RESUMO
COVID-19 is a primary respiratory illness that is frequently complicated by systemic involvement of the vasculature. Vascular involvement leads to an array of complications ranging from thrombosis to pulmonary edema secondary to loss of barrier function. This review will address the vasculopathy of COVID-19 with a focus on the role of the endothelium in orchestrating the systemic response to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. The endothelial receptor systems and molecular pathways activated in the setting of COVID-19 and the consequences of these inflammatory and prothrombotic changes on endothelial cell function will be discussed. The sequelae of COVID-19 vascular involvement at the level of organ systems will also be addressed, with an emphasis on the pulmonary vasculature but with consideration of effects on other vascular beds. The dramatic changes in endothelial phenotypes associated with COVID-19 has enabled the identification of biomarkers that could help guide therapy and predict outcomes. Knowledge of vascular pathogenesis in COVID-19 has also informed therapeutic approaches that may control its systemic sequelae. Because our understanding of vascular response in COVID-19 continues to evolve, we will consider areas of controversy, such as the extent to which SARS-CoV-2 directly infects endothelium and the degree to which vascular responses to SARS-CoV-2 are unique or common to those of other viruses capable of causing severe respiratory disease. This conceptual framework describing how SARS-CoV-2 infection affects endothelial inflammation, prothrombotic transformation, and barrier dysfunction will provide a context for interpreting new information as it arises addressing the vascular complications of COVID-19.
Assuntos
COVID-19 , Trombose , Doenças Vasculares , COVID-19/complicações , Humanos , Inflamação , SARS-CoV-2 , Trombose/etiologia , Doenças Vasculares/etiologiaRESUMO
Extracellular diphosphate and triphosphate nucleotides are released from activated or injured cells to trigger vascular and immune P2 purinergic receptors, provoking inflammation and vascular thrombosis. These metabokines are scavenged by ectonucleoside triphosphate diphosphohydrolase-1 (E-NTPDase1 or CD39). Further degradation of the monophosphate nucleoside end products occurs by surface ecto-5'-nucleotidase (NMPase) or CD73. These ectoenzymatic processes work in tandem to promote adenosinergic responses, which are immunosuppressive and antithrombotic. These homeostatic ectoenzymatic mechanisms are lost in the setting of oxidative stress, which exacerbates inflammatory processes. We have engineered bifunctional enzymes made up from ectodomains (ECDs) of CD39 and CD73 within a single polypeptide. Human alkaline phosphatase-ectodomain (ALP-ECD) and human acid phosphatase-ectodomain (HAP-ECD) fusion proteins were also generated, characterized, and compared with these CD39-ECD, CD73-ECD, and bifunctional fusion proteins. Through the application of colorimetrical functional assays and high-performance liquid chromatography kinetic assays, we demonstrate that the bifunctional ectoenzymes express high levels of CD39-like NTPDase activity and CD73-like NMPase activity. Chimeric CD39-CD73-ECD proteins were superior in converting triphosphate and diphosphate nucleotides into nucleosides when compared with ALP-ECD and HAP-ECD. We also note a pH sensitivity difference between the bifunctional fusion proteins and parental fusions, as well as ectoenzymatic property distinctions. Intriguingly, these innovative reagents decreased platelet activation to exogenous agonists in vitro. We propose that these chimeric fusion proteins could serve as therapeutic agents in inflammatory diseases, acting to scavenge proinflammatory ATP and also generate anti-inflammatory adenosine.
Assuntos
5'-Nucleotidase/farmacologia , Anti-Inflamatórios/farmacologia , Apirase/farmacologia , Inibidores da Agregação Plaquetária/farmacologia , Agregação Plaquetária/efeitos dos fármacos , Engenharia de Proteínas , 5'-Nucleotidase/química , 5'-Nucleotidase/genética , 5'-Nucleotidase/metabolismo , Nucleotídeos de Adenina/metabolismo , Anti-Inflamatórios/química , Anti-Inflamatórios/metabolismo , Apirase/química , Apirase/genética , Apirase/metabolismo , Proteínas Ligadas por GPI/química , Proteínas Ligadas por GPI/genética , Proteínas Ligadas por GPI/metabolismo , Proteínas Ligadas por GPI/farmacologia , Células HEK293 , Humanos , Concentração de Íons de Hidrogênio , Hidrólise , Cinética , Inibidores da Agregação Plaquetária/química , Inibidores da Agregação Plaquetária/metabolismo , Conformação Proteica , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Relação Estrutura-Atividade , Especificidade por SubstratoRESUMO
Purinergic signaling is important in the activation and differentiation of macrophages, which play divergent roles in the pathophysiology of liver fibrosis. The ectonucleotidase CD39 is known to modulate the immunoregulatory phenotype of macrophages, but whether this specifically impacts cholestatic liver injury is unknown. Here, we investigated the role of macrophage-expressed CD39 on the development of biliary injury and fibrosis in a mouse model of sclerosing cholangitis. Myeloid-specific CD39-deficient mice (LysMCreCd39fl/fl) were generated. Global CD39 null (Cd39-/-), wild-type (WT), LysMCreCd39fl/fl, and Cd39fl/fl control mice were exposed to 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC) to induce biliary fibrosis. Hepatic hydroxyproline levels, liver histology, immunohistochemistry, mRNA expression levels, and serum biochemistry were then assessed. Following 3 weeks of DDC-feeding, Cd39-/- mice exhibited more severe fibrosis, when compared to WT mice as reflected by morphology and increased liver collagen content. Myeloid-specific CD39 deletion in LysMCreCd39fl/fl mice recapitulated the phenotype of global Cd39-/-, after exposure to DDC, and resulted in similar worsening of liver fibrosis when compared to Cd39fl/fl control animals. Further, DDC-treated LysMCreCd39fl/fl mice exhibited elevated serum levels of transaminases and total bilirubin, as well as increased hepatic expression of the profibrogenic genes Tgf-ß1, Tnf-α, and α-Sma. However, no clear differences were observed in the expression of macrophage-elaborated specific cytokines between LysMCreCd39fl/fl and Cd39fl/fl animals subjected to biliary injury. Our results in the DDC-induced biliary type liver fibrosis model suggest that loss of CD39 expression on myeloid cells largely accounts for the exacerbated sclerosing cholangitis in global CD39 knockouts. These findings indicate that macrophage expressed CD39 protects from biliary liver injury and fibrosis and support a potential therapeutic target for human hepatobiliary diseases.
