAP/IL-18R1/IL-1R1 expression and

TE D

signaling through innate receptor-initiated pathways. J Immunol, 192(12), 5924-5932.

EP

Heneka, M. T., Kummer, M. P., Stutz, A., Delekate, A., Schwartz, S., Vieira-

CC

Saecker, A., . . . Golenbock, D. T. (2013). NLRP3 is activated in

A

Alzheimer's disease and contributes to pathology in APP/PS1 mice.

Nature, 493(7434), 674-678.

Herbert, B. A., Novince, C. M., & Kirkwood, K. L. (2016). Aggregatibacter actinomycetemcomitans, a potent immunoregulator of the periodontal host defense system and alveolar bone homeostasis. Mol Oral Microbiol,

29

30

31(3), 207-227. Hoeberichts, F. A., Perez-Valle, J., Montesinos, C., Mulet, J. M., Planes, M. D., Hueso, G., . . . Serrano, R. (2010). The role of K(+) and H(+) transport during

glucose-

and

H(2)O(2)-induced

Saccharomyces cerevisiae. Yeast, 27(9), 713-725.

cell

death

in

IP T

systems

Hoffman, H. M., Mueller, J. L., Broide, D. H., Wanderer, A. A., & Kolodner, R.

SC R

D. (2001). Mutation of a new gene encoding a putative pyrin-like protein

syndrome. Nat Genet, 29(3), 301-305.

U

causes familial cold autoinflammatory syndrome and Muckle-Wells

A

N

Hsu, L. C., Park, J. M., Zhang, K., Luo, J. L., Maeda, S., Kaufman, R. J., . . .

M

Karin, M. (2004). The protein kinase PKR is required for macrophage

345.

TE D

apoptosis after activation of Toll-like receptor 4. Nature, 428(6980), 341-

Huang, C. F., Chen, L., Li, Y. C., Wu, L., Yu, G. T., Zhang, W. F., & Sun, Z. J.

EP

(2017). NLRP3 inflammasome activation promotes inflammation-

CC

induced carcinogenesis in head and neck squamous cell carcinoma. J

Exp Clin Cancer Res, 36(1), 116.

A

Hung, S. C., Choi, C. H., Said-Sadier, N., Johnson, L., Atanasova, K. R., Sellami, H., . . . Ojcius, D. M. (2013). P2X4 assembles with P2X7 and pannexin-1 in gingival epithelial cells and modulates ATP-induced reactive oxygen species production and inflammasome activation. PLoS

30

31

One, 8(7), e70210. Ivanov, II, McKenzie, B. S., Zhou, L., Tadokoro, C. E., Lepelley, A., Lafaille, J. J., . . . Littman, D. R. (2006). The orphan nuclear receptor RORgammat

cells. Cell, 126(6), 1121-1133.

IP T

directs the differentiation program of proinflammatory IL-17+ T helper

Iwasaki, A., & Medzhitov, R. (2015). Control of adaptive immunity by the innate

SC R

immune system. Nat Immunol, 16(4), 343-353.

Jang, J. H., Shin, H. W., Lee, J. M., Lee, H. W., Kim, E. C., & Park, S. H. (2015).

U

An Overview of Pathogen Recognition Receptors for Innate Immunity in

A

N

Dental Pulp. Mediators Inflamm, 2015, 794143.

M

Jefferson, J. A., Escudero, E., Johnson, R. J., Swenson, E. R., & Hurtado, A. (2014). Increased oxidative stress at altitude. Chest, 145(2), 423.

TE D

Jiang, W., Lv, H., Wang, H., Wang, D., Sun, S., Jia, Q., . . . Ni, L. (2015). Activation of the NLRP3/caspase-1 inflammasome in human dental pulp

EP

tissue and human dental pulp fibroblasts. Cell Tissue Res, 361(2), 541-

CC

555.

A

Jiang, X., Hu, C., Arnovitz, S., Bugno, J., Yu, M., Zuo, Z., . . . Chen, J. (2016). miR-22 has a potent anti-tumour role with therapeutic potential in acute myeloid leukaemia. Nat Commun, 7, 11452.

Jin, C., Frayssinet, P., Pelker, R., Cwirka, D., Hu, B., Vignery, A., . . . Flavell, R. A. (2011). NLRP3 inflammasome plays a critical role in the pathogenesis

31

32

of hydroxyapatite-associated arthropathy. Proc Natl Acad Sci U S A, 108(36), 14867-14872. Johnson, L., Atanasova, K. R., Bui, P. Q., Lee, J., Hung, S. C., Yilmaz, O., & Ojcius, D. M. (2015). Porphyromonas gingivalis attenuates ATP-

IP T

mediated inflammasome activation and HMGB1 release through

expression of a nucleoside-diphosphate kinase. Microbes Infect, 17(5),

SC R

369-377.

