key: cord-0953465-2s1io2fg
authors: Vanderheiden, Abigail; Ralfs, Philipp; Chirkova, Tatiana; Upadhyay, Amit A.; Zimmerman, Matthew G.; Bedoya, Shamika; Aoued, Hadj; Tharp, Gregory M.; Pellegrini, Kathryn L.; Lowen, Anice C.; Menachery, Vineet D.; Anderson, Larry J.; Grakoui, Arash; Bosinger, Steven E.; Suthar, Mehul S.
title: Type I and Type III IFN Restrict SARS-CoV-2 Infection of Human Airway Epithelial Cultures
date: 2020-05-20
journal: bioRxiv
DOI: 10.1101/2020.05.19.105437
sha: 3601c5ec44494eb32661986c89dde1b833cd2644
doc_id: 953465
cord_uid: 2s1io2fg

The newly emerged human coronavirus, SARS-CoV-2, has caused a pandemic of respiratory illness. The innate immune response is critical for protection against Coronaviruses. However, little is known about the interplay between the innate immune system and SARS-CoV-2. Here, we modeled SARS-CoV-2 infection using primary human airway epithelial (pHAE) cultures, which are maintained in an air-liquid interface. We found that SARS-CoV-2 infects and replicates in pHAE cultures and is directionally released on the apical, but not basolateral surface. Transcriptional profiling studies found that infected pHAE cultures had a molecular signature dominated by pro-inflammatory cytokines and chemokine induction, including IL-6, TNFα, CXCL8. We also identified NF-κB and ATF4 transcription factors as key drivers of this pro-inflammatory cytokine response. Surprisingly, we observed a complete lack of a type I or III IFN induction during SARS-CoV-2 infection. Pre-treatment or post-treatment with type I and III IFNs dramatically reduced virus replication in pHAE cultures and this corresponded with an upregulation of antiviral effector genes. Our findings demonstrate that SARS-CoV-2 induces a strong pro-inflammatory cytokine response yet blocks the production of type I and III IFNs. Further, SARS-CoV-2 is sensitive to the effects of type I and III IFNs, demonstrating their potential utility as therapeutic options to treat COVID-19 patients. IMPORTANCE The current pandemic of respiratory illness, COVID-19, is caused by a recently emerged coronavirus named SARS-CoV-2. This virus infects airway and lung cells causing fever, dry cough, and shortness of breath. Severe cases of COVID-19 can result in lung damage, low blood oxygen levels, and even death. As there are currently no vaccines or antivirals approved for use in humans, studies of the mechanisms of SARS-CoV-2 infection are urgently needed. SARS-CoV-2 infection of primary human airway epithelial cultures induces a strong pro-inflammatory cytokine response yet blocks the production of type I and III IFNs. Further, SARS-CoV-2 is sensitive to the effects of type I and III IFNs, demonstrating their potential utility as therapeutic options to treat COVID-19 patients.

