key: cord-0707956-6w41nic8
authors: Zhang, Min; Wang, Peng; Luo, Ronghua; Wang, Yaqing; Li, Zhongyu; Guo, Yaqiong; Yao, Yulin; Li, Minghua; Tao, Tingting; Chen, Wenwen; Han, Jianbao; Liu, Haitao; Cui, Kangli; zhang, Xu; Zheng, Yongtang; Qin, Jianhua
title: Biomimetic Human Disease Model of SARS‐CoV‐2 Induced Lung Injury and Immune Responses on Organ Chip System
date: 2020-10-24
journal: Adv Sci (Weinh)
DOI: 10.1002/advs.202002928
sha: 772c08e8763431bec8bbd8345b6a9f6cbbb5a8e5
doc_id: 707956
cord_uid: 6w41nic8

Coronavirus disease 2019 (COVID‐19) is a global pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2). The models that can accurately resemble human‐relevant responses to viral infection are lacking. Here, we create a biomimetic human disease model on chip that allows to recapitulate lung injury and immune responses induced by SARS‐CoV‐2 in vitro at organ level. This human alveolar chip reproduced the key features of alveolar‐capillary barrier by co‐culture of human alveolar epithelium, microvascular endothelium and circulating immune cells under fluidic flow in normal and disease. Upon SARS‐CoV‐2 infection, the epithelium exhibited higher susceptibility to virus than endothelium. Transcriptional analyses showed activated innate immune responses in epithelium and cytokine‐dependent pathways in endothelium at 3 days post‐infection, revealing the distinctive responses in different cell types. Notably, viral infection caused the immune cell recruitment, endothelium detachment, and increased inflammatory cytokines release, suggesting the crucial role of immune cells involving in alveolar barrier injury and exacerbated inflammation. Treatment with remdesivir could inhibit viral replication and alleviate barrier disruption on chip. This organ chip model can closely mirror human‐relevant responses to SARS‐CoV‐2 infection, which is difficult to be achieved by in vitro models, providing a unique platform for COVID‐19 research and drug development. This article is protected by copyright. All rights reserved

The purpose of this work is to create a human disease model of SARS-CoV-2 infection and study human responses in vitro using microengineered lung chip device. Initially, we designed and fabricated the microfluidic lung chip, which consists of two channels separated by a thin and porous PDMS membrane coated with ECM ( Figure 1B) . The ECM coated PDMS membrane allows the co-culture of different cell types on the opposite sides of the membrane, mimicking the tissue interface in vivo. Moreover, the membrane with many pores (5 m in diameter) distribution is beneficial to substance diffusion and the interactions between the upper and lower cell layers. The culture chamber permits the perfusion of media flow, which can promote nutrients exchange and waste removal. This article is protected by copyright. All rights reserved. 6 Human alveolar epithelial type II cell (AT II) line (HPAEpiC) and lung microvasculature cell line (HULEC-5a) were seeded on the upper and lower sides of the porous membrane, respectively. These two types of cells were cultured for 3 days until confluent into monolayers under continuous media flow (50 l/h) in upper and bottom channels, thus forming the alveolus epithelium-endothelium tissue interface. The integrity of the formed tissue barrier was assessed by the expression of adherent junction proteins in both human epithelium and endothelium. Immunostaining analysis showed that epithelial cells can form adherent junctions identified by E-cadherin, and endothelial cells formed conjunctions identified by VE-cadherin, respectively (supplementary Figure S2) . Furthermore, the integrity of barrier under different culture conditions was assessed by the diffusion rate of FITC-dextran between the two parallel channels (supplementary Figure S3 ). The barrier permeability under fluid flow is lower than that in static cultures, indicating the important role of flow in maintaining the function and integrity of the alveolar-capillary barrier. These results suggest that the established human alveolus chip could effectively mimic the physiological alveolar-capillary barrier.

