Optimized human intestinal organoid model reveals interleukin-22-dependency of paneth cell formation

Abstract

Opposing roles have been proposed for IL-22 in intestinal pathophysiology. We have optimized human small intestinal organoid (hSIO) culturing, constitutively generating all differentiated cell types while maintaining an active stem cell compartment. IL-22 does not promote the expansion of stem cells but rather slows the growth of hSIOs. In hSIOs, IL-22 is required for formation of Paneth cells, the prime producers of intestinal antimicrobial peptides (AMPs). Introduction of inflammatory bowel disease (IBD)-associated loss-of-function mutations in the IL-22 co-receptor gene IL10RB resulted in abolishment of Paneth cells in hSIOs. Moreover, IL-22 induced expression of host defense genes (such as REG1A, REG1B, and DMBT1) in enterocytes, goblet cells, Paneth cells, Tuft cells, and even stem cells. Thus, IL-22 does not directly control the regenerative capacity of crypt stem cells but rather boosts Paneth cell numbers, as well as the expression of AMPs in all cell types.

Keywords: IL-22; IL10RB; Paneth cells; anti-microbial proteins; enterocytes; inflammatory bowel disease; intestinal stem cells; mTOR; organoids; regeneration.

Graphical abstract

Figure 1

Optimal culture of human intestinal organoids with multi-differentiation capacity (A) Schematics of optimal culture model for hSIOs. hSIOs are patterned from conventional expansion culture and maintained in maturation media. (B) Medium composition comparison between conventional culture and optimal culture of hSIOs. (C–E) Representative confocal images of hSIOs cultured in conventional expansion medium (C), patterning medium for 14 days (D), and maturation medium with IL-22 (E). Representative marker genes for Paneth (DEFA5, red), enteroendocrine (CHGA, white/magenta), and goblet (MUC2, green) cells are highlighted by fluorescent reporters. Right: zoom-in image of the budding crypt. The black arrowheads highlight the Paneth cells located at the crypt bottom. Scale bars (C and D), 50 μm; (E) right scale bar, 50 μm; left scale bar, 200 μm. (F) Paneth (LYZ), enteroendocrine (CHGA), and goblet cells (PAS) in maturation medium (+IL-22)-cultured organoids detected by immunohistochemistry staining and transmission electron microscopy. Scale bars, 5 μm. See also Figure S2.

Figure 2

IL-22 regulates cellular diversity of hSIOs (A) UMAP of maturation medium (+IL-22)-cultured organoids for 14 days from scRNA-seq analysis (n = 1,283 cells). Descriptive cluster labels are shown. ISC, intestinal stem cell; TA1, transit-amplifying cell stage 1; TA2, transit-amplifying cell stage 2; EC, enterocyte; EEC, enteroendocrine cell. (B) Dot plot showing the relative expression and the percentage of cells expressing selected markers across scRNA-seq clusters. Two representative markers for each cluster are plotted. (C) Bar plot showing the relative Log₂ fold change of cell populations in maturation medium-cultured organoids with or without the addition of IL-22. (D) MA plot showing differentially expressed genes between maturation medium-cultured organoids with or without the addition of IL-22 by pseudobulk analysis of scRNA-seq. Representative marker genes for the different cell types are color-coded as in (C). See also Figures S3 and S4 and Tables S2 and S3.

Figure 3

IL-22 inhibits growth of human intestinal organoids (A) Bar plot showing the absolute total cell numbers and estimated ISC numbers of maturation medium-cultured organoids with or without the addition of IL-22 for 14 days. Data are shown as mean ± SEM. ^∗∗p < 0.01; two-tailed unpaired t test, n = 3. (B) Bar plot showing the relative clonal formation efficiency from singe cells of maturation medium-cultured organoids in response to addition, or upon withdrawal, of IL-22 for 14 days. Data are shown as mean ± SEM. ^∗∗∗∗p < 0.0001; one-way ANOVA compared with maturation medium-cultured organoids (−IL-22), n = 12 for each condition. (C) Representative images of long-term maturation medium-cultured organoids (4 passages) with or without the addition of IL-22 for 28 days. Scale bars, 500 μm. (D) Quantification of the percentage of budding and dying organoids. Data are shown as mean ± SEM. ^∗∗∗∗p < 0.0001; multiple t tests using two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli compared with maturation medium-cultured organoids (+IL-22) with Q = 5%, images n = 10 for each condition. (E) Cell proliferation as determined by FACS analysis of EdU positive cells. Data are shown as mean ± SEM. ns, not significant, multiple t tests using two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli, with Q = 5%, n = 3. (F) Cell death as determined by FACS analysis of propidium iodide positive cells. Data are shown as mean ± SEM. ^∗p < 0.05; multiple t tests using two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli, with Q = 5%, n = 3. (G) Stacked bar plot showing the comparison of the early enterocyte and enterocyte cell cluster proportions identified in the integrative scRNA-seq datasets from maturation medium-cultured organoids with or without IL-22. (H) Stacked bar plot showing the comparison of enterocyte-related gene expression (ANPEP, CYP3A4) in different cell clusters identified in the integrative scRNA-seq datasets from maturation medium-cultured organoids with or without the addition of IL-22. See also Figure S1.

