key: cord-0765049-rk6grjj2
authors: Schimmel, Lilian; Chew, Keng Yih; Stocks, Claudia; Yordanov, Teodor; Essebier, Patricia; Kulasinghe, Arutha; Monkman, James; Miggiolaro, Anna Flavia Ribeiro dos Santos; Cooper, Caroline; de Noronha, Lucia; Lagendijk, Anne K.; Schroder, Kate; Labzin, Larisa I.; Gordon, Emma J.; Short, Kirsty R.
title: Endothelial cells elicit a pro-inflammatory response to SARS-CoV-2 without productive viral infection
date: 2021-05-06
journal: bioRxiv
DOI: 10.1101/2021.02.14.431177
sha: d8db25f0dfe81b41ef33881e43b6e74b09f5aad1
doc_id: 765049
cord_uid: rk6grjj2

Objectives Thrombotic and microvascular complications are frequently seen in deceased COVID-19 patients. However, whether this is caused by direct viral infection of the endothelium or inflammation-induced endothelial activation remains highly contentious. Methods Here, we use patient autopsy samples, primary human endothelial cells and an in vitro model of the pulmonary epithelial-endothelial cell barrier to show that primary human endothelial cells express very low levels the SARS-CoV-2 receptor ACE2 and the protease TMPRSS2. Results Accordingly, endothelial cells can only be infected when SARS-CoV-2 is present at very high concentrations. However, this is not a productive infection (i.e. no infectious virus is produced) and viral entry induces an inflammatory response. We also show that SARS-CoV-2 does not infect endothelial cells in 3D vessels under flow conditions. We further demonstrate that in a co-culture model endothelial cells are not infected with SARS-CoV-2. They do however sense and respond to infection in the adjacent epithelial cells, increasing ICAM-1 expression and releasing pro-inflammatory cytokines. Conclusions Taken together, these data suggest that in vivo, endothelial cells are unlikely to be infected with SARS-CoV-2 and that infection is only likely to occur if the adjacent pulmonary epithelium is denuded (basolateral infection) or a high viral load is present in the blood (apical infection). In such a scenario, whilst SARS-CoV-2 infection of the endothelium can occur, it does not contribute to viral amplification. However, endothelial cells are still likely to play a key role in SARS-CoV-2 pathogenesis by sensing adjacent infection and mounting a pro-inflammatory response to SARS-CoV-2.

also shown SARS-CoV-2 RNA to be enriched in pulmonary endothelial cells 33 When and how endothelial cells may be exposed to SARS-CoV-2 in vivo also remains unclear. As a 86 respiratory virus, SARS-CoV-2 encounters pulmonary epithelial cells prior to any interaction with the 87 endothelium. SARS-CoV-2 infects pulmonary epithelial cells apically and new viral particles are also 88 released apically into the lumen of the lung 39 . It is possible that the infected epithelial layer loses barrier 89 function, allowing endothelial infection from the basolateral side of epithelia that is adjacent to alveolar 90 endothelium. Alternatively, SARS-CoV-2 may infect endothelial cells apically via the blood, although 91 viremia appears to be rare in COVID-19 patients 40 . The question therefore remains whether endothelial 92 dysfunction in COVID-19 is the result of direct viral infection of the endothelium. 93

Here, using patient autopsy samples and in vitro models, we show that whilst SARS-CoV-2 can enter 95 endothelial cells if high viral titres are present, this infection is not productive. Rather, in response to 96 direct or indirect viral exposure, endothelial cells mount a pro-inflammatory response characterised by 97 increased expression of ICAM-1 and secretion of CXCL-10 and IL-6. Taken together, our results 98 provide clarification on the intensely debated topic of endothelial infection by SARS-CoV-2, to reveal 99 that the endotheliopathy and thrombocytopathy in patients with severe COVID-19 is likely to be due to 100 the inflammatory response, rather than direct viral infection. or NucleoZOL (BIOKÉ, The Netherlands) according to manufacturer's guidelines. cDNA was 197 synthesized using random hexamers and the high-capacity cDNA reverse transcription kit (Life 198 Technologies) according to the manufacturer's guidelines. 199

Quantitative PCR (qPCR): qPCR was performed using SYBR Green reagent (Applied Biosystems). 201

All primer sequences are listed in Table 1 . qPCR conditions were applied according to the 202 manufacturer's instructions using QuantStudio 6 Flex Real-Time PCR System (ThermoFisher 203

Scientific, Waltham, Massachusetts, MA). HPRT or Glyceraldehyde 3-phosphate dehydrogenase 204

