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The Journal of Immunology, 2008, 180, 1382 -1389
Copyright © 2008 by The American Association of Immunologists, Inc.
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Stimulation by TLR5 Modulates Osteoclast Differentiation through STAT1/IFN-β1

Hyunil Ha*, Jong-Ho Lee*, Ha-Neui Kim*, Han Bok Kwak*, Hyun-Man Kim*, Shee Eun Lee{dagger}, Joon Haeng Rhee{dagger}, Hong-Hee Kim* and Zang Hee Lee2,*

* Department of Cell and Developmental Biology, Dental Research Institute, School of Dentistry, Seoul National University, Seoul; and {dagger} Research Institute of Vibrio Infection and Genome Research Center for Enteropathogenic Bacteria, Chonnam National University Medical School, Gwangju, Republic of Korea


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Osteoclasts are bone-resorbing cells that are differentiated from hemopoietic precursors of the monocyte-macrophage lineage. Stimulation of TLRs has been shown to positively or negatively modulate osteoclast differentiation, depending on the experimental condition. However, the molecular mechanism by which this modulation takes place remains unclear. In the present study, we examined the effects of flagellin, a specific microbial ligand of TLR5, on the receptor activator of NF-{kappa}B ligand (RANKL)-stimulated osteoclastogenesis. Flagellin suppressed RANKL induction of c-Fos protein expression in bone marrow-derived macrophages without affecting c-Fos mRNA expression. Ectopic overexpression of c-Fos and a constitutively active form of NFATc1 reversed the flagellin-induced anti-osteoclastogenic effect. The inhibitory effect of flagellin was mediated by IFN-β production. Flagellin stimulated IFN-β expression and release in bone marrow-derived macrophages, and IFN-β-neutralizing Ab prevented the flagellin-induced c-Fos down-regulation and the anti-osteoclastogenic effect. IFN-β gene induction by flagellin, LPS, or RANKL was dependent on STAT1 activation. Treatment with flagellin or RANKL stimulated STAT1 activation, and STAT1 deficiency or the JAK2 inhibitor AG490 dramatically prevented IFN-β induction in response to flagellin or RANKL. In addition, STAT1 deficiency abolished the anti-osteoclastogenic effect induced by flagellin or LPS. In contrast, flagellin stimulated osteoclast differentiation in cocultures of osteoblasts and bone marrow cells without inducing IFN-β. Thus, IFN-β acts as a critical modulator of osteoclastogenesis in response to TLR5 activation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 Disclosures
 References
 
Osteoclasts are multinucleated bone-resorbing cells derived from the monocyte-macrophage lineage under the presence of both a TNF-related factor known as receptor activator of NF-{kappa}B ligand (RANKL)3 and M-CSF (1, 2, 3, 4). RANKL is a member of the TNF ligand family and is expressed on the surface of osteoblasts, stromal cells, and vascular endothelial cells and is secreted by activated T cells (5). Binding of RANKL to its receptor RANK, which is expressed on the osteoclast precursor, leads to receptor trimerization and the activation of multiple intracellular signaling pathways that stimulate osteoclastic gene expression, bone resorbing function, and survival (5, 6). RANKL-RANK interaction recruits adaptor molecules such as TNF receptor-associated factors and stimulates downstream signaling cascades, including three well-known MAPK pathways, PI3K, and NF-{kappa}B (7). RANKL also leads to the induction of osteoclastogenic transcription factors, including c-Fos, Fra-1, and cytoplasmic, calcineurin-dependent NFAT 1 (NFATc1) (8, 9, 10, 11). c-Fos deficiency causes severe osteopetrosis with a lack of osteoclasts (8). NFATc1, which is up-regulated in RANKL-stimulated osteoclast precursors, is a critical transcription factor downstream of c-Fos during osteoclast differentiation (5, 11). Overexpression of NAFATc1 in osteoclast precursors causes efficient induction of osteoclast differentiation even without RANKL stimulation (11).

The innate immune system is the first line of defense against invading pathogens. Immune competent cells, such as macrophages, dendritic cells, neutrophils, and endothelial cells, recognize pathogen-associated molecular patterns that have been evolutionarily conserved in specific classes of microbes (12). TLRs are a family of pattern-recognition receptors that can be grouped according to the types of ligands they recognize. Lipid-based structures are recognized by TLR2 (in combination with TLR1 or TLR6) and TLR4; peptidoglycan and lipoteichoic acid act as ligands of TLR2. Bacterial LPS is recognized by TLR4. Viral or bacterial nucleic acids are recognized by TLR3, TLR7, TLR8, and TLR9; dsRNA and the CpG motif of unmethylated DNA act as ligands of TLR3 and TLR9, respectively. Finally, TLR5 senses a protein, flagellin, from both Gram-positive and Gram-negative bacteria (13, 14). TLR5 activation is associated with monomeric flagellin but not the filamentous molecule (15). Recently, it was shown that proinflammatory monomeric flagellin produced by salmonella during infection of host cells is not derived from polymeric bacterial cell wall-associated flagellum but instead is synthesized and secreted de novo by the bacterium after direct sensing of host-produced lysophospholipids (16). Stimulation of TLRs activates several signaling pathways and host defense responses through their intracellular domain, the Toll/IL-1R homology domain, and the downstream adaptor proteins, including MyD88, MyD88 adaptor-like, Toll/IL-1R-related adaptor protein inducing IFN-β (TRIF), and TRIF-related adaptor molecule. Different TLRs activate specific signals via different combinations of these adaptor proteins (13, 14).

