Regenerative Endodontics

Toll-like Receptor Expression Profile of Human Dental PulpStem/Progenitor CellsKarim M. Fawzy El-Sayed, MSc, PhD, MFDS-RCSEd,*† Pauline Klingebiel, DDS,*and Christof E. D€orfer, DDS, PhD*

Abstract

Introduction: Human dental pulp stem/progenitor cells(DPSCs) show remarkable regenerative potentialin vivo. During regeneration, DPSCs may interactwith their inflammatory environment via toll-like recep-tors (TLRs). The present study aimed to depict for thefirst time the TLR expression profile of DPSCs.Methods:Cells were isolated from human dental pulp, STRO-1–immunomagnetically sorted, and seeded out to obtainsingle colony-forming units. DPSCs were characterizedfor CD14, CD34, CD45, CD73, CD90, CD105, andCD146 expression and for their multilineage differentia-tion potential. After incubation of DPSCs in basic or in-flammatory medium (interleukin-1b, interferon-g,interferon-a, tumor necrosis factor-a), TLR expressionprofiles were generated (DPSCs and DPSCs-i). Results:DPSCs showed all characteristics of stem/progenitorcells. In basic medium DPSCs expressed TLRs 1–10 indifferent quantities. The inflammatory medium upregu-lated the expression of TLRs 2, 3, 4, 5, and 8, downregu-lated TLRs 1, 7, 9, and 10, and abolished TLR6.Conclusions: The current study describes for the firsttime the distinctive TLR expression profile of DPSCs inuninflamed and inflamed conditions. (J Endod2016;42:413–417)

Key WordsFlow cytometry, polymerase chain reaction, pulp, stemcells, TLR

From the *Clinic for Conservative Dentistry and Periodon-tology, School of Dental Medicine, Christian Albrechts Univer-sity, Kiel, Germany; and †Oral Medicine and PeriodontologyDepartment, Faculty of Oral and Dental Medicine, Cairo Univer-sity, Cairo, Egypt.

Address requests for reprints to Dr Karim M. Fawzy El-Sayed, Clinic for Conservative Dentistry and Periodontology,School of Dental Medicine, Christian Albrechts-Universit€at zuKiel, Arnold-Heller-Straße 3, Haus 26, 24105 Kiel, Germany.E-mail address: karim.fawzy@gmail.com0099-2399/$ - see front matter

Copyright ª 2016 American Association of Endodontists.http://dx.doi.org/10.1016/j.joen.2015.11.014

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The pulp-dentin complex stems embryonically from the multipotent neural crest–ec-tomesenchyme and constitutes a functional and physiological unit (1). As a highly

vascularized and innervated connective tissue, it is composed of diverse cell popula-tions, among which dental pulp stem/progenitor cells (DPSCs) are anticipated tocontinuously replenish odontoblasts to form secondary and tertiary/reparative dentinthroughout adult life as well as in response to various insults (2). DPSCs are consideredan alternative and readily available source of multipotent stromal cells (MSCs) for tissueregeneration; they are characterized by their clonogenicity, their highly proliferative po-tential, their capability of self-renewal, and their multilineage differentiation aptitude(3) and are reported to hold promising attributes in the field of ameliorating ischemic,cardiac, and neurologic diseases (4, 5).

Toll-like receptors (TLRs), key molecules connecting innate and adaptive immu-nity, are germline encoded pattern recognition receptors sensing specific pathogen-associated molecular patterns (PAMPs), thereby promoting activation of immune cells,and are pivotal in pathogenesis of chronic inflammatory, autoimmune, and infectiousdiseases (6). To date, 10 functional human TLRs have been described. Depending ontheir cellular localization and PAMP ligands, TLRs are divided into extracellular ones,mostly identifying microbial membrane constituents including lipids and lipoproteins(TLR1, TLR2, and TLR6), lipopolysaccharide (LPS) (TLR4), and flagellin (TLR5),and intracellular ones, which recognize double-stranded RNA (TLR3), single-stranded viral RNA (TLR7 and TLR8), and unmethylated CpG-DNA of viruses and bac-teria (TLR9) (7).