Assuntos
Antígenos CD/metabolismo , Apirase/metabolismo , Colangite Esclerosante/metabolismo , Animais , Colangite Esclerosante/induzido quimicamente , Colangite Esclerosante/patologia , Modelos Animais de Doenças , Cirrose Hepática/metabolismo , Macrófagos , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Piridinas/toxicidadeRESUMO
In Crohn's disease, pathogenic Th17-cells express low levels of CD39 ectonucleotidase and are refractory to the immunosuppressive effects of unconjugated bilirubin (UCB), an endogenous ligand for aryl-hydrocarbon-receptor (AhR). This resistance to AhR ligation might be associated with alterations in responses to hypoxia. Limited exposure to hypoxia appears beneficial in acute tissue injury. However, in protracted inflammation, hypoxemia may paradoxically result in Th17-cell activation. We report here that in vitro exposure of Th17-cells from Crohn's disease patients to hypoxia limits responsiveness to AhR stimulation by UCB, as reflected by lower CD39 levels. Blockade of hypoxia-inducible-factor-1alpha (HIF-1α) upregulates CD39 and favors Th17-cell regulatory responses. Resistance of Th17-cells to AhR signaling results, in part, from HIF-1α-dependent induction of ATP-binding cassette (ABC) transporters: multidrug-resistance-protein-1 (MDR1) and multidrug-resistance-associated-protein-4 (MRP4). Increased ABC transporters promote efflux of suppressive AhR ligands, such as UCB, from Th17-cells. Inhibition of MDR1, MRP4 and/or HIF-1α with ritonavir (RTV) reconstitutes AhR function in Th17-cells, enhancing therapeutic effects of UCB in dextran-sulfate-sodium-induced experimental colitis. Deleterious effects of hypoxia on Th17-cells in Crohn's disease can be ameliorated either by inhibiting HIF-1α or by suppressing ABC transporters to increase UCB availability as an AhR substrate. Targeting HIF-1α-ABC transporters could provide innovative therapeutic pathways for IBD.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/imunologia , Colite/imunologia , Doença de Crohn/imunologia , Subunidade alfa do Fator 1 Induzível por Hipóxia/imunologia , Proteínas Associadas à Resistência a Múltiplos Medicamentos/imunologia , Receptores de Hidrocarboneto Arílico/imunologia , Subfamília B de Transportador de Cassetes de Ligação de ATP/antagonistas & inibidores , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/imunologia , Animais , Anti-Inflamatórios/imunologia , Anti-Inflamatórios/farmacologia , Apirase/genética , Apirase/imunologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Bilirrubina/imunologia , Bilirrubina/farmacologia , Hipóxia Celular , Colite/induzido quimicamente , Colite/tratamento farmacológico , Colite/genética , Doença de Crohn/genética , Doença de Crohn/patologia , Sulfato de Dextrana/administração & dosagem , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/antagonistas & inibidores , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Ativação Linfocitária , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Mucosa/imunologia , Mucosa/patologia , Proteínas Associadas à Resistência a Múltiplos Medicamentos/antagonistas & inibidores , Proteínas Associadas à Resistência a Múltiplos Medicamentos/genética , Cultura Primária de Células , Ligação Proteica , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/imunologia , Receptores de Hidrocarboneto Arílico/genética , Ritonavir/farmacologia , Células Th17/efeitos dos fármacos , Células Th17/imunologia , Células Th17/patologiaRESUMO
Cd39 scavenges extracellular ATP and ADP, ultimately generating adenosine, a nucleoside, which has anti-inflammatory effects in the vasculature. We have evaluated the role of Cd39 in the development of atherosclerosis in hyperlipidemic mice. ApoE KO (Cd39+/+/ApoE-/-) and Cd39/ApoE double KO (DKO) (Cd39-/-/ApoE-/-) mice were maintained on chow or Western diet for up to 20 weeks before evaluation of atherosclerotic lesions. We found that DKO mice exhibited significantly fewer atherosclerotic lesions than ApoE KO mice, irrespective of diet. Analyses of plaque composition revealed diminished foam cells in the fatty streaks and smaller necrotic cores in advanced lesions of DKO mice, when compared with those in ApoE KO mice. This atheroprotective phenotype was associated with impaired platelet reactivity to ADP in vitro and prolonged platelet survival, suggesting decreased platelet activation in vivo. Further studies with either genetic deletion or pharmacological inhibition of Cd39 in macrophages revealed increased cholesterol efflux mediated via ABCA1 to ApoA1. This phenomenon was associated with elevated plasma HDL levels in DKO mice. Our findings indicate that complete deletion of Cd39 paradoxically attenuates development of atherosclerosis in hyperlipidemic mice. We propose that this phenotype occurs, at least in part, from diminished platelet activation, increased plasma HDL levels, and enhanced cholesterol efflux and indicates the complexity of purinergic signaling in atherosclerosis.