Jontell, M., Okiji, T., Dahlgren, U., & Bergenholtz, G. (1998). Immune defense

U

mechanisms of the dental pulp. Crit Rev Oral Biol Med, 9(2), 179-200.

A

N

Jourdan, T., Godlewski, G., Cinar, R., Bertola, A., Szanda, G., Liu, J., . . . Kunos,

M

G. (2013). Activation of the Nlrp3 inflammasome in infiltrating macrophages by endocannabinoids mediates beta cell loss in type 2

TE D

diabetes. Nat Med, 19(9), 1132-1140. Jun, H. K., Lee, S. H., Lee, H. R., & Choi, B. K. (2012). Integrin alpha5beta1

EP

activates the NLRP3 inflammasome by direct interaction with a bacterial

CC

surface protein. Immunity, 36(5), 755-768.

A

Kahlenberg, J. M., & Dubyak, G. R. (2004). Mechanisms of caspase-1 activation by P2X7 receptor-mediated K+ release. Am J Physiol Cell

Physiol, 286(5), C1100-1108.

Karthikeyan, B., Talwar, Arun, K. V., & Kalaivani, S. (2015). Evaluation of transcription factor that regulates T helper 17 and regulatory T cells

32

33

function in periodontal health and disease. J Pharm Bioallied Sci, 7(Suppl 2), S672-676. Kato, S., Kowashi, Y., & Demuth, D. R. (2002). Outer membrane-like vesicles secreted by Actinobacillus actinomycetemcomitans are enriched in

IP T

leukotoxin. Microb Pathog, 32(1), 1-13.

Kawamoto, D., Ando, E. S., Longo, P. L., Nunes, A. C., Wikstrom, M., & Mayer,

SC R

M. P. (2009). Genetic diversity and toxic activity of Aggregatibacter

actinomycetemcomitans isolates. Oral Microbiol Immunol, 24(6), 493-

U

501.

A

N

Kelk, P., Abd, H., Claesson, R., Sandstrom, G., Sjostedt, A., & Johansson, A.

M

(2011). Cellular and molecular response of human macrophages exposed to Aggregatibacter actinomycetemcomitans leukotoxin. Cell

TE D

Death Dis, 2, e126.

Kelk, P., Johansson, A., Claesson, R., Hanstrom, L., & Kalfas, S. (2003).

EP

Caspase 1 involvement in human monocyte lysis induced by

CC

Actinobacillus actinomycetemcomitans leukotoxin. Infect Immun, 71(8), 4448-4455.

A

Kigerl, K. A., de Rivero Vaccari, J. P., Dietrich, W. D., Popovich, P. G., & Keane, R. W. (2014). Pattern recognition receptors and central nervous system repair. Exp Neurol, 258, 5-16. Kim, E. H., Park, M. J., Park, S., & Lee, E. S. (2015). Increased expression of

33

34

the NLRP3 inflammasome components in patients with Behcet's disease.

J Inflamm (Lond), 12, 41. Kim, J. J., & Jo, E. K. (2013). NLRP3 inflammasome and host protection against bacterial infection. J Korean Med Sci, 28(10), 1415-1423.

IP T

Kolb, R., Liu, G. H., Janowski, A. M., Sutterwala, F. S., & Zhang, W. (2014).

Inflammasomes in cancer: a double-edged sword. Protein Cell, 5(1), 12-

SC R

20.

Kong, H., Wang, Y., Zeng, X., Wang, Z., Wang, H., & Xie, W. (2015). Differential

N

A

Tumour Biol, 36(10), 7501-7513.

U

expression of inflammasomes in lung cancer cell lines and tissues.

M

Krause, K. H. (2004). Tissue distribution and putative physiological function of NOX family NADPH oxidases. Jpn J Infect Dis, 57(5), S28-29.

TE D

Kuo, H. C., Chang, L. C., Chen, T. C., Lee, K. C., Lee, K. F., Chen, C. N., & Yu, H. R. (2016). Sterol Regulatory Element-Binding Protein-1c Regulates

EP

Inflammasome Activation in Gingival Fibroblasts Infected with High-

CC

Glucose-Treated Porphyromonas gingivalis. Front Cell Infect Microbiol, 6, 195.

