Influenza-Induced Oxidative Stress Sensitizes Lung Cells to Bacterial-Toxin-Mediated Necroptosis

SUMMARY Pneumonias caused by influenza A virus (IAV) co- and secondary bacterial infections are characterized by their severity and high mortality rate. Previously, we have shown that bacterial pore-forming toxin (PFT)-mediated necroptosis is a key driver of acute lung injury during bacterial pneumonia. Here, we evaluate the impact of IAV on PFT-induced acute lung injury during co- and secondary Streptococcus pneumoniae (Spn) infection. We observe that IAV synergistically sensitizes lung epithelial cells for PFT-mediated necroptosis in vitro and in murine models of Spn co-infection and secondary infection. Pharmacological induction of oxidative stress without virus sensitizes cells for PFT-mediated necroptosis. Antioxidant treatment or inhibition of necroptosis reduces disease severity during secondary bacterial infection. Our results advance our understanding on the molecular basis of co- and secondary bacterial infection to influenza and identify necroptosis inhibition and antioxidant therapy as potential intervention strategies.


INTRODUCTION
Influenza A virus (IAV) is the most common cause of human influenza (flu) (Morens et al., 2008), infecting 4%-8% of the United States population annually (Tokars et al., 2018). Worldwide, the World Health Organization estimates that flu affects approximately 1 billion individuals annually, with 3 to 5 million cases of severe disease and a resulting 300,000 to 500,000 deaths (Clayville, 2011). IAV alone is capable of considerable morbidity and mortality, and clinical and molecular epidemiology have shown that the most serious infections are frequently associated with co-infections or a secondary infection with a bacterial pathogen. Streptococcus pneumoniae (Spn; the pneumococcus) is the leading cause of community-acquired pneumonia and by far the most common bacterium associated with IAV infections (van der Sluijs et al., 2010). Highlighting the seriousness of IAV/Spn coinfections, previous studies have shown that 34%-55% of the deaths linked to the 2009 IAV pandemic were associated with bacterial infections, with Spn being the most common bacterium identified (Gill et al., 2010;Louie et al., 2009).
Over the past 20 years, many seminal discoveries have helped to explain, at the molecular level, the synergy observed during IAV/Spn super-infection. Key findings include the observation that IAV neuraminidase cleaves terminal sialic acid on host cell glycoconjugates, exposing normally cryptic antigens for bacterial attachment (McCullers and Bartmess, 2003). Viralneuraminidase-cleaved sialic acid serves as a nutrient for Spn and promotes bacterial outgrowth (Hentrich et al., 2016). IAV-induced downregulation of ion channels in bronchial epithelial cell results in dysregulated pulmonary fluid homeostasis that favors bacterial replication (Brand et al., 2018). Cytokines and alarmins released from IAVinfected dying cells elicit a transcriptional response from Spn that causes it to disperse from biofilms and enhances its virulence (Pettigrew et al., 2014). In addition, IAV-induced interferon (IFN) gamma downregulates the expression of scavenger receptors on macrophages, such as MARCO, that are required for uptake of Spn in the absence of a capsule-specific antibody (Sun and Metzger, 2008). Finally, the immune response induced by IAV is inappropriate for the clearance of bacteria and enhances pulmonary injury (Shahangian et al., 2009;van der Sluijs et al., 2006). It is noteworthy that most of this work has not focused on events that occur within lung epithelial cells (LECs), which are the nexus of co-infection.