Assuntos
Antígenos CD/genética , Apolipoproteínas E/deficiência , Apirase/deficiência , Apirase/genética , Aterosclerose/genética , Técnicas de Inativação de Genes , Animais , Aterosclerose/metabolismo , Aterosclerose/patologia , Aterosclerose/fisiopatologia , Transporte Biológico/genética , Movimento Celular/genética , Proliferação de Células/genética , Colesterol/metabolismo , Macrófagos/metabolismo , Masculino , Camundongos , Músculo Liso Vascular/patologia , Necrose/genética , Fenótipo , Ativação Plaquetária/genéticaRESUMO
Nerve cells are continuously generated from stem cells in the adult mammalian subventricular zone (SVZ) and hippocampal dentate gyrus. We have previously noted that stem/progenitor cells in the SVZ and the subgranular layer (SGL) of the dentate gyrus express high levels of plasma membrane-bound nucleoside triphosphate diphosphohydrolase 2 (NTPDase2), an ectoenzyme that hydrolyzes extracellular nucleoside diphosphates and triphosphates. We inferred that deletion of NTPDase2 would increase local extracellular nucleoside triphosphate concentrations perturbing purinergic signaling and boosting progenitor cell proliferation and neurogenesis. Using newly generated mice globally null for Entpd2, we demonstrate that NTPDase2 is the major ectonucleotidase in these progenitor cell-rich areas. Using BrdU-labeling protocols, we have measured stem cell proliferation and determined long-term survival of cell progeny under basal conditions. Brains of Entpd2 null mice revealed increased progenitor cell proliferation in both the SVZ and the SGL. However, this occurred without noteworthy alterations in long-term progeny survival. The hippocampal stem cell pool and the pool of the intermediate progenitor type-2 cells clearly expanded. However, substantive proportions of these proliferating cells were lost during expansion at around type-3 stage. Cell loss was paralleled by decreases in cAMP response element-binding protein phosphorylation in the doublecortin-positive progenitor cell population and by an increase in labeling for activated caspase-3 levels. We propose that NTPDase2 has functionality in scavenging mitogenic extracellular nucleoside triphosphates in neurogenic niches of the adult brain, thereby acting as a homeostatic regulator of nucleotide-mediated neural progenitor cell proliferation and expansion.
Assuntos
Adenosina Trifosfatases/metabolismo , Encéfalo/citologia , Células-Tronco Neurais/citologia , Nicho de Células-Tronco/fisiologia , Animais , Encéfalo/enzimologia , Encéfalo/metabolismo , Proliferação de Células/fisiologia , Imuno-Histoquímica , Camundongos , Células-Tronco Neurais/enzimologia , Células-Tronco Neurais/metabolismo , Neurogênese/fisiologia , Transdução de SinaisRESUMO
Sepsis remains the leading cause of morbidity and mortality in critically ill patients. Excessive inflammation is a major cause of organ failure and mortality in sepsis. Ectonucleoside triphosphate diphosphohydrolase 1, ENTPDase1 (CD39) is a cell surface nucleotide-metabolizing enzyme, which degrades the extracellular purines ATP and ADP, thereby regulating purinergic receptor signaling. Although the role of purinergic receptor signaling in regulating inflammation and sepsis has been addressed previously, the role of CD39 in regulating the host's response to sepsis is unknown. We found that the CD39 mimic apyrase (250 U/kg) decreased and knockout or pharmacologic blockade with sodium polyoxotungstate (5 mg/kg; IC50 ≈ 10 µM) of CD39 increased mortality of mice with polymicrobial sepsis induced by cecal ligation and puncture. CD39 decreased inflammation, organ damage, immune cell apoptosis, and bacterial load. Use of bone marrow chimeric mice revealed that CD39 expression on myeloid cells decreases inflammation in septic mice. CD39 expression is upregulated during sepsis in mice, as well as in both murine and human macrophages stimulated with Escherichia coli. Moreover, E. coli increases CD39 promoter activity in macrophages. Altogether, these data indicate CD39 as an evolutionarily conserved inducible protective pathway during sepsis. We propose CD39 as a novel therapeutic target in the management of sepsis.
