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We aim to understand the mechanisms controlling the immune system, focusing on the development and function of tissue microenvironments in lymphoid organs, such as bone and thymus, and molecular basis for lymphocyte development and activation. We also focus on the interdisciplinary filed called osteoimmunology dealing with the interaction and shared mechanisms of bone and the immune system. The final goal of our study is to develop epoch-making strategies for the treatment of immune disorders such as autoimmune diseases and severe infectious diseases in addition to bone and joint diseases.

1. The osteoimmune system in health and diseases

The bone and immune systems share a variety of molecules and interact with each other under physiological and pathological conditions. When aquatic vertebrates moved onto land, the skeletal system evolved to support locomotive activity in the terrestrial environment. At the same time, the immune system rapidly evolved to cope with terrestrial pathogens, and the location of hematopoiesis moved to the bone marrow to protect hematopoietic stem cell (HSC) against higher levels of UV light in land. Thus, bone and immune systems developed synchronously during vertebrate evolution, constituting the “osteoimmune system” which functions as a locomotor organ and a mineral reservoir as well as a primary lymphoid organ (Tsukasaki et al, Nat Rev Immunol, 2019). We have contributed to the creation and development of osteoimmunology, an interdisciplinary research field focusing on the molecular understanding of the interplay between the immune and skeletal systems.

  Bone is a dynamic organ that continuously undergoes a remodeling process involving resorption and formation, which are mediated by osteoclasts and osteoblasts, respectively. Osteoclasts are large, multinucleated cells formed by the fusion of precursor cells of monocyte/macrophage lineage. Mature osteoclasts degrade bone matrix proteins by secreting proteolytic enzymes and decalcify the inorganic components of bone by releasing hydrochloric acid. An imbalance of bone resorption and formation is often related to bone and joint pathologies such as metabolic bone diseases, bone destruction in RA, postmenopausal osteoporosis, bone tumors and osteopetrosis. Elucidation of the molecular mechanisms underlying the differentiation and function of osteoclasts is important for the development of therapeutic strategies for the treatment of various bone diseases.

  RANKL is an essential cytokine for osteoclast differentiation. RANKL is produced by the osteoclastogenesis-supporting mesenchymal cells including osteoblasts, osteocytes and synovial fibroblasts. RANKL binds to its receptor RANK expressed on osteoclast precursor cells. We studied the signal transduction pathway for RANKL and identified nuclear factor of activated T cells c1 (NFATc1) as the master regulator for osteoclast differentiation (Takayanagi et al, Dev Cell, 2002; Asagiri et al, J Exp Med, 2005). We also identified immunoreceptor tyrosine-based activation motif (ITAM)-harboring adaptor-associated costimulatory receptors for RANK (Koga et al, Nature, 2004) and the importance of signal cascade linking RANK and ITAM via Btk/Tec for osteoclastogenesis (Shinohara et al. Cell, 2008). Studies on RANKL signaling have revealed that a number of immune-related signaling molecules and transcription factors to be involved in the regulation of osteoclastogenesis. We showed that cathepsin K, a cysteine protease that is important for osteoclastic bone resorption, has a crucial role in TLR signaling pathway in dendritic cells (Asagiri et al, Science, 2008). Moreover, RANKL was originally identified as a T cell-derived cytokine, and has essential roles in the immune system, including lymph node development, thymic epithelial cell differentiation and microfold (M) cell in the gut. We recently identified a distinct type of subepithelial mesenchymal cell as an essential source of RANKL for M-cell differentiation (M cell inducer cells: MCi) that regulates IgA production and diversifies the gut microbiota (Nagashima et al, Nat Immunol, 2017). These findings have highlighted the close relationship between the bone and immune systems.

  Furthermore, we have revealed the roles of Semaphorin4D on osteoblast differentiation (Negishi-Koga et al, Nature Med, 2011) and Semaphorin3A on inhibition of bone resorption as well as promotion of bone formation (Hayashi et al, Nature, 2012). We further showed that the reduction of Sema3A induced by estrogen deficiency and aging contributes to postmenopausal osteoporosis and age-related osteoporosis (Hayashi et al, Cell Metab, 2019). Sema4D and Sema3A may serve as new druggable targets for osteopenic diseases.

Related review:
Tsukasaki M and Takayanagi H. Osteoimmunology: evolving concepts in bone-immune interactions in health and disease. Nat Rev Immunol. 19(10): 626-642 (2019)
Okamoto K et al, Osteoimmunology: The Conceptual Framework Unifying the Immune and Skeletal Systems. Physiol Rev. 97(4): 1295-1349. (2017)