The most prevalent diseases of the skeleton are periodontal diseases such as dental cysts, osteitis, and osteomyelitis, all of which are due to the actions of bacteria on bone (17). TLR activation by bacterial components has been shown to modulate osteoclastogenesis. Engagement of TLR4 and TLR2/6 by LPS and diacyl-lipopeptide, respectively, induces osteoclastogenesis in cocultures of osteoblasts and hemopoietic cells by stimulating RANKL expression in osteoblasts through MyD88-mediated signals (18). In the cocultures, CpG DNA, which acts as a TLR9 ligand, also stimulates osteoclastogenesis by RANKL expression in osteoblasts as a result of the more efficient TNF-{alpha} induction (19). In contrast, peptidoglycan, poly(I:C) RNA, LPS, and CpG DNA, the ligands for TLR2, TLR3, TLR4, and TLR9, respectively, strongly inhibit RANKL-induced osteoclast differentiation from precursors (20). Therefore, stimulation of TLRs appears to exert opposite effects on osteoclastogenesis depending on the culture conditions.

Unlike the other TLRs, the effect of TLR5 activation on osteoclast differentiation has not yet been studied. In this study, we investigate the action of STAT1-dependent IFN-β production in modulating osteoclastogenesis in response to TLR5 activation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 Disclosures
 References
 
Mice

Male C57BL/6 and C3H/HeJ mice aged 5 to 7 wk were obtained from Central Laboratory Animal. STAT1-deficient (STAT1–/–) mice (129/S6 mice) were purchased from Taconic Farms. Mice with a frameshift mutation in the TRIF gene (TRIFLps2 mice) were obtained from The Jackson Laboratory. All mice were maintained under pathogen-free conditions, and all procedures were reviewed and approved by the Seoul National University School of Dentistry Animal Care Committee.

Cytokines and reagents

{alpha}-MEM and FBS were purchased from Invitrogen Life Technologies. Human RANKL and M-CSF were from PeproTech. LPS (Escherichia coli 0111:B4) and 1,25(OH)2D3 were purchased from Sigma-Aldrich. AG490 was from Calbiochem. FliC derived from Salmonella typhimurium was obtained from Alexis Biochemicals. Purified, endotoxin-free recombinant FlaB derived from Vibrio vulnificus was prepared as described previously (21). Abs were purchased from the following sources: anti-phospho-ERK (Thr202/Tyr204), phospho-JNK (Thr183/Tyr185), phospho-p38 (Thr180/Tyr182), I{kappa}B{alpha}, and phosphotyrosine-STAT1 (Tyr701) Abs were from Cell Signaling Technology. Anti-c-Fos, phosphoserine-STAT1 (Ser727), and STAT1 Abs were from Upstate Biotechnology. Anti-β-actin Ab was from Sigma-Aldrich. Anti-TLR5 Ab was from IMGENEX Corporation. Neutralizing Abs against IFN-β and IFNAR1 were from R&D Systems.

Cell culture and osteoclast formation

Primary osteoblasts from calvariae of newborn C57BL/6 mice and bone marrow cells from the long bone of 5 to 7-wk-old mice were isolated as previously described (22). In brief, mouse calvariae were dissected aseptically and the connective tissue was removed. Cells were liberated by five sequential 15-min incubations of calvariae with {alpha}-MEM containing 0.1% collagenase and 0.2% dispase. Cells obtained from digestions 2–5 were pooled, cultured to confluence in 10-cm culture dishes in {alpha}-MEM containing 10% FBS and antibiotics (penicillin and streptomycin), and then used as primary osteoblasts. Mouse bone marrow cells were incubated overnight on 10-cm culture dishes in {alpha}-MEM containing 10% FBS and antibiotics. Nonadherent bone marrow cells were transferred to bacterial culture dishes and were cultured in the presence of M-CSF (50 ng/ml) for 3 days. Adherent cells were used as bone marrow-derived macrophages (BMMs) after the nonadherent cells were washed out. To generate osteoclasts, BMMs (4 x 104 cells) were cultured in 48-well culture plates in the presence of M-CSF (30 ng/ml) plus RANKL (100 ng/ml) with or without flagellin for 4 days. For some experiments, primary osteoblasts (2.5 x 104 cells) and bone marrow cells (3 x 105 cells) were cocultured in 48-well tissue culture plates with or without flagellin and 1,25(OH)2D3 for 6 days. Cells that stained positively for tartrate-resistant acid phosphatase (TRAP) and that contained more than three nuclei were counted as osteoclasts.

Measurement of IFN-β production

Cocultures of osteoblasts (2 x 105 cells) and bone marrow cells (3 x 106 cells), bone marrow cells (3 x 106 cells), and BMMs (5 x 105 cells) were cultured with the indicated reagents in {alpha}-MEM containing 10% FBS in 24-well culture plates. After the cultures were incubated for 24 h, concentrations of IFN-β in the medium were measured with the use of a murine IFN-β ELISA kit (R&D Systems) according to the manufacturer’s instructions.

Retroviral gene transduction in BMMs

To introduce ectopic gene expression in BMMs, a retroviral vector carrying c-Fos and a constitutively active form of NFATc1 was used. pMX-IRES-EGFP was used as a control vector. Retrovirus packaging was performed by transient transfection of these retroviral vectors into the Plat-E retroviral packaging cell line. After incubation in fresh medium for 2 days, supernatant fluid was collected from the retrovirus-producing cells. BMMs were incubated for 8 h with the viral supernatants in the presence of M-CSF (30 ng/ml) and polybrene (8 µg/ml). After the viral supernatant fluid was removed, BMMs were further cultured for 1 day in the presence of M-CSF (30 ng/ml).

