key: cord-0725993-igj7ooip
authors: Andreyev, A. Y.; Kushnareva, Y. E.; Starkova, N. N.; Starkov, A. A.
title: Metabolic ROS Signaling: To Immunity and Beyond
date: 2020-12-28
journal: Biochemistry (Mosc)
DOI: 10.1134/s0006297920120160
sha: 4d73fe53922d01035ed2760fca757f31e60c4c85
doc_id: 725993
cord_uid: igj7ooip

Metabolism is a critical determinant of immune cell functionality. Immunometabolism, by definition, is a multidisciplinary area of immunology research that integrates the knowledge of energy transduction mechanisms and biochemical pathways. An important concept in the field is metabolic switch, a transition of immune cells upon activation to preferential utilization of select catabolic pathways for their energy needs. Mitochondria are not inert in this process and contribute to the metabolic adaptation by different mechanisms which include increasing ATP production to match dynamic bioenergetic demands and serving as a signaling platform. The latter involves generation of reactive oxygen species (ROS), one of the most intensively studied mitochondrial processes. While the role of mitochondrial ROS in the context of oxidative stress is well established, ROS signaling in immunity is an emerging and quickly changing field. In this review, we discuss ROS signaling and immunometabolism concepts from the standpoint of bioenergetics. We also provide a critical insight into the methodology for ROS assessment, outlining current challenges in the field. Finally, based on our analysis of the literature data, we hypothesize that regulatory ROS production, as opposed to oxidative stress, is controlled by mitochondrial biogenesis rather than metabolic switches.

During the past decades an entire literature dedicat ed to mitochondrial ROS (mitoROS) has been generated, signifying the importance of the topic in biology research. In 2002, we stated a simple kinetic model of ROS gener ation as a bimolecular reaction between oxygen and mito chondrial redox centers prone to auto oxidation (ROS BIOCHEMISTRY (Moscow) Vol. 85 Nos. 12 13 2020 centers) [1] . According to the mass action law, the rate of such reaction is proportional to quantities (concentra tions) of both reactants. The model also takes into account a dynamic nature of the ROS centers constantly cycling between reduced state capable of reacting with oxygen and a nonreactive oxidized state. ROS generation is therefore proportional both to the net concentration of ROS centers and to the fraction of their reduced form.

It is generally accepted that a major source of mitoROS production in the electron transport chain (ETC) is Complex I in its highly reduced state that can be produced by the specific Complex I inhibitor, rotenone, or other ETC inhibitors and conditions (such as cytochrome c depletion). Alternatively, rotenone can inhibit mitoROS production in a reverse electron trans port (RET) scenario in hyperpolarized mitochondria [2 4 ]. This phenomenon is manifested under pathological conditions promoting excessive succinate accumulation and activation of proximal ROS producing sites. Notably, RET induced oxidative stress was reported in ischemia reperfusion injury [5 7 ]. However, as discussed below, the contribution of RET to ROS signaling seems unlikely under normal physiological conditions. Another canoni cal ROS center is Complex III, as was described in pio neering studies in 1972 [8, 9] . Complex III derived ROS production is remarkably robust but typically manifests only in the presence of the specific inhibitor, antimycin A, that artificially elevates the level of unstable semi quinone form of coenzyme Q (an electron donor) on the matrix side of the Q cycle. It is unclear whether such spe cific Complex III modification can be produced by natu ral effectors, and therefore, the role of this ROS center in physiological milieu remains debatable [2, 4, 10, 11 ]. An often overlooked aspect of mitoROS production is their mitochondrial matrix sources as opposed to ETC. Previous studies suggested that mitochondrial matrix dehydrogenases contribute to ROS production in nor mally functioning mitochondria (in the absence of ETC inhibitors). In particular, a major ROS producing activi ty is associated with dihydrolipoyl dehydrogenase (Dld) component of α ketoglutarate and pyruvate dehydroge nase complexes [12, 13] .

Historically, ROS production was associated with oxidative stress by definition, a plethora of damaging effects to the cell. Evidence for regulatory roles of ROS currently lead to a paradigm shift towards "moderate" ROS as important signaling entities. Metabolic repro gramming of activated immune cells, specifically, T cells and macrophages, is associated with major bioenergetic changes. This is expected to affect both the mitochondri al mass/net number of ROS centers and their redox sta tus, i.e., both components in the ROS generation model noted above [1] . These factors need to be considered to delineate ROS signaling pathways.

This review aims to provide critical insights into selected metabolic ROS signaling mechanisms. We intro duce key immunometabolism concepts, such as glycolyt ic switch and "broken" TCA cycle (tricarboxylic acid cycle), and discuss the role of metabolites with prominent immunomodulatory effects ("immunometabolites"). This constitutes the necessary background for a more in depth analysis of bioenergetics of immune cells and their propensity to generate mitoROS. Further, we review pro posed mechanisms of ROS signaling in immunity, in par ticular, the link between mitoROS and HIF1 function. Other highlights include mitoROS as damage associated molecular patterns (DAMPs), the intrinsic mediators of proinflammatory responses. An interplay of mitoROS and non mitoROS (primarily, NOX derived H 2 O 2 pro duction in macrophages) is briefly discussed as well. The narrative is concluded with critical assessment of research tools extensively used in the field to ward off potential misinterpretation of experimental results. A broader bib liography on discussed topics can be found in specialized reviews referenced in each section.

The main conclusions drawn from the analysis of concepts outlined above are the following: (i) the regula tory mitoROS signaling, most likely, involves "mild" changes associated with mitochondrial biogenesis, as opposed to drastic changes in mitochondrial metabolic state typical for oxidative stress conditions. (ii) The meta bolic changes in immune response generally favor increased energy transduction via the most effective mechanism, oxidative phosphorylation, as opposed to switch to glycolysis. (iii) Classically activated macro phages represent an exception due to their bactericidal function that requires massive production of reactive sub stances (NO and H 2 O 2 ) that damage their own mitochon dria.

An unprecedented interest in metabolic regulation of innate and adaptive immunity along with the develop ment of multi parameter metabolic flux analysis and high precision "omic" profiling of immune cells shaped a new multidisciplinary subject of research, immunometab olism. A number of recent reviews have reintroduced fun damental metabolic pathways in the context of special ized immune cell functions and highlighted advances in the field (e.g. [14 19] ). Here, we outline some of the recent immunometabolism concepts with an emphasis on glycolytic shift and concomitant changes in mitochondr ial energy transduction.

Glycolytic switch concept. Reminiscent of the classi cal Warburg effect in cancer cells, normal immune cells (specifically, T cells and macrophages) rapidly shift from mitochondrial oxidative phosphorylation toward aerobic glycolysis when exposed to antigens, cytokines, and other stimuli. Although this metabolic change is well docu mented (with early reports of increased glycolysis activity BIOCHEMISTRY (Moscow) Vol. 85 Nos. 12 13 2020 in stimulated lymphocytes dating back to the 60 70s [20, 21] ), the obligatory requirement of aerobic glycolysis for immune cell activation and effector responses is still not completely understood. Since effector functions of immune cells, in particular cytokine production in lym phocytes, are generally associated with enhanced prolif erative capacity [22] , increased glycolytic capacity is thought to be necessary for various biosynthetic reactions supporting cell growth [14, 23 25] . Paradoxically, a hall mark of immune cell activation is increased lactate pro duction that formally precludes utilization of upstream metabolites for de novo biosynthesis of macromolecules [15] . Moreover, a "switch" from the more efficient mito chondrial ATP production to aerobic glycolysis seems inconsistent with higher energy demand imposed by ana bolic reactions, proliferation, and effector function per se.

