IL-33 Induces Murine Intestinal Goblet Cell Differentiation Indirectly via Innate Lymphoid Cell IL-13 Secretion This information is current as Amanda Waddell, Jefferson E. Vallance, Amy Hummel, of September 29, 2021. Theresa Alenghat and Michael J. Rosen J Immunol published online 7 December 2018 http://www.jimmunol.org/content/early/2018/12/06/jimmun ol.1800292 Downloaded from Supplementary http://www.jimmunol.org/content/suppl/2018/12/06/jimmunol.180029 Material 2.DCSupplemental http://www.jimmunol.org/ Why The JI? Submit online. • Rapid Reviews! 30 days* from submission to initial decision • No Triage! 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Published December 7, 2018, doi:10.4049/jimmunol.1800292 The Journal of Immunology IL-33 Induces Murine Intestinal Goblet Cell Differentiation Indirectly via Innate Lymphoid Cell IL-13 Secretion Amanda Waddell,* Jefferson E. Vallance,* Amy Hummel,* Theresa Alenghat,†,‡ and Michael J. Rosen*,‡ Regulation of the intestinal mucus layer by goblet cells is important for preventing inflammation and controlling infection. IL-33, a cytokine upregulated in inflammatory bowel disease and helminth infection, induces intestinal goblet cells, but the mechanism remains unclear. Enteroids are three-dimensional structures of primary small intestinal epithelial cells that contain all differen- tiated intestinal epithelial cell types. We developed an enteroid–immune cell coculture model to determine the mechanism through which IL-33 affects intestinal goblet cell differentiation. We report that IL-33 does not directly induce goblet cell differentiation in murine enteroids; however, IL-13, a cytokine induced by IL-33, markedly induces goblet cells and gene expression consistent with goblet cell differentiation. When enteroids are cocultured with CD90+ mesenteric lymph node cells from IL-33–treated mice, IL-33 Downloaded from then induces IL-13 secretion by group 2 innate lymphoid cells and enteroid gene expression consistent with goblet cell differen- tiation. In cocultures, IL-33–induced Muc2 expression is dependent on enteroid Il4ra expression, demonstrating a requirement for IL-13 signaling in epithelial cells. In vivo, IL-33–induced intestinal goblet cell hyperplasia is dependent on IL-13. These studies demonstrate that IL-33 induces intestinal goblet cell differentiation not through direct action on epithelial cells but indirectly through IL-13 production by goup 2 innate lymphoid cells. The Journal of Immunology, 2019, 202: 000–000. http://www.jimmunol.org/ oblet cells generate the first line of defense at the intestinal associated with ulcerative colitis (4). Mucins are also upregulated mucosa, primarily through secretion of the mucin MUC2, during helminth infections and are important for their expulsion (5). G which generates the intestinal mucus layer (1). The mucus IL-33 is a member of the IL-1 family of cytokines that signals layer prevents the luminal contents, particularly intestinal micro- through the IL-33R (also called ST2) (6). Many cell types express biota, from coming into contact with intestinal epithelial cells. IL-33R, including immune cells, epithelial cells, and stromal cells However, when the mucus becomes penetrable, this leads to colitis (7–10). Mucosal IL-33 is increased during helminth infection and in mice and is associated with disease in ulcerative colitis patients colitis. IL-33 augments type 2 cytokine (IL-4, IL-5, IL-13) pro- (2, 3). In fact, rare variants in MUC2 were recently found to be duction from T cells and innate lymphoid cells (ILCs), which is important for helminth expulsion (11–13). Injection of supra- by guest on September 29, 2021 *Division of Gastroenterology, Hepatology and Nutrition, Cincinnati Children’s physiological levels of IL-33 induces goblet cells in the healthy Hospital Medical Center, Cincinnati, OH 45229; †Center for Inflammation and mouse intestine (6). We and others have demonstrated that IL-33 Tolerance, Division of Immunobiology, Cincinnati Children’s Hospital Medical and IL-33R are protective in murine models of ulcerative colitis, Center, Cincinnati, OH 45229; and ‡Department of Pediatrics, University of Cincinnati College of Medicine, Cincinnati, OH 45229 in part through preservation of goblet cells (14, 15). However, the ORCIDs: 0000-0002-0418-253X (A.H.); 0000-0003-4514-3710 (T.A.); 0000-0002- mechanism through which IL-33 regulates intestinal goblet cells, 5149-718X (M.J.R.). whether direct or indirect, and the important secreted intermedi- Received for publication February 27, 2018. Accepted for publication November 6, aries remains under debate (8, 14). 