Oncostatin M-driven macrophage-fibroblast circuits as a drug target in autoimmune arthritis.

BACKGROUND: Recent single-cell RNA sequencing (scRNA-seq) analysis revealed the functional heterogeneity and pathogenic cell subsets in immune cells, synovial fibroblasts and bone cells in rheumatoid arthritis (RA). JAK inhibitors which ameliorate joint inflammation and bone destruction in RA, suppress the activation of various types of cells in vitro. However, the key cellular and molecular mechanisms underlying the potent clinical effects of JAK inhibitors on RA remain to be determined. Our aim is to identify a therapeutic target for JAK inhibitors in vivo. METHODS: We performed scRNA-seq analysis of the synovium of collagen-induced arthritis (CIA) mice treated with or without a JAK inhibitor, followed by a computational analysis to identify the drug target cells and signaling pathways. We utilized integrated human RA scRNA-seq datasets and genetically modified mice administered with the JAK inhibitor for the confirmation of our findings. RESULTS: scRNA-seq analysis revealed that oncostatin M (OSM) driven macrophage-fibroblast interaction is highly activated under arthritic conditions. OSM derived from macrophages, acts on OSM receptor (OSMR)-expressing synovial fibroblasts, activating both inflammatory and tissue-destructive subsets. Inflammatory synovial fibroblasts stimulate macrophages, mainly through IL-6, to exacerbate inflammation. Tissue-destructive synovial fibroblasts promote osteoclast differentiation by producing RANKL to accelerate bone destruction. scRNA-seq analysis also revealed that OSM-signaling in synovial fibroblasts is the main signaling pathway targeted by JAK inhibitors in vivo. Mice specifically lacking OSMR in synovial fibroblasts (Osmr∆Fibro) displayed ameliorated inflammation and joint destruction in arthritis. The JAK inhibitor was effective on the arthritis of the control mice while it had no effect on the arthritis of Osmr∆Fibro mice. CONCLUSIONS: OSM functions as one of the key cytokines mediating pathogenic macrophage-fibroblast interaction. OSM-signaling in synovial fibroblasts is one of the main signaling pathways targeted by JAK inhibitors in vivo. The critical role of fibroblast-OSM signaling in autoimmune arthritis was shown by a combination of mice specifically deficient for OSMR in synovial fibroblasts and administration of the JAK inhibitor. Thus, the OSM-driven synovial macrophage-fibroblast circuit is proven to be a key driver of autoimmune arthritis, serving as a crucial drug target in vivo.

Synchronized development of thymic eosinophils and thymocytes.

The thymus is an organ required for T cell development and is also an eosinophil-rich organ; however, the nature and function of thymic eosinophils remain unclear. Here, we characterized the gene expression and differentiation mechanism of thymic eosinophils in mice. Thymic eosinophils showed a distinct gene expression profile compared with other organ-resident eosinophils. The number of thymic eosinophils was controlled by medullary thymic epithelial cells. In Rag-deficient mice, the unique gene expression signature of thymic eosinophils was lost but restored by pre-T cell receptor signaling, which induces CD4+ CD8+ thymocyte differentiation, indicating that T cell differentiation beyond the CD4- CD8- stage is necessary and sufficient for the induction of thymic eosinophils. These results demonstrate that thymic eosinophils are quantitatively and qualitatively regulated by medullary thymic epithelial cells and developing thymocytes, respectively, suggesting that thymic eosinophils are a distinct, thymus-specific cell subset, induced by interactions with thymic cells.

The neutrophil-osteogenic cell axis promotes bone destruction in periodontitis.

