RNA-seq profiling of mammary duct macrophages from virgin, pregnant, lactating and post-weaning mice. RNA-seq profiling of mammary duct macrophages from virgin, pregnant, lactating and post-weaning mice

Macrophages are diverse immune cells that reside in all tissues. Although macrophages have been implicated in mammary gland function, their diversity has not been fully addressed. By exploiting high-­resolution 3D imaging and flow cytometry, we have identified a unique population of tissue-­resident ductal macrophages (DMs) that form a contiguous network between the luminal and basal layers of the entire mammary gland throughout post-­natal development. DMs are long-­lived and constantly survey the epithelium though dendrite movement based on advanced 3D intravital imaging. While they initially originate from embryonic precursors, DMs derive from monocytes as they expand during puberty. Moreover, they undergo proliferation in pregnancy to maintain complete coverage of the epithelium in lactation, where they are poised to phagocytose milk-­producing cells post-­lactation and facilitate remodelling. Interestingly, DMs strongly resemble mammary tumour macrophages and form a network that pervades the tumour epithelium. Thus, the mammary epithelium programs specialised resident macrophages in both physiological and tumorigenic contexts. To explore expression changes as DMs profilerate in pregnancy and lactation, we sorted DMs from the mouse mammary glands of virgin, pregnant, lactating and post-weaning mice and undertook RNA-seq profiling. Results from this data series are shown in Figure 5 of Dawson et al (2020). Overall design: Three biological replicates were prepared of ductal macrophages from the breast tissue of female mice at four developmental time points: virgin, 16.5 days pregnant, lactating and 1 month post weaning.

巨噬细胞是一类广泛分布于全身各组织的异质性免疫细胞。尽管已有研究表明巨噬细胞参与乳腺功能调控，但其异质性仍未被完全阐明。本研究借助高分辨率三维成像与流式细胞术，鉴定出一类独特的组织驻留导管巨噬细胞（tissue-resident ductal macrophages, DMs）：该类细胞在整个产后发育过程中，于全乳腺的腔层与基底层之间形成连续的网状结构。借助先进的三维活体成像技术可见，DMs寿命较长，并可通过树突运动持续监测上皮组织状态。尽管DMs最初起源于胚胎前体细胞，但在青春期扩增阶段则由单核细胞分化而来。此外，DMs在妊娠阶段发生增殖，以确保泌乳期上皮组织完全被其覆盖；在泌乳结束后，它们可及时吞噬产乳细胞并促进乳腺组织重塑。值得注意的是，DMs与乳腺肿瘤巨噬细胞表型高度相似，并可形成贯穿肿瘤上皮组织的网状结构。由此可见，乳腺上皮可在生理及肿瘤发生两种情境下，定向塑造具有特定功能的组织驻留巨噬细胞。

为探究DMs在妊娠与泌乳阶段增殖过程中的基因表达变化，本研究从未生育、妊娠、泌乳及断奶后小鼠的乳腺组织中分选得到DMs，并开展RNA测序（RNA-seq）表达谱分析。本数据集的分析结果已展示于Dawson等人（2020）的图5中。

实验设计：针对雌性小鼠乳腺组织中的导管巨噬细胞，在4个发育时间点（未生育期、妊娠16.5天、泌乳期及断奶后1个月）分别设置3次生物学重复。