RESUMO
Solid organ transplantation mobilizes myeloid cells, including monocytes and macrophages, which are central protagonists of allograft rejection. However, myeloid cells can also be functionally reprogrammed by perioperative costimulatory blockade to promote a state of transplantation tolerance. Transplantation tolerance holds promise to reduce complications from chronic immunosuppression and promote long-term survival in transplant recipients. We sought to identify different mediators of transplantation tolerance by performing single-cell RNA sequencing of acute rejecting or tolerized cardiac allografts. This led to the unbiased identification of the transcription factor, hypoxia inducible factor (HIF)-2α, in a subset of tolerogenic monocytes. Using flow cytometric analyses and mice with conditional loss or gain of function, we uncovered that myeloid cell expression of HIF-2α was required for costimulatory blockade-induced transplantation tolerance. While HIF-2α was dispensable for mobilization of tolerogenic monocytes, which were sourced in part from the spleen, it promoted the expression of colony stimulating factor 1 receptor (CSF1R). CSF1R mediates monocyte differentiation into tolerogenic macrophages and was found to be a direct transcriptional target of HIF-2α in splenic monocytes. Administration of the HIF stabilizer, roxadustat, within micelles to target myeloid cells, increased HIF-2α in splenic monocytes, which was associated with increased CSF1R expression and enhanced cardiac allograft survival. These data support further exploration of HIF-2α activation in myeloid cells as a therapeutic strategy for transplantation tolerance.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos , Transplante de Coração , Macrófagos , Monócitos , Tolerância ao Transplante , Animais , Camundongos , Macrófagos/metabolismo , Macrófagos/imunologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Tolerância ao Transplante/imunologia , Monócitos/imunologia , Monócitos/metabolismo , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/metabolismo , Receptores de Fator Estimulador das Colônias de Granulócitos e Macrófagos/genética , Rejeição de Enxerto/imunologia , Rejeição de Enxerto/prevenção & controle , Rejeição de Enxerto/genética , Camundongos Endogâmicos C57BL , Regulação da Expressão Gênica/efeitos dos fármacos , Sobrevivência de Enxerto/imunologia , Sobrevivência de Enxerto/efeitos dos fármacos , MasculinoRESUMO
Survivors of myocardial infarction are at increased risk for vascular dementia. Neuroinflammation has been implicated in the pathogenesis of vascular dementia, yet little is known about the cellular and molecular mediators of neuroinflammation after myocardial infarction. Using a mouse model of myocardial infarction coupled with flow cytometric analyses and immunohistochemistry, we discovered increased monocyte abundance in the brain after myocardial infarction, which was associated with increases in brain-resident perivascular macrophages and microglia. Myeloid cell recruitment and activation was also observed in post-mortem brains of humans that died after myocardial infarction. Spatial and single cell transcriptomic profiling of brain-resident myeloid cells after experimental myocardial infarction revealed increased expression of monocyte chemoattractant proteins. In parallel, myocardial infarction increased crosstalk between brain-resident myeloid cells and oligodendrocytes, leading to neuroinflammation, white matter injury, and cognitive dysfunction. Inhibition of monocyte recruitment preserved white matter integrity and cognitive function, linking monocytes to neurodegeneration after myocardial infarction. Together, these preclinical and clinical results demonstrate that monocyte infiltration into the brain after myocardial infarction initiate neuropathological events that lead to vascular dementia.
Assuntos
Encéfalo , Disfunção Cognitiva , Monócitos , Infarto do Miocárdio , Substância Branca , Animais , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Infarto do Miocárdio/complicações , Substância Branca/metabolismo , Substância Branca/patologia , Disfunção Cognitiva/metabolismo , Disfunção Cognitiva/etiologia , Monócitos/metabolismo , Camundongos , Masculino , Humanos , Encéfalo/metabolismo , Encéfalo/patologia , Receptores CCR2/metabolismo , Camundongos Endogâmicos C57BL , Modelos Animais de Doenças , Macrófagos/metabolismo , Microglia/metabolismo , Doenças Neuroinflamatórias/metabolismo , Demência Vascular/metabolismo , Demência Vascular/patologia , Oligodendroglia/metabolismoRESUMO
Femoral atherosclerotic plaques are less inflammatory than carotid plaques histologically, but limited cell-level data exist regarding comparative immune landscapes and polarization at these sites. We investigated intraplaque leukocyte phenotypes and transcriptional polarization in 49 patients undergoing femoral (n = 23) or carotid (n = 26) endarterectomy using single-cell RNA-Seq (scRNA-Seq; n = 13), flow cytometry (n = 24), and IHC (n = 12). Comparative scRNA-Seq of CD45+-selected leukocytes from femoral (n = 9; 35,265 cells) and carotid (n = 4; 30,655 cells) plaque revealed distinct transcriptional profiles. Inflammatory foam cell-like macrophages and monocytes comprised higher proportions of myeloid cells in carotid plaques, whereas noninflammatory foam cell-like macrophages and LYVE1-overexpressing macrophages comprised higher proportions of myeloid cells in femoral plaque (P < 0.001 for all). A significant comparative excess of CCR2+ macrophages in carotid versus plaque was observed by flow cytometry in a separate validation cohort. B cells were more prevalent and exhibited a comparatively antiinflammatory profile in femoral plaque, whereas cytotoxic CD8+ T cells were more prevalent in carotid plaque. In conclusion, human femoral plaques exhibit distinct macrophage phenotypic and transcriptional profiles as well as diminished CD8+ T cell populations compared with human carotid plaques.
