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Keywords:

  • Mesenchymal stem cells;
  • Monocyte;
  • Oncostatin M;
  • Bone

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Bone resorption by osteoclasts and bone formation by osteoblasts are tightly coupled processes implicating factors in TNF, bone morphogenetic protein, and Wnt families. In osteoimmunology, macrophages were described as another critical cell population regulating bone formation by osteoblasts but the coupling factors were not identified. Using a high-throughput approach, we identified here Oncostatin M (OSM), a cytokine of the IL-6 family, as a major coupling factor produced by activated circulating CD14+ or bone marrow CD11b+ monocytes/macrophages that induce osteoblast differentiation and matrix mineralization from human mesenchymal stem cells while inhibiting adipogenesis. Upon activation of toll-like receptors (TLRs) by lipopolysaccharide or endogenous ligands, OSM was produced in classically activated inflammatory M1 and not M2 macrophages, through a cyclooxygenase-2 and prostaglandin-E2 regulatory loop. Stimulation of osteogenesis by activated monocytes/macrophages was prevented using neutralizing antibodies or siRNA to OSM, OSM receptor subunits gp130 and OSMR, or to the downstream transcription factor STAT3. The induced osteoblast differentiation program culminated with enhanced expression of CCAAT-enhancer-binding protein δ, Cbfa1, and alkaline phosphatase. Overexpression of OSM in the tibia of mice has led to new bone apposition with no sign of bone resorption. Two other cytokines have also a potent role in bone formation induced by monocytes/macrophages and activation of TLRs: IL-6 and leukemia inhibitory factor. We propose that during bone inflammation, infection, or injury, the IL-6 family signaling network activated by macrophages and TLR ligands stimulates bone formation that is largely uncoupled from bone resorption and is thus an important target for anabolic bone therapies. STEM CELLS2012; 30:762–772


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Bone is a dynamic mineralized tissue that is continuously resorbed by osteoclasts and rebuilt by osteoblasts. Osteoclast precursors are hematopoietic cells of the monocyte/macrophage lineage whereas osteoblast precursors are multipotent mesenchymal stem cells (MSCs) that can also give rise to adipocytes and chondrocytes. Under physiological conditions, bone resorption is precisely replaced by new bone formation, and therefore osteoclast and osteoblast activities are tightly coupled. Indeed, osteoblasts control osteoclast differentiation mainly through receptor activator of nuclear factor κ-B ligand (RANKL), and inversely osteoclast control osteoblast differentiation through Wnt10b and bone morphogenetic protein 6 (BMP6) [1, 2]. Immune cells also control bone homeostasis or pathology in a field of research called osteoimmunology. For example, T lymphocytes produce the inflammatory cytokines tumor necrosis factor α (TNFα), transforming growth factor β (TGFβ), interferon γ (IFNγ), and interleukin (IL)-17 to induce osteogenesis on MSC [3]. More recently, bone resident macrophages, termed osteomacs, have been described as another critical immune cell population regulating bone formation by osteoblasts [4]. However, the coupling factors produced by osteomacs were not identified.

Despite their role as osteoclast precursors, monocytes/macrophages play major roles in innate immunity and in the regulation of the adaptive immune response. Once activated by microbial products such as the prototypical toll-like receptor (TLR) agonist lipopolysaccharide (LPS), they acquire microbicidal competence and can produce huge amount of proinflammatory mediators such as IL-1β, TNFα, IL-6, or prostaglandin E2 (PGE2) [5]. In culture, classically activated macrophages termed M1 macrophages can be obtained from peripheral monocytes when stimulated with IFNγ whereas alternative activation of macrophages (M2) induced by IL-4 or IL-10 leads to tissue repair and suppression of inflammation [5]. Increased number of activated macrophages is a prominent feature of inflammatory lesions, and in the case of rheumatoid arthritis (RA), the presence of macrophages in the synovial tissue correlates with joint erosions [6]. The key role of macrophages in RA is also supported by the successful treatment with TNFα blockers, considering that TNFα is mainly produced by activated macrophages [6]. Implication of proinflammatory mediators in periarticular bone erosions and systemic osteoporosis can be direct through induction of osteoclast differentiation and/or indirect through enhanced RANKL production, the key osteoclastogenic cytokine [1, 7].

Although inflammation is invariably related to increased bone resorption, new bone formation can be also observed. In RA, penetration of cortical bone and bone marrow by inflammatory cells is associated with increased bone or osteoid formation, suggesting an attempt to repair bone from the medullar cavity [8]. In spondyloarthropathies, inflammation induces excess periosteal bone formation independently of osteoclastic resorption [9]. During fracture repair, inflammatory cytokines such as TNFα and IL-6 induce recruitment and osteogenic differentiation of muscle-derived MSC [10]. Interestingly, bone macrophages are required for fracture repair and bone healing [11]. In atherosclerosis, activated macrophages produce TNFα and Oncostatin M (OSM) to promote vascular calcification from vascular smooth muscle cells [12]. Macrophages thus appear to have an essential role in the regulation of bone formation during inflammation and bone injury, and identification of the factors controlling osteogenesis represent an important step to develop new bone anabolic strategies.

Cytokines of the IL-6 family, such as OSM or leukemia inhibitory factor (LIF) share the gp130 subunit [13–15]. IL-6 first binds to a specific receptor subunit (IL-6R, either membranous or soluble) that lacks intrinsic signaling properties and then interacts with gp130 to transduce a signal. Mouse OSM first binds to gp130 and then recruits the OSM receptor subunit (OSMR). In humans, OSM is exceptional because it interacts with gp130 and with either LIF receptor subunit (LIFR) or OSMR to form a signaling-competent receptor complex. IL-6-type cytokines are overexpressed systemically and/or locally in several inflammatory diseases associated with bone loss, such as RA, inflammatory bowel disease, or psoriasis [13, 14, 16–19]. They can be produced by activated T lymphocytes, monocytes, macrophages, and neutrophils. In cocultures containing both osteoblasts/stromal cells and osteoclasts precursors, they stimulate osteoclast differentiation and activity [20]. In fact, they induce osteoblastic production of various downstream effectors that activate osteoclast differentiation or activity, such as RANKL, IL-1, parathyroid hormone-related protein, and PGE2 [7, 13]. In vivo, IL-6−/− mice are significantly protected from joint inflammation and destruction in mouse models of arthritis, leading to the tocilizumab IL-6R antibody actually used in chronic inflammatory diseases [13, 14, 21]. However, numerous reports also indicated that these cytokines, especially IL-6+sIL-6R and OSM enhance differentiation of osteoblasts through the transcription factor signal transducer and activator of transcription 3 (STAT3), leading to increased bone formation [13, 22–24]. Therefore, these cytokines control both bone resorption and formation.

