Single-cell RNA sequencing reveals dysregulated cellular programs in the inflamed epithelium of Crohn's disease patients.

Crohn’s disease (CD) is a complex inflammatory disorder of incompletely understood molecular aetiology. We generated a large single-cell RNA sequencing dataset from the terminal ileal biopsies of two independent cohorts comprising a total of 50 CD patients and 71 healthy controls. We performed transcriptomic analyses to reveal genes, cell types and mechanisms perturbed in CD, leveraging the power of the two cohorts to confirm our findings and assess replicability. In addition to mapping widespread alterations in cytokine signalling, we provide evidence of pan-epithelial upregulation of MHC class I genes and pathways in CD. Using non-negative matrix factorization we revealed intra- and inter-cellular upregulation of expression programs such as G-protein coupled receptor signalling and interferon signalling, respectively, in CD. We observed an enrichment of CD heritability among marker genes for various activated T cell types and myeloid cells, supporting a causal role for these cell-types in CD aetiology. Comparisons between our discovery and replication cohort revealed significant variation in differential gene-expression replicability across cell types. B, T and myeloid cells showed particularly poor replicability, suggesting caution should be exercised when interpreting unreplicated differential gene-expression result in these cell types. Overall, our results provide a rich resource for identifying cell-type specific biomarkers of Crohn’s disease and identifying genes, cell types and pathways that are causally and replicably associated with disease.克罗恩病（Crohn’s disease, CD）是一种发病分子机制尚未完全阐明的复杂炎症性疾病。本研究从两个独立队列的末端回肠活检组织中构建了大规模单细胞RNA测序（single-cell RNA sequencing, scRNA-seq）数据集，队列共纳入50名克罗恩病患者与71名健康对照个体。本研究通过转录组分析，揭示了克罗恩病中发生异常的基因、细胞类型与分子机制，并借助双队列的优势验证研究结果、评估结果的可重复性。除了绘制细胞因子信号通路的广泛异常变化图谱外，本研究还证实克罗恩病中存在全上皮细胞的主要组织相容性复合体I类（MHC class I）基因及通路上调现象。本研究通过非负矩阵分解（non-negative matrix factorization）分析，揭示了克罗恩病中细胞内与细胞间的表达程序上调现象：前者以G蛋白偶联受体信号通路为代表，后者则以干扰素信号通路为典型。本研究发现，多种活化T细胞亚型与髓系细胞的标记基因中富集克罗恩病遗传力，这为这些细胞类型在克罗恩病发病机制中的因果作用提供了支持依据。通过对发现队列与验证队列的比较分析，本研究发现不同细胞类型的差异基因表达结果可重复性存在显著差异。B细胞、T细胞与髓系细胞的差异基因表达结果可重复性尤其低下，这提示在解读这些细胞类型中未经验证的差异基因表达结果时需格外谨慎。综上，本研究结果为鉴定克罗恩病的细胞类型特异性生物标志物，以及筛选与疾病存在因果关联且可重复的基因、细胞类型与通路提供了宝贵的研究资源。