Modulation of the IL-33/IL-13 Axis in Obesity by IL-13R α2 Jennifer Duffen, Melvin Zhang, Katherine Masek-Hammerman, Angela Nunez, Agnes Brennan, Jessica This information is current as E. C. Jones, Jeffrey Morin, Karl Nocka and Marion Kasaian of September 27, 2021. J Immunol 2018; 200:1347-1359; Prepublished online 5 January 2018; doi: 10.4049/jimmunol.1701256

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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. The Journal of Immunology

Modulation of the IL-33/IL-13 Axis in Obesity by IL-13Ra2

Jennifer Duffen,* Melvin Zhang,* Katherine Masek-Hammerman,† Angela Nunez,‡ Agnes Brennan,* Jessica E. C. Jones,x Jeffrey Morin,‡ Karl Nocka,* and Marion Kasaian*

In obesity, IL-13 overcomes insulin resistance by promoting anti-inflammatory differentiation in . En- dogenous IL-13 levels can be modulated by the IL-13 decoy , IL-13Ra2, which inactivates and depletes the . In this study, we show that IL-13Ra2 is markedly elevated in adipose tissues of obese mice. Mice deficient in IL-13Ra2 had high expression of IL-13 response markers in adipose tissue, consistent with increased IL-13 activity at baseline. Moreover, exposure to the type 2 cytokine-inducing alarmin, IL-33, enhanced serum and tissue IL-13 concentrations and elevated tissue , , and type 2 innate lymphoid cells. IL-33 also reduced body weight, fat mass, and fasting blood glucose levels. Strikingly, however, the IL-33–induced protection was greater in IL-13Ra2–deficient mice compared with wild-type littermates, Downloaded from and these changes were largely attenuated in mice lacking IL-13. Although IL-33 administration improved the metabolic profile in the context of a high fat diet, it also resulted in diarrhea and perianal irritation, which was enhanced in the IL-13Ra2–deficient mice. Weight loss in this group was associated with reduced food intake, which was likely related to the gastrointestinal effects. These findings outline both potentially advantageous and deleterious effects of a type 2–skewed immune response under conditions of metabolic stress, and identify IL-13Ra2 as a critical checkpoint in adipose tissues that limits the protective effects of the IL-33/ IL-13 axis in obesity. The Journal of Immunology, 2018, 200: 1347–1359. http://www.jimmunol.org/

besity is a growing epidemic, with high body mass index Whereas IL-13Ra1 can mediate cellular responses to both IL-4 (BMI) contributing to disease burden worldwide (1). and IL-13, an additional receptor form, IL-13Ra2, interacts with O Emerging evidence suggests that obesity is supported IL-13 but not IL-4. It is thought to act primarily as a decoy, and maintained by immune activation in metabolic tissues. The binding IL-13 with higher affinity than IL-13Ra1, and seques- lean state is characterized by a protective anti-inflammatory mi- tering IL-13 from the signaling receptor, thereby reducing Stat6- lieu in adipose tissue, consisting of eosinophils, type 2 innate mediated signaling activity (9). IL-13Ra2 also mediates efficient lymphoid cells (ILC2), T regulatory cells (Treg), and anti- internalization and depletion of extracellular IL-13 (10). Although

