1. Modeling The Early Events of Severe Acute Respiratory Syndrome Coronavirus Infection in Vitro. Mechanistic Study of the Immunopathogenesis of Hemorrhage in a Dengue Mouse Model

嚴重急性呼吸道症候群(severe acute respiratory syndrome, SARS)是世界衛生組織於92年3月15日新公布之名稱，在這之前稱非典型肺炎。嚴重急性呼吸道症候群之特點為瀰漫性肺炎及呼吸衰竭，因較過去所知病毒、細菌所引起的非典型肺炎更嚴重，因此取名為嚴重急性呼吸道症候群。 嚴重急性呼吸道症候群的主要症狀為發高燒（>38℃）、咳嗽、呼吸急促或呼吸困難。可能伴隨其他的症狀，包括：頭痛、肌肉僵直、食慾不振、倦怠、意識紊亂、皮疹及腹瀉，胸部X光檢查可發現肺部病變。 嚴重急性呼吸道症候群最嚴重時會出現瀰漫性肺炎，氧氣交換下降，導致肺部缺氧，所以病人會呼吸困難、缺氧，甚至導致死亡。引起SARS的是一新種冠狀病毒，而SARS的致死原因主要為瀰漫性肺泡損傷導致急性呼吸窘迫症。SARS病毒(SARS-CoV)在感染早期如何刺激肺部上皮細胞及造成嚴重發炎反應是急待釐清的重要問題。們研究SARS病人在感染早期肺部組織切片，發現肺部的上皮細胞與單核球細胞是SARS-CoV的標的細胞。我們並觀察到在病人在感染SARS-CoV的早期就有嗜中性球ヽ巨嗜細胞及T 細胞浸潤到肺部，且在肺部上皮及巨嗜細胞上有P-selectin與DC-SIGN表現。我們利用各種不同的肺部表皮細胞與單核球細胞株進行體外SARS-CoV的感染ヽ發現了其中兩株細胞株(A549 和 THP-1)對於SARS-CoV有感受性，更近一步分析我們發現上皮細胞株(A549)感染SARS-CoV會表現P-selectin，VCAM-1，CXCL8/IL-8 與CCL2/MCP-1；且單核球細胞株(THP-1)被SARS–CoV感染後會釋放細胞趨化激素(CCL2/MCP-1, CXCL8/IL-8, CCL3/MIP-1, CXCL10/IP-10, CCL4/MIP-1與CCL5/RANTES)。在細胞趨化實驗中可見SARS-CoV感染之單核球細胞的上清液會吸引嗜中性球ヽ單核球細胞及活化T細胞游移。本實驗結果顯示出，SARS-CoV感染肺部上皮細胞及單核球細胞後，會引發黏著分子(adhesion molecules)的表現與細胞激素大量的釋放，創造出一個使免疫細胞浸潤的環境，而SARS-CoV也因為引起大量免疫細胞浸潤的免疫反應而導致肺部的損傷。The clinical picture of SARS is characterized by pulmonary inflammation and respiratory failure, resembling that of ARDS. However, the events that lead to the recruitment of leukocytes are poorly understood. To study the cellular response in the acute phase of SARS-CoV-host cell interaction, we investigated the induction of chemokines, adhesion molecules and DC-SIGN by SARS-CoV. Immunohistochemistry analysis of autopsiyed specimens revealed neutrophil, macrophage and CD8 T cell infiltration in the lungs of a SARS patient who died during the acute phase of the illness. Additionally, pneumocytes and macrophages in the patient’s lung expressed P-selectin and DC-SIGN. In in vitro study, we showed that A549 and THP-1 cell lines were susceptible to SARS-CoV. A549 cells produced CCL2/MCP-1 and CXCL8/IL-8 after interaction with SARS-CoV and expressed P-selectin and VCAM-1. Moreover, SARS-CoV induced THP-1 cells to express CCL2/MCP-1, CXCL8/IL-8, CCL3/MIP-1, CXCL10/IP-10, CCL4/MIP-1 and CCL5/RANTES that attracted neutrophils, monocytes and activated T cells in a chemotaxis assay. We also demonstrated that DC-SIGN was inducible in THP-1 as well as A549 cells after SARS-CoV infection. Based on our results, we proposed that there are two waves of cell migration during the acute phase of SARS: (i) Pulmonary epithelial cells infected by SARS-CoV express adhesion molecules, upregulate DC-SIGN and produce CCL2/MCP-1 and CXCL8/IL-8 which are conducive for the recruitment of monocytes and neutrophils. (ii) The recruited monocytes interacting with SARS-CoV produce chemokines that attract a second wave of cells including neutrophils, activated T cells and more monocytes. The production of CCL2/MCP-1 by epithelial cells is key to the migration of two waves of cells.Table of Contentsbstract………………………………………………………………….............. ibstract (Chinese)…...………………………………………………….…......... iiable of Contents…………………………………………….………………….. iiihapter I. Introduction……………………………………………………….... 1. The disease and the virus…………………………………………….…... 1I. The host response the SARS-CoV infection……………………………… 2II. Cellular Entry of the Virus……………………………….……………... 2hapter II. Materials and Methods…………………………………………….. 4art I. Materials and Methods……………………………………………………... 4. Immunohistochemisty……………..………………………………….. 4. Cell lines……………………………………………………………… 5. SARS-CoV infection……………...…...………………………….….. 5. Immunofluorescence staining………………………….……….. 6. RNA preparation and RT-PCR……..………………………........ 7. Quantitative real –time RT-PCR…………………..……….…... 7. Ribonuclease protection assay (RPA)………….………..……. 8. Isolation of perileral blood leukocytes………………………... 9. Chemotaxis assay………………………………………….…... 90. Protein chip assay………………………...................................... 101. Western blot…………………………………………………... 10hapter III. Results……………………………..……………………………..... 12. Early events occurring in the lung of SARS patients…………….…… 12. Susceptibility of A549 and THP-1 cells to SARS-CoV infeciton…..... 13. SARS-CoV infection induces pneumocytic epithelial cell production of CCL2/MCP-1 and CXCL8/IL-8 and expression of adhesion moldcules............................................................................................... 13. SARS-CoV interaction with pneumocytic epithelial cell and monocytic cells induces DC-SIGN expression…………………….…. 15. SARS-CoV-infected monocytic THP-1 cells produce chemoines that attract neutrophils, moncytes and activated T cells…………………. 16hapter IV. Discussion…………………………………………………………... 18. Clinical manifestations of SARS……………………………………… 18I. SARS induces cytokine and chemokines upregulation………..………. 19II. MCP-1 is important to lung injury……………....................................... 20V. SARS induce adhstion molecules in pneumocytes…………………..… 21. DC-SIGN expression in SARS patients………………….…………….. 21I. The model of SARS immunopathogenesis……………………..……… 22eference………………………………………………………………………... 23ables…………...………………….…………………………………………... 30igures……………………………………………………………………………. 3