Shiga toxin 2 from EHEC produce inframmatory response through microgial reactivity and Gb-3pERK signaling

BLAKE MG; GOLDSTEIN J; PINTO A; GIRONACCI MM; BERDASCO C; ROSATO-SIRI MV

Mendoza

Congreso; Congreso de la Sociedad de Biología de Cuyo; 2018

Sociedad de Biología de Cuyo

Shiga Toxin 2 (Stx2) is released during enterohemorrhagic Escherichia coli (EHEC) infection. Stx2 causes fever, hemorrhagic colitis, hemolytic uremic syndrome and neurological dysfunctions through its cell membrane receptor globotriaosylceramide (Gb3). Previous reports from our group have confirmed that proinflamatory agents play a critical role in the development of the pathology. The aim of this study was to determine the cell signalling mechanisms that occur in an in vivo and in vitro rodent experimental inflammatory model triggered by Stx2, validated by different methodological approaches. Male Swiss mice were injected intravenously with control (saline solution) or Stx2 (1ηg per mouse).After 2 days, each group was submitted to hole-board test to measure anxiety, stress, neophilia and emotionality. Besides, fixed hippocampi were subjected to immunofluorescence (IF) with anti-GFAP and anti-Iba1 to determine reactive astrocytes and microglial score activation respectively. In addition, another set of hippocampi were homogenized for western blot analysis to determine pERK activation at 2, 6, 12 and 24 hours. On the other hand, primary microglial cell (MG) cultures were incubated with either DMEM or 100ηg/ml of Stx2to test by IF the uptake of Stx2, microglial reactivity and the expression profile of Gb3 receptor at day 2. Hippocampal deterioration was observed after Stx2 treatment, which was correlated with memory failure. GFAP expression levels were increased in Stx2 treated mice in comparison to the control ones (0.19±0.02 control vs 0.52±0.02 Stx2, in IOD, p<0.05), while microglial activation score was increased in Stx2 treated mice in comparison to control ones. The expression of pERKwas decreased in Stx2 treated mice in comparison to controls at 24 hours (0.96±0.02 control vs 0.49±0.02 Stx2, p<0.05). Finally, MG resulted to be sensitive (12.53±0.89 Stx2, in IOD, p<0.05)and responsiveness (1±0.00 control vs52.01±1.02 Stx2, in IOD, p<0.05)to the toxin as it was determined by Iba1 expression level, and Stx2 incubation changed the distribution of Gb3 expressionin MG. In conclusion, Stx2 produced an inflammatory response both in vivo as in vitro through Gb3-pERK signaling pathway.