Histone H3 Cleavage in Severe COVID-19 ICU Patients

Joram Huckriede, Femke de Vries, Michael Hultström, Kanin Wichapong, Chris Reutelingsperger, Miklos Lipcsey, Pablo Garcia de Frutos, Robert Frithiof, Gerry A F Nicolaes, Joram Huckriede, Femke de Vries, Michael Hultström, Kanin Wichapong, Chris Reutelingsperger, Miklos Lipcsey, Pablo Garcia de Frutos, Robert Frithiof, Gerry A F Nicolaes

Abstract

The severity of coronavirus disease 19 (COVID-19) is associated with neutrophil extracellular trap (NET) formation. During NET formation, cytotoxic extracellular histones are released, the presence of which is linked to the initiation and progression of several acute inflammatory diseases. Here we study the presence and evolution of extracellular histone H3 and several other neutrophil-related molecules and damage-associated molecular patterns (DAMPs) in the plasma of 117 COVID-19-positive ICU patients. We demonstrate that at ICU admission the levels of histone H3, MPO, and DNA-MPO complex were all significantly increased in COVID-19-positive patients compared to control samples. Furthermore, in a subset of 54 patients, the levels of each marker remained increased after 4+ days compared to admission. Histone H3 was found in 28% of the patients on admission to the ICU and in 50% of the patients during their stay at the ICU. Notably, in 47% of histone-positive patients, we observed proteolysis of histone in their plasma. The overall presence of histone H3 during ICU stay was associated with thromboembolic events and secondary infection, and non-cleaved histone H3 was associated with the need for vasoactive treatment, invasive ventilation, and the development of acute kidney injury. Our data support the validity of treatments that aim to reduce NET formation and additionally underscore that more targeted therapies focused on the neutralization of histones should be considered as treatment options for severe COVID-19 patients.

Keywords: COVID-19; DAMPs (damage-associated molecular patterns); ICU - intensive care unit; NETosis; extracellular histones.

Conflict of interest statement

CR, RF and GN are scientific advisor at Matisse Pharmaceuticals B.V. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Copyright © 2021 Huckriede, de Vries, Hultström, Wichapong, Reutelingsperger, Lipcsey, Garcia de Frutos, Frithiof and Nicolaes.

Figures

Figure 1
Figure 1
Histone H3 and NET components increased in COVID-19 ICU patients on admission (A–C), are increased in COVID-19 histone H3 positive patients (D–G) and subsequent stay (H–L) at the ICU department. Plasma from COVID-19 (n=117), non-COVID-19 ICU patients (n=11), and healthy control (n=15) was tested for the presence, represented as median (IQR) or box and whisker plot, of histone H3 (A), myeloperoxidase (MPO) (B), and the MPO-DNA complex (C). COVID-19 samples were compared to control groups with the Kruskal-Wallis test with Dunn’s post-hoc test. The level of cfDNA (D), NE (E), MPO (F), MPO-DNA (G) in the COVID-19 histone H3 positive and negative groups. Samples were compared by Mann-Whitney U test. The level of histone H3 (H), cfDNA (I), NE (J), MPO (K), and MPO-DNA (L) of a subset of COVID-19 patients were followed in time on early (1-3) and late (≥ 4) days at the ICU department. Samples Early-Late were compared by Wilcoxon matched-pairs signed-rank test. P-values were considered significant if p < 0.05; *0.05, **0.01, ***0.001.
Figure 2
Figure 2
Histone H3 cleavage in COVID-19 patients. The fraction of patients with detectable cleaved or non-cleaved histone H3 (A). Western blot analysis using a polyclonal anti-histone H3 antibody allowed detection of proteolytic cleavage of histone H3, distinguishing between full-sized histone H3 (15 kDa) and a histone H3 fragment (12 kDa) (B). The concentration of histone H3 (C) or NE (D) in cleaved or non-cleaved histone H3 positive samples. The percentage (%) for the positive amount of significantly different clinical events during the stay at the ICU (E). Acute kidney injury (AKI). P-values were considered significant if p < 0.05; *0.05, **0.01, ***0.001.