Using a high-throughput approach, here we identified OSM as a major cytokine produced by classically activated monocytes/macrophages that induce osteoblast differentiation and matrix mineralization from MSC. Together with IL-6 and LIF, these three cytokines appeared to form a regulatory network controlling bone apposition.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Cell Isolation

Ethical approval for the use of bone marrow and peripheral blood from healthy donors was obtained from the Nantes University Hospital Ethics Committee. Samples were obtained from the orthopedic department and the “Etablissement Français du Sang” with informed consent.

For MSC, 10–20 ml of bone marrow was harvested by iliac crest aspiration from six donors (age = 46 ± 12; range = 36–67). MSCs were obtained as previously described [25] and cultured in proliferation medium composed of Dulbecco's modified Eagle's medium (Lonza, Basel, Switzerland, http://www. lonzabio.com), 10% fetal bovine serum (FBS; Hyclone Perbio, Bezons, France, http://www.perbio.com), 1 ng/ml basic fibroblast growth factor (bFGF; R&D systems, Minneapolis, MN, http://www.rndsystems.com), 100 U/ml penicillin, 100 U/ml streptomycin, and 2 mM L-glutamine. Adherent cells were frozen at passage 2 after characterization by flow cytometry (CD45, CD34, CD105+, CD73+, and CD90+, purity ≥99%) prior to further experiments. In addition, these cells were able to differentiate into osteoblast, adipocyte, or chondrocyte (see below).

For CD14+ monocytes, peripheral blood mononuclear cells from eight different donors (age = 58 ± 8; range = 45–67) were isolated by centrifugation over Ficoll gradient (Sigma Chemicals Co., St. Louis, MO, http://www.sigmaaldrich.com). CD14+ cells were magnetically labeled with CD14 microbeads and positively selected by MACS technology (Miltenyi Biotec, Bergisch Gladbach, Germany, http://www.miltenyibiotec.com). For CD11b+ monocytes, the bone marrow of one donor (age = 55) was harvested, and mononuclear cells were isolated and sorted using CD11b microbeads. CD14+ and CD11b+ cells were CD3 by flow cytometry (purity ≥95%; 5%–10% of the CD14+ cells were CD16+ nonclassic monocytes) and were frozen prior to further experiments.

Osteoblast Differentiation

Before passage 5, MSCs were seeded at 104 cells per centimeter square in 96-well plates in proliferation medium and allowed to attach and reach confluence. After 3 days, the medium was changed without bFGF but supplemented with vitamin D3 (10−8 M; Hoffmann-La Roche, Basel, Switzerland, http://www.roche-applied-science.com) and dexamethasone (10−7 M; Sigma), with or without treatments as indicated (this represented day 0 for MSC osteogenic differentiation). Three days later, freshly prepared ascorbic acid (50 μg/ml; Sigma) and β-glycerophosphate (10 mM; Sigma) was added to allow mineralization, this medium being changed every 2–3 days. Alizarin red-S staining was used to detect the mineralized nodules formed in vitro as described previously [26]. Briefly, between day 12 and 15, cells were fixed in ice-cold 70% ethanol for 1 hour and incubated with Alizarin red-S (40 mM, pH 7.4; Sigma) for 10 minutes at room temperature. After extensive washing, images were captured using a stereo microscope (Stemi 2000-C; Zeiss, Oberkochen, Germany, http://www.zeiss.com), and mineralized surfaces were quantified using the Qwin software (Leica, Nussloch, Germany, http://www.leica-microsystems.com).

Adipocyte Differentiation

Approximately 20,000 MSC were seeded in Lab-Tek Chamber Slides (Nunc, Rochester, NY, http://www.nuncbrand.com) in proliferation medium. After 3 days, when the confluence was reached, cells were cultured in adipogenic medium without bFGF but containing 3-isobutyl-1-methylxanthine (0.5 mM; Sigma), indomethacin (60 μM; Sigma), and dexamethasone (1 μM; Sigma) with medium replenishment every 2–3 days. After 19 days, cells were fixed with paraformaldehyde 4% for 10 minutes and intracellular lipid droplets and nuclei were stained using, respectively, Nile red (Sigma) and dapi (Invitrogen, Cergy Pontoise, France, http://www.invitrogen.com). Cells with lipid droplets were counted manually using a fluorescent DMRXA microscope (Leica).

Monocyte/Macrophage Conditioned Media

Activated CD14+ and CD11b+ monocyte/macrophage conditioned media (CM) were generated from 2.5 × 106 freshly resuscitated cells cultured for 3 days in 2 ml of (minimum essential medium α; Lonza) supplemented with 10% FBS and antibiotics and treated with LPS (100 ng/ml), poly I:C (25 μg/ml), Zymosan A (5 μg/ml), hyaluronic acid from human umbilical cord (10 μg/ml), heparan sulfate from bovine kidney (100 μg/ml), and/or meloxicam (all from Sigma). For M1 and M2 macrophages, CD14+ monocytes were cultured with granulocyte-macrophage colony-stimulating factor (GM-CSF) (20 ng/ml) together with IFNγ (50 ng/ml; M1), or IL-4 (50 ng/ml; M2a), or IL-10 (50 ng/ml; M2c) for 2 days and then treated with LPS (100 ng/ml) for 3 additional days. Cells and debris were removed by centrifugation (5 minutes, 1,600 rpm), and supernatants were aliquoted and stored at −20°C.

