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

Abstract

Antiviral strategies to inhibit Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV2) and the pathogenic consequences of COVID-19 are urgently required. Here, we demonstrate that the NRF2 antioxidant gene expression pathway is suppressed in biopsies obtained from COVID-19 patients. Further, we uncover that NRF2 agonists 4-octyl-itaconate (4-OI) and the clinically approved dimethyl fumarate (DMF) induce a cellular antiviral program that potently inhibits replication of SARS-CoV2 across cell lines. The inhibitory effect of 4-OI and DMF extends to the replication of several other pathogenic viruses including Herpes Simplex Virus-1 and-2, Vaccinia virus, and Zika virus through a type I interferon (IFN)-independent mechanism. In addition, 4-OI and DMF limit host inflammatory responses to SARS-CoV2 infection associated with airway COVID-19 pathology. In conclusion, NRF2 agonists 4-OI and DMF induce a distinct IFN-independent antiviral program that is broadly effective in limiting virus replication and in suppressing the pro-inflammatory responses of human pathogenic viruses, including SARS-CoV2.

Fig. 1. Expression of NRF2-driven genes is suppressed in COVID-19 patient biopsies.

a, b, c Reanalysis of data published by Blanco-Melo et al. (a) Bar-chart of the number of transcripts showing significant differential expression (adjusted p value < 0.05 and log2(FC) > 1.0). Data from COVID19 lung biopsies was normalized against healthy lung biopsies, and in cell lines Calu3, NHBE, and A549 infected with either SARS-CoV2, Influenza A virus (IAV), Respiratory Syncytial virus (RSV), or human parainfluenza virus type 3 (HPIV3) against mock treated cells. Expression and p values were calculated with DESeq2 using Wald test statistic and Benjamini-Hochberg correction for multiple testing. b Heat map of the subset of genes significantly differentially expressed in COVID19 biopsies and simultaneously differentially expressed in at least 3 of the other conditions tested. The genes in each cluster were used for pathway enrichment analysis. Genes in cluster 1 are dominantly down-regulated in COVID19 biopsies while a sub-cluster of genes in cluster 1 are up-regulated in the cell lines. Conversely, genes in cluster 2 are predominantly up-regulated in biopsies and in most other test-samples. A subcluster of the genes in cluster 2 are down regulated in the cell lines. For each cluster, the significantly enriched pathways are listed (EnrichR). c Cloud analysis of NRF2-driven differentially expressed genes. Subsets annotated as inflammation/NFκB signaling and Type I IFN signaling exhibit different expression patterns. The experiment is a reanalysis of data from Blanco-Melo et al. [10.1101/2020.03.24.004655]. d Reanalysis of the data from Desai et al. GEO accession code GSE150316. Heat map of NRF2 target genes of the lung autopsies from the five COVID19 patients. Healthy lung samples were used as negative control.

Fig. 2. 4-Octyl-itaconate (4-OI) and dimethyl fumarate (DMF) inhibit SARS-CoV2 replication.

(a–f) h TMPRSS2-Vero cells treated with 4-OI (48 h) and infected with SARS-CoV-2 (48 h). Viral replication by qPCR (a, b), TCID50-assay (c–e) or plaque assay (f). Are pooled data from four (a) and three (e) independent experiments in duplicates and triplicates. Data-points represent one biological sample. Data in (b), (c), and (d) are representative of two independent experiments. (f), supernatants from (e) analysed by plaque assay. Data are pooled from two independent experiments in duplicates. Data-points represent one biological sample. g TMPRSS2 cells with 4-OI and infected with SARS-CoV-2 for 48 h before LDH release-assay. Data pooled from two independent experiments in sextuplicates. h cells from (g) immunoblotted. Blot representative of two independent experiments. Calu3 cells with 4-OI and infected with SARS-CoV-2. Replication by qPCR (i) and TCID50 (j + k). Data pooled from two independent experiment in triplicates. l-m, NuLi cells treated with 4-OI and infected with SARS-CoV-2 for 48 h. Viral replication by TCID50. Data representative of two independent experiments in duplicates. n, HAE-culture graphic. o, HAE-cultures were treated with 4-OI overnight and infected with SARS-CoV-2. Viral replication by qPCR. p–r, Calu-3 and Vero hTMPRSS2 cells treated with DMF (150-200 μM) and infected with SARS-CoV-2. p is pooled data from three independent experiments in duplicates and triplicates. q is pooled data from two independent experiments in duplicates and triplicates. r is pooled data from two independent experiments in duplicates. siRNA treated Calu-3 cells infected with SARS-CoV2. Viral replication by qPCR (s), TCID50 (t), or immunoblotting (u). s is one representative of two independent experiments. t is pooled data from two independent experiments in duplicates. v, w, Vero cells treated with 4-OI at 125 µM and infected with SARS-CoV2 from Japan. Data in (u) is representative of two independent experiments, and (v) is pooled data from two independent experiments in duplicates and triplicates. Data in (w) is data from one experiment with two biological samples. Bars indicate mean ± s.e.m. Unless otherwise stated, statistical analyses by two-tailed Mann–Whitney (*p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001). P values: (a: p < 0.0001, e: p = 0.0002, f: 0.0286, g: p < 0.0001, i: p = 0.0022, k: p = 0.0022, m: p < 0.0001(two-tailed t test), o- p = 0.0268, p: p = 0.0002, q: p = 0.0079, r: p = 0.0286, t: p = 0.0286, v- p = 0.0079. Source data are provided as a Source Data file.

