The Protective Role of DDIT4 in Helicobacter pylori-induced Gastric Metaplasia Through Metabolic Regulation of Ferroptosis

Abstract

Background & aims: Helicobacter pylori (H pylori) infection is a significant factor leading to gastric atrophy, metaplasia and cancer development. Here, we investigated the role of the stress response gene DDIT4 in the pathogenesis of H pylori infection.

Methods: Cell lines, transgenic mice, and human tissue samples were implemented. Proteomics were performed on Ddit4^+/+ and Ddit4^-/- mice infected with H pylori strain PMSS1. C57BL/6 mice were administered with tamoxifen to induce gastric metaplasia. Stomach tissues were analyzed for histopathologic features, reactive oxygen species, Fe²⁺, lipid peroxidation, expression of DDIT4, and ferroptosis-related proteins.

Results: DDIT4 expression was upregulated at 6 hours but significantly decreased at 24 hours in response to H pylori infection in gastric epithelial cells. Gastric DDIT4 were downregulated in INS-GAS mice at 4 months post H pylori infection. Notably, H pylori infection led to more severe gastric metaplasia lesion in Ddit4-knockout mice. The proteomic profiling revealed an increase in ferroptosis in the gastric tissues of infected Ddit4-deficient mice, compared with infected wild-type mice. Mechanistically, knockout of DDIT4 promoted H pylori-induced ferroptosis through the accumulation of lipid peroxides and ROS levels, and alterations in proteins such as GPX4, ALOX15, and HMOX1. Overexpression of DDIT4 counteracted H pylori-induced stem cell marker CD44V9 through modulation of ferroptosis. Similarly, in another mouse model of gastric metaplasia treated with tamoxifen, as well as in human GIM tissues, we observed the loss of DDIT4 and induction of ferroptosis.

Conclusions: Our results indicate that DDIT4 serves as a protective factor against H pylori-induced gastric metaplasia by metabolic resistance to ferroptosis.

Keywords: DDIT4; Ferroptosis; Gastric Metaplasia; H pylori; Metabolic Reprogramming.

Graphical abstract

Figure 1

H pylori infection dynamic regulates DDIT4 expression in gastric epithelial cells. (A) The workflow of RNA-seq. The gastric epithelial AGS and GES-1 cells were infected with H pylori 43504 strain for 6 hours. Then total RNA was extracted from cells, and RNA-seq was performed for transcriptomic profile. (B) The Venn plot showing common upregulated DEGs in cells following H pylori infection. (C) Heatmap plot showing the significantly upregulated genes after H pylori infection based on fold change and P value. (D) The qRT-PCR analysis indicating the mRNA levels of DDIT4 at different time points, following H pylori infection in AGS cells. (E) Western blot analysis showing the protein levels of DDIT4 at different timepoints after infection with H pylori PMSS1 (upper panels) and 7.13 strains (lower panels) in AGS cells (Scale bar, 10 μm). (F) Immunofluorescence data of DDIT4 staining in AGS cells infected with PMSS1 strains at different MOI for different time points. (G) The fluorescence intensity analysis of DDIT4 for panel F. (H) IHC staining for DDIT4 expression in gastric tissues of INS-GAS mice infected with PMSS1 H pylori for 4 months, compared with non-infected mice (Scale bar, 10 μm). ∗P < .05; ∗∗P < .01; and ∗∗∗P < .001 were considered significant. Data were expressed as the means ± SD.

Figure 2

Verification of DDIT4 expression and H pylori colonization in the gastric tissues of Ddit4^+/+and Ddit4^-/-mice. (A) The qRT-PCR analysis indicating the mRNA levels of DDIT4 in the stomach tissues of Ddit4^+/+ and Ddit4^-/- mice. (B) The ratio of p-mTOR to total mTOR protein levels in infected WT and Ddit4-knockout mice. (C) Silver staining showing H pylori colonization in the gastric tissues of mice (Scale bar, 10 μm). (D) Colony-forming units (CFUs) per gram stomach in animals were evaluated for H pylori colonization by culture at 4 days after sacrifice. (E) IHC scores of ATP4A and ATP4B in infected gastric tissues of Ddit4^+/+ and Ddit4^-/- mice. (F) Immunofluorescence staining for MUC2 (left panel) or FABP1 (right panel) on infected gastric tissues from Ddit4^+/+ and Ddit4^-/- mice (Scale bar, 20 μm). ns, no significance. ∗P < .05; ∗∗P < .01; and ∗∗∗P < .001 were considered significant.

