RESUMO
BACKGROUND AND AIMS: Oxidative stress plays a key role in the development of metabolic complications associated with obesity, including insulin resistance and the most common chronic liver disease worldwide, nonalcoholic fatty liver disease. We have recently discovered that the microRNA miR-144 regulates protein levels of the master mediator of the antioxidant response, nuclear factor erythroid 2-related factor 2 (NRF2). On miR-144 silencing, the expression of NRF2 target genes was significantly upregulated, suggesting that miR-144 controls NRF2 at the level of both protein expression and activity. Here we explored a mechanism whereby hepatic miR-144 inhibited NRF2 activity upon obesity via the regulation of the tricarboxylic acid (TCA) metabolite, fumarate, a potent activator of NRF2. METHODS: We performed transcriptomic analysis in liver macrophages (LMs) of obese mice and identified the immuno-responsive gene 1 (Irg1) as a target of miR-144. IRG1 catalyzes the production of a TCA derivative, itaconate, an inhibitor of succinate dehydrogenase (SDH). TCA enzyme activities and kinetics were analyzed after miR-144 silencing in obese mice and human liver organoids using single-cell activity assays in situ and molecular dynamic simulations. RESULTS: Increased levels of miR-144 in obesity were associated with reduced expression of Irg1, which was restored on miR-144 silencing in vitro and in vivo. Furthermore, miR-144 overexpression reduces Irg1 expression and the production of itaconate in vitro. In alignment with the reduction in IRG1 levels and itaconate production, we observed an upregulation of SDH activity during obesity. Surprisingly, however, fumarate hydratase (FH) activity was also upregulated in obese livers, leading to the depletion of its substrate fumarate. miR-144 silencing selectively reduced the activities of both SDH and FH resulting in the accumulation of their related substrates succinate and fumarate. Moreover, molecular dynamics analyses revealed the potential role of itaconate as a competitive inhibitor of not only SDH but also FH. Combined, these results demonstrate that silencing of miR-144 inhibits the activity of NRF2 through decreased fumarate production in obesity. CONCLUSIONS: Herein we unravel a novel mechanism whereby miR-144 inhibits NRF2 activity through the consumption of fumarate by activation of FH. Our study demonstrates that hepatic miR-144 triggers a hyperactive FH in the TCA cycle leading to an impaired antioxidant response in obesity.
Assuntos
Fígado Gorduroso/enzimologia , Fumarato Hidratase/metabolismo , Resistência à Insulina , Fígado/enzimologia , Macrófagos/enzimologia , MicroRNAs/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Obesidade/enzimologia , Animais , Carboxiliases/genética , Carboxiliases/metabolismo , Ciclo do Ácido Cítrico , Modelos Animais de Doenças , Fígado Gorduroso/genética , Fumarato Hidratase/genética , Fumaratos/metabolismo , Humanos , Hidroliases/genética , Hidroliases/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Fator 2 Relacionado a NF-E2/genética , Obesidade/genética , Estresse Oxidativo , Células RAW 264.7 , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Succinatos/metabolismoRESUMO
Inflammation is a critical component of cardiovascular disease (CVD), encompassing coronary artery disease (CAD), cerebrovascular events and heart failure and is the leading cause of mortality worldwide. In recent years, metabolism has been placed centrally in the governance of the immune response. Termed immunometabolism, immune cells adapt cellular metabolic pathways to meet demands of activation and thus function. This rewiring influences not only the bioenergetics of the cell but altered metabolites act as signalling molecules to regulate cellular response. In this review, we focus on the TCA cycle derivative, itaconate, as one such metabolite with promising immunomodulatory and therapeutic potential in inflammatory cardiovascular disease.
Assuntos
Doenças Cardiovasculares/metabolismo , Mediadores da Inflamação/metabolismo , Succinatos/metabolismo , Biomarcadores/metabolismo , Metabolismo Energético , Glicólise , Humanos , Inflamação/metabolismo , Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , Succinato Desidrogenase/antagonistas & inibidoresRESUMO
Macrophages are mononuclear phagocytes derived from haematopoietic progenitors that are widely distributed throughout the body. These cells participate in both innate and adaptive immune responses and lie central to the processes of inflammation, development, and homeostasis. Macrophage physiology varies depending on the environment in which they reside and they exhibit rapid functional adaption in response to external stimuli. To study macrophages in vitro, cells are typically cultured ex vivo from the peritoneum or alveoli, or differentiated from myeloid bone marrow progenitor cells to form bone marrow-derived macrophages (BMDMs). BMDMs represent an efficient and cost-effective means of studying macrophage biology. However, the inherent sensitivity of macrophages to biochemical stimuli (such as cytokines, metabolic intermediates, and RNS/ROS) makes it imperative to control experimental conditions rigorously. Therefore, the aim of this study was to establish an optimised and standardised method for the isolation and culture of BMDMs. We used classically activated macrophages isolated from WT and nitric oxide (NO)-deficient mice to develop a standardised culture method, whereby the constituents of the culture media are defined. We then methodically compared our standardised protocol to the most commonly used method of BMDM culture to establish an optimal protocol for the study of nitric oxide (NO)-redox biology and immunometabolism in vitro.
