Chronic Granulomatous Lung Inflammation Elicited by Carbon Nanotubes

描述(申请人提供)：在制造的消费产品中使用纳米材料是一个迅速扩大的领域，但潜在的毒性尚未确定。燃烧生成的多壁碳纳米管(MWCNT)或纳米颗粒在非制造环境中普遍存在，并可在柴油、甲烷、丙烷和天然气的蒸气中检测到。碳纳米管在一些实验动物中会导致肉芽肿或炎症。人类疾病中的肺肉芽肿可能是对环境刺激如细胞内病原体、惰性物质和有机抗原的反应而形成的。结节病是一种典型的肉芽肿性疾病，其病因尚不清楚。多种环境风险因素与结节病有关，包括暴露在烧柴的炉子、壁炉和灭火中--这些条件可能有利于在环境空气中形成碳纳米管。有关纳米管环境因素与结节病相关的研究尚未使用动物模型进行。这项建议将利用一种新的小鼠多壁碳纳米管诱导的慢性肉芽肿模型来研究碳纳米管对特定宿主受体--过氧化物酶体增殖物激活受体？的影响。(PPAR？)趋化细胞因子受体5(CCR5)，据报道在结节病肺中表达异常。PPAR？是一种炎症的负性调节因子，在健康的肺泡巨噬细胞中有结构性表达，但在严重的结节病中缺乏。相反，促炎性CCR5配体在健康肺中没有发现，但在结节病中升高。我们在MWCNT灌输的小鼠的肺中发现了类似的发现：PPAR？被抑制，CCR5配体升高。基于这些数据，我们假设MWCNT抑制PPAR？上调CCR5通路，形成伴有慢性炎症的肺肉芽肿。具体目标1将决定PPAR的作用？在MWCNT模型中，通过监测以下两组小鼠的肉芽肿大小和数量：(A)巨噬细胞特异性PPAR？缺失与野生型小鼠；(B)慢病毒-PPAR治疗的小鼠？质粒，PPAR？激动剂罗格列酮；或PPAR？敌手徽章与未经治疗的小鼠。特异性目标2将通过定量检测来自支气管肺泡灌洗(BAL)的肺泡巨噬细胞、BAL液中的CCR5受体和趋化因子，以及通过激光捕获显微解剖[LCM]分离的肉芽肿灶、未经处理的野生型和PPAR？来研究CCR5在MWCNT肉芽肿中的作用。空白小鼠与CCR5阻滞剂治疗的小鼠。这项研究将为学生提供机会获得肺部疾病动物模型的经验，了解疾病中基因缺失的影响，并与医生互动，了解环境导致人类肺部疾病的原因以及人类肺部疾病是如何诊断和治疗的。学生还将学习用于基因转导的慢病毒质粒构建、定量RT-PCR、LCM、Luminex分析和成像方法。总之，这项关于多壁碳纳米管引起的肉芽肿性肺部疾病的独特研究非常适合学生参与一个对人类疾病有环境影响的领域。

DESCRIPTION (provided by applicant): Use of nanomaterials in manufactured consumer products is a rapidly expanding area but potential toxicities have not been established. Combustion-generated multiwall carbon nanotubes (MWCNT) or nanoparticles are ubiquitous in non-manufacturing environments and detectable in vapors from diesel fuel, methane, propane and natural gas. Carbon nanotubes induce granulomas or inflammation in some experimental animals. Pulmonary granulomas in human disease may form in response to environmental stimuli such as intracellular pathogens, inert materials, and organic antigens. In sarcoidosis, a prototypical granulomatous disease, etiology remains obscure. Multiple environmental risk factors have been linked to sarcoidosis, including exposure to wood-burning stoves, fireplaces, and firefighting - conditions that might favor carbon nanotube formation in ambient air. Investigation of putative nanotube environmental factors linked to sarcoidosis has not been done using animal models. This proposal will utilize a novel murine MWCNT-elicited, chronic granuloma model to investigate the impact of carbon nanotubes on specific host receptors, peroxisome proliferator-activated receptor? (PPAR?) and chemotactic cytokine receptor 5 (CCR5), reported to be dysregulated in sarcoidosis lung. PPAR?, a negative regulator of inflammation, is constitutively expressed in healthy alveolar macrophages but deficient in severe sarcoidosis. In contrast, proinflammatory CCR5 ligands are not found in healthy lung but are elevated in sarcoidosis. We noted similar findings in lungs of MWCNT-instilled mice: PPAR? is depressed and CCR5 ligands are elevated. Based on these data, we hypothesize that MWCNT repress PPAR? and upregulate CCR5 pathways to form pulmonary granulomas with chronic inflammation. Specific Aim 1 will determine the role of PPAR? in the MWCNT model by monitoring granuloma size and numbers in: (a) macrophage-specific PPAR?-null versus wild-type mice; and (b) mice treated with lentivirus-PPAR? plasmids, PPAR? agonist rosiglitazone; or PPAR? antagonist BADGE versus untreated mice. Specific Aim 2 will examine CCR5 involvement in MWCNT granulomas by quantifying CCR5 receptor and chemokines in bronchoalveolar lavage (BAL) derived alveolar macrophages, BAL fluids, and in granulomatous foci isolated by laser-capture microdissection [LCM], from untreated wild-type and PPAR? null mice versus mice treated with a CCR5 blocker. This study will afford students opportunities to gain experience with animal models of lung disease, learn effects of gene deletion in disease, and interact with physicians to learn environmental causes of human lung disease and how human lung disease is diagnosed and treated. Students will also learn lentivirus plasmid construction for gene transduction, quantitative RTPCR, LCM, Luminex assays, and imaging methodology. In summary, this unique investigation of MWCNT-elicited granulomatous lung disease is well suited for student participation in an area with environmental impact on human disease.