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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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Related in: MedlinePlus

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).
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pntd-0002083-g003: 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).

Mentions: Our results above showed for the first time that CaCo2 cells exposed to Entamoeba histolytica trophozoites induced the expression of HBD2 mRNA and a peptide with similar molecular features to HBD2 produced by CaCo2 cells, as previously reported in an ETEC-induction model [30]. Therefore, a functional assay was carried out to demonstrate that trophozoite-induced HBD2 has the capability to permeabilize bacteria and amebas. For this, propidium iodide penetration into pathogens was used as the indicator of permeabilization. This fluorescent dye enters to membrane-damaged cells and binds to double strand DNA, but it is excluded from cells with intact membranes. Staphylococcus aureus bacteria, known to be sensitive to HBD2 [24], [26], were utilized to test the antimicrobial activity of CM obtained from pathogen-exposed Caco2 cells. As synthetic HBD2 could not be used in this assay, because the antimicrobial activity requires of three functional S-S bonds that are present in the native molecule, we prepared an enriched fraction of the ETEC-induced HBD2 (CECE-HBD2) to be used as the positive control for cell permeabilization activity. Figure 3A shows the results obtained by flow cytometry quantification of propidium iodide inside bacteria incubated in CM in the different conditions. Conditioned media from cells exposed to trophozoites caused permeabilization of 38% of bacteria. Conditioned media from ETEC-exposed CaCo2 cells caused permeabilization of 63.8% of bacteria. CECE-HBD2 (10 ng/ml) caused permeabilization of 54% of them, while bacteria incubated with CM from control cells (not exposed to pathogens) internalized propidium iodide at low levels (18%). A similar value was determined for bacteria incubated in CM from cells exposed to pathogens in the presence of inhibitors of the classic TLR pathway. These results demonstrated that HBD2 induced by trophozoites has the capacity to permeabilize sensitive bacteria. The higher levels of permeabilization caused by CM obtained from ETEC-exposed CaCo2 cells seem related to the higher HBD2 levels found in these media.


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)

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).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3585038&req=5

pntd-0002083-g003: 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).
Mentions: Our results above showed for the first time that CaCo2 cells exposed to Entamoeba histolytica trophozoites induced the expression of HBD2 mRNA and a peptide with similar molecular features to HBD2 produced by CaCo2 cells, as previously reported in an ETEC-induction model [30]. Therefore, a functional assay was carried out to demonstrate that trophozoite-induced HBD2 has the capability to permeabilize bacteria and amebas. For this, propidium iodide penetration into pathogens was used as the indicator of permeabilization. This fluorescent dye enters to membrane-damaged cells and binds to double strand DNA, but it is excluded from cells with intact membranes. Staphylococcus aureus bacteria, known to be sensitive to HBD2 [24], [26], were utilized to test the antimicrobial activity of CM obtained from pathogen-exposed Caco2 cells. As synthetic HBD2 could not be used in this assay, because the antimicrobial activity requires of three functional S-S bonds that are present in the native molecule, we prepared an enriched fraction of the ETEC-induced HBD2 (CECE-HBD2) to be used as the positive control for cell permeabilization activity. Figure 3A shows the results obtained by flow cytometry quantification of propidium iodide inside bacteria incubated in CM in the different conditions. Conditioned media from cells exposed to trophozoites caused permeabilization of 38% of bacteria. Conditioned media from ETEC-exposed CaCo2 cells caused permeabilization of 63.8% of bacteria. CECE-HBD2 (10 ng/ml) caused permeabilization of 54% of them, while bacteria incubated with CM from control cells (not exposed to pathogens) internalized propidium iodide at low levels (18%). A similar value was determined for bacteria incubated in CM from cells exposed to pathogens in the presence of inhibitors of the classic TLR pathway. These results demonstrated that HBD2 induced by trophozoites has the capacity to permeabilize sensitive bacteria. The higher levels of permeabilization caused by CM obtained from ETEC-exposed CaCo2 cells seem related to the higher HBD2 levels found in these media.

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