RESUMEN
Alternatively activated macrophages (AAMs) contribute to the resolution of inflammation and tissue repair. However, molecular pathways that govern their differentiation have remained incompletely understood. Here, we show that uncoupling protein-2-mediated mitochondrial reprogramming and the transcription factor GATA3 specifically controlled the differentiation of pro-resolving AAMs in response to the alarmin IL-33. In macrophages, IL-33 sequentially triggered early expression of pro-inflammatory genes and subsequent differentiation into AAMs. Global analysis of underlying signaling events revealed that IL-33 induced a rapid metabolic rewiring of macrophages that involved uncoupling of the respiratory chain and increased production of the metabolite itaconate, which subsequently triggered a GATA3-mediated AAM polarization. Conditional deletion of GATA3 in mononuclear phagocytes accordingly abrogated IL-33-induced differentiation of AAMs and tissue repair upon muscle injury. Our data thus identify an IL-4-independent and GATA3-dependent pathway in mononuclear phagocytes that results from mitochondrial rewiring and controls macrophage plasticity and the resolution of inflammation.
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Metabolismo Energético , Inflamación/inmunología , Inflamación/metabolismo , Interleucina-33/metabolismo , Activación de Macrófagos/inmunología , Macrófagos/inmunología , Macrófagos/metabolismo , Biomarcadores , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Inflamación/etiología , Activación de Macrófagos/genética , Mitocondrias/genética , Mitocondrias/inmunología , Mitocondrias/metabolismo , Fagocitos , Transducción de SeñalRESUMEN
Arthritis typically involves recurrence and progressive worsening at specific predilection sites, but the checkpoints between remission and persistence remain unknown. Here, we defined the molecular and cellular mechanisms of this inflammation-mediated tissue priming. Re-exposure to inflammatory stimuli caused aggravated arthritis in rodent models. Tissue priming developed locally and independently of adaptive immunity. Repeatedly stimulated primed synovial fibroblasts (SFs) exhibited enhanced metabolic activity inducing functional changes with intensified migration, invasiveness and osteoclastogenesis. Meanwhile, human SF from patients with established arthritis displayed a similar primed phenotype. Transcriptomic and epigenomic analyses as well as genetic and pharmacological targeting demonstrated that inflammatory tissue priming relies on intracellular complement C3- and C3a receptor-activation and downstream mammalian target of rapamycin- and hypoxia-inducible factor 1α-mediated metabolic SF invigoration that prevents activation-induced senescence, enhances NLRP3 inflammasome activity, and in consequence sensitizes tissue for inflammation. Our study suggests possibilities for therapeutic intervention abrogating tissue priming without immunosuppression.
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Proteínas del Sistema Complemento/inmunología , Fibroblastos/inmunología , Inflamación/inmunología , Membrana Sinovial/inmunología , Inmunidad Adaptativa/inmunología , Animales , Artritis Reumatoide/inmunología , Línea Celular , Perros , Humanos , Mediadores de Inflamación/inmunología , Células de Riñón Canino Madin Darby , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Endogámicos NOD , Ratones SCID , Ratas Wistar , Transducción de Señal/inmunologíaRESUMEN
Glucocorticoids represent the mainstay of therapy for a broad spectrum of immune-mediated inflammatory diseases. However, the molecular mechanisms underlying their anti-inflammatory mode of action have remained incompletely understood1. Here we show that the anti-inflammatory properties of glucocorticoids involve reprogramming of the mitochondrial metabolism of macrophages, resulting in increased and sustained production of the anti-inflammatory metabolite itaconate and consequent inhibition of the inflammatory response. The glucocorticoid receptor interacts with parts of the pyruvate dehydrogenase complex whereby glucocorticoids provoke an increase in activity and enable an accelerated and paradoxical flux of the tricarboxylic acid (TCA) cycle in otherwise pro-inflammatory macrophages. This glucocorticoid-mediated rewiring of mitochondrial metabolism potentiates TCA-cycle-dependent production of itaconate throughout the inflammatory response, thereby interfering with the production of pro-inflammatory cytokines. By contrast, artificial blocking of the TCA cycle or genetic deficiency in aconitate decarboxylase 1, the rate-limiting enzyme of itaconate synthesis, interferes with the anti-inflammatory effects of glucocorticoids and, accordingly, abrogates their beneficial effects during a diverse range of preclinical models of immune-mediated inflammatory diseases. Our findings provide important insights into the anti-inflammatory properties of glucocorticoids and have substantial implications for the design of new classes of anti-inflammatory drugs.
