Toll-like Receptor Signaling Activation by <em>Entamoeba histolytica</em> Induces Beta Defensin 2 in Human Colonic Epithelial Cells: Its Possible Role as an Element of the Innate Immune Response

<div><p>Background</p><p><i>Entamoeba histolytica</i>, a protozoan parasite of humans, produces dysenteric diarrhea, intestinal mucosa damage and extraintestinal infection. It has been proposed that the intestinal microbiota composition could be an important regulatory factor of amebic virulence and tissue invasion, particularly if pathogenic bacteria are present. Recent <i>in vitro</i> studies have shown that <i>Entamoeba histolytica</i> trophozoites induced human colonic CaCo2 cells to synthesize TLR-2 and TLR-4 and proinflammatory cytokines after binding to the amebic Gal/GalNac lectin carbohydrate recognition domain. The magnitude of the inflammatory response induced by trophozoites and the subsequent cell damage were synergized when cells had previously been exposed to pathogenic bacteria.</p> <p>Methodology/Principal Findings</p><p>We show here that <i>E. histolytica</i> activation of the classic TLR pathway in CaCo2 cells is required to induce β defensin-2 (HBD2) mRNA expression and production of a 5-kDa cationic peptide with similar properties to the antimicrobial HBD2 expressed by CaCo2 cells exposed to enterotoxigenic <i>Escherichia coli</i>. The induced peptide showed capacity to permeabilize membranes of bacteria and live trophozoites. This activity was abrogated by inhibition of TLR2/4-NFκB pathway or by neutralization with an anti-HBD2 antibody.</p> <p>Conclusions/Significance</p><p><i>Entamoeba histolytica</i> trophozoites bind to human intestinal cells and induce expression of HBD2; an antimicrobial molecule with capacity to destroy pathogenic bacteria and trophozoites. HDB2's possible role as a modulator of the course of intestinal infections, particularly in mixed ameba/bacteria infections, is discussed.</p> </div

Permeabilizing effect of HBD2 released by CaCo2 cells after exposure to <i>Entamoeba histolytica</i> trophozoites.

<p>A. Permeabilization of <i>Staphylococcus aureus</i> 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 <i>E. histolytica</i> 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 <i>S. aureus</i> bacteria or <i>E. histolytica</i> 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, <i>S. aureus</i> bacteria or <i>E. histolytica</i> 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 (<i>P</i> value<0.01).</p

HBD2 mRNA expression is induced by <i>Entamoeba histolytica</i> trophozoites in CaCo2 cells by activation of TLR2/4 classic pathway.

<p>A. CaCo2 cells were exposed to PFA-fixed <i>E. histolytica</i> trophozoites (Eh) in a 1∶2 ratio for 2 h in culture media containing only 1% serum. Cells exposed to Enterotoxigenic <i>Escherichia coli</i> (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 <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002083#s2" target="_blank">Methods</a> 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 <a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0002083#s2" target="_blank">Methods</a> 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, <i>P</i> 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. <i>P</i> value<0.001. B. Translocation of NFκB p65 subunit to nuclei of CaCo2 cells after exposure to <i>Entamoeba histolytica</i> 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 <i>E. histolytica</i> (+Eh), cells exposed to ETEC (+ETEC), cells exposed to pathogens in presence of inhibitors BAY117085 (+Bay) or IMG-2005-5 (+IMG). Bar = 20 µm.</p