Zheng-An Wang,Qi-Ya Fu

School of Stomatology,Hainan Medical College,Haikou 570100,China

Keywords:Pattern recognition receptor Toll like receptor C-type lectin receptor Nucleotide-binding oligomeric domain Chronic periodontitis

ABSTRACT Pattern recognition receptor (PRR) is a kind of sensor which is mainly expressed on the surface of innate immune cells.It can recognize pathogen related molecular patterns (PAMPs)or damage related molecular patterns (DAMPs).The innate immune system uses pattern recognition receptors to recognize pathogenic microorganisms in periodontal tissues and transmit signals to downstream pathways in time,thus triggering immune responses and then eliminating them.PRR has many family members,including toll like receptor family (TLRs),C-type lectin receptor family (CLRs),retinoic acid induced gene I (RIG-I) like receptor family(RLRs) and nucleotide binding oligomer domain (NOD) like receptor family (NLRs).Among them,RLRs are cytoplasmic receptors that recognize dsRNA from RNA viruses and have little association with chronic periodontitis.In this paper,the classification and structure of TLRs,CLRs,NLRs and the role of signal transduction pathway in chronic periodontitis are reviewed.In order to enrich the pathogenesis of periodontitis,provide new ideas for the treatment and prevention of chronic periodontitis.

Chronic periodontitis is one of the two major oral diseases,affecting a large number of people all over the world.Periodontitis is a chronic inflammatory state,leading to the destruction of hard and soft tissues supporting teeth [1].Its etiology involves many factors,such as periodontal pathogens,local promotion factors,genetic factors and smoking habits [2].The development of periodontitis is not only caused by periodontal pathogens,but also mediated by the host's immune inflammatory response.However,host inflammation is controlled by signaling pathways.Therefore,the disorder of signaling pathway is the potential mechanism of periodontitis[1].

PRRS are a class of pattern recognition receptors with multiple family members,including toll like receptor family (TLRs),C-type lectin receptor family (CLRs),retinoic acid inducible gene I (RIG-I)-like receptor family (RLRs) and nucleotide binding oligomeric domain (NOD) -like receptor family (NLRs) [3].PRRS play an important role in regulating the host's innate immune response to periodontal pathogens by recognizing periodontal pathogens,activating downstream signaling pathways,and ultimately affecting the production of cytokines and defensins.Therefore,this paper reviews the role of PRRS in periodontitis.Among them,RLRs are cytoplasmic receptors that recognize dsRNA from RNA viruses,and are less associated with chronic periodontitis,so they are not elaborated [4,5].

1.TLR and chronic periodontitis

1.1 Classification and expression of TLR

Toll like receptors (TLRs) are type I transmembrane molecules that can sense pathogen associated molecular patterns (PAMPs) and activate host immune response.So far,10 TLRs (TLR1 to TLR10)have been found in human and 12 in mice.Each TLR resides in a specific part of the cell and can sense different PAMPs [6].Based on the composition of PAMPS identified by them,TLRs can be divided into two categories.TLR1,TLR2,TLR4,TLR5,TLR6 and TLR10 exist in the cell membrane and can recognize a variety of PAMPS,including lipopolysaccharide,phospholipid,yeast polysaccharide,flagellin and peptidoglycan.On the contrary,TLR3,TLR7,TLR8 and TLR9 exist in the cytoplasm and mainly recognize PAMPs composed of nucleic acids [7].

TLR11 has been identified in the human genome,but it has not been translated into a protein.TLR2 forms a heterodimer with TLR1 or TLR6 and recognizes peptidoglycan,lipopeptide and lipoprotein,while TLR4 recognizes lipopolysaccharide of gram negative bacteria.TLR3 can recognize double stranded RNA,TLR5 can detect bacterial flagellin,TLR7 and tlr8 can recognize viral single stranded RNA,and TLR9 can recognize bacterial and viral DNA through cytosine and guanine base pairing [8].

1.2 Structure of TLR

Toll like receptor consists of a horseshoe shaped extracellular leucine rich repeat (LRR) and a transmembrane and intracellular domain (TIR).LRR domain is responsible for ligand recognition,and TIR domain is responsible for intracellular signal transmission[9].

