研究登革病毒感染小鼠動物模式中引發肝臟損傷及出血之免疫致病機轉

Study of Immunopathogenesis of Liver Damage and Hemorrhage in Dengue Mouse Models

陳萱靜(Hsuen-Chin Chen)

指導教授 ： 伍安怡

國立臺灣大學/醫學院/免疫學研究所/博士(2007年)

https://doi.org/10.6342/NTU.2007.02486

摘要

登革疾病為重要的病媒蚊傳染疾病。在過去的五十年間，登革疾病的發生率在熱帶及亞熱帶區增加了30倍。登革病毒在人身上會造成各種型式的疾病症狀：從輕微的登革熱到嚴重威脅生命的登革出血熱/登革休克症候群。然而，造成嚴重的登革疾病之致病機轉，截至目前為止還是不清楚。目前，也沒有一個合適的動物模式可作為研究工具，來幫助了解登革疾病的免疫致病機轉。因此，發展小鼠動物模式對於了解登革疾病的致病機轉是迫切需要的。
過去研究證據顯示，在登革病毒感染的病人體內，發現對於病毒有專一性的T細胞有活化及增生的現象，且活化的T細胞增生與造成病人有嚴重疾病症狀有正相關。文獻中也指出，在嚴重登革病症的病人血清中，其肝功能指數上昇，表示病人有不正常的肝功能現象。然而，對於T細胞是否在登革疾病中，參予造成肝臟受損的角色仍需進ㄧ步探討。在我第一部分的研究中，將免疫健全的C57BL/6老鼠，以靜脈注射的方式，給予小鼠感染10^8 PFU的第二型登革病毒16681病毒株。在感染小鼠的血清、肝臟、脾臟及大腦組織中，以反轉錄-聚合反應方法，可偵測到病毒外殼RNA的存在。在病毒感染後的第一天，小鼠脾臟中大量的B及T細胞表面上會表現CD69，顯示細胞有活化的現象。在感染後第三天的脾臟組織中，活化的T細胞也表現O-glycosylated CD43。這些表現O-glycosylated CD43的T細胞量與IFN-g的產生在感染後的第五天達到最高峰。在達到大量活化T細胞產生最高峰的那ㄧ天，同時可以觀察到肝臟組織中有淋巴球細胞的浸潤及肝功能指數增加。利用流式細胞儀儀器分析顯示，浸潤肝臟的T淋巴球細胞族群，其多為CD8 T細胞(CD8 T細胞/CD4 T細胞 = 5/3)。浸潤肝臟組織中的T細胞及肝功能指數，在同血清型登革病毒二次感染後的第三天可觀察到快速上升。這些結果顯示，在登革病毒感染小鼠體內，T細胞的活化、肝臟組織中有細胞浸潤及肝功能指數的上升，有一個很強的相關性。此結果也暗指活化T細胞可能在登革病毒感染的宿主中，扮演致病角色。
