The Role Of IL-23 In The Development Of Hepatocellular Carcinoma

? The role of IL-23 in the development of HCCObjective: To explore the function of IL-23 in the development of HCC.Methods: Generation of murine HCC model:(a)hepa1-6 cells stably expressing IL-23 or vector control(1×106/2 ml)were injected into mice using hydrodynamic gene transfer(HGT)method,3 weeks later,mice were sacrificed and the number of tumor nodes were counted(b)Mice were given drinking water with antibiotic(80000 U/250 ml)before the surgery.hepa1-6 cells stably expressing IL-23 or control vector(1×106/25 ?l)were in situ transplanted into the mice by surgery.2 weeks later,mice were sacrificed and the tumor volumes were measured;(c)14 days-old mice were injected with diethylmitrosamine DEN(25 mg/kg)intra-peritoneally.IL-23 or vector micro-plasmid were injected hydropdymically every other month.10 months later,mice were sacrificed and the maxium tumor volumes and the number of tumor nodes were measured.Results: The promoting role of IL-23 in the development of HCC was confirmed by three kinds of orthotopic HCC models.In an orthotopic HCC model established by hydrodynamic injection of HCC cell lines,the numbers of tumor nodes in the liver at 3 weeks after tumor-cell injection were greater in the hepa1-6-IL-23 mice than in mice injected with hepa1-6-vector cells.We also created another orthotopic HCC model by surgical injection of hepa1-6-IL-23 or hepa1-6-vector cells intrahepatically.Measurement of tumor size 2 weeks later showed that overexpression of IL-23 led to increased tumor volumes.To confirm this result in a more physiologically relevant spontaneous tumor model,we used DEN to induce HCC and hydrodynamically injected minicircle-IL-23 every other month to express IL-23 in the liver during the development of HCC.Eight months later,mice given minicircle-IL-23 had more tumor nodes in the liver than did those given control vectors.Conclusion: IL-23 promoted the development of HCC.? IL-23 promoting HCC development depends on IL-17AObjective: To explore the mechanism of IL-23 promoting HCC development.Methods:(a)Flow cytometric analysis the the regulation of IL-23 to T cells mediated adaptive immune responses in the development of HCC.intracellular staining analysis the ability of CD4+ or CD8+ T cells secreting IFN-?,TNF-? or IL-17 A.(b)Using C BA assay analysis the expression level of IL-6,IL-9,IL-12 or IL-17 A.(c)Using IL-17A-/-and WT mice to confirm the function of IL-17 A during IL-23 promoting HCC development.Results: The data demonstrated that IL-23 promoted T cells secreting IL-17 A and inhibited CD8+ T cells secreting TNF-?.The expression level of IL-17 A in the serum confirmed the ability of IL-23 promoted IL-17 producing.Moreover,overexpressing IL-23 didn't affact the expression of IL-12 and IL-9,whereas increased IL-6 expressing during the late phase of HCC development.Further study using WT and IL-17A-/-mice confirmed that IL-23 promoted HCC development in an IL-17-dependent manner.Conclusion: The role of IL-23 promoting HCC development relied on IL-17 A pathway.? NCR-ILC3 cells play an important role in the development of HCCObjective: Further explore the cellular source of IL-17 A and the role of ILC3 in the development of HCC.Methods:(a)In the different stages of HCC development,flow cytometry was performed to study the source of IL-17 A.Intracellular staining to confirm the expression of IL-17 A in C D4+ T cells,CD8+ T cells,??T cells,and CD3-cells in liver cells during different stages of HCC development.(b)In the presence and absence of PMA/Ionomycin stimulation,the phenotypes of NCR-ILC3 cells were determined.(c)The proliferation ability of NCR-ILC3 cells in the presence of IL-23 was explored by Ed U assay in vivo.(d)The role of NCR-ILC3 cells in the development of HCC was determined by NCR-ILC3 cell adoptive transfer experiment.(e)NCR-ILC3 cells were co-cultured with C D4+ T cells,CD8+ T cells or ??T cells In vitro to examine the regulatory effects of NCR-ILC3 cells on CD4+ T cells,CD8+ T cells or ??T cells.(f)The percentage of ILC1 in the liver was measured in PBS group,mice bearing hepa1-6-vector or hepa1-6-IL-23 tumors.(g)The ability of ILC1 cells differentiating into ILC3 cells was examined in vitro by culturing ILC1 in the presence of IL-2 combined with IL-12,IL-1?,IL-23 or IL-1? and IL-23.(h)ILC1 cells were sorted from CD45.1 mice and transferred into mice bearing hepa1-6-vector or hepa1-6-IL-23 tumor to study the ability of ILC1 differentiating into ILC3 in the presence of IL-23 in vivo.Results: During the early phase of HCC development,IL-23 mainly promoted CD3-cells secreting IL-17 A.Later during HCC development,IL-23 mainly promoted ??T cells and CD4+ T cells secreting IL-17 A.And in the late phase of HCC development IL-23 mainly promoted CD8+ T cells producing IL-17 A.Furthermore,IL-17A-producing-CD3-cells were identified as NCR-ILC3 cells with and without PMA/Ionomycin stimulation.In vivo Ed U assay showed that IL-23 promoted the proliferation of NCR-ILC3 cells.The adoptive transfer experiment of ILC3 confirmed the pro-tumor role of NCR-ILC3 cells in the development of HCC.Furthermore,the coculture experiments of NCR-ILC3 cells and CD4+ T cel s,CD8+ T cel s or ??T cells revealed that NCR-ILC3 cells suppressed the secretion of IFN-? in CD4+ T cells,and promoted the apoptosis and inhibited the proliferation of CD8+ T cells.Moreover,the secretion of IFN-? and TNF-? was inhibited by NCR-ILC3 cells.The ability of IL-23 promoting ILC1 differention into ILC3 was established by both in vitro and in vivo experiments.Conclusion: we demonstrated in the current study that NCR-ILC3 cells were the initial responders to IL-23 to promote HCC development.They are critical in establishing IL-17-rich immune-suppressive tumor microenvironment...