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IL-33 Induces Murine Intestinal Differentiation Indirectly via IL-13

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

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The Journal of Immunology is published twice each month by The American Association of Immunologists, Inc., 1451 Rockville Pike, Suite 650, Rockville, MD 20852 Copyright © 2018 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. 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 layer by goblet cells is important for preventing inflammation and controlling . IL-33, a 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). are also upregulated mucosa, primarily through secretion of the MUC2, during helminth 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 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 , Hepatology and , 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 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 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 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 -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 in TBS-Tween 20 age 6–12 wk at the start of the experiments. The generation of the Il132/2 2/2 (0.05%) for 1 h and incubated overnight at 4˚C with primary Abs against mice and Il1rl1 mice was previously described (19, 20). Mice were actin (Seven Hills Bioreagents, Cincinnati, OH) and Phospho-p44/42 given PBS or 0.4 mg rIL-33 daily i.p. for 4 d. The study was carried out MAPK (ERK1/2) (Cell Signaling Technology, Danvers, MA). Mem- following recommendations in the Guide for the Care and Use of Labo- branes were incubated with HRP-linked secondary Abs, anti- or anti- ratory of the National Institutes of Health. The Cincinnati mouse, and visualized using ECL Prime and Fujifilm LAS 4000 Gel Children’s Hospital Medical Center Institutional Care and Use Documentation system (GE Healthcare, Piscataway, NJ). Band densitom- Committee approved the protocol. etry was determined using ImageJ (National Institutes of Health), and Generation of murine enteroids and cocultures the ratio of the intensity of Phospho-p44/42 MAPK to actin was used to determine fold activation of signaling. Mouse was dissected and flushed with ice-cold PBS. The ileum was opened longitudinally, cut into 1-cm pieces, and incubated in 2 mM Flow cytometric analysis and cell sorting

EDTA for 30 min at 4˚C with rocking. The was transferred into a new For coculture experiments, MLN cells from IL-33–treated mice were Downloaded from tube containing 5 ml of shaking buffer (PBS, 43.3 mM , 54.9 mM stained with PerCp-Cy5.5–conjugated CD90.2 (30-H12) and APC-Cy7– ) and shaken gently by hand for 2 min. Dispersed crypts were plated conjugated CD45 (30-F11; BioLegend), followed by cell sorting with a overnight in Matrigel (Corning, Corning, NY) with enteroid growth media FACSAria II (BD Biosciences, San Jose, CA). For intracellular cytokine (Advanced DMEM/F12, 2 mM GlutaMax, 10 mM HEPES, 100 U/ml pen- 3 3 analyses, after restimulation with PMA /ionomycin and treatment with icillin, 100 mg/ml streptomycin, 1 N2 supplement, 1 B27 supplement [all Golgi blocker, MLN cells were stained with biotinylated anti–T1/ST-2 from Invitrogen]) containing EGF (50 ng/ml; Sigma-Aldrich, St. Louis, (DJ8; MD Biosciences, Oakdale, MN), followed by Streptavidin-BV650, MO), 20% L-WRN conditioned media (L-WRN cells from American Type APC-conjugated anti-CD4 (GK1.5), FITC-conjugated anti-CD3 (17A2), Culture Collection, Manassas, VA). The following day, 100 ng/ml IL-33 and PerCp-Cy5.5–conjugated B220 (RA3-6B2), FCεRI (MAR-1), CD11c http://www.jimmunol.org/ (PeproTech, Rocky Hill, NJ) was added daily to the cultures for up to 5 d. (N418), NK1.1 (PK136), and CD11b (M1/70) (BioLegend). Stained cells Growth factors were replenished every other day. For cocultures, enteroids were fixed and permeabilized for intracellular cytokine staining by using were plated on hanging transwells (Corning). MLN cells from IL-33–treated PE-conjugated anti–IL-13 (eBio13A; eBioscience, Waltham, MA). MLN or naive mice (400,000 cells) were plated in the well and were stimulated and LP cells were then analyzed with an LSR II (BD Biosciences). with IL-2 (PeproTech), IL-7 (PeproTech), and anti–IFN-g (BioLegend, San Diego, CA) with or without IL-33 for 4 d. WEHI-YH2 cells (kindly provided Statistical analysis by A. Burgess) (21) were plated to confluency in 24-well plates. Primary murine colon myofibroblasts (CMF) were generated using the methods For all data from experiments with three or more groups, the nonparametric previously described (22). In brief, mouse colons were treated with EDTA Kruskal–Wallis test was performed, followed by the two-stage step-up followed by with collagenase, and single cells were plated onto method of Benjamini, Krieger, and Yekutieli for false discovery rate. tissue culture plates for 3 h before nonadherent cells were removed. Passage Data from experiments with two groups were analyzed using the non- by guest on September 29, 2021 2 was plated to confluency in 24-well plates for coculture experiments. For parametric Mann–Whitney U test. Individual data points and medians are each experiment, a separate mouse was used to generate enteroids, and each plotted on all graphs. The analysis was performed on Prism software experiment was performed in triplicate. (version 7.0b; GraphPad Software, La Jolla, CA).

