Regulation of bone by the adaptive immune system in arthritis

Studies on the immune regulation of osteoclasts in rheumatoid arthritis have promoted the new research field of 'osteoimmunology', which investigates the interplay between the skeletal and immune systems at the molecular level. Accumulating evidence lends support to the theory that bone destruction associated with rheumatoid arthritis is caused by the enhanced activity of osteoclasts, resulting from the activation of a unique helper T cell subset, 'Th17 cells'. Understanding the interaction between osteoclasts and the adaptive immune system in rheumatoid arthritis and the molecular mechanisms of Th17 development will lead to the development of potentially effective therapeutic strategies.


Introduction
Th e bony skeleton enables locomotive activity, the storage of calcium, and the harboring of hematopoietic stem cells (HSCs). Th is multifunctional organ is characterized by calcifi ed hard tissue composed of type I collagen and highly organized deposits of calcium phosphate [1]. Although bone superfi cially seems to be metabolically inert, it is, in fact, restructured at such a high speed that approximately 10% of the total bone content is replaced each year in adult vertebrates. Th is process, called bone remodeling, is dependent on the dynamic balance of bone formation and resorption, which are mediated by osteoblasts and osteoclasts, respectively. A delicate regulation of this process is requisite for normal bone homeostasis, and an imbalance is often related to metabolic bone diseases in humans [2].
Accumulating evidence has indicated that the immune and skeletal systems share a number of regulatory molecules, including cytokines, receptors, signaling molecules, and transcription factors. Furthermore, immune cells are formed and HSCs are maintained in the bone marrow, where they interact with bone cells. Th erefore, the evidence that the physiology and pathology of one system might aff ect those of the other is compelling and the term osteoimmunology was coined to cover these overlapping scientifi c fi elds. Th e most typical example of the interaction between the skeletal and immune systems is seen in the abnormal or prolonged activation of the immune system (or both) in autoimmune diseases such as rheumatoid arthritis (RA), which is characterized by progressive multiple joint destruction. Since autoreactive T lymphocytes are considered to play a key role in the pathogenesis of RA, attention must be paid to the relationship between osteoclast-mediated bone destruction and aberrant adoptive immune responses in order to develop eff ective therapeutic strategies against RA. Here, we summarize recent progress in the understanding of the relationship between bone and the adaptive immune system in arthritis by focusing mainly on osteoclasts and osteoclastogenic helper T cells, Th 17 cells.

The role of RANK/RANKL in osteoclastogenesis
Osteoclasts are large, multinucleated cells formed by the fusion of precursor cells of monocyte/macrophage lineage [2]. Mature osteoclasts degrade bone matrix proteins by secreting proteolytic enzymes, such as cathepsin K and matrix metalloproteinase, and decalcify the inorganic components of bone by releasing hydrochloric acid. In the late 1980s, an in vitro osteoclast formation system that uses a system of culturing bone marrow-derived cells of monocyte/macrophage lineage together with osteoclastogenesis-supporting cells such as osteoblasts was established [3,4]. Th ese supporting mesenchymal cells provide certain factors that are necessary for osteoclast diff erentiation [5]. Analysis of op/op mice with osteo petrosis revealed one of these essential factors to be macrophage colony-stimulating factor (M-CSF) [6]. M-CSF stimulation alone, however, does not induce the diff erentiation of osteoclasts. Forced expression of antiapoptotic molecule Bcl-2 partially rescues the osteopetrotic phenotype of the op/op mice [7], suggesting that M-CSF is a survival factor for osteoclast precursor cells. Ultimately, in 1998, Yasuda and colleagues [8] and Lacey and colleagues [9] did clone the long-sought ligand mediat ing the essential signal for osteoclast diff erentiation; this ligand was called ODF and osteoprotegerin ligand, respectively. Interestingly, this cytokine, which belongs to the tumor necrosis factor (TNF) family, was shown to be identical to receptor activator of nuclear factor-κB ligand (RANKL) and TNF-related activationinduced cytokine (TRANCE), both of which had been cloned in the immune system [10,11]. Th e cloning of ODF (RANKL, hereafter) enabled investi gation of the diff erentiation process in a sophisticated culture system employing recombinant RANKL and M-CSF [12].
