背景:成纤维细胞在克罗恩病(CD)的狭窄形成中起关键作用,但了解其发病机制需要系统水平的研究以发现新的治疗目标。我们研究了全厚度CD组织,通过生成第一个单细胞RNA测序(scRNAseq)狭窄肠图谱来表征成纤维细胞的异质性和功能,并为治疗靶标验证提供原理证据。
方法:我们进行了13例新鲜全厚度CD切除的scRNAseq,发炎不狭窄,和狭窄的节段以及7个正常的非CD肠段。将每个节段分成粘膜/粘膜下层或固有肌层,并分别分析总共99个组织样品和409,001个细胞。我们通过使用整个组织验证了cadherin-11(CDH11)作为潜在的治疗靶点,分离的肠细胞,NanoStringnCounter,下一代测序,蛋白质组学和动物模型。
结果:我们的综合数据集揭示了狭窄CD中的成纤维细胞异质性,在粘膜/粘膜下层检测到大多数狭窄选择性变化,但不是肌肉层。细胞-细胞相互作用建模显示CXCL14+以及MMP/WNT5A+成纤维细胞在CD狭窄中显示出中枢信号传导作用。CDH11,一种成纤维细胞-细胞粘附分子,被广泛表达和上调,NanoStringnCounter验证了其促纤维化功能,RNA测序,组织靶表达,体外功能增益和丧失实验,蛋白质组学,以及敲除和抗体介导的CDH11阻断在实验性结肠炎中的作用。
结论:全厚肠scRNAseq图谱揭示了以前未认识到的CD狭窄中的成纤维细胞异质性和相互作用,CDH11被验证为潜在的治疗靶标。这些结果为更好地理解CD狭窄的形成提供了新的资源,并开辟了潜在的治疗发展。
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.