Indeed, metabolic phenotypes of different types of activated immune cells (most notably, T cells and macrophages) and their functionally distinct subsets are not uniform. While glycolytic switch in activated T cells occurs concomitantly with rapid stimulation of mito chondrial respiration, stimulation of macrophages leads to iNOS upregulation and NO mediated inhibition of ETC activity [26] . (This is further discussed in the section focused on the bioenergetics assessment of glycolytic switch).

Glycolytic enzymes control effector functions. Recent implications of glycolytic enzymes and metabolites in epigenetic and post transcriptional control of specific effector functions suggest a more decisive role of glycoly sis in immune response [27 30 ]. Specifically, interferon gamma (IFNγ) expression in T cells was shown to be neg atively regulated by glyceraldehyde 3 phosphate dehy drogenase (GAPDH) binding to IFNγ mRNA; the trans lational constrain was relieved upon T cell activation induced glycolytic shift and GAPDH engagement in energy production [27] . The inverse relationship between GAPDH availability and cytokine translation suggested an additional non enzymatic role of GAPDH as a consti tutive translation inhibitor in quiescent (non activated) cells. More recently, GAPDH was implicated in an anal ogous mechanism of metabolic regulation of tumor necrosis factor α (TNFα) translation in macrophage pro inflammatory response [31] . These studies are consistent with earlier work highlighting GAPDH propensity to bind AU rich elements (AREs) of mRNA and alter protein expression [32] . It has been shown that AREs are dis placed from binding to GAPDH by both substrates, glyc eraldehyde 3 phosphate (G 3P) and NAD + [33, 34] . Accordingly, G 3P potentiates production of IFNγ in glycolytically deficient T cells growing on slowly metab olized galactose but not in cells growing on glucose that already have higher levels of G 3P [27] (Fig. 1, a c) . It should be noted, however, that this is the scenario in which glycolytic flux is restricted upstream of GAPDH. If glycolytic flux is restricted downstream of GAPDH one should expect an exactly opposite effect upon lifting the restriction; the buildup of all intermediates, including G 3P, should cease. This is the hypothetical scenario of "metabolic switch" from oxidative phosphorylation (when glycolytic ATP producing enzymes downstream of GAPDH are partially suppressed by mitochondrial com petition) to glycolytic ATP production (Fig. 1d) .

Another unresolved issue with this mechanism is that both the reaction product NADH and its structural ana log ATP are also effective in displacing the AREs from GAPDH in micromolar concentrations [27] . Since both pyridine and adenine nucleotides are present intracellu larly at millimolar levels they should effectively compete off any AREs under all conditions. To resolve this para dox, it has been speculated that there might be compart ments with much lower nucleotide levels, either in nucle us or parts of cytosol [27] , but this presumption will require testing.

An alternative mechanism coordinating glycolytic shift and cytokine production involves lactate dehydroge nase A (LDHA), the enzyme catalyzing pyruvate conver sion to lactate. Based on genetic LDHA depletion exper iments, it was reported that under conditions of aerobic glycolysis, IFNγ expression is controlled by LDHA via acetyl coA dependent epigenetic mechanism (histone acetylation) but not by the GAPDH linked translational regulation [28] . Manifestation of either mechanism might be profoundly influenced by other activities of these housekeeping proteins, in particular in loss of function paradigms. GAPDH has been predicted to be a rate lim iting glycolytic enzyme, in addition to the classical rate limiting enzymes (hexokinase, phosphofructokinase, and pyruvate kinase) [35] , while LDHA defines the end point reaction of aerobic glycolysis. As such, both GAPDH and LDHA are important determinants of steady state levels of glycolytic metabolites, NAD + /NADH redox balance, and cell survival capacities. Explored as potential cancer therapy targeting Warburg metabolism, LDHA inhibition compromises serine and aspartate biosynthesis. However, in malignant cells, this effect is counteracted by activa tion of major pro survival gene expression program medi ated by ATF4, a transcription factor from cAMP response element binding protein (CREB) family [36] . Reminiscent of this phenomenon, proliferation and effector function of activated Th1 cells involves ATF4 linked transcriptional response, in particular under con ditions of amino acid deficiency [37] . Furthermore, lac tate per se directly modulates immune cell functions [38 40] tending to decrease cytokine production at high extracellular concentrations. A recent study explored a novel lactate driven epigenetic mechanism (histone lysine lactylation) linked to metabolic reprogramming in macrophages exposed to bacterial pathogens. Lactylation was increased by the mitochondrial inhibitor rotenone demonstrating a link between oxidative phosphorylation (mitochondrial) activity and transcriptional regulation.

Indeed, lactate abundance inversely depends on mito chondrial pyruvate utilization that stops once pyridine nucleotide pool is fully reduced in the presence of rotenone [29] . LDHA dependent epigenetic modulation of cytokine expression is also directly linked to mitochon drial metabolism, specifically citrate production in the Krebs cycle. Stimulated CD4 + T cells deficient in LDHA showed increased mitochondrial respiration and TCA cycle activity, while cytosolic acetyl coA was decreased [28] . This suggested that, in wild type cells, glycolytic shift diverted citrate from intramitochondrial utilization and enhanced its export from mitochondria, thereby pro moting cytosolic acetyl coA generation from citrate, selective histone acetylation and IFNγ transcription [28] . Indeed, acetyl CoA is also utilized for other cell func tions, such as fatty acid and cholesterol biosynthesis, and both histone and non histone protein acetylation in vari ous signaling pathways [41, 42] .

With regard to citrate metabolism, mitochondrial citrate malate antiporter (SLC25A1) emerged as a poten tially important regulator of cytokine production (in par ticular in macrophages) and cellular NADP + /NADPH balance [43, 44] . These and other recent studies consti tute a growing subtopic of immunometabolism focused on the role of TCA cycle metabolites (reviewed in [44] ) that we briefly discuss below.

"Broken cycle" concept. Based on metabolic repro gramming patterns in macrophages and dendritic cells, it is postulated that under conditions of "glycolytic switch" and/or impaired mitochondrial respiration (e.g., due to nitric oxide [NO] production in activated macrophages), TCA cycle is no longer coupled to ETC activity and this leads to accumulation of immune modulatory intermedi ates, most notably citrate and succinate [44, 45] . However, suppression of electron transfer through ETC via the mechanism of respiratory control or by direct inhibition, is expected to cause nearly complete reduction of NAD + to NADH. This should terminate TCA cycle reactions due to deficit of NAD + , the substrate of four reactions, including pyruvate dehydrogenase at the entry to the cycle. In this scenario, neither citrate nor succinate could be produced (Fig. 2, a and b) . To allow anabolic reactions through TCA cycle go forward, the excess of reducing equivalents would need to be transported from mitochondria via malate/aspartate shuttle (Fig. 2c ) or otherwise utilized [46] whether these activities are suffi Mechanisms of regulation of ARE mRNA abundance by glycolytic flux. Enzymes may be regulated by fluxes only indirectly through changes in metabolite abundances. Accordingly, the moonlighting mRNA binding function of GAPDH is regulated by abundance of its sub strates, G 3P and pyridine nucleotides. GAPDH, mRNA and a skeleton scheme of glycolytic pathway are shown in green, red and blue, respectively; basal lactate production is inconsequential and omitted for simplicity (a c). Width of the arrows depicts the magnitude of the fluxes and the size of the font depicts the abundance of metabolites. (X) Indicates the restriction point in the pathway; under normal condi tions (a) the restriction points are ATP producing enzymes that are suppressed by competition from mitochondrial ATP production. Galactose shares most of classical glycolytic pathway but is slow to enter it (b and c). This leads to GAPDH binding, and depleting, of ARE mRNAs, including IFNγ (b), the process reversible by exogenous G 3P (c) [27] . A hypothetical glycolytic switch scenario (d) could lead to a decreased IFNγ mRNA (and protein), in apparent discrepancy with observed IFNγ production. This discrepancy would require additional research to resolve. (Colored versions of Figs. 1 and 2 are available in online version of the article and can be accessed at: https://www.springer.com/journal/10541) BIOCHEMISTRY (Moscow) Vol. 85 Nos. 12 13 2020 cient for the release of TCA cycle function needs to be determined experimentally.

