Human Immunology 74 (2013) 861–866

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Secreted osteoclastogenic factor of activated T cells (SOFAT), a novelosteoclast activator, in chronic periodontitis

0198-8859/$36.00 - see front matter � 2013 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.http://dx.doi.org/10.1016/j.humimm.2013.04.013

Abbreviations: Th, t helper; RANKL, receptor activator of nuclear factor-kBligand; IL, interleukin; TNF-a, tumor necrosis factor-a; PG, prostaglandin; OPG,osteoprotegerin; SOFAT, secreted osteoclastogenic factor of activated T cells; PD,probing depth; CAL, clinical attachment level; BoP, bleeding on probing; PI, Plaqueindex; SUP, suppuration; MB, marginal bleeding; ELISA, enzyme linked immuno-sorbent assay; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; qPCR, quan-titative real-time polymerase chain reaction; PBS, phosphate-buffered saline;DMEM, dulbecco’s Modified Eagle Medium; TRAP, tartrate-resistant acid phospha-tase; EDTA, ethylenediamine tetraacetic acid.⇑ Corresponding author. Address: Laboratory of Immunology and Molecular

Biology, São Leopoldo Mandic Institute and Research Center, R. José RochaJunqueira, 13, Campinas, São Paulo 13045-755, Brazil. Fax: +55 19 3211 3636.

E-mail addresses: napimogamh@yahoo.com, marcelo.napimoga@gmail.com(M.H. Napimoga).

Christian Rado Jarry a,b, Poliana Mendes Duarte c, Fabiana Furtado Freitas d, Cristina Gomes de Macedo d,Juliana Trindade Clemente-Napimoga d, Eduardo Saba-Chujfi b, Fabricio Passador-Santos e,Vera Cavalcanti de Araújo e, Marcelo Henrique Napimoga a,⇑a Laboratory of Immunology and Molecular Biology, São Leopoldo Mandic Institute and Research Center, Campinas/SP, Brazilb Periodontal Medicine Research Group, Department of Periodontology, São Leopoldo Mandic Institute and Research Center, Campinas/SP, Brazilc Department of Periodontology, Dental Research Division, Guarulhos University, Guarulhos/SP, Brazild Laboratory of Orofacial Pain, Department of Physiology, Piracicaba Dental School, State University of Campinas, Piracicaba/SP, Brazile Laboratory of Oral Pathology, São Leopoldo Mandic Institute and Research Center, Campinas/SP, Brazil

a r t i c l e i n f o a b s t r a c t

Article history:Received 8 January 2013Accepted 12 April 2013Available online 22 April 2013

A novel activated human T cell-secreted cytokine, referred as secreted osteoclastogenic factor of activatedT cells (SOFAT), that induce osteoclastogenesis in a RANKL-independent manner was recently described.This study evaluated the role of SOFAT in periodontal tissues and periodontitis. Gingival biopsies wereharvested from systemically healthy non-periodontitis (n = 15) and chronic periodontitis patients(n = 15). The mRNA and protein levels of SOFAT were measured by qPCR and by enzyme-linked immuno-sorbent assay, respectively. Moreover, RAW 264.7 cells were cultured with SOFAT or Receptor activator ofnuclear factor-kB ligand (RANKL) and stained for tartrate-resistant acid phosphatase (TRAP). Also, micereceived a palatal injection between the first and second upper molar of SOFAT (100 ng/ml) or salinesolution (0.9%). The upper jaw was removed, histologically processed and stained with hematoxilinand eosin to observe the presence of osteoclast-like cells. The mRNA and protein levels of SOFAT weresignificantly higher in the gingival tissue of the periodontitis group when compared to non-periodontitisone (p < 0.05). In addition, SOFAT potently induced TRAP-positive multinucleated cell formation by RAW264.7 cells as well as induced the formation of osteoclast-like cells in the periodontal ligament in mice.The present study demonstrated that SOFAT may play an important role in periodontitis.� 2013 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights

reserved.

1. Introduction

Periodontal diseases are infectious-inflammatory conditions[1], which induce the destruction of the tooth-supporting struc-tures and possible changes in systemic health [2]. Interestingly,only the presence of periodontal pathogens is not enough fordisease initiation and development [3]. Indeed, a series of studies

has demonstrated that a determined host immune-inflammatoryresponse against pathogens results in the destruction of periodon-tal tissues [3,4].