2018. Enteroids are structures of primary small intestinal epithelium This work was supported by the National Institute of Diabetes and Digestive and grown from isolated crypt stem cells that contain the full com- Kidney Diseases of the National Institutes of Health under Awards K23DK094832, R03DK110487, and R01DK117119 to M.J.R., R01DK114123 to T.A., and plement of differentiated intestinal epithelial cell types, including P30DK078392 for the Gene Analysis and Integrated Morphology Cores of the Di- goblet cells (16). The application of enteroids to the study of the gestive Disease Research Core Center in Cincinnati. This work was also supported by a Crohn’s & Colitis Foundation Research Fellowship Award to A.W. and funding intestinal epithelium overcomes many of the limitations of con- from CURE for IBD. ventional transformed cell lines, which may behave differently A.W.: obtaining funding, experimental design, execution of experiments, analysis than primary cells and have limited capacity for differentiation. and interpretation of data, and drafting of the manuscript; J.E.V.: experimental de- The refinement of techniques to coculture enteroids with other cell sign, execution of experiments, analysis and interpretation of data, and drafting of the manuscript; A.H.: execution of experiments, critical review of the manuscript for types holds promise for advancing studies of the interaction be- important intellectual content; T.A.: contribution of critical reagents; M.J.R.: obtain- tween the intestinal epithelium and other cellular compartments, ing funding, study concept and supervision, experimental design, analysis and inter- pretation of data, and critical revision of the manuscript for important intellectual such as mucosa-associated immune cells (17, 18). content. In this study, we modeled intestinal immune–epithelial inter- Address correspondence and reprint requests to Dr. Michael J. Rosen, Division of actions by coculturing murine enteroids with mesenteric lymph Gastroenterology, Hepatology, and Nutrition, Cincinnati Children’s Hospital Medical node (MLN) cells enriched for group 2 ILCs (ILC2s) to demon- Center, 3333 Burnet Avenue, MLC 2010, Cincinnati, OH 45229. E-mail address: [email protected] strate that IL-33 induces epithelial goblet cell differentiation The online version of this article contains supplemental material. through stimulation of ILC2s to produce IL-13. IL-13, but not Abbreviations used in this article: Areg, amphiregulin; CMF, colon myofibroblast; IL-33, directly induced goblet cell differentiation in enteroids ILC, innate lymphoid cell; ILC2, group 2 ILC; MLN, mesenteric lymph node; PAS, cultured alone. IL-33 induction of goblet cell differentiation was periodic acid Schiff; UEA I, Ulex europaeus agglutinin I; WT, wild-type. dependent on the presence of ILC2-enriched MLN cells and Copyright Ó 2018 by The American Association of Immunologists, Inc. 0022-1767/18/$37.50 enteroid IL-13 signaling in vitro and on IL-13 in vivo. www.jimmunol.org/cgi/doi/10.4049/jimmunol.1800292 2 IL-33 AND GOBLET CELL DIFFERENTIATION Materials and Methods Carlsbad, CA) for Spdef (Mm00600221_m1), Atoh1 (Mm00476035_s1), Mice and in vivo treatment Muc2 (Mm00524818_m1), amphiregulin (Areg; Mm01354339_m1), Il13 (Mm00434204_m1), Il1rl1 (Mm01233982_m1), Retnlb Il132/2 (BALB/C), Il4ra2/2 (BALB/C) (strain 003514; The Jackson Lab- (Mm00445845_m1), and Gapdh (Mm99999915_g1). All reactions oratory), Il1rl1 (IL33-R)2/2 (C57BL/6), Lgr5-EGFP-IRES-creERT2 (strain were performed on a StepOnePlus Real-Time PCR System (Thermo Fisher Scientific). Relative mRNA levels were determined using the 008875; The Jackson Laboratory), and wild-type (WT) C57BL/6 and BALB/C 2 mice were bred at Cincinnati Children’s Hospital Medical Center under threshold cycle (2 DDCT) method with Gapdh as the reference. specific pathogen-free conditions and maintained on a standard laboratory chow diet in a half-day light cycle exposure and temperature-controlled Western blot environment. Male and female strain-matched mice were used and were Membranes were blocked with 5% nonfat dry milk in TBS-Tween 20 age 6–12 wk at the start of the experiments. The generation of the Il132/2 2/2 (0.05%)