Reactive species and redox imbalance may dysregulate the immune response and account for disease progression in SARS-CoV-2 infection. This aspect suggests treatment options that could hinder disease progression and prevent multiple features of severe illness, which include clotting predisposition, cytokine storm and organ damage.
- COVID-19
- immune response
- redox biology
1. COVID-19, Redox Balance and Immunity
1.1. COVID-19 Is Characterized by Impaired Redox Homeostasis, Redox Balance and Immunity
1.1. COVID-19 Is Characterized by Impaired Redox Homeostasis
Due to its crucial role in response to infections, oxidative stress is considered a key determinant in COVID-19 pathogenesis [1][2][98,99]. Pathological changes underlying pulmonary damage induced by SARS-CoV-2 include exudative proteinaceous injury and inflammatory lymphocytic infiltrates, diffuse alveolar damage with hyaline membranes and wall thickening [3][100]. Severe COVID-19 is further characterized by hypercoagulation and hypoxia in several organs [4][101]. Such massive induction of tissue damage can be related to a defective redox balance [5][102].
Excess of reactive species and consequent dysregulated redox homeostasis were described in several respiratory viral infections. Following activation of innate immunity and pro-inflammatory cytokines, infection by respiratory syncytial virus induces overproduction of reactive species, increasing lipid peroxidation, depleting GSH and inhibiting NRF2 in respiratory epithelial cells [6][7][103,104]. Influenza virus leads to reactive species excess in several tissues, particularly in lungs, inducing apoptosis and cytotoxicity, but activating NRF2 to counteract oxidative injury in alveolar epithelial cells [8][9][10][105–107]. Several pre-clinical studies suggest that severe lung damage in SARS-CoV infection relies on both oxidative stress and innate immunity, with consequent activation of NF-κB and enhanced pro-inflammatory host response [11][12][13][108–110]. In convalescent patients, an upregulation of mitochondrial and redox-sensitive genes occurs, supporting the association between redox imbalance, inflammation and the pathogenesis of SARS-CoV infection [14][111].
According to previous evidence from similar viral infections, impairment in redox balance may deeply impact COVID-19 pathogenesis, even though reports sustaining this hypothesis are still limited. Several radical scavengers, such as GSH, NADPH or Trx, may regulate the cellular disulfide–thiol balance, which is crucial for SARS-CoV-2 entry and fusion into the host cell [1][98]. Deficiency of G6PD may be associated with severe COVID-19, since redox homeostasis mediated by this enzyme is involved in the immune response to viral infections [15][21]. This is strongly suggested by the following evidence: (1) G6PD deficiency enhances several viral infections; (2) G6PD variants may impact COVID-19 severity; and (3) higher incidence of COVID-19 in African-Americans, whose G6PD deficiency is characterized by higher oxidative stress [16][17][18][19][112–115]. A pilot study on COVID-19 patients hospitalized in intensive care unit showed reduced circulating levels of several antioxidants (such as vitamin C, thiol proteins, GSH, γ-tocopherol, β-carotene), as well increased lipid peroxides and the oxidative stress index copper/zinc ratio [20][116]. When the total oxidant status, total antioxidant capacity and level of glutathione were compared in hospitalized COVID-19 patients with different disease severity, increased oxidative stress indices and reduced antioxidant markers were related to serious clinical presentation and outcomes [21][117]. Nevertheless, another study showed no correlation observed between the oxidative stress parameters and the degree of COVID-19 severity in hospitalized patients, suggesting that disease severity may not contribute to redox changes in SARS-CoV-2 infection [22][118]. A preliminary report on a small sample of critically ill COVID-19 patients described higher levels of protein adducts of the lipid peroxidation product 4-hydroxynonenal in the deceased, as compared to survivors [23][119]. Compared to healthy controls, circulating SOD and CAT activity, as well as carbonyl and lipid peroxidation (LPO) levels, were higher, while total antioxidant capacity levels were lower in COVID-19 patients [23][120]. Moreover, higher LPO levels were independently associated with a higher risk of intubation or death at 28 days [23][120]. A further study demonstrated that neutrophils are the main source of reactive species in severe COVID-19, and circulating H2O2 levels are increased in dead patients [24][121].
