BMP feed-forward loop promotes terminal differentiation in gastric glands and is interrupted by H. pylori-driven inflammation

Abstract

Helicobacter pylori causes gastric inflammation, gland hyperplasia and is linked to gastric cancer. Here, we studied the interplay between gastric epithelial stem cells and their stromal niche under homeostasis and upon H. pylori infection. We find that gastric epithelial stem cell differentiation is orchestrated by subsets of stromal cells that either produce BMP inhibitors in the gland base, or BMP ligands at the surface. Exposure to BMP ligands promotes a feed-forward loop by inducing Bmp2 expression in the epithelial cells themselves, enforcing rapid lineage commitment to terminally differentiated mucous pit cells. H. pylori leads to a loss of stromal and epithelial Bmp2 expression and increases expression of BMP inhibitors, promoting self-renewal of stem cells and accumulation of gland base cells, which we mechanistically link to IFN-γ signaling. Mice that lack IFN-γ signaling show no alterations of BMP gradient upon infection, while exposure to IFN-γ resembles H. pylori-driven mucosal responses.

Fig. 1. H. pylori infection reduces BMP signaling in Myh11+ myofibroblasts.

a Confocal microscopy images of stomach antrum from Myh11CreErt2/Rosa26-mTmG mice. b Quantification of the signal overlap between α-SMA+ and Myh11-eGFP signal from Myh11CreErt2/Rosa26-mTmG mice (n = 3). c Heat map for selected genes expressed in sorted Myh11-eGFP cells from Myh11CreErt2/Rosa26-mTmG mice (n = 3). The gene expression level is presented as the read count. d Confocal microscopy images of gland hyperplasia (left) and Myh11+ myofibroblasts (right) in uninfected and 2-month H. pylori-infected Myh11CreErt2/Rosa26-mTmG mice. e Quantification of actin and Myh11 area per picture in uninfected (n = 3) and 2-month H. pylori-infected (n = 3) Myh11CreErt2/Rosa26-mTmG mice. f Heat map of RNAseq data with genes differentially expressed in uninfected (n = 3) and 2-month H. pylori-infected (n = 3) Myh11+ myofibroblasts. g Highlight of RNAseq data for differentially expressed genes involved in BMP signaling in uninfected (n = 3) and 2-month H. pylori-infected (n = 3) Myh11+ myofibroblasts. h qPCR validation of RNAseq data for BMP genes (uninfected: n = 3; infected: n = 3). Images are representative of at least three biological replicates. Scale bar: 100 µm. Data are presented as mean ± SEM. Statistical analyses were performed using Student’s t-test (two-tailed) for (e) and (h). RNAseq data are from three biological replicates per group. Source data are provided as a Source Data file.

Fig. 2. BMP signaling shows highly organized distribution in gastric glands, with strong expression in the pit cells and absence or inhibition in the gland base.

a–c Single-molecule in situ hybridization (sm-ISH) for Bmp2, Bmp4, Bmp6, and Bmp7 in antrum tissue (n = 3). d, e High magnification and quantification for Bmp2 and Bmp4 signal area per picture divided into top, middle, and base of the gland (n = 3). f Sm-ISH for Grem1, Grem2, and Chrdl1 in antrum tissue with high magnification of the gland base (n = 3). g Sm-ISH for Noggin in antrum tissue. h Quantification for Grem1, Grem2, and Chrdl1 signal area per picture divided for top, middle, and base (n = 3). Images are representative of at least three biological replicates. Scale bar: 100 µm. Data are presented as mean ± SEM. Statistical analyses were performed using one-way ANOVA, followed by Tukey’s multiple comparisons test for (d, e, h). Source data are provided as a Source Data file.

Fig. 3. Infection with H. pylori reduces BMP ligand at the surface and enhances BMP inhibitors in the base of the gland.

a Sm-ISH for Bmp2 and Bmp4 in antrum tissue of uninfected (n = 3) and 2-month H. pylori-infected (n = 3) C57BL/6 mice. b Quantification for Bmp2 and Bmp4 signal area per picture in antrum tissue of uninfected (n = 3) and 2-month H. pylori-infected (n = 3) C57BL/6 mice. c Sm-ISH for Grem1, Grem2, and Chrdl1 in antrum tissue of uninfected (n = 3) and 2-month H. pylori-infected (n = 3) C57BL/6 mice. d Quantification for Grem1, Grem2, and Chrdl1 signal area per picture in antrum tissue of uninfected (n = 3) and 2-month H. pylori-infected (n = 3) C57BL/6 mice. e, f Sm-ISH and quantification for Grem1 and Grem2 signal area per picture in the transitional zone between antrum and corpus of uninfected (n = 3) and 2-month H. pylori-infected (n = 3) C57BL/6 mice. g, h Sm-ISH for Id1 and quantification of the Id1-free compartment in the gland base of uninfected (n = 3) and 2-month H. pylori-infected (n = 3) C57BL/6 mice. Images are representative of at least three biological replicates. Scale bar: 100 µm. Data are presented as mean ± SEM. Statistical analyses were performed using Student’s t-test (two-tailed) for (b, d–f, h). Source data are provided as a Source Data file.

