Research ArticleMICROBIOLOGY

Helicobacter pylori–induced matrix metallopeptidase-10 promotes gastric bacterial colonization and gastritis

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Science Advances  03 Apr 2019:
Vol. 5, no. 4, eaau6547
DOI: 10.1126/sciadv.aau6547
  • Fig. 1 MMP-10 is increased in gastric mucosa of H. pylori–infected patients and mice.

    (A) Representative MMP array results for human primary gastric mucosa of H. pylori–infected patients and paired uninfected donors using Ab microarrays. Profiles of mean spot pixel density were created using a transmission-mode scanner and image analysis software (ImageJ, National Institutes of Health). (B) MMP-10 mRNA expression in gastric mucosa of H. pylori–infected (n = 65) and uninfected donors (n = 40) was compared. (C) The correlation between MMP-10 expression and H. pylori colonization in gastric mucosa of H. pylori–infected patients was analyzed. (D) MMP-10 mRNA expression in gastric mucosa of cagA+ H. pylori–infected (n = 34), cagA H. pylori–infected (n = 31),and uninfected donors (n = 40) was compared. (E) Dynamic changes of MMP-10 mRNA expression in gastric mucosa of WT H. pylori–infected, ΔcagA-infected, and uninfected mice. n = 5 per group per time point in (E). (F and G) MMP-10 protein in gastric mucosa of cagA+ H. pylori–infected, cagA H. pylori–infected, and uninfected donors or in gastric mucosa of WT H. pylori–infected, ΔcagA-infected, and uninfected mice at 9 weeks p.i. was analyzed by Western blot (F) and immunohistochemical staining (G). Scale bars, 100 μm. (H) MMP-10 mRNA expression and MMP-10 protein in/from human primary gastric mucosa from uninfected donors infected with WT H. pylori or ΔcagA ex vivo analyzed by real-time polymerase chain reaction (PCR), Western blot, or enzyme-linked immunosorbent assay (ELISA) (n = 8). The horizontal bars in (B), (D), and (H) represent mean values. Each ring or dot in (B) to (D) and (H) represents one patient or donor. *P < 0.05, **P < 0.01 for groups connected by horizontal lines or compared with uninfected mice. GAPDH, glyceraldehyde phosphate dehydrogenase.

  • Fig. 2 H. pylori stimulates gastric epithelial cells express MMP-10.

    (A) Representative immunofluorescence staining images showing MMP-10–expressing (red) H+/K+ ATPase+ parietal cells (green) in gastric mucosa of H. pylori–infected patients or H. pylori–infected mice. Scale bars, 100 μm. (B) Representative immunofluorescence staining images showing MMP-10–expressing (red) pepsinogen II+ chief cells (green) in gastric mucosa of H. pylori–infected patients or H. pylori–infected mice. Scale bars, 100 μm. DAPI, 4′,6-diamidino-2-phenylindole. (C) MMP family mRNA expression in WT H. pylori–infected and uninfected AGS cells [multiplicity of infection (MOI) = 100 for 6 hours] were compared by microarray (n = 3). (D and E) MMP-10 mRNA expression and MMP-10 protein in/from WT H. pylori–infected and uninfected AGS cells at different time points (MOI = 100) (D) or with different MOI (24 hours) (E) were analyzed by real-time PCR, Western blot, or ELISA (n = 3). n.s., not significant. (F) MMP-10 mRNA expression and MMP-10 protein in/from WT H. pylori–infected, ΔcagA-infected, and uninfected human primary gastric epithelial cells (MOI = 100 for 24 hours) were analyzed by real-time PCR, Western blot, or ELISA (n = 5). The horizontal bars in (F) represent mean values. *P < 0.05, **P < 0.01 for groups connected by horizontal lines.

  • Fig. 3 H. pylori and IL-22 synergistically induce MMP-10.

