key: cord-1005237-fhimlarx
authors: Alfi, Or; Yakirevitch, Arkadi; Wald, Ori; Wandel, Ori; Izhar, Uzi; Oiknine-Djian, Esther; Nevo, Yuval; Elgavish, Sharona; Dagan, Elad; Madgar, Ory; Feinmesser, Gilad; Pikarsky, Eli; Bronstein, Michal; Vorontsov, Olesya; Jonas, Wayne; Ives, John; Walter, Joan; Zakay-Rones, Zichria; Oberbaum, Menachem; Panet, Amos; Wolf, Dana G.
title: Human nasal and lung tissues infected ex vivo with SARS-CoV-2 provide insights into differential tissue-specific and virus-specific innate immune responses in the upper and lower respiratory tract
date: 2021-03-08
journal: bioRxiv
DOI: 10.1101/2021.03.08.434404
sha: 041975b748c93bc0bef5f807f2e7d0c9259e5fb0
doc_id: 1005237
cord_uid: fhimlarx

The nasal-mucosa constitutes the primary entry site for respiratory viruses including SARS-CoV-2. While the imbalanced innate immune response of end-stage COVID-19 has been extensively studied, the earliest stages of SARS-CoV-2 infection at the mucosal entry site have remained unexplored. Here we employed SARS-CoV-2 and influenza virus infection in native multi-cell-type human nasal turbinate and lung tissues ex vivo, coupled with genome-wide transcriptional analysis, to investigate viral susceptibility and early patterns of local-mucosal innate immune response in the authentic milieu of the human respiratory tract. SARS-CoV-2 productively infected the nasal turbinate tissues, predominantly targeting respiratory epithelial cells, with rapid increase in tissue-associated viral sub-genomic mRNA, and secretion of infectious viral progeny. Importantly, SARS-CoV-2 infection triggered robust antiviral and inflammatory innate immune responses in the nasal mucosa. The upregulation of interferon stimulated genes, cytokines and chemokines, related to interferon signaling and immune-cell activation pathways, was broader than that triggered by influenza virus infection. Conversely, lung tissues exhibited a restricted innate immune response to SARS-CoV-2, with a conspicuous lack of type I and III interferon upregulation, contrasting with their vigorous innate immune response to influenza virus. Our findings reveal differential tissue-specific innate immune responses in the upper and lower respiratory tract, that are distinct to SARS-CoV-2. The studies shed light on the role of the nasal-mucosa in active viral transmission and immune defense, implying a window of opportunity for early interventions, whereas the restricted innate immune response in early-SARS-CoV-2-infected lung tissues could underlie the unique uncontrolled late-phase lung damage of advanced COVID-19. IMPORTANCE In order to reduce the late-phase morbidity and mortality of COVID-19, there is a need to better understand and target the earliest stages of SARS-CoV-2 infection in the human respiratory tract. Here we have studied the initial steps of SARS-CoV-2 infection and the consequent innate immune responses within the natural multicellular complexity of human nasal-mucosal and lung tissues. Comparing the global innate response patterns of nasal and lung tissues, infected in parallel with SARS-CoV-2 and influenza virus, we have revealed distinct virus-host interactions in the upper and lower respiratory tract, which could determine the outcome and unique pathogenesis of SARS-CoV-2 infection. Studies in the nasal-mucosal infection model can be employed to assess the impact of viral evolutionary changes, and evaluate new therapeutic and preventive measures against SARS-CoV-2 and other human respiratory pathogens.

authentic milieu of the human respiratory tract. SARS-CoV-2 productively infected 48 the nasal turbinate tissues, predominantly targeting respiratory epithelial cells, with 49 rapid increase in tissue-associated viral sub-genomic mRNA, and secretion of 50 infectious viral progeny. Importantly, SARS-CoV-2 infection triggered robust antiviral 51 and inflammatory innate immune responses in the nasal mucosa. The upregulation 52 of interferon stimulated genes, cytokines and chemokines, related to interferon 53 signaling and immune-cell activation pathways, was broader than that triggered by 54 influenza virus infection. Conversely, lung tissues exhibited a restricted innate 55 immune response to SARS-CoV-2, with a conspicuous lack of type I and III 56 interferon upregulation, contrasting with their vigorous innate immune response to 57 influenza virus. Our findings reveal differential tissue-specific innate immune 58 responses in the upper and lower respiratory tract, that are distinct to The studies shed light on the role of the nasal-mucosa in active viral transmission 60 and immune defense, implying a window of opportunity for early interventions, 61 whereas the restricted innate immune response in early-SARS-CoV-2-infected lung 62 tissues could underlie the unique uncontrolled late-phase lung damage of advanced 63 . 64