A

Kurita-Ochiai, T., Jia, R., Cai, Y., Yamaguchi, Y., & Yamamoto, M. (2015). Periodontal Disease-Induced Atherosclerosis and Oxidative Stress.

Antioxidants (Basel), 4(3), 577-590. Lalor, S. J., Dungan, L. S., Sutton, C. E., Basdeo, S. A., Fletcher, J. M., & Mills,

34

35

K. H. (2011). Caspase-1-processed cytokines IL-1beta and IL-18 promote IL-17 production by gammadelta and CD4 T cells that mediate autoimmunity. J Immunol, 186(10), 5738-5748. Lamkanfi, M., & Dixit, V. M. (2014). Mechanisms and functions of

IP T

inflammasomes. Cell, 157(5), 1013-1022.

Lazaridis, L. D., Pistiki, A., Giamarellos-Bourboulis, E. J., Georgitsi, M.,

SC R

Damoraki, G., Polymeros, D., . . . Triantafyllou, K. (2017). Activation of NLRP3 Inflammasome in Inflammatory Bowel Disease: Differences

U

Between Crohn's Disease and Ulcerative Colitis. Dig Dis Sci, 62(9),

A

N

2348-2356.

M

Lee, M. C. (2014). [Oxidative stress and periodontal disease--periodontal disease as a life-related disease and vascular disease]. Nihon

TE D

Yakurigaku Zasshi, 144(6), 281-286. Lee, S., Suh, G. Y., Ryter, S. W., & Choi, A. M. (2016a). Regulation and

EP

Function of the Nucleotide Binding Domain Leucine-Rich Repeat-

CC

Containing Receptor, Pyrin Domain-Containing-3 Inflammasome in Lung Disease. Am J Respir Cell Mol Biol, 54(2), 151-160.

A

Lee, S., Suh, G. Y., Ryter, S. W., & Choi, A. M. K. (2016b). Regulation and Function of the Nucleotide Binding Domain Leucine-Rich RepeatContaining Receptor, Pyrin Domain-Containing-3 Inflammasome in Lung Disease. Am J Respir Cell Mol Biol, 54(2), 151-160.

35

36

Lee, S. I., Kang, S. K., Jung, H. J., Chun, Y. H., Kwon, Y. D., & Kim, E. C. (2015). Muramyl dipeptide activates human beta defensin 2 and proinflammatory mediators through Toll-like receptors and NLRP3 inflammasomes in human dental pulp cells. Clin Oral Investig, 19(6),

IP T

1419-1428.

Lenertz, L. Y., Gavala, M. L., Zhu, Y., & Bertics, P. J. (2011). Transcriptional

SC R

control mechanisms associated with the nucleotide receptor P2X7, a

critical regulator of immunologic, osteogenic, and neurologic functions.

U

Immunol Res, 50(1), 22-38.

A

N

Linden, G. J., Lyons, A., & Scannapieco, F. A. (2013). Periodontal systemic

M

associations: review of the evidence. J Clin Periodontol, 40 Suppl 14, S8-19.

TE D

Liston, A., & Masters, S. L. (2017). Homeostasis-altering molecular processes as mechanisms of inflammasome activation. Nat Rev Immunol, 17(3),

EP

208-214.

CC

Liu, C., Mo, L., Niu, Y., Li, X., Zhou, X., & Xu, X. (2017). The Role of Reactive

A

Oxygen Species and Autophagy in Periodontitis and Their Potential Linkage. Front Physiol, 8, 439.

Liu, D., Zeng, X., Li, X., Mehta, J. L., & Wang, X. (2017). Role of NLRP3 inflammasome in the pathogenesis of cardiovascular diseases. Basic

Res Cardiol, 113(1), 5.

36

37

Liu, X., Nie, S., Huang, D., & Xie, M. (2015). Nonylphenol regulates cyclooxygenase-2 expression via Ros-activated NF-kappaB pathway in sertoli TM4 cells. Environ Toxicol, 30(10), 1144-1152. Llambes, F., Arias-Herrera, S., & Caffesse, R. (2015). Relationship between

IP T

diabetes and periodontal infection. World J Diabetes, 6(7), 927-935.

Love, R. M., & Jenkinson, H. F. (2002). Invasion of dentinal tubules by oral

SC R

bacteria. Crit Rev Oral Biol Med, 13(2), 171-183.

Lozhkin, A., Vendrov, A. E., Pan, H., Wickline, S. A., Madamanchi, N. R., &

U

Runge, M. S. (2017). NADPH oxidase 4 regulates vascular inflammation

A

N

in aging and atherosclerosis. J Mol Cell Cardiol, 102, 10-21.

