Innate Immune Mechanisms in Emphysema

描述（由申请人提供）：Toll样受体（TLR）已在病原体激发背景下进行了广泛研究，但其在未激发肺中的作用尚不清楚。阐明肺在复杂的内外环境中保持结构完整性的机制对我们理解肺部疾病至关重要。肺气肿的特征在于肺弹性的逐渐丧失和不可逆的空气空间扩大，在正常老化过程中在一定程度上发生，但随着长期暴露于污染物或香烟烟雾而病理性加速。事实上，只有10 - 20%的吸烟者发展为临床上显著的肺气肿，这表明其他因素对其发病机制也很重要。最近的人类研究表明，抑制的TLR 4功能与COPD的严重程度相关，这使我们的发现与人类相关，即TLR 4-/-小鼠在2个月大后肺发育完成时发生自发性肺气肿。我们发现，TLR 4缺乏诱导一种新的NADPH氧化酶（Nox），Nox 3和过量的氧化剂产生，也是人类肺气肿的一个特征。用Nox抑制剂或Nox 3 siRNA治疗TLR 4-/-小鼠或肺内皮细胞逆转了表型。我们产生了可诱导的、肺靶向的Nox 3转基因小鼠，这些小鼠发生肺气肿，并且还检测到COPD患者的Nox 3水平升高，这表明Nox 3在体内肺气肿发病机制中起重要作用。为了研究负责这些TLR 4介导的反应的细胞类型，我们产生了骨髓嵌合体，并发现非造血细胞上的TLR 4表达是维持正常肺结构所必需的。TLR 4对气道上皮的重建仅部分地防止TLR 4-/-中的肺气肿，并且不抑制Nox 3表达或氧化剂产生，表明替代结构细胞中的TLR 4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了内皮TLR 4的重要作用，我们将在本提案中探讨。我们的假设是，内皮TLR 4通过抑制Nox 3介导的氧化剂产生来维持肺的完整性。我们将在以下目的中检验这一假设：1）确定内皮TLR 4在体内预防氧化剂介导的肺气肿中的特异性贡献，2）阐明TLR 4抑制内皮细胞中Nox 3基因表达的机制，和3）表征参与抑制Nox 3表达和预防体内和肺内皮细胞中氧化剂介导的肺气肿的TLR 4配体。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会继续发展;然而，对于这种毁灭性的疾病，目前还没有有效的治疗方法。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大我们最近发现的新途径参与肺气肿，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.