157 : Dynamin inhibition interferes with cytokine responses in streptococcus pyogenes-infected human macrophages

Abstract

Streptococcus pyogenes (GAS) is an important human pathogen, causing a wide range of diseases from mild superficial infections to severe invasive infections. In the present study we have analysed the ability of GAS to activate cytokine responses in human monocyte-derived macrophages. We focused on analysing the role of dynamic-dependent endocytosis and major streptococcal virulence factors streptolysin O (SLO) and streptolysin S (SLS) in these responses. One of the fundamental mechanisms of immune evasion is the ability of streptococcal virulence factors to inhibit phagocytosis or phagosome formation. Streptolysins have several functions in GAS pathogenesis. They form pores in host cell membranes and are involved in the escape of GAS from the endosome-lysosome pathway. Expression of SLO is higher in GAS isolates from severe invasive clinical cases than in serotype-matched non-invasive infections. We analysed cytokine expression in macrophages after stimulation with live, inactivated, and SLO or SLS defective bacteria. IL-1β, IL-10, and TNF-α cytokines were induced after bacterial stimulation and no differences in cytokine levels between live, inactivated, or mutant bacteria were seen. The inhibition of dynamin-dependent endocytosis with dynasore attenuated the induction of IL-1β, TNF-α, IFN-β, and CXCL-10 mRNAs in macrophages stimulated with GAS. We also showed that pro-IL-1β is expressed and cleaved into mature-IL-1β via inflammasome activation after bacterial stimulation. Interestingly, streptolysin mutants induced higher levels of mature-IL-1β, which might indicate lower pathogenesis of the mutant bacteria and more efficient responses of the innate immune system. Furthermore, we demonstrated the involvement of p38 MAPK and PI3K signalling pathways in GAS induced cytokine responses in human monocyte-derived macrophages. In conclusion, dynamin-dependent endocytosis is obligatory for the induction of the immune responses in macrophages and the bacteria deficient in streptolysin genes promote the activation of inflammasome-mediated inflammatory responses.