Shiga Toxin 2 (Stx2) causes hemorrhagic colitis, Hemolytic Uremic Syndrome (HUS) and neurological dysfunctions. In the hippocampus, Stx2 produces cognitive deficits in patients. Enterohemorrhagic E. Coli (EHEC) secretes Stx2 and releases LPS. The aim of this study was to study microglial cell (MG) activation profile upon
Stx2 exposure, exploring the hypothesis that Stx2-intracellullar allocation depends on the systemic status (fever and inflammatory mechanism) during infection. Rat MG primary cultures (99.6% purity) were designed in order to evaluate: i) the effect of Stx2 in these cells; ii) the cellular environment that maximized Stx2 effect; iii) MG sensitivity and responsiveness to Stx2 challenge; iv) the regulation of the Stx2- globotriaosylceramide (Gb3) receptor under different conditions. Heat shock (HS) treatment and Lipopolysaccharide (LPS) challenge were used to mimic fever and inflammatory reaction, respectively. Our results showed that MG sensitivity, described by intracellular Stx2 quantitation was higher in HS context than in LPS one (70.87±1.63, 34.12±2.81 respectively, in IOD, p<0.05.), as MG was more sensitive to low toxin concentrations in the HS context (1ƞg/ml for HS versus
100ƞg/ml for LPS). However, maximal MG activation depicted by Iba-1 expression levels (a MG marker used to identify cell activation) was increased in the LPS condition (189±2.57 LPS, 143±2.21 HS, 74.56±2.31 without context, in IOD, p<0.05). In accordance to these data, the Gb3 MG distribution changed upon cell context. While Gb3 was localized in cytoplasm vesicles under LPS context, it was localized in the cell membrane in the HS one. In conclusion, the present in vitro model seems to be useful in order to establish the sequence of events during EHEC infection: HS (fever) sensitizes MG to low toxin concentrations but the highest response profile (measured by Iba-1 expression level) results to LPS context (inflammatory reaction). MG, being reactive to Stx2, could play a pivotal role in inflammatory mechanisms underlining the cellular damage associated with neurological the dysfunction. Also, LPS activated microglia, and it may trigger an inflammatory reaction that may contribute to the observed damage.