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BACKGROUND & AIMS: Apoptosis generates plenty of membrane-bound nanovesicles, the apoptotic vesicles (apoVs), which show promise for biomedical applications. The liver serves as a significant organ for apoptotic material removal. Whether and how the liver metabolizes apoptotic vesicular products and contributes to liver health and disease is unrecognized. METHODS: apoVs were labeled and traced after intravenous infusion. Apoptosis-deficient mice by Fas mutant (Fasmut) and Caspase-3 knockout (Casp3-/-) were used with apoV replenishment to evaluate the physiological apoV function. Combinations of morphologic, biochemical, cellular, and molecular assays were applied to assess the liver while hepatocyte analysis was performed. Partial hepatectomy and acetaminophen liver failure models were established to investigate liver regeneration and disease recovery. RESULTS: We discovered that the liver is a major metabolic organ of circulatory apoVs, in which apoVs undergo endocytosis by hepatocytes via a sugar recognition system. Moreover, apoVs play an indispensable role to counteract hepatocellular injury and liver impairment in apoptosis-deficient mice upon replenishment. Surprisingly, apoVs form a chimeric organelle complex with the hepatocyte Golgi apparatus through the soluble N-ethylmaleimide-sensitive factor attachment protein receptor machinery, which preserves Golgi integrity, promotes microtubule acetylation by regulating α-tubulin N-acetyltransferase 1, and consequently facilitates hepatocyte cytokinesis for liver recovery. The assembly of the apoV-Golgi complex is further revealed to contribute to liver homeostasis, regeneration, and protection against acute liver failure. CONCLUSIONS: These findings establish a previously unrecognized functional and mechanistic framework that apoptosis through vesicular metabolism safeguards liver homeostasis and regeneration, which holds promise for hepatic disease therapeutics.
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Apoptosis , Hepatocitos , Homeostasis , Regeneración Hepática , Hígado , Ratones Noqueados , Animales , Hepatocitos/metabolismo , Hepatocitos/patología , Hígado/metabolismo , Hígado/patología , Caspasa 3/metabolismo , Ratones , Hepatectomía , Modelos Animales de Enfermedad , Receptor fas/metabolismo , Receptor fas/genética , Aparato de Golgi/metabolismo , Endocitosis , Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Enfermedad Hepática Inducida por Sustancias y Drogas/metabolismo , Enfermedad Hepática Inducida por Sustancias y Drogas/etiología , Enfermedad Hepática Inducida por Sustancias y Drogas/genética , Ratones Endogámicos C57BL , Acetaminofén , MasculinoRESUMEN
Apoptotic vesicles (apoVs) play a vital role in various pathological conditions; however, we have yet to fully understand their precise biological effects in rescuing impaired mesenchymal stem cells (MSCs) and regulating tissue homeostasis. Here, we proved that systemic infusion of bone marrow MSCs derived from wild-type (WT) mice effectively improved the osteopenia phenotype and hyperimmune state in ovariectomized (OVX) mice. Importantly, the WT MSCs rescued the impairment of OVX MSCs both in vivo and in vitro, whereas OVX MSCs did not show the same efficacy. Interestingly, treatment with apoVs derived from WT MSCs (WT apoVs) restored the impaired biological function of OVX MSCs and their ability to improve osteoporosis. This effect was not observed with OVX MSCs-derived apoVs (OVX apoVs) treatment. Mechanistically, the reduced miR-145a-5p expression hindered the osteogenic differentiation and immunomodulatory capacity of OVX MSCs by affecting the TGF-ß/Smad 2/3-Wnt/ß-catenin signaling axis, resulting in the development of osteoporosis. WT apoVs directly transferred miR-145a-5p to OVX MSCs, which were then reused to restore their impaired biological functions. Conversely, treatment with OVX apoVs did not produce significant effects due to their limited expression of miR-145a-5p. Overall, our findings unveil the remarkable potential of apoVs in rescuing the biological function and therapeutic capability of MSCs derived from individuals with diseases. This discovery offers a new avenue for exploring apoVs-based MSC engineering and expands the application scope of stem cell therapy, contributing to the maintenance of bone homeostasis through a previously unrecognized mechanism.