Real-Time Polymerase Chain Reaction

Total RNA was extracted from MSC cultures using NucleoSpin RNA II (Macherey-Nagel, Hoerd, France, http://www.mn-net. com). First-strand cDNA was synthesized from 1 μg total RNA using ThermoScript RT-PCR System (Invitrogen). The real-time polymerase chain reaction (PCR) contained 10 ng reverse-transcribed total RNA, 300 nM primers, and SYBR green buffer (Bio-Rad, Marnes-la-Coquette, France, http://www.bio-rad.com) [27]. Quantitative PCRs (qPCRs) were carried out on a CFX96 Real-Time PCR Detection System (Bio-Rad). Analysis was performed using human β2 microglobuline (β2m), cytochrome c-1 (cyc1), and hypoxanthine guanine phosphoribosyl transferase as invariant controls. For the high-throughput quantitative RT-PCR on monocytes/macrophages, see the supporting information Table S1 legend.

RNA Interference

MSCs were transfected with Interferin (PolyPlus-transfection, Illkirch, France, http://www.polyplus-transfection.com) and annealed small interfering RNA (siRNA) (10 nM; Ambion, Applied Biosystems, Courtabouef, France, http://www.appliedbiosystems.com) according to the manufacturer's recommendations. The siRNA references were LIFR (s8170), OSMR (s17542), and STAT3 (s743).

Cytokines, Neutralizing Antibodies, ELISA, and Multiplex Assays

We measured IL-1α, IL-1β, IL-1RA, IL-7, IL-15, IL-6, LIF, IL-12p70, IL-23, IL-10, chemokine (C-C motif) ligand 2 (CCL2), CCL3, CCL4, CCL5, CCL7, CCL24, chemokine (C-X-C motif) ligand 8 (CXCL8), CXCL9, CXCL10, CXCL11, IFNβ, IFNγ, Lta, vascular endothelial growth factor A (VEGFA), granulocyte colony-stimulating factor (G-CSF), GM-CSF, and FGF2 levels in CM using the Luminex technology (Bio-Plex Pro Assays from Bio-Rad and Milliplex MAP kits from Millipore, Molsheim, France, http://www.millipore.com). Additionally, PGE2, OSM, IL6, sIL6R, TNF-related apoptosis-inducing ligand (TRAIL), and macrophage colony-stimulating factor (M-SCF) release in CM were quantified using ELISA assays (R&D Systems) according to the manufacturers' instructions. Unless otherwise stated, all cytokines and neutralizing antibodies were from R&D Systems, except IL-6R (clone BR6) and gp130 (clone BK5) antibodies from Diaclone (Besançon, France, http://www.gen-probe.com) (supporting information Table S1 legend).

Western Blot

Cells were lysed in RIPA buffer and analyzed as described [27]. The membranes were blotted with antibodies to P-STAT3 (Tyr705), STAT3 (Cell Signaling Technologies, Beverly, MA, http://www.cellsignal.com), or actin (Sigma).

In Vivo Experiments

All researches involving animals were conducted following the guidelines of and have been approved by the French ethical committee CEEA.PdL.06 and by local veterinary services (license n°C44015). Researches involving viruses have been approved by the French Ministry of Research “commission de Génie Génétique” (license n°5698). Replication-deficient adenovirus encoding mouse OSM (AdOSM) has been described previously [27, 28] and was produced, together with adenovirus encoding green fluorescent protein (AdGFP), in the vector facility of the INSERM U649 Laboratory (Nantes, France). Eight-week-old male C57BL/6 mice (Janvier, Le Genest-Saint-Isle, France, http://www.janvier-europe.com) were treated with intratibial injection with either AdOSM or AdGFP at a dose of 5 × 107 pfu. After sacrifice by CO2 inhalation, tibias were fixed in 10% buffered formaldehyde for 1 week. Bone architecture was analyzed using the high-resolution SkyScan-1076 X-ray microcomputed tomography system for small animal imaging (SkyScan, Kartuizersweg, Belgium, http://www.skyscan.be). The relative trabecular bone volume (BV/TV) and other trabecular variables (number and thickness) were quantified in the metaphyseal spongiosa using the SkyScan CtAn software.

Histology

Injected tibias were decalcified with 4.13% EDTA and 0.2% paraformaldehyde in phosphate buffered saline (PBS) for 96 hours using the KOS microwave histostation (Milestone, Kalamazoo, MI, http://www.milestonemed.com) before embedding in paraffin. Sections (5 μm thick) were analyzed by Masson trichrome and tartrate-resistant acid phosphatase (TRAP) staining as described previously [27, 28]. Immunostaining for osterix was performed as described [27] with a rabbit anti-osterix antibody (1/25; Abcam, Cambridge, MA, http://www.abcam.com). Quantification of relative osteoclast surface (TRAP+ cells) and osteoblast number (osterix+ cells) in the metaphyseal spongiosa was evaluated by Qwin (Leica) and ImageJ (NIH, Bethesda, MD) softwares. All analyses were assessed by light microscopy using a DMRXA microscope (Leica).

Statistical Analysis

Results were analyzed with unpaired t test or one-way analysis of variance followed by Fisher's post hoc test using GraphPad InStat v3.02 software. Results are given as mean ± SD for in vitro experimentations) or mean ± SEM (for in vivo experimentations) and results with p < .05 were considered significant.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Activated Monocytes/Macrophages Secrete OSM to Induce Matrix Mineralization by MSC

We first compared, using cocultures of human circulating CD14+ monocytes and human bone marrow MSC, the effect of RANKL (to obtain osteclast) and LPS (to obtain activated monocytes/macrophages) on the mineralization by MSC. Both treatments similarly induce matrix mineralization (Fig. 1A). To discriminate between soluble and membranous mediators, MSCs were treated with CM from LPS-activated CD14+ monocytes (LPS CM). We observed significant induction of mineralization using the CM from eight different CD14+ donors whereas the control CM obtained without LPS treatment (CT) reduced mineralization (four donors are presented in Fig. 1B). These results indicated that control CM contained inhibitory soluble mediators whereas LPS CM contained stimulatory ones. Identical results were obtained with human bone marrow CD11b+ monocytes treated with LPS (supporting information Fig. S2A). Similarly, MSC from six different donors responded to a CD14+ CM (four donors are presented in supporting information Fig. S1A). To be active, the CD14+ CM must be added during almost all the culture time, with a minimum of nine on 12 days (supporting information Fig. S1B). Interestingly, the LPS-activated CD14+ CM inhibited differentiation of MSC into adipocytes as revealed by the reduced number of cells with lipid droplets (Figs. 1C and 2G), suggesting an inverse effect of activated monocytes/macrophages on osteogenesis and adipogenesis.