The immune-stromal cell interactions play a key role in health and diseases. In periodontitis, the most prevalent infectious disease in humans, immune cells accumulate in the oral mucosa and promote bone destruction by inducing receptor activator of nuclear factor-κB ligand (RANKL) expression in osteogenic cells such as osteoblasts and periodontal ligament cells. However, the detailed mechanism underlying immune-bone cell interactions in periodontitis is not fully understood. Here, we performed single-cell RNA-sequencing analysis on mouse periodontal lesions and showed that neutrophil-osteogenic cell crosstalk is involved in periodontitis-induced bone loss. The periodontal lesions displayed marked infiltration of neutrophils, and in silico analyses suggested that the neutrophils interacted with osteogenic cells through cytokine production. Among the cytokines expressed in the periodontal neutrophils, oncostatin M (OSM) potently induced RANKL expression in the primary osteoblasts, and deletion of the OSM receptor in osteogenic cells significantly ameliorated periodontitis-induced bone loss. Epigenomic data analyses identified the OSM-regulated RANKL enhancer region in osteogenic cells, and mice lacking this enhancer showed decreased periodontal bone loss while maintaining physiological bone metabolism. These findings shed light on the role of neutrophils in bone regulation during bacterial infection, highlighting the novel mechanism underlying osteoimmune crosstalk.

Effect of JAK inhibitors on the three forms of bone damage in autoimmune arthritis: joint erosion, periarticular osteopenia, and systemic bone loss.

BACKGROUND: The types of bone damage in rheumatoid arthritis (RA) include joint erosion, periarticular osteoporosis, and systemic osteoporosis. Janus kinase (JAK) inhibitors ameliorate inflammation and joint erosion in RA, but their effect on the three types of bone loss have not been reportedly explored in depth. We aimed to clarify how JAK inhibitors influence the various types of bone loss in arthritis by modulating osteoclastic bone resorption and/or osteoblastic bone formation. METHODS: Collagen-induced arthritis (CIA) mice were treated with a JAK inhibitor after the onset of arthritis. Micro-computed tomography (µCT) and histological analyses (bone morphometric analyses) on the erosive calcaneocuboid joint, periarticular bone (distal femur or proximal tibia), and vertebrae were performed. The effect of four different JAK inhibitors on osteoclastogenesis under various conditions was examined in vitro. RESULTS: The JAK inhibitor ameliorated joint erosion, periarticular osteopenia and systemic bone loss. It reduced the osteoclast number in all the three types of bone damage. The JAK inhibitor enhanced osteoblastic bone formation in the calcaneus distal to inflammatory synovium in the calcaneocuboid joints, periarticular region of the tibia and vertebrae, but not the inflamed calcaneocuboid joint. All the JAK inhibitors suppressed osteoclastogenesis in vitro to a similar extent in the presence of osteoblastic cells. Most of the JAK inhibitors abrogated the suppressive effect of Th1 cells on osteoclastogenesis by inhibiting IFN-γ signaling in osteoclast precursor cells, while a JAK inhibitor did not affect this effect due to less ability to inhibit IFN-γ signaling. CONCLUSIONS: The JAK inhibitor suppressed joint erosion mainly by inhibiting osteoclastogenesis, while it ameliorated periarticular osteopenia and systemic bone loss by both inhibiting osteoclastogenesis and promoting osteoblastogenesis. These results indicate that the effect of JAK inhibitors on osteoclastogenesis and osteoblastogenesis depends on the bone damage type and the affected bone area. In vitro studies suggest that while JAK inhibitors inhibit osteoclastic bone resorption, their effects on osteoclastogenesis in inflammatory environments vary depending on the cytokine milieu, JAK selectivity and cytokine signaling specificity. The findings reported here should contribute to the strategic use of antirheumatic drugs against structural damages in RA.

Identification of an intronic enhancer regulating RANKL expression in osteocytic cells.

The bony skeleton is continuously renewed throughout adult life by the bone remodeling process, in which old or damaged bone is removed by osteoclasts via largely unknown mechanisms. Osteocytes regulate bone remodeling by producing the osteoclast differentiation factor RANKL (encoded by the TNFSF11 gene). However, the precise mechanisms underlying RANKL expression in osteocytes are still elusive. Here, we explored the epigenomic landscape of osteocytic cells and identified a hitherto-undescribed osteocytic cell-specific intronic enhancer in the TNFSF11 gene locus. Bioinformatics analyses showed that transcription factors involved in cell death and senescence act on this intronic enhancer region. Single-cell transcriptomic data analysis demonstrated that cell death signaling increased RANKL expression in osteocytic cells. Genetic deletion of the intronic enhancer led to a high-bone-mass phenotype with decreased levels of RANKL in osteocytic cells and osteoclastogenesis in the adult stage, while RANKL expression was not affected in osteoblasts or lymphocytes. These data suggest that osteocytes may utilize a specialized regulatory element to facilitate osteoclast formation at the bone surface to be resorbed by linking signals from cellular senescence/death and RANKL expression.

