PROJECT SUMMARY / ABSTRACT Due to the lungs’ constant exposure to environmental irritants and airborne pathogens, polymorphonuclear neutrophils (PMN) play an essential role in the pulmonary host defense system to rapidly respond to pathogens such as bacteria and fungi. The lungs are the organ responsible for gas exchange between blood and air, and the minimum functional unit, alveoli, have a characteristic structure with thin capillaries stretched in close proximity. This proposal is based on the postulate that these characteristic lung micro- environmental structures spatio-temporally regulate neutrophil plasticity during acute lung inflammatory injury. To support this proposal, we have developed a Computer-Vision Stabilized intravital imaging (CoVSTii) system for highly efficient mouse lung imaging and have quantified PMN motility and activation dynamics during acute inflammatory lung injury. My supporting data show that PMNs recruited into the lungs extrude their nuclei and become PMN cytoplasts (PMNcyts) without nuclei. Surprisingly, those cells efficiently deform their cell shape due to the absence of nuclei and migrate faster than normal PMNs in spatially restricted infected areas of the lung (PMN rapid response). During this process increased calcium influx and mitochondrial polarization, probably as result of cell deformation, were also directly observed. On the other hand, PMNcyt activations disappeared during prolonged injury condition (PMN paralysis), leading to the fundamental question of whether they can be restored by intervention at the molecular level. I posit that these findings are due to lung-specific PMN dynamics caused by the characteristic structure of the lung and that understanding this phenomena may provide an interesting perspective on the paradox of acute lung injury (ALI)/acute respiratory distress syndrome (ARDS), which is characterized by excessive uncontrolled PMN. In Aim 1, I will elucidate the spatio-temporal regulatory mechanisms of pulmonary PMN nuclear extrusion and rapid PMNcyt activation. Here, I will carry out quantitative lung intravital imaging to visualize PMN motility and organellar dynamics such as nucleus/mitochondria and signaling dynamics such as role of calcium in controlling these functions. I will focus on the involvement of Piezo1 (mechanosensitive calcium channel present in myeloid cells) and Protein Arginine Deiminase 4 (PAD4) (a critical calcium dependent enzyme mediating NETosis, resulting in formation of neutrophil extracellular traps (NETs) composing of genomic DNA and bactericidal components) pathway. I will define the role of these pathways in lung using dynamics imaging system/drug modulation/genetically engineered mice. In Aim 2, I will address the spatio-temporal regulatory mechanisms of paralyzed PMNcyts found in distal lung microvessels and ways to reactivate these cells. Here, I will carry out quantitative lung intravital imaging of paralyzed PMNcyts. I wi...