key: cord-0951698-ddymdec1
authors: Hui, Kenrie Pui-Yan; Cheung, Man-Chun; Lai, Ka-Ling; Ng, Ka-Chun; Ho, John Chi-Wang; Peiris, Malik; Nicholls, John Malcolm; Chan, Michael Chi-Wai
title: Role of epithelial-endothelial cell interaction in the pathogenesis of SARS-CoV-2 infection
date: 2021-05-06
journal: Clin Infect Dis
DOI: 10.1093/cid/ciab406
sha: 3759487bf9782b57b4b49d14df128a075e9de72b
doc_id: 951698
cord_uid: ddymdec1

BACKGROUND: The COVID-19 pandemic caused by the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) continues to threaten public health globally. Patients with severe COVID-19 disease progress to acute respiratory distress syndrome, with respiratory and multiple organ failure. It is believed that dysregulated production of pro-inflammatory cytokines and endothelial dysfunction contribute to the pathogenesis of severe diseases. However, the mechanisms of SARS-CoV-2 pathogenesis and the role of endothelial cells are poorly understood. METHODS: Well-differentiated human airway epithelial cells were used to explore the cytokine and chemokine production after SARS-CoV-2 infection. We measured the susceptibility to infection, immune response, and expression of adhesion molecules, in human pulmonary microvascular endothelial cells (HPMVECs) exposed to conditioned medium from infected epithelial cells. The effect of imatinib on HPMVECs exposed to conditioned medium was evaluated. RESULTS: We demonstrated the production of IL-6, IP-10 and MCP-1 from the infected human airway cells after infection with SARS-CoV-2. Although human pulmonary microvascular endothelial cells (HPMVECs) did not support productive replication of SARS-CoV-2, treatment of HPMVECs with conditioned medium collected from infected airway cells induced an up-regulation of pro-inflammatory cytokines, chemokines and vascular adhesion molecules. Imatinib inhibited the up-regulation of these cytokines, chemokines and adhesion molecules in HPMVECs treated with conditioned medium. CONCLUSIONS: This study evaluates the role of endothelial cells in the development of clinical disease caused by SARS-CoV-2, and the importance of endothelial cell-epithelial cell interaction in the pathogenesis of human COVID-19 diseases.

A novel coronavirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) emerged and caused human infection from December of 2019. Clinical manifestations of severe COVID-19 disease include sepsis, acute respiratory distress syndrome (ARDS), and multiple organ failure [1, 2] . Autopsy analysis demonstrates a correlation between inflammation, diffuse alveolar and endothelial dysfunction [2] indicating there is an uncontrolled innate immune response contributing to the severity of COVID-19. Published data shows low induction of IFNs by SARS-COV-2 infection in susceptible cells and in post-mortem lung tissues [3] . Since immune cells such as macrophages, which are potent cytokine inducers, do not support productive replication of SARS-CoV-2 virus in vitro, the source of pro-inflammatory cytokine and chemokine remains unclear. Endothelial cell damage has been reported in autopsy studies of COVID-19 [2] . Furthermore, plasma collected from mild and severe COVID-19 patients leads to damaged endothelial integrity [4] . Although a large number of treatment regimens have being tried, there is no approved specific therapeutic interventions for COVID-19 yet. A better understanding on the pathophysiology of COVID-19 in terms of the induction of pro-inflammatory immune responses thus provides important information to better identify targeted therapeutic strategies and reveal potential intervention targets.

Imatinib, (also known as Gleevec), is a FDA-approved drug for the treatment of patients with various types of leukemia and tumors [5] and also an effective treatment for edema, inflammation, vascular leakage in mouse models of acute lung injury [6, 7] . It also inhibits SARS-CoV and MERS-CoV with low cytotoxicity [8] . There are now two Phase III clinical trials involved the use of imatinib to treat COVID-19 patients in progress [9, 10] , but there is not much information on the role of imatinib in SARS-CoV-2 infection, except for a single study that identified imatinib as an entry inhibitor of SARS-CoV-2 in lung and colonic organoids [11] .