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Antiinflamatorios , Glucocorticoides , Inflamación , Macrófagos , Mitocondrias , Succinatos , Animales , Femenino , Humanos , Masculino , Ratones , Antiinflamatorios/farmacología , Carboxiliasas/metabolismo , Carboxiliasas/antagonistas & inhibidores , Ciclo del Ácido Cítrico/efectos de los fármacos , Ciclo del Ácido Cítrico/genética , Citocinas/inmunología , Citocinas/metabolismo , Glucocorticoides/farmacología , Glucocorticoides/metabolismo , Hidroliasas/deficiencia , Hidroliasas/genética , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Macrófagos/citología , Macrófagos/efectos de los fármacos , Macrófagos/inmunología , Macrófagos/metabolismo , Ratones Endogámicos C57BL , Mitocondrias/metabolismo , Mitocondrias/efectos de los fármacos , Complejo Piruvato Deshidrogenasa/metabolismo , Receptores de Glucocorticoides/metabolismo , Succinatos/metabolismo , Activación Enzimática/efectos de los fármacosRESUMEN
The presence of gallstones (cholelithiasis) is a highly prevalent and severe disease and one of the leading causes of hospital admissions worldwide. Due to its substantial health impact, we investigated the biological mechanisms that lead to the formation and growth of gallstones. We show that gallstone assembly essentially requires neutrophil extracellular traps (NETs). We found consistent evidence for the presence of NETs in human and murine gallstones and describe an immune-mediated process requiring activation of the innate immune system for the formation and growth of gallstones. Targeting NET formation via inhibition of peptidyl arginine deiminase type 4 or abrogation of reactive oxygen species (ROS) production, as well as damping of neutrophils by metoprolol, effectively inhibit gallstone formation in vivo. Our results show that after the physicochemical process of crystal formation, NETs foster their assembly into larger aggregates and finally gallstones. These insights provide a feasible therapeutic concept to prevent cholelithiasis in patients at risk.
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Trampas Extracelulares/inmunología , Cálculos Biliares/inmunología , Neutrófilos/inmunología , Animales , Femenino , Humanos , Inmunidad Innata/inmunología , Masculino , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Especies Reactivas de Oxígeno/inmunologíaRESUMEN
Innate immunity is the first line of defense against infectious intruders and also plays a major role in the development of sterile inflammation. Direct microscopic imaging of the involved immune cells, especially neutrophil granulocytes, monocytes, and macrophages, has been performed since more than 150 years, and we still obtain novel insights on a frequent basis. Initially, intravital microscopy was limited to small-sized animal species, which were often invertebrates. In this review, we will discuss recent results on the biology of neutrophils and macrophages that have been obtained using confocal and two-photon microscopy of individual cells or subcellular structures as well as light-sheet microscopy of entire organs. This includes the role of these cells in infection defense and sterile inflammation in mammalian disease models relevant for human patients. We discuss their protective but also disease-enhancing activities during tumor growth and ischemia-reperfusion damage of the heart and brain. Finally, we provide two visions, one experimental and one applied, how our knowledge on the function of innate immune cells might be further enhanced and also be used in novel ways for disease diagnostics in the future.