Different molecular and cellular mechanisms/pathways under-lying periodontal tissue destruction have been recently describedand, promising therapeutic targets to arrest disease progressionhave been proposed [5]. In general, pro-inflammatory mediatorshave been associated with tissue destruction while anti-inflamma-tory proteins can counteract and attenuate disease progression[6–8]. With the discovery of T-cell subsets with variety ofimmune-regulatory properties, this pro- vs. anti-inflammatorysetting became multifaceted, and a series of studies has postulatedprotective or destructive roles according to the polarization of lym-phocyte subpopulations (T helper [Th]-1, Th2, Th17, and T regula-tory cells) [1,4]. Recent studies have demonstrated that analogousimmune response patterns considered harmful for tissue destruc-tion may play important roles in the control of periodontal infection[9,10].

The receptor activator of nuclear factor-kB ligand (RANKL) wasdiscovered as a cytokine that directly induces osteoclastogenesis

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[11]. Metabolic bone diseases such as rheumatoid arthritis sharesimilar pathways to induce the activation and differentiation ofosteoclasts via RANKL [12–15]. Thus, the discovery of the increasedRANKL levels in periodontal lesions [16], led to a possible explana-tion for the mechanism underlying alveolar bone resorption inperiodontitis [17]. However, several molecules besides RANKL areable to induce osteoclast activation, including interleukin (IL)-1b,tumor necrosis factor (TNF)-a, IL-6, IL-11, IL-17 and prostaglandin(PG) E2 [18]. In accordance, activated T cells may also induce osteo-clast formation by a mechanism that is independent of RANKL,since elevated levels of the RANKL inhibitor osteoprotegerin(OPG) were not able to counterbalance the osteoclast formation[19]. In 2009, a study using innumerous recombinant DNA technol-ogies, identified and analyzed the expression of a novel activatedhuman T cell-secreted cytokine, called secreted osteoclastogenicfactor of activated T cells (SOFAT). This unique cytokine seems toprovoke RANKL- and osteoblast-independent osteoclast inductionas well as stimulate IL-6 production by osteoblasts [20].

Several studies have focused on the development of differenttherapies and drugs able to modulate the host response and, con-sequently, to reduce the severity of inflammation, connective tis-sue degradation, and bone loss in periodontal diseases [21–25]. Aprevious study in rats, demonstrated that OPG prevents alveolarbone loss in experimental periodontitis, being a promising thera-peutic agent for the treatment of periodontal disease [26]. How-ever, to date, no conclusive therapy has been able to avoidperiodontal destruction completely. The challenge in discoveringeffective therapies for periodontal diseases may be the lack ofknowledge regarding the abundant possible pathways that activatethe bone resorbing cells. Thus, since SOFAT is a novel T cell cyto-kine that may activate osteoclasts independent of RANKL, theaim of this study was to evaluate the role of SOFAT in periodontaltissues and chronic periodontitis.

2. Materials and methods

2.1. Clinical phase

2.1.1. SubjectsFifteen systemically-healthy non-periodontitis individuals

(mean age 39.1 ± 5.2 years) and 15 systemically-healthy individu-als with chronic periodontitis (mean age 48.1 ± 13.6 years) wereselected from the population referred to the Periodontal Clinic ofGuarulhos University, from January 2009 until July 2010. Subjectswho fulfilled the following described inclusion/exclusion criteriawere invited to participate in the study. All eligible subjects wereinformed of the nature, potential risks and benefits of their partic-ipation in the study and signed their informed consent. This studyprotocol was previously approved by the Guarulhos University’sEthics Committee in Clinical Research (protocol # 100/2007).

2.1.2. Inclusion and exclusion criteriaAll individuals should be >30 years old and present at least 15

teeth (excluding third molars). Periodontitis subjects should bediagnosed with generalized chronic periodontitis [27]. All peri-odontitis individuals presented >30% of the sites with concomitantprobing depth (PD) and clinical attachment level (CAL) P 4 mmand at least one tooth indicated to extraction due to advanced peri-odontitis [sites with PD and CAL > 7 mm with bleeding on probing(BoP)]. Non-periodontitis individuals required periodontal estheticsurgery for the correction of gingival discrepancies and asymme-tries [28].

Exclusion criteria were pregnancy, lactation, current smokingand smoking within the past 5 years, periodontal or/and antibiotictherapies in the previous 6 months, use of mouthrinses containing

antimicrobials in the preceding 2 months, systemic condition thatcould affect the progression of periodontal disease (e.g. immunolog-ical disorders, diabetes), bone-related diseases (e.g. osteoporosis,rheumatoid arthritis) and long-term administration of anti-inflam-matory, immunosuppressive medications and hormone replace-ment therapy [28].

