Innate Immune Mechanisms in Emphysema

描述(由申请人提供)：Toll样受体(TLRs)在病原体挑战的背景下已被广泛研究，但它们在未受挑战的肺中的作用尚不清楚。阐明肺在面对复杂的内外环境时保持结构完整性的机制对于我们理解肺部疾病是至关重要的。肺气肿的特征是肺弹性逐渐丧失和不可逆转的肺泡扩大，在正常的衰老过程中会发生一定程度的肺气肿，但随着长期接触污染物或香烟烟雾，肺气肿的病理加速。事实上，只有10%到20%的吸烟者会患上临床上有意义的肺气肿，这表明其他因素在其发病机制中也很重要。最近的人类研究表明，TLR4功能低下与COPD的严重程度相关，这与我们的发现相关联，即TLR4-/-小鼠在2个月后肺发育完成时会出现自发性肺气肿。我们发现，TLR4缺乏会导致一种新的NADPH氧化酶(NOX)，即NOX3，以及过量的氧化剂生成，这也是人类肺气肿的一个特征。用Nox抑制剂或Nox3 siRNA处理TLR4-/-小鼠或肺内皮细胞，逆转了这种表型。我们建立了可诱导的、肺靶向的Nox3转基因小鼠，这些小鼠患上了肺气肿，并在COPD患者中检测到Nox3水平升高，这表明Nox3在体内肺气肿的发病机制中发挥了重要作用。为了研究TLR4介导的这些反应的细胞类型，我们产生了骨髓嵌合体，发现TLR4在非造血细胞上的表达是维持正常肺结构所必需的。在TLR4-/-中，TLR4在呼吸道上皮细胞上的重建只能部分防止肺气肿，而不能抑制Nox3的表达或氧化剂的产生，这表明另一种结构细胞中的TLR4也参与了对肺氧化剂的调节。我们使用原代肺内皮细胞的数据指出了内皮细胞TLR4的重要作用，我们将在本提案中探讨这一点。我们的假设是，内皮细胞TLR4通过抑制Nox3介导的氧化剂的产生来维持肺的完整性。我们将从以下几个方面对这一假说进行验证：1)确定内皮细胞TLR4在体内预防氧化剂介导的肺气肿中的具体作用；2)阐明TLR4抑制内皮细胞NOX3基因表达的机制；3)鉴定体内和肺内皮细胞中参与抑制NOX3表达和预防氧化剂介导的肺气肿的TLR4配体(S)。与公共卫生相关：肺气肿是一种危及生命的肺部破坏，即使在停止吸烟等可识别的沉淀物后也可能发展；然而，目前还没有有效的治疗方法来治疗这种毁灭性的疾病。因此，我们必须了解肺如何维持其正常的结构和功能。我们拟议的研究将扩展我们最近发现的与肺气肿有关的新途径，我们的结果将确定新的治疗靶点。

DESCRIPTION (provided by applicant): Toll-like receptors (TLRs) have been studied extensively in the context of pathogen challenges, yet their role in the unchallenged lung is unclear. Elucidating the mechanisms whereby the lung maintains structural integrity in the face of complex external and internal environments is essential to our understanding of lung disease. Emphysema is characterized by gradual loss of lung elasticity and irreversible airspace enlargement, occurring to a certain extent in the normal aging process but pathologically accelerated with chronic exposure to pollutants or cigarette smoke. The fact that only 10 to 20% of smokers develop clinically significant emphysema indicates that other factors are important to its pathogenesis. Recent human studies indicate that depressed TLR4 function is correlated with the severity of COPD, which lends human relevance to our finding that TLR4-/- mice develop spontaneous emphysema after 2 months of age, when lung development is complete. We found that TLR4 deficiency induces a novel NADPH oxidase (Nox), Nox3, and excessive oxidant generation, also a feature of human emphysema. Treatment of TLR4-/- mice or lung endothelial cells with Nox inhibitors or Nox3 siRNA reversed the phenotype. We generated inducible, lung-targeted Nox3 transgenic mice that develop emphysema and also detected elevated Nox3 levels in patients with COPD, indicating an important role for Nox3 in the pathogenesis of emphysema in vivo. In order to investigate the cell type responsible for these TLR4-mediated responses, we generated bone marrow chimeras and found that TLR4 expression on non-hematopoietic cells is required to maintain normal lung architecture. Reconstitution of TLR4 to the airway epithelium only partially prevented emphysema in TLR4-/- and did not suppress Nox3 expression or oxidant generation, indicating that TLR4 in an alternative structural cell is also involved in regulating lung oxidants. Our data using primary lung endothelial cells point to an important role for endothelial TLR4, which we will explore in this proposal. Our hypothesis is that endothelial TLR4 maintains lung integrity by suppressing Nox3-mediated oxidant generation. We will test this hypothesis in the following Aims: 1) Determine the specific contribution of endothelial TLR4 in preventing oxidant-mediated emphysema in vivo, 2) Elucidate the mechanisms whereby TLR4 inhibits Nox3 gene expression in endothelial cells, and 3) Characterize the TLR4 ligand(s) involved in inhibiting Nox3 expression and preventing oxidant-mediated emphysema in vivo and in lung endothelial cells. PUBLIC HEALTH RELEVANCE: Emphysema is a life-threatening destruction of the lungs and can progress even after cessation of identifiable precipitants such as cigarette smoking; however, effective therapies do not yet exist for this devastating disease. Therefore, it is imperative that we understand ways in which the lung maintains its normal structure and function. Our proposed studies will expand upon our recent findings of novel pathways involved in emphysema and our results will identify new therapeutic targets.