key: cord-0818020-v12nf95c authors: Chirumbolo, Salvatore; Valdenassi, Luigi; Simonetti, Vincenzo; Bertossi, Dario; Ricevuti, Giovanni; Franzini, Marianno; Pandolfi, Sergio title: Insights on the mechanisms of action of ozone in the medical therapy against COVID-19 date: 2021-05-11 journal: Int Immunopharmacol DOI: 10.1016/j.intimp.2021.107777 sha: 63cb71adc0adeb7af482faa5b8102776972e3a15 doc_id: 818020 cord_uid: v12nf95c An increasing amount of reports in the literature is showing that medical ozone (O3) is used, with encouraging results, in treating COVID-19 patients, optimizing pain and symptoms relief, respiratory parameters, inflammatory and coagulation markers and the overall health status, so reducing significantly how much time patients underwent hospitalization and intensive care. To date, aside from mechanisms taking into account the ability of O3 to activate a rapid oxidative stress response, by up-regulating antioxidant and scavenging enzymes, no sound hypothesis was addressed to attempt a synopsis of how O3 should act on COVID-19. The knowledge on how O3 works on inflammation and thrombosis mechanisms is of the utmost importance to make physicians endowed with new guns against SARS-CoV2 pandemic. This review tries to address this issue, so to expand the debate in the scientific community. Ozone (O 3 ) is an unstable molecule, a chemical allotrope of O 2 , which was recently used in a standardized mixture with oxygen to successfully treat COVID-19 alongside with usual antiinflammation pharmacology [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] , so representing a possible encouraging approach to address COVID-19 [12] . To date, the molecular mechanisms with which O 3 is able to act against COVID- 19 are yet far to be fully elucidated, though several attempts on how O 3 might work in biological systems were recently reported [13] [14] [15] [16] [17] [18] [19] . Although O 3 can easily remove SARS-CoV2 from inert surfaces, an evidence assessing its well known virucidal potential [20] [21] [22] [23] , the activity of O 3 in the human organism is radically different respect to the gaseous O 3 used for environmental disinfection. As a matter of fact, despite some reported evidence showing the direct pro-oxidant use of O 3 against microbial infections [24] , medical O 3 usually hampers virus spreading via O 3 -generated mediators, such as lipid-derivatives (aldehydes and oxysterols), which are potent SARS-CoV2 inhibitors [25] [26] [27] . More generally, when talking about O 3 in medicine, it should be mandatory to distinguish a "pollutant airborne O 3 ", usually toxic and which may directly interacts with airway epithelia, from a "medical gaseous O 3 ", which is usually administered in a balanced O 2 /O 3 mixture via autohemotherapy, or rectal insufflation or, in laboratory animals, also as peritoneal injection [28] . The long history of O 3 in medicine, used to treat a wide plethora of illnesses, dates back to Dr Walls' in 1915 and has gradually faded off its pioneering empirical hallmark to come to an excellent, straightforward expertise in employing O 3 successfully, even in chronic and degenerative disorders [29] [30] [31] [32] [33] [34] . Very recently, Franzini patients (mean age 75 yrs ± 11.4 SD), from a median of 22.13 ± 3.44 days to 13.45 ± 2.33 days (41% reduction), an effect probably due to the concurrent effect of O 3 on the usually recommended therapy protocol for COVID-19 [3] . Many further research groups are confirming these results, though with different protocols. Table 1 summarizes the more recent evidence in using O 3 to treat COVID-19. Despite the several efforts to assess the role of O 3 in reducing inflammation and prothrombotic mechanisms, the paucity of clinical papers exerts a relevant impact on the effect size when the major parameters reported in those papers are gathered for meta-analytic investigation. Table 1 reports the statistics of the effect power for IL-6 and CRP (as main markers of inflammation) and D-dimer (as a major marker of thrombotic events), resulting that inflammation is reduced by 80% (p<0.05), whereas thrombosis is reduced by 50% (p >0.05), yet these data need to be further assessed. This review has the objective to gather the bulk of evidence regarding the ability of O 3 to reduce inflammation and pro-thrombotic events, throughout the literature on the latest 30 years, in order to give insights about how ozone can exert its positive action on COVID-19 patients, currently emerging in the clinical reports. Clinical success suggests that the role of O 3 in human physiology may be much more important than expected, as O 3 , or at least its major oxidative byproducts, are physiologically present in the organism. As a matter of fact, past reports addressed the intriguing hypothesis that natural immune