This proposal describes a two-year training plan designed to provide the PI with the technical skills, scientific models and knowledge to facilitate earning a K08 award on epigenetic mechanisms of the altered host response to lung injury in sepsis survival. Applicant: Dr. Denstaedt holds an MD degree and has completed specialty training in Internal Medicine and Pulmonary and Critical Care Medicine. He has previous experience several basic science research labs. He has had an intensive research experience during the last 18 months of Pulmonary and Critical Care fellowship and is currently a T32 post-doctoral research trainee. The training outlined is designed to develop his skills in assessments of lung injury, model development, epigenetics and bioinformatics. The lab science, mentorship team, and didactics were selected to specifically advance his training toward becoming and independent physician-scientist. Research plan: Despite substantial advances in medical care, recovery from sepsis is associated with increased risk for long-term organ injury and death. Nearly 1 in 10 patients with sepsis will be re-hospitalized within 90-days with respiratory conditions including pneumonia, respiratory failure and aspiration pneumonitis. Other than supportive care and rehabilitation, there are no targeted therapies to prevent lung-related complications in sepsis survivors. Interestingly, survivors of murine sepsis have enhanced (primed) innate immune responses suggesting an activated immune program is experienced in sepsis survival. Innate immune priming has been associated with persistent elevations of the danger signal protein S100A8/A9. Given that sepsis survival is associated with significant long term morbidity and mortality, the central hypothesis of this proposal is that primed innate immune responses, mediated by persistent danger signal proteins, are associated with a predisposition to secondary lung injury. This project focuses on examining epigenetic mechanisms of persistent immune priming through modeling of secondary lung injury to mimic a clinically relevant reason for re-admission in the post-septic patient.
In Aim 1, a murine model of polymicrobial sepsis survival will be used to evaluate the effect of sepsis on secondary inflammatory lung injury. Lung macrophage/monocytes will be isolated by flow cytometry and cell sorting, and stimulated ex vivo to evaluate for a primed phenotype two weeks after sepsis. Chromatin immunoprecipitation (ChIP)-seq and assessment of chromatin modifying enzyme expression via quantitative PCR will be performed to assess dynamic changes in the epigenome and its machinery.
In Aim 2, an in vitro model of S100A8/A9 mediated priming of wild-type bone marrow derived macrophage (BMDM) will be developed with subsequent ChIP and chromatin modifying enzyme measurement as in Aim 1. These experiments will be compared in BMDM from post-septic wild-type and S100A9-/- mice.
Every year nearly 21 million people survive sepsis worldwide. One in ten will be readmitted with pneumonia, aspiration pneumonitis or respiratory failure over the next 90-days. The goal of this project is to understand the biological mechanisms of predisposition to these complications in order to prevent and treat them.
