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Keywords:

  • OSTEOCLAST DIFFERENTIATION;
  • BONE RESORPTION;
  • BACTERIAL LIPOPROTEIN;
  • LIPOPEPTIDE;
  • TLR2

ABSTRACT

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgments
  9. References

Bacterial infection can cause inflammatory bone diseases accompanied by the bone destruction resulting from excess generation of osteoclasts. Although lipoproteins are one of the major immunostimulating components of bacteria, little is known about their effects on bone metabolism. In this study, we investigated the role of lipoproteins in bacteria-induced bone destruction using Staphylococcus aureus wild type, its lipoprotein-deficient mutant, and synthetic lipopeptides Pam2CSK4 and Pam3CSK4 known to mimic bacterial lipoproteins. Formaldehyde-inactivated S. aureus or the synthetic lipopeptides induced severe bone loss in the femurs of mice after intraperitoneal administration and in a calvarial bone implantation model, whereas the lipoprotein-deficient S. aureus did not show such effects. Mechanism studies further identified three action mechanisms for the lipopeptide-induced osteoclast differentiation and bone resorption via (i) enhancement of osteoclast differentiation through Toll-like receptor 2 and MyD88-dependent signaling pathways; (ii) induction of pro-inflammatory cytokines, TNF-α and IL-6; and (iii) upregulation of RANKL expression with downregulation of osteoprotegerin expression in osteoblasts. Taken together, these results suggest that lipoprotein might be an important bacterial component responsible for bone destruction during bacterial infections through augmentation of osteoclast differentiation and activation. © 2013 American Society for Bone and Mineral Research.


Introduction

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgments
  9. References

Bone metabolism is regulated by bone-resolving osteoclasts and bone-forming osteoblasts. Osteoclasts are specialized multinucleated cells (MNCs) differentiated from the hematopoietic cell lineages by macrophage-colony stimulating factor (M-CSF) and receptor activator of nuclear factor κB ligand (RANKL).[1] Osteoblasts are differentiated from mesenchymal cell lineages and are also able to modulate the bone-resorptive capacity of osteoclasts by producing RANKL and its inhibitory competitor, osteoprotegerin (OPG).[2] Because the regulation of osteoblasts and osteoclasts is pivotal for bone homeostasis, disruption of the balance between osteoclasts and osteoblasts can result in a variety of bone diseases. For instance, bone and joint infections enhance osteoclastogenesis but reduce osteoblastogenesis, subsequently leading to bone loss.[3, 4]

Bacterial infections often cause inflammatory bone diseases, such as osteomyelitis, septic arthritis, septic bursitis, and periodontitis, and these diseases are often accompanied by excessive bone destruction.[5] Bacteria possess various pathogen-associated molecular patterns (PAMPs), including lipoprotein, lipopolysaccharide (LPS), lipoteichoic acid (LTA), and peptidoglycans in the cell wall, which are involved in bacterial adherence, host invasion, induction of inflammatory responses, and regulation of immune responses.[6] Upon infection, the host recognizes and responds to the PAMPs through pattern-recognition receptors, including Toll-like receptors (TLRs).[7] Osteoclasts and osteoblasts express various TLRs and, therefore, their differentiation and activation can be regulated in response to PAMPs.[8, 9]

Among PAMPs, bacterial lipoproteins are considered as a major virulence factor because of their strong immunostimulating activity.[10] Bacterial lipoproteins are classified into diacylated and triacylated structures, which are preferentially expressed by Gram-positive and Gram-negative bacteria, respectively, with some exceptions.[11] Diacylated and triacylated lipoproteins are recognized by TLR2/TLR6 and TLR2/TLR1, respectively,[12] triggering MyD88-dependent signaling pathways to induce the innate immune responses.[13]

Because bacteria express many lipoproteins with various amino acid sequences, most studies have focused on diacylated lipopeptide, Pam2CSK4, and triacylated lipopeptide, Pam3CSK4, which mimic bacterial lipoproteins for their potent immunostimulatory activities.[14] Although bacterial lipoproteins and the model lipopeptides have been used extensively to study the innate immune responses, little is known about their effects on bone metabolism. In the present study, we investigated how bacterial lipoproteins and their mimetic lipopeptides, Pam2CSK4 and Pam3CSK4, affect bone metabolism.

