new identification of sars-cov2-mediated suppression of nrf2 … · 2020. 7. 16. · 1 1...

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Identification of SARS-CoV2-mediated suppression of NRF2 signaling reveals a potent 1

antiviral and anti-inflammatory activity of 4-octyl-itaconate and dimethyl fumarate 2

David Olagnier1,*, Ensieh Farahani1,†, Jacob Thyrsted1,†, Julia B. Cadanet1,†, Angela Herengt1,†, Manja Idorn1, Alon 3 Hait1,7, Bruno Hernaez2, Alice Knudsen1, Marie Beck Iversen1, Mirjam Schilling3, Sofie E. Jørgensen1,7, Michelle 4

Thomsen1,7, Line Reinert1, Michael Lappe11, Huy-Dung Hoang4, Victoria H. Gilchrist4, Anne Louise Hansen1, 5 Rasmus Ottosen10, Camilla Gunderstofte1, Charlotte Møller1, Demi van der Horst1, Suraj Peri13, Siddarth 6

Balachandran13, Jinrong Huang9,12, Martin Jakobsen1, Esben B. Svenningsen10, Thomas B. Poulsen10, Lydia 7 Bartsch6, Anne L. Thielke1, Yonglun Luo1,9, Tommy Alain4, Jan Rehwinkel3, Antonio Alcamí2, John Hiscott5, Trine 8

Mogensen1,7,8, ††, Søren R. Paludan1, ††, Christian K. Holm1,*. 9 Affiliations: 10 1Department of Biomedicine, Aarhus Research Center for Innate Immunology, Aarhus University, Aarhus, Denmark 11 12 2Centro de Biología Molecular Severo Ochoa (Consejo Superior de Investigaciones Científicas - Universidad 13 Autónoma de Madrid), Nicolás Cabrera 1, 28049 Madrid, Spain. 14 15 3Medical Research Council Human Immunology Unit, Medical Research Council Weatherall Institute of Molecular 16 Medicine, Radcliffe Department of Medicine, University of Oxford, Oxford OX3 9DS, UK. 17 18 4Children’s Hospital of Eastern Ontario Research Institute, Department of Biochemistry Microbiology and 19 Immunology, University of Ottawa, Ottawa, Ontario, Canada, K1H 8L1. 20 21 5Istituto Pasteur Italia-Cenci Bolognetti Foundation, Viale Regina Elena 291, 00161, Rome, Italy. 22 23 6Department of Pediatrics and Adolescent Medicine, Division of Pediatric Neurology, University Medical Center 24 Göttingen, 37075 Göttingen, Germany. 25 26 7Department of Infectious Diseases, Aarhus University Hospital, Aarhus, Denmark 27 28 8Department of Clinical Medicine, Aarhus University, Aarhus, Denmark 29 30 9Lars Bolund Institute of Regenerative Medicine, BGI-Shenzhen, Shenzhen 518083, China 31 32 10Department of Chemistry, Aarhus University, Aarhus, Denmark 33 34 11Omiics ApS, Åbogade 15, 8200 Aarhus N, Denmark 35 36 12Department of Biology, University of Copenhagen, 2100 Copenhagen, Denmark 37

13Fox Chase Cancer Center, 333 Cottman Avenue, Philidelphia, PA 19111-2497, USA 38

39 *Correspondence to: Christian Kanstrup Holm, [email protected] and David Olagnier, [email protected] 40 †These authors contributed equally 41 †† These authors contributed equally 42 43

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was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprint (whichthis version posted July 17, 2020. ; https://doi.org/10.1101/2020.07.16.206458doi: bioRxiv preprint

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Abstract: Antiviral strategies to inhibit Severe Acute Respiratory Syndrome Coronavirus 2 49

(SARS-CoV2) and the pathogenic consequences of COVID-19 are urgently required. Here we 50

demonstrate that the NRF2 anti-oxidant gene expression pathway is suppressed in biopsies 51

obtained from COVID-19 patients. Further, we uncover that NRF2 agonists 4-octyl-itaconate (4-52

OI) and the clinically approved dimethyl fumarate (DMF) induce a cellular anti-viral program, 53

which potently inhibits replication of SARS-CoV2 across cell lines. The anti-viral program 54

extended to inhibit the replication of several other pathogenic viruses including Herpes Simplex 55

Virus-1 and-2, Vaccinia virus, and Zika virus through a type I interferon (IFN)-independent 56

mechanism. In addition, induction of NRF2 by 4-OI and DMF limited host inflammatory 57

responses to SARS-CoV2 infection associated with airway COVID-19 pathology. In conclusion, 58

