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Cited by 238Immune cells in cancerNitric Oxide and Endothelin EffectsRedox biology and oxidative stress

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Nitric Oxide in Macrophage Immunometabolism: Hiding in Plain Sight

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Palmieri, Erika M., McGinity, Christopher, Wink, David A., McVicar, Daniel W.·2020·Metabolites
DOI 10.3390/metabo10110429PMC PMC7693038
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Abstract

Nitric Oxide (NO) is a soluble endogenous gas with various biological functions like signaling, and working as an effector molecule or metabolic regulator. In response to inflammatory signals, immune myeloid cells, like macrophages, increase production of cytokines and NO, which is important for pathogen killing. Under these proinflammatory circumstances, called “M1”, macrophages undergo a series of metabolic changes including rewiring of their tricarboxylic acid (TCA) cycle. Here, we review findings indicating that NO, through its interaction with heme and non-heme metal containing proteins, together with components of the electron transport chain, functions not only as a regulator of cell respiration, but also a modulator of intracellular cell metabolism. Moreover, diverse effects of NO and NO-derived reactive nitrogen species (RNS) involve precise interactions with different targets depending on concentration, temporal, and spatial restrictions. Although the role of NO in macrophage reprogramming has been in evidence for some time, current models have largely minimized its importance. It has, therefore, been hiding in plain sight. A review of the chemical properties of NO, past biochemical studies, and recent publications, necessitates that mechanisms of macrophage TCA reprogramming during stimulation must be re-imagined and re-interpreted as mechanistic results of NO exposure. The revised model of metabolic rewiring we describe here incorporates many early findings regarding NO biochemistry and brings NO out of hiding and to the forefront of macrophages immunometabolism.