1.3 Signal transduction of TLR

After TLRs bind to ligands,the conformational changes of TIR domain in cells lead to homotypic TIR-TIR interaction and recruitment of adaptor proteins,thus promoting downstream signal transduction.At present,five TLR adaptor proteins are known:myeloid differentiation factor 88 (MyD88),TIR domain containing adaptor molecule (TRIF),MyD88 adaptor like protein (mal,also known as Tirap containing adaptor protein),TRIF related adaptor molecule (TRAM),and SARM [10].Generally speaking,TLRs mainly transmit signals through two ways,depending on the adapter MyD88 and TRIF signal axis.MyD88 signaling mainly leads to the production of proinflammatory cytokines such as TNF,IL-6,IL-1 and chemokines (such as C-C motif ligand 4,CCl4),while TRIF axis mainly induces the expression of type I IFNs (such as IFN α/ β).Most TLRs transmit signals through MyD88 dependent mechanism,while TLR3 only transmits through TRIF signal axis [11].

The classic MyD88 signaling pathway is that the C-terminal of MyD88 binds to the TIR region in TLR cells.The N-terminal of MyD88 recruits IL-1R-related kinase 4 (irak4) and activates irak1 and irak2 through autophosphorylation of its central kinase domain (KD).Thus,MyD88,irak4 and irak1/ 2 combine to form a receptor complex.This heterogeneous structure is composed of MyD88 molecule and irak4 and irak1/ 2 members,which is called myddosome.Subsequently,it induces the activation of E3 ubiquitin(UB) ligase TNF receptor associated factor 6 (TRAF6),triggers three major transduction pathways,and finally activates three key transcription factors (such as NF -κ B,interferon regulatory factor(IRF) and mitogen activated protein kinase MAPK).

In the first pathway,the activated TRAF6 forms new complexes with transforming growth factor -β activated kinase (Tak) -1,transforming growth factor -β activated kinase-1 binding protein(TAB) -1 and TAB2/ 3,and enters the cytoplasm.It stimulates the activation of the complex of IKK -α,IKK -βand Nemo(also known as IKK -γ).It can promote the phosphorylation and degradation of the inhibitor of NF -κB (IKB ),release and transfer NF -κB (including P50 and p65) to the nucleus.Transcription regulates the release of cytokines and chemokines,induces inflammation and activates innate immunity.The second pathway,MAPK pathway,induces the activation of transcription factor activator protein-1 (AP-1) and CREB downstream of TAK1 complex.In the third pathway,TRAF6 activates IFN regulator 5(IRF5) through TAK1-IKK-β pathway [12.13].TLRs are not only the most important mechanism of immune defense against fungal,bacterial and viral pathogens,but also one of the earliest mechanisms.Therefore,the expression and function of TLRs are essential in maintaining oral tissue homeostasis under the condition of a large number of microbial symbiosis [14].

1.4 The role of TLR in chronic periodontitis

The dense oral symbiotic microbial environment keeps the gingiva in a slightly activated state,and it is of great significance to maintain the homeostasis of the internal environment in order to directly deal with the challenge of pathogenicity.In oral cavity,TLRs are not only involved in the construction and maintenance of host microbial homeostasis.Moreover,under the stimulation of oral symbiotic microorganisms,TLR signal leads to the production and activation of signal transduction proteins such as AP-1 (activator protein 1) and NF -κB,and then induces the expression of neutrophil chemokines cxcl2 and CXCL8,and recruits granulocytes to resist microbial invasion,so as to maintain oral health [15].

In periodontal tissue,TLRs recognize PAMPs and produce a large number of pro-inflammatory factors and chemokines,which can cause periodontal tissue inflammation.TLR2 and TLR4 in oral epithelium can react to most periodontal pathogens,and their soluble forms,stlr-2 and stlr-4,can also play a regulatory role by binding microbial ligands.By collecting the unstimulated saliva of 40 patients with periodontitis and gingivitis,alqallaf h and other teams found that the level of stlr-2 was negatively correlated with the clinical parameters in the gingivitis cohort,while the level of stlr-4 was positively correlated.However,in the periodontitis cohort,both correlations disappeared,indicating that the host response was maladjusted [16].A Wang et al.Also found that [17]:compared with healthy saliva group,the number of SECs increased,GSI/sec increased,and soluble toll like receptor 2 (stlr-2) decreased in periodontitis saliva group.After non periodontal surgery,the number of SECs decreased,GSI/ sec and stlr-2 increased.Therefore,it is suggested that stlr-2 can be used as an indirect indicator of periodontal disease.Kim wh et al.[18] showed that pglps could increase the expression of TLR4 and pro-inflammatory factors such as tumor necrosis factor (TNFα),IL-6,IL-8 and interferon -γ(IFN -γ) in HaCaT human keratinocyte line treated with melittin LPS.Melittin inhibits the expression of proinflammatory cytokines by inhibiting the activation of NF -κ B signaling pathway,ERK and Akt.

