肝臟浸潤免疫細胞造成登革病毒引起之肝臟損傷

Abstract

血液中肝臟酵素上升在登革病人中是一個普遍的現象，但造成肝損傷的病理機制尚不清楚。在此研究中，利用老鼠模式發現了登革病毒引起之肝損傷的免疫病理機制。免疫健全的C56BL/6小鼠靜脈注射感染登革病毒16681株後，可在血液中短暫的偵測到病毒，也可在肝臟中偵測到病毒表殼基因以及切割的凋亡蛋白酶3，自然殺手細胞及T細胞分別在感染後第一天及第五天浸潤至肝臟中，中和趨化素CXCL10或消除Asialo GM1表現的細胞後，肝臟酵素下降、肝臟中切割的凋亡蛋白酶3表現及TUNEL表現的細胞皆明顯減少，在NK 細胞毒殺能力缺乏的STAT1基因剃除小鼠中減少了早期的肝臟細胞死亡，此外，NK細胞引起的細胞死亡的機制中是部份依賴perforin的；CD4+ 及CD8+ T細胞在晚期浸潤至肝臟，此時肝臟內浸潤免疫細胞對登革病毒感染的標的細胞有毒殺能力，在TCRβ 基因剃除小鼠或消除CD8+ T細胞後，肝臟中切割的凋亡蛋白酶3表現及TUNEL表現的細胞皆明顯減少，從去除CD8的小鼠中取出的肝臟內浸潤免疫細胞喪失對感染標的細胞的毒殺能力。此外，肝臟內與脾臟內CD8 T細胞皆辨認登革病毒抗原決定位NS4B99-107，CD8+ T細胞引起的細胞死亡是需要細胞直接接觸的，但不經由perforin。因此，肝臟浸潤之自然殺手細胞及CD8 T細胞是造成肝損傷的原因，自然殺手細胞在感染後早期，而CD8 T細胞在晚期使肝臟細胞死亡，有毒殺能力的CD8 T細胞中包含了可辨認NS4B99-107胜肽鍵的CD8 T細胞。此研究提供了登革病人肝損傷的病理機制。

Elevated liver enzyme level is an outstanding feature in patients with dengue. However, the pathogenic mechanism of liver injury has not been clearly demonstrated. In this study, employing a mouse model, we uncovered the immunopathogenic mechanism in dengue virus-induced liver injury. Immunocompetent C57BL/6 mice were infected intravenously with dengue virus strain 16681. Infected mice had transient viremia, detectable viral capsid gene and cleaved caspase 3 in the liver. In the mean time, NK cell and T cell infiltrations peaked at days 1 and 5, respectively. Neutralizing CXCL10 or depletion of Asialo GM1+ cells reduced levels of liver enzymes, cleaved caspase 3 and TUNEL+ cells in the liver at day 1 after infection. Liver cell death is reduced in NK cell cytotoxic function-deficient STAT1 KO mice at day 1 after infection. Perforin is partially involved in NK cell-mediated liver cell death. Both CD4+ and CD8+ T cells infiltrated into the liver at later time point and at which time intrahepatic leukocytes (IHL) exhibited antigen-specific cytotoxicity against DENV-infected targets. Cleaved caspase 3 and TUNEL+ cells were diminished in mice with TCRβ deficiency and in those depleted of CD8+ T cells, respectively, at day 5 after infection. IHLs isolated from CD8-depleting mice lost cytotoxicity against infected targets. Moreover, intrahepatic CD8+ T cells were like their splenic counterparts recognized DENV NS4B99-107 peptide. Intrahepatic CD8+ T cell-mediated DENV-induced liver cell death is cell-cell contact-dependent but perforin-independent. Together, these results show that infiltrating NK and CD8+ T cells cause liver cell death. While NK cells were responsible for cell death at early time point of infection, CD8+ T cells were for later. CD8+ T cells that recognize NS4B99-107 constitute at least one of the major intrahepatic cytotoxic CD8+ T cell populations. This study provokes the new pathogenic role in dengue virus-infected patients.