MSCs of different origin express functional TLRs in characteristic patterns, makingthem particularly sensitive to certain microbial compounds. When activated by their li-gands, TLRsmodulate themigratory, proliferative, differentiation, and immunosuppres-sive potentials of MSCs (8). Varied expressions of TLRs 1, 2, 3, 4, 5, and 6 were reportedon human and mural adipose MSCs and bone marrow–derived MSCs (BM-MSCs) aswell as on human Wharton jelly MSCs (WJ-MSCs) (9). This distinctive TLR expressionpattern could affect the therapeutic potential of MSCs during transplantation in vivo(10). To date, solely the expression of TLRs 2, 3, and 4 was described on DPSCs(11–14). The aim of the present investigation was to characterize for the first time acomplete TLR expression profile of DPSCs under inflamed and uninflamed conditions.

Materials and MethodsIsolation and Culture of DPSCs

Human dental pulp tissue was obtained from patients (age, 15–20 years) under-going extraction of non-carious third molars (n = 6) (Institutional Review Boardapproval number D-444/10). Teeth were disinfected and mechanically fractured,and the dental pulp was gently isolated with sterile forceps, rinsed several times in basicmedium, and placed into 75-mL culture flasks (Sarstedt AG, N€umbrecht, Germany) for30 minutes to adhere. Subsequently, the basic medium was carefully added, flasks wereincubated in 5% carbon dioxide at 37�C, and cells were left to grow out.

After reaching 80%–85% confluence, cells were detached with 0.10% trypsin-EDTA (Biochrom Ltd, Cambridge, UK) and counted; their viability was tested by usingtrypan blue (Sigma-Aldrich, St Louis, MO) and finally seeded out at 30 cells/cm2 densityin basic medium. After the first passage cells reached 80%–85% confluence, they wereimmunomagnetically sorted by using anti-STRO-1 (BioLegend, San Diego, CA) and anti-

TLRs of Dental Pulp Stem/Progenitor Cells 413

TABLE 1. Primer Names and ID Used for rt-PCR (as supplied by Roche)

Assay ID Gene symbol Roche accession ID

111000 TLR1 H. sapiens ENST00000308979145617 TLR2 H. sapiens ENST00000260010111008 TLR3 H. sapiens ENST00000296795135752 TLR4 H. sapiens ENST00000355622103674 TLR5 H. sapiens ENST00000366881111018 TLR6 H. sapiens ENST00000381950111012 TLR7 H. sapiens ENST00000380659103816 TLR8 H. sapiens ENST00000218032143252 TLR9 H. sapiens ENST00000360658141065 TLR10 H. sapiens NM_001017388102083 PGK1 H. sapiens ENST00000373316

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immunoglobulin M MicroBeads (Miltenyi-Biotec, Bergisch Gladbach,Germany) antibodies according to the manufacturer’s instructions(MACS; Miltenyi-Biotec). The STRO-1+–sorted cells (DPSCs) wereseeded out to form colony-forming units (CFUs).

CFUsDPSCs were cultured in basic medium at 1.63 cells/cm2 density.

Aggregates of 50 or more cells were scored as CFUs. On day 12, repre-sentative cultures were fixed with 4% formalin and stained with 0.1%crystal violet. The remainder of the CFUs were detached by using cellscrapers and seeded in new 75-mL flasks.

Flow Cytometric AnalysisAfter reaching confluence, DPSCs were characterized flow cyto-

metrically for the MSCs’ surface markers: CD14, CD34, CD45, CD73,CD90, CD105, and CD146 (all from Becton Dickinson Co, Canaan,CT). Binding of primary antibodies and isotype controls was performedby using FcR Blocking Reagent (Miltenyi-Biotec), and their expressionwas evaluated with FACSCalibur E6370 and FACSComp 5.1.1 software(Becton Dickinson Co).

