The Role Of Toll Like Receptor-2in Murine Hepatocellular Carcinoma

Hepatocellular carcinoma (HCC) is a risk to human health. HCC, as a kind of high mortality disease, is thought to be closely related to the chronic inflammation. Chronic viral infections, metabolic abnormalities and toxic ingestion are thought to be responsible to liver cancer. Tumor development and progression are not only due to DNA mutations but also to the tumor microenvironment. The tumor microenvironment include expression of cytokines and chemokines, infiltration of surpressor cells(regular T cell, M2and MDSC) and exhaustion of tumor-killing cells (NK, CTL). The common phenomenon observed in tumor development is up-regulation of inhibitory cytokines (IL-10, TGF-β) and accumulation of surpressor cells. We wonder to know how to suppress these and we think this will be the key of tumor immunotherapy.Toll like receptors are well studied in tumor development. Identification of the role for each TLR is important for tumor immunotherapy. Agonists of TLRs (e.g., TLR3,7,8and9) have been successful in treating a variety of tumors in mice and are currently being tested in clinical trials. In our study, we also find that TLR2agonist can be used in immunotherapy of HCC. First, we found TLR2expression was down-regulated by flow cytometry analysis and western blot analysis in DEN-induced tumor model. Then, we found that Tlr2-/-mice exhibited more aggressive HCC development. By tumor determination, flow cytometry analysis and western blot analysis, we came to the conclusion that caspase-8-IL-18-MDSC-CTL interaction was responsible for the tumor susceptibility of Tlr2-/-mice. Silencing IL-18in Tlr2-/-mice via hepatocyte-specific shRNA or systemic administration of the TLR2agonist Pam3CSK4in WT mice can reverse the caspase-8-IL-18-MDSC-CTL interaction and inhibit tumor growth. These results may provide new insight for HCC immunotherapy targeting TLR2. Based on the research and experimental method, we have the conclusion as flows:1. TLR2is downregulated in hepatocarcinogenesis, and tumor growth is inhibited after TLR2activation.To explore the role of TLR2in hepatocarcinogenesis, we established a tumor model by a single injection of DEN to14-day-old male mice. All mice developed HCC8months after DEN induction. Meanwhile, we observed reduced TLR2 expression on hepatocytes in the livers of DEN-treated mice. To examine the effect of TLR2on hepatocarcinogenesis, the extensively used TLR2agonist Pam3CSK4was administered to DEN-induced mice. This treatment attenuated HCC development with reduced tumor number and size. Thus, we conclude that TLR2plays a role in the development of liver cancer.2. TLR2deficiency promotes HCC.To further investigate the role of TLR2in HCC development, WT and Tlr2-/-mice were subjected to DEN treatment. Tumor number and maximum tumor size were dramatically increased in Tlr2-/-mice compared to WT mice. Meanwhile, tumor incidence in Tlr2-/-mice was greater than that in WT mice at different time points. Pathological analysis revealed that Tlr2-/-mice had larger tumor areas than WT mice. Tlr2-/-mice with HCC displayed increased PCNA levels. Moreover, Tlr2-/-mice had much higher Afp levels than WT mice. Therefore, TLR2normally plays a protective role against hepatocarcinogenesis.3. CD8+T cell function is impaired in Tlr2-/-mice.It is well accepted that tumor development and progression are not only due to DNA mutations but also to the tumor microenvironment. Although T cells are reportedly involved in DEN-induced HCC in immunodeficient mice, the mechanism by which T cells become dysfunctional in this model remains unknown. We found that hepatic and splenic CD8+T cell expression of PD-1, but not TIM-3, was much higher in Tlr2-/-mice, and that TNF-a production was significantly lower. Moreover, CD8+T cell perforin production was decreased in Tlr2-/-mice following DEN injection. These data indicate that CD8+T cell function is impaired in Tlr2-/-mice following DEN-induced carcinogenesis, suggesting that carcinogenesis is accompanied by immunosuppression.4. Enhanced MDSC accumulation and function in DEN-treated Tlr2-/-mice.In our model, we found that Ly6Chigh MDSCs were much more prevalent in Tlr2-/-mice