(GAPDH) was used as a housekeeping gene and relative gene expression was determined using the 205 comparative cycle threshold (ΔΔCt) method (Livak, 2001) . 206 207 Table 1 : Primers used in the present study 208 NAME SEQUENCE (5'-3') staining. Within the mask, Raw Integrated Density of either ACE2, NP, ICAM-1 was measured, and 220 value was corrected for total mask area resulting in RawIntDen/Area. For dsRNA, MaxEntropy 221 threshold was performed on dsRNA staining to remove background speckles before measuring Raw 222

Integrated Density within the mask. Analysis of Western blot images was performed using Gel Analyzer 223

Tool of ImageJ version 2.0.0-rc-69/1.52n. The background signal was subtracted and values were 224 normalized to corresponding GAPDH loading control. Imaris (Bitplane) version 8 was used to create 225 the XZ projection in Figure 4B ''. Extrusion of cells was quantified in Supplemental Figure 1C using 226 orthogonal views in ImageJ version 2.0.0-rc-69/1.52n. 227 228 Statistical analysis: All statistical analysis was performed using Graphpad Prism version 9.0. Data are 229 presented as mean±s.e.m. with individual data points indicated and colour coded per independent 230 experimental replicate. Statistical significance was determined using Kruskal Wallis test for multiple 231 comparisons or Mann-Whitney test between mock and SARS-CoV-2 treated conditions. *P<0.05, 232 **P<0.01, ***P<0.001 and ****P<0.0001. 233

To determine the prevalence of in vivo SARS-CoV-2 endothelial cell infection, autopsy lung sections 237 were obtained from 10 deceased COVID-19 patients and probed for SARS-CoV-2 spike mRNA using 238

RNAscope. Two of the 10 patients were positive for SARS-CoV-2 RNA in the lungs. In these 239 individuals, spike mRNA could not be detected in pulmonary endothelial cells (endothelial cells defined 240 by H & E staining ( Figure 1 ). These data suggest that the endothelium is not generally the primary site 241 of viral replication in vivo in COVID-19 patients (patient data available in Supplemental Table 1) . 242 243

To further investigate the contribution of endothelial cells to the pathogenesis of SARS-CoV-2, we 245 established in vitro cultures of primary human umbilical vein endothelial cells (HUVECs) and human 246 microvascular endothelial cells from the lung (HMVEC-L). Multiple studies have reported that 247 endothelial cells across different vascular beds express ACE2 and TMPRSS2, the host cell receptor and 248 protease that are required for efficient cell infection by SARS-CoV-2 10, 11 . We found that both HUVECs 249 and HMVEC-Ls expressed comparable ACE2 and TMPRSS-2 mRNA to that observed in immortalised 250 epithelial cells known to be susceptible to SARS-CoV-2 infection (Figure 2A ). Neuropilin-1, 251

which enhances viral entry through its interaction with the spike multibasic cleavage site 7, 8 , was readily 252 detected in endothelial cells by qPCR ( Figure 2A ) in line with established data 19 . The expression of 253 ACE2 protein detected by immunofluorescence ( Figure 2B ) was significantly reduced compared to 254 epithelial cells (Calu-3) ( Figure 2C ). We also detected ACE2 and TMPRSS2 protein in HUVECs and 255 HMVEC-Ls by western blot (Figure 2D -E). The specificity of our ACE2 antibody was confirmed by 256 western blot of BHK-21 cells (which are known to be negative for ACE2) 43 .These data suggest that 257 while vascular endothelial cells can theoretically be infected by SARS-CoV-2, infection is likely to be 258 inefficient compared to epithelial cells, because of weak receptor and protease expression in endothelial 259

SARS-CoV-2 may theoretically enter endothelial cells via the apical surface (should the virus enter the 263 blood stream) or the basolateral surface (should the virus be present at the basolateral surface of the 264 adjacent epithelial cells). We therefore assessed whether endothelial cells were susceptible to SARS-265

CoV-2 infection when exposed either apically (Supplemental Figure 1A ) or basolaterally (Supplemental 266 

To determine whether endothelial cells can be infected in 3D vessels under flow, we cultured both 299 HMVEC-L and Vero cells in microfabricated tubes 42 and added SARS-CoV2 to the luminal surface 300 ( Figure 4A ). After 24h, Vero cells were readily infected and viral replication was observed, in contrast 301

to HMVEC-L which shows viral input levels ( Figure 4B ). In agreement, NP was not detected in 

