Dependent Upon IL-13 Activation of STAT6

IL-25 or IL-17E Protects against High-Fat Diet−Induced Hepatic Steatosis in Mice Dependent upon IL-13 Activation of STAT6

This information is current as An-Jiang Wang, Zhonghan Yang, Viktoriya Grinchuk, Allen of September 28, 2021. Smith, Bolin Qin, Nonghua Lu, Duan Wang, Hongbing Wang, Thirumalai R. Ramalingam, Thomas A. Wynn, Joseph F. Urban, Jr., Terez Shea-Donohue and Aiping Zhao J Immunol published online 30 September 2015

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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 © 2015 by The American Association of Immunologists, Inc. All rights reserved. Print ISSN: 0022-1767 Online ISSN: 1550-6606. Published September 30, 2015, doi:10.4049/jimmunol.1500337 The Journal of Immunology

IL-25 or IL-17E Protects against High-Fat Diet–Induced Hepatic Steatosis in Mice Dependent upon IL-13 Activation of STAT6

An-Jiang Wang,*,†,‡,1 Zhonghan Yang,*,†,x,1 Viktoriya Grinchuk,*,† Allen Smith,{ Bolin Qin,* Nonghua Lu,‡ Duan Wang,‖ Hongbing Wang,‖ Thirumalai R. Ramalingam,# Thomas A. Wynn,# Joseph F. Urban, Jr.,{ Terez Shea-Donohue,*,† and Aiping Zhao*,†

IL-25 or IL-17E is a member of IL-17 cytokine family and has immune-modulating activities. The role of IL-25 in maintaining lipid metabolic homeostasis remains unknown. We investigated the effects of exogenous IL-25 or deficiency of IL-25 on hepatic lipid accumulation. IL-25 expression was examined in paraffin-embedded tissue sections of liver from patients or in the livers from mice. Mouse model of steatosis was induced by feeding a high-fat diet (HFD). Extent of steatosis as well as expression of cytokines, key Downloaded from enzymes for lipid metabolic pathways, markers for Kupffer cells/macrophages, and lipid droplet (LD) proteins, were analyzed. Our results show that hepatic steatosis in mice was accompanied by increased LD proteins, but decreased IL-25 in the liver. Decreased hepatic IL-25 was also observed in patients with fatty liver. Administration of IL-25 to HFD-fed wild-type mice led to a significant improvement in hepatic steatosis. This effect was associated with increased expression of IL-13, development of alternatively activated Kupffer cells/macrophages, and decreased expression of LD proteins in the liver. In contrast, administration of IL-25 to HFD-fed mice deficient in STAT6 or IL-13 had no effects. In addition, stimulation of primary hepatocytes with IL-13, but not IL-25, http://www.jimmunol.org/ resulted in downregulation of LD proteins. Finally, mice deficient in IL-25 had exacerbated hepatic lipid accumulation when fed the HFD. These data demonstrate that dysregulated IL-25 expression contributes to lipid accumulation, whereas exogenous IL-25 protects against hepatic steatosis through IL-13 activation of STAT6. IL-25 and IL-13 are potential therapeutic agents for hepatic steatosis and associated pathologies. The Journal of Immunology, 2015, 195: 000–000.

onalcoholic fatty liver disease (NAFLD) represents dysregulated lipid metabolic pathways are the important deter- a spectrum of liver diseases ranging from simple steatosis minants of hepatic steatosis (1). Inﬂammation is a core component

N and steatohepatitis to cirrhosis without excessive con- linking obesity and associated metabolic diseases including by guest on September 28, 2021 sumption of alcohol (1). Major etiological factors that contribute to NAFLD (2), and proinﬂammatory cytokines such as IL-1b, TNF-a, the development of NAFLD include obesity, type 2 diabetes, and and IL-6 are believed to be key mediators for metabolic dysreg- dyslipidemia. As the incidence of obesity reaches endemic levels ulation. worldwide, the prevalence of NAFLD has been increasing steadily IL-25, also called IL-17E, is a member of the IL-17 cytokine in recent decades, with a current estimate of 20% of all adults family (3). Although other members of the family, especially being affected (1). The hallmark of NAFLD is steatosis charac- IL-17A and IL-17F, have biological activities similar to Th1 terized by a histologic manifestation of intracytoplasmic lipid inﬂammatory cytokines, IL-25 promotes type 2 immunity. The re- accumulation in the form of triglycerides (TG) in hepatocytes. ceptor for IL-25 is a heterodimer consisting of IL-17RB and IL- Excessive lipid accumulation/storage in the liver arises from an 17RA (4). The downstream signaling pathway of IL-25 remains imbalance between lipid acquisition and disposal; therefore, to be fully elucidated, although transcription factors Act1 and