inflammatory macrophages. In contrast, obesity is associated IL-13Ra2 lacks any known cytoplasmic signaling motif, its ability by guest on September 27, 2021 with a shift toward reduced eosinophilic inflammation, with ele- to mediate cellular activation responses is debated (11). In addi- vated proinflammatory macrophages and CD8+ T cells (2, 3). In tion to the membrane-bound receptor, mice but not humans have a mouse models of obesity, the Th2 IL-4 (4), IL-13 (5), soluble form of IL-13Ra2 (sIL-13Ra2) that is abundant in the and IL-5 (6) can promote glucose tolerance and insulin sensitivity. circulation (12, 13). In contrast to the IL-13Ra1/IL-4Ra signaling IL-4 and IL-13 polarize macrophages to an anti-inflammatory receptor, which is widely expressed, tissue expression of phenotype through the IL-13Ra1/IL-4R receptor (2), whereas IL-13Ra2 is normally very low, and is induced under conditions IL-5 signaling drives the development and recruitment of eosin- of high IL-13 release (14). In the absence of IL-13Ra2, circulating ophils (7). The association of type 2 cytokines with metabolic levels of IL-13 are reduced, suggesting that the soluble form acts regulation suggests that glycemic control may be achieved as a carrier of IL-13 in murine blood (15). Mice lacking IL-13Ra2 through cytokine manipulation (8). have elevated tissue IL-13, consistent with impaired cytokine clearance, and have enhanced IL-13 bioactivity due to impaired cytokine neutralization (15), leading to exacerbated fibrotic re- sponses (16, 17), increased smooth muscle contractility and epi- *Inflammation and Immunology Research Unit, Pfizer, Inc., Cambridge, MA 02139; thelial resistance (18), and aggravated atopic responses (19, 20) †Drug Safety Research and Development, Pfizer, Inc., Cambridge, MA 02139; driven by IL-13. Although the role of IL-13 in metabolic ho- ‡Comparative Medicine, Pfizer, Inc., Andover, MA 01810; and xInternal Medicine Research Unit, Pfizer, Inc., Cambridge, MA 02139 meostasis is becoming appreciated (21–23), the contribution of IL-13Ra2 to regulation of this response has not been explored. ORCID: 0000-0002-1656-296X (J.M.). The alarmin IL-33 is stored in epithelial cells, endothelial cells, Received for publication August 31, 2017. Accepted for publication December 11, 2017. and fibroblasts, and is released upon tissue damage to mediate local Address correspondence and reprint requests to Dr. Marion Kasaian, Pfizer Research, immune activation (24). A key effector mechanism is the IL-33– 1 Portland Street, Cambridge, MA 02139. E-mail address: marion.kasaian@pfizer. induced release of cytokines, principally IL-5 and IL-13, from com ILC2, Th2, mast cells, , and other cell types expressing The online version of this article contains supplemental material. the cell surface IL-33 receptor, ST2 (24). Administration of IL-33 Abbreviations used in this article: BMI, body mass index; EMH, extramedullary to mice triggers a range of IL-13–dependent responses (25–28), hematopoiesis; eWAT, epididymal white adipose tissue; GI, gastrointestinal; HDL, but may also induce production of IL-4 and IL-5 (25, 28–31), high-density lipoprotein; HFD, high fat diet; ILC2, type 2 ; scWAT, s.c. WAT; sIL-13Ra2, soluble form of IL-13Ra2; Treg, T regulatory cell; either of which could modulate inflammation in adipose tissue VAT, visceral adipose tissue; wt, wild type. (4, 6, 32). IL-33 has been shown to protect mice from the meta- bolic consequences of obesity (29), but the extent to which this is Copyright Ó 2018 by The American Association of Immunologists, Inc. 0022-1767/18/$35.00 www.jimmunol.org/cgi/doi/10.4049/jimmunol.1701256 1348 MODULATION OF THE IL-33/IL-13 AXIS IN OBESITY BY IL-13Ra2 dependent on induction of IL-13, as opposed to other cytokines, CD3e2CD52CD192CD42NK1.12CD11c2CD11b2F4/802FcεRla2. Eo- + + + has not been evaluated. sinophils were gated as CD45 CD11b SiglecF . In this study, we manipulated tissue IL-13 levels by blocking Analysis of circulating lipids depletion through IL-13Ra2, both in untreated mice and in mice driven to express high levels of endogenous IL-13 by adminis- Blood was collected by cardiac puncture and immediately following eu- thanasia by carbon dioxide. For serum measurements, blood was allowed to tration of IL-33. We examined effects on glucose homeostasis clot at room temperature for 2 h before centrifugation to separate the serum under conditions of a normal chow diet and high fat diet (HFD), for further analysis. Total cholesterol and high-density lipoprotein (HDL) and explored the IL-13 dependence of these responses. Our find- fraction were quantified in serum using the Siemens Advia 1800 Chemistry ings support that IL-33 regulation of metabolic homeostasis is Analyzer (Malvern, PA). Whole blood in EDTA was rocked at room temperature to mix thoroughly, then total cell differential was determined largely IL-13 dependent, and that extreme type 2 skewing fol- using the Siemens Advia 2120 hematology system. lowing IL-33 administration in the presence of IL-13Ra2 defi- ciency ameliorates the metabolic consequences of HFD. Although quantification these observations suggest that manipulation of tissue IL-13 re- Flash-frozen adipose and liver tissue were weighed then homogenized in sponses could be beneficial in restoring glycemic control on HFD, Tissue Protein Extraction Reagent (Thermo Fisher Scientific, Waltham, these effects were accompanied by increased risk of gastrointes- MA) + protease inhibitors ( Technology, Danvers, MA) using tinal (GI) toxicity. the TissueLyser II (Qiagen, Germantown, MD). The samples were then centrifuged at 4˚C and the resulting supernatant used in subsequent assays. Mouse IL-13Ra2, leptin (R&D Systems), and insulin (Crystal Chem, Materials and Methods Downers Grove, IL) measurements were performed on serum and/or tissue homogenate samples by ELISA according to the manufacturer’s protocol. Downloaded from Mouse model of diet-induced obesity Mouse IL-13 was measured by ELISA (R&D Systems) following over- Male il13ra22/2 and il132/2 mice on a C57BL/6N background were night incubation at 4˚C with 0.5% BSA and an anti–IL-13Ra2Abto maintained in heterozygous colonies and cohoused with littermate wild- dissociate IL-13 from IL-13Ra2 (15). type (wt) controls. Animals were healthy and had not undergone any RNA isolation and quantitative RT-PCR previous procedures. Starting at 8 wk of age, mice were fed an HFD (60% kcal from fat, D12492; Research Diets, New Brunswick, NJ) or RNA was isolated from adipose tissue using the RNeasy Plus Universal Kit

