Dectin-1 signaling inhibits osteoclastogenesis via IL-33-induced inhibition of NFATc1

Abnormal osteoclast activation contributes to osteolytic bone diseases (OBDs). It was reported that curdlan, an agonist of dectin-1, inhibits osteoclastogenesis. However, the underlying mechanisms are not fully elucidated. In this study, we found that curdlan potently inhibited RANKL-induced osteoclast differentiation and the resultant bone resorption. Curdlan inhibited the expression of nuclear factor of activated T-cells, cytoplasmic 1 (NFATc1), the key transcriptional factor for osteoclastogenesis. Notably, dectin-1 activation increased the expression of MafB, an inhibitor of NFATc1, and IL-33 in osteoclast precursors. Mechanistic studies revealed that IL-33 enhanced the expression of MafB in osteoclast precursors and inhibited osteoclast precursors to differentiate into mature osteoclasts. Furthermore, blocking ST2, the IL-33 receptor, partially abrogated curdlan-induced inhibition of NFATc1 expression and osteoclast differentiation. Thus, our study has provided new insights into the mechanisms of dectin-1-induced inhibition of osteoclastogenesis and may provide new targets for the therapy of OBDs.


INTRODUCTION
Osteolytic bone diseases (OBDs) are a common complication in rheumatoid arthritis [1], osteoporosis [1] and Paget's disease [2], as well as in malignancies, such as multiple myeloma (MM) [3]. OBDs can adversely affect the quality of life and survival of patients due to severe bone pain, pathological fractures and hypercalcemia [1,4]. Bisphosphonates are widely used in the treatment of OBDs [5][6][7]. New therapeutic reagents have been reported to treat OBDs [8]. However, current therapies rarely halt the progression of OBDs. OBDs are caused mainly by abnormal osteoclast activation and osteoblast inhibition [9,10]. Therefore, further investigation of new strategies to inhibit the formation and function of osteoclasts will be important for the therapy of OBDs.

Research Paper
(Ctsk) and TRAP, which are responsible for osteoclastinduced bone resorption. MafB, IRF8 and BCL6 are inhibitors of NFATc1expression.
In this study, we showed that dectin-1 potently inhibited the differentiation and bone resorption of osteoclasts induced by RANKL plus M-CSF. Dectin-1 activation by curdlan in osteoclast precursors increased MafB expression and decreased NFATc1 expression, suggesting that dectin-1 inhibits NFATc1 through the stimulation of MafB. Interestingly, dectin-1 increased IL-33 expression in osteoclast precursors. Mechanistic studies revealed that IL-33 also increased MafB expression and decreased NFATc1 expression in osteoclast precursors and inhibited osteoclast precursors to differentiate into mature osteoclasts. Furthermore, blocking ST2 (IL-33 receptor) partially abrogated curdlan-induced inhibition of NFATc1 expression and osteoclast differentiation. Thus, our study has provided new insights into the mechanisms of dectin-1-induced inhibition of osteoclastogenesis and may provide new targets for the therapy of OBDs. days and with MCSF plus RANKL for 3 days. (A) curdlan at the indicated dosages was added at day 2. Cultures without addition of curdlan were used as controls. Cultures were stained for TRAP + cells. TRAP + cells with more than three nuclei were counted as osteoclasts (OCs).