Assuntos
Placa Aterosclerótica , Humanos , Placa Aterosclerótica/patologia , Artérias Carótidas/patologia , Leucócitos/patologia , Monócitos/patologia , MacrófagosRESUMO
Myocardial infarction is associated with increased risk for vascular dementia. In both myocardial infarction and vascular dementia, there is evidence that elevated inflammatory biomarkers are associated with worsened clinical outcomes. Myocardial infarction leads to a systemic inflammatory response, which may contribute to recruitment or activation of myeloid cells, including monocytes, microglia, and perivascular macrophages, within the central nervous system. However, our understanding of the causative roles for these cells linking cardiac injury to the development and progression of dementia is incomplete. Herein, we provide an overview of inflammatory cellular and molecular links between myocardial infarction and vascular dementia and discuss strategies to resolve inflammation after myocardial infarction to limit neurovascular injury.
Assuntos
Demência Vascular , Infarto do Miocárdio , Humanos , Demência Vascular/etiologia , Monócitos , Macrófagos , InflamaçãoRESUMO
Clearance of dying cells by efferocytosis is necessary for cardiac repair after myocardial infarction (MI). Recent reports have suggested a protective role for vascular endothelial growth factor C (VEGFC) during acute cardiac lymphangiogenesis after MI. Here, we report that defective efferocytosis by macrophages after experimental MI led to a reduction in cardiac lymphangiogenesis and Vegfc expression. Cell-intrinsic evidence for efferocytic induction of Vegfc was revealed after adding apoptotic cells to cultured primary macrophages, which subsequently triggered Vegfc transcription and VEGFC secretion. Similarly, cardiac macrophages elevated Vegfc expression levels after MI, and mice deficient for myeloid Vegfc exhibited impaired ventricular contractility, adverse tissue remodeling, and reduced lymphangiogenesis. These results were observed in mouse models of permanent coronary occlusion and clinically relevant ischemia and reperfusion. Interestingly, myeloid Vegfc deficiency also led to increases in acute infarct size, prior to the amplitude of the acute cardiac lymphangiogenesis response. RNA-Seq and cardiac flow cytometry revealed that myeloid Vegfc deficiency was also characterized by a defective inflammatory response, and macrophage-produced VEGFC was directly effective at suppressing proinflammatory macrophage activation. Taken together, our findings indicate that cardiac macrophages promote healing through the promotion of myocardial lymphangiogenesis and the suppression of inflammatory cytokines.
Assuntos
Traumatismos Cardíacos , Infarto do Miocárdio , Fator C de Crescimento do Endotélio Vascular/metabolismo , Animais , Traumatismos Cardíacos/metabolismo , Inflamação/genética , Inflamação/metabolismo , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/metabolismo , Fagocitose , Fator C de Crescimento do Endotélio Vascular/genéticaRESUMO
Hypoxia-inducible factors (HIFs) are activated in parenchymal cells in response to low oxygen and as such have been proposed as therapeutic targets during hypoxic insult, including myocardial infarction (MI). HIFs are also activated within macrophages, which orchestrate the tissue repair response. Although isoform-specific therapeutics are in development for cardiac ischemic injury, surprisingly, the unique role of myeloid HIFs, and particularly HIF-2α, is unknown. Using a murine model of myocardial infarction and mice with conditional genetic loss and gain of function, we uncovered unique proinflammatory roles for myeloid cell expression of HIF-1α and HIF-2α during MI. We found that HIF-2α suppressed anti-inflammatory macrophage mitochondrial metabolism, while HIF-1α promoted cleavage of cardioprotective MerTK through glycolytic reprogramming of macrophages. Unexpectedly, combinatorial loss of both myeloid HIF-1α and HIF-2α was catastrophic and led to macrophage necroptosis, impaired fibrogenesis, and cardiac rupture. These findings support a strategy for selective inhibition of macrophage HIF isoforms and promotion of anti-inflammatory mitochondrial metabolism during ischemic tissue repair.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Células Mieloides/metabolismo , Idoso , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Cardiomiopatias/fisiopatologia , Modelos Animais de Doenças , Feminino , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Pessoa de Meia-Idade , Células Mieloides/patologia , Infarto do Miocárdio/fisiopatologia , Isquemia Miocárdica/fisiopatologia , Miocardite/metabolismo , Miocardite/patologiaRESUMO
Cardiac Allograft Vasculopathy (CAV) is a leading contributor to late transplant rejection. Although implicated, the mechanisms by which bone marrow-derived cells promote CAV remain unclear. Emerging evidence implicates the cell surface receptor tyrosine kinase AXL to be elevated in rejecting human allografts. AXL protein is found on multiple cell types, including bone marrow-derived myeloid cells. The causal role of AXL from this compartment and during transplant is largely unknown. This is important because AXL is a key regulator of myeloid inflammation. Utilizing experimental chimeras deficient in the bone marrow-derived Axl gene, we report that Axl antagonizes cardiac allograft survival and promotes CAV. Flow cytometric and histologic analyses of Axl-deficient transplant recipients revealed reductions in both allograft immune cell accumulation and vascular intimal thickness. Co-culture experiments designed to identify cell-intrinsic functions of Axl uncovered complementary cell-proliferative pathways by which Axl promotes CAV-associated inflammation. Specifically, Axl-deficient myeloid cells were less efficient at increasing the replication of both antigen-specific T cells and vascular smooth muscle cells (VSMCs), the latter a key hallmark of CAV. For the latter, we discovered that Axl-was required to amass the VSMC mitogen Platelet-Derived Growth Factor. Taken together, our studies reveal a new role for myeloid Axl in the progression of CAV and mitogenic crosstalk. Inhibition of AXL-protein, in combination with current standards of care, is a candidate strategy to prolong cardiac allograft survival.