Necroptosis is a programmed form of cell death that results in host cell membrane loss of integrity and cell lysis, i.e., necrosis. It is inflammatory due to the release of cytoplasmic contents that serve as alarmins. Canonically, necroptosis is regulated by receptor-interacting serine-threonine kinase 1 (RIPK1), which activates RIPK3. Subsequently, RIPK1/RIPK3 activates the necroptosis effector molecule MLKL through phosphorylation, p-MLKL, which targets cell membranes leading to cell rupture and death (Vandenabeele et al., 2010;Moreno-Gonzalez et al., 2016). Importantly, both IAV and bacterial pore-forming toxins (PFTs), such as pneumolysin produced by Spn, have recently been shown to induce necroptosis of LECs in a death-receptor-independent manner (González-Juarbe et al., 2017Nogusa et al., 2016;Wang et al., 2019;Thapa et al., 2016). For IAV, this has been shown to be the result of viral RNA interactions with DNA-dependent activator of IFN regulatory factors (DAI) (also known as Zbp or DLM-1), a sensor for cytoplasmic nucleic acid, which activates RIPK3. Necroptosis of virally infected LECs is thought to be beneficial, as RIPK3 knockout (KO) and MLKL/FADD double-KO mice were considerably more susceptible to IAV, with the latter showing inhibited death of virus-infected cells. Along such lines, RIPK3 activation is necessary to establish an efficient response to the virus. (Nailwal and Chan, 2019;Nogusa et al., 2016;Balachandran and Rall, 2020). Our research group has shown that during bacterial pneumonia, initiation of necroptosis (i.e., MLKL-activity) is detrimental and exacerbates bacterial outgrowth, pulmonary injury, and loss of alveolar-capillary integrity (González-Juarbe et al., 2015a;González-Juarbe et al., 2017). Together, these observations suggest that inhibition of MLKL may have a possible protective role for viral and bacterial infections alike and extend to co-bacterial infections and secondary bacterial infections (SBIs). Critically and up to this point, the role of necroptosis on disease severity during IAV/bacterial co-infection was not known. Here, we determined its consequence and identified IAV-induced oxidative stress as a sensitizing agent for PFT-mediated cell death.

Necroptosis Is Synergistically Increased during IAV/Spn Co-infection
Using an established mouse model of co-infection (McCullers and Hayden, 2012;McCullers and Tuomanen, 2001), we recapitulated the synergy known to occur between IAV and Spn. Briefly, we observed a >50-fold increase in the amount of Spn present in bronchoalveolar lavage fluid (BALF) and blood ( Figures 1A and 1B), as well as a significant decrease in time to death following IAV/Spn challenge versus Spn or IAV alone ( Figure 1C). Importantly, ongoing IAV infection synergistically enhanced the number of lung cells undergoing necroptosis after Spn challenge; necroptosis activity in frozen lung sections was inferred by immunofluorescent detection of p-MLKL ( Figures 1D and 1E).
To validate this in vivo observation and to begin to dissect the molecular mechanisms underlying IAV-enhanced bacteria-induced necroptosis, we used an established in vitro coinfection model (Hoffmann et al., 2016). Briefly, A549 type II alveolar epithelial cells were infected with either pdmH1N1 or PR8 at a MOI of 2 for 2 h and then challenged with Spn at a MOI of 10 for another 4 h. Importantly, A549 cytotoxicity was synergistically increased in cells challenged with both pathogens (Figures 2A and 2B). Similar results were also observed with MH-S murine alveolar macrophages ( Figure 2C), indicating influenzamediated sensitization to necroptosis is not restricted to airway epithelial cells. Notably, the enhanced death of A549 co-infected cells occurred without significant differences in bacterial titers versus control ( Figure S1A), indicating that the increased levels of necroptosis observed in vivo were not solely due to increased bacterial burden. Tumor necrosis factor (TNF) and IFN responses have been shown to promote necroptosis during viral infection (Upton et al., 2017). Along such lines, inhibition of TNF receptor 1 or blocking of TNF-α by pre-treatment of cells with R7050 or SPD304, respectively, did not reduce influenza-induced cell death potentiation in A549 cells in vitro ( Figure S1B). Moreover, the time frame of the in vitro model did not lead to significant increases in the IFN response ( Figure S1C). Altogether, our results show that there is no evidence supporting a role for the synergistic initiation of receptor-mediated apoptosis in vitro or in vivo under the conditions tested ( Figure S2).

