Respiratory syncytial virus (RSV) can cause severe respiratory illness in infants, immunocompromised, and older adults. Despite its burden, no vaccine or specific treatment is available. RSV infection is associated with increased reactive oxygen species (ROS) production, degradation of the transcription factor nuclear factor erythroid 2-related factor 2 (NRF2), and decreased antioxidant enzymes (AOEs), leading to oxidative damage and lung injury. Hydrogen sulfide (H2S) is an endogenous gaseous molecule that plays a physiological role in numerous cellular processes and a protective role in multiple pathological conditions, displaying vasoactive, cytoprotective, anti-inflammatory, and antioxidant activities. H2S can promote NRF2 activation through the sulfhydration of Kelch-like ECH-associated protein 1, the cytoplasmic repressor of NRF2. Here we investigated whether increasing cellular H2S levels could rescue NRF2 and NRF2-dependent gene expression in RSV-infected primary airway epithelial cells. We found that treatment with the H2S donor GYY4137 significantly increased NRF2 levels and AOEs gene expression by decreasing KEAP1 levels, and by modulating pathways involved in RSV-induced NRF2 degradation, such as NRF2 ubiquitination, and promyelocytic leukemia (PML) protein levels. These results suggest that the administration of exogenous H2S can positively impact the altered redox balance associated with RSV infection, which represents an important determinant of RSV-induced lung disease.

Several viruses have been shown to modulate the transcription factor nuclear factor erythroid 2-related factor 2 (NRF2), the master regulator of redox homeostasis. The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for the COVID-19 pandemic, also seems to disrupt the balance between oxidants and antioxidants, which likely contributes to lung damage. Using in vitro and in vivo models of infection, we investigated how SARS-CoV-2 modulates the transcription factor NRF2 and its dependent genes, as well as the role of NRF2 during SARS-CoV-2 infection. We found that SARS-CoV-2 infection downregulates NRF2 protein levels and NRF2-dependent gene expression in human airway epithelial cells and in lungs of BALB/c mice. Reductions in cellular levels of NRF2 seem to be independent of proteasomal degradation and the interferon/promyelocytic leukemia (IFN/PML) pathway. Furthermore, lack of the Nrf2 gene in SARS-CoV-2-infected mice exacerbates clinical disease, increases lung inflammation, and is associated with a trend toward increased lung viral titers, indicating that NRF2 has a protective role during this viral infection. In summary, our results suggest that SARS-CoV-2 infection alters the cellular redox balance by downregulating NRF2 and its dependent genes, which exacerbates lung inflammation and disease, therefore, suggesting that the activation of NRF2 could be explored as therapeutic approach during SARS-CoV-2 infection. IMPORTANCE The antioxidant defense system plays a major function in protecting the organism against oxidative damage caused by free radicals. COVID-19 patients often present with biochemical characteristics of uncontrolled pro-oxidative responses in the respiratory tract. We show herein that SARS-CoV-2 variants, including Omicron, are potent inhibitors of cellular and lung nuclear factor erythroid 2-related factor 2 (NRF2), the master transcription factor that controls the expression of antioxidant and cytoprotective enzymes. Moreover, we show that mice lacking the Nrf2 gene show increased clinical signs of disease and lung pathology when infected with a mouse-adapted strain of SARS-CoV-2. Overall, this study provides a mechanistic explanation for the observed unbalanced pro-oxidative response in SARS-CoV-2 infections and suggests that therapeutic strategies for COVID-19 may consider the use of pharmacologic agents that are known to boost the expression levels of cellular NRF2.