2. T cell differentiation and function in autoimmune diseases

The immune system protects hosts from various microorganisms and other foreign substances. However, if dysregulated, the immune system reacts to self-antigens and mistakenly attack the self-tissues, causing autoimmune diseases. In recent years, it has become clear that IL-17-producing CD4+ helper T cells “Th17 cells” plays a critical role in host defense against certain extracellular pathogens and also contributes to the pathogenesis of various autoimmune diseases, including rheumatoid arthritis, multiple sclerosis and psoriasis. Thus, a better understanding of the mechanism of Th17 cell differentiation and function is required for development of effective therapeutic strategies against autoimmune diseases. We have identified IκBζ as an essential transcription factor for Th17 cell differentiation (Okamoto et al, Nature, 2010). In cooperation with RORγt, IκBζ enhanced IL-17 expression by directly binding to the regulatory region of the Il17a gene. Furthermore, we found that RANKL on T cells are crucial for the pathogenesis of experimental autoimmune encephalomyelitis (EAE), a mouse model of multiple sclerosis (Guerrini et al, Immunity, 2015). RANKL on T cells stimulates the chemokine production by astrocytes, leading to the chronic inflammation in the CNS. Pharmacological inhibition of RANKL prevented the development of EAE, indicating that RANKL is a potential therapeutic target for treatment of multiple sclerosis.

  We have recently found that the arginine methyltransferase PRMT5 is essential for the expression of the cytokine-signal-transducing components, the common cytokine receptor γ-chain (γc) and JAK3. PRMT5 is required for the development of iNKT cells and the proliferation and survival of peripheral T cells. PRMT5 induced the arginine methylation of the spliceosomal component SmD3 that promoted the splicing of pre-mRNA encoding γc and JAK3 (Inoue et al, Nat Immunol, 2018). In recent years, Jak inhibitors have emerged as potential therapeutic reagents for the autoimmune diseases such as rheumatoid arthritis. This study presented a novel regulatory mechanism governing γc family cytokine-Jak3 signaling in T cells.

Related review:
Okamoto K and Takayanagi H. Regulation of bone by the adaptive immune system in arthritis. Arthritis Res Ther. 13(3): 219 (2011)

3. Mechanism of inflammation-mediated bone destruction

Inflammation-mediated bone destruction observed in diseases such as rheumatoid arthritis (RA) and periodontitis, occurs as a pathological consequence of immune-bone interplay. We identified Th17 cells as osteoclastogenic T cells in the context of arthritis (Sato et al, J. Exp. Med, 2006). Treg cells express Foxp3, a transcriptional factor which is indispensable for function of Treg cells. Exploration of the origin of pathogenic Th17 cells clarified that Foxp3+T cells convert into Th17 cells (termed exFoxp3Th17 cells) in autoimmune arthritis. exFoxp3Th17 cells serve as the most potent bone-damaging T cells in arthritis by promoting osteoclastogenic function of the synovial fibroblasts (Komatsu et al, Nat Med, 2014). RANKL expressed on synovial fibroblasts is primarily responsible for bone erosions during joint inflammation (Danks et al, Ann Dis Rheu, 2016). We also found exFoxp3Th17 cells contribute to the pathogenesis of periodontitis, one of the most common infectious diseases. They play a role in clearance of oral bacteria by inducing anti-bacterial products and removing infected teeth through resorbing teeth-supporting bone, revealing a beneficial role of exFoxp3Th17 cells in host defense against oral bacteria (Tsukasaki et al, Nat Commun. 2018). These studies elucidated that plastic Foxp3+T cells and tissue-resident mesenchymal cells cooperatively induces bone damage in inflammation-mediated bone destruction. We also found that immune-complexes directly induce osteoclast differentiation by Fcγ receptors, leading to the systemic bone loss in RA (Negishi-Koga et al, Nat Commun, 2015). These studies will contribute to the establishment of novel therapies against inflammation-mediated bone destruction.

  We also reported that, using a bone-fracture model in mouse, IL-17 from γδT cells promotes bone fracture healing via stimulating the osteoblastic differentiation of mesenchymal progenitor cells (Ono et al, Nat Commun, 2016). Although it has been known that IL-17 enhances osteoclastic bone resorption in certain pathological situations, the results clearly show the IL-17 exerts a positive effect on bone formation in a context dependent way.

Related review:
Komatsu N, Takayanagi H. Immune-bone interplay in the structural damage in rheumatoid arthritis. Clin Exp Immunol. 194(1): 1-8 (2018)

4. T cell repertoire formation in the thymus

The acquired immune system does not function without T lymphocytes (T cells) that develop in the thymus. Thymus has a unique microenvironment composed of various stromal cells such as thymic epithelial cells and fibroblasts. These thymic stromal cells provide signals for selection of useful T cells (positive selection) and deletion of autoreactive T cells (negative selection) so that diverse yet self-tolerant T cell repertoire is generated. Our group has been focusing on the cellular and molecular basis of how thymic stromal cells control T cell development and repertoire selection. Recently we reported that the genetic variations of human PSMB11 gene, which encodes β5t, a proteasome subunit specifically expressed in thymic epithelial cells, significantly affected positive selection of CD8 T cells(Nitta et al, Sci Immunol. 2017). One of the PSMB11 polymorphisms, G49S, detected in the Japanese population at a high frequency, was associated with a higher risk of Sjögren’s syndrome. These results suggested that genetic variation of thymus-specific proteasome influence T cell repertoire and susceptibility to autoimmunity. We have also been working on thymic development of γδT cells. We showed that the Syk-PI3K signaling pathway is essential for thymic development of pro-inflammatory γδT cells, suggesting that the Syk-PI3K pathway might be a therapeutic target of inflammatory diseases(Muro et al, J Clin Invest. 2018).

