Baricitinib treatment resolves lower-airway macrophage inflammation and neutrophil recruitment in SARS-CoV-2-infected rhesus macaques

  1. Timothy N. Hoang
  2. Maria Pino
  3. Arun K. Boddapati
  4. Elise G. Viox
  5. Carly E. Starke
  6. Amit A. Upadhyay
  7. Sanjeev Gumber
  8. Michael Nekorchuk
  9. Kathleen Busman-Sahay
  10. Zachary Strongin
  11. Justin L. Harper
  12. Gregory K. Tharp
  13. Kathryn L. Pellegrini
  14. Shannon Kirejczyk
  15. Keivan Zandi
  16. Sijia Tao
  17. Tristan R. Horton
  18. Elizabeth N. Beagle
  19. Ernestine A. Mahar
  20. Michelle Y.H. Lee
  21. Joyce Cohen
  22. Sherrie M. Jean
  23. Jennifer S. Wood
  24. Fawn Connor-Stroud
  25. Rachelle L. Stammen
  26. Olivia M. Delmas
  27. Shelly Wang
  28. Kimberly A. Cooney
  29. Michael N. Sayegh
  30. Lanfang Wang
  31. Peter D. Filev
  32. Daniela Weiskopf
  33. Guido Silvestri
  34. Jesse Waggoner
  35. Anne Piantadosi
  36. Sudhir P. Kasturi
  37. Hilmi Al-Shakhshir
  38. Susan P. Ribeiro
  39. Rafick P. Sekaly
  40. Rebecca D. Levit
  41. Jacob D. Estes
  42. Thomas H. Vanderford
  43. Raymond F. Schinazi
  44. Steven E. Bosinger
  45. Mirko Paiardini

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Abstract

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  1. Version published to 10.1016/j.cell.2020.11.007
  2. ScreenIT

    SciScore for 10.1101/2020.09.16.300277: (What is this?)

