Hypoxia inducible factors regulate infectious SARS-CoV-2, epithelial damage and respiratory symptoms in a hamster COVID-19 model
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Abstract
Understanding the host pathways that define susceptibility to Severe-acute-respiratory-syndrome-coronavirus-2 (SARS-CoV-2) infection and disease are essential for the design of new therapies. Oxygen levels in the microenvironment define the transcriptional landscape, however the influence of hypoxia on virus replication and disease in animal models is not well understood. In this study, we identify a role for the hypoxic inducible factor (HIF) signalling axis to inhibit SARS-CoV-2 infection, epithelial damage and respiratory symptoms in the Syrian hamster model. Pharmacological activation of HIF with the prolyl-hydroxylase inhibitor FG-4592 significantly reduced infectious virus in the upper and lower respiratory tract. Nasal and lung epithelia showed a reduction in SARS-CoV-2 RNA and nucleocapsid expression in treated animals. Transcriptomic and pathological analysis showed reduced epithelial damage and increased expression of ciliated cells. Our study provides new insights on the intrinsic antiviral properties of the HIF signalling pathway in SARS-CoV-2 replication that may be applicable to other respiratory pathogens and identifies new therapeutic opportunities.
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SciScore for 10.1101/2022.03.15.484379: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics Euthanasia Agents: Viral inocula were made in sterile phosphate buffered saline (PBS) and delivered via intranasal instillation (200μL total with 100μL per nare) with animals sedated using isoflurane.
IACUC: UKHSA) Porton Down by the Animal Welfare and Ethical Review Body (AWERB) as required by the Home Office Animals (Scientific Procedures) Act 1986.Sex as a biological variable The animals were randomly assigned into groups and individually housed, with equal allocation of male and female animals to each study. Randomization The animals were randomly assigned into groups and individually housed, with equal allocation of male and female animals to each study. Blinding All slides were evaluated … SciScore for 10.1101/2022.03.15.484379: (What is this?)
Please note, not all rigor criteria are appropriate for all manuscripts.
Table 1: Rigor
Ethics Euthanasia Agents: Viral inocula were made in sterile phosphate buffered saline (PBS) and delivered via intranasal instillation (200μL total with 100μL per nare) with animals sedated using isoflurane.
IACUC: UKHSA) Porton Down by the Animal Welfare and Ethical Review Body (AWERB) as required by the Home Office Animals (Scientific Procedures) Act 1986.Sex as a biological variable The animals were randomly assigned into groups and individually housed, with equal allocation of male and female animals to each study. Randomization The animals were randomly assigned into groups and individually housed, with equal allocation of male and female animals to each study. Blinding All slides were evaluated subjectively by a qualified pathologist, blinded to treatment details and were randomised prior to examination to limit bias (blind evaluation). Power Analysis not detected. Cell Line Authentication not detected. Table 2: Resources
Antibodies Sentences Resources Staining was performed with the BOND Polymer Refine Detection kit, a rabbit anti-SARS-CoV-2 nucleocapsid antibody (Sinobiological; clone: #001; dilution: 1:5000) and counterstained with haematoxylin. anti-SARS-CoV-2suggested: (Leinco Technologies Cat# LT5000, RRID:AB_2893962)Membranes were blocked in 5% milk in PBS/0.1% Tween-20 and incubated with anti-HIF-1α (BD Biosciences), anti-β-Actin (Sigma) or SARS-CoV-2 nucleocapsid (EY-2A, a kind gift from Prof Alain Townsend) primary antibodies and appropriate HRP-conjugated secondary antibodies (DAKO). anti-HIF-1αsuggested: (Cayman Chemical Cat# 10006421, RRID:AB_409037)anti-HIF-1α (BD Biosciences), anti-β-Actin (Sigma)suggested: Noneanti-β-Actinsuggested: NoneExperimental Models: Cell Lines Sentences Resources Virus infectivity was determined by plaque assay on Vero-TMPRSS2 cells as previously reported (Wing et al., 2021a) Vero-TMPRSS2suggested: JCRB Cat# JCRB1818, RRID:CVCL_YQ48)Calu-3 cells were infected with the above strain of SARS-CoV-2 at an MOI of 0.01 for 2h. Calu-3suggested: KCLB Cat# 30055, RRID:CVCL_0609)Software and Algorithms Sentences Resources FPKM values were enumerated, and differential expression quantified using the DeSeq2 package (Love et al., 2014). DeSeq2suggested: (DESeq2, RRID:SCR_015687)Membranes were blocked in 5% milk in PBS/0.1% Tween-20 and incubated with anti-HIF-1α (BD Biosciences), anti-β-Actin (Sigma) or SARS-CoV-2 nucleocapsid (EY-2A, a kind gift from Prof Alain Townsend) primary antibodies and appropriate HRP-conjugated secondary antibodies (DAKO). BD Biosciencessuggested: (BD Biosciences, RRID:SCR_013311)P values were determined using the Mann-Whitney test (two group comparisons) or with the Kruskal–Wallis ANOVA (multi group comparisons) using PRISM version 8. PRISMsuggested: (PRISM, RRID:SCR_005375)Results from OddPub: Thank you for sharing your data.
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 did not find any issues relating to the usage of bar graphs.
Results from JetFighter: We did not find any issues relating to colormaps.
Results from rtransparent:- No conflict of interest statement was detected. If there are no conflicts, we encourage authors to explicit state so.
- 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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