國立成功大學「邁向頂尖大學計畫」
延攬優秀人才工作報告表
NCKU’s “Aim for the Top University Project”
Work Report Form for Distinguished Scholars
□續聘continuation of employment ■離職resignation
受聘者姓名
Name of the Employee 姚蕙雯 男 █女
Male Female
聘 期 Period of Employment
from 104 年(y) 7 月(m) 1 日(d) to 104 年(y) 12 月(m) 31 日(d) 研究或教學或科技研發與
管理計畫名稱 The project title of research,
teaching, technology development and management
利用小鼠模式研究調控腸病毒71 型 感染的免疫反應
計畫主持人
（申請單位主管）
Project Investigator (Head of Department/Center)
陳舜華
補助延聘編號
Grant Number HUA 104-3-22-177
一、 研究、教學、科技研發與管理工作全程經過概述。（由受聘人填寫）
Please summarize the entire research, teaching, or science and technology R&D and management work process (To be completed by the employee)
EV71, a neurotropic picornavirus, can infect the human CNS to cause fatal neurological manifestations. Severe symptoms, such as brain stem encephalitis with cardiopulmonary complications, often cause death or long-term neurological sequelae, especially in young children. In Taiwan, the largest and most severe outbreak occurred in 1998 with 129,106 cases of benign symptoms likes herpangina or hand, foot, and mouth disease, 405 cases of neurological and cardiopulmonary complications, and 78 deaths reported. There are no effective antiviral drugs currently available to treat EV71-infected patients because viral pathogenesis is not completely understood. Only supportive care and intravenous immune globulin are available for severe cases in Taiwan, but the therapeutic efficacy of intravenous immune globulin seems limited. Overt immunity is suspected to exacerbate infection because high levels of lymphocytes, antibody titer, cytokines, such as IL-20, and chemokines are detected in patients with severe symptoms. However, the functions and roles of these lymphocytes, cytokines, and chemokines in EV71 pathogenesis are not clearly understood. In this grant, we further addressed the functions of these immune factors in EV71 infection because their roles in virus infections are controversial. In this grant we investigated the role of IL-20 receptor signaling in EV71 infection using a neonatal mouse model. We found that EV71 infection increased IL-20R1 expressing in mouse brains, especially in the neurons (Figure 1). The survival rate, mean body weight, of infected mice deficient in IL-20 receptor I (IL-20R1-/-) because of genetic mutations was significantly higher that of than wild-type mice (Figure 2). The disease score of infected IL-20R1-/- mice were also lower than those of infected wild-type mice. To further investigate how IL-20R1 signaling affects the survival of EV71-infected mice, we harvested mouse tissues to determine viral titers. The mean viral titers in the spinal cord of IL-20R1-/- mice were lower than those of wild-type mice at days 3 and 5 post-infection with a significant difference at day 5 post-infection (Figure 3). At day3 post-infection, the mean viral titers in the brain and brainstem of IL-20R1-/- mice were significantly lower than those of wild-type mice, but at day 5 post-infection, the mean viral titers in the brain and brainstem of IL-20R1-/- and wild-type mice were comparable (Figure 3). IL-20R signaling has been implicated in inflammatory diseases; therefore we compared the expression of three known ligands, IL-19, IL-20, and IL-24, in the serum and brain. We found that a basal level of IL-19 expression was detected in the mouse brain but not in the serum (Figure 4). After infection, the expression of IL-19 in the brain and serum were increased. A basal level of IL-20 expression was detected in the serum, and the expression of IL-20 was increase after infection (Figure 4). Ligand binding od IL-20R1 induces proinflammatory cytokine expression and control inflammatory responses, therefore we compared the immune infiltrates in the mouse brain. Figure 5 shows that CD4+, CD8+, CD19+, and Ly6G+ cells were increased in the brain of infected wild-type mice but not in the brain of IL-20R1-/- mice. To further determine the role
of IL-20R signaling in EV71 infection, we compared the cytokines regulated by IL-20R1 signaling, such as IL-1β, IL-6, and TNF-α by ELISAs. We found that IL-1β expression was significantly reduced in the brain of IL-20R1-/- mice when compared to wild-type mice (Figure 6A). The levels of IL-1β, TNF-α, and IL-6 in the serum were comparable between IL-20R1-/- and wild-type mice (Figure 6B and C). To determine the major ligand(s) recognized by IL-20R signaling in EV71 infection, we used IL-20-specific antibodies to neutralize IL-20 expression in infected mice. Figure 7 shows that treatment with antibodies against IL-20 failed to protect mice from EV71 infection suggesting that IL-19 and IL-24, the other two ligands of IL-20R1 signaling, may be the candidates aggravating infection. In the future we will generate antibodies against IL-19 and IL-24 to evaluate the roles of IL-19 and IL-24 in EV71 infection. We will also study how EV71 infection enhances IL-19 and IL-20R1 expression using neuronal cell lines treated with inhibitors of MAPKs, NF-κB, or STAT6 those are activated in EV71 infection.
