key: cord-0951226-g599enn0
authors: Kazmierski, Julia; Friedmann, Kirstin; Postmus, Dylan; Fischer, Cornelius; Jansen, Jenny; Richter, Anja; de Jarcy, Laure Bosquillon; Schüler, Christiane; Sohn, Madlen; Sauer, Sascha; Drosten, Christian; Saliba, Antoine-Emmanuel; Sander, Leif Erik; Niemeyer, Daniela; Goffinet, Christine
title: Non-productive exposure of PBMCs to SARS-CoV-2 induces cell-intrinsic innate immunity responses
date: 2022-02-15
journal: bioRxiv
DOI: 10.1101/2022.02.15.480527
sha: 7a0e79b0c2127e7319b48d2a78be90d2bcd45337
doc_id: 951226
cord_uid: g599enn0

Cell-intrinsic responses mounted in vivo in PBMCs during mild and severe COVID-19 differ quantitatively and qualitatively. Whether they are triggered by signals emitted by productively infected cells of the respiratory tract or are, at least partially, resulting from physical interaction with virus particles, remains unclear. Here, we analyzed susceptibility and expression profiles of PBMCs from healthy donors upon ex vivo exposure to SARS-CoV and SARS-CoV-2. In line with the absence of detectable ACE2 receptor expression, human PBMCs were refractory to productive infection. Bulk and single cell RNA-sequencing revealed JAK/STAT-dependent induction of interferon-stimulated genes, but not pro-inflammatory cytokines. This SARS-CoV-2-specific response was most pronounced in monocytes. SARS-CoV-2-RNA-positive monocytes displayed a lower ISG signature as compared to bystander cells of the identical culture. This suggests a preferential invasion of cells with a low ISG base-line profile or delivery of a SARS-CoV-2-specific sensing antagonist upon efficient particle internalization. Together, non-productive physical interaction of PBMCs with SARS-CoV-2-but not SARS-CoV particles stimulates JAK/STAT-dependent, monocyte-accentuated innate immune responses that resemble those detected in vivo in patients with mild COVID-19.

The current SARS-CoV-2 pandemic represents a global medical, societal and economical 54 emergency of increasing importance. Arising at the end of 2019 in the Hubei province in China, 55 the causative agent of the coronavirus disease 2019 , SARS-CoV-2, has to date 56 infected more than 395 million individuals world-wide (World Health Organization). Owing to 57 SARS-CoV-2 infection, more than 5,7 million deaths were reported up to today (as of 2022, 58

February 6th). The predominant symptoms of symptomatic COVID-19 are fever, cough, and 59 shortness of breath, however, in severe cases disease can progress to pneumonia, acute 60 respiratory distress syndrome, and multiple organ failure Wölfel et al. 2020) . 61

The management of the pandemic is complicated by a relatively low manifestation index, a 62 large inter-individual spectrum of clinical courses ranging from asymptomatic to fatal 63 outcomes, pre-and asymptomatic infectious phases (Jones et al. 2021; Rothe et al. 2020) , 64 and the ongoing emergence of variants with increased transmissibility and/or immune escape. 65

The reasons for the high inter-individual outcome of infection are insufficiently understood and 66 may include different degrees of cross-reactive background immunity at the level of humoral 67 (Anderson et al. 2021 ; K. W. Ng et al. 2020) and T-cell-mediated immunity (Braun et al. 2020; 68 Bacher et al. 2020; Nelde et al. 2021; Schulien et al. 2021) , polymorphisms in genes related 69 to innate immunity ) and autoimmunity (Bastard et al. 2020) . Currently, 70 specific treatment regimen must be administered early post-infection. They include the RNA 71 polymerase inhibitor Remdesivir that may reduce hospitalization time but not mortality (Y. 72 Wang et al. 2020 ) and monoclonal anti-spike antibodies with variant-specific neutralization 73 potencies (Weinreich et al. 2021; RECOVERY Collaborative Group, Horby, Mafham, et al. 74 2021) . In the late phase of infection, the administration of the immune modulator 75 dexamethasone (RECOVERY Collaborative Group, Horby, Lim, et al. 2021) dampens 76 hyperactivation of cytokine-driven immune responses. While several effective vaccines are 77 available, the necessity for specific treatment options will likely persist given the expected 78