Assuntos
Antígenos CD/metabolismo , Apirase/metabolismo , Inflamação/prevenção & controle , Sepse/metabolismo , 5'-Nucleotidase/metabolismo , Animais , Antígenos CD/genética , Apirase/deficiência , Apirase/genética , Quimiocinas/metabolismo , Citocinas/metabolismo , Escherichia coli/patogenicidade , Humanos , Inflamação/metabolismo , Interleucina-10/biossíntese , Interleucina-12/biossíntese , Macrófagos/metabolismo , Macrófagos/microbiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Regiões Promotoras Genéticas , Sepse/microbiologia , Quimeras de TransplanteRESUMO
Taste buds are unusual in requiring ATP as a transmitter to activate sensory nerve fibers. In response to taste stimuli, taste cells release ATP, activating purinergic receptors containing the P2X2 and P2X3 subunits on taste nerves. In turn, the released ATP is hydrolyzed to ADP by a plasma membrane nucleoside triphosphate previously identified as nucleoside triphosphate diphosphohydrolase-2 (NTPDase2). In this paper we investigate the role of this ectonucleotidase in the function of taste buds by examining gene-targeted Entpd2-null mice globally lacking NTPDase2. RT-PCR confirmed the absence of NTPDase2, and ATPase enzyme histochemistry reveals no reaction product in taste buds of knockout mice, suggesting that NTPDase2 is the dominant form in taste buds. RT-PCR and immunocytochemistry demonstrated that in knockout mice all cell types are present in taste buds, even those cells normally expressing NTPDase2. In addition, the overall number and size of taste buds are normal in Entpd2-null mice. Luciferin/luciferase assays of circumvallate tissue of knockout mice detected elevated levels of extracellular ATP. Electrophysiological recordings from two taste nerves, the chorda tympani and glossopharyngeal, revealed depressed responses to all taste stimuli in Entpd2-null mice. Responses were more depressed in the glossopharyngeal nerve than in the chorda tympani nerve and involved all taste qualities; responses in the chorda tympani were more depressed to sweet and umami stimuli than to other qualities. We suggest that the excessive levels of extracellular ATP in the Entpd2-knockout animals desensitize the P2X receptors associated with nerve fibers, thereby depressing taste responses.
Assuntos
Adenosina Trifosfatases/metabolismo , Papilas Gustativas/enzimologia , Papilas Gustativas/fisiologia , Paladar/fisiologia , Adenosina Trifosfatases/genética , Trifosfato de Adenosina/metabolismo , Análise de Variância , Animais , Nervo da Corda do Tímpano/fisiologia , Expressão Gênica , Nervo Glossofaríngeo/fisiologia , Imuno-Histoquímica , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Papilas Gustativas/metabolismoRESUMO
Phosphohydrolysis of extracellular ATP and ADP is an essential step in purinergic signaling that regulates key pathophysiological processes, such as those linked to inflammation. Classically, this reaction has been known to occur in the pericellular milieu catalyzed by membrane bound cellular ecto-nucleotidases, which can be released in the form of both soluble ecto-enzymes as well as being associated with exosomes. Circulating ecto-nucleoside triphosphate diphosphohydrolase 1 (NTPDase 1/CD39) and adenylate kinase 1 (AK1) activities have been shown to be present in plasma. However, other ecto-nucleotidases have not been characterized in depth. An in vitro ADPase assay was developed to probe the ecto-enzymes responsible for the ecto-nucleotidase activity in human platelet-free plasma, in combination with various specific biochemical inhibitors. Identities of ecto-nucleotidases were further characterized by chromatography, immunoblotting, and flow cytometry of circulating exosomes. We noted that microparticle-bound E-NTPDases and soluble AK1 constitute the highest levels of ecto-nucleotidase activity in human plasma. All four cell membrane expressed E-NTPDases are also found in circulating microparticles in human plasma, inclusive of: CD39, NTPDase 2 (CD39L1), NTPDase 3 (CD39L3), and NTPDase 8. CD39 family members and other ecto-nucleotidases are found on distinct microparticle populations. A significant proportion of the microparticle-associated ecto-nucleotidase activity is sensitive to POM6, inferring the presence of NTPDases, either -2 or/and -3. We have refined ADPase assays of human plasma from healthy volunteers and have found that CD39, NTPDases 2, 3, and 8 to be associated with circulating microparticles, whereas soluble AK1 is present in human plasma. These ecto-enzymes constitute the bulk circulating ADPase activity, suggesting a broader implication of CD39 family and other ecto-enzymes in the regulation of extracellular nucleotide metabolism.
Assuntos
Antígenos CD/metabolismo , Apirase/metabolismo , Micropartículas Derivadas de Células/enzimologia , Difosfato de Adenosina/metabolismo , Antígenos CD/análise , Apirase/análise , Western Blotting , Cromatografia em Gel , Citometria de Fluxo , HumanosRESUMO
Ectonucleoside triphosphate diphosphohydrolase-1 hydrolyzes extracellular ATP and ADP to AMP. Previously, we showed that CD39 is expressed at several sites within the kidney and thus may impact the availability of type 2 purinergic receptor (P2-R) ligands. Because P2-Rs appear to regulate urinary concentrating ability, we have evaluated renal water handling in transgenic mice (TG) globally overexpressing hCD39. Under basal conditions, TG mice exhibited significantly impaired urinary concentration and decreased protein abundance of AQP2 in the kidney compared with wild-type (WT) mice. Urinary excretion of total nitrates/nitrites was significantly higher in TG mice, but the excretion of AVP or PGE(2) was equivalent to control WT mice. There were no significant differences in electrolyte-free water clearance or fractional excretion of sodium. Under stable hydrated conditions (gelled diet feeding), the differences between the WT and TG mice were negated, but the decrease in urine osmolality persisted. When water deprived, TG mice failed to adequately concentrate urine and exhibited impaired AVP responses. However, the increases in urinary osmolalities in response to subacute dDAVP or chronic AVP treatment were similar in TG and WT mice. These observations suggest that TG mice have impaired urinary concentrating ability despite normal AVP levels. We also note impaired AVP release in response to water deprivation but that TG kidneys are responsive to exogenous dDAVP or AVP. We infer that heightened nucleotide scavenging by increased levels of CD39 altered the release of endogenous AVP in response to dehydration. We propose that ectonucleotidases and modulated purinergic signaling impact urinary concentration and indicate potential utility of targeted therapy for the treatment of water balance disorders.
