Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 7 (1), 1-17
eCollection

IL22 Inhibits Epithelial Stem Cell Expansion in an Ileal Organoid Model

Affiliations

IL22 Inhibits Epithelial Stem Cell Expansion in an Ileal Organoid Model

Bailey Zwarycz et al. Cell Mol Gastroenterol Hepatol.

Abstract

Background & aims: Crohn's disease is an inflammatory bowel disease that affects the ileum and is associated with increased cytokines. Although interleukin (IL)6, IL17, IL21, and IL22 are increased in Crohn's disease and are associated with disrupted epithelial regeneration, little is known about their effects on the intestinal stem cells (ISCs) that mediate tissue repair. We hypothesized that ILs may target ISCs and reduce ISC-driven epithelial renewal.

Methods: A screen of IL6, IL17, IL21, or IL22 was performed on ileal mouse organoids. Computational modeling was used to predict microenvironment cytokine concentrations. Organoid size, survival, proliferation, and differentiation were characterized by morphometrics, quantitative reverse-transcription polymerase chain reaction, and immunostaining on whole organoids or isolated ISCs. ISC function was assayed using serial passaging to single cells followed by organoid quantification. Single-cell RNA sequencing was used to assess Il22ra1 expression patterns in ISCs and transit-amplifying (TA) progenitors. An IL22-transgenic mouse was used to confirm the impact of increased IL22 on proliferative cells in vivo.

Results: High IL22 levels caused decreased ileal organoid survival, however, resistant organoids grew larger and showed increased proliferation over controls. Il22ra1 was expressed on only a subset of ISCs and TA progenitors. IL22-treated ISCs did not show appreciable differentiation defects, but ISC biomarker expression and self-renewal-associated pathway activity was reduced and accompanied by an inhibition of ISC expansion. In vivo, chronically increased IL22 levels, similar to predicted microenvironment levels, showed increases in proliferative cells in the TA zone with no increase in ISCs.

Conclusions: Increased IL22 limits ISC expansion in favor of increased TA progenitor cell expansion.

Keywords: BSA, bovine serum albumin; EGFP, enhanced green fluorescent protein; FACS, fluorescence-activated cell sorter; IBD, inflammatory bowel disease; IL, interleukin; IL22RA1, IL22 receptor A1; IL22TG, IL22 transgenic; ILC, innate lymphoid cell; ILC3, IL22-secreting lymphocyte; ISC, intestinal stem cell; Inflammatory Bowel Disease; Interleukin-22; Intestinal Stem Cells; OFE, organoid forming efficiency; STAT3, signal transducer and activator of transcription 3; TA, transit-amplifying; TBS, Tris-buffered saline; cDNA, complementary DNA; mRNA, messenger RNA.

