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

GABELLONI ML; SABBIONE F; JANCIC C; GEFFNER JR; TREVANI AS

Quebec

Simposio; International Symposium “The neutrophil in immunity”; 2012

Interleukin-1β (IL-1β) is a major pro-inflammatory cytokine synthesized in the cytoplasm as a precursor that has to be proteolytically processed to become biologically active. We have previously determined that human neutrophil IL-1β processing is dependent on caspase-1 and on elastase and/or proteinase-3. Since the role of ROS in IL-1b processing is still controversial and has not been studied in neutrophils, this study was undertaken to address this issue. Neutrophils differentiated from a myelomonocytic cell line deficient in the NADPH oxidase’s gp91phox subunit (PLB-KO) released less IL-1β in response to LPS and LPS+ATP than their wild type counterparts (PLB-985; n=4, p<0.05, ELISA). However, PLB-KO cells showed a greater content of intracellular IL-1β by flow cytometry (n=3), which corresponded to the processed form of the cytokine, as shown by western blot analysis (n=3). Moreover, PLB-KO neutrophils induced caspase-1 activation and did not exhibit differences in NALP3 expression compared to PLB-985 neutrophils, as determined by Flica probe staining and flow cytometry (n=3), indicating that ROS are neither required for inflammasome activation nor for its priming. The addition of the superoxide anion generating system xanthine/xanthine oxidase (X/XO) to PLB-KO neutrophils that had been stimulated to accumulate IL-1β induced the release of the cytokine (p<0.001; n= 4). Taken together, our results suggest that ROS produced by the NADPH oxidase are not required for neutrophil inflammasome activation but are necessary for the release of mature IL-1β from the cell, a role never previously described.