Figure 4

IL-22 induces differentiation of human Paneth cells (A) Representative image of budding crypts of maturation medium-cultured organoids with or without the addition of IL-22 for 14 days. The black arrowheads highlight the Paneth cells located at the crypt bottom. Scale bars, 20 μm. (B) Representative confocal images of maturation medium-cultured organoids with or without adding IL-22. Scale bars, 200 μm. Representative marker genes for Paneth (DEFA5, red) cells are highlighted by fluorescent reporters. (C) Proportions of secretory lineages (Paneth cell, goblet cell, and EEC) as determined by FACS analysis in maturation medium-cultured (+IL-22) organoids without, with or upon withdrawal of IL-22. Data are shown as mean ± SEM. ^∗∗∗∗p < 0.0001; one-way ANOVA compared with maturation medium (+IL-22), n = 3 for each condition. (D) Proportion of Paneth cells as determined by FACS analysis of DEFA5 reporter in maturation medium-cultured organoids with addition of different dosages of IL-22 for 7 days. Data are shown as mean ± SEM. ns, not significant, ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001; one-way ANOVA compared with maturation medium (−IL-22), n = 3 for each condition. (E) Proportion of Paneth cells as determined by FACS analysis of DEFA5 reporter in maturation medium-cultured organoids in response to different cytokines for 7 days. Data are shown as mean ± SEM, n = 2. IL-22 (2 ng/mL); IL-6 (10 ng/mL); IL-10 (10 ng/mL); IL-12 (10 ng/mL); IL-17C (10 ng/mL); IL-20 (10 ng/mL); IL-23 (10 ng/mL); IL-26 (10 ng/mL); IL-27 (10 ng/mL); IL-29 (10 ng/mL); IL-33 (10 ng/mL). (F and G) Live imaging tracking Paneth cell and goblet cell reporter signals, an average with standard error (shadow area) of normalized fluorescence intensity from 3 organoids, 8 Paneth cells (F), and 7 goblet cells (G). 0 h in (F): Appearance of DEFA5 signals; 0 h in (G): starting timepoint of tracking. See also Figure S5.

Figure 5

IL10RB is required for IL-22-induced Paneth cell differentiation (A) Representative confocal image of maturation medium-cultured (+IL-22) organoids with wild-type (WT) genotype or IL10RB variants for 14 days. Scale bars, 200 μm. Representative marker genes for Paneth (DEFA5, red), enteroendocrine (CHGA, magenta), and goblet (MUC2, green) cells are highlighted by fluorescent reporters. (B) Proportions of secretory lineages (Paneth cell, goblet cell, and EEC) as determined by FACS analysis in maturation medium-cultured (+IL-22) organoids with WT genotype or IL10RB variants for 7 days. Data are shown as mean ± SEM. ^∗∗∗∗p < 0.0001; one-way ANOVA compared with WT, n = 3 for each line. (C) Representative images of immunohistochemistry staining of LYZ. Scale bars, 125 μm. (D) Quantification of LYZ-positive cells per image. Data are shown as mean ± SEM. ^∗∗∗∗p < 0.0001; one-way ANOVA compared with maturation medium (+IL-22), n = 5 for each condition. (E) RT-qPCR quantification of cell markers expression. Data are shown as mean ± SEM. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001; one-way ANOVA compared with maturation medium-cultured organoids (−IL-22), n = 3 for each treatment. See also Figure S5.

Figure 6

PI3K-mTOR signaling mediates Paneth cell differentiation downstream of IL-22 (A) Representative images of maturation medium-cultured organoids with different treatments described in the legends for 7 days. Scale bars, 500 μm. The treatments include the presence (+) or absence (−) of IL-22 (2 ng/mL). Small molecules BP-1-102 (5 μM), LY294002 (1 μM), MK-2206 (100 nM), and rapamycin (100 nM) were added to the organoids in combination with IL-22 (2 ng/mL). (B) Proportions of Paneth cells as determined by FACS analysis in maturation medium-cultured organoids by DEFA5-IRES-DsRed reporter with different treatments for 7 days. Data are shown as mean ± SEM. ns, not significant, ^∗∗∗p < 0.001, ^∗∗∗∗p < 0.0001; one-way ANOVA compared with maturation medium culture (+IL-22), n = 3 for each treatment. (C) Heat map representation of the proteins upregulated by IL-22 treatment for 3 days but reversed by rapamycin. Scale represents scaled protein abundance and ranges from low (blue) to high (red). (D) Schematics of IL-22 signaling in regulating human Paneth cell differentiation. Small molecule inhibitors targeting different signaling components are highlighted in red. See also Figure S6.

Figure 7

IL-22 induces host defense gene expression (A) Violin plots of IL22-induced module score in maturation medium-cultured organoids with or without IL-22 (left, average; right, split by cluster). (B) Stacked bar plots showing the ubiquitous upregulation of gene expression levels of REG1A, REG1B, DMBT1, CFI, and MUC1, the Paneth-specific induction of gene expression levels of DEFA6 and ITLN2, split by cell clusters, in maturation medium-cultured organoids with or without IL-22 by scRNA-seq. (C) RT-qPCR quantification of REG1A, REG1B, and DMBT1 expression in maturation medium-cultured organoids in response to IL-22 treatment after 24 h. Data are shown as mean ± SEM. ^∗p < 0.05, ^∗∗p < 0.01, ^∗∗∗p < 0.001; multiple t tests using two-stage linear step-up procedure of Benjamini, Krieger, and Yekutieli, with Q = 5%, n = 3. (D) Immunohistochemistry staining of REG1A/B (left: HPA045579, Human Protein Atlas) and DMBT1 (right: HPA040778, Human Protein Atlas) in human small intestine and colon. Scale bars, 200 μm. See also Figure S7.