PFT Activity Is Required for Spn-Induced Necroptosis during Co-infection
Spn-mediated cytotoxicity of LECs was found to require the pore-forming activity of its PFT pneumolysin (Figures 2A and 2B). Furthermore, when A549 cells were treated with inhibitors of MLKL, necrosulfonamide (NSA) (Figure 2A), RIPK3, or GSK' 872 ( Figure  S3A), the enhanced sensitivity of these cells to Spn killing was lost. Challenge of IAVinfected A549 cells with recombinant pneumolysin (rPly) or α-toxin (the PFT of Staphylococcus aureus, the second most common isolate during SBIs to influenza; Morris et al., 2017), recapitulated the potentiation of cell cytotoxicity observed with live bacterial infection ( Figure S3B-S3C). Potentiation of necroptosis by IAV was confirmed by immunoblot and immunofluorescent staining, which both showed enhanced amounts of p-MLKL in A549 cells ( Figures S3D-S3F). Further supporting a key role for necroptosis was the observation that A549 cells deficient in MLKL were protected against exacerbated PFTmediated cell death after influenza infection ( Figure 2C). Moreover, the same results were observed with primary normal human bronchiolar epithelial cells (nHBEs) ex vivo ( Figure   S3G). Together, our data provide evidence that necroptosis is the main contributor of cell necrosis during coand secondary infection. Notably, caspase inhibition ( Figure S2) did not confer significant protection, suggesting a smaller role for TLR and inflammasome signaling (apoptosis and pyroptosis) in the potentiation of cell death. In support of these results, we have previously demonstrated that pneumolysin alone leads to death-receptor-independent necroptosis in pulmonary epithelial cells (González-Juarbe et al., 2017).

IAV-Induced Oxidative Stress Sensitizes Cells In Vitro for PFT-Mediated Necroptosis
IAV-mediated oxidative stress has potent effects on pulmonary epithelial cells and the immune system (Liu et al., 2017). Therefore, it seemed plausible that the oxidative stress induced by the virus may be contributing toward the potentiation of pneumolysin-mediated necroptosis. In support of this notion, we observed that respiratory epithelial cells challenged in vitro with pdmH1N1or PR8 showed increased levels of lipid peroxidation ( Figure 3A; Figure S4A), as measured by malondialdehyde (MDA), and cellular reactive oxygen species (ROS) ( Figure 3B; Figure S4B), as measured using cell-permeant 2', 7'dichlorodihydrofluorescein diacetate (H2-DCF). Importantly, and despite not having an effect on viral titers during the course of infection ( Figure 3C), pre-treatment of A549 cells with the su-peroxide dismutase mimetic Tempol (Brissac et al., 2017) before viral challenge reduced cell death and MLKL activation in co-infected cells ( Figures 3D and 3E). Directly implicating oxidative stress as a primer for PFT-induced necroptosis, treatment of cells with paraquat (González-Juarbe et al., 2015a) enhanced the toxicity of rPly toward LECs, and the observed potentiating effect of paraquat was abolished by treatment with Tempol ( Figure  3F). Identical results were observed using nHBEs ex vivo ( Figure 3G) and were replicated by addition of exogenous H 2 O 2 in place of paraquat to A549 epithelial cells prior to rPly challenge ( Figure S4C). Note that Spn also produces H 2 O 2 by its metabolic enzyme SpxB (Brissac et al., 2017). Yet, IAV potentiation of cell death was also observed in A549 cells challenged with Spn ΔspxB ( Figure S4D), indicating that the priming effect of viral-induced ROS was sufficient. Importantly, inhibition of ROS in A549 cells with rotenone + thallium trifluoroacetate (mitochondria-dependent ROS inhibitor), apocynin (nicotinamide adenine dinucleotide phosphate [NADPH]-dependent ROS inhibitor), allopurinol (xanthine-oxidasedependent ROS inhibitor), or mefenamic acid (cyclooxygenase-dependent ROS inhibitor) all conferred protection against death caused by co-infection ( Figure 3H). These results suggested that ROS potentiation of necroptosis may come from multiple cellular sources. Last, and to further probe the specificity of oxidative stress as a primer for necroptosis, we tested whether blockage of viral neuraminidase activity with oseltamivir (Takahashi et al., 2003) or treatment of cells with Pimodivir (VX-787) (Byrn et al., 2015), a non-nucleoside polymerase basic protein 2 subunit inhibitor, impacted cell death. Neither oseltamivir nor Pimodivir affected cell death ( Figures S5A and S5B), even though viral titers were decreased by Pimodivir treatment ( Figure S5C).