Materials and Methods

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgments
  9. References

Reagents and chemicals

Pam2CSK4 and Pam3CSK4 were purchased from InvivoGen (San Diego, CA, USA). Recombinant murine RANKL was obtained from Peprotech (Rocky Hill, NJ, USA), and M-CSF and OPG were purchased from R&D Systems (Minneapolis, MN, USA). Normal goat IgG, anti-mouse TNF RI/TNFRSF1A, and anti-mouse IL-6 receptor antibodies were obtained from R&D Systems, and Armenian hamster IgG antibody was purchased from BioLegend (San Diego, CA, USA). Primary antibodies specific to p38 kinase, phospho-p38 kinase, JNK, phospho-JNK, ERK, phospho-ERK, and HRP-conjugated secondary antibodies to rabbit IgG or mouse IgG were purchased from Cell Signaling Technology (Beverly, MA, USA).

Preparation of formaldehyde-inactivated S. aureus

S. aureus RN4220 and the lipoprotein-deficient mutant (Δlgt)[11] were grown in Luria-Bertani (LB) media and LB containing erythromycin, respectively, at 30°C until it reached the mid-log phase (A600, 0.6). Bacterial cells were harvested, washed with phosphate-buffered saline (PBS), and inactivated with 0.5% formaldehyde for 5 hours. After inactivation, bacterial cell pellets were extensively washed twice with PBS. The complete inactivation was confirmed by plating and culturing onto LB agar plates (data not shown).

Animals and cells

Animal experiments were approved by the Institutional Animal Care and Use Committee of Seoul National University (SNU-100526-4). Six- to 12-week-old C57BL/6 mice were purchased from Orient Bio (Seongnam, Korea). The TLR2- or MyD88-deficient C57BL/6 mice were provided by Dr Shizuo Akira (Osaka University, Osaka, Japan). Bone marrow cells (BMs), bone marrow-derived macrophages (BMMs), and committed osteoclast precursors were prepared as described previously.[15] Briefly, to generate committed osteoclast precursors, stroma-free bone marrow cells were incubated with M-CSF for 3 days to differentiate into macrophages. Then, the cells were further incubated in the presence of M-CSF (20 ng/mL) and RANKL (20 ng/mL) for additional 2 days to differentiate into committed osteoclast precursors. After washing with fresh media, the cells were stimulated with the lipopeptides in the presence of M-CSF without RANKL. Mouse osteoblast precursors were isolated from the calvaria of 1-day-old C57BL/6 mice as previously described.[16] For BMM-osteoblast co-culture, osteoblasts were plated at 1 × 104 cells/0.5 mL/well in 48-well plates with BMMs (1 × 105 cells) and stimulated with 1 µg/mL of Pam2CSK4 or 10 µg/mL of Pam3CSK4 in the culture media containing 10 mM β-glycerophosphate, 50 µM ascorbic acid, and 100 ng/mL 1α,25-dihydroxyvitamin D3 for 48 hours to 8 days.

Tartrate-resistant acid phosphatase (TRAP) staining

Cells were fixed with a fixative solution (26% citrate, 66% acetone, and 8% formaldehyde) and stained with a leukocyte acid phosphatase staining kit (Sigma-Aldrich, St. Louis, MO, USA). Dark-reddened TRAP-stained cells with three or more nuclei were enumerated as osteoclasts under an inverted phase-contrast microscope.[17]

In vitro bone resorption assay

Stroma-free BM cells (1.8 × 105 cells/mL) were plated on 48-well calcium phosphate-coated plates (Osteogenic Core Technologies, Chunan, Korea) or dentine discs (Immunodiagnostic Systems, Boldon, UK) and differentiated into the committed osteoclast precursors. After washing, the committed osteoclasts were treated with Pam2CSK4 or Pam3CSK4 in fresh media containing M-CSF for 2 days (calcium phosphate-coated plates) or 6 days (dentine discs). Then, the areas of resorption pits were photographed by an inverted microscope and analyzed with Scion image software (Scion Corp., Frederick, MD, USA) for the calcium phosphate-coated plate and by a confocal laser scanning microscope (LSM) and analyzed with LSM Image Browser software (Carl Zeiss, Jena, Germany) for the dentine disc.

