IL28B associated polymorphism, RS12979860, controls the activity of liver lymphocytes

Introduction The single nucleotide polymorphisms (rs12979860), near the IL28B gene, is correlated with a sustained virological response (SVR) in Hepatitis C Virus (HCV) infected patients treated with Pegylated Interferon-α combined with Ribavirin (1,2). However, the association of rs12979860 polymorphism with immune function of the liver, the site of HCV production, and the mechanism of SVR remain still undefined. Methods Patients chronically HCV-infected patients were genotyped for rs12979860 defining C/T polymorphism. C allele is associated to SVR. Liver samples were collected from the needle biopsy achieved for the diagnosis prior any treatment. Single cell suspensions were prepared by mechanical disruption. Liver lymphocytes (T, Treg, NK and NKT) were identified by flow cytometry for the expression of CD45, CD3, CD4, CD8, CD56 and FoxP3 as markers and CD107a for degranulation activity. Expression of CD8β, FoxP3, IL10 and HPRT genes was measured by PCR. Immunohistochemistry were performed on in paraffin sections for the detection of CD8 and FoxP3. Statistical analysis wwas done with Mann–Whitney U test and Wilcoxon matched-t test. Results Lymphocytes from 52 fresh liver biopsies displayed similar distributions of T (CD3), NKT (CD3, CD56) and NK (CD56) among CD45 cells by flow cytometry multi parametric analysis, whatever the IL28B genotype of the patients. Strikingly, higher degranulation activity, revealed by CD107a surface expression, was observed in T (p = 0.000), NKT (p = 0.002) and NK cells (p = 0.015) of patients with CC genotype (n = 17) compared to patients with CT or TT genotypes (n = 35); patients with CC genotype displayed two fold higher degranulation activity than patients with CT genotype in T (p = 0.001), NKT (p = 0.002) and NK cells (p = 0.011); no significant difference was observed between patients with CT and TT genotypes. Sections of liver from 19 patients showed the frequency of CD4-FoxP3 lymphocytes two fold higher (p = 0.004) in patients with CC genotype (n = 11) as compared to patients with CT genotype (n = 8) supporting the presence of Treg. Previous study demonstrated that the ratio between the number of CD8 cells and FoxP3 cells in parenchymatous necro-inflammatory areas is maintained in the early stage of the chronic hepatitis (3). This is found only in patients with CC genotype, whereas this ratio is reduced in patients with CT genotype due to lower number of FoxP3 cells. Transcriptional analyses confirmed these data and further showed two strong correlations between: one between FoxP3 and CD8β, another between FoxP3 and IL-10 expressions in patients with CC genotype. Conclusion Collectively these data provide new insights into the role of IL28B polymorphism related to SVR in treatment of HCV infected patients. CC genotype, which is linked to good response, is associated to higher efficiency of effector lymphocytes (T, NK and NKT) of the liver. The liver immune response appears tightly regulated as suggested by the links between CD8 cells and CD4-FoxP3 cells, and between IL10 and FoxP3 gene expression.