system and SARS-CoV-2. Here, we modeled SARS-CoV-2 infection using primary 28 human airway epithelial (pHAE) cultures, which are maintained in an air-liquid interface. 29 We found that SARS-CoV-2 infects and replicates in pHAE cultures and is directionally 30 released on the apical, but not basolateral surface. Transcriptional profiling studies 31 found that infected pHAE cultures had a molecular signature dominated by pro-32 inflammatory cytokines and chemokine induction, including IL-6, TNFα, CXCL8. We 33 also identified NF-κB and ATF4 transcription factors as key drivers of this pro-34 inflammatory cytokine response. Surprisingly, we observed a complete lack of a type I 35 or III IFN induction during SARS-CoV-2 infection. Pre-treatment or post-treatment with 36 type I and III IFNs dramatically reduced virus replication in pHAE cultures and this 37 corresponded with an upregulation of antiviral effector genes. Our findings demonstrate 38 that SARS-CoV-2 induces a strong pro-inflammatory cytokine response yet blocks the 39 production of type I and III IFNs. Further, SARS-CoV-2 is sensitive to the effects of type 40 I and III IFNs, demonstrating their potential utility as therapeutic options to treat COVID-41 19 patients. 44 The current pandemic of respiratory illness, COVID-19, is caused by a recently 45 emerged coronavirus named SARS-CoV-2. This virus infects airway and lung cells 46 causing fever, dry cough, and shortness of breath. Severe cases of COVID-19 can 47 result in lung damage, low blood oxygen levels, and even death. As there are currently 48 no vaccines or antivirals approved for use in humans, studies of the mechanisms of 49 SARS-CoV-2 infection are urgently needed. SARS-CoV-2 infection of primary human 50 airway epithelial cultures induces a strong pro-inflammatory cytokine response yet 51 blocks the production of type I and III IFNs. Further, SARS-CoV-2 is sensitive to the 52 effects of type I and III IFNs, demonstrating their potential utility as therapeutic options 53 to treat COVID-19 patients. 55 In December 2019, a novel human coronavirus, SARS-CoV-2, emerged in Wuhan, 56 China, causing an outbreak of severe respiratory disease (1, 2). In the span of several 57 months, SARS-CoV-2 rapidly escalated to a pandemic, with over 4 million infections 58 and 275,000 deaths worldwide (3). There are currently no vaccines or antivirals 59 approved for use in humans that can prevent or treat the infection. SARS-CoV-2 60 infection manifests as an upper and lower respiratory disease (named COVID-19 by the 61 World Health Organization), characterized by fever, dry cough, and shortness of breath. 62 SARS-CoV-2 targets lower respiratory tract cells, with one study finding 93% of their 63 patients' bronchial lavages were qRT-PCR positive for SARS-CoV-2 (4). 64 Correspondingly, lung abnormalities have been observed in several patients with 65 COVID-19, and severe infection can lead to respiratory failure and lung tissue 66 destruction (5). Severe disease has also been associated with a low level of 67 lymphocytes in the blood and high levels of pro-inflammatory cytokines, such as IL-6 68 and TNF-α (6). How the host innate immune system responds to SARS-CoV-2 infection 69 is not well understood. The SARS-CoV-2 genome is 29.8 kb in length and predicted to contain 12 open reading 72 frames. This includes 15 putative non-structural proteins, envelope and capsid genes, 73 an RNA-dependent RNA polymerase (RDRP), and a spike protein (7, 8). SARS-CoV-2 74 virus is closely related to the β-coronavirus SARS-CoV, which caused an outbreak of 75 acute respiratory distress syndrome in China in 2003 (9). β-coronaviruses use the spike 76 protein receptor-binding domain to gain entry to target cells, and recent studies have 77 found that SARS-CoV-2 and SARS-CoV utilize the same receptor, ACE-2 (10). SARS-78 CoV-2 entry was also found to require the expression of the cellular protease, 79 TMPRSS2 (10). ACE-2 and TMPRSS2 are expressed in epithelial tissue from the lung 80 and gut, with the highest expression in ciliated cells from the nasal cavity (11) In this study, we seek to address some of these unanswered questions about the innate 100 immune response to SARS-CoV-2. Here, we find that SARS-CoV-2 infects and 101 replicates in pHAE cultures and is released exclusively from the apical surface. We 102 performed transcriptional profiling and found that infection triggers a robust 103 inflammatory cytokine response characterized by the induction of IL-6, CXCL8, TNF-α, 104 and IL-1 family cytokines. In contrast, we observed a lack of induction of type I IFN and 105 IFN-stimulated genes despite the expression of both type I and III IFN receptors. We 