Western blot analysis

Western blotting analyses were performed as described (22). In brief, total cells were washed twice with cold PBS and then lysed in lysis buffer containing 20 mM Tris-HCl, 150 mM NaCl, 1% Triton X-100, and protease and phosphatase inhibitors. Protein concentrations of cell lysates were determined by using the DC Protein Assay Kit (Bio-Rad), and equal amounts of protein for each sample were resolved by SDS-PAGE and were then transferred to a polyvinylidene difluoride membrane. After blocking with 5% skim milk in Tris-buffered saline containing 0.05% Tween 20, the blots were probed with anti-c-Fos (1/500 dilution), anti-TLR5 (1/500), anti-β-actin (1/10000), anti-phospho-ERK (1/1000), anti-phospho-JNK (1/1000), anti-phospho-p38 (1/1000), anti-I{kappa}B{alpha} (1/1000), anti-phosphotyrosine-STAT1 (1/1000), anti-phosphoserine-STAT1 (1/1000), and anti-STAT1 (1/1000) Abs. The blots were developed by using HRP-conjugated secondary Abs and were visualized by ECL (Amersham Biosciencess).

Preparation of nuclear extracts

Cells were washed twice with cold PBS, resuspended in cold buffer A (50 mM Tris-Cl (pH 8.0), 2 mM EDTA, 0.1% Nonidet P-40, 10% glycerol, and protease inhibitors), and incubated for 5 min at 4°C. Cytosol and nuclei were separated by brief centrifugation (5 min at 500 x g). The nuclear pellet was resuspended and lysed in ice-cold buffer B (20 mM HEPES-KOH (pH 7.6), 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 10% glycerol, 25 mM β-glycerophosphate, and protease inhibitors). Clear nuclear extract was collected after centrifugation (10 min at 15,000 x g).

RT-PCR analysis

Total RNA was prepared by using TRIzol reagent (Invitrogen Life Technologies) according to the manufacturer’s instructions. cDNA was synthesized from 2 µg total RNA by reverse transcriptase (Superscript II Preamplification System; Invitrogen Life Technologies). The following primers were used: mouse TLR5: sense, 5'-GCTCACTACAGTTCCCGAAA-3', and anti-sense, 5'-AAATGACTCCAGGGAAGGAC-3'; mouse c-Fos: sense, 5'-CTGGTGCAGCCCACTCTGGTC-3', and anti-sense, 5'-CTTTCAGCAGATTGGCAATCTC-3'; mouse IFN-β: sense, 5'-CTTCTCCACCACAGCCCTCTC-3', and anti-sense, 5'-CCCACGTCAATCTTTCCTCTT-3'; mouse universal IFN-{alpha}s to detect all known IFN-{alpha} transcripts: sense, 5'-ATGGCTAGGCYCTGTGCTTTC-3', and anti-sense, 5'-TCTGAYCACCTCCCAGGCACA-3'; mouse inducible NO synthase (iNOS): sense, 5'-ACGGAGAAGCTTAGATCTGGAGCAGAAGTG-3', and anti-sense, 5'-CTGCAGGTTGGACCACTGGATCCTGCCGAT-3'; mouse IL-1{alpha}: sense, 5'-AATCTCACTGTGCACCTTCG-3', and anti-sense, 5'-GGGCTCCCAAGTACAGGAAT-3'; mouse I-TAC: sense, 5'-GATGAACAGGAAGGTCACAGCCA-3', and anti-sense, 5'-AGGTTCTGTCGTCTCCCAGTCC-3'; and mouse β-actin: sense, 5'-AACCCTAAGGCCAACCGTGA-3', and anti-sense, 5'-ATGGATGCCACAGGATTCCA-3'. The amplified cDNA fragments were run on a 1.5% agarose gel, stained with ethidium bromide, and detected under UV light.

Statistical analysis

All quantitative data are presented as means ± SDs. Each experiment was performed three to four times, and the results from one representative experiment are shown. Statistical differences were analyzed by use of Student’s t test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 Disclosures
 References
 
Flagellin inhibits RANKL-induced osteoclast differentiation from its precursors

To examine the effect of TLR5 stimulation on osteoclast differentiation, we treated osteoclast precursors (BMMs) with flagellin in the presence of M-CSF plus RANKL. TLR5 was expressed on both primary osteoblasts and BMMs (Fig. 1A). Addition of M-CSF plus RANKL to the cultures for 4 days efficiently differentiated osteoclast precursors into multinucleated TRAP-positive osteoclasts. When recombinant FlaB derived from V. vulnificus was added at the start of culture, it dose-dependently suppressed RANKL-induced osteoclast formation. Inhibition of 87% of osteoclast differentiation was observed with 500 ng/ml FlaB. As previously reported (20), LPS (1 ng/ml) sufficiently inhibited RANKL-induced osteoclastogenesis (Fig. 1, B and C). To determine at which stage flagellin inhibits osteoclast formation, FlaB was added to the cultures on days 0–3. FlaB had an anti-osteoclastogenic effect only when added at the start of culture, which implies that it targets early osteoclastogenesis (Fig. 1D).


