Lung Injury Repair by Regulatory T cell Dhx58/LGP2 The overall objective of this R01 proposal is to investigate mechanisms that direct the immune system to resolve severe acute lung inflammation. Our team, 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. Our preliminary data identified in Tregs the Dhx58 locus, which encodes the immune regulatory protein LGP2, as a novel site that augments Treg responses to inflammation. We determined that DNA methylation at this site is dynamic and contributes to repression of the Dhx58 locus following lung injury. Thus, we hypothesize that Treg DNA hypomethylation at the Dhx58 locus will increase LGP2 protein levels, enhance Treg pro-resolution function, and promote resolution of acute lung inflammation. To test this hypothesis we propose the following Specific Aims: 1. To define the role of the DNA methylating complex Dnmt1-Uhrf1 in promoting DNA methylation at the Dhx58 locus to regulate LGP2 protein expression in Tregs during lung injury resolution; 2. To examine the role of LGP2 in regulating Treg suppressive phenotype and function; and 3. To determine the requirement for Treg LGP2 and its therapeutic potential in resolution of lung injury. 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 project 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.
Acute respiratory distress syndrome (ARDS) is a serious inflammatory lung disease without any specific therapies. Regulatory T cells (Tregs) orchestrate repair of lung damage, but mechanisms that boost Tregs to heal the lung are unknown. We hope to identify novel mechanisms that lead to new therapeutic options for ARDS by establishing how regulation of a gene called Dhx58 and its product - LGP2 - affect Tregs during lung repair