IAV-Induced Oxidative Stress Remains Beyond Viral Clearance and Maintains Susceptibility to Bacterial-Toxin-Mediated Necroptosis
We examined whether residual oxidative stress induced by IAV helped to explain the enhanced susceptibility to bacterial infection that occurs even after IAV is cleared, i.e., in a secondary infection model. Lung sections from pdmH1N1-challenged mice 10 days post-IAV infection showed considerable evidence of oxidative damage to DNA, as well as lipid peroxidation (immunofluorescence [IF] 8-hydroxydeoxyguanosine and 4-hydroxynonenal staining, respectively) (Helbock et al., 1999;Kruman et al., 1997) in pulmonary tissue ( Figures 4A-4D). Notably, these mice were confirmed to not have detectable virus ( Figure  4E). Similar to co-infection results (see Figure 1), if these mice were challenged with Spn, we observed a >100-fold increase in bacterial lung titers 2 days after Spn challenge ( Figure   5A). This finding was concomitant with greater lung consolidation, immune cell infiltration ( Figure S6), and substantially enhanced levels of lung necroptosis in co-infected mice versus those with Spn alone (Figures 5B-5D). Importantly, mice challenged with TIGR4 Δply in our secondary infection model had MLKL activation levels and bacterial titers equivalent to our negative control, i.e., mice infected with wild-type TIGR4 but also receiving the necroptosis inhibitor Nec-1s (Figures 5E-5G; Figure S7). Furthermore, TIGR4 Δplychallenged IAV-infected mice had decreased mortality versus controls ( Figure 5H). Interestingly, Tempol treatment at 12 and 24 h post-Spn infection reduced the amount of necroptosis in the airway in our secondary IAV/Spn infection model, which happened despite Tempol not having an observed effect on in vivo levels of lipid oxidation ( Figures   6A-6E). Tempol treatment also reduced bacterial burden within the airway of infected mice ( Figure 6F). Thus, necroptosis sensitizing ROS is primarily due to the virus, persisted beyond detectable IAV infection, and acted directly to sensitize the cell for necroptosis.

In Vivo Necroptosis Inhibition Reduces the Severity of SBIs to Influenza
Although no changes in oxidative-stress-induced DNA damage were observed (Figures 7A and 7B), MLKL-deficient mice with secondary Spn infection had reduced bacterial titers, reduced lung consolidation, and a reduction in overall TUNEL-positive staining in lung sections (a general marker of cell death) ( Figures 7C-7G). In addition, lungs of MLKL KO mice showed decreased levels of IFN-α and -β, suggesting a possible role for necroptosis in the IFN response during secondary infections ( Figures 7H and 7I). Most importantly, MLKL KO mice had greater survival than the control mice in our secondary infection model ( Figure  7J). Altogether, our results implicate oxidative-stress-enhanced PFT-mediated necroptosis activity as a major driver of disease severity and lung injury during co-and secondary infections to influenza.

DISCUSSION
The molecular mechanisms of IAV subversion of cellular defenses and programmed cell death continue to be investigated (Yeganeh et al., 2013;Balachandran and Rall, 2020). Only recently has it become apparent that necroptosis is essential for the control of virus replication during infection . Herein, we demonstrate that oxidative stress triggered by IAV infection plays a role in the potentiation of PFT-induced necroptosis in respiratory cells and thereby worsens injury. Furthermore, pharmacological blocking of ROS from distinct sources conferred protection against IAV-induced sensitization to PFTinduced necroptosis.
