RESUMEN
FMS-related tyrosine kinase 3 ligand (FLT3L), encoded by FLT3LG, is a hematopoietic factor essential for the development of natural killer (NK) cells, B cells, and dendritic cells (DCs) in mice. We describe three humans homozygous for a loss-of-function FLT3LG variant with a history of various recurrent infections, including severe cutaneous warts. The patients' bone marrow (BM) was hypoplastic, with low levels of hematopoietic progenitors, particularly myeloid and B cell precursors. Counts of B cells, monocytes, and DCs were low in the patients' blood, whereas the other blood subsets, including NK cells, were affected only moderately, if at all. The patients had normal counts of Langerhans cells (LCs) and dermal macrophages in the skin but lacked dermal DCs. Thus, FLT3L is required for B cell and DC development in mice and humans. However, unlike its murine counterpart, human FLT3L is required for the development of monocytes but not NK cells.
Asunto(s)
Células Asesinas Naturales , Proteínas de la Membrana , Animales , Femenino , Humanos , Masculino , Ratones , Linfocitos B/metabolismo , Linfocitos B/citología , Médula Ósea/metabolismo , Linaje de la Célula , Células Dendríticas/metabolismo , Hematopoyesis , Células Madre Hematopoyéticas/metabolismo , Células Madre Hematopoyéticas/citología , Células Asesinas Naturales/metabolismo , Células Asesinas Naturales/inmunología , Células de Langerhans/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/genética , Monocitos/metabolismo , Piel/metabolismo , Ratones Endogámicos C57BLRESUMEN
Memory B cells play a fundamental role in host defenses against viruses, but to date, their role has been relatively unsettled in the context of SARS-CoV-2. We report here a longitudinal single-cell and repertoire profiling of the B cell response up to 6 months in mild and severe COVID-19 patients. Distinct SARS-CoV-2 spike-specific activated B cell clones fueled an early antibody-secreting cell burst as well as a durable synchronous germinal center response. While highly mutated memory B cells, including pre-existing cross-reactive seasonal Betacoronavirus-specific clones, were recruited early in the response, neutralizing SARS-CoV-2 RBD-specific clones accumulated with time and largely contributed to the late, remarkably stable, memory B cell pool. Highlighting germinal center maturation, these cells displayed clear accumulation of somatic mutations in their variable region genes over time. Overall, these findings demonstrate that an antigen-driven activation persisted and matured up to 6 months after SARS-CoV-2 infection and may provide long-term protection.
Asunto(s)
Linfocitos B/inmunología , COVID-19/inmunología , Memoria Inmunológica , Adulto , COVID-19/fisiopatología , Citometría de Flujo , Centro Germinal/citología , Humanos , Activación de Linfocitos , Persona de Mediana Edad , Índice de Severidad de la Enfermedad , Análisis de la Célula Individual , Glicoproteína de la Espiga del Coronavirus/químicaRESUMEN
Benign prostate hyperplasia (BPH) is caused by the nonmalignant enlargement of the transition zone of the prostate gland, leading to lower urinary tract symptoms. Although current medical treatments are unsatisfactory in many patients, the limited understanding of the mechanisms driving disease progression prevents the development of alternative therapeutic strategies. The probasin-prolactin (Pb-PRL) transgenic mouse recapitulates many histopathological features of human BPH. Herein, these alterations parallel urodynamic disturbance reminiscent of lower urinary tract symptoms. Single-cell RNA-sequencing analysis of Pb-PRL mouse prostates revealed that their epithelium mainly includes low-androgen signaling cell populations analogous to Club/Hillock cells enriched in the aged human prostate. These intermediate cells are predicted to result from the reprogramming of androgen-dependent luminal cells. Pb-PRL mouse prostates exhibited increased vulnerability to oxidative stress due to reduction of antioxidant enzyme expression. One-month treatment of Pb-PRL mice with anethole trithione (ATT), a specific inhibitor of mitochondrial ROS production, reduced prostate weight and voiding frequency. In human BPH-1 epithelial cells, ATT decreased mitochondrial metabolism, cell proliferation, and stemness features. ATT prevented the growth of organoids generated by sorted Pb-PRL basal and LSCmed cells, the two major BPH-associated, androgen-independent epithelial cell compartments. Taken together, these results support cell plasticity as a driver of BPH progression and therapeutic resistance to androgen signaling inhibition, and identify antioxidant therapy as a promising treatment of BPH.
