Targeted transcriptome analysis of isolated intestinal epithelial cells from mice with constitutive Ptpn2-deficiency [Distal Colon]

Loss-of-function variants in the protein tyrosine phosphatase non-receptor type 2 (PTPN2) gene are associated with increased risk of inflammatory bowel disease (IBD). PTPN2 encodes T cell protein tyrosine phosphatase (TCPTP), a negative regulator of several intracellular signaling pathways including JAK-STAT. It has been shown that Ptpn2 is critical for intestinal epithelial cell (IEC) barrier maintenance, IEC-macrophage communication, and modulation of the gut microbiome in mice. However, the mechanisms by which Ptpn2 influences the intestinal flora are unknown. In this study, we aimed to identify how Ptpn2-loss affects the expression of genes associated with function/differentiation of IECs in the small and large intestines that could contribute to higher susceptibility to infection. Intestinal epithelial cells were isolated from 21 days old mice (littermates) as Ptpn2-KO mice are normal at birth but die within 3-5 weeks of age. Total RNA was used. Results of two separate panels with a predefined set of targets ('AutoImmune Profiling’ and ‘PanCancer Pathways') from the same intestinal segment were combined for analysis, with the addition of 30 customized targets to each panel comprising of IEC markers, function and differentiation factors, host-bacteria interaction, autophagy, immune response and iron transport, totaling >1500 targets. Data from each panel was normalized separately and then consolidated using the MultiRLF function in the nSolver Analysis Software 4.0.

蛋白酪氨酸磷酸酶非受体型2（protein tyrosine phosphatase non-receptor type 2, PTPN2）基因的功能丧失性变异与炎症性肠病（inflammatory bowel disease, IBD）的发病风险升高相关。PTPN2编码T细胞蛋白酪氨酸磷酸酶（T cell protein tyrosine phosphatase, TCPTP），后者是包括JAK-STAT在内的多种细胞内信号通路的负调控因子。已有研究表明，在小鼠体内，Ptpn2对肠上皮细胞（intestinal epithelial cell, IEC）屏障维持、肠上皮细胞-巨噬细胞通信以及肠道菌群调控均至关重要。然而，Ptpn2影响肠道菌群的具体分子机制尚不明确。本研究旨在探究Ptpn2缺失如何影响小肠及大肠内肠上皮细胞功能/分化相关基因的表达，进而提升感染易感性。本研究从21日龄的同窝出生小鼠中分离肠上皮细胞——因Ptpn2敲除（Ptpn2-KO）小鼠出生时表型正常，但会在出生后3~5周内死亡。实验采用总RNA进行检测。将来自同一肠段的两个预设靶标检测板（分别为「自身免疫谱分析」和「泛癌症通路」）的结果合并分析，并为每个检测板额外添加30个定制化靶标，涵盖肠上皮细胞标志物、功能与分化因子、宿主-细菌互作、自噬、免疫应答及铁转运相关基因，总靶标数超过1500个。每组检测板的数据先分别进行标准化处理，随后通过nSolver分析软件4.0中的MultiRLF函数完成整合。