Cell migration is a complicated multistep process that is central to the pathogenesis of diverse disease processes. Although neutrophils are an essential part of the innate immune response, their inappropriate recruitment is central to chronic inflammatory disorders including asthma, arthritis and inflammatory bowel disease; and also contributes to the pathogenesis of other diseases such as cardiovascular disease, tumor progression and Alzheimer's disease. Chemotaxis, the movement of cells within a chemical gradient, is the fundamental process underlying neutrophil recruitment. Despite its importance, current tools limit progress towards understanding the molecular mechanisms that regulate cell migration. In particular, current methods are largely limited to 2D environments, require relatively large blood draws and use stimuli of questionable physiological relevance. Our goal is to use and further develop microscale methods and microscale in vitro models that overcome these challenges, enhancing our ability to identify signaling pathways that regulate cell polarization and directed motility in the context of disorders involving the innate immune system - specifically the role of Hax1 signaling in neutrophil chemotaxis in the context of severe congential neutropenia (SCN). Currently, chemotaxis studies are very time and labor intensive limiting the number of experimental conditions that can be explored. Our approach fundamentally changes the way a researcher can approach chemotaxis studies. Many more experimental conditions can be examined with the same time/effort. A key strength of this application lies in the use of novel (but simple) microfluidic devices that generate defined and stable chemical gradients that do not require laminar flow for gradient formation or maintenance and can be adapted to high throughput screening. In Aim 1, we will develop improved 3D assays that provide a more physiologically relevant context for cell migration and develop methods that allow the use of small volume finger stick samples. In Aim 2, we will develop microfluidic-based wound assays to analyze neutrophil recruitment through cell sourced gradients to determine factors that regulate cell polarization and directed cell migration in multicelluar environments In Aim 3, we propose to study how Hax1/HS1/G113 signaling modulates neutrophil motility and recruitment in more complex 3D and cell sourced gradients to mimic in vivo conditions. Using these systems, we will dissect how Hax1 regulates gradient sensing and directed cell migration with Hax1-deficient neutrophil-like PLB987 cells. Future applications will include analysis of neutrophil motility from patients presenting with neutropenia using 2D and 3D/cell sourced microfluidic systems to understand disease pathogenesis and identify novel therapeutic targets.