Interleukin-27 Inhibits Human Osteoclastogenesis byAbrogating RANKL-Mediated Induction ofNuclear Factor of Activated T Cells c1 and
Suppressing Proximal RANK Signaling
George D. Kalliolias,1 Baohong Zhao,1 Antigoni Triantafyllopoulou,1 Kyung-Hyun Park-Min,1
and Lionel B. Ivashkiv2
Objective. Interleukin-27 (IL-27) has stimulatoryand regulatory immune functions and is expressed inrheumatoid arthritis (RA) synovium. This study wasundertaken to investigate the effects of IL-27 on humanosteoclastogenesis, to determine whether IL-27 canstimulate or attenuate the osteoclast-mediated boneresorption that is a hallmark of RA.
Methods. Osteoclasts were generated from blood-derived human CD14� cells. The effects of IL-27 onosteoclast formation were evaluated by counting thenumber of tartrate-resistant acid phosphatase–positivemultinucleated cells and measuring the expression ofosteoclast-related genes. The induction of nuclear factorof activated T cells c1 (NFATc1) and the activation ofsignaling pathways downstream of RANK were mea-sured by immunoblotting. The expression of key mole-cules implicated in osteoclastogenesis (NFATc1, RANK,costimulatory receptors, and immunoreceptor tyrosine–based activation motif–harboring adaptor proteins) wasmeasured by real-time reverse transcription–polymerase chain reaction. Murine osteoclast precur-sors obtained from mouse bone marrow and synovial
fluid macrophages derived from RA patients were alsotested for their responsiveness to IL-27.
Results. IL-27 inhibited human osteoclasto-genesis, suppressed the induction of NFATc1, down-regulated the expression of RANK and triggering re-ceptor expressed on myeloid cells 2 (TREM-2), andinhibited RANKL-mediated activation of ERK, p38, andNF-�B in osteoclast precursors. Synovial fluid macro-phages from RA patients were refractory to the effects ofIL-27. In contrast to the findings in humans, IL-27 onlymoderately suppressed murine osteoclastogenesis, andthis was likely attributable to low expression of theIL-27 receptor subunit WSX-1 on murine osteoclastprecursors.
Conclusion. IL-27 inhibits human osteoclastogen-esis by a direct mechanism that suppresses the re-sponses of osteoclast precursors to RANKL. Thesefindings suggest that, in addition to its well-knownantiinflammatory effects, IL-27 plays a homeostatic rolein restraining bone erosion. This homeostatic functionis compromised under conditions of chronic inflamma-tion such as in RA synovitis.
Interleukin-27 (IL-27) is a member of the IL-12family of heterodimeric cytokines that also includesIL-12, IL-23, and IL-35 (1–3). IL-27 comprises theEpstein-Barr virus–induced protein 3 and p28 subunits,which share similarity with the p40 and p35 subunits ofIL-12, respectively (4). The IL-27 receptor is a het-erodimer composed of a WSX-1 subunit (also termed Tcell cytokine receptor), which confers ligand specificity,and the gp130 signaling subunit, which is also utilized bythe IL-6 family of cytokines (5). IL-27 activates theJAK/STAT signal transduction pathway in a context-dependent manner, since its effects vary depending on
Dr. Kalliolias’ work was supported by the Stavros NiarchosFoundation. Dr. Park-Min’s work was supported by the ArthritisFoundation. Dr. Ivashkiv’s work was supported by the NIH (grantR01-DE-19420-01).
1George D. Kalliolias, MD, Baohong Zhao, PhD, AntigoniTriantafyllopoulou, MD, Kyung-Hyun Park-Min, PhD: Hospital forSpecial Surgery, New York, New York; 2Lionel B. Ivashkiv, MD:Hospital for Special Surgery, and Cornell University, New York, NewYork.
Address correspondence and reprint requests to George D.Kalliolias, MD, Hospital for Special Surgery, 535 East 70th Street,Research Building 4th Floor, New York, NY 10021. E-mail:[email protected].
Submitted for publication March 3, 2009; accepted in revisedform October 9, 2009.
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the cell type and activation state. In resting lymphocytes,IL-27 activates STAT-1, STAT-3, STAT-5, and lowamounts of STAT-4 (6), whereas the extent of activationof STAT-1 is decreased in fully activated CD4� T cellsas compared with that in resting cells (7). In myeloidcells, IL-27 induces phosphorylation of STAT-1 andSTAT-3 (5,8,9), and we recently reported that, in humanmonocytes, IL-27 has a STAT-1–dominant effect (10).In contrast, we found that murine bone marrow–derivedmacrophages are minimally responsive to IL-27 (10).