normal chow (PicoLab Rodent Diet 20; LabDiet, St. Louis, MO) for 13 (Qiagen). Duodenum, jejunum, ileum, and colon RNA was isolated using http://www.jimmunol.org/ wk. As indicated, recombinant mouse IL-33 (R&D Systems, Minneap- the RNeasy Mini Kit (Qiagen). Both protocols were performed using the olis, MN) was administered at 0.125 or 0.0125 mg/kg doses via i.p. in- TissueLyser II and QIAcube (Qiagen). RNA concentration was determined jection every day for 1 wk or every other day for 2 wk for a total of seven on the QIAxpert (Qiagen) and RNA quality measured on the Agilent 4200 doses. During this period, body weights and food weights were recorded TapeStation (Agilent Genomics, Santa Clara, CA). RNA was reverse daily. Fasting glucose measurements, glucose tolerance tests, and insulin transcribed using Superscript VILO Mastermix (Invitrogen; Thermo Fisher tolerance tests were performed using the AlphaTrak2 glucose meter Scientific). Real-time PCR was performed using Viia7 (Thermo Fisher (Abbott Animal Health, Abbott Park, IL) following a 6 h fast. For glu- Scientific), QuantStudio 7 Flex (Applied Biosystems, Foster City, CA), or cose and insulin tolerance tests, 1.5 g/kg glucose or 1 U/kg insulin, re- Biomark HD (Fluidigm, South San Francisco, CA) systems using TaqMan spectively, were injected i.p. and blood glucose measurements were Fast Advanced Mastermix (Applied Biosystems) and expression normalized obtained at 0, 15, 30, 60, 90, and 120 min. Body composition was de- to housekeeping . For expression measurements on the Biomark termined by Magnet NMR Minispec (Bruker, Madison, WI) and HD system, cDNA was preamplified using TaqMan PreAmp Mastermix by guest on September 27, 2021 expressed relative to total body weight. Diarrhea and rectal irritation (Applied Biosystems). All oligonucleotide probes were obtained from were scored daily by an investigator blinded to the mouse genotypes as Thermo Fisher Scientific. follows: 0 = normal, 1 = minimal, 2 = mild, 3 = moderate, 4 = marked, 5 = severe. All procedures performed on animals were in accordance with Spleen and peritoneal cell differential regulations and established guidelines and were reviewed and approved by the Pfizer Institutional Animal Care and Use Committee. Peritoneal cells were isolated from euthanized mice by lavage with 2.5 ml PBS. A single-cell suspension of spleen cells was obtained by manually Human adipose tissue RNA pushing the organ through a cell filter and then lysing RBCs. Spleen and peritoneal cells were affixed to slides by cytospin (Thermo Fisher Scien- Human adipose tissue RNA was obtained from ZenBio (Research Triangle tific), and stained with Wright’s Giemsa for manual analysis of macrophage Park, NC), from tissue isolated under Institutional Review Board–approved and frequency by an investigator blinded to the treatment protocols. Omental visceral adipose tissue (VAT) was collected from conditions. subjects undergoing gastric bypass surgery. s.c. white adipose tissue (scWAT) was collected from abdomen, thighs, or hips of subjects under- Histopathology going elective liposuction or abdominal plasty. Samples of esophagus, stomach, duodenum, jejunum, colorectum, pancreas, Flow cytometry spleen, and/or bone marrow were fixed in 10% neutral buffered formalin, processed, and embedded in paraffin, and sectioned and stained with H&E Adipose tissue was collected in ice cold HBSS + 0.5% BSA, minced, and for analysis. H&E-stained slides were evaluated by a board-certified vet- digested by 1 mg/ml collagenase type I (Worthington Biochemical, erinary pathologist and endpoints were scored according to the following Lakewood, NJ) for 50 min at 37˚C. For the final 5 min of incubation, 2 mM scale: 0 = background, 1 = minimal, 2 = mild, 3 = moderate, 4 = marked, EDTA was added. Cells were washed, passed through a 100 mm filter, and 5 = severe. centrifuged to separate the stromal vascular fraction (pellet) from the floating as described previously (33). The stromal vascular Statistical analysis fraction was resuspended in RBC lysis buffer (8.3 g/l NH Cl, 1 g/l 4 Data were analyzed using GraphPad Prism 7 software (San Diego, CA) and KHCO3, 0.09 g/l EDTA, pH = 7.3), incubated for 5 min at room tem- perature then washed with FACS buffer (PBS + 0.5% BSA + 2 mM are presented as mean + SEM. Box and whisker plots represent median, EDTA). Cells were treated with rat purified anti-mouse CD16/CD32 high, and low values. Group comparisons used the two-tailed Student t test or a one-way ANOVA followed by Tukey multiple comparison test. Sta- (mouse Fc block) for 5 min on ice then stained for surface markers with , combinations of the following conjugated Abs in Brilliant Stain Buffer: tistical significance was set at p 0.05. anti-mouse CD45-BUV395, Siglec F-PE, CD11b-BB515, CD3e-PE, CD5- PE, CD19-PE, CD4-PE, NK1.1-PE, CD11c-PE, CD11b-PE, F4/80-PE, FcεRla-PE, ST2(IL-33R)-biotin, CD25-BV421, and streptavidin– Results allophycocyanin (secondary). Staining Abs were purchased from BD IL-13Ra2 expression in adipose tissue is elevated in mouse Biosciences (San Jose, CA). Stained cells were acquired on an LSRFor- models of obesity tessa (BD Biosciences) and data analyzed using FlowJo software version 10 (Tree Star, Ashland, OR). were gated as CD45+Lin2CD25+ST2+ IL-13Ra2 is expressed at low or undetectable levels in the absence as previously described (34) and lineage negative cells were defined as of tissue inflammation, but is induced in the presence of IL-4 or The Journal of Immunology 1349