NRF2 agonists 4-OI and DMF induce a distinct IFN-independent antiviral program that is 59

broadly effective in limiting virus replication and suppressing the pro-inflammatory responses of 60

human pathogenic viruses, including SARS-CoV2. 61

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One Sentence Summary: NRF2 agonists 4-octyl-itaconate (4-OI) and dimethyl fumarate 63

inhibited SARS-CoV2 replication and virus-induced inflammatory responses, as well as 64

replication of other human pathogenic viruses. 65

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Introduction: 71

The 2020 SARS-CoV2 pandemic emphasizes the urgent need to identify cellular factors and 72

pathways that can be targeted by new broad-spectrum anti-viral therapies. Viral infections 73

usually cause disease in humans through both direct cytopathogenic effects and through 74

excessive inflammatory responses of the infected host. This also seems to be the case with 75

SARS-CoV2 as COVID-19 patients develop cytokine storms that are very likely to contribute to, 76

if not drive, immunopathology and severe disease(1, 2). For these reasons, anti-viral therapies 77

must aim to not only inhibit viral replication but also to limit inflammatory responses of the host. 78

Nuclear factor (erythroid-derived 2) -like 2 (NRF2) functions as a cap´n´collar basic leucine 79

zipper family of transcription factors characterized structurally by the presence of NRF2-ECH 80

homology domains(3). At homeostasis, NRF2 is kept inactive in the cytosol by its inhibitor 81

protein KEAP1 (Kelch-like ECH-associated protein 1), which targets NRF2 for proteasomal 82

degradation(4). In response to oxidative stress, KEAP1 is inactivated and NRF2 is released to 83

induce NRF2-responsive genes. In general, the genes under the control of NRF2 protect against 84

stress-induced cell death and NRF2 has thus been suggested as the master regulator of tissue 85

damage during infection(5). Importantly, NRF2 is now demonstrated as an important regulator of 86

the inflammatory response(6, 7) and functions as a transcriptional repressor of inflammatory 87

genes, most notably interleukin (IL-) 1b, in murine macrophages(8). Recent reports have now 88

demonstrated that NRF2 is induced by several cell derived metabolites including itaconate and 89

fumarate, to limit inflammatory responses to stimulation of TLR signaling with 90

lipopolysaccharide stimulation(9). The chemically synthesized and cell-permeable derivative of 91


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itaconate, 4-octyl-itaconate (4-OI) was then demonstrated to be a very potent NRF2 inducer(9). 92

Of special interest is the derivative of fumarate, dimethyl fumarate (DMF), a US Food and Drug 93

Administration (FDA) approved drug, which is used as an anti-inflammatory therapeutic in 94

multiple sclerosis (MS) and demonstrated, at least in animal models, a potent capacity to 95

suppress pathogenic inflammation through a Nrf2-dependent mechanism(10, 11). 96

Besides limiting the inflammatory response to LPS, induction of NRF2 by 4-OI also inhibits the 97

Stimulator of Interferon Genes (STING) antiviral pathway along with interferon (IFN) 98

stimulated gene expression(12). In opposition to this anti-viral effect of NRF2 on the IFN-99

response a recent single-cell RNA-seq analysis has demonstrated that NRF2 gene expression 100

signatures correlated negatively with susceptibility to HSV1 infection (13). If NRF2 agonists can 101

be used to limit viral replication of SARS-CoV2 or other pathogenic viruses is, however, not 102

known. 103

Here we demonstrate that expression of NRF2-dependent genes is suppressed in biopsies from 104

COVID-19 patients and that treatment of cells with NRF2 agonists 4-OI and DMF induces a 105

strong anti-viral program that limits SARS-CoV2 replication. The anti-viral effect of activating 106

NRF2 extended to other pathogenic viruses including Herpes Simplex Virus-1 and-2 (HSV-1 and 107

HSV-2), Vaccinia Virus (VACV), and Zika Virus (ZIKV). Further, 4-OI and DMF limited the 108

release of pro-inflammatory cytokines in response to SARS-CoV2 infection and to virus-derived 109

ligands through a mechanism that limits IRF3 dimerization. In summary, we demonstrate that 110

NRF2 agonists are plausible broad-spectrum anti-viral and anti-inflammatory agents and we 111

suggest a repurposing of the already clinically approved DMF for the treatment of SARS-CoV2. 112

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Results 116

NRF2 dependent anti-oxidant response is suppressed in COVID-19 patient biopsies 117

To identify host factors or pathways that are important for controlling SARS-CoV2 infection, 118

publicly available transcriptome data sets including transcriptome analysis of lung biopsies from 119

COVID-19 patients were analyzed using differential expression analysis(14). Here, genes linked 120

with inflammatory and anti-viral pathways, including RIG-I receptor and Toll-like receptor 121

signaling, were highly enriched in COVID-19 lung patient samples, whereas genes associated 122

with the NRF2 dependent anti-oxidant response were highly suppressed (Fig. 1a-c). That NRF2-123

induced genes are repressed during SARS-CoV2 infections was supported by re-analysis of 124

another data-set builing on transcriptome analysis of lung autopsies obtained from five individual 125