IL-27 plays both activating and regulatory roles inimmune responses (2). IL-27 is produced early duringinnate responses and augments the induction of Th1responses (4,6). In contrast, later in the evolution of animmune response, IL-27 suppresses Th1 cell polariza-tion and inhibits Th17 and Th2 cell differentiation(6,11–14), in part by inducing IL-10 production (15–17).The role of IL-27 in arthritis and other inflammatory orinfectious diseases has been explained on the basis of itseffects on T cell differentiation (1–3). IL-27 can playeither a pathogenic or a protective role in murinemodels of inflammatory arthritis, depending on themodel and underlying pathogenic mechanisms. Inadjuvant-induced arthritis and proteoglycan-induced ar-thritis, both of which are considered Th1-mediateddiseases, IL-27 is pathogenic, consistent with its knownTh1-inducing effects (18,19). In contrast, in collagen-induced arthritis, which is Th17-mediated (and in whichTh1 responses can actually be protective), IL-27 plays aprotective role, and therefore has been proposed as apotential treatment for arthritis (20). IL-27 is expressedin human rheumatoid arthritis (RA) (20), which hasbeen considered a Th1-mediated disease, but recentevidence suggests a role for Th17 responses in diseasepathogenesis (21). Thus, it is important to determinewhether IL-27 plays a pathogenic or protective role inhuman RA.
RA is characterized by chronic synovial inflam-mation and bone destruction, and a hallmark of thedisease is bone erosions mediated by osteoclasts (22). Inthe last few years, significant breakthroughs have im-proved our understanding of the mechanisms that con-trol the activity of osteoclasts under physiologic andpathologic circumstances (23,24). Osteoclasts are large,multinucleated cells created by the differentiation andfusion of myeloid lineage precursor cells, which includecells in the blood monocyte pool (25). In the microen-vironment of the bone, myeloid-derived osteoclast pre-cursors are exposed to stimuli that commit them to theosteoclast lineage (26). The driving force of thiscommitment-differentiation process is the interaction of
RANKL (a cytokine member of the tumor necrosisfactor superfamily) with its receptor, RANK, which isexpressed on the surface of osteoclast precursors(27,28). Effective osteoclastogenesis requires costimula-tory calcium-mediated signals from immunoreceptortyrosine–based activation motif (ITAM)–coupled recep-tors that cooperate with RANK signaling to induceexpression of nuclear factor of activated T cells c1(NFATc1) and the downstream osteoclast differentia-tion program (29–32). In humans, triggering receptorexpressed on myeloid cells 2 (TREM-2) is a key costimu-latory receptor, since loss-of-function mutations inTREM-2 compromise bone remodeling and lead to thedevelopment of Nasu-Hakola disease (also called poly-cystic lipomembranous osteodysplasia with sclerosingleukoengephalopathy) (33,34).
The multistep osteoclast differentiation programis tightly controlled by hormones, cytokines, and otherregulatory factors. Cytokines that activate the JAK/STAT signal transduction pathway have been implicatedin the regulation of osteoclastogenesis, bridging immuneregulation with bone remodeling. Interferons (IFNs)(35,36), IL-4 (37), IL-6 (38), and IL-10 (39) inhibitosteoclast differentiation, while IL-23 promotes oste-oclast formation by up-regulating RANK expression inprecursor cells (40). IL-27, which belongs to the samecytokine family as IL-23, has been reported to modestlyand indirectly regulate osteoclast differentiation in mu-rine systems (40,41), but the mechanisms by which IL-27regulates osteoclastogenesis and the role of IL-27 inhuman osteoclastogenesis have not been addressed.
In this study, we wished to explore the role ofIL-27 in human osteoclastogenesis, since this could yieldinsight into one facet of the potential pathogenic role,versus protective role, of IL-27 in human RA. We foundthat IL-27 is a potent inhibitor of human osteoclasto-genesis, as indicated by its direct effect on osteoclastprecursor cells. One mechanism that mediates the anti-osteoclastogenic function of IL-27 is its ability to abro-gate RANKL-induced expression of c-Jun and NFATc1by down-regulating the expression of RANK and inhib-iting the MAPK and NF-�B signaling pathways down-stream of RANK. We also found that, in osteoclastprecursor cells, IL-27 down-regulates the expression ofthe TREM-2 costimulatory receptor. Surprisingly, wefound that synovial fluid–derived macrophages from thejoint effusions of patients with active RA were refractoryto the effects of IL-27. Our findings, combined with thereported inhibitory effects of IL-27 on Th17-mediatedinflammation, suggest that IL-27 has the capacity toregulate both chronic inflammation and associated bone
erosion, but this regulatory function is attenuated withinthe inflammatory microenvironment of the joint.
PATIENTS AND METHODS
Cell cultures. Samples of peripheral blood fromhealthy volunteers were purchased from the New York BloodCenter, and peripheral blood mononuclear cells (PBMCs)were isolated by density-gradient centrifugation. Mononuclearcells were isolated from the synovial fluid of 5 patients with RAwhose diagnosis was based on the American College of Rheu-matology (formerly, the American Rheumatism Association)criteria for RA (42). CD14� cells were purified from thePBMCs and synovial fluid–derived mononuclear cells usinganti-CD14 magnetic beads (Miltenyi Biotec, Sunnyvale, CA).Murine monocytes were obtained from C57BL/6J mice (TheJackson Laboratory, Bar Harbor, ME) and were isolated bydensity-gradient centrifugation, followed by positive selectionusing anti-CD11b magnetic beads (Miltenyi Biotec). Bonemarrow cells and splenocytes were obtained from C57BL/6Jmice, and bone marrow–derived osteoclast precursors weregenerated after 4 days of culture on petri dishes in Dulbecco’smodified Eagle’s medium supplemented with 20% fetal bovineserum (FBS) (Hyclone, Logan, UT) and recombinant murinemacrophage colony-stimulating factor (M-CSF) (20 ng/ml;PeproTech, Rocky Hill, NJ).