IL-13, in combination with TNF-a. Levels may be further en- IL-13 activity gene signature is enhanced in adipose tissue of hanced by IL-17 or other inflammatory cytokines (14, 35). Obesity il13ra22/2 mice is characterized by a state of systemic low-grade inflammation, As obesity-induced IL-13Ra2 expression was associated with which is marked by elevated levels of circulating TNF-a (36). In a reduced adipose tissue IL-13 concentrations, we asked whether model of diet-induced obesity, IL-13Ra2 mRNA expression levels IL-13Ra2 deficiency would enhance tissue IL-13 levels. Wt and in adipose tissue from mice fed an HFD for 13 wk were signifi- il13ra22/2 mice were subjected to HFD feeding for 13 wk. At the cantly increased compared with levels in adipose tissue from mice end of this time, concentrations of IL-13 in scWAT did not differ fed a normal chow diet during the same time period (Fig. 1A). between wt and il13ra22/2 mice (data not shown). Weight gain Expression of IL-13Ra1 and common g-chain (IL-2Rg) was also (Fig. 2A) and total fat mass (Fig. 2B) were similar between a 2/2 elevated, whereas expression of IL-4R was not increased in mice and littermate controls. There were no significant adipose tissue of obese mice. As has been reported previously differences in glucose tolerance or insulin sensitivity either on a (36), TNF-a mRNA expression was also elevated in adipose tissue chow diet (data not shown) or HFD (Fig. 2C, 2D). A small panel of from obese mice. Similar increases in adipose tissue IL-13Ra2 2 2 genes was differentially expressed in adipose tissue of il13ra2 / expression were seen in leptin-deficient ob/ob mice, a genetic mice compared with wt controls (Fig. 2E). Genes with elevated model of obesity (data not shown). Serum concentrations of 2 2 expression in il13ra2 / mice were known to be IL-13 responsive, sIL-13Ra2 were also higher under conditions of obesity (Fig. 1B). including the chitinase family members Ym1 and Ym2 (39), the In accordance with the elevated IL-13Ra2, which inactivates and macrophage lectin Mgl2 (40), the eosinophil eotaxin 1 depletes IL-13 in tissue (15, 37), adipose tissue levels of IL-13 (Ccl11) (41), and the repair marker, amphiregulin (42). cytokine were reduced in mice fed an HFD (Fig. 1C). Downloaded from We compared IL-13Ra2 expression in epididymal WAT (eWAT), a IL-33 promotes Th2 inflammation in chow-fed mice depot of VAT associated with insulin resistance and dyslipidemia, and HFD feeding was not sufficient to differentiate systemic metabolic re- in scWAT, a storage depot associated with adipokine secretion, en- sponses in wt and il13ra22/2 mice, as reflected in the body weight, hanced lipid oxidation, and insulin action (38). In mouse adipose tissue, percentage of fat mass, glucose tolerance, and insulin tolerance findings IL-13Ra2 protein concentration was low in both eWAT and scWAT of (Fig. 2A–D). With limiting IL-13 concentrations under resting condi- mice on a chow diet, and was significantly elevated in eWAT but not tions, lack of IL-13Ra2 may have little impact on metabolic endpoints. http://www.jimmunol.org/ scWAT of mice fed an HFD (Fig. 1D). Human adipose tissue was also IL-33 is known to be a potent inducer of IL-13, IL-5, and other cyto- examined for il13ra2 gene expression. Subjects providing omental fat kines, both in vitro and in vivo (29). Therefore, we administered IL-33 and scWAT for this analysis had average ages of 31.9 6 3.5 and 48.6 6 to mice to elevate systemic IL-13 concentrations. Recombinant IL-33 8.4 y, respectively, and all were female. Average BMI of the subjects was given by i.p. injection to wt and il13ra22/2 mice, every other day providing omental fat and those providing scWAT was 41.9 6 11.0 for seven administrations, at doses of 0.0125 or 0.125 mg/kg. We ob- (n =6)and33.36 3.6 (n = 7), respectively. The difference in BMI was served dose-dependent modulation of a range of physiological and not statistically significant, and with an average BMI .30, each group metabolic endpoints (Supplemental Fig. 1). Based on these observations, was considered obese. Expression of il13ra2 was found in both a dose of 0.125 mg/kg was chosen to induce responses of sufficient by guest on September 27, 2021 omental WAT (VAT) and in scWAT (Fig. 1E). Given that the subjects magnitude to allow interrogation of the role of IL-13Ra2. providing VAT differed in age and trended toward a higher BMI than IL-33 induced marked splenomegaly in both wt and il13ra22/2 those providing scWAT, and that gene expression values could not be mice on chow or HFD (Fig. 3). After IL-33 administration on directly compared between VATand scWAT, these findings demonstrate chow diet, IL-13 concentration was greatly elevated in serum and expression of il13ra2 in human adipose tissue, but do not allow firm in tissues, including liver, eWAT, and scWAT (Fig. 3A). Compared conclusions to be drawn concerning the relative expression of il13ra2 to wt, il13ra22/2 mice on chow diet had reduced concentrations in VAT compared with scWAT. of IL-13 in serum, but higher concentrations in liver, eWAT, and

FIGURE 1. IL-13Ra2 expression is induced with HFD, associated with depletion of tissue IL-13. C57BL/6 mice were fed normal chow or a 60% HFD for 13 wk. (A) Gene expression analysis in scWAT, normalized to housekeeping genes. (B) sIL-13Ra2in mouse serum. (C) IL-13 concentration in adipose tissue extract. (D) IL-13Ra2 protein concentration in extracts of eWAT or scWAT tissue of mice fed chow or HFD. scWAT and eWAT were collected from the same ani- mals. The dashed line represents the limit of assay detection. (E) il13ra2 transcripts in human adipose tissue. VAT denotes the omental adipose depot, corre- sponding to visceral adipose tissue. Data represent three independent experiments for (A–C)(n = 6–8 mice per group), two independent experiments for (D)(n = 11 mice per group), and one experiment for (E)(n = 7 and 12 subjects for the VAT and scWAT groups, respectively). *p , 0.05, **p , 0.01, ***p , 0.005, ****p , 0.0005. 1350 MODULATION OF THE IL-33/IL-13 AXIS IN OBESITY BY IL-13Ra2