The ongoing coronavirus disease-2019 pandemic, caused by severe 82 acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has created an immense 83 global health crisis. While the majority of infections are asymptomatic or cause mild-84 to-moderate disease, a significant proportion of COVID-19 patients progress over 85 time to display severe pneumonia with acute respiratory distress syndrome, 86 reflecting extensive late-stage viral-and inflammatory-mediated lung injury (1-5). 87 SARS-CoV-2 primarily targets the respiratory tract, utilizing the cellular receptor 88 angiotensin-converting enzyme 2 (ACE-2) and the transmembrane protease serine 2 89 (TMPRSS2), shown to be expressed in respiratory epithelial cells lining the upper 90 and lower airways (6) (7) (8) (9) (10) (11) (12) (13) . 91

The nose is the main port of entry for SARS-CoV-2. The importance of the nasal 92 mucosa as the initial site for SARS-CoV-2 infection is suggested by the observed 93 sequence of clinical manifestations (proceeding from upper-to-lower respiratory 94 involvement), and the higher expression of ACE2 gene in nasal epithelial cells 95 (compared to lower respiratory airway epithelial cells), paralleled by high infectivity of 96 these cells in vitro (7, 13) . 97

Frontline protection against respiratory viral infections is mediated by early local-98 mucosal innate immune responses, exerting antiviral defense via multiple 99 upregulated interferon stimulated genes (ISGs) and cytokines release (14, 15) . In the 100 case of SARS-CoV-2, the importance of innate immune defenses in viral control has 101 been highlighted by the finding that inborn defects in innate immunity or the 102 10 nasal turbinate and in the lung tissues -as measured by viral RNA synthesis and 184 infectious viral progeny levels over time ( To gain a global insight into the earliest tissue responses to SARS-CoV-2, we 205 employed unbiased genome-wide transcriptome analysis of infected versus mock-206 infected tissues at 24 hpi. This time point was chosen based on our demonstration 207 that SARS-CoV-2 replication in the tissues already reaches its peak at 24 hpi (see 208 indicating that the transcriptional response to SARS-CoV-2 and influenza virus in 211 infected tissues is well induced by 24 hpi. We sought to define common versus virus-212 specific and tissue-specific innate immune response patterns, by comparing the 213 transcriptional response of the nasal turbinate and lung tissues to SARS-CoV-2 and 214 influenza A(H1N1) pdm09 (upon parallel infection of the same tissues, as described 215 above). 216

Transcriptome analysis was carried out independently on 3 nasal tissues and 5 lung 217 tissues (all obtained from different donors) to gain statistical significance. We 218 detected viral gene transcripts representing coverage over the entire viral genome in 219 all infected tissues (data not shown). The percent of viral transcripts (of all the 220 sequence reads in the transcriptome data) was overall comparable upon infection by 221 SARS-CoV-2 and influenza virus in both tissues, except for a significantly higher 222 percent of SARS-CoV-2 transcripts reads in the lung tissues compared to the 223 percent of SARS-CoV-2 reads in the nasal turbinate tissues ( Figure S1 ). 224

To start evaluating the global host response pertaining to each of the conditions, the 226 samples were grouped in principal-component analysis (PCA). As shown, SARS-227

CoV-2 and influenza exerted distinct global signatures in the nasal turbinate tissues, 228 reflected by their disparate coordinates ( Figure 3A ); SARS-CoV-2 and influenza 229 transcriptional signatures differentially distributed from mock along PC2, with more 230 progressive transcriptional response observed along this axis in SARS-CoV-2 231 infected tissues. We found that SARS-CoV-2 infection substantially affected the 232 global gene expression profile in the nasal turbinate tissues, leading to differential 12 expression of 371 genes (309 upregulated and 62 downregulated following infection; 234 Figure 3B ). The most profoundly upregulated genes included antiviral ISGs, 235 cytokines and chemokines ( Figures 3C, S2A ). Of note, in addition to familiar ISGs, 236 one of the most upregulated genes in SARS-CoV-2 infected nasal turbinate tissues 237 was the long non-protein coding RNA LINC00487 ( Figure 3C ), recently identified as 238 a novel ISG (37). Employing qRT-PCR of RNA purified from independent infected 239 and mock-control nasal turbinate tissues, we validated the viral-induced upregulation 240 of selected innate immunity genes following infection ( Figure S3 ). In accordance, 241 and further defining the affected biological pathways and predicted functions, 242