Figures

None
Figure 1
Figure 1
A focused screen of IBD-related cytokines shows that IL22 causes a dose-dependent decrease in organoid survival and an increase in organoid size. (A–D) Ileal organoid screen for cytokine effects on intestinal epithelium. (A) Schematic of experimental design in which ileal organoids were treated for 6 days with 100 pmol/L IL6, IL17, IL21, or IL22. (B) Representative images of treated organoids after 6 days. Scale bar: 100 μm. (C) Percentage change in area of organoids comparing day 0 with day 6. Technical replicate n = 10+ organoids; biological N = 3 mice; significance is relative to untreated control. (D) Organoid efficiency relative to control organoids. Technical replicate n = 3 wells; biological N = 3 mice; significance is relative to untreated control. (E) Organoid response to a range of concentrations of IL22 (0, 0.8, 4, 20, 100, and 500 pmol/L), measured by change in organoid area (left Y axis, black lines, technical replicate n = 10+ organoids) and organoid survival (right Y axis, red lines, technical replicate n = 3+ wells) after 6 days. Biological N = 3 mice/treatment; significance is in relation to 0 pmol/L IL22. (F) Organoid survival with 0, 60, or 500 pmol/L IL22 treatment. Technical replicate n = 3 wells; biological N = 3 mice. Asterisks denote significance between the treatment group and control at the designated time point. (G and H) Organoids were treated with 500 pmol/L IL22 in the presence of 206 ng/mL IL22 neutralizing antibody or 206 ng/mL IgG of the same species. (G) Top: Representative images of treated organoids. Bottom: Quantification of organoid area. Technical replicate n = 10+ organoids; biological N = 3 mice; significance is relative to IgG-only control. (H) Top: Representative Western blot images for pSTAT3 and β-actin. Scale bar: 100 μm. Bottom: Quantification of intensity of Western blot bands normalized within each blot to the band with the highest intensity. Technical replicate n = 3 blots, biological N = 3 mice. Significance was calculated by 1-way analysis of variance with a Bonferroni correction for multiple comparisons. *P < .05, **P < .01, ***P < .001, ****P < .0001. pSTAT3, phosphorylated signal transducer and activator of transcription 3.
Figure 2
Figure 2
Il22ra1 is expressed heterogeneously throughout the crypt. (A) The Il22ra1 gene expression profile characterized in FACS-isolated total epithelium (CD326+), absorptive/goblet differentiated cells (Sox9-EGFPneg), TA progenitor cells (Sox9-EGFPsublow), ISCs (Sox9-EGFPlow), enteroendocrine/tuft cells (Sox9-EGFPhigh), and Paneth cells (Sox9-EGFPhigh, CD24high). Technical replicate n = 3; biological N = 3 mice. Significance was calculated by 1-way analysis of variance with Tukey multiple comparisons; bars that are not connected by the same letter are statistically significant (P < .05). (B) Left: t-SNE analysis of single-cell RNA sequence analysis of mouse small intestinal epithelium. Each color represents a different population defined in the original analysis based on lineage-specific transcriptomic signatures. Right: Table depicts the number of cells in each lineage category expressing IL22ra1. (C) Cells from the t-SNE profiles in the graph in panel B are highlighted specifically for the expression of IL22ra1 levels in all epithelial cells. Darker shades of grey represent higher expression levels. Pink circles represent no expression. (D) The same analysis in panel C except only ISCs are shown. (E) The same analysis in panel C except only TA progenitors are shown. (F) Representative immunohistochemistry of IL22RA1 (red) and cell nuclei (blue) in a mouse ileal crypt. (G) FACS analysis of fixed cell populations described in panel A stained for IL22RA1. Technical replicate n = 3; biological N = 3 mice. Bars represent parts of whole. EC, enterocyte; EE, enteroendocrine; EEC, enteroendocrine; Max, maximum; Min, minimum.
Figure 3
Figure 3
COMSOL Multiphysics simulation of IL22 diffusion. (A) Representative in vivo image of mouse ILC3 follicle with RORγt+ ILC3 immune cells (pink) and cell nuclei (blue). Parameters in table were determined empirically, n = 10+ measured values per mouse, N = 3 mice. Scale bar: 100 μm. (B) Left: Single ILC3 IL22 secretion model in which values of IL22 concentration were calculated by a computational model along a line segment with a length of 9 μm surrounding 1 ILC3 cell. Right: Concentration of IL22 over time at 4 points at 3-μm increments. (C) Left: Follicle model in which values of IL22 concentration were calculated along a line segment with a length of 21 μm. Right: Concentration of IL22 from a lymphoid follicle over time at 4 points at 7-μm increments.
Figure 4
Figure 4
Cell lineage analysis in single ISCs and ileal organoids treated with IL22. (A) Gene expression analysis of single Sox9-EGFPlow single ISCs after 6 hours with or without 500 pmol/L IL22 examining expression of differentiated cell genes. Technical replicate n = 3, biological N = 3 mice. (B) Gene expression analysis of organoids after 6 days with or without 500 pmol/L IL22 examining expression of differentiated cell genes including SI (enterocytes), Lyz2 (Paneth cells), Muc2 (goblet cells), and ChgA (enteroendocrine cells). (C and D) Immunohistochemistry staining and quantification of organoids treated for 6 days with IL22 then stained for lysozyme (LYZ, red), (EPCAM) (green), and nuclei (blue). Technical replicate n = 10+ organoids; biological N = 3 mice. (C) Left: Representative intraluminal LYZ staining (red). Right: Quantification of intraluminal stain. (D) Left: Representative organoid staining. Right: Quantification of total number of LYZ+ cells relative to total nuclei per organoid. Significance was calculated using an unpaired t test relative to the untreated control. Scale bar: 100 μm. *P < .05, **P < .01, ***P < .001, and ****P < .0001.
Figure 5
Figure 5
IL22 limits ISC expansion. (A) Representative immunohistochemistry for the proliferation marker KI67 (red) and nuclei (blue) with quantification of the proportion of KI67+ nuclei. Technical n = 10+ organoids; biological N = 3 mice. Scale bar: 100 μm. (B–D) Gene expression analysis of organoids after 6 days with or without 500 pmol/L IL22 for (B) ISC-associated genes including Lgr5, Olfm4, Ascl2, and Sox9; (C) Wnt signaling pathway-associated genes including Wnt3, Ctnnb1, and Axin2; and (D) Notch signaling pathway-associated genes including Notch1, Notch2, Dll1, Dll4, Hes1, and Atoh1. Technical replicate n = 3, biological N = 3 mice. Significance was calculated using an unpaired t test relative to the untreated control. (E) Percentage organoid increase in response to 0, 60, or 500 pmol/L IL22 at each passage compared with the number of organoids at initial plating at p0. Technical replicate n = 3, biological N = 3 mice. Significance was calculated using 1-way analysis of variance with Bonferroni correction at each time point in comparison with control. +P < .05 for 500 pmol/L IL22 compared with control at passage 1. *P < .05, **P < .01, ***P < .001, and ****P < .0001.
Figure 6
Figure 6
Increased IL22 causes an increase in TA progenitors. Quantification of (A) total number of cells per crypt, (B) total crypt height, and (C) height of TA zone in control and IL22TG mice. (D) Representative immunohistochemistry for the proliferation marker KI67 (red) and nuclei (blue). Quantification of (E) the percentage of positive KI67 cells per crypt and (F) the total number of OLFM4+ cells per crypt. (G) Representative immunohistochemistry for the ISC marker OLFM4 (green) and nuclei (blue). All quantification: Technical replicate n = 10+ crypts/mouse, N = 3 mice/treatment. Scale bar: 100 μm. **P < .01, ***P < .001. Significance was calculated using an unpaired t test relative to the untreated control.