2.1.3. Clinical examinationAll clinical examinations were performed by one examiner

(PMD) who was calibrated, as previously described [29]. The clin-ical parameters, registered dichotomously (i.e. BoP), were calcu-lated by the Kappa–Light test and the intra-examiner agreementwas >0.85. The following parameters were assessed at six sites ofall teeth, excluding third molars, using a manual periodontal probe(UNC15, Hu-Friedy, Chicago, IL, USA): plaque index (PI), BoP (pres-ence/absence), suppuration (SUP, presence/absence), marginalbleeding (MB, presence/absence), PD (mm) and CAL (mm) [28].

2.1.4. Sample collectionFor the periodontitis group, the biopsies were collected from a

tooth indicated for exodontia due to advanced periodontitis (siteswith PD and CAL > 7 mm and BoP) in order to obtain representativeareas of the periodontal inflammation. If the patient had two ormore teeth with these characteristics, gingival biopsy from onlyone tooth with the worst diagnosis was included. For the non-peri-odontitis group, gingival biopsies were collected from a single toothwithout signs of clinical inflammation, indicated for gingivoplastydue to esthetical reasons. All samples included junctional and sulcu-lar epitheliums and connective gingival tissue. The gingival tissueswere divided in two pieces. One part was stored in RNA later (Ambi-on Inc., Austin, TX, USA) at �70 oC for the evaluation of the mRNAlevels and another piece was stored in protease inhibitor buffer (Sig-ma–Aldrich, St. Louis, MO, USA) at �70 oC for the enzyme linkedimmunosorbent assay (ELISA) [30].

2.1.5. RNA extractionTotal RNA from the biopsies was isolated by the Trizol method

(Invitrogen, Life Technologies, Grand Island, NY, USA) according tothe manufacturer’s recommendation. RNA samples were resus-pended in diethylpyrocarbonate-treated water and stored at�70 oC. The RNA concentration was determined from the opticaldensity using a micro-volume spectrophotometer (Nanodrop1000, Nanodrop Technologies LLC, Wilmington, NC, USA) [30].

2.1.6. Real-time PCR reactionsFor reverse transcription total RNA was treated with DNase

(Turbo DNA-frees, Ambion Inc., Austin, TX, USA), and 1 lg wasused for cDNA synthesis. The reaction was carried out using theFirst-Strand cDNA synthesis kit (Roche Diagnostic Co., Indianapolis,IN, USA), following the manufacturer’s recommendations as previ-ously described [30].

2.1.7. Primer designPrimers were designed using the Primer Express 3.0 probe

design software (Applied Biosystem, Foster City, CA, USA). The pri-mer sequences are: (SOFAT) Forward 50-ATGGACATTATCGTTCTGCTG CCC-30 and Reverse 50-CTGGGAGGTGTTGAGGGCATG-30

and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) For-ward 50-GCACC GTCAAGGCTGAGAAC-30 and Reverse 50-CCACTT-GATTTTGGAGGGATCT-30.

2.1.8. PCR reactionsQuantitative real-time polymerase chain reaction (qPCR) was

performed using a 7300 Real Time PCR machine (Applied Biosys-tem) using the SYBR Green PCR Master Mix (Fermentas, Glen Bur-nie, MD, USA). The reaction product was quantified with the

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Relative Quantification tool, using GAPDH as the reference gene.Negative controls with SYBR Green PCR Master Mix and waterwere performed for all reactions. PCR reactions were carried outin a blind fashion [30].

2.1.9. Enzyme-linked immunosorbent assay (ELISA)The gingival tissues were triturated and homogenized in 300 ll

of the appropriate buffer containing protease inhibitors (Sigma–Al-drich) followed by a centrifugation of 10 min/10,000g. The totalamount of extracted proteins was colorimetric measured usingthe micro BCA protein assay kit (Thermo, Rockford, IL, USA). Thesupernatants were stored at �70 �C until further analysis.

