Ceramide-rich microdomains facilitate nuclear envelope budding for non-conventional exosome formation

Neutrophils migrating towards chemoattractant gradients amplify their recruitment range by releasing the secondary chemoattractant leukotriene B₄ (LTB₄) refs. ^1,2. We previously demonstrated that LTB₄ and its synthesizing enzymes, 5-lipoxygenase (5-LO), 5-LO activating protein (FLAP) and leukotriene A₄ hydrolase, are packaged and released in exosomes³. Here we report that the biogenesis of the LTB₄-containing exosomes originates 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 analysed 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 non-conventional pathway for exosome generation.