1.2. Altered Redox Balance Modulates the Immune Response
Redox biology accomplishes key regulatory functions in innate immunity. Increased production of reactive species by phagocytes is one of the first-line antimicrobial responses, defined as the oxidative/respiratory burst [25][122]. More than merely producing reactive species via NADPH oxidase, neutrophils may sense the differential localization of oxidants and finely tune IL-1β expression through selective oxidation of NF-κB [26][123]. Mitochondria-derived reactive species further regulate the differentiation process of dendritic cells [27][124]. Furthermore, mitochondrial reactive species in macrophages enter the cytosol and induce a covalent modification in NF-κB essential modulator (NEMO), an element of the inhibitor of κB kinase (IKK) complex required to activate both the ERK1/2 and NF-κB pathways and to promote secretion of pro-inflammatory cytokines [28][125]. Reactive species are also produced by NADPH oxidase in natural killer T cells (but not in CD4+ or CD8+ T cells), modulating their expression of IFN-γ and IL-17, thus playing a role in the regulation of inflammatory function [29][126].
Redox signaling and regulation are crucial in adaptive immunity (Figure 2b). Indeed, excessive production of reactive species is associated with the activation and differentiation of both T and B cells. T helper activation, required for both humoral and cell-mediated immune response, relies on the redox status of the microenvironment [30][127]. Even though a reduced microenvironment could protect from oxidative stress during T cell activation, mild concentrations of reactive species are necessary for the initiation of adaptive immune response [31][128]. Activation of CD28, costimulatory of T cell activation, induces intracellular reactive species, with consequent induction of IL-2 via NF-κB [32][129]. On the other hand, the antioxidant GSH is required to regulate the proliferation of activated T cells [33][130]. Nevertheless, low amounts of reactive species are a precondition for T cell survival, while oxidant accumulation causes apoptosis/necrosis [34][131]. Moreover, redox signaling may affect T cell commitment, and different T cells present with various redox levels [34][131]. Indeed, oxidative status induces Th1 development, while prevalence of reducing molecules shifts toward Th2 responses [35][132].
2. Targeting Impaired Redox Homeostasis in COVID-19
Several studies suggest that redox balance may be a feasible therapeutic target for COVID-19 by modulating the redox-sensitive immune response. Numerous trials have been designed to test antioxidants (such as N-acetylcysteine, ascorbic acid, resveratrol) in COVID-19, and many of them are currently ongoing while this review is being written.
N-acetylcysteine (NAC) could potentially treat COVID-19 infection by stimulating glutathione synthesis, promoting T cell response and regulating inflammation [36][133]. Other than providing an extra source of cysteine to synthetize glutathione, NAC can stop ACE2 activity and SARS-CoV-2 entry into target cells by the presence of a thiol group [37][134]. NAC was intravenously administered in patients with severe COVID-19, contributing to clinical improvement, as well as reduction of C-reactive protein [38][135]. A retrospective two-center cohort study showed that oral NAC reduces the risk of mechanical ventilation and mortality when added to standard of care in patients with moderate to severe COVID-19 [39][136]. However, a double-blind randomized trial was not able to demonstrate that intravenous administration of NAC in high doses was superior to placebo in affecting the evolution of severe COVID-19 [40][137]. Similar results were observed in a pilot study, which could not support beneficial effects of intravenous NAC in COVID-19 patients with acute respiratory distress syndrome [41][138].