cells, such as neutrophils, may produce biological ozone [35] [36] [37] , though controversial opinions were also raised about [38, 39] . Yet, recent studies have reported that the exposition of amino acids or antibodies to singlet oxygen may form biological ozone [40] . Oxygen radicals and O 3 are therefore frequent byproducts of the many oxidative processes involving bio-molecules. This evidence should suggest that O 3 may work more frequently as an inside biological molecule, rather than a chemical xenobiotic, probably acting as a chemical switcher of the complex interplay made by oxidative stress response, immunity and even vascular physiology. As we are going to address further on, O 3 is not only able to modulate oxidative stress, which is considered a leading causative factor in COVID-19 [41] , but also to work as a master regulator of the complex cross talk between oxidative stress and inflammation, including blood coagulation and endothelial physiology. Recent evidence reported that during COVID-19, genes involved in the stress response, such as TRAP-1 (expressing heat shock protein 75, hsp75) and NOX (expressing NADPH oxidases) are deregulated, alongside with SAE (encoding for protein SUMOylation), VAV1 (implicated in platelet functions and blood coagulation) [42] and the expression of several cathepsin proteases [43] . The pathogenetic scenery where O 3 should work may be summarized as follows. SARS-CoV2 induces a robust type I/III interferon response via the activation of the innate immunity, inflammation and subsequently adaptive immunity, but the dysregulation of the rennin/angiotensin system caused by disturbing the angiotensin-converting enzyme 2 (ACE2) signaling, finally leads to oxidative stress, then tissue damage and a widespread triggering of the coagulation cascade causing disseminated intravascular coagulation (DIC) and finally thrombosis [44] . Oxidative stress, inflammation and coagulation disorders are closely intertwined in COVID-19 pathogenesis and these may represent fundamental targets for O 3 -mediated therapy. However, from a pharmacological point of view, O 3 is widely considered a simple pleiotropic molecule. It should be able, therefore, to fundamentally target the complex cell machinery involved in responding to the oxidative stress and then to act in a rather aspecific way on immune modulation, yet depending on the different O 3 exposure, dosages and experimental conditions [45] [46] [47] [48] . As previously introduced, medical O 3 usually uses this gas in an oxygen-ozone mixture injected via autologous hemotransfusion and should not be mismatched with airborne O 3 coming from environmental pollution, which may be toxic for organisms when associated with long term exposure and presence of further pollutants such as NO 2 and particulate matter [49, 50] . Ozone is toxic if directly inhaled, even in relatively moderate concentration [51] , so this way of assumption is never considered in a therapy approach. Despite it is a chemical toxicant, O 3 exerts its beneficial action depending on its dosage, therapy protocol, biological microenvironment and genetic or epigenetic factors, which appear to be fundamental in the development and pathogenesis of COVID-19 [52] . This beneficial effect is mainly exerted by blood-derived byproducts. On airway and lung epithelia, gaseous O 3 is often noxious because it damages lung surfactant protein B (SPB), leading to respiratory distress [53] . SPB is a member of a group of proteins, present exclusively in the lung epithelia, with anti-microbial activity (SPA and SPD) or which interacts with phospholipids, such as di-palmitoyl phosphatidylcholine (DPPC) to ensure a surface-active airwater film and prevent lung collapse [54] . As an allotrope of oxygen, O 3 is particularly instable in aqueous solutions, such as plasma of circulating blood [55, 56] , probably because O 3 , or its major oxidative byproduct -OH* radical, are rapidly quenched by anti-oxidants such as cysteinyl-groups in proteins, uric acid, ascorbic acid, reduced glutathione (GSH) or even albumin [57] . When injected into the blood, O 3 may react with poly-unsatured fatty acids (PUFA), generating hydrogen peroxide [58] , which is produced also by the O 3 interaction with molecules containing aldehyde groups, therefore forming lipid oxidation products (LOPs). One of these products is 4-hydroxynonenal (4-HNE) [ nitrogen (RNS) species [63] . The activity of Nrf2 is switched on to reduce also an excess of oxidative stressors, whereas activators of Nrf2 can be used to pharmacologically respond to the oxidative stress. For example, the synthetic Nrf2 activator, RTA-408, suppresses, by activating Nrf2, the excess ROS production following an injury, by inhibiting also γδTh17 cells [64] . Nrf2 works therefore as a major switcher in the