Th e receptor for RANKL is RANK, a type I transmembrane protein that possesses a high homology with CD40. RANK is expressed on osteoclast precursor cells and mature osteoclasts, and the binding of RANKL to RANK is inhibited by the decoy receptor osteoprotegerin (OPG) [13,14]. In bone, RANKL is expressed by osteoclastogenesis-supporting cells, including osteoblasts, in response to osteoclastogenic factors, such as 1,25dihydroxyvitamin D 3 , prostaglandin E 2 , and para thyroid hormone, and is a crucial determinant of the level of bone resorption in vivo [5,12]. Mice with a disruption of either Rank or Rankl exhibit severe osteopetrosis accompanied by a tooth eruption defect resulting from a complete lack of osteoclasts [15][16][17]. In contrast, mice lacking Opg exhibit a severe form of osteoporosis caused by both an increased number and enhanced activity of osteoclasts [18,19]. Th ese genetic fi ndings clearly demonstrate that RANK/RANKL signaling is essential for osteoclasto genesis in vivo. Furthermore, mutations in RANK, RANKL, and OPG have been identifi ed in human patients with bone disorders such as familial expansile osteolysis, autosomal recessive osteopetrosis, and juvenile Paget's disease of bone, respectively [20][21][22][23].

RANKL signaling
Th e ligation of RANK with RANKL results in trimerization of RANK and recruitment of adaptor molecules such as the TNF receptor-associated factor (TRAF) family of proteins, among which TRAF6 has been shown to be the major adaptor molecule [24,25]. TRAF6 trimerizes upon RANK stimulation and activates nuclear factor-κB (NF-κB) and mitogen-activated protein kinases, including Jun N-terminal kinase (JNK) and p38. RANK also activates the transcription-factor complex, activator protein 1 (AP-1), through the induction of its component c-Fos [26]. Th e induction mechanism of c-Fos is dependent on the activation of Ca 2+ / calmodulin-dependent protein kinase IV (CaMKIV) and cyclic adenosine mono phosphate responsive-elementbinding protein (CREB) [27] as well as the activation of NF-κB [28]. Importantly, RANKL specifi cally and potently induces nuclear factor of activated T cells cytoplasmic 1 (NFATc1), the master regulator of osteoclast diff eren tiation, and this induction is dependent on both the TRAF6 and c-Fos pathways [29]. Th e activation of NFAT is mediated by a specifi c phosphatase, calcineurin, which is activated by calcium-calmodulin signaling. Th e NFATc1 promoter contains NFAT-binding sites, and NFATc1 specifi cally autoregulates its own promoter during osteoclasto genesis, thus enabling the robust induc tion of NFATc1 [30]. Th e essential role of NFATc1 has been conclusively demonstrated by genetic experi ments [30][31][32]. NFATc1 regulates a number of osteoclast-specifi c genes, such as cathepsin K, tartrateresistant acid phosphatase (TRAP), calcitonin receptor, osteoclast-associated receptor (OSCAR), and β3 integrin, in cooperation with other transcription factors such as AP-1, PU.1, micro phthalmia-associated transcription factor (MITF), and CREB ( Figure 1).
During osteoclastogenesis, activation of calcium signaling is dependent on costimulatory receptors for RANK, which are immunoglobulin-like receptors, such as OSCAR and triggering receptor expressed in myeloid cells-2 (TREM-2). Th ese receptors associate with the adaptor molecules Fc receptor common γ subunit (FcRγ) and DNAX-activating protein 12 (DAP12), transducing signals by the phosphorylation of immunoreceptor tyrosinebased activation motifs (ITAMs) within the adaptor proteins, which, in turn, recruit the spleen tyrosine kinase (Syk) [33,34] (Figure 1). As shown recently, Tec family tyrosine kinases (Tec and Btk) activated by RANK cooperate with Syk to induce effi cient phosphorylation of phospholipase Cγ (PLCγ), which induces the release of calcium from the endoplasmic reticulum through the generation of inositol trisphosphate [35]. Although a series of genetically modifi ed mice has clearly shown that ITAM-mediated signals are essential for osteoclastogenesis, the ligands for the costimulatory receptors remain to be identifi ed [33][34][35].