proportion of the population that will not have access to vaccines or will refuse vaccination. 79 To accelerate the establishment of immunomodulatory strategies, it is crucial to 80 characterize ex vivo systems that correlate with cellular immunophenotypes of SARS-CoV-2 81 infection in vivo and that may contribute to pre-clinical testing. Furthermore, the usage of ex 82 vivo platforms allows the systematic and comparative investigation of human cellular 83 responses to exposure with different representatives of the species SARS-related 84 coronaviruses, including SARS-CoV. Peripheral immune cells are major contributors to 85 human cellular responses upon infection. Given the recruitment of blood mononuclear cells to 86 the lung compartment (Delorey et al. 2021; Bost et al. 2020; Wendisch et al. 2021) , and the 87 reported presence of viral RNA detectable in the peripheral blood of up to 10% severely ill 88 patients (Prebensen et al. 2021; Andersson et al. 2020) , direct contact of PBMCs with SARS-89

CoV-2 virions is a likely scenario. 90

Here, we analyzed susceptibility to infection and cell-intrinsic innate responses of 91 peripheral blood cells from healthy donors upon ex vivo exposure to SARS-CoV and SARS-92

CoV-2. Although both SARS-related coronaviruses failed to detectably replicate and spread 93 in PBMCs, SARS-CoV-2 specifically triggered a JAK/STAT-dependent innate immune 94 response that was most pronounced in monocytes. Single-cell, virus-inclusive RNA 95 sequencing revealed relatively inefficient and ACE2-independent uptake of virus particles and 96 a SARS-CoV-2 exposure-specific gene expression profile. Cellular responses, consisting in 97 upregulation of expression of interferon-stimulated genes (ISGs) but not pro-inflammatory 98 cytokines, partially recapitulate expression profiles obtained by single-cell RNA-sequencing of 99

PBMCs from patients experiencing mild COVID-19 (Arunachalam et al. 2020; Schulte-100 Schrepping et al. 2020; Silvin et al. 2020) . Our data demonstrate that cells from the peripheral 101 blood, when undergoing contact to SARS-CoV-2 particles, mount cellular responses that 102 potentially contribute to control and/or pathogenesis of the infection. -5 -

To address the ability of SARS-related coronaviruses to infect and propagate in cells of the 109 peripheral blood, we exposed unstimulated PBMCs from healthy individuals to purified stocks 110 of SARS-CoV and SARS-CoV-2, respectively, using equal infectious titers as determined on 111 Vero E6 cells. As a reference, PBMCs were exposed to supernatants from uninfected Vero 112 E6 cells (mock-exposed). For both SARS-related coronaviruses, infectivity in cell culture 113 supernatants drastically decreased over time compared to the inoculum, reaching 114 undetectable levels at three days post-inoculation ( Fig. 1A) , pointing towards absence of de 115 novo production of infectious particles. Treatment of cells with the polymerase inhibitor 116

