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Abstract
Little is known about the pathogenesis of Entamoeba histolytica and how epithelial cells respond to the parasite. Herein, we characterized the interactions between E. histolytica and colonic epithelial cells and the role macrophages play in modulating epithelial cell responses. The human colonic epithelial cell lines Caco-2 and T84 were grown either as monoculture or co-cultured in transwell plates with differentiated human THP-1 macrophages for 24 h before stimulation with soluble amebic proteins (SAP). In naive epithelial cells, prolonged stimulation with SAP reduced the levels of heat shock protein (Hsp) 27 and 72. However in THP-1 conditioned intestinal epithelial cells SAP enhanced Hsp27 and Hsp72, which was dependent on the activation of ERK MAP kinase. Hsp synthesis induced by SAP conferred protection against oxidative and apoptotic injuries. Treatment with SAP inhibited NF-kappaB activation induced by interleukin-1beta; specifically, the NF-kappaB-DNA binding, nuclear translocation of p65 subunit, and phosphorylation of IkappaB-alpha were reduced. Gene silencing by small interfering RNA confirmed the role of Hsp27 in suppressing NF-kappaB activation at IkappaB kinase (IKK) level. By co-immunoprecipitation studies, we found that Hsp27 interacts with IKK-alpha and IKK-beta, and this association was increased in SAP-treated conditioned epithelial cells. Overexpression of wild type Hsp27 amplified the effects of SAP, whereas a phosphorylation-deficient mutant of Hsp27 abrogated SAP-induced NF-kappaB inhibition. In conditioned epithelial cells, Hsp27 was phosphorylated at serine 15 after prolonged exposure to SAP. This mechanism may explain the absence of colonic inflammation seen in the majority of individuals infected with E. histolytica.
Highlights
Cell-specific response to amebic infection is poorly understood
E. histolytica Suppresses NF-B in Epithelial Cells cells have been shown to influence epithelial cell responses (16, 21) and macrophages are a major source of these cytokines, we studied the effect of macrophages on epithelial cell response toward E. histolytica
soluble amebic proteins (SAP) treatment suppressed Hsp27 and Hsp72 in naıve intestinal epithelial cells (IEC) (Fig. 1A), the same two heat shock proteins (Hsp) were significantly overexpressed in macrophage-conditioned Caco-2 and T84 colonic epithelial cells (Fig. 1B)
Summary
Cell-specific response to amebic infection is poorly understood. the majority of research done to unravel the mechanism of amebic colitis has been focused on proinflammatory responses (2, 3) by epithelial cells and a role of NF-B (4) and chemokines such as IL-8 3 (5) as triggering events for inflammation. The universal response to stress has been the induction of a group of highly conserved family of proteins called heat shock proteins (Hsp) and is commonly referred to as heat shock response or stress response Several pathogens and their products have been shown to induce various Hsp in different cell types including intestinal epithelial cells (11– 13). We identified Hsp as the key mediator suppressing NF-B activation by virtue of its association with IB kinase (IKK) complex in intestinal epithelial cells (IEC) We conclude that this could be one of the mechanisms by which colonic inflammation is suppressed in the majority of E. histolyticainfected individuals and that the lack of such protective responses in a susceptible individual could lead to the symptoms associated with amebic colitis
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