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Inmunidad Innata , Neutrófilos , Animales , Humanos , Microscopía Intravital/métodos , Macrófagos , Mamíferos , MonocitosRESUMEN
Macrophages are considered to contribute to chronic inflammatory diseases such as rheumatoid arthritis1. However, both the exact origin and the role of macrophages in inflammatory joint disease remain unclear. Here we use fate-mapping approaches in conjunction with three-dimensional light-sheet fluorescence microscopy and single-cell RNA sequencing to perform a comprehensive spatiotemporal analysis of the composition, origin and differentiation of subsets of macrophages within healthy and inflamed joints, and study the roles of these macrophages during arthritis. We find that dynamic membrane-like structures, consisting of a distinct population of CX3CR1+ tissue-resident macrophages, form an internal immunological barrier at the synovial lining and physically seclude the joint. These barrier-forming macrophages display features that are otherwise typical of epithelial cells, and maintain their numbers through a pool of locally proliferating CX3CR1- mononuclear cells that are embedded into the synovial tissue. Unlike recruited monocyte-derived macrophages, which actively contribute to joint inflammation, these epithelial-like CX3CR1+ lining macrophages restrict the inflammatory reaction by providing a tight-junction-mediated shield for intra-articular structures. Our data reveal an unexpected functional diversification among synovial macrophages and have important implications for the general role of macrophages in health and disease.
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Articulaciones/citología , Macrófagos/citología , Macrófagos/fisiología , Membrana Sinovial/citología , Sinoviocitos/citología , Sinoviocitos/fisiología , Uniones Estrechas/fisiología , Animales , Artritis/inmunología , Artritis/patología , Receptor 1 de Quimiocinas CX3C/análisis , Receptor 1 de Quimiocinas CX3C/metabolismo , Rastreo Celular , Femenino , Perfilación de la Expresión Génica , Humanos , Inflamación/inmunología , Inflamación/patología , Articulaciones/patología , Macrófagos/clasificación , Macrófagos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Análisis de Componente Principal , RNA-Seq , Análisis de la Célula Individual , Sinoviocitos/clasificación , Sinoviocitos/metabolismo , Transcriptoma/genéticaRESUMEN
Filamin-A-interacting protein 1 (FILIP1) is a structural protein that is involved in neuronal and muscle function and integrity and interacts with FLNa and FLNc. Pathogenic variants in filamin-encoding genes have been linked to neurological disorders (FLNA) and muscle diseases characterized by myofibrillar perturbations (FLNC), but human diseases associated with FILIP1 variants have not yet been described. Here, we report on five patients from four unrelated consanguineous families with homozygous FILIP1 variants (two nonsense and two missense). Functional studies indicated altered stability of the FILIP1 protein carrying the p.[Pro1133Leu] variant. Patients exhibit a broad spectrum of neurological symptoms including brain malformations, neurodevelopmental delay, muscle weakness and pathology and dysmorphic features. Electron and immunofluorescence microscopy on the muscle biopsy derived from the patient harbouring the homozygous p.[Pro1133Leu] missense variant revealed core-like zones of myofibrillar disintegration, autophagic vacuoles and accumulation of FLNc. Proteomic studies on the fibroblasts derived from the same patient showed dysregulation of a variety of proteins including FLNc and alpha-B-crystallin, a finding (confirmed by immunofluorescence) which is in line with the manifestation of symptoms associated with the syndromic phenotype of FILIP1opathy. The combined findings of this study show that the loss of functional FILIP1 leads to a recessive disorder characterized by neurological and muscular manifestations as well as dysmorphic features accompanied by perturbed proteostasis and myopathology.