Remdesivir did not further reduce infectivity in the supernatant, suggesting that the infectivity 117 detectable at 24 hours post-inoculation reflects virus input (Fig. 1B) . In contrast, infection of 118 Vero E6 cells with the identical SARS-CoV-2 stock gave rise to a productive and Remdesivir-119 sensitive infection (Supplemental Fig. 1) . In our experiments, virus-containing supernatant 120 was replaced with fresh medium four hours post-inoculation. Nevertheless, viral RNA genome 121 equivalents remained detectable in the culture supernatant until the end of the experiment for 122 both SARS-CoV and SARS-CoV-2 (up to 192 hours post-exposure) (Fig. 1C) . Viral RNA was 123 abundant also in supernatants from Remdesivir-treated cultures and cultures exposed to heat-124 inactivated SARS-CoV-2 until 192 hours post-exposure, arguing for a high stability of the 125 residual viral RNA of the inoculum and against a constant replenishment of extracellular viral 126 RNA pools as a reason for the stable RNA quantities (Fig. 1D) , in line with reported longevity 127 of the incoming genomic viral RNA (Lee et al. 2022) . Notably, blunting signaling by type I 128 interferons (IFNs) through constant presence of the JAK/STAT inhibitor Ruxolitinib failed to 129 enable secretion of infectious particles and viral RNA in the supernatant, suggesting that 130 JAK/STAT-dependent cell-intrinsic innate immunity is not the underlying reason for the 131 absence of detectable virus production ( Fig. 1 A, C) . 132 133 Kazmierski and Friedmann et al. -6 -To elucidate if PBMCs, despite being non-permissive, are nevertheless susceptible to 134 SARS-related coronavirus entry and initial RNA replication, we monitored cell-associated viral 135 RNA species in the adherent and the suspension cell fractions of the cultures over time. Cell-136 associated viral genome equivalents (Fig. 1E) and subgenomic viral E RNA (Supplemental 137 Fig. 2) , the latter produced during discontinuous viral transcription, remained stable over time 138 and did not differ in quantities for both SARS-related coronaviruses. Ruxolitinib treatment did 139 not detectably facilitate RNA replication (Fig. 1E, Supplemental Fig. 2) , suggesting absence 140 of essential cofactors at the level of entry and/or RNA replication rather than the antiviral 141 activity of IFN-regulated restriction factors. In line with this idea, we failed to detect expression 142 of the SARS-coronavirus receptor, angiotensin-converting enzyme 2 (ACE2) in PBMCs, as 143 judged by immunoblotting, flow cytometry and Q-RT-PCR using ACE2-specific antibodies and 144 primer/probes, respectively (Supplemental Fig. 3 A-C) . In conclusion, freshly isolated, 145 unstimulated PBMCs seem to be devoid of ACE2 expression. Furthermore, they appear to be 146 non-susceptible and non-permissive to infection with either SARS-related coronavirus, at least 147 ex vivo. However, the continuous presence of viral RNA associated to cells and in the culture 148 supernatant suggests that virus particles attach to and/or internalize into PBMCs in an ACE2-149 independent manner and remain cell-associated for up to several days. 150 151 Exposure of PBMCs to SARS-CoV-2, but not SARS-CoV, triggers a JAK/STAT-152 dependent cell-intrinsic innate immune response 153

To identify potential cell-intrinsic innate immune responses to SARS-CoV and SARS-CoV-2 154 exposure, we analyzed IFIT1 and IL6 mRNA expression over time ( Fig. 2A-B) . We selected 155 IFIT1 and IL6 as prototypic target genes that are transcribed by IRF3 and NF-κB, respectively 156 (Honda and Taniguchi, 2006) . In contrast to SARS-CoV-inoculated cells, SARS-CoV-2-157 exposed cells displayed Ruxolitinib-sensitive, significantly upregulated IFIT1 mRNA 158 expression at 16, 24 and 48 hours post-inoculation ( Fig. 2A) . Inhibition of potential low-level 159 SARS-CoV-2 RNA replication through treatment of cells with Remdesivir, and heat-160 inactivation of the SARS-CoV-2 stock inoculum did not prevent induction of IFIT1 mRNA 161 Kazmierski and Friedmann et al. -7 -expression (Supplemental Fig. 4) , corroborating the idea that the latter is triggered by 162 exposure to virions, but not by productive infection. In contrast, IL6 expression was barely 163 induced after exposure to SARS-CoV and SARS-CoV-2 (Fig. 2B) . We next analyzed if type I 164 IFN expression preceded IFIT1 mRNA expression in SARS-CoV-2-exposed PBMCs. Despite 165 a slight trend for elevated IFNA1 and IFNB1 mRNA expression at 16 hours, levels failed to 166 reach significant upregulation at four, 16 and 24 hours, when compared to mock-exposed 167 cultures (Fig. 2C) . Together, SARS-CoV-2 exposure specifically triggered IRF3-induced 168 IFIT1, but not NF-κB-transcribed IL-6 gene expression. These results suggest that, although 169 both SARS-related coronaviruses failed to establish a productive infection in PBMCs, SARS-170

CoV-2 appears to induce cell-intrinsic, JAK/STAT-dependent responses in several cell types 171 comprised in PBMCs. 172

