- Email: [email protected]
366 LIVER STROMAL CELLS PROMOTE TUMOR GROWTH VIA RECRUITING MYELOID-DERIVED SUPPRESSOR CELLS
POSTERS Methods: Expression patterns of markers related to CAM and AAM were investigated in mice with CCL4-induced liver fibrosis and spontaneous biliary (Mdr2 KO). For functional studies, AAM were inhibited with a blocking IL-4Ralpha antisense oligonucleotide (ASO) in vitro (murine RAW macrophages) and in vivo (6 weeks CCL4-treated and Mdr2 KO mice). Fibrosis and fibrogenesis were measured by collagen quantification, qPCR and immunohistochemistry for inflammation and fibrosis related transcripts. Results: In the CCL4 model, IL-4Ralpha expression was strongly reduced 2 weeks after the last dose. In Mdr2 KO mice, expression of the AAM inducing receptors IL-4Ralpha gradually increased until age 6-wk, and decreased thereafter. In RAW macrophages the ASO significantly suppressed IL-4Ralpha. When given to CCL4treated mice twice weekly from week-2 to week-4 after gavage, the ASO suppressed IL-4Ralpha and increased the CAM markers CCL3, MMP-8 and MMP-9, and decreased expression of profibrogenic procollagen alpha1(I) and the AAM markers Arg1 and Mrc1. Mdr2 KO mice that received ASO from week 6 to week 10 of age showed a similar shift from AAM to CAM. These mice displayed a mild increase in ALT due to AAM suppression. Conclusions: Expression of profibrogenic molecules and fibrogenesis are paralleled by increased hepatic AAM and concomitant IL-4Ralpha expression, suggesting a significant contribution of AAM to hepatic fibrogenesis. Modulation of AAM macrophages by specific pharmacological intervention is a promising antifibrotic approach to inhibit fibrosis progression and induce its reversal. 365 IDENTIFICATION OF PEKIN DUCK INTERFERON LAMBDA-3 AND INITIAL ASSESSMENT OF ITS ANTIVIRAL EFFECTS IN THE DUCK HEPATITIS B MODEL Q. Yao, K.P. Fischer, K. Arnesen, L. Tyrrell, K.S. Gutfreund. University of Alberta, Edmonton, AB, Canada E-mail: [email protected] Introduction: Interferon lambda-3 (IFN-lambda;3), also known as interleukin-28B (IL-28B), belongs to the recently discovered type III interferons and IL-10 family of cytokines. Type III interferons induce antiviral responses similar to type I interferons but signal through a different heterodimeric receptor complex (IFN-lambda;R1/IL-10R2). IFN-lambda;3 inhibits replication of many viruses, especially those affecting epithelial cells of the respiratory and gastrointestinal tracts and the liver. The aim of this study was to identify and characterize duck IFN-lambda;3 (duIFN-lambda;3) to study its role in the immunopathogenesis of duck hepatitis B virus (DHBV) infection. Methods: The duIFN-l3 cDNA was obtained by RT-PCR and RACE. A homology model of duIFN-l3 was obtained using the structure of human IFN-l3 as a template. A eukaryotic expression vector was generated for expression of a C-terminal-His6 -tagged IFN-l3 protein and culture supernatants of transfected 293T cells were assessed by immunoblot using an anti-His antibody. Primary duck hepatocyte cultures generated from two-week-old DHBV-negative ducklings were infected with DHBV at a MOI of 75 for 6 hours and treated with recombinant duIFN-l3 or control-treated. Intracellular virus (ICV) and 2 -5 -oligoadenylate synthetase-like (OASL) mRNA expression were assessed by real-time PCR/RT-PCR. Results: The predicted 185 amino acid protein had an amino acid identity of 63% and 37% with chicken and human IFN-l3 proteins, respectively. The duIFN-l3 structure by homology modeling was similar to that of human IFN-l3. Mapping the duIFN-l3 cDNA with duck genomic sequences revealed a five exon-four intron gene structure similar to that of chicken and human IFN-l3 genes. Recombinant duIFN-l3 up-regulated OASL mRNA expression 100fold by 24 hours but had only a modest effect on ICV after 96 hours of treatment.
