Summary/Abstract The trafficking of leukocytes from the blood stream to the sites of inflammation to find infectious agents is a hallmark of the innate immune response. Neutrophils, myeloid leukocytes of the innate immune response, are the so-called “first responders” and will rapidly traffic to the sites of inflammatory insult. Trafficking is initiated by the leukocyte adhesion cascade, a well-characterized, stepwise sequence of events in which blood borne immune cells tether, roll, firmly arrest, and migrate on the endothelium and then enter tissues to perform immune cell functions. These events all occur within blood vessels, normally post-capillary venules, where cells encounter high shear rates while interacting with and transmigrating through the endothelium. Recently, an interesting phenomenon wherein certain cells of hematopoietic origin – such as lymphocytes (T-cells and B-cells) as well as hematopoietic stem and progenitor cells (HSPCs) – will crawl upstream, against the direction of flow, after arrest on surfaces that present the ligand intercellular adhesion molecule-1 (ICAM-1). Upstream migration is mediated by the β2 integrin, αLβ2, also known as Lymphocyte Function-associated Antigen-1 (LFA-1), which binds to ICAM-1. Neutrophils express LFA-1, as well as an additional receptor for ICAM-1, Macrophage-1 Antigen (Mac-1), which is upregulated when neutrophils are activated. Our laboratory hypothesized that neutrophils could also be induced to crawl upstream on ICAM-1 if Mac-1 was disabled or blocked, allowing LFA-1/ICAM-1 interactions to dominate. Our preliminary results show that by either blocking MAC-1 (with an antibody) or by genetically manipulating Mac-1 (using CRIPSR- Cas9 deletion), neutrophils can migrate upstream on ICAM-1 surfaces, setting the premise for this application. Here, we propose to determine the critical signals which govern the upstream migration of neutrophils, how neutrophils generate force when migrating upstream, and identify the physiological implications of upstream neutrophil migration. In Aim 1 we will determine the key molecular signals involved in upstream migration in neutrophils, by first analyzing the differential signaling that occurs on ICAM-1 vs. VCAM-1 surfaces and then identify and delete the signals that operate downstream of LFA-1 and Mac-1 using CRISPR editing. Specifically, we will focus on deleting the signals downstream of Mac-1/ICAM-1 binding to determine the signals that inhibit neutrophil upstream migration. In Aim 2, we will measure and quantify the forces generated by neutrophils engineered to migrate upstream using traction force microscopy. Using the knockouts generated in aim 1, we will measure the differences in the spatial arrangement and magnitude of force generation between upstream and downstream crawling neutrophils. Finally, in Aim 3 we will determine the physiological relevance of upstream migration in neutrophils by determining the change in time needed for altered to t...