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Síntomas del Sistema Urinario Inferior , Hiperplasia Prostática , Masculino , Humanos , Ratones , Animales , Anciano , Andrógenos/farmacología , Andrógenos/metabolismo , Próstata/patología , Hiperplasia Prostática/metabolismo , Antioxidantes/farmacología , Plasticidad de la Célula , Hiperplasia/patología , Plomo/metabolismo , Plomo/uso terapéutico , Ratones Transgénicos , Prolactina/metabolismo , Prolactina/uso terapéutico , Células Epiteliales/metabolismo , Síntomas del Sistema Urinario Inferior/metabolismo , Síntomas del Sistema Urinario Inferior/patologíaRESUMEN
Sustained response off treatment (SROT) after thrombopoietin receptor agonist (TPO-RA) discontinuation has been reported in immune thrombocytopenia (ITP). This prospective multicenter interventional study enrolled adults with persistent or chronic primary ITP and complete response (CR) on TPO-RAs. The primary end point was the proportion of patients achieving SROT (platelet count >30 × 109/L and no bleeding) at week 24 (W24) with no other ITP-specific medications. Secondary end points included the proportion of sustained CR off-treatment (SCROT, platelet count >100 × 109/L and no bleeding) and SROT at W52, bleeding events, and pattern of response to a new course of TPO-RAs. We included 48 patients with a median age of 58.5 years; 30 of 48 had chronic ITP at TPO-RA initiation. In the intention-to-treat analysis, 27 of 48 achieved SROT, 15 of 48 achieved SCROT at W24; 25 of 48 achieved SROT, and 14 of 48 achieved SCROT at W52. No severe bleeding episode occurred in patients who relapsed. Among patients rechallenged with TPO-RA, 11 of 12 achieved CR. We found no significant clinical predictors of SROT at W24. Single-cell RNA sequencing revealed enrichment of a tumor necrosis factor α signaling via NF-κB signature in CD8+ T cells of patients with no sustained response after TPO-RA discontinuation, which was further confirmed by a significant overexpression of CD69 on CD8+ T cells at baseline in these patients as compared with those achieving SCROT/SROT. Our results strongly support a strategy based on progressive tapering and discontinuation of TPO-RAs for patients with chronic ITP who achieved a stable CR on treatment. This trial was registered at www.clinicaltrials.gov as #NCT03119974.
Asunto(s)
Púrpura Trombocitopénica Idiopática , Trombocitopenia , Adulto , Humanos , Persona de Mediana Edad , Estudios Prospectivos , Púrpura Trombocitopénica Idiopática/tratamiento farmacológico , Recuento de Plaquetas , Trombocitopenia/tratamiento farmacológico , Autoinmunidad , Trombopoyetina/uso terapéutico , Proteínas Recombinantes de Fusión/uso terapéutico , Receptores Fc/uso terapéutico , Hidrazinas/uso terapéuticoRESUMEN
BACKGROUND: Allogenic hematopoietic stem cell transplantation (HSCT) and gene therapy (GT) are potentially curative treatments for severe combined immunodeficiency (SCID). Late-onset posttreatment manifestations (such as persistent hepatitis) are not uncommon. OBJECTIVE: We sought to characterize the prevalence and pathophysiology of persistent hepatitis in transplanted SCID patients (SCIDH+) and to evaluate risk factors and treatments. METHODS: We used various techniques (including pathology assessments, metagenomics, single-cell transcriptomics, and cytometry by time of flight) to perform an in-depth study of different tissues from patients in the SCIDH+ group and corresponding asymptomatic similarly transplanted SCID patients without hepatitis (SCIDH-). RESULTS: Eleven patients developed persistent hepatitis (median of 6 years after HSCT or GT). This condition was associated with the chronic detection of enteric viruses (human Aichi virus, norovirus, and sapovirus) in liver and/or stools, which were not found in stools from the SCIDH- group (n = 12). Multiomics analysis identified an expansion of effector memory CD8+ T cells with high type I and II interferon signatures. Hepatitis was associated with absence of myeloablation during conditioning, split chimerism, and defective B-cell function, representing 25% of the 44 patients with SCID having these characteristics. Partially myeloablative retransplantation or GT of patients with this condition (which we have named as "enteric virus infection associated with hepatitis") led to the reconstitution of T- and B-cell immunity and remission of hepatitis in 5 patients, concomitantly with viral clearance. CONCLUSIONS: Enteric virus infection associated with hepatitis is related to chronic enteric viral infection and immune dysregulation and is an important risk for transplanted SCID patients with defective B-cell function.
