Neutrophil extracellular traps contribute to immunothrombosis in COVID-19 acute respiratory distress syndrome

Elizabeth A. Middleton; Xue Yan He; Frederik Denorme; Robert A. Campbell; David Ng; Steven P. Salvatore; Maria Mostyka; Amelia Baxter-Stoltzfus; Alain C. Borczuk; Massimo Loda; Mark J. Cody; Bhanu Kanth Manne; Irina Portier; Estelle S. Harris; Aaron C. Petrey; Ellen J. Beswick; Aleah F. Caulin; Anthony Iovino; Lisa M. Abegglen; Andrew S. Weyrich; Matthew T. Rondina; Mikala Egeblad; Joshua D. Schiffman; Christian Con Yost

doi:10.1182/blood.2020007008

Neutrophil extracellular traps contribute to immunothrombosis in COVID-19 acute respiratory distress syndrome

Elizabeth A. Middleton, Xue Yan He, Frederik Denorme, Robert A. Campbell, David Ng, Steven P. Salvatore, Maria Mostyka, Amelia Baxter-Stoltzfus, Alain C. Borczuk, Massimo Loda, Mark J. Cody, Bhanu Kanth Manne, Irina Portier, Estelle S. Harris, Aaron C. Petrey, Ellen J. Beswick, Aleah F. Caulin, Anthony Iovino, Lisa M. Abegglen, Andrew S. WeyrichMatthew T. Rondina, Mikala Egeblad, Joshua D. Schiffman, Christian Con Yost

Internal Medicine

Research output: Contribution to journal › Article › peer-review

856 Scopus citations

Abstract

COVID-19 affects millions of patients worldwide, with clinical presentation ranging from isolated thrombosis to acute respiratory distress syndrome (ARDS) requiring ventilator support. Neutrophil extracellular traps (NETs) originate from decondensed chromatin released to immobilize pathogens, and they can trigger immunothrombosis. We studied the connection between NETs and COVID-19 severity and progression. We conducted a prospective cohort study of COVID-19 patients (n 5 33) and age- and sex-matched controls (n 5 17). We measured plasma myeloperoxidase (MPO)-DNA complexes (NETs), platelet factor 4, RANTES, and selected cytokines. Three COVID-19 lung autopsies were examined for NETs and platelet involvement. We assessed NET formation ex vivo in COVID-19 neutrophils and in healthy neutrophils incubated with COVID-19 plasma. We also tested the ability of neonatal NET-inhibitory factor (nNIF) to block NET formation induced by COVID-19 plasma. Plasma MPO-DNA complexes increased in COVID-19, with intubation (P < .0001) and death (P < .0005) as outcome. Illness severity correlated directly with plasma MPO-DNA complexes (P 5 .0360), whereas PaO₂/fraction of inspired oxygen correlated inversely (P 5 .0340). Soluble and cellular factors triggering NETs were significantly increased in COVID-19, and pulmonary autopsies confirmed NET-containing microthrombi with neutrophil-platelet infiltration. Finally, COVID-19 neutrophils ex vivo displayed excessive NETs at baseline, and COVID-19 plasma triggered NET formation, which was blocked by nNIF. Thus, NETs triggering immunothrombosis may, in part, explain the prothrombotic clinical presentations in COVID-19, and NETs may represent targets for therapeutic intervention.

Original language	English
Pages (from-to)	1169-1179
Number of pages	11
Journal	Blood
Volume	136
Issue number	10
DOIs	https://doi.org/10.1182/blood.2020007008
State	Published - Sep 3 2020

Bibliographical note

Funding Information:
Conflict-of-interest disclosure: C.C.Y. has received grant funding from PEEL Therapeutics, Inc. during the conduct of this study. In addition, C.C.Y. authored a United States patent (patent no. 10 232023 B2) held by the University of Utah for the use of NET-inhibitory peptides for the “treatment of and prophylaxis against inflammatory disorders,” for which PEEL Therapeutics, Inc. holds the exclusive license. A.I. and L.M.A. are consultants and stock option holders of PEEL Therapeutics, Inc., and A.F.C. and J.D.S. are employees and stock option holders of PEEL Therapeutics, Inc. The remaining authors declare no competing financial interests.