Levels of SOFAT were determined in capture enzyme-linkedimmunosorbent assays (ELISA) using microtiter plates (Costar3590, Corning, NY) coated for 24 h at 4 �C with 5 lg/ml of rabbitIgG anti-human SOFAT in carbonate–bicarbonate buffer, pH 9.6,following the manufacturer instructions. All antibody reagentswere affinity purified and obtained from (Rheabiotech Laboratory,Campinas, SP, Brazil). After being coated, plates were washed andblocked for 1 h at room temperature with bovine serum albumin(0.1%) in phosphate-buffered saline (PBS), pH 7.5. Homogenate gin-gival samples were applied in duplicate, and plates were incubatedfor 2 h at room temperature. All experiments included serialdilutions (500, 250, 125, 62.5, 31.25, 15.62, 7.81 and 3.90 lg/ml)of a standard sample of human SOFAT antibody (Rheabiotech).The secondary antibody was biotin-conjugated rabbit IgG anti-hu-man SOFAT (Rheabiotech) at a dilution of 1:1000. After incubationwith a solution of avidin–peroxidase (30 min) at room tempera-ture, a new series of washes was performed and 100 ll of3,30,5,50-tetramethylbenzidine substrate (TMB) was added andincubated for 15 min. To obtain the absorbance units (A450 nm),plates were read in an ELISA plate reader (Microplate Reader/Mod-el 3550, Bio Rad). Negative controls included uncoated, no gingivalhomogenate, and no primary antibody wells. For determination ofSOFAT concentrations, absorbance values were plotted against thestandard curve obtained for the serial dilutions of the purified hu-man SOFAT within a linear range. The total amounts of SOFAT weredetermined as microgram per milligrams of tissue (lg/mg). TheELISA was carried out in a blind fashion.

2.2. In vitro phase

2.2.1. In vitro osteoclastogenesis assayThe murine monocyte/macrophage cell line RAW 264.7 was

purchased from American Type Culture Collection (Manasas, VA,USA) and grown in DMEM supplemented with 10% heat-inacti-vated FBS, penicillin (40 U/ml), and gentamicin (40 lg/ml). All cellswere grown in a humidified atmosphere containing 5% CO2 at37 �C. For osteoclasts-like cells differentiation, RAW 264.7 cellswere suspended in 200 ll of DMEM containing 10% FBS and thenseeded at 5 � 103 cells/well in a 96-well culture plate and culturedwith 10, 50 or 100 ng/ml of rSOFAT (Rheabiotech) or 50 ng/ml ofsoluble RANKL (PeproTech Inc., Rocky Hill, NJ, USA) for 10 days.The culture medium was replaced with fresh medium every 2 days.After the culture, the cells were subjected to tartrate-resistant acidphosphatase (TRAP) staining (Sigma–Aldrich). TRAP-positive cellsappeared dark red, and TRAP-positive multinucleated cells con-taining P3 nuclei were counted as osteoclast-like cells by onetrained, calibrated and blinded examiner, using image analysissoftware.

2.3. Animal phase

2.3.1. In vivo osteoclastogenesis assayThe Institutional Committee for Animal Care and Use at São

Leopoldo Mandic Faculty previously approved the study protocol

(#2012/035). During the acclimatization (5 days) and experimen-tal periods (10 days), 10 C57/B6 mice (6–7 weeks of age) werehoused in groups of five in plastic cages with access to food anddrinking water ad libitum. The mice were kept in a room with a12 h light/dark cycle and a temperature between 22 �C and 24 �C.The mice received a palatal injection between the first and secondupper molar (3 ll/animal) of rSOFAT diluted from a stock solutionof 10 mg/ml (100 ng/ml; n = 5, Rheabiotech) or saline solution(n = 5) during three consecutive days. The animals were killed atday 13. The upper jaw was removed and fixed with 5% formalinin saline for 2 days. After decalcification with EDTA (pH 7.0) for2 weeks the specimens were briefly washed in running tap water,dehydrated and embedded in paraffin wax. Each sample was slicedinto 6 lm sections in sagittal directions and stained with hematox-ilin and eosin. The presence of osteoclast-like cells, i.e., TRAP-posi-tive multinucleated cells containing P3 nuclei, was observed andcounted at 100� magnification in the bone surrounding the toothroots by one trained and blinded examiner (MHN), using imageanalysis software (Image-Pro�, Media Cybernetics, Silver Spring,MD, USA).

2.4. Statistical analyses

The number of teeth selected for gingival biopsy sampling wasbased on previous studies that found differences in the levels of themRNA of various immune-inflammatory genes, when comparingdifferent clinical periodontal status [31]. The statistical analysiswas performed using a software program (GraphPad Prism 4.0,La Jolla, CA, USA). Data were first examined for normality by theKolmogorov–Smirnov test and then analyzed using parametricmethods. The mean percentage of sites with visible plaque accu-mulation, BoP, SUP, the means PD, CAL was computed for all teeth.Subsequently, the clinical parameters, the mRNA and protein levelswere averaged into the periodontitis and non-periodontitis groups.The differences between periodontitis and non-periodontitisgroups were compared using the Student t test. In addition, thenumber of TRAP-positive cells was computed for different SOFATdose groups and compared by One-way ANOVA. If significancewas detected by ANOVA, the pair-wise comparison Tukey testwas used to assess differences among SOFAT dose groups. The levelof significance was set at 5% for all analysis.