In vivo bone resorption assay

Mice were intraperitoneally injected with 8 mg (equivalent to 4 × 109 colony-forming units) of formaldehyde-inactivated S. aureus or with 2.5 mg/kg of Pam2CSK4 or Pam3CSK4 twice with a 4-day interval between injections. At day 7 after the initial administration, the right femur was removed, dissected, and fixed in 4% paraformaldehyde. Then, the femurs were scanned with high-resolution micro-computed tomography (micro-CT; Skyscan1172 scanner, Skyscan, Kontich, Belgium). Three-dimensional images of the trabecular bones were obtained with CT-volume software (Skyscan) and analyzed with a CT-analyzer program (Skyscan) to determine the bone volume, trabecular number, trabecular thickness, and trabecular separation of the femur. The fixed femurs were incubated with decalcification solution for 7 days. Decalcified femurs were embedded, sectioned, and stained for TRAP.[18] In a separate experiment, a collagen sheet containing 50 µg of Pam2CSK4 or Pam3CSK4, or Pam2CSK4 with 20 µg of OPG was implanted on the mouse calvarial bone for 7 days. Then, the calvarial bone was scanned with the micro-CT as described above to measure calvarial bone resorption.

Flow cytometric analysis

To determine cell surface or intracellular expression of TLRs, cells were stained with PE-conjugated rat anti-mouse TLR6 antibody (R&D Systems), Alexa Flour 647-conjugated rat anti-mouse TLR1 antibody, and PE-conjugated rat anti-mouse TLR2 antibody (eBioscience, San Diego, CA, USA) and analyzed with FACSCalibur and CellQuest Pro software (BD Biosciences, San Jose, CA, USA) followed by FlowJo software (Tree Star, Ashland, OR, USA).

Reverse transcription-polymerase chain reaction (RT-PCR)

The mRNA expression of IL-6, TNF-α, cathepsin K, TRAP, cyclooxygenase 2 (COX2), and β-actin was determined in the committed osteoclast precursors using RT-PCR as described previously[19] with following primers: IL-6: 5′-CCGGAGAGGAGACTTCACAG-3′ and 5′-GGAAATTGGGGTAGGAAGGA-3′; TNF-α: 5′-ATGAGCACAGAAAGCATGATC-3′ and 5′-TACAGGCTTGTCACTCGAATT-3′; cathepsin K: 5′-TCAAGGTTCTGCTGCTA-3′ and 5′-GAGCCAAGAGAGCATAT-3′; TRAP: 5′-AACCGTGCAGACGATGGGCG-3′ and 5′-GCCAGGACAGCTGAGTGCGG-3′; COX2: 5′-CCCCCACAGTCAAAGACACT-3′ and 5′-GAGTCCATGTTCCAGGAGGA-3′; and β-actin: 5′-GTGGGGCGCCCCAGGCACCA-3′ and 5′-CTCCTTAATGTCACGCACGATTTC-3′.

Transfection with small interfering RNA (siRNA) and OPG overexpression vector

Mouse calvarial osteoblast precursors (3 × 104 cells/mL) were transfected with 25 picomoles of siRNA targeting RANKL or nontargeting siRNA (Dharmacon, Lafayette, CO, USA) or with 0.5 μg of an OPG overexpression plasmid or the control (pcDNA3.1) using Lipofectamine 2000 (Invitrogen, Grand Island, NY, USA) according to the manufacturer's instructions.

Enzyme-linked immunosorbent assay (ELISA), Western blot analysis, and electrophoretic mobility shift assay (EMSA)

The protein levels of TNF-α, OPG, RANKL, and IL-6 in the cell culture supernatants, serum, and bone marrow extracellular fluids were measured using commercial ELISA kits (R&D Systems and BD Biosciences) according to the manufacturer's instructions. MAP kinase (MAPK) phosphorylation and the DNA binding activity of AP-1, NF-κB, or NF-AT were determined by Western blot analysis and EMSA as described previously.[20]

Statistical analysis

All experiments were performed three to five times. Results are expressed as mean ± standard deviation from triplicate samples in each experiment. Data were analyzed with t-test. An asterisk (*) indicates a statistically significant difference from the control group at p < 0.05.

