ABSTRACT: 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).
