Department of Inflammation and Immunity, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio; Center for Global Translational Inflammatory Bowel Disease Research, Cleveland Clinic, Cleveland, Ohio.
Department of Inflammation and Immunity, Lerner Research Institute, Cleveland Clinic, Cleveland, Ohio.
Gastroenterology. 2023 Nov;165(5):1180-1196. doi: 10.1053/j.gastro.2023.07.014. Epub 2023 Jul 26.
BACKGROUND & AIMS: Fibroblasts play a key role in stricture formation in Crohn's disease (CD) but understanding its pathogenesis requires a systems-level investigation to uncover new treatment targets. We studied full-thickness CD tissues to characterize fibroblast heterogeneity and function by generating the first single-cell RNA sequencing (scRNAseq) atlas of strictured bowel and providing proof of principle for therapeutic target validation.
We performed scRNAseq of 13 fresh full-thickness CD resections containing noninvolved, inflamed nonstrictured, and strictured segments as well as 7 normal non-CD bowel segments. Each segment was separated into mucosa/submucosa or muscularis propria and analyzed separately for a total of 99 tissue samples and 409,001 cells. We validated cadherin-11 (CDH11) as a potential therapeutic target by using whole tissues, isolated intestinal cells, NanoString nCounter, next-generation sequencing, proteomics, and animal models.
Our integrated dataset revealed fibroblast heterogeneity in strictured CD with the majority of stricture-selective changes detected in the mucosa/submucosa, but not the muscle layer. Cell-cell interaction modeling revealed CXCL14+ as well as MMP/WNT5A+ fibroblasts displaying a central signaling role in CD strictures. CDH11, a fibroblast cell-cell adhesion molecule, was broadly expressed and up-regulated, and its profibrotic function was validated using NanoString nCounter, RNA sequencing, tissue target expression, in vitro gain- and loss-of-function experiments, proteomics, and knock-out and antibody-mediated CDH11 blockade in experimental colitis.
A full-thickness bowel scRNAseq atlas revealed previously unrecognized fibroblast heterogeneity and interactions in CD strictures and CDH11 was validated as a potential therapeutic target. These results provide a new resource for a better understanding of CD stricture formation and open potential therapeutic developments. This work has been posted as a preprint on Biorxiv under doi: 10.1101/2023.04.03.534781.
成纤维细胞在克罗恩病（CD）的狭窄形成中起着关键作用，但要了解其发病机制，需要进行系统水平的研究，以发现新的治疗靶点。我们通过生成首个完整肠壁单细胞 RNA 测序（scRNAseq）图谱，并提供治疗靶点验证的原理证明，研究了全厚 CD 组织，以表征成纤维细胞的异质性和功能。
我们对 13 例新鲜全厚 CD 切除标本进行了 scRNAseq，其中包含非受累、非炎症性非狭窄和狭窄段，以及 7 例非 CD 正常肠段。每个段均分为黏膜/黏膜下层或固有肌层，并分别进行分析，共 99 个组织样本和 409,001 个细胞。我们通过使用全组织、分离的肠细胞、NanoString nCounter、下一代测序、蛋白质组学和动物模型，验证了钙黏蛋白 11（CDH11）作为潜在治疗靶点的作用。
我们的综合数据集揭示了 CD 狭窄处成纤维细胞的异质性，其中大多数狭窄选择性变化发生在黏膜/黏膜下层，但不在肌肉层。细胞-细胞相互作用模型显示，CXCL14+和 MMP/WNT5A+成纤维细胞在 CD 狭窄中发挥着核心信号作用。钙黏蛋白 11（CDH11）是一种成纤维细胞细胞间黏附分子，广泛表达并上调，其成纤维作用通过 NanoString nCounter、RNA 测序、组织靶标表达、体外获得和丧失功能实验、蛋白质组学以及在实验性结肠炎中敲除和抗体阻断 CDH11 进行了验证。
全厚肠 scRNAseq 图谱揭示了 CD 狭窄处以前未被识别的成纤维细胞异质性和相互作用，并且验证了 CDH11 是一个潜在的治疗靶点。这些结果为更好地了解 CD 狭窄形成提供了新的资源，并为潜在的治疗开发开辟了途径。这项工作已作为预印本发布在 Biorxiv 上，doi：10.1101/2023.04.03.534781。
