Interleukin 13 Promotes Maturation and Proliferation in Metaplastic Gastroids

Abstract

Background & aims: Type 2 innate lymphoid cells (ILC2s) and interleukin-13 (IL-13) promote the onset of spasmolytic polypeptide-expressing metaplasia (SPEM) cells. However, little is known about molecular effects of IL-13 in SPEM cells. We now sought to establish a reliable organoid model, Meta1 gastroids, to model SPEM cells in vitro. We evaluated cellular and molecular effects of ILC2s and IL-13 on maturation and proliferation of SPEM cells.

Methods: We performed single-cell RNA sequencing to characterize Meta1 gastroids, which were derived from stomachs of Mist1-Kras transgenic mice that displayed pyloric metaplasia. Cell sorting was used to isolate activated ILC2s from stomachs of IL-13-tdTomato reporter mice treated with L635. Three-dimensional co-culture was used to determine the effects of ILC2s on Meta1 gastroids. Mouse normal or metaplastic (Meta1) and human metaplastic gastroids were cultured with IL-13 to evaluate cell responses. Air-Liquid Interface culture was performed to test long-term culture effects of IL-13. In silico analysis determined possible STAT6-binding sites in gene promoter regions. STAT6 inhibition was performed to corroborate STAT6 role in SPEM cells maturation.

Results: Meta1 gastroids showed the characteristics of SPEM cell lineages in vitro even after several passages. We demonstrated that co-culture with ILC2s or IL-13 treatment can induce phosphorylation of STAT6 in Meta1 and normal gastroids and promote the maturation and proliferation of SPEM cell lineages. IL-13 up-regulated expression of mucin-related proteins in human metaplastic gastroids. Inhibition of STAT6 blocked SPEM-related gene expression in Meta1 gastroids and maturation of SPEM in both normal and Meta1 gastroids.

Conclusions: IL-13 promotes the maturation and proliferation of SPEM cells consistent with gastric mucosal regeneration.

Keywords: AQP5; CD44v9; ILC2; Interleukin-13; Maturation; Proliferation; SPEM; STAT6.

Graphical abstract

Figure 1

Establishment and characterization of Meta1 gastroid lines. (A) H&E staining shows the stomach mucosa used for the generation of the Meta1 and Meta4 gastroids displaying pyloric metaplasia and dysplasia, respectively. Immunofluorescence staining for SPEM cell markers (AQP5 and GSII) and a dysplastic marker (TROP2). Scale bar: 50 μm. (B) Schematic illustration of the gastroid establishment process from Mist1-Kras transgenic mice. (C) Representative bright field images of Meta1 gastroids captured at day 0, 3, and 7 days after plating. Scale bars: 50 μm for first picture and 100 μm for second and third. (D) Immunofluorescence staining of Meta1 gastroids cross sections for SPEM markers (CD44v9, AQP5, and TFF2). Yellow arrows and enlarged sections indicate presence of mature SPEM cells triple positive for CD44v9, AQP5, and TFF2 in cells. Scale bar: 100 μm.

Figure 2

Meta1 and Meta4 mouse gastroids displayed different behavior. (A) Top, representative bright field images of Matrigel domes containing Meta1 and Meta4 gastroids at 1 or 2 weeks after plating. Scale bar: 500 μm. Bottom, H&E staining shows cellular distribution in the Meta1 and Meta4 gastroids. Enlarged images show cellular organization (mono- or multilayering). Scale bar: 100 μm. (B) Quantitation of the average percentage of gastroids presenting budding formation in Meta1 and Meta4 gastroids at 1 or 2 weeks of in vitro culture. Mean ± SD (N= 8–10 from 3 independent experiments). ∗∗∗∗P < .0001. (C) Immunofluorescence staining of Meta1 and Meta4 gastroids after 2 weeks of in vitro 3D culture for SPEM cell markers (CD44v9 and AQP5) and a dysplastic cell marker (TROP2). Scale bar: 100 μm. (D) Quantitation of intensity units for different SPEM and dysplastic cell markers in gastroids on C. Mean ± SD (n = 4). ∗∗∗∗P < .0001.