Figure 3

Ddit4 deletion results in gastric metaplasia in animal model of H pylori pathogenesis. (A) Western blot analysis verifying the loss of DDIT4 and activation of p-mTOR in gastric tissues of Ddit4^-/- mice. (B) Histopathology of gastric tissues of Ddit4^+/+(n = 8) and Ddit4^-/- (n = 8) mice with or without H pylori infection (Scale bar, 10 μm). (C) Gastric pathology scores from the mice in panel B. (D) PAS staining showing prominent mucous cell metaplasia in gastric tissues (Scale bar, 10 μm). (E) Immunofluorescence staining for the expression of Ki67 in gastric tissues of WT (Ddit4^+/+) and Ddit4^-/- mice with or without H pylori infection (Scale bar, 10 μm). (F) IHC staining for ATP4A and ATP4B expression in the gastric tissues of H pylori-infected Ddit4^+/+ and Ddit4^-/- mice (Scale bar, 10 μm). (G) The Western blot analysis showing the protein levels of ATP4A and ATP4B in gastric tissues of infected Ddit4^+/+ and Ddit4^-/- mice. (H) Immunofluorescence staining for GIF and GSII on the gastric tissues from H pylori-infected Ddit4^+/+ and Ddit4^-/- mice (Scale bar, 50 μm). (I) Immunofluorescence staining for CD44V9 on gastric tissues of Ddit4^+/+ and Ddit4^-/- mice infected with H pylori. (Scale bar, 20 μm). ∗P < .05; ∗∗P < .01; and ∗∗∗P < .001 were considered significant.

Figure 4

Proteome profiling analysis of the gastric tissues from infected Ddit4^+/+and Ddit4^-/-mice. (A) PCA analysis showing the variation across different gastric tissue samples. (B, C) GO (B) and KEGG (C) analysis showing the biologic processes and signaling pathways of downregulated proteins enrichment in the gastric tissues of Ddit4^-/- mice compared with Ddit4^+/+ mice, respectively. (D) Western blot analysis verifying the knockdown of DDIT4 in AGS cells treated with DDIT4 siRNA. (E) Quantification of Western blot analysis from Figure 4E using ImageJ software.

Figure 5

Proteomic profiling revealed that Ddit4 deletion results in aberrant GSH metabolism and accumulation of lipid peroxides. (A) GSEA analysis of SDE genes revealing the negative correlated signaling pathways in infected Ddit4^-/- mice (HP_DDIT4; n = 4) compared with infected Ddit4^+/+ mice (HP; n = 4). (B) GSEA analysis showing the downregulation of GSH metabolism and peroxisome in infected Ddit4^-/- mice. (C) Heatmap plot showing the relative expression of genes from GSH metabolism pathway in each mice gastric specimens. (D, E) The histogram showing the GSH/GSSG ratio and ATP levels in the gastric tissues of H pylori-infected Ddit4^+/+ (n = 8) and Ddit4^-/- mice (n = 8), respectively. (F) The DCFH-DA fluorescence staining revealing the ROS levels in gastric tissues of infected Ddit4^+/+ and Ddit4^-/- mice. The representative fluorescence images (Scale bar, 10 μm) (left panel) and the statistical analysis of ROS levels (right panel) were shown. (G) C11-BODIPY staining measured with a fluorescence microscope showing lipid peroxidation in mice tissues. The representative images (Scale bar, 10 μm) (upper panel) and the statistical analysis (lower panel) were shown, respectively. (H, I) The GSH/GSSG ratio (H) and ATP levels (I) in AGS cells treated with DDIT4 siRNA or scrambled siRNA. ∗P < .05;∗∗P < .01; and ∗∗∗P < .001 were considered significant. Data were expressed as the means ± SDs.