M

Lu, B., Nakamura, T., Inouye, K., Li, J., Tang, Y., Lundback, P., . . . Tracey, K. J. (2012). Novel role of PKR in inflammasome activation and HMGB1

TE D

release. Nature, 488(7413), 670-674. Lu, W. L., Song, D. Z., Yue, J. L., Wang, T. T., Zhou, X. D., Zhang, P., . . .

EP

Huang, D. M. (2017). NLRP3 inflammasome may regulate inflammatory

CC

response of human periodontal ligament fibroblasts in an apoptosis-

A

associated speck-like protein containing a CARD (ASC)-dependent manner. Int Endod J, 50(10), 967-975.

Lv, J., Zhu, Y. X., Liu, Y. Q., & Xue, X. (2015). Distinctive pathways characterize A. actinomycetemcomitans and P. gingivalis. Mol Biol Rep, 42(2), 441449.

37

38

Martin, S. F. (2014). Adaptation in the innate immune system and heterologous innate immunity. Cell Mol Life Sci, 71(21), 4115-4130. Maslanik, T., Mahaffey, L., Tannura, K., Beninson, L., Greenwood, B. N., & Fleshner, M. (2013). The inflammasome and danger associated

IP T

molecular patterns (DAMPs) are implicated in cytokine and chemokine responses following stressor exposure. Brain Behav Immun, 28, 54-62.

SC R

Mearini, E., Poli, G., Cochetti, G., Boni, A., Egidi, M. G., & Brancorsini, S. (2017). Expression of urinary miRNAs targeting NLRs inflammasomes in

U

bladder cancer. Onco Targets Ther, 10, 2665-2673.

A

N

Meng, G., Zhang, F., Fuss, I., Kitani, A., & Strober, W. (2009). A mutation in the

M

Nlrp3 gene causing inflammasome hyperactivation potentiates Th17 cell-dominant immune responses. Immunity, 30(6), 860-874.

TE D

Mills, K. H., Dungan, L. S., Jones, S. A., & Harris, J. (2013). The role of inflammasome-derived IL-1 in driving IL-17 responses. J Leukoc Biol,

EP

93(4), 489-497.

CC

Mima, K., Sukawa, Y., Nishihara, R., Qian, Z. R., Yamauchi, M., Inamura, K., . . .

A

Ogino, S. (2015). Fusobacterium nucleatum and T Cells in Colorectal Carcinoma. JAMA Oncol, 1(5), 653-661.

Miyauchi, M., Takata, T., Ito, H., Ogawa, I., Kobayashi, J., Nikai, H., & Ijuhin, N. (1996). Immunohistochemical demonstration of prostaglandins E2, F2 alpha, and 6-keto-prostaglandin F1 alpha in rat dental pulp with

38

39

experimentally induced inflammation. J Endod, 22(11), 600-602. Moore, W. E., & Moore, L. V. (1994). The bacteria of periodontal diseases.

Periodontol 2000, 5, 66-77. Motohira, H., Hayashi, J., Tatsumi, J., Tajima, M., Sakagami, H., & Shin, K.

IP T

(2007). Hypoxia and reoxygenation augment bone-resorbing factor

production from human periodontal ligament cells. J Periodontol, 78(9),

SC R

1803-1809.

Ni, K., Zhao, L., Wu, J., Chen, W., HongyaYang, & Li, X. (2015). Th17/Treg

U

balance in children with obstructive sleep apnea syndrome and the

A

N

relationship with allergic rhinitis. Int J Pediatr Otorhinolaryngol, 79(9),

M

1448-1454.

Odendall, C., & Kagan, J. C. (2017). Activation and pathogenic manipulation of

237.

TE D

the sensors of the innate immune system. Microbes Infect, 19(4-5), 229-

EP

Okui, T., Aoki, Y., Ito, H., Honda, T., & Yamazaki, K. (2012). The presence of

CC

IL-17+/FOXP3+ double-positive cells in periodontitis. J Dent Res, 91(6), 574-579.

A

Olsen, I., Lambris, J. D., & Hajishengallis, G. (2017). Porphyromonas gingivalis disturbs host-commensal homeostasis by changing complement function. J Oral Microbiol, 9(1), 1340085. Olsen, I., & Yilmaz, O. (2016). Modulation of inflammasome activity by

39

40

Porphyromonas gingivalis in periodontitis and associated systemic diseases. J Oral Microbiol, 8, 30385. Orozco, A., Gemmell, E., Bickel, M., & Seymour, G. J. (2006). Interleukin-1beta, interleukin-12 and interleukin-18 levels in gingival fluid and serum of

IP T

patients with gingivitis and periodontitis. Oral Microbiol Immunol, 21(4), 256-260.