Fig. 4. BMP promotes mucous pit cell differentiation and inhibits basal marker expression.

a Schematic representation of gastric antrum markers for characteristic gland areas. b Images of organoids and quantification of the diameter of organoids from antral epithelium either untreated or treated with BMP2 (n = 3). c qPCR for the BMP target gene Id1 and markers characteristic for specific gland regions from organoids untreated or treated with BMP2 (n = 3). d qPCR for antimicrobial genes from organoids untreated or treated with BMP2 (n = 3). e Representative pictures and diameter quantification of organoids from antral epithelium cultured in a full medium, medium without Rspo, medium without Rspo, and reduced WNT or with the addition of BMP2 protein (n = 3). f qPCR for BMP target gene Id1 from organoids cultured in indicated conditions (n = 3). g qPCR for markers characteristic for specific gland regions from organoids cultured in indicated conditions (n = 3). h Immunofluorescence images of organoids grown in indicated conditions and quantification of relative abundance of Ki67+ cells, Muc5ac+ pit cells, and GSII+ gland base secretory cells (n = 3). Scale bar: 100 µm. i, j Representative organoid images and quantification of antral organoids per well treated with indicated conditions, passaged and cultured in normal medium (n = 3). Images are representative of at least three biological replicates. Scale bar: 250 µm. Data are presented as mean ± SEM. Statistical analyses were performed using Student’s t-test (two-tailed) for (b, c, d, j); using one-way ANOVA, followed by Tukey’s multiple comparisons test for (e, f, g, h). Source data are provided as a Source Data file.

Fig. 5. BMP signaling has a strong impact on gland turnover through the feed-forward loop and regulates R-spondin3 expression in myofibroblasts.

a qPCR for Bmp2 from organoids cultured in indicated conditions (n = 3). b qPCR for indicated genes and forming capacity from organoids exposed to different concentrations of rBMP2 (n = 3). c Representative images of organoids that re-grew in the full medium after exposure to the indicated rBMP2 concentrations. d qPCR for indicated genes and forming capacity from organoids grown without noggin, exposed to different concentrations of rBMP2 (n = 3). e Schematic representation: organoids were grown without noggin and treated with 5 ng/ml rBMP2 (blue) for 4 days at passage 0. Organoids were washed and passaged and either kept in full medium with noggin (dark blue) or without noggin (blue). f Organoid forming capacity at different passages from organoids treated (blue) or untreated (yellow) with rBMP. Organoids exposed to full medium with noggin (dark blue) recovered their forming capacity, while organoids without noggin progressively lost forming capacity (n = 2). g qPCR from the organoids from e at passage 2: Bmp2 expression triggered by rBMP is not observed in organoids that were re-grown in full medium, while in organoids grown without noggin, endogenous Bmp2 expression is strongly upregulated (n = 4). h Immunofluorescence image of myofibroblasts from Myh11CreErt2/Rosa26-tdTomato mice cultured in 2D. Scale bar: 100 µm. i qPCR for Id1 and Rspo3 from myofibroblasts cultured with noggin and BMP2 (n = 3). j qPCR for Id1 and Rspo3 from myofibroblasts cultured with different BMP2 concentrations (n = 2). k Immunofluorescence images of EdU proliferation assay performed with myofibroblasts untreated and treated with BMP2. Scale bar: 100 µm. l Quantification of the proportion of EdU positive cells (n = 3). Images are representative of at least three biological replicates. Scale bar: 250 µm, except where indicated. Data are presented as mean ± SEM. Statistical analyses were performed using Student’s t-test (two-tailed) for (g, i, l); using one-way ANOVA, followed by Tukey’s multiple comparisons test for (a). Source data are provided as a Source Data file.

Fig. 6. Deficiency of BMP signaling blocks the BMP feedback loop and promotes gland hyperplasia.

a Sm-ISH for Id1 and quantification of Id1 signal area per image in antrum tissue from WT (n = 3) and Axin2CreErt2/Bmpr1a^fl/fl (n = 3) mice. b Sm-ISH for Bmp2 and quantification of Bmp2 signal area per image in antrum tissue from WT (n = 3) and Axin2CreErt2/Bmpr1a^fl/fl (n = 3) mice. c Immunofluorescence images of antrum tissue from WT (n = 3) and Axin2CreErt2/Bmpr1a^fl/fl (n = 3) mice. Gland height of antrum tissue and relative abundance of the Ki67+ and GSII+ compartments were quantified. Images are representative of at least three biological replicates. Scale bar: 100 µm, data are presented as mean ± SEM. Statistical analyses were performed using Student’s t-test (two-tailed) for (a–c). Source data are provided as a Source Data file.

