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Expression of Nucleotide-Binding Oligomerization Domain 2in Normal Human Dental Pulp Cells and Dental Pulp TissuesZheng-Mei Lin, MD,* Zhi Song, MS,* Wei Qin, MS,* Juan Li, PhD,† Wen-Jie Li, PhD,‡

Hai-Yan Zhu, MS,* and Li Zhang, MS*

AbstractIntroduction: The nucleotide-binding oligomerizationdomain (NOD) proteins belong to a distinct family ofproteins that are implicated in the intracellular recogni-tion of bacterial components. NOD2 appears to bea sensor of bacterial peptidoglycans because it recog-nizes a minimal motif present in all peptidoglycans.The interaction of NOD2 with downstream signalingmolecules ultimately results in the activation of NF-kBand production of inflammatory mediators in innateimmunity. As such, NOD2 may play an important rolein the detection of bacterial pathogens and the initia-tion of inflammation within the dental pulp. This studywas designed to evaluate the expression of NOD2 innormal human dental pulp cells (HDPCs) and humanpulp tissues. Methods: Human pulp tissue sampleswere collected from freshly extracted human wisdomteeth, and HDPCs were prepared from the explants ofnormal human dental pulp tissues. Nested reverse-tran-scription polymerase chain reaction (Nested RT-PCR)and Western blotting were performed to detect theexpression of NOD2 messenger RNA and protein,respectively. Immunohistochemical staining was usedto determine the distribution of NOD2 in the pulptissues. Results: The NOD2 messenger RNA andprotein were present in normal human dental pulptissues, with most NOD2 protein expression being local-ized to odontoblasts and some pulp vascular endothe-lial cells. In contrast, HDPCs only showed a low levelof NOD2 protein expression. Conclusions: Our resultssuggest that NOD2 protein expressed in HDPCs andpulp tissues may play an important role in dentalimmune defense. (J Endod 2009;35:838–842)

Key WordsDental pulp, dental pulp cell, innate immunity, nucleo-tide-binding oligomerization domain 2

From the Departments of *Operative and Endodontics,†Human Anatomy, and ‡Histology and Embryology, Sun Yat-senCollege of Medical Sciences, Sun Yat-sen University, Guang-zhou, China.

Address requests for reprints to Dr Zhengmei Lin, Depart-ment of Operative and Endodontics, Guangzhou, Guangdongprovince020, China. E-mail address: linzhm@mail.sysu.edu.cn.0099-2399/$0 - see front matter

Copyright ª 2009 American Association of Endodontists.doi:10.1016/j.joen.2009.03.047

838 Lin et al.

Microorganisms display pathogen-associated molecular patterns (PAMPs) that arerecognized by host pattern recognition receptors (PRRs), initiating host defenses

against invasive pathogens by the innate immune system (1). Toll-like receptors (TLRs)have been shown to recognize a large number of PAMPs distributed mainly on thesurface of bacteria. Peptidoglycans and lipopeptides are primarily recognized byTLR2, whereas lipopolysaccharides (LPS) are recognized by TLR4 (2). Over the pastdecade, the important contributions of TLRs to the process of recognition by myeloidcells have been exhaustively investigated. However, the expression of these receptorsdoes not preclude the involvement of other PRRs in bacterial pathogen recognition.

Indeed, a recent study suggests that peptidoglycan detection is mediated bymembers of the novel nucleotide-binding oligomerization domain (NOD) family ofproteins rather than by TLR2 (3). Although earlier reports suggest that TLR2 detectspeptidoglycan (4), more recent studies indicate that TLR2-mediated peptidoglycansensing is caused by experimental impurities. It is concluded that peptidoglycan, devoidof contaminating lipopeptides or lipoteichoic acid, is not detected by TLR2 (3). More-over, the two components of peptidoglycans, desmuramylpeptides containing diamino-pimelic acid and muramyl dipeptide (MDP), are recognized by NOD1 and NOD2,respectively (5–8), whereas TLR2 does not recognize the monomeric structure ofthe peptidoglycan (9, 10). Although the issue remains controversial, it appears mostlikely that alternative PRRs play important roles in the initiation of inflammatory hostresponses in response to bacterial challenge.

