Role of Cytosolic Phospholipase A2? in Neutrophil Chemotaxis

Abstract

In the event of infection or injury, patrolling neutrophils directionally migrate to the inflamed or damaged site and initiate a dramatic swarm-like recruitment of distant neutrophils by secreting the secondary chemoattractant leukotriene B4 (LTB4) – a process knowns as a neutrophil signal relay. Studies from the Parent group have demonstrated that disruptions in LTB4 production, secretion, or sensing lead to attenuated neutrophil response. In this context, I first studied how LTB4 is packaged in chemotaxing neutrophils. In collaboration with Dr. Subhash Arya (a post-doctoral fellow), I showed that LTB4-containing exosomes originate at ceramide-rich lipid ordered microdomains at the nuclear envelope (NE). Additionally, I showed that these exosomes are distinct from the CD63-positive, canonical exosomes. In this dissertation, I also investigated the role of cPLA2α in neutrophil chemotaxis. cPLA2α mediated arachidonic acid (AA) release is the rate-limiting step in LTB4 biogenesis. I found that inhibition or depletion of cPLA2α from the neutrophils significantly decreases LTB4 production. Using under agarose chemotaxis assays, I found that cPLA2α regulates neutrophil chemotaxis in a chemoattractant-dependent manner. I found that cPLA2α-/- cells have no defects in migration toward fMLF but have strong defects in migration toward C5a, LTB4, and IL-8. Upon further investigation of the role of cPLA2α in neutrophil chemotaxis, I found that cPLA2α is localized to both the cytosol and nucleus of the neutrophil. Additionally, I demonstrated that cPLA2α is not involved in the generation of the ceramide-rich lipid-ordered microdomains or exosomes. It is, however, present in the exosomes and required for LTB4 generation. I also provide evidence and propose that the nuclear pool cPLA2α translocates to the immerging ILV and is required for LTB4 generation. Finally, I discovered that cPLA2α regulated nuclear morphology in chemotaxing neutrophil-like cells and observed that the nuclei of cPLA2α-/- cells are unable to squeeze through tight (≤3μm) spaces. The doctoral research presented here reveals a novel mechanism by which LTB4-containing exosomes are generated and how cPLA2α mediates LTB4 production and illuminates the multiple functions of cPLA2α in neutrophil biology.