CREB Instruction of Macrophage Fate and Lung fluid homeostasis

摘要 从严重形式的炎性血管损伤中恢复，如急性肺损伤(ALI)，取决于 肺快速激活组织修复途径的能力。巨噬细胞(Mφ)，最丰富的前哨细胞 肺中的免疫细胞类型，是损伤后恢复组织液内稳态所必需的，但 修复性Mφ亚群的身份和它们是如何产生的仍然是难以捉摸的。在这里，我们有 发现肺泡Mφ(AMφ)群体中维持肺液动态平衡所需的一个亚群 基本并诱导损伤后的组织修复。我们的支持数据表明：1)cAMP反应的丧失 元素结合蛋白在髓系细胞(CREB∆LyzM小鼠)中的表达抑制AMφ的一个亚群 AM前φ阶段的血统，导致AMφ生成减少和肺血管通透性增加 基本情况：2)∆小鼠在内毒素攻击后不能解决损伤，假单胞菌感染后死亡更快 铜绿假单胞菌感染；3)转录组测序(RNAseq)和染色质可及性分析(ATACseq) 流式分选CREB缺失的AMφ显著改变了基因表达和染色质重塑；4)CREB 抑制丙酮酸脱氢酶复合体(PDC)过量产生核乙酰辅酶A所必需的 通过合成丙酮酸脱氢酶激酶4(PDK4)。根据这些具有煽动性的初步数据， 在目标1中，我们将检验CREB转录促进这一屏障分化的假设 修复性AMφ亚群。在目标2中，我们将研究CREB调节修复性AMφ的产生 通过控制组蛋白的表观遗传修饰，从而导致AMφ基因转录。在这里，我们将 提出CREB转录活性是上调PDK4表达所必需的新概念， 这反过来又阻止了其靶标PDC从线粒体到细胞核的运输，从而抑制 过量的核乙酰化Co-A产生和限制组蛋白的表观遗传修饰，导致基因 诱导屏障修复性AMφ群体的转录。这些研究将采用严格的多项 组学方法(单细胞RNA、ATAC和芯片测序)和转基因小鼠的功能分析 包括ROSA-CrebCxcxr3-ERT小鼠(其中CREB在单核/间质巨噬细胞中有条件地缺失) 确定CREB转录活性在产生修复性AMφ群体中的作用。理解 这种屏障修复性AMɸ子集是如何产生的，应该能够增强这种分化 通过药物或遗传手段损伤时的亚群，从而降低ALI及相关疾病的死亡率 条件。

Abstract Recovery from severe forms of inflammatory vascular injury, such as acute lung injury (ALI), depends on the lung's capacity to rapidly activate tissue repair pathways. Macrophages (Mφ), the most abundant sentinel immune cell type in the lung, are required for restoration of tissue-fluid homeostasis following injury, but the identity of the reparative Mφ subpopulations and how they are generated remain elusive. Here, we have discovered a subset of the alveolar Mφ (AMφ) population that is required to maintain lung fluid homeostasis basally and induce tissue repair after injury. Our Supporting Data show that: 1) loss of cAMP Response Element Binding (CREB) protein expression in myeloid cells (Creb∆LyzM mice) arrest a subpopulation of the AMφ lineage at the pre-AMφ stage, leading to decreased AMφ generation and increased lung vascular permeability basally; 2) Creb∆LyzM mice failed to resolve injury post-LPS challenge and died more rapidly after Pseudomonas aeruginosa infection; 3) transcriptome sequencing (RNAseq) and chromatin accessibility profiling (ATACseq) of flow-sorted CREB-null AMφ have markedly altered gene expression and chromatin remodeling; and 4) CREB is required to inhibit excessive production of nuclear acetyl-CoA from the pyruvate dehydrogenase complex (PDC) through synthesis of pyruvate dehydrogenase kinase 4 (PDK4). Based on these provocative Preliminary Data, in Aim#1, we will test the hypothesis that CREB transcriptionally promotes differentiation of this barrier reparative AMφ subpopulation. In Aim#2, we will investigate that CREB regulates reparative AMφ generation by controlling epigenetic modifications of histones thereby leading to AMφ gene transcription. Here, we will address the novel concept that the transcriptional activity of CREB is required to upregulate PDK4 expression, which in turn prevents transport of its target, PDC, from mitochondria to the nucleus thereby suppressing excessive nuclear acetyl Co-A generation and limiting epigenetic modifications of histones, leading to gene transcription that induces the barrier reparative AMφ population. These studies will employ a rigorous multi- omics approach (single-cell RNA-, ATAC-and Chip-sequencing) and functional assays in genetically altered mice including Rosa-CrebCxcxr3-ERT mice (in which CREB is conditionally deleted in monocytes/interstitial macrophages) to define the role of CREB transcriptional activity in generating the reparative AMφ population. Understanding how this barrier reparative AMɸ subset is generated should make it possible to enhance differentiation of this subset during injury by pharmacological or genetic means, thereby reducing the mortality of ALI and related conditions.