Assuntos
Antígenos CD/biossíntese , Apirase/biossíntese , Água/metabolismo , Animais , Antígenos CD/genética , Apirase/genética , Western Blotting , Primers do DNA , Desamino Arginina Vasopressina/farmacologia , Ingestão de Líquidos/efeitos dos fármacos , Ingestão de Alimentos/efeitos dos fármacos , Humanos , Rim/efeitos dos fármacos , Rim/metabolismo , Capacidade de Concentração Renal/efeitos dos fármacos , Capacidade de Concentração Renal/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Nucleotidases/metabolismo , Concentração Osmolar , Reação em Cadeia da Polimerase em Tempo Real , Receptores Purinérgicos P1/biossíntese , Receptores Purinérgicos P2Y/biossíntese , Fármacos Renais/farmacologia , Privação de ÁguaRESUMO
Upregulation of tissue factor (TF) expression on activated donor endothelial cells (ECs) triggered by the immune response (IR) has been considered the main initiator of consumptive coagulopathy (CC). In this study, we aimed to identify potential factors in the development of thrombocytopenia and CC after genetically engineered pig liver transplantation in baboons. Baboons received a liver from either an α1,3-galactosyltransferase gene-knockout (GTKO) pig (n = 1) or a GTKO pig transgenic for CD46 (n = 5) with immunosuppressive therapy. TF exposure on recipient platelets and peripheral blood mononuclear cell (PBMCs), activation of donor ECs, platelet and EC microparticles, and the IR were monitored. Profound thrombocytopenia and thrombin formation occurred within minutes of liver reperfusion. Within 2 h, circulating platelets and PBMCs expressed functional TF, with evidence of aggregation in the graft. Porcine ECs were negative for expression of P- and E-selectin, CD106, and TF. The measurable IR was minimal, and the severity and rapidity of thrombocytopenia were not alleviated by prior manipulation of the IR. We suggest that the development of thrombocytopenia/CC may be associated with TF exposure on recipient platelets and PBMCs (but possibly not with activation of donor ECs). Recipient TF appears to initiate thrombocytopenia/CC by a mechanism that may be independent of the IR.
Assuntos
Coagulação Intravascular Disseminada/imunologia , Trombocitopenia/imunologia , Tromboplastina/genética , Transplante Heterólogo/imunologia , Animais , Animais Geneticamente Modificados , Micropartículas Derivadas de Células/imunologia , Galactosiltransferases/imunologia , Papio/imunologia , Sus scrofa/imunologiaRESUMO
CD39/ENTPD1 hydrolyzes proinflammatory nucleotides to generate adenosine. As purinergic mediators have been implicated in intestinal inflammation, we hypothesized that CD39 might protect against inflammatory bowel disease. We studied these possibilities in a mouse model of colitis using mice with global CD39 deletion. We then tested whether human genetic polymorphisms in the CD39 gene might influence susceptibility to Crohn's disease. We induced colitis in mice using Dextran Sodium Sulfate (DSS). Readouts included disease activity scores, histological evidence of injury, and markers of inflammatory activity. We used HapMap cell lines to find SNPs that tag for CD39 expression, and then compared the frequency of subjects with high vs. low CD39-expression genotypes in a case-control cohort for Crohn's disease. Mice null for CD39 were highly susceptible to DSS injury, with heterozygote mice showing an intermediate phenotype compared to wild type (WT). We identified a common SNP that tags CD39 mRNA expression levels in man. The SNP tagging low levels of CD39 expression was associated with increased susceptibility to Crohn's disease in a case-control cohort comprised of 1,748 Crohn's patients and 2,936 controls (P = 0.005-0.0006). Our data indicate that CD39 deficiency exacerbates murine colitis and suggest that CD39 polymorphisms are associated with inflammatory bowel disease in humans.
