PROJECT SUMMARY Macrophages are central regulators of inflammation and tissue healing following injury or infection, and during disease. While much is known about how soluble, biochemical factors in the environment regulate macrophage function, less is known about how biophysical cues regulate their response, despite the fact that these cells exist within solid tissues that are rich in mechanical cues. Furthermore, many diseases in which macrophages are involved, such as cancer and fibrosis, are characterized by changes in tissue biophysical properties. Our previous work demonstrated that adhesion to soft extracellular matrix hydrogels inhibits macrophage inflammatory activation. In preliminary work, we found that matrix rigidity influences the localization of YAP, a transcriptional co-factor involved in cell proliferation, organ size control, and cancer, but with previously undescribed role in macrophage activation. Adhesion to stiff substrates leads to YAP nuclear localization, which appears to prime macrophages for a potent inflammatory response. In addition, cytoskeletal polymerization and the mechanically-activated and calcium-permeable ion channel Piezo1 appear to be involved in YAP nuclear localization and inflammatory activation. In this study, we propose to investigate the molecular mechanisms underlying YAP signaling and Piezo1 activity in the macrophage response within different stiffness environments. In Aim 1, we will examine the effect of stiffness on cytoskeletal remodeling and associated signaling pathways on YAP activity. In Aim 2, we will probe the role of Piezo1-mediated calcium activity in stiffness sensing, YAP signaling, and macrophage function. Finally, in Aim 3, we will investigate the role of YAP and Piezo1 on macrophage-mediated wound healing in vivo using a murine subcutaneous biomaterial implant model. An improved fundamental understanding of how macrophages sense their mechanical environment may lead to new immunomodulatory strategies that control macrophage function during disease.