Immunofluorescence Results Enteroids were plated in a very thin layer of Matrigel, or myofibroblasts were IL-33 does not directly induce goblet cells in murine enteroids plated on ibidi 4-well chamber slides. Enteroids were stimulated for 5 d with rIL-33 (100 ng/ml) or rIL-13 (10 ng/ml). Enteroids or myofibroblasts were We first tested whether IL-33 directly induces goblet cells in fixed with 4% paraformaldehyde and permeabilized in PBS containing 0.1% primary murine enteroids. Fluorescence microscopy demon- Tween 20. Enteroids or myofibroblasts were stained using rabbit anti-ST2 strated that IL-33R is expressed on murine enteroids derived from (1:100, AB25877; Abcam), chicken anti-GFP (1:1000, GFP-1010; Aves mouse ileal crypts (Fig. 1A). Both goblet cells, labeled by UEA I Laboratories), rabbit anti-IL4r (1:100, PAS-38615; Invitrogen), rabbit anti- IL13RA1 (1:100, PAS-50989; Invitrogen), or rabbit anti-vimentin (1:100, staining of mucins, and stem cells,labeledbyanti-GFPinLgr5- ab45939; Abcam) followed by donkey anti-rabbit AF594 (1:200; Jackson EGFP-IRES-creERT2 reporter mice, expressed IL-33R as well ImmunoResearch) or donkey anti-chicken AF488 (1:200; Jackson Immuno- as IL-4RA and IL-13RA1 (Fig. 1A–C and Supplemental Fig. Research). Cells were also stained with FITC–Ulex europaeus agglutinin 1A–C). We then stimulated enteroids with rIL-33 (10 ng/ml, (UEA) I (Vector Labs, Burlingame, CA) and Alexa Fluor 647 Phalloidin (A22287; Thermo Fisher Scientific), and nuclei were counterstained with data not shown or 100 ng/ml) for up to 5 d to determine the Hoechst (1:1000, B2261; Sigma-Aldrich). Enteroids were visualized using a effect of IL-33 on goblet cell differentiation. By quantitative Nikon A1 inverted confocal microscope. Five-micrometer confocal optical real-time PCR, we did not detect alteration in the expression sections were opened in NIS Viewer (Nikon), and nuclei and goblet cells were of the factors Atoh1 and Spdef, which direct counted. commitment to the secretory and goblet cell lineages, respec- Histopathology tively, or Muc2, the primary mucin produced by intestinal goblet cells, or Retnlb and Tff3, secretory products of goblet cells (Fig. 1D, Mouse colon and sections were stained with periodic acid Schiff (PAS), and staining was quantified (3.14 6 0.21 mm2 of distal colon and data not shown for Tff3). 1.67 6 0.15 mm2 of jejunum) using a modified nuclear algorithm with Aperio Because IL-33 induces IL-13 production by T cells and ILCs Imagescope software (Buffalo Grove, IL) as previously described (15). (6, 11), and others have shown that IL-13 induces intestinal RNA expression goblet cell differentiation (19, 23), we tested whether IL-13 in- duces goblet cell differentiation in our primary murine enteroid RNA was isolated from tissue using the RNeasy Mini Kit (QIAGEN, cultures. As expected, IL-13 significantly induced expression of Valencia, CA) per the manufacturer’s instructions. RNA (100 ng) was re- verse transcribed using the High-Capacity cDNA Reverse Transcription Kit Atoh1, Spedf, Muc2,andRetnlb (Fig. 1E). However, Tff3 was not (Thermo Fisher Scientific, Waltham, MA). Quantitative real-time PCR was induced (data not shown). We performed confocal microscopy performed with TaqMan Gene Expression Assays (Life Technologies, analysis of goblet cells using UEA I staining, which confirmed The Journal of Immunology 3 Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 1. IL-33 and IL-13 effects on goblet cell differentiation in murine enteroids. Immunofluorescence microscopy for (A) IL-33R, (B) IL-4RA, and (C) IL-13RA1 (red) in enteroids counterstained with Hoechst (blue), phalloidin (pink), and