Chronic Granulomatous Lung Inflammation Elicited by Carbon Nanotubes

描述（由申请人提供）：纳米材料在制造消费品中的使用是一个迅速扩大的领域，但潜在的毒性尚未确定。燃烧产生的多壁碳纳米管（MWCNT）或纳米颗粒在非制造环境中无处不在，在柴油、甲烷、丙烷和天然气的蒸气中都可以检测到。碳纳米管在一些实验动物中引起肉芽肿或炎症。人类疾病中的肺肉芽肿可能形成于对环境刺激的反应，如细胞内病原体、惰性物质和有机抗原。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，包括暴露于烧木头的炉子、壁炉和消防环境中，这些条件可能有利于环境空气中碳纳米管的形成。尚未使用动物模型对与结节病相关的假定纳米管环境因素进行调查。本研究将利用一种新的小鼠mwcnt诱导的慢性肉芽肿模型来研究碳纳米管对特异性宿主受体、过氧化物酶体增殖体激活受体的影响。（PPAR?）和趋化细胞因子受体5 (CCR5)，据报道在肺结节病中失调。PPAR吗?是炎症的负调节因子，在健康的肺泡巨噬细胞中组成性表达，但在严重的结节病中缺乏。相比之下，促炎CCR5配体在健康肺中未发现，但在结节病中升高。我们注意到mwcnts灌注小鼠肺部的类似发现：PPAR？CCR5配体升高。基于这些数据，我们假设MWCNT抑制PPAR？并上调CCR5通路，形成慢性炎症的肺肉芽肿。特异性目标1将决定PPAR的作用？通过监测MWCNT模型中肉芽肿的大小和数量：(a)巨噬细胞特异性PPAR？-null与野生型小鼠；(b)慢病毒- ppar ？质粒,PPAR ?受体激动剂罗格列酮;或PPAR吗?拮抗剂BADGE与未治疗小鼠的对比。特异性目标2将通过量化支气管肺泡灌洗（BAL）衍生的肺泡巨噬细胞、BAL液体和激光捕获显微解剖[LCM]分离的肉芽肿灶中的CCR5受体和趋化因子，研究CCR5在MWCNT肉芽肿中的作用，这些肉芽肿灶来自未治疗的野生型和PPAR？与CCR5阻滞剂治疗的小鼠相比。这项研究将使学生有机会获得肺病动物模型的经验，了解基因缺失在疾病中的影响，并与医生互动，了解人类肺病的环境原因以及如何诊断和治疗人类肺病。学生还将学习用于基因转导的慢病毒质粒构建、定量RTPCR、LCM、Luminex检测和成像方法。总之，这种独特的mwcnt引发的肉芽肿性肺病的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.