Results

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgments
  9. References

Inactivated S. aureus induced bone resorption in vivo and osteoclastogenesis, but the lipoprotein-deficient mutant barely produced these effects

Mice were intraperitoneally injected with formaldehyde-inactivated S. aureus wild-type or lipoprotein-deficient mutant (Δlgt). As shown in Fig. 1A, B, wild-type S. aureus substantially augmented bone resorption, but no such effect was found with the lipoprotein-deficient mutant. We further examined the differentiation of committed osteoclast precursors into osteoclasts in the presence of the inactivated wild-type or mutant S. aureus. As shown in Fig. 1C, the wild-type S. aureus significantly increased the number of TRAP-positive MNCs in a dose-dependent manner. In contrast, the lipoprotein-deficient S. aureus barely induced TRAP-positive MNCs (Fig. 1C). These results suggest that bacterial lipoproteins are crucial for S. aureus-induced bone resorption and osteoclast differentiation.

image

Figure 1. S. aureus, but not the lipoprotein-deficient mutant, induced bone resorption and osteoclast differentiation. (A) Mice (n = 3) were intraperitoneally injected with 8 mg of formaldehyde-inactivated S. aureus wild-type or the lipoprotein-deficient mutant (Δlgt), twice with a 4-day interval. At day 7 after the first injection, micro-CT image of the mouse femurs was obtained. (B) Analysis of trabecular bone parameters in the femur. BV/TV = trabecular bone volume; Tb.N = trabecular number; Tb.Th = trabecular thickness; Tb.Sp = trabecular separation. Data were analyzed from three independent trials. *p < 0.05. (C) Committed osteoclast precursors were washed with fresh media to remove residual RANKL and stimulated with the inactivated S. aureus in the presence of 20 ng/mL of M-CSF for 48 hours. After TRAP staining, TRAP-positive, multinucleated cells (MNCs) were enumerated with an inverted phase-contrast microscope. *p < 0.05. Con = control group; WT = wild type.

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Pam2CSK4 and Pam3CSK4 induced bone resorption and osteoclast differentiation

Mice were intraperitoneally injected with Pam2CSK4 or Pam3CSK4 and the right femur was scanned with micro-CT. As shown in Fig. 2A, B, trabecular bone volume and trabecular number were significantly (p < 0.05) lower and trabecular separation was higher in the femurs of lipopeptide-treated mice than the femurs of control mice not treated with lipopeptides. Although trabecular thickness was not significantly lower in the femurs of Pam2CSK4-treated mice than in those of control mice, it was considerably reduced in the femurs of Pam3CSK4-treated mice. In addition, TRAP staining of the femurs demonstrated that lipopeptides increased osteoclast differentiation (Fig. 2C). When we implanted the collagen sheets soaked in 50 µg of each lipopeptide in mouse calvaria, massive resorption in the calvarial bone was observed, although resorption was somewhat lower in the Pam3CSK4-treated mice (Fig. 2D). These results indicate that lipopeptides induce bone resorption and osteoclast differentiation in vivo.

image

Figure 2. The lipopeptides, Pam2CSK4 and Pam3CSK4 induced bone resorption. (A) C57BL/6 mice (n = 5) were intraperitoneally injected with 2.5 mg/kg of Pam2CSK4 or Pam3CSK4 twice with a 4-day interval. (B) BV/TV, Tb.N, Tb.Th, and Tb.Sp of the mouse femurs were analyzed. *p < 0.05. (C) Sections from the femurs were stained for TRAP and photographed with an inverted phase-contrast microscope at 200 times magnification. One of two similar results is shown. (D) Collagen sheets soaked with 50 µg of Pam2CSK4 or Pam3CSK4 were implanted on mouse calvaria. The micro-CT image of the calvaria shown is one of three similar results. PAM2, Pam2CSK4. PAM3, Pam3CSK4.

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Pam2CSK4 and Pam3CSK4 augmented osteoclast differentiation

Next, to examine whether the lipopeptides directly increase osteoclast differentiation, the committed osteoclast precursors were stimulated with lipopeptides in the absence of RANKL unless otherwise stated. Both Pam2CSK4 and Pam3CSK4 increased the TRAP-positive MNCs of committed osteoclast precursors in a dose-dependent manner (Fig. 3A, B). Notably, TRAP-positive MNCs were not observed when the naïve precursors (eg, the cells without RANKL priming) were stimulated with the lipopeptides (data not shown). To further examine the bone-resorptive capacity, committed osteoclast precursors were prepared on calcium phosphate–coated plates or dentine discs, treated with Pam2CSK4 or Pam3CSK4, and analyzed for resorption pits. Treatment with these lipopeptides produced large areas of resorption pits where the effect of Pam2CSK4 was more potent than that of Pam3CSK4 (Fig. 3C, D). These results imply that the lipopeptides have the capacity to induce osteoclast differentiation, resulting in bone resorption.

