Macrophage Microvesicles Induce Macrophage Differentiation and miR-223 Transfer

Microvesicles (MV) are small membrane-bound particles comprised of exosomes and various sized extracellular vesicles. These are released by a number of cell types. Microvesicles have a variety of cellular functions from communication to mediating growth and differentiation. Microvesicles contain proteins and nucleic acids. Previously, we showed that plasma microvesicles contain microRNAs (miRNAs). Based on our previous report, the majority of peripheral blood microvesicles are derived from platelets while mononuclear phagocytes, including macrophages, are the second most abundant population. Here, we characterized macrophage-derived microvesicles and whether they influenced the differentiation of naïve monocytes. We also identified the miRNA content of the macrophage-derived microvesicles. We found that RNA molecules contained in the macrophage-derived microvesicles were transported to target cells, including monocytes, endothelial cells, epithelial cells and fibroblasts. Furthermore, we found that miR-223 was transported to target cells and was functionally active. Based on our observations, we hypothesize that microvesicles bind to and activate target cells. Furthermore, we find that microvesicles induce the differentiation of macrophages. Thus, defining key components of this response may identify novel targets to regulate host defense and inflammation. We used GeneChip microarrays to examine changes in gene expression induced by MV in primary monocyte-derived macrophages (MDM) and in THP1 cells, and compare this to cells treated with GM-CSF and PMA, respectively. All experiments were done in triplicates. Primary monocytes were collected from buffy coats (BC). The freshly isolated monocytes from three donors (Mono1-3) were either treated with GM-CSF or subjected to RNA isolation. Following treatment, MVs were isolated from the GM-CSF-treated macrophage cultures. RNA was isolated from the remaining cells for profiling (GM1-3). The isolated MVs were then used to treat new BC monocytes for 24 h (BC-GMCSF-MV24). A fraction of the new BC monocytes was subjected to RNA extraction for profiling (BC1-3). For THP1 cells, they were treated with either DMSO or PMA to produce MVs. The MVs were collected and the remaining cells lyzed for RNA extraction and profiling (DMSO1-3 and PMA1-3). The collected MVs from the DMSO or PMA-treated THP1 cells were incubated with new THP1 for 24 h and designated DMSO-MV24 or PMA-MV24. We had a total of 24 samples.

微囊泡（Microvesicles, MV）是一类由外泌体与不同尺寸细胞外囊泡共同组成的小型膜结合颗粒，可由多种细胞类型释放。微囊泡具备多种细胞功能，涵盖细胞通讯、介导生长与分化等过程。微囊泡内含蛋白质与核酸。此前本团队已证实，血浆微囊泡含有微小RNA（microRNAs, miRNAs）。基于既往研究结果，外周血中的大部分微囊泡源自血小板，而包括巨噬细胞在内的单核吞噬细胞则是第二大丰度群体。本研究对巨噬细胞源性微囊泡进行了表征，并探究其是否可影响初始单核细胞的分化；同时鉴定了巨噬细胞源性微囊泡的miRNA组分。研究发现，巨噬细胞源性微囊泡所携带的RNA分子可被转运至靶细胞，包括单核细胞、内皮细胞、上皮细胞与成纤维细胞。此外，本研究证实miR-223可被转运至靶细胞并发挥功能性活性。基于上述观察结果，本团队提出假说：微囊泡可结合并激活靶细胞。进一步研究发现，微囊泡可诱导巨噬细胞分化。因此，解析该应答过程的关键组分，有望发现调控宿主防御与炎症反应的新型靶点。本研究使用基因芯片微阵列（GeneChip microarrays），检测了MV对原代单核细胞源性巨噬细胞（monocyte-derived macrophages, MDM）与THP-1细胞的基因表达诱导变化，并分别与粒细胞-巨噬细胞集落刺激因子（GM-CSF）及佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA）处理的细胞进行对照。所有实验均设置三次生物学重复。研究人员从白膜层（buffy coats, BC）中分离原代单核细胞。将来自3名供体的新鲜分离单核细胞（标记为Mono1-3）分别用GM-CSF处理，或进行RNA提取。经GM-CSF处理后，从巨噬细胞培养物中分离MV，并从剩余细胞中提取RNA用于表达谱分析（标记为GM1-3）。将分离得到的MV用于处理新分离的BC来源单核细胞，培养24小时后标记为BC-GMCSF-MV24。取部分新分离的BC来源单核细胞进行RNA提取用于表达谱分析（标记为BC1-3）。对于THP-1细胞，分别用二甲基亚砜（DMSO）或PMA处理以诱导MV产生。收集MV后，对剩余细胞进行裂解以提取RNA并开展表达谱分析（分别标记为DMSO1-3与PMA1-3）。将来自DMSO或PMA处理的THP-1细胞的MV，与新培养的THP-1细胞共培养24小时，分别标记为DMSO-MV24与PMA-MV24。本研究共纳入24个样本。