Volume 142, Issue 2, Pages 281-291 (February 2012) Helicobacter pylori Infection Recruits Bone Marrow−Derived Cells That Participate in Gastric Preneoplasia in Mice  Christine Varon, Pierre Dubus, Frédéric Mazurier, Corinne Asencio, Lucie Chambonnier, Jonathan Ferrand, Alban Giese, Nathalie Senant–Dugot, Martina Carlotti, Francis Mégraud  Gastroenterology  Volume 142, Issue 2, Pages 281-291 (February 2012) DOI: 10.1053/j.gastro.2011.10.036 Copyright © 2012 AGA Institute Terms and Conditions

Figure 1 Disease progression during H pylori infection in mice gastric mucosa. Representative histopathologic features of noninfected (A, C) or H pylori SS1-infected (B, D, E, F) stomachs over time are shown. (A, C) Normal gastric mucosa of noninfected mice. H pylori−induced increase in mucosal height (B) and alcian blue (AB)−positive mucinous metaplasia (D) after 35 WPI, then AB-positive pseudointestinal metaplasia (E) and dysplasia with GIN penetrating the muscularis mucosa (F) after 55 WPI. The picture is representative of results obtained with all H pylori strains. Bar = 50 μm. Gastroenterology 2012 142, 281-291DOI: (10.1053/j.gastro.2011.10.036) Copyright © 2012 AGA Institute Terms and Conditions

Figure 2 Scoring of gastric histopathological criteria during Helicobacter infection. Gastric inflammation, mucosal height, oxyntic atrophy, mucinous metaplasia, pseudointestinal metaplasia, and dysplasia determined on chimera mice after 15 (white), 35 (light gray), 55 (dark gray), and 75 (black) WPI. Data represent mean of scores ± SD for control noninfected (5 < n < 8) and H felis or H pylori−infected chimera mice (5 < n < 17) over time. *P < .05 compared to noninfected controls. Gastroenterology 2012 142, 281-291DOI: (10.1053/j.gastro.2011.10.036) Copyright © 2012 AGA Institute Terms and Conditions

Figure 3 Detection of BMDC engraftment in gastric mucosa after long term H pylori infection. Representative pictures of BMDC immunodetection with anti-GFP antibodies on stomach paraffin-embedded tissue sections. (A) Noninfected WT stomach without GFP positivity after 1 year. (B) A GFP stomach with GFP detected at different intensities between glands with a diffuse and/or nuclear expression pattern in epithelial cells. (C) Noninfected chimera mouse stomach after 1 year with rare GFP-positive BMDCs corresponding to stroma cells or infiltrated leukocytes and not to epithelial cells. (D−F) H pylori SS1-infected chimera mice stomachs after 55 WPI harboring healthy areas composed of BMDCs with a mosaic pattern of GFP-positive epithelial cells along the gland units, at the base (D), the middle part (E) or the top (F) of the glands. The picture is representative of results obtained with all H pylori strains. Bars = 50 μm. Gastroenterology 2012 142, 281-291DOI: (10.1053/j.gastro.2011.10.036) Copyright © 2012 AGA Institute Terms and Conditions

Figure 4 BMDCs in metaplastic and dysplastic areas of Helicobacter-infected mouse gastric mucosa. Representative pictures of BMDC immunodetection with anti-GFP antibodies in H pylori SS1-infected chimera mouse stomach paraffin-embedded tissue sections after 55 WPI. All pictures are representative of results obtained with all H pylori and H felis strains. Areas of dedifferentiation (A), pseudointestinal metaplasia (B), and dysplasia (C) composed of GFP-positive BMDCs. (D) Mixed units of GFP-positive and GFP-negative BMDCs with dedifferentiated and pseudointestinal metaplasia features. (E) A GFP-positive dysplastic gland below the muscularis mucosa in GIN. Bar = 50 μm. Gastroenterology 2012 142, 281-291DOI: (10.1053/j.gastro.2011.10.036) Copyright © 2012 AGA Institute Terms and Conditions

Figure 5 BMDC detection in gastric epithelial glands by fluorescence and fluorescent in situ hybridization (FISH). (A) Detection of GFP fluorescence (green) combined with 4′,6-diamidino-2-phenylindole (DAPI) staining of the nuclei (blue) on frozen stomach tissue sections showing no GFP in WT mice, and GFP-fluorescent epithelial cells in both GFP mice and H pylori SS1-infected chimera mice at 55 WPI. (B) Detection of BMDCs by FISH of X (red channel) and Y (green channel) chromosomes combined with DAPI staining (blue channel) on stomach paraffin-embedded tissue sections of H pylori SS1-infected chimera mice after 55 WPI. HES staining of the same area showing dedifferentiated glands in areas of metaplasia. Enlarged pictures show some cells (white arrows) composing a gastric gland possessing one X (red arrow) and one Y (green arrow) chromosomes. (A, B) Pictures from H pylori SS1-infected mice are representative of results obtained with all H pylori and H felis strains. Bar = 10 μm. Gastroenterology 2012 142, 281-291DOI: (10.1053/j.gastro.2011.10.036) Copyright © 2012 AGA Institute Terms and Conditions

Figure 6 Frequency of BMDC-positive gastric epithelial glands and dysplasia during Helicobacter infection. (A) Percentage of chimera mice harboring GFP-positive gastric epithelial glands over time of infection; n, number of mice in each group. (B) Grade (scale 1−4) of frequency/distribution of GFP-positive gastric epithelial glands (composed of at least 1 segment of 6 consecutive GFP-positive epithelial cells) in the stomach over time of infection. Data represent mean of scores ± SD for control noninfected (5 < n < 8) and H felis or H pylori−infected chimera mice (5 < n < 17). *P < .05 compared to noninfected controls. (C) Percentage of dysplastic areas composed of GFP-positive (≥10%) epithelial cells; n, number of total dysplastic areas in each group. Gastroenterology 2012 142, 281-291DOI: (10.1053/j.gastro.2011.10.036) Copyright © 2012 AGA Institute Terms and Conditions

Figure 7 BMDCs in gastric glands of Helicobacter-infected mice express epithelial markers. BMDC detection on stomach paraffin-embedded tissue sections of H pylori SS1-infected chimera mice (one representative picture after 55 WPI) by fluorescent in situ hybridization (FISH) against the X (red) and Y (green) chromosomes combined with immunofluorescent staining of epithelial pan-cytokeratins (pCK, yellow) and CD45 (magenta) with 4′,6-diamidino-2-phenylindole (DAPI) (blue). Chromosome Y-positive, pCK-positive, and CD45-negative cells (yellow arrows) within epithelial glands correspond to BM-derived epithelial cells, instead of chromosome Y-positive, pCK-negative and CD45-positive BM-derived leukocytes (white arrows). One representative picture from an H pylori SS1-infected mouse representative of results obtained with all H pylori and H felis strains. Bar = 10 μm. Gastroenterology 2012 142, 281-291DOI: (10.1053/j.gastro.2011.10.036) Copyright © 2012 AGA Institute Terms and Conditions