Assuntos
Células da Medula Óssea/patologia , Regulação da Expressão Gênica , Rejeição de Enxerto/genética , Transplante de Coração/efeitos adversos , Músculo Liso Vascular/metabolismo , Miócitos Cardíacos/metabolismo , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , Adulto , Animais , Células da Medula Óssea/metabolismo , Proliferação de Células , Células Cultivadas , Modelos Animais de Doenças , Ecocardiografia , Citometria de Fluxo , Rejeição de Enxerto/diagnóstico , Rejeição de Enxerto/metabolismo , Sobrevivência de Enxerto , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Músculo Liso Vascular/patologia , Miócitos Cardíacos/patologia , Miócitos de Músculo Liso , Proteínas Proto-Oncogênicas/biossíntese , RNA/genética , Receptores Proteína Tirosina Quinases/biossíntese , Transplante Homólogo , Receptor Tirosina Quinase AxlRESUMO
Tyro3, AXL, and MerTK (TAM) receptors are activated in macrophages in response to tissue injury and as such have been proposed as therapeutic targets to promote inflammation resolution during sterile wound healing, including myocardial infarction. Although the role of MerTK in cardioprotection is well characterized, the unique role of the other structurally similar TAMs, and particularly AXL, in clinically relevant models of myocardial ischemia/reperfusion infarction (IRI) is comparatively unknown. Utilizing complementary approaches, validated by flow cytometric analysis of human and murine macrophage subsets and conditional genetic loss and gain of function, we uncover a maladaptive role for myeloid AXL during IRI in the heart. Cross signaling between AXL and TLR4 in cardiac macrophages directed a switch to glycolytic metabolism and secretion of proinflammatory IL-1ß, leading to increased intramyocardial inflammation, adverse ventricular remodeling, and impaired contractile function. AXL functioned independently of cardioprotective MerTK to reduce the efficacy of cardiac repair, but like MerTK, was proteolytically cleaved. Administration of a selective small molecule AXL inhibitor alone improved cardiac healing, which was further enhanced in combination with blockade of MerTK cleavage. These data support further exploration of macrophage TAM receptors as therapeutic targets for myocardial infarction.
Assuntos
Macrófagos/metabolismo , Infarto do Miocárdio/complicações , Infarto do Miocárdio/metabolismo , Miocardite/etiologia , Miocardite/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Animais , Modelos Animais de Doenças , Feminino , Humanos , Inflamassomos/metabolismo , Ativação de Macrófagos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Traumatismo por Reperfusão Miocárdica/complicações , Traumatismo por Reperfusão Miocárdica/metabolismo , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas/genética , Receptor Cross-Talk , Receptores Proteína Tirosina Quinases/deficiência , Receptores Proteína Tirosina Quinases/genética , Infarto do Miocárdio com Supradesnível do Segmento ST/metabolismo , Transdução de Sinais , Receptor 4 Toll-Like/metabolismo , c-Mer Tirosina Quinase/deficiência , c-Mer Tirosina Quinase/genética , c-Mer Tirosina Quinase/metabolismo , Receptor Tirosina Quinase AxlRESUMO
OBJECTIVE: Extracellular vesicles secreted by cardiosphere-derived cells (CDCev) polarize macrophages toward a distinctive phenotype with enhanced phagocytic capacity (MCDCev). These changes underlie cardioprotection by CDCev and by the parent CDCs, notably attenuating the no-reflow phenomenon following myocardial infarction, but the mechanisms are unclear. Here, we tested the hypothesis that MCDCev are especially effective at scavenging debris from dying cells (ie, efferocytosis) to attenuate irreversible damage post-myocardial infarction. Approach and Results: In vitro efferocytosis assays with bone marrow-derived macrophages, and in vivo transgenic rodent models of myocardial infarction, demonstrate enhanced apoptotic cell clearance with MCDCev. CDCev exposure induces sustained MerTK expression in MCDCev through extracellular vesicle transfer of microRNA-26a (via suppression of Adam17); the cardioprotective response is lost in animals deficient in MerTK. Single-cell RNA-sequencing revealed phagocytic pathway activation in MCDCev, with increased expression of complement factor C1qa, a phagocytosis facilitator. CONCLUSIONS: Together, these data demonstrate that extracellular vesicle modulation of MerTK and C1qa expression leads to enhanced macrophage efferocytosis and cardioprotection.
