Hung Chi F, Holton Sarah, Chow Yu-Hua, Liles Wayne Conrad, Gharib Sina A, Altemeier William A
Division of Pulmonary, Critical Care, and Sleep Medicine, Department of Medicine, University of Washington, Seattle, WA, USA.
Center for Lung Biology, University of Washington, Seattle, WA, USA.
FASEB J. 2021 Apr;35(4):e21323. doi: 10.1096/fj.201903192RR.
We previously reported on the role of pericyte-like cells as functional sentinel immune cells in lung injury. However, much about the biological role 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 in a mouse model of acute lung injury (ALI). Purified pericyte-like cells from lung digests using PDGFRβ as a selection marker were expanded 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 hours. RNA collected from these treatment conditions were processed for RNAseq. Targets of interest identified by pathway analysis were validated using in vitro and in vivo assays. We observed robust global transcriptional changes in pericyte-like cells following treatment with uninjured and injured BALF at 6 hours, but this response persisted for 24 hours only after exposure to injured BALF. Functional enrichment analysis of pericytes treated with injured BALF revealed the activation of pro-inflammatory, cell migration, and angiogenesis-related pathways, whereas processes associated with tissue development and cell differentiation were down-regulated. We validated select upregulated targets in the inflammatory, angiogenic, and cell migratory pathways using functional biological assays in vitro and in vivo. We conclude that 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.
我们之前报道了类周细胞作为肺损伤中功能性哨兵免疫细胞的作用。然而，关于周细胞在肺损伤中的生物学作用仍有许多未知之处。肺类周细胞因其在肺泡内的解剖位置，能够很好地感知上皮屏障的破坏并协调局部炎症反应。在本报告中，我们在急性肺损伤（ALI）小鼠模型中，对来自受伤和未受伤肺的肺泡成分激活后类周细胞的转录反应和功能变化进行了表征。使用血小板衍生生长因子受体β（PDGFRβ）作为选择标记，从肺消化物中纯化的类周细胞如前所述在培养中进行扩增（1）。我们通过重组人Fas配体（rhFasL）滴注随后进行机械通气在小鼠中诱导无菌性急性肺损伤（1）。然后我们从受伤和未受伤的小鼠中收集支气管肺泡灌洗液（BALF）。将培养中的纯化类周细胞分别暴露于仅生长培养基（对照）、未受伤小鼠的BALF和受伤小鼠的BALF中6小时和24小时。对从这些处理条件下收集的RNA进行RNA测序。通过通路分析确定的感兴趣靶点使用体外和体内试验进行验证。我们观察到在6小时用未受伤和受伤的BALF处理后，类周细胞中出现了强烈的全局转录变化，但这种反应仅在暴露于受伤的BALF后持续24小时。对用受伤的BALF处理的周细胞进行功能富集分析，发现促炎、细胞迁移和血管生成相关通路被激活，而与组织发育和细胞分化相关的过程则被下调。我们使用体外和体内功能生物学试验验证了炎症、血管生成和细胞迁移通路中选定的上调靶点。我们得出结论，肺类周细胞对来自未受伤和受伤肺的肺泡腔内容物高度敏感，但受伤的BALF会引发更持续的反应。激活的类周细胞表现出的炎症、血管生成和迁移变化突出了这些特殊基质细胞在急性肺损伤情况下的表型可塑性。
