Impaired immune response drives age-dependent severity of COVID-19

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1. Version published to 10.1084/jem.20220621

   Sep 21, 2022
2. 

   ScreenIT

   Apr 25, 2022

   SciScore for 10.1101/2022.04.21.489072: (What is this?)

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

   Table 1: Rigor

   Ethics

   Euthanasia Agents: Experimental end points when mice were euthanized by cervical dislocation were defined as ≥25 % weight loss or ≥20 % weight loss for longer than two days. IACUC: All animal work conducted at the University Medical Center Freiburg and the Francis Crick Institute followed the German animal protection law, or the Animals (Scientific Prpcedures) Act 1986, respectively, and was approved by the respective local animal welfare committee (Regierungspräsidium Freiburg #35-9185.81/G-20/91) or the UK Home Office London Consent: Written informed consent was obtained from participants and the study was conducted according to federal guidelines and local ethics committee regulations …

   SciScore for 10.1101/2022.04.21.489072: (What is this?)

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

   Table 1: Rigor

   Ethics	Euthanasia Agents: Experimental end points when mice were euthanized by cervical dislocation were defined as ≥25 % weight loss or ≥20 % weight loss for longer than two days. IACUC: All animal work conducted at the University Medical Center Freiburg and the Francis Crick Institute followed the German animal protection law, or the Animals (Scientific Prpcedures) Act 1986, respectively, and was approved by the respective local animal welfare committee (Regierungspräsidium Freiburg #35-9185.81/G-20/91) or the UK Home Office London Consent: Written informed consent was obtained from participants and the study was conducted according to federal guidelines and local ethics committee regulations (Albert-Ludwigs-Universität, Freiburg, Germany: No. F-2020-09-03-160428 and no. 322/20). IRB: Written informed consent was obtained from participants and the study was conducted according to federal guidelines and local ethics committee regulations (Albert-Ludwigs-Universität, Freiburg, Germany: No. F-2020-09-03-160428 and no. 322/20).
   Sex as a biological variable	Animals of both sexes were used.
   Randomization	not detected.
   Blinding	not detected.
   Power Analysis	not detected.
   Cell Line Authentication	not detected.