Assuntos
Antígenos CD/genética , Apirase/genética , Colite/genética , Predisposição Genética para Doença , Doenças Inflamatórias Intestinais/genética , Polimorfismo Genético , Deleção de Sequência , Animais , Antígenos CD/metabolismo , Apirase/metabolismo , Colite/patologia , Doença de Crohn/genética , Modelos Animais de Doenças , Genótipo , Humanos , Doenças Inflamatórias Intestinais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , RNA Mensageiro/metabolismoRESUMO
Immunosuppressive cells accumulating in the tumor microenvironment constitute a formidable barrier that interferes with current immunotherapeutic approaches. A unifying feature of these tumor-associated immune and vascular endothelial cells appears to be the elevated expression of ectonucleotidase CD39, which in tandem with ecto-5'-nucleotidase CD73, catalyzes the conversion of extracellular ATP into adenosine. We glycoengineered an afucosylated anti-CD39 IgG2c and tested this reagent in mouse melanoma and colorectal tumor models. We identified major biological effects of this approach on cancer growth, associated with depletion of immunosuppressive cells, mediated through enhanced Fcγ receptor-directed (FcγR-directed), antibody-dependent cellular cytotoxicity (ADCC). Furthermore, regulatory/exhausted T cells lost CD39 expression, as a consequence of antibody-mediated trogocytosis. Most strikingly, tumor-associated macrophages and endothelial cells with high CD39 expression were effectively depleted following antibody treatment, thereby blocking angiogenesis. Tumor site-specific cellular modulation and lack of angiogenesis synergized with chemotherapy and anti-PD-L1 immunotherapy in experimental tumor models. We conclude that depleting suppressive cells and targeting tumor vasculature, through administration of afucosylated anti-CD39 antibody and the activation of ADCC, comprises an improved, purinergic system-modulating strategy for cancer therapy.
Assuntos
Apirase , Neoplasias , Animais , Antígenos CD/metabolismo , Células Endoteliais/metabolismo , Camundongos , Neovascularização Patológica/genética , Microambiente TumoralRESUMO
BACKGROUND & AIMS: Adenosine mediates immune suppression and is generated by the ectonucleotidases CD39 (ENTPD1) and CD73 that are expressed on vascular endothelial cells and regulatory T cells (Tregs). Although tumor-infiltrating immune cells include Foxp3(+) Tregs, it is not clear whether local adenosine generation by Tregs promotes tumor growth in a CD39-dependent manner. In this study, we have examined the effect of CD39 expression by Tregs on effector immune cell responses to hepatic metastases in vivo. METHODS: A model of hepatic metastatic cancer was developed with portal vein infusion of luciferase-expressing melanoma B16/F10 cells and MCA38 colon cancer cells in wild-type (wt) and mutant mice null for Cd39. Chimeric mice were generated by bone marrow transplantation (BMT) using Cd39 null or wt C57BL6 donors and irradiated recipient mice. RESULTS: We demonstrate that hepatic growth of melanoma metastatic tumors was strongly inhibited in mice with Cd39 null vasculature or in wt mice with circulating Cd39 null bone marrow-derived cells. We show functional CD39 expression on CD4(+)Foxp3(+) Tregs suppressed antitumor immunity mediated by natural killer (NK) cells in vitro and in vivo. Finally, inhibition of CD39 activity by polyoxometalate-1, a pharmacologic inhibitor of nucleoside triphosphate diphosphohydrolase activity, significantly inhibited tumor growth (P < .001). CONCLUSIONS: CD39 expression on Tregs inhibits NK activity and is permissive for metastatic growth. Pharmacologic or targeted inhibition of CD39 enzymatic activity may find utility as an adjunct therapy for secondary hepatic malignancies.
Assuntos
Antígenos CD/metabolismo , Apirase/metabolismo , Neoplasias do Colo/imunologia , Fatores de Transcrição Forkhead/metabolismo , Neoplasias Hepáticas/imunologia , Linfócitos do Interstício Tumoral/imunologia , Melanoma Experimental/imunologia , Linfócitos T Reguladores/imunologia , Animais , Antígenos CD/genética , Antineoplásicos/farmacologia , Apirase/antagonistas & inibidores , Apirase/deficiência , Apirase/genética , Transplante de Medula Óssea , Linhagem Celular Tumoral , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/genética , Neoplasias do Colo/secundário , Citotoxicidade Imunológica , Inibidores Enzimáticos/farmacologia , Fatores de Transcrição Forkhead/genética , Células Matadoras Naturais/imunologia , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/secundário , Linfócitos do Interstício Tumoral/efeitos dos fármacos , Masculino , Melanoma Experimental/tratamento farmacológico , Melanoma Experimental/genética , Melanoma Experimental/secundário , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Quimera por Radiação , Linfócitos T Reguladores/efeitos dos fármacos , Fatores de Tempo , Carga Tumoral , Evasão Tumoral , Compostos de Tungstênio/farmacologiaRESUMO
P2X7 receptor is an adenosine triphosphate (ATP)-gated ion channel within the multiprotein inflammasome complex. Until now, little is known about regulation of P2X7 effector functions in macrophages. In this study, we show that nucleoside triphosphate diphosphohydrolase 1 (NTPDase1)/CD39 is the dominant ectonucleotidase expressed by murine peritoneal macrophages and that it regulates P2X7-dependent responses in these cells. Macrophages isolated from NTPDase1-null mice (Entpd1(-/-)) were devoid of all ADPase and most ATPase activities when compared with WT macrophages (Entpd1(+/+)). Entpd1(-/-) macrophages exposed to millimolar concentrations of ATP were more susceptible to cell death, released more IL-1beta and IL-18 after TLR2 or TLR4 priming, and incorporated the fluorescent dye Yo-Pro-1 more efficiently (suggestive of increased pore formation) than Entpd1(+/+) cells. Consistent with these observations, NTPDase1 regulated P2X7-associated IL-1beta release after synthesis, and this process occurred independently of, and prior to, cytokine maturation by caspase-1. NTPDase1 also inhibited IL-1beta release in vivo in the air pouch inflammatory model. Exudates of LPS-injected Entpd1(-/-) mice had significantly higher IL-1beta levels when compared with Entpd1(+/+) mice. Altogether, our studies suggest that NTPDase1/CD39 plays a key role in the control of P2X7-dependent macrophage responses.