Assuntos
Proteína ADAM17/genética , Regulação da Expressão Gênica , Glicoproteínas de Membrana/genética , Infarto do Miocárdio/patologia , Fagocitose/genética , Receptores de Complemento/genética , c-Mer Tirosina Quinase/genética , Análise de Variância , Animais , Apoptose/genética , Biópsia por Agulha , Células Cultivadas , Modelos Animais de Doenças , Vesículas Extracelulares/metabolismo , Feminino , Humanos , Imuno-Histoquímica , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Infarto do Miocárdio/genética , Ratos , Ratos Sprague-Dawley , Ratos Wistar , Receptores Proteína Tirosina Quinases/metabolismo , Análise de Sequência de RNARESUMO
There is a growing number of individuals living with heart failure (HF) with reduced ejection fraction (HFrEF) or preserved ejection fraction (HFpEF). Long-term prognosis remains poor in both cases, especially in HFpEF, which is rising in incidence and lacks effective therapeutics. In both HFrEF and HFpEF, there is evidence that elevated inflammatory biomarkers, implicating innate immune cells such as macrophages, are associated with worsened clinical outcomes. Macrophage subsets are active in both inflammatory and reparative processes, yet our understanding of the causative roles for these cells in HF development and progression is incomplete. Here, we discuss recent findings interrogating the role of macrophages in inflammation and its resolution in the context of HF, with a specific focus on HFrEF versus HFpEF.
Assuntos
Insuficiência Cardíaca/etiologia , Insuficiência Cardíaca/fisiopatologia , Macrófagos/metabolismo , Volume Sistólico , Animais , Biomarcadores , Cardiotônicos/farmacologia , Cardiotônicos/uso terapêutico , Regulação da Expressão Gênica , Insuficiência Cardíaca/tratamento farmacológico , Insuficiência Cardíaca/metabolismo , Humanos , Inflamação/complicações , Inflamação/etiologia , Inflamação/metabolismo , PrognósticoRESUMO
During wound injury, efferocytosis fills the macrophage with a metabolite load nearly equal to the phagocyte itself. A timely question pertains to how metabolic phagocytic signaling regulates the signature anti-inflammatory macrophage response. Here we report the metabolome of activated macrophages during efferocytosis to reveal an interleukin-10 (IL-10) cytokine escalation that was independent of glycolysis yet bolstered by apoptotic cell fatty acids and mitochondrial ß-oxidation, the electron transport chain, and heightened coenzyme NAD+. Loss of IL-10 due to mitochondrial complex III defects was remarkably rescued by adding NAD+ precursors. This activated a SIRTUIN1 signaling cascade, largely independent of ATP, that culminated in activation of IL-10 transcription factor PBX1. Il-10 activation by the respiratory chain was also important in vivo, as efferocyte mitochondrial dysfunction led to cardiac rupture after myocardial injury. These findings highlight a new paradigm whereby macrophages leverage efferocytic metabolites and electron transport for anti-inflammatory reprogramming that culminates in organ repair.
Assuntos
Ácidos Graxos/metabolismo , Interleucina-10/metabolismo , Macrófagos/metabolismo , Mitocôndrias/metabolismo , NAD/metabolismo , Animais , Citofagocitose , Transporte de Elétrons , Humanos , Inflamação/metabolismo , Células Jurkat , Macrófagos/citologia , Camundongos , Camundongos Endogâmicos C57BL , Oxirredução , CicatrizaçãoRESUMO
Recipient infusion of donor apoptotic cells is an emerging strategy for inducing robust transplantation tolerance. Daily clearance of billions of self-apoptotic cells relies on homeostatic engagement of phagocytic receptors, in particular, receptors of the tyrosine kinase family TAM (Tyro3, Axl, and MerTK), to maintain self-tolerance. However, an outstanding question is if allogeneic apoptotic cells trigger the same receptor system for inducing allogeneic tolerance. Here, we employed allogeneic apoptotic splenocytes and discovered that the efferocytic receptor MerTK on recipient phagocytes is a critical mediator for transplantation tolerance induced by this strategy. Our findings indicate that the tolerogenic properties of allogeneic apoptotic splenocytes require MerTK transmission of intracellular signaling to suppress the production of inflammatory cytokine interferon α (IFN-α). We further demonstrate that MerTK is crucial for subsequent expansion of myeloid-derived suppressor cells and for promoting their immunomodulatory function, including maintaining graft-infiltrating CD4+ CD25+ Foxp3+ regulatory T cells. Consequently, recipient MerTK deficiency resulted in failure of tolerance by donor apoptotic cells, and this failure could be effectively rescued by IFN-α receptor blockade. These findings underscore the importance of the efferocytic receptor MerTK in mediating transplantation tolerance by donor apoptotic cells and implicate MerTK agonism as a promising target for promoting transplantation tolerance.