The essential role of osteoclasts in bone destruction in rheumatoid arthritis
Th e bone destruction observed in the joints of patients with RA presents a challenging clinical problem. In the early 1980s, researchers observed osteoclast-like cells at the bone destruction sites [36], but it was not until RANKL was cloned that the importance of osteoclasts became generally accepted. We previously demonstrated effi cient osteoclast formation in synovial cell cultures obtained from patients with RA [37]. Moreover, the expression of RANKL was detected specifi cally in the synovium of patients with RA [38,39]. Recent studies have provided further direct genetic evidence: RANKL-defi cient mice, which lack osteoclasts, were protected from bone destruction in an arthritis model induced by serum transfer [40]. Bone erosion was not observed in osteopetrotic Fos −/− mice, even when they Receptor activator of nuclear factor-κB ligand (RANKL)-RANK binding results in the recruitment of tumor necrosis factor receptor-associated factor 6 (TRAF 6), which activates nuclear factor-κB (NF-κB) and mitogen-activated protein kinases. RANKL also stimulates the induction of c-Fos through NF-κB and Ca 2+ /calmodulin-dependent protein kinase IV (CaMKIV). NF-κB and c-Fos are important for the robust induction of nuclear factor of activated T cells cytoplasmic 1 (NFATc1). Several costimulatory receptors associate with the immunoreceptor tyrosine-based activation motif (ITAM)-harboring adaptors, Fc receptor common γ subunit (FcRγ), and DNAX-activating protein 12 (DAP12): osteoclast-associated receptor (OSCAR) and triggering receptor expressed in myeloid cells 2 (TREM2) associate with FcRγ, and signal-regulatory protein β1 (SIRPβ1) and paired immunoglobulin-like receptor-A (PIR-A) associate with DAP12. RANK signaling and ITAM signaling cooperate to phosphorylate phospholipase Cγ (PLCγ) and activate calcium signaling, the latter of which is critical for the activation and autoamplifi cation of NFATc1. Tec family tyrosine kinases (Tec and Btk) activated by RANK are important for the formation of the osteoclastogenic signaling complex composed of Tec kinases, B-cell linker (BLNK)/SH2 domain-containing leukocyte protein of 76 kDa (SLP76) (activated by ITAMspleen tyrosine kinase, or Syk), and PLCγ, all of which are essential for the effi cient phosphorylation of PLCγ. AP-1, activator protein 1; CREB, cyclic adenosine monophosphate responsive-element-binding protein; MITF, microphthalmia-associated transcription factor; TRAP, tartrate-resistant acid phosphatase.
were crossed with TNF-α transgenic mice, which develop erosive arthritis spontaneously [41]. In the two cases, a similar level of infl ammation was observed, indicating that RANKL and osteoclasts are indispensable for the bone loss but not the infl ammation. Consistent with this, anti-RANKL and anti-osteoclast therapies have been shown to be benefi cial in the treatment of bone damage in animal models of arthritis [42,43]. Infl ammatory cytokines such as TNF-α, interleukin-1 (IL-1), and IL-6 have a potent capacity to induce RANKL expression on synovial fi broblasts/osteoblasts and to facilitate RANKL signaling, thus contributing directly to the bone destruction process. In particular, TNF-α is considered of special importance since anti-TNF therapy reduces bone erosion as well as infl ammation [44].

Eff ect of T cells on osteoclastogenesis
As infi ltration of T cells into the synovium is a pathological hallmark of RA, it is vital to address how T-cell immunity is linked to the enhanced expression of RANKL and eventual osteoclastic bone resorption. More specifi cally, as RANKL is known to be expressed in activated T cells, it is important to determine whether this source of RANKL can directly induce osteoclast diff erentiation. In 1999, Kong and colleagues [42] showed that the RANKL expressed on activated T cells acts directly on osteoclast precursor cells and induces osteoclasto genesis in vitro. Horwood and colleagues [45] reported that osteoclastogenesis could be induced in vitro by activated T cells. However, it is important to note that T cells produce various cytokines, including interferon-γ (IFN-γ), IL-4, and IL-10, which exert potent inhibitory eff ects on osteoclast diff erentiation [2]. In the former study, the T cells were fi xed by formaldehyde and thus were unable to release any humoral factors [42]. In the latter study, the T cells and osteoclast precursor cells were derived from diff erent species, suggesting that the eff ect of cytokines would, in all likelihood, be much lower than that on cells of the same species [45]. Th e question then arises as to how T-cell cytokines other than RANKL aff ect osteoclast diff erentiation.