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OBJECTIVE: T cells contribute to tissue injury in systemic sclerosis (SSc), yet the specific T cell subsets expanded in patients with SSc remain incompletely defined. Here we evaluated specific phenotypes and functions of peripheral helper T (Tph) and follicular helper T (Tfh) cells, which have been implicated in autoantibody production, and assessed their associations with clinical features in a well-characterized cohort of patients with SSc. METHODS: Mass cytometry of T cells from peripheral blood mononuclear cells of patients with SSc and controls were evaluated using t-distributed stochastic neighbor embedding visualization, biaxial gating, and marker expression levels. Findings were validated with flow cytometry and in vitro assays. RESULTS: The frequencies of PD-1highCXCR5+ Tfh cells and PD-1highCXCR5- Tph cells were similar in patients with SSc and controls. t-distributed stochastic neighbor embedding visualization (tSNE) revealed distinct populations within the PD-1highCXCR5- cells distinguished by expression of HLA-DR and inducible costimulator (ICOS). Among PD-1highCXCR5- cells, only the HLA-DR+ICOS- cell population was expanded in patients with SSc. Cytometric and RNA sequencing analyses indicated that these cells expressed cytotoxic rather than B cell helper features. HLA-DR+ICOS- PD-1highCXCR5- cells were less potent in inducing B cell plasmablast differentiation and antibody production than comparator T helper cell populations. HLA-DR+ICOS-PD-1highCXCR5- cells were significantly associated with the presence and severity of interstitial lung disease among patients with SSc. CONCLUSION: Among PD-1highCXCR5- T cells, a subset of HLA-DR+ICOS- cells with cytotoxic features is specifically expanded in patients with SSc and is significantly associated with interstitial lung disease severity. This potential cytotoxicity appearing in the CD4 T cell population can be evaluated as a prognostic disease biomarker in patients with SSc.
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The study of neuroimmune crosstalk and the involvement of neurotransmitters in inflammation and bone health has illustrated their significance in joint-related conditions. One important mode of cell-to-cell communication in the synovial fluid (SF) is through extracellular vesicles (EVs) carrying microRNAs (miRNAs). The role of neurotransmitter receptors in the pathogenesis of inflammatory joint diseases, and whether there are specific miRNAs regulating differentially expressed HTR2A, contributing to the inflammatory processes and bone metabolism is unclear. Expression of neurotransmitter receptors and their correlated inflammatory molecules were identified in rheumatoid arthritis (RA) and osteoarthritis (OA) synovium from a scRNA-seq dataset. Immunohistochemistry staining of synovial tissue (ST) from RA and OA patients was performed for validation. Expression of miRNAs targeting HTR2A carried by SF EVs was screened in low- and high-grade inflammation RA from a public dataset and validated by qPCR. HTR2A reduction by target miRNAs was verified by miRNAs mimics transfection into RA fibroblasts. HTR2A was found to be highly expressed in fibroblasts derived from RA synovial tissue. Its expression showed a positive correlation with the degree of inflammation observed. 5 miRNAs targeting HTR2A were decreased in RA SF EVs compared to OA, three of which, miR-214-3p, miR-3120-5p and miR-615-3p, mainly derived from monocytes in the SF, were validated as regulators of HTR2A expression. The findings suggest that fibroblast HTR2A may play a contributory role in inflammation and the pathogenesis of RA. Additionally, targeting miRNAs that act upon HTR2A could present novel therapeutic strategies for alleviating inflammation in RA.