Studies on TLRs in periodontitis mainly focus on gene methylation and gene polymorphism.Shaddox LM et al.[19] found that compared with healthy control group,subjects with moderate local aggressive periodontitis (LAP) showed hypermethylation of genes that up regulate inflammation (such as MAP3K7,MyD88,IL6R and RIPK2) and down regulate inflammation (such as FADD,IRAK and PPARA),while those with severe lap showed hypomethylation of all the above genes.CpG methylation is associated with the increase of pro-inflammatory cytokines in patients with lap.The results show that the phenotypic changes of genes in TLR signaling pathway may balance the tissue damage in the process of disease through the coordination threshold.Therefore,moderate lap shows hypermethylation,and severe lap shows hypomethylation.T Zhang study found that [20]:it is the first time to prove that the ability of transforming growth factor β (TGF β) to inhibit TLR-NF-κB signaling pathway is mediated by Smad6 methylation induced by protein arginine methyltransferase 1 (PRMT1).However,the disorder of Smad6 methylation can aggravate inflammation and bone loss in experimental periodontitis.Therefore,the regulation of prmt1-smad6 signal can be used as a new strategy to regulate the host immune response of periodontitis.In addition,Shan C et al.[21] found that TLR-2rs1898830 and rs5743708 polymorphisms may be associated with periodontitis susceptibility through metaanalysis.Leite frm and other studies [22] also confirmed that the gene polymorphisms of TLR1-rs5743611,TLR4-rs7873784,TLR7-rs3853839 and TLR8-rs3764879 were associated with juvenile periodontitis.

2.CLR and chronic periodontitis

2.1 Nomenclature and structure of CLR

C-type lectin (C-type lectin) is a kind of animal lectin that recognizes carbohydrate ligand (Ca2+-dependent) in a Ca2+-dependent manner,which can be distinguished from other Ca2+-independent animal lectins."C" stands for "Ca2+".Regardless of carbohydrate or calcium binding capacity,we use a more common term "proteins containing c-lectin domains" (CTLDCs) to define these proteins,which are characterized by at least one or more C-type lectin like domains (CTLD) [23].

The typical feature of CTLD is a double ring structure,which is stabilized by two highly conserved disulfide bridges at the bottom of the ring,as well as a group of conserved hydrophobic and polar interactions.The whole domain is a cycle,and its N-terminal and C-terminal b-chains (b1,b5) are closely connected.The second cycle,called the long ring region,is located in the domain.The long ring region is structurally and evolutionarily flexible.It participates in calcium dependent carbohydrate binding and interaction with other ligands [24].

2.2 Classification and expression of CLR

As an important PRR,CTL has high affinity for various PAMPs on the surface of pathogens,and plays a role in host defense against microbial infection by coordinating innate and adaptive immune systems [25].In vertebrates,myeloid CTLs are mainly expressed by antigen presenting cells (APCs),such as dendritic cells and macrophages.APCs sense pathogens,phagocytize them and present pathogen derived peptides on major histocompatibility complex(MHC) to T cells,thus initiating adaptive immune response [26].

C-type lectin receptors (CLRs) are divided into transmembrane and secretory types,which contain at least one carbohydrate recognition domain (CRD) and about 120 residues.According to conserved carbohydrate recognition domains (CBRS),the transmembrane CLR was constructed,Type I CTLs include macrophage mannose receptor(MMR),mannose receptor 2 (also known as endo-180),type II CTLs include Dectin-1 and dectin-2,macrophage induced CTLs (mincle)and (DC-SIGN),which are mainly expressed on myeloid cells and participate in antifungal immunity [27].Type I CTLs usually contain multiple CRDs,but type II CTLs usually have only one CRD [28].Secretory CLR includes collectin,surfactant protein A and D (SP-A,SP-D),mannose binding lectin (MBL) [29].