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

    Table 1: Rigor

    Institutional Review Board StatementIACUC: All animal experimentation was reviewed and approved by Emory University’s Institutional Animal Care and Use Committee (IACUC) under permit PROTO202000035.
    Randomizationnot detected.
    BlindingThe regularized log expression values were obtained using the rlog function with the parameters blind =FALSE and filtType = “parametric”.
    Power Analysisnot detected.
    Sex as a biological variablenot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    The slides were cooled, rinsed twice in distilled water and 1X TBS with 0.05% Tween-20 (TBS-T), blocked (TBS-T + 0.25% casein) for 30 minutes at room temperature, then incubated at room temperature with antibodies against IL-6 (Sigma; Cat. No. SAB4301665 at 1:250 for 1 hour), Mx1 (EMD; Cat. No. MABF938 at 1:1000 for 1 hour), MPO (Dako; Cat. No. A0398 at 1:1000 for 1 hour), pSTAT3 (Cell Signaling; Cat. No. 9145L at 1:200 for 2 hours), Iba-1 (BioCare; Cat. No. CP290A at 1:500 for 1 hour) and pJAK2 (abcam; Cat. No. ab32101 1:150 for 1 hour)
    IL-6
    suggested: (Leinco Technologies Cat# I-150, RRID:AB_2830544)
    Mx1
    suggested: None
    pSTAT3
    suggested: (Fluidigm Cat# 3158005, RRID:AB_2661827)
    Immunophenotyping was performed using anti-human monoclonal antibodies (mAbs), which we (Harper et al., 2020; McGary et al., 2017; Micci et al., 2015; Pino et al., 2019) and others, including databases maintained by the NHP Reagent Resource (MassBiologics), have shown as being cross-reactive in RMs.
    anti-human monoclonal antibodies ( mAbs)
    suggested: None
    Experimental Models: Cell Lines
    SentencesResources
    Vero cells were cultured and maintained in MEM (Sigma) supplemented with 10% heat inactivated fetal bovine serum (FBS) (Gibco) and 1 mM L-glutamine (Gibco), 50 U/ml penicillin and 50 μg/ml streptomycin (Gibco).
    Vero
    suggested: None
    SARS-CoV-2 (NR-52281: BEI Resources, Manassas, VA; USA-WA/2020, Lot no. 70033175) was passaged on Vero E6 cells at a MOI of 0.01 to produce the infectious viral stock.
    Vero E6
    suggested: RRID:CVCL_XD71)
    Software and Algorithms
    SentencesResources
    Data were collected and processed by Thermo Xcalibur 3.0 software.
    Thermo Xcalibur
    suggested: (Thermo Xcalibur, RRID:SCR_014593)
    The sections were incubated with Thyroid Transcription Factor-1 (Clone 8G7G3/1) for overnight at 4°C followed by a detection polymer system (MACH 2™; Biocare Medical)
    MACH
    suggested: (MACH, RRID:SCR_009621)
    RNA quality was assessed using an Agilent Bioanalyzer and total RNA was used as input for cDNA synthesis using the Clontech SMART-Seq v4 Ultra Low Input RNA kit (Takara Bio) according to the manufacturer’s instructions.
    Agilent Bioanalyzer
    suggested: None
    Bulk RNA-Seq analysis: The quality of reads was evaluated using FastQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/).
    FastQC
    suggested: (FastQC, RRID:SCR_014583)
    The combined genomic and gtf files were used for generating the STAR index.
    STAR
    suggested: (STAR, RRID:SCR_015899)
    The VennDiagram R library was used to create the venn diagrams.
    VennDiagram
    suggested: (VennDiagram, RRID:SCR_002414)
    GSEA 4.1.0 (https://www.gsea-msigdb.org/) was used for gene set enrichment analysis with the following gene sets: Hallmark and Canonical pathways (MsigDB), NHP ISGs (Sandler et al.) and Rheumatoid arthritis (KEGG map05323).
    KEGG
    suggested: (KEGG, RRID:SCR_012773)
    GSEA was run with default parameters with the permutation type set to gene_set.
    GSEA
    suggested: (SeqGSEA, RRID:SCR_005724)
    The input for GSEA was the regularized log expression values obtained from DESeq2 which was filtered to remove genes with mean expression <=0.
    DESeq2
    suggested: (DESeq, RRID:SCR_000154)
    Differential gene expression between the clusters was assessed by MAST (Finak et al., 2015).
    MAST
    suggested: (MAST, RRID:SCR_016340)
    Heatmaps, Dot plots and Violin plots were generated using ggplot2 (Wickham, 2016) package in R.
    ggplot2
    suggested: (ggplot2, RRID:SCR_014601)
    A panel of the following mAbs was used for the longitudinal phenotyping of innate immune cells in whole blood (500 μL), as described in (Kuri-Cervantes et al., 2020), and mononuclear cells (106 cells) derived from LN biopsies, BAL, and lung: anti-CD20-BB700 (clone 2H7; 2.5 μL; cat. # 745889), anti-Ki-67-BV480 (clone B56; 5 μL; cat. # 566109), anti-CD14-BV605 (clone M5E2; 2.5 μL; cat. # 564054), anti-CD56-BV711 (clone B159; 2.5 μL; cat. # 740781), anti-CD115-BV750 (clone 9-4D2-1E4; 2.5 μL; cat. # 747093), anti-CD3-BUV395 (clone SP34-2; 2.5 μL; cat. # 564117), anti-CD8-BUV496 (clone RPA-T8; 2.5 μL; cat. # 612942), anti-CD45-BUV563 (clone D058-1283; 2.5 μL; cat. # 741414), anti-CCR2-BUV661 (clone LS132.1D9; 2.5 μL; cat. # 750472), anti-CD16-UV737 (clone 3G8; 2.5 μL; cat. # 564434), anti-CD69-BUV805 (clone FN50; 2.5 μL; cat. # 748763), and Fixable Viability Stain 700 (2 μL; cat. # 564997) all from BD Biosciences; anti-CD38-FITC (clone AT1; 2.5 μL; cat. # 60131FI) from STEMCELL Technologies; anti-CD161-BV421 (clone HP-3G10; 5 μL; cat. # 339914), anti-HLA-DR-BV650 (clone L243; 5 μL; cat. # 307650), anti-CD11c-BV785 (clone 3.9; 5 μL; cat. # 301644), anti-CD11b-PE (clone ICRF44; 2.5 μL; cat. # 301306), and anti-CD123-APC-Fire750 (clone 315; 2.5 μL; cat. # 306042) all from Biolegend; anti-GranzymeB-PE-TexasRed (clone GB11; 2.5 μL; cat. # GRB17) from Thermo Fisher; anti-CD66abce-PE-Vio770 (clone TET2; 1 μL; cat. # 130-119-849) from Miltenyi Biotec; and anti-CD27-PE-Cy5 (clone 1A4CD27; 2.5 μL; cat. # 6607107) and anti-NKG2A-APC (clone Z199; 5 μL; cat. # A60797) from Beckman Coulter (Supplementary Fig. 4).
    BD Biosciences
    suggested: (BD Biosciences, RRID:SCR_013311)
    Acquisition of data was performed on a FACSymphony A5 (BD Biosciences) driven by FACS DiVa software and analyzed with FlowJo (version 10.7; Becton, Dickinson, and Company).
    FlowJo
    suggested: (FlowJo, RRID:SCR_008520)
    We used the Phenograph clustering approach (https://github.com/jacoblevine/PhenoGraph) Determination of intracellular cytokine induction following SARS-CoV-2 S peptide pool and PMA/Ionomycin stimulation: Cryo-preserved PBMCs were thawed, resuspended in RPMI medium supplemented to contain a final concentration of 10% Fetal Bovine Serum (FBS) (Corning Life Sciences/Media Tech Inc, Manassas, VA)
    Phenograph
    suggested: (Phenograph, RRID:SCR_016919)
    Panel 1: anti-CD3 BUV395 (clone SP34-2; 2.5 μL; cat. # 564117), anti-CD8-BUV496 (clone RPA-T8; 2.5 μL; cat. # 612942), and Fixable Viability Stain 700 (2 μL; cat. # 564997) all from BD Bioscience; anti-CD4 APC/Cy7 (clone OKT4; 2.5 μL; cat. # 317418) from Biolegend; To detect intracellular expression of cytokines, mononuclear cells were fixed and permeabilized with a Cytofix/Cytoperm kit (BD Biosciences) and stained as follows: anti-IL-21 BV421 (clone 3A3-N2.1; 5 μL; cat# 564755) from BD Bioscience; anti-IL-2 BV650 (clone MQ1-17H12; 5 μL; cat# 500334) and anti-IFNγ PE/Dazzle 594 (clone B27; 5 μL; cat# 506530) both from Biolegend; anti-IL-17a Alexa Fluor 488 (clone eBio64DEC17; 5 μL; cat# 53-7179-42)
    BD Bioscience
    suggested: (BD Biosciences, RRID:SCR_013311)
    Biolegend
    suggested: (BioLegend, RRID:SCR_001134)
    anti-IL-22 APC (clone IL22JOP; 5 μL; cat# 17-7222-82), and anti-TNFα PE-Cyanine7 (clone Mab11; 0.5 μL; cat# 25-7349-82) all from Thermo Fisher Scientific; and anti-IL-4 PE (clone 7A3-3; 5 μL; cat# 130-091-647) from Miltenyl Biotech.
    Thermo Fisher Scientific
    suggested: (Thermo Fisher Scientific, RRID:SCR_008452)
    Analyses, unless otherwise noted, were performed with Prism version 8 (GraphPad).
    Prism
    suggested: (PRISM, RRID:SCR_005375)
    GraphPad
    suggested: (GraphPad Prism, RRID:SCR_002798)
    Data tables for expression counts for bulk and single-cell RNA-Seq for BAL are deposited in NCBI’s Gene Expression Omnibus and are accessible through GEO accession #####
    Gene Expression Omnibus
    suggested: (Gene Expression Omnibus (GEO, RRID:SCR_005012)