Immunometabolites: anabolic building blocks versus regulators. Metabolites can serve as precursors of building blocks (fatty acids and amino acids) for proliferation and protein biosynthesis, and as regulatory molecules. With regard to anabolic mechanisms, two considerations are important. First, it is unlikely that cells can forgo energy efficiency for a "greater good" of anabolic metabolism, as implies glycolytic switch concept. From the biochemical standpoint, immune response is primarily expenditure of vast amounts of energy, whether for protein synthesis (for proliferation and differentiation; antibody and cytokine production), action of cytoskeletal motors (for morpho logical changes) or ion pumps (for ionotropic signaling). Most cells, including immune cells, typically possess spare respiratory capacity, including excess of TCA capacity over basal energy needs, which can be used for biosyntheses without need to suppress bioenergetics. Second, cells in an organism normally receive most building blocks (amino acids, fatty acids, cholesterol, etc.) with the diet. Capacity of organism to augment that supply is quite limited. Ten amino acids out of twenty are "essential" and two more are synthesized from essential amino acids. All polyunsaturated fatty acids are either essential or synthesized from them. Ability to produce palmitate (from citrate) or serine and glycine (from 3 phosphoglycerate), typically considered in metabolic reprogramming scenarios, could serve at best a moderate correction of dietary imbalances (exclud ing liver and adipose tissue specialized in lipogenesis [47] ). For cells in vitro, this also means that standard medium might have to be supplemented if some nutrients become limiting.

Regulatory mechanisms appear more impactful than anabolic. With regard to regulatory functions, as noted above, citrate and succinate participate in post transla tional and post transcriptional regulation. Changes in their levels (in particular, in the "broken" TCA cycle model) are substantial and sufficient to elicit significant effects. Citrate in TLR4 stimulated bone marrow derived macrophages (BMDM) rises about 2 fold [48] which may lead to a similar increase in cytosolic acetyl CoA levels and the rates of histone acetylation. Additionally, acetyl CoA can be converted to malonyl CoA through the action of acetyl CoA carboxylase, and malonyl CoA levels do rise approximately 2.5 fold resulting in malonylation of multiple proteins including GAPDH [49] . Malonylation of lysine 213 (or its mimicking mutation to glutamic acid) boosts enzymatic activity roughly 3 fold and completely releases sequestered TNFα transcript [49] . Thus, both energy and effector functions are upregulated with a single modification originating from the citrate increase.

Succinate also increases more that 2 fold in TLR4 activated (Toll like receptor 4) BMDMs [48] . It inhibits prolyl hydroxylase domain enzyme (PHD) with IC 50 about 0.5 mM, well in the range of extracellular concen trations [50] . Consistent with structural similarities of the two metabolites, this inhibition occurs in competitive fashion and is counteracted by the enzyme substrate α Suppression and rescue of TCA cycle biosynthetic activity by NAD + availability. The biosynthetic activity is exemplified by citrate syn thesis considered essential for lipid biosynthesis. Pyridine nucleotide reactions are shown with blue arrows, "3x" denotes 3 NADH produces in one turn of TCA cycle. Width of the arrows depicts the magnitude of the fluxes and the size of the font depicts the abundance of metabo lites. (X) Indicates suppression of ETC activity. a) Normal citrate production in intact oxidative phosphorylation. b) Low citrate production under conditions of suppressed electron transport chain (ETC), including via respiratory control in glycolytic switch scenario. c) A hypothet ical rescue of citrate synthesis by reverse malate aspartate (Mal Asp) shuttle activity, oxidation of NADH by oxaloacetate (OAA). The arrow indicating NADH to NAD + conversion is linked to Mal Asp shuttle symbol (as opposed to OAA to malate conversion) for figure clarity. All NADH (and/or NAD + ) symbols represent a single uniform NADH (and/or NAD + ) pool. BIOCHEMISTRY (Moscow) Vol. 85 Nos. 12 13 2020 ketoglutatate [48] . Accordingly, PHD activity should depend on the ratio of these metabolites rather than their individual levels. In TLR4 stimulated RAW 264.7 macrophages α ketoglutarate remains unchanged as suc cinate rises about 3 fold shifting the ratio towards inhibi tion [51] . PHD inhibition prevents destabilization of HIF1α (reviewed in Mitochondrial ROS and HIF1 regu lation section below) and allows transcription of multiple genes including the pro inflammatory cytokine IL 1β [48] .

The total pool of TCA cycle metabolites (including succinate and citrate) increases in activated macrophages implying anaplerosis [48] . Stable isotope tracing demon strates that the largest anaplerotic contributor is gluta mine to glutamate to α ketoglutatate pathway [48] . Succinate could also accumulate as a result of inhibition of succinate dehydrogenase (SDH), for example, by ita conate (reviewed below) [51, 52] . However, there appears to be no classic crossover point at SDH, as the down stream intermediates fumarate and malate are not deplet ed and may even increase [48] suggesting a modest role of SDH in succinate accumulation. Stable isotope tracing ruled out succinate production directly from ita conate [51] .

Itaconate is arguably the archetypical immuno metabolite (reviewed in [53] ). Undetectable in resting macrophages, it exponentially rises to millimolar levels within a few hours upon TLR4 activation becoming the most abundant dicarboxylate in the cell [51, 54] . This kinetics is on par with such prominently responsive metabolites as prostaglandins [55] and NO [56] . It reflects activity of highly inducible (nearly 200 fold) enzyme cis aconitate decarboxylase (CAD; also known as immunoresponsive gene 1, IRG1, protein), that converts TCA cycle intermediate aconitate to itaconate [54] . Among mammalian cells, this reaction apparently almost exclusive for macrophages [57] . It is not exactly clear what causes aconitate which is usually presumed to be tightly bound in the active center of aconitase, to dissoci ate and react with CAD.

The effects of itaconate are a subject of debate: whether it is pro inflammatory due to SDH inhibition and accumulation of succinate [51] or anti inflammatory [50, 58] . Favoring the latter, cytokines IL 1β, IL 6, IL 18, IL 12 (but not TNFα) are slightly upregulated (by 20 30%) in CAD knockout cells suggesting existence of immune suppression by itaconate in wild type cells. However, surprisingly, hif 1α transcript is increased by order(s) of magnitude in the knockout cells resulting in the elevated HIF1α protein [52] which per se could account for the modest pro inflammatory effect. Due to presence of reactive double bond conjugated with car boxylic group, itaconic acid can participate in a reaction of generalized Michael addition type with protein thiols. Posttranslational modification of Kelch like ECH asso ciated protein 1 (KEAP1) via formation of the Michael adduct with downstream activation of anti inflammatory factor Nrf2 (nuclear factor erythroid 2 related factor 2) was suggested as mechanism of itaconate action [58] . Overall, however, given the robust itaconate response upon stimulation, it is likely that we do not know yet the full potential of this metabolite.