Assuntos
Antígenos CD/fisiologia , Apirase/fisiologia , Macrófagos Peritoneais/enzimologia , Receptores Purinérgicos P2/fisiologia , Trifosfato de Adenosina/metabolismo , Animais , Antígenos CD/genética , Apoptose/fisiologia , Apirase/deficiência , Apirase/genética , Células da Medula Óssea/enzimologia , Células da Medula Óssea/metabolismo , Caspases/fisiologia , Permeabilidade da Membrana Celular , Cruzamentos Genéticos , Inibidores de Cisteína Proteinase/farmacologia , Interleucina-1beta/metabolismo , Macrófagos Peritoneais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores Purinérgicos P2X7 , Receptor 2 Toll-Like/fisiologiaRESUMO
UNLABELLED: Natural killer (NK) cells play crucial roles in innate immunity and express CD39 (Ecto-nucleoside triphosphate diphosphohydrolase 1 [E-NTPD1]), a rate-limiting ectonucleotidase in the phosphohydrolysis of extracellular nucleotides to adenosine. We have studied the effects of CD39 gene deletion on NK cells in dictating outcomes after partial hepatic ischemia/reperfusion injury (IRI). We show in mice that gene deletion of CD39 is associated with marked decreases in phosphohydrolysis of adenosine triphosphate (ATP) and adenosine diphosphate to adenosine monophosphate on NK cells, thereby modulating the type-2 purinergic (P2) receptors demonstrated on these cells. We note that CD39-null mice are protected from acute vascular injury after single-lobe warm IRI, and, relative to control wild-type mice, display significantly less elevation of aminotransferases with less pronounced histopathological changes associated with IRI. Selective adoptive transfers of immune cells into Rag2/common gamma null mice (deficient in T cells, B cells, and NK/NKT cells) suggest that it is CD39 deletion on NK cells that provides end-organ protection, which is comparable to that seen in the absence of interferon gamma. Indeed, NK effector mechanisms such as interferon gamma secretion are inhibited by P2 receptor activation in vitro. Specifically, ATPgammaS (a nonhydrolyzable ATP analog) inhibits secretion of interferon gamma by NK cells in response to interleukin-12 and interleukin-18, providing a mechanistic link between CD39 deletion and altered cytokine secretion. CONCLUSION: We propose that CD39 deficiency and changes in P2 receptor activation abrogate secretion of interferon gamma by NK cells in response to inflammatory mediators, thereby limiting tissue damage mediated by these innate immune cells during IRI.
Assuntos
Apirase/deficiência , Células Matadoras Naturais/imunologia , Traumatismo por Reperfusão/prevenção & controle , Difosfato de Adenosina , Trifosfato de Adenosina/análogos & derivados , Trifosfato de Adenosina/farmacologia , Animais , Antígenos CD/metabolismo , Apirase/metabolismo , Células Matadoras Naturais/fisiologia , Camundongos , Camundongos Endogâmicos C57BL , Receptores Purinérgicos P2/fisiologiaRESUMO
Liver ischemia reperfusion injury is associated with both local damage to the hepatic vasculature and systemic inflammatory responses. CD39 is the dominant vascular endothelial cell ectonucleotidase and rapidly hydrolyses both adenosine triphosphate (ATP) and adenosine diphosphate to adenosine monophosphate. These biochemical properties, in tandem with 5'-nucleotidases, generate adenosine and potentially illicit inflammatory vascular responses and thrombosis. We have evaluated the role of CD39 in total hepatic ischemia reperfusion injury (IRI). Wildtype mice, Cd39-hemizygous mice (+/-) and matched Cd39-null mice (-/-); (n = 25 per group) underwent 45 min of total warm ischemia with full inflow occlusion necessitating partial hepatectomy. Soluble nucleoside triphosphate diphosphohydrolase (NTPDases) or adenosine/amrinone were administered to wildtype (n = 6) and Cd39-null mice (n = 6) in order to study protective effects in vivo. Parameters of liver injury, systemic inflammation, hepatic ATP determinations by P(31)-NMR and parameters of lung injury were obtained. All wildtype mice survived up to 7 days with minimal biochemical disturbances and minor evidence for injury. In contrast, 64% of Cd39+/- and 84% of Cd39-null mice required euthanasia or died within 4 h post-reperfusion with liver damage and systemic inflammation associated with hypercytokinemia. Hepatic ATP depletion was pronounced in Cd39-null mice posthepatic IRI. Soluble NTPDase or adenosine administration protected Cd39-deficient mice from acute reperfusion injury. We conclude that CD39 is protective in hepatic IRI preventing local injury and systemic inflammation in an adenosine dependent manner. Our data indicate that vascular CD39 expression has an essential protective role in hepatic IRI.
