SERINE PROTEASES ACTIVITY IS LINKED TO NEUTROPHIL PROINFLAMMATORY CAPACITY AND CELL FATE

KEITELMAN I; RUBATTO BIRRI P; SHIROMIZU CM; PIZZANO, MANUELA; VERA AGUILAR, DOUGLAS; VEREERTBRUGGHEN, ALEXIA; CERNUTTO A; FEDERICO FUENTES; GALLETTI J; SABBIONE F; TREVANI A

San Luis

Congreso; LXXI Reunión Anual de la Sociedad Argentina de Inmunología (SAI); 2023

Neutrophils represent the first line of defense against bacteria and fungi. Theyexert their microbicidal effects by phagocytosis and the release of extracellulartraps (NET). These granulocytes can also modulate immune responses byreleasing the potent pro-inflammatory cytokine Interleukin-1 beta (IL-1β). Wepreviously determined that in human neutrophils pro-IL-1β is processed to itsactive isoform mainly by both serine proteases (NSP) leaked from azurophilgranules into the cytosol and caspase-1. However, we found that excessiveactivation of these enzymes is detrimental to IL-1β secretion. Considering thatNETosis and pro-IL-1β processing/secretion usually involve the participation ofelastase, ROS-derived from NADPH oxidase, and pores made by Gasdermin D,we conjectured that both processes might be mutually exclusive. To test thishypothesis, we stimulated human neutrophils from healthy donors with a widerange of concentrations (2.5-25 ng/ml) of phorbol myristate acetate (PMA), thegold standard NETosis activator, together with or without LPS (150 ng/ml)+ATP(2.5 mM) to promote pro-IL-1β synthesis and inflammasome activationrespectively, and consequently caspase-1 activation. We evaluated the levels ofsecreted IL-1β by ELISA, NSP activation and reactive oxygen species productionby flow cytometry, MPO by spectrophotometry, and DNA release to thesupernatants by fluorometry. We found that neutrophils released significant levelsof IL-1β when stimulated with PMA 2,5 and 5 ng/ml (p