key: cord-0854146-qtf0sl88
authors: Anand, Gautam; Perry, Alexandra M.; Cummings, Celeste L.; Raymond, Emma St.; Clemens, Regina A.; Steed, Ashley L.
title: Surface proteins of SARS-CoV-2 drive airway epithelial cells to induce interferon-dependent inflammation
date: 2020-12-14
journal: bioRxiv
DOI: 10.1101/2020.12.14.422710
sha: e22912958c11e4c939c72f8aefaa8659893e6a0f
doc_id: 854146
cord_uid: qtf0sl88

SARS-CoV-2, the virus that has caused the COVID-19 pandemic, robustly activates the host immune system in critically ill patients. Understanding how the virus engages the immune system will facilitate the development of needed therapeutic strategies. Here we demonstrate both in vitro and in vivo that the SARS-CoV-2 surface proteins Spike (S) and Envelope (E) activate the key immune signaling interferon (IFN) pathway in both immune and epithelial cells independent of viral infection and replication. These proteins induce reactive oxidative species generation and increases in human and murine specific IFN-responsive cytokines and chemokines, similar to their upregulation in critically ill COVID-19 patients. Induction of IFN signaling is dependent on canonical but discrepant inflammatory signaling mediators as the activation induced by S is dependent on IRF3, TBK1, and MYD88 while that of E is largely MYD88 independent. Furthermore, these viral surface proteins, specifically E, induced peribronchial inflammation and pulmonary vasculitis in a mouse model. Finally we show that the organized inflammatory infiltrates are dependent on type I IFN signaling, specifically in lung epithelial cells. These findings underscore the role of SARS-CoV-2 surface proteins, particularly the understudied E protein, in driving cell specific inflammation and their potential for therapeutic intervention. Author Summary SARS-CoV-2 robustly activates widespread inflammation, but we do not understand mechanistically how the virus engages the immune system. This knowledge will facilitate the development of critically needed therapeutic strategies to promote beneficial immune responses will dampening harmful inflammation. Here we demonstrate that SARS-CoV-2 surface proteins spike and envelope alone activated innate cell function and the interferon signaling pathway. This activation occurred in both immune and epithelial cells, and mechanistic studies demonstrated dependence on known key inflammatory signaling mediators, IRF3, TBK1, and MYD88. In animal studies, we showed that these viral surface proteins induce epithelial cell IFN-dependent lung pathology, reminiscent to acute COVID-19 pulmonary infection. These findings underscore the need for further investigation into the role of SARS-CoV-2 surface proteins, particularly the understudied E protein, in driving cell specific inflammation.

While the S protein interaction with ACE2 has been the focus of vaccine design, other structural 75 proteins likely play key roles in disease pathogenesis. The coronaviral genomes also encode 76 structural proteins Nucleocapsid (N), Envelope (E), Membrane (9, 11). However, little is known 77 about these structural proteins' roles in immune activation and pathogenesis. The N protein has 78 been shown to have an immunomodulatory function in SARS-CoV infection (12, 13) . 79 Interestingly, the SARS-CoV and SARS-CoV-2 E proteins have no amino acid substitutions. 80 SARS-CoV E is essential for viral morphology, budding, and tropism (14, 15) . Importantly, the 81 SARS-CoV E was found to enhance inflammasome activation (16) (17) (18) (19) . Therefore, the conserved 82 E protein and its engagement of the host immune response could prove to be a potent therapeutic immune response. While lymphopenia has been a consistent finding in 22, 23) , 90 many patients also exhibit a cytokine storm which is associated with disease severity and 91 outcome (7-9, 21, 24-27). These patients demonstrate an increase in number of inflammatory 92 monocytes and elevated serum levels of proinflammatory chemokines and cytokines including 93 IL-2, IL-7, IL-10, IL-6, G-CSF, IP-10, MIP-1α, MCP-1 and 2, 7, 21, 25, 26, [28] [29] [30] [31] [32] [33] [34] . 94 While these chemokines and cytokines attract immune cells to mount an antiviral defense, the 95 resulting cytokine storm and cellular infiltration have been implicated in lung cell damage and 96 disease pathogenesis. 97 Given the key roles of the innate immune response in both viral clearance and disease 98 pathogenesis, understanding how SARS-CoV-2 structural proteins elicit host immunity is 99 necessary for designing optimal therapeutic strategies. Therefore, we sought to investigate the 100 innate immune response to SARS-CoV-2 antigens, independent of viral infectivity and nuclei 101 acid replication. In this report, we demonstrate that the purified structural proteins of SARS- generation in ex vivo isolated wt murine peritoneal macrophages after overnight incubation 117 compared to control samples by 2.09 ± 0.35-fold and 2.63 ± 0.95-fold, respectively. (Fig 1A) .

118 Alveolar macrophages also demonstrated increased zymosan-induced ROS production in 119 response to E-Trunc (1.71±0.19-fold) but not in response to S (Fig 1B) . MIP-1α (57-fold) and IL-8 (7-fold). S protein similarly increased the expression of CCL5 (7.6-137 fold), CCL2 (12.7-fold), MIP-1α (4.2-fold), and IL-8 (4.5-fold), albeit to a lesser magnitude than 138 increased by E-Trunc. CCL5 transcript expression by S and E-Trunc was confirmed by qRT-139 PCR (S2A Fig). Interestingly, S protein alone specifically increased IL-1Rα (2.3-fold), GM-CSF 140 (2.9-fold), and CXCL12 (1.5-fold). These findings underscore that there are shared as well as distinct immune responses to specific coronavirus surface antigens and implies unique 142 mechanisms of activation.