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Enfermedades Musculares , Proteómica , Humanos , Filaminas/genética , Mutación/genética , Enfermedades Musculares/genética , Debilidad Muscular , Proteínas Portadoras/genética , Proteínas del Citoesqueleto/genéticaRESUMEN
Molecular markers scalable for clinical use are critical for the development of effective treatments and the design of clinical trials. Here, we identify proteins in sera of patients and mouse models with Charcot-Marie-Tooth disease (CMT) with characteristics that make them suitable as biomarkers in clinical practice and therapeutic trials. We collected serum from mouse models of CMT1A (C61 het), CMT2D (GarsC201R, GarsP278KY), CMT1X (Gjb1-null), CMT2L (Hspb8K141N) and from CMT patients with genotypes including CMT1A (PMP22d), CMT2D (GARS), CMT2N (AARS) and other rare genetic forms of CMT. The severity of neuropathy in the patients was assessed by the CMT Neuropathy Examination Score (CMTES). We performed multitargeted proteomics on both sample sets to identify proteins elevated across multiple mouse models and CMT patients. Selected proteins and additional potential biomarkers, such as growth differentiation factor 15 (GDF15) and cell free mitochondrial DNA, were validated by ELISA and quantitative PCR, respectively. We propose that neural cell adhesion molecule 1 (NCAM1) is a candidate biomarker for CMT, as it was elevated in Gjb1-null, Hspb8K141N, GarsC201R and GarsP278KY mice as well as in patients with both demyelinating (CMT1A) and axonal (CMT2D, CMT2N) forms of CMT. We show that NCAM1 may reflect disease severity, demonstrated by a progressive increase in mouse models with time and a significant positive correlation with CMTES neuropathy severity in patients. The increase in NCAM1 may reflect muscle regeneration triggered by denervation, which could potentially track disease progression or the effect of treatments. We found that member proteins of the complement system were elevated in Gjb1-null and Hspb8K141N mouse models as well as in patients with both demyelinating and axonal CMT, indicating possible complement activation at the impaired nerve terminals. However, complement proteins did not correlate with the severity of neuropathy measured on the CMTES scale. Although the complement system does not seem to be a prognostic biomarker, we do show complement elevation to be a common disease feature of CMT, which may be of interest as a therapeutic target. We also identify serum GDF15 as a highly sensitive diagnostic biomarker, which was elevated in all CMT genotypes as well as in Hspb8K141N, Gjb1-null, GarsC201R and GarsP278KY mouse models. Although we cannot fully explain its origin, it may reflect increased stress response or metabolic disturbances in CMT. Further large and longitudinal patient studies should be performed to establish the value of these proteins as diagnostic and prognostic molecular biomarkers for CMT.
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Antígeno CD56 , Enfermedad de Charcot-Marie-Tooth , Factor 15 de Diferenciación de Crecimiento , Animales , Ratones , Biomarcadores , Antígeno CD56/genética , Enfermedad de Charcot-Marie-Tooth/genética , Enfermedad de Charcot-Marie-Tooth/diagnóstico , Factor 15 de Diferenciación de Crecimiento/genética , Proteínas , HumanosRESUMEN
Cancer survival rates have increased significantly because of improvements in therapy regimes and novel immunomodulatory drugs. Recently, combination therapies of anthracyclines and immune checkpoint inhibitors (ICIs) have been proposed to maximize neoplastic cell removal. However, it has been speculated that a priori anthracycline exposure may prone the heart vulnerable to increased toxicity from subsequent ICI therapy, such as an anti-programmed cell death protein 1 (PD1) inhibitor. Here, we used a high-dose anthracycline mouse model to characterize the role of the PD1 immune checkpoint signaling pathway in cardiac tissue using flow cytometry and immunostaining. Anthracycline treatment led to decreased heart function, increased concentration of markers of cell death after six days and a change in heart cell population composition with fewer cardiomyocytes. At the same time point, the number of PD1 ligand (PDL1)-positive immune cells and endothelial cells in the heart decreased significantly. The results suggest that PD1/PDL1 signaling is affected after anthracycline treatment, which may contribute to an increased susceptibility to immune-related adverse events of subsequent anti-PD1/PDL1 cancer therapy.