Figure 6

Deletion of DDIT4 triggers Ferroptosis in H pylori-infected gastric tissues. (A) GSEA analysis showing the induction of ferroptosis in infected Ddit4^-/- mice. (B) The qRT-PCR analysis showing the mRNA levels of GPX4 in gastric tissues from infected Ddit4^+/+ (n = 8) and Ddit4^-/- mice (n = 8). (C) Pearson correlation analysis showing the relationship between GPX4 and DDIT4 mRNA expression. (D) Heatmap plot showing the relative expression of genes from ferroptosis pathway in each mice gastric specimens. (E) Western blot analysis showing the expression levels of ferroptosis-related proteins (ALOX15, HMOX1, and GPX4) in gastric tissues from infected Ddit4^+/+ and Ddit4^-/- mice. (F) IHC staining (Scale bar, 10 μm) for GPX4 expression in mouse stomach tissues. (G) Representative images of immunofluorescence staining showing the colocalization of ATP4A and GXP4 in mouse gastric tissues. (H–I) The Fe²⁺ levels (H) and MMP (I) in stomach tissues from infected Ddit4^+/+ and Ddit4^-/- mice determined by FerroOrange and JC-1 staining, respectively (Left panel, representative fluorescence images; right panel, the statistical data). (I) ∗P < .05; ∗∗P < .01; and ∗∗∗P < .001 were considered significant.

Figure 7

H pylori infection induces ferroptosis in gastric epithelial cells. (A) Western blot analysis showing the expression of ferroptosis-related proteins (ALOX15, HMOX1, and GPX4) in AGS cells infected with H pylori PMSS1 or 7.13 strain at different MOI. (B) DCFH-DA staining determining the ROS levels in GES-1 and AGS cells following H pylori 7.13 or PMSS1 infection. The representative fluorescence images (scale bars, 10 μm) taken using a high-content analysis system. (C–D) FerroOrange staining showing the Fe²⁺ levels in GES-1 and AGS cells infected with H pylori infection. The representative images and statistical data shown in panel C and D, respectively. (E–F) Mitochondrial structure (E) and membrane potential (F) detected by transmission electron microscopy and JC-1 staining, respectively. (G) IHC data of GPX4 staining in uninfected and H pylori PMSS1 infected gastric tissues from INS-GAS mice (Scale bar, 10 μm). (H) The Fe²⁺ levels in stomach tissues from uninfected and H pylori-infected INS-GAS mice determined by DCFH-DA staining and FerroOrange staining, respectively. ∗P < .05; ∗∗P < .01; and ∗∗∗P < .001 were considered significant. Data were expressed as the means ± SD.

Figure 8

H pylori infection induces ferroptosis in gastric epithelial cells. (A) The statistical analysis of the ROS levels in GES-1 and AGS cells following H pylori 7.13 or PMSS1 infection. (B) TEM indicating the mitochondrial structure in GES-1 cells following H pylori 7.13 and PMSS1 infection. (C–D) JC-1 stain showing MMP levels in GES-1 cells infected with H pylori infection. The representative images (C) and statistical analysis (D) of MMP in GES-1 cells; The statistical analysis of MMP in AGS cells (E) (Scale bar, 10 μm). (F) JC-1 staining showing MMP in the gastric tissues of INS-GAS mice infected with PMSS1 strain (Scale bar, 20 μm). Data were expressed as the means ± SD. ∗P < .05; ∗∗P < .01; and ∗∗∗P < .001 were considered significant.

Figure 9

DDIT4 protects gastric epithelial cells against H pylori-induced ferroptosis. (A, B) The DCFH-DA (A) and C11-BODIPY staining (B) showing the ROS levels and lipid peroxides, respectively, in AGS cells transfected with DDIT4 siRNA (siDDIT4) or control siRNA followed by H pylori infection for 24h. (C, D) The Fe²⁺ levels (C) and MMP (D) in AGS cells following H pylori infection and transfection with siDDIT4 or control siRNA determined by FerroOrange and JC-1 staining, respectively (Scale bar, 10 μm). (E) TEM determining the structure of mitochondrial in AGS cells treated with siDDIT4 and H pylori strain. (F) Immunofluorescence staining for CD44V9 in human gastric organoid following PMSS1 infection and treatment with Flag-DDIT4 in combination with RSL3. Data were expressed as the means ± SD. ∗P < .05; ∗∗P < .01; and ∗∗∗P < .001 were considered significant.

Figure 10

DDIT4 protects gastric epithelial cells against H pylori-induced ferroptosis. (A, B) The DCFH-DA (A) and FerroOrange staining (B) showing the ROS and Fe²⁺ levels in GES-1 cells following H pylori infection and transfection with DDIT4 siRNA (siDDIT4) or control siRNA. (C) The statistical analysis of MMP data of panel D from Figure 6 (Scale bar, 10 μm). (D) The representative images and statistical analysis of MMP in GES-1 cells treated with siDDIT4 and H pylori strain (Scale bar, 10 μm). Data were expressed as the means ± SD. ∗P < .05; ∗∗P < .01; and ∗∗∗P < .001 were considered significant.

