NADPH oxidase derived reactive oxygen species are involved in human neutrophil IL-1ß secretion but not in inflammasome activation.

Neutrophils are essential players in acute inflammatory responses. Upon stimulation, neutrophils activate NADPH oxidase, generating an array of reactive oxygen species (ROS). Interleukin-1 beta (IL-1ß) is a major proinflammatory cytokine synthesized as a precursor that has to be proteolytically processed to become biologically active. The role of ROS in IL-1ß processing is still controversial and has not been previously studied in neutrophils. We report here that IL-1ß processing in human neutrophils is dependent on caspase-1 and on the serine proteases elastase and/or proteinase 3. NADPH oxidase deficient neutrophils activated caspase-1 and did not exhibit differences in NALP3 expression, indicating that ROS are neither required for inflammasome activation nor for its priming, as has been reported for macrophages. Strikingly, ROS exerted opposite effects on the processing and secretion of IL-1ß; whereas ROS negatively controlled caspase-1 activity, as reported in mononuclear phagocytes, ROS were found to be necessary for the exportation of mature IL-1ß out of the cell, a role never previously described. The complex ROS-mediated regulation of neutrophil IL-1ß secretion might constitute a physiological mechanism to control IL-1ß-dependent inflammatory processes where neutrophils play a crucial role.

Autophagy Mediates Interleukin-1ß Secretion in Human Neutrophils.

Interleukin-1ß (IL-1ß), 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-1ß in infectious conditions. Here, we analyzed if an unconventional secretory autophagy mechanism is involved in the exportation of IL-1ß by these cells. Our findings indicated that inhibition of autophagy with 3-methyladenine and Wortmannin markedly reduced IL-1ß 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-1ß secretion were due to cell death. Furthermore, VPS34IN-1, a specific autophagy inhibitor, was still able to reduce IL-1ß secretion when added after it was synthesized. Moreover, siRNA-mediated knockdown of ATG5 markedly reduced IL-1ß secretion in neutrophil-differentiated PLB985 cells. Upon LPS + ATP stimulation, IL-1ß was incorporated to an autophagic compartment, as was revealed by its colocalization with LC3B by confocal microscopy. Overlapping of IL-1ß-LC3B in a vesicular compartment peaked before IL-1ß increased in culture supernatants. On the other hand, stimulation of autophagy by cell starvation augmented the colocalization of IL-1ß and LC3B and then promoted neutrophil IL-1ß secretion. In addition, specific ELISAs indicated that although both IL-1ß and pro-IL-1ß are released to culture supernatants upon neutrophil stimulation, autophagy only promotes IL-1ß secretion. Furthermore, the serine proteases inhibitor AEBSF reduced IL-1ß secretion. Moreover, IL-1ß could be also found colocalizing with elastase, suggesting both some vesicles containing IL-1ß intersect azurophil granules content and that serine proteases also regulate IL-1ß secretion. Altogether, our findings indicate that an unconventional autophagy-mediated secretory pathway mediates IL-1ß secretion in human neutrophils.

Neutrophil Extracellular Traps Stimulate Proinflammatory Responses in Human Airway Epithelial Cells.

Tissue injury leads to the release of uric acid (UA). At high local concentrations, UA can form monosodium urate crystals (MSU). MSU and UA stimulate neutrophils to release extracellular traps (NET). Here, we investigated whether these NET could be involved in the development of inflammation by stimulating cytokine release by airway epithelial cells. We found that NET significantly increased the secretion of CXCL8/IL-8 and IL-6 by alveolar and bronchial epithelial cells. These effects were not observed when NETosis was inhibited by Diphenyleneiodonium, elastase inhibitor, or Cl-amidine. Similar findings were made with NET induced by cigarette smoke extract, suggesting that NET proinflammatory capacity is independent of the inducing stimulus. Furthermore, NET affected neither the viability and morphology of epithelial cells nor the barrier integrity of polarized cells. The epithelial stimulatory capacity of NET was not affected by degradation of DNA with micrococcal nuclease, treatment with heparin, or inhibition of the elastase immobilized to DNA, but it was significantly reduced by pretreatment with an anti-HMGB-1 blocking antibody. Altogether, our findings indicate that NET exert direct proinflammatory effects on airway epithelial cells that might contribute in vivo to the further recruitment of neutrophils and the perpetuation of inflammation upon lung tissue damage.