The Effect of Activation of Invariant NKT Cells on the Allergic Airway Inflammation

氣喘是一肺部發炎疾病，其臨床表徵為鳴喘、呼吸急促與呼吸道過度反應，許多免疫細胞都會參與發炎的過程，例如：T helper 2（Th2）cells和eosinophils。natural killer T cells（NKT cells）是一群特殊的T cells，它會表現natural killer cells和conventional T cells兩者的特性，目前在invariant natural killer cells（iNKT cells）的研究最多。當iNKT cells辨識由CD1d所呈獻的抗原如α-galactosylceramide（α-GalCer）而活化後，可在數小時內快速分泌多種且大量的細胞激素如interferon-γ（IFN-γ）和interleukin-4（IL-4），這獨特的特性使iNKT cell在許多疾病中扮演調控的角色。有關NKT cells在氣喘中扮演之角色先前研究的結果並不一致，有團隊證明NKT cells會促進氣喘的產生，但另有團隊卻證明NKT cells的活化會抑制呼吸道過度反應與呼吸道發炎反應。此外， NKT cells在某些疾病不同進程時扮演不同的角色。本研究的目的是探討是否NKT cells在氣喘不同疾病進程中亦扮演不同的角色，我們在氣喘疾病進程中不同時期以α-GalCer活化iNKT cells後測量小鼠呼吸道發炎之嚴重程度是否會不同以及其機轉。 我們利用ovalbumin（OVA）致敏引發氣喘的小鼠模式，在OVA致敏前或OVA致敏後給予α-GalCer，之後分析小鼠呼吸道發炎反應的程度。實驗結果顯示OVA致敏前給予α-GalCer的小鼠其呼吸道發炎反應高於致敏後才給予α-GalCer的小鼠，證實在氣喘疾病不同時期給予α-GalCer活化iNKT cells確實會使小鼠產生不同嚴重程度之呼吸道發炎反應。接著我們分析致敏前或致敏後給予α-GalCer兩小時與五天後小鼠的免疫反應之變化，發現在OVA致敏前給予α-GalCer可活化iNKT cells分泌大量的Th2 cytokines例如IL-4、IL-5與IL-13，及使肺部TSLP與eotaxin濃度升高，導致肺部浸潤細胞增多及IL-4的顯著上升。但若是在OVA致敏後才給予α-GalCer則造成Th2 cytokines與thymic stromal lymphopoietin （TSLP）的降低，終使小鼠呼吸道發炎反應不如OVA致敏前給予α-GalCer之小鼠嚴重。 這些結果顯示naïve環境下的iNKT cells與氣喘病Th2為主的環境下的iNKT cells其活化後產生的反應不同，最後造成呼吸道發炎反應之嚴重度不同。然而欲將α-GalCer當作呼吸道佐劑或治療性藥物的時候，需注意eosinophilia的可能性。Asthma is an inflammatory lung disease characterized by the expansion of T helper 2 (Th2) cells and eosinophils as well as the production of allergen-specific IgE. Natural killer T (NKT) cell is a unique subset of T cells that coexpresses T cell receptor (TCR) and natural killer (NK) cell markers. The most studied NKT cells are Type 1 NKT cells, which also referred to invariant natural killer T cell (iNKT cells). iNKT cells express semi-invariant CD1d-restricted αβ TCR, therefore they can recognize glycolipid antigens (like α-galactosylceramide (α-GalCer)) presented by CD1d molecules on antigen presenting cells. Once NKT cells are activated, they rapidly produce large amount of cytokines, such as interferon-γ (IFN-γ) and interleukin-4 (IL-4). Due to this unique characteristic, they have been implicated in the regulation of immune responses associated with a broad range of diseases. In allergic asthma, the roles of iNKT cells are controversial. Some studies demonstrate that NKT cells are important in the development of asthma. However, others demonstrate that NKT cell activation before challenge abolishes the airway hyperresponsiveness and airway inflammation in experimental murine asthma models. These studies imply that NKT cells may play different roles in different asthma progression. Furthermore, this theory has been proved in some autoimmune diseases and allergic conjunctivitis. The aim of this study is to investigate the effect of activation of iNKT cells on the allergic airway inflammation and its mechanism. We administered BALB/c mice with α-GalCer, a stimulator for NKT cell activation, before or after ovalbumin (OVA) immunization and measured airway inflammation of those mice after 2 times of intranasal OVA challenge. In our results, the airway inflammation was more serious in mice that administered with α-GalCer before