Related review:
Nitta T, Suzuki H. Thymic stromal cell subsets for T cell development. Cell Mol Life Sci, 73: 1021-1037 (2016)

5. Expression of tissue-restricted antigens in the thymus

Central immune tolerance functions cooperatively with peripheral immune tolerance, which limits the expansion and reactivity of autoreactive T cells in periphery. In the central immune tolerance, autoreactive T cells are eliminated or redirected into regulatory T cells. To that end, medullary thymic epithelial cells (mTECs) ectopically express an enormous variety of proteins, which are supposed to be specifically expressed in the peripheral tissues named "tissue-restricted self-antigens (TRAs)”. The transcriptional regulators Aire and Fezf2 are mainly expressed by mTECs and are independently involved in the induction of many TRAs (Takaba et al, Cell, 2015). It is likely that Aire induce TRA genes through super-enahcers, but Fezf2 positively and negatively regulates TRAs expression by directly binding to their promoters (Takaba et al, Trends in immunology, 2017). Aire- and Fezf2-deficient mice develop autoimmune phenotypes, indicating that Aire and Fezf2 are crucial for the suppression of autoimmunity. We are exploring the mechanisms underlying TRA expression induced by Fezf2, and Fezf2-dependent generation of Treg cells.

Related review:
Takaba H, Takayanagi H. The Mechanisms of T Cell Selection in the Thymus. Trends Immunol. 38(11): 805-816 (2017)

6. Bone marrow microenvironment

Bone acts as the “primary lymphoid organ” that harbors hematopoietic stem cells (HSCs), immune progenitor cells and mature immune cells including myeloid cells and B cells. HSCs have the capacity to differentiate into all immune cells, and they require extrinsic signals from the microenvironments (niches) in the bone marrow. In recent years, it has been shown that CXCL12-abundant reticular (CAR) cells and leptin receptor-expressing mesenchymal stem cells are the major cellular components of HSC niche. Whereas osteoblasts are dispensable for the HSC maintenance, we showed that osteoblasts produce IL-7 to support common lymphoid progenitors in the bone marrow (Terashima et al, Immunity, 2016). Furthermore, systemic inflammation, such as sepsis, induces osteoblast ablation in the bone marrow, leading to lymphopenia due to a breakdown of the regulation of lymphocyte differentiation by osteoblasts. By understanding various cellular interactions in the bone marrow, we aim to elucidate the regulatory mechanisms underlying immune cell differentiation in the bone marrow in the context of osteoimmunology.

Related reviews:
Terashima A, Takayanagi H. The role of bone cells in immune regulation during the course of infection. Semin Immunopathol. (2019)

7. Bone metastasis

Distant metastasis accounts for the majority of cancer-associated death, and is notoriously resistant to the treatment. Bone is one of the most common sites of tumor metastasis. Bone metastasis often results in serious complications, including bone pain, hypercalcaemia, pathological fractures and spinal cord compression, which significantly affect the quality of life. Although there have been advances in the diagnosis and treatment of cancer, such as immune checkpoint inhibitors, it is still difficult to prevent and treat bone metastasis.

In bone, tumor cells induce the RANKL expression in osteoblasts through the production of PTHrP and IL-6. RANKL promotes osteoclastic bone resorption, which provides space for tumor expansion and releases certain growth factors from the degraded bone matrices to stimulate tumor growth, forming a vicious cycle. Furthermore, RANK is expressed at high levels on many different epithelial tumor cells that preferentially metastasize to bone. RANKL acts directly on RANK- expressing tumor cells to increase tumor migration to bone. RANKL is initially synthesized as a membrane-bound form, and the soluble form is produced by the proteolytic shedding. By generating mice that selectively lack soluble RANKL, we demonstrated that soluble RANKL is dispensable for physiological regulation of bone and immune systems. However, soluble RANKL contributes to bone metastasis by exerting a chemotactic activity in tumor cells expressing RANK (Asano et al, Nat Metab, 2019). Interestingly, the recent human study indicates that breast cancer patients with high levels of serum RANKL had an increased risk of developing bone metastases. Thus, to measure the serum RANKL level may help identify the patients who have a high risk of development of bone metastasis. A fully human monoclonal anti-RANKL antibody, denosumab, is currently used for patients with bone metastasis to block skeletal-related events. Using the mouse models of bone metastasis, we demonstrated that oral administration of small molecule inhibitor against RANKL suppressed the metastatic spread of human breast cancer cell line and murine melanoma cell line to bone (Nakai et al, Bone Res, 2019). Orally available medications targeting the RANKL signaling pathway should prove beneficial in reducing the patient’s tumor burden, thus providing an attractive alternative approach for the treatment of bone metastasis.