CHANGES IN IMMUNOLOGICAL PARAMETERS AND LEUCOCYTE DNA METHYLATION IN PATIENTS WITH LIVER FIBROSIS AND CIRRHOSIS RELATED TO CHRONIC HCV INFECTION

Abstract

Introduction. The work studied the change in immunological parameters and leukocyte DNA methylation in patients with liver fibrosis and cirrhosis during chronic HCV infection. Changes in methylation and subsequent modulation of gene expression may play a role in the pathogenesis of hepatitis C virus infection. In cells susceptible to infection, hepatitis C virus activation is associated with increased DNA methyltransferase activity that suggests a shift in methylation profile. The aim of the study: to study the change in immunological parameters and leukocyte DNA methylation in patients with liver fibrosis and cirrhosis during chronic HCV infection.Material and methods. The study involved 120 subjects divided into five groups. Group 1 included 40 healthy volunteers, group 2 (n = 27) - patients with signs of previous HCV infection (post-infection status), group 3 - 15 patients with chronic HCV infection without liver fibrosis, group 4 - 16 patients with chronic HCV infection and liver fibrosis (stage F1.2 according to elastography), group 5 - 22 patients with chronic hepatitis C and verified liver cirrhosis (stage F4 according to elastography). All study participants were examined for serum level of proinflammatory cytokines, including interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) and anti-inflammatory interleukin-10 (IL-10), as well as alarmins such as HMGB1 protein, interleukin-1α (IL-1α), and interleukin-33 (IL-33) analyzed by enzyme-linked immunosorbent assay (ELISA) using standard test kits. Uric acid levels were measured by a biochemical method using diagnostic kits from the HUMAN (Germany). Leukocyte DNA methylation was assessed by the concentration of 5-methyl-2'-deoxycytidine formed due to methylation of cytosine at position C5. The supernatant of washed lysed leukocytes isolated by gradient centrifugation using the Ficoll-Verografin medium was used for the analysis. The concentration of 5-methyl-2'-deoxycytidine was determined by ELISA using a standard kit from BCM Diagnostics (USA). DNA methyltransferase 1 (DNMT1) activity was also measured in supernatantized leukocytes by ELISA using HUMAN (Germany) kit.Results: At the early stage (post-HCV), a moderately increased DNMT1 activity and 5-mdC levels in leukocyte lysates mirror a compensatory mechanism aimed at suppressing proinflammatory genes. During chronic HCV infection without fibrosis, maximum DNMT1 values point at active suppression of viral replication and inflammation through hypermethylation. In chronic HCV infection and fibrosis (F1,2), a decrease in DNMT1 and 5-mdC evidence about exhausted epigenetic regulation, which contributes to profibrotic gene activation. At the stage of liver cirrhosis (F4), coupled to HCV infection, a significant decrease in DNMT1 and 5-mdC confirms deep epigenetic dysfunction associated with irreversible liver damage. In this case, inflammatory imbalance and fibrogenesis are manifested as progressively increased serum proinflammatory cytokine levels (TNF-α, IL-1β, IL-1α, IL-33) upon transition from chronic infection to cirrhosis, forming a vicious circle of inflammation and fibrosis. Anti-inflammatory IL-10 is sharply reduced in cirrhosis, exacerbating the imbalance towards pro-inflammatory states. HMGB1 in fibrosis stimulates the activation of stellate cells via TLR4, enhancing collagen synthesis and fibrosis. Uric acid in cirrhosis correlates with the disease severity, which may reflect impaired liver and kidney function.

Conclusions: The progression of HCV disease from the post-infection stage to cirrhosis is characterized by a gradual depletion of epigenetic regulatory mechanisms (DNMT1/5-mdC), which leads to elevated proinflammatory status and activation of fibrogenesis. Study of DNA methylation markers in leukocytes together/in parallel with cytokine profile allowed to identify deeper immune mechanisms of HCV development from post-infection to cirrhosis. Levels of DNMT1 and 5-mdC in leukocytes can serve as potential markers of HCV infection stage and risk of fibrotic complications.