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.