Signal transduction notes. Much interest in the immunometabolism field has focused on post activation dynamic changes in lipid metabolism, in particular, gly colysis driven de novo fatty acid synthesis (FAS). The metabolic shift from fatty acid oxidation (FAO) to FAS is positively regulated by the mammalian target of rapamycin (mTOR) dependent signal transduction path ways that is counterbalanced by AMP activated kinase (AMPK) kinase activity fueling FAO [15, 59] . The ser ine/threonine kinase mTOR couples nutrient and oxygen availability with appropriate transcription programs. This regulatory network controls various aspects of innate and adaptive immunity, including macrophage polarization, T cell glycolytic reprogramming, cellular migration, regula tory T cell (T reg ) function, and cytokine production in dif ferent types of immune cells [15, 59 62] . Among pleiotropic effects of mTOR is enhanced mitochondrial biogenesis and oxidative metabolism [62] . In addition to the oxygen sensor HIF 1α (discussed below), other essential transcription factors that control metabolic reprogramming include the Ca 2+ /calcineurin dependent nuclear factor of activated T cells (NFAT) [63, 64] and c Myc (MYC proto oncogene), a major direct regulator of pro survival gene expression [23, 45, 60, 65] . Nuclear factor kappa B (NF κB) is a major transcriptional activa tor of inflammatory responses, with a recently emerged role of a negative regulator of excessive inflammation [66] . This mechanism involves a complex interplay between selective elimination of defective mitochondria (mitophagy) and the NLRP3 inflammasome activation [67] . These signaling network components have been a subject of intense research and discussed in detail in sig nal transduction themed reviews (e.g. [62, 66, 68] ).

A recurring theme in immunometabolism is the con cept of metabolic switch, an obligatory transition of cells to utilization of specific metabolic pathways to drive immune function. The particular case of glycolytic switch includes a set of notions, such as transition to aerobic gly colysis, ROS signaling and immunometabolite activity, that, as a whole or in parts, are applied to studies of vari ous immune cells. In its entirety, such mechanism was proposed, for example, for SARS CoV 2 infected (severe acute respiratory syndrome coronavirus 2) monocytes [69] as well as lipopolysaccharide stimulated (LPS) macrophages [70] . In this section we aim to assess these BIOCHEMISTRY (Moscow) Vol. 85 Nos. 12 13 2020 notions from biochemist's point of view. Our analysis is based largely on the dataset from a comprehensive study of the glycolytic switch in TLR4 activated macrophages [70] . This study deconvolved a network of metabolic changes accompanying pro inflammatory macrophage polarization that eventually manifested in HIF1α stabi lization and cytokine production.

The notion of glycolytic switch implies that glycolyt ic pathway is upregulated to produce ATP faster than oxidative phosphorylation thus suppressing the latter through substrate (ADP) deprivation. Accordingly, inher ent to this mechanism is the higher ATP to ADP ratio (ATP/ADP) after the switch to glycolysis. Contrary to that, in TLR4 stimulated BMDMs, ATP/ADP drops bỹ 40%, corresponding to increase in ADP levels from 5 to 8% of the total pool. ADP rise should have resulted in activation of respiration, instead, respiration decreases by about ~40%. This result implies a completely different mechanism: an active suppression of oxidative phospho rylation rather than respiratory control by substrate avail ability. NO is the most likely factor responsible for this suppression [71] . It has been known since 1989 that NO is produced by activated macrophages and causes respira tory inhibition of target cells [72] . NO reversibly inhibits cytochrome c oxidase (respiratory complex IV) in a man ner competitive to oxygen. NO is produced by inducible nitric oxide synthase (iNOS) expressed upon activation; macrophages in culture produce up to 1 μM NO, suffi cient to cause their respiratory inhibition [73] . Prolonged exposure to NO can also cause an irreversible inhibition of respiratory complex I [74] . Additional targets may include aconitase 2 and pyruvate dehydrogenase inhibi tion of which may cause observed suppression of OCR (oxygen consumption rate) upstream of the respiratory chain [75] . The volatile nature of this compound may account for significant variability in respiratory inhibition observed in different studies. Metabolic flux analysis platforms designed for simul taneous measurements of oxygen consumption and extra cellular acidification rates (OCR and ECAR [extracellu lar acidification rate], respectively) combined with advanced data analysis [76] help to quantitatively re eval uate the relative contribution of oxidative phosphoryla tion and glycolysis to overall bioenergetics capacity of immune cells. The study [70] presents sufficiently detailed data for this analysis including rarely reported proton production rate (PPR); raw ECAR read out is insufficient. The analysis is based on stoichiometries of overall oxidative phosphorylation and glycolytic path ways. ATP output of oxidative phosphorylation has been revised downward from canonical 36 ATP molecules per glucose [77] to 30 32 (based on ATP/O ratios from [78] ); accordingly, we use ATP/O 2 = 5.45 [79] . The textbook stoichiometry of glycolysis is one ATP per lactic acid (ATP/H + = 1) [77] . However, calculation of glycolytic ATP production based on raw proton production rate (PPR) gives only the upper limit estimate because of inter ference from mitochondria produced CO 2 . We converted the reported proton production rate (PPR) and OCR val ues to ATP production rates. In the quiescent state 200,000 BMDMs produce ~1500 pmoles ATP per minute; this production drops upon TLR4 stimulation by about 40%, to ~900 pmol/min. The contribution of gly colysis increases from ~3% and ~17%, for quiescent and stimulated cells, respectively. These metabolic changes do not reach an extent of a "switch" that may be perceived as preferential reliance on glycolysis for energy.

Thus, paradoxically, mitochondrial function is impaired in the TLR4 stimulated macrophages and gly colysis remains a minor contributor that cannot keep up with energy demand. This results in decreased ATP pro duction and lower ATP/ADP inevitably leading to turn ing off some ATP consumers which, in quiescent cells, are usually thought to be essential (housekeeping) processes necessary for cell survival. In a broader biolog ical sense, however, survival may well be not an essential function for inflammatory macrophages.

Treatment with glycolysis inhibitor 2 deoxyglucose suppresses macrophage immune response (cytokine pro duction) and rescues animals in the models of bacterial infection and/or hyperinflammation [48, 80, 81] . These results underscore a fine tuning of energy requirements for the immune response: it is preserved despite the exist ing energy deficiency but is strongly dependent upon the residual energy provision from glucose metabolism. The latter, obviously, is not limited to glycolysis but involves oxidative metabolism of glycolytically produced pyruvate in mitochondria, a question is to what degree other mito chondrial substrates could substitute for it.

It should be also noted that, despite upregulated gly colysis, the suppression of oxidative phosphorylation does not occur in alternatively activated (in response to IL 4) macrophages. On contrary, they undergo PGC 1β medi ated (peroxisome proliferator activated receptor gamma coactivator 1 beta) mitochondrial biogenesis resulting in increased capacity for oxidative metabolism [82, 83] cor responding to elevated energy demand [71, 84, 85] . Similar changes in bioenergetics occur in T cells in response to stimulation of T cell receptor (TCR) where both glycolysis and oxidative phosphorylation are upreg ulated [27, 86] . Thus, although activated glycolysis is a signature response of various immune cells to stimuli, glycolytic switch appears to have a limited scope.