COVID-19 patients (Desai et al., 2020) (Fig. 1d). Further, that the NRF2-pathway is repressed 126

during infection with SARS-CoV2 was also supported by in vitro experiments where the 127

expression of NRF2-inducible proteins Heme Oxygenase 1 (HO-1) and NAD(P)H quinone 128

oxydoreducatse 1 (NqO1) was repressed in SARS-CoV2 infected Vero hTMPRSS2 cells while 129

the protein levels of canonical anti-viral transcription factors such as STAT1 and IRF3 seemed 130

unaffected (Fig. S1). These data indicate that SARS-CoV2 targets the anti-oxidant NRF2 131

pathway and thus suggests that the NRF2 pathway restricts SARS-CoV2 replication. 132


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Fig. 1. Expression of NRF2-driven genes is suppressed in COVID-19 patient biopsies 134 (a, b, and c) Reanalysis of data published by Blanco-Melo et al., 2020. (a) Bar-chart of the number of transcripts 135 that show differential expression (up and down). Genes with p<0.05 and Log2 fold change in COVID-19 patient 136 lung biopsies were normalized against healthy lung biopsies, and in cell lines Calu3, NHBE and A549 infected with 137 either SARS-CoV2, Influenza A virus (IAV), Respiratory Syncytial virus (RSV), or human parainfluenza virus type 138 3 (HPIV3) were normalized against mock treated cells. (b) Cloud analysis of NRF2-driven differentially expressed 139 genes. Subsets annotated as inflammation/NFkB signaling and Type I IFN signaling exhibit different expression 140 patterns. The experiment is a re-analysis of data from Blanco-Melo et al., 141 https://doi.org/10.1101/2020.03.24.004655. (c) Heat map of the subset of genes significantly differentially 142 expressed in COVID19 biopsies and simultaneously differentially expressed in at least 3 of the other conditions 143 tested. The genes in each cluster were used for pathway enrichment analysis. Genes in cluster 1 are dominantly 144


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down-regulated in COVID19 biopsies while a sub-cluster of genes in cluster 1 are up-regulated in the cell lines. 145 Conversely, genes in cluster 2 are predominantly up-regulated in biopsies and in most other test-samples. A sub-146 cluster of the genes in cluster 2 are down regulated in the cell lines. For each cluster, the significantly enriched 147 pathways are listed (EnrichR). (d) Reanalysis of the data from Desai N et al. (May 2020), GEO accession code 148 GSE150316. Heat map of NRF2 target genes of the lung autopsies from the five COVID19 patients. Healthy lung 149 samples were used as negative control. 150 151 152

NRF2 agonists 4-OI and dimethyl fumarate are strong inhibitors of SARS-CoV2 153

replication 154

Considering that NRF2 suppresses anti-viral IFN-responses, it was surprising to discover that 155

treatment of Vero cells with 4-OI generated before infection with SARS-CoV2 (strain #291.3 156

FR-4286 isolated from a patient in Germany) resulted in a remarkable reduction in SARS-CoV2 157

RNA levels in a dose-dependent manner (Fig. 2a+b) while not affecting cell viability as 158

determined by lactate dehydrogenase (LDH) assay (Fig. S2). Further, subsequent release of 159

progeny SARS-CoV2 virus particles to the cell supernatant was equally decreased by 4-OI 160

treatment as measured by TCID50 assay to quantify virus by dilution of virus-induced 161

cytopathogenic effects and by plaque assay (Fig. 2c-f). The reduced viral replication led to 162

reduced virus-induced cytotoxicity of the infected Vero cells determined by lactate 163

dehydrogenase release assay and by immunoblotting for cleaved Caspase 3 and Poly(ADP-164

Ribose) Polymerase 1 (PARP-1), which are hallmark indicators of apoptosis(15) (Fig. 2g+h). 165

Interestingly, the observation that the NRF2 pathway is inhibited in response to SARS-CoV-2 166

infection could be recapitulated in SARS-CoV-2 infected Vero cells as demonstrated by both the 167

basal decrease in NRF2-driven proteins HO-1 and NqO1 and their incapacity to be induced by 168

Nrf2 agonist 4-OI (Fig. 2h). The effect of 4-OI was also retained in the lung cancer cell line 169

Calu-3, where SARS-CoV2 RNA levels were reduced by >2-logs (Fig. 2i), while release of 170

progeny virus was reduced by > 6-logs based on TCID50 analysis of cell supernatants (Fig. 171

2j+k). In the immortalized human epithelial cell line NuLi, total infection levels were relatively 172


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low compared to what we could observe in Calu3 and Vero cells but 4-OI treatment still reduced 173

SARS-CoV2 RNA levels and release of progeny virus (Fig. 2l+m). We further tested the anti-174

viral effect towards SARS-CoV2 in primary human airway epithelial (HAE) cultures (Fig. 2n). 175

Here, 4-OI treatment also significantly reduced viral RNA levels (Fig. 2o). Interestingly, when 176

treating Calu3 cells with DMF, another known NRF2 inducer and a clinically approved drug in 177

the first-line-of treatment of multiple sclerosis, we could also observe an anti-viral effect toward 178