A B
C
FIG 1 EV71 infection enhanced IL-20R1 expression in mouse brains. Levels of viral titers (A) and IL-20R1 protein (B) in brains of mice mock-infected (Mock) or infected with 8 × 105 plaque forming units of EV71 at the indicated days post-infection are shown. Data represent means plus SE (error bars) of more than three samples per group, with samples derived from individual mice. BD, below detection.
*, P < 0.05, via a Mann-Whitney U test (right panel). (C) Brains of mock-infected and EV71-infected mice harvested 5 days post-infection were processed and stained with Hoechst and antibodies against IL-20R1 or NeuN. Data are representative of at least three samples per group from two independent experiments.
A B
FIG 2 Absence of IL-20R1 increased mouse resistance to EV71 infection. The survival rates (A) body weights (B), and disease scores (C) of EV71-infected wild-type (WT) mice (n = 15) and IL-20R1-/- mice (n = 15) mice are shown. ***, P < 0.001, via a log-rank test in panel A; *, P < 0.05 and **, P <
0.01, via a Student t test in panel B; *, P < 0.05, via a Wilcoxon signed rank test in panel C. Data represent means ± SE values (error bars).
FIG 3 Absence of IL-20R1 reduced EV71 titers in the mouse central nervous system. The viral titers in the indicated tissues of wild-type (WT) mice and IL-20R1-/- mice at the indicated days
post-infection are shown. Data represent means ± SE values (error bars) of five to six samples per data point, with samples derived from individual mice. *, P < 0.05, via a Mann-Whitney U test.
A B C D
FIG 4 EV71 infection increased serum and brain IL-19 levels in mice. Levels of brain IL-19 (A), serum viral titers (B), serum IL-19 (C), and serum IL-20 (D) in mice mock-infected (Mock) or infected with EV71 at the indicated days post-infection are shown. Data represent means plus SE values (error bars) of more than two samples per group, with samples derived from individual mice. BD, below detection.
FIG 5 Absence of IL-20R1 decreases CD4+ T cells in the brain of infected mice. The brains of wild-type (WT) mice and IL-20R1-/- mice mock-infected (-) or infected with EV71 (+) harvested on day 3 post-infection were subjected to flow cytometry to quantify cells positive for CD4 (A), CD8 (B), CD19 (D), or Ly6G (E). Data are means plus SE values (error bars) of three samples per group, with samples derived from individual mice. **, P < 0.01, via a Student t test.
A B C D
FIG 6 Effects of IL-20R1 deficiency on cytokine expression. Levels of brain IL-1β (A), serum IL-1β (B), serum TNF-α (C), and serum IL-6 (D) of wild-type (WT) mice and IL-20R1-/- mice mock-infected (M) or infected with EV71 at the indicated days post-infection are shown. Data are means plus SE (error bars) of at least three samples per data point, with samples derived from individual mice. *, P <
0.05, via a Student t test. BD, below detection.
FIG 7 Antibody against IL-20 failed to protect mice from EV71 infection. The survival rates of infected wild-type mice treated without antibody (No Ab) or with control antibodies (Control Ab) or antibodies against IL-20 are shown (n ≥ 5 for each group).