Figure 1
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  		FIGURE 1. Flagellin suppresses RANKL-induced osteoclast differentiation from precursors. A, TLR5 expression in osteoblasts and BMMs from C57BL/6 mice. Total RNA from the cells was isolated, and the expression levels of TLR5 (29 cycles) and β-actin (18 cycles) mRNA were analyzed by RT-PCR (upper panel). Whole lysates of the cells were subjected to Western blotting (WB) with the indicated Abs (lower panel). B and C, BMMs from C57BL/6 mice were cultured in the presence of M-CSF (30 ng/ml) plus RANKL (100 ng/ml) for 4 days. FlaB (5–500 ng/ml) or LPS (1 ng/ml) was added at the start of culture. *, p < 0.01 vs untreated control. D, FlaB (500 ng/ml) was added to the cultures on days 0, 1, 2, and 3. The cells were stained for TRAP, and TRAP-positive multinucleated cells were counted as osteoclasts. *, p < 0.01 vs untreated control. All values are the means ± SD of three independent experiments.

 
Flagellin inhibits osteoclastogenesis by down-regulating c-Fos expression

To gain insights into the molecular mechanism by which flagellin inhibits osteoclastogenesis, we analyzed the effect of flagellin on the induction of transcription factors induced by RANKL. Induction and activation of c-Fos and NFATc1 are essential for RANKL-induced osteoclastogenesis (5, 8, 11). RANKL increased c-Fos mRNA and protein expression, whereas FlaB increased c-Fos mRNA expression only. Interestingly, FlaB inhibited RANKL-induced c-Fos protein but not mRNA levels (Fig. 2A). To investigate whether the reduction in c-Fos protein expression was involved in mediating the inhibitory effect of FlaB, we overexpressed c-Fos and a constitutively active form of NFATc1 in BMMs by using a retroviral vector system. Overexpression of either c-Fos or constitutively active NFATc1 prevented the anti-osteoclastogenic action of FlaB (Fig. 2B).


Figure 2
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  		FIGURE 2. Flagellin interferes with RANKL-induced c-Fos protein expression. A, BMMs from C57BL/6 mice were cultured for 24 h in the presence of M-CSF (30 ng/ml) with or without RANKL (100 ng/ml) and FlaB (500 ng/ml). c-Fos (26 cycles) and β-actin (18 cycles) mRNA expression were analyzed by RT-PCR. c-Fos protein expression was detected by Western blotting (WB). B, BMMs infected with the indicated retrovirus were cultured with or without FlaB (500 ng/ml) in the presence of M-CSF (30 ng/ml) plus RANKL (100 ng/ml) for 4 days. Cells were then stained for TRAP, and the number of TRAP-positive multinucleated cells was counted as osteoclasts. *, p < 0.01 vs vector control of FlaB-treated groups. All values are expressed as the means ± SD of three independent experiments.

 
Flagellin stimulates IFN-β expression and secretion in BMMs

Because IFN-β has been shown to inhibit osteoclastogenesis by interfering with RANKL-induced c-Fos expression (23), we next investigated whether IFN-β is involved in the inhibitory effect of flagellin. We found that FlaB dose-dependently stimulated IFN-β mRNA expression in BMMs. FliC (20 ng/ml) derived from S. typhimurium and LPS (1 ng/ml) also increased IFN-β production. Increased IFN-β mRNA expression was accompanied by stimulation of IFN-β release (Fig. 3A). IFN-β mRNA expression by flagellin was apparent at 3 h and increased further at 24 h. Although flagellin also stimulated the induction of IFN-{alpha} genes, their expression was very low compared with IFN-β expression. We also found that flagellin induced a rapid, transient up-regulation of iNOS mRNA expression (Fig. 3B). Previously, it was reported that the induction of NO synthesis by flagellin is dependent on signaling via the TLR5/4 complex (24). That study showed that TLR5 can form heteromeric complexes with TLR4 as well as homomeric complexes when TLR4 and TLR5 are overexpressed and that flagellin induces NO synthesis in HeNC2 cells, a murine macrophage cell line that express wild-type TLR4, but not in TLR4-mutant GG2EE cells. Therefore, we next examined whether TLR5/4 complex formation is required for flagellin-induced IFN-β production in BMMs. FlaB, but not LPS, stimulated induction of the IFN-β gene in BMMs derived from LPS-hyporesponsive C3H/HeJ mice (Fig. 3C). Consistent with this, FlaB, but not LPS, inhibited RANKL-induced osteoclast formation from BMMs of C3H/HeJ mice (Fig. 3D). These results indicate that flagellin-induced IFN-β production is independent of TLR4 and does not stem from the influence of LPS contamination.


Figure 3
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  		FIGURE 3. Flagellin induces IFN-β expression in BMMs. A, BMMs from C57BL/6 mice were cultured for 24 h in the presence of M-CSF (30 ng/ml) with or without FlaB (50–500 ng/ml), FliC (20 ng/ml), and LPS (1 ng/ml). IFN-β (29 cycles) and β-actin (18 cycles) mRNA expression were analyzed by RT-PCR, and the concentration of IFN-β in the medium was measured by using ELISA kits. *, p < 0.01 vs untreated control. B, BMMs were cultured with FlaB (500 ng/ml) for up to 24 h. IFN-β (29 cycles), IFN-{alpha}s (34 cycles), iNOS (28 cycles), and β-actin (18 cycles) mRNA expression were analyzed. C, BMMs from C3H/HeJ mice were cultured for 24 h in the presence of M-CSF (30 ng/ml) with or without FlaB (50–500 ng/ml) and LPS (1 ng/ml). IFN-β and β-actin mRNA expression were analyzed. D, BMMs from C3H/HeJ mice were cultured for 4 days in the presence of M-CSF (30 ng/ml) plus RANKL (100 ng/ml) with or without FlaB (50 and 500 ng/ml) and LPS (1 ng/ml). TRAP-positive multinucleated cells were counted. *, p < 0.01 vs untreated control. All values are means ± SD of three independent experiments.