Fig. 7. H. pylori induce an IFN-γ response.

a Immunofluorescence images of antrum tissue from uninfected (n = 3) and 2-month PMSS1 WT H. pylori-infected (n = 3) mice. Relative abundance of the Muc5ac+, Ki67+, and GSII+ compartments were quantified. b Representative images of H&E staining and gland height quantification of antrum tissue from C57BL/6 mice infected for two months with PMSS1 WT (n = 3) or ΔCagE H. pylori (n = 3). c Immunofluorescence images labeled for Ki67 and quantification of Ki67+ cells per gland in antrum tissue from C57BL/6 mice infected for two months with PMSS1 WT (n = 3) or ΔCagE H. pylori (n = 3). d Sm-ISH for Bmp2 and quantification of Bmp2 signal area per image in antrum tissue from C57BL/6 mice infected for 2 months with PMSS1 WT (n = 3) or ΔCagE H. pylori (n = 3). e GSEA with the hallmark of IFN-γ response genes for PMSS1 WT H. pylori-infected (n = 2) compared to uninfected C57BL/6 mice (n = 2). f qPCR for Ifnγ in the gastric antrum from C57BL/6 mice infected for 2 months with PMSS1 WT (n = 3) or ΔCagE H. pylori (n = 3). g Immunofluorescence images of antrum tissue from 6-week H. pylori-infected C57BL/6 mice (n = 4) and IfnγR KO mice (n = 4). Gland height of antrum tissue and relative abundance of the Muc5ac+, Ki67+, and GSII+ compartments were quantified. h CFUs from 6-week H. pylori-infected C57BL/6 mice (n = 4) and IfnγR KO mice (n = 4). i, j Sm-ISH images and quantification for Bmp2 and Id1 in antrum tissue of 6-week H. pylori-infected C57BL/6 mice (n = 4) and IfnγR KO mice (n = 4). Images are representative of at least three biological replicates. Scale bar: 100 µm, data are presented as mean ± SEM. Statistical analyses were performed using Student’s t-test (two-tailed) for (a–d, f–h, j). Source data are provided as a Source Data file.

Fig. 8. IFN-γ blocks BMP signaling.

a qPCR for indicated genes in untreated or IFN-γ treated organoids (n = 3). b, c GSEA with the hallmark of targets downregulated or upregulated by BMP2 (Lee) for IFN-γ treated organoids compared to untreated ones (n = 2). d qPCR for indicated genes from primary gastric antrum stromal cells untreated or treated with IFN-γ (n = 4). e Immunofluorescence images of organoids either untreated or treated with IFN-γ; relative abundance of different cell types was quantified (n = 3). f Quantification of forming capacity of organoid either untreated or pretreated with IFN-γ (n = 3). g Images and quantification of tdTomato positive organoids from Lgr5CreErt2/Rosa26-tdTomato mice untreated or treated with IFN-γ, passaged and cultured in normal medium (tamoxifen was given 24 h before passaging; n = 3). Scale bar: 250 µm. h Images and quantification of tdTomato positive organoids from Axin2CreErt2/Rosa26-tdTomato mice untreated or treated with IFN-γ, passaged and cultured in normal medium (tamoxifen was given 24 h before passaging; n = 3). Scale bar: 250 µm. i Representative images and quantification of organoids pretreated with BMP2 or with BMP2 and IFN-γ together before passaging and then cultured in normal medium (n = 3). Scale bar: 250 µm. j qPCR for indicated genes with untreated, BMP2, or BMP2 and IFN-γ treated organoids (n = 4). k Immunofluorescence images from antrum of untreated (n = 3) or IFN-γ treated mice (n = 3). l, m Quantification of gland height and relative abundance of different cell compartments in mice upon IFN-γ treatment (ctrl: n = 3; +IFN-γ: n = 3). n Sm-ISH images and quantification for Bmp2 and Id1 in mice either untreated (n = 3) or treated with IFN-γ (n = 3). Images are representative of at least three biological replicates. Scale bar: 100 µm, except where indicated. Data are presented as mean ± SEM. Statistical analyses were performed using Student’s t-test (two-tailed) for (a, d–h, I, l, m, o); using one-way ANOVA, followed by Tukey’s multiple comparisons test for (j). Source data are provided as a Source Data file.