Helicobacter pylori-Induced Angiopoietin-Like 4 Promotes Gastric Bacterial Colonization and Gastritis

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  Fig. 1.. ANGPTL4 is increased in gastric mucosa of H. pylori-infected patients and mice. (A) Analyses of the whole-transcriptome sequencing datasets and the 3 cohorts (GSE60427_Bhutan, GSE60427_DR, and GSE60662) from 2 GEO datasets. Hierarchical clustering heatmaps of significant DEGs are shown in DEG analysis. Overlapping 3 cohorts screened and identified 226 significantly up-regulated DEGs. PPI network analysis of these 226 DEGs was performed, and the DEGs with the significance levels (P < 0.01 and correlation > 0.8) are shown in PPI network, and the top 10 DEGs as candidate genes are shown in the side table. (B) ANGPTL4 protein amounts in gastric mucosa of H. pylori-infected patients (n = 131) and uninfected donors (n = 50) were compared. (C) The correlation between ANGPTL4 protein amount and H. pylori colonization in gastric mucosa of H. pylori-infected patients was analyzed. (D) ANGPTL4 protein amounts in gastric mucosa of H. pylori-infected patients with mild (n = 34), moderate (n = 45), and severe inflammation (n = 24) and with normal gastric histopathology (n = 28) were compared. (E) ANGPTL4 protein amounts in gastric mucosa of cagA+ H. pylori-infected (n = 74), cagA− H.…

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  Fig. 2.. H. pylori stimulates GECs to produce ANGPTL4. (A) Representative immunofluorescence staining images showing ANGPTL4-expressing (red) pepsinogen II/PGC+ GECs (green) in gastric mucosa of H. pylori-infected patients or H. pylori-infected mice. Scale bars, 100 μm. (B) ANGPTL family mRNA expressions in WT H. pylori-infected and uninfected AGS cells (MOI = 100, 6 h) were compared by microarray (n = 3). (C to E) ANGPTL4/Angptl4 gene and ANGPTL4 protein expressions in WT H. pylori-infected, ΔcagA-infected, and uninfected AGS cells (C), human primary GECs (D), or mouse primary GECs (E) (MOI = 100, 24 h), and ANGPTL4/Angptl4 gene and ANGPTL4 protein expressions in WT H. pylori-infected and uninfected AGS cells (C), human primary GECs (D), or mouse primary GECs (E) at different time points (MOI = 100) or infected with different MOIs (24 h) were analyzed by real-time PCR, ELISA, and Western blot (n = 5). (F and G) Angptl4/ANGPTL4 gene and ANGPTL4 protein expressions in WT H. pylori-infected, ΔcagA-infected, and uninfected mouse (F) and human (G) gastric organoids (MOI = 100, 24 h) were analyzed by real-time PCR, ELISA, Western blot, and immunofluorescence (n = 5). Scale bars, 50 μm. (H and…

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  Fig. 3.. H. pylori and IL-17A synergistically induce ANGPTL4. (A to D) ANGPTL4/Angptl4 gene and ANGPTL4 protein expressions in AGS cells (A), human primary GECs (C), or mouse primary GECs (D) infected with WT H. pylori (MOI = 100) in the presence or absence of IL-17A (100 ng/ml) (24 h), or in AGS cells infected with WT H. pylori (MOI = 100) in the presence of IL-17A (50, 100, and 200 ng/ml) (24 h) (B) were analyzed by real-time PCR, ELISA, and Western blot (n = 5). (E) IL-17A protein expression in gastric mucosa of H. pylori-infected (n = 131) and uninfected donors (n = 50) or in gastric mucosa of cagA+ H. pylori-infected (n = 74), cagA− H. pylori-infected (n = 57), and uninfected donors (n = 50) was compared. (F) IL-17A protein expression in gastric mucosa of H. pylori-infected patients with mild (n = 34), moderate (n = 45), and severe inflammation (n = 24) and with normal gastric histopathology (n = 28) was compared. (G) The correlation between ANGPTL4 protein and IL-17A protein in gastric mucosa of H. pylori-infected patients was analyzed. (H) Angptl4 gene and ANGPTL4 protein expressions in gastric mucosa of WT H. pylori-infected WT and Il17a−/− mice at 12 weeks p.i. were compared (n =…