Assuntos
Diabetes Mellitus Experimental/imunologia , Rejeição de Enxerto/etiologia , Interferon Tipo I/imunologia , Células Supressoras Mieloides/imunologia , Linfócitos T Reguladores/imunologia , Tolerância ao Transplante/imunologia , c-Mer Tirosina Quinase/fisiologia , Animais , Apoptose , Linfócitos T CD4-Positivos/imunologia , Diabetes Mellitus Experimental/terapia , Rejeição de Enxerto/patologia , Sobrevivência de Enxerto/imunologia , Transplante de Coração/efeitos adversos , Interferon Tipo I/metabolismo , Transplante das Ilhotas Pancreáticas/efeitos adversos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Doadores de TecidosRESUMO
Post-transplant immunosuppression has reduced the incidence of T cell-mediated acute rejection, yet long-term cardiac graft survival rates remain a challenge. An important determinant of chronic solid organ allograft complication is accelerated vascular disease of the transplanted graft. In the case of cardiac allograft vasculopathy (CAV), the precise cellular etiology remains inadequately understood; however, histologic evidence hints at the accumulation and activation of innate phagocytes as a causal contributing factor. This includes monocytes, macrophages, and immature dendritic cell subsets. In addition to crosstalk with adaptive T and B immune cells, myeloid phagocytes secrete paracrine signals that directly activate fibroblasts and vascular smooth muscle cells, both of which contribute to fibrous intimal thickening. Though maladaptive phagocyte functions may promote CAV, directed modulation of myeloid cell function, at the molecular level, holds promise for tolerance and prolonged cardiac graft function.
Assuntos
Transplante de Coração/efeitos adversos , Fagócitos/imunologia , Fagócitos/metabolismo , Doenças Vasculares/etiologia , Doenças Vasculares/metabolismo , Doença Aguda , Animais , Antígenos de Diferenciação/metabolismo , Antígeno CD47/imunologia , Antígeno CD47/metabolismo , Comunicação Celular , Doença Crônica , Endocitose/imunologia , Rejeição de Enxerto/imunologia , Humanos , Hipóxia/imunologia , Hipóxia/metabolismo , Imunidade Inata , Isoantígenos/imunologia , Receptores Imunológicos/metabolismo , Transdução de Sinais , Linfócitos T/imunologia , Linfócitos T/metabolismo , Doenças Vasculares/patologiaRESUMO
RATIONALE: Clinical benefits of reperfusion after myocardial infarction are offset by maladaptive innate immune cell function, and therapeutic interventions are lacking. OBJECTIVE: We sought to test the significance of phagocytic clearance by resident and recruited phagocytes after myocardial ischemia reperfusion. METHODS AND RESULTS: In humans, we discovered that clinical reperfusion after myocardial infarction led to significant elevation of the soluble form of MerTK (myeloid-epithelial-reproductive tyrosine kinase; ie, soluble MER), a critical biomarker of compromised phagocytosis by innate macrophages. In reperfused mice, macrophage Mertk deficiency led to decreased cardiac wound debridement, increased infarct size, and depressed cardiac function, newly implicating MerTK in cardiac repair after myocardial ischemia reperfusion. More notably, Mertk(CR) mice, which are resistant to cleavage, showed significantly reduced infarct sizes and improved systolic function. In contrast to other cardiac phagocyte subsets, resident cardiac MHCIILOCCR2- (major histocompatibility complex II/C-C motif chemokine receptor type 2) macrophages expressed higher levels of MerTK and, when exposed to apoptotic cells, secreted proreparative cytokines, including transforming growth factor-ß. Mertk deficiency compromised the accumulation of MHCIILO phagocytes, and this was rescued in Mertk(CR) mice. Interestingly, blockade of CCR2-dependent monocyte infiltration into the heart reduced soluble MER levels post-ischemia reperfusion. CONCLUSIONS: Our data implicate monocyte-induced MerTK cleavage on proreparative MHCIILO cardiac macrophages as a novel contributor and therapeutic target of reperfusion injury.