Upon activation, naïve CD4 + T cells diff erentiate into diff erent lineages of helper T (Th ) cells, depending on the cytokine milieu [46]. Th 1 and Th 2 cells are traditionally thought to be the major subsets generated upon antigenic stimulation. Th 1 cells, which are induced by IL-12, produce mainly IFN-γ and are involved in cellular immunity; Th 2 cells produce mainly IL-4, IL-5, and IL-10 and contribute to humoral immunity. RA was previously considered to be a disease in which the Th 1-Th 2 balance is skewed toward Th 1. However, IFN-γ is not highly expressed in the joints of patients with RA [47]. Notably, IFN-γ strongly inhibits osteoclastogenesis, even at minute concentrations, through ubiquitin-proteasome-mediated degradation of TRAF6 [48]. Moreover, the severity of collagen-induced arthritis was reported to be exaggerated in the absence of IFN-γ signaling [49,50], suggesting that Th 1 cells are not linked to bone damage in arthritis.

Th17 cells function as osteoclastogenic Th cells
It is worthwhile to defi ne what is believed to be a very rare but pathologically important Th cell subset that is responsible for abnormal bone resorption as osteo clastogenic Th cells. Previous investigations in our laboratory together with other studies on synovial T cell in RA have clarifi ed the characteristics of osteoclastogenic Th cells in autoimmune arthritis [51]. First, osteoclastogenic Th cells do not produce a large amount of IFN-γ. Second, they trigger both local infl am mation and the production of infl ammatory cytokines that induce RANKL ex pression on synovial fi broblasts. Th ird, osteoclasto genic Th cells express RANKL and might thereby participate directly in accelerated osteo clastogenesis. Because these Th cells have such osteoclastogenic characteristics, they can tip the balance in favor of osteoclastogenesis synergistically.
Th 17 cells have recently been identifi ed as a new eff ector Th cell subset characterized by the production of proinfl ammatory cytokines, including IL-17, IL-17F, IL-21, and IL-22. Th 17 cell diff erentiation is induced by the combination of IL-6 and transforming growth factorβ (TGF-β). IL-23 is dispensable for the lineage commitment of Th 17 cells but is required for the growth, survival, and eff ector functions of Th 17 cells [52,53]. Importantly, this unique subset plays a critical role in host defense against certain extracellular pathogens and also contributes to the pathogenesis of various autoimmune diseases [53]. Recent data from our laboratory indicate that Th 17 cells represent the long sought-after osteoclastogenic Th -cell subset, fulfi lling all of the criteria mentioned above [54]. IL-17 induces RANKL on osteoclastogenesis-supporting mesenchymal cells, such as osteoblasts and synovial fi broblasts [55]. IL-17 also enhances local infl ammation and increases the production of infl ammatory cytokines, which further promote RANKL expression and activity. Th erefore, the infi ltration of Th 17 cells into the infl ammatory lesion is the link between the abnormal T-cell response and bone damage ( Figure 2).