Multilineage Differentiation PotentialFor osteogenic differentiation, third passage 2� 104 DPSCs were

cultured on 6-well plates in osteogenic medium (PromoCell, Heidel-berg, Germany) and in basic medium (control). At day 14, cell cultureswere stained with alizarin red (Sigma-Aldrich) to label calcified de-posits. For adipogenic differentiation, third passage 3 � 105 DPSCswere cultured on 6-well plates in adipogenic medium (PromoCell)and in basic medium (control). The presence of lipid droplets was eval-uated after 21 days by oil red O (Sigma-Aldrich). For chondrogenic dif-ferentiation, micromasses of third passage 3 � 104 DPSCs wereincubated with chondrogenic medium (PromoCell) in 1.5-mL Eppen-dorf tubes (Eppendorf, Hamburg, Germany) and in basic medium(control). Chondrogenic differentiation was evaluated at day 35 bystaining of glycosaminoglycans with alcian blue and nuclear fast redcounterstaining (Sigma-Aldrich).

Inflammatory MediumTo test the effect of the inflammatory environment on the TLR expres-

sion profile of DPSCs, standardized inflammatory medium, composed of25 ng/mL interleukin (IL)-1b, 1� 103 U/mL interferon (IFN)-g, 50 ng/mL tumor necrosis factor (TNF)-a, and 3� 103U/mL IFN-a (inflamma-torymedium; all from PeproTech, Hamburg, Germany) (15) added to thebasic medium components, was used. DPSCs were incubated 18 hours inthe inflammatory (DPSCs-i) or basic medium (DPSCs).

mRNA Extraction and cDNA SynthesismRNA extraction was performed for DPSCs and DPSCs-i by using

RNeasy kit (Qiagen, Hilden, Germany). Obtained RNA was purified byusing RNase-free-DNase (Promega, Mannheim, Germany) and quanti-fied photometrically. Complementary cDNA was synthesized from 1 to13 mL RNA (1 mg/mL) by reverse transcription by using QuantiTectreverse transcription kit (Qiagen) in a volume of 20mL reactionmixturecontaining 4 pmol of each primer, 10mL of the LightCycler Probes Mas-ter mixture (Roche Diagnostics, Risch-Rotkreuz, Switzerland), and5 mL specimen cDNA. Real-time polymerase chain reaction (rt-PCR)(LightCycler-96 Real-Time-PCR System; Roche Molecular Biochemi-cals, Indianapolis, IN) was performed according to the manufacturer’sinstructions. Relative quantities of each transcript were normalized ac-cording to the expression of phosphoglycerate kinase 1 (PGK1).

414 Fawzy El-Sayed et al.

Primers for TLRs 1–10 and PGK1 reference gene were supplied byRoche and tested on DPSCs and DPSCs-i (Table 1).

Flow Cytometric Determination of TLR ExpressionDPSCs and DPSCs-i were characterized flow cytometrically for the

expression of TLRs 1–10 at protein level. For intracellular TLR staining,cells were fixed and permeabilized with Fix&Perm kit (Imtec, Ant-werpen, Belgium) before incubation. Antibodies used were anti-TLR1, anti-TLR3, and anti-TLR9 (eBioscience, San Diego), anti-TLR2,anti-TLR4, and anti-TLR8 (Enzo Life Sciences, L€orrach, Germany),anti-TLR5 (R&D Systems, Hessen, Germany), anti-TLR6 (BioLegend),anti-TLR7 (Perbio Science, Bonn, Germany), and anti-TLR10 (Acris An-tibodies, Herford, Germany). Binding of primary antibodies and corre-sponding isotype controls was performed by using FcR BlockingReagent and evaluated with FACSCalibur E6370 and FACSComp 5.1.1software (Becton Dickinson).

Statistical AnalysisShapiro-Wilk test tested the normal distribution of data. Differ-

ences in TLR expression on mRNA and protein levels in DPSCs andDPSCs-i were evaluated by using the Wilcoxon signed rank test (SPSSsoftware version 11.5; SPSS, Chicago, IL). The level of significancewas P = .05.