image

Figure 3. The lipopeptides enhanced the osteoclastogenesis of committed osteoclast precursors. (A, B) Committed osteoclast precursors were washed and stimulated with (A) Pam2CSK4 or (B) Pam3CSK4 in the presence of 20 ng/mL of M-CSF for 48 hours. The cells were stained for TRAP, and TRAP-positive MNCs were enumerated. *p < 0.05. (C, D) The committed osteoclast precursors were prepared on (C) calcium phosphate–coated plates or (D) dentine discs and stimulated with the lipopetides as described above. The resorbed areas were photographed (upper panels). The total resorption area and/or depth were measured with image software and plotted as mean ± standard deviation from three independent results (lower panels). One of three similar results is shown. *p < 0.05.

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Pam2CSK4- or Pam3CSK4-induced osteoclast differentiation required TLR2 and MyD88

Flow cytometry demonstrated that the committed osteoclast precursors expressed high levels of TLR2 and TLR6 but low level of TLR1 on the cell surface (Fig. 4A). In addition, Pam2CSK4 and Pam3CSK4 failed to induce differentiation of the committed osteoclast precursors derived from TLR2- or MyD88-deficient mice (Fig. 4B, C). These findings suggest that the lipopeptides-induced osteoclast differentiation requires TLR2 and MyD88.

image

Figure 4. TLR2 and MyD88 were essential for the lipopeptide-induced osteoclastogenesis. (A) Committed osteoclast precursors were stained with fluorescence-labeled antibodies specific for TLR1, TLR2, or TLR6 and subjected to flow cytometric analysis. The gray area indicates an isotype control. Mean fluorescence intensity is shown in the upper right of each histogram: the upper one indicates TLR expression, and the lower one isotype control. (B, C) Stroma-free bone marrow cells were obtained from wild-type, TLR2-deficient, or MyD88-deficient mice and differentiated into the committed osteoclast precursors. The committed osteoclast precursors were washed and treated with (B) Pam2CSK4 or (C) Pam3CSK4 in the presence of 20 ng/mL of M-CSF for 48 hours. Then, TRAP-positive MNCs were photographed under an inverted phase-contrast microscope (left panel) or enumerated to obtain mean ± standard deviation (right panel) from three independent trials. Scale bars = 200 µm. One of three similar results is shown. *p < 0.05 when compared with the wild-type groups.

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Pam2CSK4 and Pam3CSK4 induced osteoclast differentiation through MAPK pathways

MAPKs are known to be closely associated with osteoclastogenesis.[21] Coincidentally, Pam2CSK4 maximally increased phosphorylation of MAPKs, such as ERK, p38 kinase, and JNK, at 5 minutes after the treatment. Pam3CSK4 also increased phosphorylation of p38 kinase and JNK, maximally so at 15 minutes, but the maximal phosphorylation of ERK was observed at 5 minutes (Fig. 5A). Notably, the phosphorylation pattern of MAPKs in the cells under the serum-starvation condition for 5 hours before the stimulation was very similar (data not shown). In addition, the number of TRAP-positive MNCs were remarkably decreased in the presence of inhibitors of ERK (U0126), p38 kinase (SB203580), and JNK (SP600125) (Fig. 5B), suggesting that the lipopeptides induce osteoclast differentiation through MAPK signaling pathways. No change in cell viability was observed in any treatment group (data not shown).

image

Figure 5. The lipopeptides induced osteoclastogenesis through the activation of MAPK and increased the DNA-binding activity of AP-1, NF-κB, and NF-AT. (A) Committed osteoclast precursors were washed and stimulated with Pam2CSK4 (1 µg/mL) or Pam3CSK4 (10 µg/mL) in the presence of 20 ng/mL of M-CSF for various time periods. Immunoblots were performed with antibodies to phosphorylated or nonphosphorylated forms of MAPKs including ERK, p38 kinase, and JNK. (B) Committed osteoclast precursors were pretreated with specific inhibitors of ERK (U0126), p38 kinase (SB203580), or JNK (SP600125) for 1 hour and subsequently stimulated with Pam2CSK4 (1 µg/mL) or Pam3CSK4 (10 µg/mL) for 48 hours. The cells were stained for TRAP and TRAP-positive MNCs were enumerated. *p < 0.05 when compared with the lipopeptide-only groups. (C) Committed osteoclast precursors were stimulated with Pam2CSK4 (1 µg/mL) or Pam3CSK4 (10 µg/mL) for 90 minutes. Nuclear extracts were isolated and incubated with 32P-labeled AP-1, NF-κB, or NF-AT binding site oligonucleotides. Protein-DNA binding complexes were electrophoresed and subjected to autoradiography. One picomole of 32P-unlabeled probes (marked as “Cold competitor”) was used for competition assay. One of three similar results is shown.

