ACTIVATION OF PBMCS AND PMNS BY STAPHYLOCOCCAL ENTEROTOXINS N AND U

REDOLFI, DANIELA; NOLI TRUANT, SOFIA; BELEN, SARRATEA MARIA; IANNANTUONO LOPEZ, LAURA V.; VERGARA RUBIO, MARICEF; MALCHIODI, EMILIO L.; MARISA M. FERNANDEZ

San Luis

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

Sociedad Argentina de Inmunología

This study aims to analyze the effects of Staphylococcal enterotoxin N (SEN) and U (SEU) on diverse cell populations representing both the innate and adaptive immune systems. For this propose, SEN and SEU were cloned and expressed as recombinant proteins in E. coli, and then purified using Ni++/NTA column followed by size exclusion chromatography. Subsequently, peripheral blood mononuclear cells (PBMCs) from donors who had not been previously exposed to SAgs were incubated with different concentrations of SEN and SEU (0.0001-10 μg/ml) for 48 h. Proliferation was evaluated by [methyl-3H] thymidine incorporation, and supernatants were collected to quantify cytokines (IL-6, IL-12, IL-10, TNF-α, and IFN-) by ELISA. In addition, polymorphonuclear cells (PMNs), mainly neutrophils, were stimulated with 1 μM of SEN and SEU for 4 or 24 h depending on the assay. NET release was evaluated by indirect immunofluorescence (IFI), IL-6 and TNF-α production was measured in supernatants using ELISA. Hoechst staining and propidium iodide (PI) were used to assess the proportion of living and dead cells while MMPs activity was studied by zymography. Statistical analysis involved t-tests, one-way and two-way ANOVA, followed by post-hoc tests.Results showed that PBMCs exhibited significant proliferation with SEN and SEU at concentrations above 0.001 μg/mL and 0.1 μg/mL respectively, compared to the control (p < 0.05). Moreover, both SAgs induced a significant release of IL-6, IL-12, IL-10, TNF-α, and IFN- at different concentrations compared to untreated cells (p < 0.05). Otherwise, PMNs incubated with both SAgs for 4 h showed a significant production of IL-6 and TNF-α compared to basal levels (p < 0.05). Thus, NETosis was observed by confocal microscopy in PMNs exposed to SEN for 4 h and a significant increase in cell death compared to the control was found in PMNs incubated with this SAg for 24 h (p < 0.05). Additionally, both SAgs significantly enhanced MMP-9 activity at 24 h (p < 0.05). In conclusion, these findings show that SEN and SEU activate immune cells, as PBMCs, promoting their proliferation along with the release of pro-inflammatory cytokines as IFN-γ, TNF-α, IL-12, IL-6, and the anti-inflammatory cytokine IL-10. Among others cellular targets, they display activity on innate immune cells like neutrophils, leading to the release of IL-6 and TNF-α and an increment of MMP-9 activity. Here we provide the first evidence of NETosis induction by SEN, which correlates with the observed increase in cell death within this population. These effects highlight the complexity of the response elicited by these toxins in different populations of the immune system, favoring the success of the bacteria infection at different levels.