SC R

Palova-Jelinkova, L., Danova, K., Drasarova, H., Dvorak, M., Funda, D. P.,

Fundova, P., . . . Tuckova, L. (2013). Pepsin digest of wheat gliadin increases

production

of

U

fraction

IL-1beta

via

A

N

TLR4/MyD88/TRIF/MAPK/NF-kappaB signaling pathway and an NLRP3

M

inflammasome activation. PLoS One, 8(4), e62426. Pan, X., Wu, X., Yan, D., Peng, C., Rao, C., & Yan, H. (2018). Acrylamide-

TE D

induced oxidative stress and inflammatory response are alleviated by Nacetylcysteine in PC12 cells: Involvement of the crosstalk between Nrf2

EP

and NF-kappaB pathways regulated by MAPKs. Toxicol Lett, 288, 55-

CC

64.

A

Pandey, A. K., Patnaik, R., Muresanu, D. F., Sharma, A., & Sharma, H. S. (2012). Quercetin in hypoxia-induced oxidative stress: novel target for neuroprotection. Int Rev Neurobiol, 102, 107-146.

Park, E., Na, H. S., Song, Y. R., Shin, S. Y., Kim, Y. M., & Chung, J. (2014). Activation of NLRP3 and AIM2 inflammasomes by Porphyromonas

40

41

gingivalis infection. Infect Immun, 82(1), 112-123. Park, S. R., Kim, D. J., Han, S. H., Kang, M. J., Lee, J. Y., Jeong, Y. J., . . . Park, J. H. (2014). Diverse Toll-like receptors mediate cytokine production

by

Fusobacterium

nucleatum

and

Aggregatibacter

IP T

actinomycetemcomitans in macrophages. Infect Immun, 82(5), 19141920.

SC R

Parthiban, P., & Mahendra, J. (2015). Toll-Like Receptors: A Key Marker for

Periodontal Disease and Preterm Birth - A Contemporary Review. J Clin

U

Diagn Res, 9(9), Ze14-17.

Spleen

tyrosine

kinase

inhibition

ameliorates

airway

M

(2018).

A

N

Patel, D., Gaikwad, S., Challagundla, N., Nivsarkar, M., & Agrawal-Rajput, R.

inflammation through modulation of NLRP3 inflammosome and

TE D

Th17/Treg axis. Int Immunopharmacol, 54, 375-384. Paugh, S. W., Bonten, E. J., Savic, D., Ramsey, L. B., Thierfelder, W. E.,

EP

Gurung, P., . . . Evans, W. E. (2015). NALP3 inflammasome upregulation

CC

and CASP1 cleavage of the glucocorticoid receptor cause glucocorticoid resistance in leukemia cells. Nat Genet, 47(6), 607-614.

A

Prakash, S., Sunitha, J., & Hans, M. (2010). Role of coenzyme Q(10) as an antioxidant and bioenergizer in periodontal diseases. Indian J

Pharmacol, 42(6), 334-337. Ramos-Junior, E. S., Morandini, A. C., Almeida-da-Silva, C. L., Franco, E. J.,

41

42

Potempa, J., Nguyen, K. A., . . . Coutinho-Silva, R. (2015). A Dual Role for P2X7 Receptor during Porphyromonas gingivalis Infection. J Dent

Res, 94(9), 1233-1242. Reid, B. C., Winn, D. M., Morse, D. E., & Pendrys, D. G. (2000). Head and neck

IP T

in situ carcinoma: incidence, trends, and survival. Oral Oncol, 36(5), 414-420.

SC R

Roopashree, M. R., Gondhalekar, R. V., Shashikanth, M. C., George, J., Thippeswamy, S. H., & Shukla, A. (2010). Pathogenesis of oral lichen

U

planus--a review. J Oral Pathol Med, 39(10), 729-734.

M

lichenoid disease. 12(7).

A

N

Rusanen, P. (2017). TLR1-10, NF-κB and p53 expression is increased in oral

Sandoval, M., & Harris, L. (2014). Cancer stem cells. Oncology (Williston Park),

TE D

28(12), 1110-1111, 1114.

Sarode, S. C., Sarode, G. S., & Patil, A. (2014). Therapeutic aspect of oral

EP

lichen planus in context to accompanying candidal infection. Oral Oncol,

CC

50(7), e34.