3. Results

3.1. Demographic parameters

Table 1 presents the demographic characteristics of the studypopulation and the clinical parameters at sampled teeth and full-mouth levels. There were no differences in the mean age and gen-der distribution between groups (p > 0.05). As expected, the levelsof all periodontal parameters (plaque index, BoP, SUP, PD and CAL)were statistically lower in the non-periodontitis group when com-pared to the periodontitis group, at full-mouth and sampled teethlevels (p < 0.05).

3.2. mRNA and protein levels of SOFAT in the gingival tissue

We performed qPCR analysis of SOFAT gene expression in gingi-val tissue of non-periodontitis and chronic periodontitis patients.The mRNA levels of SOFAT were significantly higher for 4.75-foldin the gingival tissue of the periodontitis group when compared tonon-periodontitis one (Fig. 1A; p < 0.05). In addition, the concentra-tion of SOFAT in the gingival tissue was analyzed using ELISA. Inaddition, the protein amounts of SOFAT were also greater (2.34-fold

Table 1Demographic characteristics of the study population and clinical parameters atfull-mouth and teeth levels of gingival biopsies (mean ± SD).

Non-periodontitis(n = 15)

Periodontitis(n = 15)

Age (years) 39.1 ± 5.2 48.1 ± 13.6M/F (n) 5/10 6/9

PI (%) Full-mouth 9.0 ± 7.2* 58.0 ± 18.2Sampledteeth

5.2 ± 4.0* 84.1 ± 23.1

BoP (%) Full-mouth 5.0 ± 1.5* 53.4 ± 20.1Sampledteeth

0* 73.3 ± 5.0

SUP (%) Full-mouth 0* 6.5 ± 7.2Sampledteeth

0* 13.1 ± 6.8

PD (mm) Full-mouth 1.9 ± 0.3* 3.6 ± 0.5Sampledteeth

2.9 ± 0.5* 7.3 ± 0.6

CAL(mm)

Full-mouth 2.0 ± 0.3* 3.9 ± 0.6Sampledteeth

2.0 ± 0.1* 8.7 ± 1.4

M: male; F: female; PI: plaque index; BoP: bleeding on probing; SUP: suppuration;PD: probing depth; CAL: clinical attachment level.* Differences between non-periodontitis and periodontitis groups (Student t-test;p < 0.05).

Fig. 2. Effects of SOFAT on in vitro osteoclastogenesis using mouse monocyte/macrophage cell line RAW264.7. SOFAT induced the formation of TRAP-positivemononucleated cells compared to culture medium or sRANKL (100 ng/ml) �significantly different when compared to RAW264.7 cells with medium alone and10 ng/ml of SOFAT; �� significantly different when compared to RAW264.7 cells inthe presence of medium alone, 10 and 50 ng/ml of SOFAT (p < 0.05; ANOVAfollowed by Tukey test). The dose of 100 ng/ml of SOFAT did not differ from sRANKLadministration regarding the number of TRAP-positive cells (p > 0.05).

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increase) in the gingival tissue from the periodontitis group whencompared to the non-periodontitis group (Fig. 1B; p < 0.05).

3.3. Induction of TRAP-positive cells in mouse RAW 264.7 cells bySOFAT

Since SOFAT induces osteoclastogenesis in a manner indepen-dent of exogenously added RANKL [20], we examined whether SO-FAT may induce osteoclastogenesis in RAW 264.7 cells. Treatmentwith SOFAT potently induced TRAP-positive multinucleated cellformation by RAW 264.7 cells in a dose-dependent manner(Fig. 2; p < 0.05). In addition, the dose of 100 ng/ml of SOFAT didnot significantly differ from sRANKL group regarding the numberof TRAP-positive cells (p > 0.05), although there was a trend towardhigher number of stained cell in 100 ng/ml of SOFAT group.

3.4. In vivo induction of osteoclast cells by SOFAT

In order to evaluate the effects of SOFAT on in vivo osteoclast-like cells differentiation, SOFAT or saline solution were injectedinto the maxilla of mice. SOFAT injection induced the formation

Fig. 1. (A) Relative expression of genes encoding SOFAT. qPCR analysis of SOFAT expperiodontitis patients (n = 15/group). (B) Concentration of SOFAT in the gingival tissue(n = 15/group). The symbol � indicates significant differences between groups (p < 0.05;

of osteoclast-like cells in the periodontal ligament (Fig. 3B),whereas saline solution did not stimulate the development ofosteoclast-like cells in the maxilla (Fig. 3A). There was a significantincrease in the number of osteoclast-like cells in the SOFAT-in-jected animals (p < 0.05). As demonstrated in Fig. 3C, a significant5.55-fold increase in the number of osteoclast-like cells wasobserved in the animals that received SOFAT injections.

