PROJECT SUMMARY/ABSTRACT The lung’s ability to recover from severe inflammatory injury depends on its capacity to rapidly mobilize intrinsic tissue repair pathways. Macrophages (Mɸ), the most abundant sentinel immune cell in lungs, have different lineages and functions. A key, but poorly understood, aspect of these cells is their intrinsic property to promote repair after lung injury. In the basal state, the lung contains alveolar Mɸ (AMɸ) (CD11c+/CD11b-/SiglecF+) as well as a population of interstitial Mɸ (IMɸ) (CD11b+/CX3CR1+/SiglecF-). AMɸ are necessary for restoring lung homeostasis after lung injury but the mechanisms regulating reparative AMɸ generation remain elusive. It is clear that a reparative AMɸ population needs to be efficiently and rapidly mobilized, in particular, in the face of sharp decrease in their number during lung infection and injury. In Project 2, we will address this question based on the seminal observation that the transcription factor cAMP Response Element Binding (CREB) plays a key role in giving rise to a reparative AMɸ lineage. In support of this concept, we show that the myeloid-specific deletion of CREB in mice (Creb∆LyzM mice) resulted in the generation of immature AMɸ (CD11c+/CD11b+/SiglecFlo), which give rise to inflammatory AMɸ, thus subverting the anti-inflammatory and reparative function of mature AMɸ. These mice thereby showed clear evidence of lung injury in the basal state due to the increase in inflammatory AMɸ. Furthermore, lung injury in these mice after LPS was prolonged and agonal. They showed significantly greater mortality than controls. By studying flow-sorted Mɸ from Creb∆LyzM lungs, we also found alterations in the expression of regulatory genes such as Pparγ , an essential driver of reparative AMɸ lineage specification, as well as genes regulating AMɸ metabolism and immune responses. Further analysis showed that CREB induced the expression of pyruvate dehydrogenase kinase 4 (PDK4). PDK4 in turn suppressed the translocation of pyruvate dehydrogenase complex (PDC) from mitochondria to the nucleus, thus inhibiting the production of nuclear acetyl-CoA. In the absence of CREB and its target PDK4, PDC activity was markedly increased which resulted in excessive nuclear acetyl-CoA levels, increased histone acetylation, and the generation of AMɸ, that were incapable of promoting lung repair. Based on these observations and with the availability of powerful tools generated by the Cores, in Project 2 we will define the central role of CREB in generating a pro-resolving AMɸ population through the epigenetic regulation of Pparγ expression. Our Specific Aims are (Aim 1): to address the role of CREB in mediating the generation of a lung reparative AMɸ population following lung injury, and (Aim 2): to investigate the role of CREB in signaling the generation of AMɸ by epigenetically upregulating Pparγ expression. Based on the provocative phenotype of Creb∆LyzM mice, we believe understanding how reparative AMɸ ...