Extracellular vesicles released from human neutrophils bind Shiga toxin, the agent responsible for Hemolytic Uremic Syndrome.

CAROLINA MAIUMI SHIROMIZU; IRENE ANGÉLICA KEITELMAN; FERNANDO DANIEL GOMEZ; ALEXIA VEREERTBRUGGHEN; DOUGLAS VERA AGUILAR; MANUELA PIZZANO; AGOSTINA CERNUTTO; PAULA S PEREZ; MICAELA ROSATO; MARÍA VICTORIA RAMOS; CAROLINA JANCIC; FEDERICO FUENTES; MARÍA MARTA AMARAL; JEREMÍAS GALLETTI; MARINA PALERMO; FLORENCIA SABBIONE; ANALÍA TREVANI

San Luis

Congreso; LXXI Reunión Anual de la Sociedad Argentina de Inmunología; 2023

Sociedad Argentina de Inmunología

Infection with Shiga toxin (Stx) producing Escherichia coli (STEC) can cause fromself-limited diarrhea to a systemic life-threatening condition called HaemolyticUremic Syndrome (HUS). The STEC colonizes the intestine where it secretes theStx, its main virulence factor, which can translocate to the bloodstream and reachtarget organs. In HUS patients, Stx is not found free in circulation but is detectedassociated with blood cells and extracellular vesicles (EV). Furthermore,neutrophil (PMN) count and blood EV amounts are increased during HUS acutephase. We have previously described that human PMNs upon stimulation withStx2 release EV that bear functional Stx (EVstx). The aim of this study was tofurther characterize the release of EV by PMNs and their interaction with Stx.Human PMNs (1x107) were treated with purified Stx2 (100 ng/ml, EVstx) orvehicle (EVc). After incubation for 1 or 4 h, samples were spun down, cellulardebris was removed, and EV were isolated by differential centrifugations. Thecapacity of the EV to carry functional Stx was evaluated by a cytotoxicity assayemploying the Vero cell line. We determined that the release of EV by PMNs wasnot modulated by Stx2 stimulation, as we found similar protein concentrations(n=5) and equivalent levels of CD63 (n=10) in samples of EVc and EVstx bywestern blot. Moreover, both EV samples expressed equal amounts ofmyeloperoxidase (MPO) and elastase measured by western blot (n=3) and acomparable enzymatic activity of both proteases when analysed byspectrophotometry (n=7). Thus, we then investigated if EVc could bind Stx oncereleased from PMNs. To this end, we isolated EVc and incubated them for 1 hwithout or with Stx2 (100 ng/ml, EVc+Stx). After a washing step, EVc+Stx, EVstxand EVc were seeded onto Vero cells at different dilutions to evaluate theircytotoxic capacity. EVc+Stx and EVstx significantly reduced cell viability (n=7,p