Spatial Decoding of Immune Cell Contribution to Fibroblastic Foci in Idiopathic Pulmonary Fibrosis.
Spatial Decoding of Immune Cell Contribution to Fibroblastic Foci in Idiopathic Pulmonary Fibrosis.
复制标题
免疫细胞对特发性肺纤维化成纤维细胞灶的贡献的空间解码。
DOI：
10.1164/rccm.202303-0372le
复制
发表时间：
2023
影响因子：
24.7
通讯作者：
Chen,Ching-Hsien
中科院分区：
文献类型：
--
作者：
Yang,DavidC;Hsu,Ssu-Wei;Li,Ji-Min;Oldham,Justin;Chen,Ching-Hsien
方法和结果为了研究免疫细胞在介导纤维化中的作用，我们使用了来自IPF患者的活检样本。我们使用组织学染色和多重免疫荧光相结合的方法来识别每个样本中的纤维化病变。我们选择病变并将其分为两种类型：高水平表达CD45的周围免疫细胞的“热”纤维化病变和低数量的周围免疫细胞的“冷”纤维化病变（图1A）。然后对这些感兴趣的区域（roi）进行数字空间分析，以进行基因表达分析。为了尽量减少遗传变异的影响，我们比较了同一患者的病变。为了确保每个ROI中有等量的成纤维细胞，我们选择了免疫细胞浸润最少的区域，并在比较热损和冷损之前评估成纤维细胞标记物的水平（图1B）。在获得一组差异表达基因后，我们利用肺纤维化不同阶段的基因特征对数据进行聚类(5)，发现热病变显示早期基因特征，而冷病变显示进行性/终末期特征（图1C和1D）。基因集富集分析也发现了冷热病变之间的差异上调通路（图1E）。热损表现出促增殖和免疫相关通路的增加，如KRAS、PI3K/AKT、Myc、mTOR、IL-2、IL-6、IFN、TNF-α/NF-κB(6,7)，与增殖、细胞凋亡抵抗和免疫激活有关。这表明免疫细胞可能在这些病变中持续被激活，并与促增殖途径协同作用，导致病变迅速扩大。相比之下，冷病变表现出典型的促纤维化途径上调，包括上皮-间质转化、缺氧、Wnt、Hedgehog和TGF-β(6)。图1F进一步显示了热病变中促增殖基因的富集和冷病变中促纤维化基因表达的激增。
Methods and ResultsTo investigate the role of immune cells in mediating fibrosis, we utilized biopsy samples obtained from patients with IPF. We used a combination of histological staining and multiplex immunofluorescence to identify fibrotic lesions in each sample. We selected lesions and categorized them into two types:“hot” fibrotic lesions with high levels of surrounding immune cells expressing CD45 and “cold” fibrotic lesions with lower amounts of surrounding immune cells (Figure 1A). These regions of interest (ROIs) were then subjected to digital spatial profiling analysis for gene expression profiling. To minimize the effect of genetic variability, we compared lesions within the same patients. To ensure equivalent amounts of fibroblasts in each ROI, we selected regions with minimal immune cell infiltration and assessed the levels of fibroblast markers before comparing hot and cold lesions (Figure 1B). On obtaining a set of differentially expressed genes, we clustered the data utilizing gene signatures of various stages of pulmonary fibrosis (5) and revealed that hot lesions displayed an early-stage gene signature, whereas cold lesions demonstrated a progressive/end-stage signature (Figures 1C and 1D). Gene set enrichment analysis also identified differentially upregulated pathways between hot and cold lesions (Figure 1E). Hot lesions showed an increase in proproliferative and immune-associated pathways such as KRAS, PI3K/AKT, Myc, mTOR, IL-2, IL-6, IFN, TNF-α/NF-κB (6, 7), linked to proliferation, apoptosis resistance, and immune activation. This suggests that the immune cells may be continually activated in these lesions and, in tandem with the pro-proliferative pathways, contribute to rapidly expanding lesions. In contrast, cold lesions demonstrated the upregulation of typical profibrotic pathways, including epithelial-mesenchymal transition, hypoxia, Wnt, Hedgehog, and TGF-β (6). Figure 1F further shows an enrichment of proproliferative genes in hot lesions and a surge in profibrotic gene expression in cold lesions.