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Figure 1. Activated monocytes/macrophages induced mineralization by mesenchymal stem cell (MSC). (A): Human bone marrow MSC (20,000 cells) were cocultured with human circulating CD14+ monocytes (200,000 cells) in the osteoblast differentiation medium (Diff) and treated with LPS (100 ng/ml) or RANKL (100 ng/ml) for 15 days as indicated. (B): MSCs were treated with the CM (diluted 1/10) of LPS-activated CD14+ monocytes from four different donors. All cultures were fixed after 15 days, stained with Alizarin red-S, quantified and expressed relative to the control culture with differentiation medium only. (C): MSCs were cultured in the adipocyte differentiation medium (Diff) and treated with the CM (1/10) of LPS-activated CD14+ monocytes as indicated. Cultures were fixed after 15 days, stained with Nile red (lipid droplets) and dapi (nuclei), and photographed (original magnification, ×100). All assays were performed with n = 3–5 for each condition and are representative of at least two independent experiments. Results are expressed as the mean ± SD. *, p < .05 compared to the indicated culture or to the control with differentiation medium only if not indicated. Abbreviations: CM, conditioned media; CT, control CM obtained without LPS treatment; CT CM, control CM from CD14+ monocytes without LPS; dapi, 4′,6-diamidino-2-phenylindole; LPS, CM obtained with LPS treatment; LPS CM, CM obtained with LPS treatment; MSC, mesenchymal stem cell; RANKL, receptor activator of nuclear factor κ-B ligand.

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To identify the soluble mediators produced by LPS-activated monocytes and implicated in osteoblast maturation, we first used a high-throughput quantitative RT-PCR approach that allows the analysis of the genes encoding all known cytokines, chemokines, and growth factors [29]. On the 161 tested genes, 55 were upregulated by LPS and 1 (CCL24/eotaxin-2) was downregulated (Fig. 2A and supporting information Table S1). Mediators with known activities on osteoblasts differentiation were IL-1β, IL-6, IL-10, TNFα, VEGFA, GCSF, and FGF2. LPS also enhanced mRNA level of Wnt and BMP family members (Wnt5a and BMP6) as observed previously in monocytes treated with RANKL [2]. Using ELISA and high-throughput multiplex immunoassays (Luminex technology), we could confirm a significant induction of 22 soluble mediators by LPS and reduction of 2 other secreted proteins (eotaxin-2 and sIL-6R) (supporting information Table S1). We then used a panel of neutralizing antibodies or other inhibitors to prevent matrix mineralization induced by the CM of LPS-activated circulating CD14+ or bone marrow CD11b+ monocytes and identified the IL-6 family, sharing the gp130 receptor subunit (Fig. 2B, supporting information Fig. S1C; Table S1).

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Figure 2. OSM is a major mediator secreted by LPS-activated monocytes and implicated in the mineralization by mesenchymal stem cell (MSC). (A): CD14+ monocytes obtained from three different donors were treated or not with LPS (100 ng/ml) for 8 hours and mRNA expression was assessed by real-time polymerase chain reaction. On the 161 tested genes, the expression of 56 genes was significantly modulated by LPS above control (CT) in the three CD14+ cultures. Results are presented as normalized expression (×10−3) using the indicated color code. A list of 15 candidate coupling factors with known activities on osteoblast differentiation was then established from the literature (in red). See supporting information Table S1 legend for more details. (B–F): MSCs were treated in the osteoblast differentiation medium. (B, E): MSCs were treated with LPS-activated CD14+ monocytes CM (1/10, LPS) and neutralizing antibodies (10 μg/ml) as indicated. *, p < .05 compared to the culture treated with the control IgG. (C): MSCs were treated with indicated cytokines (100 ng/ml). *, p < .05 compared to the culture without cytokine. (D): MSCs were treated with increasing concentrations of OSM, LIF, sIL-6R in the presence of 100 ng/ml of IL-6 or IL-6 in presence of 100 ng/ml of sIL-6R. (F): MSCs were treated with three different LPS-activated CD14+ CM (LPS, 1/10) and neutralizing antibodies (10 μg/ml) as indicated. *, p < .05 compared to the control antibody (IgG). Mineralization was quantified and expressed as in Figure 1 except in (F) where it is expressed relative to the control antibody. (G): MSCs were cultured in the adipocyte differentiation medium (Diff) and treated with OSM (10 ng/ml) or the CM (1/10) of LPS-activated CD14+ monocytes as indicated for 15 days. Cultures were stained as in Figure 1(C) and adipocytes containing lipid droplets were counted in three different fields (> 300 total cells). *, p < .05 compared to the control with differentiation medium only. All assays were performed with n = 3–5 for each condition and are representative of at least two independent experiments. Results are expressed as the mean ± SD. Abbreviations: BMP, bone morphogenetic protein; CC, chemokine (C-C motif) ligand; CT, control CM from CD14+ monocytes without LPS; CM, conditioned media; CSF, colony stimulating factor; CXCL, chemokine (C-X-C motif) ligand; CXCR, C-X-C chemokine receptor; CLC, cardiotrophin-like cytokine; CNTF, ciliary neurotrophic factor; IL, interleukin; IFN, interferon; LIF, leukemia inhibitory factor; LPS, CM from CD14 monocytes with LPS; OSM, Oncostatin M; sIL-6R, soluble IL-6 receptor subunit; TGF, transforming growth factor; TNF, tumor necrosis factor; VEGF, vascular endothelial growth factor.