Ingenuity Pathway Analysis (IPA) showed that SARS-CoV-2 infection in the nasal 243 mucosal tissues primarily induced antiviral and proinflammatory pathways related to 244 interferon signaling, innate immunity, and immune cell activation ( Figure 3C ,D, see 245 also Figure 5A ). Despite similar levels of infection, we observed some transcriptional 246 response variability between the three independent nasal turbinate tissues (mainly 247 with respect to the extent, but not the direction: up-versus downregulation, of 248 differential gene response; Figure 3D ). As we have shown before, this tissue-to-249 tissues variability, reflecting the natural diversity between individuals, is expected in 250 studies involving human tissues (36). 251

Influenza virus infection of the same nasal turbinate tissues differentially affected the 252 expression of a lower number of genes compared to SARS-CoV-2 (186; 182 253 upregulated and 4 downregulated; Figure 3B ). Comparison between the nasal 254 turbinate tissue response to SARS-CoV-2 and influenza, identified 173 common and 255 198 SARS-CoV-2-distinct differentially expressed (DE) genes ( Figure 3B ). Innate 256 immunity genes related to interferon signaling, immune activation, and antiviral 257 pathways were commonly induced (albeit to a variable extent) by the 2 viruses in the 258 13 nasal mucosal tissues (see also Figure 5A ). The common and distinct response 259 patterns of the nasal mucosal tissues to SARS-CoV-2 and influenza virus could be 260 clearly delineated by a clustered heatmap analysis of all significantly DE genes, 261 which identified 4 clusters of genes, defined by the direction (upregulation vs. 262 downregulation) and/or the extent of their differential expression ( Figure 3D ). In 263 general, the nasal turbinate tissue response to SARS-CoV-2 appeared broader than 264 the response to influenza virus, and included innate immune and immune cell 265 maturation and activation pathways which were distinctively or more significantly 266 were not affected by SARS-CoV-2 infection; Figure 3B ). In fact, in agreement with 286 the PCA ( Figure 4A ), a clustered heatmap analysis of all significantly DE genes, 287 showed that the relative transcriptional profile of SARS-CoV-2 infected lung tissues 288 was closer to that of mock-infected tissues, and completely distinct from that of the 289 highly responsive influenza-infected lung tissues ( Figure 4D Employing independent qRT-PCR, we showed a low-to-absent upregulation of IFN-296 I, IFN-II, and IFN-III by SARS-CoV-2, compared to influenza virus, with the 297 differences reaching statistical significance for IFNα, IFNβ and IFNλ ( Figure 5C ). 298

Significantly, no upregulation of IFNλ1 was observed in all SARS-CoV-2 infected 299 lung tissues examined, a finding that contrasted with the upregulation of IFNλ1 in 300 both SARS-CoV-2-infected turbinate tissues and influenza-infected lung tissues 301 ( Figure 5C ). 302

Together, the combined data revealed that SARS-CoV-2 affected the nasal mucosal 303 tissues and the lung tissues in a distinct virus-specific and organ-specific manner. 304 SARS-CoV-2 triggered a robust antiviral and proinflammatory innate immune 305 response, broader than the innate response to influenza virus infection, in the nasal 306 turbinate tissues, yet induced a restricted innate immune response and no apparent 307 IFN induction in the lung tissues, which was further underscored by the vigorous 308 response of the same lung tissues to influenza virus. Our findings imply that SARS-CoV-2 successfully manipulates the innate immune 351 response in the lung tissues, which were otherwise capable of mounting a robust IFN 352 antiviral response to influenza. In this regard, SARS-CoV-2 has been shown to 353 encode synergistic innate immune antagonist genes [i.e., Nsp1-shutting down 354 cellular translation, Nsp3, Nsp5, Nsp10, Nsp13, Nsp14, ORF3, ORF6 and ORF7, 355 ORF8; (12, 38, 40, 41) ], and thus may more effectively dampen the lung antiviral 356 defence compared with influenza virus, whose IFN evasion function is mediated 357 mainly by NS1 (12, 42) . The question remains: why the same SARS-CoV-2 immune 358 manipulation strategies are rendered ineffective in the nasal mucosa tissue milieu? 359

We suggest that the nasal mucosa, being constantly exposed to environmental 360 agents and resident microflora (unlike the relatively sterile lower respiratory tract), is 361 conditioned to persistent innate immune signalling, which could override the viral 362

antagonists. In support of this hypothesis is the recently demonstrated skewed 363 expression of innate immune genes in cultured nasal epithelial cells (13). 364

A schematic illustration of the differential tissue-specific innate immune responses to 365 SARS-CoV-2 in nasal and lung tissues, as compared to influenza virus mediated 366 responses, is shown in Figure 6 

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