Funding Information:
This work was supported by grants from the National Institutes of Health (NIH)/National Institute of Children’s Health and Development (R01 HD093826 [C.C.Y.]), NIH/National Cancer Institute (5P30CA045508-30 [M.E.]), NIH/National Heart, Lung, and Blood Institute (R01 HL135265 [A.C.P.]; R35 HL145237, R01 HL142804NIA, and R61HL141783 [A.S.W.]), and NIH/National Institute on Aging (K01 AG059892 [R.A.C.]); the University of Utah Triple I Program (E.A.M.); Fonds voor Weten-schappelijk Onderzoek Vlaanderen FWO 12U7818N (F.D.); Animal Cancer Foundation (J.D.S.); Soccer For Hope Foundation (J.D.S.); Closer To Cure Foundation (J.D.S.); and PEEL Therapeutics, Inc (C.C.Y.). This work was also supported, in part, by Merit Review Award I01 CX001696 (M.T.R.) from the US Department of Veterans Affairs Clinical Sciences R&D Service. M.E., X.-Y.H., and D.N. were supported by the William C. and Joyce C. O’Neill Charitable Trust and the Linartz-Meier Family Foundation. This article is the result of work supported with resources and the use of facilities at the George E. Wahlen VA Medical Center.

Publisher Copyright:
© 2020 American Society of Hematology. All rights reserved.

ASJC Scopus subject areas

Biochemistry
Immunology
Hematology
Cell Biology

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10.1182/blood.2020007008

Cite this

Middleton, E. A., He, X. Y., Denorme, F., Campbell, R. A., Ng, D., Salvatore, S. P., Mostyka, M., Baxter-Stoltzfus, A., Borczuk, A. C., Loda, M., Cody, M. J., Manne, B. K., Portier, I., Harris, E. S., Petrey, A. C., Beswick, E. J., Caulin, A. F., Iovino, A., Abegglen, L. M., ... Yost, C. C. (2020). Neutrophil extracellular traps contribute to immunothrombosis in COVID-19 acute respiratory distress syndrome. Blood, 136(10), 1169-1179. https://doi.org/10.1182/blood.2020007008

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title = "Neutrophil extracellular traps contribute to immunothrombosis in COVID-19 acute respiratory distress syndrome",

abstract = "COVID-19 affects millions of patients worldwide, with clinical presentation ranging from isolated thrombosis to acute respiratory distress syndrome (ARDS) requiring ventilator support. Neutrophil extracellular traps (NETs) originate from decondensed chromatin released to immobilize pathogens, and they can trigger immunothrombosis. We studied the connection between NETs and COVID-19 severity and progression. We conducted a prospective cohort study of COVID-19 patients (n 5 33) and age- and sex-matched controls (n 5 17). We measured plasma myeloperoxidase (MPO)-DNA complexes (NETs), platelet factor 4, RANTES, and selected cytokines. Three COVID-19 lung autopsies were examined for NETs and platelet involvement. We assessed NET formation ex vivo in COVID-19 neutrophils and in healthy neutrophils incubated with COVID-19 plasma. We also tested the ability of neonatal NET-inhibitory factor (nNIF) to block NET formation induced by COVID-19 plasma. Plasma MPO-DNA complexes increased in COVID-19, with intubation (P < .0001) and death (P < .0005) as outcome. Illness severity correlated directly with plasma MPO-DNA complexes (P 5 .0360), whereas PaO2/fraction of inspired oxygen correlated inversely (P 5 .0340). Soluble and cellular factors triggering NETs were significantly increased in COVID-19, and pulmonary autopsies confirmed NET-containing microthrombi with neutrophil-platelet infiltration. Finally, COVID-19 neutrophils ex vivo displayed excessive NETs at baseline, and COVID-19 plasma triggered NET formation, which was blocked by nNIF. Thus, NETs triggering immunothrombosis may, in part, explain the prothrombotic clinical presentations in COVID-19, and NETs may represent targets for therapeutic intervention.",

author = "Middleton, {Elizabeth A.} and He, {Xue Yan} and Frederik Denorme and Campbell, {Robert A.} and David Ng and Salvatore, {Steven P.} and Maria Mostyka and Amelia Baxter-Stoltzfus and Borczuk, {Alain C.} and Massimo Loda and Cody, {Mark J.} and Manne, {Bhanu Kanth} and Irina Portier and Harris, {Estelle S.} and Petrey, {Aaron C.} and Beswick, {Ellen J.} and Caulin, {Aleah F.} and Anthony Iovino and Abegglen, {Lisa M.} and Weyrich, {Andrew S.} and Rondina, {Matthew T.} and Mikala Egeblad and Schiffman, {Joshua D.} and Yost, {Christian Con}",