UEA I or LGR5 (green). Scale bar, 20 mm. (D and E) Graphs of quantitative real-time PCR analysis of enteroids treated with (D) IL-33 (100 ng/ml) or (E) IL-13 (10 ng/ml). (F) Representative photomicrographs of UEA I FITC (green) immunofluorescence microscopy in enteroids treated for 4 d with PBS, IL-33, or IL-13. Enteroids are counterstained with DAPI (blue) and phalloidin (pink). Scale bar, 20 mm. (G) Quantification of goblet cells. White arrows indicate goblet cells. Seven to nine wells per condition pooled from three independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001. that IL-13, but not IL-33, directly induced goblet cells in We chose to examine both intestinal myofibroblasts and immune enteroids (Fig. 1F, 1G). cells, because both have been shown to respond to IL-33 and can make mediators that could affect goblet cell differentiation and IL-33 induction of goblet cells is dependent on + + mucus production (6, 9, 12, 14, 24, 25). To examine the role of intestine-associated CD45 CD90 immune cells in vitro secreted factors from an intermediate cell type, we developed a Because we did not observe a direct effect of IL-33 on enteroid coculture system whereby murine enteroids were suspended on a goblet cells, we hypothesized that IL-33 affects goblet cells in- semipermeable membrane over other cell types and both were directly through action on either myofibroblasts or lymphoid cells. exposed to IL-33 (Fig. 2A). We demonstrated that MLN cells, the 4 IL-33 AND GOBLET CELL DIFFERENTIATION Downloaded from

FIGURE 2. IL-33 induced goblet cells in enteroids cocultured with immune cells but not those with myofibroblasts. (A) Diagram of in vitro model + wherein enteroids in Matrigel were suspended in transwells in coculture over either CD90 MLN cells from IL-33–treated mice, WEHI-YH2 myofibroblast http://www.jimmunol.org/ cells, or primary CMF with or without IL-33 (100 ng/ml). (B) Il1rl1 expression in CMF, WEHI-YH2, and MLN cells. (C) Immunofluorescence analysis of IL-33R and vimentin in primary CMF counterstained with Hoechst. Scale bar, 25 mm. Atoh1, Spdef, and Muc2 expression in enteroids cocultured with (D) primary CMF or WEHI-YH2 cells, (E) WT CD90+,or(F) Il1rl12/2 CD90+ MLN cells was assessed by quantitative real-time PCR. Data are graphed as medians. n = 8–16 wells pooled from three or four independent experiments. *p , 0.05, ***p , 0.001. colonic subepithelial myofibroblast cell line WEHI-YH2 cells, and To identify the MLN cells producing IL-13, flow cytometry primary CMF all expressed Il1rl1 (Fig. 2B). Furthermore, immu- analysis was performed for IL-13 and markers for T cells and nofluorescence analysis demonstrated that CMF expressed IL-33R, ILC2s. All cells were negative for lineage markers B220, CD11b, and purity of this population was confirmed with vimentin staining NK1.1, FCεRI, and CD11c (data not shown). Both CD4+ and by guest on September 29, 2021 (Fig. 2C). To determine whether myofibroblasts are required for CD4– cells expressed IL-13 (Fig. 3C). However, IL-33 treatment IL-33 to affect epithelial goblet cells, we treated enteroids cocultured in vitro only induced IL-13 production in CD4– cells, and 94 6 with WEHI-YH2 cells or CMF with IL-33 for 4 d. We observed no 3.8% of the IL-13–expressing cells after IL-33 treatment were change in enteroid Muc2 expression (Fig. 2D). CD4– (Fig. 3D). Both CD4+ and CD4– IL-13–producing cells also We then sought to determine whether intestine-associated T cells expressed IL-33R, which is known to be expressed on Th2 cells or ILCs are required for IL-33 to affect enteroid goblet cells by and ILC2s (26) (Fig. 3C). As expected, CD4+IL-13+ cells