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Among the nine cytokines in the IL-6 family, only OSM, LIF, and IL-6 when associated with its soluble receptor (sIL-6R) induced mineralization by MSC (Fig. 2C). However, dose-response experiments indicated that OSM and LIF were active at lower concentrations, with a significant increased mineralization observed with 0.1–1 ng/ml (Fig. 2D). In comparison, LPS-activated CD14+ CM contained sufficient amount of OSM or IL-6 to induce mineralization, but the level of LIF or sIL-6R was too low (supporting information Table S1). When using neutralizing antibodies to IL-6, IL-6R, OSM, or LIF, we identified OSM as a major cytokine implicated in mineralization induced by all LPS-activated CD14+ donor CM, although prevention of mineralization was only partial between 30% and 75% depending on the donor (Fig. 2E, 2F). The anti-OSM antibody was validated on TF-1 cells to totally inhibit OSM activity (ND50 of 0.02–0.04 μg/ml, ref≠AF-295-NA from R&D systems) and was used here at a 5,000-fold molar excess over OSM. Moreover, the BK5 anti-gp130 antibody that preferentially inhibits OSM signaling [30] also prevented the induced mineralization by only 30% (Fig. 2E). In fact, only the combined neutralization of OSM and LIF totally prevented mineralization to the level obtained with the pan anti-gp130 antibody, indicating that both cytokines were implicated (Fig. 2F, see below for further description of the role of LIF). Inhibition of adipogenesis by LPS-activated CD14+ CM was also prevented using anti-OSM antibodies (Fig. 2G).

The COX2-PGE2 Pathway Is Implicated in OSM Production in M1 Macrophages

One of the most induced genes in CD14+ monocytes treated with LPS was cyclooxygenase 2 (COX2), and consequently PGE2 was detected in large amount in the LPS-activated CD14+ CM (supporting information Table S1). Because OSM production can be induced by PGE2 [31], we next collected the CM of LPS-activated CD14+ monocytes treated with the COX inhibitor Meloxicam, which is 300-fold more active on COX2 than on COX1 (ref≠M3935 from Sigma). Inhibition of COX2 lead to reduced production of PGE2 and OSM whereas IL-6 secretion was reduced only by 20% and sIL-6R production was not significantly affected (Fig. 3B). COX2 inhibition in monocytes also significantly prevented the mineralization induced by the CD14+ CM on MSC (Fig. 3A). In control experiments, we confirmed that (a) PGE2 does not directly induce mineralization on MSC, (b) COX2 or prostaglandin PGE2 receptor (EP receptor) inhibition in MSC does not prevent mineralization induced by the CD14+ CM (supporting information Fig. S2A), and (c) concentrations of Meloxicam as low as 5 μM were active to reduce OSM secretion by CD14+ monocytes (−54%) and the induced mineralization by MSC (−82%). Together these results indicated that LPS induced COX2 and PGE2 production in CD14+ monocytes, leading to enhanced OSM secretion and thus to enhanced OSM-driven mineralization by MSC.

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Figure 3. OSM is produced in M1 macrophages upon various toll-like receptors' activations through a cyclooxygenase 2/PGE2 regulatory loop. (A): CD14+ monocytes were treated with LPS (100 ng/ml) and Melo (100 μM, a COX2 inhibitor) as indicated. Their CM (1/10) was then used to treat mesenchymal stem cell (MSC). Mineralization was quantified and expressed as in Figure 1. (B): Levels of PGE2, OSM, IL-6, and sIL-6R in indicated CD14+ CM were determined by ELISA. (C): CD14+ monocytes were cultured with granulocyte-macrophage colony stimulating factor (GM-CSF) together with interferon γ (IFNγ) (M1), or IL-4 (M2a), or IL-10 (M2c) for 2 days and then treated with LPS (100 ng/ml) for 3 additional days. (D): CD14+ monocytes were treated with LPS (100 ng/ml), poly I:C (25 μg/ml), Zymo (5 μg/ml), HA (10 μg/ml), or HS (100 μg/ml) for 3 days. OSM secretion was determined by ELISA. (E–H) Indicated monocytes/macrophages CM (1/10) were used to treat MSC in presence or absence of OSM or gp130 neutralizing antibodies (10 μg/ml). Original images are shown in (E) and (G). Mineralization was quantified and expressed as in Figure 1 in (F) and (H). All assays were performed with n = 3–5 for each condition and are representative of at least two independent experiments. Results are expressed as the mean ± SD. *, p < .05. Abbreviations: CM, conditioned media; CT, control CM obtained without LPS treatment; HA, hyaluronic acid; HS, heparan sulfate; IL, interleukin; LPS, CM obtained with LPS treatment; Melo, Meloxicam; OSM, Oncostatin M; PGE2, prostaglandin E2; sIL-6R, soluble IL-6 receptor subunit; Zymo, zymosan.

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The cytokine/chemokine profile of LPS-activated CD14+ monocytes is typical of classically activated M1 macrophages, with high expression of TNFα, IL-1β, IL-6, IL-8, CCL2, CCL3, and CXCL10 (supporting information Table S1). To analyze the expression of OSM in other macrophage lineages, M2 macrophages were generated with IL-4 (M2a) or IL-10 (M2c) [5]. In comparison to prototypical M1 macrophages generated with IFNγ, M2 macrophages produced 5- to 10-fold less OSM at basal level or after LPS stimulation (Fig. 3C). Similarly, induction of mineralization by M1 macrophages CM was prevented by OSM or gp130 neutralizing antibodies, whereas M2a or M2c macrophages CM did not induce mineralization by MSC (Fig. 3E, 3F).

We next analyzed whether OSM was induced by other TLR ligands. Activation of TLR3 using Poly I:C (a synthetic analog of viral double-stranded RNA) or TLR2 and 6 using Zymosan from yeast cell wall also augmented OSM secretion by CD14+ cells. Similarly, endogenous TLR4 ligands released from the breakdown of extracellular matrices such as hyaluronic acid and heparan sulfate were potent inducers of OSM production (Fig. 3D). Induction of mineralization by these monocytes/macrophages CM correlated with OSM production and was reduced by OSM or gp130 neutralizing antibodies (Fig. 3G, 3H).

Role of LIF and IL-6

In contrast to OSM, activated monocytes expressed only low amount of LIF mRNA and the secreted LIF protein could never be detected (supporting information Table S1). However, LIF and OSM neutralizing antibodies synergized to prevent mineralization induced by LPS-activated CD14+ CM and the CM from several donors (two on eight tested) was sensitive to LIF neutralization alone (donor ≠2 in Fig. 2F). Interestingly, these donors CM appeared to contain lower amount of OSM (419 pg/ml for donor ≠2 vs. 858 and 1,184 pg/ml for donors ≠3 and 4, respectively). These results raised the possibility of a synergistic, compensatory effect between OSM produced by monocytes and LIF produced by MSC. To study the production of LIF directly from MSC, we treated these cells with LPS but without monocytes/macrophages. LPS directly induced LIF mRNA and protein secretion in MSC (supporting information Fig. S3A, S3B) and the LIF neutralizing antibody prevented the two- to fourfold increased mineralization observed with LPS in the absence of monocytes (supporting information Figs. S2A, S2B, and S3C).

