Shiga Toxin induces NETosis in PMN through NOX-dependent mechanism

FERNANDO NICOLÁS SOSA; FLORENCIA SABBIONE; ALAN MAURO BERNAL; ROMINA JIMENA FERNÁNDEZ-BRANDO; ANALIA SILVINA TREVANI; MARINA SANDRA PALERMO; MARÍA VICTORIA RAMOS

Congreso; Reunión Anual de Sociedades de Biociencias (SAIC-SAI-AAFE-NANOMED.AR) 2021; 2021

Hemolytic Uremic Syndrome (HUS), a vascular disease characterized by hemolytic anemia, thrombocytopenia and acute renal failure is caused by enterohemorrhagic Shiga toxins (Stx)-producing bacteria. Besides Stx, inflammatory response mediated by neutrophils (PMN) is essential to HUS outcome. PMN have the capacity to release "neutrophil extracellular traps" (NETs) which are involved in the pathogenesis of several diseases. Particularly, we have previously demonstrated that Stx type 2 (Stx2) is able to induce NETosis in PMN from healthy donors. The aim of this study was to evaluate the mechanisms involved in the NETosis triggered by Stx2 in PMN. To achieve this, PMN were purified from healthy blood donors and incubated for 4 h with RPMI medium alone (Basal), Stx2 (0.1 µg/mL) alone or with commercial inhibitors of NOX2 (DPI, 10 µM) or Elastase (EI, 10 µM). After incubation, supernatants were recovered and the DNA content was evaluated by a fluorometric assay by employing Sybr Gold and the Neutrophil Elastase (NE) activity was assessed by a spectrophotometric assay (absorbance at 405 nm). We observed a 2-fold increase value of DNA over the control (Basal) after incubation with Stx2 which was impaired by the presence of both inhibitors, (Median (IQR)= Basal: 1; Stx2:1.91(1.46-2.40)*; Stx2+DPI: 0.97 (0.75-2.36); Stx2+EI:1.16 (0.79-2.18); *p