also treating enteroids cocultured with CD45+CD90+ MLN cells from expressed CD3, whereas CD4–IL-13+ cells were CD3–, consistent IL-33–treated mice (IL-33 i.p.) with additional IL-33 in vitro with an ILC2 phenotype (Lineage–CD90+CD4–CD3–IL-33R+, (IL-33 i.p. plus IL-33). IL-33 treatment in vitro significantly in- Fig. 3C). We confirmed the finding from C57BL/6 mice in BALB/C duced Atoh1, Spdef, and Muc2 expression in enteroids cocultured mice, because BALB/C mice are known to be Th2 prone and with MLN cells (Fig. 2E). Furthermore, increases in goblet cell were required for other experiments (Fig. 3E) (27). In BALB/C markers were dependent on IL-33R expression on CD90+ MLN mice, we used mice without IL-33 i.p. (naive) because IL-33 i.p. cells. Il1rl12/2 CD90+ MLN cells treated with IL-33 and cocul- leads to high levels of IL-13 secretion in vitro even without tured with enteroids did not induce Atoh1, Spdef,orMuc2 ex- additional IL-33 added in vitro (Fig. 4A). Similarly to C57BL/6 pression compared with media alone (Fig. 2F). Collectively, these mice, BALB/C mice exhibited a large increase in ILC2s with data show that IL-33R–expressing T cells and/or ILCs are re- IL-33 stimulation in vitro, but there was not a significant increase quired for IL-33 to induce epithelial goblet cells. in IL-13+CD4+ cells from naive to IL-33 i.p. or IL-33 i.p. plus IL-33 (Fig. 3E). IL-13 produced by primarily ILC2s is required for To confirm that epithelial cell–intrinsic IL-13 signaling is re- IL-33–induced goblet cell differentiation in vitro quired for IL-33 induction of Muc2 expression, we generated Because IL-33 induces IL-13 production by T cells and ILCs, and enteroids from Il4ra2/2 BALB/C mice (IL-4RA is a component of we observed that IL-13 directly induces goblet cell differentiation the IL-13RA1 heterodimer). CD45+CD90+ MLN cells were iso- in murine enteroids (Figs. 1, 2), we tested the role of IL-13 lated from naive or IL-33 i.p. mice, cocultured with enteroids signaling in the interaction between IL-33, intestinal immune derived from WT or Il4ra2/2 mice, and, in some conditions, cells, and epithelial cells. IL-33 significantly induced Il13 ex- stimulated with additional rIL-33. MLN cells from IL-33 i.p. mice pression, as detected by quantitative real-time PCR (Fig. 3A), and secreted IL-13 in vitro (5.9 6 1.5 ng/ml in the media from IL-33 IL-13 production, as detected by ELISA (Fig. 3B), in i.p. mice versus undetectable in that of naive mice), which was MLN cells cocultured with enteroids. IL-4 was undetectable at further augmented by the addition of IL-33 to the culture media baseline, and IL-33 induced minimal IL-4 production (0.45 6 (45.7 6 13.6 ng/ml, Fig. 4A). Comparatively very small amounts 0.26 pg/ml IL-4 compared with 4297 6 698 pg/ml IL-13). of IL-4 were secreted by the MLN cells from the IL-33 i.p. mice The Journal of Immunology 5 Downloaded from http://www.jimmunol.org/

FIGURE 3. IL-33 induced IL-13 from ILC2s in MLN–enteroid cocultures. CD90+ MLN cells from C57BL/6 mice were cocultured with enteroids for 4 d with or without 100 ng/ml IL-33, and IL-13 was assessed. (A) Il13 quantitative real-time PCR. (B) IL-13 ELISA. (C) Representative flow plots for by guest on September 29, 2021 CD4+ and CD4– IL-13+ cells from cocultured MLN cells from C57BL/6 mice and expression of IL-33R and CD3 on CD4+ and CD4– IL-13+ cells. (D) Quantification of IL-13+ cells from (C). (E) Representative flow plots for CD4+ and CD4– IL-13+ cells from cocultured MLN cells from BALB/C mice. (F) Quantification of IL-13+ cells from (E). n = 6–9 wells pooled from three independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001.

