Microarray analysis of splenic reservoir monocytes and their blood counterparts. Mus musculus

A current paradigm states that monocytes circulate freely and patrol blood vessels, but differentiate irreversibly into dendritic cells or macrophages upon tissue entry. Here we show that bona fide undifferentiated monocytes reside in the spleen and outnumber their equivalents in circulation. The reservoir monocytes are relatively immotile, assemble in clusters in the cords of the subcapsular red pulp, and are distinct from macrophages and dendritic cells. In response to ischemic myocardial injury, splenic monocytes increase their motility, exit the spleen en masse, accumulate in injured tissue and participate in wound healing. These observations uncover a role for the spleen as a site for storage and rapid deployment of monocytes, and identify the splenic monocyte reservoir as a resource that the body exploits to regulate inflammation. The goal of this gene expression study was to compare the gene expression of Ly-6C hi inflammatory monocytes residing in the spleen and their circulating counterparts in the blood. Overall design: Monocyte subsets of a group of four mice (C57BL/6, 8-12 weeks) were isolated by fluorescence activated cell sorting (FACS Aria, BD biosciences) as CD11bhi (CD90/B220/CD49b/NK1.1/Ly-6G)lo (F4/80/I-Ab/CD11c)lo Ly-6Chi (all antibodies BD biosciences) cells. Samples of 1,000 Ly-6Chi blood and Ly-6Chi splenic monocytes of each mouse were collected directly into 20 µl lysis buffer of the PicoPure RNA isolation kit (Arcturus). RNA extraction was subsequently performed according to the manufacturer’s instructions (Arcturus). RNA quality was assessed using RNA pico lab chips on the Agilent Bioanalyzer. For all samples a RIN above 8 could be achieved. All further steps were performed at the UCSF Shared Microarray Core Facilities according to standard protocols (http://www.arrays.ucsf.edu and http://www.agilent.com).

当前主流范式认为，单核细胞（monocytes）可自由循环并巡逻监护血管，但在进入组织后会不可逆地分化为树突状细胞（dendritic cells）或巨噬细胞（macrophages）。本研究证实，脾脏中存在真正未分化的单核细胞，其数量多于循环中的同类单核细胞。这类储备池单核细胞相对不具运动性，聚集于被膜下红髓的索状区域中，且与巨噬细胞和树突状细胞存在显著差异。当发生缺血性心肌损伤时，脾脏单核细胞的运动性增强，会大规模迁出脾脏，聚集于受损组织并参与伤口愈合。上述发现揭示了脾脏作为单核细胞储存与快速动员位点的功能，并明确脾脏单核细胞储备池是机体用以调控炎症反应的重要资源。本基因表达研究的目标是对比脾脏内Ly-6C高表达（Ly-6C hi）炎性单核细胞与血液中循环同类单核细胞的基因表达情况。实验设计：从4只8~12周龄的C57BL/6小鼠中分离单核细胞亚群，采用荧光激活细胞分选术（fluorescence activated cell sorting，FACS Aria，碧迪生物科学（BD biosciences）），分选标记为CD11bhi(CD90/B220/CD49b/NK1.1/Ly-6G)lo(F4/80/I-Ab/CD11c)lo Ly-6Chi的细胞（所有抗体均购自碧迪生物科学）。每只小鼠的1000个Ly-6Chi血液单核细胞与脾脏单核细胞样本，均直接收集于20 μl Arcturus公司PicoPure RNA提取试剂盒配套裂解液中。随后按照Arcturus公司试剂盒说明书进行RNA提取操作。采用安捷伦生物分析仪（Agilent Bioanalyzer）搭配RNA微量实验芯片对RNA质量进行评估，所有样本的RNA完整性数（RNA Integrity Number，RIN）均达到8以上。后续所有实验步骤均在加州大学旧金山分校（University of California, San Francisco，UCSF）共享微阵列核心实验室按照标准流程完成（相关流程信息可参考http://www.arrays.ucsf.edu与http://www.agilent.com）。