OVA immunization than that of after OVA immunization, suggesting that activation of iNKT cells in different asthma progression could lead to different severity of airway inflammation. Moreover, we analyzed the cell components and cytokines expression in lung after 2 hours and 5 days of α-GalCer administration to study the mechanism. Our data demonstrated that mice administered with α-GalCer before OVA immuniztion could activate iNKT cells to secrete large amount of Th2 cytokines, such as IL-4, IL-5 and IL-13, and increase eotaxin and thymic stromal lymphopoietin (TSLP) expression in bronchoalveolar lavage fluid (BALF), which recruit cell infiltrate in lung. In contrast, administration of α-GalCer after OVA immunization decreased Th2 cytokines, TSLP, and airway inflammation when compared to mice administered with a-GalCer before OVA immunization. Our data demonstrated that different immune response was noted in activation of naïve NKT cells and Th2-dominant environment NKT cells, which led to different outcome of airway inflammation in mice. However, the potential risks of treatment with NKT cell activation in human diseases should be considered.封面試委員審定書權書謝……………………………………………………………………………….. i文摘要………………………………………………………………………….. ii文摘要………………………………………………………………………….. iv寫對照表……………………………………………………………………….. vi錄……………………………………………………………………………….. vii目錄…………………………………………………………………………..… ix一章 研究背景………………………………………………………………… 1.1 氣喘之背景介紹……………………………...………………………… 1.1.1 發炎反應 (inflammatory response)……….………………….… 1.1.2 呼吸道過度反應 (airway hyperesponsiveness；AHR)….…….. 5.2 NKT cells之背景介紹…………………………………..……………… 6.2.1 NKT cells的定義…………………………..………………...… 6.2.2 iNKT cells的活化……………………………....……………… 6.2.3 NKT cells的生物功能…………………………..…………...… 7.3 NKT cells在氣喘中扮演的角色…………………………..…………… 8.4 NKT cells在疾病進程中，會扮演不同的角色..……………………… 9.5 研究目的………………………………………………………………… 10二章 實驗材料與方法……………….…………………………………...…… 11.1 實驗用小鼠……………………………………….………………...…… 11.2 氣喘動物模式之建立………………………….…………………...….... 11.3 α-GalCer 之給予…………………….….……………………...…...…… 11.4 血清樣品的收集…………….….…………………………………..……. 11.5 抽取小鼠支氣管肺泡沖洗液(bronchoalveolar lavage fluid；BALF )… 12.6 製備BAL cell玻片………….….…………………………………….… 12.7 流式細胞儀(flow cytometry)分析細胞表面抗原…………………….… 12.8 利用ELISA測量血清中OVA-specific IgE………………..………...… 13.9 利用ELISA測量BALF中細胞激素之濃度………………………...… 13.10 小鼠肺臟細胞初代培養……………………………………..………....... 14.11 以α-GalCer體外刺激肺臟初代細胞………………..………..............… 14.12 腹腔注射小鼠α-GalCer實驗………………..………..........................… 14.13 收集小鼠腹腔沖洗液………………..……….......................................… 15.14 繪圖及統計分析………………..……………………………………...… 15三章 結果………..……………………………………….…………………...… 16.1 小鼠在OVA致敏前與致敏後注射α-GalCer對於呼吸道發炎反嚴重程度之影響………………………….…………………….…...… 16.2 探討小鼠在OVA致敏前或致敏後給予α-GalCer兩小時後，肺的iNKT cells與細胞激素有何變化….……………………….…...… 17.3 以OVA致敏小鼠兩次後，其肺部的iNKT cells升高……….….....… 19.4 探討小鼠在OVA致敏前或致敏後給予α-GalCer五天後，肺部細胞組成與細胞激素有何變化.……………....……………….…...… 20.5 研究α-GalCer是否可直接影響初代培養之肺臟細胞產生eotaxin能力……………....…………………………………….……….…..… 22.6 探討腹腔注射α-GalCer是否吸引eosinophils至腹腔中…...….…....... 23四章 討論…………....…………………………………….………….….…..… 24…………....…………………………………….……………………...….…..… 29考文獻…………………………………….……………………...….………..… 52錄…………………………………….……………………...….……………..… 6