RSV and SARS-CoV-2 are prone to co-infection with other respiratory viruses. In this study, we use RSV/SARS-CoV-2 co-infection to evaluate changes to clinical disease and viral replication in vivo. To consider the severity of RSV infection, effect of sequential infection, and the impact of infection timing, mice were co-infected with varying doses and timing. Compared with a single infection of RSV or SARS-CoV-2, the co-infection of RSV/SARS-CoV-2 and the primary infection of RSV followed by SARS-CoV-2 results in protection from SARS-CoV-2-induced clinical disease and reduces SARS-CoV-2 replication. Co-infection also augmented RSV replication at early timepoints with only the low dose. Additionally, the sequential infection of RSV followed by SARS-CoV-2 led to improved RSV clearance regardless of viral load. However, SARS-CoV-2 infection followed by RSV results in enhanced SARS-CoV-2-induced disease while protecting from RSV-induced disease. SARS-CoV-2/RSV sequential infection also reduced RSV replication in the lung tissue, regardless of viral load. Collectively, these data suggest that RSV and SARS-CoV-2 co-infection may afford protection from or enhancement of disease based on variation in infection timing, viral infection order, and/or viral dose. In the pediatric population, understanding these infection dynamics will be critical to treat patients and mitigate disease outcomes.

Poly(ADP-ribose) polymerase (PARP) is involved in the pathogenesis of cell dysfunction, inflammation and organ failure during septic shock. The goal of the current study was to investigate the efficacy and safety of the clinically approved PARP inhibitor olaparib in experimental models of oxidative stress in vitro and in sepsis in vivo. In mice subjected to cecal ligation and puncture (CLP) organ injury markers, circulating and splenic immune cell distributions, circulating mediators, DNA integrity and survival was measured. In U937 cells subjected to oxidative stress, cellular bioenergetics, viability and DNA integrity were measured. Olaparib was used to inhibit PARP. The results show that in adult male mice subjected to CLP, olaparib (1-10 mg/kg i.p.) improved multiorgan dysfunction. Olaparib treatment reduced the degree of bacterial CFUs. Olaparib attenuated the increases in the levels of several circulating mediators in the plasma. In the spleen, the number of CD4+ and CD8+ lymphocytes were reduced in response to CLP; this reduction was inhibited by olaparib treatment. Treg but not Th17 lymphocytes increased in response to CLP; these cell populations were reduced in sepsis when the animals received olaparib. The Th17/Treg ratio was lower in CLP-olaparib group than in the CLP control group. Analysis of miRNA expression identified a multitude of changes in spleen and circulating white blood cell miRNA levels after CLP; olaparib treatment selectively modulated these responses. Olaparib extended the survival rate of mice subjected to CLP. In contrast to males, in female mice olaparib did not have significant protective effects in CLP. In aged mice olaparib exerted beneficial effects that were less pronounced than the effects obtained in young adult males. In in vitro experiments in U937 cells subjected to oxidative stress, olaparib (1-100 μM) inhibited PARP activity, protected against the loss of cell viability, preserved NAD+ levels and improved cellular bioenergetics. In none of the in vivo or in vitro experiments did we observe any adverse effects of olaparib on nuclear or mitochondrial DNA integrity. In conclusion, olaparib improves organ function and extends survival in septic shock. Repurposing and eventual clinical introduction of this clinically approved PARP inhibitor may be warranted for the experimental therapy of septic shock.

Cystathionine-y-lyase (CSE) is a critical enzyme for hydrogen sulfide (H2S) biosynthesis and plays a key role in respiratory syncytial virus (RSV) pathogenesis. The transcription factor NRF2 is the master regulator of cytoprotective and antioxidant gene expression, and is degraded during RSV infection. While some evidence supports the role of NRF2 in CSE gene transcription, its role in CSE expression in airway epithelial cells is not known. Here, we show that RSV infection decreased CSE expression and activity in primary small airway epithelial (SAE) cells, while treatment with tert-butylhydroquinone (tBHQ), an NRF2 inducer, led to an increase of both. Using reporter gene assays, we identified an NRF2 response element required for the NRF2 inducible expression of the CSE promoter. Electrophoretic mobility shift assays demonstrated inducible specific NRF2 binding to the DNA probe corresponding to the putative CSE promoter NRF2 binding sequence. Using chromatin immunoprecipitation assays, we found a 50% reduction in NRF2 binding to the endogenous CSE proximal promoter in SAE cells infected with RSV, and increased binding in cells stimulated with tBHQ. Our results support the hypothesis that NRF2 regulates CSE gene transcription in airway epithelial cells, and that RSV-induced NRF2 degradation likely accounts for the observed reduced CSE expression and activity.