Assuntos
Macrófagos/citologia , Macrófagos/metabolismo , Óxido Nítrico/metabolismo , Animais , Biopterinas/metabolismo , Técnicas de Cultura de Células/métodos , Diferenciação Celular/efeitos dos fármacos , Feminino , Fator Estimulador de Colônias de Granulócitos e Macrófagos/farmacologia , Macrófagos/efeitos dos fármacos , Masculino , Camundongos Endogâmicos C57BL , Camundongos TransgênicosRESUMO
Macrophages are derived from hematopoietic progenitor cells throughout the body, are central to inflammatory processes, and participate in innate and adaptive immune responses. In vitro study of macrophages can be undertaken by ex vivo culture from the peritoneum or through differentiation of myeloid bone marrow progenitor cells to form bone marrow-derived macrophages (BMDMs). A common approach to macrophage differentiation from precursors involves the use of conditioned media from L929 cells (LCM). This media is easy to self-produce but suffers from batch variability, and its constituents are undefined. Similarly, Foetal Bovine Serum (FBS) is used to support growth but contains a vast mixture of undefined molecules that may vary between batches. These methods are not adequate for the study of nitric oxide biology and redox mechanisms as they both contain substantial amounts of small molecules that either interfere with redox mechanisms or supplement levels of cofactors, such as tetrahydrobiopterin (BH4), required for the production of NO from inducible nitric oxide synthase (iNOS). In this report, we present an optimized protocol allowing for control of the NO-redox environment by reducing the levels of exogenous biopterin while maintaining conditions suitable for cell growth and differentiation. Tight control of culture media composition helps ensure experimental reproducibility and facilitates accurate interpretation of results. In this protocol, BMDMs were obtained from a GTP cyclohydrolase (GCH)- deficient mouse model. Culture of BMDMs was performed with media containing either (i) conditioned LCM, or (ii) recombinant M-CSF and GM-CSF to produce minimal artifacts while obtaining BH4 and NO-deficient culture conditions - thus allowing for the reproducible study of NO-redox biology and immunometabolism in vitro.
Assuntos
Macrófagos , Óxido Nítrico , Animais , Biologia , Camundongos , Oxirredução , Reprodutibilidade dos TestesRESUMO
Classical activation of macrophages (M(LPS+IFNγ)) elicits the expression of inducible nitric oxide synthase (iNOS), generating large amounts of NO and inhibiting mitochondrial respiration. Upregulation of glycolysis and a disrupted tricarboxylic acid (TCA) cycle underpin this switch to a pro-inflammatory phenotype. We show that the NOS cofactor tetrahydrobiopterin (BH4) modulates IL-1ß production and key aspects of metabolic remodeling in activated murine macrophages via NO production. Using two complementary genetic models, we reveal that NO modulates levels of the essential TCA cycle metabolites citrate and succinate, as well as the inflammatory mediator itaconate. Furthermore, NO regulates macrophage respiratory function via changes in the abundance of critical N-module subunits in Complex I. However, NO-deficient cells can still upregulate glycolysis despite changes in the abundance of glycolytic intermediates and proteins involved in glucose metabolism. Our findings reveal a fundamental role for iNOS-derived NO in regulating metabolic remodeling and cytokine production in the pro-inflammatory macrophage.