To explore cell-intrinsic responses in individual cell types, we performed single-cell RNA-174 sequencing of PBMCs exposed to SARS-CoV and SARS-CoV-2, respectively. We identified 175 the five major cell types, namely B-cells, CD4 + and CD8 + T-cells, NK cells and monocytes 176 for SARS-CoV-2-, but not for SARS-CoV-exposed cultures, compared to mock-inoculated 185 cells (Fig. 3C) . Notably, SARS-CoV-2 monocytes clustered separately from the other 186 conditions in the UMAP despite library batch correction, implying a pronouncedly altered 187 transcriptome. The T-and NK cell clusters slightly and partially shifted, indicating a change in 188

Kazmierski and Friedmann et al.

-8 -their transcriptional profile (Fig. 3C) . The relative abundance of T-cells and monocytes in 189 SARS-CoV-2-exposed cells as compared to mock-exposed PBMCs remained constant, as 190 judged by flow cytometric analysis (Supplemental Fig. 6 ). Together, this analysis revealed 191 that transcriptomic changes occurred in most cell types upon SARS-CoV-2 exposure, 192 particularly in the monocytic fraction. 193

PBMCs with cell type-specific signatures 196

We next investigated in more detail the cell type-specific response to SARS-CoV-2. Based on 197 differentially expressed genes (DEGs) between mock-, SARS-CoV-and SARS-CoV-2-198 exposed PBMCs, a pseudotime cell trajectory analysis for all cell types was performed (Fig. 199 4A). For all five major cell types, cells inoculated with SARS-CoV-2 developed towards a 200 separate cell fate and branched off from mock-exposed and SARS-CoV-exposed cells, which, 201 conversely, shared a common trajectory. Interestingly, B-cell analysis resulted in four 202 branching points, from which only two (#1 and #3) were specific for SARS-CoV-2-exposed 203 cells, suggesting a high transcriptional heterogeneity of B-cells independently of virus 204 exposure. Though progression through pseudotime resulted in a distinct and highly 205 pronounced trajectory of all SARS-CoV-2-exposed cell types, this effect was most pronounced 206 in monocytes (Fig. 4A) . Analysis of expression of specific genes, including ISG15 and IFIT1, 207 confirmed that in general, all cell types contributed to gene expression changes upon SARS-208

CoV-2 challenge, and monocytes displayed the most pronounced elevation of expression of 209 both genes (Fig. 4B) . Identification of DEGs in mock-exposed compared to SARS-CoV-2-210 inoculated PBMCs revealed a significant upregulation of gene expression in all cell types, 211 especially in monocytes (Fig. 4C) . Interestingly, the majority of DEGs were identified as known 212

ISGs (defined by the interferome database; colored in green (interferome.org; v2.01)). Scoring 213 the individual cell types and conditions by their expression of an IFN-signaling module 214 revealed a SARS-CoV-2-specific upregulated expression in all cell types, though this was 215

Kazmierski and Friedmann et al.

-9 -most prominent in monocytes (Fig. 4D) . Moreover, IFN module scores were colinear with 216

Pseudotime scores along the SARS-CoV-2 trajectory, supporting the notion that SARS-CoV-217 2 exposure induces a development of PBMCs towards an antiviral phenotype. Increase of 218 expression of several ISGs, including ISG15, IFIT1, IFITM3, DDX58, IFIH, LY6E, MX2, IFI6, 219 GBP1, BST2, was detectable predominantly, but not exclusively, in monocytes ( Fig. 4E) , 220 supporting the hypothesis that monocytes play a key role in the induction of cell-intrinsic innate 221 immune response to SARS-CoV-2 stimulation. In line with our previous findings (Fig. 2) , 222 SARS-CoV-2-and SARS-CoV-exposed cells scored virtually negative for expression of 223 various cytokines, including IL6 (Fig. 4E) and IFN mRNAs (Supplemental Fig. 7) , although 224 they express IFN receptors (Supplemental Fig. 7 ). In conclusion, these data reveal a strong 225 induction of cell-intrinsic innate immunity in SARS-CoV-2-exposed PBMCs that manifests 226 predominantly in monocytes. 227 228

