key: cord-336938-03366q9t
authors: Thacker, Vivek V; Sharma, Kunal; Dhar, Neeraj; Mancini, Gian-Filippo; Sordet-Dessimoz, Jessica; Mckinney, John D
title: Rapid endothelialitis and vascular inflammation characterise SARS-CoV-2 infection in a human lung-on-chip model
date: 2020-08-10
journal: bioRxiv
DOI: 10.1101/2020.08.10.243220
sha: 
doc_id: 336938
cord_uid: 03366q9t

Background Severe manifestations of COVID-19 include hypercoagulopathies and systemic endothelialitis. The underlying dynamics of damage to the vasculature, and whether it is a direct consequence of endothelial infection or an indirect consequence of immune cell mediated cytokine storms is unknown. This is in part because in vitro infection models are typically monocultures of epithelial cells or fail to recapitulate vascular physiology. Methods We establish a vascularised lung-on-chip infection model consisting of a co-culture of primary human alveolar epithelial cells (‘epithelial’) and human lung microvascular endothelial cells (‘endothelial’), with the optional addition of CD14+ macrophages to the epithelial side. A combination of qRT-PCR, RNAscope, immunofluorescence, and ELISA measurements are used to study the dynamics of viral replication and host responses to a low dose infection of SARS-CoV-2 delivered to the apical surface of the epithelial face maintained at an air-liquid interface. Findings SARS-CoV-2 inoculation does not lead to a productive amplification of infectious virions. However, both genomic and antisense viral RNA can be found in endothelial cells within 1-day post infection (dpi) and persist upto 3 dpi. This generates an NF-KB inflammatory response typified by IL-6 secretion and a weak antiviral interferon response even in the absence of immune cells. Endothelial inflammation leads to a progressive loss of barrier integrity, a subset of cells also shows a transient hyperplasic phenotype. Administration of Tocilizumab slows the loss of barrier integrity but does not reduce the occurrence of the latter. Interpretation Endothelial infection can occur through basolateral transmission from infected epithelial cells at the air-liquid interface. SARS-CoV-2 mediated inflammation occurs despite the lack of rapid viral replication and the consequences are cell-type dependent. Infected endothelial cells might be a key source of circulating IL-6 in COVID-19 patients. Vascular damage occurs independently of immune-cell mediated cytokine storms, whose effect would only exacerbate the damage. Finding Core support from EPEL.

Organ-on-chip technologies recreate key aspects of human physiology in a bottom-up and modular manner 1 . In the context of infectious diseases, this allows for studies of cell dynamics 2,3 , infection tropism 4 , and the role of physiological factors in disease pathogenesis in more native settings 5 . This is particularly relevant for the study of respiratory infectious diseases 6 , where the vast surface area of the alveoli poses a challenge to direct experimental observation.

COVID-19, caused by the novel betacoronavirus SARS-CoV-2, first manifests as an infection of the upper airways. Severe cases are marked by progression into the lower airways and alveoli. Here, it manifests as an atypical form of acute respiratory distress syndrome (ARDS) characterized by good lung compliance measurements 7, 8 , and elevated levels of coagulation markers such as D-dimers 9 , and pro-inflammatory markers in the blood 10 . Autopsy reports show numerous microvascular thrombi in the lungs of deceased patients together with evidence of the intracellular presence of the virus in vascular cells 11, 12 . These suggest that the lung microvasculature plays a key role in COVID-19 pathogenesis 13 , yet most in vitro studies have focused on monocultures of upper airway respiratory cells. In studies with alveolar epithelial cells, SARS-CoV-2 has been shown to replicate poorly both in the A549 lung adenocarcinoma cell line 14 and in primary alveolar epithelial cells ex vivo 15 and has been reported to be unable to infect primary microvascular endothelial cells 16 , which are at odds with the reported medical literature. There is therefore an urgent need for a better understanding of the pathogenesis of SARS-CoV-2 in alveolar epithelial cells and in a more realistic model of the alveolar space that is vascularized. The lung-onchip model is well-suited to this purpose because it includes a vascular compartment maintained under flow, and infection can occur at the air-liquid interface, two key physiological features that are lacking in organoid models [17] [18] [19] . We therefore establish a human lung-on-chip model for SARS-CoV-2 infections, and probe the viral growth kinetics, cellular localization and responses to a low dose infection using qRT-PCR, ELISA, RNAscope, immunofluorescence and confocal imaging (Fig. 1J) .

