MAP2K1 AND MAP2K2 IN ACUTE LUNG INJURY AND RESOLUTION

摘要 随着与SARS-CoV-2感染相关的ARDS病例的激增，迫切需要了解 参与肺部炎症和损伤消退的新途径为新的治疗方法提供依据 接近了。比较啮齿动物和人类肺损伤基因表达特征的研究显示保守 包括MAPK/ERK在损伤过程中的激活。最近，我们发现了一种基因多态 MAP2K2与ARDS死亡相关，提示MAP2K2在ALI的恢复中起生物学作用。我们证明了这一点 巨噬细胞中MAP2K1/MAP2K2(MEK1/MEK2)活化促进促炎通路并抑制 修复性的，使其成为操纵ALI巨噬细胞表型的潜在靶点。在临床前急性 肺损伤(ALI)模型，抑制MEK1/MEK2改善ALI的措施，包括更快的身体恢复 体重，肺内中性粒细胞减少，以及更大的修复性巨噬细胞激活。这些结果表明 MEK通路可能是ALI的有效靶点。为了解决驱动这一现象的异构体和细胞来源 在结果的改善上，我们产生了髓系细胞MEK1缺陷的小鼠(LysmCre+Mek1fl)。这些老鼠 在幼稚的条件下没有表型，但在使用中等剂量的内毒素诱导的ALI时经历100%的死亡率 所有野生型小鼠都能从中恢复的脂多糖剂量。这些小鼠对脂多糖有相似的早期炎症反应 但未能在以后的时间点上消除炎症。这种表型可以用IFNAR1完全挽救 阻断，提示MEK1抑制I型干扰素反应对ALI的解决至关重要。我们也 在没有MEK1的情况下发现持续的MEK2/p-ERK激活，这表明MEK1对MEK2是关键的 停用促进ALI决议。为了支持这一假设，我们发现在全球范围内缺乏MEK2的小鼠 或在白细胞室有较快的ALI分解。以下建议的目标概述了我们的方法 确定MEK亚型如何协同调节髓系细胞的反应，以更好地定义和靶向一个新的 ALI中的调控途径。在目标1中，我们将确定MEK1和MEK2细胞特定的角色和信号 ALI和TEST OUR中的通路将检验我们的假设，即需要MEK1来去激活肺泡中的MEK2 巨噬细胞促进肺部炎症的消退。在目标2中，我们计划评估MEK1和MEK2 MEK2失活的相互作用和机制。我们将使用MEK1突变体来验证我们的假设 MEK2通过与pT292 MEK1结合而失活，我们将确定这些突变体如何改变巨噬细胞 炎性(内毒素)和修复性(IL-4)反应。在目标3中，我们计划测试MEK1和MEK2降解器作为 ALI小鼠模型的治疗学。我们将测试我们的假设，即特定于MEK2的降解将导致 更快的ALI分辨率。这些研究将增进我们对免疫系统如何停止的理解 炎症和促进ALI的解决，揭示了新的治疗靶点和方法，可以 被带到床边。

ABSTRACT With the surge of ARDS cases associated with SARS-CoV-2 infection, there is an urgent need to understand novel pathways involved in resolution of lung inflammation and injury to provide the basis for new therapeutic approaches. Studies comparing rodent and human lung injury gene expression signatures reveal conserved pathways, including MAPK/ERK activation during injury. More recently, we found a genetic polymorphism in MAP2K2 associates with death in ARDS, suggesting a biological role in ALI recovery. We demonstrated that MAP2K1/MAP2K2 (MEK1/MEK2) activation in macrophages promotes pro-inflammatory pathways and inhibits reparative ones, making it a potential target to manipulate macrophage phenotypes in ALI. In preclinical acute lung injury (ALI) models, inhibition of MEK1/MEK2 improves measures of ALI, including faster recovery of body weight, reduced pulmonary neutrophilia, and greater reparative macrophage activation. These results suggest that MEK pathways could be effective targets in ALI. To address the isoform 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. This phenotype can be completely rescued with IFNAR1 blockade, suggesting MEK1 suppression of type I interferon responses is critical for ALI resolution. We also found sustained MEK2/p-ERK activation in the absence of MEK1, suggesting that MEK1 is critical for MEK2 deactivation to promote ALI resolution. In support of this hypothesis, we found that mice lacking MEK2 globally or in the leukocytes compartment have faster ALI resolution. The proposed aims below outline our approach to identify how MEK isoforms work in concert to regulate myeloid cell responses to better define and target a novel regulatory pathway in ALI. In aim 1, we will determine MEK1 and MEK2 cell-specific roles and signaling pathways in ALI and test our will test our hypothesis that MEK1 is required to deactivate MEK2 in alveolar macrophages to promote resolution of lung inflammation. In aim 2, we plan to evaluate MEK1 and MEK2 interactions and mechanisms of MEK2 deactivation. We will use MEK1 mutants to test our hypothesis that MEK2 is deactivated by binding to pT292 MEK1, and we will determine how these mutants alter macrophage inflammatory (LPS) and reparative (IL-4) responses. In aim 3, we plan to test MEK1 and MEK2 degraders as therapeutics in murine models of ALI. We will test our hypothesis that MEK2-specific degradation will result in faster ALI resolution. These studies will advance our understanding of how the immune system stops inflammation and promotes ALI resolution, revealing new therapeutic targets and approaches that could be brought to the bedside.