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Infecciones por Enterovirus , Trasplante de Células Madre Hematopoyéticas , Hepatitis , Inmunodeficiencia Combinada Grave , Virosis , Humanos , Inmunodeficiencia Combinada Grave/terapia , Inmunodeficiencia Combinada Grave/etiología , Linfocitos T CD8-positivos , Trasplante de Células Madre Hematopoyéticas/efectos adversos , Trasplante de Células Madre Hematopoyéticas/métodos , Virosis/etiología , Hepatitis/etiologíaRESUMEN
CD4+ FOXP3+ Tregs are currently explored to develop cell therapies against immune-mediated disorders, with an increasing focus on antigen receptor-engineered Tregs. Deciphering their mode of action is necessary to identify the strengths and limits of this approach. Here, we addressed this issue in an autoimmune disease of the CNS, EAE. Following disease induction, autoreactive Tregs upregulated LAG-3 and CTLA-4 in LNs, while IL-10 and amphiregulin (AREG) were increased in CNS Tregs. Using genetic approaches, we demonstrated that IL-10, CTLA-4, and LAG-3 were nonredundantly required for the protective function of antigen receptor-engineered Tregs against EAE in cell therapy whereas AREG was dispensable. Treg-derived IL-10 and CTLA-4 were both required to suppress acute autoreactive CD4+ T-cell activation, which correlated with disease control. These molecules also affected the accumulation in the recipients of engineered Tregs themselves, underlying complex roles for these molecules. Noteworthy, despite the persistence of the transferred Tregs and their protective effect, autoreactive T cells eventually accumulated in the spleen of treated mice. In conclusion, this study highlights the remarkable power of antigen receptor-engineered Tregs to appropriately provide multiple suppressive factors nonredundantly necessary to prevent autoimmune attacks.
Asunto(s)
Autoinmunidad , Enfermedades del Sistema Inmune , Animales , Antígeno CTLA-4 , Tratamiento Basado en Trasplante de Células y Tejidos , Factores de Transcripción Forkhead/genética , Interleucina-10 , Ratones , Receptores de Antígenos , Linfocitos T ReguladoresRESUMEN
INTRODUCTION: Metabolic dysfunction is now recognized as a pivotal component of Alzheimer's disease (AD), the most common dementia worldwide. However, the precise molecular mechanisms linking metabolic dysfunction to AD remain elusive. OBJECTIVE: Here, we investigated the direct impact of soluble oligomeric amyloid beta (Aß) peptides, the main molecular hallmark of AD, on the leptin system, a major component of central energy metabolism regulation. METHODS: We developed a new time-resolved fluorescence resonance energy transfer-based Aß binding assay for the leptin receptor (LepR) and studied the effect of Aß on LepR function in several in vitro assays. The in vivo effect of Aß on LepR function was studied in an Aß-specific AD mouse model and in pro-opiomelanocortin (POMC) neurons of the hypothalamic arcuate nucleus. RESULTS: We revealed specific and high-affinity (Ki = 0.1 nM) binding of Aß to LepR. Pharmacological characterization of this interaction showed that Aß binds allosterically to the extracellular domain of LepR and negatively affects receptor function. Negative allosteric modulation of LepR by Aß was detected at the level of signaling pathways (STAT-3, AKT, and ERK) in vitroand in vivo. Importantly, the leptin-induced response of POMC neurons, key players in the regulation of metabolic function, was completely abolished in the presence of Aß. CONCLUSION: Our data indicate that Aß is a negative allosteric modulator of LepR, resulting in impaired leptin action, and qualify LepR as a new and direct target of Aß oligomers. Preventing the interaction of Aß with LepR might improve both the metabolic and cognitive dysfunctions in AD condition.