Assuntos
Ciclo do Ácido Cítrico , Inflamação/metabolismo , Macrófagos/metabolismo , Óxido Nítrico/metabolismo , Succinatos/metabolismo , Animais , Biopterinas/análogos & derivados , Biopterinas/metabolismo , Ciclo do Ácido Cítrico/efeitos dos fármacos , Transporte de Elétrons/efeitos dos fármacos , Endotoxemia/induzido quimicamente , Endotoxemia/metabolismo , GTP Cicloidrolase/genética , GTP Cicloidrolase/metabolismo , Glicólise/efeitos dos fármacos , Interferon gama/farmacologia , Interleucina-1beta/metabolismo , Isocitrato Desidrogenase/metabolismo , Lipopolissacarídeos/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Macrófagos/efeitos dos fármacos , Macrófagos/enzimologia , Camundongos , Camundongos Knockout , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/enzimologia , Mitocôndrias/metabolismo , Infecções por Mycobacterium/metabolismo , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismo , Fragmentos de Peptídeos/metabolismo , Proteoma/genética , Proteoma/metabolismo , Ácido Succínico/metabolismo , Espectrometria de Massas em TandemRESUMO
Nitric Oxide (NO) is an intracellular signalling mediator, which affects many biological processes via the posttranslational modification of proteins through S-nitrosation. The availability of NO and NOS-derived reactive oxygen species (ROS) from enzymatic uncoupling are determined by the NO synthase cofactor Tetrahydrobiopterin (BH4). Here, using a global proteomics "biotin-switch" approach, we identified components of the ubiquitin-proteasome system to be altered via BH4-dependent NO signalling by protein S-nitrosation. We show S-nitrosation of ubiquitin conjugating E2 enzymes, in particular the catalytic residue C87 of UBC13/UBE2N, leading to impaired polyubiquitylation by interfering with the formation of UBC13~Ub thioester intermediates. In addition, proteasome cleavage activity in cells also seems to be altered by S-nitrosation, correlating with the modification of cysteine residues within the 19S regulatory particle and catalytic subunits of the 20S complex. Our results highlight the widespread impact of BH4 on downstream cellular signalling as evidenced by the effect of a perturbed BH4-dependent NO-Redox balance on critical processes within the ubiquitin-proteasome system (UPS). These studies thereby uncover a novel aspect of NO associated modulation of cellular homeostasis.
Assuntos
Biopterinas/análogos & derivados , Complexo de Endopeptidases do Proteassoma/metabolismo , Enzimas de Conjugação de Ubiquitina/metabolismo , Ubiquitina/metabolismo , Animais , Biopterinas/metabolismo , GTP Cicloidrolase/deficiência , GTP Cicloidrolase/genética , Técnicas de Silenciamento de Genes , Camundongos , Células NIH 3T3 , Óxido Nítrico/metabolismo , Nitrosação , Transdução de SinaisRESUMO
Inducible nitric oxide synthase (iNOS) plays a crucial role in controlling growth of Mycobacterium tuberculosis (M.tb), presumably via nitric oxide (NO) mediated killing. Here we show that leukocyte-specific deficiency of NO production, through targeted loss of the iNOS cofactor tetrahydrobiopterin (BH4), results in enhanced control of M.tb infection; by contrast, loss of iNOS renders mice susceptible to M.tb. By comparing two complementary NO-deficient models, Nos2-/- mice and BH4 deficient Gch1fl/flTie2cre mice, we uncover NO-independent mechanisms of anti-mycobacterial immunity. In both murine and human leukocytes, decreased Gch1 expression correlates with enhanced cell-intrinsic control of mycobacterial infection in vitro. Gene expression analysis reveals that Gch1 deficient macrophages have altered inflammatory response, lysosomal function, cell survival and cellular metabolism, thereby enhancing the control of bacterial infection. Our data thus highlight the importance of the NO-independent functions of Nos2 and Gch1 in mycobacterial control.
Assuntos
Biopterinas/análogos & derivados , GTP Cicloidrolase/fisiologia , Óxido Nítrico Sintase Tipo II/fisiologia , Óxido Nítrico/biossíntese , Tuberculose/imunologia , Animais , Biopterinas/genética , Biopterinas/metabolismo , Biopterinas/fisiologia , Sobrevivência Celular , GTP Cicloidrolase/genética , GTP Cicloidrolase/metabolismo , Deleção de Genes , Perfilação da Expressão Gênica , Humanos , Macrófagos/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Óxido Nítrico Sintase Tipo II/genética , Óxido Nítrico Sintase Tipo II/metabolismoRESUMO
Glutathione (GSH), a major cellular antioxidant, is considered an inhibitor of the inflammatory response involving reactive oxygen species (ROS). However, evidence is largely based on experiments with exogenously added antioxidants/reducing agents or pro-oxidants. We show that depleting macrophages of 99% of GSH does not exacerbate the inflammatory gene expression profile in the RAW264 macrophage cell line or increase expression of inflammatory cytokines in response to the toll-like receptor 4 (TLR4) agonist lipopolysaccharide (LPS); only two small patterns of LPS-induced genes were sensitive to GSH depletion. One group, mapping to innate immunity and antiviral responses (Oas2, Oas3, Mx2, Irf7, Irf9, STAT1, il1b), required GSH for optimal induction. Consequently, GSH depletion prevented the LPS-induced activation of antiviral response and its inhibition of influenza virus infection. LPS induction of a second group of genes (Prdx1, Srxn1, Hmox1, GSH synthase, cysteine transporters), mapping to nrf2 and the oxidative stress response, was increased by GSH depletion. We conclude that the main function of endogenous GSH is not to limit inflammation but to fine-tune the innate immune response to infection.