Emerging studies suggest that innate immune cells, such neutrophils, can adopt multiple activation/differentiation states, either conducive or detrimental to host defense and inflammation resolution. The conceptual existence of diverse activation states of innate leukocytes is also evident in circulating leukocytes from healthy individuals and patients with system inflammatory syndromes such as sepsis. However, quantification and understanding of single-cell phenotypes in a spectrum of differentiation states is lacking. This bottle-neck explains the complete lack of cure for sepsis, despite decades of extensive basic and translational studies. The overarching focus of the research program in my laboratory is to define and quantify principal factors that underlie the decision-making processes of immune cell migration, differentiation and activation in response to challenges. The research funded by this award will combine technologies, experimental methods, and modeling approaches to investigate leukocyte decision-making in the context of activation state and sepsis. In the past 5 years, we have engineered microfluidic platforms to quantify neutrophil migratory and anti-microbial decision-making. Building off of our current work with neutrophils and microfluidic platforms, over the next five years we propose to: 1) Develop, improve and implement state-of-the-art multi-sensing platforms to analyze septic patient leukocyte migratory and antimicrobial phenotypes in precisely defined microenvironments. Mathematical models will be created based on known molecular pathways and will be validated by the molecular signatures from septic patients and healthy donors; 2) Quantify leukocyte migratory and anti-microbial functions in vivo using novel implantable hydrogels combined with intra-vital imaging techniques in a mouse sepsis model. By defining the fundamental processes, including differentiation state and microenvironment, that determine a leukocyte’s phenotype, we can better predict, diagnose, and eventually design effective treatments for sepsis. These fundamental processes involve general biology principles at cellular and molecular levels applicable to diverse eukaryotic cells, lending broader significance to our proposed investigation. The work proposed is highly innovative because it integrates methods from different scientific disciplines to solve the “big problem” of sepsis. Novel microfluidic platforms will be engineered to measure single-cell phenotypes in precisely defined conditions and directly connect these phenotypes to the molecular signature of the cell. Our vision is to integrate these quantitative single cell measurements with computational modeling and analysis to create intuitive descriptions of complex leukocyte decision-making processes. Finally, these devices have clear translational potential and may be used in the future in a hospital setting to assist with diagnosing or monitoring treatment of sepsis.

Narrative/Public Health Relevance Statement Caroline N. Jones, Ph.D. Sepsis is the number one cause of death in hospitals and is associated with ~$17B in annual U.S. healthcare expenditures. The overarching goal of this project is to define and quantify principal factors that underlie the decision-making processes of immune cell migration, differentiation and activation in response to challenges. The microsystems developed in this project to quantify immune cell phenotype with single-cell resolution have clear translational potential and may be used in the future in a hospital setting to assist with diagnosing or monitoring treatment of sepsis.