*Department of Radiation Oncology, University of Maryland School of Medi- This article was prepared while A.Z. was employed at the University of Maryland cine,Baltimore,MD21201;†Department of Medicine, University of Maryland School of Medicine. The opinions expressed in this article are the author’s own and School of Medicine, Baltimore, MD 21201; ‡Department of Gastroenterology do not reflect the view of the National Institutes of Health, the Department of Health and Hepatology, First Affiliated Hospital of Nanchang University, Nanchang and Human Services, or the U.S. government. Mention of trade names or commercial 330006, China; xDepartment of Biochemistry, Zhongshan Medical School, products in this publication is solely for the purpose of providing specific information Sun Yat-sen University, Guangzhou 510080, China; {Diet, Genomics, and Im- and does not imply recommendation or endorsement by the U.S. Department of munology Laboratory, Agricultural Research Service, Beltsville Human Nutri- Agriculture. The U.S. Department of Agriculture is an equal opportunity provider tion Research Center, U.S. Department of Agriculture, Beltsville, MD 20705; and employer. ‖Department of Pharmaceutical Sciences, University of Maryland School of Address correspondence and reprint requests to Dr. Aiping Zhao at the current Pharmacy, Baltimore, MD 21201; and #Division of Parasitology, National Insti- address: Division of Physiological and Pathological Sciences, Center for Scientific tute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Review, National Institutes of Health, 6701 Rockledge Drive, Bethesda, MD 20892. MD 20892 E-mail address: [email protected] 1A.-J.W. and Z.Y. contributed equally to this work. The online version of this article contains supplemental material. ORCIDs: 0000-0002-3009-4074 (Z.Y.); 0000-0001-5823-5601 (B.Q.). 2 2 Abbreviations used in this article: / , deficient; Arg1, arginase I; CIDEA, cell Received for publication February 24, 2015. Accepted for publication September 9, death–inducing DFFA-like effector A; FA, fatty acid; FIZZ, found in inflammatory 2015. zone; Hadh, hydroxyacyl–CoA dehydrogenase; HFD, high-fat diet; ILC2, type 2 innate lymphoid cell; LD, lipid droplet; M1, classically activated; M2, alternatively This work was supported by National Institutes of Health Grants R01-DK083418 (to activated; NAFLD, nonalcoholic fatty liver disease; NCD, normal control diet; PLIN, A.Z.), R01-AI/DK49316 (to T.S.-D.), and R01-DK061652 (to H.W.), U.S. Depart- perilipin; qPCR, quantitative PCR; TG, triglyceride; WT, wild-type. ment of Agriculture Current Research Information System project 8040-51000-058- 00 (to A.S. and J.F.U.), and National Natural Science Fund of China Grant 81460122 Ó (to A.-J.W.). Copyright 2015 by The American Association of Immunologists, Inc. 0022-1767/15/$25.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1500337 2 IL-25 AND HEPATIC STEATOSIS

TNFR-associated factor 6 may be involved, and the signaling Materials and Methods events initiated by binding to IL-17RB are likely to be tissue/cell Mice and diet specific (5). Recent studies demonstrated that IL-25 acts pri- C57BL/6 wild-type (WT) mice were purchased from the National Cancer marily on type 2 innate lymphoid cells (ILC2) to induce the Institute Mouse Repository (Frederick, MD). Mice deficient in STAT6 production of the type 2 cytokines IL-13 and IL-5 (6). It is (STAT62/2; The Jackson Laboratory) or IL-13 (IL-132/2; National In- critical for host protective immunity against parasitic nematode stitutes of Allergy and Infectious Diseases Taconic contract) on C57BL/6 background were bred in the U.S. Department of Agriculture/Beltsville infection and is also associated with airway allergic inflamma- 2/2 tion (7–9). Of equal importance is the ability of IL-25 to inhibit animal facility. Mice deficient in IL-25 (IL-25 ) were generated by Regeneron Pharmaceuticals (Tarrytown, NY) and backcrossed to C57BL/6 proinflammatory Th1 and Th17 cytokine responses that are im- mice for 10 generations. A commonly used high-fat diet (HFD)–fed mouse plicated in the metabolic manifestation associated with obesity model of hepatic steatosis was selected because it is considered physio- (10). Therefore, IL-25 is emerging as a key regulator of in- logically relevant. Male 5-wk-old mice were fed an HFD (60% of kcal flammation. The highest levels of IL-25 are found in the gas- from fat; Research Diet, New Brunswick, NJ) or normal control diet (NCD; 10% of kcal from fat) ad libitum. These studies were conducted trointestinal tract and lung, where it is expressed primarily by with institutional approval from both the University of Maryland, Balti- epithelial cells and certain types of immune cells (8, 9, 11–13). more and the U.S. Department of Agriculture Beltsville Area Animal Care More recently, a study showed that hepatocytes were capable of and Use Committees (protocol #13-003), in accordance with principles set producing IL-25, and decreased hepatic IL-25 was observed in forth in the Guide for Care and Use of Laboratory Animals, Institute of animals or humans with hepatitis (14). Whether IL-25 is in- Laboratory Animal Resources, National Research Council, Health and Human Services Publication. volved in regulation of lipid metabolism and associated hepatic steatosis is not known. Administration of IL-25 Downloaded from Given that inflammation affects the progression of NAFLD, After being on the respective diet for ∼14 wk, mice were separated into whereas IL-25 possesses anti-inflammatory properties, we hy- treatment or control groups. Mice in the treatment group were injected i.p. pothesized that constitutively expressed IL-25 maintains lipid with 1 mg mouse rIL-25 (R&D Systems, Minneapolis, MN) in 100 mlPBS metabolic homeostasis, whereas enhanced IL-25 production in vivo daily for 5 d in the first week followed by three injections per week for an would protect against the excessive lipid accumulation in the liver. additional 2 wk. Control mice were given injections of 35 mg BSA, which is equal to the amount of BSA carrier protein included in the IL-25 preparation. The present study was designed to investigate: 1) the effects of The amount of cytokine administered was based on the minimum dose of http://www.jimmunol.org/ exogenous IL-25 on obesity-associated hepatic steatosis in mice; IL-25 that induced a prominent Th2 immune response (8). 2) the molecular mechanisms of IL-25 regulation of lipid accumulation/storage; 3) the contribution of the IL-13–STAT6 Mouse hepatocyte preparation and treatment axis to the effects of IL-25; and 4) the impact of IL-25 deficiency Mouse primary hepatocytes were isolated from HFD-fed obese mice using on expression of hepatic steatosis. a two-step collagenase perfusion method (15). Hepatocytes were seeded at by guest on September 28, 2021

FIGURE 1. Increased expression of LD-associated proteins and decreased expression of IL-25 in the livers of mice with steatosis. Mice were fed an HFD or NCD. (A) Liver mass. (B) Liver TG levels. (C) H&E-stained liver sections (original magniﬁcation 3100) and steatosis scores. (D) Hepatic expressions of LD-associated proteins by qPCR. (E) Hepatic expressions of macrophage markers and immune mediators by qPCR. (F) Levels of IL-25 protein expression in the liver by ELISA. Data shown in bar graphs are mean 6 SEM and representatives of two independent experiments with 5–10 mice per group per experiment. *p , 0.05 versus NCD. The Journal of Immunology 3