Asunto(s)
Regulación Alostérica/fisiología , Enfermedad de Alzheimer/metabolismo , Péptidos beta-Amiloides/metabolismo , Núcleo Arqueado del Hipotálamo/metabolismo , Leptina/metabolismo , Proopiomelanocortina/metabolismo , Receptores de Leptina/metabolismo , Animales , Línea Celular , Modelos Animales de Enfermedad , Células HEK293 , Humanos , Masculino , Ratones , Transducción de Señal/fisiologíaRESUMEN
Transmission of extracellular signals by G protein-coupled receptors typically relies on a cascade of intracellular events initiated by the activation of heterotrimeric G proteins or ß-arrestins followed by effector activation/inhibition. Here, we report an alternative signal transduction mode used by the orphan GPR50 that relies on the nuclear translocation of its carboxyl-terminal domain (CTD). Activation of the calcium-dependent calpain protease cleaves off the CTD from the transmembrane-bound GPR50 core domain between Phe-408 and Ser-409 as determined by MALDI-TOF-mass spectrometry. The cytosolic CTD then translocates into the nucleus assisted by its 'DPD' motif, where it interacts with the general transcription factor TFII-I to regulate c-fos gene transcription. RNA-Seq analysis indicates a broad role of the CTD in modulating gene transcription with ~ 8000 differentially expressed genes. Our study describes a non-canonical, direct signaling mode of GPCRs to the nucleus with similarities to other receptor families such as the NOTCH receptor.
Asunto(s)
Proteínas del Tejido Nervioso/genética , Transporte de Proteínas/genética , Receptores Acoplados a Proteínas G/genética , Núcleo Celular/genética , Núcleo Celular/metabolismo , Citoplasma/genética , Citoplasma/metabolismo , Humanos , Unión Proteica/genética , Receptores Notch , Transducción de Señal/genética , Espectrometría de Masa por Láser de Matriz Asistida de Ionización DesorciónRESUMEN
Melatonin receptors play important roles in the regulation of circadian and seasonal rhythms, sleep, retinal functions, the immune system, depression, and type 2 diabetes development. Melatonin receptors are approved drug targets for insomnia, non-24-hour sleep-wake disorders, and major depressive disorders. In mammals, two melatonin receptors (MTRs) exist, MT1 and MT2 , belonging to the G protein-coupled receptor (GPCR) superfamily. Similar to most other GPCRs, reliable antibodies recognizing melatonin receptors proved to be difficult to obtain. Here, we describe the development of the first monoclonal antibodies (mABs) for mouse MT1 and MT2 . Purified antibodies were extensively characterized for specific reactivity with mouse, rat, and human MT1 and MT2 by Western blot, immunoprecipitation, immunofluorescence, and proximity ligation assay. Several mABs were specific for either mouse MT1 or MT2 . None of the mABs cross-reacted with rat MTRs, and some were able to react with human MTRs. The specificity of the selected mABs was validated by immunofluorescence microscopy in three established locations (retina, suprachiasmatic nuclei, pituitary gland) for MTR expression in mice using MTR-KO mice as control. MT2 expression was not detected in mouse insulinoma MIN6 cells or pancreatic beta-cells. Collectively, we report the first monoclonal antibodies recognizing recombinant and native mouse melatonin receptors that will be valuable tools for future studies.
Asunto(s)
Anticuerpos Monoclonales/inmunología , Especificidad de Anticuerpos/inmunología , Receptor de Melatonina MT1/análisis , Receptor de Melatonina MT2/análisis , Animales , Ratones , Dominios Proteicos , Receptor de Melatonina MT1/inmunología , Receptor de Melatonina MT2/inmunologíaRESUMEN
Meiotic recombination shapes the genetic diversity transmitted upon sexual reproduction. However, its non-random distribution along the chromosomes constrains the landscape of potential genetic combinations. For a variety of purposes, it is desirable to expand the natural repertoire by changing the distribution of crossovers in a wide range of eukaryotes. Toward this end, we report the local stimulation of meiotic recombination at a number of chromosomal sites by tethering the natural Spo11 protein to various DNA-binding modules: full-length DNA binding proteins, zinc fingers (ZFs), transcription activator-like effector (TALE) modules, and the CRISPR-Cas9 system. In the yeast Saccharomyces cerevisiae, each strategy is able to stimulate crossover frequencies in naturally recombination-cold regions. The binding and cleavage efficiency of the targeting Spo11 fusions (TSF) are variable, being dependent on the chromosomal regions and potential competition with endogenous factors. TSF-mediated genome interrogation distinguishes naturally recombination-cold regions that are flexible and can be warmed-up (gene promoters and coding sequences), from those that remain refractory (gene terminators and centromeres). These results describe new generic experimental strategies to increase the genetic diversity of gametes, which should prove useful in plant breeding and other applications.