Comment in

Similar articles

See all similar articles

Cited by 4 articles

References

    1. Fakhoury M., Negrulj R., Mooranian A., Al-Salami H. Inflammatory bowel disease: clinical aspects and treatments. J Inflamm Res. 2014;7:113–120. - PMC - PubMed
    1. van der Sloot K.W.J., Amini M., Peters V., Dijkstra G., Alizadeh B.Z. Inflammatory bowel diseases: review of known environmental protective and risk factors involved. Inflamm Bowel Dis. 2017;23:1499–1509. - PubMed
    1. Neurath M.F. Cytokines in inflammatory bowel disease. Nat Rev Immunol. 2014;14:329–342. - PubMed
    1. Wolk K., Witte E., Hoffmann U., Wolf-Dietrich D., Endesfelder S., Asadullah K., Wolfram S., Volk H., Wittig B.M., Sabat R. IL-22 induces lipopolysaccharide-binding protein in hepatocytes: a potential systemic role of IL-22 in Crohn’s disease. J Immunol. 2007;178:5973–5981. - PubMed
    1. Li J., Zhang L., Zhang J., Wei Y., Li K., Huang L., Zhang S., Gao B., Wang X., Lin P. Interleukin 23 regulates proliferation of lung cancer cells in a concentration-dependent way in association with the interleukin-23 receptor. Carcinogenesis. 2013;34:658–666. - PubMed

Publication types

MeSH terms

Feedback