ResultsMicroscopy, CFUs, and Flow Cytometric Analysis

After the initial adherence phase, fibroblast-like cells grew out ofthe pulpal tissue masses (Fig. 1A). Twelve days after seeding, STRO-1+–sorted cells (DPSCs) showed CFUs (Fig. 1B) and were CD14�, CD34�,and CD45� and CD73+, CD90+, CD105+, and CD146+ (Fig. 1C).

Multilineage DifferentiationThe osteogenic differentiation of DPSCs was demonstrated by the

formation of alizarin red–positive calcified deposits in contrast to theircontrol (Fig. 1D and E). Adipogenic differentiation of DPSCs resulted inthe formation of oil red O–positive lipid droplets in contrast to theircontrol (Fig. 1F and G). The chondrogenic differentiation of DPSCs re-sulted in the formation of alcian blue–positive glycosaminoglycans incontrast to their control (Fig. 1H and I).

TLR mRNA ExpressionOn the mRNA level, DPSCs incubated in basic medium expressed

(median gene copies/PGK1 copies, Q25/Q75) TLR1 (0.0013, 0.0008/0.0058), TLR2 (0.0002, 0.0000/0.0018), TLR3 (0.0015, 0.0009/0.0022), TLR4 (0.0067, 0.0024/0.0105), TLR5 (0.0000, 0.0000/0.0005), TLR6 (0.0017, 0.0007/0.0026), and TLR10 (0.0005,

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Figure 1. Microscopic appearance, colony formation, surface marker expression, and differentiation potential. (A) Phase contrast microscopic appearance of theadherent tissue mass with outgrowing cells (second week). (B) CFUs of DPSCs (crystal violet). (C) Flow cytometric analysis of the surface marker expression profileof DPSCs. Multilineage differentiation potential of DPSCs. Alizarin red staining of the osteogenically stimulated DPSCs (D) and their controls (E); oil red O staining ofthe adipogenically stimulated DPSCs (F) and their controls (G). Alcian blue staining of the chondrogenically stimulated DPSCs (H) and their controls (I).

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0.0002/0.0012) (Fig. 2A). Inflammation (DPSCs-i) significantly upre-gulated the expression of TLR1 (0.0027, 0.0015/0.0114; P = .019),TLR2 (0.0041, 0.0009/0.0075; P = .006), and TLR3 (0.0160,0.0049/0.0318; P = .002) in addition to significantly lower expressionof TLR6 (0.0006, 0.0004/0.0012; P = .012, Wilcoxon signed rank test)and downregulation of TLR10 (0.0003, 0.0001/0.0007). No expressionfor TLRs 7, 8, and 9 was recorded in both media on mRNA level(Fig. 2B).

Flow Cytometric TLR ExpressionDPSCs incubated in basic medium expressed (% protein expres-

sion, Q25/Q75) TLR1 (30.67, 12.07/42.71), TLR2 (33.74, 22.78/52.94), TLR3 (0.60, �0.29/2.08), TLR4 (18.90, 14.92/43.13),TLR5 (23.42, 17.40/44.70), TLR6 (3.15, �0.23/10.60), TLR7(9.14, 2.09/12.23), TLR8 (0.31, �0.62/59.59), TLR9 (11.18, 2.78/24.22), and TLR10 (37.43, 19.28/75.72) (Fig. 2C). Inflammatory me-dium upregulated TLR2 (45.45, 29.15/63.95), TLR3 (0.95, 0.12/2.34), TLR4 (19.84, 14.85/42.05), TLR5 (24.22, 7.78/42.07), andTLR8 (5.08, 2.39/34.06), downregulated TLR1 (24.40, 20.55/37.81), TLR7 (2.21, 0.60/12.27), TLR9 (3.55, 0.52/14.84), andTLR10 (23.58, 6.48/62.44), and diminished TLR6 expression(�0.91, �1.85/0.12; P = .008, Wilcoxon signed rank test) onDPSCs-i (Fig. 2D).