1 3 106 cells/well in six-well collagen-coated plates in DMEM supple- injected with BSA and using the same conditions to evaluate the samples mented with 5% FBS, 100 U/ml penicillin, 100 mg/ml streptomycin, from mice injected with IL-25. Comparisons were made only among slides 4 mg/ml insulin, and 1 mmol dexamethasone. After 4 h of attachment at prepared on the same day. 37˚C in a humidified atmosphere of 5% CO2, the medium was changed to DMEM without FBS, and cells were cultured for another 24 h before Immunohistochemical staining on human liver sections treatment with IL-25 or IL-13. Medium was replaced on a daily basis. Paraffin-embedded tissue slides of liver sections were acquired from a total RNA extraction, cDNA synthesis, real-time quantitative PCR, of 14 patients. Six of them had hepatic steatosis, diagnosed initially with ultrasound examination and later confirmed by liver biopsy evaluation. The and ELISA other eight patients had no hepatic steatosis and were used as control Total RNA was extracted with TRIzol reagent (Invitrogen, Grand Island, subjects. The medical history was reviewed retrospectively by a medical NY) per the manufacturer’s instructions. RNA samples (2 mg) were reverse- doctor specializing in gastroenterology and hepatology. All of the patients . transcribed to cDNA using the First Strand cDNA Synthase Kit (MBI were 18 y old at the time of biopsy and had no history of alcohol abuse, Fermentas, Hanover, MD) with random hexamer primer. Quantitative PCR serologic evidence of viral hepatitis, blood transfusion, or history of other (qPCR) was performed on an iCycler detection system (Bio-Rad, Hercules, competing etiologies for hepatic steatosis and coexisting causes for chronic CA) in a 25 ml volume using SYBR Green Supermix (Bio-Rad). Amplifi- liver disease (18). For immunohistochemical staining, slides were dewaxed cation conditions were: 95˚C for 3 min, 50 cycles of 95˚C for 15 s, 60˚C for with xylene, gradiently washed with ethanol, blocked with goat serum, and 15 s, and 72˚C for 20 s. The fold change in mRNA expression for targeted then incubated with anti–IL-25 mAb (1:100; catalog number MAB1258; gene was relative to the respective vehicle after normalization to 18s rRNA. R&D Systems) or the isotype control. The slides were stained with Tissue homogenates of liver samples were prepared with RIPA buffer (Cell Peroxidase/DAB kit (Dako Denmark) and then digitally photographed with Signaling Technology, Beverly, MA). IL-25 protein expression in the liver Olympus BX51WI microscope (Olympus). was measured with ELISA kit according to the manufacturer’s instructions Lipid extraction and analysis

(BioLegend, San Diego, CA). Downloaded from Tissue homogenates were prepared in lipid extraction buffer, shaken in the H&E and immunofluorescent staining on mouse liver sections dark for 2 h, and centrifuged at 3,500 rpm for 10 min. The soluble portion Tissue sections (5 mm) were cut from frozen blocks of mouse liver and was transferred to a 1.5-ml tube and dried under vacuum for 30 min. The stained with H&E for histological analysis. Slides were graded for steatosis dried lipids were made soluble in TG assay buffer by vortexing until the lipids were homogeneous. Levels of hepatic TG were determined using according to the Kleiner system by an investigator who was unaware of the a commercial kit (Cayman Chemical, Ann Arbor, MI). treatment and mouse genotype (16). A score of 0–3 was assigned to describe the extent of steatosis based on lipid accumulation in the hepatocytes Western blot analysis http://www.jimmunol.org/ (0, ,5%; 1, 5–33%; 2, 33–66%; and 3, .66%). Immunofluorescent staining of mouse liver sections was carried out as described previously Western blot analysis was performed as described previously (19). In brief, (17). Briefly, the slides were blocked with 5% normal donkey serum and tissue lysates prepared in RIPA buffer (Cell Signaling Technology) were then incubated with anti-F4/80 (BioLegend) and anti–YM-1 (R&D Sys- separated on 14% Novex Tris-glycine Mini Gels and transferred to Novex tems) Abs overnight. The slides were stained with Dylight 488–donkey Nitrocellulose Membrane (Life Technologies, Grand Island, NY). The anti-rat IgG and Dylight 659–donkey anti-goat IgG (1:400; Jackson membranes were blocked with 5% milk and incubated with primary Ab ImmunoResearch Laboratories, West Grove, PA) for 1 h and then digitally against cell death–inducing DFFA-like effector A (CIDEA; 1:2000; Cell photographed with a Nikon TE 2000-E microscope (Nikon, Melville, NY). Signaling Technology) overnight. Bands were detected using an HRP- The images were taken by establishing settings for the samples from mice conjugated goat anti-rabbit secondary Ab (Santa Cruz Biotechnology, by guest on September 28, 2021

FIGURE 2. Exogenous IL-25 ameliorates HFD-induced hepatic steatosis. Mice were fed the HFD or NCD and then injected with IL-25 or BSA. (A) Liver mass. (B) Liver TG levels. (C) H&E-stained liver sections (original magniﬁcation 3100). (D) Steatosis scores. Data shown in bar graphs are mean 6 SEM and representative of two independent experiments with ﬁve mice per group per experiment. *p , 0.05 versus respective NCD, fp , 0.05 versus respective BSA treated. 4 IL-25 AND HEPATIC STEATOSIS