A

Schwechheimer, C., & Kuehn, M. J. (2015). Outer-membrane vesicles from Gram-negative bacteria: biogenesis and functions. Nat Rev Microbiol, 13(10), 605-619.

Seymour, G. J., Ford, P. J., Cullinan, M. P., Leishman, S., & Yamazaki, K. (2007). Relationship between periodontal infections and systemic

42

43

disease. Clin Microbiol Infect, 13 Suppl 4, 3-10. Shalev, S. A., Sprecher, E., Indelman, M., Hujirat, Y., Bergman, R., & Rottem, M. (2007). A novel missense mutation in CIAS1 encoding the pyrin-like protein, cryopyrin, causes familial cold autoinflammatory syndrome in a

IP T

family of Ethiopian origin. Int Arch Allergy Immunol, 143(3), 190-193.

Shenker, B. J., Ojcius, D. M., Walker, L. P., Zekavat, A., Scuron, M. D., &

SC R

Boesze-Battaglia, K. (2015). Aggregatibacter actinomycetemcomitans

cytolethal distending toxin activates the NLRP3 inflammasome in human

N

A

Infect Immun, 83(4), 1487-1496.

U

macrophages, leading to the release of proinflammatory cytokines.

M

Socransky, S. S. (1977). Microbiology of periodontal disease -- present status and future considerations. J Periodontol, 48(9), 497-504.

TE D

Sohn, K. C., Lee, E. J., Shin, J. M., Lim, E. H., No, Y., Lee, J. Y., . . . Kim, C. D. (2014). Regulation of keratinocyte differentiation by O-GlcNAcylation. J

EP

Dermatol Sci, 75(1), 10-15.

CC

Song, Z., Lin, Z., He, F., Jiang, L., Qin, W., Tian, Y., . . . Huang, S. (2012).

A

NLRP3 is expressed in human dental pulp cells and tissues. J Endod, 38(12), 1592-1597.

Stutz, A., Kolbe, C. C., Stahl, R., Horvath, G. L., Franklin, B. S., van Ray, O., . . . Latz, E. (2017). NLRP3 inflammasome assembly is regulated by phosphorylation of the pyrin domain. J Exp Med, 214(6), 1725-1736.

43

44

Sutterwala, F. S., Haasken, S., & Cassel, S. L. (2014). Mechanism of NLRP3 inflammasome activation. Ann N Y Acad Sci, 1319, 82-95. Tao, X. A., Xia, J., Chen, X. B., Wang, H., Dai, Y. H., Rhodus, N. L., & Cheng,

correlated with disease activity. Oral Dis, 16(1), 76-82.

IP T

B. (2010). FOXP3 T regulatory cells in lesions of oral lichen planus

Taxman, D. J., Swanson, K. V., Broglie, P. M., Wen, H., Holley-Guthrie, E.,

SC R

Huang, M. T., . . . Ting, J. P. (2012). Porphyromonas gingivalis mediates inflammasome repression in polymicrobial cultures through a novel

U

mechanism involving reduced endocytosis. J Biol Chem, 287(39),

A

N

32791-32799.

M

Teles, R., Sakellari, D., Teles, F., Konstantinidis, A., Kent, R., Socransky, S., & Haffajee, A. (2010). Relationships among gingival crevicular fluid

TE D

biomarkers, clinical parameters of periodontal disease, and the subgingival microbiota. J Periodontol, 81(1), 89-98.

EP

Thi Do, T., Phoomak, C., Champattanachai, V., Silsirivanit, A., & Chaiyarit, P. of

connections between

increased O-

CC

(2018). New evidence

A

GlcNAcylation and inflammasome in the oral mucosa of patients with oral lichen planus. Clin Exp Immunol, 192(1), 129-137.

Ting, J. P., Kastner, D. L., & Hoffman, H. M. (2006). CATERPILLERs, pyrin and hereditary immunological disorders. Nat Rev Immunol, 6(3), 183-195. Ting, J. P., Lovering, R. C., Alnemri, E. S., Bertin, J., Boss, J. M., Davis, B.

44

45

K., . . . Ward, P. A. (2008). The NLR gene family: a standard nomenclature. Immunity, 28(3), 285-287. Wang, L., Wang, J., Jin, Y., Gao, H., & Lin, X. (2014). Oral administration of all-

the Th17/Treg imbalance. J Periodontol, 85(5), 740-750.

IP T

trans retinoic acid suppresses experimental periodontitis by modulating

Wang, Y., Andrukhov, O., & Rausch-Fan, X. (2017). Oxidative Stress and

SC R

Antioxidant System in Periodontitis. Front Physiol, 8, 910.