To understand the response of airway epithelial cells to SARS-CoV-2 infection, we 114 utilized pHAE cultures isolated from the bronchial or tracheal region. These cells were 115 cultured using an air-liquid interface model to create a polarized, pseudostratified 116 epithelial layer. This culture system excellently re-capitulates the unique features of the 117 human respiratory tract, including mucus production and coordinated cilia movement 118 (16). pHAE cultures were infected on the apical side with SARS-CoV-2 at an MOI of 0.1 119 and 0.25 (as determined on VeroE6 cells). In our study, we used a low cell culture-120 passaged and sequence-verified SARS-CoV-2 strain, nCoV2019/WA, which was 121 isolated in January 2020 from nasopharyngeal and oropharyngeal swab specimens 122 collected three days post-symptom onset (17). Using two different measurements, we 123 demonstrate that SARS-CoV-2 infects and replicates in pHAE cultures. On the apical 124 surface, we detected infectious SARS-CoV-2 beginning 24 hours post-infection (p.i.) 125 and increased through 48 hours p.i. as measured by plaque assay (Fig. 1A) . In 126 contrast, we were unable to detect infectious SARS-CoV-2 virus on the basolateral side 127 at any timepoint or MOI, suggesting the directional release of the virus from pHAE 128 cultures. Next, we confirmed the presence of viral RNA in the cells by qRT-PCR with 129 primer/probes that anneal to the SARS-CoV-2 RNA-dependent RNA polymerase 130 (RDRP). We observed an increase in viral RNA between MOI 0.1 and 0.25 at 48 hours 131 p.i. (Fig. 1B) . Combined, these findings demonstrate that pHAE cultures are permissive 132 for SARS-CoV-2 infection. 135 We next evaluated the innate immune response to SARS-CoV-2 infection. To this end, 136 we performed bulk mRNA-sequencing analysis on differentiated pHAE cultures infected 137 with SARS-CoV-2 (MOI= 0.25) at 48 hours p.i. Following infection, we observed 1,039 138 differentially expressed genes (DEG) (P < 0.01; 1.5-fold change cut-off), with 458 139 upregulated ( Fig. 2A in red) and 581 downregulated ( Fig. 2A in blue) DEGs. We 140 detected viral transcripts spanning most of the viral genome, although there was minor 141 variation between the three replicates ( Fig 2B) . Table 2 ). This suggests that SARS-CoV-2 does not alter mucus and 160 AMP production in pHAE cultures. (22). Accordingly, GSEA also revealed enrichment 180 of genes associated with the transition from epithelial to mesenchymal tissue (Fig. 3F) . 181 Overall, the transcriptional profiling of infected pHAE cultures revealed that the normal 182 homeostatic functions are disrupted and they induce a pro-inflammatory phenotype 183 characterized by NF-κB signaling, ER stress response and IL-6 production. (Fig. 4E) (Fig. 5A) . As compared to untreated cells, we observed significantly reduced viral 214 RNA in type I (3-fold less) and III (3-fold less) IFN treated cells by 24 hours p.i. (Fig.   215   5B) . We next evaluated infectious virus release and found that pHAE cultures pre-216 treated with type I or III IFNs significantly reduced (14-fold and 12-fold respectively) 217 SARS-CoV-2 viral burden by 24 hours p.i., resulting in greater than 90% reduction in 218 virus replication as compared to untreated SARS-CoV-2 infected cells (Fig. 5C) . the IFIT family of antiviral effector genes (Fig. 5D) . Changes in transcription factors 225 were also observed with increases in IRF-7 and to a lesser extent IRF-1. Treatment with 226 type I and III IFNs upregulated ISGs regardless of infection status, but SARS-CoV-2 227 infected samples had larger fold changes compared to mock-infected cells. In SARS-228 CoV-2 infected samples, treatment with type I IFN induced higher expression of certain 229 ISGs (IFIH1, IFIT2) than type III IFN (Fig. 5D) . These findings demonstrate that pre- CoV-2 (MOI= 0.5) and, at 24 hours p.i., we treated the basolateral side with IFNβ1 or 237 IFNλ1 (100 IU/mL). Infectious virus release was measured before and after treatment 238 (Fig. 6A) . Twenty-four hours after treatment (48 hours p.i.) there was no significant 239 difference in viral burden between treatments. However, by 72 hours p.i., treatment with 240 both type I and III IFN had reduced SARS-CoV-2 viral levels 50-fold compared to 241 untreated samples, resulting in a 98% reduction in viral burden at 72 hours p.i. (Fig.   242 6A). Analysis of RNA at 72 hours p.i. confirmed that both IFNβ1 and IFNλ1 treatment 243 reduced viral RNA compared to untreated cells, 12-fold and 20-fold, respectively (Fig.   244   6B) . Similarly, to our pre-treated samples (Fig. 5) , treatment after infection with type I 245 or III IFNs upregulated ISGs, such as RIG-I, MDA5, and IFIT family members, 246 compared to untreated (Fig. 6C) In this study, we found that human pHAE cultures, which model the air-liquid interface of 