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Artritis Reumatoide , Fibroblastos , MicroARNs , Osteoartritis , Humanos , Artritis Reumatoide/metabolismo , Artritis Reumatoide/patología , Artritis Reumatoide/genética , Vesículas Extracelulares/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patología , Regulación de la Expresión Génica , Inflamación/metabolismo , MicroARNs/genética , MicroARNs/metabolismo , Osteoartritis/metabolismo , Osteoartritis/genética , Osteoartritis/patología , Receptor de Serotonina 5-HT2A/metabolismo , Receptor de Serotonina 5-HT2A/genética , Líquido Sinovial/metabolismo , Membrana Sinovial/metabolismo , Membrana Sinovial/patologíaRESUMEN
Background: Data with fine granularity about COVID-19-related outcomes and risk factors were still limited in the idiopathic inflammatory myopathies (IIMs) population. This study aimed to investigate clinical factors associated with hospitalized and severe COVID-19 in patients with IIMs, particularly those gauged by myositis-specific antibodies. Methods: This retrospective cohort study was conducted in the Renji IIM cohort in Shanghai, China, under an upsurge of SARS-CoV-2 omicron variant infections from December 2022 to January 2023. Clinical data were collected and analyzed by multivariable logistic regression to determine risk factors. High-dimensional flow cytometry analysis was performed to outline the immunological features. Results: Among 463 infected patients in the eligible cohort (n=613), 65 (14.0%) were hospitalized, 19 (4.1%) suffered severe COVID-19, and 10 (2.2%) died. Older age (OR=1.59/decade, 95% CI 1.18 to 2.16, p=0.003), requiring family oxygen supplement (2.62, 1.11 to 6.19, 0.028), patients with anti-synthetase syndrome (ASyS) (2.88, 1.12 to 7.34, 0.027, vs. other dermatomyositis), higher IIM disease activity, and prednisone intake >10mg/day (5.59, 2.70 to 11.57, <0.001) were associated with a higher risk of hospitalization. Conversely, 3-dose inactivated vaccination reduced the risk of hospitalization (0.10, 0.02 to 0.40, 0.001, vs. incomplete vaccination). Janus kinase inhibitor (JAKi) pre-exposure significantly reduced the risk of severe COVID-19 in hospitalized patients (0.16, 0.04 to 0.74, 0.019, vs. csDMARDs). ASyS patients with severe COVID-19 had significantly reduced peripheral CD4+ T cells, lower CD4/CD8 ratio, and fewer naive B cells but more class-switched memory B cells compared with controls. Conclusion: ASyS and family oxygen supplement were first identified as risk factors for COVID-19-related hospitalization in patients with IIMs. JAKi pre-exposure might protect IIM patients against severe COVID-19 complications.
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COVID-19 , Miositis , Humanos , Estudios Retrospectivos , Ligasas , COVID-19/terapia , COVID-19/complicaciones , SARS-CoV-2 , China/epidemiología , Miositis/complicaciones , Miositis/epidemiología , OxígenoRESUMEN
Mesenchymal stem cells (MSCs) influence T cells in health, disease and therapy through messengers of intercellular communication including extracellular vesicles (EVs). Apoptosis is a mode of cell death that tends to promote immune tolerance, and a large number of apoptotic vesicles (apoVs) are generated from MSCs during apoptosis. In an effort to characterize these apoVs and explore their immunomodulatory potential, here we show that after replenishing them systemically, the apoV deficiency in Fas mutant mice and pathological lymphoproliferation were rescued, leading to the amelioration of inflammation and lupus activity. ApoVs directly interacted with CD4+ T cells and inhibited CD25 expression and IL-2 production in a dose-dependent manner. A broad range of Th1/2/17 subsets and cytokines including IFNγ, IL17A and IL-10 were suppressed while Foxp3+ cells were maintained. Mechanistically, exposed phosphatidylserine (PtdSer/PS) on apoVs mediated the interaction with T cells to disrupt proximal T cell receptor signaling transduction. Remarkably, administration of apoVs prevented Th17 differentiation and memory formation, and ameliorated inflammation and joint erosion in murine arthritis. Collectively, our findings unveil a previously unrecognized crosstalk between MSC apoVs and CD4+ T cells and suggest a promising therapeutic use of apoVs for autoimmune diseases.