FIGURE 2. HFD-induced metabolic changes do not differ between wt and il13ra22/2 mice, despite adi- pose tissue IL-13 response profile. C57BL/6 mice were fed a 60% HFD for 13 wk. (A) Body weight; (B) % fat mass, determined by nuclear magnetic resonance; (C) glucose tolerance was assessed in fasted mice after injection of 1.5 g/kg glucose i.p.; (D) insulin tolerance was assessed in fasted mice after i.p. injection of 1 U/kg human insulin; (E) relative mRNA expression of representative genes in eWAT of il13ra22/2 mice Downloaded from compared with wt, normalized to housekeeping genes. Graphs represent median, high, and low values from three independent experiments (n = 10–16 mice per group). *p , 0.05, **p , 0.01, ****p , 0.0001 by t test. http://www.jimmunol.org/ by guest on September 27, 2021 scWAT (Fig. 3A). These observations are consistent with findings gree of weight loss than wt mice (Fig. 4A). There was no effect of that sIL-13Ra2 acts as a reservoir for the cytokine, such that mice IL-33 on body weight in il132/2 mice on an HFD, confirming lacking sIL-13Ra2 have a reduced IL-13 concentration in the IL-13 dependence of the effect (Fig. 4A). Similarly, fasting blood blood (15). The cell-surface form of IL-13Ra2 acts to internalize glucose concentrations were reduced in HFD-fed wt mice ad- and deplete IL-13, such that mice lacking IL-13Ra2 have elevated ministered IL-33, and reduced to a greater extent in il13ra22/2 IL-13 concentration in tissues (15). IL-13 administration under mice, but were not reduced in il132/2 mice (Fig. 4B). Under comparable conditions failed to elevate tissue IL-13 levels (data conditions of HFD, IL-33 also significantly decreased fat mass not shown), suggesting that endogenous IL-13 production was (Fig. 4C) and size (data not shown). In the course of required to sustain the responses. these studies, we noted that the wt and il13ra22/2 mice admin- In mice on an HFD, the magnitude of IL-13 induction in response istered IL-33 on chow diet exhibited mild perianal irritation and to IL-33 in eWAT and scWAT was reduced compared with that of diarrhea, which was relatively more pronounced in the il13ra22/2 mice on a chow diet (Fig. 3). In eWAT, the IL-13 concentration was animals (Fig. 4E). The perianal irritation and diarrhea were both significantly higher in IL-33–treated il13ra22/2 mice compared increased in wt mice fed an HFD compared with chow diet, and with wt mice on chow diet, but significantly lower in il13ra22/2 heightened further in HFD-fed il13ra22/2 animals (Fig. 4E, 4F). mice compared with wt mice on an HFD. Trends toward similar This may have contributed to the markedly reduced food con- findings were seen in scWAT (Fig. 3), showing a strong effect of sumption observed in the IL-33–treated animals on an HFD diet on adipose tissue IL-13 regulation in response to IL-33. In (Fig. 4D). Mice that were deficient in IL-13 were protected from mice on a chow diet, IL-33 also increased adipose tissue eosino- the perianal irritation and diarrhea (Fig. 4E), and maintained food phils, ILC2s, and total CD45+ leukocytes in both wt and il13ra22/2 consumption (Fig. 4D) and body weight (Fig. 4A) on HFD. mice (Fig. 3A). On an HFD, IL-33 induction of leukocytes and Elevations in serum concentrations of liver enzymes alanine eosinophils was maintained in wt mice, but attenuated in il13ra22/2 aminotransferase and aspartate aminotransferase, but not alkaline mice (Fig. 3B), emphasizing that effects of IL-13Ra2 deficiency phosphatase, were seen in mice fed an HFD as compared with those became apparent under the stress of an HFD. on a chow diet (Fig. 5A–C). Concentrations of these markers were reduced to variable degrees in mice administered IL-33 (Fig. 5A–C). IL-33–induced metabolic changes are IL-13 dependent Levels of alkaline phosphatase and aspartate aminotransferase were Administration of IL-33 at 0.125 mg/kg every other day for 2 wk lower in the il132/2 mice compared with il13ra22/2 animals, sug- did not produce appreciable weight change in mice on a chow diet, gesting an influence of IL-13 on liver function. Serum levels of in- but caused weight loss in obese mice (Fig. 4A), consistent with sulin, low-density lipoprotein, HDL, triglycerides, and cholesterol previous reports (29, 43). Il13ra22/2 mice showed a greater de- were all significantly reduced in HFD-fed mice administered IL-33 The Journal of Immunology 1351 Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 3. IL-33 promotes tissue IL-13 elevation, splenomegaly, and adipose tissue inflammation in: (A) chow-fed; or (B) HFD-fed wt and il13ra22/2 mice. Mice were administered PBS (N) or IL-33 (n) i.p. at 0.125 mg/kg every other day for 2 wk. Serum and tissues were harvested 24 h after the last injection. Panels depict IL-13 concentration in serum or the indicated tissues, spleen weight, % CD45+ cells in the stromal vascular fraction (SVF) of scWATor eWAT, % eosinophils (CD45+,CD11b+, SiglecF+) in scWATor eWAT, % ILC2s (CD45+, Lin2, CD25+, ST2+) in the SVF of eWAT. Data represent mean 6 SEM of n = 5–6 mice per group from two to three independent experiments. scWAT and eWAT were collected from the same animals. *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001, by t test, for comparisons between IL-33–treatment groups.

(Fig. 5D–H). There was a trend toward greater reduction in serum il13ra22/2 and il132/2 animals (Fig. 5E–H). Taken together, these lipids in il13ra22/2 animals, and attenuated reduction in il132/2 data implicate IL-13 in mediating IL-33-induced protection from mice, producing a significant difference between lipid levels in HFD-induced changes in liver enzymes, insulin, and serum lipids. 1352 MODULATION OF THE IL-33/IL-13 AXIS IN OBESITY BY IL-13Ra2 Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 4. Differential effects of IL-33 administration in chow versus HFD for il13ra22/2 (a2KO), il132/2 (13KO), and wt mice. Mice on chow diet were administered IL-33 i.p. at 0.125 mg/kg every other day for 2 wk. (A) Body weight; (B) fasting blood glucose; (C) % fat mass; (D) food consumption; and (E)perianal irritation/diarrhea score were assessed 24 h after the final injection of IL-33; (F) perianal irritation in IL-33–treated il13ra22/2 mice fed an HFD. Data represent the mean 6 SEM of three independent experiments, with 6–12 mice per group for the chow groups, and 10–26 mice per group for the HFD groups. *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001 for the indicated comparisons, by t test.

