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.

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 pro-thrombotic disease states and regenerative medicine.

OBJECTIVES: Identify a common transcriptional shift in cultured blood clot leukocytes.

METHODS: Differential gene expression of whole blood and cultured clots (4h 37°C) was assessed by RNA sequencing (RNAseq), RT-PCR, 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 upregulation 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 pro-resolving bioactivity.

CONCLUSIONS: 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.