RESUMO
ß-catenin is required for establishment of leukemic stem cells (LSCs) in acute myeloid leukemia (AML). Targeted inhibition of ß-catenin signaling has been hampered by the lack of pathway components amenable to pharmacologic manipulation. Here we identified a novel ß-catenin regulator, GPR84, a member of the G protein-coupled receptor family that represents a highly tractable class of drug targets. High GPR84 expression levels were confirmed in human and mouse AML LSCs compared with hematopoietic stem cells (HSCs). Suppression of GPR84 significantly inhibited cell growth by inducing G1-phase cell-cycle arrest in pre-LSCs, reduced LSC frequency, and impaired reconstitution of stem cell-derived mixed-lineage leukemia (MLL) AML, which represents an aggressive and drug-resistant subtype of AML. The GPR84-deficient phenotype in established AML could be rescued by expression of constitutively active ß-catenin. Furthermore, GPR84 conferred a growth advantage to Hoxa9/Meis1a-transduced stem cells. Microarray analysis demonstrated that GPR84 significantly upregulated a small set of MLL-fusion targets and ß-catenin coeffectors, and downregulated a hematopoietic cell-cycle inhibitor. Altogether, our data reveal a previously unrecognized role of GPR84 in maintaining fully developed AML by sustaining aberrant ß-catenin signaling in LSCs, and suggest that targeting the oncogenic GPR84/ß-catenin signaling axis may represent a novel therapeutic strategy for AML.
Assuntos
Transformação Celular Neoplásica/genética , Leucemia Mieloide Aguda/genética , Células-Tronco Neoplásicas/metabolismo , Receptores de Superfície Celular/fisiologia , beta Catenina/genética , Animais , Transformação Celular Neoplásica/metabolismo , Células Cultivadas , Perfilação da Expressão Gênica , Regulação Leucêmica da Expressão Gênica , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patologia , Camundongos , Camundongos Endogâmicos C57BL , Análise em Microsséries , Células-Tronco Neoplásicas/patologia , Receptores Acoplados a Proteínas G , Transdução de Sinais/genética , beta Catenina/metabolismoRESUMO
Neutrophil extracellular traps (NETs) are major contributors to inflammation and autoimmunity, playing a key role in the development of thrombotic disorders. NETs, composed of DNA, histones, and numerous other proteins serve as scaffolds for thrombus formation and promote platelet activation, coagulation, and endothelial dysfunction. Accumulating evidence indicates that NETs mediate thrombosis in autoimmune diseases, viral and bacterial infections, cancer, and cardiovascular disease. This article reviews the role and mechanisms of immune complexes in NETs formation and their contribution to the generation of a prothrombotic state. Immune complexes are formed by interactions between antigens and antibodies and can induce NETosis by the direct activation of neutrophils via Fc receptors, via platelet activation, and through endothelial inflammation. We discuss the mechanisms by which NETs induced by immune complexes contribute to immune thrombotic processes and consider the potential development of therapeutic strategies. Targeting immune complexes and NETosis hold promise for mitigating thrombotic events and reducing the burden of immune thrombosis.
RESUMO
Platelets play an important role in hemostasis by forming clots and stopping bleeding. In immune thrombotic conditions, platelets and leukocytes are aberrantly activated by pathogenic antibodies resulting in platelet aggregates and NETosis, leading to thrombosis and thrombocytopenia. A simple assay that assesses platelet function and antibody activity is light transmission aggregometry. This assay can be used to determine antibody activity in patients with disorders such as heparin-induced thrombocytopenia (HIT) and vaccine-induced thrombotic thrombocytopenia (VITT). Briefly, for detection of pathogenic antibody, platelet-rich plasma (PRP) is treated with a specific agent (e.g., patient sera or purified patient antibodies) with constant stirring. Upon activation, platelets undergo a shape change and adhere to each other forming aggregates. This causes a reduction in opacity allowing more light to pass through PRP. Light transmission through the cuvette is proportional to the degree of platelet aggregation and is measured by the photocell over time. The advantage of this protocol is that it is a simple, reliable assay that can be applied to assess antibody activity in thrombotic conditions. Light transmission aggregometry does not require the use of radioactive reagents and is technically less demanding compared with 14C-serotonin release assay, another common assay for detecting antibody activity. Key features ⢠This protocol can be used to assess platelet function and to detect platelet activating antibodies in diseases such as HIT and VITT. ⢠Does not require radioactive reagents, requires an aggregometer; based on the light transmission aggregometry protocol, adapted for detection of VITT and other platelet-activating antibodies. ⢠Two positive controls are required for reliable detection of antibodies in diseases such as HIT/VITT, namely a weak HIT/VITT antibody and a physiological agonist. ⢠For detection of HIT/VITT antibodies, it is essential to use donors known to have platelets reactive to these antibodies to avoid false negative results.
