Whole blood coagulation in an ex vivo thrombus is sufficient to induce clot neutrophils to adopt a myeloid-derived suppressor cell signature and shed soluble Lox-1

Julia Leonard; David Kepplinger; Virginia Espina; Pat Gillevet; Yunbo Ke; Konstantin G Birukov; Allan Doctor; Caroline D Hoemann

doi:10.1016/j.jtha.2023.12.014

Whole blood coagulation in an ex vivo thrombus is sufficient to induce clot neutrophils to adopt a myeloid-derived suppressor cell signature and shed soluble Lox-1

J Thromb Haemost. 2024 Apr;22(4):1031-1045. doi: 10.1016/j.jtha.2023.12.014. Epub 2023 Dec 20.

Authors

Julia Leonard¹, David Kepplinger², Virginia Espina³, Pat Gillevet⁴, Yunbo Ke⁵, Konstantin G Birukov⁵, Allan Doctor⁶, Caroline D Hoemann⁷

Affiliations

¹ Department of Bioengineering, Institute of Biomedical Engineering, George Mason University, Manassas, Virginia, USA.
² Department of Statistics, George Mason University, Fairfax, Virginia, USA.
³ Department of Systems Biology, George Mason University, Fairfax, Virginia, USA.
⁴ Department of Biology, George Mason University, Fairfax, Virginia, USA.
⁵ Department of Anesthesiology, School of Medicine, University of Maryland at Baltimore, Baltimore, Maryland, USA.
⁶ Departments of Pediatrics & Bioengineering and Center for Blood Oxygen Transport and Hemostasis, School of Medicine, University of Maryland at Baltimore, Baltimore, Maryland, USA.
⁷ Department of Bioengineering, Institute of Biomedical Engineering, George Mason University, Manassas, Virginia, USA. Electronic address: choemann@gmu.edu.

Abstract

Background: Blood clots are living tissues that release inflammatory mediators including IL-8/CXCL8 and MCP-1/CCL2. A deeper understanding of blood clots is needed to develop new therapies for prothrombotic disease states and regenerative medicine.

Objectives: To identify a common transcriptional shift in cultured blood clot leukocytes.

Methods: Differential gene expression of whole blood and cultured clots (4 hours at 37 °C) was assessed by RNA sequencing (RNAseq), reverse transcriptase-polymerase chain reaction, proteomics, and histology (23 diverse healthy human donors). Cultured clot serum bioactivity was tested in endothelial barrier functional assays.

Results: All cultured clots developed a polymorphonuclear myeloid-derived suppressor cell (PMN-MDSC) signature, including up-regulation of OLR1 (mRNA encoding lectin-like oxidized low-density lipoprotein receptor 1 [Lox-1]), IL-8/CXCL8, CXCL2, CCL2, IL10, IL1A, SPP1, TREM1, and DUSP4/MKP. Lipopolysaccharide enhanced PMN-MDSC gene expression and specifically induced a type II interferon response with IL-6 production. Lox-1 was specifically expressed by cultured clot CD15⁺ neutrophils. Cultured clot neutrophils, but not activated platelets, shed copious amounts of soluble Lox-1 (sLox-1) with a donor-dependent amplitude. sLox-1 shedding was enhanced by phorbol ester and suppressed by heparin and by beta-glycerol phosphate, a phosphatase inhibitor. Cultured clot serum significantly enhanced endothelial cell monolayer barrier function, consistent with a proresolving bioactivity.

Conclusion: This study suggests that PMN-MDSC activation is part of the innate immune response to coagulation which may have a protective role in inflammation. The cultured blood clot is an innovative thrombus model that can be used to study both sterile and nonsterile inflammatory states and could be used as a personalized medicine tool for drug screening.

Keywords: RNA; blood clot; immunity; innate; myeloid-derived suppressor cells; polymorphonuclear neutrophils; sequence analysis.

MeSH terms

Blood Coagulation / physiology
Humans
Interleukin-8
Myeloid-Derived Suppressor Cells* / pathology
Neutrophils
Thrombosis* / pathology

Substances

Interleukin-8

Abstract

MeSH terms

Substances

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