   Table 2: Resources

   Antibodies
   Sentences	Resources
   The primary polyclonal serum was applied overnight at 4 °C (1:3000, diluted in TRIS buffer), the secondary biotinylated goat anti-rabbit antibody was applied for 30 minutes at room temperature (Vector Laboratories, Burlingame, CA, USA, 1:200).	anti-rabbit suggested: None
   Experimental Models: Cell Lines
   Sentences	Resources
   For virus growth curves, Vero E6 or Calu-3 cells seeded in 24-well plates were washed with PBS and then infected with the respective virus strain by incubating the cells with virus containing Opti-MEM 0.3 % BSA for 2 h at 37 °C with 5 % CO2 using an MOI of 0.001.	Calu-3 suggested: None
   The mixture was then added to Vero E6 cells and incubated for 1.5 h at 37 °C with 5 % CO2.	Vero E6 suggested: None
   Experimental Models: Organisms/Strains
   Sentences	Resources
   Mice: C57BL/6JRj, BALB/cJRj and 129S2/SvPasOrlRj mice were purchased from Janvier Labs.	C57BL/6JRj suggested: None 129S2/SvPasOrlRj suggested: None
   B6.A2G-Mx1, B6.A2G-Mx1-Ifnar1−/−, B6.A2G-Mx1-Ifnlr1−/−, B6.AG2-Mx1-Ifnar1−/−Ifnlr1−/−, B6.Ifngr1-/-, B6.Ifnar1-/-Ifngr1-/-, and B6.A2G-Mx1-Stat1-/- were bred and kept at the animal facilities of the University Medical Center Freiburg.	B6.A2G-Mx1 suggested: None B6.A2G-Mx1-Ifnar1−/− suggested: None B6.A2G-Mx1-Ifnlr1−/− suggested: None B6.AG2-Mx1-Ifnar1−/−Ifnlr1−/− suggested: None B6.Ifngr1-/- suggested: RRID:IMSR_JAX:007077) B6.Ifnar1-/-Ifngr1-/- suggested: None B6.A2G-Mx1-Stat1-/- suggested: None
   In addition, to identify subtle inflammation in brain and heart samples from infected 10-week (n=10) and 40-week-old (n=6) C57BL/6, hearts were evaluated for the presence of CD3-positive T cell infiltrates and brains evaluated for the presence of CD3-positive T cells and Iba-1-positive microglial cells/macrophages as described in 80.	C57BL/6 suggested: None
   Software and Algorithms
   Sentences	Resources
   De-multiplexed raw reads were subjected to a customized Galaxy pipeline based on bioinformatics pipelines on usegalaxy.eu68.	Galaxy suggested: (Galaxy, RRID:SCR_006281)
   0.20.169 and mapped to the SARS-CoV-2 Wuhan-Hu-1 reference genome (Genbank: NC_045512) using BWA-MEM v.	BWA-MEM suggested: None
   Variants (SNPs and INDELs) were called using the ultrasensitive variant caller LoFreq v2.1.571, demanding a minimum base quality of 30 and a coverage ≥ 10-fold.	LoFreq suggested: (LoFreq, RRID:SCR_013054)
   Effects of mutations were automatically annotated in vcf files using SnpEff v.4.3.172.	SnpEff suggested: (SnpEff, RRID:SCR_005191)
   Read quality trimming and adaptor removal was carried out using Trimmomatic (version 0.36).	Trimmomatic suggested: None
   All data was processed relative to the mouse GRCm38 genome downloaded from Ensembl.	Ensembl suggested: None
   Gene counts per gene per sample were obtained using the RSEM-STAR (77, 78) option of the pipeline and they were imported on DESeq (v1.28.0, 79) within R environment v4.0.2 for differential expression analysis.	DESeq suggested: (DESeq, RRID:SCR_000154)
   Gene Ontology and Gene Set Enrichment analysis (GSEA) were carried out using R package Cluster Profiler (v3.16).	Cluster Profiler suggested: (clusterProfiler, RRID:SCR_016884)
   Gene signatures were considered significant if FDR q-value ≤ 0.05. ggplot2, RColorBrewer, ComplexHeatmap were used for plotting purposes.	ComplexHeatmap suggested: (ComplexHeatmap, RRID:SCR_017270)
   Ingenuity Pathway Analysis was performed using differentially expressed genes (fold change ≥ 1.5, padj ≤ 0.05).	Ingenuity Pathway Analysis suggested: None
   Statistical analyses: Data visualization and analyses were performed using GraphPad Prism 9.0 and R version 3.5.1.	GraphPad Prism suggested: (GraphPad Prism, RRID:SCR_002798)

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   Results from OddPub: Thank you for sharing your code and data.

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

   Small animal models faithfully recapitulating characteristics of human disease are pivotal to overcome these limitations. In this study, we generated a highly pathogenic mouse-adapted SARS-CoV-2 variant that can be used to model mild, moderate or severe COVID-19 as well as the age-associated aggravation of disease in standard inbred mice. Using this small animal model, we found that the age-dependent increase in disease severity is driven by an impaired interferon response which causes a delayed, insufficient and dysregulated innate and adaptive immune response in the aged host. Transcriptome analyses of infected lungs from mature adult and middle-aged C57BL/6 mice revealed that adult mice initiated a rapid and well-coordinated innate and adaptive immune response, which was associated with high IFN-γ and low IL-10 expression levels. This effective and timely immune response in adults limited viral loads, mediated rapid viral clearance, and efficiently prevented the development of severe disease. In aged mice, by contrast, virus replication was markedly increased which correlated with the absence of effective antiviral immune responses. Instead, aged mice showed strong IL-6- and IL-1-mediated responses associated with low IFN-γ and high IL-10 expression levels. The markedly different IFN-γ to IL-10 ratio likely explains the effective immune response, including NK cell mediated immunity, efficient antigen presentation, lymphocyte activation, and immunoglobulin production in adu...

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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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3. 

   Version published to 10.1101/2022.04.21.489072v1 on bioRxiv

   Apr 22, 2022