Assuntos
Macrófagos/enzimologia , Traumatismo por Reperfusão Miocárdica/enzimologia , Miocárdio/enzimologia , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Infarto do Miocárdio com Supradesnível do Segmento ST/enzimologia , Animais , Apoptose , Citocinas/imunologia , Citocinas/metabolismo , Modelos Animais de Doenças , Feminino , Predisposição Genética para Doença , Antígenos de Histocompatibilidade Classe II/imunologia , Antígenos de Histocompatibilidade Classe II/metabolismo , Humanos , Imunidade Inata , Macrófagos/imunologia , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Pessoa de Meia-Idade , Monócitos/enzimologia , Monócitos/imunologia , Traumatismo por Reperfusão Miocárdica/imunologia , Traumatismo por Reperfusão Miocárdica/patologia , Traumatismo por Reperfusão Miocárdica/fisiopatologia , Miocárdio/imunologia , Miocárdio/patologia , Fagocitose , Fenótipo , Proteólise , Proteínas Proto-Oncogênicas/deficiência , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/imunologia , Receptores Proteína Tirosina Quinases/deficiência , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/imunologia , Receptores CCR2/genética , Receptores CCR2/imunologia , Receptores CCR2/metabolismo , Infarto do Miocárdio com Supradesnível do Segmento ST/imunologia , Infarto do Miocárdio com Supradesnível do Segmento ST/patologia , Infarto do Miocárdio com Supradesnível do Segmento ST/fisiopatologia , Transdução de Sinais , Fatores de Tempo , c-Mer Tirosina QuinaseRESUMO
BACKGROUND: Kawasaki disease (KD) is widely viewed as an acute arteritis. However, our pathologic studies show that chronic coronary arteritis can persist long after disease onset and is closely linked with arterial stenosis. Transcriptome profiling of acute KD arteritis tissues revealed upregulation of T lymphocyte, type I interferon, and allograft inflammatory factor-1 (AIF1) genes. We determined whether these immune responses persist in chronic KD arteritis, and we investigated the role of AIF1 in these responses. METHODS: Gene expression in chronic KD and childhood control arteries was determined by real-time reverse-transcriptase polymerase chain reaction, and arterial protein expression was determined by immunohistochemistry and immunofluorescence. Allograft inflammatory factor-1 small-interfering ribonucleic acid macrophage treatment was performed to investigate the role of AIF1 in macrophage and T lymphocyte activation. RESULTS: Allograft inflammatory factor-1 protein was highly expressed in stenotic KD arteries and colocalized with the macrophage marker CD68. T lymphocyte and interferon pathway genes were significantly upregulated in chronic KD coronary artery tissues. Alpha interferon-induced macrophage expression of CD80 and major histocompatibility complex class II was dependent on AIF1, and macrophage expression of AIF1 was required for antigen-specific T lymphocyte activation. CONCLUSIONS: Allograft inflammatory factor-1, originally identified in posttransplant arterial stenosis, is markedly upregulated in KD stenotic arterial tissues. T lymphocyte and type I interferon responses persist in chronic KD arteritis. Allograft inflammatory factor-1 may play multiple roles linking type I interferon response, macrophage activation, and antigen-specific T lymphocyte activation. These results suggest the likely importance of lymphocyte-myeloid cell cross-talk in the pathogenesis of KD arteritis and can inform selection of new immunotherapies for clinical trials in high-risk KD children.
Assuntos
Arterite/imunologia , Proteínas de Ligação a DNA/metabolismo , Interferons/metabolismo , Ativação de Macrófagos , Síndrome de Linfonodos Mucocutâneos/imunologia , Linfócitos T/imunologia , Adolescente , Antígenos CD/genética , Antígenos CD/metabolismo , Antígenos de Diferenciação de Linfócitos T/genética , Antígenos de Diferenciação de Linfócitos T/metabolismo , Apoptose/genética , Arterite/metabolismo , Antígeno B7-1/genética , Antígeno B7-1/metabolismo , Linfócitos T CD8-Positivos , Proteínas de Ligação ao Cálcio , Chicago , Criança , Pré-Escolar , Vasos Coronários/patologia , Proteínas de Ligação a DNA/genética , Feminino , Fibrinogênio , Imunofluorescência , Expressão Gênica , Humanos , Imuno-Histoquímica , Lactente , Recém-Nascido , Peptídeos e Proteínas de Sinalização Intercelular/genética , Interferons/genética , Lectinas Tipo C/genética , Lectinas Tipo C/metabolismo , Macrófagos/metabolismo , Masculino , Proteínas dos Microfilamentos , Síndrome de Linfonodos Mucocutâneos/genética , Síndrome de Linfonodos Mucocutâneos/metabolismo , Síndrome de Linfonodos Mucocutâneos/patologia , Receptores de Interferon/genética , Adulto JovemRESUMO
Hypoxia-inducible factor (HIF)-α isoforms regulate key macrophage (MΦ) functions during ischemic inflammation. HIF-2α drives proinflammatory cytokine production; however, the requirements for HIF-2α during other key MΦ functions, including phagocytosis, are unknown. In contrast to HIF-1α, HIF-2α was not required for hypoxic phagocytic uptake. Surprisingly, basal HIF-2α levels under nonhypoxic conditions were necessary and sufficient to suppress phagocytosis. Screening approaches revealed selective induction of the scavenger receptor MARCO, which was required for enhanced engulfment. Chromatin immunoprecipitation identified the antioxidant NRF2 as being directly responsible for inducing Marco Concordantly, Hif-2α-/- MΦs exhibited reduced antioxidant gene expression, and inhibition of mitochondrial reactive oxygen species suppressed Marco expression and phagocytic uptake. Ex vivo findings were recapitulated in vivo; the enhanced engulfment phenotype resulted in increased bacterial clearance and cytokine suppression. Importantly, natural induction of Hif-2α by IL-4 also suppressed MARCO-dependent phagocytosis. Thus, unlike most characterized prophagocytic regulators, HIF-2α can act as a phagocytic repressor. Interestingly, this occurs in resting MΦs through tempering of steady-state mitochondrial reactive oxygen species. In turn, HIF-2α promotes MΦ quiescence by blocking a MARCO bacterial-response pathway. IL-4 also drives HIF-2α suppression of MARCO, leading to compromised bacterial immunosurveillance in vivo.