Extracellular aaRSs drive autoimmune and inflammatory responses in rheumatoid arthritis via the release of cytokines and PAD4.

OBJECTIVES: Recent studies demonstrate that extracellular-released aminoacyl-tRNA synthetases (aaRSs) play unique roles in immune responses and diseases. This study aimed to understand the role of extracellular aaRSs in the pathogenesis of rheumatoid arthritis (RA). METHODS: Primary macrophages and fibroblast-like synoviocytes were cultured with aaRSs. aaRS-induced cytokine production including IL-6 and TNF-α was detected by ELISA. Transcriptomic features of aaRS-stimulated macrophages were examined using RNA-sequencing. Serum and synovial fluid (SF) aaRS levels in patients with RA were assessed using ELISA. Peptidyl arginine deiminase (PAD) 4 release from macrophages stimulated with aaRSs was detected by ELISA. Citrullination of aaRSs by themselves was examined by immunoprecipitation and western blotting. Furthermore, aaRS inhibitory peptides were used for inhibition of arthritis in two mouse RA models, collagen-induced arthritis and collagen antibody-induced arthritis. RESULTS: All 20 aaRSs functioned as alarmin; they induced pro-inflammatory cytokines through the CD14-MD2-TLR4 axis. Stimulation of macrophages with aaRSs displayed persistent innate inflammatory responses. Serum and SF levels of many aaRSs increased in patients with RA compared with control subjects. Furthermore, aaRSs released PAD4 from living macrophages, leading to their citrullination. We demonstrate that aaRS inhibitory peptides suppress cytokine production and PAD4 release by aaRSs and alleviate arthritic symptoms in a mouse RA model. CONCLUSIONS: Our findings uncovered the significant role of aaRSs as a novel alarmin in RA pathogenesis, indicating that their blocking agents are potent antirheumatic drugs.

Identification of BST2 as a conjunctival epithelial stem/progenitor cell marker.

The conjunctival epithelium consists of conjunctival epithelial cells and goblet cells derived from conjunctival epithelial stem/progenitor cells. However, the source of these cells is not well known because no specific markers for conjunctival epithelial stem/progenitor cells have been discovered. Therefore, to identify conjunctival epithelial stem/progenitor cell markers, we performed single-cell RNA sequencing of a conjunctival epithelial cell population derived from human-induced pluripotent stem cells (hiPSCs). The following conjunctival epithelial markers were identified: BST2, SLC2A3, AGR2, TMEM54, OLR1, and TRIM29. Notably, BST2 was strongly positive in the basal conjunctival epithelium, which is thought to be rich in stem/progenitor cells. Moreover, BST2 was able to sort conjunctival epithelial stem/progenitor cells from hiPSC-derived ocular surface epithelial cell populations. BST2-positive cells were highly proliferative and capable of successfully generating conjunctival epithelial sheets containing goblet cells. In conclusion, BST2 has been identified as a specific marker of conjunctival epithelial stem/progenitor cells.

Sugar transporter Slc37a2 regulates bone metabolism in mice via a tubular lysosomal network in osteoclasts.

Osteoclasts are giant bone-digesting cells that harbor specialized lysosome-related organelles termed secretory lysosomes (SLs). SLs store cathepsin K and serve as a membrane precursor to the ruffled border, the osteoclast's 'resorptive apparatus'. Yet, the molecular composition and spatiotemporal organization of SLs remains incompletely understood. Here, using organelle-resolution proteomics, we identify member a2 of the solute carrier 37 family (Slc37a2) as a SL sugar transporter. We demonstrate in mice that Slc37a2 localizes to the SL limiting membrane and that these organelles adopt a hitherto unnoticed but dynamic tubular network in living osteoclasts that is required for bone digestion. Accordingly, mice lacking Slc37a2 accrue high bone mass owing to uncoupled bone metabolism and disturbances in SL export of monosaccharide sugars, a prerequisite for SL delivery to the bone-lining osteoclast plasma membrane. Thus, Slc37a2 is a physiological component of the osteoclast's unique secretory organelle and a potential therapeutic target for metabolic bone diseases.

