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。在此资助的前一个周期中，我们发现矽肺病患者对肺移植反应较差。我们还发现，二氧化硅促进p42 MAPK/ERK的激活，并诱导TNFR1的磷酸化，这一过程调节了二氧化硅诱导的巨噬细胞的细胞毒性。巨噬细胞经历erk介导的TNFR1磷酸化，并免于凋亡。相比之下，IC21巨噬细胞不磷酸化TNFR1，并且在二氧化硅的作用下经历了增强的凋亡。二氧化硅诱导巨噬细胞凋亡与线粒体功能障碍和心磷脂（CL）氧化有关，而心磷脂氧化与TNFR1磷酸化呈负相关。二氧化硅诱导CL氧化的机制尚不清楚，但可能与细胞色素c （cyt c）过氧化物酶活性有关。磷酸化TNFR1保护巨噬细胞免受二氧化硅诱导的凋亡及其对矽肺的影响的机制尚不清楚，并且构成了本更新应用中解决的问题。我们召集了一组具有TNF1和脂质生物学专业知识的研究人员来研究TNFR1磷酸化在二氧化硅诱导的肺损伤中的重要性。具体来说，我们将研究细胞内TNFR1在矽肺病中的作用及其对半胱天冬酶激活和线粒体损伤的影响。我们假设巨噬细胞暴露于二氧化硅后，TNFR1发生磷酸化并抑制促凋亡信号复合物的形成，从而阻止细胞凋亡。我们的中心假设是，磷酸化的TNFR1抑制cyt C的过氧化物酶活性，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凋亡，并改善小鼠矽肺。为了验证这一假设，我们提出以下具体目标：1)确定磷酸化TNFR1保护RAW 264.7巨噬细胞免受二氧化硅诱导的凋亡的机制。2)在二氧化硅诱导的巨噬细胞凋亡过程中，磷酸化TNFR1是否调节细胞过氧化物酶活性，降低CL氧化。3)确定磷酸化TNFR1在体内表达对小鼠硅致肺损伤的影响。我们将从C57BL/6小鼠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巨噬细胞，以防止或模拟TNFR1磷酸化。随后，将稳定转染的巨噬细胞转移到二氧化硅暴露小鼠中，以评估这些细胞对二氧化硅诱导小鼠肺损伤发展的影响。项目简介：矽肺病是指长期吸入游离二氧化硅引起的肺纤维结节性疾病。在美国，每年有超过200万人接触到二氧化硅，最近的调查表明，这些人中有很大一部分人接触到的二氧化硅水平超过了允许的接触限度。随后，从1987年到1996年，美国估计有3600 - 7300例新确诊的矽肺病例。虽然预防措施降低了二氧化硅造成的死亡率，但现实情况是，大量接触二氧化硅的病人仍因这种疾病而死亡。美国疾病控制与预防中心的调查表明，包括宾夕法尼亚州在内的9个州报告二氧化硅引起的死亡率很高，每年每百万人中有超过3人死亡。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矽肺病造成的死亡率要高得多，匹兹堡市周围的县群（采矿是一项重要活动）对这一死亡率的贡献是不成比例的（每百万人/年有14.8人死亡）。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）。我们确定了1986-2004年间12例因该疾病接受肺移植的患者。这些患者的结果与一组接受肺移植治疗IPF的79名患者的结果进行了对比，IPF是一种肺移植可带来生存益处的疾病。我们的数据表明，矽肺患者的生存率较低（2.4年），移植后肺移植排斥反应较IPF患者早（分别为5.34年和2.4年）（0.9年）。因此，这些数据表明矽肺病仍然是一种严重且经常致命的肺部疾病，目前尚无成功的治疗方法。

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.