Fig. 3. 4-OI broadly inhibits other pathogenic viruses including HSV, VACV, and Zika Virus.

a HaCaT cells treated with 4-OI (125 μM) for 48 h and infected with HSV1-GFP (MOI 0.01). Viral titers were determined by plaque assay. Data are obtained from one experiment of at least seven independent experiments each performed in triplicates. b RNA analyzed using RNA-sequencing (n = 3) performed once. c–e HaCaT cells treated with 4-OI (125μM) and infected with HSV1-GFP (MOI 0.01). Lysates were analyzed by immunoblotting with Vinculin (VCL) as loading control and by flow cytometry (n = 7 from three independent experiments). f, g HaCaT cells were lipofected with siRNA for 72 h, subsequently challenged with 4-OI (125 μM) before HSV1-GFP infection (MOI 0.01) for 24 h. Infectivity and silencing efficiency was determined by immunoblotting (f) and flow-cytometry (g). Data obtained from one experiment representative of three independent experiments. (h–n) HaCaT cells (h-l) and BMDCs (m, n) were treated with 4-OI (125 μM) for 48 h and infected with VACV expressing either GFP or ECTV expressing mCherry for 24 h. Viral titers and infectivity were determined by plaque assay (h), flow cytometry (i-j and m-n), and visualized by confocal microscopy (k-l). Data in (i, j), and (n) are obtained from one experiment representative of two independent experiments each performed in triplicates. o-p A549 and Huh-7 cells were pre-treated with 4-OI for 48 h (150 μM) and infected with Zika virus (ZIKV) (MOI 0.1) for 4 days. Viral genome was determined by qPCR. Data were obtained from one experiment representative of two independent experiments. For all panels, bars indicate mean ±  s.e.m. Unless otherwise stated, all statistical analysis were performed using a two-tailed Mann–Whitney test to determine statistical significance where *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. Individual p values were, a: p < 0.0001, e: p < 0.0001, i: p = 0.0002(two tailed t test, MOI 0.1) and p < 0.0001(two tailed t test, MOI 0.01), j: p < 0.0001(two tailed t test, MOI 0.1) and p < 0.0001 (two tailed t test, MOI 0.01), n: p < 0.001(two tailed t test, MOI 0.1) and p = 0.0178(two tailed t test, MOI 0.01). Source data are provided as a Source Data file.

Fig. 4. 4-OI and DMF limit SARS-CoV2 and HSV induced inflammatory responses.

a–c Calu-3 treated with 4-OI (125 µM, 48 h) before SARS-CoV2 infection (MOI 0.5, 48 h) and qPCR analysis. a, b, and c are pooled data from three independent experiments in triplicates. d–f HAE-cultures treated with 4-OI before SARS-CoV2 infection for 24 h and analysis by qPCR. Data are from two donors. g–i Calu-3 cells with DMF (48 h) before SARS-CoV2 infection. Analysis by qPCR. In (g, h, and i), display pooled data from three independent experiments in duplicates and triplicates. (d–f), data are representative of four independent HAE-cultures. (j) Healthy PBMCs treated with 4-OI before infection and qPCR analysis. Data are representative of two donors. (k) PBMCs from four COVID-19 patients (k) and 2 healthy controls (HC) treated with 4-OI before qPCR. (l-m) HaCaTs treated with 4-OI before stimulation with M8 for 6 h followed by qPCR. Data represent one experiment in triplicate. n HaCaT cells with siRNAs before M8 treatment for 3 h followed by immunoblotting. Data are representative of two independent experiments. o HaCaTs treated with siRNA for 72 h before 4-OI and stimulation with M8. Analysis by immunoblotting. Data representative of two independent experiments. o-q HEK293 and HaCaTs transfected with expression-plasmids before 4-OI. In (q), HaCaTs were treated with siRNAs for 72 h before plasmid transfection. Analysis by qPCR and immunoblotting. Data in (p) are from one experiment performed in triplicate. Data displayed in (q) is representative of two independent experiments. (r–t) HaCaTs treated with 4-OI before infection with HSV1 or transfection with dsDNA. Analysis by qPCR and immunoblotting at 6 and 3 h respectively. (n–t), data are from one experiment representative of two independent experiments. Data in (r) and (s) are representative of two independent experiments in triplicates. Bars indicate mean ± s.e.m. Unless otherwise stated, all statistical analysis by two-tailed Mann–Whitney test where *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001. P values:, a: p < 0.0001(IFNB1) and p < 0.0001(CXCL10), b: p < 0.0001(CCL5) and p < 0.0001(TNFA) and p < 0.0001(IL1B), c: p < 0.0001, d: p = 0.0286(IFNB1) and p = 0.0286(CXCL10), e: p = 0.0286(TNFA) and p = 0.0286(CCL5), f: p = 0.0286, g: p = 0.0003(IFNB1) and p = 0.0002(CXCL10), h: p = 0.0002(CCL5) and p = 0.0003(TNFA), i: p = 0.0011, j: p = 0.0005 (two-tailed t test, SARS-CoV2) and p = 0.0053 (two-tailed t test, SeV), p: p = 0.0092 (two-tailed t test). Source data are provided as a Source Data file.