IL-6 mRNA and protein expression was also directly induced by LPS in MSC (supporting information Fig. S3A, S3B) whereas OSM was not produced (below the detection limits in both RT-qPCR and ELISA). Thus OSM was exclusively produced by monocytes/macrophages, LIF only by MSC, and IL-6 by both cell types. However, the neutralizing IL-6 antibody, validated in a plasmacytoma IL-6-dependent bioassay with a ND50 of 0.05–0.15 μg/ml (ref≠AB-206-NA from R&D systems) and used here at a 100-fold molar excess over IL-6, never prevented the mineralization induced by activated monocytes (Fig. 2E), presumably because expression of its soluble receptor sIL-6R is too low (supporting information Table S1; Fig. 3C). To confirm this possibility, we added the sIL-6R to MSC cultures treated with LPS-activated CD14+ CM and neutralizing antibodies to OSM and LIF (supporting information Fig. S4). In these conditions, the sIL-6R induced mineralization by MSC.

Activation of the OSMR-STAT3-C/EBPδ-Cbfa1 Differentiation Pathway by OSM in MSC

We next analyzed the osteoblast differentiation program initiated by activated monocytes in MSC. The mRNA levels of CCAAT-enhancer-binding protein δ (C/EBPδ) and core-binding factor subunit alpha-1 (Cbfa1, or Runt-related transcription factor 2), two key transcription factors implicated in osteoblast differentiation, were first induced by the LPS-activated CD14+ CM within 1–3 days of culture. Thereafter, the two osteoblast markers alkaline phosphatase (ALP) and bone sialoprotein (BSP) were induced, just before mineralization can be detected (Fig. 4A, 4B). Interestingly, mRNA levels of gp130 and OSMR were also induced, suggesting again an activation of this receptor complex. Using the gp130 neutralizing antibody, we confirmed that all these osteoblastic genes were effectively induced in a gp130-dependent manner (Fig. 4A). OSM also induced the mRNA level of C/EBPδ, Cbfa1, ALP, BSP, and OSMR in MSC with very similar kinetics (supporting information Fig. S5A). RANKL, the key osteoclastogenic cytokine, was induced only twofold and only transiently by the LPS-activated CD14+ CM at day 1 of treatment, whereas its decoy receptor Osteoprotegerin (OPG) was reduced at the same time in all the cultures containing the osteoblast differentiation medium (Fig. 4A, arrows). Longer culture times indicated a reduced RANKL/OPG ratio with the LPS-activated CD14+ CM, independently of gp130.

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Figure 4. Regulated genes in MSC. MSCs were treated as indicated in the differentiation medium (refer to legends of Figs. 1 and 2). (A): mRNA expression of indicated genes was assessed by real-time polymerase chain reaction. Assays were performed with n = 2 for each condition and are representative of two independent experiments. Arrows indicate the only time point where the RANKL/OPG ratio is induced by the LPS-activated CD14+ CM. (B): Cultures were analyzed for mineralization after 8, 12 and 15 days as in Figure 1. Results are expressed relative to the control culture with differentiation medium only at day 15. LPS, LPS-activated CD14+ CM (1/10); gp130, neutralizing anti-gp130 antibody (10 μg/ml). Abbreviations: ALP, alkaline phosphatase; asc. ac, ascorbic acid; BSP, bone sialoprotein; C/EBPδ, CCAAT-enhancer-binding protein δ; cbfa1, core-binding factor subunit alpha-1; CM, conditioned media; CT, control CM obtained without LPS treatment; dex, dexamethasone; OPG, osteoprotegerin; OSMR, OSM receptor subunit; RANKL, receptor activator of nuclear factor κ-B ligand; β-gly, β-glycerophosphate.

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As shown in Figure 5C, LPS-activated CD14+ CM or OSM induced the activation of STAT3 in MSC. Knockdown of OSMR or STAT3 using siRNA (Fig. 5A, 5B) prevented induction of ALP mRNA by LPS-activated CD14+ CM, whereas the role of LIFR was weaker (Fig. 5D). Similarly, OSMR and STAT3 but not LIFR were implicated in ALP induction by OSM (supporting information Fig. S5B).

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Figure 5. Role of OSMR, LIFR, and STAT3. MSCs were transfected with siCT or siOSMR, siLIFR, or siSTAT3. (A): mRNA expression of OSMR or LIFR was reduced by more than 70% as assessed 2 days after transfection by real time polymerase chain reaction (PCR). (B): STAT3 expression was also reduced using specific siRNA as assessed by Western blot. (C): MSCs were treated with OSM (10 ng/ml), LPS (10 ng/ml), control, or LPS-activated CD14+ CM (1/10) for 15 minutes. Phosphorylation of STAT3 was analyzed by Western blot. (D): 2 days after siRNA transfection, MSCs were treated with LPS-activated CD14+ CM in the differentiation medium. After 24 hours of treatment, MSCs were analyzed for ALP mRNA expression by real time PCR. Assays were performed with n = 2 for each condition and are representative of two independent experiments. *, p < .05 compared to the control siRNA. (E): Schematic representation of the coupling between activated monocyte-macrophage and MSC-osteoblast based on this study. See the text for a detailed description. Abbreviations: ALP, alkaline phosphatase; BSP, bone sialoprotein; C/EBPδ, CCAAT-enhancer-binding protein δ; cbfa1, core-binding factor subunit alpha-1; COX2, cyclooxygenase 2; CT, control CM obtained without LPS treatment; CT CM, control CM from CD14+ monocytes without LPS; EPR, PGE2 receptor; IL, interleukin; LIF, leukemia inhibitory factor; LPS, CM obtained with LPS treatment; LPS CM, CM obtained with LPS treatment; LIFR, LIF receptor subunit; MSC, mesenchymal stem cell; OPG, osteoprotegerin; OSM, Oncostatin M; OSMR, OSM receptor subunit; PGE2, prostaglandin E2; RANKL, receptor activator of nuclear factor κ-B ligand; sIL-6R, soluble IL-6 receptor subunit; siCT, control siRNA; siOSMR, siRNA for OSMR; siLIFR, siRNA for LIFR; siRNA, small interfering RNA; STAT, signal transducer and activator of transcription; siSTAT, siRNA for STAT3; TLR, toll-like receptor.