We established a human lung-on-chip model for SARS-CoV-2 pathogenesis (Fig. 1A) using primary human alveolar epithelial cells ('epithelial') and lung microvascular endothelial cells ('endothelial'), which form confluent monolayers on the apical and vascular sides of the porous membrane in the chip (Fig. 1B, C) . The modular nature of the technology allowed us to recreate otherwise identical chips either with ( Neuropilin-1, an integrin-binding protein 23 has recently been reported to be an alternative receptor for SARS-CoV-2 entry 24, 25 . NRP1 expression was between one and four orders of magnitude higher than ACE2 expression both in monoculture and on-chip (Fig. S1A, 1D) , with a10-fold higher expression in endothelial cells compared to epithelial cells that was retained on-chip ( Fig S1B) . Expression of cell surface receptor proteins used for SARS-CoV-2 entry in both cell types differs significantly in coculture at an air-liquid interface, and an alternative entry receptor such as NRP1 are far more expressed than ACE2. Infection of the alveolar space is characterized by a lack of productive infection, cell-to-cell transmission, and slow intracellular replication

We first characterized the progression of infection by measuring the release of viral progeny and intracellular viral RNA loads. Infected LoCs were monitored daily for the release of infected viral progeny (1) apically -on the epithelial layer and (2) basolaterally -in the cell culture media flowed through the vascular channel ('apical wash' and 'vascular effluent' in Fig. 1A) . A low number of viral genomes were released apically in the epithelial layer, and the number of genomes detected decreased over 1-3 dpi ( Fig. 2A) . Genome copy numbers were 100-fold lower than the starting inoculum 6 (100 PFU), suggesting that instances of productive infection were rare. No viral genomes were detected in the vascular effluent (Fig. 2B) , and the lack of infectious particles in the effluent was confirmed for two LoCs with and w/o macrophages by plaque forming unit assays (data not shown). Nevertheless, total RNA extracted from the apical and vascular channels of an infected LoC without macrophages at 1 dpi revealed >10 4 genomes in both epithelial and endothelial cells (Fig. 2C ) and genome copy numbers exceeded those for cellular housekeeping gene RNAseP (Fig. 2D ).

Similar numbers of viral genome copy numbers have been reported for infections of alveolar epithelial cell monoculture 15 and are modest in comparison to airway epithelial cells and Vero E6 cells 27 . Nevertheless, SARS-CoV-2 appears to disseminate rapidly from the epithelial to the endothelial layer. Basolateral transmission has not been reported to be the major mode of transmission for both SARS-CoV-2 28 and SARS-CoV-1 29 infections of monocultures of upper airway cells at the air-liquid interface.

This suggests that basolateral transmission may either be a unique feature of alveolar epithelial cells or a consequence of cell-to-cell contact between epithelial and endothelial cells in our vascularised model.

Autopsy reports show features of exudative alveolar damage 12 . The LoC model is compatible with microscopy-based assays which allowed us to assess changes in cellular physiology with high spatial resolution using confocal microscopy. We began by enumerating the number of nuclei per unit area of the membrane surface for LoCs at 1 and 3 dpi. In the epithelial layer, the density of nuclei declines progressively irrespective of the presence (p=0.010, p=9E-4) and absence (p=0.054, p=0.029) of 7 macrophages compared to uninfected controls (Fig. 3A) . In contrast, the nuclear density increases in the endothelial layer at 1 dpi before decreasing back to levels in the control samples at 3 dpi (Fig. 3B) . These results appeared counterintuitive and so we examined if infection affected the morphology of the cells. Staining for F-actin localisation revealed striking changes to the morphology of cells in the vascular channel. 3D views of the endothelial layer from an uninfected control LoC also maintained at the air liquid interface show a confluent layer of cells aligned with the long axis of the channel as expected (Fig 3C, Fig. S2A ). However, in infected LoCs there are clear signs of vascular inflammation (Fig 3D-E) . At 2 dpi, areas of cellular hyperplasia characterised by an increased cell density and stronger nucleic acid staining ( Fig 3D, yellow arrows) coexist with areas with normal nucleic acid staining levels but reduced cell-to-cell contact ( Fig 3D, white arrows) . By 3 dpi, a significant loss of tight junctions and cell confluency is observed (Fig 3E, Fig S2B-D) . Vascular cells form clusters, and a much larger proportion of the surface area of the chip has low or no actin staining compared to the uninfected control (Fig. S2E ). In stark contrast, for the same chip, the epithelial layer maintains a high degree of confluency (Fig, 3F, Fig. S2F ).