 
The anti-osteoclastogenic effect of flagellin is mediated by IFN-β production

To investigate whether IFN-β mediates the anti-osteoclastogenic activity of flagellin, we used neutralizing Abs against IFN-β and IFNAR1. Anti-IFN-β Ab completely rescued the RANKL induction of c-Fos protein expression that was inhibited by flagellin (Fig. 4A). Moreover, Ab to either IFN-β or IFNAR1 reversed the RANKL-induced osteoclastogenesis inhibited by flagellin. Unrelated IgG failed to affect the inhibitory effect of flagellin (Fig. 4B). Flagellin inhibited osteoclastogenesis by down-regulating c-Fos protein expression (Fig. 2). Thus, these results show that IFN-β production by flagellin suppresses RANKL-induced c-Fos protein expression, thereby preventing osteoclastogenesis.


Figure 4
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  		FIGURE 4. IFN-β mediates flagellin-induced anti-osteoclastogenic activity. A, BMMs from C57BL/6 mice were cultured for 24 h in the presence of M-CSF (30 ng/ml) with or without RANKL (100 ng/ml), FlaB (500 ng/ml), and anti-IFN-β Ab (4 µg/ml). c-Fos protein expression was analyzed by Western blotting. B, BMMs were cultured for 4 days in the presence of M-CSF (30 ng/ml) plus RANKL (100 ng/ml) with or without FlaB (500 ng/ml), anti-IFN-β Ab (4 µg/ml), anti-IFNAR1 Ab (4 and 8 µg/ml), and unrelated IgG (8 µg/ml). Cells were then stained for TRAP, and the number of TRAP-positive multinucleated cells was scored. *, p < 0.01 vs untreated control. #, p < 0.01 vs treatment with FlaB alone. All values are means ± SD of three independent experiments.

 
TRIF-dependent signal is not required for flagellin-induced IFN-β production

Toll/IL-1R domain-containing adapter molecules are known to be recruited to the cytoplasmic Toll/IL-1R domains of activated TLRs and to mediate downstream signaling, including the activation of NF-{kappa}B and MAPKs (13, 14). Among these adapters, MyD88 functions as a common adapter to all known TLRs, except for TLR3, whereas TRIF is specifically involved in TLR3- and TLR4-mediated MyD88-independent NF-{kappa}B activation (25). Activation of TRIF also leads to the activation of IFN regulatory factor (IRF)-3 via IKK{epsilon} and TNF receptor-associated factor family member-associated NF-{kappa}B activator-binding kinase 1 (TBK1), thereby stimulating the induction of IFN-β genes (14, 25, 26, 27, 28). Thus, we investigated whether the TRIF-mediated signal is involved in flagellin-induced IFN-β production and the anti-osteoclastogenic effect. For this purpose, we used BMMs from TRIFLps2 mice that bear a distal frameshift mutation that generates a nonfunctional TRIF (28). LPS-induced IFN-β secretion was dramatically reduced in BMMs from TRIFLps2 mice, whereas FlaB-induced IFN-β secretion was not affected (Fig. 5A). In accordance with IFN-β production, defective TRIF signaling had no effect on flagellin-induced anti-osteoclastogenic activity. LPS-induced anti-osteoclastogenic activity was slightly rescued in BMMs from TRIFLps2 mice, as shown by the generation of several mononucleated TRAP-positive cells (Fig. 5B).


Figure 5
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  		FIGURE 5. Flagellin induction of IFN-β is independent of TRIF-mediated signals. A, BMMs from wild-type (C57BL/6) and TRIFLps2 mice were cultured for 1 day in the presence of M-CSF (30 ng/ml) with or without FlaB (500 ng/ml) and LPS (10 ng/ml). The concentration of IFN-β in the medium was measured by using ELISA kits. *, p < 0.01 vs wild-type counterpart. B, BMMs from wild-type and TRIFLps2 mice were cultured for 4 days in the presence of M-CSF (30 ng/ml) plus RANKL (100 ng/ml) with or without FlaB (500 ng/ml) and LPS (10 ng/ml). Cells were then stained for TRAP, and the number of TRAP-positive multinucleated cells was scored. All values are means ± SD of three independent experiments.

 
STAT1 is required for IFN-β gene expression by flagellin and LPS

IFNAR stimulation by type I IFNs (IFN-{alpha}s and IFN-β) results in the activation of the Janus family protein tyrosine kinases Tyk2 and Jak1, which is followed by the tyrosine phosphorylation of STAT1 and STAT2. This activation elicits cellular responses through activation of IFN-stimulated transcriptional factor 3 (the heterotrimeric complex consisting of STAT1, STAT2, and IRF-9), which binds to the IFN-stimulated response element to activate IFN-inducible genes as well as IFN-{gamma}-activated factor/IFN-{alpha}-activated factor (the STAT1 homodimer), which binds to the IFN-{gamma}-activated site (GAS) and activates its target genes (29, 30). The inhibitory action of IFN-β on osteoclastogenesis is linked to the IFN-stimulated transcriptional factor 3-mediated gene induction pathway. The anti-osteoclastogenic activity of IFN-β is abrogated in BMMs of mice lacking STAT1 or IRF-9, but not IRF-1 (23). Therefore, we further confirmed whether flagellin-induced IFN-β production mediates the anti-osteoclastogenic action by using STAT1-deficient mice. STAT1 deficiency reversed the RANKL-induced osteoclastogenesis inhibited by flagellin and LPS (Fig. 6A). Interestingly, flagellin- and LPS-induced IFN-β expression and release were dramatically reduced in BMMs from STAT1–/– mice. In contrast, the expression levels of IL-1{alpha} mRNA by LPS were slightly increased in STAT1–/– mice (Fig. 6B). These results show that STAT1 is required for TLR-induced IFN-β production as well as for IFNAR signaling.