As severe COVID-19 disease is associated with an elevated cytokine response 44 , we sought to assess 308 whether the abortive SARS-CoV-2 infection observed in endothelial cells induces a pro-inflammatory 309 response. We assessed expression of the leukocyte adhesion molecule, ICAM-1, which is well 310 established to be induced under inflammatory conditions in the endothelium 45 . Immunofluorescence 311

showed that HMVEC-L exposed both basally and apically to SARS-CoV-2 significantly increased 312 ICAM-1 expression, except for in nucleocapsid protein-positive HMVEC-L (likely as they are apoptotic 313 and extruded from the monolayer) ( Figure 5A -C). The moderate basal ICAM-1 expression observed in mock infected cells is likely due to the fact that the cells are kept for 72h without a media change (as 315 seen in PBS controls ( Figure 5B ). However, this response was not as robust as when HMVEC-L were 316 exposed to the established inflammatory cytokine TNFα ( Figure 5B -C). Western blot analysis 317 confirmed that HMVEC-L exposed to SARS-CoV-2 displayed a trending increase in ICAM-1 318 expression ( Figure 5D , E). In addition to upregulating inflammatory adhesion molecules, we analysed 319 whether endothelial cells can be the source of pro-inflammatory cytokines during SARS-CoV-2 320 infection. As IL-6 and CXCL10 are elevated in patients with severe COVID-19 46 , we analysed whether 321 endothelial cells released these pro-inflammatory cytokines when infected either apically or 322 basolaterally with SARS-CoV-2 for 72h. HMVEC-Ls released modest levels of CXCL10 ( Figure 5F ) 323 and IL-6 ( Figure 5G ). While these modest levels of cytokine release from endothelial cells upon SARS-324

CoV-2 infection are lower than CXCL10 or IL-6 induced in response to high concentrations of 325 recombinant TNF or IFNß, (Supplemental Figure 2F -G), this shows that endothelial cells express high 326 basal levels of IL-6 and that both cytokines are increased during infection. Taken together, these results 327

show that endothelial cells do not support a productive SARS-CoV-2 infection but still mount a modest 328 pro-inflammatory response to the virus. 329 330

To create a more physiologically relevant in vitro model of the pulmonary endothelium, we adapted our 332 previously described co-culture model of the lung epithelial-endothelial barrier 47 , where epithelial 333 These results were confirmed by the presence of higher levels of viral RNA as measured by qPCR in 340 the Calu-3 compared to the HMVEC-L ( Fig 6G) and higher titres of infectious virions in the 341 endothelial infection, we observed that HMVEC-L cells expressed ICAM-1 expression when virus was 343 added to the upper (Calu-3) compartment, suggesting that the HMVEC-L respond to adjacent epithelial 344 infection ( Figure 6A , D). These HMVEC-L also responded to the infection in the adjacent Calu-3 cells 345 by increasing CXCL10 secretion (detected in lower compartment of the co-culture) ( Figure 6I ). Both 346

Calu-3 and HMVEC-L appeared to secrete IL-6 in response to SARS-CoV-2 infection, as IL-6 levels 347

were increased in both compartments (significantly in the upper, trending in the lower) ( Figure 6J ). In productive replication of SARS-CoV-2, but that they induce pro-inflammatory cytokines and adhesion 359 molecules upon either direct or indirect exposure to SARS-CoV-2 virions. This supports the hypothesis 360 that the endothelial dysfunction observed in COVID-19 patients is likely to be largely mediated by 361 inflammatory signalling pathways. 362

As COVID-19 case numbers rose, reports of COVID-19 coagulopathy and vascular dysfunction also 364 began to accumulate. Whether SARS-CoV-2 directly infects the vasculature to drive this vascular 365 pathology has thus remained a topic of intense debate. Multiple studies show viral particles surrounding 366 the vasculature [3, 25, 43 ], yet it is unclear whether this infection is truly specific to the endothelium or 367 occurs within the perivascular compartment. Our in vivo analyses of lung tissue samples from deceased 368 COVID-19 patients indicates the endothelium remains uninfected. Nevertheless, we showed that in 369 vitro, endothelial cells are theoretically susceptible to SARS-CoV-2 infection, as they express ACE2, TMPRSS2 and NRP1, albeit at significantly lower levels than epithelial cells. This is in line with other 371 studies that showed expression of SARS-CoV-2 receptors by the endothelium in human tissue or in 372 cultured cells 14, 17, 23, 31 . However, we did not detect endothelial cell infection following exposure to 6 373