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Pam2CSK4 and Pam3CSK4 increased DNA-binding activity of AP-1, NF-κB, and NF-AT in the committed osteoclast precursors

Transcription factors, such as AP-1, NF-κB, and NF-AT, are known to be responsible for osteoclastogenesis.[22] As shown in Fig. 5C, the DNA-binding activity of all transcription factors tested increased in the presence of lipopeptides, which suggests that activation of AP-1, NF-κB, and NF-AT is involved in the lipopeptide-induced osteoclast differentiation.

TNF-α and IL-6 contributed to the lipopeptide-induced osteoclast differentiation

When the committed osteoclast precursors were treated with Pam2CSK4 or Pam3CSK4, TNF-α and IL-6 were induced in both mRNA and protein levels (Fig. 6A, B). Furthermore, when the cells were pretreated with blocking antibodies to receptors for IL-6 or TNF-α, the lipopeptide-induced osteoclast differentiation was reduced (Fig. 6C). These results indicate that the pro-inflammatory cytokines, TNF-α and IL-6, are involved, at least partially, in the lipopeptide-induced osteoclast differentiation. Meanwhile, although the mRNA expression of COX2, a representative enzyme generating PGE2, was increased in the committed osteoclast precursors in response to the lipopeptides (Fig. 6A), the COX2 inhibitor NS398 did not affect the lipopeptide-induced osteoclast differentiation at the concentration completely inhibiting PGE2 synthesis (Fig. 6D).

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Figure 6. IL-6 and TNF-α mediated the lipopeptide-induced osteoclastogenesis. (A) Committed osteoclast precursors were washed and stimulated with Pam2CSK4 (1 µg/mL) or Pam3CSK4 (10 µg/mL) in the presence of 20 ng/mL of M-CSF for 0, 3, 6, or 12 hours. The mRNA expression levels of IL-6, TNF-α, COX2, cathepsin K, TRAP, and β-actin were analyzed by RT-PCR. β-actin was used as an internal control. (B) Committed osteoclast precursors were stimulated with Pam2CSK4 or Pam3CSK4 for 48 hours. The culture supernatants were then analyzed for IL-6 and TNF-α production using ELISA. *p < 0.05. (C) Committed osteoclast precursors were pre-incubated with blocking antibodies to receptors for TNF-α or IL-6 or an appropriate isotype control for 1 hour, followed by treatment with Pam2CSK4 (1 µg/mL) or Pam3CSK4 (10 µg/mL) for 48 hours. Hamster IgG antibody for anti-TNF RI antibody and goat IgG antibody for anti-IL6 receptor. *p < 0.05 when compared with antibody-untreated groups. (D) Committed osteoclast precursors were pretreated with a COX2 inhibitor NS398 for 1 hour followed by stimulation with the lipopeptides for additional 48 hours. TRAP-positive MNCs were enumerated following TRAP-staining (left panel), and PGE2 levels in the culture supernatants were determined by ELISA (right panel). One of three similar results is shown. N.D. = not detected.

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Upregulation of RANKL with downregulation of OPG contributed to the lipopeptides-induced osteoclast differentiation and the bone resorption

When mice were given the lipopeptide intraperitoneally, the levels of RANKL, IL-6, and TNF-α were significantly (p < 0.05) increased in the bone marrow, whereas the level of OPG was reduced (Fig. 7A). However, in the sera, the level of RANKL was significantly (p < 0.05) decreased, whereas the level of OPG was remarkably increased (Fig. 7B). These results are in agreement with previous reports that RANKL/OPG levels in the regional bone environment are correlated with the bone loss but not those in the serum.[23-25] The BMM-osteoblast co-culture system has been used as a representative in vitro model to demonstrate dynamic interaction of osteoclasts with osteoblasts and the homeostatic regulation of bone metabolism.[26] As shown in Fig. 7C, Pam2CSK4 and Pam3CSK4 substantially increased TRAP-positive MNCs in the co-culture system. Furthermore, the expression of RANKL at both cellular and secretory levels was augmented, but OPG expression was downregulated under the same conditions (Fig. 7C), all of which are indicative of osteoclast differentiation in vivo.[27] On the other hand, a significant decrease in the lipopeptide-induced TRAP-positive MNCs was observed when BMMs were co-cultured with osteoblast transfected with siRNA targeting RANKL (Fig. 7D) or with OPG overexpression plasmid (Fig. 7E). Furthermore, Fig. 7F indicated that the addition of OPG significantly blocked Pam2CSK4-induced inhibition of bone resorption. These results suggest that the lipopeptides enhance osteoclast differentiation through upregulation of RANKL and downregulation of OPG by osteoblasts.

