Enteric Coronavirus Infection and Treatment Modeled With an Immunocompetent Human Intestine-On-A-Chip
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Abstract
Many patients infected with coronaviruses, such as SARS-CoV-2 and NL63 that use ACE2 receptors to infect cells, exhibit gastrointestinal symptoms and viral proteins are found in the human gastrointestinal tract, yet little is known about the inflammatory and pathological effects of coronavirus infection on the human intestine. Here, we used a human intestine-on-a-chip (Intestine Chip) microfluidic culture device lined by patient organoid-derived intestinal epithelium interfaced with human vascular endothelium to study host cellular and inflammatory responses to infection with NL63 coronavirus. These organoid-derived intestinal epithelial cells dramatically increased their ACE2 protein levels when cultured under flow in the presence of peristalsis-like mechanical deformations in the Intestine Chips compared to when cultured statically as organoids or in Transwell inserts. Infection of the intestinal epithelium with NL63 on-chip led to inflammation of the endothelium as demonstrated by loss of barrier function, increased cytokine production, and recruitment of circulating peripheral blood mononuclear cells (PBMCs). Treatment of NL63 infected chips with the approved protease inhibitor drug, nafamostat, inhibited viral entry and resulted in a reduction in both viral load and cytokine secretion, whereas remdesivir, one of the few drugs approved for COVID19 patients, was not found to be effective and it also was toxic to the endothelium. This model of intestinal infection was also used to test the effects of other drugs that have been proposed for potential repurposing against SARS-CoV-2. Taken together, these data suggest that the human Intestine Chip might be useful as a human preclinical model for studying coronavirus related pathology as well as for testing of potential anti-viral or anti-inflammatory therapeutics.
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SciScore for 10.1101/2021.06.03.446968: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IRB: De-identified human patient-derived apheresis collars (a by-product of platelet isolation) were obtained from the Crimson Biomaterials Collection Core Facility under approval obtained from the Institutional Review Board at Harvard University (#22470); informed written consent was not required.
Consent: De-identified human patient-derived apheresis collars (a by-product of platelet isolation) were obtained from the Crimson Biomaterials Collection Core Facility under approval obtained from the Institutional Review Board at Harvard University (#22470); informed written consent was not required.Sex as a biological variable not detected. Randomization not detected. Blinding not detected. Power … SciScore for 10.1101/2021.06.03.446968: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics IRB: De-identified human patient-derived apheresis collars (a by-product of platelet isolation) were obtained from the Crimson Biomaterials Collection Core Facility under approval obtained from the Institutional Review Board at Harvard University (#22470); informed written consent was not required.
Consent: De-identified human patient-derived apheresis collars (a by-product of platelet isolation) were obtained from the Crimson Biomaterials Collection Core Facility under approval obtained from the Institutional Review Board at Harvard University (#22470); informed written consent was not required.Sex as a biological variable not detected. Randomization not detected. Blinding not detected. Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources The fixed samples were permeabilized in PBS containing 0.1% Triton X-100 and 1% Fetal Bovine Serum for 30 min at room temperature before filling the channels with staining buffer (1.5% BSA in PBS) containing primary antibodies directed against cleaved caspase-3 (Cell Signaling Technology, 9661S) or VE-cadherin (Thermo Fisher Scientific, 14-1449-82), and incubating them overnight at 4°C with gentle shaking. cleaved caspase-3 ( Cell Signaling Technology ,suggested: NoneVE-cadherinsuggested: (Thermo Fisher Scientific Cat# 14-1449-82, RRID:AB_467495)Experimental Models: Cell Lines Sentences Resources Virus infection of human Intestine Chips: OC43 virus (ATCC, VR-1558) was propagated in HCT-8 cells in RPMI with 3% horse serum at 34°C and quantified by TCID50 assays. HCT-8suggested: NoneSoftware and Algorithms Sentences Resources Sequencing libraries were demultiplexed using bcl2fastq (v2.20.0.422) with default settings (mask_short_adapter_reads = 10, minimum_trimmed_read_length = 10) on Cumulus14 (snapshot 4). bcl2fastqsuggested: (bcl2fastq , RRID:SCR_015058)Libraries were aligned using STAR implemented on Cumulus (snapshot 9). STARsuggested: (STAR, RRID:SCR_004463)Dimensionality reduction, cell clustering, and differential gene analysis were performed in Seurat v3.1 and differential gene expression analysis in Seurat v4.016,17. Seuratsuggested: (SEURAT, RRID:SCR_007322)Fluorescence imaging was performed using a confocal laser-scanning microscope (Leica SP5 X MP DMI-6000) and the images obtained were processed by Imaris software (Bitplane) and analyzed by Image J. Imarissuggested: (Imaris, RRID:SCR_007370)Image Jsuggested: (ImageJ, RRID:SCR_003070)Statistical analysis: Unpaired Student’s t-test was performed in GraphPad Prism using the Welch correction for different standard deviations and differences were considered statistically significant when *P<0.05, **P < 0.01, and ***P < 0.001. GraphPad Prismsuggested: (GraphPad Prism, RRID:SCR_002798)Results from OddPub: We did not detect open data. We also did not detect open code. Researchers are encouraged to share open data when possible (see Nature blog).
Results from LimitationRecognizer: An explicit section about the limitations of the techniques employed in this study was not found. We encourage authors to address study limitations.Results from TrialIdentifier: No clinical trial numbers were referenced.
Results from Barzooka: We found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
- Thank you for including a funding statement. Authors are encouraged to include this statement when submitting to a journal.
- No protocol registration statement was detected.
Results from scite Reference Check: We found no unreliable references.
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