Santa Cruz, CA). The Western blot was reprobed with anti-GAPDH as Exogenous IL-25 ameliorates HFD-induced hepatic steatosis a loading control. We next determined if exogenous IL-25 ameliorates HFD-induced Statistical analysis hepatic steatosis. To this end, mice were fed the HFD for ∼14 wk Statistical analysis was performed using one-way ANOVA followed by and then administered either IL-25 or BSA. After a 3-wk treat- Newman-Keuls test to compare the difference among three or more ment, mice receiving IL-25 had significantly reduced hepatic treatment groups or the Student t test to compare the difference between steatosis indicated by decreased liver mass and hepatic TG levels , two groups. The p values 0.05 were considered significant. (Fig. 2A, 2B). The amelioration was further confirmed by histological evaluation of H&E-stained liver sections showing de- Results creased size/number of fat vacuoles and lower steatosis score HFD-induced hepatic steatosis is associated with decreased (Fig. 2C, 2D). In addition, IL-25 treatment did not alter signifi- expression of IL-25 in the liver cantly the liver mass or TG levels in mice fed the NCD (Fig. 2). Of Hepatocytes are capable of producing IL-25, and decreased hepatic note, IL-25 decreased food intake in both groups of NCD- and IL-25 expression has been reported in humans or mice with HFD-fed mice in the first week of injection, but induced loss of hepatitis (14). We established an HFD-induced model of hepatic body weight and decrease in hepatic lipid accumulation only in steatosis in mice and evaluated a role of IL-25 in NAFLD. As HFD-fed mice (Supplemental Fig. 1). This suggests that the expected, C57BL/6 mice fed the HFD for 14 wk had significantly transient decrease in food intake was unlikely a major factor for enlarged livers and increased levels of hepatic TG (Fig. 1A, 1B), the loss of body weight or the amelioration of hepatic steatosis in characteristics of steatosis, when compared with age- and sex- IL-25–treated HFD-fed mice. matched mice fed an NCD. Evaluation of the H&E-stained liver To dissect the lipid metabolic pathways that might contribute to Downloaded from sections indicated that HFD-fed mice had a high degree of IL-25–induced improvement in hepatic steatosis, we analyzed steatosis accompanied by the presence of both large and small fat expression of genes encoding key enzymes for lipid metabolism. vacuoles (Fig. 1C). In addition, steatotic livers from HFD-fed IL-25 treatment did not alter hepatic expression of key lipogenic mice had significantly upregulated gene expression of lipid enzymes (Fasn, Acly,andAcaca), lipid transporters (Cd36, Fatp2, droplet (LD)–associated proteins including the CIDE proteins and Fatp5), hepatic lipase (Lipc), or carnitine palmitoyltransfer- Cidea, Cideb, Cidec, as well as perilipin 2 (Plin2) (Fig. 1D). ase 1a, whereas it significantly increased the expression of http://www.jimmunol.org/ Hepatic expression of F4/80 (Emr1) and Cd11b (Fig. 1E), the hydroxyacyl–CoA dehydrogenase (Hadh), an enzyme important markers for Kupffer cells (20, 21), were comparable between mice for fatty acid (FA) oxidation (Fig. 3A, 3B, and data not shown). In fed the NCD and HFD. A significant upregulation of Tnfa and addition, IL-25 treatment significantly decreased expressions of Ccl2, but not Il6, was also observed in the livers of HFD-fed mice the CIDEs as well as Plin2 in the livers of HFD-fed mice (Fig. 1E). Interestingly, HFD-fed mice had a significantly de- (Fig. 3C). The decrease in CIDEA protein in the livers of IL-25– creased IL-25 protein in the livers when compared with NCD-fed treated mice was further confirmed by Western blot (Fig. 3D). mice (Fig. 1F). Hepatic expression of these LD-associated proteins was not by guest on September 28, 2021

FIGURE 3. Administration of IL-25 to HFD-fed mice alters the expressions of key enzyme for lipid metabolic pathway and LD-associated proteins in the liver. Mice were fed the HFD or NCD and then injected with IL-25 or BSA. qPCR was carried out to examine gene expression of lipogenic enzymes (A), FA oxidation proteins (B), and LD-associated proteins (C). CIDEA protein was detected by Western blot, and the density represents average of the bands after normalization to GAPDH (D). Data shown in bar graphs are mean 6 SEM and representative of two independent experiments with five mice per group per experiment. *p , 0.05 versus respective BSA treated. The Journal of Immunology 5 significantly altered by IL-25 treatment in NCD-fed mice, possi- transcription factor retinoic acid–related orphan receptor a in the bly due to the low constitutive expression of these genes (data not liver (Fig. 4A). Other type 2–related cytokines—Il4, Il25,andIl33— shown). These data suggest that increased FA oxidation and al- were expressed either at relatively low levels in the liver or unaf- tered LD formation may contribute to the effects of IL-25 on fected by treatment with IL-25 (data not shown). In contrast, IL-25 hepatic steatosis. treatment significantly decreased the expression of Ccl2, but not Tnfa or Il6, in the livers of HFD-fed mice (Fig. 4B). Upregulation Exogenous IL-25 induces expression of type 2 cytokine and of Il5 and Il13 as well as a downregulation of Ccl2 were also de- alternative activation of Kupffer cell/macrophage in the liver tected in the livers of IL-25–treated NCD-fed mice (Supplemental Consistent with a role of IL-25 in promoting type 2 immunity, there Fig. 2A). was an upregulation of major type 2 cytokine gene expression of Kupffer cells in the liver are derived from either intrahepatic Il13 and Il5 in the livers of HFD-fed mice receiving IL-25 precursors or infiltrating monocytes (23) and play an important (Fig. 4A). ILC2 are the major IL-25–responsive/IL-13–produc- role in obesity-induced hepatic steatosis (24). IL-25 treatment did ing cells in the liver (22). Accordingly, IL-25 injection increased not affect the gene expression of F4/80 (Emr1), but significantly the expression of major ILC2 markers (6) including CD25 (Il2ra), upregulated the expression of alternatively activated (M2) Kupffer CD90 (Thy1), CD127 (Il7r), and Sca-1 (Ly6a) as well as the cell markers arginase I (Arg1), YM-1 (Chi3l3), and found in Downloaded from http://www.jimmunol.org/ by guest on September 28, 2021

