SARS-CoV2-mediated suppression of NRF2-signaling reveals potent antiviral and anti-inflammatory activity of 4-octyl-itaconate and dimethyl fumarate

The study addresses the urgent need for broad-spectrum antiviral strategies against SARS-CoV-2 that also mitigate excessive host inflammation. The authors focus on the NRF2 pathway, a master regulator of antioxidant and anti-inflammatory responses that is normally restrained by KEAP1 and activated during oxidative stress to induce cytoprotective genes. Prior observations suggested NRF2 can modulate inflammatory signaling and disease severity during viral infections. The central research questions are: (1) whether NRF2 signaling is altered in COVID-19 patient tissues; (2) whether pharmacologic NRF2 agonists—4-octyl-itaconate (4-OI) and the clinically approved dimethyl fumarate (DMF)—can inhibit SARS-CoV-2 replication; (3) whether such effects extend to other human pathogenic viruses; and (4) whether NRF2 activation dampens virus-induced inflammatory responses via IFN-independent mechanisms. The purpose is to identify repurposable host-directed therapeutics that both suppress viral replication and limit harmful inflammation in COVID-19.

Existing literature indicates NRF2 regulates antioxidant and anti-inflammatory gene networks and protects against stress-induced cell death. NRF2 has been implicated as a master regulator of tissue damage during infection and an important modulator of inflammatory responses. Reports have shown NRF2 induction in response to certain viral infections can limit inflammation, and NRF2 deficiency worsens influenza severity. DMF, an FDA-approved drug for multiple sclerosis, suppresses inflammation via multiple mechanisms, including NRF2 activation. Prior work showed 4-OI can inhibit LPS-induced inflammation and suppress STING- and NF-κB/IFN-stimulated gene expression via NRF2 induction. Single-cell RNA-seq studies suggested an NRF2 antioxidant signature correlates with resistance to viral infection. However, before this work it was unclear whether NRF2 agonists could inhibit SARS-CoV-2 or other pathogenic viruses and through what mechanisms.

- Patient transcriptomics: Public RNA-seq datasets, including lung biopsies/autopsies from COVID-19 patients, were reanalyzed (e.g., Blanco-Melo et al.; Desai et al.). Differential expression used DESeq2 (Wald test, Benjamini–Hochberg correction; adjusted p < 0.05, |log2FC| > 1). Pathway enrichment used EnrichR; clustering assessed patterns in inflammatory/IFN vs NRF2 target gene sets. - Cell lines and primary cells: TMPRSS2-expressing Vero E6 cells, Calu-3 lung epithelial cells, A549, HaCaT keratinocytes, HEK293T, and others were cultured under standard conditions. Peripheral blood mononuclear cells (PBMCs) from healthy donors and from four ICU-admitted COVID-19 patients were used ex vivo. An air–liquid interface nasal epithelium model was also described. - Compounds: NRF2 agonists 4-octyl-itaconate (4-OI) and dimethyl fumarate (DMF) were used; dosing commonly 25–125 µM and pre-treatment for ~48 h before infection/stimulation. - Viral infections: SARS-CoV-2 infection in TMPRSS2-Vero E6 and Calu-3 cells; readouts included qPCR for viral RNA, TCID50, and plaque assays. Additional infections included HSV-1/HSV-2 in HaCaT cells, vaccinia virus (VACV; GFP-expressing) and ectromelia virus (ECTV; mCherry) in HaCaT and BMDCs, and Zika virus (ZIKV) in A549 and Huh7-derived cells. HSV-1 entry assays used cold binding protocols with qPCR quantification of bound viral DNA. - Genetic perturbations: siRNA silencing of NRF2 to test dependence of the antiviral effect; overexpression and silencing approaches to probe IRF3 signaling components. - Inflammatory readouts: qPCR of cytokine/chemokine genes (e.g., IFNB1, TNF, CXCL10, CCL5, IL1B) in infected or ligand-stimulated cells (e.g., RIG-I agonist, dsDNA, cGAMP). Western blotting for NRF2 targets (HO-1, NQO1, SQSTM1), signaling proteins (TBK1, IRF3), and loading controls. - IRF3 dimerization assay: Native gel-based assays assessed IRF3 dimer formation upon stimulation, with and without 4-OI/DMF treatment, alongside assessment of TBK1 and IRF3 phosphorylation. - Statistics: Typically two-tailed Mann–Whitney tests (and some two-tailed t-tests), with significance thresholds *p < 0.05, **p < 0.01, ***p < 0.001; data reported as mean ± s.e.m. with biological replicates and independent experiments as indicated.