Eff ects of regulatory T cells on osteoclastogenesis
CD4 + CD25 + regulatory T (Treg) cells are a specialized T-cell subset that engages in the maintenance of immunological self-tolerance and immune homeostasis, as evidenced by the development of severe autoimmune disease, allergy, and immunopathology in humans and mice with a mutation of forkhead box P3 (Foxp3), a master regulator for the Treg cell lineage [56]. Treg cells can be classifi ed into two main populations: FoxP3 + naturally occurring Treg cells generated in the thymus and FoxP3 + Treg cells induced by antigen stimulation in a milieu rich in TGF-β in the periphery. Although the diff erences and similarities between these two popu lations are yet to be fully elucidated, both have been considered to be essential for immune homeostasis. Notably, Th 17 cells and Treg cells are reciprocally regulated during diff erentiation but exert the opposite eff ects on autoimmunity, and the balance between these populations is associated with infl ammation and autoimmune diseases [53,56]. In many studies, Treg cells were found in high numbers within joint fl uid from patients with RA [57][58][59]. However, Treg cells in joint fl uid from patients with RA failed to suppress eff ector T-cell proliferation or cytokine production. Th is is because infl ammatory cytokines, includ ing IL-6 and TNF-α, attenuate Treg function. Eff ector T cells in joint fl uid were also reported to be resistant to suppression by Treg cells. In addition, serum markers of bone resorption such as C-terminal telo peptide of type I collagen inversely correlated with the number of CD4 + CD25 + Treg cells in peripheral blood of healthy control and RA patients [60]. Th us, it is of key interest whether Treg cells aff ect infl ammatory-associated bone destruction. Several groups have reported the inhibitory eff ect of Treg cells on osteoclastogenesis and bone resorp tion, but no consensus regarding their inhibitory mechanisms has been established. Kim and colleagues [61] reported that the human CD4 + CD25 + Treg cells isolated from peripheral blood mononuclear cells (PBMCs) suppress osteoclast diff erentiation in a cytokine-dependent manner and proposed that TGF-β and IL-4 are required for the suppressive function of Treg cells. Zaiss and colleagues [62] demonstrated the inhi bitory eff ect of CD4 + CD25 + Treg cells purifi ed from mouse spleen on osteoclast diff erentiation. However, the authors showed that CD4 + CD25 + Treg cells inhibit osteoclastogenesis partially via IL-4 and IL-10 production but mainly through cell-to-cell contact via cytotoxic T lympho cyte antigen 4. It is notable that wild-type Treg cells failed to inhibit the diff erentiation of osteoclasts from CD80/86 −/− monocytes [63]. A decrease in osteo clast number and bone resorption was observed after transfer of CD4 + CD25 + Treg cells into Rag1-defi cient mice, indicating that Treg cells could directly block osteoclastogenesis without engaging eff ector T cells [63]. Furthermore, Luo and colleagues [64] recently reported that human PBMCderived CD4 + CD25 + Treg cells suppress osteoclastogenesis and bone resorption in a TGF-β1 and IL-10 cytokine-dependent manner. Since TGF-β, IL-10, and IL-4 are cytokines that are well known to inhibit osteoclastogenesis, these cytokines produced by Treg cells may be involved, at least partially, in the suppressive function of Treg cells on osteoclastogenesis. In all studies by these three groups, Treg cells were activated before coculture experiments, but their culture conditions varied, and this may cause the diff erence among their results. Zaiss and colleagues [63] also reported increased bone mass and partial protection from bone loss after ovariectomy in Foxp3 transgenic mice. Foxp3 + Treg cells have been shown to protect against local and systemic bone destruction in the mouse model of TNF-α-induced arthritis [60]. It is likely that, taken as a whole, Foxp3 + Treg cells exert inhibitory eff ects on infl ammatoryassociated bone destruction, but it is important to consider the possibility that the charac ter istics of Treg cells are aff ected by the specifi c micro environment such as autoimmune infl ammation, as described above. Additional studies would be needed to determine how Treg cells aff ect osteoclast-mediated bone destruction under infl ammatory conditions.