FIGURE 4. Administration of IL-25 to HFD-fed mice induces a type 2 cytokine (Cyto.) response and alternative activation of Kupffer cell/macrophage in the liver. Mice were fed an HFD and then received injections of IL-25 or BSA. (A) Hepatic expression of type 2 cytokines and molecular markers for ILC2. (B and C) Hepatic expression of proinflammatory cytokines/mediators and markers for Kupffer cell/macrophage activation. (D) Immunofluorescent staining of liver sections with anti-F4/80 (green) and anti–YM-1 (red). The images are representatives of five mice in each group (original magnification 3200). Data shown in bar graphs are mean 6 SEM and representatives of two independent experiments with five mice per group per experiment. *p , 0.05 versus respective BSA-treated. 6 IL-25 AND HEPATIC STEATOSIS inflammatory zone (FIZZ) 1 (Retnla) (Fig. 4C). Increased number (data not shown). In addition, the IL-25–induced alterations in of M2 Kupffer cells/macrophages in the liver was validated further hepatic expression of Cideb, Cidec, Plin2, Hadh,orCcl2 in WT by immunofluorescent staining with anti–YM-1, as more F4/80+/ mice (Figs. 3C, 4A) were absent in STAT62/2 mice, with the YM-1+ cells could be visualized in the livers from IL-25–treated exception of Cidea, which remained downregulated (Fig. 5E, HFD-fed mice than in those from BSA-treated mice (Fig. 4D). Supplemental Fig. 3). Likewise, M2 markers were upregulated by IL-25 administration in Exogenous IL-25 can increase the expression of IL-4 and IL-13 the livers of NCD-fed mice (Supplemental Fig. 2B). (9, 10), both of which activate the STAT6 pathway; therefore, parallel experiments were carried out to determine the contribu- Amelioration of hepatic steatosis induced by IL-25 depends tion of IL-13 to the effects of IL-25 on hepatic steatosis. In IL- upon STAT6 and IL-13 132/2 mice fed the HFD for 14 wk, IL-25 treatment for 3 wk had The increased expression of IL-13 and number of M2 Kupffer cells/ no effect on liver mass, hepatic TG level, or lipid accumulation macrophages in the livers of HFD-fed mice injected with IL-25 (Fig. 6A, 6B). There was also no increase in the number of M2 prompted us to investigate whether the IL-13–STAT6 axis medi- Kupffer cells in the livers of IL-25–treated IL-132/2 mice, indi- ated the beneficial effects of IL-25. Thus, WT and STAT62/2 mice cated by the similar transcript levels of the markers (Arg1 and were fed the HFD or NCD for 14 wk and then treated with IL-25 Retnla) between mice injected with BSA and IL-25 (Fig. 6C) and or BSA. IL-25 administration to either NCD- or HFD-fed confirmed by immunofluorescent staining of liver section with STAT62/2 mice had no effect on liver mass or hepatic TG lev- anti–YM-1 (data not shown). Among the LD-associated proteins els (Fig. 5A, 5B), whereas HFD-fed WT mice treated with IL-25 in the livers of IL-132/2 mice, IL-25 did not affect the expression showed a marked reduction in liver mass and hepatic TG levels of Cideb and Plin2, but surprisingly induced a modest upregula- Downloaded from (data not shown and Fig. 2). IL-25 injection also did not impact tion of the expression of Cidea and Cidec (Fig. 6D) contrary to histological steatosis grade of HFD-fed STAT62/2 mice (Fig. 5C). that observed in WT mice (Fig. 3C). qPCR showed that IL-25 injection induced upregulation of Il5 and The in vivo results indicated that the IL-13–STAT6 axis plays IL13 in the livers of HFD-fed STAT62/2 mice (Fig. 5D) compa- a critical role in the IL-25–induced amelioration of hepatic steatosis, rable to that of WT mice (Fig. 4A). However, the IL-25–induced likely through inhibiting expression of LD-associated proteins.

upregulation of M2 Kupffer cell markers Arg1 and YM-1 (Chi3l3) Whether IL-13 directly acts on hepatocytes was examined further in http://www.jimmunol.org/ in WT mice (Fig. 4B, 4C) was almost absent in STAT62/2 mice primary culture of mouse hepatocytes. qPCR analysis showed that (Fig. 5D) consistent with a STAT6 dependence. Immunofluores- IL-13 treatment of hepatocytes significantly downregulated the cent staining with anti–YM-1 confirmed no increase in the number expression of Cidea, Cideb,andPlin2, whereas it had no effect on of M2 Kupffer cells in the livers of IL-25–treated-STAT62/2 mice the expression of Cidec (Fig. 6E). Of note, IL-13 treatment did not by guest on September 28, 2021

FIGURE 5. STAT6 dependence of IL-25–induced changes in hepatic steatosis and gene expression in the liver. STAT62/2 mice were fed an HFD or NCD diet and then treated with IL-25 or BSA. (A) Liver mass. (B) Liver TG levels. (C) H&E-stained liver sections from HFD-fed mice (original magniﬁcation 3100) and steatosis scores. (D) Expression of cytokines or M2 Kupffer cell markers in the livers of HFD-fed mice. (E) Expression of LD-associated proteins in the livers of HFD-fed mice. Data shown in bar graphs are mean 6 SEM and representatives of two independent experiments with ﬁve mice per group per experiment. *p , 0.05 versus respective STAT62/2-NCD (A and B)orSTAT62/2-BSA (D and E). The Journal of Immunology 7 Downloaded from http://www.jimmunol.org/