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Figures

Figure 1 Biochemical mechanisms of Nitric Oxide synthesis and inhibition of mitochondrial targets. (a) Nitric oxide (NO) is synthesized from l-arginine by nitric oxide synthase (NOS) enzymes. The reaction involves the incorporation of oxygen (O2) into the unstable intermediate N omega-hydroxy-l-arginine and, subsequently, into l-citrulline, co-product of the generation of NO. N G -monomethyl-l-arginine (L-NMMA), N omega-Nitro-l-arginine (L-NAME), s-ethylisothiourea (SEITU), n-(3-(Aminomethyl)benzyl)acetamidine (1400W), and aminoguanidine (AG) are competitive inhibitor of NOS, the last two specific for inducible NOS (iNOS or NOS2), and block the pathway at the points indicated. (b) Nanomolar concentrations of NO immediately, specifically, and reversibly inhibit cytochrome c oxidase (Complex IV of mitochondrial electron transport chain, ETC) in competition with oxygen. NO reacts with the metals of the binuclear site of cytochrome c oxidase, binding tightly to the reduced iron in the distal pocket with the N closest to the Fe and the O close to, but not directly bonding, the CuB. (c) NO can target aconitase: NO is capable of oxidizing the cubane iron-sulfur (Fe-S) cluster that participates in the catalysis of the non-redox reaction of reversible isomerization of citrate to isocitrate with the intermediate cis-aconitate. This leads to iron release and consequent loss of the catalytic activity.
Figure 1 Biochemical mechanisms of Nitric Oxide synthesis and inhibition of mitochondrial targets. (a) Nitric oxide (NO) is synthesized from l-arginine by nitric oxide synthase (NOS) enzymes. The reaction involves the incorporation of oxygen (O2) into the unstable intermediate N omega-hydroxy-l-arginine and, subsequently, into l-citrulline, co-product of the generation of NO. N G -monomethyl-l-arginine (L-NMMA), N omega-Nitro-l-arginine (L-NAME), s-ethylisothiourea (SEITU), n-(3-(Aminomethyl)benzyl)acetamidine (1400W), and aminoguanidine (AG) are competitive inhibitor of NOS, the last two specific for inducible NOS (iNOS or NOS2), and block the pathway at the points indicated. (b) Nanomolar concentrations of NO immediately, specifically, and reversibly inhibit cytochrome c oxidase (Complex IV of mitochondrial electron transport chain, ETC) in competition with oxygen. NO reacts with the metals of the binuclear site of cytochrome c oxidase, binding tightly to the reduced iron in the distal pocket with the N closest to the Fe and the O close to, but not directly bonding, the CuB. (c) NO can target aconitase: NO is capable of oxidizing the cubane iron-sulfur (Fe-S) cluster that participates in the catalysis of the non-redox reaction of reversible isomerization of citrate to isocitrate with the intermediate cis-aconitate. This leads to iron release and consequent loss of the catalytic activity.CC BY · PubMed Central
Figure 2 Mitochondrial bioenergetics following acute and prolonged NO injury. (a) Cytochrome c oxidase exert considerable control over the rate of respiration because NO increases the Km of the enzyme for O2. Inhibition of cytochrome c oxidase by NO leads to a drop in the electron flux and consequently in proton extrusion. This changes the ETC to a more reduced state and leads to a defense response where reversal of the ATP synthase will hydrolyze cytoplasmic glycolytic ATP extruding protons (H+). This will re-establish the proton leak transiently lost during initial gradient dissipation. The effect of these changes is a hyperpolarization of the inner mitochondrial membrane. (b) Prolonged exposure to NO and, therefore, persistent inhibition of respiration by NO favors the formation of superoxide anions (O2−) and peroxynitrite (ONOO–), from the reaction between superoxide and NO, resulting from changes in intracellular redox conditions and depletion of the glutathione pool. These species can activate mechanisms that lead to irreversible damage, like deleterious modifications of CI. This situation facilitates the induction of the permeability transition pore (PTP), which leads to the collapse of the membrane potential and cell death. The passage of electrons (e–) along the ETC is depicted. Cyt c, cytochrome c; FADH2, flavin-adenine dinucleotide reduced; NADH, nicotinamide adenine dinucleotide reduced; Q, ubiquinone; I, II, III, and IV refer to the complexes of ETC. ANT, adenosine nucleotide translocator.
Figure 2 Mitochondrial bioenergetics following acute and prolonged NO injury. (a) Cytochrome c oxidase exert considerable control over the rate of respiration because NO increases the Km of the enzyme for O2. Inhibition of cytochrome c oxidase by NO leads to a drop in the electron flux and consequently in proton extrusion. This changes the ETC to a more reduced state and leads to a defense response where reversal of the ATP synthase will hydrolyze cytoplasmic glycolytic ATP extruding protons (H+). This will re-establish the proton leak transiently lost during initial gradient dissipation. The effect of these changes is a hyperpolarization of the inner mitochondrial membrane. (b) Prolonged exposure to NO and, therefore, persistent inhibition of respiration by NO favors the formation of superoxide anions (O2−) and peroxynitrite (ONOO–), from the reaction between superoxide and NO, resulting from changes in intracellular redox conditions and depletion of the glutathione pool. These species can activate mechanisms that lead to irreversible damage, like deleterious modifications of CI. This situation facilitates the induction of the permeability transition pore (PTP), which leads to the collapse of the membrane potential and cell death. The passage of electrons (e–) along the ETC is depicted. Cyt c, cytochrome c; FADH2, flavin-adenine dinucleotide reduced; NADH, nicotinamide adenine dinucleotide reduced; Q, ubiquinone; I, II, III, and IV refer to the complexes of ETC. ANT, adenosine nucleotide translocator.CC BY · PubMed Central
Figure 3 A/D transition of mitochondrial Complex I. The A-form of CI is capable of catalysis, but D-form is not. Divalent cations and free fatty acids influence the dynamics of the A/D transition. Majorly, substrate availability depicted by NAD+/NADH ratio in the matrix, together with presence of NO or hypoxic conditions strongly favor the kinetic of the A/D transition. Exposure of Cys39 of ND3 subunit of CI makes the D-form susceptible for thiol modifications that define deactivation reversibility, with nitrosation being the lead for further oxidation and potentially irreversible modifications and destruction of the enzyme.
Figure 3 A/D transition of mitochondrial Complex I. The A-form of CI is capable of catalysis, but D-form is not. Divalent cations and free fatty acids influence the dynamics of the A/D transition. Majorly, substrate availability depicted by NAD+/NADH ratio in the matrix, together with presence of NO or hypoxic conditions strongly favor the kinetic of the A/D transition. Exposure of Cys39 of ND3 subunit of CI makes the D-form susceptible for thiol modifications that define deactivation reversibility, with nitrosation being the lead for further oxidation and potentially irreversible modifications and destruction of the enzyme.CC BY · PubMed Central
Figure 4 Orchestration of macrophage metabolism by NO. Macrophage metabolism switches to a high glycolytic status upon pro-inflammatory stimulation with increase lactate production, which is independent on NO and NOS2. Accumulation of metabolites like succinate and itaconate is a hallmark of pro-inflammatory macrophages. Treatment of Bone Marrow Derived Macrophages (BMDMs) with cytokines that elicit low NO fluxes (es. IFNγ+IL-1β) or the beginning of a strong signal does not result in further metabolic characteristics. With IFNγ+TNFα or cytokine combination that produce medium NO concentrations or with time after a strong stimulation, macrophages undergo oxidative shortage most likely from an interrupted flux through PDH, underlined by decreased oxygen consumption rate (OCR)/extracellular acidification rates (ECAR), with responsive compensatory anaplerotic pathways for TCA supply, like glutaminolysis. The presence of high concentration of NO in LPS+IFNγ-stimulated macrophages results in further multiple metabolic rewirings. Entrance of carbon into TCA via PDH is halted and ACO2 is inhibited. Compensatory carboxylation and glutaminolysis are enhanced, leading to accumulation of citrate and limitation in itaconate. The lack of NADH and reduced substrates generated by the broken TCA cycle leads to inactive mitochondrial complexes and suppressed OXPHOS, with significant appreciable drops in OCR and full commitment to glycolysis. In turn, β -oxidation of fatty acids (FA) is strongly decreased, leading to esterification of intracellular FA with glycolytic-derived glycerol for the synthesis of triglycerides and neutral lipids. In absence of NO (a situation represented by Nos2−/− LPS+IFNγ -stimulated BMDMs), higher glycolysis is maintained, PDH and ACO2 are intact and mitochondrial ETC displays full function. In this scenario, production of itaconate is strongly increased. The arrows represent the general direction of the metabolic flow in the system with the specific contribution of glucose and glutamine. The red color for arrows/ metabolites represents higher flux of depicted pathway/ increase in metabolite levels upon respective condition, compared to macrophage resting state. PDH pyruvate dehydrogenase; ACO2 mitochondrial aconitase; IDH isocitrate dehydrogenase; β -ox, β -oxidation; α-KG, α-KG, alpha-ketoglutarate; Acetyl-CoA, Acetyl-coenzyme A.
Figure 4 Orchestration of macrophage metabolism by NO. Macrophage metabolism switches to a high glycolytic status upon pro-inflammatory stimulation with increase lactate production, which is independent on NO and NOS2. Accumulation of metabolites like succinate and itaconate is a hallmark of pro-inflammatory macrophages. Treatment of Bone Marrow Derived Macrophages (BMDMs) with cytokines that elicit low NO fluxes (es. IFNγ+IL-1β) or the beginning of a strong signal does not result in further metabolic characteristics. With IFNγ+TNFα or cytokine combination that produce medium NO concentrations or with time after a strong stimulation, macrophages undergo oxidative shortage most likely from an interrupted flux through PDH, underlined by decreased oxygen consumption rate (OCR)/extracellular acidification rates (ECAR), with responsive compensatory anaplerotic pathways for TCA supply, like glutaminolysis. The presence of high concentration of NO in LPS+IFNγ-stimulated macrophages results in further multiple metabolic rewirings. Entrance of carbon into TCA via PDH is halted and ACO2 is inhibited. Compensatory carboxylation and glutaminolysis are enhanced, leading to accumulation of citrate and limitation in itaconate. The lack of NADH and reduced substrates generated by the broken TCA cycle leads to inactive mitochondrial complexes and suppressed OXPHOS, with significant appreciable drops in OCR and full commitment to glycolysis. In turn, β -oxidation of fatty acids (FA) is strongly decreased, leading to esterification of intracellular FA with glycolytic-derived glycerol for the synthesis of triglycerides and neutral lipids. In absence of NO (a situation represented by Nos2−/− LPS+IFNγ -stimulated BMDMs), higher glycolysis is maintained, PDH and ACO2 are intact and mitochondrial ETC displays full function. In this scenario, production of itaconate is strongly increased. The arrows represent the general direction of the metabolic flow in the system with the specific contribution of glucose and glutamine. The red color for arrows/ metabolites represents higher flux of depicted pathway/ increase in metabolite levels upon respective condition, compared to macrophage resting state. PDH pyruvate dehydrogenase; ACO2 mitochondrial aconitase; IDH isocitrate dehydrogenase; β -ox, β -oxidation; α-KG, α-KG, alpha-ketoglutarate; Acetyl-CoA, Acetyl-coenzyme A.CC BY · PubMed Central