Wang, Y., Viscarra, J., Kim, S. J., & Sul, H. S. (2015). Transcriptional regulation

U

of hepatic lipogenesis. Nat Rev Mol Cell Biol, 16(11), 678-689.

A

N

Warnakulasuriya, S. (2009). Global epidemiology of oral and oropharyngeal

M

cancer. Oral Oncol, 45(4-5), 309-316.

Wei, S., Kawashima, N., Suzuki, N., Xu, J., Takahashi, S., Zhou, M., . . . Suda,

TE D

H. (2013). Kinetics of Th17-related cytokine expression in experimentally induced rat periapical lesions. Aust Endod J, 39(3), 164-170.

EP

Wolfert, M. A., Murray, T. F., Boons, G. J., & Moore, J. N. (2002). The origin of

CC

the synergistic effect of muramyl dipeptide with endotoxin and peptidoglycan. J Biol Chem, 277(42), 39179-39186.

A

Wu, C. S., Chang, K. P., OuYang, C. N., Kao, H. K., Hsueh, C., Chen, L. C., . . . Chang, Y. S. (2016a). ASC contributes to metastasis of oral cavity squamous cell carcinoma. Oncotarget, 7(31), 50074-50085. Wu, C. S., Chang, K. P., OuYang, C. N., Kao, H. K., Hsueh, C., Chen, L. C., . . .

45

46

Chang, Y. S. (2016b). ASC contributes to metastasis of oral cavity squamous cell carcinoma. Oncotarget, 7(31), 50074-50085. Xie, S. X., Feng, L., Zhu, S. R., & Ding, L. (2014). [Expressions of RORgammaT; and FOXP3 and clinical significance in patients with oral lichen planus].

IP T

Shanghai Kou Qiang Yi Xue, 23(4), 472-476.

Xue, F., Shu, R., & Xie, Y. (2015). The expression of NLRP3, NLRP1 and AIM2

SC R

in the gingival tissue of periodontitis patients: RT-PCR study and immunohistochemistry. Arch Oral Biol, 60(6), 948-958.

U

Yamaguchi, Y., Kurita-Ochiai, T., Kobayashi, R., Suzuki, T., & Ando, T. (2015).

A

N

Activation of the NLRP3 inflammasome in Porphyromonas gingivalis-

M

accelerated atherosclerosis. Pathog Dis, 73(4). Yamaguchi, Y., Kurita-Ochiai, T., Kobayashi, R., Suzuki, T., & Ando, T. (2017).

TE D

Regulation of the NLRP3 inflammasome in Porphyromonas gingivalisaccelerated periodontal disease. Inflamm Res, 66(1), 59-65.

EP

Yang, S., Zhu, L., Xiao, L., Shen, Y., Wang, L., Peng, B., & Haapasalo, M.

CC

(2014). Imbalance of interleukin-17+ T-cell and Foxp3+ regulatory T-cell dynamics in rat periapical lesions. J Endod, 40(1), 56-62.

A

Yilmaz, Ö. (2015). The Inflammasome and Danger Molecule Signaling: At the Crossroads of Inflammation and Pathogen Persistence in the Oral Cavity. 69(1), 83-95. Yilmaz, O., & Lee, K. L. (2015). The inflammasome and danger molecule

46

47

signaling: at the crossroads of inflammation and pathogen persistence in the oral cavity. Periodontol 2000, 69(1), 83-95. Yilmaz, O., Yao, L., Maeda, K., Rose, T. M., Lewis, E. L., Duman, M., . . . Ojcius, D. M. (2008). ATP scavenging by the intracellular pathogen

IP T

Porphyromonas gingivalis inhibits P2X7-mediated host-cell apoptosis.

Cell Microbiol, 10(4), 863-875.

SC R

Yoneda, T., Tomofuji, T., Ekuni, D., Azuma, T., Endo, Y., Kasuyama, K., . . .

Morita, M. (2013). Anti-aging effects of co-enzyme Q10 on periodontal

U

tissues. J Dent Res, 92(8), 735-739.

A

N

Yoshida, K., Okamura, H., Hiroshima, Y., Abe, K., Kido, J. I., Shinohara, Y., &

the

NF-kappaB

M

Ozaki, K. (2017). PKR induces the expression of NLRP3 by regulating pathway

in

Porphyromonas

gingivalis-infected

TE D

osteoblasts. Exp Cell Res, 354(1), 57-64. Yu, J. W., & Lee, M. S. (2016). Mitochondria and the NLRP3 inflammasome:

EP

physiological and pathological relevance. Arch Pharm Res, 39(11),

CC

1503-1518.

