RESUMO
Mutations in the IDH1 and IDH2 genes encoding isocitrate dehydrogenases are frequently found in human glioblastomas and cytogenetically normal acute myeloid leukaemias (AML). These alterations are gain-of-function mutations in that they drive the synthesis of the 'oncometabolite' R-2-hydroxyglutarate (2HG). It remains unclear how IDH1 and IDH2 mutations modify myeloid cell development and promote leukaemogenesis. Here we report the characterization of conditional knock-in (KI) mice in which the most common IDH1 mutation, IDH1(R132H), is inserted into the endogenous murine Idh1 locus and is expressed in all haematopoietic cells (Vav-KI mice) or specifically in cells of the myeloid lineage (LysM-KI mice). These mutants show increased numbers of early haematopoietic progenitors and develop splenomegaly and anaemia with extramedullary haematopoiesis, suggesting a dysfunctional bone marrow niche. Furthermore, LysM-KI cells have hypermethylated histones and changes to DNA methylation similar to those observed in human IDH1- or IDH2-mutant AML. To our knowledge, our study is the first to describe the generation and characterization of conditional IDH1(R132H)-KI mice, and also the first report to demonstrate the induction of a leukaemic DNA methylation signature in a mouse model. Our report thus sheds light on the mechanistic links between IDH1 mutation and human AML.
Assuntos
Epigênese Genética/genética , Células-Tronco Hematopoéticas/citologia , Isocitrato Desidrogenase/genética , Isocitrato Desidrogenase/metabolismo , Proteínas Mutantes/metabolismo , Mutação/genética , Envelhecimento , Animais , Medula Óssea/patologia , Linhagem da Célula , Ilhas de CpG/genética , Metilação de DNA , Modelos Animais de Doenças , Feminino , Técnicas de Introdução de Genes , Glioma/patologia , Hematopoese , Células-Tronco Hematopoéticas/metabolismo , Histonas/metabolismo , Humanos , Leucemia Mieloide Aguda/genética , Masculino , Camundongos , Proteínas Mutantes/genética , Células Mieloides/citologia , Células Mieloides/metabolismo , Baço/patologiaRESUMO
Interactions between acute myeloid leukemia (AML) and the bone marrow microenvironment (BMME) are critical to leukemia progression and chemoresistance. Altered metabolite levels in the tumor microenvironment contribute to immunosuppression in solid tumors, while this has not been studied yet in the leukemic BMME. Metabolomics of AML patient bone marrow serum detected elevated metabolites, including lactate, compared to age- and sex-matched controls. Excess lactate has been implicated in solid tumors for inducing suppressive tumor-associated macrophages (TAMs) and correlates with poor prognosis. We describe the role of lactate in the polarization of leukemia-associated macrophages (LAMs) using a murine model of blast crisis chronic myelogenous leukemia (bcCML) and mice genetically lacking the lactate receptor GPR81. LAMs were CD206hi and suppressive in transcriptomics and cytokine profiling. Yet, LAMs had a largely unique expression profile from other types of TAMs. We demonstrate GPR81 signaling as a mechanism of both LAM polarization and the direct support of leukemia cell growth and self-repopulation. Furthermore, LAMs and elevated lactate diminished the function of hematopoietic progenitors and stromal support, while knockout of GPR81 had modest protective effects on the hematopoietic system. We report microenvironmental lactate as a critical driver of AML-induced immunosuppression and leukemic progression, thus identifying GPR81 signaling as an exciting and novel therapeutic target for treating this devastating disease.
