TNF-alpha Signaling in Silica-induced Lung Fibrosis

描述（由申请人提供）：矽肺仍然是一种严重的炎症性肺部疾病，目前尚无成功的治疗方法。在本基金的上一个周期中，我们发现矽肺患者对肺移植的反应很差。我们还发现，二氧化硅促进p42 MAPK/ERK的活化，并诱导TNFR1的磷酸化，这是一个调节二氧化硅诱导的巨噬细胞细胞毒性的过程。RAW 264.7巨噬细胞经历ERK介导的TNFR1磷酸化并被保护免于凋亡。相比之下，IC21巨噬细胞不磷酸化TNFR1，并且响应于二氧化硅而经历增强的凋亡。二氧化硅诱导的巨噬细胞凋亡与线粒体功能障碍和心磷脂（CL）的氧化有关，后者与TNFR 1磷酸化呈负相关。二氧化硅诱导的CL氧化的机制尚不清楚，但可能涉及细胞色素c（cyt c）的过氧化物酶活性。磷酸化TNFR 1保护巨噬细胞免受二氧化硅诱导的细胞凋亡的机制及其对硅肺的影响尚不清楚，构成了本更新申请中解决的问题。我们组建了一个具有TNF1和脂质生物学专业知识的研究团队，研究TNFR1磷酸化对二氧化硅诱导的肺损伤的重要性。具体而言，我们将研究细胞内TNFR1在矽肺中的作用及其对caspase激活和线粒体损伤的影响。我们推测，在巨噬细胞暴露于二氧化硅后，TNFR1经历磷酸化并抑制促凋亡信号复合物的形成，从而防止凋亡。我们的中心假设是，磷酸化TNFR 1抑制细胞色素C的过氧化物酶活性，并限制二氧化硅诱导的CL氧化，从而保护巨噬细胞免受二氧化硅诱导的细胞凋亡，改善小鼠矽肺。为了验证这一假设，我们提出了以下具体目标：1）确定磷酸化TNFR 1保护RAW 264.7巨噬细胞免受二氧化硅诱导的凋亡的机制。2)确定磷酸化TNFR 1是否调节cyt c的过氧化物酶活性，并减少二氧化硅诱导的巨噬细胞凋亡过程中的CL氧化。3)确定磷酸化TNFR 1在小鼠体内表达对二氧化硅诱导的肺损伤的影响。我们将从C57 BL/6小鼠的骨髓中分离巨噬细胞，用编码TNFR1突变体的病毒构建体稳定地转染它们，以防止或模拟TNFR1磷酸化。随后，将稳定转染的巨噬细胞转移至暴露于二氧化硅的小鼠中，以评价这些细胞对小鼠中二氧化硅诱导的肺损伤的发展的影响。项目叙述：术语硅肺是指由长期吸入游离二氧化硅引起的纤维结节性肺病。在美国，每年有超过200万人暴露于二氧化硅，最近的调查表明，这些人中有很高比例的人暴露于高于允许暴露限度的二氧化硅水平。随后，从1987年到1996年，美国估计有3，600 - 7，300例新发现的矽肺病例。尽管预防措施降低了二氧化硅引起的死亡率，但现实情况是，大量接触二氧化硅的患者仍因这种疾病而死亡。CDC调查表明，包括宾夕法尼亚州在内的9个州报告了二氧化硅诱导的高死亡率，超过3例死亡/百万人/年。这些报告还指出，与全国其他地区相比，宾夕法尼亚州西部因硅肺病而死亡率要高得多，匹兹堡市周围的县群（采矿是一项重要活动）可确定为造成这一死亡率的原因（> 14.8例死亡/百万/年）。强调这些报告的重要性是我们在匹兹堡大学对矽肺患者进行肺移植的经验（在我们的初步数据中描述）。我们从1986年至2004年确定了12例因这种疾病接受肺移植的患者。这些患者的结果与一组79例接受肺移植治疗IPF的患者进行了对比，IPF是一种肺移植可带来生存获益的疾病。我们的数据表明，矽肺患者的生存率（2.4年）较低，移植后肺移植物发生排斥反应（0.9年）的时间早于IPF患者（生存期和移植物排斥反应分别为5.34年和2.4年）。因此，这些数据表明，矽肺仍然是一种严重的，经常致命的肺部疾病，没有成功的治疗。

DESCRIPTION (provided by applicant): Silicosis remains a serious inflammatory lung disease for which no successful treatment is available. During the previous cycle of this grant we showed that patients with silicosis have poor response to lung transplantation. We also showed that silica promotes activation of p42 MAPK/ERK and induces phosphorylation of TNFR1, a process that modulates silica-induced cytotoxicity in macrophages. RAW 264.7 macrophages experience ERK-mediated phosphorylation of TNFR1 and are protected from apoptosis. In contrast, IC21 macrophages do not phosphorylate TNFR1, and experience enhanced apoptosis in response to silica. Silica induction of macrophage apoptosis is associated with mitochondrial dysfunction and the oxidation of cardiolipin (CL) that inversely correlate with TNFR1 phosphorylation. The mechanisms responsible for the silica-induced oxidation of CL are unknown but may involve the peroxidase activity of cytochrome c (cyt c). The mechanism(s) by which phosphorylated TNFR1 protects macrophages from silica-induced apoptosis and its effect on silicosis is unknown and constitutes the question addressed in this renewal application. We have assembled a team of researchers with expertise in TNF1 and lipid biology to study the importance of TNFR1 phosphorylation on silica-induced lung injury. Specifically, we will study the role of the intracellular TNFR1 in silicosis and its effects on caspase activation and mitochondrial damage. We hypothesize that following exposure of macrophages to silica TNFR1 undergoes phosphorylation and inhibits the formation of the pro-apoptotic signaling complex thus preventing apoptosis. Our central hypothesis is that phosphorylated TNFR1 inhibits the peroxidase activity of cyt C and limits the silica-induced oxidation of CL thus protecting macrophages from silica-induced apoptosis and ameliorating silicosis in mice. To test this hypothesis we propose the following specific aims: 1) To determine the mechanism(s) by which phosphorylated TNFR1 protects RAW 264.7 macrophages from silica-induced apoptosis. 