出血在登革病人中是常見的臨床症狀。然而，登革病毒如何引發出血的致病機轉仍需釐清。在我的第二部分研究中，利用免疫健全的C47BL/6小鼠以真皮間注射方式，給予小鼠第二型登革病毒16681病毒株感染，來建立出血的小鼠動物模式。當小鼠給予3 × 10^9 PFU的病毒感染，會造成所有病毒感染小鼠全身系統性的出血；而給予小鼠4-8 × 10^7 PFU的病毒感染，則只會造成1/3的病毒感染小鼠有皮下組織的出血。將出血的皮下組織以H&E染色的方式，可觀察到組織中有血管滲漏的的現象。為了進一步探討是什麼樣的因子造成登革出血的現象，因此，以注射4-8 × 10^7 PFU登革病毒量到小鼠中，比較沒有出血現象的小鼠和有出血現象的小鼠互相比較分析，來找出其有差異的因子，其因子可能是造成登革出血的重要因子。在感染後的1-3天，感染小鼠顯示高病毒量與出血發展有正相關。血小板的數量，在感染後第三天的出血及沒出血的小鼠血液中，皆有減少的現象。在感染後的第7天，血小板數量在出血小鼠的血液中有相較於第三天更低，而非出血的小鼠血液中的血小板數目，仍維持跟第三天一樣。當有出血現象發生同時即感染後第三天，在出血的皮下組織中，也可觀察到大量巨噬細胞浸潤、高量TNF-α的產生及大量細胞死亡。免疫螢光染色結果顯示內皮細胞及巨噬細胞均是登革病毒的目標細胞，而內皮細胞中超過90%的細胞是死亡的。綜合以上的結果，高病毒量、巨噬細胞浸潤及TNF-α在局部組織的產生是造成出血的三個重要因子。除此之外，在組織中存在高量的TNF-α與內皮細胞的死亡及出血發展有相關性。
在出血發生同時，出血組織中也可偵測到補體3 的存在、血清中有對病毒專一性的抗體產生且淋巴結的T 細胞有活化的現象。為了釐清TNF-α或其他免疫因子是否參予登革病毒引發出血的免疫致病機轉，我將TNF-α -/-、TCRβ-/-、IgH-/-、STAT1-/-、IFN-γ-/-、A/HeJ (C5-/-)小鼠感染病毒，且比較這些基因缺陷小鼠與免疫健全小鼠的出血發生率。結果顯示，只有在TNF-α缺陷小鼠的出血率(5%)相較於登革感染的其他基因缺陷小鼠(29-40%)及免疫健全小鼠(33-35%)低。這個發現顯示TNF-α在於出血的發展是個重要的因子。
在體外細胞培養實驗中，老鼠的微血管內皮細胞對於登革病毒的感受性很高，且在有TNF-α的存在下，會增加登革病毒對內皮細胞造成死亡的情形。登革病毒感染巨噬細胞後，收集細胞培養上清液，此上清液可以增加登革病毒引發內皮細胞的死亡比率，且在有中和性TNF-α的抗體存在下，會中止TNF-α增加登革病毒引發內皮細胞死亡的現象。這些結果顯示登革病毒感染巨噬細胞，引發產生TNF-α，而TNF-α的產生增加了登革病毒引發內皮細胞的死亡。我以真皮間注射病毒方式所建立的小鼠動物模式，用來探討登革出血機轉，研究結果顯示登革病毒會感染內皮細胞，在有TNF-α存在之下，內皮細胞會死亡，進而造成內皮層受損及出血。這個發現顯示說明了天生的免疫反應在登革出血中的貢獻。
綜合以上研究，我成功的建立兩種動物模式，其ㄧ可用來探討登革病毒引發肝臟受損的免疫致病機轉，其二可探討登革病毒引發出血的免疫致病機轉。此二動物模式，將來均可被運用作為更進ㄧ步探討登革病毒與宿主的免疫反應之交互作用與疾病之相關性。

