Analysis of transcriptomes in intestinal stem cells in response to Interferon gamma

There is limited understanding of how immune-mediated damage impacts intestinal stem cells (ISCs). We found that stem cell compartment injury is a shared feature of both alloreactive and autoreactive intestinal immunopathology, reducing ISCs and impairing their recovery in T-cell-mediated injury models. Modeling with ex vivo epithelial cultures indicated ISC depletion and impaired human as well as murine organoid survival upon co-culture with activated T cells, and screening of effector pathways identified Interferon-γ (IFNγ) as a principal mediator of ISC compartment damage. Flow cytometry analysis confirmed expression of IFNγ receptor on ISCs. To investigate if the receptor was functional, we performed transcriptome analysis of ISCs treated with IFNγ. In brief, mRNA profiles of sorted Lgr5-high cells treated with/without IFNγ were generated by deep sequencing, in triplicate, using the TruSeq SBS Kit V4 (Illumina). RNA profiles confirmed a robust IFNγ transcriptional signature in the stem cells, as several IFNγ-related genes were upregulated shortly after exposure, indicating direct activity of IFNγ in ISCs. Epithelial cultures with IFNγ-receptor-deficient ISCs and purified stem cell colonies also indicated direct targeting of the ISCs. This study showed that dysregulated T cell activation and Interferon-γ production are potent mediators of ISC injury. mRNA profiles of isolated Lgr5-high intestinal stem cells treated w/wo interferon gamma were generated by deep sequencing, in triplicate, using the TruSeq SBS Kit V4(Illumina).

目前学界对免疫介导的损伤如何影响肠道干细胞（intestinal stem cells，ISCs）的认知仍较为有限。我们的研究发现，干细胞龛损伤是同种反应性与自身反应性肠道免疫病理的共同特征：在T细胞介导的损伤模型中，该损伤会导致肠道干细胞数量减少，并损害其修复能力。通过离体上皮细胞培养建模的实验显示，与活化T细胞共培养时，人类及小鼠类器官的存活能力受损，且肠道干细胞发生耗竭；对效应通路的筛选则确认，干扰素γ（Interferon-γ，IFNγ）是导致干细胞龛损伤的主要介导因子。流式细胞术分析证实，肠道干细胞表面表达干扰素γ受体。为探究该受体是否具备功能，我们对经干扰素γ处理的肠道干细胞进行了转录组分析。简言之，我们使用TruSeq SBS Kit V4（Illumina）试剂盒，通过深度测序对经/未经干扰素γ处理的分选得到的Lgr5高表达细胞进行了三次生物学重复的mRNA转录组分析。转录组分析结果证实，肠道干细胞中存在显著的干扰素γ转录特征：暴露于干扰素γ后不久，多个干扰素γ相关基因即出现上调，这表明干扰素γ可直接作用于肠道干细胞。使用干扰素γ受体缺陷型肠道干细胞进行的上皮细胞培养实验，以及纯化的干细胞集落实验，同样证实了干扰素γ可直接靶向肠道干细胞。本研究证实，T细胞活化失调及干扰素γ过量产生，是导致肠道干细胞损伤的强效介导因素。本研究通过深度测序，使用TruSeq SBS Kit V4（Illumina）试剂盒，对经/未经干扰素γ处理的分离得到的Lgr5高表达肠道干细胞进行了三次生物学重复的mRNA转录组分析。