Dectin-1 activation inhibits osteoclastogenesis in vitro
To examine the effects of dectin-1 signaling on osteoclastogenesis, we cultured bone marrow cells (BMCs) with RANKL plus M-CSF in the presence or absence of a selective dectin-1 agonist Curdlan. Curdlan treatment inhibited RANKL-induced osteoclast formation by decreasing the number and size of TRAP + multinucleated (> 3 nuclei) osteoclasts in a dose-dependent manner ( Figure 1A-1D). Concomitant to the inhibition of osteoclast formation, mRNA expression of Dcstamp for osteoclast fusion and Ctsk for bone resorption and the protein levels of bone resorption-related gene TRAP5b in the culture supernatants were also decreased by curdlan treatment ( Figure 1E,1F).
To explore the function of dectin-1 in curdlaninduced inhibition of osteoclast formation, we generated osteoclasts from dectin-1 knockout (dectin-1 -/-) mice with or without addition of curdlan. As shown in Figure 1G, curdlan treatment failed to inhibit dectin-1 -/osteoclast formation as compared to the untreated controls.
To assess the effects of curdlan on osteoclast bone resorption, we performed resorption pit formation assay. As compared to untreated controls, curdlan treatment remarkably diminished RANKL-induced osteoclast bone resorption ( Figure 4F,4G). Together, these results demonstrated that dectin-1 activation in osteoclast precursors inhibits osteoclast differentiation and bone resorptive function.

Dectin-1 signaling inhibits NFATc1 in osteoclast precursors
To explore the molecular mechanisms of dectin-1-induced inhibition of osteoclast differentiation, we performed gene expression profiling (GEP) analyses in osteoclast precursors with (Cur-pre-OC) or without (pre-OC) curdlan treatment. We found that Cur-pre-OCs expressed lower levels of Nfatc1, the master transcription factor for osteoclast differentiation [12], than pre-OCs ( Figure 2A); whereas the transcription factors Mafb, Bcl6, Irf7, Irf8 and Irf9 were upregulated in Cur-pre-OCs compared to pre-OCs ( Figure 2A). Interestingly, Curpre-OCs expressed higher levels of Clec7a (the gene for dectin-1) as compared to pre-OCs ( Figure 2A). The decrease of NFATc1 in Cur-pre-OCs compared to pre-OCs was confirmed by quantitative real-time PCR (qPCR) ( Figure 2B) and Western-blot analysis ( Figure 2C). The up-regulation of Mafb, Bcl6, Irf7 and Irf8 in Cur-pre-OCs compared to pre-OCs was confirmed by qPCR ( Figure 2D).

IL-33 inhibits osteoclastogenesis in vitro
Dectin-1 signaling stimulates the production of some inflammatory cytokines [19,20], which may be involved in osteoclast differentiation. To address this issue, microarray data were examined. Cur-pre-OCs expressed higher levels of Tnf, Il1b and Il33 as compared to pre-OCs ( Figure 3A). qPCR and ELISA further confirmed the increased expression of IL-33 in Cur-pre-OCs compared to pre-OCs ( Figure 3B,3C). These results demonstrated that dectin-1 activation increased IL-33 expression in osteoclast precursors.
TNF-α and IL-1β were shown to promote but not inhibit osteoclast differentiation [21,22]. We next examined the role of IL-33 in dectin-1-induced inhibition of osteoclast differentiation. Osteoclasts were generated in vitro in the presence of M-CSF plus RANKL with or without addition of curdlan or IL-33. IL-33 potently inhibited the development of osteoclasts ( Figure 4A) by decreasing the cell number and size of osteoclasts as compared with untreated control (Figure 4B,4C); while IL-33 induced comparable inhibition on osteoclastogenesis as compared to curdlan ( Figure 4A-4C).

Blocking ST2 partially abrogates curdlaninduced inhibition of osteoclastogenesis
To examine the role of IL-33 in dectin-1-induced inhibition of osteoclastogenesis, a ST2 (the IL-33 receptor) blocking antibody (αST2) was used during osteoclast culture. The addition of αST2 compared to control IgG increased the generation of osteoclasts in curdlan-treated cultures ( Figure 5A), as demonstrated by significantly higher osteoclast cell number and size in the cultures treated with curdlan plus αST2 compared to curdlan alone ( Figure 5B,5C), while lower cell number and size of osteoclasts were obtained in the cultures treated with curdlan plus αST2 compared to untreated controls ( Figure  5A-5C), indicating that blocking ST2 partially abrogated curdlan-induced inhibition of osteoclast differentiation. Though as compared to untreated cells, cells treated with Curdlan plus αST2 expressed lower levels of Nfatc1 and Ctsk ( Figure 5D,5E), these cells expressed higher levels of Nfatc1 and Ctsk than cells treated with curdlan alone (Figure 5D,5E). Furthermore, cells treated with Curdlan plus αST2 slightly increased the expression of TRAP5b as compared to curdlan-treated cells ( Figure 5F). Collectively, these results demonstrated the important role of IL-33 in mediating dectin-1-induced inhibition of osteoclastogenesis.