The involvement of B cells in bone destruction
B cells and antibodies make up the body's humoral immune response. B cells develop within bone marrow with the support of the stromal cells and the osteoblast lineage cells via various growth factors and cytokines, and are released into the blood and lymphatic systems. In the sera of most patients with RA, a variety of autoantibodies such as rheumatoid factor and anti-cyclic citrullinated peptide antibodies can be detected [65]. Th e clinical benefi t of the treatment of anti-CD20 antibody, rituximab, supports the notion that B cell-mediated immune responses contribute to the pathogenesis of RA [65,66]. However, there are confl icting data on the role of B cells on bone remodeling: whereas some reported that activated B cells have the potential to promote osteoclastogenesis via RANKL expression [67,68], others insisted that B cells have an inhibitory eff ect on osteoclastogenesis through TGF-β or IFN-γ production [69,70]. Weitzmann and colleagues [71] reported that μMT heavy chain-defi cient mice, which lack mature B cells, are osteoporotic. Th e authors proposed that B cells are critical regulators of physiological bone turnover by secreting OPG and that T cells promote enhanced OPG secretion by activated B cells via CD40/CD40L costimulation. Interestingly, T cell-defi cient nude mice, CD40defi cient mice, and CD40L-defi cient mice displayed osteo porosis and diminished bone marrow OPG production [71]. However, the other group reported that neither μMT-defi cient mice nor Rag1-defi cient mice have an obvious bone phenotype [72]. Th us, the role of B-cell lineages in physiological bone remodeling has not been fi rmly established. IL-7, a major growth factor for B cells, has been reported to be upregulated under infl ammatory conditions and during estrogen defi ciency [73,74]. Suda and colleagues [75] reported that systemic administration of IL-7 induced bone loss, which was similar to that of ovariectomized mice, and that IL-7Ra-defi cient mice had increased bone mass. Th e authors proposed that increased B lymphopoiesis due to induction of IL-7 by estrogen defi ciency may be involved in the elevated osteoclastogenesis. On the other hand, Weitzmann and colleagues [76] reported the other eff ect of IL-7 on bone metabolism; IL-7 promotes osteoclastogenesis by upregu lating T cell-derived osteoclastogenic cytokines, including RANKL. Indeed, IL-7 administration did not induce bone loss in T cell-defi cient nude mice [77]. In contrast, Lorenzo and colleagues [78] reported that IL-7 inhibited osteoclast formation in bone marrow culture and that IL-7 defi ciency caused increased osteoclastogenesis and decreased trabecular bone mass in vivo [79]. Wild-type and IL-7-defi cient mice lose similar amounts of trabecular bone mass after ovariectomy. Consideration of the various eff ects of IL-7 on diff erent target cells will be required to defi ne the precise role of IL-7-mediated B lymphopoiesis on bone remodeling.
Kawai and colleagues [80] reported that, in case of bone destruction in periodontal disease, RANKL was highly expressed by activated B cells isolated from gingival tissues of patients. Furthermore, it has been recently reported that, after injection of lipopolysaccharide (LPS) into mouse gingival, alveolar bone destruction was more highly induced in B cell-reconstituted severe combined immunodefi ciency (SCID) mice than in SCID mice and that LPS-stimulated B cells enhanced osteoclast diff erentiation by producing TNF-α in vitro [81]. Th ese reports suggested that activated B cells have stimu latory eff ects on bone destruction under infl ammatory conditions such as periodontitis, but further studies are needed to determine how B cell-mediated immune responses are directly involved in the osteoclast activation in RA.

Mechanisms involved in Th17 cell diff erentiation
Th e Th 17 cell subset has emerged as an attractive therapeutic target for both infl ammation and bone destruc tion. It is therefore important to understand the molecular mechanism underlying Th 17 development in order to develop novel therapeutic strategies.

ROR nuclear receptors in Th17 development
Th cell diff erentiation is initiated by the T-cell receptor signal in combination with other cytokine receptor signals. Th ese signals induce the activation of specifi c transcription factors to promote lineage-specifi c cytokine production [46]. For example, the T-box-containing protein expressed in T cells, which is activated by IL-12 and IFN-γ, is required for Th 1 cell diff erentiation. Th 2 cell diff erentiation requires the function of the GATAbinding protein 3, which is induced by the IL-4-activated signal transducer and activator of transcription (Stat) 6.
Soon after the discovery of Th 17 cells, Littman and colleagues [82] reported that retinoid-related orphan receptor (ROR) γt is selectively expressed in Th 17 cells and is required for Th 17 cell diff erentiation. RORγt expression is induced by the combination of IL-6 and TGF-β through Stat3. Furthermore, RORγt defi ciency was shown to lead to an impairment of Th 17 cell diff erentiation both in vitro and in vivo. A subsequent study by Dong and colleagues [83] showed that another ROR family member, RORα, is highly induced during Th 17 cell diff erentiation in a Stat3-dependent manner. Although RORα deletion in mice had only a minimal eff ect on IL-17 production, the defi ciency of both RORα and RORγt completely abolished IL-17 production and protected mice from experimental autoimmune encephalo myelitis (EAE), a mouse model of multiple sclerosis. Th us, RORγt and RORα have redundant functions, but RORγt seems to be the major player in Th 17 cell diff erentiation. Although the mecha nisms by which the ROR nuclear receptors drive Th 17 development and production of Th 17-related cytokines such as IL- 17 have not yet been fully elucidated, they are considered to be essential factors for Th 17 development.