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Antraciclinas , Neoplasias , Animales , Ratones , Antraciclinas/farmacología , Antraciclinas/uso terapéutico , Células Endoteliales/metabolismo , Inmunoterapia/métodos , Transducción de Señal , Antígeno B7-H1/metabolismoRESUMEN
High-resolution X-ray microscopy (XRM) is gaining interest for biological investigations of extremely small-scale structures. XRM imaging of bones in living mice could provide new insights into the emergence and treatment of osteoporosis by observing osteocyte lacunae, which are holes in the bone of few micrometres in size. Imaging living animals at that resolution, however, is extremely challenging and requires very sophisticated data processing converting the raw XRM detector output into reconstructed images. This paper presents an open-source, differentiable reconstruction pipeline for XRM data which analytically computes the final image from the raw measurements. In contrast to most proprietary reconstruction software, it offers the user full control over each processing step and, additionally, makes the entire pipeline deep learning compatible by ensuring differentiability. This allows fitting trainable modules both before and after the actual reconstruction step in a purely data-driven way using the gradient-based optimizers of common deep learning frameworks. The value of such differentiability is demonstrated by calibrating the parameters of a simple cupping correction module operating on the raw projection images using only a self-supervisory quality metric based on the reconstructed volume and no further calibration measurements. The retrospective calibration directly improves image quality as it avoids cupping artefacts and decreases the difference in grey values between outer and inner bone by 68-94%. Furthermore, it makes the reconstruction process entirely independent of the XRM manufacturer and paves the way to explore modern deep learning reconstruction methods for arbitrary XRM and, potentially, other flat-panel computed tomography systems. This exemplifies how differentiable reconstruction can be leveraged in the context of XRM and, hence, is an important step towards the goal of reducing the resolution limit of in vivo bone imaging to the single micrometre domain.
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Procesamiento de Imagen Asistido por Computador , Microscopía , Animales , Calibración , Procesamiento de Imagen Asistido por Computador/métodos , Ratones , Microscopía/métodos , Estudios Retrospectivos , Rayos XRESUMEN
OBJECTIVES: Eosinophils possess pro-inflammatory functions in asthma. However, our recent studies have suggested that innate lymphoid cells type 2 (ILC2s) and eosinophils have proresolving properties in rheumatoid arthritis (RA). Nothing is known yet about the mechanisms determining the double-edged role of eosinophils. Therefore, we investigated whether asthma, a paradigm eosinophilic disease, can elicit resolution of chronic arthritis. METHODS: Ovalbumin-triggered eosinophilic asthma was combined with K/BxN serum-induced arthritis, where lung and synovial eosinophil subsets were compared by single-cell RNA sequencing (scRNA-seq). To investigate the involvement of the ILC2-interleukin-5 (IL-5) axis, hydrodynamic injection (HDI) of IL-25 and IL-33 plasmids, IL-5 reporter mice and anti-IL-5 antibody treatment were used. In patients with RA, the presence of distinct eosinophil subsets was examined in peripheral blood and synovial tissue. Disease activity of patients with RA with concomitant asthma was monitored before and after mepolizumab (anti-IL-5 antibody) therapy. RESULTS: The induction of eosinophilic asthma caused resolution of murine arthritis and joint tissue protection. ScRNA-seq revealed a specific subset of regulatory eosinophils (rEos) in the joints, distinct from inflammatory eosinophils in the lungs. Mechanistically, synovial rEos expanded on systemic upregulation of IL-5 released by lung ILC2s. Eosinophil depletion abolished the beneficial effect of asthma on arthritis. rEos were consistently present in the synovium of patients with RA in remission, but not in active stage. Remarkably, in patients with RA with concomitant asthma, mepolizumab treatment induced relapse of arthritis. CONCLUSION: These findings point to a hitherto undiscovered proresolving signature in an eosinophil subset that stimulates arthritis resolution.
