CREB Instruction of Macrophage Fate and Lung fluid homeostasis

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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.