    Results from OddPub: Thank you for sharing your code.

    Results from LimitationRecognizer: We detected the following sentences addressing limitations in the study:
    Being performed in an animal model, this study has some key advantages and some important limitations. Advantages include the ability to correct for parameters that may impact clinical outcome and treatment readout, including using the same virus inoculum, dose, and route of infection, and starting baricitinib at the same phase of infection in all subjects. Furthermore, the NHP model permits longitudinal collection of BAL and lung at necropsy, which in turn allows the in-depth characterization of the impact of baricitinib on immune activation and immunologic responses, including single-cell RNA sequencing analyses of macrophages, at the foci of infection. The main limitation of the study is the small group size, with a total of eight RMs; furthermore, being that SARS-CoV-2 infected RMs is a model of moderate COVID-19 (at least in animals between 11-17 years old as in our study) and with treatment initiated early after infection, we cannot determine if the therapeutic impact of baricitinib will be the same in severe COVID-19 patients and when started later than 2 days post-infection. Mitigating that concern, in our study, treatment started at peak viremia, including in BAL, and once inflammatory signatures were already highly up-regulated, mimicking the conditions in which baricitinib would be administered clinically. Indeed, a recent small clinical trial in 20 severe COVID-19 patients indicated that baricitinib mitigated immune dysregulation by reducing plasma levels of IL6, ...

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

    IdentifierStatusTitle
    NCT04401579CompletedAdaptive COVID-19 Treatment Trial 2 (ACTT-2)
    NCT04421027RecruitingA Study of Baricitinib (LY3009104) in Participants With COVI…

    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:
    • 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.

    About SciScore

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  3. Version published to 10.1101/2020.09.16.300277v1 on bioRxiv