anti-oxidant response following an oxidative injury. Oxidative stress provides a fundamental contribution in the development and exacerbation of COVID-19 [41] . This encourages researchers to seek for novel therapeutic suggestions, targeting Nrf2 to treat COVID-19 [65] Nrf2 is linked in the cytoplasm with Kelch like-ECH-associated protein 1 (Keap1) and with Cullin-3, which can degrade Nrf2 via ubiquitination, as Cullin-3 ubiquinates Nrf2 and Keap-1 promotes this reaction by binding to the Nrf2 conserved aminoterminal Neh2 domain [66] . Mammals are endowed with several hundreds of ARE-driven genes. The genetic region containing the sequence 5'-A /G TGAC /T nnnGCA /G -3' is the core box of an AREregulating cis-acting element [67] . Many oxidative stress-derived molecules, including O 3 and its oxidized mediators, such as hydroxyl radical (OH -), carbon mono-oxyde (CO), nitric oxide (NO), peroxynitrite (ONOO-), peroxyntrous acid (ONOOH) and hypochlorite (HOCl), can directly activate ARE-dependent gene expression [68] . Moreover, Nrf2 is a component of the "cap 'n collar" (CNC) family, collecting at least six factors in mammals, i.e. p45, Bach1, Bach2, Nrf1, Nrf2 and Nrf3, representing the NF-E2 subfamily, which forms active dimers able to enhance or inhibit ARE-dependent gene expression [68] . Recent studies have demonstrated that O 3 activates Nrf2 in a dose-dependent manner [69, 70] . Actually, several reports have shown the ability of medical O 3 to reduce oxidative stress [71] [72] [73] but even to modulate the Nrf2/NF-κB interplay, probably affecting the IL-6/IL-1β rate of expression in COVID-19 [72, 73] . Furthermore, NF-κB interacts, via p65, with Keap1, so repressing the Nrf2-ARE pathway [74] . O 3 activates Nrf2 and inhibits the NF-κB pathway [69, 75] , therefore showing anti-oxidant and anti-inflammatory properties [76] . This ability is possessed also by ozonized low density lipoproteins (ozLDLs), which can inhibit NF-κB via the down-regulation of the IRAK-1 associated signaling [77] . Therefore, medical O 3 in the plasma can generate ozLDLs, which induce decrease in IκBα proteolysis, reduction in κB-dependent gene transcription and the phosphorylation and proteolysis of the IL-1 receptor associated kinase 1 (IRAK-1), so triggering an antiinflammatory pathway [77] . Chemical interaction of O 3 with peripheral blood, which should occur during its medical use via the O 2 -O 3 -AHT [1-3], generates a huge deal of biochemical mediators, which probably work on the Nrf2/NF-κB interplay via a hormetic dose-response mechanism [78] . The concept of "hormesis", firstly reported by Calabrese and Baldwin in 1998 [79] , which has been recently associated with the concept of "mild stress" or "eustress" [69] , was introduced for O 3 by Bocci and colleagues, to highlight the beneficial effect of relatively low doses (or low exposure) of O 3 , which usually, at high doses, is a pro-oxidant and potentially toxic molecule [78] . Interestingly, likewise many xenobiotics inducing benefits by a hormetic mechanism [78] [79] [80] [81] , O 3 interacts with aryl-hydrocarbon receptors (Ahr), controlling lung inflammation by modulating the IL-22-mediated signaling [82] , a way used also by plant derived phyto-chemicals, to induce an anti-inflammatory response [83] . According to some authors, the hypothesis by which O 3 should induce the Nrf2pathway activation, may involve the onset of a mild oxidative stress, able to elicit the expression of the antioxidant endowment of the cell, without causing stress-related injury [84, 85] . Oxidative stress response is an early mechanism modulating immunity and actually the Nrf2/Keap1/ARE pathway is of major importance in inflammation [86, 87] , particularly in COVID-19 [65] . As a matter of fact, recent evidence reported that SARS-CoV2 dampens the activity of Nrf2 signaling, as the Nrf2 pathway-mediated expression of the antioxidant genes is suppressed in biopsies from patients with COVID-19 [88] . Recent reports have shown that the transcriptome analysis of lung biopsies from patients with COVID-19 showed an enrichment in the expression of genes associated with inflammation, such as Toll-like receptors (TLRs) and the RIG-I like receptors RIG-I, MDA-5 and LGP2, whereas a strong reduction in the genes associated with Nrf2 was observed [88] . The activation of the Nrf2-mediated signaling appears therefore pharmacologically strategic in the COVID-19 treatment. Furthermore, cells produce molecules able to trigger the Nrf2mediated pathway, such as fumarate and itaconate [88] . While fumarate is a common citrate and urea cycle intermediate, itaconate is produced by the aconitate decarboxylase I in macrophage mitochondria, usually upon inflammatory or xenobiotic stimuli. Following Keap 1 alkylation, itaconate induces an Nrf2-mediated response [89] . Furthermore, during a chronic lung disease a metabolic reprogramming of airway macrophages does occur, as these innate immune cells use the ROS signaling to produce itaconate, which fundamentally is able to dampen bacteria infection, such as P aeruginosa, by inhibiting the microbial isocitrate lyase in the shunt of glycoxylate [90, 91] . Itaconate in airway macrophages is a leading anti-microbial molecule and its production is activated by ROS signaling, probably by molecules able to trigger an oxidative stress response such as O 3 and its mediators [92] . Many of these mediators are produced by O 3 in the blood. Macrophages highly express two fundamental receptors, i.e. the sterol receptor element binding protein (SREBP) and the liver X receptor alpha (LXRα), which regulate cell response and cytokine release [93, 94] . The role of SREBP is fundamental because is increased, via the NF-κB signaling, by an inflammasome-mediated pathway in M1-pro inflammatory macrophages, whereas the antiinflammatory M2 phenotype is activated by a LXRα-mediated pathway [92] . O 3 in the blood is able to produce a great deal of lipid oxidized products (LOPs), and O 3 itself may have a major role in modulating the response of innate immune cells and the macrophage M1/M2 phenotype switching [96] . Cholesterol may be oxidized by O 3 and its oxidant radicals in the blood, forming products generally known as oxysterols, which can interact with LXRα [97] . Oxysterols include a wide family of oxidized cholesterol byproducts, which exert an immuno-modulatory role [98] . In the lung, O 3 derived oxysterols exert primarily a pro-inflammatory activity as quite exclusively interacting with the SREBP-mediated pro-inflammatory signaling in airway type II cells, due to the presence of surfactant proteins [99, 100] . In the blood, O 3 can generate several lipid-derived mediators, besides to oxygen and nitrogen-derived radical species, even from polyunsaturated fatty acids (PUFAs), including oxysterols interacting with LXRα [101] [102] [103] . According to Bocci, the "therapeutic window" for O 3 might range from 0.21 μmol/ml (10 μg/ml O 3 for each ml of blood) to 1.68 μmol/ml (80 μg/ml O 3 for each ml of blood), as in this dosage range the anti-oxidant system is able to neutralize O 3 and to maintain its biological benefit, whereas higher doses are undoubtedly toxic, following the U-shaped paradoxical pharmacology of hormesis [104] . Besides to ROS, O 3 may produce reactive electrophilic species (RES), such as α,β-unsaturated aldheydes from PUFA and interestingly, at least from a functional point of view, itaconate too is a RES, being able to activate macrophages in responding to stress via mito-hormesis, a mechanism suggested also for O 3 [78,105.106] . The oxidation of lipids such as arachidonic and linoleic acids by O 3 may form α,βunsaturated hydroxyalkenals [78, 107] , such as the same 4-hydroxy-2-nonenal (4-HNE), which has a leading role in the anti-oxidative response via Nrf2 even in human lung cells [108, 109] . Actually, the relative short-time contact of O 3 with blood, during an O 2 -O 3 -AHT, allows O 3 to react with ω-3 PUFA, forming hydroxyl-hexaenal (HHE) or with ω-6 PUFAs forming 4-HNE [110] . This latter, enabling chemical adducts with Cys-34 residue in the albumin, may trigger, in picomolar concentrations, an oxidative Nrf2-mediated stress response [110] . Literature about 4-HNE describes this byproduct of lipid peroxidation as an inducer of oxidative stress, then involved in several oxidant-induced disorders, despite the evidence that, at low doses, 4-HNE exerts a beneficial, antioxidant and cytoprotective action via the induction of the thioredoxin reductase 1 from Nrf2 activation [111] . The anti-oxidant and anti-inflammatory role of 4-HNE has been recently reviewed [112] . Low doses of 4-HNE (5-10 μml/L) enhances the expression of heme oxygenase-1 (HO-1), contributing in protective endothelia and vascular physiology [113] . The biological activity of O 3 is mainly mediated by cholesterol-derived oxysterols, electrophiles such as α,β-unsaturated aldheydes from PUFA and modified cysteinyl (Cys) residues in proteins. None of these byproducts are beneficial per se, being, as other oxidized end products, toxic at high concentrations. Yet, they may trigger, as signaling molecules, the expression of cytoprotective and survival genes [114] . RES such as 4-hydroxy-2-hexenal, 4-HNE, 15d-Δ 12,14 -PGJ2, induce a cell adaptive response as they can disrupt the Nrf2-Keap1 complex via the modification of Cys273 and Cys288 of the at least 25 Cys residues in Keap-1, then activating Nrf2 [115] . Actually, the ability of O 3 to interact with cysteinyl residues, may tune the activity of strategic Cys-residues in the Keap1 function. Three major cysteine sensors were reported in the Keap1 involvement in