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.
Figure 2.
Production and release of HBD2 by CaCo2 cells exposed to Entamoeba histolytica trophozoites.
A. Quantification of HBD2-positive CaCo2 cells after exposure to pathogens. CaCo2 cells were exposed for 2 h to ETEC or PFA-fixed trophozoites in the presence of 1.0 µg/ml Brefeldin A. Cells were permeabilized and fixed and then labeled with a specific anti-HBD2 antibody tagged with FITC. Levels of HBD2 inside the CaCo2 cells were determined by flow cytometry and data are presented as mean fluorescence intensity (MFI). Asterisks indicate statistical differences relative to control CaCo2 cells in three experiments done in triplicate (P value<0.01). B. Detection of HBD2 in cultured media from CaCo2 cells. Culture media were analyzed by SDS-PAGE in 15% gels. A silver-stained representative gel is shown. Lane 1, synthetic HBD2 peptide. Lane 2, CM from CaCo2 cells exposed to PFA-fixed E. histolytica trophozoites (CaCo2+Eh). Lane 3, CM of ETEC-exposed CaCo2 cells (CaCo2+ETEC). Lane 4, CM from CaCo2 cells not exposed to pathogens (Control CaCo2). C. Immunodetection of HBD2 in a parallel gel to the one shown in panel B. After electrophoresis, proteins were blotted onto nitrocellulose membranes and challenged with anti-HBD2 antibody. Lane 1, synthetic HDB2 peptide. Lane 2, CM of CaCo2 cells exposed to PFA-fixed Entamoeba histolytica trophozoites (CaCo2+Eh). Lane 3, CM of CaCo2 cells exposed to ETEC (CaCo2+ETEC). Lane 4. CM of CaCo2 cell cultures not exposed to pathogens (Control CaCo2).
Figure 3.
Permeabilizing effect of HBD2 released by CaCo2 cells after exposure to Entamoeba histolytica trophozoites.
A. Permeabilization of Staphylococcus aureus bacteria, a known strain sensitive to HBD2 activity, by CM from CaCo2 cells exposed to pathogens. One hundred thousand bacteria from an overnight culture were exposed for 1 h to CM from CaCo2 cells exposed to ETEC (CaCo2+ETEC, positive control), to CM from cells exposed to PFA-fixed trophozoites (CaCo2+Eh) or incubated with 10 ng/ml of CECE-HBD2. Propidium iodide internalization into permeabilized bacteria was quantified by flow cytometry. B. Permeabilization of E. histolytica trophozoites by CM from CaCo2 cells exposed to pathogens. One hundred thousand trophozoites were exposed to CM from CaCo2 cells exposed to ETEC (CaCo2+ETEC, positive control), to CM from cells exposed to PFA-fixed trophozoites (CaCo2+Eh) or incubated with 10 ng/ml of CECE-HBD2. In parallel, CM from CaCo2 cell cultures also exposed to either pathogen and incubated with inhibitors of the TLR2/4-NFκB pathway, Bay117085 and IMG-2005-5, were used to permeabilize S. aureus bacteria or E. histolytica trophozoites. Propidium iodide internalization into trophozoites was quantified as indicated for bacteria. C and D. Neutralization of HBD2 activity present in CM. Media obtained from CaCo2 cell cultures exposed to pathogens were neutralized with anti-HBD2 antibody (2.0 µg/ml for 2 h); then, S. aureus bacteria or E. histolytica trophozoites were incubated with these neutralized media. As control, a non-related polyclonal antibody (anti-human Sirt1) was used. Permeabilization levels were evidenced by propidium iodide penetration and quantified by flow cytometry. Data for all panels are presented as percentage of permeabilized cells ± SD. * indicates differences between control cells and cells exposed to pathogens. ** Indicates statistical differences between inhibited (panels A and B) or neutralized (panels C and D) conditions versus values obtained in non-inhibited or non-neutralized conditions in three experiments done in triplicate (P value<0.01).
Figure 4.
Ultrastructural alterations of trophozoites assessed by transmission electron microscopy.
In panels A and B, E. histolytica trophozoites were incubated in: CM of CaCo2 cells not exposed to the pathogens (a); CM from cells exposed to PFA-fixed trophozoites (b); CM from cells exposed to ETEC (c) or incubated with 10 ng/ml of CECE-HBD2 (d) and then prepared for transmission electron microscopy. Panel Aa, a control trophozoite showing continuous plasma membrane without alterations (arrows), small clear zones in the cytoplasm in which glycogen localizes (g) and clear vacuoles (v). Panel Ab shows a trophozoite incubated in CM from CaCo2 cells exposed to trophozoites with many alterations in the cytoplasm, ruptured zones in the plasma membrane and extruded cellular material and a great increase in the number of vacuoles containing cellular material (v). Panel Ac shows a representative trophozoite exposed to CM from ETEC-exposed cells with discontinuities in the membrane and ruptures (arrows), increased content of glycogen (g) and increased number of vacuoles, many containing cellular material (v). Arrowheads point to extracellular material. Panel Ad, a trophozoite treated with CECE-HBD2 shows alterations of the membranes similar to those observed in trophozoites incubated in CM from pathogen-exposed cells. Bar = 0.5 µm. B. Low magnification of representative trophozoites treated as indicated in each of the panels above, shows control trophozoites with regular distribution of the chromatin (N), tethered to the nuclear membrane. Trophozoites treated with CM of pathogen-exposed cells or CECE-HBD2 show chromatin aggregated and polarized (N) and a high number of big vacuoles containing cellular material (v). Bar = 3.0 µm.