It has been reported that SARS-CoV-2 uses ACE2 as a host receptor for cellular entry, and transmembrane serine proteinase 2 (TMPRSS2) for Spike protein priming. [12, [31] [32] [33] Prior to create the SARS-CoV-2 infection model based on human alveolus chip, we sought to identify the susceptibility of alveolar epithelial cells to this virus. Initially, we examined the expression of ACE2 and TMPRSS2 proteins in HPAEpiC and HULEC-5a cells, respectively ( Figure 2A) . The western blot data showed the positive expression of ACE2 and TMPRSS2 in both cell types, [12, 34] and the expression of ACE2 in HPAEpiC cells was higher than that in HULEC-5a cells. To further determine the expression of ACE2 and TMPRSS2 proteins in HPAEpiC cells and HULEC-5a cells after viral infection, we infected these two cell types separately in monolayer cultures at a MOI of 10. At day 3 post-induction, the protein This article is protected by copyright. All rights reserved. 7 expressions were analyzed by western blot. The results showed a higher expression of viral NP protein in HPAEpiC cells as compared to that observed in HULEC-5a cells, indicating the higher susceptibility to SARS-CoV-2 infection in alveolar epithelial cells than pulmonary microvascular endothelial cells. Moreover, there are no obvious changes in the expression levels of ACE2 and TMPRSS2 in both cell types after viral infection (Figure 2B) , suggesting the less effects of viral infection on the protein levels of ACE2 and TMPRSS2 in host cells.

Similarly, upon SARS-CoV-2 infection, no significant difference was observed in the protein levels of these two proteins between the HPAEpiC cells cultured alone and co-cultures with HULEC-5a cells (supplementary Figure S4 ). The results suggest the expressions of ACE2 and TMPRSS2 proteins in HPAEpiC cells maintains stable, which are not dependent on viral infection and culture conditions. HPAEpiC were then infected with SARS-CoV-2 at a MOI of 10, and detected by immunofluorescent staining to check the infection efficiency. At day 3 post-infection, more than 20 % Spike protein-positive cells were observed ( Figure 2C ). To further examine the ultrastructure of SARS-CoV-2-infected cells, transmission electron microscope (TEM) analysis of mock-or SARS-CoV-2-infected HPAEpiC cells was carried out (Figure 2D and E). The TEM micrographs showed that mock cells exhibited primary AT II cell-like morphological characteristics, including small cellular size with square or round shape (Figure 2Di) , microvilli on free surface (Figure 2Dii ) and lamellar bodies within cell body (Figure 2Diii ). [35, 36] In the infected cells, lots of viral particles were detected and distributed in clusters within cell bodies as shown in Figure 2E , indicating the susceptibility of HPAEpiC cells to SARS-CoV-2 infection.

To mimic alveolar infection by SARS-CoV-2, the virus was inoculated into the upper alveolus channel of the chip at a MOI of 10, and the cells were cultured for 3 days. The predominated expression of spike protein was observed in epithelial cells, demonstrating viral infection and massive replication in alveolar epithelium ( Figure 3A and B), but not in endothelial cells. In addition, there are no significant changes in the organization of adherent junction proteins in HPAEpiC cells (E-cadherin) and HULEC-5a cells (VE-cadherin), as well as the confluent rate of epithelial cells and endothelial cells (Figure 3C and D). These results This article is protected by copyright. All rights reserved. 8 suggested that SARS-CoV-2 can primarily infect and replicate in the alveolar epithelial cells, but not in endothelial cells.