A c c e p t e d M a n u s c r i p t 5 Wd-Calu-3 cells possess epithelial integrity and polarity with the development of tight junction, cilia, and mucus secretion at apical surface [12] . They are susceptible for both influenza viruses and SARS-CoV infection [13] [14] . In this study, we used well differentiated (wd)-Calu-3 cells in air-liquid interface system for assessing the viral replication, cytokine and chemokine production by the three pathogenic coronaviruses. We further demonstrate the interaction between pulmonary epithelial cells and endothelial cells during SARS-CoV-2 infection. Induction of cytokine and adhesion molecules from human pulmonary microvascular endothelial cells (HPMVECs) was detected via paracrine signalling by the treatment of conditioned medium from SARS-CoV-2 infected wd-Calu-3. Treatment with imatinib significantly reduced some of the elevated cytokines, chemokines and adhesion molecules in these HPMVECs. 

Human airway epithelial Calu-3 cells (ATCC ® HTB-55 ™ ), derived from human lung adenocarcinoma, were maintained in DMEM supplemented with 15% FBS and 1% Penicillin/Streptomycin. Differentiation of Calu-3 cells was modified as reported previously [12, 16] . Cells were seeded on transwell inserts, with 0.4µm pore size at a density of 100,000. After reaching confluency, the cells were maintained at air-liquid interface (ALI) and medium was changed every 48h. The well-A c c e p t e d M a n u s c r i p t 6 differentiated Calu-3 (wd-Calu-3) cultures were subjected to virus infection after 21 days of ALI culture.

HPMVECs were isolated from human lung tissues by using anti-CD31-tagged dynabeads (Thermo Fisher) as described previously (see Supplementary Materials) [17, 18] 

Cells in transwell inserts were infected with viruses at a multiplicity of infection (MOI) of 0.1 for viral replication kinetics at the apical side or basolateral side. Viral titers in supernatant were determined by median tissue culture infectious dose (TCID 50 ) assay. Infection was done at MOI 2 and cell lysates were collected at 24 and 96 hpi for the mRNA expression using real-time PCR analysis.

Mock-infected cells served as negative controls. At 96 hpi, cleaved-caspase 3 was stained.

Culture supernatants from the basolateral side of wd-Calu-3 cells infected with SARS-CoV-2 at a MOI of 2 for 96 h were filtered. Mock-infected culture supernatants served as control medium.

HPMVECs were either treated with conditioned medium from mock (CM-MK), SARS-CoV-2 infected cells (CM-SARS2), directly infected by SARS-CoV-2 virus. HPMVECs were treated with cytomix-a mixture composed of IL-1β, TNF-α and IFN- at 50ng/ml, which impaired epithelial integrity previously [19] . 

We developed well differentiated human airway epithelial cells (wd-Calu-3) and directly compared the viral replication efficiency of three human coronaviruses-SARS-CoV-2, SARS-CoV and MERS-CoV, together with two influenza A control viruses-pandemic H1N1 and highly pathogenic avian H5N1 viruses, which replicate efficiently in wd-Calu-3. The latter shows high cytokine induction phenotype [20] . Viral titers of SARS-CoV-2 increased by 4 log10 at the apical side at 24 h postinfection, which was significantly higher than SARS-CoV and MERS-CoV, but similar to that of 

Direct infection of HPMVECs with SARS-CoV-2 or SARS-CoV led to less than 1 log10 increase in titers irrespective of apical or basolateral side infection (Figure 4a, 4c) . In contrast, the two control viruses, MERS-CoV and H5N1, had significantly higher viral titers as early as 24h post-infection Figure 9 ).

Clinical manifestations of SARS-CoV-2 infected patients have been attributed to abnormal cytokine release, the so-called "cytokine storm" especially those with severe disease [22] . The production of pro-inflammatory cytokines and chemokines has been demonstrated in airway epithelial cells after SARS-CoV-2 infection [23] . Even though there have been publications demonstrating damage to the endothelium in severe COVID-19 [24] [25] [26] [27] , the interaction between respiratory epithelial and endothelial cells during SARS-CoV-2 infection is not fully understood, and there is a lack of information on the active role of endothelial cells contributing to the disease progression.

Wd-Calu-3 cells supported more robust productive replication of SARS-CoV-2 and SARS-CoV than undifferentiated Calu-3 cells and viruses were mainly released from the apical side. The higher expression of ACE2 in wd-Calu-3 than undifferentiated Calu-3 cells may explain the differential susceptibilities of the two models to SARS-CoV-2 infection. More importantly, we found robust cytokine and chemokine production at the basolateral side including IL-6, IP-10, and MCP-1, by A c c e p t e d M a n u s c r i p t 12 SARS-CoV-2 infection at 96 h post-infection. Although production of IL-6 and IP-10 by SARS-CoV-2 was lower than that following infection by H5N1, a number of reports indicate that these cytokines and chemokines are associated with disease severity of COVID-19 [28] [29] [30] . Therefore, airway epithelial cells are most likely one of the sources of pro-inflammatory cytokines and chemokines with clinically important roles to the pathogenesis of COVID-19, in SARS-CoV-2 infection. These findings are in line with reports using primary human airway epithelial cultures [23, 31] .

