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.