SARS-CoV2 replication similar in magnitude as what we had observed with 4-OI (Fig 2p-q) as 179

well as a reduced but significant effect when using Vero cells (Fig. 2r). To further evaluate if the 180

NRF2/KEAP1 axis controls an anti-viral pathway effective in inhibiting SARS-CoV2 181

replication, we used genetic activation of NRF2 by silencing of KEAP1. This approach 182

supported the 4-OI data as silencing of KEAP1 led to suppressed replication of SARS-CoV2 in 183

Calu3 cells by qPCR analysis, immunoblotting, and TCID50 analysis (Fig. 2s-u). Finally, the 184

anti-viral effect of SARS-CoV2 was not isolated to this particular isolate as the effect of 4-OI 185

was reproduced using a different SARS-CoV2 isolate obtained in Japan(16) (Fig. 2v+x). These 186

data demonstrate that NRF2 inducers 4-OI and DMF induce potent anti-viral responses that 187

efficiently inhibit SARS-CoV2 replication across multiple cellular systems. 188


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Fig. 2. 4-Octyl-itaconate (4-OI) and dimethyl fumarate (DMF) inhibit SARS-CoV2 replication 190 (a-e) Vero E6 expressing human TMPRSS2 cells were treated with 4-OI (125µM) or lower concentrations were 191 indicated for 48h and subsequently infected with SARS-CoV-2 at a MOI of 0.1 for 48h, respectively. Infection and 192 replication were assessed by qPCR and TCID50 or plaque assay, respectively. Data displayed in a and e are means 193 and s.e.m. of pooled data from three independent experiments with each data-point representing one biological sample. 194 Data displayed in b, c, and d are represenatative of two independent experiments and indicating means and s.e.m.. In 195 (f), supernatants from (e) were also assessed for viral titers by semi-solid agarose plaque assay. Data are pooled from 196 two independent experiments with each data-point representing one biological sample. (g) Vero E6 expressing human 197 TMPRSS2 cells were treated with 4-OI (125µM) for 48h and subsequently infected with SARS-CoV-2 at a MOI of 198 0.1 for 48h. Cell death was evaluated using an LDH release assay. Data are pooled from two independent experiments 199 each performed in sextuplicate displaying the means and s.e.m. In (h), whole cell extracts from (g) were 200 immunoblotted for various Nrf2 and death associated markers. Blot is representative of two independent experiments. 201 In (i-k) Calu3 cells were treated with 4-OI (100µM) for 48h and subsequently infected with SARS-CoV-2 (MOI 0.5) 202 for 48h. Viral RNA content and progeny virus were assessed by qPCR and TCID50, respectively. Data are the means 203 and s.e.m. of pooled data from two independent experiment performed in triplicate. In (l-m), NuLi cells were treated 204 with 4-OI (100µM) for 48h and subsequently infected with SARS-CoV-2 (MOI 0.5) for 48h. Viral titers were 205 determined by TCID50. Data are the means and s.e.m. and representative of two independent experiments performed 206 in duplicate. In, (n) schematic of an HAE culture. (o) HAE cultures (n=4) were treated with 4-OI (125µM) overnight 207 and were subsequently infected with SARS-CoV-2 (MOI 0.1). Viral genome content was assessed by qPCR (mean 208 +/- s.e.m. from 4 independent primary HAE cultures). In (p-r), Calu-3 cells and Vero E6 expressing hTMPRSS2 cells 209 were treated with dimethyl fumarate (DMF) (150-200 µM) for 48h and subsequently infected with SARS-CoV-2 at a 210 MOI of 0.5 and 0.1, respectively. Data in (p and q) are pooled from two independent experiments with means and 211 s.e.m. In (s-u), Calu-3 cells were treated with indicated siRNAs for 48 hours before infection with SARS-CoV2 at a 212 MOI of 0.5. Cell pellets were then etiher collected for RNA extraction and qPCR analysis (s) or for immunoblotting 213 (u). Cell supernatants were analysed by TCID50 assay (t). Data are mean and s.e.m and representative of two 214 independent experiments. In (v-w), Vero cells were treated with 4-OI at 125µM before infection with a different 215 SARS-CoV2 isolate obtained from Japan(16). Data in u and v are obtained from on experiment performed in duplicate. 216 All statistical analysis were performed using a two-tailed Student’s t-test where **p< 0.01, ***p<0.001, and 217 ****p<0.0001. 218 219

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Activation of NRF2 with 4-OI broadly inhibits viral replication through an IFN-221

independent pathway 222

The anti-viral effect of 4-OI was not restricted to SARS-CoV2 but also extended to other human 223

pathogenic viruses. Using the human keratinocyte cell line HaCaT as a model of Herpes Simplex 224

Virus (HSV) type 1 and 2 we could observe that treatment with 4-OI reduced both the release of 225

progeny virus, the cellular content of virus RNA determined by RNA sequence analysis, and 226

viral protein as determined by both immunoblotting and flow cytometry (Fig. 3a-e and Fig. S3). 227