A role of cathepsin K in autoimmunity
Cathepsin K is a lysosomal cysteine protease that plays a pivotal role in osteoclast-mediated degradation of the bone matrices [84]. Th us, cathepsin K has been considered a potential therapeutic target for the treatment of bone diseases such as osteoporosis. We developed a new orally active cathepsin K inhibitor, NC-2300, and examined the eff ect of the inhibitor in osteoporosis as well as arthritis models [85]. We observed unexpected results that cathepsin K suppression leads to the reduction of infl ammation in the latter model. Cathepsin K, despite a low expression level in dendritic cells, plays an important role in the activation of Toll-like receptor (TLR) 9 signaling. CpG (cytosine followed by guanine) DNA (a TLR9 ligand)-induced production of cytokines such as IL-6 and IL-23 was found to be impaired in cathepsin K inhibitor-treated or cathepsin Kdefi cient dendritic cells. Th e immune function of cathepsin K was further analyzed in EAE, and the severity of the disease was markedly suppressed in cathepsin Kdefi cient mice. Th e suppression of infl ammation was associated with the reduced induction of Th 17 cells, indicating that cathepsin K contributes to autoimmune infl ammation by inducing Th 17 cells, possibly through cytokines such as IL-6 and IL-23 in dendritic cells.
Th e detailed mechanism by which cathepsin K regulates TLR9 signaling remains elusive, but it has been reported that functional maturation of TLR9 requires its proteolytic cleavage [86,87], to which cathepsin K might contribute. As cathepsin K is now known to be expressed by other cell types, including synovial cells [88], we cannot exclude the possibility that NC-2300 exerted an anti-arthritic eff ect through other cells. However, cathepsin K is an interesting example of a molecule that was originally found in bone and subsequently shown to regulate the immune system. Our study identifi ed cathepsin K as a novel dendritic cell-specifi c regulator of TLR9 signaling and as a potential target of therapeutic intervention into infl ammation-associated bone loss.

Regulation of Th17 development by IκBζ
We found that a nuclear IκB family member, IκBζ, was most highly expressed in Th 17 cells among the Th cell subsets [89]. IκBζ is a nuclear protein highly homologous to Bcl-3, which interacts with the NF-κB subunit via the ankyrin repeat domain [90]. Its expression is rapidly induced by TLR ligands or IL-1 stimulation in peritoneal macrophages. Yamamoto and colleagues [91], using IκBζdefi cient mice, demonstrated that IκBζ is essential for the LPS induction of a subset of secondary response genes, including IL-6 and the IL-12 p40 subunit, in macrophages. However, no attempt to determine the function of IκBζ in T cells was reported in their study.
IκBζ expression was shown to be upregulated by the combination of IL-6 and TGF-β. IκBζ induction was mediated by Stat3, but not by RORγt, in Th 17 cells. Importantly, not only IκBζ-defi cient mice but also Rag2defi cient mice transferred with IκBζ-defi cient CD4 + T cells were shown to be highly resistant to EAE. When naïve CD4 + T cells were activated in vitro under Th 1-and Th 2-polarizing conditions, IκBζ-defi cient naïve CD4 + T cells normally produced IFN-γ and IL-4, respectively. On the other hand, when activated under Th 17polarizing conditions, IL-17 production in IκBζ-defi cient T cells was markedly reduced compared with wild-type T cells. Since the expression of RORγt and RORα was shown to be normal in IκBζ-defi cient T cells, it is unlikely that ROR nuclear receptors function downstream of IκBζ or vice versa.