There is a lack of information on direct comparison of the innate immune responses induced by the 

CoV-2 infected differentiated HBEC [33] and Calu-3 cells [34, 35] . These discrepancies could be explained by the cell type specific phenotypes, differences of infectious dosage and time points after infection since we used the ALI-model of Calu-3 cells and we aimed at infecting the cells at synchronizing manner with a high MOI. Our findings reveal that SARS-CoV-2 induced a more robust innate immune responses than SARS-CoV and MERS-CoV as early as 24 h and the differential expression was also seen at 96 hpi in wd-Calu-3 cells.

There were minimal cytopathic effects in SARS-CoV-2 infected cells up to 96 h post-infection. This phenotype provides long period of time of virus release and cytokine and chemokine production.

Patra's report that the protein expression of ACE2 is down-regulated upon SARS-CoV-2 infection in human lung epithelial and liver epithelial cells [36] . The loss of ACE2 at the cell surface abolishes the degradation of extracellular angiotensin II, which is supported by the markedly increased level of angiotensin II in the plasma samples from COVID-19 patients [37] . A number of studies A c c e p t e d M a n u s c r i p t 13 demonstrated that SARS-CoV infection down-regulated ACE2 expressions, resulting in increased angiotensin II level in blood samples and this contributed to acute lung injury [38, 39] .

Dysfunction of endothelial cells has been frequently reported in severe COVID-19 [24] [25] [26] [27] . Virus particles are occasionally found in endothelial cells of COVID-19 patients in an autopsy study [27] . however we found that using human primary pulmonary endothelial cells [40] there was no productive replication of SARS-CoV-2 in HPMVECs following infection from the apical or basolateral side of the endothelium, nor in HPMVECs pretreated with conditioned medium from SARS-CoV-2 infected airway cells. These findings suggest that though SARS-CoV-2 virus can enter into endothelial cells, it is not able to produce infectious viral particles. This is in line with reports using immortalized human endothelial cell line and umbilical vein endothelial cells [41] .

There are several reports explore the cross-talk between lung epithelial and endothelial cells upon exposure to SARS-CoV-2 virus or protein. A recent study reported the induction of IL-6 via spike protein expression in A549 cells and the induction of MCP-1 in liver endothelial cells by supernatants from SARS-CoV-2 spike protein-transfected A549 cells [36] . Another study shows the disruption of intracellular organelles after SARS-CoV-2 infection in both human lung epithelial cells and immortalized pulmonary endothelial cells in a co-culture system, while the damage to endothelial cells is via an indirect manner [42] . Damaged barrier function after prolonged SARS-CoV-2 infection in umbilical vein endothelial cells has also been reported in a human chip model composed of nondifferentiated Calu-3 cells, endothelial and mononuclear cells [41] . In contrast to the reports mentioned above, our study applied more a physiological relevant cell model using of differentiated Calu-3 in air-liquid interface system and primary HPMVECs. Furthermore, live virus was used to infect from the apical side of the airway epithelial cells. We believe our findings reflect a more physiological interaction between airway epithelial cells and pulmonary endothelial cells. In line with the studies of Deinhardt, induction of pro-inflammatory and type I and III interferon responses by A c c e p t e d M a n u s c r i p t 15 Imatinib has been studied in two Phase III clinical trials as a treatment for COVID-19 patients [9, 10] .

However, there is limited information on the role of imatinib and SARS-CoV-2 infection. Apart from anti-viral effects, we reported here that imatinib can act as an immuno-modulator as it significantly reduced the induction of pro-inflammatory cytokines, chemokines and leukocyte adhesion molecules in endothelial cells treated with conditioned medium from SARS-CoV-2 infected airway cells. The anti-inflammatory effects observed are in line with a number of in vivo acute lung injury studies using imatinib as a therapeutic strategy [49, 50] . In LPS and ventilator-induced lung injury study, imatinib decreased bronchoalveolar lavage protein, immune cells influx and TNF-alpha levels in mice [6] . Our 

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There is no conflict of interest for all authors.