RET and ROS. Another notion that needs to be reassessed is the ROS production mediated by reverse electron transport (RET). It is plausible that the glycolyt ic shift might result in activation of this mechanism if cer tain benchmarks were met. It is known from experiments with isolated mitochondria that this mechanism is exquis itely sensitive to any depolarization of mitochondrial membrane because it is driven by membrane potential (Δψ) "uphill", against redox potential of the carriers BIOCHEMISTRY (Moscow) Vol. 85 Nos. 12 13 2020 [87, 88] . For this mechanism to function, mitochondria should be essentially in a non phosphorylating, "resting" state. This is not the case for mitochondria in stimulated BMDMs that may preserve more than half of their nor mal ATP synthetic activity (see above). Direct measure ments show that although the signal of Δψ sensitive probe tetramethylrhodamine methyl ester (TMRM) does go up upon TLR4 stimulation, it does not approach the level of actual State 4 attained with oligomycin [70] . Besides, this change is proportional to an increase in fluorescence of Δψ insensitive mitochondrial probe MitoTracker Green indicating likelihood of the response to increased mito chondrial volume rather than Δψ. Also, as originally termed, RET is actually a transport of electrons from suc cinate to NAD + and is manifested in highly reduced state of NAD + /NADH. However, in the activated BMDMs, the pyridine nucleotides remain 90% oxidized. Overall, in our opinion, stimulated BMDMs do not possess the benchmark conditions for RET and RET mediated ROS production. In the inhibitor analysis of ROS, mitochon drial inhibitors that are capable of acutely blocking RET were added 3 hours before cell stimulation [70] leaving a room for a multitude of alternative chronic effects. Thus, although ROS signaling via HIF1α stabilization may still be the mechanism, the origin of the ROS remains obscure. The abovementioned increase in MitoTracker Green staining [70] consistent with increased mitochon drial mass, as well as syntheses of additional mitochondr ial DNA in activated BMDMs [89] , implies biogenesis with concomitant increase in mitochondrial redox carri ers, including ROS producing centers. This may be per se sufficient to increase overall ROS production, that can be augmented by slightly reduced milieu (change from ~8 tõ 13% NADH reduction [70] ), possibly due to a partial block in the respiratory chain.

Succinate and ROS generation. In classical experi ments with isolated mitochondria succinate has to be provided to fuel both RET mediated and Complex III dependent ROS production. In intact cells, however, suc cinate is intrinsically present as a metabolite so, unless TCA cycle metabolites are severely depleted, it is not obvious why exogenous succinate would be required. Also, if TCA cycle is indeed depleted, any anaplerotic intermediate would suffice to replenish it, for example, provision of glutamine, precursor of α ketoglutarate, is much easier achieved than delivery of succinate that can not permeate plasma membrane. Succinate delivery from alkylated derivative (e.g., diethyl succinate) depends on its cleavage by intracellular nonspecific esterases of unknown nature and activity. Those activities are suffi cient for a trapping of various probes and/or inhibitors. However, their capacity to cleave enough of a major con sumable molecule as a TCA cycle metabolite to sustain normal flux has not been proven. In particular, the study discussed above [70] shows that diethyl succinate (referred to as simply succinate there) fails to increase intracellular succinate pool. However, it exerts a distinct pattern of biological effects: stimulation of TLR4 dependent pro inflammatory signaling (IL 1β produc tion) and suppression of anti inflammatory signaling (IL 10 and IL 1RA) with no effect on TNFα. Surprisingly, diethyl ester of butyl malonate, a dicarboxylate trans porter inhibitor, has the same biological signature, where as it is known to inhibit of succinate oxidation in isolated mitochondria by preventing its access to SDH; this dis crepancy indicates a potential off target effect for both diethyl esters. Additionally, diethyl succinate per se bare ly caused any ROS production (~40% at 5 mM) com pared to 4 fold increase from the TLR4 ligand LPS; unfortunately, a test of combination of the two effectors that gives the distinct biological signature mentioned above is not reported. Unlike diethyl succinate supple mentation, LPS elevates cellular succinate levels (in the absence of exogenous sources) to the extent 3 times over the effect of SDH inhibitor malonate. This result argues that TLR4 dependent metabolic changes go beyond redistribution within a combined pool of TCA cycle inter mediates but cause up to 10 fold increase of the pool (anaplerosis). Such an increase is entirely consistent with proposed increase in mitochondrial mass (see above).

ρ ρ 0 cells as a model of the glycolytic switch. A model of inducible respiration deficient (ρ 0 ) cells generated via expression of dominant negative DNA polymerase γ, originally described by one of us [90] , is used to mimic "metabolic switch" and probe downstream roles of mito chondria originated signals (e.g., [91, 92] ). These cells lack functional respiratory chain which undermines their ability to oxidize NADH produced in the TCA cycle and keep the cycle going. This may result in diminished biosynthesis of citrate, its product, cytosolic acetyl CoA and, therefore, reduced histone acetylation. Several spe cific sites lose histone acetylation in ρ 0 cells implying potential suppression of gene expression as the conse quence [91] . Histone deacetylation is reversed by expres sion of an alternative respiratory chain consisting of rotenone insensitive NADH ubiquinone oxidoreduc tase, mitochondrial (NDI1) and AOX, a result interpret ed as re activation of the TCA cycle via NADH oxida tion. Unexpectedly, NAD + /NADH ratio is unaffected by the ρ 0 phenotype [91] suggesting some different mecha nisms of histone deacetylation. NDI1 + AOX are not capable of generating ATP and, consistently, proliferation of ρ 0 cells is not rescued by their expression [91] showing the critical importance of bioenergetic rather than biosynthetic function. Minor fraction (10 15%) of prolif erative capacity can be rescued by normalization of Δψ in the ρ 0 cells. Based on this and on concomitant response of CellROX indicator, ROS signaling via HIF1 mediated pathway was suggested [91] . However, regulation of ROS production by Δψ is mediated by hyper reduced state of mitochondria; the latter can be detected as hyper reduced NAD + /NADH, and it is not the case in the ρ 0 BIOCHEMISTRY (Moscow) Vol. 85 Nos. 12 13 2020 cells [91] . As an alternative explanation, Δψ is necessary for protein import to mitochondria including compo nents of biosynthetic machinery critical for proliferation and/or the major ROS producing enzyme Dld [12, 13] . Overall, the ρ 0 model emphasizes importance of bioener getics for cell function while the role of the glycolyic switch remains questionable. The mechanism of ROS production is not clear, and its relevance, given ruined respiratory chain in this model, uncertain.

Growing literature is focused on specific regulatory roles of mitochondria derived ROS as opposed to their well documented pro apoptotic and/or damaging effects promoted by mitochondrial dysfunction under various pathological conditions. MitoROS acting in a signaling mode have been implicated in autophagy, mitophagy, mitochondrial biogenesis, cell cycle progression, and other specific cell functions controlled by redox sensitive signal transduction and transcription factors. Proteins that can be functionally modified by ROS are catalogued in other reviews. The list of ROS targets includes ATF4, NF κB, mitogen activated protein kinases (MAPK): extracel lular signal regulated kinases (ERKs), p38 MAPKs, and the c Jun N terminal kinases (JNKs) [93] ; protein tyro sine phosphatases, and p53 [10, 94 103] . However, the mechanisms of ROS generation and signaling in the con text of immunometabolic regulation awaits further inves tigations and perhaps re evaluation of existing concepts.