Next, we aimed at identifying viral RNA-positive cells and their specific transcriptional profile 230 that we hypothesized to differ from cells without detectable viral RNA of the identical culture. 231 SARS-CoV-2 RNA was detectable in all cell types, but predominantly in monocytes (Fig. 5A) . 232

Identified viral reads were distributed over the viral genome sequence, with a high over-233 representation of the 3´ RNA sequences that all subgenomic and genomic viral RNA have in 234 common, corresponding to the 3´ part of the N-coding sequence and polyA tail (Fig. 5B) . 235

Specifically, in SARS-CoV-and SARS-CoV-2-exposed PBMC cultures, we identified 99 236 (2.13%) and 212 (2.88%) viral RNA-positive cells, respectively (Fig. 5C ). Among those, we 237 identified 56 (7.8%) and 173 (15.3%) viral RNA-positive monocytes among all monocytes, 238 respectively. First of all, no statistically significant differences in expression of individual genes 239 of RNA-positive and RNA-negative monocytes were identified. However, the IFN module 240 score ( Fig. 4) was slightly, but statistically highly significantly elevated in SARS-CoV-2-241 exposed monocytes with undetectable viral RNA ( Fig. 5D-E) . Specifically, within the 94 genes 242 that were expressed marginally more abundantly in cells lacking detectable SARS-CoV-2 243

Kazmierski and Friedmann et al.

-10 -RNA, 18 represented ISGs, including ISG15, IFITM2, IFITM3, IFI27 and HLA genes tended to 244 be upregulated in viral RNA-negative bystander cells. Importantly, the presence of viral RNA 245 did not specifically associate with expression of BSG/CD147 and NRP1, and ACE2 and 246 TMPRSS2 expression was undetectable, suggesting that particles internalize in a manner that 247 is independent of these confirmed or proposed receptors, respectively. In SARS-CoV-2 RNA-248 positive cells as compared to SARS-CoV-2 RNA-negative cells of the identical cultures, 249 among others, CD163 reads tended to be slightly more abundant. Expression of the 250 hemoglobin-haptoglobin scavenger receptor CD163 has been associated with regulation of 251 inflammation (Kowal et al. 2011 ) and has interestingly been linked to immunological changes 252 in monocytes and monocyte-derived macrophages from SARS-CoV-2-infected individuals 253 (Gómez-Rial et al. 2020; Trombetta et al. 2021; Wendisch et al. 2021) . Looking specifically at 254 the CD163 HIGH monocyte population, we found that it displayed high expression levels of high inter-donor variability (L. F. P. Ng et al. 2004) . In vivo, in situ hybridization and electron 282 microscopy analyses reported presence of SARS-CoV material in lymphocytes and 283 monocytes derived from infected patients (Gu et al. 2005) . MERS-CoV was suggested to 284 efficiently replicate in ex vivo-infected monocytes (Chu et al. 2014 ), but to only abortively infect 285

human T-cells (Chu et al. 2016) . Of note, the confirmed receptor for SARS-CoV-2 cell entry 286 (Hoffmann et al. 2020) has been reported to be virtually absent in PBMCs (Song et al. 2020; 287 Xu et al. 2020; Zou et al. 2020; Xiong et al. 2020) , a finding that is in line with our own inability 288 to detect ACE2 mRNA and ACE2 protein expression in PBMCs by various methods. 289 Therefore, we hypothesize that virus particles attach and/or internalize in an ACE2-290 independent manner, resulting in viral RNA associated to and/or internalized into cells. Given 291 that receptor-independent phagocytosis is a hallmark of monocytes, our observation that the 292 majority of the viral reads were retrieved in monocytic cells underlines this idea. Furthermore, 293

as SARS-CoV ORF7a is a virion-associated protein (Cheng Huang et al. 2006 ) and SARS-294

CoV-2 ORF7a was reported to efficiently interact with PBMC-derived monocytes (Zhou et al. 295 2021), ORF7a may contribute to attachment to monocytes. Interestingly, the binding capability 296

Kazmierski and Friedmann et al.