DISCUSSION
Abnormal osteoclast activation is a major cause of osteolytic bone diseases (OBDs); therefore, targeting osteoclasts may have important clinical significance in the therapy of OBDs [3,6,7,23]. In this study, we found that dectin-1 activation with curdlan inhibited RANKL-induced osteoclast differentiation by reducing osteoclast cell number and size in vitro. Dectin-1 activation also decreased the expression of Dcstamp, the key regulator of osteoclast precursor differentiation and fusion, and TRAP and Cathepsin K, which are essential for osteoclastic bone resorption. In addition, in the functional tests, we found that dectin-1 activation inhibited bone resorption of RANKL-induced osteoclasts. These results are consistent with previous observations that intravenous injection with Candida albicans enhanced new bone formation in mice [24], and dectin-1 activation in osteoclast precursors inhibited RANKL-induced osteoclast differentiation in vitro [18]. Furthermore, curdlan treatment failed to suppress dectin-1 -/osteoclast differentiation. Thus our data demonstrated that dectin-1 activation inhibits osteoclast differentiation and function.
We and others found that dectin-1 activation inhibited NFATc1 expression in osteoclast precursors [18]. However, the dectin-1 downstream signals responsible for NFATc1 inhibition were not fully defined. In this study, we found that dectin-1 activation enhanced the expression of transcription factors MafB, Bcl6, IRF7, IRF8 and IRF9. MafB and Bcl6 are known inhibitors of NFATc1 and osteoclast differentiaton [12]. The IRF family member IRF8 was also reported to inhibit NFATc1 and osteoclast differentiaton [12]. These data suggested that dectin-1 signaling may inhibit NFATc1 expression through upregulation of MafB, Bcl6 and IRF8. In contrast, Yamasaki et al. reported that dectin-1 inhibited NFATc1 expression through the inhibition of syk/c-fos downstream signaling [18]. Thus, our data reveals new insights into dectin-1-induced inhibition of NFATc1 expression and osteoclast differentiation.
It was reported that dectin-1 stimulates macrophages to produce some pro-inflammatory cytokines, such as TNF-α, IL-6 and IL-1β [19,20]. However, these factors are related to the stimulation of osteoclast differentiation [21,22,25,26]. In this study, we identified IL-33 as a new cytokine that was upregulated by dectin-1 and was related to the inhibition of osteoclast differentiation. Functional tests showed that addition of IL-33 inhibited osteoclast precursors to differentiate into mature osteoclasts and reduced their bone resorptive activity. This result is consistent with previous observations that IL-33 inhibits osteoclastogenesis [27,28]. In addition, blocking IL-33/ST2 by using a ST2 blocking antibody partially abrogated dectin-1 induced inhibition of osteoclastogenesis. Mechanistic studies revealed that IL-33 increased MafB, IRF7, IRF8 and IRF9 and decreased NFATc1 expression in osteoclast precursors. And blocking ST2 increased NFATc1 expression in dectin-1-activated osteoclast precursors. Thus, we identify IL-33 as an important mediator for dectin-1-induced inhibition of osteoclastogenesis.
Notably, the concentrations of IL-33 in supernatants of curdlan-treated osteoclast precursors were much lower than those we used to efficiently inhibit osteoclast differentiation in vitro. The reasons for this discrepancy are unclear. IL-33 is a nuclear cytokine, which is released via cell necrosis. Full length IL-33 can be cleaved by a wide range of proteases, such as caspase-1, elastase, chymase and tryptase, leading to production of different IL-33 variants [29][30][31][32]. Full length IL-33 and its cleaved variants may all exhibit some bioactivities but with different intensities [29][30][31][32]. Therefore, we first suggested that the bioactivity of the natural IL-33 produced by curdlan-treated osteoclast precursors might be much higher than the commercial synthetic IL-33 that we purchased from the company. Second, after release, most of IL-33 might be captured by the adjacent cells and only little amount of IL-33 was released into the supernatant. Third, the IL-33 ELISA kit may detect only some of IL-33 variants.
In summary, our study demonstrates that dectin-1 activation potently inhibits osteoclast differentiation and bone resorption function. Dectin-1 activation increases MafB and decreases NFATc1 expression. Dectin-1 activation increases the expression of IL-33, which is an important mediator for dectin-1-induced inhibition of osteoclast differentiation and bone resorptive function. Our study has provided new insights into the mechanisms of dectin-1-induced inhibition of osteoclastogenesis and may provide new targets for the therapy of OBDs.

