Treatment of human polymorphonuclear neutrophils with platelet-derived mitochondria or damage-associated molecular patterns

Inflammation is an essential process of the host defense against infections, illness, or tissue injuries. However, unregulated inflammation has been associated with chronic inflammatory autoimmune diseases, such as rheumatoid arthritis, multiple sclerosis, and atherosclerosis. Polymorphonuclear neutrophils (PMNL) are amongst the first immune cells involved in the acute inflammatory response used to fight bacterial infections. Once activated, PMNL releases inflammatory mediators, enzymes and large quantities of microparticles in the extracellular milieu to recruit various immune cells required to fight the invading pathogens. Recent evidence also shows that platelets (PLTs), well known for their coagulation proprieties, are also implicated in the body's inflammatory response. Interestingly, activated PLTs can release fully functional mitochondria in the extracellular milieu. Known as the powerhouse of the cell, the mitochondria share similar characteristics with bacteria. Therefore, we hypothesize that PLTs-derived mitochondria present in the extracellular milieu, acting in a similar way as bacteria, induce a sterile inflammatory response that involves the PMNL. The objective of this study was to investigate the sterile inflammatory response of PMNL caused by the exposure to PLTs-derived extracellular mitochondria. Blood was obtained from healthy consenting donors, then PMNL and PLTs-derived mitochondria were isolated and purified. Following the co-incubation of PMNL with various physiological doses of PLTs-derived mitochondria, a characterization of the interaction between PMNL and PLTs-derived mitochondria and an investigation of the inflammatory proprieties of PMNL were performed using flow cytometry, transmission electron microscopy, and high-resolution respirometry. While the transcriptomic analysis indicated that several mitochondrial genes had increased expression, no mitochondrial-dependent increase in oxygen consumption was observed by high-resolution respirometry experiments. However, our data show that PLTs-derived mitochondria significantly induce, in a dose-dependent manner, the release of PMNL microparticles. This study provides new insight into the role of PLTs-derived mitochondria in the context of sterile inflammation. Overall design: PMNLs were freshly isolated and treated with either platelet-derived mitochondria or DAMPs. Controls were untreated resting PMNLs. RNA was subsequently extracted and subjected to rRNA-depletion, library preperation and RNA-sequencing.

炎症是宿主对抗感染、疾病或组织损伤的核心防御过程。然而，失控的炎症与慢性炎症性自身免疫疾病密切相关，例如类风湿关节炎（rheumatoid arthritis）、多发性硬化（multiple sclerosis）及动脉粥样硬化（atherosclerosis）。

多形核中性粒细胞（Polymorphonuclear neutrophils，PMNL）是参与对抗细菌感染的急性炎症应答的首批免疫细胞之一。一旦被激活，PMNL会向细胞外微环境释放炎症介质、酶类及大量微粒，以招募抵御入侵病原体所需的各类免疫细胞。

近期研究还表明，以凝血特性闻名的血小板（platelets，PLTs）也参与机体的炎症应答过程。有趣的是，激活的PLTs可向细胞外微环境释放功能完整的线粒体。线粒体被称为细胞的“能量工厂”，其生物学特征与细菌相似。因此，本研究提出假说：存在于细胞外微环境中的血小板源性线粒体，可通过类似细菌的方式，诱导涉及PMNL的无菌性炎症应答。

本研究的目的是探究PMNL暴露于血小板源性细胞外线粒体后引发的无菌性炎症应答。研究采集了知情同意的健康志愿者的血液，随后分离并纯化了PMNL与血小板源性线粒体。将PMNL与不同生理剂量的血小板源性线粒体共孵育后，本研究通过流式细胞术（flow cytometry）、透射电子显微镜（transmission electron microscopy）与高分辨率呼吸测定法（high-resolution respirometry），对PMNL与血小板源性线粒体的相互作用进行了表征，并对PMNL的炎症特性展开了探究。

转录组分析显示，多个线粒体基因的表达水平上调，但高分辨率呼吸测定实验未观察到线粒体依赖性的氧消耗增加。然而，本研究数据表明，血小板源性线粒体可通过剂量依赖性方式显著诱导PMNL微粒的释放。本研究为理解血小板源性线粒体在无菌性炎症中的作用提供了新的视角。

整体实验设计：将新鲜分离的PMNL分别用血小板源性线粒体或损伤相关分子模式（damage-associated molecular patterns，DAMPs）处理，以未处理的静息PMNL作为对照组。随后提取RNA，进行核糖体RNA去除、文库制备及RNA测序。