SARS-CoV-2-triggered mast cell rapid degranulation induces alveolar epithelial inflammation and lung injury

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1. Version published to 10.1038/s41392-021-00849-0

   Dec 17, 2021
2. 

   ScreenIT

   Jul 1, 2021

   SciScore for 10.1101/2021.06.24.449680: (What is this?)

   Please note, not all rigor criteria are appropriate for all manuscripts.

   Table 1: Rigor

   Ethics	IACUC: Ethics statement: All animal experiments were approved by the Institutional Animal Care and Use Committee of XXXXXX.
   Sex as a biological variable	For Ad5-hACE2-transduced BALB/c mice 51, specific pathogen-free 6-10 weeks old male and female BALB/c mice were lightly anesthetized with isoflurane and transduced intranasally with 2.5×108 fluorescence focus units (FFU) of Ad5-ACE2 in 75 μL DMEM.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   The cells were then fixed with 4% paraformaldehyde (Sigma-Aldrich) for 30 min at room temperature and stained with anti-His-tag antibodies …

   SciScore for 10.1101/2021.06.24.449680: (What is this?)

   Please note, not all rigor criteria are appropriate for all manuscripts.

   Table 1: Rigor

   Ethics	IACUC: Ethics statement: All animal experiments were approved by the Institutional Animal Care and Use Committee of XXXXXX.
   Sex as a biological variable	For Ad5-hACE2-transduced BALB/c mice 51, specific pathogen-free 6-10 weeks old male and female BALB/c mice were lightly anesthetized with isoflurane and transduced intranasally with 2.5×108 fluorescence focus units (FFU) of Ad5-ACE2 in 75 μL DMEM.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   The cells were then fixed with 4% paraformaldehyde (Sigma-Aldrich) for 30 min at room temperature and stained with anti-His-tag antibodies (Abmart, M30111S).	anti-His-tag suggested: None
   Subsequently, the cells were stained with goat anti-mouse Alexla Fluor 488-conjugated secondary antibodies (Invitrogen, A11001), and were detected with flow cytometry.	anti-mouse suggested: (Thermo Fisher Scientific Cat# A-11001, RRID:AB_2534069)
   In some experiments, LAD2 cells were prior-treated with 0.25% trypsin (without EDTA) for 10 min at 37 °C or prior-blocked with anti-ACE2 antibody (5 μg/mL, R&D Systems, AF933) for 2 h at 37 °C before the incubation with Spike-RBD protein.	anti-ACE2 suggested: None
   The anti-ACE2 antibody (Abcam, EPR4435), anti-MMP9 antibody (signal antibody, 29091), anti-GAPDH antibody (Abcam, 6C5), and the horseradish peroxidase-conjugated secondary antibody were used in Western blotting.	anti-MMP9 suggested: None anti-GAPDH suggested: (Abcam Cat# ab92412, RRID:AB_2278693)
   For detecting tight junction proteins ZO-1, Occludin, Claudin-5 and JAM2 in A549 cells, cells were blocked with 5% BSA in PBS for 1 h at room temperature then incubated with primary antibodies for 2h at 4°C.	ZO-1, Occludin, suggested: None Claudin-5 suggested: None JAM2 suggested: None
   Primary antibodies against ZO-1 (Invitrogen, 402200), Occludin (Invitrogen, OC-3F10), Claudin-5 (Invitrogen, 4C3C2) and JAM-2 (Abcam, EPR2489), were used.	ZO-1 suggested: (Thermo Fisher Scientific Cat# 40-2200, RRID:AB_2533456) Occludin suggested: None JAM-2 suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Pseudotyped virus was generated by EZ Trans cell transfection reagent-mediated co-transfection of HEK293T cells with the Spike-expressing plasmid pcDNA3.1-2019-nCoV-S-IRES (strain 2019-nCoV WIV04) and pNL4-3. Luc. ΔR ΔE 81.	HEK293T suggested: None
   For immuno-staining assay, A549 cells were added leukocyte activation cocktail containing BD GolgiPlug (BD, 550583) and cultured for 6 h.	A549 suggested: None
   Experimental Models: Organisms/Strains
   Sentences	Resources
   ACE2-humanzied mice and rhesus macaques experiments: 3-4 months old C57BL/6N-Ace2em2(hACE2-WPRE,pgk-puro)/CCLA mice were provided by Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Science 47.	C57BL/6N-Ace2em2(hACE2-WPRE,pgk-puro)/CCLA suggested: None
   For Ad5-hACE2-transduced BALB/c mice 51, specific pathogen-free 6-10 weeks old male and female BALB/c mice were lightly anesthetized with isoflurane and transduced intranasally with 2.5×108 fluorescence focus units (FFU) of Ad5-ACE2 in 75 μL DMEM.	BALB/c suggested: None
   Recombinant DNA
   Sentences	Resources
   Pseudotyped virus was generated by EZ Trans cell transfection reagent-mediated co-transfection of HEK293T cells with the Spike-expressing plasmid pcDNA3.1-2019-nCoV-S-IRES (strain 2019-nCoV WIV04) and pNL4-3. Luc. ΔR ΔE 81.	Spike-expressing suggested: None pcDNA3.1-2019-nCoV-S-IRES suggested: None pNL4-3 suggested: None
   Software and Algorithms
   Sentences	Resources
   After washing, cells were detected with BD Accuri C6 and analyzed with FlowJo.	FlowJo suggested: (FlowJo, RRID:SCR_008520)
   Raw RNA sequencing (RNA-seq) reads were filtered using Trimmomatic v0.36 82.	Trimmomatic suggested: (Trimmomatic, RRID:SCR_011848)
   The filtered reads were mapped to the human (hg38) reference genomes using HISAT v2.1 with corresponding gene annotations (GRCh38.p13) with default settings 83.	HISAT suggested: (HISAT2, RRID:SCR_015530)
   Total counts per mapped gene were determined using featureCounts function in SubReads package v1.5.3 with default parameter 84.	featureCounts suggested: (featureCounts, RRID:SCR_012919)
   Next, counts matrix obtained from featureCounts was used as input for differential expression gene analysis with the bioconductor package DESeq2 v1.26 in R v4.085.	bioconductor suggested: (Bioconductor, RRID:SCR_006442)
   Filtered counts matrix was normalized using the DESeq2 method to remove the library-specific artifacts.	DESeq2 suggested: (DESeq, RRID:SCR_000154)
   Transcription-factor enrichment analysis and functional enrichment analysis was performed using Metascape server tool 86 (https://metascape.org/gp/index.html#/main/step1).	Metascape suggested: (Metascape, RRID:SCR_016620)
   Gene set enrichment analysis (GSEA) were performed using the R package clusterProfiler v3.18.1 87.	Gene set enrichment analysis suggested: (Gene Set Enrichment Analysis, RRID:SCR_003199) clusterProfiler suggested: (clusterProfiler, RRID:SCR_016884)
   Protein-protein interaction (PPI) networks of DEGs were built using STRING v11 with a confidence score threshold of 0.7 and visualized with Cytoscape v3.8.1 88,89.	STRING suggested: (STRING, RRID:SCR_005223) Cytoscape suggested: (Cytoscape, RRID:SCR_003032)