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Macrófagos/fisiologia , Mitocôndrias/metabolismo , Infecções Estafilocócicas/imunologia , Staphylococcus aureus/imunologia , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Hipóxia Celular , Células Cultivadas , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Imunidade Inata , Interleucina-4/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fator 2 Relacionado a NF-E2/metabolismo , Fagocitose , Espécies Reativas de Oxigênio/metabolismo , Receptores Imunológicos/metabolismoRESUMO
BACKGROUND: Mobilization of the innate immune response to clear and metabolize necrotic and apoptotic cardiomyocytes is a prerequisite to heart repair after cardiac injury. Suboptimal kinetics of dying myocyte clearance leads to secondary necrosis, and in the case of the heart, increased potential for collateral loss of neighboring non-regenerative myocytes. Despite the importance of myocyte phagocytic clearance during heart repair, surprisingly little is known about its underlying cell and molecular biology. OBJECTIVE: To determine if phagocytic receptor MERTK is expressed in human hearts and to elucidate key sequential steps and phagocytosis efficiency of dying adult cardiomyocytes, by macrophages. RESULTS: In infarcted human hearts, expression profiles of the phagocytic receptor MER-tyrosine kinase (MERTK) mimicked that found in experimental ischemic mouse hearts. Electron micrographs of myocardium identified MERTK signal along macrophage phagocytic cups and Mertk-/- macrophages contained reduced digested myocyte debris after myocardial infarction. Ex vivo co-culture of primary macrophages and adult cardiomyocyte apoptotic bodies revealed reduced engulfment relative to resident cardiac fibroblasts. Inefficient clearance was not due to the larger size of myocyte apoptotic bodies, nor were other key steps preceding the formation of phagocytic synapses significantly affected; this included macrophage chemotaxis and direct binding of phagocytes to myocytes. Instead, suppressed phagocytosis was directly associated with myocyte-induced inactivation of MERTK, which was partially rescued by genetic deletion of a MERTK proteolytic susceptibility site. CONCLUSION: Utilizing an ex vivo co-cultivation approach to model key cellular and molecular events found in vivo during infarction, cardiomyocyte phagocytosis was found to be inefficient, in part due to myocyte-induced shedding of macrophage MERTK. These findings warrant future studies to identify other cofactors of macrophage-cardiomyocyte cross-talk that contribute to cardiac pathophysiology.
Assuntos
Imunidade Inata/genética , Infarto do Miocárdio/genética , Miócitos Cardíacos/metabolismo , Fagocitose/genética , Proteínas Proto-Oncogênicas/biossíntese , Receptores Proteína Tirosina Quinases/biossíntese , Animais , Apoptose/genética , Apoptose/imunologia , Linhagem Celular , Técnicas de Cocultura , Humanos , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/patologia , Camundongos , Infarto do Miocárdio/imunologia , Infarto do Miocárdio/patologia , Miócitos Cardíacos/patologia , Necrose/genética , Necrose/metabolismo , Fagocitose/imunologia , Proteínas Proto-Oncogênicas/genética , Receptores Proteína Tirosina Quinases/genética , c-Mer Tirosina QuinaseRESUMO
Elevated levels of solTNFR2 are observed in a variety of human pathophysiological conditions but regulation of TNFR2 levels during disease is not well understood. We found that solTNFR2 levels were increased following influenza infection or live-attenuated influenza virus challenge in mice and humans, respectively. As influenza-specific CD8(+) T cells up-regulated expression of TNFR2 after infection in mice, we hypothesized that CD8(+) T cells contributed, in part, to solTNFR2 production after influenza infection and were interested in the mechanisms by which CD8(+) T cells regulate TNFR2 shedding. Activation of these cells by TCR stimulation resulted in enhanced shedding of TNFR2 that required actin remodeling and lipid raft formation and was dependent on MAPK/ERK signaling. Furthermore, we identified ADAM17 as the protease responsible for TNFR2 shedding by CD8(+) T cells, with ADAM17 and TNFR2 required in "cis" for shedding to occur. We observed similar activation thresholds for TNF-α expression and TNFR2 shedding, suggesting that solTNFR2 functioned, in part, to regulate solTNF-α levels. Production of solTNFR2 by activated CD8(+) T cells reduced the availability of solTNF-α released by these cells, and TNFR2 blockade during influenza infection in mice enhanced the levels of solTNF-α, supporting this hypothesis. Taken together, this study identifies critical cellular mechanisms regulating TNFR2 shedding on CD8(+) T cells and demonstrates that TNFR2 contributes, in part, to the regulation of TNF-α levels during infection.