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Figure 7. Upregulation of RANKL with downregulation of OPG contributed to the lipopeptide-induced osteoclast differentiation and the bone resorption. (A, B) Mice were intraperitoneally injected with 2.5 mg/kg of Pam2CSK4 or PBS. Then, the (A) bone marrow extracellular fluids and (B) serum were collected at 18 hours after the injection, and the levels of RANKL, OPG, IL-6, and TNF-α were measured using ELISA. *p < 0.05. (C) Mouse calvarial osteoblasts were co-cultured with BMMs in the presence of Pam2CSK4 (1 µg/mL) or Pam3CSK4 (10 µg/mL) for 8 days to stain for TRAP and for 48 hours to analyze cellular RANKL, secreted RANKL, and secreted OPG using ELISA. *p < 0.05. (D) Calvarial osteoblast precursors (3 × 104 cells) were transfected with 25 pmol of RANKL siRNA or nontargeting siRNA as a control. BMMs (1 × 105 cells) were co-cultured with the transfected osteoblsts and treated with Pam2CSK4 (1 µg/mL) in the presence of 10 mM β-glycerophosphate, 50 µM ascorbic acid, and 100 ng/mL 1α,25-dihydroxyvitamin D3 for 6 days. After TRAP staining, TRAP-positive MNCs were enumerated. *p < 0.05. (E) Calvarial osteoblast precursors (3 × 104 cells) were transfected with 0.5 µg of OPG expression vector or empty vector as a control. Stroma-free BMs (1 × 105 cells) were co-cultured with the transfected osteoblasts and treated with or without Pam2CSK4 (1 µg/mL) for 6 days. Then, the cells were stained for TRAP, and TRAP-positive MNCs were enumerated. *p < 0.05. (F) Mouse calvaria were implanted with collagen sheets soaked with 50 µg of Pam2CSK4 in the presence or absence of 20 µg of OPG for 7 days. The calvaria were obtained and scanned by micro-CT followed by analysis of bone volume using micro-CT analyzer program. *p < 0.05. (G) Schematic illustration of the proposed action mechanism. Bacterial lipoproteins induce osteoclast differentiation and bone resorption via (i) enhancement of osteoclast differentiation through TLR2/MyD88-dependent signaling pathways, (ii) induction of pro-inflammatory cytokines, TNF-α and IL-6, and (iii) upregulation of RANKL with downregulation of OPG in osteoblasts.

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Discussion

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgments
  9. References

Here, we demonstrated that inactivated S. aureus induced bone destruction and enhanced osteoclast differentiation in mouse model, whereas lipoprotein-deficient S. aureus abrogated such effects. Furthermore, the results showed that the synthetic lipopeptides mimicking bacterial lipoproteins led to severe bone loss and induced osteoclast differentiation in both in vivo and in vitro models. This observation is in keeping with the previous report showing that diacyl lipopeptide increased the formation of TRAP-positive osteoclasts.[28] We further identified three action mechanisms for the lipopeptide-induced osteoclast differentiation and bone resorption via (i) enhancement of osteoclast differentiation through TLR2/MyD88-dependent signaling pathways, (ii) induction of pro-inflammatory cytokines, TNF-α and IL-6, and (iii) upregulation of RANKL with downregulation of OPG in osteoblasts, as summarized in Fig. 7G.

Pam2CSK4 was more potent than Pam3CSK4 in the induction of bone destruction and osteoclast differentiation. This differential potency could be the result of the different expression levels of the receptors required for recognition of Pam2CSK4 and Pam3CSK4. Although both lipopeptides require TLR2/MyD88-dependent signaling pathways, Pam2CSK4 and Pam3CSK4 are recognized by TLR2/TLR6 and TLR2/TLR1 heterodimers, respectively.[12, 13] Thus, our results showing the high surface expression of TLR6 with low TLR1 expression on the committed osteoclast precursors imply that osteoclasts are likely to detect and respond to Pam2CSK4 to a greater extent than they do with Pam3CSK4. Indeed, our results also demonstrated that MAPK phosphorylation and transcription factors activation induced by Pam2CSK4 was more potent than those induced by Pam3CSK4.

