Toll-like Receptor Signaling Activation by Entamoeba histolytica Induces Beta Defensin 2 in Human Colonic Epithelial Cells: Its Possible Role as an Element of the Innate Immune Response
Figure 1
HBD2 mRNA expression is induced by Entamoeba histolytica trophozoites in CaCo2 cells by activation of TLR2/4 classic pathway.
A. CaCo2 cells were exposed to PFA-fixed E. histolytica trophozoites (Eh) in a 1∶2 ratio for 2 h in culture media containing only 1% serum. Cells exposed to Enterotoxigenic Escherichia coli (ETEC) in a 1∶100 ratio, in the same conditions, were used as reference or positive control of HBD2 mRNA induction. After pathogen exposure, CaCo2 cells were washed extensively as indicated in Methods before been lysed for total RNA isolation. Expression of HBD2 mRNA was measured by relative quantitative RT-PCR. To investigate the participation of the classic pathway of TLR2/4 in the induction of HBD2 mRNA expression, CaCo2 cells exposed to pathogens were incubated with the inhibitors of NFκB activity, Bay117085, or MyD88 signaling, IMG-2005-5, as described in Methods section. Data are presented as fold change relative to control CaCo2 cells ± SD from two independent experiments done in triplicate. * Indicates statistical differences in CaCo2 cells exposed to pathogens in comparison to non-exposed cells, P value<0.01. ** Indicates statistical differences between expression of HBD2 mRNA in cells exposed to the same pathogen in presence or absence of the inhibitors. P value<0.001. B. Translocation of NFκB p65 subunit to nuclei of CaCo2 cells after exposure to Entamoeba histolytica trophozoites. Cells treated as indicated in A were fixed and permeabilized for detection of NFκB p65 subunit with a specific antibody and a secondary antibody tagged with FITC. Nuclei were visualized by DAPI staining. Cells exposed to E. histolytica (+Eh), cells exposed to ETEC (+ETEC), cells exposed to pathogens in presence of inhibitors BAY117085 (+Bay) or IMG-2005-5 (+IMG). Bar = 20 µm.