To measure cell viability, MTT assays were performedusing an MTT assay kit (Roche Diagnostics, Indianapolis, IN).Experiments with human cells were approved by the Hospitalfor Special Surgery Institutional Review Board, while experi-ments with mouse cells were approved by the InstitutionalAnimal Care and Use Committee.
Osteoclast differentiation. Human CD14� cells wereincubated in �-minimum essential medium (�-MEM; Invitro-gen, Carlsbad, CA) supplemented with 10% FBS (Defined;Hyclone) and 20 ng/ml of human M-CSF (PeproTech) for 2days, to generate osteoclast precursors. Osteoclast precursorswere incubated with 20 ng/ml of M-CSF and 40 ng/ml ofsoluble RANKL (PeproTech) for an additional 5 days. Cyto-kines were replenished every 3 days. On culture day 7, cellswere fixed and stained for tartrate-resistant acid phosphatase(TRAP) using the Acid Phosphatase Leukocyte diagnostic kit(Sigma, San Diego, CA). Multinucleated (�3 nuclei), TRAP-positive osteoclasts were counted in triplicate wells of 96-wellplates. Recombinant human IL-27 (R&D Systems, Minneap-olis, MN) was added either from the beginning of the cultureand before RANKL (IL-27 pretreatment), simultaneously withRANKL on culture day 3, or after RANKL (on culture day 5).Murine bone marrow–derived osteoclast precursors were in-cubated with murine M-CSF (20 ng/ml) and soluble RANKL(80 ng/ml) for 5 days in �-MEM in the same manner asdescribed above.
Pit formation assay. Human preosteoclasts were re-plated on dentin slices (5 � 104 cells/slice) in 96-well cultureplates and stimulated as described above. Cells on dentin wereremoved, and the dentin slices were immersed in 1% toluidineblue O (Sigma-Aldrich, St. Louis, MO) to stain resorption pitsformed by the mature osteoclasts.
Immunoblotting. Whole cell extracts were prepared bylysis of cells in buffer containing 20 mM HEPES (pH 7.0), 300
mM NaCl, 10 mM KCl, 1 mM MgCl2, 0.1% Triton X-100, 0.5mM dithiothreitol (DTT), 20% glycerol, and 1� proteinaseinhibitor cocktail (Roche, Basel, Switzerland). Nuclear extractswere prepared by incubating cells for 15 minutes (at 4°C) inbuffer containing 10 mM HEPES (pH 7.9), 1.5 mM MgCl2, 10mM KCl, 1� proteinase inhibitor cocktail, and 1 mM DTT.Nonidet P-40 was added to a final concentration of 0.2%, andthe lysate was centrifuged at 10,000g for 30 seconds. Finally,the nuclear pellet was lysed in sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) loadingbuffer.
For immunoblotting, 5 or 10 �g of whole cell lysateswere fractioned on polyacrylamide gels using SDS-PAGE,transferred to polyvinylidene difluoride membranes (Milli-pore, Bedford, MA), and incubated with specific antibodies.Enhanced chemiluminescence was used for detection. ERK,p-ERK, p-p38, p-I�B�, total I�B�, c-Jun, pY–STAT-1, andpY–STAT-3 antibodies were from Cell Signaling Technology(Beverly, MA). Tumor necrosis factor receptor–associatedfactor 6 (TRAF6), TATA binding protein, and p38 antibodieswere from Santa Cruz Biotechnology (Santa Cruz, CA), whileNFATc1, STAT-1, and STAT-3 antibodies were purchasedfrom BD Transduction Laboratories (Lexington, KY).
Real-time reverse transcription–polymerase chain re-action (RT-PCR) and quantitative RT-PCR. For real-timeRT-PCR and quantitative PCR, total RNA was extracted usingan RNeasy Mini kit (Qiagen, Chatsworth, CA) and 1 �g oftotal RNA was reverse transcribed using a First Strand com-plementary DNA synthesis kit (Fermentas, Hanover, MD).Quantitative PCR was performed using an iQ SYBR GreenSupermix and iCycler iQ thermal cycler (Bio-Rad, Hercules,CA). PCR (34 cycles at 95°C for 30 seconds, 60°C for 30seconds, and 72°C for 1 minute) was utilized to quantify themessenger RNA (mRNA) levels of WSX-1 in human CD14�or CD14� cells and murine osteoclast precursors or spleno-cytes.
RESULTS
Inhibition of human osteoclastogenesis by IL-27in a dose- and time-dependent manner. In the presenceof M-CSF and RANKL, myeloid lineage precursorsdifferentiate into osteoclasts (25), but activation ofSTAT-1, which occurs downstream of IFNs, has beenshown to inhibit osteoclastogenesis (43). Recently, wereported that IL-27 strongly activates STAT-1 in humanmonocytes and macrophages (10). Therefore, in thepresent study, we sought to investigate whether IL-27has any regulatory function in human osteoclastogen-esis. As expected from previous observations (44), cul-ture of human CD14� cells with M-CSF plus RANKLinduced differentiation of large, multinucleated TRAP�cells, which became readily apparent within 5 days afterthe addition of RANKL (Figures 1A and B). WhenIL-27 was added at the beginning of culture (day 1), adose-dependent inhibition of osteoclastogenesis was ob-served (Figures 1A and B), with nearly complete inhibi-
404 KALLIOLIAS ET AL
tion of the generation of multinucleated TRAP� cellsobserved in cultures with IL-27 doses of �30 ng/ml (P �0.01, by Student’s t-test). At later time points during thedifferentiation process, a substantial inhibition of oste-oclastogenesis was observed when IL-27 (100 ng/ml) wasadded (simultaneously with the addition of RANKL) onday 3, while there was only slight inhibition when IL-27(100 ng/ml) was added (along with RANKL) on day 5(Figures 1A and B).