Figure 3

Single-cell RNA-seq of Meta1 gastroid lines. (A) UMAP plot three-colored to show the cell distribution of the Meta1 and Meta4 gastroid lines. Each dot represents a single cell. (B) UMAP plots showing expression of different genes in red. Tff2, Muc6, Gkn3, and Aqp5 are SPEM-related genes and overlayed with Meta1 gastroid-derived cells. Gif and Pgc are genes expressed in chief cells and overlayed with Meta1 gastroid-derived cells. Mmp7, a dysplastic marker, overlayed with the Meta4 gastroid-derived cells and Cd44 was present in both types of gastroids. (C) Volcano plot of the differentially expressed genes between both Meta1 lines versus Meta4 gastroid line with emphasis in up-regulation of SPEM-related genes and down-regulation of dysplastic-related genes in Meta1 gastroid cells. (D) Quantitative RT-PCR showing the relative expression levels of key chief cell and SPEM cell genes. Mean ± SD (n = 4). ∗P <.05, ∗∗P < .01, ∗∗∗P < .001, ∗∗∗∗P < .0001. (E) UMAP plot three-colored to show the clusters found in the gastroid lines. There are 2 distinctive cell clusters present in the Meta1 gastroid-derived cells, Cluster A and Cluster B. Cluster C contains most of Meta4 gastroid cells. (F) Gene set enrichment analysis of the Kyoto Encyclopedia of Genes and Genomes database using the up-regulated genes in Cluster B versus Cluster A.

Figure 4

Isolation of activated ILC2s and 3D co-culture with Meta1 gastroids. (A) H&E and immunofluorescence staining of stomach tissues from untreated and L635-treated mice. Yellow arrows point to ILC2 cells double positive for GATA3 and TdTomato. Quantitation of GATA3 positive cells (ILC2s) and GATA3 and TdTomato double-positive cells (activated ILC2s). Scale bar: 100 μm. Mean ± SD (n = 6–8 from 3 different experiments). ∗∗P < .01. (B) Schematic illustration of activated ILC2s isolation from L635-treated mice using FACS. (C) FACS plots showing tdTomato positive cells (ILC2s) in untreated and L635-treated mice. Cell number quantitation shows significant increase of tdTomato positive cells (activated ILC2s) in stomachs from treated mice. Mean ± SD (N = 4). ∗P < .05. (D) Representative bright field images of Matrigel domes containing Meta1 gastroids only or co-cultured with ILC2s. Immunofluorescence imaging shows proximity of activated ILC2s (IL-13-tdTomato positive) to the Meta1 gastroid (GFP-positive membrane). Quantitation of gastroid diameters demonstrates significantly higher sizes in Meta1 gastroids co-cultured with ILC2s compared with gastroids only after 5 days of plating. Scale bar: 500 μm. Mean ± SD (N = 4 independent experiments). ∗P < .05. (E) Immunoblotting shows significant up-regulation of phosphorylated STAT6 in Meta1 gastroid co-cultured with ILC2s. Mean ± SD (N = 4 independent experiments). ∗∗∗∗P < .0001.

Figure 5

Co-culture with activated ILC2s promoted maturation and proliferation of SPEM cells in Meta1 gastroids. (A) Immunofluorescence staining of Meta1 gastroids cross sections for SPEM-related markers, AQP5 and TFF2. Quantitation of AQP5 and TFF2 double positive cells demonstrated significant increase of proportion of mature SPEM cells in co-cultured condition. Yellow arrowheads point at double positive mature SPEM cells. Scale bar: 100 μm and 50 μm for enlarged. Mean ± SD (N = 3 independent experiments). ∗P < .05. (B) Immunofluorescence staining of Meta1 gastroids cross sections for a proliferation marker, Ki67, and SPEM-related markers, MUC6 and CD44v9. Quantitation of MUC6 and CD44v9 double positive cells demonstrated significant increase of proportion of mature SPEM cells in Meta1 gastroids co-cultured with ILC2s. Quantitation of MUC6, CD44v9, and Ki67 triple positive cells demonstrated significant increase of proportion of proliferative SPEM cells. Yellow arrowheads point at triple positive mature and proliferative SPEM cells. Scale bar: 100 μm and 50 μm for enlarged. Mean ± SD (N = 3 independent experiments). ∗P < .05.