Ascorbic acid (vitamin C) is a potent antioxidant, which directly scavenges reactive species, and it is also highly concentrated in leukocytes for several immune responses [42][43][139,140]. The first suggestion on the efficacy of vitamin C in reducing susceptibility to respiratory tract infections derives from Linus Pauling [44][141]. Circulating levels of ascorbic acid were severely depleted in COVID-19 patients with acute respiratory distress syndrome [45][142]. Administration of high-dose intravenous vitamin C was associated with improved inflammatory and immune response, as well as restored organ function in severe/critical COVID-19 [46][143]. Nevertheless, a pilot study failed to demonstrate any clinical improvement in critically ill COVID-19 patients treated with intravenous high doses of ascorbic acid [47][144]. Two randomized controlled trials could not demonstrate that the addition of intravenous vitamin C to standard therapy had an impact on mortality, length of stay or the need for mechanical ventilation in COVID-19 patients [48][49][145,146].
Resveratrol is a polyphenol with several antioxidant, anti-inflammatory and immunomodulator properties [50][147]. More than being a simple scavenger of reactive species, resveratrol increases the expression of the antioxidant protein SIRT1, which in turn boosts NAD levels, improving the immune response [51][148]. Resveratrol has been demonstrated to reduce the replication of SARS-CoV-2 in vitro, showing antiviral properties in infected human bronchial epithelial cells [52][53][149,150]. In a placebo-controlled cross-over study, resveratrol supplementation in obese men reduced the expression of ACE2 in adipose tissue, suggesting that this compound could reduce SARS-CoV-2 diffusion [54][151]. A randomized placebo-controlled phase 2 trial showed that oral supplementation of resveratrol in COVID-19 outpatients presented with lower incidence of pneumonia and hospitalization [55][152].
Pharmacological activation of the transcriptional factor NRF2 has been recently suggested as a promising therapeutic strategy against COVID-19 due to restoration of redox homeostasis and resolution of inflammation [56][153]. Sulforaphane is an electrophile that modifies cysteine sensors of Keap1, inactivating its repressor functions [57][154]. This compound is able to inhibit the replication of SARS-CoV-2 in vitro and in the upper respiratory tract or lungs of SARS-CoV-2-infected mice, reducing pulmonary injury [58][155]. Furthermore, sulforaphane inhibits the expression of IL-6 and IL-8 in cultured bronchial cells exposed to the S-protein of SARS-CoV-2, supporting its anti-inflammatory effect [59][156]. Nevertheless, no clinical data on the efficacy of sulforaphane are currently available. Bardoxolone and bardoxolone methyl are electrophilic moieties able to activate the NRF2 pathway and inhibit the NF-κB pathway [60][157]. Both compounds can inhibit SARS-CoV-2 replication by specifically binding the 3C-like protease in infected Vero cells [61][158]. Hence, even these Nrf2 activators may be considered in a multifaceted antiviral treatment strategy.
Other compounds involved in the interplay with redox homeostasis were suggested to be potentially beneficial in the treatment of COVID-19. Polyphenols are natural agents with high antioxidant and anti-inflammatory properties, which could also target virus proteins or cell receptors, preventing SARS-CoV-2 entry and replication [62][159]. Acting as anti-inflammatory and antioxidant, the bioactive molecule melatonin may be effective in reducing acute lung injury caused by SARS-CoV-2 [63][160]. The trace element zinc—whose deficiency is reported in severe COVID-19—may prevent SARS-CoV-2 by improving respiratory tissue barrier and inhibiting viral replication, but also balancing immune response and redox homeostasis [64][161]. The novel antifibrotic agent pirfenidone could reduce inflammation and counteract oxidative stress, antagonizing apoptosis and downregulating ACE2 expression [65][162]. Selenium is another trace element incorporated in several selenoproteins with both anti-inflammatory and antioxidant functions; the expression of several selenoproteins is decreased by SARS-CoV-2 infection, and redox-active selenium molecules might potentially inhibit SARS-CoV-2 proteases [163]. All these redox compounds can be considered as promising in counteracting SARS-CoV-2 infection and modulating an immune response to COVID-19, even though further studies are needed.