The urgent need for antiviral strategies against SARS-CoV-2 and its associated pathology, COVID-19, is highlighted by the 2020 pandemic. Viral infections cause disease through direct cytopathic effects and excessive host inflammatory responses, a phenomenon observed in COVID-19 with its characteristic cytokine storms contributing to disease severity. Therefore, effective antiviral therapies must target both viral replication and the host's inflammatory response. Nuclear factor (erythroid-derived 2)-like 2 (NRF2), a transcription factor, plays a crucial role in cellular protection against stress and inflammation. While studies have shown NRF2's involvement in limiting inflammatory responses to other viruses and its potential regulation by DMF, a clinically approved drug for multiple sclerosis, the impact of NRF2 agonists on SARS-CoV-2 replication remained unknown. This study investigates the role of NRF2 in COVID-19 and evaluates the antiviral and anti-inflammatory potential of NRF2 agonists, specifically 4-OI and DMF.

Previous research has demonstrated the induction of NRF2 by several viruses to mitigate inflammatory responses. For instance, NRF2 deficiency exacerbated influenza infection severity. Dimethyl fumarate (DMF), an FDA-approved drug, possesses anti-inflammatory properties through various mechanisms, including the suppression of inflammation triggered by LPS. 4-OI, another NRF2 agonist, inhibited STING (stimulator of interferon genes), NF-κb, and IFN-stimulated gene expression through NRF2 induction. Single-cell RNA sequencing analysis suggested a correlation between NRF2 antioxidant gene signature and resistance to HIV-1 infection. However, the use of NRF2 agonists to inhibit SARS-CoV-2 or other pathogenic viruses remained unexplored before this current study.

The study employed several methodologies. Initially, publicly available transcriptomic data from lung biopsies of COVID-19 patients were analyzed to identify differentially expressed genes associated with inflammatory and antiviral pathways. This analysis revealed suppressed expression of NRF2-dependent genes in COVID-19 patients. In vitro experiments were conducted using various cell lines (A549, HaCaT, Calu-3, Vero E6, Hu7-T, ASsF cells, and PBMCs from healthy donors and COVID-19 patients) treated with 4-OI and DMF, followed by infection with SARS-CoV-2 or other viruses (HSV-1, HSV-2, Vaccinia, Zika). Viral replication was assessed using qPCR, TCID50 assays, plaque assays, and measurement of viral RNA and protein levels. Cytokine levels were also measured using qPCR. The role of NRF2 was further investigated using siRNA-mediated knockdown. The mechanisms of action were explored by examining IRF3 dimerization and the involvement of pathways like RIG-I and STING. Specific methods included qPCR, immunoblotting, flow cytometry, confocal microscopy, cold binding assay, RNA sequencing, and cell painting assays. The study also involved the analysis of PBMCs from COVID-19 patients to assess the impact of 4-OI on the inflammatory response ex vivo. Air-liquid interface epithelium models were also utilized. Detailed protocols for cell culture, virus infection, RNA extraction, qPCR, Western blotting, IRF3 dimerization assays, and transcriptome analysis are provided in the supplementary material.

The study's key findings include: 1. Downregulation of NRF2-dependent genes in COVID-19 patient lung biopsies. 2. Significant inhibition of SARS-CoV-2 replication by 4-OI and DMF across multiple cell lines, with several logs of magnitude reduction. 3. Broad-spectrum antiviral activity of 4-OI against various viruses (HSV-1, HSV-2, Vaccinia, Zika) through an IFN-independent mechanism, partially dependent on NRF2. 4. Reduction of pro-inflammatory cytokine release in response to SARS-CoV-2 infection by 4-OI and DMF. 5. 4-OI's mechanism of action involves inhibition of IRF3 dimerization without affecting upstream TBK1 phosphorylation. 6. Reduction of CXCL10 expression in PBMCs from COVID-19 patients after 4-OI treatment. 7. 4-OI's effect on inflammatory responses appears to be partially mediated by reduction in intracellular viral RNA and subsequent inhibition of cytokine induction through RNA sensors.

The study's findings directly address the research question by demonstrating the potent antiviral and anti-inflammatory effects of NRF2 agonists against SARS-CoV-2 and other viruses. The IFN-independent mechanism of action is significant, as it suggests a potential therapeutic strategy even in cases of impaired IFN response. The broad-spectrum activity of 4-OI highlights its potential as a pan-viral agent. The observed suppression of inflammatory responses is crucial in mitigating the severe pathology associated with COVID-19. The repurposing of DMF, an already approved drug, presents a readily available and clinically translatable strategy for treating COVID-19. These results contribute significantly to the development of novel therapies for viral infections and offer a new perspective on the therapeutic potential of NRF2 modulation.

This research demonstrates that NRF2 signaling is suppressed in COVID-19, and that NRF2 agonists 4-OI and DMF potently inhibit SARS-CoV-2 replication and inflammation. The broad-spectrum antiviral activity and IFN-independent mechanism make these compounds attractive candidates for COVID-19 and other viral infections. The clinical applicability of DMF highlights the potential for rapid translation of these findings into clinical practice. Further research could explore the precise mechanisms of action, optimize dosing strategies, and evaluate the long-term efficacy and safety of these compounds in clinical trials.

The study primarily relied on in vitro experiments, limiting the direct translation of findings to in vivo settings. While PBMCs from COVID-19 patients were used to evaluate 4-OI's impact on the inflammatory response ex vivo, a larger sample size and in vivo studies would strengthen these findings. Further investigations are needed to explore the long-term safety and efficacy of these compounds in humans. The specific mechanisms of action require further investigation.