x 10 4 PFUs of SARS-CoV-2, whereas epithelial cells were readily infected at the same viral dose. These 374 results support those of Wang et al. 31 , who showed that epithelial cells are more susceptible to infection 375 than endothelial cells. When exposed to higher viral titres, endothelial cells were positive for viral NP 376 protein, suggesting effective viral entry. However, no infectious virions were detected in the endothelial 377 cell supernatant, indicating that this infection was abortive. It is interesting to speculate as to why 378 SARS-CoV-2 infection is productive in a monoculture of epithelial, but not endothelial cells. This may 379 relate to cell-dependent differences in viral entry. For example, in epithelial cells, there may be 380 sufficient surface TMPRSS2 levels available to cleave the spike protein at the S2' site and mediate 381 viral-host cell membrane fusion. This may in turn allow for efficient release of viral RNA into the 382 cytoplasm and thus viral replication. In contrast, in cells with lower TMPRSS2 expression (such as 383 endothelial cells), SARS-CoV-2 may enter the cell via endocytosis, with S2' cleavage being mediated 384 by endosomal cathepsins 12 . Endosomal entry may not only be inefficient, but it may also activate the 385 cellular anti-viral response and thereby limit productive viral replication 48, 49 . Alternatively, a host of 386 other cellular co-factors expressed preferentially in epithelial cells may support selective SARS-CoV-387 2 replication, or endothelial-specific restriction factors may limit effective viral replication. 388

In contrast to endothelial cells in a monoculture, viral proteins could not be detected in endothelial cells 390 grown in a co-culture with SARS-CoV-2-positive epithelial cells, despite being exposed to the same 391 dose of virus. These data may reflect the fact that SARS-CoV-2 typically infects and buds from 392 epithelial cells in an apical manner, resulting in limited exposure of the endothelium to infectious 393 virions. Extrapolating these data to the in vivo situation would suggest that a basolateral (abortive) 394 infection of the endothelium by SARS-CoV-2 is only likely to occur when the epithelial barrier of the 395 lung is severely damaged, exposing the endothelium to incoming virions. 396 397 While endothelial cells (either in a monoculture or in a co-culture) did not produce infectious virions, 398 they did respond to SARS-CoV-2 infection. Analysis of infected endothelial monolayers revealed that 399 cells positive for viral proteins were extruded in an apical fashion, without any apparent disruption to 400 the monolayer, as assessed by phalloidin expression. This is in contrast to work from Buzhdygan and 401 colleagues, who demonstrated that purified SARS-CoV-2 spike protein can induce the breakdown of 402 the vascular barrier in brain microvascular endothelial cells 50 . Discrepancies between these results may 403 be due to different sources of primary endothelial cells (lung versus brain), or the differences in response 404

of the endothelium to a purified viral protein vs infectious virus 1, 3, 24, 29, 51-53 . However, if infected cells 405 are promptly removed from exposed vessels as our data suggests, then this may explain why detection 406 of viral particles in the endothelium of patients has varied between studies 4, 54 . 407

In the absence of direct endothelial infection, hyperinflammation is likely to contribute to endothelial 409 dysfunction observed in COVID-19 patients. SARS-CoV-2 triggered an upregulation of endothelial 410 ICAM-1 expression when HMVEC-Ls were directly exposed to virus, or co-cultured with infected 411 epithelial cells. ICAM-1 enables immune cells to effectively extravasate into tissues, so its upregulation 412 is in keeping with the observed influx of inflammatory monocytes, neutrophils and other immune cells 413

into the lungs in severe COVID-19 patients 55 . This suggests that locally produced pro-inflammatory 414 cytokines stimulate the endothelium to induce adhesion marker expression. Our previous studies with 415 influenza virus showed that endothelial cells can themselves be a source of these pro-inflammatory 416 cytokines 56 , and indeed we also observed SARS-CoV-2-induced release of IL-6 and CXCL10 in co-417

cultures. Precisely how SARS-CoV-2 is sensed by the endothelium in either the monoculture or co-418 culture system remains to be determined. Given that SARS-CoV-2 does not replicate in endothelial 419 cells, we speculate that endothelial cells are unlikely to sense this virus through cytosolic RNA receptors 420 such as RIG-I or MDA5, as these immune sensors would detect viral replication intermediates. A more 421 likely scenario is that the endothelial cells respond to danger signals from neighbouring infected 422 epithelial or endothelial cells. These data add to a growing body of literature that indicate that 423 endothelial cell-directed inflammation plays a key role in the pathogenesis of SARS-CoV-2 57 . It is 424 tempting to speculate that, similar to influenza virus, the SARS-CoV-2-induced pro-inflammatory state may induce endothelial cells to express tissue factor, which in turn induces a pro-coagulant state, 426 microvascular leakage and pulmonary haemorrhage 58 , all of which have been described in patients with 427

severe COVID-19. Furthermore, regardless of direct or indirect infection, elevated IL-6 correlates with 428 increased fibrinogen 59 and although still controversial, elevated fibrinogen levels have been detected 429 in critically ill patients 2 . 430