T cell highlights. Emerging body of literature suggests that mitoROS are potentially indispensable factors for T cell activation, effector functions and differentiation [42, 49, 96, 102, 104 108 ]. An earlier study of TCR activated cells described acute effects of TCR dependent ROS pro duction on signal transduction pathways regulating T cell proliferation and survival [109] . Surprisingly, ROS signal ing entities produced distinct outcomes: hydrogen perox ide promoted a proliferative signal via extracellular recep tor kinase (ERK) dependent pathway, while the effect of superoxide anion was consistent with activating a pro death pathway (involving FAS ligand expression). Other functional outcomes such NFAT activation and IL 2 expression were ROS independent [109] . Although the site of ROS generation was not identified in this study, the involvement of mitoROS could not be excluded. Based on chemical and genetic inactivation of ETC compo nents, other studies linked mitochondrial effects on T cell responses to ROS signaling originated from ETC [104, 110] . In particular, CD4 + T cells deficient in Rieske iron sulfur protein, participant in the classical ROS pro ducing site at Complex III, exhibited impaired Ca 2+ dependent NFAT IL 2 pathway, but other redox dependent signaling pathways (such as ERK activity) were not affected by the knockout. Rescue experiments with exogenously generated H 2 O 2 supported a ROS dependent factor in IL 2 induction. Interestingly, glycol ysis was dispensable for T cell activation and IL 2 pro duction in this paradigm [104] . In another report, IL 2 and IL 4 production in activated T cells was decreased by the antioxidant N acetyl cysteine (NAC) and reduced in Complex I deficient cells that led the authors to impli cate Complex I as the source of regulatory ROS required for the effector function [110] . TCR induced cytokine expression and H 2 O 2 production were also inhibited by rotenone (at concentrations significantly exceeding those typically needed to achieve Complex I inhibition) that leaves an open question as to whether the ROS signal of interest originated from Complex I. A less invasive approach (with respect to ETC functional intactness) is the use of conventional and mitochondria specific antioxidants. Although this approach alone is not suffi cient to identify molecular mechanisms of ROS signaling (such as the site of ROS generation and their recipient targets), it provides the necessary proof of principle results to substantiate ROS involvement in a particular signaling pathway. In the "tool box" section below we discuss some currently used ROS scavenging systems.

ROS as "SOS". Pathogen associated (PAMPs) and damage asociated (DAMPs) molecular patterns are rec ognized by innate immune receptors as danger signals that trigger an acute immune response culminating in pathogen clearance and tissue repair and regeneration. The immune response to DAMP is often termed sterile inflammation as it is driven by intracellular entities, in the absence of foreign pathogens [111] . Impactful studies of danger signal recognition in macrophages identified path ways to activation of the NLRP3 (nucleotide binding oligomerization domain, leucine rich repeat and pyrin domain containing 3 protein) inflammasome, a molecular platform integrating pro caspase I and pro inflammatory cytokines processing upon stress signals [67, 89, 112 115] . Inflammasome activating DAMPs include mitochon dria derived molecules, such as ROS, cardiolipin and mitochondrial DNA [67, 89, 113, 116] . Released mito chondrial DNA is also engaged in antiviral innate immu nity by triggering the cGAS STING signaling pathways and type I interferon responses [117 119] . This under scores yet another important role of mitochondria in immunity, beyond the ATP producing task and classical apoptotic signaling via cytochrome c release [120, 121] . To release and/or display these "SOS" signals, mitochon dria have to be selectively permeabilized or damaged in some way, but the conduits for DAMP release remain to be defined. Components of mitochondrial apoptotic pores have been discussed in the context of DNA and ROS release and consequent NLRP3 activation. Mitochondrial apoptotic permeabilization involves two well known distinct mechanisms: the mitochondrial per meability transition pore (MPTP) and Bax/Bak depend BIOCHEMISTRY (Moscow) Vol. 85 Nos. 12 13 2020 ent mitochondrial outer membrane permeabilization (MOMP) pore [122] . The latter mechanism does not compromise the inner mitochondrial membrane integrity and is considered immunologically inert, although it has been shown that effector caspase activation downstream of Bax/Bak induced MOMP promotes NLRP3 depend ent IL 1β production [123] . The non selective MPTP may be permissive to DNA transport [124, 125] . Translocation of mitochondrial DNA via MPTP was recently suggested in experiments with TDP 43 mediated inflammation; however, this suggestion was based largely on an effect of MPTP inhibitor cyclosporine A that was applied at a dose ~10 times higher than a specific dose [126] . An effect on an alternative target, calcineurin was also not ruled out. In addition, MPTP is generally associ ated with excessive mitochondrial dysfunction and would require control mechanisms. A recent study described a regulatory mechanism that involves protein tyrosine phosphatase 2 (SHP2) interaction with the adenine nucleotide translocase 1 (ANT1), a component of perme ability transition pore [PTP] (based on the classical PTP model). It is suggested that SHP2 mediated inhibition of ANT1 keeps DAMP release in check and prevents exces sive inflammasome activation [127] . Since the predomi nant species of reactive oxygen is freely permeating H 2 O 2 , it is reasonable to predict that mitoROS signal to inflam masome activation involves relatively subtle mitochondri al dysregulation, as opposed to the major "damage" such as the classical mitochondrial permeability transition. In this regard, previously described reversible and/or low conductance states of MPTP [10, 128, 129] deserve fur ther attention as they may be linked to ROS signal trans mission from functionally intact mitochondria.

NLRP3 inflammasome activation involves mitoROS generated by Complex I and Complex III (but not Complex II, as expected) [113] . Interestingly, both ROS generation and the downstream inflammatory immune response were inhibited by down regulation of the volt age dependent anion channel (VDAC) potentially link ing the ROS signal to metabolite transport [113] . MitoROS also convey damage signal through oxidation of mitochondrial DNA, as only oxidized DNA binds and activates NLRP3 [89, 114] . Another notable recent study identified a key role of parkin dependent mitophagy in NF κB dependent control of excessive of NLRP3 inflammasome activation [67] . This and other studies [130] highlight the importance of mitochondrial quality control system that integrates mitophagy, mitochondrial dynamics, and biogenesis in the control of innate immune response and inflammation.

Although transcriptional factor HIF1 is currently broadly implicated in a variety of cellular adaptive processes including immune response, the idea of ROS regulating the stability of its critical subunit HIF1α origi nates from the adaptation to physiological hypoxia (0.3 3% oxygen). It has been proposed that, under these con ditions, HIF1α stabilization is critically dependent on mitoROS signal, therefore positioning mitochondria as the sensor of oxygen levels [131] . Under normoxic condi tions, any expressed HIF1α is unstable as it is quickly hydroxylated at a critical proline residue by one of the family of α ketoglutarate dependent oxygenases called prolyl hydroxylase domain (PDH) proteins. The hydrox ylated form has a three orders of magnitude heightened affinity to ubiquitin ligase complex in which von Hippel Lindau tumor suppressor (pVHL) plays a role of recogni tion component. Thus, HIF1α is quickly ubiquitinilated and targeted for proteosomal degradation (see [132] for review). PDH proteins are perfect sensors for oxygen, as their K M is just above the solubility of atmospheric oxygen in aqueous phase (230 250 μM) [133] so their activity should respond nearly linearly to oxygen tensions in hypoxic range. Any alternative oxygen sensing mecha nism should therefore imply that this fundamental, mass action control is somehow lost and the activity of the enzyme remains high despite virtual lack of one of the substrates, oxygen. Devising and proving such an intricate scenario may become a challenging enzymological task for future researchers.

An argument in favor of the mitochondrial ROS mediated mechanism is increased responses of ROS sensi tive dyes under hypoxic conditions [92, 131, 134] . These data should be interpreted with some caution, especially when kinetic data are lacking [92, 134] . For example, an observed reversible change in fluorescence of dichlorofluo rescin [131] may be difficult to explain. Responses of mul tiple mitochondrial ROS producing sites to various oxygen tensions have been studied in the less complex system, iso lated mitochondria, with somewhat varying results but none of the studies detected elevated ROS production in the hypoxic range [135 137] . It was concluded that the ROS responses observed in cells are not intrinsic to mito chondria [136] and how they occur is unclear [95] .

Exogenous hydrogen peroxide appears to stabilize HIF1α in a catalase sensitive manner [92] lending some support to the idea that non mitochondrial ROS may play a role in the process. However, the underlying question as to how do ROS inhibit HIF1α hydroxylation remains to be explored.