二、研究或教學或科技研發與管理成效評估（由計畫主持人或單位主管填寫）
Please evaluate the performance of research, teaching or science and technology R&D and management Work: (To be completed by Project Investigator or Head of Department/Center)
(1)是否達到延攬預期目標？
Has the expected goal of recruitment been achieved?
受延攬人的工作內容和進度達到延攬預期目標。
(2)研究或教學或科技研發與管理的方法、專業知識及進度如何？
What are the methods, professional knowledge, and progress of the research, teaching, or R&D and management work?
姚博士過去的研究是探討在疱疹病毒感染的過程中，會與病毒產生交互作用的宿主細胞因 子，特別是那些會促進病毒複製與疾病惡化的細胞因子，因為這類的細胞因子也許可以做為疱疹 病毒腦炎與失明性角膜炎的治療標的。我們的研究結果發現，Egr-1 蛋白可以增加病毒的複製，
以及失明性角膜炎中的血管新生反應與免疫反應。在本聘期間，她利用過去利用小鼠感染模式研
究細胞因子與病毒之間交互作用的背景，應用於研究免疫因子介白素 20 (IL-20)是否參與調控腸
病毒七十一型的致病機轉。根據我們實驗室之前的研究發現，CD4 T、CD8 T、與 B 淋巴球都參 與保護幼鼠免於腸病毒七十一型的感染，因為先天缺乏淋巴球的幼鼠被腸病毒七十一型感染時，
其死亡率、疾病嚴重度、組織中的病毒量都比野生型幼鼠來得高。目前介白素 20 被發現可以調
控自體免疫疾病，如類風濕性關節炎等的發炎反應，然而介白素 20 在病毒感染症中的角色仍未
探討，因此我們利用介白素20 受體缺陷小鼠來探討此一議題。目前發現介白素 20 受體缺陷小鼠
在感染後的死亡率明顯比野生型小鼠低，而小鼠中樞神經系統的病毒量也較野生型小鼠高，同時
伴隨較少的免疫浸潤與細胞激素的表現，顯示介白素 20 受體的訊息傳遞路徑可能會影響免疫反
應的產生。我們進一步的研究發現將可中和介白素 20 的抗體以腹腔注射方式給予被感染小鼠無
法降低小鼠的死亡率，顯示介白素 19 與介白素 24 可能才是透過介白素 20 受體調控免疫反應生
成與惡化疾病的主因。
(3)受延攬人之研究或教學或科技研發與管理成果對該計畫(或貴單位)助益如何？
How have the research, teaching, or R&D and management results of the employed person given benefit to the project (or your unit)?
受延攬人姚博士除了執行本研究計畫也協助指導研究生，並且提供個人學習研究經驗，對實 驗室相當有助益，對她個人將來的工作發展也可做為訓練及有益的經驗。
(4)受延攬人於補助期間對貴單位或國內相關學術科技領域助益如何？
How has the employed person, during his or her term of employment, benefited your unit or the relevant domestic academic field?
受延攬人姚博士的專長為病毒學、免疫學及分子生物學，先前研究的主題是鑑別宿主細胞因 子與病毒感染的交互作用及機制。現在負責研究免疫因子如細胞激素等在腸病毒七十一型的 致病機制中的角色，有助於未來研發抗腸病毒藥物或是腸病毒疫苗的發展，其專長也有助於 本校腸病毒七十一型研究的發展。
(5)具體工作績效或研究或教學或科技研發與管理成果：
Please describe the specific work performance, or the results of research, teaching, or R&D and management work:
姚博士的具體工作績效包括，設計研究內容與撰寫計畫、執行研究計畫、撰寫期刊論文，以 及指導研究生。其以博士後研究員的身分對於實驗室事務的推動與長期發展可做為一股助力。
(6)是否續聘受聘人? Will you continue hiring the employed person? □續聘Yes■不續聘No
※ 此報告表篇幅以三～四頁為原則。This report form should be limited to 3-4 pages in principle.
※ 此表格可上延攬優秀人才成果報告繳交說明網頁下載。
This report form can be downloaded in http://scholar.lib.ncku.edu.tw/explain/