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Immune checkpoint inhibitor (ICI) therapies used to treat cancer, such as anti-PD-1 antibodies, can induce autoimmune conditions in some individuals. The T cell mechanisms mediating such iatrogenic autoimmunity and their overlap with spontaneous autoimmune diseases remain unclear. Here, we compared T cells from the joints of 20 patients with an inflammatory arthritis induced by ICI therapy (ICI-arthritis) with two archetypal autoimmune arthritides, rheumatoid arthritis (RA) and psoriatic arthritis (PsA). Single-cell transcriptomic and antigen receptor repertoire analyses highlighted clonal expansion of an activated effector CD8 T cell population in the joints and blood of patients with ICI-arthritis. These cells were identified as CD38hiCD127- CD8 T cells and were uniquely enriched in ICI-arthritis joints compared with RA and PsA and also displayed an elevated interferon signature. In vitro, type I interferon induced CD8 T cells to acquire the ICI-associated CD38hi phenotype and enhanced cytotoxic function. In a cohort of patients with advanced melanoma, ICI therapy markedly expanded circulating CD38hiCD127- T cells, which were frequently bound by the therapeutic anti-PD-1 drug. In patients with ICI-arthritis, drug-bound CD8 T cells in circulation showed marked clonal overlap with drug-bound CD8 T cells from synovial fluid. These results suggest that ICI therapy directly targets CD8 T cells in patients who develop ICI-arthritis and induces an autoimmune pathology that is distinct from prototypical spontaneous autoimmune arthritides.
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Artritis Psoriásica , Artritis Reumatoide , Linfocitos T CD8-positivos , Humanos , Artritis Psoriásica/metabolismo , Líquido Sinovial/metabolismo , Linfocitos T Citotóxicos/metabolismoRESUMEN
T peripheral helper (Tph) cells represent a T cell population that stimulates B cell responses within peripheral tissues. Like T follicular helper (Tfh) cells, PD-1hi CXCR5- CD4+ Tph cells produce IL-21 and CXCL13, yet these cells differ from Tfh cells in expression of both transcription factors and chemokine receptors. Originally identified in rheumatoid arthritis synovium, Tph cells are expanded in multiple autoimmune diseases. Tph cells can be identified and distinguished from Tfh cells by both flow cytometry and mass cytometry. Here, we describe detailed methods to identify Tph cells and characterize associated phenotypic features and functions. These protocols and additional notes provide a guide to identify and explore the roles of Tph cells in various human diseases.
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Linfocitos T Colaboradores-Inductores , Artritis Reumatoide , Linfocitos B , Humanos , Interleucinas , Receptores CXCR5 , Membrana SinovialRESUMEN
Mesenchymal stem cells (MSCs) are a type of immunosuppressive stromal cell found in multiple tissues and organs. However, whether MSCs possess immunosupportive characteristics remains unclear. In this study, we showed that the lymph nodes contain immunosupportive MSCs. They produce and secrete a high level of MCP-1 to promote T-cell proliferation and differentiation, in contrast to bone marrow MSCs (BMMSCs), which repress T-cell activation. Unlike BMMSCs, lymph node MSCs (LNMSCs) fail to respond to activated T-cell-induced production of PD-L1 to induce T-cell apoptosis. Mechanistically, MCP-1 activates phospho-Erk to sustain T-cell proliferation and activation while it represses NF-κB/PD-L1 pathway to avoid induction of T-cell apoptosis. Interestingly, inflammatory lymph node-derived LNMSCs abolish their immunosupportive function due to reduction of MCP-1 expression. Finally, we show that systemic infusion of LNMSCs rescues immunosuppression in cytoxan (CTX)-treated mice. This study reveals a previously unrecognized mechanism underlying MSC-based immunoregulation using the MCP-1/PD-L1 axis to energize T cells and suggests a potential to use MSCs to treat immunosuppressive disorders.
