Helicobacter pylori-induced Sonic Hedgehog expression is regulated by NFκB pathway activation: the use of a novel in vitro model to study epithelial response to infection

Abstract

Background: Helicobacter pylori (H. pylori) infection leads to acute induction of Sonic Hedgehog (Shh) in the stomach that is associated with the initiation of gastritis. The mechanism by which H. pylori induces Shh is unknown. Shh is a target gene of transcription factor Nuclear Factor-κB (NFκB). We hypothesize that NFκB mediates H. pylori-induced Shh.

Materials and methods: To visualize Shh ligand expression in response to H. pylori infection in vivo, we used a mouse model that expresses Shh fused to green fluorescent protein (Shh::GFP mice) in place of wild-type Shh. In vitro, changes in Shh expression were measured in response to H. pylori infection using 3-dimensional epithelial cell cultures grown from whole dissociated gastric glands (organoids). Organoids were generated from stomachs collected from the fundic region of control and mice expressing a parietal cell-specific deletion of Shh (PC-Shh(KO) mice).

Results: Within 2 days of infection, H. pylori induced Shh expression within parietal cells of Shh::GFP mice. Organoids expressed all major gastric cell markers, including parietal cell marker H(+) ,K(+) -ATPase and Shh. H. pylori infection of gastric organoids induced Shh expression; a response that was blocked by inhibiting NFκB signaling and correlated with IκB degradation. H. pylori infection of PC-Shh(KO) mouse-derived organoids did not result in the induction of Shh expression.

Conclusion: Gastric organoids allow for the study of the interaction between H. pylori and the differentiated gastric epithelium independent of the host immune response. H. pylori induces Shh expression from the parietal cells, a response mediated via activation of NFκB signaling.

Keywords: Helicobacter pylori; NF-kB; stomach.

Figure 1. Parietal cell-expressed Shh

(A) Fundic and antral sections were collected from stomachs of Shh::GFP mice an immunostained using antibodies specific for GFP (Shh-expressing cells, green), H⁺,K⁺-ATPase (parietal cells, red) and UEAI (surface pit cells, blue). Fundic sections collected from stomachs of Shh::GFP mice inoculated with (B–E) Brucella broth (controls) or (F–I) H. pylori were immunostained using antibodies specific for GFP (Shh-expressing cells, green), H⁺,K⁺-ATPase (parietal cells, red) and UEAI (surface pit cells, blue). (J) The number of cells co-expressing Shh::GFP and H⁺,K⁺-ATPase were quantified and expressed as cells/gland. Data is expressed as the mean ± SEM where *P<0.05 compared to controls, n = 6 mice per group.

Figure 2. Parietal cell-expressed Shh within mouse-derived fundic gastric organoids

(A) Gastric glands grow into spheroids by day 7 in culture. Fundic organoids were immunostained using antibodies specific for (B) Shh (green) and (C) H⁺,K⁺-ATPase (parietal cells, red). Merged image is shown in (D). (E) Z stack of whole mount organoid shown in D demonstrating the presence of multiple parietal cells-expressing Shh. Images were collected at 1.5 μm intervals. (F) RNA extracted from fundic organoids was used to measure the expression of major gastric cell lineages including mucin 5AC (Muc5AC), mucin 6 (Muc6), pepsinogen C (PgC), somatostatin (SST), gastrin (GAST), H⁺,K⁺-ATPase (HK) and Shh using RT-PCR.

Figure 3. H. pylori infection of fundic organoids

(A) Micro-injection of H. pylori into fundic organoids. H. pylori appears as a cloud within the organoid. H&E of organoid sections microinjected with (B) culture media or (C) H. pylori. Arrows in C show H. pylori attachment to epithelium of fundic gastric organoids. (D) Quantitative cultures of control (con) and H. pylori (HP) infected organoids.

Figure 4. H. pylori-induced NFκB activation and Shh expression

(A) NFκB status was measured using protein lysates extracted from organoids infected with H. pylori for 0, 2, 4, and 24 hours. Changes in IκBα, IKKα and GAPDH expression were measured by western blot. Quantification of Changes in IκBα relative to GAPDH is shown in B. Data are expressed as the mean + SEM, where *P<0.05 compared to time 0 hours, n = 3 individual organoid cultures. Organoids were derived from the fundus of (C) control or (D) PC-Shh^KO mice. Organoids were treated with vehicle (veh), NFκB inhibitor alone (INH) or NFκB inhibitor pre-treatment followed by H. pylori infection (HP+INH), and H. pylori infection alone (HP). Shh and Ptch expression was analyzed by qRT-PCR 24 hours post-infection. *P<0.05 compared to veh group, n=6 individual wells per group.