Mice
Balb/c mice were purchased from the Jackson Laboratory. Mice were bred and maintained in pathogenfree facilities at the First Hospital Animal Center of Jilin University. 6-8 weeks old mice were used for experiments. All animal experimental procedures were reviewed and approved by the Animal Ethical Committee of First Hospital of Jilin University.
In blocking experiments, BMCs were cultured with M-CSF (10 ng/mL) for 2 days. At day 2 and 4, cells were cultured with M-CSF/ RNAKL with or without addition of curdlan in the presence of a ST2 neutralization antibody (αST2) (5 μg/mL) or control IgG (5 μg/mL). At day 5, cells were processed for TRAP staining, or cells and culture supernatants were collected for gene expression by qPCR or ELISA.

Tartrate-resistant acid phosphatase (TRAP) staining
BMCs were cultured with M-CSF for 2 days and with MCSF plus RANKL for 3 days. In some cultures, cells were treated with curdlan (10 μg/mL) or IL-33 (50 ng/mL) at day 2 and day 4. In ST2 blocking experiments, cultures were treated with curdlan in the presence of αST2 (5 μg/mL) or a control IgG (5 μg/mL) at day 2 and day 4. At day 5, culture medium was removed and cells were fixed and stained with Acid Phosphatase, Leukocyte (TRAP) Kit (Sigma) according to the manufacturer's instructions. TRAP + cells with more than three nuclei were considered as osteoclasts. Osteoclast circumference was calculated by the formula: 3.14 × (mean diameter).

Real time-polymerase chain reaction
qPCR was performed as previously described [34]. Total RNA was extracted from cells by using an RNeasy Mini kit (Qiagen) according to the manufacturer's instructions. Primer sets used for these analyses are: Il33,

Enzyme-linked immunosorbent assay (ELISA) and western blot analyses
IL-33 and TRAP5b ELISA kits were purchased from R&D Systems and Elabscience Biotechnology Co., Ltd, respectively. ELISA assays were performed according the manufacturer's instructions.
Western blot assay was performed as previously described [34]. Anti-mouse NFATc1 and β-actin antibodies were purchased from Cell Signaling Technology (CST).

Gene-expression profiling
BMCs were cultured with M-CSF for 2 days. At day 2, culture medium was removed and replaced with fresh medium containing M-CSF/RNAKL (10ng/ml) with or without addition of curdlan (10 μg/mL) or IL-33 (50 ng/mL). At day 4, cells were collected and stored in Trizol reagent (Invitrogen) at -80 0 C. Samples were sent to OneArray (http://www.OneArray.com.cn/, Beijing, China) for transcription profiling via genome-wide microarrays, and the subsequent data analysis was also performed by OneArray.