Increased serum TNF-α has been found during 25, 26, 31) . Previous 145 work has also shown a specific increase in TNF-α expression in mouse macrophages by the 146 SARS-CoV S protein (37). Likewise, we also found that TNF-α increased in mouse monocytes 147 incubated with SARS-CoV-2 proteins E-Trunc or S (Fig 1C) . However in human myeloid cells 148 our results were inconsistent; there was no difference in the levels of TNF-α after exposure to S 149 and a small decrease (0.7-fold) in protein but increased mRNA transcript after incubation with E- Purified SARS-CoV-2 proteins induce inflammatory signaling 156 As our findings above showed that E-Trunc and S protein upregulate multiple chemokines and 157 cytokines known to be IFN responsive, we directly asked whether these antigens activate IFN 158 induction. We incubated the IFN reporter cell lines, which harbor tandem interferon stimulated 159 response elements inducing luciferase expression, with E-Trunc and S as well as the SARS-CoV-160 2 structural protein N and the full length E protein (E-Full). After 24-hour incubation, IFN 161 induction were enhanced in both murine and human monocytes, most robustly by E-full in the 162 human THP-1 reporter cells and E-Trunc in the murine RAW reporter cells (Fig 2A) . E-Full 163 enhanced luciferase expression by 3-fold in RAW cells and 6.5-fold in THP-1 cells while E-164 Trunc lead to a 5.5-fold and 2.8-fold increase, respectively. Protein S enhanced IFN signaling in 165 these cells to a lesser extent by 1.3-fold in RAW cells and 1.5-fold in THP-1 cells. A VSV-166 pseudovirus expressing the SARS-CoV-2 S protein on its virion surface also increased IFN 167 induction in both THP-1 and RAW cells compared to VSV expressing its glycoprotein (VSV-G), 168 albeit with observed cytotoxicity in THP-1 VSV-G infected cells (Fig 2B-C) . Importantly, the 169 structural protein N did not induce IFN signaling in any of the cell lines tested. Of note, the N 170 protein is contained inside the virion while E and S are displayed on the viral surface. To gain further mechanistic insight into how viral antigens independently activate IFN signaling, 173 we investigated the role of known mediators of IFN induction by pathogen recognition receptors.

THP-1 reporter cells deficient in IRF3 and TBK1 did not exhibit IFN induction in response to E-175 Trunc or Spike and had a dramatic decrease in response to E-Full (Fig 2A) , demonstrating key 176 dependence on these well described IFN inducing mediators. MyD88 also modulates IFN 177 responses, largely through TLR activation. Indeed, E-Trunc and E-Full showed partial decreases 178 in IFN induction in THP-1-MyD88 KO cells while the response to S was abolished.

Given that these THP-1 cells are also capable of reporting NFKB induction, we investigated the 181 effect of the above viral antigens in a similar fashion. NFKB induction was also increased in 182 response to viral peptide incubation with the most striking response to E-full (8.5-fold) and least 

Of note, throughout these studies we sought to assure that our findings of IFN and NFKB 190 induction were in response to the viral peptides and not LPS contamination. Using the limulus 191 amoebocyte lysate assay, we found minimal LPS (less than 0.4ng/ml) in our viral protein next studied the direct effect of these viral surface antigens in vivo. We administered E-Trunc 204 and S intranasally to C57Bl/6J wildtype mice and examined the effect on lung histology three 205 days later. Cross-sections of lungs showed significant organized peribronchial and medium-sized 206 airway pathology in those mice exposed to E-Trunc or S compared to control treated mice (Fig   207   3A , 3D). Immunostaining for CD45 demonstrated the hematopoietic origin of these 208 inflammatory infiltrates ( Fig 3B) . Furthermore, animals exposed to E-Trunc and S also showed 209 significant vascular pathology with evidence of vasculitis ( Fig 3C) , a finding that has been 

234 pulmonary epithelial cells 235 To determine the role of type I IFN signaling in SARS-CoV-2 surface protein induction of 236 pulmonary pathology, we rendered the type I IFN signaling pathway defective genetically in the 237 type I IFN receptor (Ifnar -/-) or using an Ifnar blocking monoclonal antibody (42) While the S protein is responsible for cell entry via ACE2 and is the focus of numerous 305 therapeutic strategies, the E protein of SARS-CoV-2 is understudied although recent evidence 306 points to its potential as an ion channel (53). Prior work in other coronaviruses has demonstrated 307 that E protein is indispensable for viral morphogenesis and tropism as well as enhances 308 inflammasome activation (14-18); our work further points to its crucial role on innate immune 309 activation and function. Given that E is highly conserved with SARS-CoV (9, 54) further study 310 is necessary as E may be a potent target for therapeutic strategies with broader applications, Further information and requests for resources and reagents may be directed to and will be 389 fulfilled by the corresponding author, Ashley L. Steed (steeda@wustl.edu). 

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