SUPERANTIGENS OF THE EGC OPERON SEI, SEO, SEG AND SEM INDUCE HUMAN NEUTROPHILS ACTIVATION PROMOTING DIFFERENTIAL PEPTIDOME PROFILES, MODIFICATION OF MMPS ACTIVITY, CYTOKINES AND NETS RELEASE

NOLI TRUANT SOFIA; REDOLFI, DANIELA M.; LEDESMA MARTIN MANUEL; BELEN, SARRATEA MARIA; IANNANTUONO LOPEZ, LAURA V.; CARLOS VAY; MALCHIODI, EMILIO L.; FERNÁNDEZ, MARISA MARIEL

San Luis

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

Sociedad Argentina de Inmunología

Superantigens (SAgs) are potent immunomodulatory enterotoxins primarily produced by extracellular bacteria, such as S. aureus. While extensive research has focused on SAgs activation of T cells, recent studies have unveiled their targeting of innate immune cells. Polymorphonuclear cells (PMNs), mainly neutrophils, are the first line of defense against these pathogens, being a key component of the innate immune system.Here, we investigated the effect of four natural SAgs of the egc operon (SEG, SEI, SEO and SEM) on the neutrophil population, its peptidome, and activation markers.For this purpose, SEI, SEO, SEG and SEM were cloned and produced as recombinant proteins in E. coli and purified by Ni++/NTA column. PMNs were enriched from healthy donors and incubated with 1 μM of each SAg and PMA as a positive control, during 4 and 24 h depending on the assay. To characterize the proteomic profile within the 2 to 20 kDa range, the protein extract of neutrophils challenged with different stimuli was analyzed using MALDI-TOF-MS. NET release was evaluated by IFI, metabolic activity assessed via XTT assay, and viability determined using Hoechst/IP fluorescent microscopy. Supernatants were collected to measure IL-6, IL-12, IL-10, and TNF-α by ELISA. Concurrently, MMPs activity was evaluated by zymography. Results were analyzed by one-way ANOVA with the corresponding post-hoc test. Stimulation of PMNs with 1 μM of SEI, SEO, SEG, and SEM led to distinct peptidome profiles, with SEM inducing the most unique pattern. Additionally, a noteworthy increase in metabolic activity after 24 hours (p < 0.05) was observed, accompanied by augmented cell death, primarily attributed to SEG (p < 0.005). In addition, SAgs were found to trigger NET release and enhance MMP-9 activity in both multimeric and monomeric forms (p < 0.05). Furthermore, all SAgs induced a higher production of IL-6, IL-12, and TNF-α by PMNs compared to untreated cells (p < 0.0001). In conclusion, these results show that SEI, SEO, SEG, and SEM stimulate NET release from PMNs with the associated production of pro-inflammatory cytokines, IL-6, TNF-α, and IL-12, each exhibiting a unique peptidome profile. Moreover, these toxins induced increased metabolic and MMP activity. Overall, these findings underscore the distinct modulation of PMNs by egc SAgs, highlighting their capacity to influence not only adaptive but also innate immune responses.