Gastric Sonic Hedgehog acts as a macrophage chemoattractant during the immune response to Helicobacter pylori

Abstract

Background & aims: Macrophages mediate the epithelial response to Helicobacter pylori and are involved in the development of gastritis. Sonic Hedgehog (Shh) regulates gastric epithelial differentiation and function, but little is known about its immunoregulatory role in the stomach. We investigated whether gastric Shh acts as a macrophage chemoattractant during the innate immune response to H pylori infection.

Methods: Mice with parietal cell-specific deletion of Shh (PC-Shh(KO)) and control mice were infected with H pylori. Levels of gastric Shh, cytokines, and chemokines were assayed by quantitative reverse-transcriptase polymerase chain reaction or by a Luminex-based multiplex assay 2, 7, or 180 days after infection. Circulating concentrations of Shh were measured by enzyme-linked immunosorbent assay. Bone marrow chimera experiments were performed with mice that have myeloid cell-specific deletion of the Hedgehog signal transduction protein Smoothened (LysMCre/Smo(KO)). Macrophage recruitment was measured in gastric tissue and peripheral blood by fluorescence-activated cell sorting analysis.

Results: Control mice infected with H pylori for 6 months developed an inflammatory response characterized by infiltration of CD4(+) T cells and increased levels of interferon gamma and interleukin 1β in the stomach. PC-Shh(KO) mice did not develop gastritis, even after 6 months of infection with H pylori. Control mice had increased concentrations of Shh, accompanied by the recruitment of CD11b(+)F4/80(+)Ly6C(high) macrophages 2 days after infection. Control mice that received bone marrow transplants from control mice had an influx of macrophages to the gastric mucosa in response to H pylori infection; this was not observed in H pylori-infected control mice that received bone marrow transplants from LysMCre/Smo(KO) mice.

Conclusions: H pylori induces release of Shh from the stomach; Shh acts as a macrophage chemoattractant during initiation of gastritis.

Fig. 1. Analysis of inflammation in H. pylori infected control and PC-Shh^KO mice

Representative H&E-stained sections of inflamed stomach from control mice (HKCre or Shhflx/flx) inoculated with (A) media (W/O HP) or (B) W/ HP, and or PC-Shh^KO mice inoculated with (C) media (W/O HP) or (D) W/ HP. Area of inflammatory infiltrate is shown in the stomachs of infected control mice (arrow). Images captured at 10X magnification. (E) Histological score was graded on inflammation (neutrophil and lymphocytic infiltration). A score of 1=5–25%, 2=26–50%, 3=51–75% and 4=76–100% of the total mucosa. Each data point represents the histological score given for an individual animal. CD4+ T cells were analyzed by flow cytometry and expressed as the number of cells in the gastric mucosa per mouse in uninfected and H. pylori infected control and PC-Shh^KO mice. W/O HP: without H. pylori infection, W/ HP: with H. pylori infection, *P < 0.05 compared to uninfected group as analyzed by two way ANOVA, n = 5–8 animals/group.

Fig. 2. Shh and IL-1β expression in control and PC-Shh^KO H. pylori-infected mouse stomachs

RNA was extracted from stomachs of uninfected (open bars, W/O HP) and H. pylori-infected (closed bars, W/ HP) control and PC-Shh^KO (KO) mice 2, 7 and 180 days post-inoculation. Quantitative RT-PCR was performed and average fold change in gene expression is shown for Shh from RNA collected from (A) control and (B) PC-Shh^KO; IL-1β from RNA collected from (C) control and (D) PC-Shh^KO; Data is shown as the mean + SEM, n = 4 mice/group, *P<0.05 compared to uninfected group (W/O HP).

Fig. 3. Shh protein expression in response to acute H. pylori infection in hypochlorhydric omeprazole-treated controls and gastrin-deficient mice

