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1.
J Immunol ; 209(8): 1545-1554, 2022 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-36165178

RESUMO

The murine CMV (MCMV) immunoevasin m04/gp34 escorts MHC class I (MHC I) molecules to the surface of infected cells where these complexes bind Ly49 inhibitory receptors (IRs) and prevent NK cell attack. Nonetheless, certain self-MHC I-binding Ly49 activating and inhibitory receptors are able to promote robust NK cell expansion and antiviral immunity during MCMV infection. A basis for MHC I-dependent NK cell sensing of MCMV-infected targets and control of MCMV infection however remains unclear. In this study, we discovered that the Ly49R activation receptor is selectively triggered during MCMV infection on antiviral NK cells licensed by the Ly49G2 IR. Ly49R activating receptor recognition of MCMV-infected targets is dependent on MHC I Dk and MCMV gp34 expression. Remarkably, although Ly49R is critical for Ly49G2-dependent antiviral immunity, blockade of the activation receptor in Ly49G2-deficient mice has no impact on virus control, suggesting that paired Ly49G2 MCMV sensing might enable Ly49R+ NK cells to better engage viral targets. Indeed, MCMV gp34 facilitates Ly49G2 binding to infected cells, and the IR is required to counter gp34-mediated immune evasion. A specific requirement for Ly49G2 in antiviral immunity is further explained by its capacity to license cytokine receptor signaling pathways and enhance Ly49R+ NK cell proliferation during infection. These findings advance our understanding of the molecular basis for functionally disparate self-receptor enhancement of antiviral NK cell immunity.


Assuntos
Muromegalovirus , Animais , Antivirais/metabolismo , Proteínas de Transporte/metabolismo , Antígenos de Histocompatibilidade Classe I , Evasão da Resposta Imune , Camundongos , Camundongos Endogâmicos C57BL , Subfamília A de Receptores Semelhantes a Lectina de Células NK/metabolismo , Receptores de Citocinas/metabolismo , Receptores de Células Matadoras Naturais/metabolismo
2.
J Biol Chem ; 298(12): 102706, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36395887

RESUMO

The red blood cell (RBC)-Omics study, part of the larger NHLBI-funded Recipient Epidemiology and Donor Evaluation Study (REDS-III), aims to understand the genetic contribution to blood donor RBC characteristics. Previous work identified donor demographic, behavioral, genetic, and metabolic underpinnings to blood donation, storage, and (to a lesser extent) transfusion outcomes, but none have yet linked the genetic and metabolic bodies of work. We performed a genome-wide association (GWA) analysis using RBC-Omics study participants with generated untargeted metabolomics data to identify metabolite quantitative trait loci in RBCs. We performed GWA analyses of 382 metabolites in 243 individuals imputed using the 1000 Genomes Project phase 3 all-ancestry reference panel. Analyses were conducted using ProbABEL and adjusted for sex, age, donation center, number of whole blood donations in the past 2 years, and first 10 principal components of ancestry. Our results identified 423 independent genetic loci associated with 132 metabolites (p < 5×10-8). Potentially novel locus-metabolite associations were identified for the region encoding heme transporter FLVCR1 and choline and for lysophosphatidylcholine acetyltransferase LPCAT3 and lysophosphatidylserine 16.0, 18.0, 18.1, and 18.2; these associations are supported by published rare disease and mouse studies. We also confirmed previous metabolite GWA results for associations, including N(6)-methyl-L-lysine and protein PYROXD2 and various carnitines and transporter SLC22A16. Association between pyruvate levels and G6PD polymorphisms was validated in an independent cohort and novel murine models of G6PD deficiency (African and Mediterranean variants). We demonstrate that it is possible to perform metabolomics-scale GWA analyses with a modest, trans-ancestry sample size.


