Effect Of IL-23/IL-23R In The Development Of HBV-related Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is one of the most common malignant tumors in China and more than 90% of HCC is associated with chronic infection of hepatitis B virus (HBV). The HBV-related HCC is characterized with large amounts of infiltration of different types of immune cells, and these cells displayed different functions and phenotypes.Interleukin 23 (IL-23), composed of IL-23p19 and IL-12p40 two subunits, is a member of IL-12 family. Different from IL-12, IL-23 was found mainly involved in chronic inflammation, autoimmune diseases and chronic inflammation related cancers. IL-23 is mainly expressed by myeloid cells, including dendritic cells (DCs) and macrophages in response to different stimulation, such as microbial products. IL-12Rβ1 and IL-23 R were found to interact with IL-23 and IL-23 plays its role mainly through IL-23 R. However, high concentration of IL-23 was also found to interact with IL-12Rβ1. IL-23R expression plays a critical role in the differentiation of Th17 cells and the Th17 mediated chronic inflammation. The role of IL-23 in chronic HBV infection associated HCC still remain elusive.Distinct subtypes of CD4+Th cells play critical roles in mediating the immunity to virus and tumor. Firstly, we utilized a cohort of HBsAg-positive carriers which was established in 1992 in Qidong for HCC screening. All the participants in this cohort were followed every year and their serum samples were stored at-80 ℃. By using case-control study, we selected 50 HCC cases and 150 controls and quantified their serum levels of different Th subsets related cytokines by using multiple ELISA.The results showed that Th17 immunity related cytokines were elevated significantly in chronic HBV infected individuals with HCC outcome compared to those who remained as the chronic HBV infection after 10-15 years follow-up. By comparing the cytokine levels between participants with normal and those with abnormal liver functions, we found that IL-23 concentration in those with abnormal liver function status was significantly higher than those with normal liver function, indicating the important role of injured HBV-infected hepatocytes in stimulating the production of IL-23. Subsequently, we generated the monocyte-derived macrophages from healthy volunteers and from the patients with chronic hepatitis B and stimulated the cells with the cell lysates prepared from the immortalized human liver cell line (LO2 cells) or the LO2 cells transfected with 2x HBV(2xHBV-LO2 cells). Both in the healthy volunteers and in the chronic hepatitis B patients, increased production of IL-23 was found in the cells stimulated with the cell lysates of LO2 cells and of 2xHBV-LO2 cells. However, higher IL-23 production was observed in the macrophages stimulated with cell lysates from 2xHBV-LO2 cells, especially in chronic hepatitis B patients. After the macrophages were stimulated with HBsAg which contains small S protein only and HBcAg respectively, we found that HBcAg was more potent in inducing the production of IL-23.Interacting with its receptor expressed on different types of cells is indispensable for IL-23 to exert its effect. FACS analysis showed that IL-23R was mainly expressed on CD14+macrophages in HCC tissues, and the expression of IL-23R mRNA was positively correlated with VEGF mRNA. Since HCC shows high vasculature that provides sufficient nutrients for tumor growth, we then investigated the effect of IL-23 on macropahages in the angiogenesis for HCC development. By stimulating human macrophage cell line THP-1 cellsand monocyte-derived macrophages prepared from healthy voluteers with IL-23, we found IL-23/IL-23R can promote macrophages to produce VEGF via an autocrine manner. Tube formation assay in vitro further confirmed that IL-23 treated macrophages can promote angiogenesis. Finally, we treated HBV transgenic mice with diethylnitrosamine (DEN) at 2 weeks of age to initiate the malignant transformation of hepatocytes. After blocking IL-23R at 6 weeks,10 weeks and 16 weeks of age by injection of rat anti-IL-23R neutralizing antibodie for 4 weeks, both the tumor numbers (P=0.0007) and sizes (P=0.0079) decreased significantly when IL-23R was blocked starting at 6 weeks and 10 weeks of age, compared with the mice that received the rat IgGlisotype antibodies. In the mouse liver the VEGF expression reduced massively after the mice receive the IL-23R neutralizing antibodies. In addition, we found that the percentage of liverinfiltrated CD8+IFNγ+T cells increased remarkably after blocking IL-23R. Cytotoxic assay in vitrodemonstrated that the cytotoxic function of the T cells isolated from the mice administrated with IL-23R neutralizing antibodies enhanced dramatically.In this study, we find that IL-23 plays an important role in the development of chronic HBV infection associated HCC. Firstly, we found serum levels of IL-23 elevated in the chronic hepatitis B patients with HCC outcome through a cohort study. Then we identified that HBV core antigen is the major stimulator for the production of IL-23 by macrophages. After investigating the mechanisms, we foundIL-23 can promote macrophages to produce VEGF via an autocrine manner.In vivo study further confirmed that IL-23 can promote HBV related HCC progression. Our findings may provide a new potential target for future clinical diagnosis and treatment of HCC.