T Cell ERK Signaling May Contribute To The Development Of Biliary Atresia Through Enhanceing Overexpression Of Methylation Sensitive Gene Interferon Gamma

Biliary atresia (BA) is a progressive obliterative process involving the extrahepatic and intrahepatic bile ducts in the newborn. It is characterised by worsening cholestasis, hepatic fibrosis, and cirrhosis, which lead to portal hypertension and a decline in hepatic synthetic function. The etiology of BA remains unknown. It has recently been proposed that the perinatal/acquired form of BA may be caused by a biliary trophicviral infection, leading to an initial bile duct epithelial injury that triggers a persistent immune-mediated sclerosing process resulting in obstruction of extrahepatic bile ducts. One theory suggests that it may result from a primary perinatal hepatobiliary viral infection that elicits autoimmune-mediated bile duct injury associated primarily with CD4+Th-1(T helper1) cell-mediated inflammatory processes and genome-wide association studies have identified BA susceptibility loci on several chromosomes. However, studies carried out in twins demonstrated that non-genetic factors also play an important role in mediating generalized BA pathogenesis even though specific causes of generalized BA remain obscure since no common (specific) environmental factors that trigger disease progression (either directly or via an autoimmune response) have yet been identified.To better understand and clarify the mechanism of BA, we performed a genome-wide DNA methylation study in peripheral blood mononuclear cells from infants with BA. We investigated genomic and gene specific DNA methylation levels in CD4+T cells of patients with BA and observed the methylation and expression of autoimmunity-related gene interferon gamma (IFN-y). We also evaluated the relationship between ERK transduction pathway and DNA Methyltransferase1in BA, and investigated the effects of ERK pathway in CD4+T cells and Jurkat cells in vitro. Part One Genome-wide DNA methylation patterns analysis in peripheral blood mononuclear cells from infants with biliary atresiaAims The pathogenesis of biliary atresia (BA) is still unknown. Recent evidence strongly supports an epigenetic contribution to the pathogenesis of BA. To better understand and clarify the mechanism of BA, we performed a genome-wide DNA methylation study in peripheral blood mononuclear cells (PBMCs) from infants with BA.Methods Peripheral blood was collected from patients and controls. PBMCs were isolated by Ficoll-Hypaque density gradient centrifugation. Whole genomic DNA was then prepared using a Qiagen DNEasy kit and bisulfite-treated using a Zymo EZ DNA Methylation Kit. Genome-wide PBMCs DNA methylation was measured using the Illumina Infinium HumanMethylation450BeadChip assay, which interrogates more than480,000cytosine-guanine (CpG) dinueleotide sites, selected predominantly from the promoter regions of the entire genome. Two probes were designed to each CpG site: a ’methylated’ and an ’unmethylated’ query probe. Meanwhile, bioinformatic analysis including Normalization, Background, Average and None was applied to classify the differentially methylated sites in BA. Absolute methylation values (β-values) were extracted with the GenomeStudioTM software and then subjected to detailed analysis.Results We identified5611CG loci with differential methylation in PBMCs between cases and controls. We observed over four times more hypomethylated loci compared to hypermethylated loci in BA patients:4300and1311, representing1375and704unique genes, respectively. Among the1375hypomethylated genes in BA patients,25belong to the "autoimmunity" category. Of interest, the interferon gamma gene (IFN-γ), a methylation-sensitive gene was also hypomethylated in BA patients. Among the704hypermethylated genes in BA PBMCs, nineteen were represented in the "autoimmunity" category.Conclusions Our data indicate the presence of hypomethylated and hypermethylated loci in BA PBMCs. We observed that CpG sites in the entire genome of BA were largely hypomethylated, and some for autoimmunity-related genes, including the methylation-sensitive gene IFN-y. This work provides novel insights that could help to better understand the pathogenesis of BA. Part Two Abnormal DNA methylation and interferon gamma gene expression in CD4+T cells from patients with biliary atresiaAims Biliary atresia (BA) is a virus-induced autoimmune disease. Abnormal DNA methylation patterns contribute to many autoimmune diseases. In this study, we have examined DNA methylation patterns in BA, and investigated genomic and gene specific DNA methylation levels in CD4+T cells of patients with BA, specially interferon gamma (IFN-y) gene.Methods We quantified global methylcytosine levels in CD4+T cells from15patients with BA and12healthy controls. mRNA levels of DNA methyltransferases (DNMTs), methylated CpG binding proteins (MBDs) and IFN-y were measured by real-time reverse transcriptase-PCR. Methylation of an IFN-y regulatory element domain was determined by bisulfite genomic sequencing.Results CD4+T cells genomic DNA from infants with BA were hypomethylated relative to healthy controls (p<0.0001). Compared with controls, DNMT1and DNMT3a mRNA levels were significantly lower in BA CD4+T cell (p=0.0012and p=0.0210, respectively). MBD1mRNA expression was significantly lower in CD4+T cells (p=0.0029) from BA infants than in controls, whereas MBD4mRNA expression was significantly increased in CD4+T cells (p=0.0199). DNMT1expression positively correlated with global DNA methylation in BA CD4+T cells (r=0.6290, p=0.0120). IFN-γ mRNA expression was significantly increased (p=0.0376), and an IFN-γ promoter region was hypomethylated in BA CD4+T cells relative to controls (p=0.0035) and negatively correlated with DNA methylation (r=-0.5720, p=0.026).Conclusions These data show that thelevels of DNA methylation are altered in CD4+T cells of patients with BA, which suggest that DNA methylation changes may contribute to the disease by affecting the expression of autoimmunity-related genes. Part Three ERK pathway signaling in CD4+T cells from patients with biliary atresia and effects on DNA Methyltransferase1Aims Current evidence indicates that a pathologic CD4+T cell subset, characterized by impaired extracellular signal-regulated kinase (ERK) pathway signaling, DNA hypomethylation, and consequent aberrant gene expression contributes to disease pathogenesis. To evaluate the relationship between ERK transduction pathway and DNA Methyltransferase1(DNMT1) in biliary atresia (BA). We investigate the effects of ERK pathway signaling in CD4+T cells and Jurkat cells in vitro.Patients and methods Peripheral blood was collected in from20patients and15controls. Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll-Hypaque density gradient centrifugation, and CD4+T cells were isolated by positive selection using magnetic beads as described by the manufacturer. Total proteins were extracted from CD4+T cells. We used Western-blot to compare ERK1and2, phosphorylated ERK1and2(p-ERK1/2), and DNMT1protein levels in CD4+T cells from BA patients and healthy control. In vitro, Jurkat cells were cultured, and then treated with ERK pathway signaling inhibitor PD98059. ERK1/2, p-ERK1/2and DNMT1protein levels were determined by Western-blot in Jurkat cells.Results The protein levels of ERK1/2, p-ERK1/2and DNMT1in CD4+T cells from infants with BA were significantly lower compared with controls (p<0.05). Meanwhile, the protein levels of ERK1/2, p-ERK1/2and DNMT1in Jurkat cells treated with PD98059were significantly decreased than those in the untreated groups (p<0.05).Conclusions The protein levels of ERK1/2, p-ERK1/2and DNMT1in BA CD4+T were significantly decreased. We show that decreased ERK pathway signaling in T cells results in decreased expression of DNA methyltransferase1and overexpression of the methylation-sensitive genes.