Effect of T cells on bone.

Bone and immune systems mutually influence each other by sharing a variety of regulatory molecules and the tissue microenvironment. The interdisciplinary research field "osteoimmunology" has illuminated the complex and dynamic interactions between the two systems in the maintenance of tissue homeostasis as well as in the development of immune and skeletal disorders. T cells play a central role in the immune response by secreting various immune factors and stimulating other immune cells and structural cells such as fibroblasts and epithelial cells, thereby contributing to pathogen elimination and pathogenesis of immune diseases. The finding on regulation of osteoclastic bone resorption by activated CD4+ T cells in rheumatoid arthritis was one of the driving forces for the development of osteoimmunology. With advances in research on helper T cell subsets and rare lymphoid cells such as γδ T cells in the immunology field, it is becoming clear that various types of T cells exert multiple effects on bone metabolism depending on immune context. Understanding the diverse effects of T cells on bone is essential for deciphering the osteoimmune regulatory network in various biological settings.

Osteoclast biology in the single-cell era.

Osteoclasts, the only cells that can resorb bone, play a central role in bone homeostasis as well as bone damage under pathological conditions such as osteoporosis, arthritis, periodontitis, and bone metastasis. Recent studies using single-cell technologies have uncovered the regulatory mechanisms underlying osteoclastogenesis at unprecedented resolution and shed light on the possibility that there is heterogeneity in the origin, function, and fate of osteoclast-lineage cells. Here, we discuss the current advances and emerging concepts in osteoclast biology.

ETS1 governs pathological tissue-remodeling programs in disease-associated fibroblasts.

Fibroblasts, the most abundant structural cells, exert homeostatic functions but also drive disease pathogenesis. Single-cell technologies have illuminated the shared characteristics of pathogenic fibroblasts in multiple diseases including autoimmune arthritis, cancer and inflammatory colitis. However, the molecular mechanisms underlying the disease-associated fibroblast phenotypes remain largely unclear. Here, we identify ETS1 as the key transcription factor governing the pathological tissue-remodeling programs in fibroblasts. In arthritis, ETS1 drives polarization toward tissue-destructive fibroblasts by orchestrating hitherto undescribed regulatory elements of the osteoclast differentiation factor receptor activator of nuclear factor-κB ligand (RANKL) as well as matrix metalloproteinases. Fibroblast-specific ETS1 deletion resulted in ameliorated bone and cartilage damage under arthritic conditions without affecting the inflammation level. Cross-tissue fibroblast single-cell data analyses and genetic loss-of-function experiments lent support to the notion that ETS1 defines the perturbation-specific fibroblasts shared among various disease settings. These findings provide a mechanistic basis for pathogenic fibroblast polarization and have important therapeutic implications.

Simultaneous augmentation of muscle and bone by locomomimetism through calcium-PGC-1α signaling.

Impaired locomotion has been extensively studied worldwide because those afflicted with it have a potential risk of becoming bedridden. Physical exercise at times can be an effective remedy for frailty, but exercise therapy cannot be applied in all clinical cases. Medication is safer than exercise, but there are no drugs that reinforce both muscle and bone when administered alone. Multiple medications increase the risk of adverse events; thus, there is a need for individual drugs targeting both tissues. To this end, we established a novel sequential drug screening system and identified an aminoindazole derivative, locamidazole (LAMZ), which promotes both myogenesis and osteoblastogenesis while suppressing osteoclastogenesis. Administration of this drug enhanced locomotor function, with muscle and bone significantly strengthened. Mechanistically, LAMZ induced Mef2c and PGC-1α in a calcium signaling-dependent manner. As this signaling is activated upon physical exercise, LAMZ mimics physical exercise. Thus, LAMZ is a promising therapeutic drug for locomotor diseases, including sarcopenia and osteoporosis.

Reduced dynamic loads due to hip dislocation induce acetabular cartilage degeneration by IL-6 and MMP3 via the STAT3/periostin/NF-κB axis.

