The broad objectives of this K08 proposal are two-fold: 1) to foster the development of essential scientific and professional skills that will allow he candidate, Dr. Benjamin Singer, to achieve his long-term goal of becoming an independent physician-scientist concentrating on epigenetic modifications as therapeutic targets in lung pathology, and 2) to investigate mechanisms that direct the immune system to resolve a severe lung disease. Through laboratory experience, coursework in the Bloomberg School of Public Health, and the peer review process, Dr. Singer will gain expertise in experimental design, laboratory procedures, data analysis, and scientific communication. Dr. Singer and his mentors at Johns Hopkins University, Drs. Srinivasan Yegnasubramanian and Franco D'Alessio, have designed a specific training plan that will afford Dr. Singer new knowledge and research skills in the pathobiology of acute lung inflammation and acute respiratory distress syndrome (ARDS), which is a lung condition that causes tremendous morbidity and mortality in the United States. Despite extensive research into the initial injury and inflammation that drive ARDS, no targeted therapies accelerate its resolution. Experimental studies established that committed regulatory T cells (Tregs)-immune system cells that limit inflammation and orchestrate repair of damaged tissues-resolve inflammation in mouse models of lung injury. However, the mechanisms that cause Tregs to execute their pro-repair program following lung injury remain unknown. Our preliminary data identify DNA methylation, which involves a DNA methyltransferase adapter protein known as Uhrf1, as a critical phenomenon limiting expression of the main protein that directs Treg pro- repair function: Foxp3. Thus, we hypothesize that Uhrf1 deficiency in committed Tregs will lead to Foxp3 locus hypomethylation, increased Foxp3 expression, and enhanced Treg pro-repair function that facilitates resolution of acute lung injury. To test this hypothesis we propose the following Specific Aims: 1) define the role of Uhrf1 in promoting DNA methylation at the Foxp3 locus in committed Tregs following lung injury, and 2) define the role of Uhrf1 on committed Treg pro-repair function and immunoregulatory phenotype. To specifically test our hypothesis we are breeding novel mice that have Uhrf1 deficiency only within Tregs. We have also designed an RNA interference strategy to acutely knock down Uhrf1 in cultured Tregs. Major methods for this proposal include an established mouse model of acute lung injury (intratracheal lipopolysaccharide administration), DNA methylation sequencing techniques, and multicolor flow cytometry. Accomplishment of these aims will provide a rigorous training program for Dr. Singer and uncover mechanisms controlling Treg function during resolution of acute lung injury that could be translated for therapeutic benefit in ARDS.

Public Health Relevance

Acute respiratory distress syndrome (ARDS) is a common, oftentimes fatal inflammatory lung condition without any specific treatments. Regulatory T cells (Tregs) promote repair of lung damage, but mechanisms that encourage Tregs to heal the lung are unknown. We hope to identify novel mechanisms that ultimately lead to new therapeutic options for ARDS by establishing how a protein called Uhrf1 changes DNA and affects Tregs during lung injury and repair.

National Institute of Health (NIH)
National Heart, Lung, and Blood Institute (NHLBI)
Clinical Investigator Award (CIA) (K08)
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Special Emphasis Panel (MCBS (MA))
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Reineck, Lora A
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Northwestern University at Chicago
Internal Medicine/Medicine
Schools of Medicine
United States
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Walter, James M; Helmin, Kathryn A; Abdala-Valencia, Hiam et al. (2018) Multidimensional assessment of alveolar T cells in critically ill patients. JCI Insight 3:
Morales-Nebreda, Luisa; McLafferty, Fred S; Singer, Benjamin D (2018) DNA methylation as a transcriptional regulator of the immune system. Transl Res :
Kurihara, Chitaru; Walter, James M; Singer, Benjamin D et al. (2018) Extracorporeal Membrane Oxygenation Can Successfully Support Patients With Severe Acute Respiratory Distress Syndrome in Lieu of Mechanical Ventilation. Crit Care Med 46:e1070-e1073
McGrath-Morrow, Sharon A; Ndeh, Roland; Collaco, Joseph M et al. (2018) Inflammation and transcriptional responses of peripheral blood mononuclear cells in classic ataxia telangiectasia. PLoS One 13:e0209496
Wang, Lu; Ozark, Patrick A; Smith, Edwin R et al. (2018) TET2 coactivates gene expression through demethylation of enhancers. Sci Adv 4:eaau6986
Reyfman, Paul A; Walter, James M; Joshi, Nikita et al. (2018) Single-Cell Transcriptomic Analysis of Human Lung Provides Insights into the Pathobiology of Pulmonary Fibrosis. Am J Respir Crit Care Med :
McGrath-Morrow, Sharon A; Ndeh, Roland; Helmin, Kathryn A et al. (2018) DNA methylation regulates the neonatal CD4+ T-cell response to pneumonia in mice. J Biol Chem 293:11772-11783
Walter, James M; Corbridge, Thomas C; Singer, Benjamin D (2018) Invasive Mechanical Ventilation. South Med J 111:746-753
Morales-Nebreda, Luisa; Alakija, Omolola; Ferguson, Keith T et al. (2018) Systemic lupus erythematosus-associated diffuse alveolar hemorrhage: A case report and review of the literature. Clin Pulm Med 25:166-169
Khan, Sadiya S; Singer, Benjamin D; Vaughan, Douglas E (2017) Molecular and physiological manifestations and measurement of aging in humans. Aging Cell 16:624-633

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