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Metadata

Authors
Palmieri, E. M., McGinity, C., Wink, D. A., & McVicar, D. W.
Affiliations
National Cancer Institute (US)
Corresponding author
McVicar, Daniel W.
ORCID
Venue
Metabolites
Publisher
MDPI AG
Published
2020-10-26
Volume
10
Issue
11
Pages
429
ISSN
2218-1989 (ISSN-L)
DOI
10.3390/metabo10110429
PMC
PMC7693038
OpenAlex
W3096340664
Cited by
237
References
252
Citations by year
2026: 40 · 2025: 59 · 2024: 56 · 2023: 46 · 2022: 15 · 2021: 20 · 2020: 1
FWCI
2.00
Type
journal article
Language
en
Open access
Licence
Locations
  • Metabolites (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
National Cancer Institute (ZIA BC 010300, ZIA BC 010899)
Topics
  • Life Sciences › Immunology and Microbiology › Immunology › Immune cells in cancer
  • Health Sciences › Medicine › Physiology (medical) › Nitric Oxide and Endothelin Effects
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Redox biology and oxidative stress
Keywords
nitric oxide, macrophage immunometabolism, M1 macrophages, reactive nitrogen species, electron transport chain, heme proteins, cellular respiration, macrophage metabolic reprogramming, inflammation

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