Division of Rheumatology and Clinical Immunology, University of Pittsburgh, Pittsburgh, Pennsylvania.
Department of Human Genetics, University of Pittsburgh, Pittsburgh, Pennsylvania.
Arthritis Rheumatol. 2022 Dec;74(12):2003-2014. doi: 10.1002/art.42286. Epub 2022 Oct 31.
Systemic sclerosis-associated interstitial lung disease (SSc-ILD) is the leading cause of death in patients with SSc with unclear pathogenesis and limited treatment options. Evidence strongly supports an important role for profibrotic secreted phosphoprotein 1 (SPP1)-expressing macrophages in SSc-ILD. This study was undertaken to define the transcriptome and chromatin structural changes of SPP1 SSc-ILD macrophages in order to better understand their role in promoting fibrosis and to identify transcription factors associated with open chromatin driving their altered phenotype.
We performed single-cell RNA sequencing (scRNA-Seq) on 11 explanted SSc-ILD and healthy control lung samples, as well as single-cell assay for transposase-accessible chromatin sequencing on 5 lung samples to define altered chromatin accessibility of SPP1 macrophages. We predicted transcription factors regulating SPP1 macrophages using single-cell regulatory network inference and clustering (SCENIC) and determined transcription factor binding sites associated with global alterations in SPP1 chromatin accessibility using Signac/Seurat.
We identified distinct macrophage subpopulations using scRNA-Seq analysis in healthy and SSc-ILD lungs and assessed gene expression changes during the change of healthy control macrophages into SPP1 macrophages. Analysis of open chromatin validated SCENIC predictions, indicating that microphthalmia-associated transcription factor, transcription factor EB, activating transcription factor 6, sterol regulatory element binding transcription factor 1, basic helix-loop-helix family member E40, Kruppel-like factor 6, ETS variant transcription factor 5, and/or members of the activator protein 1 family of transcription factors regulate SPP1 macrophage differentiation.
Our findings shed light on the underlying changes in chromatin structure and transcription factor regulation of profibrotic SPP1 macrophages in SSc-ILD. Similar alterations in SPP1 macrophages may underpin fibrosis in other organs involved in SSc and point to novel targets for the treatment of SSc-ILD, specifically targeting profibrotic macrophages.
系统性硬化症相关间质性肺病（SSc-ILD）是导致系统性硬化症患者死亡的主要原因，其发病机制尚不清楚，治疗选择有限。有证据强烈支持促纤维化的分泌磷酸化蛋白 1（SPP1）表达的巨噬细胞在 SSc-ILD 中发挥重要作用。本研究旨在确定 SSc-ILD 巨噬细胞中 SPP1 的转录组和染色质结构变化，以便更好地了解其在促进纤维化中的作用，并确定与开放染色质相关的转录因子，从而驱动其表型改变。
我们对 11 例肺活检的 SSc-ILD 患者和健康对照者的肺组织进行了单细胞 RNA 测序（scRNA-Seq），对 5 例肺组织进行了单细胞转座酶可及性染色质测序，以确定 SPP1 巨噬细胞染色质可及性的改变。我们使用单细胞调控网络推断和聚类（SCENIC）预测调控 SPP1 巨噬细胞的转录因子，并使用 Signac/Seurat 确定与 SPP1 染色质可及性全局改变相关的转录因子结合位点。
我们使用 scRNA-Seq 分析在健康和 SSc-ILD 肺组织中鉴定了不同的巨噬细胞亚群，并评估了健康对照者的巨噬细胞向 SPP1 巨噬细胞转变过程中的基因表达变化。开放染色质分析验证了 SCENIC 预测，表明小眼畸形相关转录因子、转录因子 EB、激活转录因子 6、固醇调节元件结合转录因子 1、碱性螺旋-环-螺旋家族成员 E40、 Kruppel 样因子 6、ETS 变体转录因子 5 和/或激活蛋白 1 家族的转录因子调节 SPP1 巨噬细胞分化。
我们的研究结果揭示了 SSc-ILD 中促纤维化的 SPP1 巨噬细胞染色质结构和转录因子调节的潜在变化。SPP1 巨噬细胞的类似改变可能是 SSc 相关其他器官纤维化的基础，并为 SSc-ILD 的治疗提供了新的靶点，特别是针对促纤维化的巨噬细胞。
