Innate Immune Mechanisms in Emphysema

描述（由申请人提供）：toll样受体（TLRs）已经在病原体挑战的背景下进行了广泛的研究，但它们在未挑战的肺中的作用尚不清楚。阐明肺在面对复杂的外部和内部环境时保持结构完整性的机制对我们了解肺部疾病至关重要。肺气肿的特征是肺弹性的逐渐丧失和不可逆的空气空间扩大，在一定程度上发生在正常的衰老过程中，但随着长期暴露于污染物或香烟烟雾而病理加速。事实上，只有10%到20%的吸烟者会出现临床显著的肺气肿，这表明其他因素对其发病也很重要。最近的人类研究表明，TLR4功能的抑制与COPD的严重程度相关，这与我们发现TLR4-/-小鼠在肺发育完成2月龄后发生自发性肺气肿具有人类相关性。我们发现TLR4缺陷诱导一种新的NADPH氧化酶（Nox）， Nox3和过量的氧化剂生成，这也是人类肺气肿的一个特征。用Nox抑制剂或Nox3 siRNA处理TLR4-/-小鼠或肺内皮细胞可逆转表型。我们培育了可诱导的、肺靶向的Nox3转基因小鼠，这些小鼠发生肺气肿，并在COPD患者中检测到Nox3水平升高，表明Nox3在体内肺气肿发病机制中发挥重要作用。为了研究负责这些TLR4介导反应的细胞类型，我们生成了骨髓嵌合体，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。TLR4对气道上皮的重建仅部分阻止了TLR4-/-中的肺气肿，并没有抑制Nox3的表达或氧化剂的产生，这表明TLR4在另一种结构细胞中也参与调节肺氧化剂。我们使用原代肺内皮细胞的数据指出内皮TLR4的重要作用，我们将在本提案中对此进行探讨。我们的假设是内皮TLR4通过抑制nox3介导的氧化剂生成来维持肺的完整性。我们将在以下几个方面验证这一假设：1)确定内皮细胞TLR4在体内预防氧化介导的肺气肿中的具体作用；2)阐明TLR4在内皮细胞中抑制Nox3基因表达的机制；3)表征TLR4配体在体内和肺内皮细胞中抑制Nox3表达和预防氧化介导的肺气肿的作用。公共卫生相关性：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继续发展；然而，目前还没有针对这种毁灭性疾病的有效疗法。因此，我们必须了解肺维持其正常结构和功能的方式。我们提出的研究将扩展我们最近发现的涉及肺气肿的新途径，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.