By contrast, the expression of NRF2-inducible HO-1, NqO1, and Sequestosome 1 (SQSTM1) 228

was highly increased in response to 4-OI treatment (Fig. 2c and Fig. S3). The anti-viral effect of 229


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4-OI was at least partially dependent on NRF2 as silencing hereof by siRNA clearly reduced the 230

suppression of HSV1 infection by 4-OI (Fig. 2f-g). 231

Vaccinia virus (VACV) belongs to the family of human pathogenic poxviruses. We used HaCaT 232

cells, but also bone marrow derived dendritic cells (BMDCs), to test if the anti-viral effect of 4-233

OI extended to these viruses. Here we could observe that both HaCaT cells and BMDCs became 234

highly resistant to infection with VACV when these were pre-treated with 4-OI as measured by 235

plaque assay and flow cytometry (Fig. 2h-n). This seemed also to be the case for another 236

poxvirus Ectromelia virus (ECTV) as assesses by confocal imaging (Fig. 2k-l). For both HSV1 237

and VACV the anti-viral effect of 4-OI was extended to other cell type including murine cancer 238

cell line 4T1 and human renal carcinoma 786-O cells (Fig. S4). Interestingly, the anti-viral effect 239

of 4-OI was not extended to infection with vesicular stomatitis virus (VSVd51M) emphasizing 240

that the anti-viral program induced by 4-OI effectively inhibits replication of many, but not all, 241

viruses (Fig. S4). The anti-viral effect of 4-OI relied on intracellular restriction of replication, 242

since viral entry was not affected by 4-OI treatment – if anything it seemed to be slightly 243

increased (Fig. S5). 244

To determine if the anti-viral effect of 4-OI extended to an in vivo model of viral pathogenesis, 245

female C57BL6J mice were treated with 4-OI prior to vaginal inoculation with HSV; pre-treatment 246

with 4-OI decreased disease progression (Fig. S6), an effect that was enhanced in mice deficient 247

in STING (TMEM173–/–) most likely to due to the pro-viral effect 4-OI has on the STING signaling 248

pathway(12, 17), which is eliminated in these mice. 249

Finally, we tested the efficacy of 4-OI on Zika virus, an important human pathogenic virus 250

causing mild symptoms in the competent adult but severe disease when transmitted in utero(18). 251

Here, we could demonstrate that the anti-viral program induced by 4-OI reduced replication of 252


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Zika virus in the human lung cancer cell line A549 and in the human liver cell line Huh-7 (Fig. 253

2o-p). Given that Vero cells are deficient in type I IFN(19), this suggested that the inhibitory 254

effect of 4-OI was actually independent of type I IFN signaling. To address this possibility, we 255

used either HaCaT cells deficient in IFN alpha receptor 2 (IFNAR2), Signal Transducer and 256

Activator of Transcription 1 (STAT1), both of which is necessary for type I IFN-signaling(20); 257

or deficient in STING, which is central to type I IFN-response to DNA viruses. Here, cells were 258

treated with 4-OI, followed by infection with HSV1 and VACV. Replication of both viruses was 259

inhibited by 4-OI in STAT1 KO cells and for HSV1 also in STING KO cells as measured by 260

plaque assay and expression of viral proteins by immunoblotting and flow cytometry (Fig. S7). 261

In conclusion, 4-OI induces an anti-viral program that operates independently of IFN signaling 262

(Fig. S7). 263

To examine what general pathways are affected by 4-OI treatment either alone or during 264

infection that could predict the antiviral mode of action itaconate, we treated HaCaT cells with 4-265

OI before infection with HSV-1. RNA was then collected and analysed by RNA sequencing. 266

Pathway analysis was then used to compare untreated cells to 4-OI treated cells either with or 267

without infection with HSV-1. This analysis identified several pathways that were either induced 268

or repressed by 4-OI treatment while re-confirming that IFN-signaling pathways was repressed 269

by 4-OI (Fig. S8). Amongst the top up-regulated genes induced by 4-OI was the heme 270

oxygensase-1, an enzyme canonicaly involved in stress detoxification, also reported to have 271

antiviral activity against amongst others Zika, Dengue and Ebola viruses (21-23). To assess 272

whether, HO-1 had any antiviral activity in our cellular system, Vero hTMPRSS2 and Calu-3 273

cells were either transfected with an overexpression plasmid encoding HO-1 or genetically 274

silenced for KEAP1 and HO-1 by siRNA, respectively before infection with SARS-CoV-2. None 275


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of the treatments (HO-1 overexpression or silencing) really altered SARS-CoV-2 276

infection/replication suggesting of an HO-1-independent antiviral program induced by NRF2 277