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   Results from OddPub: Thank you for sharing your data.

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   Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:

   The limitation of our study is that the administration of Eba or Lor significantly reduced SARS-CoV-2-induced inflammation and lung injury, but showed limitation in suppressing viral replication in lungs of ACE2 humanized mice. A combination of MC stabilizers with antiviral drugs such as the RNA polymerase inhibitors Remdesivir and Favipiravir 6,78–80, may provide more optimal treatment strategy that dampening inflammation and clearing viruses at the same time. In summary, we demonstrate that SARS-CoV-2 triggers lung MCs degranulation, which induces the remodeling of various cellular signalings in human alveolar epithelial cells, particularly, MCs degranulation induces the alveolar epithelial inflammation and leads to the consequent disruption of tight junction proteins; importantly, we find that the clinically used MC degranulation stabilizers Eba and Lor are potent agents at reducing virus-induced production of pro-inflammatory factors and preventing lung injury. Our finding uncovers a potentially novel mechanism of SARS-CoV-2 infection initiates alveolar epithelial inflammation and induces lung Injury. Significantly, our results suggest a potential off-label use of MC stabilizers as immunomodulators to treat the severe cases of COVID-19.

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   Results from TrialIdentifier: We found the following clinical trial numbers in your paper:
   

   Identifier	Status	Title
   NCT04324021	Terminated	Efficacy and Safety of Emapalumab and Anakinra in Reducing H…
   NCT04338958	Recruiting	Ruxolitinib in Covid-19 Patients With Defined Hyperinflammat…

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   Results from Barzooka: We did not find any issues relating to the usage of bar graphs.

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   Results from JetFighter: We did not find any issues relating to colormaps.

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   Results from rtransparent:

   • Thank you for including a conflict of interest statement. Authors are encouraged to include this statement when submitting to a journal.
   • No funding statement was detected.
   • No protocol registration statement was detected.

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   Results from scite Reference Check: We found no unreliable references.

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3. 

   Version published to 10.1101/2021.06.24.449680v1 on bioRxiv

   Jun 24, 2021