江伯倫臺灣大學：免疫學研究所傅麒玲Fu, Chi-LingChi-LingFu2007-11-292018-07-092007-11-292018-07-092006http://ntur.lib.ntu.edu.tw//handle/246246/63340過敏性氣喘肇因於不當的Th2 反應，過敏原專一性 Th2 細胞失控地大量的製造細胞激素，引發肺部發炎反應，嗜酸性白血球聚集，呼吸道黏液大量分泌，氣管劇烈收縮等氣喘症狀。 因此，藉由免疫調節性細胞激素的作用達成抑制過度的 Th2 發炎反應應是合理的治療策略。目前已知 Th1 細胞激素(例如︰ IL-12) 和免疫抑制型細胞激素(例如︰ TGF-β 和 IL-10)，都具有降低 Th2 反應並且在調節肺部發炎反應上扮演重要角色。 因此，在本論文中，我們在同一個氣喘動物模式的實驗中，應用四種不同細胞激素(IL-12, IL-10, TGF-β and IL-4) 的DNA 基因質體分別在不同的過敏老鼠身上觀察其對治療氣喘的功效。我們的結果顯示，這些抑制發炎型的細胞激素，特別是 IL-10，可以有效抑制呼吸道的發炎反應。並且，我們也證明這種抑制療效與所給予的細胞激素 DNA 載體的劑量是正相關的。因此，為了 IL-10 基因的表現量與作用時間可以增加，我們進一步的用重組腺病毒載體取代 DNA 質體去評估 IL-10 的抑制效果。如我們所預期，在呼吸道區域表現的 IL-10 是一個可直接抑制過度發炎反應的方法。 然而，更好的治療方法應該是設法增進免疫調節功能去調控 Th2 反應進而能完全治癒過敏性氣喘。 由於，樹突細胞在啟動發炎反應及影響 T 細胞反應發展方向上扮演了舉足輕重的角色; 因此，調控樹突細胞功能會是一個極有潛力的治療策略。於是，接下來我們就藉由DC-IL-10 與 na?ve DO11.10 CD4+ T 細胞的共同培養實驗來評估所培養出來的細胞株之功能分析。結果證明此類細胞株具有類似調節性 T 細胞的功能。總結實驗結果說明了，抑制發炎型的細胞激素 IL-10，不只可以應用在發炎反應部位直接抑制發炎現象，也可以藉由 IL-10 基因改造後的DC 來達成調控免疫反應的治療策略。Allergic asthma strongly correlates with airway inflammation caused by the unregulated production of cytokines secreted by allergen-specific type-2 T helper (Th2) cells. The inappropriate Th2 response causes airway eosinophilic inflammation, mucus hypersecretion and airway hyperreactivity (AHR) result in the symptoms of asthma. The logical resolution of inflammation in the lung occurs through the inhibition of exaggerated Th2-mediated responses by immuno-regulatory cytokines. Both Th1-relatived cytokines, such as IL-12 , and immunosuppressive cytokines, including TGF-β and IL-10, are the candidate cytokines for the treatment of allergic diseases as they downregulate Th2 responses. In current study, we had compared the effect of several cytokines (IL-12, IL-10, TGF-β and IL-4) simultaneously on disease development in a murine model of asthma. Our data demonstrated that anti- inflammatory cytokines, particularly IL-10, have the therapeutic potential for the alleviation of airway inflammation in the asthma model in a dose-dependent manner. Furthermore, in order to achieve high local concentration of IL-10, we used IL-10-expressing adenovirus (Ad-IL-10) to elucidate the therapeutic effect of prolonged homologous IL-10 administration on airway inflammation. As our respect, localized expression of IL-10 may provide a more direct therapeutic approach for diseases of exaggerated proinflammation. However, a more attracting strategy is to enhance immune regulation, which can suppress or modulate Th2 response and cure allergic disease finally. Dendritic cells (DCs) play a crucial role in directing T helper cells differentiation and could be a good candidate of this therapeutic approach. Thus, we had examined whether Ad-IL-10 infected DCs (DC-IL-10) have the potential to induce the differentiation of Tr1-like cells by in vitro repetitive stimulation of na?ve DO11.10 CD4+ T cells with DC-IL-10 and suggest their therapeutic use. Taken together, these results suggested that application of immune-suppressive cytokine, such as IL-10, not only can suppress airway inflammation locally, but also has potential to modulate immune response by genetically modified DCs.Abstract………………………………………………………………….. i Abstract in Chinese….…………………………………………………. iii Contents…………………………………………………………………. v List of Figures…………………………………………………………… xi Chapter I. General Introduction…………………………………….. 1 1. An overview of the pathogenesis of asthma…………………. 2 2. How allergens induce and maintain allergy…………………..3 2.1 Sensitization and memory…………………………………..…………….3 2.2 Challenge phase……………………………………………..…………….4 2.3 Late phase response………………………………………..……………..4 3. The pathogenesis of asthma…………………………………….5 3.1 T-helper cells in allergic asthma………………………………………….5 3.2 Th1/Th2 paradigm…………………………………………………….……8 3.3 Immunomodulatory cytokines in asthmatic inflammation………….9 3.3.1 IL-10…………………………………………………..….10 3.3.2 TGF-β…………………………………………………….11 3.4 Role of dendritic cells (DCs) in the sensitization process to inhaled antigen……………………………………………………………….…. 13 3.4.1 Lung dendritic cells preferentially induce Th2 differentiation 15 3.4.2 Lung dendritic cells and tolerance….……………………...16 3.4.3 Dendritic cells as targets for therapy……………………....17 3.4.4 The therapeutic potential of modified DCs ………….….….18 3.4.5 IL-10 expressing dendritic cells………………………..…..19 3.5 T regulatory cells………………………………………………………..….20 3.5.1 Type 1 regulatory cells…………………………………....21 3.5.2 CD4+CD25+TRegcells………………………………….…..22 3.5.3 Th3 cells………………………………….......................23 3.5.4 Other TRegcells…………………………………...............23 3.5.5 Role of regulatory T cells in peripheral T-cell tolerance in the healthy immune response to allergens and allergen-SIT..........................24 4. Interleukin-10…………….……..................................................25 4.1 Biological activities…………………………...........................26 4.2 Interleukin-10 Receptor Signaling…………………………............27 5. Aims of the study………………………….................................. 30 Chapter II. Effects of overexpression of IL-10, IL-12, TGF-β and IL-4 on allergen-induced change in bronchial responsiveness……………….................................... 