- 學位論文
白血球菸醯胺腺嘌呤二核甘酸磷酸氧化脢與自然免疫細胞於二異氰酸甲苯所造成的肺部發炎反應中扮演免疫活化角色
The role of phagocytic NADPH oxidase and innate immune cells in the immune activation in toluene diisocyanate-induced lung inflammation
摘要
二異氰酸甲苯(TDI)屬於低分子量高活性的化合物，通常被使用於工業中，如製作聚氨酯泡棉、油漆混和物、清潔劑…等等，易揮發於空氣中而被工業環境工作者吸入肺部，是導致職業性氣喘的主要原因之一。職業性氣喘的病理特徵與常見的氣喘病徵相似，主要是由於特定職業環境的原因和狀況引起的。我們先前的研究發現，菸醯胺腺嘌呤二核苷酸磷酸氧化酶(NADPH氧化酶)是TDI誘導的肺部組織發炎反應的重要調節者，其免疫反應特徵在於具其調控了Th17和Th2免疫反應而造成嚴重的肺部發炎。我們研究認為NADPH缺陷的吞噬細胞氧化酶的氧化還原失衡的變化可能會影響早期自然免疫細胞聚集和其後伴隨的特異性免疫反應所造成的肺部發炎。在此研究中，我們對ROS和自然免疫類淋巴球細胞（ILC）之間對於TDI誘發的肺部發炎所調控的免疫機制感興趣。已知自然免疫類性淋巴球細胞在淋巴組織的發育及誘發早期免疫反應中起重要作用。在本研究中，我們假設白血球NADPH氧化酶會藉由影響自然免疫類性淋巴球細胞活化進而引發下游一連串免疫反應機制，最終導致TDI誘發肺部發炎的病徵。我們使用野生型和白血球NADPH氧化酶2缺陷型（NOX2缺陷型; Ncf1 - / -）小鼠並用3％TDI進行引起致敏以誘導肺部發炎反應。首先，我們測試了TDI刺激是否引起野生型或NOX2缺乏（Ncf1 - / -）小鼠過敏性氣喘病徵。結果發現TDI刺激會引起野生型小鼠的高反應性的氣喘病徵，但Ncf1 - / -小鼠則無此症狀。而在檢測TDI是否誘發與免疫反應相關細胞因子產生中，我們發現包括IL-33和IL-5在內的第2型炎症細胞因子，在TDI刺激的組別中，野生型和Ncf1 - / -小鼠中均會增加。此外，促炎細胞因子IL-1β和IL-17A在TDI刺激的野生型小鼠中更有顯著的增加，在Ncf1 - / -小鼠中則無此現象。接著，我們想進一步了解自然免疫類淋巴球細胞是否存在於TDI刺激的野生型和Ncf1 - / -小鼠肺部中並參與了免疫調控。比較TDI刺激下的野生型小鼠和Ncf1 - / -小鼠之間自然免疫類淋巴球細胞的結果顯示在野生型小鼠中的第二型自然免疫類淋巴球細胞(ILC2s)和第三型自然免疫類淋巴球細胞(ILC3s)的數量顯著更高。我們也透過給予刺激第三型自然免疫類淋巴球細胞群的上游細胞因子，介白素-1β(IL-1β)及介白素-23(IL-23)，發現ROS會透過影響自然免疫類淋巴球細胞中特定子群體(NCR-ILC3)，進而促進免疫反應在TDI刺激下誘發的嚴重肺部發炎。因此，這些結果表明ROS可能會經由調節ILC2s及ILC3s的活化，並促使產生許多會使TDI刺激下產生嚴重肺部發炎反應的第2型及第17型炎症細胞因子。我們的研究結果表明ROS會透過調節上游特定自然免疫類淋巴球細胞群進而調控下游免疫反應的機制，在TDI所誘發的嚴重肺部發炎反應中是重要的上游免疫機制調節者。
並列摘要
Isocyanates are low-molecular-weight compounds noted for inducing an oxidant stress-dependent pulmonary inflammation, which is the leading cause of occupational asthma. The occupational asthma is characterized by variable airflow limitation and airway hyperresponsiveness due to causes and conditions attributable to a particular occupational environment and not to stimuli encountered outside the workplace. Oxidant stress mediated by reactive oxygen species (ROS), including superoxide, singlet oxygen (O.), H2O2, and hydroxyl radical (-OH), has been known to be important in the immunopathogenesis of the lung, which is exposed to a higher concentration of oxygen than is most other tissue. The phagocyte NADPH oxidase (NOX2), which is highly expressed in phagocytes such as neutrophils and monocytes, is usually the most active producer of ROS in inflammatory tissues. Our previous studies indicated that phagocytic NADPH oxidase is an essential regulator in TDI-induced airway inflammation characterized by a server lung inflammation with both Th17 and Th2 immune components. Change of redox balance by antioxidants or defective phagocyte oxidase may affect the lung inflammation in terms of early innate immune cell activation/ recruitment and the subsequent specific immune responses. Innate lymphoid cells (ILCs) have been known to play an important role in the development of lymphoid tissues and in the initiation of inflammation at barrier surfaces in response to infection or tissue damage. In this study, we hypothesized that ILCs activation may play an important role in phagocytic NADPH oxidase-mediated downstream immune mechanism in TDI-induced airway inflammation. The wild type and phagocytic NADPH oxidase 2 deficient (NOX2 deficiency; Ncf1-/-) mice were sensitized and challenged with 3% TDI to induce lung inflammation. First, we tested the effects of TDI on allergic airway inflammation in wild type and NOX2 deficiency (Ncf1-/-) mice. We found that TDI exposure induced airway hyperresponsiveness in wild type but not in Ncf1-/- mice. In the effects of TDI on the induction of cytokines production, type 2 inflammatory cytokines, including IL-33 and IL-5, increased in both WT and Ncf1-/- mice but more significantly in TDI-induced lung inflammation. Moreover, the pro-inflammatory cytokine IL-1β and IL-17A production was only induced by TDI-exposure in wild type but not in Ncf1-/- mice. The relative abundance of ILC subsets tended to change in TDI-induced wild type and Ncf1-/- mice. We identified the population of lineage negative and CD45 positive cells in inflammation lung tissue. Comparison of ILCs population between TDI-induced wild type mice and Ncf1-/- mice showed that the number of ILC2s and ILC3s were significantly higher in wild type mice. To investigate whether ROS affect more NCR- ILC3 population in TDI-induced lung inflammation through stimulating ILC3 upstream cytokines production, we purified the NCR- ILC3, which were sorted from the inflammatory lung tissue of TDI-induced wild type and Ncf1-/- mice, and stimulated with recombined protein IL-1β in cell culture. We found that IL-1β stimulation induced change of exILC3 phenotype to NCR- ILC3 which produce more IL-17A. Moreover, in the case of IL-1β and IL-23 combined stimulation, the change of exILC3 phenotype to NCR- ILC3 was more pronounced. These results suggested that ROS may modulate the activation of ILC2s, ILC3s and may determine the Th2 and Th17 cytokines production in the TDI-induced lung inflammation.