A similar pattern of dose- and time-dependentinhibition by IL-27 was observed with regard toRANKL-induced expression of the osteoclast-relatedgenes cathepsin K (Figure 1C) and integrin �3 (resultsnot shown). Cathepsin K and integrin �3 were stillexpressed in the cells (mostly mononuclear cells) when30 ng/ml of IL-27 was used, but gene expression wasstrongly down-regulated by saturating concentrations ofIL-27, which nearly completely suppressed osteoclasto-genesis.
The functional consequence of the observed IL-27–mediated inhibition of osteoclastogenesis was as-
sessed by resorption pit formation assay using dentinslices. As expected, the observed inhibition of osteoclas-togenesis by IL-27 was also reflected by the absence ofresorption pits (Figure 1D, left). Interestingly, whenIL-27 was added later, on day 5, the formed osteoclastswere capable of creating resorption pits (Figure 1D,left). This finding implies that IL-27 is unable to inhibitthe late stages of osteoclast differentiation and function.In addition, with the use of the MTT assay, we foundthat IL-27 mediated antiosteoclastogenic effects withoutaffecting the viability of the osteoclast precursors (Fig-ure 1D, right). Taken together, these observations sug-gest that IL-27 is a potent inhibitor of the early stages ofhuman osteoclastogenesis, by exerting a direct effect onosteoclast precursors.
Abrogation of RANKL-mediated induction ofNFATc1 by IL-27. We next wished to investigate themechanisms by which IL-27 inhibits osteoclast differen-tiation. It is well established that NFATc1 is a masterregulator of osteoclastogenesis, driving the expression ofgenes that are crucial for the commitment and differen-
Figure 1. Interleukin-27 (IL-27) inhibits human osteoclastogenesis in a dose- and time-dependent manner. Freshly isolated human CD14�cells were cultured with macrophage colony-stimulating factor (M-CSF) (20 ng/ml) for 48 hours, and on culture day 3, RANKL (40 ng/ml) wasadded. IL-27 (3, 10, 30, or 100 ng/ml) was added at the initiation of cultures (day 1) or later (days 3 or 5). A and B, Tartrate-resistant acidphosphatase–positive (TRAP�) multinucleated (�3 nuclei) cells (MNCs) (A) were counted 5 days after the addition of RANKL (B). Barsshow the mean and SD results from triplicate wells of 96-well plates. � � P � 0.05; �� � P � 0.05; ��� � P � 0.01, versus control, by Student’st-test. C, Cathepsin K mRNA was measured using real-time polymerase chain reaction, with results normalized to the values for GAPDH. Barsshow the mean and SD results of triplicate experiments; small standard deviations are not readily apparent because of large inductions. D (left),Freshly isolated human monocytes were cultured with RANKL (40 ng/ml) in the presence or absence of IL-27 (100 ng/ml) up to 5 days, andosteoclast function was measured by a resorption pit formation assay. D (right), Human CD14� monocytes were cultured with M-CSF (20ng/ml) in the presence or absence of IL-27 (100 ng/ml) for 2 days, and then cell viability was measured by MTT assay. In A and D,representative results from 1 of at least 3 independent experiments are shown.
IL-27 INHIBITION OF HUMAN OSTEOCLASTOGENESIS 405
tiation of precursor cells to osteoclasts (45,46). Wetherefore tested the effects of IL-27 on NFATc1 expres-sion. As expected, within 24 hours of culture, RANKL(40 ng/ml) induced NFATc1 protein expression (Figure2A, lane 4), and the levels of NFATc1 protein remainedelevated for at least 72 hours following RANKL stimu-lation (Figure 2A, lanes 4–6). Addition of IL-27 resultedin the abrogation of RANKL-induced NFATc1 proteinexpression for the entire 72-hour period of RANKLstimulation (Figure 2A, lanes 7–9).
We also investigated whether IL-27 inhibits NF-ATc1 gene expression. The levels of NFATc1 mRNAincreased following 24 hours and 48 hours of stimulationwith RANKL (Figure 2B). Pretreatment with IL-27resulted in substantial suppression of the NFATc1mRNA levels (Figure 2B).
A similar striking abrogation of RANKL-inducedNFATc1 protein expression was observed when IL-27was added together with RANKL (on culture day 3)(Figure 2C, lanes 4–9). Moreover, the simultaneousaddition of IL-27 had a similar suppressive effect onNFATc1 mRNA levels (Figure 2D). These results indi-
cate that the abrogation of NFATc1 induction is amechanism that mediates the inhibitory effects of IL-27on human osteoclastogenesis.