- Suppressed NRF2 pathway in COVID-19 tissues: RNA-seq reanalysis of lung biopsies/autopsies from COVID-19 patients showed enrichment of inflammatory/antiviral pathways (RIG-I, TLR) and suppression of NRF2-dependent antioxidant response genes (Fig. 1). Heat maps of NRF2 target genes confirmed downregulation in patient samples. - Potent inhibition of SARS-CoV-2 replication by NRF2 agonists: 4-OI and DMF markedly inhibited SARS-CoV-2 replication in TMPRSS2-Vero E6 and Calu-3 cells, reducing viral RNA and infectious titers by several logs (multiple assays: qPCR, TCID50, plaque). Reported p-values include 0.0001, 0.0002, 0.026, 0.0079 depending on assay and condition (Fig. 2), indicating robust statistical significance. - Broad-spectrum antiviral activity via IFN-independent mechanism: 4-OI inhibited HSV-1 and HSV-2 in HaCaT cells, lowering viral titers, intracellular viral RNA, and viral protein accumulation while strongly inducing NRF2 targets HO-1, NQO1, and SQSTM1. Silencing NRF2 by siRNA diminished the antiviral effect, indicating NRF2 dependence. 4-OI also reduced VACV/ECTV infectivity and ZIKV replication in A549 and Huh7-T cells. Antiviral effects occurred despite suppression of type I IFN responses, demonstrating an IFN-independent antiviral program. - Anti-inflammatory effects during SARS-CoV-2 infection and stimulation: In Calu-3 and other models, 4-OI and DMF significantly reduced expression of IFNB1, TNF, and CXCL10 during SARS-CoV-2 infection (e.g., IFNB1 p = 0.0010; CXCL10 p < 0.0001; TNF p < 0.0001 in various panels). In healthy donor PBMCs, SARS-CoV-2 induced modest CXCL10, which was reduced by 4-OI; in PBMCs from four severe COVID-19 patients, elevated CXCL10 levels were reduced to near-basal after 4-OI treatment in all four donors. - Mechanism involves inhibition of IRF3 dimerization without blocking upstream phosphorylation: 4-OI reduced RIG-I-induced IFN responses by blocking IRF3 dimerization while not inhibiting upstream TBK1 or IRF3 phosphorylation. 4-OI also inhibited responses to STING agonists (dsDNA, cGAMP) and HSV-1, consistent with prior observations that 4-OI can downregulate STING expression. Silencing of NRF2 impaired these inhibitory effects, supporting an NRF2-driven mechanism.

The findings demonstrate that SARS-CoV-2 infection is associated with suppression of NRF2-dependent antioxidant genes in patient lung tissues, implicating NRF2 dysregulation in COVID-19 pathogenesis. Pharmacologic activation of NRF2 with 4-OI or DMF restores a host-protective program that simultaneously suppresses viral replication and dampens excessive inflammatory and type I IFN responses. Notably, the antiviral activity remains effective despite attenuation of IFN signaling, indicating an IFN-independent antiviral state that likely involves metabolic and redox reprogramming and interference with IRF3 dimerization. The broad-spectrum efficacy against HSV-1/2, VACV, and ZIKV suggests NRF2 activation targets host pathways essential for replication of diverse viruses. Importantly, the anti-inflammatory effects (e.g., reduced IFNB1, TNF, CXCL10) align with therapeutic goals to mitigate cytokine-driven pathology in COVID-19. Given DMF’s established clinical use and safety profile in MS, repurposing it for COVID-19 could expedite translation, although careful evaluation of timing, dosing, and patient selection will be required to balance antiviral benefits with controlled immune responses.

This study identifies suppression of NRF2 signaling as a feature of COVID-19 lung pathology and shows that NRF2 agonists 4-OI and DMF elicit a robust, IFN-independent antiviral program that inhibits SARS-CoV-2 and other pathogenic viruses while concurrently reducing pro-inflammatory cytokine responses. These results highlight NRF2 activation as a promising host-directed, broad-spectrum antiviral and anti-inflammatory strategy. The clinically approved status of DMF supports rapid clinical evaluation in COVID-19. Future research should include in vivo efficacy and safety studies, optimization of dosing and treatment windows, mechanistic dissection of NRF2-mediated antiviral pathways (including IRF3 dimerization control and metabolic effects), and randomized clinical trials assessing clinical outcomes and biomarkers of inflammation.

- Most efficacy data derive from in vitro cell lines and ex vivo PBMC assays with small sample sizes (e.g., four severe COVID-19 patients), limiting generalizability to in vivo human disease. - The precise molecular mechanism of the IFN-independent antiviral state is only partially defined; while IRF3 dimerization is inhibited, upstream signaling and additional NRF2-regulated pathways likely contribute. - Potential immunomodulatory effects (suppression of IFN responses) could be context-dependent; inappropriate timing or dosing might impair necessary antiviral immunity in some settings. - Lack of animal model data and clinical trial evidence in this study precludes conclusions about therapeutic efficacy, safety, and optimal regimens in patients.