47.    SAGS of the ECG operon: impact on the innate immune response

Bacterial superantigens (SAgs) are enterotoxins that bind to MHC-II and TCR, activating as much as 20% of T cells and promoting a cytokine storm which enhances endotoxic shock and produces immunosuppression, hindering the immune response against bacterial infection. In this work, we investigated the effect on the innate immune response of four natural variants of SAgs (SEG, SEI, SEO and SEM), encoded by the egc operon in autochthonous S. aureus strains. First, we analyzed the effect of SAgs on monocytic THP-1 cells finding a strong inhibition of the proliferation determined by 3H-thymidine. Thus, 100 μg/ml of SEO, SEG and SEI inhibited proliferation up to 70% while SEM inhibited ~30% (p<0.0001). We confirmed cell death by flow cytometry (FC) and fluorescent microscopy. We also found in THP-1 cells supernatant high concentration of IL-6, IL-8, IL-12 and TNF-α after stimulation with these SAgs(p<0.05). Each of these four SAgs had differential capacity to stimulate human PBMCs to proliferate (p<0.0001). As with THP-1 cell line, these SAgs induced production of IL-6 and IL-12 in human PBMCs, but in this case we found also IL-10. To analyze whether other cells of the innate immunity were responsible for the pro-inflammatory profile, we evaluated the SAgs effect on human purified γδ T cells from healthy donors by FC and ELISA. We found that, in contrast to the effect on αβ T cells, only some SAgs differentially activated γδ T cells from 0.1 μM (p<0.05), but became toxic at 10 μM. We also found a significant
production of IFN-γ and TNF-α starting at 0.1 μM (p<0.05), without production of IL- 17. Notably, such activity was not related to the αβ TCR binding site, since mutant SAg lacking the ability to bind αβ TCR, activated γδ T cells as much as wild type SAgs. Considering that CD1 molecules are targets of γδ TCR we analyzed their expression on PBMCs upon SAgs stimulation by FC. We found a differential expression of monocytic CD1a and b in response to these SAgs(p<0.1). In addition, we evaluated the ability of these SAgs to bind to the IL-6 signal transducer (gp130) by Surface Plasmon Resonance. SEO and SEG showed the higher affinity (KD= ~1.10-6), while SEI and SEM interacted with lower affinity (KD=~1.10-5). In contrast, mutant SAg in TCR binding site did not display interaction with gp130. In summary, we described a M1 profile induced by SAgs on PBMCs and monocytic cells and the capacity of SAgs to stimulate γδ T cells to produce IFN-γ and TNF-α, which strongly suggest an early pro inflammatory activity of SAgs. The interaction of SAgs with gp130, known to be involved in inflammation, immune regulation and the activation of the Th17 arm, may explain part of the wide effects that these toxins show in vivo. In addition, the reduction of the pool of phagocytic and effector cells and the generation of a non-efficient Th1 profile accompanied with anti-inflammatory cytokines as IL-10, would conspire against the successful eradication of this extracellular bacteria.