(Fig. S9). 278

In an attempt to pin-point the anti-viral mode of action of 4-OI, we also used microscope-based 279

analysis of morphology by cell-paint technology (Fig. S10). With this analysis we are able to 280

compare morphological changes in cells treated with 4-OI to cells treated with compounds that 281

have known cellular targets and with cells treated with other compounds with reported anti-viral 282

activity towards SARS-CoV2 including Remdesivir and Hydroxychloroquine(24, 25). In this 283

analysis, 4-OI was determined to have an low but significant morphological activity whitout loss 284

of cell viability. Interestingly, the activity of 4-OI did not seem to overlap with other compound 285

with known perturbation in cell morphology including Rapamycin, Bafilomycin, Tunicamycin, 286

Cyclohexamide, Emetine, Mitomycin, or Doxorubicin. Interestingly, there was also no 287

observable overlab with the activity profile of Remdesivir or hydroxychloroquine indicating that 288

the anti-viral mode of action of 4-OI is distinct from known anti-viral mechanisms. 289


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Fig. 3. 4-OI broadly inhibits other pathogenic viruses including HSV, VACV, and Zika Virus 291 (a) HaCaT cells treated with 4-OI (125µM) for 48h and infected with HSV1-GFP (MOI 0.01). Viral titers were 292 determined by plaque assay. Data are obtained from one experiment of at least seven independent experiments 293 duplicates (b) RNA analyzed using RNA-sequencing (n=3) performed once. (c-e) HaCaT cells treated with 4-OI 294 (125µM) and infected with HSV1-GFP (MOI 0.01). Lysates were analyzed by immunoblotting with Vinculin (VCL) 295 as loading control and by flow cytometry (n=7 from three independent experiments, mean +/- s.e.m.). (f-g) HaCaT 296 cells were lipofected with siRNA for 72h, subsequently challenged with 4-OI (125µM) before HSV1-GFP infection 297 (MOI 0.01) for 24h. Infectivity and silencing efficiency was determined by immunoblotting (f) and flow-cytometry 298 (g). Data obtained from one experiment representative of three independent experiments. (h-n) HaCaT cells (h-l) and 299 BMDCs (m-n) were treated with 4-OI (125µM) for 48h and infected with VACV expressing either GFP or ECTV 300 expressing mCherry for 24h. Viral titers and infectivity were determined by plaque assay (h), flow cytometry (i-j and 301 m-n), and visualized by confocal microscopy (k-l). Data are obtained from one experiment representative of two 302 independent experiments. (o-p) A549 and Huh-7 cells were pre-treated with 4-OI for 48h (150µM) and infected with 303 Zika virus (ZIKV) (MOI 0.1) for 4 days. Viral genome was determined by qPCR. Data were obtained from one 304


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experiment representative of two independent experiments. All statistical analysis were performed using a two-tailed 305 Student’s t-test to determine statistical significance where **p< 0.01, ***p<0.001, and ****p<0.0001. 306 307 308

4-OI and DMF suppress the inflammatory response to SARS-CoV2 309

In COVID-19, an uncontrolled pro-inflammatory cytokine storm contributes to disease 310

pathogenesis and lung damage (26). For this reason, we investigated if 4-OI and DMF could 311

inhibit expression of pro-inflammatory cytokines induced by SARS-CoV2. In Calu-3 cells, 312

infection with SARS-CoV2 increased the expression of IFNB1, C-X-C motif chemokine 10 313

(CXCL10), Tumor Necrosis Factor alpha (TNFA), IL-1B and C-C chemokine ligand 5 (CCL5). 314

Interestingly, this was abolished by pretreatment with 4-OI thus severely reducing the pro-315

inflammatory response to SARS-CoV2 (Fig. 4a-b). By contrast, expression of the NRF2 316

inducible gene HMOX1 was highly increased in response to 4-OI treatment (Fig. 4c). The 317

potential anti-inflammatory effect of 4-OI in this context was supported when using HAE 318

cultures. Here, treatment with 4-OI also reduced the expression of IFNB1, CXCL10, TNFa, and 319

CCL5 in the context of SARS-CoV2 infection (Fig. 4d-e), while increasing the expression of the 320

NRF2 inducible gene HMOX1 (Fig. 4f). A similar pattern was seen in experiments where Calu3 321

cells were treated with DMF before SARS-CoV2 infection. Here, IFNB1, CXCL10 and CCL5 322

mRNA levels were highly reduced in DMF treated cells while TNFA mRNA levels seemed 323

unaffected (Fig. 4g+h). By contrast, treatment with DMF increased the mRNA expression levels 324

of NRF2 inducible gene HMOX1 (Fig. 4i). As inflammatory responses often stem from immune 325

cells we also tested the effect of 4-OI on Peripheral Blood Mononuclear Cells (PBMCs) 326

harvested from healthy donors. Although stimulation of PBMCs with SARS-CoV2 yielded a 327

very weak induction of CXCL10 compared to sendai virus (SeV) infection, and no detectable 328

induction of other cytokines, 4-OI treatment also reduced CXCL10 mRNA levels in this context 329


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(Fig. 4j). Further, when using PBMCs harvested from four individual patients with severe 330

