Cytosolic myeloid related protein 8/14 (cytosolic MRP8/14) is a critical regulator of beta2 integrin outside-in signaling during leukocyte recruitment in vivo

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Myeloid related protein 8/14 (MRP8/14, S100A8/A9, Calprotectin) belongs to the family of Ca2+ binding S100 proteins. Upon release, the protein acts as an alarmin with potent immune modulatory properties. Elevated levels of extracellular MRP8/14 are present in a broad spectrum of inflammatory disorders, like inflammatory bowel disease, rheumatoid arthritis, dermatitis or cardio-vascular diseases, thereby qualifying the molecule as a reliable serum biomarker in the clinics. Recently, we were able to show that the interaction of neutrophils with inflamed endothelium induces MRP8/14 release, leading to dependent activation of beta2 integrins, resulting in slow leukocyte rolling and firm leukocyte adhesion on inflamed endothelium. Little is known about the intracellular function of MRP8/14 in neutrophils, although the dimer represents 40% of the cytosolic protein content in these cells. However, a few studies propose an additional intracellular function of the protein, which is independent on its extracellular role as an alarmin. The so far published data suggest a role of cytosolic MRP8/14 in in the Ca2+ dependent interplay of the plasma membrane and the cytoskeleton. Cytoskeletal rearrangement is a prerequisite for post arrest modifications in leukocytes during leukocyte recruitment and hence indispensable for efficient leukocyte migration. Within this project, we intent to identify the function of intracellular MRP8/14 in leukocyte recruitment in vivo. Based on our preliminary findings we assume a role of cytosolic MRP8/14 in the so-called “outside-in” signalling, leading to a defective adhesion strengthening and thus resulting in a defective transition from rolling to firm adhesion. In order to prove this hypothesis we will perform in vivo experiments using the TNF- induced inflammation model of the mouse cremaster muscle and ex vivo flow chamber assays utilizing C57BL/6 und Mrp14-/- mice (functional MRP8/14 double knock out mice). We will investigate the role of cytosolic MRP8/14 in inside-out signaling and outside-in signaling via different functional assays. Finally, we will elucidate the underlying molecular mechanism of MRP8/14 dependent outside-in signalling by visualizing stimulation dependent adhesion clusters and Ca2+ localization and by analyzing phosphorylation patterns of intracellular molecules and GTPase activation in C57Bl/6 and Mrp14-/- neutrophils.