2.3 Signal transduction of CLR

CTL mediated signaling triggers various effector functions,such as the production of proinflammatory cytokines,thus affecting the activation and polarization of T cells,as well as the interaction with other receptors (such as TLRs) [30].There are immune receptor tyrosine activating motifs (ITAMs) or inhibitory motifs (ITIMs) in the cytoplasmic tail of CTL.The connection with these receptors will cause signal cascade reaction,leading to the activation of transcription factors.However,the activation of transcription factors is inhibited by binding to ITIMs receptor [31].

One of the typical CTL signaling pathways is mediated by the direct or indirect interaction between Syk kinase and ITAMs or ITAMs in the cytoplasmic tail of CTLs.CLRs recruit Syk with Fc receptor gamma (FCR gamma) chain containing ITAM,or a single YXXL motif in the cytoplasm of CTL tail,called hemitam,to bind Syk directly without involving additional adaptor proteins.Therefore,Syk is phosphorylated to initiate the formation of a ternary protein complex composed of caspase recruitment domain protein 9 (CARD9),B-cell lymphoma/ leukemia 10 (BCL10)and mucosa associated lymphoid tissue lymphoma translocation protein 1 (malt-1).It is called CARD9/ BCL10/ malt-1 complex,which causes the activation of transcription factors (such as NF -κB,NFAT or AP-1) and induces the expression of cytokine genes.The activation of Syk kinase may also lead to the production of ROS,thus inducing the activation of inflammatory bodies and the production of IL-1 β.However,CTL signal can also be independent of Syk pathway.DC-SIGN activates Raf-1 kinase through different pathways,which eventually leads to the activation of NF -κ B[32,33].

2.4 The role of CLR in chronic periodontitis

Macrophage induced C-type lectin receptors,also known as clec4e and clecsf9,belong to type II transmembrane receptors and are fcry coupled pathogen recognition receptors.Mincle recognizes different sugar containing ligands,including trehalose bicyclate glycolipids of mycobacteria,mannose or glucose containing sugar conjugates of fungal pathogens,mannose or new sugar conjugates containing fucose,and LPS of Helicobacter pylori [34].Forsythia is a kind of oral gram-negative anaerobic bacteria,which is located in subgingival tissue and participates in the development of periodontitis.Chinthamani s et al.[35] found that mincle specifically binds to S-glycoprotein of Forsythia through direct ligand binding test,leading to the secretion of pro-inflammatory cytokine TNF-a and anti-inflammatory cytokine IL-10 in macrophages.Therefore,TNF-a and IL-10 produced by mincle mediated signaling pathway may have a direct impact on the pathogenesis of periodontitis.

Mannose binding lectin (MBL) is a soluble glycoprotein in human blood.It activates the lectin pathway of the complement system by forming complexes with MBL related serine proteases.This complex can bind to the surface of gram-positive and Gram-negative bacteria,thus activating the complement system [36].MBL mutation will weaken its affinity to bacteria,thus affecting its ability to activate the complement system [37].Liukkonen a et al.[38] showed that periodontal actinomycetes are related to periodontal tissue inflammation and destruction and genetic variation of human MBL2.

3.NLR and chronic periodontitis

3.1 Nomenclature and structure of NLR

The nucleotide binding domain (NBD) and leucine rich repeat(LRR) of NLR protein are a family of intracellular receptors,which play an important role in regulating innate immune response [39].NLR protein is a conserved tripartite structure,one of which is the leucine rich repeat (LRR) at the C-terminal,responsible for ligand recognition,one is the central nucleotide binding oligomeric domain (NOD) and one is the N-terminal protein-protein interaction domain (commonly known as the effector domain),responsible for intracellular signal transduction [40].Nacht domain (also known as NBD or nod domain) belongs to a large superfamily of NTP enzyme domain,which hydrolyzes ATP or GTP.LRRs,known as proteinprotein interaction domains,can interact with many other molecules.They are ligand recognition domains that define the specificity of NLR proteins for specific ligands [41].

