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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

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.
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pntd-0002083-g004: 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.

Mentions: HBD2 has been reported to damage other protozoan parasites initiating cell destruction by making perforations in their membrane [23]. Considering this antecedent and the above results, we analyzed the ultrastructural integrity of trophozoites incubated in CM from pathogen-exposed CaCo2 cells, from control trophozoites and trophozoites incubated with CECE-HBD2. Figure 4A (panel a) depicts a representative trophozoite incubated in CM from control CaCo2 cells. It shows a continuous plasma membrane, the typical small accumulations of cytoplasmic glycogen (g) and well-formed vacuoles with clear content and continuous membranes (v). In contrast, trophozoites incubated in CM from CaCo2 cells exposed to Entamoeba histolytica (panel b) or to ETEC (panel c), as well as trophozoites incubated with 10 ng/ml of CECE-HBD2 (panel d) showed membranes, both plasmatic and vacuolar with multiple discontinuous zones and irregular width that could correspond to pores (arrows); together with several ruptured points and release of cellular material can be seen (arrowheads). Also evident, is the increased accumulation of glycogen in the cytoplasm (g). Figure 4B shows low magnification micrographs of trophozoites treated as indicated in figure 4A. In panels b, c and d, it is possible to appreciate a notorious increase in the number of vacuoles (v) and glycogen accumulation (g), plus chromatin aggregation and its polarization inside the nucleus (N), and other signs of cell lysis, such as the extruded cytoplasm in one side of the trophozoite (Figure 4Bb, arrowhead). These alterations were not observed in trophozoites incubated in CM from CaCo2 cells not exposed to pathogens (panel a) nor in those incubated in CM from cells exposed to TLR classical pathway inhibitors (not shown). These results corroborated that trophozoite-induced HBD2 in CaCo2 cells has the capacity to permeabilize bacteria and trophozoite membranes. Membrane damage in trophozoites leads to loss of cell integrity and their destruction.


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)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
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pntd-0002083-g004: 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.
Mentions: HBD2 has been reported to damage other protozoan parasites initiating cell destruction by making perforations in their membrane [23]. Considering this antecedent and the above results, we analyzed the ultrastructural integrity of trophozoites incubated in CM from pathogen-exposed CaCo2 cells, from control trophozoites and trophozoites incubated with CECE-HBD2. Figure 4A (panel a) depicts a representative trophozoite incubated in CM from control CaCo2 cells. It shows a continuous plasma membrane, the typical small accumulations of cytoplasmic glycogen (g) and well-formed vacuoles with clear content and continuous membranes (v). In contrast, trophozoites incubated in CM from CaCo2 cells exposed to Entamoeba histolytica (panel b) or to ETEC (panel c), as well as trophozoites incubated with 10 ng/ml of CECE-HBD2 (panel d) showed membranes, both plasmatic and vacuolar with multiple discontinuous zones and irregular width that could correspond to pores (arrows); together with several ruptured points and release of cellular material can be seen (arrowheads). Also evident, is the increased accumulation of glycogen in the cytoplasm (g). Figure 4B shows low magnification micrographs of trophozoites treated as indicated in figure 4A. In panels b, c and d, it is possible to appreciate a notorious increase in the number of vacuoles (v) and glycogen accumulation (g), plus chromatin aggregation and its polarization inside the nucleus (N), and other signs of cell lysis, such as the extruded cytoplasm in one side of the trophozoite (Figure 4Bb, arrowhead). These alterations were not observed in trophozoites incubated in CM from CaCo2 cells not exposed to pathogens (panel a) nor in those incubated in CM from cells exposed to TLR classical pathway inhibitors (not shown). These results corroborated that trophozoite-induced HBD2 in CaCo2 cells has the capacity to permeabilize bacteria and trophozoite membranes. Membrane damage in trophozoites leads to loss of cell integrity and their destruction.

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