Oxidative stress is pleiotropic and capable of oxidizing proteins and lipid membranes, damaging nucleic acids, and potentially altering cellular energy levels or ion homeostasis of the cell (Sies et al., 2017). The latter were shown to be triggers for non-canonical activation of necroptosis within bacteria-infected cells (González-Juarbe et al., 2015a. Importantly, in this study, increased susceptibility to PFT-mediated necroptosis was still observed even when IAV replication was blocked with Pimodivir. Moreover, Tempolmediated protection against priming for PFT killing was applicable to both the co-infection and secondary infection scenario, namely, the latter when virus is no longer present. This suggests the mechanism responsible for IAV-mediated necroptosis potentiation is directly affected by acute intracellular ROS levels and is independent of viral replication. Whether and how the latter pathways are sensitized as a result of IAV-induced ROS or if an independent mechanism is responsible remains unclear, and detailed studies are now warranted to discern key similarities and differences between these events.
Necroptosis is inflammatory and therefore a "dual-edged sword" important for instigating a robust immune response, but it is also detrimental under conditions in which it is excessive (Yeganeh et al., 2013;Balachandran and Rall, 2020). Along such lines, sensitization to necroptosis most likely contributes to a variety of clinical problems during co-and secondary pneumonia marked by excessive inflammation, such as acute respiratory distress syndrome and sepsis, which are both a direct consequence of the enhanced level of cell death and release of pro-inflammatory alarmins. It is noteworthy that IAV has been specifically demonstrated to drive Spn development of otitis media (Tong et al., 2000), which is recognized to be a chronic inflammatory state. Critically, it is unknown if other viruses enhance permissiveness for PFT-mediated necroptosis, and this is an important avenue of future investigation. In support of this notion, a wide variety of viruses have been shown to induce oxidative stress in host cells by a variety of means (Schwarz, 1996). For example, respiratory syncytial virus does so by modulating levels of antioxidant enzymes (Hosakote et al., 2009) and has recently been shown to induce necroptosis in children and in a neonatal mouse model of infection (Simpson et al., 2020). Thus, it is likely that this phenomena is not restricted to IAV. Our prior published work (González-Juarbe et al., 2015a and that with S. aureus α-toxin herein, which showed a wide variety of PFTproducing bacteria can instigate necroptosis of LECs, suggest viral-enhanced PFT-mediated necroptosis is not restricted to the pathogen Spn. Thus, this synergy may be an important contributor to enhanced disease severity at other anatomical sites in which virus and bacteria can co-infect. Here, we demonstrate the critical role of pneumolysin in inducing cell necroptosis during secondary infections to influenza. Interestingly, Wolf et al. (2014) have shown that prior exposure of mice to a pneumolysin overexpressing Spn strain conferred protection against virus-induced morbidity and lung pathology. The authors concluded that immunity mounted against pneumolysin and its modulation of macrophage activity by arginase-1 reduce the severity of infection after re-challenge with pneumococcus and/or influenza infection. A recent publication by our group also demonstrated that pneumolysin induction of necroptosis during bacterial colonization initiated low levels of inflammation required for the generation of an antibody-based response against Spn (Riegler et al., 2019). These findings suggest that the order in which the host is exposed to virus and bacteria has a profound impact on outcomes. Along such lines, McCullers and Rehg (2002) showed that Spn infection preceding IAV conferred protection against secondary bacterial challenge, whereas the reverse order of infection resulted in the dramatic sensitization to Spn that is studied herein.
We now recognize IAV-induced susceptibility to necroptosis by oxidative stress as one contributing factor for this sequence-dependent sensitization.
