Respiratory viruses such as influenza A virus (IAV) are responsible for annual seasonal epidemics and have the potential to cause pandemic outbreaks, depending on the viral pathogenicity and level of pre-existing host immunity. IAV infection can cause debilitating respiratory illness, which poses a major concern for patients, especially high risk individuals, including the young (>age of 5), elderly (>65 years of age), pregnant, and those that are immunocompromised; notwithstanding the ongoing burden it has on global healthcare systems (Hsieh et al., 2006; La Gruta et al., 2007; Lewnard and Cobey, 2018). Currently, vaccination is the best available preventative measure for controlling and containing the spread of IAV. However, antigenic mismatches between the circulating strains and vaccines reduce the effectiveness of the vaccination approach (La Gruta et al., 2007; Houser and Subbarao, 2015; Agor and Özaltın, 2018). Prophylactic treatments include antivirals that target the viral envelope, however drug resistance is likely to limit their use in the future (Abed et al., 2005; La Gruta et al., 2007; McKimm-Breschkin, 2013; Hussain et al., 2017). Therefore, in addition to vaccination strategies, it is imperative to have therapeutic strategies that target the critical cellular machinery and signaling pathways that the virus utilizes in the host to promote inflammation and viral replication.

Reactive oxygen species (ROS), including superoxide anion and its derivatives, are highly reactive molecules that are generated by respiratory burst enzymes such as the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase family of enzymes (NOX1-5, DUOX1-2), or as a consequence of cellular metabolism from the mitochondria (Wang et al., 2009; Drummond et al., 2011). ROS have important physiological roles in cellular signaling, proliferation and programmed cell death (Kannan and Jain, 2000; Redza-Dutordoir and Averill-Bates, 2016). ROS are produced by a myriad of inflammatory cells during IAV infection, including epithelial cells, monocytes, macrophages and neutrophils (Bedard and Krause, 2007; Khomich et al., 2018). Excessive ROS production to IAV infection can contribute to lung inflammation and disease pathogenesis (Imai et al., 2008; Vlahos et al., 2011). Elevations in ROS production in influenza patients were associated with higher levels of sterol oxidation products and nitro-tyrosine content, indicating changes in redox homeostasis (Nin et al., 2012; Ng et al., 2014). The specific subcellular localization of these ROS has recently emerged, with evidence showing a role for both NOX2-derived ROS from an endosomal location and mitochondrial ROS, in the pathogenesis of IAV (To et al., 2020). ROS generated from these subcellular compartments during IAV infection augment the downstream signaling pathways involved in promoting inflammation (To et al., 2017; To et al., 2019). For example, the initial internalization of virus into endosomes drives a rapid burst of ROS using a TLR7-PKC and NOX2 oxidase-dependent mechanism. This endosomal ROS response occurs within 2–5 min of virus addition to macrophages in vitro (To et al., 2017). This elevation in endosomal ROS also results in a marked suppression of Type I IFN that likely promotes IAV replication, and thereby exacerbating lung inflammation and oxidative stress. The molecular target of endosome ROS appears to be a single, evolutionary conserved and unique cysteine residue, C98 on TLR7, which is crucial for forming a disulphide bond with C475 and for TLR7 activation. Notably, a targeted endosomal NOX2 inhibitor, protected mice against both low and high pathogenic IAV, limiting viral replication and dramatically reducing pulmonary inflammation and alveolitis (To et al., 2017; To et al., 2019) when it was delivered to mice prior to IAV infection. In addition to this rapid endosomal ROS response to IAV, alterations in mitochondrial function increase mtROS (Wang et al., 2009; Makino et al., 2010; West et al., 2011; Koshiba, 2013). The temporal features of mtROS production appear to be slower in onset compared to the endosomal ROS response and are dependent on an alteration in the overall metabolic state of the immune cell(s). For instance, stimuli such as LPS stimulate an immunometabolic switch from oxidative phosphorylation to glycolysis, hours after exposure, resulting in succinate accumulation from the TCA cycle and mtROS production by Complex 1 (O’Neill and Pearce, 2016; Langston et al., 2017). Mitochondrial ROS are then required for the activation of the NLRP3 inflammasome complex (Allen et al., 2009), which is necessary for IL-1β processing and release, which promotes inflammation during IAV infection. We have recently shown that administration of the mitochondrial ROS scavenger (mitoTEMPO 36) prior to IAV infection reduced airway and lung inflammation, reduced viral mRNA and improved the overall morbidity to both low and high pathogenic influenza virus strains (To et al., 2020). In a similar fashion to endosomal NOX2 oxidase inhibition, mitoTEMPO treatment resulted in an elevation in Type I IFN and a concomitant reduction in IL-1β production. Therefore, we envisage a complex regulation of Type I IFN expression by subcellular specific ROS, involving endosome NOX2-derived ROS suppressing TLR7 directly via oxidation of C98, and mtROS targeting IRF7, a critical downstream regulator of TLR7 function.