Immunostaining for the viral S protein at 3 dpi ( In some cases, S proteins appear to co-localize with the periphery of the nucleus ( Interestingly, IL-6 levels were not significantly different between these two categories of LoCs, which suggested a non-immune cell source for this cytokine (Fig. 5A ). ELISA assays for IL-1B and IP10 did not detect these cytokines in the effluent (data not shown). Expression levels of pro-inflammatory genes (TNFA, IL6, IL1B) at 1 dpi was an order of magnitude higher than that of interferon genes in both cell types, and little to no interferon stimulatory gene expression was detected in both cell types ( Expression at 3 dpi was higher than that at 1 dpi, the epithelial layer at 3 dpi, and the uninfected controls, both on a per-cell and per-field-of-view basis (Fig. 5L, Fig. S9A p=0.004, p=0.014, p=0.008 respectively). Unlike viral RNA levels (Fig. S5B , D) IL6 expression in the endothelial layer does not diminish over 1-3 dpi and would therefore appear to be the major contributor to IL-6 secretion in the vascular effluent.

Given the pleiotropic nature of IL-6 31 , we also examined expression of the IL6R receptor and the metallopeptidase ADAM17 which sheds the TNF-alpha receptor and the IL-6R receptor 32 in the endothelial layer at 1 dpi. IL6R expression was low ( Fig.   5M ) and was not altered by infection, whereas ADAM17 expression was high (Fig. 5M) and was increased 40-fold over uninfected controls (Fig. 5N) . ADAM17 has been shown to enhance vascular permeability 33 , and so we reasoned that targeting trans IL-6 signalling using the anti-IL-6R monoclonal antibody Tocilizumab 34 , that is also undergoing clinical trials as a repurposed therapeutic for COVID-19, might ameliorate the vascular inflammation observed. Tocilizumab administration at 56 g/mL via continuous perfusion per se did not abrogate IL-6 secretion (Fig. 5A ). An investigation of cell morphologies in the endothelial layer also showed that the perfusion did not prevent the occurrence of hyperplasia (Fig. 6A, 6E ). To quantify this, we compared regions of interest (ROIs) that excluded areas of cellular hyperplasia across at least six fields of view from the endothelial layer of LoCs with and without Tocilizumab perfusion, and identified areas with low or no Factin staining as those with reduced confluence, as in Fig. S2E . A plot of the proportion of pixels with intensities below a defined cut-off threshold (Fig. 6G) showed that the untreated LoC had a significantly higher proportion of pixels with intensities lower than 5% (p=0.032) of the maximum intensity (p=0.022 for 3% and p=0.038 for 2%).

Inhibition of IL-6 signalling through Tocilizumab is able to ameliorate some but not all of the vascular damage observed. into the dynamics and mechanisms that underlie these observations that are difficult to obtain in other experimental models and that we hope will improve our understanding of the pathogenesis of this multi-organ disease.

We gratefully acknowledge assistance from Dr Muhammet Fatih Gulen for the ELISA assays. We are also grateful to the members of the BioImaging Core Facility (BIOP) for assistance with confocal microscopy. conditions. The next day, the chip was washed and a reduced medium for the airliquid interface (ALI) was flowed through the vascular channel using syringe pumps (Aladdin-220, Word Precision Instruments) at 60 l/hour as described 41 .

The composition of the ALI media used was as described in 41 

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Representative 232 x 232 m 2 fields of view of the epithelial (A) and endothelial layer. (C) Fold-change in the markers in (B) relative to uninfected controls at the same timepoint. The bar represents the mean and the error bars the standard deviation. (D-G) 3D views of representative 232 x 232 m 2 fields of view of the epithelial (D, F) and endothelial layer (E, G) from infected LoCs reconstituted with macrophages at 1 (D, E) and 3 dpi (F, G). IL6 mRNA

K) Zooms corresponding to the regions in (G) highlighted with white (H) and yellow boxes with solid (I) and dashed lines (J, K) respectively. In these panels, orf1abantisense RNA (pink) and S RNA (amber) are also shown. The panels show examples of cells with similar levels of viral infection but with no (H), intermediate (I) and high levels (J, K) of IL6 expression. (L) Quantification of IL6 expression in epithelial

Plots show the total number of spots normalized by the number of cells in 4-6 fields of view detected using RNAscope assay using identical imaging conditions for all chips. Bars represent the mean value, the solid line represents the median, and error bars represent the standard deviation. Data from uninfected controls for 3 days at air-liquid interface is indicated by *. (M) Expression relative to GAPDH and (N) fold-change in expression relative to uninfected controls for IL6R and ADAM17 expression in the endothelial layer. The bar represents the mean and the error bars the standard deviation