Of note and during infection, other cells, particularly innate immune cells, also produce copious amounts of ROS (Chen et al., 2018), and they too may contribute to sensitization of LECs. Along such lines, To et al. (2020) recently showed that influenza infection promotes mitochondrial-derived ROS and that this in itself is detrimental to the lung epithelium, as measured by increased histopathological damage. Detailed studies are thereby required to better define the role of the bystander cells into these mechanisms as well as their own sensitivity to PFT-mediated killing during ROS production. Our results showing no differences in caspase activation suggest the responsible mechanism is also independent of canonical apoptotic and pyroptotic pathways, although it is likely these mechanisms are contributory to overall disease and occur in parallel during natural infection. This is in agreement with our previous observation that showed pneumolysin is able to initiate necroptosis in a death-receptor (TNFr and TLR4)-independent manner and that caspase activity may occur in parallel with necroptosis to modulate inflammation (González-Juarbe et al., 2017. Finally, our results suggest that inhibition of the necroptosis pathway may be a viable therapeutic treatment during IAV-mediated co-or secondary infections, although the possibility remains that necroptosis inhibition may promote viral replication during coinfection, an aspect that needs to be studied (unpublished results with primary NHBEs suggest it does not). It is also important to consider that inhibition of RIPK1 and RIPK3 will have effects on aspects other than necroptosis, as the pathway is linked to apoptosis, nuclear factor κB (NF-κB) signaling, inflammation, and responses to ion changes (He and Wang, 2018). For this reason, targeting MLKL alone may be preferable, as it will not inhibit the ability of the cell to drive other responses beneficial against infection and reduce necrotic tissue damage and excess inflammation (Moreno-Gonzalez et al., 2016). Altogether, our results provide a molecular explanation for how influenza infection enhances permissiveness for SBIs. We demonstrate that PFT-mediated necroptosis is enhanced as a result of oxidative stress caused by prior or ongoing viral replications. Increased sensitivity to PFT-mediated necroptosis in turn worsens pulmonary damage and creates an environment that is further permissive for bacterial replication. The fact that oxidative stress induced by virus and PFT production are common across a wide range of viral and bacterial pathogens, respectively, suggests this is an important aspect of human infectious disease pathogenesis.
Cell Infections-A549 type II alveolar epithelial cells (Fulcher et al., 2005), MH-S mouse alveolar macrophages (Saxena et al., 2008), and primary normal human bronchiolar epithelial cells (Fulcher et al., 2005), were infected with IAV at MOI 2 for 2 hours, and subsequently challenged with Spn at an MOI 10 for 4 hours. As our purpose was to characterize the cellular responses to influenza infection in regard to potentiation of bacteria induced cell death without the complication of cell lysis caused by late stage replication we used a high infectious dose and short time period. Other investigators have used the same approach (Hoffmann et al., 2016). The majority of chemical inhibitors were obtained from Sigma-Aldrich. Exceptions include necrosulfonamide (Tocris Bioscience, QL, UK), GSK'872 and Nec1s (BioVision, Milpitas, CA), oseltamivir carboxylate (MCE, Monmouth, NJ), TNFR inhibitor R-7050 and TNF-α inhibitor SPD-304 (Cayman Chemicals, Ann Arbor, MI) and Pimodivir (AdooQ Bioscience, Irvine, CA). Cells receiving inhibitors were treated continuously beginning 1-hour prior to IAV infection and inhibitor was maintained in the media until the end of the experiment. Pimodivir treated cells received the drug 2-hours prior to IAV challenge. A549 cells deficient in MLKL have been previously described (González-Juarbe et al., 2018). Cell death was evaluated by detection of lactate dehydrogenase (LDH) in culture supernatants (González-Juarbe et al., 2015a). The presence of reactive oxygen species (ROS) was measured with the H2-DCF assay (Thermo Fisher Scientific, Waltham, MA). Lipid peroxidation was detected with the lipid peroxidation malondialdehyde (MDA) assay (Abcam). Antibodies against 8-hydroxydeoxyguanosine, an oxidative stress-mediated DNA damage marker, and HNE-J, a lipid peroxidation marker, were purchased (Abcam).

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.
(F) Cytotoxicity was measured in A549 cells pre-treated with Tempol and then treated with Paraquat (10 μM) for an additional 2 h, followed by challenge with rPly (0.1 μg) for 2 h. (G) Cytotoxicity of ex-vivo-cultured primary normal human bronchial epithelial cells pretreated with Tempol for 1 h, infected with pdmH1N1 at a MOI of 2 for 2 h, and challenged with Spn at an MOI of 10 for 4 additional h.