Stx2 from enterohemorrhagic E. coli induces NF-kappaB activation in reactive astrocytes

ALEJANDRO VILLAREAL ; JORGE GOLDSTEIN; ELIZAGARAY LUCAS; ALBERTO JAVIER RAMOS; CLARA VALENTINA BERDASCO

Congreso; LXIV Reunión Anual de la Sociedad Argentina de Investigación Clínica; 2019

Shiga toxin 2 (Stx2) from enterohemorrhagic E. coli causes hemolytic uremic syndrome (HUS) and acute encephalopathy, which may lead to fatal outcomes in patients. When neurological symptoms are evidenced mortality rate may rise up to 40%. The mechanism by which the encephalopathy emerges in patients with HUS is still unknown. Reactive astrogliosis is a widespread glial response to brain injury and NF-kappa B activation was related to the proinflammatory-neurodegenerative astroglial polarization. The aim of this study was to determine whether Stx2, LPS or a combination of both produce astrocyte reactivity in vitro, and whether this reactivity involves the activation of NF-kappaB pathway. Primary cortical astrocytes were obtained from P3-P5 C57 mice. Confluent astroglial cultures were incubated either with control (saline solution), LPS (50 ng/ml), Stx2 (50 or 200 ng/ml), or a combination of both toxins. GFAP expression and astroglial cell morphology was evaluated by immunocytochemistry. Reactive astrogliosis was observed following the treatment with 200 ng of Stx2 plus 50 ng of LPS in comparison to the control (0. 058 ± 0.006 control vs 0.088 ± 0.006 Stx2+LPS, measured as the number of filamentous astrocytes per total number of astrocytes). Nuclear translocation of p65 NF-kappaB subunit was measured as an index of NF-kappaB activation. The 3h treatment with 50 ng/ml LPS, 200 ng/ml Stx2, and 200 ng/ml Stx2 plus LPS showed a significantive NF-kappa B activation in primary astrocytes when compared with controls (63.19±4.51, 23.77±1.97, 55.30±4.2, 1.33±0.51 respectively; expressed as a ratio of nuclear p65 vs. total number of astrocytes). We conclude that Stx2 causes reactive gliosis in vitro and NF-kappaB activation which it may be involved in the proinflammatory astroglial polarization known to produce neurodegenerative effects. Supported by grants UBACYT (AJR); PICT 2015-1451 (AJR); PICT-2016-1175 (JG); UBACyT 20020160100135BA (JG)