(0.006 6 0.001 ng/ml), and this was not increased with additional cell hyperplasia in vivo. We administered IL-33 i.p. for 4 d to WT IL-33 in vitro (Fig. 4A). and Il132/2 mice and sacrificed mice to examine intestinal goblet Muc2 and Atoh1 expression was significantly increased in WT cells. In the colon, IL-33 significantly increased the number of PAS+ enteroids cocultured with MLN cells from IL-33 i.p. mice both goblet cells in WT mice but had no effect on goblet cell numbers in with and without IL-33 in culture (Fig. 4B). There were numerical Il132/2 mice (Fig. 6A, 6B). In line with this finding, colon mucosal but not statistically significant increases in Spedf in cocultures Atoh1, Spdef,andMuc2 expression was increased in IL-33–treated with MLN cells from IL-33 i.p. mice both with and without IL-33 WT mice but not in Il132/2 mice (Fig. 6B). in culture. In contrast, no increase in Atoh1, Spdef,orMuc2 was We observed similar effects in the , with IL-33 observed in Il4ra2/2 enteroids cocultured with MLN cells from inducing increased goblet cells in WT but not Il132/2 mice IL-33 i.p. mice with or without IL-33 (Fig. 4C). These studies (Fig. 6C, 6D). There were also numerical increases in mean small indicate that goblet cell differentiation induced by IL-33 in intestinal mucosal Atoh1, Spdef, and Muc2 expression with IL-33 enteroids is dependent on IL-13 signaling in the enteroids. treatment in WT but not Il132/2 mice in vivo. These differences MLN cells cultured with IL-33 also expressed increased levels of did not reach statistical significance, likely because of high vari- mRNA for the ligand Areg (Fig. 5A), which ability in the expression of these mRNAs in the small intestine has been shown to be important for maintaining goblet cells during (Fig. 6D). Interestingly, there was some signal, albeit inconsistent, dextran sodium sulfate–induced colitis (14). Although Areg (50 ng/ml) for increased goblet cell markers in Il132/2 mice at baseline did induce phopho-ERK1/2 in enteroids (Fig. 5B), it did not alter compared with WT, which decreased with IL-33 treatment. These Atoh1, Spdef,orMuc2 expression (Fig. 5C), further supporting the numerical differences were only statistically significant in the hypothesis that IL-13 secreted predominantly from ILC2s is pri- cases of increased small intestinal Spdef expression in Il132/2 marily responsible for the effect of IL-33 on intestinal goblet cells. mice compared with WT mice and decreased Atoh1 expression in IL-33–treated compared with untreated Il132/2 mice. Induction of goblet cells by IL-33 in vivo is dependent on IL-13 Because others have demonstrated that Areg is required for Although is known that IL-13 induces goblet cell hyperplasia in vivo IL-33 induction of goblet cells during inflammation, we examined (19) and that IL-33 potentiates IL-13 secretion by T cells and ILCs, both Il13 and Areg expression in the large and small intestine it is not known whether IL-13 is required for IL-33 to induce goblet following IL-33 i.p. As expected, Il13 was significantly increased 6 IL-33 AND GOBLET CELL DIFFERENTIATION Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 4. IL-33–exposed CD90+ MLN cells induce gene expression consistent with goblet cell differentiation in WT but not Il4ra2/2 enteroids. CD90+ MLN cells were purified from naive mice or mice given IL-33 (0.4 mg) i.p. daily for 4 d. (A) IL-13 and IL-4 secretion in cocultures was determined by ELISA. ND, not detected. MLN cells were cocultured with (B)WTor(C) Il4ra2/2 enteroids with 100 ng/ml IL-33 added in vitro to some cultures, and enteroid mRNA expression was assessed by quantitative real-time PCR. n = 9 wells pooled from three independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001. in both the colon and the jejunum (Fig. 6E, 6F). However, there goblet cells in mice in the large and small intestine was dependent was no increase in Areg in the large or small intestine with IL-33 on IL-13. i.p. compared