Asunto(s)
Intercambio Genético , Endodesoxirribonucleasas/genética , Meiosis/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Sistemas CRISPR-Cas , Cromosomas Fúngicos/genética , Roturas del ADN de Doble Cadena , Fusión Génica , Marcación de Gen/métodos , Recombinación Genética , Reproducibilidad de los Resultados , Efectores Tipo Activadores de la Transcripción/genética , Efectores Tipo Activadores de la Transcripción/metabolismoRESUMEN
An uncontrolled exaggerated Th17 response can drive the onset of autoimmune and inflammatory diseases. In this study, we show that, in T cells, Foxo1 is a negative regulator of the Th17 program. Using mixed bone marrow chimeras and Foxo1-deficient mice, we demonstrate that this control is effective in vivo, as well as in vitro during differentiation assays of naive T cells with specific inhibitor of Foxo1 or inhibitors of the PI3K/Akt pathway acting upstream of Foxo1. Consistently, expressing this transcription factor in T cells strongly decreases Th17 generation in vitro as well as transcription of both IL-17A and IL-23R RORγt-target genes. Finally, at the molecular level, we demonstrate that Foxo1 forms a complex with RORγt via its DNA binding domain to inhibit RORγt activity. We conclude that Foxo1 is a direct antagonist of the RORγt-Th17 program acting in a T cell-intrinsic manner.
Asunto(s)
Factores de Transcripción Forkhead/metabolismo , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/metabolismo , Subgrupos de Linfocitos T/metabolismo , Células Th17/metabolismo , Animales , Diferenciación Celular/genética , Línea Celular , Proteína Forkhead Box O1 , Factores de Transcripción Forkhead/deficiencia , Factores de Transcripción Forkhead/genética , Humanos , Inmunofenotipificación , Interleucina-17/genética , Interleucina-17/metabolismo , Recuento de Linfocitos , Ratones , Ratones Noqueados , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/antagonistas & inhibidores , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/química , Fenotipo , Fosfatidilinositol 3-Quinasas/metabolismo , Regiones Promotoras Genéticas , Dominios y Motivos de Interacción de Proteínas , Proteínas Proto-Oncogénicas c-akt/metabolismo , Transducción de Señal , Subgrupos de Linfocitos T/citología , Subgrupos de Linfocitos T/inmunología , Células Th17/citología , Células Th17/inmunología , Transcripción GenéticaRESUMEN
Inasmuch as the neurohormone melatonin is synthetically derived from serotonin (5-HT), a close interrelationship between both has long been suspected. The present study reveals a hitherto unrecognized cross-talk mediated via physical association of melatonin MT2 and 5-HT2C receptors into functional heteromers. This is of particular interest in light of the "synergistic" melatonin agonist/5-HT2C antagonist profile of the novel antidepressant agomelatine. A suite of co-immunoprecipitation, bioluminescence resonance energy transfer, and pharmacological techniques was exploited to demonstrate formation of functional MT2 and 5-HT2C receptor heteromers both in transfected cells and in human cortex and hippocampus. MT2/5-HT2C heteromers amplified the 5-HT-mediated Gq/phospholipase C response and triggered melatonin-induced unidirectional transactivation of the 5-HT2C protomer of MT2/5-HT2C heteromers. Pharmacological studies revealed distinct functional properties for agomelatine, which shows "biased signaling." These observations demonstrate the existence of functionally unique MT2/5-HT2C heteromers and suggest that the antidepressant agomelatine has a distinctive profile at these sites potentially involved in its therapeutic effects on major depression and generalized anxiety disorder. Finally, MT2/5-HT2C heteromers provide a new strategy for the discovery of novel agents for the treatment of psychiatric disorders.