-12 -of SARS-CoV ORF7a protein was reported to be significantly weaker as compared to SARS-297

CoV-2 ORF7a (Zhou et al. 2021) , which is consistent with the observed two-fold reduced 298

proportion of virus RNA-positive monocytes in SARS-CoV-exposed PBMCs as compared to 299 SARS-CoV-2. 300

In vivo, a multitude of cytokines, including IL-1β, IL-1RA, IL-7, IL-8, IL-9, IL-10, 301 Genes associated to fibrosis, migration and integrin binding were mildly upregulated in cells 340

with detectable viral RNA when compared to bystander cells, defined as cells of the SARS-341

CoV-2-exposed cell culture which lacked detectable viral reads. Interestingly, monocytes 342 developing profibrotic functions have recently been established in the context of COVID-19 in 343 vivo (Wendisch et al. 2021) . Bystander cells displayed enhanced ISG expression, suggesting 344 either a more efficient and more probable internalization into cells with a low ISG profile or, 345 alternatively, delivery of a SARS-CoV-2-specific sensing antagonist in the context of efficient 346 particle internalization. Multiple SARS-related coronaviruses-encoded IFN antagonists, 347 including structural components of the incoming virion that do not require productive infection 348 for being expressed and functional, dampen innate immune responses when ectopically 349 expressed, including membrane and nucleocapsid proteins (Lei et al. 2020 ). In addition, virion 350 components including ORF3 and ORF6 (Bai et al. 2021; Cheng Huang, Peters, and Makino 351 2007; Ito et al. 2005) have type I IFN evasion properties (Li et al. 2020; Schroeder et al. 2021; 352 Kazmierski and Friedmann et al. -14 -Lei et al. 2020) ). Interestingly, among those, ORF6 from SARS-CoV-2 was described to be 353 inferior in counteracting phospho-IRF3 nuclear translocation in infected cells, compared to 354 SARS-CoV ORF6, resulting in higher ISG induction (Schroeder et al. 2021) . Therefore, 355 incoming viral RNA sensing may be less efficiently prevented by SARS-CoV-2 ORF6 as 356 compared to SARS-CoV ORF6. Finally, the large absence of a detectable ISG expression 357 profile in SARS-CoV-exposed PBMCs is consistent with a previous report analyzing abortively 358 infected monocyte-derived macrophages (Cheung et al. 2005) . 359

Together, our study provides analysis of gene expression in ex vivo-exposed PBMCs 360 at the cell type and individual cell´s level. Our data suggest that direct stimulation of monocytes 361 through physical contact to SARS-CoV-2 particles is followed by strong ISG induction, despite 362 absence of detectable productive infection. -20 -significance was calculated by performing Student's t-test using GraphPad Prism. P values 518 <0.05 were considered significant and marked accordingly: P<0.05 (*),P <0.01 (**) or P< 519 0.001; n.s. = not significant (≥0.05). 520 521

The raw sequencing datasets generated during this study will be made available at the NCBI 523 Gene Expression Omnibus. PBMCs isolated from two donors were exposed to SARS-CoV, SARS-CoV-2 or mock-580 IFITM1  LGALS3BP  IFI27  IFITM3  OASL  MX2  IFIT1  STAT2  IFI35  DDX60L  IFIT2  PARP9  IFI44  OAS1  RSAD2  IFIH1  DDX60  DDX58  ISG15  LY6E   IFI6   EIF2AK2  STAT1  IFI44L  XAF1  MX1  ISG20  PARP14  TRIM22  IFITM2  BST2 

Antibodies Are Boosted upon SARS-CoV-2 Infection but Not Associated with 625 Protection

SARS-CoV-2 RNA 628 Detected in Blood Products from Patients with COVID-19 Is Not Associated with 629 Infectious Virus

Systems Biological 632 Assessment of Immunity to Mild versus Severe COVID-19 Infection in Humans

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China: A Descriptive Study

Sin-Fun Sia

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Monocytes in COVID-19 Immunopathology

Multiple Organ Infection and the Pathogenesis of SARS

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Coronavirus-Infected Pneumonia in Wuhan, China

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