In order to fully understand the transcriptional responses to SARS-CoV-2 infection, we performed RNA-seq analysis of HPAEpiC and HULEC-5a cells following viral infection in the alveolus chip. Briefly, 3 days after infection, HPAEpiC cells and HULEC-5a cells were collected separately and analyzed by RNA-sequencing. Firstly, the ratio of virus-aligned reads over total reads in each sample was calculated to estimate the viral replication levels in these two cell types. The results showed that the ratio of viral reads in HPAEpiC cells is much higher than HULEC-5a cells ( Figure 4A and B), which are consistent with the western blot ( Figure 2B ) and immunostaining analysis ( Figure 3B ). It revealed that human alveolar epithelial cells were more permissive to SARS-CoV-2 infection than microvascular endothelial cells, similar to the histopathological findings from autopsy reports. [37] Volcano plots showed that SARS-CoV-2 infection induced global transcriptome modulations in both HPAEpiC cells and HPAEpiC cells ( Figure 4C ). To identify the differentially expressed genes (DEGs) in the two cell types, the cut-off values for the fold change and P value were set to 2.0 and 0.05, respectively. Among the DEGs, 972 genes (324 down-regulated genes and 648 up-regulated genes) were significantly modulated in HPAEpiC cells, while 735 genes (495 down-regulated genes and 240 up-regulated genes) were significantly modulated in HULEC-5a cells. By combining the two data sets, we found the two cell types only shared 52 overlapping up-regulated DEGs (6.2% of total up-regulated DEGs) and only 43 overlapping down-regulated DEGs (5.5% of total down-regulated DEGs) ( Figure 4D) . These results suggested that the alveolar epithelial and pulmonary microvascular endothelial cells showed distinct transcriptional responses to SARS-CoV-2

infection. This article is protected by copyright. All rights reserved. 9 To better understand the host responses to SARS-CoV-2 infection, Gene Ontology (GO) enrichment analysis was performed to identify biological processes enriched among significantly regulated genes. It appeared the distinctive GO terms were enriched between HPAEpiC cells and HULEC-5a cells following viral infection. Notably, among the top 20 enriched terms, the biological processes associated with epithelial cell apoptosis, cell division and mitotic cell cycle were particularly enriched in HPAEpiC cells ( Figure 4E) . While, in HULEC-5a cells, the biological processes involved in cytokinesis, transcriptional factor activity, chemotaxis were significantly modulated ( Figure 4F ). 

The accumulation and extensive infiltration of immune cells in the lungs may contribute significantly to the pathogenesis in patients infected with respiratory viruses. [38] We next Figure 6D and E), which was similar to the inflammatory cell This article is protected by copyright. All rights reserved. 11 infiltration in lungs as observed clinically. [9, 39] In addition, we also applied the non-specific stimulants (e.g., IL-2 and IL-6) on the alveolar chip to identify the response of human immune cells to these stimulators. The immune cells were introduced into the vascular channel of the chip after IL-2 or IL-6 (30 ng/mL) treatment for 2 days. More immune cells were observed to be attached to the pulmonary microvascular endothelium induced by IL-6, 

To assess the potential therapeutics against SARS-CoV-2, we treated the virus infected human alveolus chip with remdesivir. Remdesivir is recognized as a promising antiviral compound against many RNA viruses (e.g., SARS, MERS-CoV), including SARS-CoV-2. [40] [41] [42] In this study, an indicated dose of remdesivir (1μM) was added into the monolayer culture of HPAEpiC cells at 1h post-infection of SARS-CoV-2. After administration for 3 days, the culture supernatants were collected for virus titers determination by qRT-PCR. A marked decrease of virus titers was detected in the infected-HPAEpiC cells following remdesivir treatment ( Figure 7A ). Furthermore, we tested the antiviral efficacy of remdesivir in the infected chip model with the addition of PBMCs in the vascular channel. As shown in Figure 6B and C, remdesivir treatment could restore the damage of epithelial layers and endothelial layer to some extent ( Figure 7B and C). These results indicated the potential role of remdesivir in suppressing SARS-CoV-2 replication and alleviating the virus-induced injury of alveolar-capillary barrier. In later work, this alveolar infection model may also be used to explore the effects of remdesivir administration in combination with other repurposing drugs as therapeutics against COVID-19.

In this study, we created a human disease model of SARS-CoV-2 infection on chip that can recapitulate the key pathophysiology and immune response of lung tissue associated with COVID-19. The biomimetic human alveolus chip can resemble the alveolar-capillary barrier injury and inflammatory response of lung after SARS-CoV-2 infection, such as viral replication in human alveolar epithelium, vascular dysfunction, recruitment of immune cells, and release of inflammatory cytokines in a physiological relevant manner (Figure 8) . In particular, we found circulating immune cells may largely contribute to the exacerbation of inflammatory responses and the injury of alveolar-capillary barrier induced by SARS-CoV-2.