the stress response, namely Cys151, Cys273, and Cys288, which work as sensing stressors to activate the anti-oxidant Nrf2-mediated machinery [116, 117] . The chemical modification exerted by O 3 on Keap-1 crucial Cys residues in their thiol groups, should inhibit the Nrf2 shut-down by Keap1, so prolonging the oxidative stress response by O 3 [110] . The interaction of O 3 with blood, forming important metabolites able to trigger an anti-oxidant response at low doses, should be a crucial issue to be further expanded and investigated in pharmacology [118] . In rheumatoid arthritis purified synovial fibroblasts, 3-5% v/v of gaseous O 3 reduced cell expression of TNF-α, IL-1β and IL-6 [119] , pro-inflammatory cytokines actively participating in COVID-19 pathogenesis [120] . O 3 -derived metabolites such as 4-HNE, are able to inhibit IL-6 production in liver macrophages by acting on NF-κB, i.e. preventing its activation and suppressing the phosphorylation of IκBα [121] , and noteworthy 4-HNE inhibits both TNF-α and IL-1β expression in the human monocytic cell line THP-1 in response to LPS [122] . As described before, O 3 may activate an anti-oxidant response via the Nrf2/Keap-1/ARE pathway either eliciting a ROS-mediated signaling by lipid oxidized products, such as oxysterols and α,βunsaturated aldheydes from PUFA or by eliciting other mediators such as heme oxygenase-1 (HO-1) [123] . Both O 3 and 4-HNE induce the production of HO-1, connecting the Nrf2/NF-κB cross talk with endothelia physiology and coagulation [113, 123] . The oxidative stress has been suggested as a leading issue in the COVID-19 pathogenesis [124, 125] , therefore any pharmacological strategy to dampen oxidative stress in SARS-CoV2 infected subjects is of the utmost importance. In this context, some authors have suggested that targeting HO-1 may be a promising step in controlling SARS-CoV2 infection and addressing a successful COVID-19 treatment [126] . Heme oxygenases, i.e. heme oxygenase 1 (HO-1) and heme oxygenase 2 (HO-2), not only degrade physiologic heme then releasing CO, biliverdin and iron, but act as oxygen sensors during hypoxia [127] . Actually, one of the leading causes of COVID-19 exacerbation, often associated with co-morbidities such as obesity, is hypoxia [128] . Obesity, which is a major comorbidity in COVID-19, may enhance the production of the hypoxia-inducible factor-1α (HIF-1α), shifting an existing cytokine storm to a fulminant event [128] . As HO-1 is one of the genes expressed by the activation of the Nrf2/Keap1/ARE pathway and being a major tuner of blood O 2 level, its induction by O 3 and O 3derivatives may be particularly crucial for successfully treating COVID-19 patients. Mammalian cells must regulate their oxygen levels to the proper homeostatic balance. In this sense, HIF-1α is a molecular oxygen sensor, a subunit of the heterodimeric gene transcription factor HIF-1 together with HIF-1β, and encompasses the analogs HIF-2α and HIF-3α [129] . As like as Nrf2, also HIF-1α has a DNA binding motif, called hypoxia response element (HRE) [130] . The role of O 3 towards HIF-1α has been recently addressed in experimental animals with diabetic nephropathy, resulting in a decrease of the apoptotic signal by inhibiting the expression of caspases 1,3, and 9 and modulating the activity of HIF-1α [131] . Fundamentally O 3 seems to inhibit HIF-1α expression [132] , so reducing the hypoxic stimulus. Moreover, the interplay Nrf2-NF-κB and HO-1 regulates the expression of the vascular cell adhesion molecule-1 (VCAM-1), inhibiting their expression, which is normally up-regulated in COVID-19 [133, 134] . A complex interrelated functional network can be described involving the cross talk Nrf2/NF-κB in the activity of hormetic doses of O 3 and its derivatives in the blood [73] . In the blood O 3 may form ROS from water, reactive nitrogen species (RNS) from oxidized nitrogen and RES from lipoproteins, membrane lipids and other PUFA derivatives such as 15deoxy-Δ 12,14 -PGJ2 [135, 136] . These byproducts trigger an anti-oxidant mechanism via the Nrf2/Keap1/ARE activation, so inhibiting the pro-inflammatory machinery led by the NF-κB pathway. Moreover, the interaction of RES with Cys residues in Keap1, reduces the proteasome-dependent degradation of Nrf2, enhancing its activated state and by inhibiting the apoptotic pathways, both the FAS/TNFR/caspase 8 signaling and the mitochondria-mediated apoptosis, induce the activation of survival genes [137] . Briefly speaking, the activation of an anti-oxidant mechanism via Nrf2 induces an inhibition of the pro-inflammatory mechanism suppressing NF-κB activation. Moreover, the activation of the NRf2/Keap1/ARE triggers the production of HO-1, which is induced by O 3 directly and inhibits platelets-dependent thrombosis [138] [139] [140] [141] . HIF-2α