RESUMO
Chronic viral infections are associated with hematopoietic suppression, bone marrow (BM) failure, and hematopoietic stem cell (HSC) exhaustion. However, how persistent viral challenge and inflammatory responses target BM tissues and perturb hematopoietic competence remains poorly understood. Here, we combine functional analyses with advanced 3D microscopy to demonstrate that chronic infection with lymphocytic choriomeningitis virus leads to (1) long-lasting decimation of the BM stromal network of mesenchymal CXCL12-abundant reticular cells, (2) proinflammatory transcriptional remodeling of remaining components of this key niche subset, and (3) durable functional defects and decreased competitive fitness in HSCs. Mechanistically, BM immunopathology is elicited by virus-specific, activated CD8 T cells, which accumulate in the BM via interferon-dependent mechanisms. Combined antibody-mediated inhibition of type I and II IFN pathways completely preempts degeneration of CARc and protects HSCs from chronic dysfunction. Hence, viral infections and ensuing immune reactions durably impact BM homeostasis by persistently decreasing the competitive fitness of HSCs and disrupting essential stromal-derived, hematopoietic-supporting cues.
Assuntos
Medula Óssea/virologia , Células-Tronco Hematopoéticas/patologia , Células-Tronco Hematopoéticas/virologia , Coriomeningite Linfocítica/patologia , Animais , Medula Óssea/metabolismo , Medula Óssea/patologia , Linfócitos T CD8-Positivos/metabolismo , Linfócitos T CD8-Positivos/patologia , Linfócitos T CD8-Positivos/virologia , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Doença Crônica , Regulação da Expressão Gênica , Transplante de Células-Tronco Hematopoéticas , Interferons/metabolismo , Coriomeningite Linfocítica/metabolismo , Coriomeningite Linfocítica/virologia , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Receptor de Interferon alfa e beta/genética , Receptor de Interferon alfa e beta/imunologia , Receptor de Interferon alfa e beta/metabolismoRESUMO
Hematopoietic stem cells are maintained and regulated in spatially confined microenvironments within the bone marrow, in which oxygen availability is hypothesized to be very limited. The hypoxic nature of HSC niches is proposed to play a fundamental role in the preservation of fundamental stem cell properties through the induction of a distinct glycolytic metabolic profile in HSCs. Thus, the capacity to determine oxygen levels or cellular oxygenation status in specific tissue locations is essential to deepen our understanding of HSC biology. We here describe a methodology to indirectly quantify the hypoxic status of individual cells in situ within histological sections of bone marrow tissues. We employ the well-characterized nitroimidazole probe, pimonidazole, which acts as an oxygen mimetic and irreversibly incorporates into cellular proteins only under hypoxic conditions. The use of fluorescently labeled antibodies that recognize pimonidazole epitopes then enables the indirect assessment of the intracellular hypoxic status and its relationship to cell positioning within the complex tissue topography of the bone marrow.
Assuntos
Células da Medula Óssea/citologia , Células-Tronco Hematopoéticas/citologia , Oxigênio/análise , Animais , Materiais Biomiméticos/farmacologia , Células da Medula Óssea/metabolismo , Hipóxia Celular , Glicólise , Células-Tronco Hematopoéticas/metabolismo , Humanos , Camundongos , Nitroimidazóis/farmacologia , Nicho de Células-TroncoRESUMO
B cell acute lymphoblastic leukemia (B-ALL) is the most common childhood cancer, with cure rates of â¼80%. MLL-rearranged (MLLr) B-ALL (MLLr-B-ALL) has, however, an unfavorable prognosis with common therapy refractoriness and early relapse, and therefore new therapeutic targets are needed for relapsed/refractory MLLr-B-ALL. MLLr leukemias are characterized by the specific expression of chondroitin sulfate proteoglycan-4, also known as neuron-glial antigen-2 (NG2). NG2 was recently shown involved in leukemia invasiveness and central nervous system infiltration in MLLr-B-ALL, and correlated with lower event-free survival (EFS). We here hypothesized that blocking NG2 may synergize with established induction therapy for B-ALL based on vincristine, glucocorticoids, and L-asparaginase (VxL). Using robust patient-derived xenograft (PDX) models, we found that NG2 is crucial for MLLr-B-ALL engraftment upon intravenous (i.v.) transplantation. In vivo blockade of NG2 using either chondroitinase-ABC or an anti-NG2-specific monoclonal antibody (MoAb) resulted in a significant mobilization of MLLr-B-ALL blasts from bone marrow (BM) to peripheral blood (PB) as demonstrated by cytometric and 3D confocal imaging analysis. When combined with either NG2 antagonist, VxL treatment achieved higher rates of complete remission, and consequently higher EFS and delayed time to relapse. Mechanistically, anti-NG2 MoAb induces neither antibody-dependent cell-mediated not complement-dependent cytotoxicity. NG2 blockade rather overrides BM stroma-mediated chemoprotection through PB mobilization of MLLr-B-ALL blasts, thus becoming more accessible to chemotherapy. We provide a proof of concept for NG2 as a therapeutic target for MLLr-B-ALL.
Assuntos
Antígenos/metabolismo , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Linfócitos B/patologia , Resistencia a Medicamentos Antineoplásicos , Rearranjo Gênico , Histona-Lisina N-Metiltransferase/genética , Proteína de Leucina Linfoide-Mieloide/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/prevenção & controle , Proteoglicanas/metabolismo , Animais , Antígenos/genética , Asparaginase/administração & dosagem , Dexametasona/administração & dosagem , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Invasividade Neoplásica , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Proteoglicanas/genética , Indução de Remissão , Taxa de Sobrevida , Vincristina/administração & dosagem , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Sinusoidal endothelial cells and mesenchymal CXCL12-abundant reticular cells are principal bone marrow stromal components, which critically modulate haematopoiesis at various levels, including haematopoietic stem cell maintenance. These stromal subsets are thought to be scarce and function via highly specific interactions in anatomically confined niches. Yet, knowledge on their abundance, global distribution and spatial associations remains limited. Using three-dimensional quantitative microscopy we show that sinusoidal endothelial and mesenchymal reticular subsets are remarkably more abundant than estimated by conventional flow cytometry. Moreover, both cell types assemble in topologically complex networks, associate to extracellular matrix and pervade marrow tissues. Through spatial statistical methods we challenge previous models and demonstrate that even in the absence of major specific interaction forces, virtually all tissue-resident cells are invariably in physical contact with, or close proximity to, mesenchymal reticular and sinusoidal endothelial cells. We further show that basic structural features of these stromal components are preserved during ageing.