FIGURE 6. Critical role of IL-13 in IL-25–induced changes in hepatic steatosis and gene expression in the liver. IL-132/2 mice were fed the HFD and then treated with IL-25 or BSA. (A) Liver mass and liver TG levels. (B) H&E-stained liver sections from HFD-fed mice (original magnification 3100). (C) Expression of M2 Kupffer cell markers in the livers of HFD-fed mice. (D) Expression of LD-associated proteins in the livers of HFD-fed mice. (E) Expression of LD-associated proteins in cultures of mouse primary hepatocytes. Data are mean 6 SEM and from one experiment with five mice per group (A–D) or representative of two independent experiments in triplicates (E). *p , 0.05 versus IL-132/2-BSA or vehicle. by guest on September 28, 2021 alter the gene expression of the LD-associated proteins described without fatty liver. IL-25–positive hepatocytes were visualized above in the hepatocytes isolated from STAT62/2 mice (data not clearly in the liver sections from both groups of patients (Fig. 8). shown). In contrast, IL-25 had no direct effect on the expression However, IL-25 staining was dramatically less in patients with of any of the LD-associated proteins examined (Fig. 6E). fatty liver compared with those without. This is consistent with the ELISA results (Fig. 1F) showing decreased IL-25 protein in the Genetic deletion of IL-25 increases susceptibility to livers of HFD-fed mice. HFD-induced steatosis The potent effects of exogenous IL-25 on HFD-induced hepatic Discussion steatosis led us to examine further whether endogenous IL-25 is Immune components play an important role in the development important for maintaining lipid metabolic homeostasis. Thus, WT of hepatic steatosis, yet much of the focus has been on proin- 2/2 and IL-25 mice were fed the HFD or NCD for ∼16 wk. When flammatory cytokines with less attention to anti-inflammatory compared with age- and sex-matched WT mice, both NCD- and immune mediators. Type 2–related cytokines, including IL-25, 2/2 HFD-fed IL-25 mice had lower body weights (Fig. 7A). No are capable of counterregulating Th1 and Th17 immunity. Results significant differences in the liver mass or hepatic TG levels were from our current study indicate that exogenous IL-25 potently detected between the two strains of mice fed the NCD (Fig. 7B, ameliorates the hepatic steatosis induced by HFD feeding through 2/2 7C). However, HFD-fed IL-25 mice had enlarged livers that IL-13 activation of STAT6, whereas genetic deletion of IL-25 renders were macroscopically pale in color (Fig. 7B, 7D) and increased mice more susceptible to HFD-induced lipid accumulation in the levels of hepatic TG (Fig. 7C) as compared with their WT coun- liver. These results reveal a previously unrecognized role for IL-25 in terparts, although histological analysis was unable to reveal a sig- the regulation of lipid metabolism. nificant difference statistically in steatosis (Fig. 7E) because of IL-25 belongs to the IL-17 cytokine family (3). IL-17A, an- insufficient sensitivity of the grading system. The exacerbated other key member of the family, was identified as a central 2/2 hepatic steatosis in HFD-fed IL-25 mice was associated with player to the development and progression of NAFLD to steato- upregulated expressions of Cidea,Cideb, Cidec, as well as Plin2 hepatitis (25–27). Our results, however, show that IL-25 has (Fig. 7F). beneficial effects against NAFLD, which is consistent with the established anti-inflammatory role for IL-25. Gut and lung ep- Hepatic IL-25 expression is reduced in patients with fatty liver ithelial cells are the recognized cellular sources for IL-25 To investigate the clinical relevance of our findings from mouse (9, 10). Several types of immune cells may also produce IL-25 studies, we performed immunohistochemical staining with anti–IL- under certain circumstances, including Th2 cells, mast cells, 25 on paraffin-embedded liver sections from patients with or and macrophages (8, 11–13). A recent study further identified 8 IL-25 AND HEPATIC STEATOSIS Downloaded from http://www.jimmunol.org/

FIGURE 7. Mice with genetic deletion of IL-25 are more susceptible to HFD-induced hepatic steatosis. WT and IL-252/2 mice were fed an HFD or NCD diet. (A) Body weight. (B) Liver mass. (C) Liver TG level. (D) Gross appearance of the liver; representative H&E-stained liver sections (original magnification 3100). (E) Steatosis scores. (F) Hepatic expression of LD-associated proteins by qPCR. Data are mean 6 SEM and are representatives of two independent experiments with five to eight mice per group. *p , 0.05 versus respective NCD (A–C) or WT-HFD (F), fp , 0.05 versus respective WT. by guest on September 28, 2021 hepatocytes as IL-25–expressing cells (14). Our current study accompanied by decreased adiposity (31). Consistent with previ- showed that lipid accumulation in the liver was accompanied by ous observations, we observed that HFD-induced hepatic steatosis decreased levels of IL-25 protein indicative of a role in regula- was associated with increased expression of all members of the tion of lipid metabolism. Indeed, administration of IL-25 to CIDE family and PLIN2. More importantly, treatment with IL-25 HFD-fed WT mice resulted in a significant improvement in he- ameliorated hepatic steatosis and was accompanied by signifi- patic steatosis. Lipid accumulation in the liver arises from the cantly decreased gene expression of Cidea, Cideb, Cidec, and imbalance of lipid acquisition and disposal; therefore, impaired Plin2. Conversely, IL-252/2 mice had increased hepatic levels of FA oxidation, decreased lipid export, and increased de novo li- LD-associated proteins. These results strongly support that IL-25 pogenesis in the liver are considered the key determinants of regulates the expression of LD-associated protein, thereby con- hepatic steatosis. Our results showed that IL-25 treatment did not trolling lipid accumulation in the liver. affect hepatic expressions of key enzymes for lipogenesis or lipid Kupffer cells/macrophages in the liver represent ∼10% of the export, but increased the expression of Hadh, an enzyme critical resting total liver cell population and .80% of all tissue macro- for FA oxidation, implicating one of the potential mechanisms phages in the body (32). Traditional roles of Kupffer cells include for amelioration of steatosis by IL-25. launching biochemical attack, recruiting immune cells to the liver, LDs are cytosolic lipid storage organelles present in nearly all eliminating cell debris and microorganisms, as well as Ag pre- cell types, and abnormal LD formation contributes to various sentation. Kupffer cells are implicated in the pathogenesis of human diseases including NAFLD (28). In hepatocytes, the size various liver diseases including hepatitis, alcoholic liver disease, and number of TG-containing LDs are regulated by several types and liver fibrosis (32). Like all macrophages, Kupffer cells un- of proteins referred to as LD-associated proteins including dergo distinct pathways of activation and display different phe- members of CIDE and PLIN families. Both CIDEs and PLINs, notypes depending on the cytokine microenvironment. Classically especially the highly expressed PLIN2 in the liver, are believed to activated (M1) Kupffer cells are induced by Th1 cytokines and be the important regulators of lipid storage and formation of LDs characterized by upregulation of inducible NO synthase, whereas in hepatocytes (29). Multiple lines of research demonstrated that M2 Kupffer cells are induced by Th2 cytokines IL-4 and IL-13 mice or humans with hepatic steatosis have increased expression and feature highly upregulated Arg1 as well as the secretion of of LD-associated proteins in the liver (28). In addition, mice de- chitinase and FIZZ family members such as YM1, FIZZ1, and ficient in either one of the CIDEs exhibit decreased lipid accu- FIZZ2 (33). Emerging evidence indicates that differentially acti- mulation and reduced hepatic steatosis when fed an HFD. In vated Kupffer cells play distinctive roles in the progression of contrast, overexpression of CIDEA in mouse liver leads to in- NAFLD. Specifically, M1 Kupffer cells produce proinflammatory creased hepatic lipid accumulation (30). Mice with PLIN2 defi- cytokines, such as IL-1b and TNF-a, which inhibit peroxisome ciency are protected also from HFD-induced fatty liver disease proliferator–activated receptor a activity, leading to decreased The Journal of Immunology 9