Figure 6
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  		FIGURE 6. STAT1 is required for the induction of IFN-β by flagellin or LPS. A, BMMs from wild-type (129/S6) and STAT1–/– mice were cultured for 4 days in the presence of M-CSF (30 ng/ml) plus RANKL (100 ng/ml) with or without FlaB (500 ng/ml) and LPS (10 ng/ml). Cells were then stained for TRAP, and the number of TRAP-positive multinucleated cells was scored. *, p < 0.01 vs wild-type counterpart. B, BMMs from wild-type and STAT1–/– mice were cultured for 1 day in the presence of M-CSF (30 ng/ml) with or without FlaB (500 ng/ml) and LPS (10 ng/ml). IFN-β (29 cycles), IL-1{alpha} (26 cycles), and β-actin (18 cycles) mRNA expression were analyzed by RT-PCR, and the concentration of IFN-β in the medium was measured by using ELISA kits. *, p < 0.01 vs wild-type counterpart. All values are means ± SD of three independent experiments.

 
JAK2/STAT1 pathway is involved in the induction of IFN-β in response to flagellin or RANKL

RANKL has been shown to stimulate IFN-β induction as an autocrine negative feedback signal that limits RANKL-induced osteoclastogensis (23). We further investigated whether STAT1 is also required for RANKL-induced IFN-β expression. Treatment with either RANKL or flagellin for 3 h stimulated induction of IFN-β mRNA in BMMs from wild-type (129/S6) mice, whereas this induction was abrogated in STAT1–/– mice, which implies that STAT1 is also required for the RANKL-induced transient elevation of IFN-β expression (Fig. 7A).


Figure 7
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  		FIGURE 7. Preventing STAT1 activation inhibits IFN-β expression induced by flagellin or RANKL. A, BMMs from wild-type (129/S6) and STAT1–/– mice were cultured for 3 h in the presence of M-CSF (30 ng/ml) with or without FlaB (500 ng/ml) and RANKL (RL; 100 ng/ml). IFN-β (29 cycles), iNOS (28 cycles), I-TAC (26 cycles), and β-actin (18 cycles) mRNA expression were analyzed by RT-PCR. B, BMMs from C57BL/6 mice were stimulated with FlaB (500 ng/ml) for the indicated times. Whole-cell lysates were subjected to Western blotting with the indicated Abs. STAT1 blots served as the loading control. PY, phosphotyrosine; PS, phosphoserine. C, As in B, except RANKL (100 ng/ml) was used instead of FlaB. D and E, BMMs from C57BL/6 mice were pretreated with or without AG490 (20 µM) for 1 h followed by a 3 h treatment with RANKL or FlaB. Nuclear extracts were analyzed by Western blotting with Abs to PY-STAT1 and STAT1 (D). RT-PCR was performed (E).

 
A recent study showed that IFN-β inhibits the differentiation of human monocytes into osteoclasts, at least in part through the chemokine CXCL11, which is also called IFN-inducible T cell {alpha} chemoattractant (I-TAC) (31). It was also reported that RANKL up-regulates iNOS mRNA expression and NO production in murine osteoclast precursors through NF-{kappa}B-dependent IFN-β induction (32). Consistent with these reports, either RANKL or flagellin stimulated the induction of iNOS and I-TAC. Like IFN-β induction, the induction of the two genes was also dependent on the presence of STAT1 (Fig. 7A).

We next analyzed several flagellin-induced signaling pathways in BMMs. Activation of ERK, JNK, and p38 MAPK was transiently induced after 15 min of flagellin stimulation. I{kappa}B{alpha} degradation, an event required for NF-{kappa}B activation, was stimulated at 15 min and delayed time points (Fig. 7B). Next, we examined STAT1 activation in response to flagellin. STAT tyrosine phosphorylation is generally considered to be an essential prerequisite for SH2 domain-mediated dimerization, subsequent translocation into the nucleus, and binding to target DNA (33). In addition, STAT serine phosphorylation also has profound effects on target gene transcription in certain cases (34). In contrast with the activation of MAPKs, STAT1 tyrosine phosphorylation by flagellin increased at a relatively delayed time point (2 h) and was maintained for at least 4 h. STAT1 serine phosphorylation maximally increased at the earliest time point (15 min) and was maintained for up to 4 h. Total levels of STAT1 were maintained throughout the time course (Fig. 7B). We also investigated RANKL-induced STAT1 activation. Like flagellin, RANKL also stimulated STAT1 tyrosine phosphorylation at delayed time points (2–4 h). In contrast, serine phosphorylation increased from 15 to 30 min and then declined (Fig. 7C). AG490, a pharmacologic inhibitor of JAK2, prevented the nuclear translocation of tyrosine-phosphorylated STAT1 in response to RANKL or flagellin (Fig. 7D). In addition, AG490 inhibited RANKL- or flagellin-induced gene induction of IFN-β, I-TAC, and iNOS, which implies that a JAK2/STAT1-mediated signal is required for IFN-β induction by RANKL or flagellin (Fig. 7E).

