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.
OBJECTIVE: 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.
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.
OBJECTIVE: 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.
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.
摘要：
背景：血凝块是释放炎症介质（包括IL-8/CXCL8和MCP-1/CCL2）的活组织。需要对血凝块有更深入的了解，以开发血栓前疾病状态和再生医学的新疗法。
目的：确定培养的血凝块白细胞中常见的转录变化。
方法：通过RNA测序（RNAseq）评估全血和培养凝块（4h37°C）的差异基因表达，RT-PCR,蛋白质组学，和组织学（23个不同的健康人类供体）。在内皮屏障功能测定中测试培养的凝块血清生物活性。
结果：所有培养的凝块都形成了多形核髓样抑制细胞（PMN-MDSC）特征，包括OLR1（编码凝集素样氧化低密度脂蛋白受体1，Lox-1的mRNA）的上调，IL-8/CXCL8、CXCL2、CCL2、IL10、IL1A、SPP1、TREM1和DUSP4/MKP。脂多糖增强了PMN-MDSC基因表达，并特异性诱导了II型干扰素反应，产生IL-6。Lox-1由培养的凝块CD15+嗜中性粒细胞特异性表达。培养的凝块中性粒细胞，但不是活化的血小板,脱落大量可溶性Lox-1（sLox-1），具有供体依赖性振幅。sLox-1脱落被佛波醇酯增强，被肝素和β-甘油磷酸酯抑制，磷酸酶抑制剂.培养的血块血清显着增强内皮细胞单层屏障功能，符合促分解的生物活性。
结论：这项研究表明，PMN-MDSC激活是对凝血的先天免疫反应的一部分，可能在炎症中具有保护作用。培养的血凝块是一种创新的血栓模型，可用于研究无菌和非无菌炎症状态，并可用作药物筛选的个性化医学工具。
目的：确定培养的血凝块白细胞中常见的转录变化。
方法：通过RNA测序（RNAseq）评估全血和培养凝块（4h37°C）的差异基因表达，RT-PCR,蛋白质组学，和组织学（23个不同的健康人类供体）。在内皮屏障功能测定中测试培养的凝块血清生物活性。
结果：所有培养的凝块都形成了多形核髓样抑制细胞（PMN-MDSC）特征，包括OLR1（编码凝集素样氧化低密度脂蛋白受体1，Lox-1的mRNA）的上调，IL-8/CXCL8、CXCL2、CCL2、IL10、IL1A、SPP1、TREM1和DUSP4/MKP。脂多糖增强了PMN-MDSC基因表达，并特异性诱导了II型干扰素反应，产生IL-6。Lox-1由培养的凝块CD15+嗜中性粒细胞特异性表达。培养的凝块中性粒细胞，但不是活化的血小板,脱落大量可溶性Lox-1（sLox-1），具有供体依赖性振幅。sLox-1脱落被佛波醇酯增强，被肝素和β-甘油磷酸酯抑制，磷酸酶抑制剂.培养的血块血清显着增强内皮细胞单层屏障功能，符合促分解的生物活性。
结论：这项研究表明，PMN-MDSC激活是对凝血的先天免疫反应的一部分，可能在炎症中具有保护作用。培养的血凝块是一种创新的血栓模型，可用于研究无菌和非无菌炎症状态，并可用作药物筛选的个性化医学工具。
