Characterization of the subcellular compartment involved in human neutrophil interleukin-1beta (IL-1b) secretion

FUENTES, FEDERICO; MIGLIO, MAXIMILIANO; ANALÍA, TREVANI; SABBIONE, FLORENCIA; GUZMÁN, MAURICIO ; JANCIC, CAROLINA C; KEITELMAN, IRENE A; SHIROMIZU, CAROLINA MAIUMI; GALLETTI, JEREMÍAS

CABA

Congreso; Reunión Conjunta de Sociedades de Biociencias; 2017

IL-1beta is a major proinflammatory cytokine synthesized in the cytoplasmas an inactive precursor that is activated by proteolytic cleavage. It is a leaderless cytosolic protein that is secreted by unconventional mechanisms different from the classical RE-Golgi pathway. We previously determined that human neutrophil IL-1beta processing is dependent on caspase-1 and elastase and/or proteinase-3. Our studies also indicated that neutrophil IL-1beta is secreted by an unconventional secretory autophagic mechanism. Here we aim to getinsights of the subcellular mechanisms that control IL-1b secretion from human neutrophils by employing immunofluorescence staining and confocal microscopy. We determined that after 3.5 h post-LPS+ATP stimulation part of the neutrophil IL-1beta colocalized with elastase and myeloperoxidase in a vesicular compartment (n=3). Neutrophil stimulation with LPS+ATP also induced colocalization of caspase-1 with IL-1beta (Mander´s coefficient -MC-: 0.52; n=2; at least 80 cells analyzed/experiment), and this effect was potentiated when neutrophils were also subjected to starvation (MC: 0.59). We also found colocalization of IL-1beta with the chaperone protein HSP90 (MC: 0.55 and 0.47; full nutrient and starvation, respectively; n=2). Noteworthy, wide vesicles with IL-1beta overlapping with HSP90 signals were found protruding from the cell membrane. By employing the fluorescent probe FLICA to track the presence of activated caspase-1 in live cell imaging assays, we detected FLICA signal as defined spots inside cells with a pattern similar to that observed with caspase-1. Additionally, IL-1beta did not colocalize with LAMP-2 at either 3.5 or 5 h post-LPS+ATP stimulation (MC: 0.21 and 0.22; n=4, at least 50 cells analyzed/experiment). Our results suggest that IL-1beta is packaged in vesicular compartments which can also include caspase-1 and/or elastase where it might be processed or degraded. HSP90 might contribute to IL-1beta entry to vesicles different to lysosomes.