Targeting reactive oxygen species for respiratory infection: Fact or fancy?

Reactive oxygen species (ROS) is a generic term that describes a series of molecules often misconceived as being solely undesirable toxic molecules, which damage DNA, cause cancer and a myriad of other diseases such as respiratory (COPD, asthma and idiopathic pulmonary fibrosis to name a few), cardiovascular, metabolic, neurodegenerative and autoimmune diseases. Therefore, it has been assumed that strategies that reduce ROS levels should in theory have beneficial properties and represent a means for alleviating symptoms of these pathologies. However, this has clearly not been the case. Despite the huge body of evidence demonstrating that excessive ROS are associated with many diseases including respiratory-related pathologies, clinical evidence is still lacking.1 For example, oral therapy with antioxidants, such as vitamins C and E, failed to prevent the development or progression of cardiovascular disease. Several large-scale antioxidant clinical trials yielded disappointing results regarding all-cause mortality and, in some cases, oral antioxidants had detrimental effects. Moreover, several small molecule thiol antioxidants including N-acetylcysteine have generally yielded mixed outcomes for the treatment of chronic respiratory diseases.2 Hence, no antioxidant is in clinical use. To some degree, the lack of effectiveness of antioxidants in these clinical trials has tarnished the idea that ROS or oxidative stress are a viable target for disease therapy. Once again, this is another misconception in this field and there are at least three possible explanations for this often-called oxidative stress paradox. First, it is important to note that ROS are a family of molecules with a great deal of species and biological variability. The commonality is oxygen, and whilst it is essential for life on earth, it also gives rise to some of the most aggressive, indiscriminately toxic molecules with the capacity to cause significant alterations in cellular function by oxidative modifications. To counterbalance this, cells have evolved a large suite of antioxidant systems that act to buffer ROS and under normal physiological circumstances, there is an intricate and fine balance in ROS production and ROS metabolism via antioxidant processes. Critically, under these conditions, ROS play roles in redox signalling including in processes leading to cell proliferation, apoptosis and, in perhaps the best-known function of ROS, pathogen clearance. Indeed, phagocytes such as neutrophils generate huge bursts of ROS within the confines of the phagosome following engulfment of invading bacteria or fungi. This large ROS insult together with a variety of proteases provide a highly toxic and unfavourable environment for the pathogen within the phagosome that ultimately kills it. Therefore, it is critical that our antioxidant strategy does not modify beneficial redox signalling and preserves host defence mechanisms. Second, it is important to consider the chemistry of ROS, which governs the spatiotemporal and biological properties of ROS. As the term reactive oxygen species implies, ROS are highly reactive molecules with rapid rates of production and with short half-lives. For example, superoxide anion (O2.−), which is often defined as the ‘parent’ ROS, is produced in either a deliberate fashion by NADPH oxidase enzymes or inadvertently as a consequence of mitochondrial metabolism or by a dysfunctional enzyme variant, such as uncoupled nitric oxide (NO) synthase. Superoxide is then rapidly converted to hydrogen peroxide (H2O2) via an extremely rapid (rate constant of ~108 mol/L/s) enzymatic catalysis using the superoxide dismutase (SOD) family of enzymes. Superoxide also reacts with NO in the fastest known biological reaction (~5 × 109 mol/L/s) to give rise to peroxynitrite (ONOO−), which is a very powerful oxidant with pleiotropic actions. If one considers the rate constant for ROS scavenging by vitamins E and C, which is approximately 103 mol/L/s, then there is very little chance of these antioxidants outcompeting the endogenous reactions. Thus, the kinetics need to be carefully considered and favourable, and these properties govern the efficacy of antioxidants in vivo. Third, due to the rapid rates of ROS production and removal, most ROS are highly diffusion-limited and therefore their subcellular site of generation will influence their site of action, their biological action and their sensitivity to an exogenous antioxidant strategy. It is increasingly becoming recognized that cells have evolved ways to compartmentalize ROS production that aids in signalling that is localized and restricted such that inadvertent effects of ROS on other critical cellular machinery are minimized. For example, ROS production occurs in specific compartments of the cell in response to invading microorganisms, such as bacteria, viruses and fungi. As already mentioned, bacteria and fungi are phagocytosed by neutrophils and macrophages resulting in a NOX2-containing NADPH oxidase-dependent ROS production in the phagosomal compartment. We have recently shown that viruses that enter cells by endocytosis trigger ROS production within the confines of endosomes via a NOX2-containing NADPH oxidase.3 We also know that invading pathogens are likely to drive alterations in metabolism and mitochondrial function.4 This has been shown exquisitely in macrophages where there is a switch from oxidative phosphorylation to glycolysis resulting in mitochondrial ROS generation. This mitochondrial ROS serves a powerful stimulus for inflammasome activation and thereby inflammation. Therefore, non-specific antioxidants such as vitamins C and E, as well as N-acetylcysteine, which raises cellular glutathione, are unlikely to target subcellular compartmentalized ROS production. Our recent study demonstrates how knowledge of organelle-specific ROS production can be exploited for the treatment of respiratory viral infections.3 It demonstrates that viruses including low to highly pathogenic influenza, respiratory syncytial virus and rhinovirus are internalized by endocytosis and activate an endosomally located NOX2 oxidase that generates ROS within the endosome. Consistent with the spatial restrictions mentioned above, the H2O2 generated by the virus in the endosome suppressed the activity of TLR7 (toll-like receptor 7), also located on the endosome, by targeting its cysteine 98, resulting in a decrease in antiviral Type I interferon (IFN) expression. As proof of concept of endosome targeting, we developed an innovative molecular targeting system to deliver one of the most specific NOX2 oxidase inhibitors available, gp91ds-TAT,5 which comprised of gp91ds-TAT conjugated via a polyethylene glycol (PEG) linker to cholestanol. Excitingly, cholestanol-conjugated gp91ds-TAT caused a substantial reduction in airway inflammation, oxidative stress, viral replication and overall disease severity caused by influenza A virus infection in a mouse model.3 On a final note, how do we overcome these major limitations of current antioxidant strategies and can we utilize an antioxidant strategy to treat respiratory infections? In the first instance, we need to increase our understanding of the physiological and pathophysiological roles of ROS in the cells of the airways. It is also important to decipher what are the critical enzymatic sources of ROS, how are they activated by pathogens and in which organelles are these ROS generated within. Having this information will allow for a more targeted approach which might involve the activation of intrinsic antioxidant processes such as glutathione peroxidase (GPX) and nuclear factor erythroid 2–related factor 2 (NRF2)-dependent pathways or the inhibition of ROS production by NADPH oxidases and mitochondria by organelle-targeted inhibitors or antioxidants. So in conclusion, ‘Targeting reactive oxygen species to treat respiratory infections: Fact’. The author wishes to thank Professor Ross Vlahos (School of Health and Biomedical Sciences, RMIT University) for proofreading the manuscript.