REGULATION OF THE MACROPHAGE INFLAMMATORY PHENOTYPE IN ARDS

我们已发表的数据表明，巨噬细胞中NFATc 3的基因消融和药理学抑制是一种重要的机制。 有益于维持肺泡-毛细血管屏障功能，防止炎性细胞因子释放， LPS和盲肠结扎穿刺中的嗜酸性炎症、动脉氧合改善和存活率 小鼠ARDS模型。在这里，我们建议确定下游分子靶标的颗粒细节 NFATc 3的表达，使用2次打击小鼠模型、人肺巨噬细胞和来自ARDS患者的BALF。我们的团队 已经开发了一种新的无毒细胞渗透性钙调磷酸酶抑制（CNI）肽（CNI 103）， 活化巨噬细胞中的NFATc 3并减轻小鼠中的ARDS。我们建议确定分子 使用临床前2次打击小鼠模型、人肺巨噬细胞和患者BALF的NFATc 3靶点 符合ARDS的柏林标准。我们的中心假设是钙调神经磷酸酶依赖性NFAT的激活 调节ALI/ARDS的发展，并通过一种新的 肽钙调磷酸酶抑制剂（CNI）减轻疾病的严重程度。我们提出两个具体目标： 具体目标1：描述NFATc 3-钙调神经磷酸酶激活途径的下游分子靶点， 肺巨噬细胞在ALI/ARDS期间。新的初步数据表明，细胞外的脂质含量 BALF中的囊泡（EV）是NFATc 3依赖性的，并介导肺微血管屏障功能的破坏 内皮细胞（MVEC）。在SA 1中，我们将1）确定NFAT调节的脂质介质的谱， 参与脂质代谢的酶，2）确定这些介质是否被包装在细胞外 囊泡，3）评估EV是否介导导致渗透性的细胞间通讯 肺水肿和4）确定阻断NFAT活化是否防止肺损伤和炎症， 临床相关的小鼠和ARDS细胞模型。 具体目的2：确定优化的细胞可渗透钙调磷酸酶抑制剂的有效性和安全性， 在临床相关的感染性和非感染性小鼠模型中， ALI/ARDS。我们将测试细胞渗透性钙调磷酸酶肽抑制剂是否能预防和逆转肺损伤 和炎症。我们还将评估药代动力学和药效学 (PK/PD）特性、细胞选择性、安全性、功效和在预防和逆转肺损伤中的效力。 ARDS的临床前小鼠模型。 这些研究将进一步了解NFATc 3激活在巨噬细胞中的重要作用。 和其他肺细胞类型在ALI/ARDS发病机制中的作用。我们预计， 这些研究将确立NFATc 3作为一种新的治疗靶点， 通过控制生物活性脂质和蛋白质介体的组成来调节相互作用。

Our published data show that genetic ablation and pharmacologic inhibition of NFATc3 in macrophages is beneficial in maintaining alveolar-capillary barrier function, prevents inflammatory cytokine release and neutrophilic inflammation, improved arterial oxygenation and survival in the LPS and cecal ligation puncture mouse models of ARDS. Here, we propose to determine the granular details of the downstream molecular targets of NFATc3 using a 2-hit mouse model, human lung macrophages, and BALF from patients with ARDS. Our team has developed a novel non-toxic cell permeable calcineurin inhibitory (CNI) peptide (CNI103) that blocks activation of NFATc3 in macrophages and mitigates ARDS in mice. We propose to determine the molecular targets of NFATc3 using a pre-clinical 2-hit mouse model, human lung macrophages, and BALF from patients meeting the Berlin criterion for ARDS. Our central hypothesis is that activation of calcineurin-dependent NFAT in macrophages regulates the development of ALI/ARDS, and inhibition of NFAT activation by a novel peptide calcineurin inhibitor (CNI) lessens disease severity. We propose two specific aims: Specific Aim 1: To delineate the downstream molecular targets of NFATc3-Calcineurin activation pathway in pulmonary macrophages during ALI/ARDS. Novel preliminary data show that the lipid content of extracellular vesicles (EVs) in BALF are NFATc3 dependent and mediate disruption of barrier function in lung microvascular endothelial cells (MVEC). In SA1, we will 1) determine the spectrum of NFAT regulated lipids mediators and enzymes involved in lipid metabolism, 2) determine whether these mediators are packaged in extracellular vesicles, 3) assess whether EVs mediate the the cell-to-cell communication that results in permeability pulmonary edema and 4) determine whether blocking NFAT activation prevents lung injury and inflammation in clinically relevant mouse and cellular models of ARDS. Specific Aim 2: To determine the efficacy and safety of an optimized cell permeable calcineurin inhibitor, which prevents NFAT activation, in clinically relevant infectious and non-infectious mouse models of ALI/ARDS. We will test whether cell permeable calcineurin peptide inhibitors prevent and reverse lung injury and inflammation in mouse models of ARDS. We will also assess the pharmacokinetic and pharmacodynamics (PK/PD) properties, cellular selectivity, safety, efficacy, and potency in preventing and reversing lung injury in preclinical mouse models of ARDS. These studies will advance knowledge about the essential role of NFATc3 activation in macrophages and other lung cell types in the pathogenesis of ALI/ARDS. We anticipate that knowledge gained from these studies will establish NFATc3 as a novel therapeutic target that regulates EV-mediated cell-cell interactions by governing the composition of biologically active lipid and protein mediators.