Dysregulated alveolar epithelial maintenance in Hermansky-Pudlak syndrome pulmonary fibrosis

Hermansky-Pudlak syndrome (HPS) is a genetic disorder of intracellular endosomal trafficking defects associated with pulmonary fibrosis. Double mutant HPS1/2 mice exhibit spontaneous fibrosis and single mutant HPS1 and HPS2 mice display increased fibrotic sensitivity. To identify mechanisms of AT2 cell dysfunction contributing to fibrosis in HPS, we examined lungs from aged HPS1/2 mice and observed regions of AT2 cell loss adjacent to areas of marked AT2 cell hyperplasia as compared to wild type (WT). Overall, there was accelerated loss of AT2 cells in HPS1/2, HPS1, and HPS2 mice with aging based on immunohistochemistry and lineage tracing studies. Lung organoids generated with HPS2 AT2 cells with WT fibroblasts were smaller in size and displayed significantly decreased colony forming efficiency compared to organoids with WT AT2 cells. Utilizing an H1N1 PR8 influenza model to examine AT2 cell regeneration after injury, there was a significant decrease in the percentage of proliferating AT2 cells in HPS1 and HPS2 mice compared to WT mice. RNA sequencing of AT2 cells isolated from unchallenged mice demonstrated upregulation of genes associated with proliferation and cellular senescence in HPS AT2 cells. Collectively, AT2 cells in HPS mice exhibit dysregulated proliferation with transcriptomic features suggesting subpopulations of senescent and hyperproliferative cells. Dysregulated maintenance of the alveolar epithelium with accelerated senescence and aberrant proliferation of AT2 cells appears to be a shared pathway underlying HPS and other sporadic and genetic forms of pulmonary fibrosis. To investigate age and genotype-dependent mRNA expression differences in AT2 cells captured from the lungs of WT and HPS-affected mouse lungsHermansky-Pudlak综合征（Hermansky-Pudlak syndrome, HPS）是一类与肺纤维化相关的细胞内内体运输缺陷性遗传病。双突变HPS1/2小鼠可出现自发性肺纤维化，而单突变HPS1、HPS2小鼠则表现出纤维化易感性升高。为明确介导HPS相关肺纤维化的肺泡II型（AT2）细胞功能障碍机制，我们对老年HPS1/2小鼠的肺部组织进行检测，结果发现相较于野生型（WT）小鼠，其肺部可见AT2细胞缺失区域与显著AT2细胞增生区域相邻的病理改变。通过免疫组织化学与谱系示踪实验证实，随年龄增长，HPS1/2、HPS1及HPS2小鼠的AT2细胞均出现加速丢失。将HPS2小鼠的AT2细胞与野生型成纤维细胞共培养构建的肺类器官，其尺寸显著小于野生型AT2细胞构建的肺类器官，且集落形成效率明显降低。利用H1N1 PR8流感模型检测损伤后AT2细胞的再生能力时，相较于野生型小鼠，HPS1、HPS2小鼠体内增殖性AT2细胞的占比显著下降。对未受干预小鼠分离得到的AT2细胞进行RNA测序分析发现，HPS小鼠的AT2细胞中与细胞增殖及细胞衰老相关的基因表达显著上调。综上，HPS小鼠的AT2细胞存在增殖失调现象，其转录组特征提示体内存在衰老与过度增殖的AT2细胞亚群。AT2细胞介导的肺泡上皮维持功能失调，伴随细胞衰老加速与AT2细胞异常增殖，似乎是HPS及其他散发性、遗传性肺纤维化共有的发病通路。为探究野生型及HPS受累小鼠肺部分离得到的AT2细胞中，年龄与基因型依赖的mRNA表达差异。