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We also observed that OSM can induce mineralization by MSC in a minimal medium containing β-glycerophosphate but not vitamin D3, dexamethasone, or ascorbic acid (supporting information Fig. S6). However, the highest level of mineralization was observed when all factors were used.

Adenoviral Gene Transfer of OSM Induces Bone Formation in Mice

Overexpression of OSM in the joint or injection of recombinant OSM in the calvaria can lead to induced bone resorption, bone formation, or both, depending on the experimental context [13, 32, 33]. We developed a new approach based on injection of AdOSM in the tibia of C57BL/6 mice. After 7 days, X-ray micro-CT scan analysis and histology indicated that a bone healing reaction was initiated in the control animals injected with PBS or AdGFP (Fig. 6A, 6D). OSM overexpression significantly induced woven bone formation locally at the injury site with increased metaphyseal trabecular bone volume (BV/TV, Fig. 6B), trabecular thickness (Fig. 6C), collagen deposition (Fig. 6D), and osteoblast number (Fig. 6G, 6H). In contrast, osteoclasts were grossly absent in the bone healing zone and the metaphyseal osteoclast surface in the AdOSM groups was reduced, although not significantly (p = .08, Fig. 6E, 6F).

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Figure 6. OSM induces bone formation in mice. Indicated adenoviruses (5 × 107 pfu) were injected in the tibia of C57BL/6 mice. After 7 days, animals were sacrificed and tibias were analyzed by X-ray micro-CT scan. Representative sagittal sections are shown in (A), BV/TV and Tb.Th in (B and C) (n = 6 in each group, representative of two independent experiments). The same tibias were fixed, included, sectioned, and stained as described in Materials and Methods section with Masson's trichrome (D, collagen fibers stained in green) or for TRAP (E, osteoclasts stained in red) and osterix (G, osteoblasts stained in red). Positive cells in the metaphyseal spongiosa were quantified using the Qwin and ImageJ softwares. Results are expressed as relative TRAP+ Oc.S (F) or osterix+ Ob.N (H) with n = 6–7 per group. Columns, mean; bars, SEM; *, p < .05 between AdOSM and AdGFP group; arrows, passage of the syringe; #, new woven bone induced by the AdOSM. Original magnification: ×50 (D and E); ×200 (G). Abbreviations: AdGFP, adenovirus encoding green fluorescent protein; AdOSM, adenovirus encoding mouse OSM; BM, bone marrow; BV/TV, relative trabecular bone volume; Ob.N, osteoblast number; Oc.S, osteoclast surface; OSM, Oncostatin M; Tb.Th, trabecular thickness; TRAP, tartrate-resistant acid phosphatase; v, blood vessel.

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DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

In this study, we report that activated monocytes/macrophages secrete coupling factors to induce mineralization by MSC. Whereas osteoclasts induce osteoblast differentiation through the Wnt and BMP pathways [2], T lymphocytes through TNFα, TGFβ, IFNγ, and IL-17 [3], activated monocytes/macrophages appear to do so mainly through OSM. Some Wnt and BMP members are induced by LPS in monocytes but their neutralization using a similar approach as the one described for osteoclasts (Dkk1 and BMP6 antibodies) [2] does not prevent mineralization induced by LPS-activated monocytes/macrophages. Similarly, LPS induces TNFα and IFNγ (but not TGFβ or IL-17) production in monocytes/macrophages, but their neutralization is ineffective in our culture model. We consider it likely that macrophages are also implicated in MSC migration and chemoattraction to the inflammatory site, but it remains to determine the role of OSM, Wnt, BMP, and chemokines in this initial step of osteogenesis.

OSM is a known product of activated macrophages where its expression is regulated by a PGE2-cyclic adenosine monophosphate-protein kinase A pathway [18, 31] in contrast to IL-6 which is directly induced through the TLR-nuclear factor k-B pathway [34] and IL-6R protein which is known to be reduced by LPS [35]. We propose here that insufficient sIL-6R production renders IL-6 unable to stimulate osteoblast differentiation in our culture model, whereas OSM is produced in sufficient amount, in a COX2-PGE2 dependent manner. Interestingly, nonsteroidal anti-inflammatory drugs (NSAIDs) such as COX2 inhibitors have an inhibitory effect on fracture repair and spinal fusion [36, 37]. Our results suggest that NSAIDs could inhibit osteogenesis in part through reduction of OSM production by macrophages. Moreover, OSM production and hence regulation of matrix mineralization appeared largely restricted to classically activated inflammatory M1 macrophages, with various exogenous (from bacteria, viruses, or fungi) or endogenous (from breakdown of the extracellular matrices) TLR ligands being similarly active. These results suggest that bone formation could be induced by macrophage-derived OSM in numerous conditions of inflammation, infection, or injury and is not associated with M2 macrophages, despite their known role in tissue repair [5, 6]. These observations are in line with recent studies showing that osteal macrophages implicated in bone formation or healing are inflammatory and produced high amount of TNFα upon TLR activation [4, 11].

Addition of sIL-6R to MSC cultures allows activated monocytes to stimulate mineralization and high concentrations of sIL-6R are normally found in the circulation [35, 38]. Therefore, IL-6 could be also implicated in bone formation in vivo if its soluble receptor is provided by other cell sources such as liver cells. The third cytokine with a significant role in bone formation appeared to be LIF, but its production is restricted to MSC. Indeed, LPS can directly stimulate mineralization on MSC through enhanced LIF synthesis. This pathway of bone formation is rather weak when considered alone but it presumably acts additively or synergistically with OSM or IL-6, forming a complex network of autocrine/paracrine regulations between M1 macrophages and MSC/osteoblasts (Fig. 5E). Monocytes from few donors (two on eight tested) appear to produce lower amount of OSM, and in that case the production of LIF from MSC appears important to stimulate matrix mineralization, maybe to compensate the insufficient secretion of OSM. These donors could have a particular inflammatory status, such as increased nonclassic CD16+ monocytes or skewed differentiation into M2 macrophages, but a larger cohort is needed to ascertain this possibility.

