Caspase-4/11 exacerbates disease severity in SARS–CoV-2 infection by promoting inflammation and immunothrombosis

  1. Mostafa M. Eltobgy
  2. Ashley Zani
  3. Adam D. Kenney
  4. Shady Estfanous
  5. Eunsoo Kim
  6. Asmaa Badr
  7. Cierra Carafice
  8. Kylene Daily
  9. Owen Whitham
  10. Maciej Pietrzak
  11. Amy Webb
  12. Jeffrey Kawahara
  13. Adrian C. Eddy
  14. Parker Denz
  15. Mijia Lu
  16. Mahesh KC
  17. Mark E. Peeples
  18. Jianrong Li
  19. Jian Zhu
  20. Jianwen Que
  21. Richard Robinson
  22. Oscar Rosas Mejia
  23. Rachael E. Rayner
  24. Luanne Hall-Stoodley
  25. Stephanie Seveau
  26. Mikhail A. Gavrilin
  27. Xiaoli Zhang
  28. Jeronay Thomas
  29. Jacob E. Kohlmeier
  30. Mehul S. Suthar
  31. Eugene Oltz
  32. Andrea Tedeschi
  33. Frank H. Robledo-Avila
  34. Santiago Partida-Sanchez
  35. Emily A. Hemann
  36. Eman Abdelrazik
  37. Adriana Forero
  38. Shahid M. Nimjee
  39. Prosper N. Boyaka
  40. Estelle Cormet-Boyaka
  41. Jacob S. Yount
  42. Amal O. Amer

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Abstract

We report the discovery of fundamental roles for the noncanonical inflammasome molecule Caspase-4/11 in promoting pathological inflammatory and prothrombotic pathways in severe acute respiratory syndrome coronavirus 2 (SARS–CoV-2) infections. Our work demonstrates that Caspase-11 has a broader role in immune responses beyond its previously appreciated effects in bacterial infections. Further, we show that Caspase-11–deficient mice infected with SARS–CoV-2 fare significantly better in terms of overall illness, lung inflammation, and thrombosis than wild-type (WT) mice, thus implicating Caspase-11 as a new therapeutic target for preventing or treating COVID-19.

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  1. Version published to 10.1073/pnas.2202012119
  2. ScreenIT

    SciScore for 10.1101/2021.09.24.461743: (What is this?)

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

    Table 1: Rigor

    EthicsIACUC: All procedures were approved by the OSU BSL3 Operations/Advisory Group, the OSU Institutional Biosafety Officer, and the OSU Institutional Biosafety Committee.
    Sex as a biological variablenot detected.
    Randomizationnot detected.
    Blindingnot detected.
    Power Analysisnot detected.
    Cell Line Authenticationnot detected.

    Table 2: Resources

    Antibodies
    SentencesResources
    Membranes were incubated overnight with antibodies against CASP11 (Cell Signaling Technology, 14340), VWF (
    antibodies against CASP11 ( Cell Signaling Technology , 14340) , VWF
    suggested: None
    Sections were then transferred to blocking solution containing the primary antibody against IL1β (GeneTex, GTX74034), and incubated overnight at 4ºC.
    IL1β
    suggested: None
    GTX74034
    suggested: None
    For IHC, Ly6G (Abcam, ab25377) and SARS-CoV-2 nucleocapsid protein (GeneTex, GTX635686) primary antibodies were used.
    Ly6G
    suggested: (Abcam Cat# ab25377, RRID:AB_470492)
    SARS-CoV-2 nucleocapsid protein ( GeneTex , GTX635686
    suggested: None
    The tissues were first incubated with peroxide block buffer (Leica Microsystems), followed by incubation with the rabbit Caspase 4 antibody (Novus Bio NBP1-87681) at 1:700 dilution for 30mins, followed by DAB rabbit secondary reagents: polymer, DAB refine and hematoxylin (Leica Microsystems).
    rabbit Caspase 4
    suggested: (Novus Cat# NBP1-87681, RRID:AB_11012097)
    Experimental Models: Cell Lines
    SentencesResources
    Non-mutated clones were propagated on Vero E6 cells stably expressing TMPRSS2 (provided by Dr. Shan-Lu Liu, The Ohio State University)
    Vero E6
    suggested: RRID:CVCL_XD71)
    Experimental Models: Organisms/Strains
    SentencesResources
    Mice: C57BL/6 wild-type (WT) mice were obtained from the Jackson Laboratory (Bar Harbor, ME, USA).
    C57BL/6
    suggested: None
    Casp11-/- mice were generously given by Dr.
    Casp11-/-
    suggested: None
    Gsdmd-/- mice were a gift from Dr. Thirumala-Devi Kanneganti at St. Jude Children’s Research Hospital, Memphis, TN, USA.
    Gsdmd-/-
    suggested: None
    K18-hACE2 mice40 were purchased from Jackson Laboratories.
    K18-hACE2
    suggested: RRID:IMSR_GPT:T037657)
    Software and Algorithms
    SentencesResources
    Densitometry analyses were performed by normalizing target protein bands to their respective loading control (β-Actin) using ImageJ software as previously described 19,42. ELISAs: Cytokine/chemoking ELISAs were performed on lung homogenates or macrophage supernatants using R&D Systems Duoset ELISA kits (IL-6, DY406;
    ImageJ
    suggested: (ImageJ, RRID:SCR_003070)
    Finally, Opal dyes (Opal 520 and 570 were used, Fisher Scientific; cat# NC1601877 and cat#NC601878) was then applied, 520 (Fisher Scientific; cat#NC1601877) diluted in RNAscope TSA buffer (Advanced Cell Diagnostics, cat# 322809) for 30 min at 40 C.
    Fisher Scientific
    suggested: (Thermo Fisher Scientific, RRID:SCR_008452)
    Flt1 mRNA expression was quantified using spot function in IMARIS.
    IMARIS
    suggested: (Imaris, RRID:SCR_007370)
    Briefly, raw RNAseq data (fastq) were aligned to mouse reference genome (GRCh38) using hisat2 (v2.1.0) 47 and converted to counts using the ‘subread’ package (v1.5.1) 48 in R.
    hisat2
    suggested: (HISAT2, RRID:SCR_015530)
    For data visualization, DESeq2 rlog transformation was used for principal component analysis (PCA).
    DESeq2
    suggested: (DESeq, RRID:SCR_000154)
    Functional enrichment performed with Ingenuity Pathway Analysis (Qiagen) to enrich for IPA Canonical Pathways, ‘clusterProfiler’ to generate enrichment maps 46, and EnrichR 50.
    Ingenuity Pathway Analysis
    suggested: (Ingenuity Pathway Analysis, RRID:SCR_008653)
    EnrichR
    suggested: (Enrichr, RRID:SCR_001575)

    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 found bar graphs of continuous data. We recommend replacing bar graphs with more informative graphics, as many different datasets can lead to the same bar graph. The actual data may suggest different conclusions from the summary statistics. For more information, please see Weissgerber et al (2015).

    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.

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

    About SciScore

    SciScore is an automated tool that is designed to assist expert reviewers by finding and presenting formulaic information scattered throughout a paper in a standard, easy to digest format. SciScore checks for the presence and correctness of RRIDs (research resource identifiers), and for rigor criteria such as sex and investigator blinding. For details on the theoretical underpinning of rigor criteria and the tools shown here, including references cited, please follow this link.

  3. Version published to 10.1101/2021.09.24.461743v1 on bioRxiv