THE SECRETION OF HUMAN NEUTROPHIL IL-1β TRIGGERED BY SHIGA TOXIN-PRODUCING ESCHERICHIA COLI IS REGULATED BY THE ACTIVITY OF NEUTROPHIL SERINE PROTEASES WHICH IS CONTROLLED BY GASDERMIN D

SABBIONE F; KEITELMAN I; SHIROMIZU CM; VEREERTBRUGGHEN, ALEXIA; VERA AGUILAR, DOUGLAS; RUBATTO BIRRI P; PIZZANO, MANUELA; RAMOS MV; FEDERICO FUENTES; SAPOSNIK L; CASSATARO J; CERNUTTO A; JANCIC C; GALLETTI J; PALERMO M; TREVANI A

San Luis

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

Shiga toxin (Stx)-producing Escherichia coli (STEC) is an enteric pathogen thatcauses self-limited gastrointestinal infections and bloody diarrhea, but also asevere systemic condition known as Hemolytic Uremic Syndrome (HUS).Neutrophils are recruited to the intestine upon STEC infection. Previously wedetermined that STEC stimulated neutrophils to release elevated levels of IL-1βthrough a mechanism that involved the activation of caspase-1 driven by theNLRP3-inflammasome. Based on the effect of a PAN-neutrophil serine proteases(NSPs) inhibitor, we also found that NSPs’ activity is required for IL-1β processingand release. Other studies showed that active Gasdermin D (GSDMD) producedby caspase-1 processing form pores on the membranes of neutrophil azurophilgranules that allow the leakage of the NSP elastase (NE) to the cytosol,explaining its capacity to process cytosolic substrates, among them the IL-1βprecursor and GSDMD. We also found that neutrophils IL-1β secretion washigher at lower STEC multiplicities of infection (MOI) and it was independent ofthe capacity of the bacteria to secrete Stx. The aim of this study was to investigatethe molecular mechanism that underlies this secretory profile. We first evaluatedthe effect of Ecotin, another serine protease inhibitor, capable of inhibiting NE, onSTEC-induced IL-1β secretion. Ecotin significantly reduced IL-1β secretion in aconcentration-dependent manner (p< 0,0001) but neither affected IL-8 secretionnor induced neutrophil lytic death (n=4). These findings confirmed the role ofNSPs in neutrophil IL-1β secretion induced by STEC. We then evaluated theeffect of Disulfiram (DSF), an inhibitor of GSDMD pore formation. DSFsignificantly reduced the activation of NSPs induced by STEC (n=6, p< 0,05)supporting a role of GSDMD pore in NSPs cytosolic leakage and activation. DSFalso reduced the total levels (intracellular and extracellular) of mature IL-1β (n=3,p< 0,05) and increased the intracellular pro-IL-1β levels (n=5, p< 0,05). Also, preactivation of caspase-1 which would be expected to increase GSDMD cleavageand pore formation, led to a reduction in IL-1β secretion when challenging cellswith STEC compared to cells without pre-activated caspase-1 (n=7, p< 0,05).None of these treatments modulated neutrophil lytic death. We previouslydetermined that the higher the MOI, the greater was the activation of bothcaspase-1 and NSPs. These findings together with those of this study, and theability of NE to cleave IL-1β at numerous sites, suggest that higher STEC MOIsinduce more NSPs leakage to the cytosol, which instead of contributing only toIL-1β processing could lead to its degradation. Overall, our findings suggest thatNSPs are key regulators of human neutrophil proinflammatory capacity inresponse to STEC.