and HIF-3α for ubiquitination and degradation by the 26S-proteasome [142] . With low oxygen levels HIF-1α cannot be longer degraded, due to impairment in PHDs activation, so accumulating HIF-1α, which is translocated to the nucleus where activates HRE-dependent genes, such as TNF-family death receptors inducing apoptosis [142] . In this sense, the reduction of HIF-1α level by O 3 may be explained as a counteracting action to reduce the pro-inflammatory and proapoptotic signal led by the HIF-1α on HREs. Recent reports have outlined a major cross-talk between HIF-1α pathway and Nrf2 signaling, suggesting that the role of O 3 in this context may be tunable and intertwined with the Nrf2/Keap1/ARE signaling [142, 143] . During hypoxemic stimuli caused by COVID-19 associated pneumonia, the role of HIF-1α appears particularly intriguing, because HIF-1α up-regulates ACE-1 receptors, therefore reducing the expression of ACE-2 ones. As a balance ACE-1/ACE-2 receptors exists in physiological conditions, HIF-1α therefore reduces SARS-COV2 spreading in the organism [144] . Stabilization of HIF-1α is considered, therefore, fundamental to dampen SARS-CoV2 infection [144] and HO-1 stabilizes HIF-1α, protecting the organism from the ischemia-reperfusion injury [145] . Moreover, HIF-1α, in mild stress conditions, promotes the expression of HO-1, so enhancing the anti-thrombotic and cardiovascular protective mechanisms [146] . Low oxygen promotes SARS-CoV2 replication [147] . In this sense, the role of O 3 in restoring optimal oxygen availability may be crucial also in reducing viral spread in multiple organs and tissues. In this respect past investigations have reported that O 3 , when in contact with human blood, is able to elicit a remarkable up-regulation of HO-1. This should emphasize the role of O 3 in counteracting oxidative stress, inflammation and ischemicthrombogenic processes. Actually, HO-1 has been recently considered a pharmaceutical target for COVID-19 treatment [149] . During SARS-CoV2 infection HO-1 increases in the blood alongside with heme, anemia and desaturation [150] . The evidence reporting that O 3 can activate HO-1 may represent a promising approach to treat COVID-19, for which targeting HO-1 is an issue of increasing interest [151, 152] . HO-1 and Nrf2 plays a fundamental interplay in the oxidative stress response, as HO-1 is a Nrf2-regulated downstream gene [150] , therefore it is tempting to speculate that the activity of O 3 on HO-1 may be indirectly tuned by a much more direct effect on the Nrf2/Keap-1/ARE pathway, as suggested above. Elucidating the action of O 3 and its derived ROS and RES on immune cells is fundamental, due the involvement of immunity in COVID-19 [153] [154] [155] . Ozone modulates the differential expression of pro-inflammatory M1 and anti-inflammatory M2 macrophages, therefore participating in their balance in COVID-19 affected tissues such as airway and lung epithelia [95] . In an experimental model of rheumatoid arthritis, O 3 reduced the level of TNF-α and IL-12 from synovial immune cells and increased the anti-inflammatory cytokine IL-10 [156] . The anti-inflammatory property of O 3 is more often exerted by its oxidized phospholipids, such as 4-HNE, which is a powerful activator of Nrf2 and HO-1 synthesis, as reported in BV-2 microglial cells [157] . [163] [164] [165] . The evidence that O 3 modulates immunity was reported also by its anti-microbial activity, for example against Klebsiella pneumoniae by enhancing innate immunity and MIP-2 production and for its anti-parasite activity in vivo [166] . Cabral The same Nrf2/Keap-1/ARE signaling has a leading role in the modulation of cytokine storm, as outlined by some authors suggesting Nrf2 as an issue of pharmacological targeting [169] . In this perspective, it is conceivable to suggest the hypothesis that O 3 may exert a major anti-inflammatory effect via the activation of the Nrf2/Keap-1/ARE signaling. Actually, Nrf2 is able to suppress proinflammatory cytokine expression in macrophages, particularly IL-6 and IL-1β, as reported also in recent clinical studies where medical O 3 , used to treat elderly people hospitalized in ITUs with COVID-19, significantly decreased IL-6 levels in the bloodstream [3, 170] . Inhibition of inflammation by O 3 regards therefore medical hematological O 3 in the clinical course of an illness. As O 3 is a highly reactive substance, its anti-inflammatory potential can be retrieved only with proper and sound protocols, as empirical attempts usually fail in giving encouraging outcomes [171] [172] [173] . On the other hand, the immune pathogenesis of COVID-19 appears particularly complex. In this sense, the immunological context in which O 3 At least in obese patients and in adipocytes, 4-HNE was reported to regulate the genetic expression of TNF-α