The NOD proteins, including NOD1 and NOD2, are members of a distinct family ofproteins that have been implicated in the intracellular recognition of bacterial compo-nents. NOD1 appears to interact with motifs found in peptidoglycans from gram-nega-tive bacteria (5, 7). In contrast, NOD2 is suggested to be a more general sensor ofbacterial peptidoglycans because it recognizes a minimal motif present in all peptido-glycans (6, 8, 11). Upon interaction with PAMPs, NOD2 initiates a signaling cascade,ultimately leading to the activation of NF-kB, a pivotal transcription factor in the produc-tion of proinflammatory mediators of innate immunity (12) (Fig. 1).

We speculate that NOD2 may play an important role in the detection of bacterialpathogens and the initiation of inflammation within the dental pulp. The pulp-dentincomplex responds to exogenous stimuli or injuries by triggering defensive activitiessuch as inflammation (13). As the major cells of the pulp-dentin complex, normalhuman dental pulp cells (HDPCs) play a pivotal role in maintaining the structural integ-rity of connective tissues during pathological alterations. In addition, HDPCs producevarious inflammatory cytokines, including interleukin (IL)-1, IL-6, and IL-8, upon stim-ulation with LPS (14–16). Moreover, TLR2 and TLR4 are expressed in inflammatorymurine pulp cells (17). These findings indicate that dental pulp cells actively participatein inflammatory processes and immune responses. However, it is not known whether ornot HDPCs express the NOD2 receptor and recognize and respond to invading bacteriathrough NOD2.

MDP is the minimal essential structural unit responsible for the immunoadjuvantactivity of peptidoglycans produced by gram-negative and gram-positive bacteria (18).Previous reports have shown that MDP can stimulate murine and human macrophagesand monocytes to produce cytokines and chemokines (19) and act synergistically withLPS to stimulate the release of both pro- and anti-inflammatory cytokines by myeloidcells (20). Moreover, MDP and low-dose LPS synergistically amplify dendritic cell(DC) maturation, indicating that cooperative engagement of TLR4 and NOD2 can prime

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DCs for the onset of adaptive immunity (20). As a key receptor medi-ating MDP signal transduction and operating in a cooperative mannerwith TLRs to initiate immune responses, NOD2 has an important rolein the priming of innate and adaptive immunity. The present studywas performed to elucidate the possible expression and distributionof NOD2 in human dental pulp and pulp cells, providing a basis forfurther investigations on the role of NOD2 in immune responses.

Materials and MethodsTissue Samples

The subjects in the study were recruited from the Department ofOral and Maxillofacial Surgery, Sun Yat-Sen University Dental Hospital,Guangzhou, China. Sixteen healthy human third molars, free fromcaries, were used for the preparation of dental pulp specimens (8women and 8 men, 18-25 years old). Written informed consent was ob-tained from each of the volunteers, and routine surgical procedureswere used. The specimens were used for nested reverse-transcriptionpolymerase chain reaction (RT-PCR) (n = 4), Western blot analysis(n = 6), and immunohistochemical staining (n = 6), respectively.

Dental Pulp Cell CultureAll tooth extractions were conducted under approved guidelines

set out by the Office of Human Subject Research at our institution.HDPCs were prepared from healthy premolars extracted from patients(aged 9-25 years) who required tooth extraction for orthodontic treat-ment. HDPCs were established by growing the minced explants asdescribed previously (21). In brief, teeth were split, and the dentalpulp tissue was aseptically removed from the crown and root by using

Figure 1. A schematic overview of the possible role of NOD2 in innate immu-nity. When bacteria are phagocytosed, peptidoglycan (PGN) present in thegram-positive and gram-negative bacterial wall is degraded to produce muro-peptides, which are similar to MDP. Subsequently, these peptides (or MDP)are translocated into the cytoplasm (by an unknown mechanism). Recognitionof MDP through the leucine-rich repeat (LRR) domain activates the NOD2protein, which then recruits receptor-interacting serine/threonine kinase(RICK) through caspase-recruitment domain (CARD)-CARD interaction. Theresulting receptor-interacting serine/threonine kinase (RICK) signalingcascade eventually leads to NF-kB activation and the secretion of proinflamma-tory cytokines, which are prerequisites for innate immune responses.

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a dental probe and barbed broach. The pulp was rinsed with Dulbecco’smodified Eagle’s Medium; placed in a 35-mm Petri dish; minced intosmall fragments; and grown in Dulbecco’s modified Eagle’s Mediumsupplemented with 10% fetal bovine serum, 100 U/mL of penicillin,100 mg/mL of streptomycin, and 1 mg/mL of amphotericin B (Gibco,Grand Island, NY). Cultures were maintained in an incubator with5% CO2 humidified atmosphere at 37�C.

