Role of GM-CSF in Myeloid Cell Function and Innate Immunity

描述（由申请人提供）：肺泡蛋白沉积症（PAP）是一种以肺泡巨噬细胞（AMs）和肺泡中表面活性剂积累导致呼吸衰竭和感染死亡率增加为特征的综合征。近40年来，唯一可用的治疗方法是全肺灌洗，这是一种高度侵入性的治疗方法，在少数中心进行，其中一只肺机械通气，另一只肺反复充满生理盐水，并大力敲击胸部以物理去除表面活性剂。直到在GM-CSF-/-小鼠中发现了PAP，这一发现改变了我们对GM-CSF生物学作用的概念，并导致了PAP的新诊断和治疗，由于缺乏致病的认识，在药物治疗方面没有任何进展。我的实验室对我们的理解做出了重大贡献，即GM-CSF对表面活性剂稳态、AM个体发生、中性粒细胞和AM功能以及先天免疫至关重要，并且在约90%的患者中，PAP是由高水平的GM-CSF自身抗体（GMAbs）引起的。目前的证据表明GM-CSF通过转录因子PU.1和PPAR？通过刺激脂质转运体ABCG1的表达：在gm - csf缺陷小鼠和PAP患者中，这三种物质都缺乏AMs。尽管如此，关于1)PAP的自然历史，2)GM-CSF信号丢失导致PAP的机制，以及3)PU.1和PPAR3在GM-CSF调节AMs中表面活性剂清除和免疫功能的机制中的作用和关系仍然存在疑问。我们将使用我们的新型灵长类自身免疫PAP， AM细胞系和PAP生物标志物模型；现有小鼠遗传性PAP模型；自身免疫性和遗传性PAP患者验证我们的中心假设：PAP是由GM-CSF?PU.1?PPAR3？abcg1依赖性中性脂从AMs中排泄，这损害了它们清除表面活性剂的能力。这一假设将在三个特定的目标中得到解决，重点是GM-CSF对髓细胞的调节。在Aim 1中，我们将在灵长类动物模型和PAP患者中确定自身免疫性PAP的自然史、GMAbs的临界阈值及其对骨髓免疫功能的影响。在Aim 2中，PU.1、PPAR？ABCG1在CSF2RA或B突变引起的遗传性PAP中的作用将在体外通过慢病毒介导的巨噬细胞在遗传性PAP小鼠或人中的表达进行评估，在体内通过将ABCG1转导的骨髓移植到CSF2RB-/-小鼠中进行评估。在Aim 3中，我们将确定GM-CSF是否通过PPAR的pu .1依赖性调节AMs中的表面活性剂清除和免疫功能？使用不自发表达PU.1或也对GM-CSF有反应的新型AM细胞系。GM-CSF在AMs中调控的转录程序将通过我们的灵长类动物模型在体内无表面活性剂二次效应的情况下进行研究。我们将确定PPAR？激动剂吡格列酮在体外恢复PAP小鼠和人细胞以及体内CSF2RB-/-小鼠对AM表面活性剂的清除。预期的结果对PAP的发病机制和治疗、表面活性剂的稳态和常见炎症性疾病的GMAb治疗具有重要意义。

DESCRIPTION (provided by applicant): Pulmonary alveolar proteinosis (PAP) is a syndrome characterized by accumulation of surfactant in alveolar macrophages (AMs) and alveoli resulting in respiratory failure and increased mortality from infection. For nearly 4 decades, the only available therapy was whole lung lavage, a highly invasive procedure performed at few centers in which one lung is mechanically ventilated while the other is repeatedly filled with saline and the chest is percussed vigorously to physically remove surfactant. No advances in pharmacologic therapy occurred due to a lack of pathogenic insight until PAP was discovered in GM-CSF-/- mice, a finding that transformed our concepts of the biological role of GM-CSF and led to novel diagnostics and therapy for PAP. My laboratory has contributed significantly to our understanding that GM-CSF is critical for surfactant homeostasis, AM ontogeny, neutrophil and AM functions, and innate immunity, and that in ~90% of patients, PAP is caused by a high level of GM-CSF autoantibodies (GMAbs). Current evidence suggests GM-CSF regulates surfactant homeostasis via the transcription factors PU.1 and PPAR? by stimulating expression of the lipid transporter, ABCG1: all three are deficient in AMs in GM-CSF-deficient mice and PAP patients. Notwithstanding, questions remain regarding the 1) natural history of PAP, 2) mechanism by which loss of GM-CSF signaling causes PAP, and 3) roles and relationship of PU.1 and PPAR3 in mechanisms by which GM-CSF regulates surfactant clearance and immune functions in AMs. We will use our novel primate model of autoimmune PAP, AM cell lines and PAP biomarkers; an existing murine model of hereditary PAP; and autoimmune and hereditary PAP patients to test our central hypothesis: PAP is caused by reduced GM-CSF?PU.1?PPAR3?ABCG1-dependent excretion of neutral lipids from AMs, which impairs their ability to clear surfactant. This hypothesis will be addressed in 3 specific aims focusing to GM-CSF regulation of myeloid cells. In Aim 1, we will determine the natural history of autoimmune PAP, critical threshold of GMAbs and their effects on myeloid immune functions in our primate model and PAP patients. In Aim 2, the roles of PU.1, PPAR?, and ABCG1 in hereditary PAP caused by CSF2RA or B mutations will be evaluated in vitro using lentiviral-mediated expression in macrophages from mice or humans with hereditary PAP, and in vivo by transplanting ABCG1-transduced bone marrow into CSF2RB-/- mice. In Aim 3, we will determine if GM-CSF regulates surfactant clearance and immune functions in AMs via the PU.1-dependent regulation of PPAR? using novel AM cell lines that do not spontaneously express PU.1, or that also respond to GM-CSF. The transcriptional program that GM-CSF regulates in AMs will be examined in vivo free of secondary effects of surfactant by using our primate model. We will determine if the PPAR? agonist pioglitazone restores AM surfactant clearance in vitro in cells from mice and humans with PAP and in vivo using CSF2RB-/- mice. Anticipated results have implications for PAP pathogenesis and therapy, surfactant homeostasis, and GMAb therapy of common inflammatory diseases. PUBLIC HEALTH RELEVANCE: GM-CSF is a cytokine regulator of alveolar macrophage function, surfactant homeostasis, and host defense. Disruption of GM-CSF signaling, due to GM-CSF autoantibodies or GM-CSF receptor mutations, causes pulmonary alveolar proteinosis (PAP), a syndrome of respiratory insufficiency due to surfactant accumulation in alveoli caused by reduced clearance by alveolar macrophages. In Aim 1, we will use a novel primate model of PAP and PAP patients to determine the natural history and level of GM-CSF autoantibodies causing PAP. In Aim 2, we will use gene transfer to determine the role PU.1, PPAR?, and ABCG1, in the pathogenesis of hereditary PAP and surfactant clearance by alveolar macrophages in vitro and in vivo (in mice). In Aim 3, we will determine if GM-CSF regulates PPAR3 in a PU.1-dependent manner and test whether pioglitazone, an FDA-approved PPAR? activator, is effective as therapy of hereditary PAP in vitro and in vivo (in mice). Anticipated results are relevant to surfactant homeostasis, pathogenesis and therapy of PAP, and the use of GM-CSF autoantibodies to treat common diseases like asthma and rheumatoid arthritis.