via the activation of the transcription factor ETS1 and the microRNA miRNA29b [175] . The role of 4-HNE in reducing inflammation, by inhibiting NF-κB, is well known [176] , and furthermore 4-HNE can inhibit the production of TNF-α and IL-1β from monocytes activated by bacterial LPS, via the inhibition of the p38MAPK and ERK1/ERK2 signaling [177] . It is possible to speculate, therefore, that the anti-inflammatory activity of O 3 in COVID-19, may be fundamentally exerted by aldehydes derived from O 3 -oxidized lipids in cells and the blood. Moreover, it has to be taken into account that innate immune cells may produce endogenous O 3 , though in particular conditions [36, 178, 179] . Endogenous O 3 and O 3 -caused lipid mediators are powerful antiinflammatory tools. Cyclo-pentenone isoprostanes, which may be produced by O 3 interactions with lipids, are strong inhibitors of the inflammatory response in macrophages [180] . Some oxysterols, such as 25-hydroxycholesterol, have anti-inflammatory properties, being able to dampen the IL-1 mediated inflammation, downstream of TNF-α activation [181] . So far, we have outlined that the main way by which O 3 would act against SARS-CoV2 infection and more exactly towards COVID-19 reducing its clinical impact, involves the activation of the anti-oxidant endowment of infected cells, i.e. scavenging enzymes and transcription factors, probably via a mild stress, which in turn activates ROS signaling and triggers the expression of a survival and anti-inflammatory response [182] . This may result a good hypothesis, as several reports have shown the ability of little or moderate dosages of gaseous O 3 into the blood to promote an anti-oxidant response in the organism, fundamentally targeting the Nrf2/keap-1/ARE pathway and HO-1 expression. Furthermore, O 3 may target also the complex nitric oxide/inducible nitric oxide synthase (NO/iNOS) pathway [183] . The highly widespread belief that COVID-19 may be fundamentally an endothelial-pro-thrombotic disease, as reported by the observation that NO, statins and ACE inhibitors are able to induce a less severe manifestation of COVID-19, reducing exacerbation, has currently set on the spotlight the role of NO in treating COVID-19 [184] . It is well known that NO is released by endothelia to prevent platelet-mediated thrombosis and to hamper new platelets recruitment in the thrombus formation [185] . Recent reports showed that, standing GSH depletion and hypoxic stimuli, NO simulates HO-1 production [186] , an evidence strengthening the result that HO-1, the major anti-thrombotic mediator, is released by the Nfr2mediated anti-oxidant activity and by NO, which in turn cross talk with HIF-1α signaling. Actually, while mild NO levels (usually ≤ 400 nmoles/L) are reported to promote HIF-1α proteasomemediated degradation, impairing HIF-1α signaling, high NO doses (≥ 1.0 moles/L) stabilize HIF-1α, even during normoxic conditions [187] . It is tempting to speculate that during COVID-19 a finely regulated interplay NO-HO-1-HIF-1α, more that a gigantic income of O 2 , is the leading mechanism preventing vascular disorders and thrombosis reported in severe COVID-19. Table 2 summarizes some of the major evidence on the effect of O 3 on ischemia/reperfusion injury models in laboratory animals and in vitro studies [187] [188] [189] [190] [191] [192] [193] [194] [195] [196] . Besides to exacerbation in the prooxidant and pro-inflammatory status, COVID-19 is characterized by severe disorders in the endothelia-coagulation and pro-thrombotic system [197] [198] [199] [200] [201] . The ischemia/reperfusion (I/R) injury model represents a reliable experimental bench to investigate the activity of O 3 and its mediators on vascular-endothelial and thrombotic processes [202] . Pioneering studies conducted by Rokitansky et al., in 1981 , evaluated the role of O 3 in modulating the production of 2,3-diphosphoglycerate (2,3-DPG), a fundamental factor for platelet thrombogenic function [78, 203, 204] . Factors enhancing blood oxygenation affect many mechanisms involving the physiology of vascular endothelia. A synergistic action exists between the Nrf2 activity and the role of HO-1, particularly in the cardiovascular function, therefore the antithrombotic and vascular-protective activity of HO-1 is potentiated by a positive synergistic loop from the O 3 -mediated action on Nrf2 [205] . Several research studies, in laboratory animals, showed that the administration of O 3 in I/R models, often reduced the impact of the I/R-mediated damage and triggering an anti-oxidant response. However, the condition and O 3 dosages used in I/R models may be largely limitant, as O 3 exerts its action in a very complex system. Elsurer [191] . Rats treated with O 3 also in the I/R model, showed a marked increase in MDA, protein carbonyl (PCO), total antioxidant capacity (TAC), SOD, GSH-Px and catalase [191] . Onal and colleagues induced an I/R injury in male Wistar rats occluding the superior mesenteric artery for 60 min (ischemia), followed by 2 hours of reperfusion. In the group pre-treated with O 3 , using 3 L/min of an O 2 (97%)-O 3 (3%) gas mixture with 60 μg/mL O 3 , volume 3.2-4.2 ml, the animals exhibited a decreased intestine mucosa injury and increased total antioxidant capacity (TAC), SOD, glutathione peroxidase and catalase [206] . The role of NO in I/R injury models is supported by the observation that administering NO-donors or compounds able to enhance NO production before inducing ischemia, the injury following I/R is greatly reduced [207] . The O 2 /O 3 mixture used to inject O 3 in the blood is able to activate the endothelial nitric oxide synthase (eNOS) [208, 209] , therefore the release of NO is directly triggered by O 3 , or by its end products [210] . also Table 1 ) [206] . The correct O 3 protocol is mandatory to earn positive outcome in the use of this gas. O 2 -O 3 -AHT may lead to the formation of NO and oxidants causing the production of 3-nitrotyrosine as a fundamental signal to activate a mild oxidative stress and inhibit thrombotic events, as NO is in itself an anti-thrombotic signal [213, 214] . NO has a role in aspirin-induced thrombolysis [215] and moreover NO regulates tissue factor (TF) for coagulation, which is Furthermore, in ischemia/reperfusion (I/R) injury models, O 3 proved to reduce and prevent the IRcaused damage, usually by activating the oxidative stress response. Endothelial dysfunction plays a major role in I/R injury [219] . Wistar rats, undergoing superior mesenteric artery occlusion for 1 hour and reperfusion for two hours, when administered with 1.0 mg/kg of O 3 increased intestinal tissue levels of SOD, glutathione peroxidase and catalase [206] . Moreover, tissue protection from I/R injury by O 3 may involve the activation of iNOS. Foglieni et al., recovered the kidney functional activity in rats undergoing unilateral nephrectomy by using 1 ml autologous blood with 5 ml of an O 2 /O 3 mixture (50 μg/ml O 3 ) and observed that the production of iNOS by β-NADPH diaphorase in glomerular capillaries increased significantly, following ischemic injury [220] . Although many data about ozone in I/R injury refer to the gaseous airborne O 3 as a pollutant, inhaled O 3 reduces the plasminogen activator inhibitor-1 (PAI-1), therefore enhancing the presence of tissue plasminogen (tPA), then enabling the reduction of clots and thrombi and promoting fibrinolysis [221] , i.e. disrupting intravascular thrombi, particularly in microcirculation [222] . Interestingly, PAI-1 increases also when SOD and glutathione peroxydase (GSH-Px) are enhanced, as occurring following treatment with O 3 [223] . It is tempting to speculate that RES produced by O 3 , such as 4-HNE, may play a crucial role in the complex balance pro-thrombotic/anti-thrombotic signals following O 3 exposure, as its dosage has a particular stringency in this context [224] . The anti-oxidant property of O 3 is therefore fundamental to prevent ROS formation in venous thrombosis [225] . Streptozotocin-induced diabetic male [227] . Furthermore, the expression of caspase 3 in myocardial tissue decreased at 150 μg/ml O 3 and much more at 300 μg/ml O 3 [227] . In addition, using the same I/R injury models on rats, this research team observed that in animals treated with the O 2 /O 3 mixture, an increase in the expression of CD34+ and CD117/c-kit in myorcardial tissue and of eNOS, rapidly occurred, whereas the pre-treatment with a known eNOS inhibitor (30 mg/kg N5-(1-Iminoethyl)-L-ornithine dihydrochloride (L-NIO) subcutaneous injection), suppressed the protective role of O 3 in inducing eNOS [228] . The numerous I/R injury models suggest altogether the fundamental role of O 3 as a small molecule able to target fundamental genes involved in I/R injury, such as LCN2, CCL2, HP, HMOX1, CCL7, CCL4, and S100A8 and several micro-RNAs, as O 3 dampens the pro-inflammatory machinery in the I/R injury model by inhibiting the NLRP3mediated inflammation and enhancing the Nrf2/Keap1/ARE pathway [229, 230] . Which is the potential of O 3 in treating COVID-19? 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Ferritin, CRP, D-dimer, LDH [5]  CRP  Liver protection via mechanisms producing NO [191]  Mitochondria-mediated apoptosis SH-SY5Y cell lineIn vitro neuroblastoma cell line SH-SY5Y in erebral I/R model 4 × 10 5 SH-SY5Y cells in 1 mL medium with 1 ml O 3 in 10 mL  SOD, CAT, GSH-Px [192] ADA; adenosine deaminase; CAT: catalase; GSH-Px: glutathione peroxydase; MDA: malonyldialdheyde; MPO: myeloperoxidase; PCO: protein carbonyl; SOD: superoxide dismutase; TAC: total antioxidant capacity; TOS: total oxidant status.