3.1.1 Structure of NOD

NOD1 (CARD4) and NOD2 (CARD15) are the first nucleotide binding domain and leucine rich repeat receptor (NLR) family to be described and studied.They include 22 human genes and more than 30 mouse genes.They are characterized by a recruitment domain(card) at the N-terminal.Both NOD1 and NOD2 are members of the card subfamily of NLRs,in which Nod1 contains a card domain at the N-terminal and NOD2 contains two card domains at the N-terminal [42].

3.1.2 Structure of NLRP3

Nod like receptor protein 3 (NLRP3) inflammasome is an inflammatory complex composed of NLRP3,ASC and caspase-1 in card domain.The upstream receptor molecule nrlp3 belongs to the nod like receptor family,which can regulate the inflammatory response by recruiting ASC and caspase-1 to gather and activate after binding with pathogenic microorganisms [43].

3.2 Classification and expression of NLR

NLR protein family can be divided into subfamilies according to N-terminal effector domain.There are two main subfamilies:the card subfamily (called NLRC or NACHT,LRR and CARD domain proteins) and the pyrin subfamily (called NLRP or NACHT,LRR and PYD proteins) [44].

Both Nod1 and NOD2 can recognize the peptidoglycan part of bacterial cell wall.Specifically,it recognizes γ-d-glutaminediaminobenzoic acid (iEDAP) and cell wall acyl dipeptide (MDP)respectively.Iedap structure mainly exists in the peptidoglycan(PGN) of gram negative bacteria,while MDP exists in almost all bacterial PGNs [45].

3.3 Signal transduction of NLR

3.3.1 Signal transduction of NOD

After recognizing bacterial peptidoglycan (PGN),NOD1 and NOD2 can rapidly form oligomer,and then recruit receptor interacting protein 2 (RIP2) to form nod-rip2 complex through card-card interaction [46].This complex can cause the aggregation of inhibitor of NF -κB kinase complex (IKK),and then degrade the inhibitor of NF -κB (Iκb) through ubiquitination and phosphorylation of protease,separate NF -κB from its inhibitor,and lead to the activation of NF -κB [47].Studies have shown that the deletion of card in Nod1 or NOD2 can affect the activation of NF-κ B [48].Therefore,N-terminal cards of Nod1 and NOD2 are signal effect domains.Once activated,RIP2 opens downstream signaling cascades,such as NF -κB,through NF -κB kinase complex and MAPK cascade inhibitors,thereby producing proinflammatory cytokines,such as IL-6,TNF-a,IL-12 or IL-8 [49].

3.3.2 Signal transduction of NLRP3

The formation of NLRP3 inflammasome is a response to cell infection,cell stress or tissue damage [50].Its activation requires two steps (start and trigger).In the first step,damps or PAMPs are recognized by toll like receptors (TLRs),which induce the production of inactive NLRP3,pro-IL-1 β and pro-IL-18 through the MyD88/ NF-κB dependent signaling pathway,and lead to the de ubiquitination of NLRP3 and the phosphorylation of ASC by activating NF -κ B [51].The second step involves damps mediated (including potassium (k) efflux,calcium (Ca2) influx,lysosomal instability,mitochondrial reactive oxygen species (ROS)and mitochondrial DNA damage,etc.),which can promote the oligomerization of inactive NLRP3,ASC and pro-caspase-1,and assemble them into high molecular weight inflammatory complex.In the oligomer,pro-caspase-1 is transformed into caspase-1 with biological activity,and then can be activated Pro-IL-1 β and pro-IL-18 were cleaved into bioactive forms of IL-1 β and IL-18 [52,53].

Extracellular ATP is the most effective stimulator in the second step of the formation of NLRP3 inflammasome.By cell stress or injury,extracellular ATP is released to activate purinergic receptor 7 to transmit pathogen invasion signal [54].New evidence suggests that extracellular ATP plays an important role in regulating NLRP3 inflammasome by activating purinergic receptor 7,which is an important promoter of inflammatory response [55].Purinergic receptor 7 is a cation permeable ligand gated ion channel,which forms a pore on the membrane by recruiting the hemichannel protein pannexin-1,leading to K+efflux.The connection between extracellular ATP and purinergic receptor 7 can activate phospholipase D,lead to the production of reactive oxygen species,and eliminate intracellular pathogens [56].The pathogenesis of periodontitis is related to the imbalance between reactive oxygen species and antioxidant defense system.Reactive oxygen species(ROS) are part of a variety of cellular pathways,which play the roles of antibacterial effector molecules,signaling molecules,and regulating NF-κB transcription [57].

