PROJECT SUMMARY Hyperinflammatory gene expression upon tissue injury is a major health risk and the cause of significant morbidity and mortality, particularly if it occurs in the lung. The polarization and injury-responsiveness of macrophages (MF) is tightly regulated to ensure proper control of homeostatic, inflammatory, anti-inflammatory and regenerative processes. The transcriptional and epigenomic determinants of (hyper)responsiveness are poorly understood. We have recently determined that the nuclear hormone receptor, Peroxisome Proliferator Activated Receptor gamma (PPAR) is part of a transcription factor cascade in Interleukin (IL)4 polarized MFs, downstream of the Signal Transducer and Activator of Transcription (STAT)6 induced Early Growth Regulator (EGR)2. Remarkably, PPAR proved to have ligand-dependent, but also apparently ligand-independent, epigenomic activities controlling alternative polarization. In addition, we can show that IL-4 polarized MFs establish a unique epigenome and inflammatory gene expression program upon Toll-Like Receptor (TLR)-ligand exposure. A newly identified part of this interaction, we termed extended synergy, is dependent on the expansion of Nuclear Factor (NF)κB-p65 cistrome and increased enhancer activity. The previously alternatively polarized MFs produce immune-modulatory factors, including Chemokine (C-C) Ligand (CCL) 2, IL6 at an extremely high level in vitro and in vivo in a murine Th2-type airway inflammation and lung injury model upon lipopolysaccharide (LPS) exposure leading to exacerbation of the response. The extended synergy and the LPS response of several genes, including Ccl2 and Il6, depend on the presence of PPAR and some can be modulated by its synthetic and selective ligand, Rosiglitazone. We hypothesize that PPAR acts as signal-dependent epigenomic bookmark, selecting and safeguarding enhancers during MF-extended synergistic inflammatory gene expression by modulating the amplitude of the response. The receptor acts via two distinct mechanisms (1) actively selecting and inducing enhancers genome-wide and (2) also as epigenomic bookmarker and architectural factor altering gene expression only upon non-cognate inflammatory signals. We are examining these hypotheses by systematically mapping the transcriptional and epigenomic changes requiring PPAR, the receptor’s role in the dynamically changing 3D chromatin architecture and in mouse models of lung injury, Th2 inflammation and the combination of the two leading to a hyperinflammatory response leading to exacerbation of these disease processes. Using cutting-edge epigenomics and transcriptomics technologies, with innovative genetic and chimeric mouse models analyzed by high dimensional flow cytometry and single cell technologies will provide mechanistic insights into this novel mode of action of the nuclear receptor PPAR and may lead to the identification of novel pathways to alleviate lung injury and disease progression.