Assuntos
Estudo de Associação Genômica Ampla , Locos de Características Quantitativas , Humanos , Animais , Camundongos , Doadores de Sangue , Eritrócitos/metabolismo , Voluntários , 1-Acilglicerofosfocolina O-Aciltransferase/metabolismo
3.
J Pharmacol Exp Ther ; 386(3): 323-330, 2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-37348965

RESUMO

Glucose 6-phosphate dehydrogenase (G6PD) deficiency is the most common enzymopathy in humans (∼5% of all individuals). G6PD deficiency (G6PDd) is caused by an unstable enzyme and manifests most strongly in red blood cells (RBCs) that cannot synthesize new protein. G6PDd RBCs have decreased ability to mitigate oxidative stress due to lower levels of NADPH, as a result of a defective pentose phosphate pathway. Accordingly, oxidative drugs can result in hemolysis and potentially life-threatening anemia in G6PDd patients. Dapsone is a highly useful drug for treating a variety of pathologies but oral dapsone is contraindicated in patients with G6PDd due to oxidative stress-induced anemia. Dapsone must be metabolized to become hemolytic. Dapsone hydroxylamine (DDS-NOH) has been implicated as the major hemolytic dapsone metabolite, but this has never been tested on G6PDd RBCs with in vivo circulation as a metric. Moreover, the metabolic lesion caused by DDS-NOH is unknown. We report that RBCs from a novel humanized mouse expressing the human Mediterranean G6PD-deficient variant have increased sensitivity to DDS-NOH. In addition, we show that DDS-NOH damaged RBCs can either undergo sequestration (with subsequent return to circulation) or permanent removal in a dose-dependent manner, with G6PD-sufficient RBCs mostly being sequestered, and G6PDd RBCs mostly being permanently removed. Finally, we characterize the metabolic lesion caused by DDS-NOH in G6PDd RBCs and report a blockage in terminal glycolysis resulting in a cellular accumulation of pyruvate. These findings confirm DDS-NOH as a hemolytic metabolite and elucidate metabolic effects of DDS-NOH on G6PDd RBCs. SIGNIFICANCE STATEMENT: These findings confirm that dapsone hydroxylamine, an active metabolite of dapsone, causes in vivo clearance of murine red blood cells expressing a human variant of deficient glucose 6-phosphate dehydrogenase (G6PD), an enzymopathy that affects half a billion individuals (G6PD deficiency). Both cellular mechanisms of clearance (sequestration versus destruction) and specific metabolic disturbances caused by dapsone hydroxylamine are elucidated, providing novel mechanistic understanding.


Assuntos
Deficiência de Glucosefosfato Desidrogenase , Hemólise , Animais , Humanos , Camundongos , Dapsona/farmacologia , Dapsona/metabolismo , Eritrócitos/metabolismo , Glucose/metabolismo , Deficiência de Glucosefosfato Desidrogenase/complicações , Deficiência de Glucosefosfato Desidrogenase/metabolismo , Fosfatos/metabolismo
4.
Transfusion ; 61(6): 1867-1883, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33904180

RESUMO

BACKGROUND: Increases in the red blood cell (RBC) degree of fatty acid desaturation are reported in response to exercise, aging, or diseases associated with systemic oxidant stress. However, no studies have focused on the presence and activity of fatty acid desaturases (FADS) in the mature RBC. STUDY DESIGN AND METHODS: Steady state metabolomics and isotope-labeled tracing experiments, immunofluorescence approaches, and pharmacological interventions were used to determine the degree of fatty acid unsaturation, FADS activity as a function of storage, oxidant stress, and G6PD deficiency in human and mouse RBCs. RESULTS: In 250 blood units from the REDS III RBC Omics recalled donor population, we report a storage-dependent accumulation of free mono-, poly-(PUFAs), and highly unsaturated fatty acids (HUFAs), which occur at a faster rate than saturated fatty acid accumulation. Through a combination of immunofluorescence, pharmacological inhibition, tracing experiments with stable isotope-labeled fatty acids, and oxidant challenge with hydrogen peroxide, we demonstrate the presence and redox-sensitive activity of FADS2, FADS1, and FADS5 in the mature RBC. Increases in PUFAs and HUFAs in human and mouse RBCs correlate negatively with storage hemolysis and positively with posttransfusion recovery. Inhibition of these enzymes decreases accumulation of free PUFAs and HUFAs in stored RBCs, concomitant to increases in pyruvate/lactate ratios. Alterations of this ratio in G6PD deficient patients or units supplemented with pyruvate-rich rejuvenation solutions corresponded to decreased PUFA and HUFA accumulation. CONCLUSION: Fatty acid desaturases are present and active in mature RBCs. Their activity is sensitive to oxidant stress, storage duration, and alterations of the pyruvate/lactate ratio.


