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研究生： 黃名毅
Huang, Ming-Yi
論文名稱： 白血球菸鹼醯胺腺嘌呤二核苷酸磷酸氧化酶的缺損加重塵螨誘發的肺纖維化
Defective NADPH oxidase 2 leads to more severe Dermatophagoides pteronyssinus-induced lung fibrosis
指導教授： 謝奇璋
Shieh, Chi-Chang
學位類別： 碩士
Master
系所名稱： 醫學院 - 臨床醫學研究所
Institute of Clinical Medicine
論文出版年： 2021
畢業學年度： 109
語文別： 英文
論文頁數： 51
中文關鍵詞： 過敏性肺纖維化 、 塵蟎 、TH2/17免疫反應 、活性氧化物質 、白血球NADPH 氧化酶
外文關鍵詞： Allergic lung fibrosis, Dermatophagoides pteronyssinus (Der p1), TH2/17 inflammatory response, Reactive oxygen species (ROS), NADPH oxidase 2(NOX2)
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    • 肺纖維化是一種不可逆轉的疾病，且在受損的部位會因為成纖維細胞的分化，產生胞外基質 (ECM) 的堆積造成組織硬化。目前發現過敏原造成的肺纖維化主要發生在重度的氣喘患者。與TH2 免疫反應為主的輕至中度氣喘患者相比，重度氣喘患者通常伴隨著TH2和TH17混和型的免疫反應，並藉由分泌促纖維化的細胞因子進一步調節成纖維細胞的分化。在纖維化過程中，成纖維細胞分化為肌成纖維細胞並在組織中分泌細胞外基質(ECM)造成組織硬化。先前的研究表明，白血球NADPH 氧化酶 (NOX2) 會產生活性氧化物質 (ROS)，進而調節氣喘患者的肺部發炎反應。在我們之前的研究中，我們發現有缺陷的 NOX2 會透過增強第2 型先天淋巴細胞（ILC2）的活性，進而提升TH2和TH17兩者的免疫反應，造成過敏原塵蟎（Der p1）所誘導的氣喘症狀較為嚴重。然而，尚不清楚有缺陷的 NOX2 是否會同樣促進Der p1所誘導的肺纖維化。因此，透過之前的證據，我們假設有缺陷的 NOX2 會導致更嚴重的 Der p1 誘導的肺纖維化。在這項研究中，我們使用過敏原Der p1 刺激野生型 (WT) 和 NOX2 缺陷型 (Ncf1-/-) 小鼠。首先，我們發現 NOX2 缺陷型小鼠藉由檢測出動態順應性的降低，說明其有更嚴重的肺僵硬跡象。以及透過病理切片發現有較多的胞外基質，例如第I 型膠原蛋白，累積在細支氣管周圍。接著我們分析了成纖維細胞群，發現在沒有 NOX2 的情況下，刺激後小鼠的肺中肌成纖維細胞的比例有增加的趨勢。第三，我們檢測TH2/17免疫反應相關的促纖維化細胞因子。我們發現這些促纖維化細胞因子的表現在NOX2 缺陷型小鼠的肺中呈現減少的趨勢，目前推測可能的原因是在NOX2缺少的情況下，這些促纖維化細胞因子被過度消耗以激活第2和17型免疫反應的下游成員，間接促進 TGF-β 的表現造成纖維化加劇。此外，在FGF2方面，我們發現相較於野生型小鼠，NOX2 缺陷型小鼠的肺部組織在不受Der p1刺激的情況下，就已經有表現有較高的趨勢。這說明NOX2 缺陷型小鼠可能較容易發生肺纖維化。最後，從我們的結果看到與 WT 小鼠相比，NOX2 缺陷小鼠在Der p1刺激後，肺中的 ILC2 數量有增加的趨勢。因此，我們推論NOX2 的缺陷會加劇過敏原塵蟎所引起的ILC2 活化，調節免疫細胞因子，從而加速成纖維細胞的分化，造成更嚴重肺纖維化的病變，證明白血球NADPH 氧化酶 (NOX2) 在過敏性纖維化的的重要性。