IL-33–induced gene expression changes are modulated by Among cytokine receptor genes, expression of il13ra2 was IL-13Ra2 greatly reduced in eWAT of il132/2 mice, below the baseline level Following IL-33 administration, increased expression of numerous genes seen in the absence of IL-33 (dashed line, Fig. 6B), suggesting that was noted in adipose tissue, including those encoding cytokines, cytokine an active inhibitory pathway exists to block IL-13Ra2 expression receptors, and metabolic markers (Fig. 6, Supplemental Fig. 2). Whether in mice lacking IL-13. The IL-13Ra1 and IL-4Ra signaling these changes signified altered expression per cell, or reflected altered chains were not regulated by IL-33. The IL-33–associated ex- cellular composition of the adipose tissue, could not be determined. In pression of both IL-2Rg and IL-1RL1 (ST2) was reduced in 2/2 2/2 eWAT, the expression of genes encoding Th2 cytokines IL-13, IL-4, and il13ra2 mice, but elevated in il13 mice (Fig. 6B). Among 2/2 IL-5 was increased 30–50-fold following administration of IL-33 to wt markers of cell differentiation, il13ra2 mice also displayed mice on an HFD, and was reduced in eWAT of il13ra22/2 mice (Fig. reduced expression of genes encoding the Th2 transcription factor, 6A), consistent with the relative concentrations of IL-13 cytokine in Gata3, the costimulatory molecule, PD1, the ILC2 transcription eWAT (Fig. 3B). In contrast to the effect of IL-13Ra2 deficiency, ex- factor, Rora, the Treg marker, Foxp3, and the Th17 transcription pression of IL-5 was elevated in eWAT of il132/2 mice, suggesting that factor, Rorc. For all but Rorc, the transcripts were elevated in 2 2 IL-13 normally functions to limit IL-5 elevation (Fig. 6A). Among il13 / mice (Fig. 6C). Collectively, these gene expression other Th2-associated cytokines, IL-25 expression was not induced changes in mice lacking IL-13Ra2 displayed an inverse profile to in eWAT. IL-33 expression was mildly increased following IL-33 those seen in mice lacking IL-13. A similar gene expression administration, but the response did not differ between wt, profile was seen in scWAT (Supplemental Fig. 2). Overall, the il13ra22/2,andil132/2 mice (Fig. 6A). reduced expression of cellular differentiation genes in adipose The Journal of Immunology 1353 Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 5. Effects of IL-33 administration on serum metabolic markers in HFD-fed il13ra22/2 (a2KO), il132/2 (13KO), and wt mice after 13 wk of HFD. (A) Alanine aminotransferase (ALT; units per milliliter); (B) alkaline phosphatase (ALP; units per liter); (C) aspartate aminotransferase (AST; units per liter); (D) insulin (picograms per milliliter); (E) low density lipoprotein (LDL; milligrams per deciliter); (F) HDL (milligrams per deciliter); (G)TG (triglyceride; milligrams per deciliter); and (H) cholesterol (milligrams per deciliter). The dashed line indicates concentration in untreated wt mice on chow diet. Data represent the mean 6 SEM of three independent experiments, with 5–19 mice per group for the HFD groups. *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001 for the indicated comparisons, by t test. tissue of il13ra22/2 mice reflected the reduced level of CD45+ eWAT of il132/2 mice given IL-33, expression of most IL-13 re- leukocyte infiltration in the stromal vascular fraction of IL-33–treated sponse markers was reduced in il132/2 animals (Fig. 6D, il13ra22/2 mice compared with wt on an HFD (Fig. 3B). In contrast Supplemental Fig. 2), and did not differ between wt and to the elevated expression of lymphocyte differentiation markers in il13ra22/2 mice. 1354 MODULATION OF THE IL-33/IL-13 AXIS IN OBESITY BY IL-13Ra2 Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 6. Gene expression changes in eWATof il13ra22/2 or il132/2 mice with matched wt controls, administered 0.125 mg/kg IL-33 every other day for 2 wk. Expression of each gene was determined relative to housekeeping controls. The dashed line represents the average expression level in all PBS- treated groups (il13ra22/2 mice and wt controls, il132/2 mice and wt controls), normalized to a value of one. Genes were grouped into those encoding: (A) cytokines; (B) cytokine receptors; (C) cell differentiation markers; (D) IL-13 response markers. Graphs represent median, high, and low values from two to three independent experiments, with 5–10 mice per group. *p , 0.05, **p , 0.01, ***p , 0.001, ****p , 0.0001 compared with matched wt,byt test.

The expression of a panel of metabolism-related genes was ex- scWAT, expression of most genes in the panel was reduced in re- amined in the adipose tissue of mice administered IL-33 compared sponse to IL-33 administration, and was not significantly affected with PBS-treated control animals (Supplemental Fig. 3A). In eWAT, by IL-13Ra2 deficiency. Unlike in eWAT, however, IL-13 defi- expression of most genes in the panel was reduced following IL-33 ciency did not exacerbate the IL-33–associated gene expression treatment, in both wt and il13ra22/2 mice. In all cases, expression changes in scWAT (Supplemental Fig. 3B). These observations was further reduced in mice lacking IL-13, suggesting that IL-33– suggest that although IL-13 plays a key regulatory role in IL-33– induced IL-13 normally functions in eWAT to regulate expression induced metabolic changes in eWAT, additional regulatory path- of metabolic response genes (Supplemental Fig. 3A). Similarly, in ways exist in scWAT, abrogating their IL-13 dependence. The Journal of Immunology 1355

