The Role Of IL-1α In Acute Liver Inflammation And Hepatocellular Carcinogenesis

Part I The differential roles of secreted and membrane IL-lα in carbon tetrachloride-induced acute liver injuryObjective:Interleukin-la is mainly expressed as the membrane form, while it can be released as the secreted form during inflammation. The roles of secreted and membrane IL-la in acute liver inflammation are still not known. Here we examined the functions of secreted and membrane IL-la in carbon tetrachloride-induced acute live injury,Methods:First, we constructed the minicircle plasmids which could express secreted or membrane IL-lα, and injected the plasmids into mice by hydrodynamic gene transfer method to induce their expression in the liver. Then the mice were injected with Carbon tetrachloride (CCl4) to induce acute liver injury. We examined the immune cell by flow cytometry and detected the cytokines levels in serum by CBA detection kits.Results:We showed that secreted IL-la aggravated acute liver damage and membrane IL-la slightly protected mice from liver injury. Further studies showed secreted IL-lα promoted T cell activation. It also significantly increased the expansion of MDSCs, and depletion of MDSC could aggravate acute liver injury. Therefore, MDSC may serve as the negative regulator of acute liver inflammation. Moreover, secreted IL-la induced IL-6 production from hepatocytes. IL-6 neutralization reduced the proliferation of MDSCs in vivo. CCL2 and CXCL5 expression was increased by secreted IL-la in vitro and in vivo. The antagonists of chemokine receptors for CCL2 and CXCL5 significantly reduced the migration of MDSCs.Conclusion:These results demonstrated that the secreted and membrane IL-la played different roles in acute liver injury. Secreted IL-la could promote T cell activation and the recruitment and expansion of MDSCs through the induction of CCL2, CXCL5 and IL-6. The controlled release of IL-la could be a critical regulator during acute liver inflammation.Part II The role of membrane IL-la in hepatocellular carcinoma developmentObjective:Hepatocellular carcinoma (HCC) is a worldwide health problem with limited treatment options and poor prognosis. Inflammation is associated with liver injury and hepatocyte regeneration, which leads to fibrosis, cirrhosis and eventually HCC. IL-la belonging to dual-function cytokines is one of the most important inflammatory cytokines involved in the inflammation and tumor development. IL-la presents as multiple forms in vivo, including precursor, propiece, membrane and secreted forms, and their functions were thought to be different. The role of membrane IL-la in HCC is still not clear. Here we examined the functions of membrane IL-la in hepatocellular carcinoma.Methods:Hepal-6 cells stably expressing membrane IL-la or vector control were constructed and three murine HCC models (subcutaneous HCC model, Orthotopic HCC model and DEN induced HCC model) were established. The expression of membrane IL-la was confirmed by confocal microscopy and western blot analysis. CCK-8, Ki67 and cell cycle methods were used to evaluate the bioactivity of hepal-6 expressing membrane IL-la. Subcutaneous HCC model and orthotopic HCC model were established by injecting hepal-6 cells. In DEN induced HCC model, mice were injected with minicircle plasmids by hydrodynamic gene transfer method. FACS analysis was used to examine the immnune cell subsets in tumor bearing mice. Intracellular staining was performed to examine the TNF-a and IFN-y expression by CD4+T, CD8+T or NK cells. Nonradioactive cytotoxicity assay was performed to evaluate the cytotoxic T cell activity. The serum levels of cytokines were measured by CBA detection kits.Results:Hepal-6 cells stably expressing membrane IL-la were established. Membrane IL-la promoted tumor cells proliferation. We showed that membrane IL-la potently inhibited tumor growth in all three HCC models. Further studies showed membrane IL-1α promoted T cell activation in a cell contact dependment manner. Moreover, membrane IL-1α increased the numbers of NK, DC and macrophages in tumor microenvironment. IFN-γ production by CD8+T and NK cells was increased by membrane IL-1α in vivo. Membrane IL-1α could also enhance cytotoxic T lymphocytes (CTL) activity.Conclusion:Our studies demonstrated that membrane IL-1α can promote anti-tumor responses through activation of T and NK cells. Thus, our finding offers new insights of IL-1α functions on tumor immunity, indicating its potential implications in tumor immunotherapy. membrane IL-1α promoted T cell activation in a cell contact dependment manner. Moreover, membrane IL-1α increased the numbers of NK, DC and macrophages in tumor microenvironment. IFN-γ production by CD8+T and NK cells was increased by membrane IL-1α in vivo. Membrane IL-1α could also enhance cytotoxic T lymphocytes (CTL) activity.Conclusion:Our studies demonstrated that membrane IL-1α can promote anti-tumor responses through activation of T and NK cells. Thus, our finding offers new insights of IL-1α functions on tumor immunity, indicating its potential implications in tumor immunotherapy.Part III The role of secreted IL-la in hepatocellular carcinoma developmentObjective:IL-la is a pro-inflammatory cytokine, which can stimulate the gene expression associated with inflammation. IL-la can be cleaved by calpain into secreted form. Secreted IL-la could be released from necrotic cells passively or actively secrete from cells. Secreted IL-la may play important role in inflammatory disease and tumor development. The role of tumoral secreted IL-la and systemic injection of secreted IL-la protein in hepatocellular carcinogensis is still not clear. Here we examined the functions of secreted IL-la in HCC.Methods:Hepal-6 cells stably expressing secreted IL-la or vector control were constructed and two murine HCC models (subcutaneous HCC model and Orthotopic HCC model DEN induced HCC model) were used. The expression of secreted IL-la was confirmed by confocal microscopy, western blot analysis and ELISA. CCK-8, Ki67 and cell cycle methods were used to evaluate the bioactivity of hepal-6 transfected cells. Subcutaneous HCC model and orthotopic HCC model, mice were injected with hepal-6 cells stably expressing secreted IL-la to study the role of tumoral secreted IL-lα. In orthotopic HCC model, mice were injected with hepal-6 cells and recombinant secreted IL-la protein to study the role of systemicaly secreted IL-la. FACS analysis was used to examine the immnune cell subsets in tumor bearing mice. Nonradioactive cytotoxicity assay was performed to evaluate the cytotoxic T cell activity. The serum levels of cytokines were measured by CBA detection kits.Results:We constructed hepal-6 cells stably expressing secreted IL-la. Tumoral secreted IL-lα almost had no significant effect on tumor cell bioactivity in vitro. Tumoral secreted IL-la significantly promoted tumor development in HCC models. Further studies showed tumoral secreted IL-la inhibited T and NK cell activation, and attenuated the CTL cytotoxic activity. Tumoral secreted IL-la aslo decreased the number of DC and neutropils in tumor microenvironment. Moreover, MDSCs were increased by tumoral secreted IL-la, and depletion of MDSCs could inhibit tumor growth. On the contrary, systemic injection of IL-la protein significantly inhibited the tumor development. Systemic administration of IL-la promoted T cell activation in vivo and in vitro. Secreted IL-la also could enhance cytotoxic T lymphocytes (CTL) activity in vitro.Conclusion:Our studies demonstrated that the roles of tumoral secreted IL-lα and systemicaly injected recombinant IL-la protein were different in HCC development. Tumoral secreted IL-la promoted HCC development through recruiting MDSCs to suppress T cell activation. Systemic injection of recombinant IL-la promoted anti-tumor responses through activation of T cells. Thus, our finding provides new insights of IL-la functions for tumor immunity, indicating its potential implications for tumor immunotherapy.