Assuntos
Proteínas ADAM/metabolismo , Linfócitos T CD8-Positivos/imunologia , Influenza Humana/imunologia , Influenza Humana/virologia , Infecções por Orthomyxoviridae/imunologia , Receptores Tipo II do Fator de Necrose Tumoral/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Proteína ADAM17 , Animais , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Humanos , Vacinas contra Influenza/imunologia , Camundongos Endogâmicos C57BL , Infecções por Orthomyxoviridae/virologia , Receptores de Antígenos de Linfócitos T/metabolismo , Transdução de Sinais , Solubilidade , Vacinas Atenuadas/imunologiaRESUMO
Virus infection triggers a CD8(+) T cell response that aids in virus clearance, but also expresses effector functions that may result in tissue injury. CD8(+) T cells express a variety of activating and inhibiting ligands, though regulation of the expression of inhibitory receptors is not well understood. The ligand for the inhibitory receptor, NKG2A, is the non-classical MHC-I molecule Qa1(b), which may also serve as a putative restricting element for the T cell receptors of purported regulatory CD8(+) T cells. We have previously shown that Qa1(b)-null mice suffer considerably enhanced immunopathologic lung injury in the context of CD8(+) T cell-mediated clearance of influenza infection, as well as evidence in a non-viral system that failure to ligate NKG2A on CD8(+) effector T cells may represent an important component of this process. In this report, we examine the requirements for induction of NKG2A expression, and show that NKG2A expression by CD8(+) T cells occurs as a result of migration from the MLN to the inflammatory lung environment, irrespective of peripheral antigen recognition. Further, we confirmed that NKG2A is a mediator in limiting immunopathology in virus infection using mice with a targeted deletion of NKG2A, and infecting the mutants with two different viruses, influenza and adenovirus. In neither infection is virus clearance altered. In influenza infection, the enhanced lung injury was associated with increased chemoattractant production, increased infiltration of inflammatory cells, and significantly enhanced alveolar hemorrhage. The primary mechanism of enhanced injury was the loss of negative regulation of CD8(+) T cell effector function. A similar effect was observed in the livers of mutant mice infected intravenously with adenovirus. These results demonstrate the immunoregulatory role of CD8(+) NKG2A expression in virus infection, which negatively regulates T cell effector functions and contributes to protection of tissue integrity during virus clearance.
Assuntos
Linfócitos T CD8-Positivos/imunologia , Lesão Pulmonar/patologia , Subfamília C de Receptores Semelhantes a Lectina de Células NK/genética , Subfamília C de Receptores Semelhantes a Lectina de Células NK/metabolismo , Infecções por Orthomyxoviridae/imunologia , Adenoviridae/patogenicidade , Infecções por Adenoviridae/imunologia , Infecções por Adenoviridae/patologia , Infecções por Adenoviridae/veterinária , Transferência Adotiva , Animais , Técnicas de Inativação de Genes , Vírus da Influenza A/patogenicidade , Lesão Pulmonar/imunologia , Lesão Pulmonar/virologia , Camundongos , Infecções por Orthomyxoviridae/patologia , Infecções por Orthomyxoviridae/veterináriaRESUMO
TNF-α is a pleotropic cytokine that has both proinflammatory and anti-inflammatory functions during influenza infection. TNF-α is first expressed as a transmembrane protein that is proteolytically processed to release a soluble form. Transmembrane TNF-α (memTNF-α) and soluble TNF-α (solTNF-α) have been shown to exert distinct tissue-protective or tissue-pathologic effects in several disease models. However, the relative contributions of memTNF-α or solTNF-α in regulating pulmonary immunopathology following influenza infection are unclear. Therefore, we performed intranasal influenza infection in mice exclusively expressing noncleavable memTNF-α or lacking TNF-α entirely and examined the outcomes. We found that solTNF-α, but not memTNF-α, was required to limit the size of the immune response and the extent of injury. In the absence of solTNF-α, there was a significant increase in the CD8(+) T cell response, including virus-specific CD8(+) T cells, which was due in part to an increased resistance to activation-induced cell death. We found that solTNF-α mediates these immunoregulatory effects primarily through TNFR1, because mice deficient in TNFR1, but not TNFR2, exhibited dysregulated immune responses and exacerbated injury similar to that observed in mice lacking solTNF-α. We also found that solTNF-α expression was required early during infection to regulate the magnitude of the CD8(+) T cell response, indicating that early inflammatory events are critical for the regulation of the effector phase. Taken together, these findings suggest that processing of memTNF-α to release solTNF-α is a critical event regulating the immune response during influenza infection.