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Artritis Experimental , Artritis Reumatoide , Asma , Animales , Artritis Experimental/tratamiento farmacológico , Artritis Reumatoide/tratamiento farmacológico , Asma/tratamiento farmacológico , Eosinófilos , Humanos , Inmunidad Innata , Interleucina-5/farmacología , Linfocitos , RatonesRESUMEN
OBJECTIVE: Cancer-associated fibroblasts (CAFs) influence the tumour microenvironment and tumour growth. However, the role of CAFs in colorectal cancer (CRC) development is incompletely understood. DESIGN: We quantified phosphorylation of STAT3 (pSTAT3) expression in CAFs of human colon cancer tissue using a tissue microarray (TMA) of 375 patients, immunofluorescence staining and digital pathology. To investigate the functional role of CAFs in CRC, we took advantage of two murine models of colorectal neoplasia and advanced imaging technologies. In loss-of-function and gain-of-function experiments, using genetically modified mice with collagen type VI (COLVI)-specific signal transducer and activator of transcription 3 (STAT3) targeting, we evaluated STAT3 signalling in fibroblasts during colorectal tumour development. We performed a comparative gene expression profiling by whole genome RNA-sequencing of fibroblast subpopulations (COLVI+ vs COLVI-) on STAT3 activation (IL-6 vs IL-11). RESULTS: The analysis of pSTAT3 expression in CAFs of human TMAs revealed a negative correlation of increased stromal pSTAT3 expression with the survival of colon cancer patients. In the loss-of-function and gain-of-function approach, we found a critical role of STAT3 activation in fibroblasts in driving colorectal tumourigenesis in vivo. With different imaging technologies, we detected an expansion of activated fibroblasts in colorectal neoplasias. Comparative gene expression profiling of fibroblast subpopulations on STAT3 activation revealed the regulation of transcriptional patterns associated with angiogenesis. Finally, the blockade of proangiogenic signalling significantly reduced colorectal tumour growth in mice with constitutive STAT3 activation in COLVI+ fibroblasts. CONCLUSION: Altogether our work demonstrates a critical role of STAT3 activation in CAFs in CRC development.
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Neoplasias Colorrectales/etiología , Interleucina-11/metabolismo , Interleucina-6/metabolismo , Factor de Transcripción STAT3/metabolismo , Animales , Colon/metabolismo , Neoplasias Colorrectales/diagnóstico , Fibroblastos/metabolismo , Humanos , Ratones , Fosforilación , Pronóstico , Análisis de Matrices Tisulares , TranscriptomaAsunto(s)
Neoplasias del Colon/diagnóstico por imagen , Diagnóstico por Imagen/métodos , Imagenología Tridimensional/métodos , Microscopía/métodos , Procesamiento de Lenguaje Natural , Algoritmos , Animales , Biología Computacional/métodos , Ratones , Movimiento (Física) , Lenguajes de Programación , Reproducibilidad de los Resultados , Rotación , Programas InformáticosRESUMEN
Breast cancer often metastasizes to bone, causing osteolytic lesions. Structural and biophysical changes are rarely studied yet are hypothesized to influence metastasis. We developed a mouse model of early bone metastasis and multimodal imaging to quantify cancer cell homing, bone (re)modeling, and onset of metastasis. Using tissue clearing and three-dimensional (3D) light sheet fluorescence microscopy, we located enhanced green fluorescent protein-positive cancer cells and small clusters in intact bones and quantified their size and spatial distribution. We detected early bone lesions using in vivo microcomputed tomography (microCT)-based time-lapse morphometry and revealed altered bone (re)modeling in the absence of detectable lesions. With a new microCT image analysis tool, we tracked the growth of early lesions over time. We showed that cancer cells home in all bone compartments, while osteolytic lesions are only detected in the metaphysis, a region of high (re)modeling. Our study suggests that higher rates of (re)modeling act as a driver of lesion formation during early metastasis.