2) To determine whether phosphorylated TNFR1 modulates the peroxidase activity of cyt c and reduces CL oxidation during silica-induced macrophage apoptosis. 3) To determine the consequences of in vivo expression of phosphorylated TNFR1 on silica-induced lung injury in mice. We will isolate macrophages from the bone marrow of C57BL/6 mice, to stably transfect them with viral constructs encoding for TNFR1 mutants to prevent or mimic TNFR1 phosphorylation. Subsequently, stably transfected macrophages will be transferred to silica-exposed mice to evaluate the effect of these cells on the development of silica induced lung injury in mice. PROJECT NARRATIVE: The term silicosis refers to the fibronodular lung disease caused by long-term inhalation of free silica. In the United States, more than 2 million people are exposed to silica every year and recent surveys indicate that a high percentage of these individuals are exposed to levels of silica above the permissible exposure limits. Subsequently, there were an estimated 3,600-7,300 newly recognized silicosis cases in the US from 1987 to 1996. Although preventive measures have decreased mortality attributable to silica the reality is that a large number of silica-exposed patients are still dying as a result of this disease. CDC surveys indicate that nine states, including Pennsylvania, report high rates of silica- induced mortality, more than 3 deaths/million people/year. These reports also point out that Western Pennsylvania exhibits a much higher mortality due to silicosis when compared to the rest of the country and clusters of counties (where mining is an important activity) around the city of Pittsburgh are identifiable that contribute disproportionably (> 14.8 deaths/million/year) to this mortality. Emphasizing the importance of these reports is our experience (described in our preliminary data) at the University of Pittsburgh with lung transplantation in patients afflicted with silicosis. We identified 12 patients that received lung transplantation for this disease from 1986-2004 . The outcome of these patients was contrasted with that of a group of 79 patients that received lung transplantation for IPF, a disease for which lung transplantation confers survival benefit. Our data indicate that patients with silicosis have poor survival rates (2.4 years) and experienced earlier rejection of their lung grafts (0.9 years) following transplantation than patients with IPF (5.34 and 2.4 years for survival and graft rejection respectively). Consequently, these data indicate that silicosis still is a serious and frequently lethal lung disease for which no successful treatment is available.