4. Discussion

Alveolar bone loss is one of the most important clinical manifes-tations in periodontitis and one of the major causes of tooth loss.Therefore, this study evaluated, for the first time, the role of SOFAT,a novel activated T cell-secreted cytokine that induces osteoclasto-genesis independent of the RANKL/RANK system, on periodontaltissues and in chronic periodontitis. The present results demon-strated that mRNA and protein levels of SOFAT were up-regulatedin the gingival tissues of chronic periodontitis patients. In addition,SOFAT administration induced the formation of osteoclast-likecells in vitro in a dose dependent-manner and, the appearance ofosteoclast-like cells in the periodontal ligament of mice. Together,these findings suggest that alveolar bone destruction in periodon-

ression on mRNA extracted from gingival tissue of non-periodontitis and chronicwas analyzed using ELISA. Results are expressed as means (lg/mg of tissue) ± SDStudent t test).

Fig. 3. In vivo induction of osteoclast-like cells. Histological sections of the upper jaw between first and second molars sampled of mice (staining with HE). (a) Mice thatreceived saline solution and (b) mice that received SOFAT injection. The presence of osteoclast-like cells (asterisks) and erosion bone sites were observed in the group treatedwith SOFAT. T: tooth; B: bone; PL: periodontal ligament. Magnification of 40�. Scale bar represents 5 lm. (C) SOFAT induce increasing number of osteoclast-like cells. Thesymbol ⁄indicates significant differences between groups (p < 0.05; Student t test).

C.R. Jarry et al. / Human Immunology 74 (2013) 861–866 865

titis may be mediated by SOFAT. These data support the hypothesisthat SOFAT may be involved in bone loss associated withperiodontitis.

Several studies have tried to explain the pathogenesis of boneloss in inflammatory disease by invoking the RANK/RANKL/OPGsystem, since this pathway is generally considered to be a key mech-anism for the differentiation and activation of osteoclasts under ba-sal conditions [32,33]. In fact, it has also been demonstrated thatRANKL is up-regulated, whereas OPG is down-regulated, in peri-odontitis, resulting in an imbalance in the RANKL/OPG ratio [16].Despite the importance of the RANK/RANKL/OPG system, evidenceindicates that there are other RANKL-independent pathways thatlead to the differentiation and activation of osteoclasts, especiallyunder inflammatory conditions [20,34]. In fact, an activity has beenreported in the culture medium of activated T cells, which stronglyinduced the production of IL-6 by osteoblastic cells [35] and, stimu-late osteoclastogenesis by a RANKL-independent pathway [19]. Re-cently in 2009, the SOFAT, which may represent the first in apotential family of novel cytokines was identified [20]. The discov-ery of the SOFAT has increased the list of molecules linked with theregulation of bone metabolism and opened an avenue for possiblenovel therapies to ameliorate the bone destruction in diseases asso-ciated with inflammation, including rheumatoid arthritis, osteopo-rosis, and periodontitis.

It has been demonstrated that the exposure of osteoclast precur-sors (monocytes) to SOFAT induced osteoclastogenesis by a mecha-nism that was not mediated by cell-antonymous RANKL productionsince the addition of OPG did not prevent osteoclast formation. Itwas previously demonstrated that SOFAT signaling involves activa-tion of the nuclear factor of activated T cells (NFAT) signal transduc-tion pathway and up-regulates the expression of TRAP. In addition,like RANKL, SOFAT-induced active osteoclasts that were able to pro-duce significant pit formation on BioCoat, an artificial bone surro-gate, demonstrating that the osteoclasts induced by SOFAT arefunctional [20]. Similarly to the present investigation, the authorsassessed the effect of recombinant SOFAT on osteoclast formationby RAW 264.7 cells and demonstrate that treatment with recombi-

nant SOFAT (100 ng/ml) potently induced formation of TRAP posi-tive multinucleated cell formation. Our present study ratifiesthese data and further demonstrates that SOFAT stimulated the for-mation of TRAP-positive multinucleated cells in a dose-dependentmanner.

Interestingly, in the present study, the gene and protein expres-sion of SOFAT was significantly increased in the gingival tissues ofpatients with periodontitis, when compared to non-periodontitisindividuals. In addition, SOFAT administration in vivo was able tostimulate the appearance of osteoclast-like cells in the periodontalligament of mice. Therefore, based on the biological activities ofSOFAT, our preliminary findings suggest that this novel cytokinemay play an important role in periodontitis, in addition to theosteoclastogenic activity of RANKL, amplifying alveolar bone loss.However, further studies are necessary to accurately elucidatethe mechanisms of SOFAT and its importance in the bone loss ofperiodontitis.