Assuntos
Preservação de Sangue/métodos , Eritrócitos/enzimologia , Ácidos Graxos Dessaturases/metabolismo , Estearoil-CoA Dessaturase/metabolismo , Animais , Doadores de Sangue , Dessaturase de Ácido Graxo Delta-5 , Eritrócitos/metabolismo , Ácidos Graxos Insaturados/metabolismo , Humanos , Ácido Láctico/metabolismo , Metabolômica , Camundongos , Estresse Oxidativo , Ácido Pirúvico/metabolismo
5.
Blood Transfus ; 21(1): 50-61, 2023 01.
Artigo em Inglês | MEDLINE | ID: mdl-36346885

RESUMO

BACKGROUND: The Red blood cell (RBC) storage lesion results in decreased circulation and function of transfused RBCs. Elevated oxidant stress and impaired energy metabolism are a hallmark of the storage lesion in both human and murine RBCs. Although human studies don't suffer concerns that findings may not translate, they do suffer from genetic and environmental variability amongst subjects. Murine models can control for genetics, environment, and much interventional experimentation can be carried out in mice that is neither technically feasible nor ethical in humans. However, murine models are only useful to the extent that they have similar biology to humans. Hypoxic storage has been shown to mitigate the storage lesion in human RBCs, but has not been investigated in mice. MATERIALS AND METHODS: RBCs from a C57BL6/J mouse strain were stored under normoxic (untreated) or hypoxic conditions (SO2 ~ 26%) for 1h, 7 and 12 days. Samples were tested for metabolomics at steady state, tracing experiments with 1,2,3-13C3-glucose, proteomics and end of storage post transfusion recovery. RESULTS: Hypoxic storage improved post-transfusion recovery and energy metabolism, including increased steady state and 13C3-labeled metabolites from glycolysis, high energy purines (adenosine triphosphate) and 2,3-diphospholgycerate. Hypoxic storage promoted glutaminolysis, increased glutathione pools, and was accompanied by elevation in the levels of free fatty acids and acyl-carnitines. DISCUSSION: This study isolates hypoxia, as a single independent variable, and shows similar effects as seen in human studies. These findings also demonstrate the translatability of murine models for hypoxic RBC storage and provide a pre-clinical platform for ongoing study.


Assuntos
Transfusão de Eritrócitos , Eritrócitos , Camundongos , Humanos , Animais , Metabolismo Energético , Hipóxia/metabolismo , Glicólise , Preservação de Sangue/métodos
6.
JCI Insight ; 6(14)2021 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-34138756

RESUMO

Deficiency of glucose-6-phosphate dehydrogenase (G6PD) is the single most common enzymopathy, present in approximately 400 million humans (approximately 5%). Its prevalence is hypothesized to be due to conferring resistance to malaria. However, G6PD deficiency also results in hemolytic sequelae from oxidant stress. Moreover, G6PD deficiency is associated with kidney disease, diabetes, pulmonary hypertension, immunological defects, and neurodegenerative diseases. To date, the only available mouse models have decreased levels of WT stable G6PD caused by promoter mutations. However, human G6PD mutations are missense mutations that result in decreased enzymatic stability. As such, this results in very low activity in red blood cells (RBCs) that cannot synthesize new protein. To generate a more accurate model, the human sequence for a severe form of G6PD deficiency, Med(-), was knocked into the murine G6PD locus. As predicted, G6PD levels were extremely low in RBCs, and deficient mice had increased hemolytic sequelae to oxidant stress. Nonerythroid organs had metabolic changes consistent with mild G6PD deficiency, consistent with what has been observed in humans. Juxtaposition of G6PD-deficient and WT mice revealed altered lipid metabolism in multiple organ systems. Together, these findings both establish a mouse model of G6PD deficiency that more accurately reflects human G6PD deficiency and advance our basic understanding of altered metabolism in this setting.