with control (Fig. 6E, 6F). Large doses of IL-33 administered to mice leads to goblet cell hyperplasia, both in the and the intestines (6), but the Discussion mechanisms by which IL-33 induces goblet cells in the intestines We have applied an enteroid–immune cell coculture system as a have not been fully delineated. IL-33 increases production of the model of epithelial–immune cross-talk in the intestine to dem- Th2 cytokine IL-13 both in vitro and in vivo in T cells and ILC2s onstrate that IL-33 induces goblet cell differentiation indirectly by (6, 11, 12), and IL-13 is able to induce intestinal and goblet stimulating primarily ILCs to produce IL-13, rather than through cells (19, 23, 28, 29). We now tie together this circuit by dem- direct action on epithelial cells. Although intestinal epithelial cells onstrating the dependence of IL-33–induced goblet cell differen- express the IL-33R and activate ERK1/2 in response to IL-33, tiation on primarily IL-13–producing ILC2s and epithelial IL-13 IL-33 did not directly induce goblet cell differentiation, whereas signaling in vitro and IL-13 in vivo. IL-13 did. In cocultures with CD90+ MLN cells from IL-33–treated Because IL-33–treated mice have goblet cell hyperplasia, for mice, IL-33 induced Muc2 and Atoh1 expression, indicating in- cocultures with immune cells, we used MLN from IL-33–treated creased goblet cell differentiation. CD3–CD90+ST2+ ILC2s from mice as a relevant source of immune cells for the intestine, as MLN of IL-33–treated mice produce large amounts of IL-13 in other studies have previously done (14). Because IL-33 is known cocultures. The IL-33–induced Muc2 response in vitro was depen- to induce a type 2 immune response in both T cells and ILCs dent on enteroid Il4ra expression. Furthermore, IL-33 induction of (6, 11, 12), we isolated CD90+ cells from the MLN to capture both The Journal of Immunology 7

FIGURE 5. Areg does not induce gene expression consistent with goblet cell differentiation in enteroids. (A) Areg expression in CD90+ MLN cocultured with enteroids with or without IL-33 (100 ng/ml) for 4 d. (B) Representative Western blot and (C) quantification of ERK1/2 activation following Areg stimulation for 30 min. (D) Quantitative real-time PCR analysis of enteroids stimulated with Areg (100 ng/ml) for 4 d. n = 9 wells pooled from three independent experiments. **p , 0.01, ***p , 0.001. Downloaded from

cell types. When stimulated further with IL-33 in vitro, CD90+ MLN further differentiation of epithelial tuft cells (34). The same study cells made substantial amounts of IL-13. Importantly, in Trichuris showed that IL-33 similarly induces tuft cells through ILC2 IL-13

muris (13) and Nippostrongylus brasiliensis (12) infections, nano- production. Tuft cells branch off from a common secretory pro- http://www.jimmunol.org/ gram amounts of IL-13 are produced, which is what we saw fol- genitor cell as goblet cells and require Atoh1 but not Spdef (35). We lowing IL-33 stimulation of MLN cells in vitro. Although both ILCs now build on these findings by demonstrating the requirement of and T helper cells produced IL-13, over 90% of the IL-13–producing ILC2s and IL-13 for induction of goblet cell differentiation by cells were ILC2s. This is consistent with the findings of others that IL-33 using an in vitro model of epithelial–immune cross-talk. IL-33 treatment leads to accumulation of ILC2s in the MLN (14). Interestingly, we show that in IL-13–deficient mice, there is a Furthermore, in the setting of N. brasiliensis infection, ILC2s are the signal for increased goblet cell markers in mouse intestine, which predominant cell type producing IL-13, and T cells are not required decreased following IL-33 treatment. These differences were, for for IL-13 production to be induced (12, 30). However, a limitation of the most part, not statistically significant. It is possible that at this study is that in an unchallenged mouse, the majority of T cells in