Finally, we @examined expression of a panel of genes in the GI IL-33–induced extramedullary hematopoiesis and tract of wt and il13ra22/2 mice to identify correlates of the inflammation are modulated by IL-13Ra2 metabolic changes seen with HFD feeding and IL-33 adminis- As previously reported (30, 45), IL-33 administration induced tration. The mice expressed IL-13Ra2 throughout the GI tract, infiltration of inflammatory cells in multiple tissues, and resulted progressively increasing from duodenum to jejunum to ileum to in splenomegaly (Fig. 7A, 7B), associated with microscopic evi- colon (Supplemental Fig. 4A). As has been reported previously dence of disrupted splenic architecture and increased extra- (44), IL-13Ra2 expression in the gut was increased following medullary hematopoiesis (EMH) (Fig. 7C, data not shown). IL-33 administration, and this was seen in the duodenum, jejunum, Increased cellularity was also observed in bone marrow in IL-33– and ileum (Supplemental Fig. 4A). Because the greatest difference be- administered mice compared with PBS-treated controls, consistent tween expression of IL-13Ra2 in IL-33–treated mice compared with PBS-treated mice was seen in the jejunum, we focused on this region for with the splenic EMH (data not shown). Marginally elevated eo- analysis of type 2 markers that could differ in wt versus il13ra22/2 mice. sinophil numbers were found in the circulation, spleen, and Neither IL-13Ra1norIL-4Ra expression was induced in the jejunum peritoneal lavage of wt mice in response to IL-33 (Fig. 7D–F). In a 2/2 following IL-33 dosing (Supplemental Fig. 4B). Expression of IL-1RL1 contrast, the peritoneal lavage of IL-33–treated wt and il13r 2 (ST2) was increased with IL-33 administration, but was not modulated in mice was characterized by large numbers of vacuolated macro- the absence of IL-13Ra2 (Supplemental Fig. 4B). Several genes were phages, comprising up to 70% of total leukocytes (Fig. 7G–I). 2 2 induced with IL-33 in wt mice, and displayed significantly lower ex- Because they were found in il13ra2 / mice that displayed re- pression in mice lacking IL-13Ra2, including those encoding the M2/ duced body weight on an HFD, the macrophages were examined ILC2 marker Arg1, the Th2 transcription factor, Gata3, the costimulatory for lipid in the vacuoles by Oil Red O staining, and found to be Downloaded from molecule, PD1, the Treg marker, Foxp3, the eosinophil chemokine, negative (data not shown). Ccl24, and the inflammatory marker, IL-12Rb1 (Supplemental Fig. 4C). In IL-13–deficient mice, eosinophilic inflammation was dra- Additional markers were elevated throughout the GI tract with IL-33, matically increased, with elevations of up to 40–50% total including IL-5, IL-13, and Ym1, but the expression level did not differ leukocytes in the circulation, spleen, peritoneal lavage (Fig. 7D–F, between wt versus il13ra22/2mice (data not shown). 7J), and bone marrow (data not shown). The increased eosinophils http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 7. Exacerbated splenomegaly, EMH, and eosinophilia in HFD-fed il132/2 mice treated with IL-33. (A)Spleensize;(B) spleen weight; (C)EMHscore; (D) % circulating eosinophils; (E) % eosinophils in spleen; (F) % eosinophils in peritoneal lavage; (G) % macrophages in peritoneal lavage; Wright’s Giemsa staining of peritoneal cells from IL-33–treated: (H)wt;(I) il13ra22/2;(J) il132/2 mice. Data are representative of three independent experiments. (B–G)Data plotted as mean 6 SEM of five to six mice per group. ****p , 0.0001 compared with matched wt,byt test. 1356 MODULATION OF THE IL-33/IL-13 AXIS IN OBESITY BY IL-13Ra2 drove marked splenomegaly (Fig. 7A, 7B). Increased numbers of mice. These observations suggest that any elevations in IL-13 granulocyte precursors were observed in the bone marrow (data concentration resulting from IL-13Ra2 deficiency alone were in- not shown), and were accompanied by a reduction in the relative sufficient to affect metabolic responses. To further elevate tissue percentage of vacuolated macrophages in the peritoneal lavage IL-13 concentrations, we administered IL-33 to the mice. The (Fig. 7G, 7J). The striking increase in eosinophil number seen metabolic protective effects of IL-33 have been well described. in mice lacking IL-13 suggests that IL-13 acts to limit eo- Administration of exogenous IL-33 protects mice against obesity sinophil development and/or infiltration in response to IL-33 and type 2 diabetes (29). In obese mice, IL-33 induces Th2 cy- administration. tokines, polarizes adipose tissue macrophages toward an anti- inflammatory phenotype, and reduces adipose tissue mass and IL-13–dependent GI toxicity develops in wt mice following fasting blood glucose levels (29). IL-33–responsive ILC2 have IL-33 administration and is exacerbated in il13ra22/2 mice emerged as an important regulatory cell type in adipose tissue under HFD conditions (47, 48), influencing obesity, and browning and beiging of fat As described above, diarrhea and perianal irritation were observed (34, 49, 50). Through release of IL-13, IL-5, and other mediators, 2 2 in mice administered IL-33, and were more severe in il13ra2 / ILC2 orchestrate macrophage phenotype, eosinophilia, and Treg mice compared with wt littermates (Fig. 4E, 4F). IL-13–deficient in adipose tissue (51, 52). In accordance with this, we observed mice were protected from these GI effects, suggesting IL-13 de- increased ILC2 in adipose tissue of both wt and il13ra22/2 mice pendence. Microscopic examination showed that IL-33 adminis- administered IL-33. tration increased eosinophilic and mononuclear cell infiltrates in IL-33 administration to mice on HFD elevated adipose tissue the jejunum and colorectum (Fig. 8A–D), with increased goblet expression of genes encoding IL-13–response markers Ym1, Ym2, Downloaded from cell size and increased size and secretory material within Paneth Mgl2, Ccl11 (eotaxin 1), and Ccl2/Mcp1, to a similar extent in cells in the jejunum (Fig. 8E, 8K, 8L). In the pancreatic ducts, il13ra22/2 and wt mice. In contrast, markers associated IL-33 induced epithelial cell hypertrophy with accumulation of with lymphocyte subsets, including the Th2 differentiation factor eosinophilic material within epithelial cells (hyalinosis), periductal Gata3, the costimulatory marker, PD1, the ILC2/M2 differentia- infiltration of inflammatory cells, and decreased zymogen content in tion factor, Rora, the Th17 differentiation factor, Rorc, and the 2/2 the pancreas, which may have been related to the decreased food Treg marker, Foxp3, were reduced in il13ra2 mice compared http://www.jimmunol.org/ intake (Fig. 8F–J). Accumulation of similar eosinophilic material with wt under these conditions. Overall, the expression of genes was also present within epithelial cells in the esophagus and associated with leukocyte infiltration was proportional to the 2 2 stomach (data not shown). Although il13ra2 / mice had more percentage of CD45+ cells detected in the adipose tissue. Because severe diarrhea and perianal irritation (Fig. 4E, 4F), the microscopic the gene expression analysis was carried out with whole tissue changes throughout the GI tract were similar in severity and dis- extracts, however, the current findings do not allow us to distin- 2 2 tribution between wt and il13ra2 / mice. Taken together, these guish expression level per cell compared with changes in infil- findings show that IL-33 induced diarrhea and perianal irritation in trating cell types, or whether the genes of interest were expressed the context of HFD, which was exacerbated in IL-13Ra2–deficient in the immune infiltrate or in the adipose tissue itself. For ex- mice, associated with reduced food intake, but was not associated ample, Rora can be expressed in adipocytes under conditions of by guest on September 27, 2021 with an identifiable gene expression pattern or signature in the gut. inflammation and obesity, leading to endoplasmic reticulum stress and supporting macrophage infiltration (53). IL-13 deficiency Discussion produced the opposite profile, resulting in increased expression of The infiltration of metabolic tissues by macrophages, eosinophils, markers of immune infiltration, along with reduced expression of and lymphocytes establishes a cytokine milieu that influences IL-13 response genes. These findings support a regulatory role for glucose regulation and insulin responsiveness. A type 1 environ- IL-13 in limiting tissue inflammation in response to IL-33. ment of classically activated macrophages, reactive oxygen spe- Along with the gene expression changes, IL-33 administration cies, and proinflammatory cytokines contributes to insulin reduced body weight on HFD, and reduced circulating levels of resistance, whereas a type 2 environment of alternatively activated insulin, cholesterol, and fasting blood glucose. In both eWAT and macrophages and eosinophils promotes glucose homeostasis in scWAT, IL-33 administration reduced expression of genes adipose tissue (46). Shifting this balance by cytokine manipulation encoding the adipokine adiponectin, the insulin-sensitive glucose could alleviate insulin resistance and re-establish glucose ho- transporter, Glut4 (Slc2A4), and the metabolic hormone leptin. It meostasis under conditions of metabolic stress. IL-13 may play a also reduced expression of the beige adipocyte-associated markers role, as IL-13–deficient mice have been reported to have hyper- UCP1 and Cidea. Beige adipocytes regulate caloric expenditure, glycemia on HFD (22), and IL-13 overexpression protects against and their induction by IL-33–activated ILC2 is thought to promote HFD-induced obesity (5). IL-13Ra2 modulates endogenous IL-13 metabolic homeostasis (34, 47). Although IL-33 administration concentration and activity by regulating IL-13 neutralization and induces beiging of WAT under conditions of a low-fat or chow depletion (10). IL-13Ra2 is inducibly expressed on fibroblasts, diet, this effect is abrogated under conditions of HFD (47). In smooth muscle cells, and other cell types, but its expression has response to HFD, UCP1 can be transiently induced in brown ad- not been previously described in adipose tissue. The finding that ipose tissue, but not WAT (47, 54). Thus, we did not observe in- IL-13Ra2 was induced in adipose tissue under conditions of HFD creased expression of UCP1 in WAT of mice on HFD in response suggests that blockade or deficiency of IL-13Ra2 could elevate to IL-33. Although the current study was focused on changes local IL-13 concentrations, which in turn could drive anti- induced by obesity, the effects of IL-33 administration on beiging inflammatory macrophage polarization, and help to maintain in il13ra22/2 mice under conditions of caloric restriction or metabolic homeostasis. temperature modulation will be of importance to examine. We found that IL-13Ra2 deficiency ameliorated the metabolic Whereas IL-33 administration reduced expression of these consequences of HFD, but only when a type 2 environment was metabolic markers in adipose tissue, IL-13Ra2 deficiency did not established by IL-33 administration. In the absence of IL-33 ad- have any further effect. In eWAT, IL-13 deficiency produced a ministration, a small number of IL-13 response genes was ele- strong additional reduction in expression of these markers, vated, but metabolic parameters were unchanged in il13ra22/2 whereas in scWAT, neither IL-13Ra2 deficiency nor IL-13 The Journal of Immunology 1357 Downloaded from http://www.jimmunol.org/ by guest on September 27, 2021

