Autophagy Mediates Interleukin-1β Secretion in Human Neutrophils

Abstract

Interleukin-1β (IL-1β), a major pro-inflammatory cytokine, is a leaderless cytosolic protein whose secretion does not follow the classical endoplasmic reticulum-to-Golgi pathway, and for which a canonical mechanism of secretion remains to be established. Neutrophils are essential players against bacterial and fungi infections. These cells are rapidly and massively recruited from the circulation into infected tissues and, beyond of displaying an impressive arsenal of toxic weapons effective to kill pathogens, are also an important source of IL-1β in infectious conditions. Here, we analyzed if an unconventional secretory autophagy mechanism is involved in the exportation of IL-1β by these cells. Our findings indicated that inhibition of autophagy with 3-methyladenine and Wortmannin markedly reduced IL-1β secretion induced by LPS + ATP, as did the disruption of the autophagic flux with Bafilomycin A1 and E64d. These compounds did not noticeable affect neutrophil viability ruling out that the effects on IL-1β secretion were due to cell death. Furthermore, VPS34IN-1, a specific autophagy inhibitor, was still able to reduce IL-1β secretion when added after it was synthesized. Moreover, siRNA-mediated knockdown of ATG5 markedly reduced IL-1β secretion in neutrophil-differentiated PLB985 cells. Upon LPS + ATP stimulation, IL-1β was incorporated to an autophagic compartment, as was revealed by its colocalization with LC3B by confocal microscopy. Overlapping of IL-1β-LC3B in a vesicular compartment peaked before IL-1β increased in culture supernatants. On the other hand, stimulation of autophagy by cell starvation augmented the colocalization of IL-1β and LC3B and then promoted neutrophil IL-1β secretion. In addition, specific ELISAs indicated that although both IL-1β and pro-IL-1β are released to culture supernatants upon neutrophil stimulation, autophagy only promotes IL-1β secretion. Furthermore, the serine proteases inhibitor AEBSF reduced IL-1β secretion. Moreover, IL-1β could be also found colocalizing with elastase, suggesting both some vesicles containing IL-1β intersect azurophil granules content and that serine proteases also regulate IL-1β secretion. Altogether, our findings indicate that an unconventional autophagy-mediated secretory pathway mediates IL-1β secretion in human neutrophils.

Leptospiraspecies promote a pro‐inflammatory phenotype in human neutrophils

“…Unlike macrophages, neutrophils did not undergo pyroptosis upon NLRC4 inflammasome activation triggered by Salmonella infection (Chen et al, ). Interestingly, it has been recently shown that an unconventional autophagy‐mediated secretory pathway mediates IL‐1β secretion in human neutrophils (Iula et al, ). Because autophagy usually blocks apoptosis (Marino, Niso‐Santano, Baehrecke, & Kroemer, ), it could be that this or a similar mechanism may be involved in the enhanced IL‐1β and reduced apoptosis triggered by Leptospira in human neutrophils, a fact that future studies might clarify.…”

TFEB activation in macrophages attenuates postmyocardial infarction ventricular dysfunction independently of ATG5-mediated autophagy

“…Several reports have highlighted an additional role for autophagy in IL-1β secretion. Specifically, an unconventional autophagy-mediated secretory pathway regulates IL-1β secretion in both neutrophils and macrophages (15,19,54), where ATG5 was demonstrated to have a critical role. In the setting of myocardial IR injury, macrophages encounter hypoxia/reoxygenation that we showed can impair autophagy, concomitant with the release of damage-associated molecular patterns that can activate NLRP3 through direct and indirect mechanisms.…”

“…mitochondria and reducing ROS, which can leak DNA into the cytosol and thus serve as a stimulus for inflammasome activation (15)(16)(17)(18). Paradoxically, autophagy also plays a role in unconventional secretion of IL-1β, a mechanism that has now been demonstrated in macrophages and neutrophils (15,19,20).…”

miR-146b-5p Plays a Critical Role in the Regulation of Autophagy in ∆per Brucella melitensis-Infected RAW264.7 Cells

“…RNA preparation and quantitation were performed as previously described [7]. Small RNA was extracted from RAW264.7-M5 and RAW264.7-M5-∆per, at an equal amount compared with RAW264.7 cells infected with B. melitensis M5-90 and B. melitensis M5-90 ∆per for 4 h, multiplicity of infection (MOI) is 10 : 1, a mirVanaTM microRNA Isolation kit (Ambion, Life technologies, USA) was used to extract the miRNA as described previously [7,26]. miRNA microarray analyses were performed by LC-Bio Co (Hangzhou, China) as described previously [28].…”

“…Brucella abortus can activate the autophagy pathway by promoting a fibrotic phenotype in hepatic stellate cells [25]. Upon LPS + ATP stimulation, IL-1β was incorporated to an autophagic compartment, which indicated that an unconventional autophagy-mediated secretory pathway mediates IL-1β secretion in human neutrophils [26].…”