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Letters to the Editor
tones demonstrated that rising histone levels were asso- ciated with mortality (odds ratio =1.031 (95% CI=1.013- 1.049, P=0.001). Using categorical data where patients were stratified based on a ≥30 μg/mL threshold,3,7 similar results were obtained (Figure 1C, OR=4.875 (95% CI=1.879-12.649, P=0.001), demonstrating that patients with high circulating histone levels on admission had a higher risk of mortality. Subsequent multivariate analysis demonstrated that histones were independently associat- ed with mortality after adjustment for age, gender, eth- nicity and co-morbidity when histone levels were treated as either continuous (odds ratio=1.032; 95% CI=1.013- 1.051, P=0.001) or categorical variables (odds ratio=5.404; 95% CI=1.852-15.770, P=0.002). ROC curve analysis shows an area under the curve [AUC] of 0.708 (95% CI=0.589-0.827, P=0.002). A Kaplan-Meier survival curve demonstrated a significant increase in the probabil- ity of mortality during the 28-day period in patients with histones ≥30 μg/mL (Figure 1D, P<0.001).
Coagulopathy has emerged as a key feature of severe COVID-19 and has been linked to increased mortality.13 It has been documented that extracellular histones, released following cell death, are drivers of coagulation by activating platelets,7 generating thrombin2 and damag- ing endothelial cells8 to induce coagulopathy in critical ill- ness.3 This is the first report to demonstrate high levels of circulating histones in SARS-CoV-2 infection, with levels strongly associated with coagulopathy. This suggests their involvement in thrombosis in severe cases.14
High levels of circulating histones reflect the extent of cellular death, such as lymphopenia or NETosis,15 which may be a major source of circulating histones in COVID-19. Histone release following cell death triggers IL-6 release to induce an acute-phase response.8 We found that circulating histone levels significantly correlat- ed with IL-6 and acute-phase protein levels, including fib- rinogen and CRP, indicating histone-induced acute phase response in patients with COVID-19.
Extracellular histones disrupt cell membranes through phospholipid binding to induce cytotoxic effects on cells, including endothelial cells8 and cardiomyocytes.12 This study demonstrates circulating histones associated with cardiac injury, which is frequently observed in severe COVID-19 and associated with poor outcomes.5 Therefore, the cytotoxic and pro-coagulant properties of circulating histones may be an underlying molecular mechanism contributing to disease severity and poor out- comes (Figure 1E).
In conclusion, this is the first report to quantify high levels of circulating histones in viral infection and demonstrate that extracellular histones play a central role in the development of immunothrombosis and critical ill- ness in COVID-19.
Rebecca J. Shaw,1,2* Simon T. Abrams,1* James Austin,1* Joseph M. Taylor,3 Steven Lane,4 Tina Dutt,2 Colin Downey,3 Min Du,1 Lance Turtle,1,5 J. Kenneth Baillie,6,7 Peter J.M. Openshaw,8 Guozheng Wang,1 Malcolm G. Semple1,9# and Cheng-Hock Toh1,2# on behalf of the ISARIC4C investigators
*RJS, STA and JA contributed equally as co-first authors. #MGS and C-HT contributed equally as-senior authors.
1Department of Clinical Infection, Microbiology and Immunology, University of Liverpool, Liverpool; 2Roald Dahl Haemostasis and Thrombosis Centre, Liverpool University Hospitals NHS Foundation Trust, Liverpool; 3Liverpool Clinical Laboratories, Liverpool University Hospitals NHS Foundation Trust, Liverpool; 4Department of Biostatistics, University of Liverpool, Liverpool; 5Infectious Diseases Unit, Royal Liverpool University Hospital, Liverpool; 6Roslin Institute,
University of Edinburgh, Edinburgh; 7Intensive Care Unit, Royal Infirmary Edinburgh, Edinburgh; 8National Heart and Lung Institute, Imperial College London, London and 9Respiratory Medicine, Alder Hey Children’s Hospital NHS Foundation Trust, Liverpool, UK
Correspondence:
CHENG-HOCK TOH - toh@liverpool.ac.uk doi:10.3324/haematol.2021.278492
Received: February 5, 2021.
Accepted: April 1, 2021.
Pre-published: April 8, 2021.
Disclosures: all authors have completed and submitted the ICMJE Form for Disclosure of Potential Conflicts of Interest and none were reported.
Contributions: RJS, JT, TD, CD and STA collected and interpreted the clinical data and performed data analysis. RJS, STA and SL per- formed statistical analysis. JA, STA, RJS and MD measured the levels of circulating histones. RJS, STA, TD, GW and CHT wrote, edited and reviewed the manuscript and figures. PJMO, JKB, LT and MS edited and reviewed the manuscript. STA, GW and CHT designed and supervised the work. STA and CHT had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. STA, GW, CHT, PJMO, JKB, and MS obtained funding for this work.
Acknowledgments: this work uses data provided by patients and col- lected by the NHS as part of their care and support #DataSavesLives. We are extremely grateful to the 2,648 frontline NHS clinical and research staff and volunteer medical students, who collected this data in challenging circumstances; and the generosity of the participants and their families for their individual contributions in these difficult times.
We also acknowledge the support of Jeremy J Farrar and Nahoko Shindo.
Funding: this work was funded by University of Liverpool COVID-19 strategic funding, the British Heart Foundation [PG/16/65/32313], Bayer AG (Germany) and the Royal Liverpool & Broadgreen University Hospitals NHS Trust. It was funded in whole, or in part, by the Wellcome Trust [205228/Z/16/Z]. This research is supported by grants from: the National Institute for Health Research (NIHR) [award CO-CIN-01]; the Medical Research Council [grant MC_PC_19059] and by the NIHR Health Protection Research Unit (HPRU) in Emerging and Zoonotic Infections at University of Liverpool, in partnership with Public Health England (PHE), in collaboration with the Liverpool School of Tropical Medicine and the University of Oxford [award 200907]; NIHR HPRU in Respiratory Infections at Imperial College London with Public Health England (PHE) [award 200927]; the Wellcome Trust and Department for International Development [215091/Z/18/Z]; the Bill and Melinda Gates Foundation [OPP1209135]; the Liverpool Experimental Cancer Medicine Centre (Grant Reference: C18616/A25153); the NIHR Biomedical Research Centre at Imperial College London [IS-BRC- 1215-20013]; the EU Platform foR European Preparedness Against (Re-) emerging Epidemics (PREPARE) [FP7 project 602525] and NIHR Clinical Research Network for providing infrastructure support for this research. PJMO is supported by an NIHR Senior Investigator Award [award 201385]. The views expressed are those of the authors and not necessarily those of the DHSC, DID, NIHR, MRC, Wellcome Trust or PHE. The sponsors had no role in the design and conduct of the study; collection, management, analysis, and interpreta- tion of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication.
Data-sharing: this work uses data provided by patients and collected by the NHS as part of their care and support #DataSavesLives. The CO-CIN data was collated by ISARIC4C Investigators. ISARIC4C welcomes applications for data and material access through our Independent Data and Material Access Committee (https://isaric4c.net).
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