FIGURE 8. IL-33 increases mononuclear and eosinophilic infiltrates in the GI tract and pancreas, with perianal irritation. HFD-fed il13ra22/2 and wt control mice were administered PBS or 0.125 mg/kg IL-33 every other day for 2 wk. (A) Mononuclear infiltrates in jejunum; (B) eosinophilic infiltrates in jejunum; (C) mononuclear infiltrates in colorectum; (D) eosinophilic infiltrates in colorectum; (E) goblet cells in jejunum; (F) epithelial hyalinosis/ hypertrophy in pancreatic duct; (G) pancreatic periductal inflammatory infiltrates; (H) reduction in pancreatic zymogen content. For (A)–(H), data plotted are median of two to five mice per group. For (F), only one mouse was scored in the PBS-treated il13ra22/2 group, representative of no detectable in this group. Pancreatic ductal from: (I) wt mice administered PBS; or (J) il13ra22/2 mice administered IL-33, showing increased eosinophilic material in ductal epithelial cells (*), decreased zymogen granules within acinar cells (+), and periductal inflammatory cell infiltrates (↑); jejunum from: (K) wt mice administered PBS; or (L) il13ra22/2 mice administered IL-33 showing increased goblet cell numbers (*), increased inflam- matory cell infiltrates in lamina propria (+), and increased size/secretory material in Paneth cells (↑). deficiency affected expression. Both adipose tissues responded to reduced metabolic risk, improved glucose tolerance, and insulin IL-33 administration by skewing toward a type 2 profile, with sensitivity (38, 56). Lymphocytes homing to eWAT and scWAT increased expression of tissue IL-5, IL-13, Gata3, and Ym1. The may differ in their responsiveness to IL-33. Recent studies have observation that IL-33–induced gene expression changes were defined the critical role of IL-33 in expansion and maintenance of altered in eWAT but not in scWAT of il132/2 mice suggests that tissue-resident Foxp3+ Treg in VAT, which prevent obesity- IL-13 regulation of the IL-33 response is more prominent in associated inflammation and preserve insulin sensitivity and glu- eWAT, consistent with the higher expression of IL-13Ra2 found in cose tolerance (57–59). In VAT of obese mice, Treg numbers are that tissue. Compared to scWAT, the VAT depot eWAT displays reduced, contributing to inflammation and insulin resistance (60), increased inflammation (55), and is associated with insulin resis- and this deficit could be corrected by administration of exogenous tance and dyslipidemia (38). In contrast, scWAT is associated with IL-33 (43). 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Notari, J. A. Bohl, Disclosures L. P. McLean, T. R. Ramalingam, T. A. Wynn, J. F. Urban, Jr., et al. 2013. IL-33- All authors are employees of Pfizer. induced alterations in murine intestinal function and cytokine responses are The Journal of Immunology 1359

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