These findings provide new insights into the pathogenesis of SARS-CoV-2, in which virus-induced lung injury may be mediated by the complex and intrinsic cross-talk among epithelium-endothelium interface and immune cells.

This article is protected by copyright. All rights reserved. 13 As we know, alveolar epithelial type II cells have been proved to be the primary target of SARS-CoV-2 infection by histopathological studies. [43] In this study, we found that human alveolar epithelial cells are more susceptible to SARS-CoV-2 infection than endothelial cells identified on the chip. Moreover, transcriptomic analysis demonstrated the distinctive responses of alveolar epithelial and endothelial cells to SARS-CoV-2 infection, in which epithelium exhibited much higher viral load than that of endothelium. This may partially explain why SARS-CoV-2 cannot be easily detected in blood samples of COVID-19

patients. [37] In addition, compared with pulmonary microvascular endothelial cells, alveolar epithelial cells displays a broader innate immune response after viral infection, such as IFN-I signaling pathway. A recent study reported the IFN-I responsive gene sets were up-regulated in lung tissues from severe COVID-19 cases, which may be associated with exacerbated lung inflammation. [44] Our results are similar to these clinical findings, indicating the advantage of this disease model to reflect the human relevant immune responses of lung. Meanwhile, we detected the activation of JAK-STAT signaling pathway in the infected pulmonary microvascular endothelial cells as compared to non-infected cells. It has been recognized that cytokines can activate JAK-STAT pathway and regulate different cellular and immune processes. [45, 46] Clinical studies reported Ruxolitinib, a JAK inhibitor can effectively relieve the symptoms of patients with severe COVID-19. [47, 48] In combination with these findings, it might suggest the feasibility to develop new therapeutics against SARS-CoV-2 by targeting JAK-STAT signaling pathway in microvascular endothelium.

Clinically, severe COVID-19 patients often show inflammatory cytokine storms, which are associated with excessive immune responses, and may aggravate respiratory failure and cause multi-organ damage. Circulating cytokines, including IL-1β, IL-6, IL-8 and TNF-α were significantly elevated in patients with severe COVID-19. [49, 50] As such, we compared the secretion of these cytokines in this alveolus chip under different treatment conditions. Our results showed that SARS-CoV-2 infection triggered the increased secretions of cytokines IL-6, and IL-8 on the alveolus chip. In particular, the addition of circulating immune cells caused the increased level of all these cytokines secretion, accompanied by recruitment of immune cells, detachment of endothelial cells and severe disruption of intercellular junctions in both epithelium and endothelium. These findings are highly relevant with the pathological manifestations observed in severe COVID-19 patients clinically, [51, 52] verifying the role of immune cells in mediating alveolar injury, microvascular endothelial dysfunction and excessive inflammatory response. Notably, the microvascular endothelial injury might explain the pathogenesis of microvascular thrombosis existed in the lung of severe COVID-19 patients. [53, 54] This work still has some limitations. In vivo, human lung alveolus consisted of multiple cell types of pneumocytes (e.g. alveolar epithelial type I and type II cells). Here, we used only one cell type of alveolus epithelium, which may not fully simulate native alveolar tissues. The primary alveolar tissue from human biopsy can be selected for further studies. In addition, we use this model to assess only one type of antiviral candidates, remdesivir.

Actually, the advantages and capabilities of this chip model system make it possible to evaluate other candidate antiviral agents, inflammatory cytokine inhibitors or repurposing potential drugs against COVID-19 in future. Due to the limited time and challenging conditions, in this work, we are still not able to conduct very deep research of SARS-CoV-2 pathogenesis or drug pharmacology. Nevertheless, the great value of this disease model on chip is that it is capable to reflect the human relevant pathophysiology of human lung and host-immune response to this novel virus at organ level, which is difficult to be obtained by existing cell-based system. This bioengineered lung infection model could provide a complement to animal models to evaluate drug candidates and repurpose approved drugs to face the crisis of SARS-CoV-2 epidemic.