(A) Representative western blot of Shh protein (ShhNp, 19kDa processed Shh) expression in stomach homogenates collected from vehicle- or omeprazole-treated mice without H. pylori (−HP) or with H. pylori (+HP) 6 hours post-infection. Acid levels measured as μEq/kg of H⁺ are shown. (B) Quantification of Shh protein expression. Data are shown as means ± SEM for 3 individual experiments and expressed as Shh (pixels/mm²), *P < 0.05 compared to uninfected mice. Tissue KC concentrations measured by Luminex® multiplex assay in stomach collected from vehicle- or omeprazole-treated mice without H. pylori (−HP) or with H. pylori (+HP) 6 hours (C) and 2 days (D) post-infection. (E) Representative western blot of ShhNp expression in gastrin-deficient (G^KO) and PC-Shh^KO/G^KO mice without H. pylori (−HP) or with H. pylori (+HP) 6 hours post-infection. Also shown are changes in Shh protein expression in 1 and 3 month old G^KO and 3 month old wild type (WT) mice. (F) Tissue KC concentrations measured by Luminex® multiplex assay in stomach collected from gastrin-deficient (G^KO) and PC-Shh^KO/G^KO mice without H. pylori (−HP) or with H. pylori (+HP) 6 hours and 2 days post-infection. n = 4–7 per group, *P < 0.05 compared to uninfected mice analyzed by two-way ANOVA.

Fig. 4. Generation and verification of mice expressing a deletion of Smoothened in the myeloid cell lineage (LysMCre/Smo^KO mice)

(A) A mouse model expressing a myeloid cell-specific deletion of Smoothened (LysMCre/Smo^KO) was generated using transgenic mice bearing loxP sites flanking exon 1 of the Smo gene (Smo loxP) and mice expressing a Cre recombinase transgene from the Lysozyme M locus (LysMCre). (B) Genotyping was based on polymerase chain reaction primers specific for Cre, mutant Smo and wild type Smo (Smo WT). Shown are the fragments amplified from LysMCre/Smo^KO, WT control and LysMCre/Smo^het (heterozygous for WT and mutant Smo) mice. (C) Representative flow cytometric contour plots of the gating scheme for CD11b^highF4/80^highLy6C^highLy6G^low macrophages in peripheral blood collected from control recipients transplanted with control donor (cont^cont) or LysMCre/Smo^KO donor (cont^SmoKO) bone marrow cells. (D) RNA was extracted from FACS sorted macrophages and analyzed for expression of smoothened. Shown is the fold change relative to the cont^cont group.

Fig. 5. Changes in gastric macrophage number in response to H. pylori infection

Flow cytometric dot plots showing changes in CD11b^highF4/80^highLy6C^highLy6G^low cell distribution in gastric tissue control recipients transplanted with control donor (cont^cont) (A) without H. pylori (W/O H. pylori) or (B) with H. pylori (W/ H. pylori) infection 2 days post-inoculation. (C) Quantification of gastric CD11b^highF4/80^highLy6C^highLy6G^low cells collected from control recipients transplanted with control donor (cont^cont) or LysMCre/Smo^KO donor (cont^SmoKO) bone marrow cells and LysMCre/Smo^KO recipients transplanted with control bone marrow cells (LysMSmo^cont) without H. pylori (−HP) or with H. pylori (+HP) infection. N = 4–8 per group, *P < 0.05 compared to uninfected analyzed by one-way ANOVA.

Fig. 6. Changes in peripheral macrophage number in response to H. pylori infection

Flow cytometric dot plots showing changes in CD11b^highF4/80^highLy6C^highLy6G^low cell distribution in peripheral blood collected from control recipients transplanted with control donor (cont^cont) (A) without H. pylori (W/O H. pylori) or (B) with H. pylori (W/ H. pylori) infection 2 days post-inoculation. (C) Quantification of peripheral CD11b^highF4/80^highLy6C^highLy6G^low cells collected from control recipients transplanted with control donor (cont^cont) or LysMCre/Smo^KO donor (cont^SmoKO) bone marrow cells and LysMCre/Smo^KO recipients transplanted with control bone marrow cells (LysMSmo^cont) without H. pylori (−HP) or with H. pylori (+HP) infection. n = 4–8 per group, *P < 0.05 compared to uninfected analyzed by one-way ANOVA.

Fig. 7. Circulating Shh concentrations in control and PC-Shh^KO mice

Circulating Shh concentrations were measured by ELISA using plasma collected from (A) control recipients transplanted with control donor (cont^cont) or LysMCre/Smo^KO donor (cont^SmoKO) bone marrow cells and LysMCre/Smo^KO recipients transplanted with control bone marrow cells (LysMSmo^cont) or (B) PC-Shh^KO mice without H. pylori (−HP) or with H. pylori (+HP) infection. *P < 0.05 compared to uninfected group as analyzed by one-way ANOVA, n = 3–6 animals/group.