RESUMO
Despite improvements in prevention and management of colorectal cancer (CRC), uncontrolled tumor growth with metastatic spread to distant organs remains an important clinical concern. Genetic deletion of CD39, the dominant vascular and immune cell ectonucleotidase, has been shown to delay tumor growth and blunt angiogenesis in mouse models of melanoma, lung and colonic malignancy. Here, we tested the influence of CD39 on CRC tumor progression and metastasis by investigating orthotopic transplanted and metastatic cancer models in wild-type BALB/c, human CD39 transgenic and CD39 deficient mice. We also investigated CD39 and P2 receptor expression patterns in human CRC biopsies. Murine CD39 was expressed by endothelium, stromal and mononuclear cells infiltrating the experimental MC-26 tumors. In the primary CRC model, volumes of tumors in the subserosa of the colon and/or rectum did not differ amongst the treatment groups at day 10, albeit these tumors rarely metastasized to the liver. In the dissemination model, MC-26 cell line-derived hepatic metastases grew significantly faster in CD39 over-expressing transgenics, when compared to CD39 deficient mice. Murine P2Y2 was significantly elevated at both mRNA and protein levels, within the larger liver metastases obtained from CD39 transgenic mice where changes in P2X7 levels were also noted. In clinical samples, lower levels of CD39 mRNA in malignant CRC tissues appeared associated with longer duration of survival and could be linked to less invasive tumors. The modulatory effects of CD39 on tumor dissemination and differential levels of CD39, P2Y2 and P2X7 expression in tumors suggest involvement of purinergic signalling in these processes. Our studies also suggest potential roles for purinergic-based therapies in clinical CRC.
RESUMO
CD39, the endothelial ecto-nucleoside triphosphate diphosphohydrolase (NTPDase), regulates vascular inflammation and thrombosis by hydrolyzing ATP and ADP. Although ecto-NTPDase activities have been used as a marker of epidermal dendritic cells (DCs) known as Langerhans cells, the identity and function of these activities remain unknown. Here we report that Langerhans cells in CD39-/- mice express no detectable ecto-NTPDase activity. Irritant chemicals triggered rapid ATP and ADP release from keratinocytes and caused exacerbated skin inflammation in CD39-/- mice. Paradoxically, T cell-mediated allergic contact hypersensitivity was severely attenuated in CD39-/- mice. As to mechanisms, T cells increased pericellular ATP concentrations upon activation, and CD39-/- DCs showed ATP unresponsiveness (secondary to P2-receptor desensitization) and impaired antigen-presenting capacity. Our results show opposing outcomes of CD39 deficiency in irritant versus allergic contact dermatitis, reflecting its diverse roles in regulating extracellular nucleotide-mediated signaling in inflammatory responses to environmental insults and DC-T cell communication in antigen presentation.
Assuntos
Adenosina Trifosfatases/fisiologia , Antígenos CD/fisiologia , Inflamação/enzimologia , Inflamação/imunologia , Células de Langerhans/enzimologia , Células de Langerhans/imunologia , Adenosina Trifosfatases/deficiência , Adenosina Trifosfatases/genética , Trifosfato de Adenosina/farmacologia , Animais , Antígenos CD/genética , Apirase , Feminino , Imunidade , Inflamação/etiologia , Inflamação/patologia , Irritantes/toxicidade , Células de Langerhans/patologia , Masculino , Camundongos , Camundongos Knockout , Receptores Purinérgicos P2/fisiologia , Transdução de SinaisRESUMO
BACKGROUND: Dysregulation of immune responses in inflammatory bowel diseases (IBD) results in intestinal inflammation and vascular injury while exacerbating systemic disease. CD39 is an ectonucleotidase, expressed by T regulatory cells and dendritic cells, that hydrolyzes extracellular nucleotides to modify those cellular immune responses implicated in IBD. Genetic polymorphisms of CD39 have been linked to Crohn's disease while gene deletion in mice exacerbates dextran sodium sulphate-induced colitis. AIM: The aim of this study was to test how global deletion of CD39 in mice impacts other models of experimental colitis. METHODS: Colitis was induced in CD39-null and -wt mice, using trinitrobenzene sulfonic acid (TNBS, 125 mg/kg) administered intrarectally. Oxazolone colitis (1.5% oxazolone in 50% alcohol) was induced in comparable groups. Morphology, clinical and molecular parameters, and FACS analyses of lamina propria mononuclear cells (LPMC) were examined in CD39-null mice. CD39 expression was analyzed in human IBD biopsies. RESULTS: Paradoxically, TNBS colitis in CD39-null mice was characterized by improved survival, favorable clinical scores, and decreased MPO activity, when compared to wt mice (P < 0.05). LPMC from TNBS colitis contained significantly increased amounts of T-cells (CD3(+) and CD4(+)) and TNF-α mRNA expression were increased over those in CD39 null mice (P < 0.05). In contrast, oxazolone treated CD39-null and wt mice had comparable outcomes. In both ulcerative colitis and Crohn's disease, CD39 is present at high levels in intestinal tissue biopsies. CONCLUSIONS: TNBS colitis was attenuated in CD39-null mice whereas oxazolone-induced colitis was not impacted. Impaired adaptive cellular immune reactivity in the CD39-null environment appears protective in hapten-mediated Th1-type colitis. CD39 is expressed at high levels in clinical IBD tissues.