Ceramide-Rich Microdomains Facilitate Nuclear Envelope Budding during the Biogenesis of LTB4-containing Exosomes

Abstract

Neutrophils migrating towards chemoattractant gradients amplify their recruitment range by releasing the secondary chemoattractant leukotriene B4 (LTB4)1,2. We previously demonstrated that LTB4 and its synthesizing enzymes, the 5-lipoxygenase (5-LO), 5-LO activating protein (FLAP), and leukotriene A4 hydrolase (LTA4H), are packaged and released in exosomes3. We now report that the biogenesis of the LTB4-containing exosomes is initiated at the nuclear envelope (NE) of activated neutrophils. We show that the neutral sphingomyelinase 1 (nSMase1)-mediated generation of ceramide enriched lipid-ordered microdomains initiates the clustering of the LTB4-synthesizing enzymes on the NE. We isolated and analyzed exosomes from activated neutrophils and established that the FLAP/5-LO-positive exosome population is distinct from that of the CD63-positive exosome population. Furthermore, we observed a strong co-localization between ALIX and FLAP at the periphery of nuclei and within cytosolic vesicles. We propose that the initiation of NE curvature and bud formation is mediated by nSMase1-dependent ceramide generation, which leads to FLAP and ALIX recruitment. Together, these observations elucidate the mechanism for LTB4 secretion and identify a novel pathway for exosome generation.