Porcine nasal and lung macrophage subsets isolated by FACS and LCM show different transcriptomic profiles depending on tissue origin and location

Macrophages have a pivotal role during viral infections in pigs. By expressing cell surface receptors, macrophages become viral targets and reservoirs. In the case of upper respiratory tract infections, many viruses target the peripheral nasal mucosa. Recent in vivo and in vitro (primary nasal cell cultures) porcine reproductive and respiratory syndrome virus (PRRSV) studies demonstrated that several macrophage subsets exist in the porcine nasal mucosa, and that virus strains with different virulence showed altered tropism for these different macrophage populations. To further investigate these macrophage subsets, total RNA sequencing was performed in parallel on two subsets from the nasal mucosa and lung macrophages. Macrophages were isolated by either fluorescent activated cell sorting (FACS; cf. bulk RNAseq) or laser capture microdissection (LCM; cf. LCM RNAseq) in combination with immunofluorescence staining against two macrophage markers, CD163 and Sialoadhesin (Sn). With both RNAseq methods, nasal macrophages showed a different transcriptomic profile compared to lung macrophages. Differentially expressed genes were identified in the two subsets of nasal macrophages. Gene set enrichment analysis on the three macrophage populations showed that GO terms and KEGG pathways on LCM RNAseq data were more specific to the spatial location of macrophage subsets than bulk RNAseq data. Cell type signature analysis revealed that nasal CD163+Sn- cells resemble squamous epithelial cells (LCM RNAseq) or antigen presenting cells (bulk RNAseq) while nasal CD163+Sn+ cells are more like fibroblasts/stromal cells (LCM RNAseq) or vascular endothelial cell (bulk RNAseq). Our results confirmed that not only macrophages in different tissues but also macrophages in different areas within the same tissue have different transcriptional programs, suggesting their differential roles in their interaction with pathogens and corresponding immune responses.

巨噬细胞（Macrophages）在猪病毒感染过程中发挥关键作用。它们通过表达细胞表面受体，成为病毒的靶细胞与储存库。就上呼吸道感染而言，多种病毒会靶向外周鼻黏膜。近期针对猪繁殖与呼吸综合征病毒（PRRSV）开展的体内外（原代鼻细胞培养）研究显示，猪鼻黏膜中存在多种巨噬细胞亚群，且不同毒力的病毒株对这些巨噬细胞亚群的嗜性存在差异。为进一步探究这些巨噬细胞亚群，研究团队对鼻黏膜的两个巨噬细胞亚群以及肺脏巨噬细胞并行开展了总RNA测序。巨噬细胞的分离采用荧光激活细胞分选（FACS；参见批量RNA测序）或激光捕获显微切割（LCM；参见LCM RNA测序）联合针对两种巨噬细胞标志物CD163与唾液酸黏附素（Sn）的免疫荧光染色方法。两种RNA测序方法均显示，鼻黏膜巨噬细胞与肺脏巨噬细胞的转录组谱存在差异。研究人员在鼻黏膜巨噬细胞的两个亚群中鉴定出了差异表达基因。针对三类巨噬细胞群的基因集富集分析结果显示，相较于批量RNA测序数据，激光捕获显微切割RNA测序数据所对应的GO条目与KEGG通路更贴合巨噬细胞亚群的空间定位特征。细胞类型特征分析揭示：鼻黏膜CD163+Sn-细胞在激光捕获显微切割RNA测序结果中类似鳞状上皮细胞，在批量RNA测序结果中则对应抗原呈递细胞；而鼻黏膜CD163+Sn+细胞在激光捕获显微切割RNA测序结果中更接近成纤维细胞/基质细胞，在批量RNA测序结果中则更近似血管内皮细胞。本研究结果证实，不仅不同组织中的巨噬细胞，同一组织内不同区域的巨噬细胞也拥有独特的转录程序，这提示它们在与病原体互作及相应免疫应答中发挥着不同的作用。