Although ROR nuclear receptors have been proposed as essential regulators for Th 17 development as described above, several groups have reported that the ectopic expression of RORγt or RORα leads to only modest IL-17 production in the absence of IL-6 and TGF-β [83,92]. Th e ectopic expression of IκBζ in naïve CD4 + T cells did not induce IL-17 production in the absence of IL-6 and TGF-β. Interestingly, however, even in the absence of IL-6 and TGF-β, the ectopic expression of IκBζ, together with RORγt or RORα, potently induced IL-17 production. A reporter assay system showed that IκBζ moderately activated the promoter of the mouse Il17 gene as well as RORγt and RORα. When the ROR nuclear receptor was expressed, IκBζ highly activated the Il17 promoter. Previous studies showed that an evolutionarily conserved noncoding sequence 2 (CNS2) region in the Il17 locus is associated with histone H3 acetylation in a Th 17 lineagespecifi c manner and that the ROR nuclear receptor is recruited to the CNS2 region during Th 17 development [83,93,94]. In combination with RORγt and RORα, IκBζ potently induced the CNS2 enhancer activity. IκBζ was recruited to the CNS2 region in Th 17 cells, and recruitment of IκBζ to the CNS2 region was dependent on RORγt function (Figure 3). Moreover, the expression of IL-17F, IL-21, and IL-23 receptor was decreased in IκBζ-defi cient T cells. IκBζ also bound to the promoter or the enhancer region of these genes in Th 17 cells. Collectively, these fi ndings indicate that IκBζ is critical for the transcriptional program in Th 17 cell lineage commitment [89].

Conclusions
Th e new fi eld of osteoimmunology originated from studies on bone destruction in RA. Increasing evidence has made it evident that the skeletal and immune systems are connected in complex ways; in fact, it would be diffi cult to understand either system in depth without the insights aff orded by studying their interaction in an osteo immunological context [44]. Th e fi ndings in RA might be applicable to numerous infl am ma tory or neoplastic diseases, such as periodontitis, infec tious diseases, and primary or metastatic bone tumors.
Clearly, the Th 17 cell subset is an auspicious target for future therapeutic investigation, and cytokines related to Th 17 cell diff erentiation and function will be of great clinical importance. Antibodies against IL-17 or IL-23 would be expected to exert benefi cial eff ects in autoimmune diseases, and antibodies targeting the IL-6 receptor might not only inhibit Th 17 development in RA but also eff ect a direct inhibition of local infl ammation Interleukin (IL)-6 and transforming growth factor-β (TGF-β) induce Th17 cell diff erentiation, in which the ROR nuclear receptors, RORγt and RORα, have an indispensable role. The expression of IκBζ is induced by the combination of IL-6 and TGF-β. IκBζ induction is mediated by signal transducer and activator of transcription 3 (Stat3), but not RORγt. IκBζ and ROR nuclear receptor bind directly to the CNS2 region of the Il17 promoter and cooperatively activate the Il17 promoter. Notably, recruitment of IκBζ to the CNS2 region was dependent on RORγt, suggesting that the binding of both IκBζ and ROR nuclear receptors to the Il17 promoter leads to an effi cient recruitment of transcriptional coactivators having histone acetylase activity. CNS2, conserved noncoding sequence 2; MHC II, major histocompatibility complex class II; ROR, retinoid-related orphan receptor; TCR, T-cell receptor; Th, helper T.
Okamoto and Takayanagi Arthritis Research & Therapy 2011, 13:219 http://arthritis-research.com/content/13/3/219 and osteoclastogenesis [95,96]. Th e mechanism of Th 17 development is currently one of the most important subjects in immunology. In recent years, several transcriptional regulators of Th 17 development, including IRF4, BATF, Ahr, and Runx1, have been reported [92,93,[97][98][99]. Although further studies will be required to determine whether or how IκBζ synergizes with other transcriptional regulators of Th 17 cells, our results raise the possibility that the targeting of IκBζ may prove eff ective in the treatment of autoimmune diseases.
Importantly, Th 17 cells are also implicated in host defense against a number of microorganisms. Inhibition of Th 17 cells might thus carry a risk of increasing the susceptibility to infection. Th erefore, great care will be required to eff ectively treat autoimmune diseases without compromising the host defense system. Understanding the precise role of Th 17 cells in human autoimmune disorders therefore will be required for the development of eff ective therapeutic applications.