Inhibition of RANKL-induced activation of theMAPK and NF-�B pathways and suppression of RANKexpression by IL-27. The induction of NFATc1 expres-sion by RANK is dependent on RANKL-induced acti-vation of the MAPK and NF-�B pathways (23,24), andwe therefore investigated whether IL-27 inhibits RANKsignaling. Consistent with the findings reported in theliterature, we found that stimulation of osteoclast pre-cursors with RANKL (40 ng/ml) induced rapid phos-phorylation of the ERK and p38 MAPKs (Figure 3A,lanes 2–4). With the addition of IL-27 to the cultures, weobserved a strong inhibition of the RANKL-inducedphosphorylation of ERK and p38 (Figure 3A, lanes6–8). Furthermore, as expected, RANKL induced thephosphorylation and rapid degradation of I�B� (Figure3B, left, lanes 2–4), indicating activation of the classicNF-�B pathway, while IL-27 prevented RANKL-induced I�B� phosphorylation and degradation (Figure3B, left, lanes 6–8).
Figure 2. IL-27 abrogates RANKL-mediated induction of nuclear factor of activated T cells c1 (NFATc1). A and B, Freshly isolated humanCD14� cells were cultured with M-CSF (20 ng/ml) in the presence or absence of IL-27 (100 ng/ml) for 48 hours, and then were stimulatedwith RANKL. C and D, Freshly isolated human CD14� cells were cultured with M-CSF (20 ng/ml) for 48 hours, and then were stimulatedwith RANKL (40 ng/ml) in the presence or absence of IL-27 (100 ng/ml). NFATc1 protein expression 24, 48, and 72 hours following RANKLstimulation was measured by immunoblotting (A and C); STAT-3 expression was used as a loading control. NFATc1 mRNA was measuredusing real-time polymerase chain reaction, with results normalized to the values for GAPDH (B and D). Bars show the mean and SD resultsof triplicate measurements from 1 of at least 3 independent experiments. See Figure 1 for other definitions.
IFN�, the prototypic STAT-1–activating cyto-kine, inhibits RANKL signaling in murine osteoclastprecursors by inducing rapid degradation of the adaptorprotein TRAF6, which lies upstream of NF-�B andMAPK activation (35). In our system, IL-27 had noeffect on the expression of TRAF6 protein (Figure 3B,right).
RANKL-mediated induction of activator protein1 (AP-1) proteins, including Fos and Jun, is importantfor osteoclastogenesis. Induction of Fos by RANKL wasnot consistently observed in our human osteoclastogen-esis system (results not shown). However, RANKLconsistently induced c-Jun protein expression, and thisinduction was detectable in nuclear extracts for at least
Figure 3. IL-27 inhibits RANKL-mediated activation of the MAPK and NF-�B pathways and induction of c-Jun. Freshly isolatedhuman CD14� cells were cultured with M-CSF (20 ng/ml) in the presence or absence of IL-27 (100 ng/ml) for 48 hours. Control andIL-27–treated cells were stimulated with RANKL (40 ng/ml) for 5, 10, and 15 minutes (A and B) or for 0.5, 1, and 3 hours (C).Immunoblotting was used to measure threonine-202/tyrosine-204 phosphorylation of ERK-1/ERK-2 and threonine-180/tyrosine-182phosphorylation of p38 (A), serine-32 phosphorylation of I�B� and total I�B� (left) and expression of total tumor necrosis factorreceptor–associated factor 6 (TRAF6) (right) (B), and expression of nuclear c-Jun protein (C). In A and B, p38 was used as the loadingcontrol, while in C, TATA binding protein (TBP) was the loading control. Representative results from 1 of at least 5 independentexperiments are shown. D, RANK mRNA was measured at 24 and 48 hours in control and IL-27–treated cells using real-timepolymerase chain reaction, with results normalized to the values for GAPDH. Bars show the mean and SD results of triplicateexperiments; small standard deviations are not readily apparent because of large inductions. See Figure 1 for other definitions.
IL-27 INHIBITION OF HUMAN OSTEOCLASTOGENESIS 407
3 hours following RANKL stimulation (Figure 3C, lanes2–4). In the presence of IL-27, the RANKL-mediatedinduction of c-Jun was abrogated (Figure 3C, lanes 6–8).
We then investigated whether IL-27 inhibits ex-pression of RANK, which could explain the IL-27–mediated inhibition of several RANK signaling path-ways. Culture with M-CSF induced the expectedincrease in levels of RANK mRNA, which peaked at 24hours and remained elevated at 48 hours of culture(Figure 3D). Addition of IL-27 partially suppressed theM-CSF–mediated induction of RANK mRNA expres-sion in all donors tested (n � �10) (Figure 3D). Whenwe tested the effects of IL-27 on the expression of theM-CSF receptor c-Fms, there was variability amongdonors in the effects of IL-27 on c-Fms mRNA levelsand cell surface expression (results not shown), whereasthere was strong inhibition of osteoclastogenesis byIL-27 in all donors tested (n � �10). This observationthat IL-27 inhibited osteoclastogenesis effectively evenin donors in whom c-Fms expression was not affectedsuggests that an effect of IL-27 on c-Fms expression isnot a major mechanism to explain the observed inhibi-tion of osteoclastogenesis. In summary, the results dem-onstrate that IL-27 inhibits the RANK-induced signaling
that is required for NFATc1 expression, and suggest thatthis inhibition may be mediated, in part, by down-regulation of RANK expression.