關鍵字

登革 ； 動物模式 ； 肝臟受損 ； 出血

並列摘要

Dengue is one of the most important mosquito-borne diseases in the world. In the past 50 years, its incidence has increased 30-fold with significant outbreaks occurring in tropical and sub-tropical regions of the world. Dengue virus (DV) causes a wide range of disease in humans, from dengue fever (DF) to life-threatening dengue hemorrhagic fever/dengue shock syndrome (DHF/DSS). The pathogenesis of severe dengue disease is not well understood. So far, there is no suitable animal model to study the immunopathogenesis of dengue. Therefore, to develop mouse model is necessary and urgent to the understanding of the pathogenesis of dengue.
Evidences show that dengue virus-specific T cells are activated and expanded in patients with dengue and the extent of T cell activation correlates with development of severe disease. Elevated liver enzyme levels in the sera of patients with severe dengue disease have been documented, indicating that dengue patients have abnormal liver functions. However, it remains to be investigated whether T cells are involved in the pathogenic mechanism of liver injury in dengue. In the first part of my study, immunocompetent C57BL/6 mice were inoculated intravenously with 108 PFU DV serotype 2 strain 16681. Dengue viral capsid RNA was detectable in the serum, liver, spleen, and brain by RT-PCR at different time points after infection. Splenic B and T cells are activated as evidenced by the expression of CD69 at as early as day 1 of infection. Activated T cells also expressed O-glycosylated CD43 at day 3 of infection. T cell expression of O-glycosylated CD43 and IFN-γ peaked at day 5. Coincided with the peak of splenic T cell activation was hepatic lymphocyte infiltration and elevation of liver enzymes. Flow cytometric analysis revealed that infiltrating cells included CD8 and CD4 T cells and their ratio was 5 to 3. Hepatic T cell infiltration and liver enzyme levels increased sharply at day 3 after second noculation of DV. The results demonstrated a strong correlation between T cell activation, hepatic cellular infiltration, and elevation of liver enzymes in mice infected with DV. It implied that activated T cells may be pathogenic to the host and cause liver injury in DV infection.
Hemorrhage is a common clinical manifestation in dengue patients. Thepathogenic mechanism of DV-induced hemorrhage still awaits clarification. In the second part of my study, a dengue hemorrhage mouse model was established in
immunocompetent C57BL/6 mice by intradermal inoculation of DV-2 strain 16681. While inoculation of 3 × 109 PFU of DV induced systemic hemorrhage in all of the mice by day 3 of infection, 1/3 of those injected with 4-8 × 107 PFU developed hemorrhage in the subcutaneous tissues. H & E stain of the hemorrhagesubcutaneous tissue sections revealed signs of vascular leakage. The mice that wereinoculated with 4-8 × 107 PFU but did not develop hemorrhage were used as a basisfor comparison to explore the pathogenic mechanism of dengue hemorrhage. Itappeared that high viral titer with in 1-3 days after infection had positive correlationwith hemorrhage development. Results showed that the platelet counts weresignificantly reduced in both hemorrhage and non hemorrhage mice at 3 days afterinfection. The counts were further reduced in hemorrhagic mice but remained at thesame level as that on day 3 in nonhemorrhage mice at day 7. At day 3, the time ofhemorrhage development, there was massive macrophage infiltration, high level ofTNF-α production, and overwhelming presence of apoptotic cells in the hemorrhagetissue. Immunofluorescence staining revealed that DV targeted both endothelialcells and macrophages became apoptotic, and >90% of the endothelial cells wereapoptotic. Taken together, high viral titer, macrophage infiltration, TNF-αproduction in the local tissues are three important events that lead to hemorrhage. In addition,production of high TNF-α in tissues correlated with endothelial cell apoptosis and hemorrhage.
At the time of hemorrhage development, there was also deposition of complement 3 in the hemorrhage tissue, DV-specific antibody production and lymph node T cell activation. To clarify the role of TNF-α and the involvement of other immune factor(s) in the immunopathogenesis of DV-induced hemorrhage, I infected TNF-α -/- to TCRβ-/-, IgH-/-, STAT1-/-, IFN-γ-/-, A/HeJ (C5-/-) mice with DV and ompared to wild type mice for hemorrhage development. The percentage of immune deficient mice that developed hemorrhage was not different from the wild type mice except those with TNF-α deficiency. This finding indicates that TNF-α but not other immune factors is important to hemorrhage development.
In vitro studies showed that primary mouse microvascular endothelial cells were susceptible to DV and TNF-α enhanced DV-induced apoptosis. Supernatants from DV-infected macrophage cultures also enhanced DV-induced endothelial cell death and anti-TNF-α antibody treatment abolished the effect. These results demonstrated
that DV infection of macrophages induces TNF-α production and TNF-α enhanced DV-induced endothelial cell death. The mouse model established in the present study illustrates that intradermal inoculation of high titers of DV predisposes endothelial cells to TNF-α-induced cell death which leads to endothelium damage and hemorrhage development. This finding highlights the contribution of innate immune response to dengue hemorrhage.
In summary, I have successfully established two mouse models to study the immunopathogenic mechanisms of DV-induced liver damage and hemorrhage. Each of them will be useful for further dissection of the pathogenic mechanisms of immune response to disease development in hosts infected by dengue virus.

並列關鍵字

Dengue ； animal model ； liver damage ； hemorrhage

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