note = "Funding Information: Conflict-of-interest disclosure: C.C.Y. has received grant funding from PEEL Therapeutics, Inc. during the conduct of this study. In addition, C.C.Y. authored a United States patent (patent no. 10 232023 B2) held by the University of Utah for the use of NET-inhibitory peptides for the “treatment of and prophylaxis against inflammatory disorders,” for which PEEL Therapeutics, Inc. holds the exclusive license. A.I. and L.M.A. are consultants and stock option holders of PEEL Therapeutics, Inc., and A.F.C. and J.D.S. are employees and stock option holders of PEEL Therapeutics, Inc. The remaining authors declare no competing financial interests. Funding Information: This work was supported by grants from the National Institutes of Health (NIH)/National Institute of Children{\textquoteright}s Health and Development (R01 HD093826 [C.C.Y.]), NIH/National Cancer Institute (5P30CA045508-30 [M.E.]), NIH/National Heart, Lung, and Blood Institute (R01 HL135265 [A.C.P.]; R35 HL145237, R01 HL142804NIA, and R61HL141783 [A.S.W.]), and NIH/National Institute on Aging (K01 AG059892 [R.A.C.]); the University of Utah Triple I Program (E.A.M.); Fonds voor Weten-schappelijk Onderzoek Vlaanderen FWO 12U7818N (F.D.); Animal Cancer Foundation (J.D.S.); Soccer For Hope Foundation (J.D.S.); Closer To Cure Foundation (J.D.S.); and PEEL Therapeutics, Inc (C.C.Y.). This work was also supported, in part, by Merit Review Award I01 CX001696 (M.T.R.) from the US Department of Veterans Affairs Clinical Sciences R&D Service. M.E., X.-Y.H., and D.N. were supported by the William C. and Joyce C. O{\textquoteright}Neill Charitable Trust and the Linartz-Meier Family Foundation. This article is the result of work supported with resources and the use of facilities at the George E. Wahlen VA Medical Center. Publisher Copyright: {\textcopyright} 2020 American Society of Hematology. All rights reserved.",

year = "2020",

month = sep,

day = "3",

doi = "10.1182/blood.2020007008",

language = "English",

volume = "136",

pages = "1169--1179",

number = "10",

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Middleton, EA, He, XY, Denorme, F, Campbell, RA, Ng, D, Salvatore, SP, Mostyka, M, Baxter-Stoltzfus, A, Borczuk, AC, Loda, M, Cody, MJ, Manne, BK, Portier, I, Harris, ES, Petrey, AC, Beswick, EJ, Caulin, AF, Iovino, A, Abegglen, LM, Weyrich, AS, Rondina, MT, Egeblad, M, Schiffman, JD & Yost, CC 2020, 'Neutrophil extracellular traps contribute to immunothrombosis in COVID-19 acute respiratory distress syndrome', Blood, vol. 136, no. 10, pp. 1169-1179. https://doi.org/10.1182/blood.2020007008

TY - JOUR

T1 - Neutrophil extracellular traps contribute to immunothrombosis in COVID-19 acute respiratory distress syndrome

AU - Middleton, Elizabeth A.

AU - He, Xue Yan

AU - Denorme, Frederik

AU - Campbell, Robert A.

AU - Ng, David

AU - Salvatore, Steven P.

AU - Mostyka, Maria

AU - Baxter-Stoltzfus, Amelia

AU - Borczuk, Alain C.

AU - Loda, Massimo

AU - Cody, Mark J.

AU - Manne, Bhanu Kanth

AU - Portier, Irina

AU - Harris, Estelle S.

AU - Petrey, Aaron C.

AU - Beswick, Ellen J.

AU - Caulin, Aleah F.

AU - Iovino, Anthony

AU - Abegglen, Lisa M.

AU - Weyrich, Andrew S.

AU - Rondina, Matthew T.

AU - Egeblad, Mikala

AU - Schiffman, Joshua D.

AU - Yost, Christian Con

N1 - Funding Information: Conflict-of-interest disclosure: C.C.Y. has received grant funding from PEEL Therapeutics, Inc. during the conduct of this study. In addition, C.C.Y. authored a United States patent (patent no. 10 232023 B2) held by the University of Utah for the use of NET-inhibitory peptides for the “treatment of and prophylaxis against inflammatory disorders,” for which PEEL Therapeutics, Inc. holds the exclusive license. A.I. and L.M.A. are consultants and stock option holders of PEEL Therapeutics, Inc., and A.F.C. and J.D.S. are employees and stock option holders of PEEL Therapeutics, Inc. The remaining authors declare no competing financial interests. Funding Information: This work was supported by grants from the National Institutes of Health (NIH)/National Institute of Children’s Health and Development (R01 HD093826 [C.C.Y.]), NIH/National Cancer Institute (5P30CA045508-30 [M.E.]), NIH/National Heart, Lung, and Blood Institute (R01 HL135265 [A.C.P.]; R35 HL145237, R01 HL142804NIA, and R61HL141783 [A.S.W.]), and NIH/National Institute on Aging (K01 AG059892 [R.A.C.]); the University of Utah Triple I Program (E.A.M.); Fonds voor Weten-schappelijk Onderzoek Vlaanderen FWO 12U7818N (F.D.); Animal Cancer Foundation (J.D.S.); Soccer For Hope Foundation (J.D.S.); Closer To Cure Foundation (J.D.S.); and PEEL Therapeutics, Inc (C.C.Y.). This work was also supported, in part, by Merit Review Award I01 CX001696 (M.T.R.) from the US Department of Veterans Affairs Clinical Sciences R&D Service. M.E., X.-Y.H., and D.N. were supported by the William C. and Joyce C. O’Neill Charitable Trust and the Linartz-Meier Family Foundation. This article is the result of work supported with resources and the use of facilities at the George E. Wahlen VA Medical Center. Publisher Copyright: © 2020 American Society of Hematology. All rights reserved.