Suppression of TREM-2 mRNA expression byIL-27. Because the strong inhibition of osteoclastogen-esis and NFATc1 expression by IL-27 could not beexplained solely by its inhibition of RANK expression,we investigated the effects of IL-27 on other pathwaysimplicated in the induction of NFATc1 expression. Theimmunoreceptors TREM-2, osteoclast-associated recep-tor (OSCAR), signal-regulatory protein �1 (SIRP�1),and immunoglobulin-like transcript 7 (ILT-7; homologof murine paired immunoglobulin-like receptor A) andthe integrin �v�3 provide signaling via the ITAM-containing adaptor proteins DNAX-activating protein of12 kd (DAP-12) and Fc receptor � (FcR�) and cooper-ate with RANK signaling pathways to induce NFATc1expression and osteoclastogenesis (29–32). We there-fore investigated the effects of IL-27 on the expressionof these costimulatory receptors and DAP-12/FcR�.
The expression of TREM-2 increased duringthe early differentiation of osteoclast precursors, andthis increase in TREM-2 expression was strongly sup-pressed by IL-27 (Figure 4A). In contrast, expression of
Figure 4. IL-27 inhibits the expression of triggering receptor expressed on myeloid cells 2 (TREM-2). Freshly isolated humanCD14� cells were cultured with M-CSF (20 ng/ml) in the presence or absence of IL-27 (100 ng/ml) for 24 and 48 hours. Expressionof TREM-2 (A), osteoclast-associated receptor (OSCAR), signal-regulatory protein �1 (SIRP�1), and immunoglobulin-liketranscript 7 (ILT-7) (B), and DNAX-activating protein of 12 kd (DAP-12) and Fc receptor � (FcR�) (C) mRNA was measuredusing real-time polymerase chain reaction, with results normalized to the values for GAPDH. Bars show the mean and SDresults of triplicate measurements from 1 of at least 4 independent experiments. See Figure 1 for other definitions.
408 KALLIOLIAS ET AL
OSCAR, SIRP�1, DAP-12, and FcR� did not substan-tially change during differentiation of osteoclast precur-sors, and was not suppressed by IL-27 (Figures 4B andC). The levels of ILT-7 mRNA were moderately in-creased by IL-27 (Figure 4B). Since costimulatory sig-naling is important for NFATc1 expression, andTREM-2 plays a key role in costimulation of humanosteoclastogenesis (33,34), these observations suggestdown-regulation of TREM-2 as a mechanism by whichIL-27 suppresses NFATc1 induction and inhibits humanosteoclastogenesis.
Nonresponsiveness of RA synovial fluid macro-phages to IL-27. Given the above-described inhibitoryeffects of IL-27 in human osteoclastogenesis, we won-dered whether IL-27 exerts these regulatory functions inhuman CD14� cells derived from the synovial fluid ofpatients with active RA. Synovial fluid–derived CD14�cells from RA patients responded to IFN� stimulation,as shown by the activation of STAT-1 (Figure 5A, lane2) and a robust induction of CXCL10 gene expression(Figure 5B). Surprisingly, following stimulation withIL-27, we observed a very faint activation of STAT-1(Figure 5A, lane 3), which was inadequate to induceSTAT-1 target gene expression, including the expressionof CXCL10 (P � 0.001, by Student’s t-test) (Figure 5B),CXCL9, IRF1, and STAT1 (results not shown). Theseresults were consistently observed in synovial macro-
phages from all 5 patients tested, and suggest that cellsderived from the joints of many patients with active RAare only minimally responsive to IL-27. The latter im-plies that, within the microenvironment of an inflamedjoint, IL-27 may not exert its regulatory functions.
Association of moderate inhibition of murineosteoclastogenesis by IL-27 with low expression ofWSX-1. We recently reported that murine macrophagesare minimally responsive to IL-27 (10). Therefore, inthis study, we sought to determine whether IL-27 has aneffect on the differentiation of murine osteoclasts. Be-cause we wished to use murine monocytes as osteoclastprecursors (to directly compare with human monocytes),we first tested the responsiveness of murine monocytesto IL-27. Following stimulation of murine monocyteswith IFN� (used as a positive control), we observed theexpected activation of STAT-1 and STAT-3 (Figure 6A,lanes 2–4) and the induction of classic STAT-1 targetgenes, including IP10, Mig, and IRF1 (results notshown). Similar to our previous findings in murinemacrophages (10), following IL-27 stimulation of mu-rine monocytes, there was no detectable activation ofSTAT-1 and STAT-3 (Figure 6A, lanes 5–7) and mini-mal induction of STAT-1 target genes (results notshown).
Because murine monocytes did not differentiateefficiently into osteoclasts in our system, we used bone
Figure 5. Synovial fluid macrophages derived from patients with rheumatoid arthritis (RA) are refractory to the regulatoryeffects of interleukin-27 (IL-27). Freshly isolated CD14� cells derived from the synovial fluid of 5 RA patients were stimulatedfor 15 minutes (A) or 3 hours (B) with human interferon-� (IFN�) (100 units/ml) or human IL-27 (100 ng/ml). A, Tyrosine-701phosphorylation of STAT-1 was measured by immunoblotting. Representative results from 1 of 5 independent experiments areshown. B, CXCL10 mRNA expression (n � 5 patients) was measured using real-time polymerase chain reaction, with resultsnormalized to the values for GAPDH. P values were calculated using Student’s paired t-test.
