Pathogenesis-Based Diagnostics and Pharmacotherapeutics for PAP

摘要 尽管我们对肺泡蛋白沉积症（PAP）的认识有了很大的提高， 在多种不同疾病中发生的积聚和呼吸衰竭;临床上，PAP仍被诊断为 通过不能鉴定致病疾病的方法（例如，肺活检），并且没有药物被FDA批准用于治疗它。 虽然存在许多引起PAP的疾病，但原发性PAP（由GM-CSF自身抗体或CSF 2 RA/B引起 突变）占病例的90%以上。在上一个资助期，我们开发了“研究测试”， 包括目前唯一可用于特异性诊断原发性PAP的手段。GM-CSF信号传导的破坏 在原发性PAP的肺泡巨噬细胞（AM）中，GM-CSF依赖性表面活性剂通过AM的清除受损。 肺GM-CSF对其他功能也至关重要，包括AM成熟、自我更新和种群数量。 因此，它对表面活性物质体内平衡、肺泡稳定性、肺功能和宿主防御至关重要。它 普遍认为GM-CSF信号传导的缺失通过降低AM的内在能力而导致PAP， 分解代谢磷脂，但还没有发现这种机制。根据初步数据，我们 确定了一种新的机制，挑战了目前的PAP发病机制的概念，并确定了 我们现在正在利用这些分子靶点来开发新的诊断和治疗方法。这项建议 试图测试以下中心假设：胆固醇毒性，而不是减少磷脂催化剂，驱动 原发性PAP中AM表面活性物质清除受损的发病机制。我们还假设GM-CSF 是增强AM通过PU.1/ CEBPβ介导的 不饱和脂肪酸对过氧化物酶体增殖物激活受体γ（及其下游靶点ABCG 1）的翻译后激活作用 由表面活性剂磷脂代谢产生的酸。在目标1中，我们将确定AM的机制 在患有原发性PAP的人、猴和小鼠中由GM-CSF信号传导的丧失引起的功能障碍。在目标2中， 我们将评估肺胆固醇：胆碱比率用于支气管镜诊断PAP，血清β-胡萝卜酸用于 监测PAP疾病的严重程度，和全血中的GM-CSF信号传导作为多功能诊断测试。在 目的3为PAP的药物治疗提供新的分子靶点。我们希望1）确定 在>90%的患者中引起PAP的疾病的分子和细胞发病机制， AM调节健康和疾病中的表面活性剂稳态; 2）开发新的基于生物标志物的研究 检测有助于PAP的诊断，监测疾病的严重程度，加速临床研究，并提供新的 为临床医生提供了新的治疗工具; 3）验证了PAP药物治疗的新靶点。结果预计将 改善了执业医生的医疗服务，提高了PAP患者的生活质量， 并将PAP研究重新聚焦于分子靶点，FDA批准的药物可以重新用于 PAP治疗结果可能对胆固醇代谢在肺部疾病中的作用具有更广泛的意义 以及动脉粥样硬化性心血管疾病的发病机制。

ABSTRACT Despite our vastly improved understanding of pulmonary alveolar proteinosis (PAP) – a syndrome of surfactant accumulation and respiratory failure that occurs in multiple distinct diseases; clinically, PAP remains diagnosed by methods unable to identify the causative disease (e.g., lung biopsy) and no drug is FDA-approved to treat it. While numerous PAP-causing diseases exist, Primary PAP (caused by GM-CSF autoantibodies or CSF2RA/B mutations) accounts for more than 90% of cases. In the prior funding period, we developed `research tests' that comprise the only means now available to specifically diagnose Primary PAP. Disruption of GM-CSF signaling in alveolar macrophages (AMs) in Primary PAP impairs GM-CSF-dependent surfactant clearance by AMs. Pulmonary GM-CSF is also critical for other functions including AM maturation, self-renewal, and population size and, consequently, is vital to surfactant homeostasis, alveolar stability, lung function, and host defense. It is widely-believed that the loss of GM-CSF signaling causes PAP by reducing the intrinsic ability of AMs to catabolize phospholipids but no such mechanism has ever been identified. Based on our Preliminary Data, we identified a novel mechanism that challenges the current concept of PAP pathogenesis and has identified molecular targets that we are now exploiting to develop novel diagnostics and therapeutics. This proposal seeks to test the following central hypothesis: cholesterol toxicity, not reduced phospholipid catabolism, drives the pathogenesis of impaired surfactant clearance of AMs in Primary PAP. We also hypothesize that GM-CSF is required constitutively to enhance cholesterol clearance by AMs via PU.1/ CEBPβ-mediated expression of PPARγ (and its downstream target ABCG1) and post-translational activation of PPARγ by unsaturated fatty acids arising from surfactant phospholipid metabolism. In Aim 1 we will determine the mechanism of AM dysfunction caused by the loss of GM-CSF signaling in man, monkeys, and mice with Primary PAP. In Aim 2, we will evaluate lung cholesterol:choline ratio for bronchoscopic diagnosis of PAP, serum cholestenoic acid for monitoring PAP disease severity, and GM-CSF signaling in whole blood as a multifunctional diagnostic test. In Aim 3, we will validate a novel molecular target for pharmacotherapy of PAP. We expect to 1) determine molecular and cellular pathogenesis of diseases causing PAP in >90% of patients and inform mechanisms by which AMs regulate surfactant homeostasis in health and disease; 2) develop novel biomarker-based `research tests' to facilitate diagnosis of PAP, monitor disease severity, accelerate clinical research, and provide new tools to practicing clinicians; and 3) validate new targets for pharmacotherapy of PAP. Results are expected to led to improved healthcare delivery by practicing physicians, improving quality of life for people living with PAP, and to refocus PAP research to molecular targets for which FDA-approved drugs could be repurposed as therapy of PAP. Results may have broader implications for role of cholesterol metabolism in lung diseases beyond PAP, and for the pathogenesis of atherosclerotic cardiovascular disease.