Developmental dysplasia of the hip (DDH) is characterized by anatomical abnormalities of the hip joint, ranging from mild acetabular dysplasia to hip subluxation and eventually dislocation. The mechanism underlying the cartilage degeneration of the hip joints exposed to reduced dynamic loads due to hip dislocation remains unknown. We established a rodent hip dislocation (disarticulation; DA) model of DDH (DA-DDH rats and mice) by swaddling. Expression levels of periostin (Postn) and catabolic factors, such as interleukin-6 (IL-6) and matrix metalloproteinase 3 (Mmp3), increased and those of chondrogenic markers decreased in the acetabular cartilage of the DA-DDH models. Postn induced IL-6 and Mmp3 expression in chondrocytes through integrin αVß3, focal adhesion kinase, Src, and nuclear factor-κB (NF-κB) signaling. The microgravity environment created by a random positioning machine induced Postn expression in chondrocytes through signal transducer and activator of transcription 3 (STAT3) signaling. IL-6 stimulated Postn expression via STAT3 signaling. Furthermore, cartilage degeneration was suppressed in the acetabulum of Postn-/- DA-DDH mice compared with that in the acetabulum of wild type DA-DDH mice. In summary, reduced dynamic loads due to hip dislocation induced acetabular cartilage degeneration via IL-6 and MMP3 through STAT3/periostin/NF-κB signaling in the rodent DA-DDH models.

Periosteal stem cells control growth plate stem cells during postnatal skeletal growth.

The ontogeny and fate of stem cells have been extensively investigated by lineage-tracing approaches. At distinct anatomical sites, bone tissue harbors multiple types of skeletal stem cells, which may independently supply osteogenic cells in a site-specific manner. Periosteal stem cells (PSCs) and growth plate resting zone stem cells (RZSCs) critically contribute to intramembranous and endochondral bone formation, respectively. However, it remains unclear whether there is functional crosstalk between these two types of skeletal stem cells. Here we show PSCs are not only required for intramembranous bone formation, but also for the growth plate maintenance and prolonged longitudinal bone growth. Mice deficient in PSCs display progressive defects in intramembranous and endochondral bone formation, the latter of which is caused by a deficiency in PSC-derived Indian hedgehog (Ihh). PSC-specific deletion of Ihh impairs the maintenance of the RZSCs, leading to a severe defect in endochondral bone formation in postnatal life. Thus, crosstalk between periosteal and growth plate stem cells is essential for post-developmental skeletal growth.

Mechanisms of joint destruction in rheumatoid arthritis - immune cell-fibroblast-bone interactions.

Rheumatoid arthritis (RA) is characterized by inflammation and destruction of bone and cartilage in affected joints. Autoimmune responses lead to increased osteoclastic bone resorption and impaired osteoblastic bone formation, the imbalance of which underlies bone loss in RA, which includes bone erosion, periarticular bone loss and systemic osteoporosis. The crucial role of osteoclasts in bone erosion has been demonstrated in basic studies as well as by the clinical efficacy of antibodies targeting RANKL, an important mediator of osteoclastogenesis. Synovial fibroblasts contribute to joint damage by stimulating both pro-inflammatory and tissue-destructive pathways. New technologies, such as single-cell RNA sequencing, have revealed the heterogeneity of synovial fibroblasts and of immune cells including T cells and macrophages. To understand the mechanisms of bone damage in RA, it is important to clarify how the immune system promotes the tissue-destructive properties of synovial fibroblasts and influences bone cells. The interaction between immune cells and fibroblasts underlies the imbalance between regulatory T cells and T helper 17 cells, which in turn exacerbates not only inflammation but also bone destruction, mainly by promoting RANKL expression on synovial fibroblasts. An improved understanding of the immune mechanisms underlying joint damage and the interplay between the immune system, synovial fibroblasts and bone will contribute to the identification of novel therapeutic targets in RA.

Long-term survival in non-human primates of stem cell-derived, MHC-unmatched corneal epithelial cell sheets.

