Neutrophils mount an early and critical defense in the innate immune response to pathogens, but are also associated with chronic inflammatory diseases such as atherosclerosis, autoimmune disease, and cancer. Recruitment of leukocytes to sites of acute inflammation is a finely orchestrated process initiated by membrane expression and functional activation of leukocyte and endothelial cell adhesion molecules (CAMs) including selectins, integrins, and Ig-super family ligands. A set of unifying themes have emerged over the tenure of this R01 which provide molecular scale insight into how PMNs integrate force as a spatio-mechanical cue in the transition from rolling to firm arrest and transendothelial migration: 1) Selectins are endowed with mechanical and biochemical properties that allow their dissociation lifetime to increase with the rate of tensile loading; 2) Shear stress and transmembrane calcium release-activated Ca2+ (CRAC) channels regulate calcium flux which functions to synchronize integrin mediated arrest and shape orientation under shear; 3) allosteric upregulation in affinity of both LFA-1 and L-selectin in binding ICAM-1 and E-selectin, respectively, facilitates tensile force buildup to trigger key outside-in signals that are cooperative during PMN recruitment. In this competitive renewal we apply innovative vascular mimetic microfluidic channels combined with real-time immunofluorescence imaging to pursue the following specific aims: 1) Establish the ligand binding and mechanotransduction events by which human neutrophils are activated to arrest on E-selectin/ICAM-1 via signaling through L-selectin. 2) Define the mechanotransduction mechanism by which LFA-1 bonds to ICAM-1 elicits outside-in signaling via Kindlin-3 and other as yet unidentified adaptors. 3) Develop and test small molecule antagonists to allosteric domains that interrupt neutrophil activation and recruitment using vascular mimetic microfluidic screening technology. Our strategy entails the use of freshly isolated human neutrophils with the overarching goal of identifying regulatory pathways and molecular targets for prognosis and treatment of granulocytic inflammatory diseases. Keywords Neutrophils, Tensile bond force, Integrins, Selectins, Inflammation, Adhesion, Signaling
White blood cell emigration into inflamed tissue may be likened to a double edged sword because it is critical to innate immune defense against bacterial infection, but if unchecked contributes to autoimmune disease and tissue destruction. This proposal will delve the molecular basis of neutrophil emigration from blood vessels to sites of inflammation with a goal of designing more effective anti-inflammatory therapeutics.
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