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Identification of Coronavirus Isolated from a Patient in Korea with COVID-19

A cluster of 481 cases of severe acute respiratory syndrome in Hong Kong

CoV-2 Cell Entry Depends on ACE2 and TMPRSS2 and Is Blocked by a Clinically 485

SARS-CoV-2 493 entry factors are highly expressed in nasal epithelial cells together with innate immune 494 genes

Severe acute 496 respiratory syndrome coronavirus infection of human ciliated airway epithelia: role of 497 ciliated cells in viral spread in the conducting airways of the lungs

Shared and Distinct Functions of Type I 499 and Type III Interferons

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CX3CR1 is an 511 important surface molecule for respiratory syncytial virus infection in human airway 512 epithelial cells

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Dysregulated Type I Interferon and Inflammatory Monocyte-565

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ACE2 receptor expression and severe acute 569 respiratory syndrome coronavirus infection depend on differentiation of human airway 570 epithelia

Differential Activation of the Transcription Factor IRF1 Underlies the Distinct Immune 574

Responses Elicited by Type I and Type III Interferons

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Epithelial-mesenchymal transition and its implications for 589 fibrosis

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STAR: ultrafast universal RNA-seq aligner

A) Healthy, 603 differentiated pHAE cultures were infected by adsorption to the apical side at the 604 indicated MOI. The supernatant was collected from the apical or basolateral side of the 605 epithelial layer, and the virus was measured by plaque assay. B) Viral RNA was 606 measured by probing for the SARS-CoV-2 RDRP RNA at 48 hours p.i. by qRT-PCR. C T 607 values are represented as relative fold change over mock (log 10 )

Bulk RNA-Seq analysis of SARS-CoV-2 infected pHAE cultures. pHAE 611 cultures were infected apically with SARS-CoV-2 (MOI= 0.25) for 48 hours, at which 612 point mock and SARS-CoV-2 infected (n=3) samples were harvested for bulk RNA-Seq 613 analysis. A) Volcano plot demonstrating DEGs. Lines indicate cut-offs

fold-change < -1.5). B) Normalized read counts (log 2 ) of SARS-CoV-2 RNA 617 products, using the MT246667.1 reference sequence

SARS-CoV-2 infection promotes a pro-inflammatory and ER stress 620 response in pHAE cultures. pHAE cultures were infected apically with SARS

25) for 48 hours, at which point mock and SARS-CoV-2 infected (n=3) samples 622 were harvested for bulk RNA-Seq analysis. For global DEG analysis see figure 2

Volcano plot with all DEGs in grey and the indicated gene set highlighted (purple= pro-624 inflammatory signaling). B) GSEA plots of the enrichment score plotted against gene 625 rank. Individual gene hits are indicated by the solid black line below the enrichment 626 score curve. NES and p-value are indicated on the plot

Hallmarks gene set from MSigDB. C) Heatmap illustrating z-scores for the indicated 628 genes in mock and SARS-CoV-2 infected samples D) Volcano plot illustrating barrier 629 immunity associated genes in orange. E) Network map illustrating regulatory nodes for 630 our DEGs. F) GSEA plots for the indicated gene sets