References

    1. Abrams S. T., Zhang N., Manson J., Liu T., Dart C., Baluwa F., et al. . (2013). Circulating Histones are Mediators of Trauma-Associated Lung Injury. Am. J. Respir. Crit. Care Med. 187 (2), 160–169. doi: 10.1164/rccm.201206-1037OC
    1. Barnes B. J., Adrover J. M., Baxter-Stoltzfus A., Borczuk A., Cools-Lartigue J., Crawford J. M., et al. . (2020). Targeting Potential Drivers of COVID-19: Neutrophil Extracellular Traps. J. Exp. Med. 217 (6), e20200652. doi: 10.1084/jem.20200652
    1. Breitbach S., Tug S., Helmig S., Zahn D., Kubiak T., Michal M., et al. . (2014). Direct Quantification of Cell-Free, Circulating DNA From Unpurified Plasma. PLoS One 9 (3), e87838. doi: 10.1371/journal.pone.0087838
    1. Brinkmann V., Reichard U., Goosmann C., Fauler B., Uhlemann Y. (2004). Neutrophil Extracellular Traps Kill Bacteria. Science 303 (5663), 1532–1535. doi: 10.1126/science.1092385
    1. Busch M. H., Timmermans S., Nagy M., Visser M., Huckriede J., Aendekerk J. P., et al. . (2020). Neutrophils and Contact Activation of Coagulation as Potential Drivers of COVID-19. Circulation 142 (18), 1787–1790. doi: 10.1161/CIRCULATIONAHA.120.050656
    1. Cheng Z., Abrams S. T., Alhamdi Y., Toh J., Yu W., Wang G., et al. . (2019). Circulating Histones Are Major Mediators of Multiple Organ Dysfunction Syndrome in Acute Critical Illnesses. Crit. Care Med. 47 (8), e677–ee84. doi: 10.1097/CCM.0000000000003839
    1. Chen R., Kang R., Fan X. G., Tang D. (2014). Release and Activity of Histone in Diseases. Cell Death Dis. 5, e1370. doi: 10.1038/cddis.2014.337
    1. Cicco S., Cicco G., Racanelli V., Vacca A. (2020). Neutrophil Extracellular Traps (NETs) and Damage-Associated Molecular Patterns (DAMPs): Two Potential Targets for COVID-19 Treatment. Mediat. Inflamm. 1 (2), e7527953. doi: 10.1155/2020/7527953
    1. Hoeksema M., van Eijk M., Haagsman H. P., Hartshorn K. L. (2016). Histones as Mediators of Host Defense, Inflammation and Thrombosis. Future Microbiol. 11 (3), 441–453. doi: 10.2217/fmb.15.151
    1. Huckriede J., Anderberg S. B., Morales A., de Vries F., Hultström M., Bergqvist A., et al. . (2021). Evolution of NETosis Markers and DAMPs Have Prognostic Value in Critically Ill COVID-19 Patients. Sci. Rep. 11 (1), e15701. doi: 10.1038/s41598-021-95209-x
    1. Kano H., Aminul Huq M., Tsuda M., Noguchi H., Takeyama N. (2017). Sandwich ELISA for Circulating Myeloperoxidase- and Neutrophil Elastase-DNA Complexes Released From Neutrophil Extracellular Traps. Adv. Tech. Biol. Med. 05 (01), e1000196. doi: 10.4172/2379-1764.1000196
    1. Leest P. V., Boonstra P. A., Elst A. T., Kempen L. C. V., Tibbesma M., Koopmans J., et al. . (2020). Comparison of Circulating Cell-Free DNA Extraction Methods for Downstream Analysis in Cancer Patients. Cancers (Basel) 12 (5), e1222. doi: 10.3390/cancers12051222
    1. Middleton E. A., He X.-Y., Denorme F., Campbell R. A., Ng D., Salvatore S., et al. . (2020). Neutrophil Extracellular Traps Contribute to Immunothrombosis in COVID-19 Acute Respiratory Distress Syndrome. Blood 136 (10), 1169 –11 79. doi: 10.1182/blood.2020007008
    1. Moreno R. P., Metnitz P. G., Almeida E., Jordan B., Bauer P., Campos R. A., et al. . (2005). SAPS 3–From Evaluation of the Patient to Evaluation of the Intensive Care Unit. Part 2: Development of a Prognostic Model for Hospital Mortality at ICU Admission. Intensive Care Med. 31 (10), 1345–1355. doi: 10.1007/s00134-005-2763-5
    1. Ng H., Havervall S., Rosell A., Aguilera K., Parv K., von Meijenfeldt F. A., et al. . (2020). Circulating Markers of Neutrophil Extracellular Traps Are of Prognostic Value in Patients With COVID-19. Arterioscler. Thromb. Vasc. Biol. 41(2):988–994. doi: 10.1161/ATVBAHA.120.315267
