PROJECT SUMMARY Lung Injury Repair by Regulatory T cell LGP2 The overall objective of this R01 proposal is to investigate mechanisms that direct the immune system to resolve severe acute lung inflammation. The team for this proposal, comprising expert investigators from two leading universities, has designed a research strategy that will validate new therapeutic targets acute respiratory distress syndrome (ARDS), which is a devastating condition that causes significant morbidity and mortality in the U.S. Despite thorough investigation into the injury and inflammation that drive ARDS, no targeted therapies promote its resolution. Regulatory T cells (Tregs) ? a subset of CD4+ lymphocytes that suppress exuberant immune system activation?resolve inflammation in mouse models of lung injury. However, the mechanisms that promote Treg function following lung injury remain unknown. Preliminary data identified in Tregs the Lgp2 locus, which encodes the immune regulatory protein LGP2, as a novel site that augments Treg responses to inflammation. DNA methylation at this site is dynamic and contributes to repression of the Dhx58 locus following lung injury. Thus, the hypothesis of Treg DNA hypomethylation at the Lgp2 locus will increase LGP2 protein levels, enhance Treg pro-repair function, and promote resolution of acute lung inflammation. To test this hypothesis the following Specific Aims are proposed: 1. Define the role of the Dnmt-Uhrf1 complex in promoting methylation of Lgp2 in Tregs during ALI resolution; 2. Determine the role of LGP2 in regulating Treg pro-repair function during ALI resolution; and 3. Evaluate the role of JHU848 in promoting Treg-mediated ALI resolution. To specifically test our hypothesis we will employ transgenic mice including strains with Dhx58 deficiency as well as Treg-specific Uhrf1 deficiency. We have also designed an RNA interference strategy to acutely knock down Dhx58 in cultured Tregs and boost Dhx58 expression with mutant DNA plasmids. Major methods for this proposal include established mouse models of acute lung injury (intratracheal lipopolysaccharide and Pseudomonas aeruginosa administration), DNA methylation sequencing techniques, and multicolor flow cytometry. Accomplishment of these aims will uncover mechanisms controlling Treg function during resolution of acute lung injury that could be translated for therapeutic benefit in ARDS.