Figure 4. pHAE cultures fail to upregulate type I or III IFNs in response to SARS-633

RNA-Seq analysis of the IFN response, for global DEG analysis see 634 figure 2. A) Normalized read counts (log 2 ) in mock (white) and SARS-CoV-2 infected 635 (grey samples) of type I and type III IFN

B) genes associated with IFN production in green C) genes associated with IFN 637 signaling in pink. D) Bar graphs indicating the normalized read count (log 2 ) for 638 interferon-stimulated genes and E) type I and type III IFN receptors for mock and 639 SARS-CoV-2

Pre-treatment with type I or III IFNs restricts SARS-CoV-2 replication in 642 pHAE cultures. pHAE cultures were pre-treated from the basolateral side with IFNβ1 or 643

Cultures were then infected apically (MOI= 0.25) and 644 harvested at 24 hours p.i. A) Experimental schematic. B) SARS-CoV-2 viral burden in 645 untreated, IFNβ1, and IFNλ1 treated cultures as assessed via focus forming assay

Percent reduction was calculated as the percent of the untreated sample at 24 hours p.i. 647 qRT-PCR analysis was performed at 24 hours p.i. for C) viral RNA, or D) ISGs. qRT-648 PCR data are represented as fold change over mock, untreated pHAE samples. Data is 649 representative of two independent experiments performed in biological triplicate. All 650 data were analyzed using one-way ANOVA

Post-treatment with type I or III IFNs decreases viral burden in pHAE 653 cultures. pHAE cultures were infected with SARS-CoV-2 (MOI = 0.5) apically. Twenty-654 four hours p.i., cultures were treated from the basolateral side with IFNβ1 or IFNλ1

SARS-CoV-2 burden was assessed via FFA for the apical side of 657 untreated, IFNβ1, and IFNλ1 treated cultures. Percent reduction was calculated for the 658 72-hour timepoint. qRT-PCR analysis at 72 hours p.i. as compared to mock, untreated 659 samples, measuring B) viral RNA, or C) ISGs. qRT-PCR data are represented as fold 660 change over mock. Results are representative of two independent experiments 661 performed in triplicate. Growth curves were analyzed using a two-way ANOVA, qRT-662 PCR data were analyzed using one-way ANOVA

Supplemental Table 1. Pro-inflammatory associated gene expression in pHAE 668 cultures. List of the pro-inflammatory genes surveyed in pHAE cultures. Fold-changes 669 (log 2 ) are calculated over mock

Barrier immunity associated with gene expression in pHAE 672 cultures. List of the barrier immunity associated genes surveyed in pHAE cultures

Fold-changes (log 2 ) are calculated over mock

ATF-4 and CEBPB associated genes. List of genes 676 promoted by the NF-kB, ATF-4 and CEBPB transcription factors. Fold-changes (log 2 ) 677 are calculated over mock

Genes associated with the induction of interferon in pHAE 680 cultures. List of the PRR signaling genes surveyed in pHAE cultures. Fold-changes 681 (log 2 ) are calculated over mock

Interferon stimulated and IFN receptor-associated genes in 684 pHAE cultures. List of the genes associated with IFN signaling and ISGs surveyed in 685 pHAE cultures. Fold-changes (log 2 ) are calculated over mock

We would like to thank Dr. C. U. Cotton (Case Western Reserve 421 University) for generously providing our pHAE specimens, Natalie Thornburg (CDC, 422 Atlanta, GA) for providing our SARS-CoV-2 viral stock, and Drs. Jens Wrammert and

Robert Kauffman (Emory University, Atlanta, GA) for making the cross-reactive anti-424 SARS spike CR3022 biotin-conjugated monoclonal antibody. We also thank the Yerkes 425 Genomics Core (Emory University, Atlanta, GA) for help with the RNA-Seq analysis.