It is conceivable that targeting both endosomal and mitochondrial ROS production might be an effective way of limiting the exacerbated host inflammation in response to IAV infection. Treating mice prior to IAV infection with either Cgp91ds-TAT or mitoTEMPO reduced the overall morbidity with reduced lung inflammation and improved function (To et al., 2017; To et al., 2019). However, we have up until now, no evidence that these compounds show efficacy during an established IAV infection. Therefore, in the present study we investigated the effect of the endosomal and mitochondrial ROS inhibitor 1 day after an established IAV infection. To achieve this, we delivered both Cgp91ds-TAT and mitoTEMPO simultaneously or individually 1 day post inoculation with IAV. We demonstrate that a combination of Cgp91ds-TAT and mitoTEMPO resulted in a significant reduction in airway and pulmonary inflammation, lowered the lung viral burden and elevated the number of virus-specific CD8⁺ T cells in the lung, which are important for the promotion of viral clearance. Overall, this study unravels new molecular mechanisms by which ROS promote IAV pathology and shows that subcellular specific inhibition of endosomal ROS and mtROS is a promising immunomodulatory approach against IAV infection.

IAV infections trigger respiratory tract complications, which feature heightened oxidative burden due to excessive ROS generation, resulting in exacerbated lung inflammation and injury (Vlahos et al., 2011). NOX2 oxidase-derived ROS contributes to lung pathology during IAV infection and endosomes are a major site for this ROS generation (Snelgrove et al., 2006; Vlahos et al., 2011; To et al., 2017; To et al., 2019). ROS produced by the mitochondria is also a major contributor to IAV pathogenesis (Dikalova et al., 2010; Kim et al., 2015). In this present study, we demonstrated that acute inhibition of ROS in both subcellular compartments (mitochondria and endosomes) was effective in reducing airway and pulmonary inflammation and in limiting viral load following IAV infection. Collectively, these data indicate that the targeting of ROS in these distinct compartments is a novel immunomodulatory approach for the treatment of IAV infections.

