PERICYTE-LIKE CELLS UNDERGO TRANSCRIPTIONAL REPROGRAMMING AND DISTINCT FUNCTIONAL ADAPTATIONS IN ACUTE LUNG INJURY

Background: We previously reported on the role of pericyte-like cells as functional sentinel immune cells in lung injury. However, much about the biology of pericytes in lung injury remains unknown. Lung pericyte-like cells are well-positioned to sense disruption to the epithelial barrier and coordinate local inflammatory responses due to their anatomic niche within the alveoli. In this report, we characterized transcriptional responses and functional changes in pericyte-like cells following activation by alveolar components from injured and uninjured lungs. Methods: We purified pericyte-like cells from lung digests using PDGFR_ as a selection marker and expanded them in culture as previously described (1). We induced sterile acute lung injury in mice with recombinant human Fas ligand (rhFasL) instillation followed by mechanical ventilation (1). We then collected bronchoalveolar lavage fluid (BALF) from injured and uninjured mice. Purified pericyte-like cells in culture were exposed to growth media only (control), BALF from uninjured mice, and BALF from injured mice for 6 and 24 h. RNA collected from pericyte-like cells for each of these treatment conditions underwent genome-wide sequencing using RNA-seq. Targets of interest identified by bioinformatics analysis of RNA-seq data were validated using in vitro and in vivo assays. Results: We observed robust global transcriptional changes in pericyte-like cells following treatment with uninjured and injured BALF at 6 h, but this response persisted for 24 h only after exposure to injured BALF. Functional enrichment analysis of pericytes treated with injured BALF revealed activation of immuno-inflammatory, cell migration and angiogenesis-related pathways, whereas processes associated with tissue development and remodeling were down-regulated. We validated select targets in the inflammatory, angiogenesis-related, and cell migratory pathways using functional assays in vitro and in vivo. Conclusion: Lung pericyte-like cells are highly responsive to alveolar compartment content from both uninjured and injured lungs, but injured BALF elicits a more sustained response. The inflammatory, angiogenic, and migratory changes exhibited by activated pericyte-like cells underscore the phenotypic plasticity of these specialized stromal cells in the setting of acute lung injury. Mouse lung pericyte-like cell mRNA profiles from culutre after exposure to injured BALF (6h, 24h; n = 6 per time point), uninjured BALF (6h, 24h; n = 6 per time point) or media only (6h, 24h; n = 6 per time point) were generated by deep sequencing using Illumina HiSeq.

**背景**：本课题组此前已报道类周细胞（pericyte-like cells）作为功能性哨兵免疫细胞在肺损伤中的作用，但目前对于肺损伤中周细胞的生物学特性仍有诸多未知。鉴于类周细胞在肺泡内的解剖定位，其具备精准感知上皮屏障破坏、协调局部炎症应答的天然优势。本研究旨在明确损伤肺与未损伤肺的肺泡成分激活后，类周细胞的转录应答与功能变化特征。

**方法**：本研究参照既往研究[1]，以血小板衍生生长因子受体（PDGFR）为筛选标记，从肺组织消化产物中纯化类周细胞并进行体外扩增。通过向小鼠气道滴注重组人Fas配体（rhFasL）并联合机械通气，构建无菌性急性肺损伤模型[1]。随后分别收集损伤组与未损伤组小鼠的支气管肺泡灌洗液（bronchoalveolar lavage fluid, BALF）。将体外培养的纯化类周细胞分为三组：仅添加生长培养基（对照组）、添加未损伤小鼠BALF、添加损伤小鼠BALF，分别培养6小时与24小时。收集各处理组类周细胞的总RNA，采用RNA测序（RNA-seq）进行全基因组转录组分析。通过生物信息学分析RNA-seq数据筛选得到的目标靶点，采用体外与体内实验进行验证。

**结果**：在处理6小时后，未损伤与损伤BALF处理组的类周细胞均出现显著的全基因组转录水平变化，但仅损伤BALF处理组的转录应答可维持至24小时。对损伤BALF处理的类周细胞进行功能富集分析显示，免疫炎症、细胞迁移及血管生成相关通路被激活，而组织发育与重塑相关进程则被下调。本研究通过体外与体内功能实验，对炎症、血管生成及细胞迁移通路中的部分靶点进行了验证。

**结论**：肺类周细胞对未损伤与损伤肺的肺泡腔内容物均具有高度应答性，但损伤BALF可诱导更为持久的转录应答。活化类周细胞所表现出的炎症、血管生成及细胞迁移相关变化，凸显了这类特化基质细胞在急性肺损伤中的表型可塑性。本数据集包含经Illumina HiSeq平台深度测序得到的小鼠肺类周细胞mRNA表达谱，样本分别为：暴露于损伤BALF（6小时、24小时，每个时间点n=6）、未损伤BALF（6小时、24小时，每个时间点n=6）或仅添加培养基（6小时、24小时，每个时间点n=6）的体外培养细胞。