PY - 2020/9/3

Y1 - 2020/9/3

N2 - COVID-19 affects millions of patients worldwide, with clinical presentation ranging from isolated thrombosis to acute respiratory distress syndrome (ARDS) requiring ventilator support. Neutrophil extracellular traps (NETs) originate from decondensed chromatin released to immobilize pathogens, and they can trigger immunothrombosis. We studied the connection between NETs and COVID-19 severity and progression. We conducted a prospective cohort study of COVID-19 patients (n 5 33) and age- and sex-matched controls (n 5 17). We measured plasma myeloperoxidase (MPO)-DNA complexes (NETs), platelet factor 4, RANTES, and selected cytokines. Three COVID-19 lung autopsies were examined for NETs and platelet involvement. We assessed NET formation ex vivo in COVID-19 neutrophils and in healthy neutrophils incubated with COVID-19 plasma. We also tested the ability of neonatal NET-inhibitory factor (nNIF) to block NET formation induced by COVID-19 plasma. Plasma MPO-DNA complexes increased in COVID-19, with intubation (P < .0001) and death (P < .0005) as outcome. Illness severity correlated directly with plasma MPO-DNA complexes (P 5 .0360), whereas PaO2/fraction of inspired oxygen correlated inversely (P 5 .0340). Soluble and cellular factors triggering NETs were significantly increased in COVID-19, and pulmonary autopsies confirmed NET-containing microthrombi with neutrophil-platelet infiltration. Finally, COVID-19 neutrophils ex vivo displayed excessive NETs at baseline, and COVID-19 plasma triggered NET formation, which was blocked by nNIF. Thus, NETs triggering immunothrombosis may, in part, explain the prothrombotic clinical presentations in COVID-19, and NETs may represent targets for therapeutic intervention.

AB - COVID-19 affects millions of patients worldwide, with clinical presentation ranging from isolated thrombosis to acute respiratory distress syndrome (ARDS) requiring ventilator support. Neutrophil extracellular traps (NETs) originate from decondensed chromatin released to immobilize pathogens, and they can trigger immunothrombosis. We studied the connection between NETs and COVID-19 severity and progression. We conducted a prospective cohort study of COVID-19 patients (n 5 33) and age- and sex-matched controls (n 5 17). We measured plasma myeloperoxidase (MPO)-DNA complexes (NETs), platelet factor 4, RANTES, and selected cytokines. Three COVID-19 lung autopsies were examined for NETs and platelet involvement. We assessed NET formation ex vivo in COVID-19 neutrophils and in healthy neutrophils incubated with COVID-19 plasma. We also tested the ability of neonatal NET-inhibitory factor (nNIF) to block NET formation induced by COVID-19 plasma. Plasma MPO-DNA complexes increased in COVID-19, with intubation (P < .0001) and death (P < .0005) as outcome. Illness severity correlated directly with plasma MPO-DNA complexes (P 5 .0360), whereas PaO2/fraction of inspired oxygen correlated inversely (P 5 .0340). Soluble and cellular factors triggering NETs were significantly increased in COVID-19, and pulmonary autopsies confirmed NET-containing microthrombi with neutrophil-platelet infiltration. Finally, COVID-19 neutrophils ex vivo displayed excessive NETs at baseline, and COVID-19 plasma triggered NET formation, which was blocked by nNIF. Thus, NETs triggering immunothrombosis may, in part, explain the prothrombotic clinical presentations in COVID-19, and NETs may represent targets for therapeutic intervention.

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U2 - 10.1182/blood.2020007008

DO - 10.1182/blood.2020007008

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EP - 1179

JO - Blood

JF - Blood

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ER -

Neutrophil extracellular traps contribute to immunothrombosis in COVID-19 acute respiratory distress syndrome

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