Plasma Gradient of Soluble Urokinase-Type Plasminogen Activator Receptor Is Linked to Pathogenic Plasma Proteome and Immune Transcriptome and Stratifies Outcomes in Severe COVID-19

  1. Jafar Sarif
  2. Deblina Raychaudhuri
  3. Ranit D’Rozario
  4. Purbita Bandopadhyay
  5. Praveen Singh
  6. Priyanka Mehta
  7. Md. Asmaul Hoque
  8. Bishnu Prasad Sinha
  9. Manoj Kushwaha
  10. Shweta Sahni
  11. Priti Devi
  12. Partha Chattopadhyay
  13. Shekhar Ranjan Paul
  14. Yogiraj Ray
  15. Kausik Chaudhuri
  16. Sayantan Banerjee
  17. Debajyoti Majumdar
  18. Bibhuti Saha
  19. Biswanath Sharma Sarkar
  20. Prasun Bhattacharya
  21. Shilpak Chatterjee
  22. Sandip Paul
  23. Pramit Ghosh
  24. Rajesh Pandey
  25. Shantanu Sengupta
  26. Dipyaman Ganguly

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Abstract

Disease caused by SARS-CoV-2 coronavirus (COVID-19) led to significant morbidity and mortality worldwide. A systemic hyper-inflammation characterizes severe COVID-19 disease, often associated with acute respiratory distress syndrome (ARDS). Blood biomarkers capable of risk stratification are of great importance in effective triage and critical care of severe COVID-19 patients. Flow cytometry and next-generation sequencing were done on peripheral blood cells and urokinase-type plasminogen activator receptor (suPAR), and cytokines were measured from and mass spectrometry-based proteomics was done on plasma samples from an Indian cohort of COVID-19 patients. Publicly available single-cell RNA sequencing data were analyzed for validation of primary data. Statistical analyses were performed to validate risk stratification. We report here higher plasma abundance of suPAR, expressed by an abnormally expanded myeloid cell population, in severe COVID-19 patients with ARDS. The plasma suPAR level was found to be linked to a characteristic plasma proteome, associated with coagulation disorders and complement activation. Receiver operator characteristic curve analysis to predict mortality identified a cutoff value of suPAR at 1,996.809 pg/ml (odds ratio: 2.9286, 95% confidence interval 1.0427–8.2257). Lower-than-cutoff suPAR levels were associated with a differential expression of the immune transcriptome as well as favorable clinical outcomes, in terms of both survival benefit (hazard ratio: 0.3615, 95% confidence interval 0.1433–0.912) and faster disease remission in our patient cohort. Thus, we identified suPAR as a key pathogenic circulating molecule linking systemic hyperinflammation to the hypercoagulable state and stratifying clinical outcomes in severe COVID-19 patients with ARDS.

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  1. Version published to 10.3389/fimmu.2021.738093
  2. ScreenIT

    SciScore for 10.1101/2021.06.19.21259125: (What is this?)

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

    Table 1: Rigor

    NIH rigor criteria are not applicable to paper type.

    Table 2: Resources

    Software and Algorithms
    SentencesResources
    The stained cells were acquired in a FACS ARIA III flow cytometer and data were analyzed on FlowJo™ software.
    FlowJo™
    suggested: (FlowJo, RRID:SCR_008520)
    We analysed the sequencing data from all 3 GEO Datasets using the Seurat R package version 4.0 [30].
    Seurat
    suggested: (SEURAT, RRID:SCR_007322)
    wiff format files generated in DDA mode against UniProtKB human FASTA database (Swissprot and TrEMBL; 74,255 entries) using Proteinpilot™Software 5.0.1 (SCIEX).
    UniProtKB
    suggested: (UniProtKB, RRID:SCR_004426)
    Proteomics data are submitted to the PRIDE database [33].
    PRIDE
    suggested: (Pride-asap, RRID:SCR_012052)
    The final library was quantified using Qubit™ dsDNA High Sensitivity Assay Kit (Catalog number: Q32851) and the library size was determined using Agilent High Sensitivity DNA Kit (Catalog number: 5067-4626) on Agilent Bioanalyzer 2100 platform.
    Agilent Bioanalyzer
    suggested: None
    The mapping-based mode of Salmon was used for quantification [35].
    Salmon
    suggested: (Salmon, RRID:SCR_017036)
    The reference transcriptome (Ensembl GRCh38, release 103) was used for indexing and quantification of the individual genes.
    Ensembl
    suggested: (Ensembl, RRID:SCR_002344)
    The Limma tool [37] was used to find out the differentially expressed genes (DEGs) between the 3 different groups having 3 patients each, categorised according to the concentration of sUPAR in their plasma as described in the figure legend.
    Limma
    suggested: (LIMMA, RRID:SCR_010943)
    The list of DEGs (transcripts) (p<=0.05) provided by Limma was divided into two groups-(1) upregulated genes (having all differentially expressed transcripts upregulated) and (2) downregulated genes (having all differentially expressed transcripts downregulated) before being entered into the online NetworkAnalyst software [38] to obtain the list of enriched pathways (p<=0.05) from the Reactome database, separately for upregulated and downregulated genes.
    NetworkAnalyst
    suggested: (NetworkAnalyst, RRID:SCR_016909)
    Reactome
    suggested: (Reactome, RRID:SCR_003485)
    Statistics: All statistical analyses, as depicted in the results as well in appropriate figures and their legends, were performed using Graphpad Prism 8 or in some cases using R.
    Graphpad Prism
    suggested: (GraphPad Prism, RRID:SCR_002798)

    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:
    • 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.

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  3. Version published to 10.1101/2021.06.19.21259125v1 on medRxiv