In conclusion, the results of the present study demonstrateupregulated expression and production of SOFAT in human gingi-vae from patients with periodontal tissues and show for the firsttime osteoclastogenic activity of SOFAT in an animal model in vivo.Our data suggest that SOFAT may play an important role in peri-odontal bone loss.

Acknowledgments

This work was supported by grant from Conselho Nacional deDesenvolvimento Científico e Tecnológico, Brazil (CNPq # 471305/2009-0).

References

[1] Krauss JL, Potempa J, Lambris JD, Hajishengallis G. Complementary Tolls in theperiodontium: how periodontal bacteria modify complement and Toll-likereceptor responses to prevail in the host. Periodontol 2000 2010;52:141–62.

[2] Tonetti MS, Claffey N. European Workshop in Periodontology group C.Advances in the progression of periodontitis and proposal of definitions of aperiodontitis case and disease progression for use in risk factor research. Group

866 C.R. Jarry et al. / Human Immunology 74 (2013) 861–866

C consensus report of the 5th European Workshop in Periodontology. J ClinPeriodontol 2005;32:210–3.

[3] Graves DT, Fine D, Teng YT, Van Dyke TE, Hajishengallis G. The use of rodentmodels to investigate host-bacteria interactions related to periodontaldiseases. J Clin Periodontol 2008;35:89–105.

[4] Liu YC, Lerner UH, Teng YT. Cytokine responses against periodontal infection:protective and destructive roles. Periodontol 2000 2010;52:163–206.

[5] Garlet GP. Destructive and protective roles of cytokines in periodontitis: a re-appraisal from host defense and tissue destruction viewpoints. J Dent Res2010;89:1349–63.

[6] Graves DT. The potential role of chemokines and inflammatory cytokines inperiodontal disease progression. Clin Infect Dis 1999;28:482–90.

[7] Teng YT, Nguyen H, Gao X, Kong YY, Gorczynski RM, Singh B, et al. Functionalhuman T-cell immunity and osteoprotegerin ligand control alveolar bonedestruction in periodontal infection. J Clin Invest 2000;106:1259–67.

[8] Graves DT, Li J, Cochran DL. Inflammation and uncoupling as mechanisms ofperiodontal bone loss. J Dent Res 2011;90:143–53.

[9] Garlet GP, Cardoso CR, Campanelli AP, Ferreira BR, Avila-Campos MJ, Cunha FQ,et al. The dual role of p55 tumour necrosis factor-alpha receptor inActinobacillus actinomycetemcomitans-induced experimental periodontitis:host protection and tissue destruction. Clin Exp Immunol 2007;147:128–38.

[10] Garlet GP, Cardoso CR, Campanelli AP, Garlet TP, Avila-Campos MJ, Cunha FQ,et al. The essential role of IFN-gamma in the control of lethal Aggregatibacteractinomycetemcomitans infection in mice. Microbes Infect 2008;10:489–96.

[11] Kong YY, Feige U, Sarosi I, Bolon B, Tafuri A, Morony S, et al. Activated T cellsregulate bone loss and joint destruction in adjuvant arthritis throughosteoprotegerin ligand. Nature 1999;402:304–9.

[12] Lacey DL, Timms E, Tan HL, Kelley MJ, Dunstan CR, Burgess T, et al.Osteoprotegerin ligand is a cytokine that regulates osteoclast differentiationand activation. Cell 1998;93:165–76.

[13] Boyle WJ, Simonet WS, Lacey DL. Osteoclast differentiation and activation.Nature 2003;423:337–42.

[14] Hofbauer LC, Kühne CA, Viereck V. The OPG/RANKL/RANK system in metabolicbone diseases. J Musculoskelet Neuronal Interact 2004;4:268–75.

[15] Lewiecki EM. Treatment of osteoporosis with denosumab. Maturitas2010;66:182–6.

[16] Crotti T, Smith MD, Hirsch R, Soukoulis S, Weedon H, Capone M, et al. Receptoractivator NF kappaB ligand (RANKL) and osteoprotegerin (OPG) proteinexpression in periodontitis. J Periodontal Res 2003;38:380–7.

[17] Kawai T, Matsuyama T, Hosokawa Y, Makihira S, Seki M, Karimbux NY, et al. Band T lymphocytes are the primary sources of RANKL in the bone resorptivelesion of periodontal disease. Am J Pathol 2006;169:987–98.

