Macrophage Signaling Pathways in Acute Lung Injury and Resolution

摘要 我们一直有兴趣了解巨噬细胞活化的新决定因素作为治疗工具， 操纵肺部炎症及其解决方案。我们确定MEK途径作为新的抑制剂， 巨噬细胞修复功能。“通过使用市售的MEK 1和MEK 2（MEK 1/2） 抑制剂，我们已经能够增强巨噬细胞清除凋亡的中性粒细胞的能力，并响应于IL- 4.重要的是，我们在2种小鼠肺损伤模型中显示了MEK 1/2抑制的治疗益处：无菌急性肺损伤模型， 使用LPS的肺损伤（ALI）和使用铜绿假单胞菌的细菌性肺炎。在这些ALI模型中， 在LPS或细菌暴露后24和72小时用MEK抑制剂处理的小鼠经历了改善的活性， 体重恢复更快，肺嗜中性粒细胞减少，M2极化更强。“为了解决 机制和细胞来源驱动这一结果的改善，我们产生了小鼠缺乏MEK 1， 骨髓细胞（LysMCre+ MEK 1fl）。这些小鼠在初始条件下没有表型，但经历了100%的 使用中等LPS剂量的LPS诱导的ALI的死亡率，所有野生型小鼠从该剂量恢复。这些小鼠 对LPS有类似的早期炎症反应，但在以后的时间点不能关闭炎症。 有趣的是，在第1天和第3天给予MEK 1/2抑制剂可以完全挽救这种表型， 这表明MEK 2是不消退的ALI的重要促成因素。“重要的是，我们还发现了一个SNP， MEK 2与ARDS患者死亡相关，提示MEK通路可能在ARDS患者中具有临床意义。 ARDS。这些结果强调了巨噬细胞中炎症控制的潜在新机制， 在小鼠肺损伤模型中的显著结果。以下拟议目标概述了我们的方法， MEK 1和MEK 2如何协同调节巨噬细胞对LPS的反应，以更好地定义一种新的 MEK途径和巨噬细胞在ALI消退中的调节作用。在目标1中，我们将确定角色和 细胞来源的MEK 1在控制肺部炎症，并测试我们的假设， 细胞导致LPS诱导的ALI中来自骨髓腔室的炎症信号失调， 有助于增强炎症细胞向肺的募集。在目标2a中，我们将确定MEK 2的作用 在急性肺部炎症及其消退中的作用，并验证我们的假设，即MEK 1缺失导致 持续的MEK 2活性作为延长对LPS的炎症反应的驱动力。在目标2b中，我们计划 评估巨噬细胞中MEK 1和MEK 2的相互作用和MEK 2失活的机制。这些 研究将推进我们对免疫系统如何阻止炎症的理解，揭示新的 治疗目标和方法，可以带到床边。

ABSTRACT We have been interested in understanding novel determinants of macrophage activation as therapeutics tools in manipulating lung inflammation and its resolution. We identified MEK pathways as novel suppressors of macrophage reparative function."By targeting both MEK1 and MEK2 (MEK1/2) using commercially available inhibitors, we have been able to enhance the ability of macrophages to clear apoptotic PMNs and respond to IL- 4. Importantly, we showed a therapeutic benefit of MEK1/2 inhibition in 2 murine lung injury models: sterile acute lung injury (ALI) using LPS, and bacterial pneumonia using Pseudomonas aeruginosa. In these ALI models, mice treated with MEK inhibitors at 24 and 72 h post-LPS or bacteria exposure experienced improved activity, faster recovery of body weight, reduced pulmonary neutrophilia, and greater M2 polarization."To address the mechanism and cell source driving this improvement in outcome, we generated mice deficient in MEK1 in myeloid cells (LysMCre+MEK1fl). These mice have no phenotype in naïve conditions, but experience 100% mortality with LPS-induced ALI using a moderate LPS dose from which all wild-type mice recover. These mice have a similar early inflammatory response to LPS, but fail to turn off inflammation at later time-points. Interestingly, this phenotype can be completely rescued with MEK1/2 inhibitors given at days 1 and 3, suggesting that MEK2 is an important contributor to non-resolving ALI."Importantly, we also identified a SNP in MEK2 associated with death in ARDS patients, suggesting that MEK pathways may be clinically significant in ARDS. These results highlight a potentially novel mechanism of inflammatory control in the macrophage with striking consequences in murine lung injury models. The proposed aims below outline our approach to identify how MEK1 and MEK2 work in concert to regulate macrophage responses to LPS to better define a novel regulatory role of both MEK pathways and macrophages in ALI resolution. In aim 1, we will determine role and cell source of MEK1 in controlling lung inflammation and test our hypothesis that loss of MEK1 in myeloid cells leads to dysregulated inflammatory signals from the myeloid compartment in LPS-induced ALI and contributes to enhanced inflammatory cell recruitment to the lung. In aim 2a, we will determine role of MEK2 in acute lung inflammation and its resolution and test our hypothesis that absence of MEK1 leads to sustained MEK2 activity as the driver of prolonged inflammatory responses to LPS.!In aim 2b, we plan to evaluate MEK1 and MEK2 interactions and mechanisms of MEK2 deactivation in macrophages. These studies would advance our understanding of how the immune system stops inflammation, revealing new therapeutic targets and approaches that could be brought to the bedside.