baseline, in the chronic absence of IL-13, other cytokines known by guest on September 29, 2021 the MLN are naive, and there are only a minority of IL-33R– to induce goblet cells, such as IL-22, could be increased (36). expressing Th2 cells able to respond to IL-33 (31). We now show Furthermore, without the strong induction of IL-13 by IL-33 in that IL-33–exposed MLN cells can increase goblet cell differentia- IL-13–deficient mice, other cytokines induced by IL-33, such as tion in murine enteroids in vitro. Furthermore, we used Il4ra2/2 IFN-g (37), which is known to decrease goblet cells, may take the enteroids to show that this effect is dependent on epithelial-intrinsic lead in regulating goblet cells (38, 39). IL-13 signaling. In contrast to our findings, others have proposed that IL-33 This study is one of a small number of emerging reports of in- directly induces goblet cell differentiation in the intestinal epi- testinal epithelial organoids cocultured with other cell types as a thelium. One group of investigators similarly treated murine more complex model of the intestinal mucosa. Intestinal sub- enteroids with IL-33 and observed marked increases in epithelial myofibroblasts support the growth of enteroids and colon numbers and the Paneth cell marker Ang4, with more modest organoids (colonoids) when cocultured together (21, 32, 33). increases in goblet cells and Muc2 expression (8). They went on to Macrophages cocultured on the basolateral surface of enteroid show that the induction of Paneth cell differentiation was inde- monolayers enhance epithelial barrier function and maturation and pendent of enteroid Il4ra expression. Using the same IL-33 con- phagocytose apical (17). Cocultures of T lymphocytes and centration and exposure duration, we did not observe any effect of enteroids have also been reported. One group cocultured group 3 IL-33 alone on enteroid goblet cell numbers, the secretory lineage ILCs from Il22-deficient mice in Matrigel with enteroids to dem- differentiation marker Atoh1, or the goblet cell differentiation onstrate that group 3 ILC augmentation of enteroid growth is IL-22 markers Spdef and Muc2 across multiple experiments. Only with dependent (18). Our approach differed in that enteroids in Matrigel the addition of MLN cells enriched for IL-13–producing ILC2s were on a standing semipermeable insert so that ILC2-enriched did we observe effects of IL-33 on enteroid goblet cell numbers lymphoid cells were physically separated from the enteroids. This and marker expression, leading us to conclude that IL-33 pri- approach further supported the hypothesis that an ILC2 secreted marily acts on ILCs to indirectly induce goblet cell differentiation. factor, rather than direct intercellular interactions, was responsible IL-33 may directly activate other important pathways in intestinal for enteroid goblet cell differentiation in our system. epithelial cells. Recently, it has been demonstrated that IL-33 acts Several studies have begun to dissect the role of IL-33 in regu- directly on the Caco2 intestinal epithelial cell line to increase lating intestinal secretory cell differentiation, including goblet cells proliferation through upregulation of miR-320a (40), and further (8, 34). With the discovery of IL-33, it was demonstrated that IL-13 studies are warranted to explore direct effects of IL-33 in is required for IL-33–induced goblet cell hyperplasia in the lung intestinal epithelial cells. in vivo (6). A more recent study demonstrated a positive feedback Goblet cell depletion is a pathologic hallmark of inflammatory circuit in vivo whereby intestinal tuft cells produce IL-25, which bowel disease. We and others previously demonstrated that in the potentiates IL-13 production by ILC2s, which then stimulates the setting of colitis, IL-33 and IL-33R reduce histopathologic severity 8 IL-33 AND GOBLET CELL DIFFERENTIATION Downloaded from http://www.jimmunol.org/ by guest on September 29, 2021