Flagellin stimulates osteoclast formation in bone marrow cell-osteoblast cocultures

Engagement of TLRs by several ligands can also stimulate osteoclastogenesis in cocultures of osteoblasts and bone marrow cells by stimulating RANKL expression (18). Thus, depending on the experimental condition, TLR activation seems to inhibit or stimulate osteoclast differentiation. To clarify this issue, we next investigated how TLR5 activation affects osteoclastogenesis in cocultures. As shown in Fig. 8A, flagellin stimulated osteoclast formation and strongly augmented the osteoclastogenic effect of 1,25(OH)2D3. Because flagellin inhibited RANKL-induced osteoclast formation from BMMs via IFN-β production (Fig. 4), we investigated IFN-β production in cocultures of osteoblasts and bone marrow cells. Neither flagellin nor 1,25(OH)2D3 stimulated IFN-β production in cocultures. Furthermore, even in the bone marrow cell cultures without osteoblasts, flagellin had no effect on IFN-β production (Fig. 8B). We also found that flagellin increased the RANKL/osteoprotegerin ratio in osteoblasts (data not shown). Thus, our findings show that flagellin stimulates osteoclast differentiation in cocultures by up-regulating the RANKL/osteoprotegerin ratio as well as by not inducing IFN-β production, which implies that IFN-β production acts as a critical determinant of the effects of flagellin on osteoclast differentiation.


Figure 8
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  		FIGURE 8. Flagellin stimulates osteoclast formation in bone marrow cell-osteoblast cocultures. A, Primary osteoblasts and bone marrow cells were cocultured with or without FlaB (500 ng/ml) and 1,25(OH)2D3 (VD3; 1 nM) for 6 days. The number of TRAP-positive multinucleated cells was scored. *, p < 0.01 vs untreated control. #, p < 0.01 vs a group treated with 1,25(OH)2D3 alone. B, Cocultures of osteoblasts and BMs, BMs, and BMMs were cultured with or without 1,25(OH)2D3 (1 nM), FlaB (500 ng/ml), and M-CSF (30 ng/ml) for 1 day. The concentration of IFN-β in the culture medium was measured by using ELISA kits. All values are means ± SD of three independent experiments.

 

   Discussion
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
Disclosures
 References
 
Various factors, including cytokines, chemokines, and hormones, have been shown to modulate osteoclast differentiation by their direct action on osteoclast precursors. Osteoprotegerin, a soluble decoy receptor for RANKL, is produced by osteoblasts and inhibits osteoclast formation by blocking RANKL binding to RANK (35, 36). IL-4 (37), GM-CSF (38), IFN-β (23), and IFN-{gamma} (39) also inhibit osteoclastogenesis by inhibiting RANKL-induced MAPK and NF-{kappa}B signaling, down-regulating c-Fos expression, diverting the cells to a macrophage or dendritic cell lineage, or promoting proteasomal degradation of TNF receptor-associated factor 6. In contrast, IL-1 (40), TNF-{alpha} (40, 41), and the pituitary-derived hormone follicle-stimulating hormone (42) have stimulatory effects on RANKL-induced osteoclastogenesis. In addition, TLR activation by bacterial components may also regulate osteoclast differentiation by direct action on osteoclast precursors as well as by indirect action via osteoclast supporting cells (18, 19, 20).

The results presented in this study show that stimulation of TLR5 modulates osteoclastogenesis by regulating IFN-β production. TLR5 activation by flagellin inhibited RANKL-induced osteoclast differentiation from BMMs (Fig. 1). This anti-osteoclastogenic activity stemmed from suppression of c-Fos protein expression (Fig. 2). IFN-β induction by RANKL negatively limits osteoclastogenesis by suppression of c-Fos protein expression (23). We also found that IFN-β production mediated the flagellin-induced reduction in c-Fos expression and anti-osteoclastogenic action in BMMs (Figs. 3 and 4). Although the heteromeric TLR5/4 complex has been shown to be required for flagellin-induced iNOS expression (24), this complex is not necessary for flagellin induction of IFN-β (Figs. 3C and 5A).

The promoter region of the IFN-β gene contains at least four regulatory cis-elements: AP-1, NF-{kappa}B, and the two elements activated by members of the IRF family (43). Among the TLR family members, TLR3- and TLR4-mediated signals have been shown to induce IFN-β induction through TRIF-dependent signals (14, 25, 28). IFN-β induction via TLR4 or TLR3 is mediated by the homodimer of IRF-3, which is activated by a TRIF-, NAK-associated protein 1-, and TNF receptor-associated factor family member-associated NF-{kappa}B activator-binding kinase 1-dependent signaling pathway (43). However, TRIF-mediated IFN gene induction via TLR4 and TLR3 differs. TLR3 activation can stimulate the induction of IFN-{alpha} as well as IFN-β, whereas TLR4 induces IFN-β only. In addition, IRF-3-mediated activation of the IFN-stimulated response element by TLR4 but not TLR3 requires the p65 subunit of NF-{kappa}B (43, 44). In this study, LPS-induced IFN-β production was dramatically suppressed in TRIFLps2 BMMs, whereas flagellin-induced IFN-β production was insensitive (Fig. 5). Moreover, LPS but not flagellin stimulated IRF-3 activation, as determined by phosphorylation and dimerization (at 1, 2, and 4 h; data not shown). Thus, these results imply that TRIF-mediated IRF-3 activation is not required for flagellin induction of IFN-β. TLR5 activation by flagellin can stimulate the activation of MAPKs and NF-{kappa}B, leading to the induction of IL-8 and expression of the macrophage inflammatory protein 3{alpha} gene (45). TLR5 also activates the JAK/STAT pathway through a protein-synthesis-dependent mechanism in epithelial cells. IL-6 production mediates flagellin-induced STAT3 activation, whereas STAT1 activation is independent of IL-6, IFN-β, and IFN-{gamma} (46). In the present study, flagellin-induced STAT1 activation exhibited delayed kinetics and was suppressed by the pharmacological JAK2 inhibitor AG490 (Fig. 7). Interestingly, flagellin-induced IFN-β expression required STAT1 activation. In STAT1–/– BMMs, flagellin- and LPS-induced IFN-β production was dramatically suppressed (Fig. 6). In addition, AG490 significantly suppressed flagellin-stimulated IFN-β induction (Fig. 7).

