The broad objectives of this NRSA Individual Fellowship are two-fold: 1) to facilitate development of essential skills that will allow the candidate to become an effective physician-scientist, and 2) to investigate the mechanisms that direct the immune system to resolve a severe lung disease. The candidate and his mentors have designed a specific training plan. The plan includes a rigorous research component that lays the foundation for a successful career. The proposal concerns acute lung injury (ALI) and its clinical counterpart, the acute respiratory distress syndrome (ARDS). ALI and ARDS are diffuse lung diseases that cause tremendous morbidity and mortality in the United States. Despite decades of research into the inflammation that drives ALI, no specific therapies have been found to accelerate its resolution and save the lives of afflicted patients. There is a paucit of information about how ALI resolves. However, regulatory T cells (Tregs)- immune system cells that decrease inflammation and promote repair of damaged tissues-resolve ALI in a mouse model of the disease. Tregs also increase in the lungs of patients with ARDS, suggesting that they may be important in the human immune response to ALI. Despite having the same DNA code as all cells in the body, only certain genes are turned """"""""on"""""""" within cells-including Tregs-at any given time. The specific genes that are """"""""on"""""""" regulate Treg development and function. Epigenetics is the study of how genes are turned """"""""on"""""""" or """"""""off"""""""" by changes to chromatin: DNA and the proteins attached to DNA known as histones. The candidate has preliminary data demonstrating that a chromatin-modifying drug (5-aza-2'-deoxycytidine [AZA]) enhances resolution of ALI in mice and increases the number of Tregs in their lungs. AZA blocks activity of DNA methyltransferase, an enzyme known to turn """"""""off"""""""" genes that induce Treg development and function. The focus of this proposal is to further examine how epigenetic phenomena mediated via DNA methyltransferase affect Tregs following ALI. The long-term hope of the proposal is to identify novel mechanisms that may lead to treatment options for patients with ARDS.
In Specific Aim 1, the candidate will examine the effect of DNA methyltransferase inhibition on Tregs following lung injury in mice. Multicolor flow cytometry and adoptive transfer of AZA-treated Tregs to injured lymphocyte-deficient mice will be used to define the effect of DNA methyltransferase inhibition on Treg function and phenotype during ALI resolution. A suppression assay will measure ex vivo Treg function.
In Specific Aim 2, the effect of DNA methyltransferase inhibition on a critical Treg gene (Foxp3) will be determined following lung injury using chromatin immunoprecipitation and real-time PCR. These experiments will explore the mechanisms controlling Treg function following ALI with the ultimate goal of improving patient outcomes in this oftentimes fatal disease.
Acute lung injury (ALI) is a common, oftentimes fatal lung condition without any specific treatments. Regulatory T cells (Tregs) are important in the resolution of ALI, but the mechanisms that cause Tregs to increase and function following ALI are unknown. We hope to identify novel mechanisms that ultimately lead to new therapeutic options for this devastating disease by studying how changes to DNA and the proteins associated with DNA affect Tregs during ALI resolution.
|D'Alessio, F R; Craig, J M; Singer, B D et al. (2016) Enhanced resolution of experimental ARDS through IL-4-mediated lung macrophage reprogramming. Am J Physiol Lung Cell Mol Physiol 310:L733-46|
|Singer, Benjamin D; Mock, Jason R; D'Alessio, Franco R et al. (2016) Flow-cytometric method for simultaneous analysis of mouse lung epithelial, endothelial, and hematopoietic lineage cells. Am J Physiol Lung Cell Mol Physiol 310:L796-801|
|Singer, Benjamin D; D'Alessio, Franco R (2016) Histone/protein deacetylase 3 dictates critical aspects of regulatory T cell development and function. Cell Mol Immunol 13:415-7|
|Singer, Benjamin D; Mock, Jason R; Aggarwal, Neil R et al. (2015) Regulatory T cell DNA methyltransferase inhibition accelerates resolution of lung inflammation. Am J Respir Cell Mol Biol 52:641-52|
|Singer, Benjamin D; D'Alessio, Franco R (2014) Regulatory T cell Itch reins in Th2 inflammation. Cell Mol Immunol 11:126-8|
|Singer, Benjamin D; King, Landon S; D'Alessio, Franco R (2014) Regulatory T cells as immunotherapy. Front Immunol 5:46|
|Singer, Benjamin D; D'Alessio, Franco R (2014) Comment on Adamzik et al.: An increased alveolar CD4 + CD25 + Foxp3 + T-regulatory cell ratio in acute respiratory distress syndrome is associated with increased 30-day mortality. Intensive Care Med 40:1604|