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Neoplasias Óseas , Osteólisis , Animales , Ratones , Microtomografía por Rayos X/métodos , Neoplasias Óseas/complicaciones , Neoplasias Óseas/secundario , Huesos/diagnóstico por imagen , Osteólisis/diagnóstico por imagen , Osteólisis/etiología , Osteólisis/patología , Modelos Animales de Enfermedad , Línea Celular TumoralRESUMEN
Efficient cellular fusion of mononuclear precursors is the prerequisite for the generation of fully functional multinucleated bone-resorbing osteoclasts. However, the exact molecular factors and mechanisms controlling osteoclast fusion remain incompletely understood. Here we identify RANKL-mediated activation of caspase-8 as early key event during osteoclast fusion. Single cell RNA sequencing-based analyses suggested that activation of parts of the apoptotic machinery accompanied the differentiation of osteoclast precursors into mature multinucleated osteoclasts. A subsequent characterization of osteoclast precursors confirmed that RANKL-mediated activation of caspase-8 promoted the non-apoptotic cleavage and activation of downstream effector caspases that translocated to the plasma membrane where they triggered activation of the phospholipid scramblase Xkr8. Xkr8-mediated exposure of phosphatidylserine, in turn, aided cellular fusion of osteoclast precursors and thereby allowed generation of functional multinucleated osteoclast syncytia and initiation of bone resorption. Pharmacological blockage or genetic deletion of caspase-8 accordingly interfered with fusion of osteoclasts and bone resorption resulting in increased bone mass in mice carrying a conditional deletion of caspase-8 in mononuclear osteoclast precursors. These data identify a novel pathway controlling osteoclast biology and bone turnover with the potential to serve as target for therapeutic intervention during diseases characterized by pathologic osteoclast-mediated bone loss. Proposed model of osteoclast fusion regulated by caspase-8 activation and PS exposure. RANK/RANK-L interaction. Activation of procaspase-8 into caspase-8. Caspase-8 activates caspase-3. Active capase-3 cleaves Xkr8. Local PS exposure is induced. Exposed PS is recognized by the fusion partner. FUSION. PS is re-internalized.
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Caspasa 8 , Fusión Celular , Osteoclastos , Fosfatidilserinas , Proteínas de Transferencia de Fosfolípidos , Caspasa 8/metabolismo , Caspasa 8/genética , Animales , Osteoclastos/metabolismo , Fosfatidilserinas/metabolismo , Proteínas de Transferencia de Fosfolípidos/metabolismo , Proteínas de Transferencia de Fosfolípidos/genética , Ratones , Ratones Endogámicos C57BL , Resorción Ósea/metabolismo , Resorción Ósea/patología , Resorción Ósea/genética , Diferenciación Celular , Ligando RANK/metabolismoRESUMEN
The ecosystem of brain tumors is considered immunosuppressed, but our current knowledge may be incomplete. Here we analyzed clinical cell and tissue specimens derived from patients presenting with glioblastoma or nonmalignant intracranial disease to report that the cranial bone (CB) marrow, in juxtaposition to treatment-naive glioblastoma tumors, harbors active lymphoid populations at the time of initial diagnosis. Clinical and anatomical imaging, single-cell molecular and immune cell profiling and quantification of tumor reactivity identified CD8+ T cell clonotypes in the CB that were also found in the tumor. These were characterized by acute and durable antitumor response rooted in the entire T cell developmental spectrum. In contrast to distal bone marrow, the CB niche proximal to the tumor showed increased frequencies of tumor-reactive CD8+ effector types expressing the lymphoid egress marker S1PR1. In line with this, cranial enhancement of CXCR4 radiolabel may serve as a surrogate marker indicating focal association with improved progression-free survival. The data of this study advocate preservation and further exploitation of these cranioencephalic units for the clinical care of glioblastoma.
RESUMEN
Clodronate liposomes (Clo-Lip) have been widely used to deplete mononuclear phagocytes (MoPh) to study the function of these cells in vivo. Here, we revisited the effects of Clo-Lip together with genetic models of MoPh deficiency, revealing that Clo-Lip exert their anti-inflammatory effects independent of MoPh. Notably, not only MoPh but also polymorphonuclear neutrophils (PMN) ingested Clo-Lip in vivo, which resulted in their functional arrest. Adoptive transfer of PMN, but not of MoPh, reversed the anti-inflammatory effects of Clo-Lip treatment, indicating that stunning of PMN rather than depletion of MoPh accounts for the anti-inflammatory effects of Clo-Lip in vivo. Our data highlight the need for a critical revision of the current literature on the role of MoPh in inflammation.