A

Yu, X. J., Xiao, C. J., Du, Y. M., Liu, S., Du, Y., & Li, S. (2015). Effect of hypoxia on the expression of RANKL/OPG in human periodontal ligament cells in vitro. Int J Clin Exp Pathol, 8(10), 12929-12935.

Zamanian-Daryoush, M., Mogensen, T. H., DiDonato, J. A., & Williams, B. R. (2000).

NF-kappaB

activation

47

by

double-stranded-RNA-activated

48

protein kinase (PKR) is mediated through NF-kappaB-inducing kinase and IkappaB kinase. Mol Cell Biol, 20(4), 1278-1290. Zhai, S., Wang, Y., Jiang, W., Jia, Q., Li, J., Wang, W., . . . Ni, L. (2013).

cells migration. Exp Cell Res, 319(10), 1544-1552.

IP T

Nemotic human dental pulp fibroblasts promote human dental pulp stem

Zhang, A., Wang, P., Ma, X., Yin, X., Li, J., Wang, H., . . . Ni, L. (2015).

SC R

Mechanisms that lead to the regulation of NLRP3 inflammasome

expression and activation in human dental pulp fibroblasts. Mol Immunol,

U

66(2), 253-262.

A

N

Zhang, C., Li, H., Zhou, G., Zhang, Q., Zhang, T., Li, J., . . . Yin, D. (2007).

M

Transcriptional silencing of the TMS1/ASC tumour suppressor gene by an epigenetic mechanism in hepatocellular carcinoma cells. J Pathol,

TE D

212(2), 134-142.

Zhang, Y., Liu, W., Zhang, S., Dan, H., Lu, R., Wang, F., . . . Zhou, Y. (2012).

EP

Salivary and serum interleukin-18 in patients with oral lichen planus: a

CC

study in an ethnic Chinese population. Inflammation, 35(2), 399-404.

A

Zhao, P., Liu, J., Pan, C., & Pan, Y. (2014). NLRP3 inflammasome is required for apoptosis of Aggregatibacter actinomycetemcomitans-infected human osteoblastic MG63 cells. Acta Histochem, 116(7), 1119-1124.

Zhou, R., Yazdi, A. S., Menu, P., & Tschopp, J. (2011). A role for mitochondria in NLRP3 inflammasome activation. Nature, 469(7329), 221-225.

48

49

Zhou, Y., Qian, M., Liang, Y., Liu, Y., Yang, X., Jiang, T., & Wang, Y. (2011). Effects of leukemia inhibitory factor on proliferation and odontoblastic differentiation of human dental pulp cells. J Endod, 37(6), 819-824.

Figure legend：

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The activation of the NLRP3 inflammasome requires a two-step signal. Signal 1 is “priming”, which is mainly mediated by TLRs and TNF-α and aims to upregulate proIL-1β, pro-IL-18 and NLRP3 in an NF-κB-dependent manner. Caspase-8 and FADD are engaged in the NF-κB signalling pathway. Inflammasome priming also occurs via non-transcriptional pathways. For example, BRCC3, a deubiquitinating enzyme, can regulate NLRP3 inflammasome activation via transcription-independent priming. Signal 2 is “triggering”, which involves the recruitment and assembly of NLRP3, ASC and pro-caspase-1 to form the active NLRP3 inflammasome complex. Three models have been proposed to lead to Signal 2: (1) eATP induces K+ efflux via a purogenic P2X7-dependent pore; (2) PAMPs /DAMPs lead to mitochondrial dysfunction, ROS production, and oxidative stress; and (3) lysosomal rupture induced by phagocytosed crystalline or particulate structures releases lysosomal contents, such as cathepsin B. In addition, NDK induces the expression of ecto-ATPases, resulting in the cleavage of eATP, which prevents P2X7 activation. The inflammasome selfcleaves and activates caspase-1, which in turn contributes to the production and secretion of bio-active IL-1β and IL-18.

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FADD: FAS-associated death domain protein; OMV, Outer membrane vesicles, MDP: muramyl dipeptide; LTX: leukotoxin; CDT: cytolethal distending toxin; ER, endoplasmic reticulum, IL-1R: IL-1β receptor; TNFR: tumor necrosis factor receptor; BRCC3: Lys-63-specific deubiquitinase BRCC36; PAMPs/DAMPs, pathogen /damage-associated molecular patterns; DNK: Nucleoside-diphosphate-kinase

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