CD44 Plays a Functional Role in Helicobacter pylori-induced Epithelial Cell Proliferation

The cytotoxin-associated gene (Cag) pathogenicity island is a strain-specific constituent of Helicobacter pylori (H. pylori) that augments cancer risk. CagA translocates into the cytoplasm where it stimulates cell signaling through the interaction with tyrosine kinase c-Met receptor, leading cellular proliferation. Identified as a potential gastric stem cell marker, cluster-of-differentiation (CD) CD44 also acts as a co-receptor for c-Met, but whether it plays a functional role in H. pylori-induced epithelial proliferation is unknown. We tested the hypothesis that CD44 plays a functional role in H. pylori-induced epithelial cell proliferation. To assay changes in gastric epithelial cell proliferation in relation to the direct interaction with H. pylori, human- and mouse-derived gastric organoids were infected with the G27 H. pylori strain or a mutant G27 strain bearing cagA deletion (ΔCagA::cat). Epithelial proliferation was quantified by EdU immunostaining. Phosphorylation of c-Met was analyzed by immunoprecipitation followed by Western blot analysis for expression of CD44 and CagA. H. pylori infection of both mouse- and human-derived gastric organoids induced epithelial proliferation that correlated with c-Met phosphorylation. CagA and CD44 co-immunoprecipitated with phosphorylated c-Met. The formation of this complex did not occur in organoids infected with ΔCagA::cat. Epithelial proliferation in response to H. pylori infection was lost in infected organoids derived from CD44-deficient mouse stomachs. Human-derived fundic gastric organoids exhibited an induction in proliferation when infected with H. pylori, that was not seen in organoids pre-treated with a peptide inhibitor specific to CD44. In the wellestablished Mongolian gerbil model of gastric cancer, animals treated with CD44 peptide inhibitor Pep1, resulted in the inhibition of H. pylori-induced proliferation and associated atrophic gastritis. The current study reports a unique approach to study H. pylori interaction with the human gastric epithelium. Here, we show that CD44 plays a functional role in H. pyloriinduced epithelial cell proliferation

The Development of Spasmolytic Polypeptide/TFF2-Expressing Metaplasia (SPEM) During Gastric Repair Is Absent in the Aged StomachSummary

Background & Aims: During aging, physiological changes in the stomach result in more tenuous gastric tissue that is less capable of repairing injury, leading to increased susceptibility to chronic ulceration. Spasmolytic polypeptide/trefoil factor 2âexpressing metaplasia (SPEM) is known to emerge after parietal cell loss and during Helicobacter pylori infection, however, its role in gastric ulcer repair is unknown. Therefore, we sought to investigate if SPEM plays a role in epithelial regeneration. Methods: Acetic acid ulcers were induced in young (2â3 mo) and aged (18â24 mo) C57BL/6 mice to determine the quality of ulcer repair with advancing age. Yellow chameleon 3.0 mice were used to generate yellow fluorescent proteinâexpressing organoids for transplantation. Yellow fluorescent proteinâpositive gastric organoids were transplanted into the submucosa and lumen of the stomach immediately after ulcer induction. Gastric tissue was collected and analyzed to determine the engraftment of organoid-derived cells within the regenerating epithelium. Results: Wound healing in young mice coincided with the emergence of SPEM within the ulcerated region, a response that was absent in the aged stomach. Although aged mice showed less metaplasia surrounding the ulcerated tissue, organoid-transplanted aged mice showed regenerated gastric glands containing organoid-derived cells. Organoid transplantation in the aged mice led to the emergence of SPEM and gastric regeneration. Conclusions: These data show the development of SPEM during gastric repair in response to injury that is absent in the aged stomach. In addition, gastric organoids in an injury/transplantation mouse model promoted gastric regeneration. Keywords: Epithelial Regeneration, Gastric Cancer, Human Gastric Organoids, CD44

<i>H. pylori</i>-induced epithelial-to-mesenchymal transition (EMT).

<p>Immunofluorescence of EdU (red) and E-cadherin (green) using (<b>A</b>) uninfected control (CON), (<b>B</b>) <i>H</i>. <i>pylori</i> (HP) or (<b>C</b>) ∆CagA infected mFGOs. (<b>D</b>) EMT marker (αSMA, SNAIL, TWIST, N-cadherin and Zeb) expression measured by qRT-PCR using RNA isolated from mFGOs uninfected (control), <i>H</i>. <i>pylori</i> (HP) or ∆CagA infected. *P<0.05 compared to CON group, n = 4 individual experiments/group. (<b>E</b>) Immunofluorescence of EdU (red) and E-cadherin (green) using uninfected control (CON), <i>H</i>. <i>pylori</i> (HP) and ∆CagA infected mFGOs derived from stomachs of CD44-deficient mice. (<b>F</b>) (<b>D</b>) EMT marker (αSMA, SNAIL, TWIST, N-cadherin and Zeb) expression measured by qRT-PCR using RNA isolated from mFGOs derived from CD44-deficient mouse stomachs uninfected (control), <i>H</i>. <i>pylori</i> (HP) or ∆CagA infected.</p

H. pylori microinjection and colonization of mFGOs.

<p>Organoids were microinjected with <i>H</i>. <i>pylori</i> shown are mFGOs (<b>A</b>) before and (<b>B</b>) after injection. Arrow indicates injection needle. ‘Cloud’ of <i>H</i>. <i>pylori</i> observed after injection is shown. Twenty-four hours after luminal microinjection, <i>H</i>. <i>pylori</i> (HP) attach to the luminal surface demonstrated by Warthin-Starry stain of (<b>C</b>) control and (<b>D</b>) HP infected organoids. (<b>E</b>) Quantitative cultures of HP of control and HP infected organoids. *P<0.05 compared to bacterial numbers at day 1 after infection.</p

Changes in proliferation in <i>H</i>. <i>pylori</i> infected mFGOs.

<p>(<b>A</b>) Immunofluorescence of EdU+ cells in control (CON), <i>H</i>. <i>pylori</i> (HP) and HP ∆CagA infected and HP infected fundic organoids pretreated with c-Met inhibitor (c-MetI+HP). (<b>B</b>) Quantification of EdU+ cells/total cells. *P<0.05 compared to CON group, n = 6 individual organoid preps per group. (<b>C</b>) Protein lysates were prepared from mFGOs that were uninfected (CON), HP or ∆cagA infected for 24 hours. Lysates were immunoprecipitated using an anti-c-Met antibody and immunoblotted for TyrP, c-Met, CagA, CD44 and HGF. (<b>D</b>) Proposed model of the CD44 co-receptor function in response to <i>H</i>. <i>pylori</i>. (<b>E</b>) Immunofluorescence of EdU+ cells in CON, and HP infected organoids derived from CD44KO mouse fundus. Quantification of EdU+ cells in CON, HP and or ∆CagA infected and Wnt agaonist-treated (Wnt) organoids derived from CD44KO mouse fundus. *P<0.05 compared to CON group, n = 3 individual organoid preps per group. (<b>F</b>) Protein lysates were prepared from mFGOs derived from CD44-deficient mice that were uninfected (CON), HP or ∆CagA infected for 24 hours. Lysates were immunoprecipitated using an anti-c-Met antibody and immunoblotted for TyrP, c-Met, CagA, CD44 and HGF.</p