Asunto(s)
Melatonina/metabolismo , Multimerización de Proteína , Receptor de Melatonina MT2/química , Receptor de Serotonina 5-HT2C/química , Serotonina/metabolismo , Transducción de Señal , Acetamidas/farmacología , Arrestinas/metabolismo , Sinergismo Farmacológico , Regulación de la Expresión Génica/efectos de los fármacos , Células HEK293 , Células HeLa , Humanos , Melatonina/farmacología , Multimerización de Proteína/efectos de los fármacos , Estructura Cuaternaria de Proteína , Transporte de Proteínas/efectos de los fármacos , Receptor de Melatonina MT1/metabolismo , Receptor de Melatonina MT2/genética , Receptor de Melatonina MT2/metabolismo , Receptor de Serotonina 5-HT2C/genética , Receptor de Serotonina 5-HT2C/metabolismo , Serotonina/farmacología , Transducción de Señal/efectos de los fármacos , Activación Transcripcional/efectos de los fármacos , Activación Transcripcional/genética , Fosfolipasas de Tipo C/metabolismo , beta-ArrestinasRESUMEN
Proliferative glomerulonephritis is a severe condition that often leads to kidney failure. There is a significant lack of effective treatment for these disorders. Here, following the identification of a somatic PIK3CA gain-of-function mutation in podocytes of a patient, we demonstrate using multiple genetically engineered mouse models, single-cell RNA sequencing, and spatial transcriptomics the crucial role played by this pathway for proliferative glomerulonephritis development by promoting podocyte proliferation, dedifferentiation, and inflammation. Additionally, we show that alpelisib, a PI3Kα inhibitor, improves glomerular lesions and kidney function in different mouse models of proliferative glomerulonephritis and lupus nephritis by targeting podocytes. Surprisingly, we determined that pharmacological inhibition of PI3Kα affects B and T lymphocyte populations in lupus nephritis mouse models, with a decrease in the production of proinflammatory cytokines, autoantibodies, and glomerular complement deposition, which are all characteristic features of PI3Kδ inhibition, the primary PI3K isoform expressed in lymphocytes. Importantly, PI3Kα inhibition does not impact lymphocyte function under normal conditions. These findings were then confirmed in human lymphocytes isolated from patients with active lupus nephritis. In conclusion, we demonstrate the major role played by PI3Kα in proliferative glomerulonephritis and show that in this condition, alpelisib acts on both podocytes and the immune system.
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Fosfatidilinositol 3-Quinasa Clase I , Modelos Animales de Enfermedad , Nefritis Lúpica , Podocitos , Animales , Femenino , Humanos , Ratones , Linfocitos B/inmunología , Linfocitos B/patología , Fosfatidilinositol 3-Quinasa Clase I/genética , Fosfatidilinositol 3-Quinasa Clase I/antagonistas & inhibidores , Fosfatidilinositol 3-Quinasa Clase I/metabolismo , Glomerulonefritis/patología , Glomerulonefritis/inmunología , Glomerulonefritis/genética , Glomerulonefritis/enzimología , Glomerulonefritis/tratamiento farmacológico , Nefritis Lúpica/patología , Nefritis Lúpica/inmunología , Nefritis Lúpica/genética , Nefritis Lúpica/enzimología , Inhibidores de las Quinasa Fosfoinosítidos-3/farmacología , Podocitos/patología , Podocitos/inmunología , Podocitos/metabolismo , Linfocitos T/inmunología , Linfocitos T/patología , TiazolesRESUMEN
Congenital pseudarthrosis of the tibia (CPT) is a severe pathology marked by spontaneous bone fractures that fail to heal, leading to fibrous nonunion. Half of patients with CPT are affected by the multisystemic genetic disorder neurofibromatosis type 1 (NF1) caused by mutations in the NF1 tumor suppressor gene, a negative regulator of RAS-mitogen-activated protein kinase (MAPK) signaling pathway. Here, we analyzed patients with CPT and Prss56-Nf1 knockout mice to elucidate the pathogenic mechanisms of CPT-related fibrous nonunion and explored a pharmacological approach to treat CPT. We identified NF1-deficient Schwann cells and skeletal stem/progenitor cells (SSPCs) in pathological periosteum as affected cell types driving fibrosis. Whereas NF1-deficient SSPCs adopted a fibrotic fate, NF1-deficient Schwann cells produced critical paracrine factors including transforming growth factor-ß and induced fibrotic differentiation of wild-type SSPCs. To counteract the elevated RAS-MAPK signaling in both NF1-deficient Schwann cells and SSPCs, we used MAPK kinase (MEK) and Src homology 2 containing protein tyrosine phosphatase 2 (SHP2) inhibitors. Combined MEK-SHP2 inhibition in vivo prevented fibrous nonunion in the Prss56-Nf1 knockout mouse model, providing a promising therapeutic strategy for the treatment of fibrous nonunion in CPT.
