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CAG STUDENT PRIZE. SUPPRESSION OF NF-kB IN INTESTINAL EPITHELIAL CELLS BY E HISTOLYTICA IS MEDIATED BY HSP 27

S Kammanadiminti, K Chadee
Department of Microbiology and Infectious Diseases, University of Calgary, Calgary, Alberta

Amebiasis is one of the leading parasitic causes of mortality around the world resulting in 100,000 deaths annually. It is not known why only 10% of the infected individuals show symptoms of amebic colitis. We hypothesize that the epithelial cell responses towards E histolytica can modulate the outcome of infection. Immune cell conditioning was done by coculturing human colonic epithelial cells (Caco-2 or T84) in transwells in the presence of differentiated THP-1 for 24 h. Heat shock protein (HSP) expression was checked by Western blot. NF-kB activation was studied by Western blot, electrophoretic mobility shift assay and in vitro kinase assay. Role of specific HSP in inhibiting NF-kB was confirmed by siRNA and co-immunoprecipitation. Prolonged stimulation of naïve cells with soluble amebic proteins (SAP) reduced the levels of Hsp27 and 72 while in THP-conditioned epithelial cells, SAP enhanced Hsp27 and 72 expressions. Inhibiting HSF-1 by Quercetin or by siRNA abrogated SAP-induced Hsp72 expression. Pretreatment of conditioned epithelial cells with SAP for 12 h resulted in increased survival following oxidative stress induced by hydrogen peroxide and also decreased apoptosis induction by Fas L. Amebic pretreatment also reduced the activation of NF-kB induction by IL-1b. We observed decreased p65 nuclear translocation, IkB phosphorylation and found that amebic proteins inhibit IKK activity. By silencing specific Hsp genes, we identified that Hsp27 mediates this IKK suppression. Consistent with this, IKK-a coimmunoprecipitated with Hsp27 and this interaction was increased in SAP treated conditioned epithelial cells. We conclude that stress protein mediated NF-kB suppression by amebic proteins could be a potential mechanism for the absence of colonic inflammation in the majority of infected people and deduce that lack of such protective response could result in colitis.
Supported by CIHR

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