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Dec 16, 2017 - HLA-DQA1-positive cells in the liver sinusoid were positive for CD68 (macrophages or Kupffer cells); those in the lymphocytic infiltrates were ...
Archives of Virology (2018) 163:855–865 https://doi.org/10.1007/s00705-017-3675-8

ORIGINAL ARTICLE

Contradictory intrahepatic immune responses activated in high‑load hepatitis C virus livers compared with low‑load livers Mariko Ishibashi1,4 · Hiromi Yamaguchi1 · Yukari Hirotani2 · Akihisa Sakurada1 · Toshihide Endo1 · Masahiko Sugitani2 · Tadatoshi Takayama3 · Makoto Makishima1 · Mariko Esumi1 Received: 3 April 2017 / Accepted: 26 November 2017 / Published online: 16 December 2017 © Springer-Verlag GmbH Austria, part of Springer Nature 2017

Abstract We found a HLA class II histocompatibility antigen gene, DQ alpha 1 chain (HLA-DQA1), that was expressed more than 9-fold higher in high-load hepatitis C virus (HCV) livers than low-load HCV livers using transcriptomics of chronic HCVinfected livers. To further investigate this finding, we examined which cells were positive for HLA-DQA1 and what liver immune responses were different between HCV-high and -low livers. HLA-DQA1-positive cells were significantly increased in the HCV-high group, and most positive cells were identified as non-parenchymal sinusoid cells and lymphocytic infiltrates in the portal area. Parenchymal hepatocytes were negative for HLA-DQA1. HLA-DQA1-positive cells in the liver sinusoid were positive for CD68 (macrophages or Kupffer cells); those in the lymphocytic infiltrates were positive for CD20 (B cells) or CD3 (T cells). mRNA levels of antigen-presenting cell (APC) markers such as CD68 and CD11c were significantly upregulated in the HCV-high group and were correlated with HLA-DQA mRNA levels. CD8B mRNA (­ CD8+ T cells) was upregulated in both HCV-positive livers compared with HCV-negative livers, whereas CD154 mRNA (­ CD4+ T helper cell) was upregulated in the HCV-high group compared with the HCV-low group. The immune regulatory molecules FOXP3 mRNA (regulatory T cell, T reg) and programmed cell death ligand-1 (PD-L1) mRNA were significantly increased in the HCV-high group. HCV-high livers had two molecular immune responses: increased APC numbers and adaptive immunity and the induction of immune tolerance. The local hepatic imbalance of contradictory immune responses might be responsible for high HCV loads.

Introduction Handling Editor: Michael A. Purdy. Electronic supplementary material  The online version of this article (https://doi.org/10.1007/s00705-017-3675-8) contains supplementary material, which is available to authorized users. * Makoto Makishima makishima.makoto@nihon‑u.ac.jp * Mariko Esumi esumi.mariko@nihon‑u.ac.jp 1



Division of Biochemistry, Department of Biomedical Sciences, Nihon University School of Medicine, 30‑1, Ohyaguchikami‑cho, Itabashi‑ku, Tokyo 173‑8610, Japan

2



Department of Pathology, Nihon University School of Medicine, Tokyo, Japan

3

Department of Digestive Surgery, Nihon University School of Medicine, Tokyo, Japan

4

Present Address: Department of Microbiology and Immunology, Nippon Medical School, Tokyo, Japan



Chronic infection develops in 70-85% of patients with hepatitis C virus (HCV) infection without viral clearance [1] and causes serious liver diseases, i.e., chronic hepatitis, liver cirrhosis, and hepatocellular carcinoma (HCC). Chronic infection appears to be due to severe T cell dysfunction, that is, immune tolerance in patients with chronic hepatitis C [1, 20]. For example, immune suppresser cells, including regulatory T (Treg) cells and myeloid-derived suppressor cells (MDSCs), are increased in peripheral blood mononuclear cells (PBMCs) from HCV-infected patients compared with healthy controls [4, 28, 30]. These cells might inhibit ­CD4+ effector T cells and HCV-specific C ­ D8+ cytotoxic T-lymphocytes (CTLs). Further, T cells were found to be exhausted by immune negative regulators, e.g., programmed death 1 (PD-1) and cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) in patients with chronic HCV infection [20]. However, these immune responses in chronic HCV infection were investigated in PBMCs but not in liver tissues from

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patients. To explore the precise host responses in chronic infection of HCV in human liver, we investigated differences in gene expression between two groups of liver samples with high and low HCV loads (>100-fold difference) using oligonucleotide microarrays. We identified 26 genes that were upregulated in the livers with high HCV loads, of which HLA class II histocompatibility antigen DQ alpha 1 chain (HLA-DQA1) was the most differentially expressed [6]. To clarify the role of HLA-DQA1 in the HCV-high livers, we examined what cells were positive for HLA-DQA1 and differences in the liver immune responses between the HCVhigh and -low groups. The HLA-DQA1 gene is located in a short arm of chromosome 6 (6p21.3) and encodes the α-chain subunit of the HLA-DQ molecule. MHC class II molecules (HLA-DR, -DQ and -DP) are primarily expressed in APCs such as macrophages, dendritic cells and B cells and play a central role in the antigen-presenting process. MHC class II polymorphisms are well known to influence immune responses against HCV. The HLA-DQB1*0301 allele has been associated with spontaneous clearance of HCV viremia [5, 11, 14], and the HLA-DRB1*0101, DRB1*0401, DRB1*1101 and DRB1*1501 alleles in HCV-infected patients have been associated with HCV viral clearance [11, 15]. These alleles are assumed to present the HCV epitope to ­CD4+ lymphocytes more efficiently than other alleles. However, HLA-DQA1 gene upregulation in human livers with high HCV viral loads does not lead to HCV clearance. In the present study, we examined and compared the immunological responsiveness of livers between the HCV-high and -low groups.

Materials and methods Liver specimens HCV-positive liver samples were obtained from non-tumorous liver tissues of patients with either HCC stage I or stage II, except for a case with stage IIIA. We measured HCV RNA concentrations in 59 liver samples and classified the 59 samples into three groups, high (>30,000 arbitrary units of HCV RNA), middle, and low (