RESUMO
Platelets are immune responsive in many diseases as noted by changes in platelet mRNA in conditions such as sepsis1, atherosclerosis2, COVID-193,4, and many other inflammatory and infectious etiologies5. The malaria causing Plasmodium parasite is a persistent public health threat and significant evidence shows that platelets participate in host responses to infection. Using a mouse model of non-lethal/uncomplicated malaria, P. yoelii XNL (PyNL), infected, but not control mouse platelets expressed Ido1, a rate limiting enzyme in tryptophan metabolism that increases kynurenine at the expense of serotonin. Interferon-gamma (IFNï§) is a potent inducer of Ido1 and mice treated with recombinant IFNï§ had increased platelet Ido1 and IDO1 activity. PyNL infected mice treated with anti-IFNï§ antibody had similar platelet Ido1 and metabolic profiles to that of uninfected controls. PyNL infected mice become thrombocytopenic by day 7 post-infection and transfusion of platelets from IFNï§ treated wild type mice, but not Ido1-/- mice, increased the plasma kynurenine to tryptophan ratio, indicating platelets are a source of post-infection IDO1 activity. We generated platelet specific Ido1 knockout mice to assess the contribution of platelet Ido1 during PyNL infection. Platelet specific Ido1-/- mice had increased death and evidence of lung thrombi which were not present in infected WT mice. Platelet Ido1 may be a significant contributor to plasma KYN in IFNï§ driven immune processes and the loss of platelets may limit total Ido1, leading to immune and vascular dysfunction.
RESUMO
Raf1 is a key player in growth factor receptor signaling, which has been linked to multiple viral infections, including Human Cytomegalovirus (HCMV) infection. Although HCMV remains latent in most individuals, it can cause acute infection in immunocompromised populations such as transplant recipients, neonates, and cancer patients. Current treatments are suboptimal, highlighting the need for novel treatments. Multiple points in the growth factor signaling pathway are important for HCMV infection, but the relationship between HCMV and Raf1, a component of the mitogen-activated protein kinase (MAPK) cascade, is not well understood. The AMP-activated protein kinase (AMPK) is a known regulator of Raf1, and AMPK activity is both induced by infection and important for HCMV replication. Our data indicate that HCMV infection induces AMPK-specific changes in Raf1 phosphorylation, including increasing phosphorylation at Raf1-Ser621, a known AMPK phospho-site, which results in increased binding to the 14-3-3 scaffolding protein, an important aspect of Raf1 activation. Inhibition of Raf1, either pharmacologically or via shRNA or CRISPR-mediated targeting, inhibits viral replication and spread in both fibroblasts and epithelial cells. Collectively, our data indicate that HCMV infection and AMPK activation modulate Raf1 activity, which are important for viral replication.
RESUMO
Succinate accumulates during ischemia, and its oxidation at reperfusion drives injury. The mechanism of ischemic succinate accumulation is controversial and is proposed to involve reversal of mitochondrial complex II. Herein, using stable-isotope-resolved metabolomics, we demonstrate that complex II reversal is possible in hypoxic mitochondria but is not the primary succinate source in hypoxic cardiomyocytes or ischemic hearts. Rather, in these intact systems succinate primarily originates from canonical Krebs cycle activity, partly supported by aminotransferase anaplerosis and glycolysis from glycogen. Augmentation of canonical Krebs cycle activity with dimethyl-α-ketoglutarate both increases ischemic succinate accumulation and drives substrate-level phosphorylation by succinyl-CoA synthetase, improving ischemic energetics. Although two-thirds of ischemic succinate accumulation is extracellular, the remaining one-third is metabolized during early reperfusion, wherein acute complex II inhibition is protective. These results highlight a bifunctional role for succinate: its complex-II-independent accumulation being beneficial in ischemia and its complex-II-dependent oxidation being detrimental at reperfusion.