Osteoclast differentiation is regulated by inflammatory mediators including IL-6,[29] TNF-α,[30] IL-1[29, 30], and PGE2.[31] We found that both lipopeptides induced IL-6, TNF-α, and COX-2. However, IL-6 and TNF-α appeared to be more closely associated with lipopeptide-induced osteoclast differentiation because this differentiation was attenuated by the antibodies blocking receptors for IL-6 and TNF-α, but antibodies to IL-1β (data not shown) or PGE2 inhibitor failed to do so. IL-6 and TNF-α are known to promote osteoclastogenesis by directly potentiating RANKL-induced osteoclast differentiation and also by indirectly enhancing the expression of RANKL and M-CSF from osteoblast/stromal cells.[32] Concordantly, both lipopeptides significantly induced RANKL expression with inhibition of OPG expression in the mouse bone marrow and in the BMM-osteoblast co-culture. The role of RANKL and OPG in the mediation of the lipopeptide-induced osteoclast differentiation and bone resorption was further confirmed with silencing of RANKL and overproduction of OPG. Therefore, the lipopeptide-induced osteoclast differentiation results not only from the direct effect of the lipopeptides on the osteoclasts but also from activation of other cell types (including osteoblasts) by the lipopeptides, leading to the production of osteoclastic factors.

Bacterial lipoproteins are not exclusively expressed on pathogens; rather, they are a universal component of both pathogens and commensals.[33] However, commensals possessing lipoproteins are not likely to induce osteoclast differentiation or bone resorption because lactic acid bacteria inhibit osteoclast differentiation and even facilitate bone formation.[34] One possible explanation is a difference in the structure of lipoproteins that microbes have. We found that Pam2CSK4 was at least 10 times more potent than Pam3CSK4 with regards to the induction of osteoclast differentiation. Notably, Pam3CSK4 has the same molecular structure as Pam2CSK4, with the exception of one additional fatty acid chain. Thus, a subtle difference in lipid moiety could potentially exist between pathogens and nonpathogens. Interestingly, expression of diacylated and triacylated lipoproteins can be changed by environmental conditions.[35] In addition, peptide sequences of lipoproteins also affect their biological properties in the view of the fact that diacyl lipopeptides with various amino acid sequences designed for S. aureus lipoproteins produce differential potency in the stimulation of TLR2, dendritic cells, and natural killer cells.[36] Another possible explanation is the combinatorial effects of various PAMPs of bacteria. For example, LTA is also a TLR2 ligand,[37] but LTAs are less potent than lipoproteins in the immunostimulating potential.[10] Furthermore, the LTAs of commensal bacteria seem to have weak immunostimulating potential.[38] Thus, although commensal bacteria may have lipoproteins that positively contribute to osteoclast differentiation and bone destruction, other PAMPs, including LTA, may compensate for these lipoproteins.

In summary, our in vivo and in vitro results suggest that diacylated and triacylated lipoproteins might be an important bacterial component responsible for bone destruction provoked by bacterial infections. These findings call for further research to develop immunoregulatory therapeutics targeting bacterial lipoproteins that could be an efficient treatment or prevention of bone diseases occurring at bacterial infections.

Acknowledgments

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgments
  9. References

We are grateful to Prof B Brett Finlay at the University of British Columbia for critical reading of the manuscript. This work was supported by grants from the National Research Foundation of Korea funded by the Ministry of Education, Science, and Technology (2012-0008693, 2012-0000492, and 2012-0007883) and the R&D Convergence Center Support Program, Ministry for Food, Agriculture, Forestry and Fisheries, Republic of Korea.

Authors' roles: JK, JY, OJP, WSK, and KK performed the experiments and analyzed or interpreted the data. SSK, CHY, HHK, and BLL contributed important intellectual input in the study. SHH contributed experimental design, important intellectual input, and supervision of the study. All authors contributed to discussion of the results followed by writing and reviewing the manuscript.

References

  1. Top of page
  2. ABSTRACT
  3. Introduction
  4. Materials and Methods
  5. Results
  6. Discussion
  7. Disclosures
  8. Acknowledgments
  9. References
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