In this study, we report for the first time that TL1A methylation levels were significantly reduced in PBMC consistent with the result that mRNA expression level in serum and PBMC were significantly increased in patients with HBV- associated cirrhosis. In LC group, TL1A methylation level markedly reduced in patients more than 50 years old, CTP score more than C stage, LSM more than 12.5Kpa, with positive HBV DNA and symptoms of decompensation compared with the other groups. We further observed that there was a significant positive correlation between TL1A methylation level and MELD score and LSM of patients. Methylation, as a high-throughput quantitative methylation detection method has high sensitivity and specificity. In addition, CpG islands refer to DNA fragments containing large amounts of CpG, which usually leads to gene silencing. In this study, ROC curves of TL1A methylation level showed significantly higher than that of LSM and MELD score in discriminating HBV- associated LC from CHB.
For patients with HBV-associated cirrhosis, early detection and anti-viral treatment is the key to delay the development of liver fibrosis and improve the survival. At present, the gold standard for diagnosis of cirrhosis was liver biopsy. However, due to its invasiveness, clinical diagnosis is mostly based on decompensated symptoms of cirrhosis, combined with imaging examination such as transient elastography (TE) but the early diagnosis of cirrhosis is difficult. TE test liver stiffness measurement (LSM) was use as diagnostic marker for LC is limited owing to image examination is easy affected by patient positions and instrument. In this research, we quantitatively assessed the methylation level of TL1A and ROC curve was performed to further evaluate its diagnostic value. As shown above, TL1A methylation level had a significantly better performance than LSM. These results might indicate that TL1A methylation level have the potential to be one of the non-invasive biomarker for LC diagnosis.
Previous reports have suggested elevated serum TL1A levels in chronic liver inflammatory diseases such as PBC and in mice overexpression of TL1A can accelerate the process of liver fibrosis and worsen liver function.[9, 11] However, the expression and methylation level of TL1A in human HBV-associated cirrhosis has not been clearly defined. Therefore, in this study, we firstly quantitatively assessed TL1A promoter methylation status in HBV -associated LC patients using blood samples and came to the conclusion that TL1A was hypomethylated in LC. It is also noteworthy that the methylation status of the TL1A promoter was detected only in PBMC, but not in liver tissue, mainly because the present study was designed to evaluate the diagnostic value of TL1A methylation levels as a non-invasive biomarker. In addition, peripheral blood mononuclear cells including monocytes, T cells, B cells and natural killer cells, are considered the first line of the immune system to defend against inflammation, and previous studies have reported that various diseases can influence gene expression in PBMC through host immune or inflammatory responses. [22, 23]
Meanwhile, the result that mRNA level was significantly higher in LC group than CHB and HC groups and PMR value of TL1A was significantly negatively correlated with mRNA expression level in PBMC. However, it is noteworthy that a large reduce in TL1A methylation was observed while mRNA increased not as much as reduced methylation level. The degree of inconsistency may be due to the fact that not only promoter methylation can influence the transcription process, but also microRNAs, histone modifications, and transcription factors.[24, 25] Previous studies have shown that the expression level of serum TL1A and mRNA can also be increased in other liver diseases, such as the late-stage CHC. In addition, elevated serum TL1A levels have been reported in various chronic inflammatory diseases such as IBD [7, 8] and RA . Previous studies were also identified TL1A in intrahepatic small bile ducts as well as infiltrating mononuclear cells. In addition, up-regulation of TL1A and its decoy receptor has been demonstrated in PBMC and plasma of sickle cell anemias.[8, 26] These results suggest that TL1A is involved in the pathogenesis of these chronic inflammatory diseases. In this study, we found that TL1A mRNA expression levels in HBV-associated LC groups and CHB groups were significant increase in PBMC. These results may indicate that TL1A is not only involved in the pathogenesis of LC, but also serves as a common feature of chronic liver inflammation in CHB, although there was no statistically significant difference(p = 0.065).
The important mechanism for macrophages to participate in liver fibrosis is to secrete a series of proinflammatory and profibrotic factor such as IL-1β and TNF-α to perpetuate the proinflammatory pro-fibrotic stimulus, and overexpressed TL1A can up-regulate the expression of TNF-α, and IL-1β in liver tissues and macrophages. In vitro, TL1A is induced in endothelial cells by TNF-α and IL-1β, and in monocytes and dendritic cells by stimulation with TLR ligands. Previous data showed that cultured biliary epithelial cells (BECs) constitutionally express TL1A and various human TLRS and produce inflammatory cytokines and chemokines when stimulated by TLR ligands in vitro.[28, 29] In additional, LPS binding to TLR4 also induces activation of NF-κB and stimulates up-regulation of TNF-α, IL-6 and IL-1β, all of which have been implicated in inflammatory diseases. Therefore, we tested the ability of TLR ligands to induce expression of TL1A in PBMC and found that LPS could in higher concentrations induced TL1A at the mRNA level and detected the expression levels of TNF-α, IL-1β and IL6 in patients with HBV-associated cirrhosis. We found that the expression levels of TNF-α, IL-1β were significantly increased than the other groups, the increase of IL-6 expression level was not statistically significant. These results may indicate that TL1A is involved in the development of HBV-associated cirrhosis inflammation and fibrosis, and that after LPS stimulation can up-regulate the expression of hepatocirrhosis related inflammatory factors such as TNF-α, IL-1β.
Interaction between TL1A and DR3 in-vitro can activate both NF-κB pathways which being pro-inflammatory results in cytokine secretion, cell proliferation and cell activation. In this study, we demonstrated that serum TL1A and DcR3 levels was significant increases in serum TL1A and DcR3 levels were observed in HBV-associated LC than CHB and HC groups. This observation is consistent with previous studies in patients with other cirrhosis and autoimmune diseases such as IBD [7, 8], SLE , and RA .
There were some limitations to this study. First, the sample size was relatively small and all patients were from a single center, which might lead to selection bias. In addition, the possibility of false positive clinicopathological features were not negligible. Secondly, we only assessed the changes in the expression levels of relevant inflammatory factors before and after LPS stimulation, and the regulatory relationship between them and TL1A has been poor know.
In conclusion, TL1A promoter methylation level was hypomethylated in patients with HBV-associated LC. PMR value of TL1A promoter was showed significantly higher diagnostic value in distinguishing patients with HBV-associated LC from those with CHB than LSM testing, suggesting that TL1A methylation level in PBMCs might serve as a promising non-invasive diagnostic biomarker for HBV-associated LC in the future. In additional we showed that serum and PBMC TL1A and DcR3 levels were increased in HBV-associated LC patients, these changes indicating that TL1A and DcR3 might be involved in the pathogenesis of HBV-associated liver cirrhosis. The exact role of TL1A in the pathogenesis of LC as well as its potential as a therapeutic target and prognosis need to be further studied.