We demonstrated either no effect or a rather modest reduction in the disease course in mice treated with a combination of Cgp91ds-TAT and mitoTEMPO following IAV infection at Day 3 post infection. However, the effects of Cgp91ds-TAT and mitoTEMPO treatment were significantly more pronounced at Day 6 post infection. The phenotype of the treated mice was characterised by a reduction in BALF inflammation and neutrophilia, suppression in bodyweight loss, inhibition of lung oedema and a reduction in viral load signifying an overall improved disease course following treatment. The delayed improvement in disease course by ROS inhibition exemplifies that ROS are important regulators of the immune response to IAV infection, and that the dampening of their actions can be an effective means in re-shaping the inflammatory response to IAV. Often the inflammatory response to IAV is uncontrolled and exacerbated resulting in a significant lung immunopathology that governs the overall degree of sickness with impacts on both the innate and adaptive immune control of the viral burden. Our study demonstrates that both endosomal and mitochondrial ROS inhibition resulted in a reduced lung inflammatory response and a more effective anti-viral CD8⁺ T cell response. It is important to indicate that our study is only a proof of concept that the dual inhibition of endosomal ROS and mitochondrial ROS is effective at reducing IAV pathogenesis. The study is not a dose-finding study for either mitoTEMPO or Cgp91ds-TAT. Each drug on its own demonstrated very modest effects at re-shaping the immune response to IAV. This could signify a great deal of redundancy in the cellular effects of mtROS and endosomal ROS and that effective control can only occur when both pools of ROS are suppressed. Alternatively, it might signify that the dosing regimen of drugs chosen i.e., 3 days treatment protocol might be sub-optimal. Another important observation is that the endosomal targeted Cgp91ds-TAT did not suppress the PDB-dependent NOX2 activity indicating that Cgp91ds-TAT allows for intact extracellular superoxide production by NOX2 but rather provides a site specific targeting of superoxide in endosomes. This is critical, as elements of NOX2 activity remaining functional, is important for clearance of bacteria. The co-operative effects of the two drugs suggest a potential interplay between mitochondrial and endosomal ROS. Indeed, in support of such interplay, NOX2 has been shown to induce mitochondrial superoxide production in angiotensin-II mediated endothelial oxidative stress during hypertension (Dikalov et al., 2014). Certainly, crosstalk between these important sources of ROS including with other sources such as NOX4 and Duox enzymes warrants further investigation.

It is interesting that when using a prophylactic drug approach with either Cgp91ds-TAT or mitoTEMPO alone, the pathology to IAV infection was significantly dampened (To et al., 2017; To et al., 2019). Thus, suppression of either endosomal ROS or mitochondrial prior to IAV infection effects a different immune response that improves the disease course in mice. Intriguingly in both previous studies, we showed that either Cgp91ds-TAT or mitoTEMPO treatment alone in mice prior to IAV infection resulted in a significantly greater anti-viral Type I IFN, i.e., IFN-β response to IAV. In contrast in the present study, the administration of either Cgp91ds-TAT or mitoTEMPO alone post-IAV infection had no effect on Type I IFN, however, only with the combination treatment did we observe a significant elevation in Type I IFN. Again, this indicates the complex nature and timing of IFN signaling in response to IAV and how modifiable this signaling is to ROS suppression.

The Type I IFN system is upregulated during IAV infection and regarded as one of the most powerful innate defenses against viral pathogens (Khaitov et al., 2009; Crotta et al., 2013). Here we demonstrate that ROS inhibition causes an elevation in IFN-β expression at the early stages of infection. Pattern recognition receptors, such as toll-like receptors have been implicated in the induction of IFNs during IAV infection. In particular, TLR7 detects single stranded RNA components of IAV in endosomes and induces IFN secretion (Lund et al., 2004; Diebold et al., 2015; To et al., 2017). We have previously unveiled a feedback loop where endosomal NOX2-derived ROS suppressed TLR7 activity (To et al., 2017). Similarly, an elevation in mtROS reduced type I IFN production markedly via inhibiting the phosphorylation of interferon regulatory factor 7 (IRF7), which is a key transcription factor for TLR7 signaling (Agod et al., 2017). Based on these findings, it appears that there is a synergistic effect in targeting both endosomal and mtROS to boost a protective IFN response at the early phase of infection. Despite an increase in type I IFN responses with the dual ROS inhibition strategy, this did not correlate with a reduction in viral load at day 3 pi but it was significantly suppressed at Day 6 pi. This may be due to a delayed manifestation of the anti-viral effects of the increased IFN response. The reduction in viral titres was associated with a reduction in IAV-induced bodyweight loss, which is likely due to the overall reduced lung inflammation. From these observations, it can be postulated that both mitochondrial and endosomal ROS negatively regulate anti-viral cytokine generation. Although the exact mechanisms underlying this phenomenon are not yet characterized, it is tempting to propose that ROS causes oxidation of respiratory chain proteins that affect metabolism and pattern recognition receptors, such as endosomal TLR7, which have been implicated in ROS biology (To et al., 2017).