COVID-19 and admitted to hospital Intensive Care Units (ICUs), we could conclude that in three 331

out of four patients, expression levels of CXCL10 were increased when compared to healthy 332

controls; and that in all four patients, these levels were strongly reduced to or below normal 333

when treating the PBMCs with 4-OI (Fig. 4k) indicating that 4-OI is able to relieve 334

inflammatory responses induced by SARS-CoV2 in PBMCs in vivo. That CXCL10 levels is a 335

relevant readout in SARS-CoV2 was recently supported by a report by Cheemarla et al., 2020 336

(https://doi.org/10.1101/2020.06.04.20109306) demonstrating that CXCL10 expression is increased 337

in the upper airways of patients infected with SARS-CoV2. 338

The observed decrease in anti-viral and pro-inflammatory responses could possibly be explained 339

by the 4-OI mediated reduction in cellular viral RNA with subsequent reduced induction of 340

cytokines through cellular RNA sensors such as RIG-I. We therefore investigated the effect of 4-341

OI on the induction of IFN and of IFN stimulated genes (ISGs) responses activated by a sequence 342

optimized RIG-I agonist M8(27). Interestingly, 4-OI treatment reduced IFN-responses induced by 343

this RIG-I agonist M8 (Fig 4l-m), through an effect linked to the inhibition of Interferon 344

Regulatory Factor 3 (IRF3) dimerization but not of upstream phosphorylation of Tank Binding 345

Kinase 1 (TBK1) or of IRF3 expression itself (Fig 4n). Importantly, NRF2 expression itself was 346

closely associated with the inhibition of IRF3 dimerization and host antiviral gene expression, 347

since NRF2 silencing by siRNA was sufficient to restore IRF3 dimerization and limit the inhibitory 348

effect of 4-OI (Fig. 4n-o). When using the constitutively active form of IRF3, IRF3(5D) (28), 4-349

OI was still able to block IRF3 dimerization, and again, this effect was eliminated when NRF2 350

expression was suppressed by siRNA (Fig 4p-q). These data indicate that an NRF2 inducible and 351

dependent mechanism targets the induction of IFN by inhibition of IRF3 dimerization. This 352


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phenomenon is likely to add to the inhibition of SARS-CoV2 induced cytokine release we could 353

observe when using NRF2 agonists. We have previously reported that 4-OI inhibits the expression 354

of STING, which is important for the induction of the IFN-response in cells stimulated with 355

cytosolic DNA(12). In line, 4-OI inhibited the IFN-response to HSV1 infection and to stimulation 356

with STING agonists dsDNA and cGAMP (Fig. 4R-T). 357

358


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359 Fig. 4. 4-OI and DMF limit SARS-CoV2 and HSV induced inflammatory responses. 360 (a-c) Calu-3 treated with 4-OI (125µM) for 48h before infection with SARS-CoV2 (MOI 0.5) for 48 hours. RNA was 361 extracted for analysis by qPCR. Graphs display pooled data from three independent experiments with each data-point 362


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representing one biological sample where means and s.e.m. are displayed. (d-f) HAE cultures (n=4,) were treated 363 overnight with 4-OI at 125µM before SARS-CoV2 infection (MOI 0.1) for 24 and analysis by qPCR. Data are 364 representative of two independent experiments with two donors. (g-i) Calu-3 treated with DMF (200 µM) for 48h 365 before infection with SARS-CoV2 (MOI 0.5) for 48 hours. RNA was extracted for analysis by qPCR. In 366 (a+b+c+g+h+i) Graphs display pooled data from three independent experiments with each data-point representing one 367 biological sample where means and s.e.m. are displayed. In (d-f) data are representative of four independent primary 368 HAE cultures where means and s.e.m. are displayed. (j) Healthy PBMCs were pre-treated overnight with 4-OI 369 (100µM) before a challenge with SARS-CoV-2 (MOI 10) or sendai virus (SeV) (50 HAU) for an additional 24h. 370 CXCL10 mRNA levels were determined by qPCR. Data are from one healthy donor in triplicate representative of two 371 independent healthy donors. (k) PBMCs from four COVID-19 patients (k) and 2 healthy controls (HC) treated with 372 4-OI at 100µM overnight before analysis by qPCR. (l-m) HaCaT cells were treated with 4-OI (125µM) before 373 stimulation with the sequence optimized RIG-I agonist M8 (10ng/mL) for 6 hours followed by qPCR gene expression 374 analysis. Data represent the means and s.e.m. of one experiment performed in triplicate. (n) HaCaT cells were 375 lipofected with indicated siRNAs for 72h before treatment with M8 (10 ng/mL) for 3 hours followed by analysis by 376 immunoblotting. (o) HaCaT cells were lipofected with indicated siRNAs for 72h before treatment with 4-OI (125µM) 377 for 48h and stimulation with M8 (10 ng/mL) for 3 hours followed by analysis by immunoblotting. (p-q) HEK293(p) 378 and HaCaT(q) cells were transfected with indicated plasmids before treatment with 4-OI at 125µM. In (q), HaCaT 379 cells were lipofected with siRNAs for 72h before plasmid transfection. Cells were then collected for analysis by qPCR 380 (p) and immunoblotting (q). (r-t) HaCaT cells were treated with 4-OI at 125µM before infection with HSV1 at MOI 381 0.01 or transfection with dsDNA (4 µg.mL-1). Cell pellets were collected for qPCR and immunoblotting at 6 and 3 382 hours respectively. In (n-t), data display data from one experiment representative of at least to independent 383 experiments. All statistical analysis were performed using a two-tailed Student’s t-test to determine statistical 384 significance where **p< 0.01, ***p<0.001, and ****p<0.0001. 385 386