Figure 6

IL-13 caused higher phosphorylation ratio of STAT6 than IL-4 in Meta1 gastroids. (A) Representative bright field images of Matrigel domes containing Meta1 gastroids treated with rIL-13, rIL-4, or vehicle solution (BSA). Quantitation of gastroid diameters showed significantly higher sizes in Meta1 gastroids treated with rIL-13 compared with rIL-4 and BSA starting at 2 days of treatment. There was no significant difference between rIL-4- and BSA-treated Meta1 gastroids. Scale bar: 1000 μm. Mean ± SD (N = 4 independent experiments). ∗P < .05, ∗∗P < .01, ∗∗∗P < .001, ∗∗∗∗P < .0001. (B) Immunoblotting shows significant up-regulation of phosphorylated STAT6 in Meta1 gastroids treated with rIL-13 when compared with rIL-4 and BSA. Mean ± SD (N = 4 independent experiments). ∗∗P < .01, ∗∗∗P < .001.

Figure 7

IL-13 alone promoted phosphorylation of STAT6 and maturation and proliferation of SPEM cells. (A) Representative bright field images of Matrigel domes containing Meta1 gastroids treated with either rIL-13 or vehicle solution (BSA). Enlarged images from H&E staining show cellular distribution and height differences between the rIL–13-treated and the BSA-treated Meta1 gastroids. Quantitation of matched gastroid diameters showed significantly higher sizes in Meta1 gastroids treated with rIL-13 compared with control after 2 days of treatment. Scale bar: 1000 μm for brightfield and 100 μm for H&E. Mean ± SD (N = 3 independent experiments). ∗∗P < .01, ∗∗∗∗P < .0001. (B) Immunoblotting shows significant up-regulation of phosphorylated STAT6 in Meta1 gastroids treated with different concentrations of rIL-13 when compared with BSA. Quantitation showed rIL-13–dose-dependent increase. Mean ± SD (N = 3 independent experiments). ∗P < .05. (C) Immunofluorescence staining of Meta1 gastroids cross sections for SPEM-related markers, AQP5 and TFF2. Quantitation of AQP5 and TFF2 double positive cells demonstrated significant increase of proportion of mature SPEM cells in rIL-13–treated Meta1 gastroids. Yellow arrowheads point at double positive mature SPEM cells. Scale bar: 100 μm. Mean ± SD (N = 3 independent experiments). ∗∗∗P < .001. (D) Immunofluorescence staining of Meta1 gastroids cross sections for a proliferation marker, Ki67, and SPEM-related markers, MUC6 and CD44v9. Quantitation of MUC6 and CD44v9 double positive cells demonstrated significant increase of proportion of mature SPEM cells in rIL-13–treated Meta1 gastroids. Moreover, quantitation of MUC6, CD44v9, and Ki67 triple positive cells demonstrated significant increase of proportion of proliferative SPEM cells. Yellow arrowheads point at triple positive mature and proliferative SPEM cells. Scale bar: 100 μm. Mean ± SD (N = 3 independent experiments). ∗∗P < .01, ∗∗∗P < .001.

Figure 8

IL-13 maintained the mature SPEM phenotype in Meta1 gastroid cells in long-term ALI culture. (A) Schematic illustration of ALI culture procedure. (B) Representative bright field images of top view of ALI filters. Secretion and accumulation of mucins increase after 14 days of culture with rIL-13. At day 28, there was a tenacious mucus layer on top of the Meta1 gastroid cells that were treated with rIL-13. Scale bar: 200 μm. (C) Whole mount immunofluorescence staining for SPEM-related markers, GSII and CD44v9, showed significant increase of GSII coverage proportion in the rIL-13 treatment. Scale bar: 100 μm. Quantitation of GSII staining per cell area is shown on the right. Mean ± SD (N = 3 independent experiments). ∗∗P < .01.

