Chronic Granulomatous Lung Inflammation Elicited by Carbon Nanotubes

描述（由申请人提供）：在制成品中使用纳米材料是一个迅速扩大的领域，但尚未确定潜在毒性。燃烧产生的多壁碳纳米管（MWCNT）或纳米颗粒在非制造环境中普遍存在，并且在柴油燃料、甲烷、丙烷和天然气的蒸气中可检测到。碳纳米管在一些实验动物中诱导肉芽肿或炎症。人类疾病中的肺肉芽肿可能是对环境刺激的反应，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，包括暴露于燃木炉，壁炉和消防-可能有利于环境空气中碳纳米管形成的条件。尚未使用动物模型研究与结节病相关的假定纳米管环境因素。这项建议将利用一种新的小鼠MWCNT引起的，慢性肉芽肿模型，研究碳纳米管对特定的宿主受体，过氧化物酶体增殖物激活受体？(PPAR?)和趋化性细胞因子受体5（CCR 5），据报道在结节病肺失调。PPAR？，炎症的负调节因子，在健康肺泡巨噬细胞中组成性表达，但在严重的结节病中缺乏。相反，促炎性CCR 5配体在健康肺中未发现，但在结节病中升高。我们注意到类似的发现，在肺的多壁碳纳米管灌注小鼠：过氧化物酶体增殖物激活受体？降低，CCR 5配体升高。基于这些数据，我们假设，多壁碳纳米管抑制过氧化物酶体增殖反应？并上调CCR 5通路以形成具有慢性炎症的肺肉芽肿。具体目标1将决定的作用，过氧化物酶体增殖物激活受体？在MWCNT模型中，通过监测以下方面的肉芽肿大小和数量：（a）巨噬细胞特异性PPAR？空与野生型小鼠;和（B）用慢病毒-PPAR？质粒，过氧化物酶体增殖物激活受体？激动剂罗格列酮;或过氧化物酶体增殖物激活受体？拮抗剂BADGE与未处理小鼠的比较。具体目标2将通过量化CCR 5受体和趋化因子在支气管肺泡灌洗（BAL）衍生的肺泡巨噬细胞，BAL液，并在肉芽肿病灶分离激光捕获显微切割[LCM]，从未经处理的野生型和PPAR？空白小鼠与用CCR 5阻断剂处理的小鼠。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，了解人类肺部疾病的环境原因以及如何诊断和治疗人类肺部疾病。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测定和成像方法。总之，这种独特的调查多壁碳纳米管引起的肉芽肿性肺病是非常适合学生参与的一个地区与环境对人类疾病的影响。

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.