IL-27 INHIBITION OF HUMAN OSTEOCLASTOGENESIS 409
marrow–derived osteoclast precursors, a standard ap-proach to study murine osteoclastogenesis, to test theeffects of IL-27. As expected, addition of RANKL tomurine bone marrow–derived osteoclast precursors re-sulted in the formation of large, multinucleated TRAP�cells within 5 days (Figure 6B). When IL-27 was added(prior to or simultaneously with RANKL), in dosesranging from 1 ng/ml to 100 ng/ml, there was a minimal-to-moderate inhibition of osteoclast formation (Figure6B). Similarly, the number of TRAP� multinucleatedcells (Figure 6C) and the expression of osteoclastmarker genes (results not shown) were only moderatelylower in the presence of IL-27. In stark contrast to itseffects in the human system, IL-27 had a minimal effecton RANKL-mediated induction of NFATc1 expressionin murine osteoclast precursors (results not shown),further supporting the notion that murine osteoclastprecursors are moderately responsive to IL-27.
In our recent study (10), we demonstrated thatIL-6 induces a substantial activation of STAT-3 inmurine myeloid cells, indicating that gp130, the sharedreceptor subunit for IL-6 and IL-27 receptors, is ex-
pressed on these cells. We next investigated the expres-sion levels of the WSX-1 subunit of the IL-27 receptor inhuman and murine cells. We found that human lympho-cytes and human osteoclast precursors expressed WSX-1mRNA at comparable levels (Figure 6D, lanes 1 and 2),whereas WSX-1 expression was low in murine osteoclastprecursors compared with that in murine splenocytes(predominantly, lymphocytes) (Figure 6D, lanes 3 and4). The latter suggests that low expression of WSX-1 inmurine osteoclast precursors is a potential explanationfor the modest responsiveness of these cells to theeffects of IL-27.
DISCUSSION
In this study, we have shown that IL-27 is a potentinhibitor of human osteoclastogenesis. This effect ismediated by IL-27 via its direct targeting of the earlystages of differentiation of precursor cells into oste-oclasts. In the presence of IL-27, we observed down-regulation of the expression of RANK and TREM-2,inhibition of the MAPK and NF-�B pathways down-
Figure 6. IL-27 is a moderate inhibitor of in vitro osteoclastogenesis in murine systems. A, Murine monocytes were stimulated for 5, 10, and 20minutes with murine IFN� (100 units/ml) or murine IL-27 (100 ng/ml). Immunoblotting was used to measure tyrosine-701 phosphorylation ofSTAT-1 and tyrosine-705 phosphorylation of STAT-3. B and C, Bone marrow–derived osteoclast precursors obtained from C57BL/6J mice werecultured in the presence of macrophage colony-stimulating factor (20 ng/ml), followed by the addition of RANKL (80 ng/ml). IL-27 (1, 10, or 100ng/ml) was added 1 day before the addition of RANKL (pretreatment) or simultaneously with RANKL. Tartrate-resistant acid phosphatase–positive(TRAP�) multinucleated (�3 nuclei) cells (MNCs) (B) were counted 5 days after the addition of RANKL (C). Bars show the mean and SD intriplicate wells of 96-well plates. Representative results from 1 of 3 independent experiments are shown. � � P � 0.05; �� � P � 0.05, versus control,by Student’s t-test. D, The expression of WSX-1 mRNA was measured by polymerase chain reaction in freshly isolated human (h) CD14� andCD14� cells and in murine (m) bone marrow–derived macrophages (BMDMs) and splenocytes. See Figure 5 for other definitions.
410 KALLIOLIAS ET AL
stream of RANK, and, most importantly, abrogation ofRANKL-induced expression of c-Jun and NFATc1. Instark contrast to the responsiveness of blood-derivedCD14� cells from healthy donors, synovial fluid–derived CD14� cells from RA patients were refractoryto the regulatory effects of IL-27. Our observations,combined with the well-characterized antiinflammatoryfunctions of IL-27 (3), suggest that IL-27 has the poten-tial to limit the extent of bone destruction in the settingof infection or inflammation. However, this homeostaticfunction of IL-27 can be compromised during chronicinflammation, such as occurs in RA.
NFATc1 is essential for osteoclast differentiationin vitro and in vivo (45,46). We observed a remarkablesuppression of the levels of NFATc1 mRNA and proteinby IL-27 in our system. Signaling pathways downstreamof RANK and immunoreceptors (including TREM-2)act in a cooperative manner, leading to the activation ofcalcium signaling, the activation of NFATc1, and, finally,strong induction of NFATc1 itself through an autoam-plification loop (24). In our study, the attenuation of theMAPK and NF-�B signaling pathways downstream ofRANK appeared to represent a mechanism that contrib-utes to the IL-27–mediated suppression of NFATc1expression. IL-27 also appeared to regulate the levels ofc-Jun protein, which is induced downstream of theMAPK pathways. The c-Jun protein is the partner ofc-Fos in the formation of AP-1, a transcription factoressential for the induction of NFATc1 (47). A potentialexplanation for the inhibition of the MAPK and NF-�Bsignaling pathways observed in our system is the down-regulation of RANK expression by IL-27 in humanosteoclast progenitors. This down-regulation of RANKwas partial, suggesting that it contributes to diminishedresponses to RANKL only under conditions in whichosteoclast precursors are exposed to low concentrationsof RANKL (as in the case of the inflamed synovium inRA). In contrast, TREM-2 expression was effectivelyinhibited by IL-27, indicating that IL-27 suppressescostimulation of RANK signaling.