The primary focus of this review is mitoROS, howev er, the topic of ROS signaling would be incomplete with out touching upon other sources of ROS that may be even more potent. In response to pathogen invasion, phago cytic cells release enormous amounts of ROS in the process called respiratory burst. This process is executed BIOCHEMISTRY (Moscow) Vol. 85 Nos. 12 13 2020 by enzymes of the NADPH oxidase (NOX) family (the seven known members of the family are reviewed in [138] ). The main player involved in immune response, NOX2 is a multimeric complex that assembles on the plasma membrane of the cell in a tightly regulated process in response to extracellular signals. It utilizes intracellular reducing equivalents in the form of NADPH to reduce oxygen with release of superoxide (and, eventually, its product hydrogen peroxide) into extracellular milieu [138, 139] . In the case of phagocytic cells, macrophages and neutrophils, these ROS play a major role in killing invading pathogens but may also signal back to the cell or to the other cells. For example, they suppress T cells responses to TCR activation in co cultures of neutrophils and T cells [140] . In the absence of phagocytes, endoge nous NOX derived ROS may also play a role in activa tion induced cell death in previously activated T cells (reviewed in [106] ). In addition to these immunosuppres sive mechanisms, there are reports of ROS involvement in activation of T cells. TCR activation causes extracellular superoxide generation, apparently via NOX2, but surpris ingly it is not required for T cell activation, proliferation and cytokine production [141] . Based on these and other data, it was concluded that NOX2 is required for proper differentiation to Th2, and away from Th17, phenotype but not the activation of T cells. Analysis of effects of antioxidants led to a hypothesis that lipid soluble peroxi dation products, or their enzymatically derived counter parts, leukotrienes, rather than ROS per se are the acting entities in T cell activation [106] . NOX generated ROS have been implicated in NLRP3 inflammasome activa tion in macrophages (reviewed in [142] ). There are sever al lines of evidence supporting this notion. Whereas specificity of NOX inhibitors can be questioned [143] , complexity of NOX responses, involving translocation and assembly of multiple subunits, paradoxically lend support to the model through genetic manipulations and localization studies (reviewed in [142] ). Activation of inflammasome in response to particulate matter also fits in the model of phagocytosis driven NOX activation and ROS production, analogous to the respiratory burst [144] . Thus, the NOX enzymes appear to be a viable alternative to mitochondria as the source of ROS signaling. There is no apparent reason why a ROS signal coming from NOX would be any different from a one originated in mito chondria, as in both cases it would most likely be the same relatively stable and freely permeating H 2 O 2 . The relative contributions of the two sources were recently determined in cells not specializing in phagocytosis, myoblasts, for which there is no apparent reason to expect extra high activity of NOXs. However, their contribution was sub stantial, around 40% [145] . A similar analysis in different types of immune cells would significantly enhance our understanding of the immune ROS signaling.

Reciprocally, as NOXs contribute to signaling, mito chondria may contribute to oxidative burst and bacterici dal activity. This has been reported for both macrophages [146] and neutrophils [147] . Importantly, in the process mitochondria are actively recruited to the sites of bacteri cidal activity in NOX2 containing phagosomes [146] . A positive feedback mechanism between mitochondrial and NOX dependent ROS production may occur leading to amplification of bactericidal activity [147 149 ].

As the results of any study are only as good as the methods employed, we will briefly touch upon some key tools used in the studies of ROS and metabolic signaling.

ROS probes. Some of the methods to assess produc tion rates and resulting levels of ROS along with potential missteps and pitfalls in their application were discussed in our previous review on the subject of ROS with emphasis on dichlorofluorescin and its esterified analog (H2 DCF DA or DCF DA) [11] . In recent years, however, a posi tively charged tri phenyl phosphonium derivative of dihydroethydium (DHE), MitoSOX, gained popularity in the ROS research as a supposedly specific sensor of intramitochondrial superoxide. It should be noted how ever, that it is a subject to all limitations of Δψ sensitive fluorescent indicators [150, 151] . MitoSOX fluorescence depends not only on ROS reactivity but also on reversible compartmentalization between mitochondrial matrix, cytosol and external medium that is determined by Δψ on both mitochondrial and plasma membranes; any changes in Δψ will change probe levels and fluorescence in matrix and cytosol. Another potential variable could be differen tial binding to mitochondrial versus nuclear DNA [151] . Besides, the probe uncouples mitochondria and inhibits cytochrome c oxidase (Complex IV) at very low concen trations [152] making control for those parameters oblig atory for meaningful interpretation of the data. Existence of more than one fluorescent oxidation product makes this probe purely qualitative unless measured with HPLC [153] . Although there are no very reliable probes for ROS, MitoSOX appears to be particularly difficult for use.

Antioxidants. NAC. Although broadly employed as a generalized antioxidant it should be rather considered as a reducing agent and moderate sulfhydryl protector; it can also play a role in glutathione replenishment but only under conditions of severe depletion [154, 155] . The free radical scavenging mechanism was strongly questioned based on the low rate constants of reaction with superox ide [156 158] . NAC is weakly reactive with H 2 O 2 and superoxide [157] . Reaction of NAC with superoxide is a chain reaction in which superoxide is regenerated; the chain reaction terminates with conversion of superoxide to another ROS, H 2 O 2 [157] . Metabolism to produce more reductive low molecular weight sulfur compounds has been described [158] but its relevance to ROS detox ification requires further validation. BIOCHEMISTRY (Moscow) Vol. 85 Nos. 12 13 2020 SkQ and MitoQ. Mitochondria targeted quinones MitoQ and, to a lesser degree, its close structural analog SkQ [159, 160] also became widely used to probe ROS sig naling with implied specificity for mitochondrially gener ated ROS [159, 161] . It should be noted, however, that the spatial selectivity of these compounds was inferred based of proximity and compartmentalization considerations [161] . On the other hand, SkQ and MitoQ are effective scavengers of free radicals in membranes, including the ones that are generated from the exogenous peroxide [162] . Given that the bulk of cellular ROS is the freely dif fusible hydrogen peroxide, protection afforded by mito chondrially targeted quinones may be a questionable pre dictor of the ROS origin. Both compounds were developed with an idea of catalytic antioxidant in mind [159, 160] as both are structural analogs of a natural redox carrier coen zyme Q which is undergoing redox cycling in the respira tory chain. The MitoQ is a close coenzyme Q mimic, whereas structural features of SkQ were optimized for best reactivity and membrane permeation [160, 161] . The abil ity of SkQ to be reduced by complexes I and II, and there fore regenerate, was directly demonstrated [160] . It should be noted that both compounds are actually strong prooxi dants at micromolar concentrations [160] , so special care should be taken to not overdose. The submicromolar con centrations do show antioxidant properties with SkQ demonstrating broader therapeutic window than MitoQ [160] . Overall, the SkQ appears to have advantages over MitoQ and, in our opinion, should be a compound of choice in studies of oxidative stress and ROS signaling. On the other hand, any conclusions about the source of ROS based on its effects would be speculative at best.