Several inflammatory cytokines such as IL-1β, TNFα, IL-6, and OSM are inhibitors of adipocyte differentiation and play a role in atrophy of adipose tissue connected to cancer cachexia and chronic inflammatory diseases [39]. We show here that osteoblast differentiation induced by activated monocytes occurs at the expense of adipocyte differentiation and that OSM dictates this lineage commitment. Indeed, OSM inhibits C/EBPα and peroxisome proliferator-activated receptor γ expression, the two key transcription factors implicated in adipogenesis [32, 39]. In contrast, OSM induces expression of C/EBPδ in MSC, as observed previously in other cell types [40]. This basic leucine zipper transcription factor is transcriptionally controlled by STAT3 and acts synergistically with Cbfa1 to induce genes that are important for osteoblast replication and differentiation such as Osteocalcin and insulin-like growth factor 1 [41, 42]. We demonstrate here that OSM induces osteoblast differentiation and matrix mineralization through STAT3, with induced expression of C/EBPδ, Cbfa1, ALP, and BSP. All these events are also induced by activated monocytes in a gp130-dependent manner and are schematically presented in Figure 5E.

In human, OSM can bind to two types of receptor complexes, the gp130+OSMR and gp130+LIFR complexes, whereas in mice OSM has long been thought to signal only through the gp130+OSMR complex. However, this concept has recently been challenged. Indeed, OSMR−/− mice have reduced bone formation and increased adipogenesis but recombinant OSM is still able to induce bone formation in OSMR−/− mice apparently through the gp130+LIFR complex in osteocytes by downregulation of sclerostin, an inhibitor of bone formation [32]. Using knockdown experiments, we show here that OSM or activated monocytes induce osteoblast differentiation in human MSC mainly through the gp130+OSMR complex, in a STAT3-dependent manner, leading to accelerated matrix mineralization. The smaller effect of LIFR knockdown presumably reflects the smaller role of LIF produced by MSC cells. We also provide evidence that overexpression of murine OSM in the tibia of mice leads to enhanced woven bone formation in the medullar cavity, expanding previous reports showing that murine OSM induces bone formation in the calvaria [32]. Together, these results indicate that activated monocytes/macrophages produce OSM to form new bone, by stimulating MSC differentiation through the OSMR-STAT3-C/EBPδ-Cbfa1 pathway (Fig. 5E). We cannot, however, exclude the possibility that activated monocytes/macrophages also induce bone formation in vivo through the gp130+LIFR complex in osteocytes.

In addition to bone formation, OSM is known to stimulate bone resorption by osteoclasts indirectly through induction of the RANKL/OPG ratio in bone stromal cells [20, 32]. We show here that induction of RANKL expression in MSC by activated monocytes is small and very transient and the RANKL/OPG ratio is rather reduced in long-term cultures. In addition, overexpression of OSM in the tibia of mice does not show any sign of induced osteoclast formation or bone resorption. Therefore, in the experimental contexts presented here, OSM appears as a much more active cytokine for bone formation than for bone degradation, in line with the known role of osteal macrophages in anabolic bone modeling independently of bone resorption [11]. One previous study described that macrophages produce OSM to induce ALP expression and mineralization by vascular smooth muscle cells, suggesting a key role for this cytokine in vascular calcification associated with atherosclerosis [12]. OSM is also well-known to be produced by macrophages and neutrophils locally in the synovium of RA patients where it participates to joint destruction [18, 19]. However, overexpression of OSM in the joint of mice also leads to periosteal bone formation, suggesting that OSM could have a role in bone apposition observed at varying degree and anatomic sites in RA, juvenile arthritis, and spondyloarthropathies such as psoriatic arthritis and ankylosing spondylitis [33, 43]. Similarly, IL-6 was shown to stimulate fracture healing and bone mechanical resistance [44]. LIFR−/− mice are osteopenic with induced osteoclast formation and reduced bone formation [45], and null mutations in LIFR leads to the Stüve-Wiedemann syndrome in humans characterized by skeletal defects [46].

CONCLUSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Monocytes/macrophages classically activated with TLR ligands such as LPS produce OSM through a COX2-PGE2 regulatory loop. On MSC, OSM mainly recruits its type II receptor, activates the transcription factor STAT3, and induces the osteoblast differentiation program, leading to bone formation. This bone anabolic signal overwhelms the bone resorption signal mediated by RANKL. Also, LPS stimulates osteogenesis in MSC directly through enhanced production of LIF. The third cytokine presumably implicated in this regulatory network is IL-6, produced by LPS-activated monocytes/macrophages and MSC. We propose that during bone inflammation, infection, or injury, this IL-6 family signaling network activated by macrophages and TLR ligands stimulates osteogenesis and bone formation that is largely uncoupled from bone resorption and is thus an important target for anabolic bone therapies.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

We thank the Vector Core of the University Hospital of Nantes (France) supported by the Association Française contre les Myopathies (AFM) for producing the adenovirus vectors. We are very grateful to Maria Cristina Cuturi, Claire Usal, Emmanuel Merieau, and Ignacio Anegon (Inserm U643, Nantes, France) for animal care and their help in the experimental design. This work was supported by Inserm and La Ligue Contre le Cancer (comité Grand Ouest). P.G. is a recipient of a fellowship from le Ministère de la Recherche. B.B. is currently affiliated with Division of Bone Diseases, Department of Rehabilitation and Geriatrics, Geneva University Hospital, Geneva, Switzerland.

REFERENCES

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. CONCLUSION
  8. Acknowledgements
  9. DISCLOSURE OF POTENTIAL CONFLICTS OF INTEREST
  10. REFERENCES
  11. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
STEM_1040_sm_SuppFig1-4.pdf1235KSupplementary Figure 1-4
STEM_1040_sm_SuppFig5-6.tif2493KSupplementary Figure 5-6
STEM_1040_sm_SuppTab1.pdf1861KSupplementary Table 1
STEM_1040_sm_SuppInfo.pdf160KSupplementary Data

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