    Lung fibrosis is well-known as an irreversible process which is characterized by accumulation of extracellular matrix (ECM) produced by fibroblasts after tissue damage. In the fibrotic progression, fibroblasts differentiate into myofibroblasts and secrete the ECM, such as collagen, in the tissues. Lung fibrosis is a severe symptom in asthmatic patients. Comparing to TH2-predominant inflammation in mild-to-moderate asthmatic patients, severe asthmatic patients usually have the sign of TH2/17-mediated inflammation and further modulates fibroblast differentiation through pro-fibrotic cytokines. Previous studies indicated that phagocytic NADPH oxidase (NOX2) produces reactive oxygen species (ROS) which regulate lung inflammation in asthma. In our previous researches, we found that defective NOX2 elevated Dermatophagoides pteronyssinus (Der p1)-induced asthmatic inflammation induced by TH2/17-mediated phenotypes due to enhancing type 2 innate lymphoid cell (ILC2) activity. However, it’s not clear whether defective NOX2 promotes allergen-induced lung fibrosis. Therefore, we hypothesized that defective NOX2 leads to more severe Der p1-induced lungs fibrosis. In this study, we challenged wild-type (WT) and NOX2-deficient (Ncf1-/-) mice with intranasal instillation of Der p1. Firstly, we found that more severe lung stiffness as measured by dramatic decrease of dynamic compliance and more ECM (including type I collagen) accumulation around the bronchioles in NOX2-deficient mice after challenging with Der p1. Next, we analyzed fibroblasts populations and found that myofibroblast population increased significantly in NOX2-deficient mice after Der p1 stimulation. Thirdly, we detected the IL-33, IL-13, IL-1β, IL-17, TGF-β, and FGF2. Type 2 inflammatory cytokines tended to increase in the NOX2-deficient mice after challenging with Der p1. Also, higher basic level of FGF2 was observed in NOX2-deficient mice, which could be prone to the process of fibrosis. Finally, our data indicated that ILC2 population increased in fibrotic lungs of NOX2-deficient mice after Der p1 stimulation. Therefore, we concluded that Der p1-induced ILC2 and type 2 inflammatory cytokines may contribute to the more severe lung fibrosis in defective NOX2 mice, causing the severe fibrotic phenotypes. This study supports the importance of phagocyte NADPH in allergic fibrosis.

    Abstract I 中文摘要 III 誌 謝 V Abbreviations IX Chapter. 1 Introduction 1 1.1 Lung fibrosis 2 1.1.1 The phenotypes of lung fibrosis 2 1.1.2 The current treatment of lung fibrosis 3 1.2 The process of allergic lung fibrosis 3 1.2.1 Allergic lung fibrosis 3 1.2.2 Dermatophagoides pteronyssinus (Der p) initiates allergic inflammation in lungs 4 1.2.3 Type 2 and type 17 immunity are involved in allergic lung fibrosis 5 1.3 The NADPH oxidase system (NOX system) 6 1.3.1 Reactive oxygen species (ROS) and NADPH oxidases 2 (NOX2) 6 1.3.2 NOX-derived ROS is an essential regulator in allergic inflammation 8 Chapter 2. Materials and methods 10 2.1 Experimental animals 11 2.2 Der p1 exposure experiment 11 2.3 Measurement of lung function 12 2.4 Lung tissue collection, staining, and histology examination 12 2.4.1 Masson’s trichrome stain 13 2.4.2 Immunohistochemistry (IHC) stain 14 2.5 Flow cytometric analysis 15 2.5.1 Fibroblasts detection by flow cytometric analysis 15 2.5.2 ILCs detection by flow cytometric analysis 15 2.6 Measurement of cytokines 16 2.7 Statistical analysis 16 Chapter 3. Results 17 3.1 The dramatic decrease of dynamic compliance in Ncf1-/- mice after challenging with Der p1 18 3.2 Cell infiltration and ECM increased in Ncf1-/- mice after challenging with Der p1 19 3.3 Myofibroblast population increased significantly in Ncf1-/- mice after Der p1 stimulation 20 3.4 More collagen accumulation in Ncf1-/- mice after challenging with Der p1 21 3.5 Type 2 inflammatory cytokines tended to increase in the Ncf1-/- mice after challenging with Der p1 22 3.6 ILC2 population increased in fibrotic lungs of Ncf1-/- mice after Der p1 stimulation 23 Chapter 4. Discussion 24 4.1 Respiratory burst is a multifaceted factor to regulate the lung fibrosis 25 4.2 NOX2 plays a protective role in Der p1-induced lung fibrosis. 26 4.3 NOX2 deficiency upregulates more type 2 inflammation and further exacerbate allergic fibrosis 27 4.4 ROS-mediated host defense and Nrf2 pathway are necessary to recover the homeostasis 29 Chapter 5. Figures and Table 31 Figure 1. The dramatic decrease of dynamic compliance in Ncf1-/-mice after challenging with Der p1 33 Figure 2. Cell infiltration and ECM increased in Ncf1-/- mice after challenging with Der p1 35 Figure 3. Myofibroblast population increased significantly in Ncf1-/- mice after Der p1 stimulation. 37 Figure 4. More collagen accumulation in Ncf1-/- mice after challenging with Der p1 39 Figure 5. Type 2 inflammatory cytokines tended to increase in the Ncf1-/- mice after challenging with Der p1 41 Figure 6. ILC2 population increased in fibrotic lungs of Ncf1-/- mice after Der p1 stimulation 43 Table 1. Antibodies for depletion of lineage-positive cells and flow cytometry analysis of fibroblast and ILC2. 44 Chapter 6. References 46

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