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Antígeno B7-H1 , Células Madre Mesenquimatosas , Animales , Antígeno B7-H1/genética , Antígeno B7-H1/metabolismo , Proliferación Celular , Ganglios Linfáticos/metabolismo , Activación de Linfocitos , Células Madre Mesenquimatosas/metabolismo , Ratones , Linfocitos TRESUMEN
OBJECTIVE: Amyopathic dermatomyositis (ADM) is a heterogeneous and life-threatening autoimmune disease with a high mortality rate. In particular, anti-melanoma differentiation-associated protein 5 antibody-positive patients are at a high risk of developing rapidly progressive interstitial lung disease (RPILD). This study was undertaken to identify immunologic signatures among patients who have ADM with ILD (ADM-ILD) and to discover the biomarkers predicting prognosis. METHODS: The landscape of 42 immune cell phenotypes in the peripheral blood of 82 ADM-ILD patients and 82 age- and sex-matched healthy donors was assessed by multicolor flow cytometry. Patients were stratified using an unsupervised machine learning method (hierarchical clustering analysis) by immune cell subsets. Multiple Wilcoxon's signed rank tests and supervised machine learning methods were performed to identify important immune cell subsets. Kaplan-Meier survival analysis with log rank tests was used to create survival curves. RESULTS: We identified 2 distinct clusters correlating with different disease activities and clinical outcomes in ADM-ILD. Cluster 1 was enriched in the activated CD45RA+HLA-DR+CD8+ T cells with decreased CD56dim natural killer cell proportions and showed a higher prevalence of RPILD and higher mortality. In contrast, the other subgroup, cluster 2 (the nonactivated T cell-dominant cluster), displayed favorable clinical outcomes with high survival rates. Our data also revealed that immunophenotype was an independent risk factor associated with 1-year survival. CONCLUSION: Peripheral immunologic features may have the potential to stratify patients with ADM-ILD according to different disease severity and clinical outcomes, which may have implications for outcome prediction, pathogenesis study, and therapy selection.
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Dermatomiositis , Enfermedades Pulmonares Intersticiales , Humanos , Progresión de la Enfermedad , Dermatomiositis/tratamiento farmacológico , Helicasa Inducida por Interferón IFIH1 , Enfermedades Pulmonares Intersticiales/etiología , Pronóstico , Estudios Retrospectivos , AutoanticuerposRESUMEN
T cell-derived pro-inflammatory cytokines are a major driver of rheumatoid arthritis (RA) pathogenesis. Although these cytokines have traditionally been attributed to CD4 T cells, we have found that CD8 T cells are notably abundant in synovium and make more interferon (IFN)-γ and nearly as much tumor necrosis factor (TNF) as their CD4 T cell counterparts. Furthermore, using unbiased high-dimensional single-cell RNA-seq and flow cytometric data, we found that the vast majority of synovial tissue and synovial fluid CD8 T cells belong to an effector CD8 T cell population characterized by high expression of granzyme K (GzmK) and low expression of granzyme B (GzmB) and perforin. Functional experiments demonstrate that these GzmK+ GzmB+ CD8 T cells are major cytokine producers with low cytotoxic potential. Using T cell receptor repertoire data, we found that CD8 GzmK+ GzmB+ T cells are clonally expanded in synovial tissues and maintain their granzyme expression and overall cell state in blood, suggesting that they are enriched in tissue but also circulate. Using GzmK and GzmB signatures, we found that GzmK-expressing CD8 T cells were also the major CD8 T cell population in the gut, kidney, and coronavirus disease 2019 (COVID-19) bronchoalveolar lavage fluid, suggesting that they form a core population of tissue-associated T cells across diseases and human tissues. We term this population tissue-enriched expressing GzmK or TteK CD8 cells. Armed to produce cytokines in response to both antigen-dependent and antigen-independent stimuli, CD8 TteK cells have the potential to drive inflammation.