than in WT mice in different organs after DEN injection. Evaluating the kinetics of Ly6Chigh MDSC, Ly6Chigh MDSCs were significantly increased at5months post-DEN injection in Tlr2-/-mice compared to WT mice. Since Nos2(iNOS) were increased in the liver after DEN treatment, we found that iNOS inhibition could attenuate the inhibitory effects of Ly6Chigh MDSCs on IFN-y production and CD8+T cell proliferation. Meanwhile, WT and Tlr2-/-MDSCs similarly inhibited IFN-y production and CD8+T cell proliferation, and we also found that Tlr2-/-and WT MDSCs showed a similar phenotype and activation status. The only observed difference between Tlr2-/-and WT MDSCs was increased Ki-67in Tlr2-/-Ly6Chigh MDSC after DEN treatment. These data indicate that Ly6Chigh MDSCs may promote HCC via iNOS-mediated inhibition of anti-tumor immunity, and the enhanced MDSC accumulation in Tlr2-/-mice may be due to their augmented proliferative ability.5. Hepatocyte-derived IL-18stimulates the generation of hepatic MDSCs.To identify factors that contribute to MDSC accumulation in the liver, we examined a set of inflammatory cytokines and found that only Il18mRNA was increased in the livers of Tlr2-/-mice. Immunohistochemistry and western blot demonstrated that hepatocytes were the main IL-18producers. The increased IL-18expression and greater monocytic MDSC accumulation we observed here after DEN injection raised the possibility that hepatocyte-derived IL-18might be linked to MDSC accumulation. To test this hypothesis, we detected the IL-18receptor IL-18Ra on MDSCs and found that it was upregulated on MDSCs compared to normal controls. Ly6Chigh MDSCs but not Ly6G+MDSCs, from Tlr2-/-mice expressed higher IL-18Ra than WT mice, indicating that IL-18might drive Ly6Chigh MDSC accumulation in the livers of Tlr2-/-mice. As expected, recombinant IL-18induced Tlr2-/-Ly6Chigh MDSC accumulation when MNCs were cultured with IL-18in vitro, especially following DEN treatment. When IL-18was overexpressed in hepatocytes by hydrodynamically injecting an IL-18-expressing plasmid in vivo, we found a dose-dependent increase in MDSC accumulation in the liver.6. Silencing IL-18inhibits MDSC accumulation and tumor growth.We knocked down IL-18in hepatocytes by hydrodynamically injecting an shIL-18plasmid once every2weeks for7months. Although no difference in tumor growth was observed between WT/mock and WT/shIL-18mice after8months of DEN treatment, silencing IL-18in hepatocytes significantly attenuated tumor progression in Tlr2-/-mice, which was accompanied by decreased accumulation of Ly6Chigh MDSCs and recovered TNF-a production by CD8+T cells.7. DEN-induced IL-18processing is caspase-8-dependent.We then examined the relationship between TLR2and IL-18. We found that Casp8mRNA was significantly increased after DEN treatment in Tlr2-/-mice. Pam3CSK4agonist-stimulated TLR2activation inhibited caspase-8expression, raising the possibility that IL-18correlated with TLR2-mediated activation of caspase-8. Therefore, we designed a caspase-8shRNA to explore the effect of caspase-8on IL-18synthesis. Tlr2-/-shcaspase-8mice exhibited decreased serum ALT and IL-18levels compared to Tlr2-/-mock mice48hours after DEN injection, suggesting that IL-18expression might be caspase-8-dependent in Tlr2-/-mice.8. Pam3CSK4stimulation reduces MDSCs and restores immunity against HCC.Based on the results mentioned above, TLR2agonist Pam3CSK4alleviated HCC progression. We further found that Pam3CSK4treatment reduced both Ly6Chigh and Ly6G+MDSCs, and this treatment also importantly reduced serum IL-18levels. Pam3CSK4stimulation also upregulated the frequency and number of activated CD8+T cells as assessed by CD69expression. Moreover, inflammatory cytokines known to play crucial roles in anti-tumor activity were increased after Pam3CSK4treatment in WT mice. Together, these results demonstrate that TLR2activation by Pam3CSK4may inhibit HCC progression by inhibiting the IL-18-MDSC-CTL interaction.Conclusion:Our findings show how TLR2deficiency accelerates IL-18-mediated immunosuppression during liver carcinogenesis, providing new insights into immune control that may assist the design of effective immunotherapies to treat hepatocellular carcinoma.