RANKL induces transient IFN-β mRNA expression via an NF-{kappa}B-dependent pathway in osteoclast precursors (32). The induction of IFN-β mRNA by RANKL was also abolished in osteoclast precursor cells from Fos–/– mice but not from IRF-3/IRF-9–/– mice (23). In our study, STAT1 deficiency also prevented RANKL-induced IFN-β expression (Fig. 7). Thus, STAT1 activation may be considered an essential prerequisite for IFN-β expression in response to TLR signals and RANKL. However, the exact molecular mechanism of this STAT1-dependent IFN-β induction remains to be elucidated.

Recently, I-TAC was identified as a mediator of IFN-β-induced anti-osteoclastogenic action in human osteoclast precursors (31). In this study, we showed that flagellin and RANKL induce I-TAC expression through a STAT1-dependent pathway. However, in contrast with the case in human osteoclast precursors, I-TAC (20–200 ng/ml) had no effect on RANKL-induced osteoclast formation from murine BMMs (data not shown), which suggests that I-TAC does not mediate IFN-β-induced anti-osteoclastogenic action in murine osteoclast precursors. It was also reported that RANKL-induced IFN-β triggers iNOS/NO as an important negative signal during osteoclastogenesis (32). The interaction of STAT1 with the GAS site has been shown to be necessary for full expression of the mouse iNOS gene in response to IFN-{gamma} and LPS in murine macrophages (47). Thus, our finding of STAT1-dependent iNOS expression in response to flagellin or RANKL could stem from a reduction in IFN-β expression or a deficiency of STAT1 binding on the GAS site of the iNOS gene. We also found that flagellin-induced IFN-β expression is dominant compared with IFN-{alpha}s (Fig. 3B). NO has been shown to inhibit IFN-{alpha} production of plasmacytoid dendritic cells partly via a cGMP-dependent mechanism (48). Thus, this NO production may negatively regulate flagellin-induced IFN-{alpha} production. However, whether blockade of iNOS/NO reveres the flagellin- or IFN-β-induced anti-osteoclastogenic effect remains to be studied.

In contrast with the case in BMMs, flagellin did not induce IFN-β expression in bone marrow cells or in cocultures of osteoblasts and bone marrow cells. Rather, flagellin stimulated osteoclastogenesis in cocultures (Fig. 8). Stimulation of TLRs exerts a dual effect on osteoclastogenesis depending on the culture condition (18, 19, 20). In addition, a previous report showed that the effects of TLRs and TNF-{alpha} on osteoclastogenesis are differently regulated, depending on osteoclast precursors in the bone marrow and extramedullary organs (49). Thus, our finding of flagellin-induced IFN-β production in BMMs but not in bone marrow cells or in cocultures may shed light on this discrepancy.

Mammalian cells can sense not only extracellular flagellin through TLR5 but also intracellular flagellin through Ipaf. Recent reports have shown that Ipaf, a cytosolic pattern-recognition receptor in the family of nucleotide-binding oligomerization domain-leucine-rich repeat proteins, recognizes cytosolic flagellin and thereby triggers caspase-1-dependent cell death and secretion of IL-1β (50, 51, 52). Therefore, during bacterial infection, flagellin may modulate osteoclastogensis and the innate immune system by these two sensory pathways. In summary, we have shown that TLR5 activation by flagellin modulates RANKL-induced c-Fos protein expression and osteoclast differentiation by regulating IFN-β production, which is dependent on STAT1 activation.


   Disclosures
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 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 Disclosures
References
 
The authors have no financial conflict of interest.


   Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This study was supported by a grant from the Korea Health 21 R&D Project, Ministry of Health and Welfare, Republic of Korea (A060480). Back

2 Address correspondence and reprint requests to Dr. Zang Hee Lee, Department of Cell and Developmental Biology, College of Dentistry, Seoul National University, 28 Yeongon-Dong, Jongro-Gu, Seoul 110-749, Republic of Korea. E-mail address: zang1959{at}snu.ac.kr Back

3 Abbreviations used in this paper: RANKL, receptor activator of NF-{kappa}B ligand; RANK, receptor activator of NF-{kappa}B; NFATc1, cytoplasmic, calcineurin-dependent NFAT 1; TRIF, TIR-related adaptor protein inducing interferon-β; BMM, bone marrow-derived macrophage; TRAP, tartrate-resistant acid phosphatase; iNOS, inducible NO synthase; IRF, IFN regulatory factor; I-TAC, IFN-inducible T cell {alpha} chemoattractant; GAS, IFN-{gamma}-activated site. Back

Received for publication February 13, 2007. Accepted for publication November 18, 2007.


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S.-H. Lee, J. S. Kim, H.-K. Jun, H.-R. Lee, D. Lee, and B.-K. Choi
The Major Outer Membrane Protein of a Periodontopathogen Induces IFN-{beta} and IFN-Stimulated Genes in Monocytes via Lipid Raft and TANK-Binding Kinase 1/IFN Regulatory Factor-3
J. Immunol., May 1, 2009; 182(9): 5823 - 5835.
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