The Collection of Human Normal Pulp Tissues and HDPCsDental pulp tissues were obtained by using a previously described

method (22). In brief, the crowns of human wisdom teeth weredissected, and the pulp was gently removed by using a sterile spoonexcavator and dental probe. Confluent cells were detached by incuba-tion with 0.25% trypsin and 0.05% EDTA for 5 minutes, and aliquotsof separated cells were subcultured. Pulp cell cultures at passage 4and HDPCs isolated as described earlier were dissolved in lysis bufferfor Western blot analyses. In addition, the pulps from six teeth werefixed in 4% paraformaldehyde and used for immunohistochemistry.

The Detection of NOD2 Messenger RNA in Human PulpTissues

The total RNA was extracted from pulp tissues using the TRIzolReagent (Invitrogen, CA) according to the manufacturer’s instructions.Complementary DNAs were synthesized by using the Thermoscript RT-PCR System Kit (Qiagen, Studio City, CA). Nested RT-PCR amplificationswere performed in a 50-mL volume containing 5 mL 10� KCL buffer, 1mL each primer (10 mmol/L), 1 mL dNTPs (10 mmol/L), 3 mL of TaqDNA-polymerase (1 U/mL), 5 mL complementary DNA, and 35 mLdistilled water. The nested PCR primer sequences were as follows:outside sense primer 50-CAGGATGAAGGTGTATGTTCTCTC-30, outsideantisense primer 50-CTTCAAAGCAAGAGTCTGGTGTC-30 for NOD2 (224bp), inside sense primer 50-CAGGATGAAGGTGTATGTTCTCTC-30, insideantisense primer 50-CCTAGGTAGGTGATGCAGTTATTG-30 for NOD2 (95bp), 50-GACTCATGACCACAGTCCATG-30(forward), and 50-CCATCACGC-CACAGTTTC-30 (reverse) for GAPDH (79 bp). The first cycle reactionconditions were denaturation at 93�C for 3 minutes, followed by 30cycles of 93�C for 30 seconds, 55�C for 30 seconds, 72�C for 45seconds, and 72�C for 7 minutes. The second PCR was performedwith 2 mL of the first PCR reaction product as a template under similarreaction conditions as the first cycle (93�C for 3 minutes, followed by 30cycles at 93�C for 30 seconds, 55�C for 30 seconds, 72�C for 30 seconds,and 72�C for 7 minutes). GAPDH was amplified using 30 cycles and thesame conditions as the second cycle. PCR products were separated byelectrophoresis in 1.5% agarose gels and stained with the fluorescentdye ethidium bromide. Sterilized water was used as a negative control.

Western BlottingThe total protein was extracted from the dental pulp tissues and

HDPCs using cell lysis buffer (0.1 mol/L NaCl, 0.01 mol/L Tris$HCl[pH = 7.6], 0.001 mol/L EDTA [pH = 8.0], 1 mg/L aprotinin, and100 mg/L benzyl sulfuryl fluoride) or tissue lysis buffer (50 mmol/LTris$HCl, 150 mmol/L NaCl, 0.2 g/L NaN3, 1 g/L sodium dodecyl sulfate(SDS), 1 mmol/L PMSF, 5 mg/L aprotinin, 1 g/L NP-40, and 5.0 g/Lsodium deoxycholate, pH = 8.0). Equal amounts of total protein ex-tracted from the cell or tissue samples were resolved by SDS–polyacryl-amide gel electrophoresis and transferred to a nitrocellulosemembrane. Proteins were detected with antibodies against anti-NOD2(IMGENEX, Inc, San Diego, CA) and anti-GAPDH (Santa Cruz Biotech-nology, Inc, Santa Cruz, CA) as an internal loading control. Relativeband intensities were determined by densitometry by using Scion imagesoftware (version 4.0; Scion Corporation, Frederick, Maryland). The

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results are presented as mean � standard error of the mean, anda Student-Newman-Keuls test was performed to evaluate statisticalsignificance. A p value of less than 0.05 was defined to be statisticallysignificant.

