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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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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).
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pntd-0002083-g002: 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).

Mentions: The above results demonstrated that exposure of CaCo2 cells to PFA-fixed E. histolytica trophozoites induced a significant augment of HBD2 mRNA expression. To determine whether the corresponding peptide was present in CaCo2 cells exposed to PFA-fixed trophozoites or to ETEC, Brefeldin A (BFA) was added to the cultures at a final concentration of 1.0 µg/ml at the same time as cells were exposed to pathogens to inhibit protein transport and release into CM. Cells not exposed to pathogens were the negative control. After incubation for 2 h and removal of pathogens, CaCo2 cells were washed several times with medium without BFA and treated with the permeabilizing solution as indicated in Methods. Cells were then incubated with anti-HBD2 antibody and a FITC-tagged secondary antibody and analyzed by flow cytometry. Results in Figure 2A show the mean fluorescence intensity (MFI) measured as arbitrary units in 10,000 cells in each condition. HBD2-positive cells exposed to trophozoites showed an MFI value of 15, whereas cells exposed to ETEC showed a value of 70 and control cells (not exposed to pathogens) showed a value of 4.


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)

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).
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getmorefigures.php?uid=PMC3585038&req=5

pntd-0002083-g002: 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).
Mentions: The above results demonstrated that exposure of CaCo2 cells to PFA-fixed E. histolytica trophozoites induced a significant augment of HBD2 mRNA expression. To determine whether the corresponding peptide was present in CaCo2 cells exposed to PFA-fixed trophozoites or to ETEC, Brefeldin A (BFA) was added to the cultures at a final concentration of 1.0 µg/ml at the same time as cells were exposed to pathogens to inhibit protein transport and release into CM. Cells not exposed to pathogens were the negative control. After incubation for 2 h and removal of pathogens, CaCo2 cells were washed several times with medium without BFA and treated with the permeabilizing solution as indicated in Methods. Cells were then incubated with anti-HBD2 antibody and a FITC-tagged secondary antibody and analyzed by flow cytometry. Results in Figure 2A show the mean fluorescence intensity (MFI) measured as arbitrary units in 10,000 cells in each condition. HBD2-positive cells exposed to trophozoites showed an MFI value of 15, whereas cells exposed to ETEC showed a value of 70 and control cells (not exposed to pathogens) showed a value of 4.

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