Northwestern University Feinberg School of Medicine

Benjamin Singer Lab

Research

We have a specific interest in epigenetic control of lung lymphocytes and how epigenetic phenomena determine the lymphocyte pro-resolution and pro-repair response to lung injury induced by influenza as well as other injurious agents. One specific lymphocyte subset, regulatory T cells, plays a key role in coordinating resolution and repair following lung injury; we seek to understand the epigenetic basis of lung regulatory T cell phenotype and function in response to lung inflammation and injury. We use transgenic mouse models, primary cell culture, and multiparameter flow cytometry paired with cutting-edge next-generation sequencing techniques to help reveal the elegant molecular networks controlling the balance between inflammation/injury and repair/resolution.

Acute respiratory distress syndrome (ARDS) is a severe inflammatory lung disease that occurs following an acute lung injury and affects over 190,000 patients in the United States each year with close to 75,000 deaths. Unremitting lung inflammation portends a poor prognosis in ARDS, but pharmacotherapies designed to sup­press inflammation have failed to im­prove outcomes. Management remains supportive. However, the immuno­regulatory system boasts tar­gets that could be manipulated to promote resolution of lung damage. Regulatory T cells (Tregs) comprise a sub­set of CD4+ lymphocytes that coordinate resolution and repair following acute lung injury. While cell-based therapy using Tregs repre­sents a potential therapeutic strategy for ARDS, exploiting endogenous Treg control mecha­nisms to aug­ment their pro-repair function embodies a more feasible approach to enhance resolution and improve ARDS outcomes.

Tregs express the forkhead box protein 3 (Foxp3) transcription factor; stable Foxp3 expression identifies com­mitted Tregs in mice and the major Treg population in humans. Increasing Foxp3 protein expres­sion aug­ments Treg pro-repair activity, but Foxp3 expression decreases during acute lung inflammation. Thus, mecha­nisms that promote and stabilize Foxp3 expression could accelerate and enhance resolution of lung injury. DNA methylation acts as a stabilizing rheostat that controls Foxp3 expression at the transcriptional level. Meth­ylation of cytosine-phospho-guanine (CpG) residues within critical non-coding regions at the Foxp3 locus re­press­es Foxp3 transcription. DNA methyltransferases (DNMTs) maintain these methyl-CpG marks. As DNMTs become si­lenced or inhibited, methylation is progressively lost and a hypomethylation pattern emerges that facili­tates Foxp3 transcription. Further hypomethylation increases and stabilizes Foxp3 transcription and Treg function. Maintenance of CpG methylation involves a complex of proteins including the epigenetic regulator Uhrf1, which recruits DNA methyl­transferase 1 (Dnmt1) to DNA.

We aim to determine the role of epigenetic regulators in com­mitted Foxp3+ Tregs with respect to DNA methylation, gene expres­sion, and Treg pro-repair func­tion and immuno­regulatory phe­notype that enhance lung injury resolution and repair. To that end, we use transgenic mice exposed to influenza and other injurious agents to model the acute lung injury seen in ARDS. Multiparameter flow cytometry and next-generation sequencing techniques aid in our search for molecular mechanisms that direct the lung’s immune system to resolve inflammation and promote repair of lung damage.