Immunohistochemical StainingImmediately upon extraction, each tooth was halved lengthwise

by using a diamond instrument and a hammer. The dental pulp wasthen carefully extracted by using a sterile explorer and a spoon exca-vator. Immunohistochemistry was conducted as follows: after a 24-hour fixation in 4% paraformaldehyde, the pulp tissue was immersedin a 30% sucrose solution for 48 hours. Fixed tissues were thenembedded in paraffin. After deparaffinization, five randomly chosen,5-mm-thick sections with intact structure were treated with 3% H2O2

in ice-cold methanol for 20 minutes to block endogenous peroxi-dase. Next, slide-mounted sections were immersed in 0.01 mol/Lsodium citrate buffer (pH = 6.0) at 95� to 100�C in a microwaveoven and incubated for 20 minutes for antigen retrieval. Sectionswere blocked with 5% bovine serum albumin for 20 minutes atroom temperature, washed in phosphate-buffered saline, rabbit poly-clonal anti-NOD2 antibody (1:1500, IMGENEX Inc) or phosphate-buffered saline solution (as a negative control) was then added,and the samples were incubated overnight at 4�C. Subsequently,sections were incubated with biotinylated goat antirabbit immuno-globulin G secondary antibodies (Bo Shi De Tech, Co, Ltd, Wuhan,PR China). Finally, sections were stained by using the Streptavidin-Biotin Complex (Bo Shi De Tech, Co, Ltd) according to the manufac-turer’s instructions. The reaction was visualized by using diamino-benzidine. Stained slides were counterstained with hematoxylin,dehydrated, and mounted with Eukitt Mounting Medium (Specimenand Model Factory, Shanghai, PR China). Finally, slides wereobserved under a light microscope. Samples prepared from inflam-matory dental pulp tissues were used as a positive control.

Figure 2. The expression of NOD2 messenger RNA. NOD2 messenger RNAexpression (95 bp) was detected using nested RT-PCR in dental pulp (lanes1-4) after the second amplification. No NOD2 was detected in the negativecontrol. M, marker; DP, dental pulp; CN, negative control.

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ResultsNOD2 Is expressed in the HDPCs and Dental Pulps

As shown in Figure 2, the expression of NOD2 messenger RNA wasdetected in all the four specimens of human dental pulp tissues. No posi-tive bands were detected in the negative control lanes (Fig. 2). Theexpression of NOD2 protein was detected in both pulp tissue specimensand HDPCs by Western blotting (Fig. 3). Of note, two distinct NOD2protein isoforms were present in both pulp tissue specimens and HDPCsin our study. The levels of NOD2 protein in the dental pulp tissues weresignificantly higher than those in the HDPCs after normalization by thelevels of GAPDH in corresponding samples (Fig. 3).

Immunohistochemical Staining in Pulp TissueAs shown in Figure 4, the NOD2 protein was distinctly expressed in

the cytoplasm of odontoblasts, but the staining was not obvious in pulptissue fibroblasts. Strongly positive NOD2 staining was observed in thepulp vascular endothelial cells. Confirming the validity of the study, theNOD2 protein expression was not detected in the negative control butwas strong in the positive control (Fig. 4).

DiscussionPrevious studies have suggested that NOD2 is actively involved in

the innate immune response and acts as an activator of adaptive immu-nity (23, 24). It is mainly expressed in cells on the front lines of the hostdefense system, guarding against invasion of pathogenic microorgan-isms, including macrophages, monocytes, leukocytes, dendritic cells,and epithelial cells (25–27). Vascular endothelial cells have recentlybeen reported to express NOD2 and are increasingly thought to activelycontribute to immune responses through activation-dependent secre-tion of a large number of cytokines, including IL-6 (28, 29). Theseobservations, in conjunction with the fact that gingival fibroblasts

Figure 3. The expression of NOD2 protein. NOD2 protein expression (36-55kd) was detected by Western blot analysis in dental pulp (lanes 1-6) andHDPCs (lane 7 and 8). Blots were stripped and reprobed for GAPDH asa loading control. Densitometric values for each group are shown in thebar graph as mean � standard error of the mean. The level of NOD2 protein(compared with GAPDH) in the dental pulp group was significantly higher thanthat in the HDPCs group (*indicates significance at p < 0.05). All data arerepresentative of three separate experiments. CN, positive control MCF-7;DP, dental pulp; HDPCs, human dental pulp cells.

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Figure 4. Immunohistochemical detection of NOD2 in the dental pulp. (A) Positive anti-NOD2 staining was detected in the cytoplasm of odontoblasts situated inthe outermost layer of the dental pulp, but positive anti-NOD2 staining was not observed in dental pulp fibroblasts (SABC�400). (B) Positive circular staining wasobserved within the pulpal vessels (SABC�400). (C) Positive control: NOD2 was strongly expressed in inflammatory cells (SABC�400). (D) Negative control: nostaining was observed (SABC �400). OD, odontoblasts; V, vessel.

constitutively express NOD2 (30), raised the question as to whetherNOD2 might also be expressed in human dental pulp tissues and HDPCs.