We have previously reported that IL-27 has aSTAT-1–dominant, IFN�-like effect on human mono-cytes (10). IFN� is also a powerful suppressor of oste-oclast differentiation by a STAT-1–mediated mechanism(43). Similar to our observations on the effects of IL-27,IFN� is also known to inhibit the activation of theMAPK and NF-�B pathways downstream of RANK(35). IFN� inhibits RANK signaling in murine osteoclastprecursors by inducing rapid degradation of a signalingadaptor, TRAF6, that functions downstream of RANK(35). In our system, the protein levels of TRAF6 were
not reduced in IL-27–treated cells prior to or afterRANKL stimulation, indicating that there was no overtdegradation of TRAF6. This observation suggests thatIL-27 and IFN� inhibit osteoclastogenesis by differentmechanisms, although it is possible that differences inTRAF6 protein regulation and stability between murineand human osteoclast precursors contributes to theobserved differences.
In addition, in the present study, we found thatsynovial fluid macrophages derived from the joint effu-sions of patients with active RA retained their respon-siveness to IFN� but were refractory to regulation byIL-27. We have recently reported that resistance toIL-27 can be induced with lipopolysaccharide by ap38-dependent mechanism (10). These findings suggestthat inflammatory factors that activate p38 can induce astate of refractoriness to IL-27, and in this context, wehypothesize that, within the inflammatory microenviron-ment of the joint, in which p38 is known to be activated,synovial fluid macrophages are exposed to stimuli thatrender them refractory to the regulatory effects of IL-27.The level of refractoriness is likely to wax and wane withdisease activity, but the data suggest that patients withactive disease would be refractory to IL-27 therapy.However, greater understanding of the mechanisms thatrender cells refractory to IL-27 could potentially lead totherapeutic manipulations to restore cell responsivenessto the regulatory functions of IL-27. Under such condi-tions, endogenous IL-27 would have a beneficial effectin suppressing bone resorption, and exogenous IL-27might represent an effective therapy.
Recently, several differences in the regulation ofosteoclastogenesis and bone remodeling between hu-mans and mice have become apparent (48). For exam-ple, TREM-2 deficiency in humans is associated withimpaired osteoclastogenesis, aberrant bone remodeling,and Nasu-Hakola disease (33,34). In contrast, micedeficient in TREM-2 do not have a clear bone pheno-type in vivo and have increased osteoclastogenesis invitro (49). Our observations add to the understanding ofthese species differences by showing substantially lesseffective inhibition of osteoclast differentiation by IL-27in mouse osteoclast precursors as compared with humanosteoclast precursors. One potential explanation of thisdifference is that murine osteoclast precursors are mod-erately responsive to IL-27 because of the low expressionof WSX-1. An additional explanation is likely related todifferences in TREM-2 regulation and function in hu-mans and mice, and the contribution of TREM-2 down-regulation to the suppressive effects of IL-27 in humancells. In addition, we have shown that, in human cells,
IL-27 INHIBITION OF HUMAN OSTEOCLASTOGENESIS 411
IL-27 is a powerful inhibitor of osteoclastogenesis thatdirectly targets osteoclast progenitors, whereas our find-ings with murine bone marrow–derived osteoclast pro-genitors, as well as evidence from other groups, suggestthat, in murine systems, IL-27 regulates osteoclast dif-ferentiation mainly in an indirect manner, by affectingcell populations other than the osteoclast precursors.
It has been reported that IL-27 induces activationof STAT-1 and STAT-3 in murine osteoblasts, but noprofound functional consequences were observed (41).In that same study, IL-27 inhibited osteoclastogenesisindirectly, possibly by regulating the functions of acti-vated CD4� T cells. Along the same lines, another studyidentified murine Th17 cells as the exclusive osteoclas-togenic subset among the known CD4� T cell lineages(50), and results from several studies suggest that IL-27suppresses the polarization of Th17 cells (12,13,51).
The findings of the present study provide evi-dence that IL-27 is a powerful inhibitor of humanosteoclastogenesis. Nevertheless, the prospect of usingIL-27 as a potential treatment in human diseases, includ-ing RA, needs further investigation and careful evalua-tion.
AUTHOR CONTRIBUTIONS
All authors were involved in drafting the article or revising itcritically for important intellectual content, and all authors approvedthe final version to be published. Dr. Kalliolias had full access to all ofthe data in the study and takes responsibility for the integrity of thedata and the accuracy of the data analysis.Study conception and design. Kalliolias, Zhao, Triantafyllopoulou,Park-Min, Ivashkiv.Acquisition of data. Kalliolias, Zhao, Triantafyllopoulou, Park-Min,Ivashkiv.Analysis and interpretation of data. Kalliolias, Zhao, Triantafyllopou-lou, Park-Min, Ivashkiv.
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