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Gliadin Intake Causes Disruption of the Intestinal Barrier and an Increase in Germ Cell Apoptosis in A Caenorhabditis Elegans Model

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Gliadin Intake Causes Disruption of the Intestinal Barrier and an Increase in Germ Cell Apoptosis in A Caenorhabditis Elegans Model

Hyemin Min et al. Nutrients.

Abstract

Gliadin is a major protein component of gluten and causes gluten toxicity through intestinal stress. We previously showed that gliadin intake induces oxidative stress in the intestine and reduces fertility in a Caenorhabditis elegans model. To elucidate the possible link between intestinal stress and reproduction, changes in the intestine and germ cells of C. elegans after gliadin intake were examined at the molecular level. Gliadin intake increased reactive oxygen species (ROS) production in the intestine, decreased intestinal F-actin levels, and increased germ cell apoptosis. These gliadin-triggered effects were suppressed by antioxidant treatment. These results suggest that ROS production in the intestine induced by gliadin intake causes disruption of intestinal integrity and increases germ cell apoptosis. Gliadin-induced germ cell apoptosis (GIGA) was suppressed by depletion of cep-1, ced-13, egl-1, or mpk-1. However, HUS-1 was not activated, suggesting that GIGA is activated through the mitogen-activated protein kinase (MAPK) pathway and is CEP-1-dependent but is a separate pathway from that controlling the DNA damage response. Taken together, our results suggest that gliadin causes intestinal barrier disruption through ROS production and interacts with the germ cells to reduce fertility through GIGA.

