Human galectin-9 potently enhances SARS-CoV-2 replication and inflammation in airway epithelial cells

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1. Version published to 10.1093/jmcb/mjad030

   Apr 1, 2023
2. 

   Version published to 10.1101/2022.03.18.484956v3 on bioRxiv

   May 16, 2022
3. 

   Version published to 10.1101/2022.03.18.484956v2 on bioRxiv

   Mar 29, 2022
4. 

   ScreenIT

   Mar 23, 2022

   SciScore for 10.1101/2022.03.18.484956: (What is this?)

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

   Table 1: Rigor

   Ethics	Field Sample Permit: All the studies involving live viruses were conducted in the Vitalant Research Institute BSL-3 under approved safety protocols.
   Sex as a biological variable	not detected.
   Randomization	Image Analysis: To measure the frequency of infected cells, randomly-selected areas were imaged.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	Contamination: All cells had been previously tested for mycoplasma contamination and incubated at 37°C in a humidified atmosphere with 5% CO2.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Cells were then incubated with a primary antibody (monoclonal rabbit anti-SARS-CoV-2 nucleocapsid antibody (GeneTex, GTX135357)) in 1 X PBS …

   SciScore for 10.1101/2022.03.18.484956: (What is this?)

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

   Table 1: Rigor

   Ethics	Field Sample Permit: All the studies involving live viruses were conducted in the Vitalant Research Institute BSL-3 under approved safety protocols.
   Sex as a biological variable	not detected.
   Randomization	Image Analysis: To measure the frequency of infected cells, randomly-selected areas were imaged.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	Contamination: All cells had been previously tested for mycoplasma contamination and incubated at 37°C in a humidified atmosphere with 5% CO2.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   Cells were then incubated with a primary antibody (monoclonal rabbit anti-SARS-CoV-2 nucleocapsid antibody (GeneTex, GTX135357)) in 1 X PBS (1:1,000) overnight at 4°C.	anti-SARS-CoV-2 suggested: None
   The following day, cells were washed three times with 1 X PBS and incubated with a secondary antibody (Goat anti-Rabbit IgG (H+L) secondary antibody, FITC (Thermo Fisher, 65-6111)) in 1 X PBS (1:200) for 1 h at 37°C.	anti-Rabbit IgG suggested: (Thermo Fisher Scientific Cat# 65-6111, RRID:AB_2533966)
   An unrelated anti-goat IgG antibody was used as a control.	anti-goat IgG suggested: None
   The amount of captured ACE2, which is proportional to ACE2 binding activity, is then recognized by an ACE2 detection antibody and measured by reading the absorbance at a wavelength of 450 nm.	ACE2 suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   Viruses were propagated in Vero E6-TMPRSS2 cells in DMEM with 2% FBS and 1% penicillin/streptomycin and viral stocks were stored at −80°C.	Vero E6-TMPRSS2 suggested: None
   Virus titer was measured in Vero E6 cells by TCID50 assay.	Vero E6 suggested: None
   In brief, Calu-3 cells cultured in 96-well cell culture plates were incubated with different concentrations of Gal-9 (0-5,000 nM).	Calu-3 suggested: None
   Pseudovirus production: VSVΔG-luciferase-based viruses, in which the glycoprotein (G) gene has been replaced with luciferase, were produced by transient transfection of viral glycoprotein expression plasmids (pCG SARS-CoV-2 Spike and pCAGGS VSV-G) or no glycoprotein control into HEK293T cells by TransIT-2020 as previously described (Ng et al., 2020).	HEK293T suggested: None
   Recombinant DNA
   Sentences	Resources
   Pseudovirus production: VSVΔG-luciferase-based viruses, in which the glycoprotein (G) gene has been replaced with luciferase, were produced by transient transfection of viral glycoprotein expression plasmids (pCG SARS-CoV-2 Spike and pCAGGS VSV-G) or no glycoprotein control into HEK293T cells by TransIT-2020 as previously described (Ng et al., 2020).	pCG suggested: RRID:Addgene_51476) pCAGGS VSV-G suggested: None
   Software and Algorithms
   Sentences	Resources
   The data from three independent experiments was used to calculate the CC50 by nonlinear regression using GraphPad Prism 8.0 software.	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798)
   RNA-seq and Ingenuity Pathway Analysis (IPA): RNA concentration and quality was measured using High Sensitivity RNA ScreenTape Analysis (Agilent, 5067-1500). cDNA libraries were prepared using the Illumina TruSeq Stranded total RNA library prep kit (Illumina, 20020597) and sequencing was performed on the Illumina Nextseq 550 platform generating 75 bp paired-end reads.	Ingenuity Pathway Analysis suggested: (Ingenuity Pathway Analysis, RRID:SCR_008653)
   The quality of raw sequencing reads was assessed using FastQC.	FastQC suggested: (FastQC, RRID:SCR_014583)
   Differentially-expressed genes were identified by GSA or ANOVA in Partek® Flow® imported into the QIAGEN Ingenuity Pathway Analysis (IPA) software application.	QIAGEN Ingenuity Pathway Analysis suggested: None Ingenuity suggested: (Ingenuity Pathway Analysis, RRID:SCR_008653)
   Data was analyzed using FlowJo.	FlowJo suggested: (FlowJo, RRID:SCR_008520)
   Data analyses were performed using Bio-Plex Manager™ 6.1.1 (Bio-Rad Laboratories, Hercules, CA, USA).	Bio-Plex suggested: None Bio-Rad Laboratories suggested: (Bio-Rad Laboratories, RRID:SCR_008426)
   Quantitative analysis of synergy: The combinatorial effects of Gal-9 treatment and SARS-CoV-2 infection on pro-inflammatory cytokine expression were analyzed using the SynergyFinder web application, implementing the Bliss Independence model.	SynergyFinder suggested: (SynergyFinder, RRID:SCR_019318)

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   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).

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

   Our study has limitations that must be considered. Firstly, we focused exclusively on the airway epithelium, and it is now established that SARS-CoV-2 is capable of infecting other cell lineages including monocytes, monocyte-derived macrophages, and microglia (Boumaza et al., 2021; Jeong et al.; Liu et al., 2021). It is possible that Gal-9 does not exert similar effects on viral replication or immune signaling in other target cell types. Secondly, our studies were all performed in vitro or in transplant tissue-derived primary epithelial cells ex vivo. As it is well-established that Gal-9 exerts conditional, pleiotropic immunomodulatory effects, the net effect of Gal-9 signaling on SARS-CoV-2 pathogenesis cannot be fully appreciated outside of an animal model with a functional immune system. Validation and extension of our observations in murine, hamster or nonhuman primate models of SARS-CoV-2 infection will help in evaluating the clinical relevance of our reported findings. In relevance to the implementation of animal models, our study did not elucidate the principal cell or tissue sources responsible for secreting Gal-9 in the setting of SARS-CoV-2 infection. Leveraging an animal model to identify these source compartments will be critical in developing interventions to manipulate Gal-9 signaling as a therapeutic approach. To our knowledge, the data presented here are the first to show that Gal-9 is directly involved in enhancing SARS-CoV-2 infection and virus-induced pro-i...

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   Results from TrialIdentifier: No clinical trial numbers were referenced.

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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.
   • 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.

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

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5. 

   Version published to 10.1101/2022.03.18.484956v1 on bioRxiv

   Mar 20, 2022