Conclusively, this work made the first attempt to build a human alveolar infection model by SARS-CoV-2 using organ chip that allows to recapitulate the lung injury and immune This work provides the proof-of-concept to create a human disease model on chip to study host-virus interactions and human relevant responses at organ level. The obvious advantages of this chip platform lie in its accessibility to study responses of various cells to virus in real time simultaneously, and to rapidly test candidate drugs with low cost and short time, which cannot be easily achieved by existing in vitro experimental models or animal models.

Moreover, it provides a synthetic strategy with flexibility for incorporating varying elements, thus can be adapted to emerging needs in the case of the current COVID-19 pandemic. Immunostaining: HPAEpiC cells cultured on well plate were washed with PBS and fixed with 4% PFA at 4°C overnight. Cells were then permeabilized with 0.2% Triton X-100 in PBS (PBST buffer) for 5 min and blocked with PBST buffer containing 5% normal goat serum for 30 minutes at room temperature. Antibodies were diluted with PBST buffer. Cells were stained with corresponding primary antibodies at 4°C overnight and with secondary antibodies (supplementary Table S1 ) at room temperature for 1 hour. After staining with the secondary antibodies, the cell nuclei were counterstained with DAPI.

For immunofluorescent imaging of alveolus chip, cells were washed with PBS through the upper and bottom channels and fixed with 4% PFA. The fixed tissues were subjected to This article is protected by copyright. All rights reserved. 19 immunofluorescence staining by the same procedure as described above. All images were acquired using a confocal fluorescent microscope system (Carl zeiss LSM880). Image processing was done using ImageJ (NIH). 

were collected separately from the chips, and total RNAs were extracted from samples using TRIzol (Invitrogen) following the methods by Chomczynski et al. [55] DNA digestion was carried out after RNA extraction by DNaseI. RNA quality was determined by examining A260/A280 with NanodropTM OneCspectrophotometer (Thermo Fisher Scientific Inc). RNA Integrity was confirmed by 1.5% agarose gel electrophoresis. Qualified RNAs were finally RNA-seq data analysis: Raw sequencing data was first filtered by Trimmomatic (version 0.36), low-quality reads were discarded and the reads contaminated with adaptor sequences were trimmed. Clean reads were further treated with in-house scripts to eliminate duplication bias introduced in library preparation and sequencing. In brief, clean reads were first clustered according to the UMI sequences, in which reads with the same UMI sequence were grouped into the same cluster, resulting in 65,536 clusters. Reads in the same cluster were compared to each other by pairwise alignment, and then reads with sequence identity over 95% were extracted to a new sub-cluster. After all sub-clusters were generated, multiple sequence alignment was performed to get one consensus sequence for each sub-cluster. After these steps, any errors and biases introduced by PCR amplification or sequencing were eliminated.

The de-duplicated consensus sequences were used for standard RNA-seq analysis. They were mapped to the reference genome of Homo sapiens from Ensembl database Statistical analyses: Data were collected in Excel (Microsoft). The difference between two groups was analyzed using Student's t-test. Multiple group comparison was performed using one-way analysis of variance (ANOVA) followed by post-hoc test. The bar graphs with error This article is protected by copyright. All rights reserved. 22 bars represent mean ± standard deviation (SD). Significance is indicated by asterisks: *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Data availability: All relevant data are available in the manuscript or supporting information.

All of the RNA-seq raw data have been deposited on SRA under the accession number PRJNA647532.

Supporting Information is available from the Wiley Online Library or from the author. Data were presented as mean ± SD. Three chips were quantified for each group.

This article is protected by copyright. All rights reserved. The cytokines or chemokines released from infected cells can recruit circulating immune cells (such as CD14 + monocytes) to infected sites and initiate inflammatory responses. This process further exacerbates the disruption of alveolus-capillary barrier integrity, leading to lung injury.

Emerg Infect Dis

This article is protected by copyright. All rights reserved.

The authors declare no conflict of interest.This article is protected by copyright. All rights reserved.