    1. Papayannopoulos V., Metzler K. D., Hakkim A., Zychlinsky A. (2010). Neutrophil Elastase and Myeloperoxidase Regulate the Formation of Neutrophil Extracellular Traps. J. Cell Biol. 191 (3), 677–691. doi: 10.1083/jcb.201006052
    1. Schumski A., Ortega-Gomez A., Wichapong K., Winter C., Lemnitzer P., Viola J. R., et al. . (2021). Endotoxinemia Accelerates Atherosclerosis Through Electrostatic Charge-Mediated Monocyte Adhesion. Circulation 143 (3), 254–266. doi: 10.1161/CIRCULATIONAHA.120.046677
    1. Silk E., Zhao H., Weng H., Ma D. (2017). The Role of Extracellular Histone in Organ Injury. Cell Death Dis. 8 (5), e2812. doi: 10.1038/cddis.2017.52
    1. Silvestre-Roig C., Braster Q., Wichapong K., Lee E. Y., Teulon J. M., Berrebeh N., et al. . (2019). Externalized Histone H4 Orchestrates Chronic Inflammation by Inducing Lytic Cell Death. Nature 569 (7755), 236–240. doi: 10.1038/s41586-019-1167-6
    1. Veras F. P., Pontelli M. C., Silva C. M., Toller-Kawahisa J. E., de Lima M., Nascimento D. C., et al. . (2020). SARS-CoV-2-Triggered Neutrophil Extracellular Traps Mediate COVID-19 Pathology. J. Exp. Med. 217 (12), e20201129. doi: 10.1084/jem.20201129
    1. Vincent J. L., Moreno R., Takala J., Willatts S., De Mendonca A., Bruining H., et al. . (1996). The SOFA (Sepsis-Related Organ Failure Assessment) Score to Describe Organ Dysfunction/Failure. On Behalf of the Working Group on Sepsis-Related Problems of the European Society of Intensive Care Medicine. Intensive Care Med. 22 (7), 707–710. doi: 10.1007/BF01709751
    1. Voicu S., Ketfi C., Stepanian A., Chousterman B. G., Mohamedi N., Siguret V., et al. . (2020). Pathophysiological Processes Underlying the High Prevalence of Deep Vein Thrombosis in Critically Ill COVID-19 Patients. Front. Physiol. 11, e608788. doi: 10.3389/fphys.2020.608788
    1. Wang J., Jiang M., Chen X., Montaner L. J. (2020). Cytokine Storm and Leukocyte Changes in Mild Versus Severe SARS-CoV-2 Infection: Review of 3939 COVID-19 Patients in China and Emerging Pathogenesis and Therapy Concepts. J. Leukoc. Biol. 108 (1), 17–41. doi: 10.1002/JLB.3COVR0520-272R
    1. World Health Organization . (2021). Weekly Operations Update on COVID-19. [Accessed March 10, 2021].
    1. Wildhagen K. C., Garcia de Frutos P., Reutelingsperger C. P., Schrijver R., Areste C., Ortega-Gomez A., et al. . (2014). Nonanticoagulant Heparin Prevents Histone-Mediated Cytotoxicity In Vitro and Improves Survival in Sepsis. Blood 123 (7), 1098–1101. doi: 10.1182/blood-2013-07-514984
    1. Wildhagen K. C., Wiewel M. A., Schultz M. J., Horn J., Schrijver R., Reutelingsperger C. P., et al. . (2015). Extracellular Histone H3 Levels are Inversely Correlated With Antithrombin Levels and Platelet Counts and are Associated With Mortality in Sepsis Patients. Thromb. Res. 136 (3), 542–547. doi: 10.1016/j.thromres.2015.06.035
    1. Xu J., Zhang X., Pelayo R., Monestier M., Ammollo C. T., Semeraro F., et al. . (2009). Extracellular Histones are Major Mediators of Death in Sepsis. Nat. Med. 15 (11), 1318–1321. doi: 10.1038/nm.2053
    1. Zhou F., Yu T., Du R., Fan G., Liu Y., Liu Z., et al. . (2020). Clinical Course and Risk Factors for Mortality of Adult Inpatients With COVID-19 in Wuhan, China: A Retrospective Cohort Study. Lancet 395 (10229), 1054–1062. doi: 10.1016/S0140-6736(20)30566-3
    1. Zuo Y., Yalavarthi S., Shi H., Gockman K., Zuo M., Madison J. A., et al. . (2020). Neutrophil Extracellular Traps in COVID-19. JCI Insight 5 (11), e138999. doi: 10.1172/jci.insight.138999

Source: PubMed

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