Conclusion: Our observations suggest evolutionary conservation of genomic organization and structural features implicated in receptor binding of IFN-l3 and demonstrated bioactivity of the expressed duIFN-l3 protein. The identification and expression of duIFN-l3 will allow the study of the role of type III interferon in the immunopathogenesis of DHBV infection and may facilitate the exploration of novel immunotherapeutic strategies in the duck hepatitis B infection model. 366 LIVER STROMAL CELLS PROMOTE TUMOR GROWTH VIA RECRUITING MYELOID-DERIVED SUPPRESSOR CELLS G. He1 , H. Zhang1 , Y. Chen1 , B. Wang2 , Y. Kong2 , B. Jia2 , P. Miao2 , X. Xie1 , L. Wei1 , H. Chen1 . 1 Peking University People’s Hospital, Peking University Hepatology Institute, Beijing Key Laboratory of Hepatitis C and Immunotherapy for Liver Diseases., 2 Beijing Ditan Hospital, China Capital Medical University, Institute of Infectious Diseases, Beijing, China E-mail: [email protected] Background and Aims: Immune suppressor mechanisms such as Tregs or MDSCs during hepatocellular carcinoma (HCC) progression are being uncovered. We showed here the critical role of liver stroma in the process of MDSCs accumulation and tumor progression in HCC. Methods: Murine hepatoma cells were injected subcutaneously to BALB/c mice with or without liver stromal cells to obtain tumorbearing mice. Purified MDSCs were labeled with CFSE or DiR for in vivo migration assay. Liver stromal cells were used to mimic the liver microenvironment for in vitro chemotaxis assay. Neutralizing antibodies were employed for in vivo and in vitro blockade. Immunohistochemistry was performed to detect pathological injury and inflammation in liver and tumor tissue. Results: Adoptively transferred MDSCs accumulated mainly into liver and partially in spleen both in normal mice and tumorbearing mice. Interestingly, much more adoptively transferred MDSCs accumulated into liver site in tumor-bearing mice than that in tumor-free mice as confirmed by flow cytometry and living image. Adoptively transferred MDSCs also accumulated in tumor tissues in tumor-bearing mice. MCP-1 and SDF-1 expressed at high level in liver stromal cells. We examined the expression of MCP-1 and SDF-1 in liver stromal cells after exposure to heaptoma cellcultured supernatant and found an increased expression of the two chemokines. In vitro chemotaxis assay showed liver stromal cell derived factors promote MDSCs migration which could be reversed by MCP-1 and SDF-1 neutralizing antibodies. Liver stromal cells also promote hepatoma growth when co-injected with tumor cells, which may be attributed to increased MDSCs accumulation as detected by living image. Tumor growth could be significantly reduced in vivo by MCP-1 and SDF-1 neutralization. Besides, in HCC patients, tumor associated stroma cells and CD11b+ myeloid cells are highly enriched in peri-tumor tissues, which is accompanied by high expression of MCP-1 and SDF-1. Conclusions: These results demonstrate that liver stromal cells promote MDSCs migration via SDF-1 and MCP-1 in hepatomabearing mice and thus foster tumor progression. Understanding the cross-talk between liver stroma and MDSCs in HCC may provide a promissing breakthrough for further immunotherapeutic strategies. Gaixia He and Henghui Zhang contributed equally to this work.
Journal of Hepatology 2013 vol. 58 | S63–S227
S151
Conclusion: Our observations suggest evolutionary conservation of genomic organization and structural features implicated in receptor binding of IFN-l3 and demonstrated bioactivity of the expressed duIFN-l3 protein. The identification and expression of duIFN-l3 will allow the study of the role of type III interferon in the immunopathogenesis of DHBV infection and may facilitate the exploration of novel immunotherapeutic strategies in the duck hepatitis B infection model. 366 LIVER STROMAL CELLS PROMOTE TUMOR GROWTH VIA RECRUITING MYELOID-DERIVED SUPPRESSOR CELLS G. He1 , H. Zhang1 , Y. Chen1 , B. Wang2 , Y. Kong2 , B. Jia2 , P. Miao2 , X. Xie1 , L. Wei1 , H. Chen1 . 1 Peking University People’s Hospital, Peking University Hepatology Institute, Beijing Key Laboratory of Hepatitis C and Immunotherapy for Liver Diseases., 2 Beijing Ditan Hospital, China Capital Medical University, Institute of Infectious Diseases, Beijing, China E-mail: [email protected] Background and Aims: Immune suppressor mechanisms such as Tregs or MDSCs during hepatocellular carcinoma (HCC) progression are being uncovered. We showed here the critical role of liver stroma in the process of MDSCs accumulation and tumor progression in HCC. Methods: Murine hepatoma cells were injected subcutaneously to BALB/c mice with or without liver stromal cells to obtain tumorbearing mice. Purified MDSCs were labeled with CFSE or DiR for in vivo migration assay. Liver stromal cells were used to mimic the liver microenvironment for in vitro chemotaxis assay. Neutralizing antibodies were employed for in vivo and in vitro blockade. Immunohistochemistry was performed to detect pathological injury and inflammation in liver and tumor tissue. Results: Adoptively transferred MDSCs accumulated mainly into liver and partially in spleen both in normal mice and tumorbearing mice. Interestingly, much more adoptively transferred MDSCs accumulated into liver site in tumor-bearing mice than that in tumor-free mice as confirmed by flow cytometry and living image. Adoptively transferred MDSCs also accumulated in tumor tissues in tumor-bearing mice. MCP-1 and SDF-1 expressed at high level in liver stromal cells. We examined the expression of MCP-1 and SDF-1 in liver stromal cells after exposure to heaptoma cellcultured supernatant and found an increased expression of the two chemokines. In vitro chemotaxis assay showed liver stromal cell derived factors promote MDSCs migration which could be reversed by MCP-1 and SDF-1 neutralizing antibodies. Liver stromal cells also promote hepatoma growth when co-injected with tumor cells, which may be attributed to increased MDSCs accumulation as detected by living image. Tumor growth could be significantly reduced in vivo by MCP-1 and SDF-1 neutralization. Besides, in HCC patients, tumor associated stroma cells and CD11b+ myeloid cells are highly enriched in peri-tumor tissues, which is accompanied by high expression of MCP-1 and SDF-1. Conclusions: These results demonstrate that liver stromal cells promote MDSCs migration via SDF-1 and MCP-1 in hepatomabearing mice and thus foster tumor progression. Understanding the cross-talk between liver stroma and MDSCs in HCC may provide a promissing breakthrough for further immunotherapeutic strategies. Gaixia He and Henghui Zhang contributed equally to this work.
Journal of Hepatology 2013 vol. 58 | S63–S227
S151