FIGURE 8. IL-25 expression is decreased in the livers of patients with hepatic steatosis. Immunohistochemical staining with anti–IL-25 Ab was performed on parafﬁn-embedded tissue slides of human liver sections. The images are representatives of six patients with hepatic steatosis and eight patients without hepatic steatosis as control (original magniﬁ- cation 3200). One image from the staining with anti–IL-25 isotype control is also shown. Downloaded from

expression of genes involved in FA oxidation (24, 34). Depletion the immune mediators upregulate members of the CIDE family, of Kupffer cells with clodronate liposomes reduces HFD-induced similar to that by dietary FA shown previously (30), but the effects

steatosis (24, 34). In contrast, M2 Kupffer cells promote M1 are counterregulated when IL-13 signaling is intact. Despite the http://www.jimmunol.org/ Kupffer cell apoptosis through activation of arginase, thereby vital importance of IL-13 in the effects of IL-25, we were unable protecting against NAFLD (35). Our results showed that mice to detect significant differences in liver mass, hepatic TG level, treated with IL-25 exhibited a polarized type 2 immunity char- and steatosis grade among HFD-fed WT, STAT62/2, and IL-132/2 acterized by upregulations of IL-5 and IL-13, which led to de- mice. There were also no significant differences in mRNA ex- velopment of M2 Kupffer cells in the liver. Thus, it is conceivable pression for most of genes encoding LD-associated proteins that the beneficial effects of IL-25 on steatosis are derived, at least among HFD-fed WT, STAT62/2, and IL-132/2 mice (data not in part, through its ability to promote a type 2 environment, par- shown). These results suggest that the low constitutive level of IL- ticularly increasing IL-13 that can lead to development of M2 13 is not pivotal in controlling hepatic lipid accumulation. The Kupffer cells, as demonstrated previously (36, 37). It should be major cellular source of IL-13 in the liver could be ILC2 that are by guest on September 28, 2021 noted that Kupffer cells/macrophages in the liver are a heteroge- known to respond to IL-25 (42). Several other types of immune neous population (23). It remains to be determined whether the IL- cells are also capable of producing IL-13, including Th2 cells, 25–induced M2 development occurred in the resident Kupffer eosinophils, NKT cells, and B cells (41); however, none of them cells, the infiltrated monocytes/macrophages, or both. are known to respond to IL-25 in vivo (42). Thus, it is likely that IL-25 induces increased expression of IL-4 and IL-13 that ILC2 are the IL-13–producing cells that mediate the IL-25 effects primarily activate the STAT6 pathway, as well as IL-5 that elicits on hepatic steatosis. eosinophilia (9, 38). Consistent with these observations, mice Mice deficient in IL-25 develop intestinal inflammation in re- treated with IL-25 had increased gene expression of il5 and il13 sponse to enteric nematode infection, characterized by increased but negligible levels of il4 in the liver. Notably, the amelioration of expression of IL-17A as well as defective type 2 immune response hepatic steatosis induced by exogenous IL-25 depended on STAT6 (10). Hepatocytes can make IL-25, and decreased expression of or IL-13, indicating that IL-25 acts through IL-13 activation of IL-25 in the liver is observed in animals or human with hepatitis STAT6 signaling to regulate lipid accumulation in the liver. He- (14). Our results show that there is an increased severity of stea- patocytes are known to express receptors for IL-13, and IL-13 tosis in mice with IL-25 deficiency accompanied by high levels of treatment of hepatocyte in culture can induce STAT6 activation LD-associated proteins. Finally, the observation that patients with (39–41). Our current studies showed that IL-13, but not IL-25, NAFLD had decreased immunostaining of IL-25 in the livers decreased the expression of LD-associated proteins in culture of demonstrates the clinical importance of hepatic expression of hepatocytes from WT but not STAT62/2 mice, indicating that at IL-25. The mechanisms by which IL-25 was decreased in steatosis least part of the effects of IL-25 on steatosis were mediated by its remain to be investigated; however, the decrease in IL-25 was downstreamIL-13actingonhepatocytesthroughSTAT6- accompanied by an increase in TNF-a in steatotic livers, and dependent pathways. In addition, in vivo IL-25 treatment TNF-a was shown to negatively regulate IL-25 expression in the induced development of M2 Kupffer cells in the livers of colon or brain (43). More studies are needed to ascertain whether HFD-fed WT but not in STAT62/2 