[18] Teng YT. Protective and destructive immunity in the periodontium: Part 1–innate and humoral immunity and the periodontium. J Dent Res2006;85:198–208.

[19] Weitzmann MN, Cenci S, Rifas L, Haug J, Dipersio J, Pacifici R. T cell activationinduces human osteoclast formation via receptor activator of nuclear factor jBligand-dependent and -independent mechanisms. J Bone Miner Res2001;16:328–37.

[20] Rifas L, Weitzmann MN. A novel T cell cytokine, secreted osteoclastogenicfactor of activated T cells, induces osteoclast formation in a RANKL-independent manner. Arthritis Rheum 2009;60:3324–35.

[21] Hassumi MY, Silva-Filho VJ, Campos-Júnior JC, Vieira SM, Cunha FQ, Alves PM,et al. PPAR-gamma agonist rosiglitazone prevents inflammatory periodontalbone loss by inhibiting osteoclastogenesis. Int Immunopharmacol2009;9:1150–8.

[22] Kostenuik PJ, Nguyen HQ, McCabe J, Warmington KS, Kurahara C, Sun N, et al.Denosumab, a fully human monoclonal antibody to RANKL, inhibits boneresorption and increases BMD in knock-in mice that express chimeric (murine/human) RANKL. J Bone Miner Res 2009;24:182–95.

[23] Napimoga MH, Benatti BB, Lima FO, Alves PM, Campos AC, Pena-Dos-SantosDR, et al. Cannabidiol decreases bone resorption by inhibiting RANK/RANKLexpression and pro-inflammatory cytokines during experimental periodontitisin rats. Int Immunopharmacol 2009;9:216–22.

[24] Rodrigues WF, Madeira MF, da Silva TA, Clemente-Napimoga JT, Miguel CB,Dias-da-Silva VJ, et al. Low dose of propranolol down-modulates boneresorption by inhibiting inflammation and osteoclast differentiation. Br JPharmacol 2012;165:2140–51.

[25] Napimoga MH, da Silva CA, Carregaro V, Farnesi-de-Assunção TS, Duarte PM,de Melo NF, et al. Exogenous administration of 15d-PGJ2-loaded nanocapsulesinhibits bone resorption in a mouse periodontitis model. J Immunol2012;189:1043–52.

[26] Jin Q, Cirelli JA, Park CH, Sugai JV, Taba Jr M, Kostenuik PJ, et al. RANKLinhibition through osteoprotegerin blocks bone loss in experimentalperiodontitis. J Periodontol 2007;78:1300–8.

[27] Armitage GC. Development of a classification system for periodontal diseasesand conditions. Ann Periodontol 1999;4:1–6.

[28] Duarte PM, Napimoga MH, Fagnani EC, Santos VR, Bastos MF, Ribeiro FV, et al.The expression of antioxidant enzymes in the gingivae of type 2 diabetics withchronic periodontitis. Arch Oral Biol 2012;57:161–8.

[29] Araujo MW, Hovey KM, Benedek JR, Grossi SG, Dorn J, Wactawski-Wende J,et al. Reproducibility of probing depth measurement using a constant-forceelectronic probe: analysis of inter- and intra- examiner variability. JPeriodontol 2003;74:1736–40.

[30] Napimoga MH, Nunes LHAC, Maciel AAB, Demasi APD, Benatti BB, Santos VR,et al. Possible involvement of IL-21 and IL-10 on salivary IgA levels in chronicperiodontitis subjects. Scand J Immunol 2011;74:596–602.

[31] Duarte PM, Miranda TS, Lima JA, Dias-Gonçalves TE, Santos VR, Bastos MF,et al. Expression of immune-inflammatory markers in sites of chronicperiodontitis in patients with type 2 diabetes. J Periodontol 2012;83:426–34.

[32] Tanaka S, Nakamura K, Takahasi N, Suda T. Role of RANKL in physiological andpathological bone resorption and therapeutics targeting the RANKL-RANKsignaling system. Immunol Rev 2005;208:30–49.

[33] Koide M, Kinugawa S, Takahashi N, Udagawa N. Osteoclastic bone resorptioninduced by innate immune responses. Periodontol 2000 2010;54:235–46.

[34] Kobayashi K, Takahashi N, Jimi E, Udagawa N, Takami M, Kotake S, et al. Tumornecrosis factor alpha stimulates osteoclast differentiation by a mechanismindependent of the ODF/RANKL-RANK interaction. J Exp Med2000;191:275–86.

[35] Rifas L, Avioli LV. A novel T cell cytokine stimulates interleukin-6 in humanosteoblastic cells. J Bone Miner Res 1999;14:1096–103.