Both mtROS and NOX2 oxidase-derived ROS were significantly blunted in lung inflammatory cells with daily drug administration of a combination of Cgp91ds-TAT or mitoTEMPO, validating the effectiveness of this approach in dampening ROS production. This is likely due to a reduction in airway neutrophils, as they are a major source of ROS (Dupré-Crochet et al., 2013). Mechanistically, ROS inhibition reduces the recruitment of neutrophils, most likely by lowering the expression of the chemokine receptor CXCL2. Indeed, CXCL2 antagonism protected mice against respiratory IAV and pneumococcal infections, suggesting neutrophilia contributes to a worsening of the pathological condition during influenza infection (Perrone et al., 2008; Tavares et al., 2017). Moreover, another study revealed that the lack of the antioxidant defense enzyme nuclear factor erythroid 2–related factor 2 (NRF-2) caused persistent inflammatory cell infiltration into the lung with elevated levels of CXCL2 expression during hyperoxia-induced acute lung injury (Reddy et al., 2009). However, there is evidence to demonstrate that neutrophils are necessary for host recovery (Fujisawa, 2008). In fact, neutrophil-depleted mice exhibited exacerbated levels of pulmonary inflammation and respiratory dysfunction during IAV infection (Tate et al., 2008; Tate et al., 2009), indicating that an excessive and uncontrolled neutrophilic inflammation precedes mortality in mice. These findings indicate a balance of the pro-oxidative capacity and antioxidant defense system needs to be tightly regulated to avoid complete ablation of neutrophils, as basal levels are necessary for pathogen killing, but too many neutrophils contribute to pathological inflammation owing to their high capacity for ROS production.

Airway and lung parenchymal inflammation are pathological characteristics of IAV and in the present study, IAV infection caused significant and extensive alveolitis, peribronchiolar inflammation and infiltrating inflammatory cells, together with airway epithelial denudation and peri-vascular inflammation. The combination of Cgp91ds-TAT and mitoTEMPO treatment was more effective than monotherapy treatment in attenuating alveolitis and to a lesser degree peribronchiolar inflammation, indicative that ROS produced from these subcellular compartments synergistically exacerbate the resultant pulmonary damage during influenza virus infection. Concurrently, this correlated with an improvement in lung oedema. A key characteristic that perpetuates the formation of lung oedematous tissue during influenza virus challenge is the impairment of the alveolar fluid clearance (Wolk et al., 2008). Interestingly, this deterioration in alveolar fluid clearance was reversed with a protein kinase C inhibitor, which phosphorylates the subunits on NADPH NOX enzymes, thus preventing the culmination of ROS production. This suggests that ROS are a major contributor to the deleterious parenchymal inflammation during IAV infection and that suppression of ROS might be an effective means of minimizing pathological parenchymal inflammation, whilst retaining some level of peri-bronchial inflammation for effective viral clearance. Importantly, targeting these two ROS compartments did not result in a global immunosuppressive phenotype but rather specific elements of the inflammatory response were modulated(Dikalov et al., 2014).

This is the first study to investigate intranasal delivery of a combination of an endosomal and mitochondrial ROS inhibitor as a therapeutic strategy against IAV infection. Intranasal delivery of ROS inhibitors to effectively reduce influenza immunopathology provides a potential therapeutic strategy to reduce the severity of IAV infection. The advent of this novel technology in inhibiting compartmentalized ROS not only advances the fundamental knowledge of how ROS-dependent processes influence IAV pathogenesis, but also highlights exciting and novel interventional strategies for the treatment of IAV infections, which is independent of the infecting viral strain, and appropriate for when annual vaccinations and antivirals fail to offer protection.