3.4 The role of NLR in chronic periodontitis

3.4.1 Role of NOD in chronic periodontitis

NOD1 and NOD2 receptors were found to be involved in the recognition of periodontal pathogens,indicating their importance in periodontitis [60].Chaves de Souza JA et al.[61] found that:bone marrow-derived macrophages from wild-type mice and Nod1 gene knockout mice were used for in vitro experiments.The results showed that the deletion of NOD1 significantly aggravated bone resorption induced by gram-negative bacteria,accompanied by an increase in the number of osteoclasts,indicating that NOD1 played a role in protecting bone in periodontitis model.Polymorphonuclear neutrophils (PMN) can fight against infectious microorganisms through phagocytosis and neutrophil extracellular traps (NETs)[62].When stimulated by microorganisms,neutrophils release cellular contents,such as particles,DNA and proteins,forming an extracellular structure that resists the "neutrophil extracellular traps" (NETs) of microorganisms during infection,which is called"reticular structure" [63].The data of alyami HM et al.[64] showed that:bacilli with nucleic acids activated neutrophils and induced the formation of their network structure by up regulating NOD1 and NOD2.Therefore,CRISPR/ Cas9 knockout HL-60 cells and ligand inhibitors confirmed that NOD1 and NOD2 receptors were involved in the formation of nuclease mediated network structure.

3.4.2 The role of NLRP3 in chronic periodontitis

NLRP3 has been reported to play an important role in the development of chronic periodontitis.Among them,Garc í a-hern á ndez al et al.[65] found that the activation of NLRP3 inflammasome may lead to more serious destruction of periodontal tissue in patients with periodontitis and uncontrolled type 2 diabetes.Lian D et al.[66] also found that NLRP3 inflammasome was highly expressed in mouse periodontal ligament fibroblasts (mPDLFs).The results showed that ROS/ TXNIP/ NLRP3 inflammasome pathway was the key mechanism of chronic periodontitis induced by mpdlfs induced by p.g-lps.Yoon y et al.[67] showed that intracellular antioxidant enzyme SOD2 plays a protective role by inhibiting the NLRP3 inflammasome caspase-1-il-1 β signaling pathway under inflammatory conditions.Tan y et al.[68] found that metformin can significantly inhibit the inflammatory response of human periodontal ligament cells (hPDLCs) induced by gingival p.g-lps,which is characterized by the decreased secretion of pro-inflammatory factors IL-1 β and IL-18.Metformin also significantly reduced the expression of nucleotide binding domain,leucine rich family,pyrin domain 3 (NLRP3) and caspase-1 in hPDLCs.Isaza Guzm á n DM et al.[69] also showed that the levels of NLRP3,ASC and IL-1 β in periodontitis group were significantly higher than those in healthy control group.The concentration of NLRP3 in saliva of patients with aggressive periodontitis was significantly higher than that of patients with chronic periodontitis.However,there are few studies on the relationship between NLRP3 and the pathogenesis of chronic periodontitis,which is also a new research hotspot in the future.

4.Expectation

Chronic periodontitis is a common oral disease.Its progress is mediated by the interaction between host immunity and periodontal bacteria.Although periodontal bacteria can directly destroy periodontal tissue,the dysfunction of host immune response caused by bacteria will lead to more serious and lasting damage.Pattern recognition receptors (such as TLRs,CLRs,NLRs) exist in various oral tissues,which has become a research hotspot in recent years.PRRS play an important role in the regulation of innate immunity by forming the first defense line of host defense,and further act as a bridge between innate immunity and adaptive immunity.By recognizing PAMPs and damps,PRRS trigger signal transduction pathways,leading to the activation of various transcription factors,inducing the production of pro-inflammatory factors and chemokines,thus aggravating the tissue inflammatory response.Therefore,in-depth study of pattern recognition receptors (PRRS)can provide new ideas for exploring the pathogenesis of chronic periodontitis in the future,which is of great significance for clinical prevention and treatment.