RESUMO
Cardiovascular disease, including myocardial infarction (MI), is the leading cause of death globally. Current antithrombotic medications used during MI treatment are predominantly directed towards platelet inhibition and, to a lesser extent, anticoagulation. Bleeding is a major risk of such treatment and could be circumvented by targeting other causative factors essential for arterial thrombus formation. We sought to re-evaluate the cellular composition of arterial thrombus in order to better understand mechanisms that lead to coronary artery thrombosis in acute MI. We performed detailed histological and immunohistochemical analysis of coronary artery thrombi aspirated from 26 patients undergoing emergency percutaneous coronary intervention for acute ST elevated myocardial infarction (STEMI). Coronary arterial thrombi had an unanticipated cellular heterogeneity. Neutrophil extracellular traps (NETs) were observed in thrombi as identified by anti-citrullinated histone 3 and anti-myeloperoxidase staining. Increased abundance of NETs was seen directly surrounding erythrocytes. Extracellular iron and erythrocyte fragments were also associated with areas of NETs suggesting a possible link. Our results shed light on potential involvement of erythrocytes in coronary arterial thrombosis through activation of platelets and induction of NETs. If supported by further in vitro and in vivo studies, novel therapies to inhibit NET formation or coagulation activation by erythrocyte release products, could bolster current myocardial infarction treatment.
Assuntos
Trombose Coronária , Eritrócitos , Armadilhas Extracelulares , Infarto do Miocárdio/complicações , Neutrófilos/patologia , Idoso , Coagulação Sanguínea , Plaquetas/patologia , Trombose Coronária/etiologia , Trombose Coronária/patologia , Vasos Coronários/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Infarto do Miocárdio/patologiaRESUMO
Vaccine-induced immune thrombotic thrombocytopenia (VITT) is a rare yet serious adverse effect of the adenoviral vector vaccines ChAdOx1 nCoV-19 (AstraZeneca) and Ad26.COV2.S (Janssen) against COVID-19. The mechanisms involved in clot formation and thrombocytopenia in VITT are yet to be fully determined. Here we show neutrophils undergoing NETosis and confirm expression markers of NETs in VITT patients. VITT antibodies directly stimulate neutrophils to release NETs and induce thrombus formation containing abundant platelets, neutrophils, fibrin, extracellular DNA and citrullinated histone H3 in a flow microfluidics system and in vivo. Inhibition of NETosis prevents VITT-induced thrombosis in mice but not thrombocytopenia. In contrast, in vivo blockage of FcγRIIa abrogates both thrombosis and thrombocytopenia suggesting these are distinct processes. Our findings indicate that anti-PF4 antibodies activate blood cells via FcγRIIa and are responsible for thrombosis and thrombocytopenia in VITT. Future development of NETosis and FcγRIIa inhibitors are needed to treat VITT and similar immune thrombotic thrombocytopenia conditions more effectively, leading to better patient outcomes.
Assuntos
COVID-19 , Armadilhas Extracelulares , Púrpura Trombocitopênica Idiopática , Trombocitopenia , Trombose , Vacinas , Ad26COVS1 , Animais , ChAdOx1 nCoV-19 , Armadilhas Extracelulares/metabolismo , Humanos , Camundongos , Púrpura Trombocitopênica Idiopática/induzido quimicamente , Trombocitopenia/induzido quimicamente , Trombose/prevenção & controle , Vacinas/metabolismoRESUMO
Heparin-induced thrombocytopenia (HIT) is associated with severe and potentially lethal thrombotic complications. NETosis was recently shown to be an important driver of thrombosis in HIT. We investigated the role of reactive oxygen species (ROS) and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase 2 (NOX2) and their contributions to thrombus development in HIT. We showed that neutrophil activation by HIT immune complexes induced ROS-dependent NETosis. Analysis of thrombi formed in a microfluidics system showed ROS production in both platelets and neutrophils, and abundant neutrophil extracellular traps (NETs) and ROS distributed throughout the clot. Neutrophil-targeted ROS inhibition was sufficient to block HIT-induced NETosis and thrombosis using human blood. Inhibition of NOX2 with diphenyleneiodonium chloride or GSK2795039 abrogated HIT-induced thrombi in vivo using FcγRIIa+/hPF4+-transgenic mice. Thrombocytopenia in mice remained unaffected by ROS inhibition. Increased ROS production in activated neutrophils was also confirmed using fresh blood from patients with active HIT. Our findings show that ROS and NOX2 play a crucial role in NETosis and thrombosis in HIT. This enhances our understanding of the processes driving thrombosis in HIT and identifies NOX2 as a potential new therapeutic target for antithrombotic treatment of HIT.