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Ácido Clodrónico , Liposomas , Humanos , Ácido Clodrónico/farmacología , Neutrófilos , Inflamación , Antiinflamatorios/farmacologíaRESUMEN
OBJECTIVE: Spondyloarthritis (SpA) is characterized by pathologic osteogenesis, inflammation, and extensive angiogenesis in axial and peripheral tissues. Current therapies effectively target inflammation, but these therapies lack efficacy in preventing pathologic osteogenesis. Transgenic mice overexpressing transmembrane tumor necrosis factor (tmTNF-Tg mice) exhibit SpA-like features. We hypothesized that type H blood vessels, which are implicated in osteogenesis, are increased and contribute to pathology in this experimental SpA model. METHODS: We analyzed ankles, femora, and vertebrae of tmTNF-Tg mice and nontransgenic littermates and tmTNF-Tg mice on either a TNF receptor type I (TNFRI)-deficient or TNF receptor type II (TNFRII)-deficient background for osteogenesis, angiogenesis, and inflammation using advanced imaging technologies at various stages of disease. RESULTS: Compared to nontransgenic littermates, tmTNF-Tg mice exhibited an increase in vertebral type H vessels and osteoprogenitor cells in subchondral bone. These features of increased angiogenesis and osteogenesis were already present before onset of clinical disease symptoms. Type H vessels and osteoprogenitor cells were in close proximity to inflammatory lesions and ectopic lymphoid structures. The tmTNF-Tg mice also showed perivertebral ectopic type H vessels and osteogenesis, an increased number of vertebral transcortical vessels, and enhanced entheseal angiogenesis. In tmTNF-Tg mice crossed on a TNFRI- or TNFRII-deficient background, no clear reduction in type H vessels was shown, suggesting that type H vessel formation is not exclusively mediated via TNFRI or TNFRII. CONCLUSION: The contribution of type H vessels to pathologic osteogenesis in experimental SpA advances our knowledge of the pathophysiology of this disease and may also provide a novel opportunity for targeted intervention.
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Osteogénesis , Espondiloartritis , Ratones , Animales , Inflamación , Espondiloartritis/tratamiento farmacológico , Ratones Transgénicos , Factor de Necrosis Tumoral alfaRESUMEN
PPP1R21 acts as a co-factor for protein phosphatase 1 (PP1), an important serine/threonine phosphatase known to be essential for cell division, control of glycogen metabolism, protein synthesis, and muscle contractility. Bi-allelic pathogenic variants in PPP1R21 were linked to a neurodevelopmental disorder with hypotonia, facial dysmorphism, and brain abnormalities (NEDHFBA) with pediatric onset. Functional studies unraveled impaired vesicular transport as being part of PPP1R21-related pathomechanism. To decipher further the pathophysiological processes leading to the clinical manifestation of NEDHFBA, we investigated the proteomic signature of fibroblasts derived from the first NEDHFBA patient harboring a splice-site mutation in PPP1R21 and presenting with a milder phenotype. Proteomic findings and further functional studies demonstrate a profound activation of the ubiquitin-proteasome system with presence of protein aggregates and impact on cellular fitness and moreover suggest a cross-link between activation of the proteolytic system and cytoskeletal architecture (including filopodia) as exemplified on paradigmatic proteins including actin, thus extending the pathophysiological spectrum of the disease. In addition, the proteomic signature of PPP1R21-mutant fibroblasts displayed a dysregulation of a variety of proteins of neurological relevance. This includes increase proteins which might act toward antagonization of cellular stress burden in terms of pro-survival, a molecular finding which might accord with the presentation of a milder phenotype of our NEDHFBA patient.