The immunoregulatory effects of CD8 T cell-derived perforin on diet-induced nonalcoholic steatohepatitis

Method: To better understand the mechanism of perforin in regulating liver CD8 T cell-mediated inflammation during NASH, we sorted CD8 T cells from the livers of WT or Prf1null mice fed the MCD diet for 4 weeks and performed mRNA sequencing of these hepatic CD8 T cells. Total RNA was isolated from FACS-separated hepatic CD8 T cells. Transcriptome sequencing libraries were generated using NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB, USA) following manufacturer’s recommendations and sequenced on an Illumina Hiseq platform (Illumina, San Diego, CA). Sequences were aligned to the reference genome with TopHat and processed with Cufflinks, which quantifies each transcript in each sample using reference annotations produced bythe University of California Santa Cruz UCSC. Differentially expressed genes with a fold change of >=2.0 and padj < 0.05 between CD8 T cells from perforin knockout mice or WT mice fed the MCD were submitted to GO and KEGG enrichment analysis, which uses unbiased methods to assess pathway enrichment. Result: Gene Ontology categories (biological processes) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analyses revealed that the DEGs in hepatic CD8 T cells from MCD-fed Prf1null mice are involved in cell adhesion, apoptotic process, cell migration, T cell proliferation, regulation of cytokine secretion, complement and coagulation cascades, etc. Compared with CD8 T cells from the steatotic livers of WT mice, hepatic CD8 T cells from MCD-fed perforin knockout mice showed significantly upregulated levels of anti-apoptotic genes (Ei24, Tnfrsf22, Tnfrsf23, Maf and Siah2), chemokines (Cxcl3), chemokine receptor (Ccr5), genes linked to cell activation (Sfpq and P2rx7), proinflammatory cytokine secretion (C1qtnf1, C1qtnf6, CD36, and Tek) and proinflammatory signaling pathways (Tnfsf12, Tnfsf14, Ptgs2, Mapk3, Mapk7, Mapk12, Il12rb2 and Grb2), and downregulated levels of apoptosis-promoting genes (Dffb and Tnfrsf21). The hepatic CD8 T cells mRNA profiles of MCD-fed WT mice and perforin konckout mice were generated by deep sequencing in duplicate, using Illumina HiSeq.

方法：为深入阐明穿孔素（perforin）在非酒精性脂肪性肝炎（NASH，Non-Alcoholic Steatohepatitis）进程中调控肝脏CD8+ T细胞介导炎症的分子机制，我们对喂食甲硫氨酸-胆碱缺乏（MCD，Methionine-Choline Deficient）饲料4周的野生型（WT，Wild Type）或穿孔素敲除（Prf1null）小鼠的肝脏中分离CD8+ T细胞，并对这些肝源性CD8+ T细胞进行mRNA测序。总RNA从经荧光激活细胞分选（FACS，Fluorescence-Activated Cell Sorting）分离的肝源性CD8+ T细胞中提取得到。转录组测序文库按照制造商操作指南，使用NEBNext® Ultra™ RNA文库制备试剂盒（适配Illumina®测序平台，NEB，美国）构建完成，并在Illumina Hiseq测序平台（Illumina，美国加利福尼亚州圣地亚哥）上完成测序。测序序列通过TopHat比对至参考基因组，随后使用Cufflinks进行数据分析；Cufflinks依托加州大学圣克鲁兹分校（UCSC，University of California Santa Cruz）生成的参考基因组注释信息，对每个样本中的各转录本进行定量。将喂食MCD饲料的穿孔素敲除小鼠与野生型小鼠的肝脏CD8+ T细胞间，满足差异倍数≥2.0且校正后P值（padj）<0.05的差异表达基因（DEGs）进行基因本体（GO，Gene Ontology）和京都基因与基因组百科全书（KEGG，Kyoto Encyclopedia of Genes and Genomes）富集分析，该分析采用无偏方法评估通路富集情况。
结果：基因本体（GO，Gene Ontology）生物学过程分类及京都基因与基因组百科全书（KEGG）通路富集分析结果显示，喂食MCD饲料的Prf1null小鼠肝源性CD8+ T细胞中的差异表达基因，参与细胞黏附、细胞凋亡过程、细胞迁移、T细胞增殖、细胞因子分泌调控、补体与凝血级联反应等生物学过程。与野生型小鼠脂肪变性肝脏中的CD8+ T细胞相比，喂食MCD饲料的穿孔素敲除小鼠肝源性CD8+ T细胞中，抗凋亡基因（Ei24、Tnfrsf22、Tnfrsf23、Maf及Siah2）、趋化因子（Cxcl3）、趋化因子受体（Ccr5）、细胞活化相关基因（Sfpq与P2rx7）、促炎细胞因子分泌相关基因（C1qtnf1、C1qtnf6、CD36及Tek）以及促炎信号通路相关基因（Tnfsf12、Tnfsf14、Ptgs2、Mapk3、Mapk7、Mapk12、Il12rb2及Grb2）的表达水平显著上调，而促凋亡基因（Dffb与Tnfrsf21）的表达水平显著下调。本研究通过Illumina HiSeq平台进行双重复深度测序，获取了喂食MCD饲料的野生型小鼠与穿孔素敲除小鼠的肝源性CD8+ T细胞mRNA表达谱。