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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.

Ayala-Sumuano JT, Téllez-López VM, Domínguez-Robles Mdel C, Shibayama-Salas M, Meza I - PLoS Negl Trop Dis (2013)

Bottom Line: Recent in vitro studies have shown that Entamoeba histolytica 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.We show here that E. histolytica 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 Escherichia coli.This activity was abrogated by inhibition of TLR2/4-NFκB pathway or by neutralization with an anti-HBD2 antibody.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biomedicine, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, México DF, México.

ABSTRACT

Background: Entamoeba histolytica, 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 in vitro studies have shown that Entamoeba histolytica 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.

Methodology/principal findings: We show here that E. histolytica 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 Escherichia coli. 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.

Conclusions/significance: Entamoeba histolytica 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.

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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.
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pntd-0002083-g001: 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.

Mentions: To test this hypothesis, CaCo2 cells were exposed to PFA-fixed trophozoites in a 1∶2 ratio for 2 h or to ETEC bacteria in a 1∶100 ratio for the same length of time. As induction of HBD2 in CaCo2 cells has already been reported to occur when they are exposed to ETEC [30], cells exposed to these bacteria were used as positive control for HBD2 induction by amebas. Total RNA was isolated from cells either not exposed to pathogens (control cells) or from cells exposed to pathogens and HBD2 mRNA expression evaluated by real time quantitative RT-PCR. Expression of constitutive rplp0 mRNA was evaluated as normalizing factor. Primers used and controls for the quantitative RT-PCR assays are shown in supplementary material (Supplemental Figure S1). Figure 1A shows that when CaCo2 cells were exposed to ETEC (positive control), the relative expression of HBD2 mRNA increased close to 100-fold, compared to its baseline expression in cells not exposed to pathogens. Exposure of CaCo2 cells to PFA-fixed E. histolytica trophozoites increased expression of HBD2 mRNA close to 38-fold, compared to the baseline expression of control cells. When the expression of HBD2 mRNA was evaluated in presence of inhibitors that block the TLR-NFκB pathway, a strong negative effect was evident in the expression of the defensin mRNA.


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.

Ayala-Sumuano JT, Téllez-López VM, Domínguez-Robles Mdel C, Shibayama-Salas M, Meza I - PLoS Negl Trop Dis (2013)

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.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3585038&req=5

pntd-0002083-g001: 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.
Mentions: To test this hypothesis, CaCo2 cells were exposed to PFA-fixed trophozoites in a 1∶2 ratio for 2 h or to ETEC bacteria in a 1∶100 ratio for the same length of time. As induction of HBD2 in CaCo2 cells has already been reported to occur when they are exposed to ETEC [30], cells exposed to these bacteria were used as positive control for HBD2 induction by amebas. Total RNA was isolated from cells either not exposed to pathogens (control cells) or from cells exposed to pathogens and HBD2 mRNA expression evaluated by real time quantitative RT-PCR. Expression of constitutive rplp0 mRNA was evaluated as normalizing factor. Primers used and controls for the quantitative RT-PCR assays are shown in supplementary material (Supplemental Figure S1). Figure 1A shows that when CaCo2 cells were exposed to ETEC (positive control), the relative expression of HBD2 mRNA increased close to 100-fold, compared to its baseline expression in cells not exposed to pathogens. Exposure of CaCo2 cells to PFA-fixed E. histolytica trophozoites increased expression of HBD2 mRNA close to 38-fold, compared to the baseline expression of control cells. When the expression of HBD2 mRNA was evaluated in presence of inhibitors that block the TLR-NFκB pathway, a strong negative effect was evident in the expression of the defensin mRNA.

Bottom Line: Recent in vitro studies have shown that Entamoeba histolytica 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.We show here that E. histolytica 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 Escherichia coli.This activity was abrogated by inhibition of TLR2/4-NFκB pathway or by neutralization with an anti-HBD2 antibody.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biomedicine, Centro de Investigación y de Estudios Avanzados del Instituto Politécnico Nacional, México DF, México.

ABSTRACT

Background: Entamoeba histolytica, 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 in vitro studies have shown that Entamoeba histolytica 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.

Methodology/principal findings: We show here that E. histolytica 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 Escherichia coli. 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.

Conclusions/significance: Entamoeba histolytica 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.

Show MeSH
Related in: MedlinePlus