DiscussionThe innate immune system, of which TLRs are fundamental com-

ponents, is the host’s first defense line interacting with invading patho-logic components. Inflammation follows most tissue injuries; it is anintegral part of early healing processes, affecting in multiple ways thesucceeding tissue reparative/regenerative phase (11). DPSCs may be

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exposed to such stimuli in many clinical and therapeutic conditions.Clinically active deep caries may result in localized odontoblastic dam-age or death through bacteria and their by-products, ultimately result-ing in pulpitis (16). Through their sensing receptors (17), some of thesurviving odontoblasts may participate in the pulpal response, whereasunder the infected dentin, undifferentiated DPSCs are activated to extendthe inflammation via their immunomodulatory properties and initiatetertiary/reparative dentin formation (16). In the field of tissue engineer-ing, therapeutic approaches may further use a direct DPSC transplanta-tion into inflamed environment, resulting ultimately in an interactionbetween DPSCs and PAMPs through their TLRs. The cell-specific TLRexpression profile and its alteration in inflammatory environmentplay a decisive role in their aptitude for such interactions. Therefore,the aim of the present study was to characterize the distinctive TLRexpression profile of DPSCs in inflamed and in uninflamed conditionsas a first stage of exploring this possible communication.

The putative STRO-1 stem cell marker has been exploited in thepresent study to isolate the DPSCs immunomagnetically. The sortedDPSCs showed all classic features for MSCs; they were CD73+,CD90+, CD105+, and CD146+ and CD14�, CD45�, and CD45� anddemonstrated remarkable CFU ability, plastic adherence, and multiline-age differentiation potential.

PAMPs of gram-positive/gram-negative bacteria involved in pulpalpathologic conditions can induce an inflammatory reaction to whichmost MSCs are sensitive (9, 18), with an upregulation of multipleproinflammatory cytokines in the surrounding environment. Tomimic such inflammatory condition, DPSCs were cultured in astandardized medium supplemented with IL-1b, IFN-g, TNF-a, andIFN-a, the cytokines mostly present at inflammatory sites (15). Earlierinvestigations demonstrated multiple profound effects of these inflam-matory cytokines on TLR signaling pathways. The same intracellular

TLRs of Dental Pulp Stem/Progenitor Cells 415

Figure 2. mRNA and protein expression of TLRs in DPSCs and DPSCs-i. (A) mRNA expression of TLRs 1–10 in uninflamed condition. (B) mRNA expression ofTLRs 1–10 in inflamed condition (n = 6, box and whisker plots with medians and quartiles). The green boxes show increased mRNA expression after stimulation bythe inflammatory medium (Wilcoxon signed rank test, statistical significance marked with asterisk: **P < .01, ***P < .001). (C) Protein expression of TLRs 1–10in uninflamed condition. (D) Protein expression of TLRs 1–10 in inflamed condition (n = 6, box and whisker plots with medians and quartiles) (Wilcoxon signedrank test, statistical significance marked with asterisk: **P < .01).

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signaling proteins activated through binding of IL-1b to IL-1 receptor(IL-1R) participate in signaling through other receptors with toll-IL-1receptor domains (19). IL-1R, TNF-receptor (TNF-R), and TLR4signaling pathways produce intracellular nuclear factor kappa Bthrough converging on a common IkB kinase complex phosphorylatingthe nuclear factor kappa B inhibitory protein (IkBa), the inhibitor ofnuclear factor kappa B kinase (20). TNF-R–associated factor 6, apivotal signaling molecule regulating a diverse array of physiologicalprocesses including adaptive and innate immunity, is essential for thesignaling downstream of the IL-1-R/TLR superfamily (21). Finally, apositive feedback loop exists between IFN levels and the expressionof multiple TLRs (22).

On the protein level, the DPSCs in uninflamed environment ex-pressed TLRs 1–10 in different quantities. According to their medianexpression values, TLR10 was the highest expressed, followed byTLRs 2, 1, 5, 4, 9, 7, 6, 3, and finally 8 in a descending order. The in-flammatory medium upregulated TLRs 2, 3, 4, 5, and 8, downregulatedTLRs 1, 7, 9, and 10, and abolished TLR6 expression in DPSCs-i. Itfurther altered the quantitative order of TLR expression; TLR2 was

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the highest expressed, followed by TLRs 1, 5, 10, 4, 9, 8, 7, and 3 ina descending order. The mRNA level of most DPSC TLRs in inflamedand uninflamed conditions correlated mostly with their protein expres-sion, with a statistically significant upregulation reached for TLRs 1, 2,and 3 and a down-regulation in TLR6. On mRNA level no expression ofTLRs 7, 8, and 9 was noted.