Asunto(s)
Ratones Noqueados , Neurofibromina 1 , Proteína Tirosina Fosfatasa no Receptora Tipo 11 , Seudoartrosis , Células de Schwann , Animales , Femenino , Humanos , Masculino , Ratones , Diferenciación Celular/efectos de los fármacos , Fibrosis , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Neurofibromatosis 1/patología , Neurofibromatosis 1/metabolismo , Neurofibromatosis 1/complicaciones , Neurofibromina 1/metabolismo , Neurofibromina 1/genética , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Proteína Tirosina Fosfatasa no Receptora Tipo 11/antagonistas & inhibidores , Seudoartrosis/patología , Seudoartrosis/metabolismo , Seudoartrosis/congénito , Células de Schwann/metabolismo , Células de Schwann/efectos de los fármacos , Células de Schwann/patología , Células Madre/metabolismo , Células Madre/efectos de los fármacos , Tibia/patologíaRESUMEN
Acquired mutations in the UBA1 gene were recently identified in patients with severe adult-onset auto-inflammatory syndrome called VEXAS (vacuoles, E1 enzyme, X-linked, autoinflammatory, somatic). However, the precise physiological and clinical impact of these mutations remains poorly defined. Here we study a unique prospective cohort of VEXAS patients. We show that monocytes from VEXAS are quantitatively and qualitatively impaired and display features of exhaustion with aberrant expression of chemokine receptors. In peripheral blood from VEXAS patients, we identify an increase in circulating levels of many proinflammatory cytokines, including IL-1ß and IL-18 which reflect inflammasome activation and markers of myeloid cells dysregulation. Gene expression analysis of whole blood confirms these findings and also reveals a significant enrichment of TNF-α and NFκB signaling pathways that can mediate cell death and inflammation. This study suggests that the control of the nflammasome activation and inflammatory cell death could be therapeutic targets in VEXAS syndrome.
Asunto(s)
Inflamasomas , Monocitos , Síndromes Mielodisplásicos , Enfermedades Cutáneas Genéticas , Adulto , Humanos , Inflamasomas/genética , Estudios Prospectivos , Células Mieloides , MutaciónRESUMEN
Aggregates of the tau protein are a well-known hallmark of several neurodegenerative diseases, collectively referred to as tauopathies, including frontal temporal dementia and Alzheimer's disease (AD). Monitoring the transformation process of tau from physiological monomers into pathological oligomers or aggregates in a high-throughput, quantitative manner and in a cellular context is still a major challenge in the field. Identifying molecules able to interfere with those processes is of high therapeutic interest. Here, we developed a series of inter- and intramolecular tau biosensors based on the highly sensitive Nanoluciferase (Nluc) binary technology (NanoBiT) able to monitor the pathological conformational change and self-interaction of tau in living cells. Our repertoire of tau biosensors reliably reports i. molecular proximity of physiological full-length tau at microtubules; ii. changes in tau conformation and self-interaction associated with tau phosphorylation, as well as iii. tau interaction induced by seeds of recombinant tau or from mouse brain lysates of a mouse model of tau pathology. By comparing biosensors comprising different tau forms (i.e. full-length or short fragments, wild-type, or the disease-associated tau(P301L) variant) further insights into the tau transformation process are obtained. Proof-of-concept data for the high-throughput suitability and identification of molecules interfering with the pathological tau transformation processes are presented. This novel repertoire of tau biosensors is aimed to boost the disclosure of molecular mechanisms underlying pathological tau transformation in living cells and to discover new drug candidates for tau-related neurodegenerative diseases.
Asunto(s)
Enfermedad de Alzheimer , Tauopatías , Ratones , Animales , Proteínas tau/genética , Proteínas tau/metabolismo , Enfermedad de Alzheimer/metabolismo , Tauopatías/patología , Microtúbulos/metabolismo , Neuronas/fisiología , Encéfalo/metabolismoRESUMEN
Gain-of-function mutations in the PIK3CD gene result in activated phosphoinositide 3-kinase δ syndrome type 1 (APDS1). This syndrome is a life-threatening combined immunodeficiency and today there are neither optimal nor long-term therapeutic solutions for APDS1 patients. Thus, new alternative treatments are highly needed. The aim of the present study is to explore one therapeutic avenue that consists of the correction of the PIK3CD gene through gene editing. Our proof-of-concept shows that TALEN-mediated gene correction of the mutated PIK3CD gene in APDS1 T cells results in normalized phospho-AKT levels in basal and activated conditions. Normalization of PI3K signaling was correlated to restored cytotoxic functions of edited CD8+ T cells. At the transcriptomic level, single-cell RNA sequencing revealed corrected signatures of CD8+ effector memory and CD8+ proliferating T cells. This proof-of-concept study paves the way for the future development of a gene therapy candidate to cure activated phosphoinositide 3-kinase δ syndrome type 1.
