EXTRACELLULAR VESICLES RELEASED FROM HUMAN NEUTROPHILS BIND SHIGA TOXIN, THE AGENT RESPONSIBLE FOR HAEMOLYTIC UREMIC SYNDROME

SHIROMIZU CM; KEITELMAN I; GOMEZ, F; VEREERTBRUGGHEN, A; VERA AGUILAR, DOUGLAS; PIZZANO, MANUELA; CERNUTTO A; PEREZ PS; ROSATO, M; RAMOS MV; JANCIC C; F. FUENTES; AMARAL MM; GALLETTI J; PALERMO M; SABBIONE F; TREVANI A

San Luis

Congreso; LXXI REUNIÓN CIENTÍFICA ANUAL DE LA SOCIEDAD ARGENTINA DE INMUNOLOGÍA (SAI); 2023

InfectionwithShigatoxin(Stx)producingEscherichiacoli(STEC)cancausefromself-limited diarrhea to a systemic life-threatening condition called HaemolyticUremicSyndrome(HUS).TheSTECcolonizestheintestinewhereitsecretestheStx,itsmainvirulencefactor,whichcantranslocatetothebloodstreamandreachtarget organs. In HUS patients, Stx is not found free in circulation but is detectedassociatedwithbloodcellsandextracellularvesicles(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 assayemployingtheVerocellline.WedeterminedthatthereleaseofEVbyPMNswasnot 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 bywesternblot.Moreover,bothEVsamplesexpressedequalamountsofmyeloperoxidase (MPO) and elastase measured by western blot (n=3) and acomparableenzymaticactivityofbothproteaseswhenanalysedbyspectrophotometry (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 hwithoutorwithStx2(100ng/ml,EVc+Stx).Afterawashingstep,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