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  Fig. 4.. ANGPTL4 increases bacterial burden, inflammation, and Treg accumulation but decreases neutrophil accumulation and CLDN1 expression in gastric mucosa during H. pylori infection. (A and B) The bacteria colonization (A) and histological scores of inflammation (B) in gastric mucosa of WT H. pylori-infected WT, Il17a−/−, Angptl4−/−, and Il17a−/−Angptl4−/− mice or in gastric mucosa of WT H. pylori-infected BM chimera mice at 12 weeks p.i. were compared (n = 5). (C and E) The levels of neutrophils (C) and Tregs (E) in gastric mucosa of uninfected WT mice (red) and WT H. pylori-infected WT, Il17a−/−, Angptl4−/−, and Il17a−/−Angptl4−/− mice or in gastric mucosa of WT H. pylori-infected BM chimera mice at 12 weeks p.i. were compared (n = 5). Results are expressed as the percentage of neutrophils in CD45+ cells and the percentage of Tregs among CD4+ T cells. (D and F) Representative dot plots of neutrophils (D) or Tregs (F) (defined as described in Fig. S4A) in gastric mucosa of uninfected WT mice (red) and WT H. pylori-infected WT, Il17a−/−, Angptl4−/−, and Il17a−/−Angptl4−/− mice at 12 weeks p.i. (G) CLDN1 proteins in gastric mucosa of uninfected WT mice (red) and WT H. pylori-infected…

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  Fig. 5.. ANGPTL4 suppresses CLDN1 and CXCL1 in GECs by binding to ITGAV to inhibit ERK, leading to decreased neutrophil accumulation and increased bacterial burden during H. pylori infection. (A) The members of the 4 GO terms in “Cellular Component,” “Biological Process,” “Molecular Function,” and “KEGG” (described in Fig. S13B) have overlaps by MS analysis of AGS cells expressing the Flag-tagged ANGPTL4 (ANGPTL4-Flag) immunoprecipitated (IP) with anti-Flag Abs. ITGAV is the core member of each GO terms. (B) AGS cells expressing ANGPTL4-Flag were lysed and IP with IgG or anti-Flag Abs. The IP samples were subjected to Western blot analysis with the indicated Abs to detect the interaction with ITGAV. (C) The optimally predicted protein–protein complex obtained from HADDOCK’s easy interface: left, an overarching schematic of ANGPTL4 (represented as a blue cartoon model) binding with ITGAV (indicated as a red surface model); middle, the key active residues involved in the binding of ANGPTL4 to ITGAV, denoted as yellow sticks within the blue structural domain of ANGPTL4; right, the key active residues crucial for ITGAV binding with ANGPTL4, denoted as yellow sticks within the red structural…

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  Fig. 6.. ANGPTL4 promotes CCL5 production in monocytes by binding to ITGAV to activate NF-κB, leading to increased Treg accumulation during H. pylori infection. (A) The expressions of chemokine family members in gastric mucosa of WT H. pylori-infected WT and Angptl4−/− mice at 12 weeks p.i. were analyzed by real-time PCR (n = 5). (B) CCL5 proteins in gastric mucosa of uninfected WT mice (red) and WT H. pylori-infected WT, Il17a−/−, Angptl4−/− and Il17a−/−Angptl4−/− mice, in gastric mucosa of WT H. pylori-infected BM chimera mice, or in gastric mucosa of WT mice injected with ANGPTL4, cANGPTL4, or PBS control at 12 weeks p.i. were compared (n = 5). (C) The correlations between ANGPTL4 expression and CCL5 expression, and between CCL5 expression and Foxp3 expression in gastric mucosa of H. pylori-infected patients were analyzed. Results are expressed as log10(fold change). (D) Representative immunofluorescence staining images showing CCL5-expressing (green) CD14+ monocytes (red) in gastric mucosa from H. pylori-infected patients. (E) Human (left panel) or mouse (right panel) monocytes were pretreated with wortmannin or BAY 11-7082 and then stimulated with ANGPTL4 or cANGPTL4 (1 μg/ml) for 24…

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  Fig. 7.. ANGPTL4 promotes Treg proliferation by binding to ITGAV to activate NF-κB, aggravating inflammation during H. pylori infection. (A) The correlations between ANGPTL4 expression and CD25 expression, and between ANGPTL4 expression and Foxp3 expression in gastric mucosa of H. pylori-infected patients were analyzed. Results are expressed as log10(fold change). (B) Tregs were pretreated with anti-ITGAV Abs and then stimulated with ANGPTL4 or cANGPTL4 (1 μg/ml) for 6 h. AKT and p-AKT and p65 and p-p65 proteins were analyzed by Western blot. (C) Tregs were pretreated with anti-ITGAV Abs and then stimulated with ANGPTL4 (1 μg/ml) for 6 h. AKT and p-AKT and p65 and p-p65 proteins were analyzed by Western blot. (D to F) CFSE-labeled Tregs from H. pylori-infected patients were pretreated with anti-ITGAV Abs and cultured with ANGPTL4 (1 μg/ml) for 5 d (D), or were cultured with ANGPTL4 or cANGPTL4 (1 μg/ml) for 5 d (E), or were pretreated with wortmannin or BAY 11-7082 and cultured with ANGPTL4 (1 μg/ml) for 5 d (F). Representative data and statistical analysis of Treg proliferation were shown (n = 5). (G to I) CFSE-labeled Tregs from WT H. pylori-infected mice (12 weeks p.i.) were pretreated…