Discussion 387

Altogether, this study demonstrated that the expression of NRF2 dependent anti-oxidant genes was 388

significantly inhibited in COVID-19 patients, and that the NRF2 agonists 4-OI and DMF inhibited 389

both SARS-CoV2 replication, as well as the expression of associated inflammatory markers. The 390

ability of these NRF2 inducers to also reduce potentially pathogenic IFN- and inflammatory 391

responses while retaining their anti-viral properties is unique to these compounds and promotes 392

their applicability to prevent virus-induced pathology. That NRF2 might be a natural regulator of 393

IFN-responses in the airway epithelium is supported by a recent report demonstrating that NRF2 394

activity is high while IFN activity is low in the bronchial epithelium(29). As DMF is currently 395

used as an anti-inflammatory drug in relapsing-remitting MS, this drug could be easily repurposed 396

and tested in clinical trials to test its ability to limit SARS-CoV2 replication and inflammation-397

induced pathology in COVID-19 patients. Our observation that 4-OI strongly inhibits the IFN-398


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response to both cytosolic DNA and d RNA, which are canonical anti-viral pathways, but still 399

retain its ability to block viral replication also suggests a spectrum of unidentified cellular 400

programs that are inducible through NRF2 and efficiently restrict viral replication independently 401

of IFNs. This is supported by already mentioned negative correlation between expression of 402

NRF2-inducible genes and infection with HSV1 discovered by Wyler et al., through single cell 403

transcriptome analysis(13). Similarly to how IFNs block viral replication through the induction of 404

hundreds of effector IFN-stimulated effector genes, the NRF2 controlled anti-viral program might 405

also consists of a myriad of mechanisms that restrict viral replication each by targeting distinct 406

stages of viral replication. 407

Future studies will determine if patients developing severe SARS-CoV2 pathology also have an 408

underlying NRF2 deficiency, leading to reduced control of viral replication, coupled with excess 409

inflammatory responses. Further, it could be valuable to investigate if patients already in DMF 410

therapy have altered susceptibility to SARS-CoV2 infection and if those infected have milder 411

symptoms and reduced cytokine load. Finally, the fact that 4-OI effectively limited replication of 412

several human pathogenic viruses demonstrated that 4-OI, or related chemically modified 413

compounds such as DMF, could be evaluated as broad-spectrum anti-viral agents for protection 414

against seasonal and pandemic viral infections in general. 415

416

Acknowledgments: 417

Funding: This research work was supported by Ester M og Konrad Kristian Sigurdssons Dyreværnsfond, Beckett-418

Fonden, Kong Christian IX og Dronning Louises Jubilæumslegat, Læge Sofus Carl Emil Friis og Hustru Olga Doris 419

Friis´ legat, Købmand I Odense Johan og Hanne Weimann Født Seedorffs Legat, Hørslev Fonden, UK Medical 420

Research Council (MRC core funding of the MRC Human Immunology Unit; JR), Lundbeck foundation (R303-421

2018-3379 and R219-2016-878, and R268-2016-3927), and Independent Research Fund Denmark – Medical 422


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21

Sciences (9039-00078B, 4004-00047B, and 0214-00001B). CarlsbergFoundation (Semper Ardens) 423

and European Research Council (ERC-AdG ENVISION; 786602). Marie Skłodowska-Curie Action of the 424

European Commission # 813343 and Italian Cancer Research Society #22891 to JH. Author contributions: DO 425

and CH conceived the project and prepared figures. DO, ALH, JT, JC, BH, MI, EBS, AK, LR, MI, MS, SF, MT, 426

ML, HH, VG, AH, AK, CG, DVDH, CM, LB, AT, TA, JH, RO, and AA performed experiments, analyzed data, and 427

prepared figures. EF performed bioinformatics and prepared figures. TM was responsible for including COVID-19 428

patients and achieving patient material. TA, JR, AA, TM, SP, and CH planned experiments and analyzed data. CH 429

and DO drafted and finalized the manuscript. JH planned experiments and edited the manuscript. EF, SP, SB and 430

ML performed bioinformatics and prepared figures. MJ facilitated SARS-CoV2 laboratories. Authors declare no 431

competing interests. Data and materials availability: All data is available in the main text or the supplementary 432

materials, 433


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