In the present study, we provide evidence of NOD2 expression inhuman dental pulp tissues and HDPCs. The NOD2 messenger RNA andprotein expression were clearly shown in all dental pulp tissues bynested RT-PCR and Western blotting. It may be argued that this expres-sion could be because of the presence of immunological cells orvascular endothelial cells in the normal pulp. However, immunohisto-chemical staining has clearly shown that NOD2 is expressed in the cyto-plasm of odontoblasts, providing further evidence of a defensive role forNOD2 against invasive foreign bodies (31). In addition, immunohisto-chemical staining revealed that NOD2 was also expressed in normalpulp tissue in the vascular endothelial cells within and around vessels,suggesting an important role for NOD2 in the recognition of invadingpathogenic microorganisms and in the initiation of an immuneresponse to activate immunological cells within the dental pulp.

In our study, the NOD2 protein was distinctly shown to be ex-pressed in the cytoplasm of odontoblasts when immunohistochemistrywas used but was not visibly present in pulp fibroblasts. In contrast,HDPCs showed NOD2 expression by Western blotting. It is possiblethat NOD2 is more readily detected by Western blotting than by immu-nohistochemistry. However, we cannot rule out the possibility that thesedifferences may be caused by variations in individual expression (32),which may be one of the key factors explaining differences in pulp resis-tance to dental caries. Although the NOD2 expression level variedbetween specimens, two different NOD2 protein isoforms were notablypresent in human dental pulp tissues and HDPCs by Western blotting.Also, it has been reported that through alternative splicing, NOD2 canproduce different transcripts, all with introns, putatively encodingdifferent protein isoforms. Therefore, it is not surprising that the molec-ular weights of NOD2 observed by Western blotting can vary (33). More-

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over, these findings showed that HDPCs express NOD2, indicating thatHDPCs have the potential to defend against invading pathogenic micro-organisms and may actively participate in the innate immune response.

As the characteristic cells of the pulp-dentin complex, odonto-blasts are the first pulp cells to come in contact with dental pathogens(34). In addition to dentinal formation, odontoblasts play a pivotal rolein dental defense during microbial invasion (35). Osteoblasts andodontoblasts are cells that contribute to mineral deposition in thebone and dentin, respectively. NOD2 expressed in osteoblasts canmediate MDP-enhancing osteoclast formation induced by LPS (36).Moreover, osteoblasts have been proven to express TLR4 as part ofimmune and inflammatory responses (37). Therefore, we speculatethat NOD2 expression by odontoblasts also mediates the productionof inflammatory factors such as IL-8 as part of the dental defense mech-anisms.

HDPCs are the major constituent of pulp tissue and are capable ofplaying a pivotal role in healing processes, pathological alterations, andthe production of various inflammatory cytokines. It has been proposedthat HDPCs actively participate in the inflammatory processes associatedwith pulp diseases. In the initial studies on the innate immune responsesof pulp, HDPCs were found to produce various inflammatory cytokines,including IL-6 and IL-8, upon stimulation with LPS from pathogenicbacteria (16, 38–40). Recently, it has been reported that TLR2 andTLR4 are expressed in the inflammatory murine pulp cells (17). Theseresults indicate that dental pulp cells actively participate in inflammatoryprocesses and immune responses. We hypothesized that HDPCs are en-dowed with receptors for bacterial components and can inducesignaling through innate immune pathways in response to invadingbacteria.

Both HDPCs and gingival fibroblasts have a common fibroblastphenotype. Furthermore, it has been reported that gingival fibroblasts

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constitutively express NOD2 (30). In the present study, NOD2 was notobviously present in pulp fibroblasts when observed using immunohis-tochemical staining. HDPCs were found to have low NOD2 expression byWestern blotting. One explanation for the low level of NOD2 expressionin HDPCs could be their physiological isolation from pathogenic micro-organisms, suggesting that only after situations involving tissue injury dothey interact with microbes that would induce inflammatory reactions.Therefore, we speculate that NOD2 expressed in HDPCs might be upre-gulated during inflammation, rather than being constitutively expressedat a high level as it is in other cells. In conclusion, we have shown that theNOD2 protein is expressed in the dental pulp and HDPCs, indicating thatNOD2 may play an important role in the immune defense of the pulpdentin complex.

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