    (A and B) MMP-10 mRNA and protein expression in/from AGS cells stimulated with WT H. pylori (MOI = 100) and/or IL-22 (100 ng/ml) (24 hours) or stimulated with WT H. pylori (MOI = 100) and IL-22 (100, 200, and 400 ng/ml) (24 hours) were analyzed by real-time PCR, ELISA (A), or Western blot (B) (n = 3). (B and C) MMP-10 mRNA and protein expression in/from human primary gastric epithelial cells stimulated with WT H. pylori (MOI = 100) and/or IL-22 (100 ng/ml) (24 hours) were analyzed by real-time PCR and ELISA (C) or Western blot (B) (n = 3). (D) MMP-10 mRNA expression in gastric mucosa of uninfected or WT H. pylori–infected WT and IL-22−/− mice at 9 weeks p.i. was compared (n = 5). (E) The correlation between MMP-10 expression and IL-22 expression in gastric mucosa of H. pylori–infected patients was analyzed. (F) AGS cells were pretreated with signal pathway inhibitors and then stimulated with WT H. pylori (MOI = 100) and IL-22 (100 ng/ml) for 24 hours. MMP-10 mRNA expression in AGS cells was compared (n = 3). DMSO, dimethyl sulfoxide; GSK, glycogen synthase kinase. (G) AGS cells were stimulated with WT H. pylori (MOI = 100) and/or IL-22 (100 ng/ml) for 24 hours or pretreated with U0126 (an ERK inhibitor) and then stimulated with WT H. pylori (MOI = 100) and IL-22 (100 ng/ml) for 24 hours. ERK1/2 and p-ERK1/2 proteins were analyzed by Western blot. (H) AGS cells were pretreated with anti–IL-22 and/or IL-22R1 neutralizing Abs and then stimulated with WT H. pylori (MOI = 100) and/or IL-22 (100 ng/ml) for 24 hours. MMP-10, ERK1/2, and p-ERK1/2 proteins in/from AGS cells were analyzed by ELISA or Western blot (n = 3). Each dot in (E) represents one patient. *P < 0.05; **P < 0.01; n.s., P > 0.05 for groups connected by horizontal lines.

  • Fig. 4 MMP-10 increases bacterial burden, inflammation, and CD8+T cell accumulation in gastric mucosa during H. pylori infection.

    (A and B) The bacteria colonization in gastric mucosa of WT H. pylori–infected IL-22−/−, MMP-10−/−, IL-22−/−MMP-10−/−, and WT mice (A) or in gastric mucosa of WT H. pylori–infected BM chimera mice (B) at 9 weeks p.i. was compared (n = 6). (C and D) Histological scores of inflammation in gastric antra of WT H. pylori–infected IL-22−/−, MMP-10−/−, IL-22−/−MMP-10−/−, and WT mice (C) or in gastric antra of WT H. pylori–infected BM chimera mice (D) at 9 weeks p.i. was compared (n = 6). (E and F) CD3+CD8+ cell level in gastric mucosa of uninfected WT mice and WT H. pylori–infected IL-22−/−, MMP-10−/−, IL-22−/−MMP-10−/−, and WT mice (E) or in gastric mucosa of WT H. pylori–infected BM chimera mice (F) at 9 weeks p.i. was compared (n = 5 to 6). The horizontal bars in (A) to (F) represent mean values. Each dot in (A) to (F) represents one mouse. *P < 0.05; **P < 0.01; n.s., P > 0.05 for groups connected by horizontal lines.

  • Fig. 5 MMP-10 promotes CD8+T cell accumulation in gastric mucosa in vivo and migration in vitro during H. pylori infection via CXCL16.

    (A) The correlation between MMP-10 expression and CXCL16 expression in gastric mucosa of H. pylori–infected patients was analyzed. CXCL16 mRNA expression in gastric mucosa of H. pylori–infected patients with mild (n = 22), moderate (n = 20), and severe inflammation (n = 18) was compared. (B) MMP-10 siRNA, NC siRNA, or Lipo3000 only (Mock) pretreated AGS cells or AGS cells without treatment and primary gastric epithelial cells from uninfected IL-22−/−, MMP-10−/−, IL-22−/−MMP-10−/−, and WT mice were stimulated with WT H. pylori (MOI = 100) for 24 hours. CXCL16 production was measured in cell culture supernatants by ELISA (n = 3). (C) Representative dot plots of CD3+CD8+ T cells by gating on CD45+ cells and CXCR6 expression on CD3+CD8+ T cells in blood of H. pylori–infected patients or WT H. pylori–infected mice at 9 weeks p.i. CXCR6 levels on CD3+CD8+ T cells in blood of H. pylori–infected patients (n = 15) and uninfected donors (n = 19) or WT H. pylori–infected and uninfected mice (n = 6) at 9 weeks p.i. were compared. MFI, mean fluorescence intensity. (D) CD3+CD8+ T cell level in gastric mucosa of WT H. pylori–infected mice injected with CXCL16 or PBS control or Abs against CXCL16 or corresponding isotype control Ab at 9 weeks p.i. was compared (n = 5). (E and F) CD8+ T cell migration was assessed by a transwell assay, as described in Materials and Methods, and was statistically analyzed (n = 5). The horizontal bars in (A), (C), and (D) represent mean values. Each dot in (A), (C), and (D) represents one mouse or donor. *P < 0.05; **P < 0.01; n.s., P > 0.05 for groups connected by horizontal lines. sup, supernatant.