33 1. Background……………………………............................ 34 2. Materials & Methods……………………………....................... 36 2.1 Animals……………………………......................................................... 36 2.2 Plasmids and preparation of lipid-plasmid DNA complexes …….... 37 2.3 Administration of DNA-lipid complexes……………………………… 38 2.4 Administration of cytokine plasmid into allergen-sensitized mice… 38 2.5 Measurement of airway hyper-responsiveness………..................... 39 2.6 Analysis of bronchoalveolar lavage (BAL) fluid and lung histology. 40 2.7 Eotaxin level in BAL fluid……………………..………………...…….... 41 2.8 Measurement of cytokines……………………………........................ 41 2.9 Quantification of PGE2 and leukotriene B4 (LTB4)………………..... 42 2.10 Statistical analysis……………………………................................. 42 3. Results……………………………............................................. 42 3.1 The effect of different cytokine gene plasmids on methacholine-induced increase in AHR and airway eosinophilia………………..…. 42 3.2 The effect of different cytokine gene plasmids on eotaxin and LTB4 levels in BAL fluid…………………………………………..……. 45 3.3 Dose-dependent effect of IL-10 gene plasmid in the suppression of AHR and airway eosinophilic inflammation in OVA-sensitized mice………………………………………………………………….….. 46 3.4 Combination effect of IL-10 and IL-12 gene plasmids in the suppression of AHR and airway eosinophilic inflammation in OVA-sensitized mice………………………………………………………….. 47 4. Discussion………….……………………………........................ 48 5. Conclusions………………………………................................. 55 Chapter III. Alleviating effects of adenovirus-expressing IL-10 on airway inflammation in a murine model of asthma……………………………............................... 57 1. Introduction…………………………….....................................58 2. Materials and methods……………………………...................59 2.1 Animals……………………………....................................................59 2.2 Recombinant adenovirus construct………………………………..…..59 2.3 Detection of IL-10 in lungs and BAL fluid after the administration of adenoviral vector…………………………………………………… 60 2.4 Sensitization, treatment, and challenge…………………………..…..61 2.5 Measurement of airway hyper-responsiveness…………………..….62 2.6 Collection of BAL fluid and lung histology……………......................62 2.7 Statistical analysis ……………………………....................................62 3. Results…………………………….............................................63 3.1 IL-10 expression in BALF and lung tissues after intra-tracheal delivery of IL-10- expressing adenovirus……....………………..……63 3.2 Intra-tracheal delivery of Ad-IL-10 alleviated the severity of AHR and Ag-induced eosinophilic airway inflammation……………...……63 3.3 The levels of OVA-specific serum antibodies in OVA-sensitized mice after treatment with adenovirus-expressing IL-10 gene……... 65 4. Discussion……………………………………………………….. 65 Chapter IV. Induction of IL 10- producing CD4+ T cells with regulatory properties by repetitive stimulation with IL-10 gene-modified bone marrow derived DCs…………………………………………………….... 71 1. Introduction……………………………………………….. 72 2. Materials and methods…………….…………………………… 73 2.1 Mice……………………………..........................................................73 2.2 Reagents and Abs…………………………….....................................73 2.3 Cell preparations……………………………....................................74 2.3.1 Transduction of bone marrow derived dendritic cells with recombinant adenovirus………………………………………. 74 2.3.2 Isolation and purification of na?ve CD4+ T cells………..……75 2.3.3 Generation of regulatory T cells in vitro ……………….……75 2.4 In vitro stimulating ability of adenovirus infected DCs…………..…76 2.5 Characterization of T cell lines………………………………..………..76 2.5.1 Proliferation and cytokine secreting profiles of T cell lines..76 2.5.2 In vitro suppression assays………………………….77 2.6 Measurement of cytokine production…………………77 2.7 Statistical analysis……………………………....................77 3. Results…………………………….............................................78 3.1 Determination of the optimal ratio of infectious Ad-IL-10 particles to DCs……………………………...........…………………………………….78 3.2 Phenotype of Ad-infected DCs………………………………78 3.3 In vitro stimulatory ability of na?ve CD4+ T cells with adenovirus infected DCs…………………………….................................................79 3.4 Stimulation with DC-IL-10 further enhances the differentiation of IL-10-secreting, Tr1-like T-cells……………………………………………..79 4. Discussion………………………………………………………... 81 Chapter V. Conclusion and Perspectives…………………….... 85 Figures……………………………..............……………………….… 91 References……………………………............................................ 121 Appendix……………………………............................................... 1678544923 bytesapplication/pdfen-US氣喘介白質10基因治療調節性 T 細胞樹突細胞asthmainterleukin-10gene therapyregulatory T cellsdendritic cellsotherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/63340/1/ntu-95-D89449001-1.pdf