Figure 9

IL-13 promoted maturation of SPEM cells in normal gastroids and mimicked gastroids derived from L635-treated mice. (A) Schematic illustration of gland-derived gastroids from untreated wild-type mice. (B) Representative bright field images of Matrigel domes containing normal gastroids cultured with either rIL-13 or vehicle (BSA). Enlarged images show distinct cell heights of the gastroid cell layer. H&E staining shows cell organization and height differences between rIL-13–treated and BSA-treated normal gastroids. Scale bar: 100 μm. (C) Immunofluorescence staining normal gastroids cross sections for SPEM-related markers, MUC6 and AQP5. Quantitation of MUC6 and AQP5 double positive cells demonstrated significant increase of proportion of mature SPEM cells in rIL-13–treated normal gastroids. Scale bar: 100 μm and 50 μm for enlarged. Normalized with gastroid area. Mean ± SD (N = 3 independent experiments). ∗∗P < .01. (D) Immunofluorescence staining for SPEM-related markers, MUC6 and CD44v9, of normal gastroids treated with BSA (left), or with rIL-13 (middle), and of gastroids derived from L635-treated mice (right). rIL-13–treated normal gastroids and L365-treatment derived gastroids showed similarities in increased expression of MUC6 when compared with BSA-treated normal gastroids. (E) Immunoblotting shows significant up-regulation of phosphorylated STAT6 in rIL-13–treated normal gastroids compared with BSA. Quantitation showed rIL-13–dose-dependent increase. Mean ± SD (N = 3 independent experiments). ∗P < .05, ∗∗∗P < .001. (F) Schematic illustration of gland-derived gastroids from untreated and L365-treated wild-type mice. Exposure of normal gastroids to rIL-13 promoted the transdifferentiation and maturation toward a SPEM cell phenotype as seen in gastroids derived from mucosa with pyloric metaplasia after L635 treatment.

Figure 10

rIL-13–treated normal gastroids displayed both chief cell and SPEM cell markers. (A) Immunofluorescence staining for MIST1 (chief cell), PDX1 (an antral lineage marker), and CD44v9 (SPEM cell) in normal gastroids and Meta1 gastroids. Scale bar: 100 μm. (B) Quantitation of average percentage of MIST1-positive cells and PDX1-positive cells. Mean ± SD (N = 6–7 from 3 independent experiments). ∗∗P < .01, ∗∗∗P < .001.

Figure 11

STAT6 can up-regulate transcription of SPEM-related genes and the protein expression of ATF3. (A) Consensus sequence of the DNA motif recognized by STAT6. (B) Positions (P1-P4) of the STAT6 biding sites predicted with the JASPAR database. These sites were located upstream -2000 base pairs of the Transcription Start Site (TSS) of SPEM-related genes including Muc6, Tff2, Gkn3, Cd44, He4, and Atf3. (C) Quantitative RT-PCR showing the relative expression levels of SPEM-related genes (Muc6, Tff2, Gkn3, Cd44v9, and He4). Mean ± SD (n = 4 independent experiments). ∗P < .05. (D) Immunoblotting shows significant up-regulation of ATF3 protein in Meta1 gastroids treated with rIL-13 compared with vehicle (BSA). Mean ± SD (N = 3 independent experiments). ∗∗P < .01.

