Contribution of Toll-like Receptors in the Pulmonary Response to Nanoparticles an

描述（由申请人提供）：尽管广泛使用，但我们对与纳米颗粒（NP）暴露相关的生物毒性的了解有限，这导致了对潜在不良健康影响的担忧。吸入可能是主要的接触途径，而呼吸的颗粒物与肺部疾病之间的联系强调了了解NP如何影响肺部的迫切需要。我们对单壁碳纳米管（SWNT）特别感兴趣，因为它们被广泛使用，并且与石棉表面相似，这可能与其长期健康后果有关。另外的关注点围绕着NP调节感染因子行为的能力。由于NP暴露而增加的对感染的易感性可以具有巨大的后果，特别是对于病毒，例如甲型流感（IAV），其因引起全球大流行而臭名昭著。由于现实的暴露情景可能涉及多种因子，触发保守的信号传导机制可能导致有害反应增强，从而导致更严重的健康后果。基于这些概念，本申请的总体目标是表征控制暴露于单独和组合的SWNT和IAV的肺上皮细胞的初级免疫应答的机制。我们的机制重点将集中在Toll样受体（TLR）上，因为它们是进入体内的外来因子的早期防线。我们将测试不同手性/直径的SWNT刺激TLR导致通过激活转录因子NF-κ B和IRF产生促炎细胞因子的假设。此外，SWNT和IAV的组合暴露将协同激活TLR驱动的途径，导致增强的炎症和损伤。这一前提将在3个全面的具体目标中进行测试，这些目标采用最先进的技术来（1）确定具有不同手性包裹角和直径的SWNT以及IAV对不同TLR的刺激，（2）评估SWNT和IAV的直接相互作用，并研究这些试剂在肺上皮细胞中选择TLR调节的作用，以及（3）检查在体内部分由TLR-NF-κ B和/或TLR-IRF介导的单次和连续暴露SWNT和IAV的肺部炎症和清除反应。体外研究将采用肺上皮细胞作为体内暴露的主要靶点，使我们能够确定导致器官功能障碍的分子途径。我们将利用一系列基于人类细胞的TLR筛选试验、创新的结合研究和转基因小鼠来实现这些目标。此外，我们将采用定制的近红外荧光（NIRF）成像系统来跟踪肺组织中的SWNT，并在SWNT暴露后的活体动物中进行系统性研究。这项工作的结果将全面了解NP的多个方面如何影响细胞功能，并将提供可靠的体外模型系统来评估和设计安全的纳米材料。

DESCRIPTION (provided by applicant): Despite wide-scale use, we have limited understanding of the biotoxicity associated with nanoparticle (NP) exposures which has led to concerns regarding potential adverse health effects. Inhalation is likely a primary exposure route and the association between respired particulate matter with pulmonary disease, underscores the critical need to comprehend how NP impact the lungs. We have a particular interest in single-walled carbon nanotubes (SWNT) as they are widely used and possess a superficial resemblance to asbestos which may be relevant to their long term health consequences. Additional concerns surround the ability of NP to modulate the behavior of infectious agents. Increased susceptibility to infections as a result of NP exposure can have immense consequences particularly for viruses, such as influenza A (IAV), that are notorious for causing global pandemics. As realistic exposure scenarios are likely to involve multiple agents, triggering conserved signaling mechanisms may lead to enhanced detrimental responses that contribute to more severe health outcomes. Based on these notions, the overall objective of this application is to characterize the mechanisms controlling the primary immune response of lung epithelial cells exposed to SWNT and IAV singly and in combination. Our mechanistic focus will center on toll-like receptors (TLRs) as they are an early line of defense to foreign agents that enter in the body. We will test the hypothesis that SWNT of distinct chirality/diameter stimulate TLRs resulting in the production of pro-inflammatory cytokines through activation of the transcription factors NF-k¿ and IRFs. Furthermore, combined exposures of SWNT and IAV will synergistically activate TLR-driven pathways leading to enhanced inflammation and injury. This premise will be tested in 3 comprehensive specific aims which employ state-of-the-art technologies to (1) determine stimulation of distinct TLRs by SWNT having different chiral wrapping angles and diameters as well as by IAV, (2) assess direct interactions of SWNT and IAV and investigate the role of select TLR modulation by these agents in lung epithelial cells and (3) examine the lung inflammatory and clearance response of single and sequential exposures of SWNT and IAV mediated, in part, by TLR-NF-k¿ and/or TLR-IRFs in vivo. In vitro studies will employ lung epithelial cells as a primary target of in vivo exposures, permitting us t define the molecular pathways which lead to organ dysfunction. We will utilize a series of human cell-based TLR screening assays, innovative binding studies and genetically modified mice to address these aims. In addition, we will employ a custom near-infrared fluorescence (NIRF) imaging system to track SWNT in lung tissues and systemically in live animals following SWNT exposures. Results of this work will generate a comprehensive understanding of how multiple aspects of NP affect cell function and will provide reliable in vitro model systems to evaluate and engineer safe nanomaterials.