FIGURE 6. IL-33–induced intestinal goblet cell hyperplasia is dependent on IL-13 in vivo. Representative photomicrographs of PAS-stained goblet cells in (A) colon and (C) small intestine. Scale bars, 100 mm. Quantification of PAS staining using a modified nuclear algorithm and quantitative real-time PCR analysis of tissue RNA from the (B) colon and (D) small intestine. Quantitative real-time PCR for Il13 and Areg from the (E) colon and (F) small intestine. n = 6–10 mice per group across three independent experiments. *p , 0.05, **p , 0.01, ***p , 0.001. and preserve goblet cells (14, 15). In this study, we show that in did not increase goblet cells, despite activation of signaling. unchallenged mice and primary intestinal epithelial cells, IL-33 We found that IL-33 robustly increased intestinal mucosal Il13 induces goblet cell differentiation through IL-13 produced mainly expression but did not affect Areg expression in vivo, and that by ILC2s. Others have shown that in the setting of epithelial IL-33–induced goblet cell hyperplasia was IL-13–dependent. injury and acute colitis induced by dextran sodium sulfate, IL-33 Together these studies indicate that, although Areg is important protection and preservation of goblet cells are dependent on for epithelial repair and preservation of goblet cells during colitis, ILC-intrinsic Areg (14). Although we corroborate that ILC2s in IL-13, and not Areg, induced by IL-33 directly stimulates goblet culture expressed Areg, direct stimulation of enteroids with Areg cell differentiation in unchallenged primary epithelium. The Journal of Immunology 9

IL-33 induction of goblet cells through promoting ILC2s and 6. Schmitz, J., A. Owyang, E. Oldham, Y. Song, E. Murphy, T. K. McClanahan, G. Zurawski, M. Moshrefi, J. Qin, X. Li, et al. 2005. IL-33, an interleukin-1-like IL-13 expression may be important for immune regulation in cytokine that signals via the IL-1 receptor-related protein ST2 and induces T multiple settings. IL-33 expression is increased in the colon of helper type 2-associated cytokines. Immunity 23: 479–490. patients with ulcerative colitis as well as in various mouse models 7. Matta, B. M., J. M. Lott, L. R. Mathews, Q. Liu, B. R. Rosborough, B. R. Blazar, and H. R. Turnquist. 2014. IL-33 is an unconventional Alarmin that stimulates of colitis and parasitic infection (13, 15, 41–44). We acknowledge IL-2 secretion by dendritic cells to selectively expand IL-33R/ST2+ regulatory that the levels of IL-33 we used in vitro and in vivo were higher T cells. J. Immunol. 193: 4010–4020. than reported serum levels in during health or disease; 8. Mahapatro, M., S. Foersch, M. Hefele, G. W. He, E. Giner-Ventura, T. Mchedlidze, M. Kindermann, S. Vetrano, S. Danese, C. Gu¨nther, et al. 2016. however, it is difficult to ascertain the levels of IL-33 that cells are Programming of intestinal epithelial differentiation by IL-33 derived from per- exposed to in the tissue microenvironment. IL-13 is required for icryptal fibroblasts in response to systemic infection. Cell Rep. 15: 1743–1756. expulsion of N. brasiliensis and Heligmosomoides polygyrus adult 9. Maywald, R. L., S. K. Doerner, L. Pastorelli, C. De Salvo, S. M. Benton, E. P. Dawson, D. G. Lanza, N. A. Berger, S. D. Markowitz, H. J. Lenz, et al. parasites from the intestinal by inducing intestinal epithe- 2015. IL-33 activates tumor stroma to promote intestinal polyposis. Proc. Natl. lial cells to differentiate into goblet cells that secrete -like Acad. Sci. 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A WT Il1rl1–/– B WT Il4ra–/– C WT 2o alone IL33R IL33R IL4RA IL4RA IL13RA1 2o alone

Merge Merge Merge Merge Merge Merge

SUPPLEMENTAL FIGURE 1. IL-33R, IL-4RA and IL-13RA1 are expressed on enteroids. Immunofluorescence for (A) IL-33R in WT and Il1rl1–/– enteroids, (B) IL-4RA in WT and Il- 4ra–/– enteroids and (C) IL-13RA1 in WT or secondary alone. Enteroids are counterstained with Hoechst nuclear stain. Scale bars = 20 μM.