Figure 12

AS1517499, a STAT6 inhibitor, impacted Meta1 gastroid growth and gene expression. (A) Time-course diagram of the treatment to Meta1 gastroids with rIL-13 and/or AS1517499. (B) Representative bright field images of Matrigel domes containing Meta1 gastroids at different times of treatment. Quantitation of gastroid diameters showed a significant difference in Meta1 gastroids treated with AS1517499. Scale bar: 500 μm. Mean ± SD (N = 3 independent experiments). ∗P < .05, ∗∗∗P < .001, ∗∗∗∗P < .0001. (C) Quantitative RT-PCR showing relative expression levels of SPEM-related genes (Muc6, Tff2, Gkn3, Cd44v9, Atf3, and He4). Mean ± SD (N = 3 independent experiments). ∗P < .05, ∗∗P < .01, ∗∗∗P < .001. (D) Immunoblotting shows significant down-regulation of phosphorylated STAT6 and ATF3 in Meta1 gastroids treated with STAT6 inhibitor (AS). Mean ± SD (N = 4 independent experiments). ∗∗∗P < .001.

Figure 13

AS1517499 attenuated maturation of transdifferentiating cells into SPEM mucinous cells. (A) Time-course diagram of treatment of normal gastroids with rIL-13 and/or AS1517499 for immunoblotting experiments. (B) Immunoblotting shows down-regulation of phosphorylated STAT6 in normal gastroids treated with both rIL-13 and AS1517499. The phosphorylation decrease was evident beginning at 1 hour of AS1517499 addition. Quantitation showed that this decrease occurred in a time-dependent manner. Mean ± SD (N = 2 independent experiments). (C) Immunofluorescence staining of normal gastroids cross sections treated with rIL-13 and/or AS1517499 for SPEM-related markers, MUC6 and AQP5. Quantitation of MUC6 and AQP5 double positive cells demonstrated significant decrease in proportion of mature SPEM cells as seen by reduction of MUC6 in the gastroids exposed to the STAT6 inhibitor. Scale bar: 100 μm and 50 μm for enlarged. Mean ± SD (N = 4 independent experiments). ∗P < .05, ∗∗P < .01, ∗∗∗P < .001.

Figure 14

AS1517499 attenuated development of SPEM in stomachs of mice treated with L635. (A) Immunoblotting shows down-regulation of phosphorylated STAT6 and SOX9, a SPEM cell marker, in stomachs from mice treated with both L635 and AS1517499. Quantitation shows decreasing trend in STAT6 and SOX9 band intensity. Mean ± SD (N = 2 independent experiments). (B) Immunofluorescence staining of stomach tissues for H-K-ATPase, a parietal cell marker, and SPEM cell markers including AQP5 and GSII. Yellow arrowheads indicate GSII+/AQP5 positive SPEM cells, while GSII or AQP5 negative cells are indicated by an empty arrowhead. Quantitation of average percentage of gland bases expressing both AQP5 and GSII markers showed significant decrease under L635 and AS1517499 combined treatment. Mean ± SD (N = 16–17 images from 3 mice of each group). ∗∗∗∗P < .0001.

Figure 15

Human rIL-13 promotes phosphorylation of STAT6 and maturation and proliferation in SPEM cells in human metaplastic gastroids. (A) Representative bright field images of Matrigel domes containing human metaplastic gastroids treated with either rIL-13 or control (BSA). Quantitation of matched gastroid diameters showed significantly higher sizes in gastroids treated with rIL-13 compared with control after 5 days of treatment. Scale bar: 500 μm for brightfield. Mean ± SD (N = 3 independent experiments). ∗∗∗∗P < .0001. (B) Immunoblotting shows up-regulation of pSTAT6 in human metaplastic gastroids treated with rIL-13. Mean ± SD (N = 3 independent experiments). ∗∗∗P < .001. (C) Immunofluorescence staining of human metaplastic gastroids cross sections for Ki67, a proliferation marker, and SPEM-related markers, MUC6 and CD44v9. Quantitation of MUC6 and CD44v9 double positive cells demonstrated significant increase in proportion of mature SPEM cells in rIL-13–treated gastroids. (D) Quantitation of MUC6, CD44v9, and Ki67 triple positive cells demonstrated significant increase in proportion of proliferative SPEM cells. Yellow arrowheads indicate triple positive mature and proliferative SPEM cells. Scale bar: 50 μm. Mean ± SD (N = 8 from 3 independent experiments). ∗∗∗∗P < .0001.