Assuntos
Armadilhas Extracelulares , Trombocitopenia , Trombose , Animais , Humanos , Camundongos , NADPH Oxidases/genética , Ativação de Neutrófilo , Trombocitopenia/induzido quimicamente , Trombose/induzido quimicamente , Trombose/tratamento farmacológicoRESUMO
Heparin-induced thrombocytopenia/thrombosis (HIT) is a serious immune reaction to heparins, characterized by thrombocytopenia and often severe thrombosis with high morbidity and mortality. HIT is mediated by IgG antibodies against heparin/platelet factor 4 antigenic complexes. These complexes are thought to activate platelets leading to thrombocytopenia and thrombosis. Here we show that HIT immune complexes induce NETosis via interaction with FcγRIIa on neutrophils and through neutrophil-platelet association. HIT immune complexes induce formation of thrombi containing neutrophils, extracellular DNA, citrullinated histone H3 and platelets in a microfluidics system and in vivo, while neutrophil depletion abolishes thrombus formation. Absence of PAD4 or PAD4 inhibition with GSK484 abrogates thrombus formation but not thrombocytopenia, suggesting they are induced by separate mechanisms. NETs markers and neutrophils undergoing NETosis are present in HIT patients. Our findings demonstrating the involvement of NETosis in thrombosis will modify the current concept of HIT pathogenesis and may lead to new therapeutic strategies.
Assuntos
Plaquetas/imunologia , Armadilhas Extracelulares/imunologia , Heparina/efeitos adversos , Neutrófilos/imunologia , Receptores de IgG/genética , Trombocitopenia/imunologia , Trombose/imunologia , Animais , Complexo Antígeno-Anticorpo/biossíntese , Plaquetas/efeitos dos fármacos , Citrulinação , Inibidores Enzimáticos/farmacologia , Armadilhas Extracelulares/química , Armadilhas Extracelulares/efeitos dos fármacos , Regulação da Expressão Gênica , Histonas/genética , Histonas/imunologia , Humanos , Imunoglobulina G/biossíntese , Camundongos , Camundongos Transgênicos , Técnicas Analíticas Microfluídicas , Ativação de Neutrófilo/efeitos dos fármacos , Ativação de Neutrófilo/imunologia , Neutrófilos/efeitos dos fármacos , Ativação Plaquetária/efeitos dos fármacos , Ativação Plaquetária/imunologia , Fator Plaquetário 4/genética , Fator Plaquetário 4/imunologia , Proteína-Arginina Desiminase do Tipo 4 , Desiminases de Arginina em Proteínas/antagonistas & inibidores , Desiminases de Arginina em Proteínas/genética , Desiminases de Arginina em Proteínas/imunologia , Receptores de IgG/imunologia , Transdução de Sinais , Trombocitopenia/induzido quimicamente , Trombocitopenia/patologia , Trombose/induzido quimicamente , Trombose/patologia , Trombose/prevenção & controleRESUMO
Platelet production occurs principally in the bone marrow in a process known as thrombopoiesis. During thrombopoiesis, hematopoietic progenitor cells differentiate to form platelet precursors called megakaryocytes, which terminally differentiate to release platelets from long cytoplasmic processes termed proplatelets. Megakaryocytes are rare cells confined to the bone marrow and are therefore difficult to harvest in sufficient numbers for laboratory use. Efficient production of human megakaryocytes can be achieved in vitro by culturing CD34+ cells under suitable conditions. The protocol detailed here describes isolation of CD34+ cells by magnetic cell sorting from umbilical cord blood samples. The necessary steps to produce highly pure, mature megakaryocytes under serum-free conditions are described. Details of phenotypic analysis of megakaryocyte differentiation and determination of proplatelet formation and platelet production are also provided. Effectors that influence megakaryocyte differentiation and/or proplatelet formation, such as anti-platelet antibodies or thrombopoietin mimetics, can be added to cultured cells to examine biological function.