Assuntos
Eritrócitos/metabolismo , Deficiência de Glucosefosfato Desidrogenase/metabolismo , Glucosefosfato Desidrogenase/genética , Hemólise/genética , Animais , Modelos Animais de Doenças , Feminino , Técnicas de Introdução de Genes , Glucosefosfato Desidrogenase/metabolismo , Deficiência de Glucosefosfato Desidrogenase/sangue , Deficiência de Glucosefosfato Desidrogenase/genética , Humanos , Masculino , Camundongos , Mutação , Estresse Oxidativo/genética
7.
Curr Hematol Malig Rep ; 15(2): 103-112, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32062772

RESUMO

PURPOSE OF REVIEW: The past decade in LGL leukemia research has seen increased pairing of clinical data with molecular markers, shedding new insights on LGL leukemia pathogenesis and heterogeneity. This review summarizes the current standard of care of LGL leukemia, updates from clinical trials, and our congruent improved understanding of LGL pathogenesis. RECENT FINDINGS: Various clinical reports have identified associations between stem, bone marrow, and solid organ transplants and incidence of LGL leukemia. There is also a potential for underdiagnosis of LGL leukemia within the rheumatoid arthritis patient population, emphasizing our need for continued study. Preliminary results from the BNZ-1 clinical trial, which targets IL-15 along with IL-2 and IL-9 signaling pathways, show some evidence of clinical response. With advances in our understanding of LGL pathogenesis from both the bench and the clinic, exciting avenues for investigations lie ahead for LGL leukemia.


Assuntos
Antineoplásicos/uso terapêutico , Imunossupressores/uso terapêutico , Leucemia Linfocítica Granular Grande/diagnóstico , Leucemia Linfocítica Granular Grande/tratamento farmacológico , Terapia de Alvo Molecular/tendências , Animais , Antineoplásicos/efeitos adversos , Difusão de Inovações , Previsões , Humanos , Imunossupressores/efeitos adversos , Leucemia Linfocítica Granular Grande/imunologia , Leucemia Linfocítica Granular Grande/mortalidade , Terapia de Alvo Molecular/efeitos adversos , Valor Preditivo dos Testes , Fatores de Risco , Transdução de Sinais/efeitos dos fármacos , Resultado do Tratamento
8.
Best Pract Res Clin Haematol ; 32(3): 196-206, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31585620

RESUMO

Genomic analysis of cancer offers the hope of identifying new treatments or aiding in the selection of existing treatments. Rare leukemias pose additional challenges in this regard as samples may be hard to acquire and when found the underlying pathway may not be attractive to drug development since so few individuals are affected. In this case, it can be useful to identify common mutational overlap among subsets of rare leukemias to increase the number of individuals that may benefit from a targeted therapy. This chapter examines the current mutational landscape of large granular lymphocyte (LGL) leukemia with a focus on STAT3 mutations, the most common mutation in LGL leukemia to date. We examined the linkage between these mutations and autoimmune symptoms and disorders, in cases of obvious and suspected LGL leukemia. We then summarized and compared mutations in a set of other rare leukemias that also have JAK/STAT signaling pathway activation brought about by genomic changes. These include T-cell acute lymphoblastic leukemia (T-ALL), T-cell prolymphocytic leukemia (T-PLL), cutaneous T-cell lymphoma (CTCL), select peripheral T-cell lymphoma (PTCL), and adult T-cell leukemia/lymphoma (ATLL). Though STAT3 activation is common in these leukemias, the way in which it is achieved, such as the activating cytokine pathway and/or the co-mutational background, is quite diverse.


Assuntos
Genômica , Leucemia Linfocítica Granular Grande , Mutação , Doenças Raras , Humanos , Leucemia Linfocítica Granular Grande/classificação , Leucemia Linfocítica Granular Grande/genética , Leucemia Linfocítica Granular Grande/metabolismo , Leucemia Linfocítica Granular Grande/patologia , Leucemia-Linfoma de Células T do Adulto/classificação , Leucemia-Linfoma de Células T do Adulto/genética , Leucemia-Linfoma de Células T do Adulto/metabolismo , Leucemia-Linfoma de Células T do Adulto/patologia , Doenças Raras/classificação , Doenças Raras/genética , Doenças Raras/metabolismo , Doenças Raras/patologia , Fator de Transcrição STAT3/genética , Fator de Transcrição STAT3/metabolismo
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