RESUMEN
Loss of NBEAL2 function leads to grey platelet syndrome (GPS), a bleeding disorder characterized by macro-thrombocytopenia and α-granule-deficient platelets. A proportion of patients with GPS develop autoimmunity through an unknown mechanism, which might be related to the proteins NBEAL2 interacts with, specifically in immune cells. Here we show a comprehensive interactome of NBEAL2 in primary T cells, based on mass spectrometry identification of altogether 74 protein association partners. These include LRBA, a member of the same BEACH domain family as NBEAL2, recessive mutations of which cause autoimmunity and lymphocytic infiltration through defective CTLA-4 trafficking. Investigating the potential association between NBEAL2 and CTLA-4 signalling suggested by the mass spectrometry results, we confirm by co-immunoprecipitation that CTLA-4 and NBEAL2 interact with each other. Interestingly, NBEAL2 deficiency leads to low CTLA-4 expression in patient-derived effector T cells, while their regulatory T cells appear unaffected. Knocking-down NBEAL2 in healthy primary T cells recapitulates the low CTLA-4 expression observed in the T cells of GPS patients. Our results thus show that NBEAL2 is involved in the regulation of CTLA-4 expression in conventional T cells and provide a rationale for considering CTLA-4-immunoglobulin therapy in patients with GPS and autoimmune disease.
Asunto(s)
Síndrome de Plaquetas Grises , Humanos , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Plaquetas/metabolismo , Proteínas Sanguíneas/genética , Antígeno CTLA-4/genética , Antígeno CTLA-4/metabolismo , Síndrome de Plaquetas Grises/genética , Síndrome de Plaquetas Grises/metabolismoRESUMEN
Gain-of-function mutations in stimulator of interferon gene 1 (STING1) result in STING-associated vasculopathy with onset in infancy (SAVI), a severe autoinflammatory disease. Although elevated type I interferon (IFN) production is thought to be the leading cause of the symptoms observed in patients, STING can induce a set of pathways, which have roles in the onset and severity of SAVI and remain to be elucidated. To this end, we performed a multi-omics comparative analysis of peripheral blood mononuclear cells (PBMCs) and plasma from SAVI patients and healthy controls, combined with a dataset of healthy PBMCs treated with IFN-ß. Our data reveal a subset of disease-associated monocyte, expressing elevated CCL3, CCL4, and IL-6, as well as a strong integrated stress response, which we suggest is the result of direct PERK activation by STING. Cell-to-cell communication inference indicates that these monocytes lead to T cell early activation, resulting in their senescence and apoptosis. Last, we propose a transcriptomic signature of STING activation, independent of type I IFN response.
Asunto(s)
Interferón Tipo I , Enfermedades Vasculares , Humanos , Monocitos/metabolismo , Leucocitos Mononucleares/metabolismo , Enfermedades Vasculares/genética , Enfermedades Vasculares/metabolismo , Interferón Tipo I/metabolismo , ARNRESUMEN
Bone regeneration involves skeletal stem/progenitor cells (SSPCs) recruited from bone marrow, periosteum, and adjacent skeletal muscle. To achieve bone reconstitution after injury, a coordinated cellular and molecular response is required from these cell populations. Here, we show that SSPCs from periosteum and skeletal muscle are enriched in osteochondral progenitors, and more efficiently contribute to endochondral ossification during fracture repair as compared to bone-marrow stromal cells. Single-cell RNA sequencing (RNAseq) analyses of periosteal cells reveal the cellular heterogeneity of periosteum at steady state and in response to bone fracture. Upon fracture, both periosteal and skeletal muscle SSPCs transition from a stem/progenitor to a fibrogenic state prior to chondrogenesis. This common activation pattern in periosteum and skeletal muscle SSPCs is mediated by bone morphogenetic protein (BMP) signaling. Functionally, Bmpr1a gene inactivation in platelet-derived growth factor receptor alpha (Pdgfra)-derived SSPCs impairs bone healing and decreases SSPC proliferation, migration, and osteochondral differentiation. These results uncover a coordinated molecular program driving SSPC activation in periosteum and skeletal muscle toward endochondral ossification during bone regeneration. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).