Disparate interferon signaling and shared aberrant basaloid cells in single-cell profiling of idiopathic pulmonary fibrosis and systemic sclerosis-associated interstitial lung disease

Idiopathic pulmonary fibrosis (IPF) and systemic sclerosis-associated interstitial lung disease (SSc-ILD) differ in the predominant demographics and identified genetic risk alleles of affected patients, however both diseases frequently progress to respiratory failure and death. Contrasting advanced SSc-ILD to IPF provides insight to the role dysregulated immunity may play in pulmonary fibrosis. To analyze cell-type specific transcriptome commonalities and differences between IPF and SSc-ILD, we compared single-cell RNA-sequencing (scRNA-seq) of 21 explanted lung tissue specimens from patients with advanced IPF, SSc-ILD, and organ donor controls. Comparison of IPF and SSc-ILD tissue identified divergent patterns of interferon signaling, with interferon-gamma signaling upregulated in the SPP1hi and FABP4hi macrophages, cytotoxic T cells, and natural kill cells of IPF, while type I interferon signaling and production was upregulated in the corresponding SSc-ILD populations. Plasmacytoid dendritic cells were found in diseased lungs only, and exhibited upregulated cellular stress pathways in SSc-ILD compared to IPF. Alveolar type I cells were dramatically decreased in both IPF and SSc-ILD, with a distinct transcriptome signature separating these cells by disease. KRT5-/KRT17+ aberrant basaloid cells exhibiting markers of cellular senescence and epithelial-mesenchymal transition were identified in SSc-ILD for the first time. In summary, our study utilizes the enriched capabilities of scRNA-seq to identify key divergent cell types and pathways between IPF and SSc-ILD, providing new insights into the shared and distinct mechanisms between idiopathic and autoimmune interstitial lung diseases. Overall design: To reduce batch effects in analyzing multiple samples, all control, IPF, and SSc-ILD samples were combined into 3 objects by disease status using Seurat's IntegrateData function, followed by integration of these 3 objects. Following clustering and visualization with uniform manifold approximation and projection (UMAP), cell populations were classified using multiple gene markers in the transcriptome. Doublet cells were manually identified and subsequently removed. To better define individual cell types, the myeloid, lymphoid, epithelial, and stromal populations were separately reclustered. Differential gene expression analysis for IPF versus SSc-ILD cells for each cluster was performed using the Wilcoxon rank-sum statistical test.

特发性肺纤维化（IPF）与系统性硬化症相关间质性肺疾病（SSc-ILD）在受累患者的主要人口统计学特征及已明确的遗传风险等位基因方面存在差异，但两种疾病均常进展为呼吸衰竭乃至死亡。对比晚期SSc-ILD与IPF，有助于解析免疫失调在肺纤维化中的作用。为分析IPF与SSc-ILD间细胞类型特异性转录组的共性与差异，我们对21例晚期IPF患者、晚期SSc-ILD患者及器官供体对照者的切除肺组织标本开展了单细胞RNA测序（scRNA-seq）分析。对比IPF与SSc-ILD组织标本，发现二者干扰素信号通路模式存在显著差异：IPF患者的SPP1高表达（SPP1hi）巨噬细胞、FABP4高表达（FABP4hi）巨噬细胞、细胞毒性T细胞及自然杀伤细胞中，γ干扰素信号通路显著上调；而SSc-ILD患者的对应细胞群中，I型干扰素信号通路及干扰素产生过程则呈激活状态。浆细胞样树突状细胞仅在病变肺组织中被检出，且相较于IPF患者，SSc-ILD患者的此类细胞中细胞应激通路表达显著上调。I型肺泡细胞在IPF与SSc-ILD患者中均显著减少，且两类细胞的转录组特征可依据疾病类型明确区分。本研究首次在SSc-ILD患者中检出了表达细胞衰老与上皮间质转化标志物的KRT5阴性/KRT17阳性异常基底样细胞。综上，本研究借助单细胞RNA测序的强大分析能力，鉴定出IPF与SSc-ILD间关键的差异细胞类型与通路，为特发性与自身免疫性间质性肺疾病的共享及独特发病机制提供了全新见解。整体实验设计：为降低多样本分析中的批次效应，我们依据疾病状态将所有对照、IPF及SSc-ILD样本整合为3个数据集，采用Seurat软件的IntegrateData函数完成整合。随后通过均匀流形近似和投影（UMAP）进行聚类与可视化，并利用转录组中的多个基因标志物对细胞群进行分类注释。双重态细胞经人工识别后予以移除。为进一步明确各类细胞类型，我们对髓系、淋巴系、上皮及基质细胞群分别开展再聚类。针对每个细胞簇，采用威尔科克森秩和检验完成IPF与SSc-ILD细胞的差异基因表达分析。