Ester forms of metabolites. Since many of immuno metabolites are plasma membrane impermeable, use of alkylated derivatives became a matter of such a routine in the studies of immunometabolism that even the name of original metabolite is used in place of its derivative [70, 163] . It should not be overlooked, however, that those derivatives are distinct compounds with their unique chemistries rather than mere precursors of desired metabolites. For example, di methyl itaconate is much more reactive than itaconate in formation of Michael adducts with proteins [58] as is another esterified dicar boxylic acid derivative dimethyl fumarate in comparison to fumarate [164] . On the other hand, di methyl itaconate is not metabolized to itaconate inside the cells, so its use would completely defeat the purpose [165] . To circumvent this, 4 octyl itaconate was proposed as an alternative [58] . However, it gives completely discordant effects versus endogenous itaconate on particulate matter induced inflammatory response in BMDM. The authors noted important differences between endogenous itaconate and the exogenously applied octly ester [163] . There is no detectable increase in the desired metabolite succinate in the BMDM supplemented with dimethyl succinate [70] . Remarkably, the use of the esters may be even unnecessary in the specific case of macrophages. It is possible to increase intracellular itaconate to millimolar range with just prolonged incubation with unmodified compound [51] . It is also seems possible to effectively deliver canoni cal membrane impermeant molecules, such as coenzyme A and carboxyatractlyoside [84] , apparently owing to the phagocytic nature of macrophages. Obviously, other strategies may be required for T cells and other immune cells. Thus, it is advisable to monitor changes in the desired metabolite levels upon application of a derivative and control for the effects of the cleaved groups (convert ed to methanol or ethanol for methyl and ethyl derivatives, respectively). A useful strategy would be to use control non hydrolysable analogs (as is done to distinguish between enzymatic and non enzymatic effects of ATP).

The conceptual distinction between ROS signaling and oxidative stress is primarily quantitative: "moderate" ROS produce regulatory signals, while excessive ROS overwhelm ROS defense systems and damage cell con stituents. Somewhere in between, ROS may not be the direct damaging agents, but initiate a regulated pathway for cell self destruction in the form of apoptosis.

The steep dependence of ROS production on redox status of ETC carriers provides a direct link to the meta bolic state of the cells. This underlying notion prompted us to extend this review of ROS signaling related data to include bioenergetic aspects of immunometabolism. It appears that the harmless mode of ROS signaling that safeguards against oxidative stress is the basal ROS pro duction. It is augmented, upon stimulation, by mito chondrial biogenesis rather than activation through a sharp metabolic switch. Consistently, bioenergetic func tion remains sustained or upregulated for energy demanding functions, like proliferation. However, for macrophages, the sentinel cells, upon discovery of an invader, production of cytotoxic molecules (NO and H 2 O 2 ) apparently takes precedence over energy efficiency resulting in self injury and suppressed bioenergetics.

In our analysis, we highlighted some controversial mechanisms (such as RET) not to downplay the data but rather to evoke further interest in the matter. The area of research, at crossroads of immunometabolism and ROS signals, is full of unresolved mysteries and stunning para doxes. It presents an unplowed field for researchers will ing to devote themselves to resolving finest details of immensely complex and extremely important mecha nisms of immunity.

Funding. This work was supported in part by a grant from the National Institute of Health (P01AG014930/ AG/NIA NIH HSS). BIOCHEMISTRY (Moscow) Vol. 85 Nos. 12 13 2020 Ethics declarations. The authors declare that they have no conflict of interest. This article does not contain description of studies with the involvement of humans or animal subjects.

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2020) DAMP sensing receptors in sterile inflammation and inflammato ry diseases

Intracellular pattern recognition receptors in the host response

A role for mitochondria in NLRP3 inflammasome activa tion

Oxidized mitochondrial DNA activates the NLRP3 inflammasome during apoptosis

Autophagy proteins regulate innate immune responses by inhibiting the release of mito chondrial DNA mediated by the NALP3 inflammasome

Mitochondria in innate immune signaling

Mitochondrial DNA stress primes the antiviral innate immune response

Apoptotic caspases suppress mtDNA induced STING mediated type I IFN produc tion

Apoptotic caspases prevent the induc tion of type I interferons by mitochondrial DNA

Induction of apoptotic program in cell free extracts: requirement for dATP and cytochrome c

The release of cytochrome c from mitochondria: a primary site for Bcl 2 regulation of apop tosis

Bioenergetics and cell death

The mitochondrial apoptotic effec tors BAX/BAK activate caspase 3 and 7 to trigger NLRP3 inflammasome and caspase 8 driven IL 1beta activation

Mitochondrial damage as a source of diseases and aging: a strategy of how to fight these

Mitochondrial per meability transition triggers the release of mtDNA frag ments

TDP 43 triggers mito chondrial DNA release via mPTP to activate cGAS/STING in ALS

Tyrosine phosphatase SHP2 negatively regulates NLRP3 inflammasome activation via ANT1 dependent mitochon drial homeostasis

Prooxidants open both the mitochondrial permeability transition pore and a low conductance channel in the inner mitochondrial membrane

Mitochondria are excitable organelles capable of generat ing and conveying electrical and calcium signals

Emerging views of mitophagy in immunity and autoimmune diseases

Mitochondrial reactive oxygen species trigger hypoxia induced transcription

Oxygen sensing by HIF hydroxylases

Characterization of the human prolyl 4 hydroxylases that modify the hypoxia inducible factor

Mitochondrial reactive oxygen species trigger hypoxia inducible factor dependent extension of the replicative life span during hypoxia

The properties of hydrogen peroxide production under hyper oxic and hypoxic conditions of perfused rat liver

Response of mitochondrial reactive oxygen species gener ation to steady state oxygen tension: implications for hypoxic cell signaling

The dependence of brain mitochondria reactive oxygen species production on oxy gen level is linear, except when inhibited by antimycin A

NADPH oxidases: an overview from structure to innate immunity associated pathologies

Nox proteins in signal transduction, Free Radic

Reactive oxygen species differential ly affect T cell receptor signaling pathways

TCR triggered extracellular superoxide production is not required for T cell activation

Signaling by ROS drives inflamma some activation

12 13 2020 inhibitors of NOX NAD(P)H oxidases are not specific

Innate immune activation through Nalp3 inflammasome sensing of asbestos and silica

Mitochondrial and cytosolic sources of hydrogen peroxide in resting C2C12 myoblasts, Free Radic

TLR signalling aug ments macrophage bactericidal activity through mito chondrial ROS

Mitochondrial reactive oxygen species are involved in chemoattractant induced oxidative burst and degranu lation of human neutrophils in vitro

Cross talk between mitochondria and NADPH oxidases, Free Radic

The role of mitochondrial ROS in antibacterial immu nity

Fluorescence measurement of mitochondrial membrane potential changes in cultured cells

Use of potentiometric fluorophores in the measurement of mitochondrial reactive oxygen species

Low micromolar concentrations of the superoxide probe MitoSOX uncouple neural mitochondria and inhibit complex IV, Free Radic

Hydroethidine and MitoSOX derived red fluorescence is not a reliable indicator of intracellular superoxide forma tion: another inconvenient truth, Free Radic

Thiol redox transitions in cell signaling: a lesson from N acetylcysteine

The chemistry and biological activities of N acetyl cysteine

The reaction of superoxide radical with N acetyl cysteine, Free Radic

Reactivity of biologically important thiol compounds with superoxide and hydrogen peroxide, Free Radic

Acetyl cysteine functions as a fast acting antioxidant by triggering intracellular H 2 S and sul fane sulfur production

Selective targeting of a redox active ubiquinone to mitochondria within cells: antioxidant and antiapoptotic properties

A biochemical approach to the prob lem of aging: "megaproject" on membrane penetrating ions. The first results and prospects

Cationic antioxidants as a powerful tool against mitochondrial oxidative stress

Protective effects of mitochondria targeted antioxidant SkQ in aqueous and lipid membrane environments

Endogenous itaconate is not required for particulate matter induced NRF2 expression or inflam matory response

Dimethyl fumarate and monoethyl fumarate exhibit differential effects on KEAP1, NRF2 activation, and glutathione depletion in vitro

Dimethyl itaconate is not metabolized into itaconate intracellularly