Keywords: Caenorhabditis elegans; germ cell apoptosis; gliadin intake; gluten toxicity; intestinal barrier; reactive oxygen species.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Gliadin intake-induced glutathione S-transferase 4 (GST-4), cytochrome P450 oxidase (CYP-35), and reactive oxygen species (ROS) production in adult-stage Caenorhabditis elegans worms. (A) Pgst-4::GFP transgenic populations synchronized at the L4 larval stage were treated with gliadin for 24 h at 20 °C. Pictures show representative images of Pgst-4::GFP expression after 24 h of gliadin treatment. The bar graph (right panel) shows quantified induction levels of the Pgst-4::GFP reporter in the intestine categorized as low (L), medium (M), and high (H) based on a previously established scale [31]. Scale bar, 100 μm. (B) cyp-35::GFP transgenic populations synchronized at the L4 larval stage were treated with gliadin for 24 h at 20 °C. Pictures show representative images of cyp-35::GFP expression after 24 h of gliadin treatment. The bar graph shows the distribution of GFP expression levels after gliadin treatments. Scale bar, 100 μm. (C) Wild-type N2 animal populations synchronized at the L4 larval stage were treated with gliadin or left untreated as controls. ROS production was measured by 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) staining after 24 h of treatment. Pictures show representative images of ROS production after treatment as visualized by DCFDA staining (left panels). (D) Bar graph showing pixel intensities from DCFDA fluorescence per worm. Error bars represent s.d. **p < 0.005 (Student’s t-test).
Figure 2
Figure 2
Effects of treatment with synthetic gliadin peptides or wheat gluten hydrolysate (WGH) on reactive oxygen species (ROS) production in adult-stage C. elegans worms. (A) Schematic of α-gliadin motifs with respective peptide sequences. Three kinds of synthetic α-gliadin peptides were generated that either possess cytotoxic activity (red) or gut-permeating activity (green or blue) as previously reported [7]. (B,C) Wild-type N2 animal populations synchronized at the L4 larval stage were treated with synthetic gliadin peptides and ROS production was measured by 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) staining 24 h after treatment. Pictures show representative images of DCFDA staining indicating ROS production after treatment (B). Bar graph (C) shows the pixel intensities from DCFDA fluorescence per worm. Error bars represent s.d.; n.s., not significant; ** p < 0.005 (Student’s t-test). (D,E) Wild-type N2 animal populations synchronized at the L4 larval stage were treated with WGH and ROS production measured by DCFDA staining after 24 h of treatment. Pictures show representative images by DCFDA staining indicating ROS production after treatment (D). Bar graph (E) shows the pixel intensities from DCFDA fluorescence per worm. Error bars represent s.d. n.s., not significant. (Student’s t-test).
Figure 2
Figure 2
Effects of treatment with synthetic gliadin peptides or wheat gluten hydrolysate (WGH) on reactive oxygen species (ROS) production in adult-stage C. elegans worms. (A) Schematic of α-gliadin motifs with respective peptide sequences. Three kinds of synthetic α-gliadin peptides were generated that either possess cytotoxic activity (red) or gut-permeating activity (green or blue) as previously reported [7]. (B,C) Wild-type N2 animal populations synchronized at the L4 larval stage were treated with synthetic gliadin peptides and ROS production was measured by 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) staining 24 h after treatment. Pictures show representative images of DCFDA staining indicating ROS production after treatment (B). Bar graph (C) shows the pixel intensities from DCFDA fluorescence per worm. Error bars represent s.d.; n.s., not significant; ** p < 0.005 (Student’s t-test). (D,E) Wild-type N2 animal populations synchronized at the L4 larval stage were treated with WGH and ROS production measured by DCFDA staining after 24 h of treatment. Pictures show representative images by DCFDA staining indicating ROS production after treatment (D). Bar graph (E) shows the pixel intensities from DCFDA fluorescence per worm. Error bars represent s.d. n.s., not significant. (Student’s t-test).
Figure 3
Figure 3
Effects of the synthetic gliadin peptide GP151–170 on reactive oxygen species (ROS) production in mammalian cell cultures. (A,B) Cell viability in RAW264.7 macrophages after treatment with either the synthetic gliadin peptide GP110–130 or GP151–170. (C) Intracellular ROS levels were evaluated by measuring the intensity of 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence using a microplate reader. A bar graph showing the pixel intensities of DCFDA fluorescence is shown. Error bars represent s.d. ** p < 0.01. *** p < 0.001 (Student’s t-test). C, control.
Figure 3
Figure 3
Effects of the synthetic gliadin peptide GP151–170 on reactive oxygen species (ROS) production in mammalian cell cultures. (A,B) Cell viability in RAW264.7 macrophages after treatment with either the synthetic gliadin peptide GP110–130 or GP151–170. (C) Intracellular ROS levels were evaluated by measuring the intensity of 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence using a microplate reader. A bar graph showing the pixel intensities of DCFDA fluorescence is shown. Error bars represent s.d. ** p < 0.01. *** p < 0.001 (Student’s t-test). C, control.
Figure 4
Figure 4
Effects of gliadin treatment on intestinal F-actin in adult-stage wild-type N2 and mev-1 mutant C. elegans worms. (A,B) Wild-type N2 and mev-1 mutants synchronized at the L4 larval stage were treated with or without gliadin. Detection of phalloidin staining of intestinal F-actin reveals a significant reduction in fluorescence after gliadin treatment in either N2 and mev-1 mutants based on quantification by image J analysis (B). Error bars represent s.d. ** p < 0.005 (Student’s t-test).