The present study was subject to several limitations. Firstly, coagulation, thrombosis and induction of 432 angiogenesis in the lungs of deceased COVID-19 patients has been described in addition to endothelial 433 dysfunction 3 . This angiogenic response is thought to be predominantly mediated through 434 intussusceptive angiogenesis, where a new blood vessel is formed by splitting of an existing vessel. 435

This effect appears to be specific to COVID-19 patients, as lungs from decreased influenza patients do 436 not display increased angiogenic features. Whether this is due to relative hypoxia in the lungs remains Accordingly, our data may best reflect the initial stages of infection when only a limited number of 451 tissue-resident leukocytes may be present. Future studies will necessitate the addition of leukocytes to 452 our co-culture system, to determine whether their presence results in a further induction of inflammation 453 and markers of severe disease. 454 455 Here, we have conclusively shown that in vitro, endothelial cells are not productively infected by SARS-456

CoV-2 but that they mount an inflammatory response after direct or indirect exposure to the virus, 457 characterised by increased cytokine secretion and expression of adhesion molecules. Our results thus 458 suggest a key role of the endothelium in the pathogenesis of COVID-19, and that targeting the 459 inflammatory response may present the best opportunity to prevent endothelial dysfunction. 460

We thank Dr. Fernando Guimaraes for assisting with acquiring samples necessary for this study. 

Chen L, Li X, Chen M, Feng Y, Xiong C. The ACE2 expression in human heart indicates new 538 potential mechanism of heart injury among patients infected with SARS-CoV-2. 

Activation of coagulation and tissue 619 fibrin deposition in experimental influenza in ferrets

The procoagulant pattern of patients with COVID-621 19 acute respiratory distress syndrome

Dynamics of airway blood vessels and lymphatics: lessons 623 from development and inflammation

Pathological inflammation in patients with COVID-19: a key role for 625 monocytes and macrophages

Neutrophil extracellular traps contribute to 627 immunothrombosis in COVID-19 acute respiratory distress syndrome

Neutrophil extracellular traps in COVID-19

G) qPCR shows presence of viral RNA (MPRO) in SARS-CoV-2 infected Calu-3 and 718

HMVEC-L co-cultured cells represented as fold change relative to mock infection at 72h after infection

n=3 independent experiments. H) Viral replication shown as number of PFU per ml of supernatant from 720

SARS-CoV-2 infected Calu-3 and HMVEC-L co-cultured cells at 72h after infection. n=3 independent 721 experiments. Measurement of cytokines with AlphaLISA Immunoassay kit for I) CXCL10 and J) IL6

in the supernatant of Calu-3 and HMVEC-L co-cultured cells with mock or SARS-CoV-2 infection at 723 72h after infection. n=4 independent experiments. Data are presented as mean±s.e.m. with individual 724 data points indicated and colour coded per independent experimental replicate. Statistical significance 725 was determined using Mann-Whitney test between mock and + SARS-CoV-2

Supplemental Table 1. Patient information. Additional information on cohort of COVID-19 patients

Supplemental Figure 1. Endothelial cells are not productively infected with 6 x 10 4 PFU of SARS-731

Schematic of A) apical and B) basolateral infection of cells cultured on transwell membranes

C) Viral replication shown as number of PFU per ml of supernatant from SARS-CoV-2 infected 733

Calu-3 cells at 24h, 48h and 72h after infection. n=2 (HUVEC and HMVEC

Calu-3) independent experiments. Representative immunofluorescent images of D) HUVEC

-3 or F) HMVEC-L stained for NP (shown as single channel in top panel) (green), phalloidin 736 (magenta) and DAPI (blue) with mock or SARS-CoV-2 infection from either apical or basolateral side 737 of the cells at 48h after infection. Scalebar 50 µm. G) Quantification of NP staining

n=9 images from 3 independent experiments. H) Western blot 739 analysis showing NP protein levels in HUVEC, HMVEC-L and Calu-3 cells after 48h of infection. I)

Quantification of protein levels for NP in HUVEC, HMVEC-L and Calu-3. n=2 independent 741 experiments. Data are presented as mean±s.e.m. with individual data points indicated and colour coded 742 per independent experimental replicate. Statistical significance was determined using Kruskal Wallis 743 test between 24h and other time points (C) or Mann-Whitney test between mock and + SARS-CoV-2 744 (G, I)

reprobe P a g e R u le r P re s ta in e d p ro te in la d d e r X X H U V E C B H K -2 1 H M V E C -L C a lu -3 H u m a n n a s a l e p it h e li u m c e ll s H U V E C + A C E 2 X X X X X Actin X