Assuntos
Ciclo do Ácido Cítrico , Isquemia Miocárdica/metabolismo , Ácido Succínico/metabolismo , Animais , Ácido Aspártico/metabolismo , Autofagia , Complexo II de Transporte de Elétrons/metabolismo , Metabolismo Energético , Glicogenólise , Glicólise , Masculino , Camundongos Endogâmicos C57BL , Oxirredução , Espécies Reativas de Oxigênio/metabolismo , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Transaminases/metabolismoRESUMO
The phagocytosis of apoptotic cells (efferocytosis) shifts macrophages to an anti-inflammatory state through a set of still poorly understood soluble and cell-bound signals. Apoptosis is a common feature of inflamed tissues, and efferocytosis by tissue macrophages is thought to promote the resolution of inflammation. However, it is not clear how the exposure of tissue macrophages to inflammatory cues (e.g., PAMPs, DAMPs) in the early stages of inflammation affects immune outcomes of macrophage-apoptotic cell interactions occurring at later stages of inflammation. To address this, we used low-dose endotoxin conditioning (LEC, 1 ng/ml LPS 18 h) of mouse resident peritoneal macrophages (RPMФ) to model the effects of suboptimal (i.e., non-tolerizing), antecedent TLR activation on macrophage inflammatory responses to apoptotic cells. Compared with unconditioned macrophages (MФ), LEC-MФ showed a significant enhancement of apoptotic cell-driven suppression of many inflammatory cytokines (e.g., TNF, MIP-1ß, MCP-1). We then found that enzymatic depletion of adenosine or inhibition of the adenosine receptor A2a on LEC-MФ abrogated apoptotic cell suppression of TNF, and this suppression was entirely dependent on the ecto-enzyme CD73 (AMPî²adenosine) but not CD39 (ATPî²AMP), both of which are highly expressed on RPMФ. In addition to a requirement for CD73, we also show that Adora2a levels in macrophages are a critical determinant of TNF suppression by apoptotic cells. LEC treatment of RPMФ led to a ~3-fold increase in Adora2a and a ~28-fold increase in adenosine sensitivity. Moreover, in RAW264.7 cells, ectopic expression of both A2a and CD73 was required for TNF suppression by apoptotic cells. In mice, mild, TLR4-dependent inflammation in the lungs and peritoneum caused a rapid increase in macrophage Adora2a and Adora2b levels, and CD73 was required to limit neutrophil influx in this peritonitis model. Thus immune signaling via the CD73-A2a axis in macrophages links early inflammatory events to subsequent immune responses to apoptotic cells.
Assuntos
5'-Nucleotidase/metabolismo , Apoptose/efeitos dos fármacos , Lipopolissacarídeos/toxicidade , 5'-Nucleotidase/deficiência , 5'-Nucleotidase/genética , Adenosina/metabolismo , Animais , Antígenos CD/genética , Antígenos CD/metabolismo , Apirase/deficiência , Apirase/genética , Apirase/metabolismo , Células Cultivadas , Quimiocina CXCL10/metabolismo , Quimiocina CXCL2/metabolismo , Técnicas de Cocultura , Citocinas/metabolismo , Pulmão/metabolismo , Macrófagos Peritoneais/citologia , Macrófagos Peritoneais/efeitos dos fármacos , Macrófagos Peritoneais/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Knockout , Células RAW 264.7 , Receptor A2A de Adenosina/genética , Receptor A2A de Adenosina/metabolismo , Receptor A2B de Adenosina/genética , Receptor A2B de Adenosina/metabolismo , Transdução de Sinais/efeitos dos fármacos , Receptor 4 Toll-Like/metabolismoRESUMO
In addition to dopaminergic neuron loss, it is clear that Parkinson disease includes other pathological changes, including loss of additional neuronal populations. As a means of addressing multiple pathological changes with a single therapeutically-relevant approach, we employed delayed transplantation of a unique class of astrocytes, GDAs(BMP), that are generated in vitro by directed differentiation of glial precursors. GDAs(BMP) produce multiple agents of interest as treatments for PD and other neurodegenerative disorders, including BDNF, GDNF, neurturin and IGF1. GDAs(BMP) also exhibit increased levels of antioxidant pathway components, including levels of NADPH and glutathione. Delayed GDA(BMP) transplantation into the 6-hydroxydopamine lesioned rat striatum restored tyrosine hydroxylase expression and promoted behavioral recovery. GDA(BMP) transplantation also rescued pathological changes not prevented in other studies, such as the rescue of parvalbumin(+) GABAergic interneurons. Consistent with expression of the synaptic modulatory proteins thrombospondin-1 and 2 by GDAs(BMP), increased expression of the synaptic protein synaptophysin was also observed. Thus, GDAs(BMP) offer a multimodal support cell therapy that provides multiple benefits without requiring prior genetic manipulation.