When corneal epithelial stem cells residing in the corneal limbus become dysfunctional, called a limbal stem cell deficiency (LSCD), corneal transparency is decreased, causing severe vision loss. Transplantation of corneal epithelial cell sheets (CEPS) derived from stem cells, including induced pluripotent stem cells, is a promising treatment for LSCD. However, the potential effect of human leukocyte antigen (HLA) concordance on CEPS transplantation has not been addressed. Here, we show that there is no difference in the immune response to CEPS between HLA-matched and -unmatched peripheral blood mononuclear cells in mixed lymphocyte reactions. CEPS transplantation in cynomolgus monkeys revealed that the immune response to major histocompatibility-unmatched CEPS was not strong and could be controlled by local steroid administration. Furthermore, programmed death ligand 1 was identified as an immunosuppressive molecule in CEPS under inflammatory conditions in vitro. Our results indicate that corneal epithelium has low immunogenicity and allogeneic CEPS transplantation requires mild immunosuppression.

Spleen tyrosine kinase mediates the γδTCR signaling required for γδT cell commitment and γδT17 differentiation.

The γδT cells that produce IL-17 (γδT17 cells) play a key role in various pathophysiologic processes in host defense and homeostasis. The development of γδT cells in the thymus requires γδT cell receptor (γδTCR) signaling mediated by the spleen tyrosine kinase (Syk) family proteins, Syk and Zap70. Here, we show a critical role of Syk in the early phase of γδT cell development using mice deficient for Syk specifically in lymphoid lineage cells (Syk-conditional knockout (cKO) mice). The development of γδT cells in the Syk-cKO mice was arrested at the precursor stage where the expression of Rag genes and αßT-lineage-associated genes were retained, indicating that Syk is required for γδT-cell lineage commitment. Loss of Syk in γδT cells weakened TCR signal-induced phosphorylation of Erk and Akt, which is mandatory for the thymic development of γδT17 cells. Syk-cKO mice exhibited a loss of γδT17 cells in the thymus as well as throughout the body, and thereby are protected from γδT17-dependent psoriasis-like skin inflammation. Collectively, our results indicate that Syk is a key player in the lineage commitment of γδT cells and the priming of γδT17 cell differentiation.

The transcription factor Sox4 is required for thymic tuft cell development.

Medullary thymic epithelial cells (mTECs) help shape the thymic microenvironment for T-cell development by expressing a variety of peripheral tissue-restricted antigens (TRAs). The self-tolerance of T cells is established by negative selection of autoreactive T cells that bind to TRAs. To increase the diversity of TRAs, a fraction of mTECs terminally differentiates into distinct subsets resembling atypical types of epithelial cells in specific peripheral tissues. As such, thymic tuft cells that express peripheral tuft cell genes have recently emerged. Here, we show that the transcription factor SRY-box transcription factor 4 (Sox4) is highly expressed in mTECs and is essential for the development of thymic tuft cells. Mice lacking Sox4 specifically in TECs had a significantly reduced number of thymic tuft cells with no effect on the differentiation of other mTEC subsets, including autoimmune regulator (Aire)+ and Ccl21a+ mTECs. Furthermore, Sox4 expression was diminished in mice deficient in TEC-specific lymphotoxin ß receptor (LTßR), indicating a role for the LTßR-Sox4 axis in the differentiation of thymic tuft cells. Given that Sox4 promotes differentiation of peripheral tuft cells, our findings suggest that mTECs employ the same transcriptional program as peripheral epithelial cells. This mechanism may explain how mTECs diversify peripheral antigen expression to project an immunological self within the thymic medulla.

Osteoimmunology as an intrinsic part of immunology.

Osteoimmunology has emerged as a field linking immunology and bone biology, but it has yet to be recognized as belonging to mainstream immunology. However, the extent of the research fields immunology actually covers has been enormously widened, and it is now ready to include such an interdisciplinary subject. One of the most obvious examples of an interaction between the immune and bone systems is the pathogenesis of rheumatoid arthritis, where bone resorption is increased by the autoimmune response. Moreover, the regulation of the immune system by bone cells has been clearly demonstrated by the finding that osteoprogenitor cells contribute to hematopoietic stem cell maintenance as well as the suppression of hematopoietic malignancy. Thus, the bidirectional dialogue has been established and inevitably will lead to the union of bone and immunity. Here, I summarize the history and concept of osteoimmunology, providing a perspective on the future of immunology.