Figure 5
Figure 5
Effects of gliadin treatment and age on intestinal barrier function in wild-type N2 and mev-1 mutant C. elegans worms. (A,B) Wild-type N2 and mev-1 mutants synchronized at the L4 larval stage were treated with or without gliadin then soaked in blue food dye for 3 h on respective hours of gliadin treatment at adulthood. Differential interference contrast (DIC) images of wild-type N2 and mev-1 mutants (A) after soaking in blue food dye for 3 h after the indicated hours of gliadin treatment. Quantification of body-cavity leakage in wild-type N2 and mev-1 mutants (B) after indicated lengths of gliadin treatment during adulthood. Error bars represent s.d. ** p < 0.005 (Student’s t-test).
Figure 6
Figure 6
Effects of combining N-acetyl-L-cysteine (NAC) treatment with treatment by gliadin or synthetic gliadin peptides on intestinal F-actin in adult-stage wild-type N2 C. elegans. (A,B) Wild-type N2 worms synchronized at the L4 larval stage were treated with or without NAC and either no fed (control), fed gliadin, wheat gluten hydrolysate (WGH), or synthetic gliadin peptides (GP31–43, GP111–130, or GP151–170) for 24 h. Detection of intestinal F-actin by phalloidin staining reveals a significant increase in fluorescence (quantification by image J analysis in (B)) in worms treated with both NAC and either gliadin, GP111–130, or GP151–170). Error bars represent s.d. n.s., not significant. * p < 0.05. ** p < 0.005 (Student’s t-test).
Figure 7
Figure 7
Effect of gliadin intake on germ cell proliferation and apoptosis in adult wild-type N2 C. elegans worms. (A) The brood size of gliadin-treated mothers compared to control mothers after 4 days. ** p < 0.005. (B,C) Germ cell proliferation was not affected by gliadin treatment. Wild-type N2 worms synchronized at the L4 larval stage were treated with or without gliadin for 24 h, and dissected gonads were DNA-stained using TO-PRO-3. Bar graph (C) shows relative levels of germ cell proliferation of gliadin-treated worms compared to control worms. (D,E) Germ cell apoptosis increases with gliadin treatment as indicated by staining apoptotic germ cells with acridine orange (AO). *, Distal end of each gonad arm. Bar, 20 μm. ** p < 0.005 (Student’s t-test).
Figure 8
Figure 8
Effects of CEP-1 activity and mitogen-activated protein kinase (MAPK) signaling on gliadin-induced germ cell apoptosis (GIGA) in adult C. elegans worms. (A) Average numbers of acridine orange (AO)-positive germ cells per gonad arm in wild-type N2, ced-4, ced-3, cep-1, egl-1, ced-13, lip-1, and mpk-1 mutants (n = 30–40 per group) after gliadin treatment for 24 h of treatment with N-acetyl-L-cysteine (NAC) (+) or without NAC (–) starting at the L4 larval stage. (B) CEP-1::GFP was observed by immunostaining in CEP-1::GFP transgenic animals (n = 30 per group) fed with or without gliadin for 24 h starting in the L4 larval stage. UV-treatment condition was used as a positive control. Bar, 10 μm. (C) HUS-1::GFP transgenic animals (n = 30 per group) were synchronized at the L4 stage then treated with or without gliadin. HUS-1::GFP aggregates were observed after UV-treatment (positive control) by immunostaining using an anti-GFP antibody. Bar, 20 μm. (D) Phospho-MPK was observed by immunostaining using an anti-Phospho-p44/42 MAPK antibody in wild-type N2 worms (n = 32 per group) after gliadin treatment for 24 h starting during the L4 larval stage. Bar, 10 μm. (E) Wild-type N2, ced-4, ced-3, cep-1, egl-1, ced-13, lip-1, and mpk-1 mutants (n = 30–40 per group) after gliadin treatment for 24 h treated with NAC (+) or without NAC (–) starting at the L4 larval stage. Reactive oxygen species (ROS) production was measured by 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) staining. The bar graph shows the pixel intensities from DCFDA fluorescence per worm. Error bars represent s.d.; n.s., not significant. * p < 0.05 (Student’s t-test).
Figure 9
Figure 9
cep-1 and mpk-1 function in different tissues is required for increases in germ cell apoptosis triggered by gliadin intake. (A,B) After RNA interference (RNAi) treatment of control, cep-1, or mpk-1 worms at the L1 stage, worms were grown in nematode growth medium (NGM) plates and treated with or without gliadin for starting at the L4 stage for 24 h. Acridine orange (AO)-positive germ cells were observed in either RNAi-treated rrf-1 or ppw-1 mutant backgrounds 24 h after gliadin treatment. n.s., not significant. * p < 0.05. ** p < 0.005 (Student’s t-test).
Figure 10
Figure 10
Effect of N-acetyl-L-cysteine (NAC) treatment on germ cell apoptosis triggered by intake of gliadin or synthetic gliadin peptides in adult wild-type N2 C. elegans worms. (A) Average numbers of acridine orange (AO)-positive germ cells per gonad arm in gliadin-treated wild-type N2 worms fed with or without NAC. (B) Average numbers of AO-positive germ cells per gonad arm in wild-type N2 after treatment with synthetic gliadin peptides (GP31–43, GP111–130, or GP151–170). (C) Average numbers of AO-positive germ cells per gonad arm in synthetic gliadin peptide (GP111–130 and GP151–170)-treated wild-type N2 worms fed with or without NAC. n.s., not significant. ** p < 0.005 (Student’s t-test).
Figure 11
Figure 11
Effects of antioxidants on mev-1 mutants after intake of either gliadin or a synthetic gliadin peptide. (A) Bar graph showing the pixel intensities of 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence per worm for gliadin-treated wild-type N2 and mev-1 mutant worms fed either with or without N-acetyl-L-cysteine (NAC). (B) Average numbers of acridine orange (AO)-positive germ cells per gonad arm in gliadin-treated wild-type N2 and mev-1 mutant worms fed with or without NAC. Error bars represent s.d. * p < 0.05. ** p < 0.005 (Student’s t-test).

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