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COVID-19 , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/metabolismo , Citocinas/metabolismo , Granzimas/metabolismo , HumanosRESUMEN
Mesenchymal stem cell transplantation (MSCT) has been applied to treat a variety of autoimmune and inflammatory diseases. Psychosocial stress can aggravate disease progression in chronic inflammatory patients. Whether psychological stress affects MSCT is largely unknown. In this study we show that psychological stress attenuates therapeutic effects of MSCT in a DSS-induced colitis mouse model by elevating the levels of exosomal Mir7k/mmu-let-7 k (microRNA 7 k) in circulation. Mechanistically, Mir7k inhibits STAT3 pathway in donor MSCs, leading to upregulated expression of BECN1 (beclin 1, autophagy related) and, thus, activation of macroautophagy/autophagy. Inhibition of autophagy by blocking Mir7k or activating STAT3 signaling can restore MSCT-mediated therapy in psychologically stressed colitis mice. Our study identifies a previously unknown role of autophagy in regulating MSCT therapy via exosomal miRNA Mir7k.Abbreviations: BafA1: bafilomycin A1; BECN1: beclin 1, autophagy related; DAI: disease activity index; DAPI: 4',6-diamidino-2-phenylindole; DSS: dextran sulfate sodium; GFP: green fluorescent protein; HAI: histological activity index; IFNG/IFN-γ: interferon gamma; IL10: interleukin 10; IL1RN/IL-1Rra: interleukin 1 receptor antagonist; KD: knockdown; miRNA: microRNA; MSCs: mesenchymal stem cells; MSCT: mesenchymal stem cell transplantation; NTA: nanoparticle tracking analysis; PGE2: prostaglandin E2; SD: standard deviation; siRNA: small-interfering RNA; STAT3: signal transducer and activator of transcription 3; TEM: transmission electron microscopy; TGFB1/TGF-ß1: transforming growth factor, beta 1; Th17 cell: T helper cell 17; TNF/TNF-α: tumor necrosis factor; TNFAIP6/TSG6: tumor necrosis factor alpha induced protein 6; Tregs: regulatory T cells.
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Colitis , Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas , MicroARNs , Animales , Autofagia/genética , Colitis/inducido químicamente , Colitis/terapia , Sulfato de Dextran/efectos adversos , Sulfato de Dextran/metabolismo , Modelos Animales de Enfermedad , Humanos , Ratones , Ratones Endogámicos C57BL , MicroARNs/genética , MicroARNs/metabolismo , Estrés PsicológicoRESUMEN
Mesenchymal stem cells (MSCs) closely interact with the immune system, and they are known to secrete inflammatory cytokines in response to stress stimuli. The biological function of MSC-derived inflammatory cytokines remains elusive. Here, we reveal that even under physiological conditions, MSCs produce and release a low level of tumor necrosis factor alpha (TNFα), which is unexpectedly required for preserving the self-renewal and differentiation of MSCs via autocrine/paracrine signaling. Furthermore, TNFα critically maintains MSC function in vivo during bone homeostasis. Mechanistically, we unexpectedly discovered that physiological levels of TNFα safeguard MSC homeostasis in a receptor-independent manner through mechanical force-driven endocytosis and that endocytosed TNFα binds to mammalian target of rapamycin (mTOR) complex 2 and restricts mTOR signaling. Importantly, inhibition of mTOR signaling by rapamycin serves as an effective osteoanabolic therapeutic strategy to protect against TNFα deficiency and mechanical unloading. Collectively, these findings unravel the physiological framework of the dynamic TNFα shuttle-based mTOR equilibrium that governs MSC and bone homeostasis.
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In the human body, 50-70 billion cells die every day, resulting in the generation of a large number of apoptotic bodies. However, the detailed biological role of apoptotic bodies in regulating tissue homeostasis remains unclear. In this study, we used Fas-deficient MRL/lpr and Caspase 3-/- mice to show that reduction of apoptotic body formation significantly impaired the self-renewal and osteo-/adipo-genic differentiation of bone marrow mesenchymal stem cells (MSCs). Systemic infusion of exogenous apoptotic bodies rescued the MSC impairment and also ameliorated the osteopenia phenotype in MRL/lpr, Caspase 3-/- and ovariectomized (OVX) mice. Mechanistically, we showed that MSCs were able to engulf apoptotic bodies via integrin αvß3 and reuse apoptotic body-derived ubiquitin ligase RNF146 and miR-328-3p to inhibit Axin1 and thereby activate the Wnt/ß-catenin pathway. Moreover, we used a parabiosis mouse model to reveal that apoptotic bodies participated in the circulation to regulate distant MSCs. This study identifies a previously unknown role of apoptotic bodies in maintaining MSC and bone homeostasis in both physiological and pathological contexts and implies the potential use of apoptotic bodies to treat osteoporosis.