  • Fig. 6 MMP-10 impairs host defense and promotes the damage of gastric mucosa during H. pylori infection.

    (A and B) Concentration of Reg3a protein in gastric mucosa of uninfected WT mice and WT H. pylori–infected IL-22−/−, MMP-10−/−, IL-22−/−MMP-10−/−, and WT mice (A) or in gastric mucosa of WT H. pylori–infected BM chimera mice (B) at 9 weeks p.i. was compared (n = 6). (C) The bacteria colonization in gastric mucosa of WT H. pylori–infected WT mice injected with Reg3a or PBS control or in gastric mucosa of WT H. pylori–infected MMP-10−/− mice injected with anti-Reg3a or isotype control Abs at 9 weeks p.i. was compared (n = 6). (D) MMP-10 expression in patients with gastritis (n = 65) and in patients with gastric ulcer (n = 45) was compared. (E) E-cadherin and ZO-1 proteins in gastric mucosa of uninfected WT mice and WT H. pylori–infected IL-22−/−, MMP-10−/−, IL-22−/−MMP-10−/−, and WT mice at 9 weeks p.i. were analyzed by Western blot. (F) A proposed model of cross-talk among H. pylori, IL-22, gastric epithelial cells, MMP-10, and CD8+ T cells leading to MMP-10–mediated procolonization and proinflammation in gastric mucosa during H. pylori infection. The horizontal bars in (A) to (D) represent mean values. Each dot or ring in (A) to (D) represents one mouse or donor. **P < 0.01; n.s., P > 0.05 for groups connected by horizontal lines.

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/5/4/eaau6547/DC1

    Fig. S1. MMP-10 is increased in gastric mucosa of H. pylori–infected patients and mice.

    Fig. S2. H. pylori and IL-22 synergistically induce gastric epithelial cells to express MMP-10.

    Fig. S3. MMP-10 increases bacterial burden and inflammation in gastric mucosa during H. pylori infection.

    Fig. S4. MMP-10 promotes CD8+ T cell accumulation in gastric mucosa in vivo during H. pylori infection.

    Fig. S5. MMP-10 promotes CD8+ T cell accumulation in gastric mucosa in vivo during H. pylori infection.

    Fig. S6. MMP-10 promotes CD8+ T cell accumulation in gastric mucosa in vivo and migration in vitro during H. pylori infection by CXCL16.

    Fig. S7. MMP-10 impairs host defense and promotes the damage of gastric mucosa during H. pylori infection.

    Table S1. Clinical characteristics of patients.

    Table S2. Antibodies and other reagents.

    Table S3. Primer and probe sequences for real-time PCR analysis.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. MMP-10 is increased in gastric mucosa of H. pylori–infected patients and mice.
    • Fig. S2. H. pylori and IL-22 synergistically induce gastric epithelial cells to express MMP-10.
    • Fig. S3. MMP-10 increases bacterial burden and inflammation in gastric mucosa during H. pylori infection.
    • Fig. S4. MMP-10 promotes CD8+ T cell accumulation in gastric mucosa in vivo during H. pylori infection.
    • Fig. S5. MMP-10 promotes CD8+ T cell accumulation in gastric mucosa in vivo during H. pylori infection.
    • Fig. S6. MMP-10 promotes CD8+ T cell accumulation in gastric mucosa in vivo and migration in vitro during H. pylori infection by CXCL16.
    • Fig. S7. MMP-10 impairs host defense and promotes the damage of gastric mucosa during H. pylori infection.
    • Table S1. Clinical characteristics of patients.
    • Table S2. Antibodies and other reagents.
    • Table S3. Primer and probe sequences for real-time PCR analysis.

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