Diverse TLR expression profiles have been described in humanMSCs originating from different tissues. TLRs 1, 2, 3, 4, 5, 6, and 9were expressed in umbilical cord MSCs (23). BM-MSCs demonstrateda wider expression profile with added TLRs 8 and 10 expression (9,15). WJ-MSCs showed a comparable pattern with marginal/deficientTLR4 expression (9). Dental tissue–derived MSCs demonstrated theexpression of TLRs 2, 3, and 4 in dental follicle MSCs (11) and DPSCs(11, 24), whereas TLRs 1, 2, 3, 4, 5, 6, 8, 9, and 10 were expressed inperiodontal ligament MSCs (25). Similar to the current study’s results,inflammation upregulated the expression of TLR2 and TLR4 and down-regulated the expression of TLR6 in BM-MSCs (15).

Earlier studies proved direct relationships between levels ofcellular TLR expression and responsiveness to their corresponding

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ligands (26). The outlined TLR expression profile, especially underinflammation, may influence the DPSCs’ response to the correspondingligands in vivo. An upregulation of the lipoteichoic acid–sensing TLR2(27) and LPS-sensing TLR4 (28) could increase the DPSCs’ ability torecognize both gram-positive and gram-negative pathogens, respec-tively, under inflammation, whereas the inflammatory upregulation ofTLR5 expression could favor the recognition of bacterial flagellin(28). A downregulation of TLR1 and TLR6 reduces the ability for recog-nition of lipoprotein (29), and a downregulation of TLR7 and TLR9lessens the aptitude for recognition of viral pathogens (30).

A coactivation of multiple TLRs makes interpretation of this directligand-receptor relationship more complex. Studies described that co-transfection of different TLRs could either augment or inhibit the recog-nition of specific PAMPs (31, 32), suggesting that cellular responses toPAMPs are dependent on the entire repertoire of TLRs expressed on acell, necessary cofactors, and levels of each PAMP present (26). Furthercomplexity arises from the fact that some TLRs may in combined stimu-lation act as co-receptors (eg, TLR1, TLR6) for other TLRs (eg, TLR2)and can thereby stimulate or inhibit cellular responsiveness to their stim-ulating ligands (31, 33). To date, only effects of TLR 2, 3, and 4 activationhave been characterized on DPSCs. Earlier studies reported onproduction of distinctive inflammatory cytokines by DPSCs, includingan increase in IL-8 (12–14), IL-1 and IL-6 (13, 14) production inresponse to TLR4 activation. Activation of the expressed/upregulatedTLRs during inflammatory conditions could further affect themigration, proliferation, and differentiation potentials of DPSCs. TLR4activation could boost cell migration, downregulate their proliferation(13, 14), and enhance osteogenesis and suppress adipogenic potentialof DPSCs through increased Wnt-5a expression (13). TLR4 activationfurther lowers the endogenous TGF-b and the immunosuppressive factorindolamine-2,3-dioxygenase production, affecting combined the immu-nosuppressive phenotype of DPSCs (11).

The current study describes for the first time the distinctive TLRexpression profiles of DPSCs in inflamed and uninflamed conditions,which could impact their immunomodulatory and therapeutic poten-tials in vivo (10). Studies are further needed to test the effect of eachof the outlined TLRs’ activation, solely or in combination, on the variouscellular attributes of DPSCs.

AcknowledgmentsThe authors thank Dr Mohamed Mekhemar, Mrs Mojgan

Paymard-Stoltz, Mrs Regina Marquart, and Mrs Kerstin Marx fortheir technical assistance and Dr Andreas Kerscher for his supportin sample collection.

The authors deny any conflicts of interest related to this study.

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