Figure 11

DDIT4 protects gastric epithelial cells against H pylori-induced ferroptosis. (A, B) Western blot analysis of DDIT4, ALOX15, HMOX1, and GPX4 expression transfection with siDDIT4 or control siRNA in GES-1 (A) and AGS cells (B), respectively. (C, D) Western blot analysis of DDIT4, ALOX15, HMOX1, and GPX4 expression following PMSS1 infection and treatment with Flag-DDIT4 in GES-1 (C) and AGS cells (D), respectively. Data were expressed as the means ± SD.

Figure 12

Tamoxifen induces metaplasia in mouse stomach, eliciting the loss of DDIT4 and induction of ferroptosis. (A) Histopathology of gastric tissues from tamoxifen (TAM)-treated and untreated control mice. (Scale bar, 10 μm). (B) PAS staining showing prominent mucous cell metaplasia in gastric tissues of TAM-treated or untreated mice (Scale bar, 10 μm). (C) IHC staining for ATP4A and ATP4B expression in gastric mouse tissues treated or untreated with TAM (Scale bar, 10 μm). (D) IHC analysis showing the expression of DDIT4 in stomach tissues with TAM treatment. (E–F) The MMP (E) and Fe²⁺ levels (F) in mouse stomach tissues after TAM treatment determined by JC-1 and FerroOrange staining, respectively (Scale bar, 10 μm). (G) Immunofluorescence staining for GPX4⁺ATP4A⁺ cells in mouse stomach tissues with TAM treatment. (H) Western blot analysis showing the expression levels of DDIT4, ALOX15, HMOX1, and GPX4 in mouse gastric tissues with or without TAM treatment. N = 6. ∗P < .05; ∗∗P < .01; and ∗∗∗P < .001 were considered significant.

Figure 13

DDIT4 expression and ferroptosis sensitivity in mouse gastric metaplasia tissues induced by tamoxifen. (A–D) Gastric pathology scores of tamoxifen (TAM)-treated mice included mucous metaplasia (A), GIM (B), inflammation (C), and atrophy (D). (E) Immunofluorescence staining for MUC2 and CD44V9 in stomach tissues of TAM-treated mice. (F) The ATP levels in mouse gastric tissues from TAM-treated mice. (G) The Western blot data from Figure 7H was quantified by densitometric analysis using ImageJ software. (H–I) IHC analysis showing the expression of ALOX15 (H) and HMOX1 (I) in mouse gastric tissues with or without TAM treatment.

Figure 14

Clinical relevance of DDIT4 and ferroptosis in human gastric intestinal metaplasia tissues. (A) Western blot data for the expression levels of DDIT4, ALOX15, HMOX1 and GPX4 in human GIM and non-atrophic gastritis (GS) tissues. (B–C) IHC staining showing DDIT4 and GPX4 expression in human GS and GIM tissues. Representative images (Scale bar, 10 μm) shown in (B). Quantification of staining shown in (C). (D–E) C11-BODIPY and DCFH-DA staining measured with a fluorescence microscope showing lipid peroxidation (D) and ROS levels (E) in human GS and GIM tissues. (F) Diagram of the mechanism by which loss of DDIT4 promoted H pylori-induced gastric metaplasia lesions through metabolic regulation of ferroptosis. Chronic H pylori infection causes the loss of DDIT4 and mTOR signaling pathway activation, which in turn inhibits GSH synthesis and increases ROS levels and lipid peroxides, inducing ferroptosis in the gastric microenvironment. As a result, gastric metaplasia lesions develop following parietal cell loss. ∗P < .05; ∗∗P < .01; and ∗∗∗P < .001 were considered significant.

Figure 15

DDIT4 expression and ferroptosis sensitivity in human GS and IM tissues. (A) The Western blot data from Figure 8A was quantified by densitometric analysis using ImageJ software. (B–C) Quantification of IHC staining of DDIT4 (B) and GPX4 (C) in H pylori-positive or -negative GS and IM tissues. (D) FerroOrange staining measured with a fluorescence microscope showing the Fe²⁺ levels in human GS and IM tissues (Scale bar, 10 μm). ns, no significance. ∗P < .05; ∗∗P < .01; and ∗∗∗P < .001 were considered significant.