and IL-132/2 mice. Coupled this same mechanism of TNF-a regulating IL-25 occurs in the with the well-documented role of IL-13 in promoting M2 mac- liver. rophages, it is plausible that IL-25 in the liver regulates the lipid Taken together, our study is the first, to our knowledge, to show accumulation/storage primarily through the direct action of IL- that IL-25 is important in maintaining lipid metabolic homeostasis 13 on Kupffer cells as well as on hepatocytes. What is somewhat in the liver. Even though the detailed mechanism remains to be surprising is that IL-25 administration to IL-132/2 mice induced fully understood, it is believed that IL-25 in the liver stimulated a modest upregulation of Cidea and Cidec. It is well recognized ILC2 to release IL-13 that in turn acted on hepatocytes as well as that IL-25 has multifaceted biological activities through the in- Kupffer cells via STAT6-dependent pathways, which then altered duction of different immune mediators. It is possible that some of the expression of proinflammatory mediators, LD-associated 10 IL-25 AND HEPATIC STEATOSIS proteins, and factors that control lipid metabolic pathways. These critical to Th2 immunity against enteric nematode infection. J. Immunol. 190: 5779–5787. combined effects of IL-25 led to attenuated hepatic steatosis 20. Kinoshita, M., T. Uchida, A. Sato, M. Nakashima, H. Nakashima, S. Shono, (Supplemental Fig. 4). Conversely, IL-25 deficiency caused ex- Y. Habu, H. Miyazaki, S. Hiroi, and S. Seki. 2010. Characterization of two F4/ acerbated hepatic lipid accumulation in HFD-fed mice. These 80-positive Kupffer cell subsets by their function and phenotype in mice. J. Hepatol. 53: 903–910. results implicate IL-25 and the downstream type 2 cytokines as 21. Ikarashi, M., H. Nakashima, M. Kinoshita, A. Sato, M. Nakashima, H. Miyazaki, potential therapeutic agents for control of human hepatic steatosis K. Nishiyama, J. Yamamoto, and S. Seki. 2013. Distinct development and and associated pathologies. functions of resident and recruited liver Kupffer cells/macrophages. J. Leukoc. Biol. 94: 1325–1336. 22. Price, A. E., H. E. Liang, B. M. Sullivan, R. L. Reinhardt, C. J. Eisley, D. J. Erle, Disclosures and R. M. Locksley. 2010. Systemically dispersed innate IL-13-expressing cells in type 2 immunity. Proc. Natl. Acad. Sci. USA 107: 11489–11494. The authors have no financial conflicts of interest. 23. Klein, I., J. C. Cornejo, N. K. Polakos, B. John, S. A. Wuensch, D. J. Topham, R. H. Pierce, and I. N. Crispe. 2007. Kupffer cell heterogeneity: functional properties of bone marrow derived and sessile hepatic macrophages. Blood 110: References 4077–4085. 1. Tiniakos, D. G., M. B. Vos, and E. M. Brunt. 2010. Nonalcoholic fatty liver 24. Stienstra, R., F. Saudale, C. Duval, S. Keshtkar, J. E. M. Groener, N. van Rooijen, B. Staels, S. Kersten, and M. Muller.€ 2010. Kupffer cells promote disease: pathology and pathogenesis. Annu. Rev. Pathol. 5: 145–171. hepatic steatosis via interleukin-1beta-dependent suppression of peroxisome 2. Jin, C., J. Henao-Mejia, and R. A. Flavell. 2013. Innate immune receptors: key proliferator-activated receptor alpha activity. Hepatology 51: 511–522. regulators of metabolic disease progression. Cell Metab. 17: 873–882. 25. Harley, I. T. W., T. E. Stankiewicz, D. A. Giles, S. Softic, L. M. Flick, 3. Iwakura, Y., H. Ishigame, S. Saijo, and S. Nakae. 2011. Functional specialization M. Cappelletti, R. Sheridan, S. A. Xanthakos, K. A. Steinbrecher, R. B. Sartor, of interleukin-17 family members. Immunity 34: 149–162. et al. 2014. IL-17 signaling accelerates the progression of nonalcoholic fatty 4. Rickel, E. A., L. A. Siegel, B. R. P. Yoon, J. B. Rottman, D. G. Kugler, liver disease in mice. Hepatology 59: 1830–1839. D. A. Swart, P. M. Anders, J. E. Tocker, M. R. Comeau, and A. L. Budelsky. Downloaded from 26. Xu, R., A. Tao, S. Zhang, and M. Zhang. 2013. Neutralization of interleukin-17 2008. Identification of functional roles for both IL-17RB and IL-17RA in me- attenuates high fat diet-induced non-alcoholic fatty liver disease in mice. Acta diating IL-25-induced activities. J. Immunol. 181: 4299–4310. Biochim. Biophys. Sin. (Shanghai) 45: 726–733. 5. Chang, S. H., and C. Dong. 2011. Signaling of interleukin-17 family cytokines in 27.Tang,Y.,Z.Bian,L.Zhao,Y.Liu,S.Liang,Q.Wang,X.Han,Y.Peng, immunity and inflammation. Cell. Signal. 23: 1069–1075. X. Chen, L. Shen, et al. 2011. Interleukin-17 exacerbates hepatic steatosis and 6. Hammad, H., and B. N. Lambrecht. 2015. Barrier epithelial cells and the control inflammation in non-alcoholic fatty liver disease. Clin. Exp. Immunol. 166: of type 2 immunity. Immunity 43: 29–40. 281–290. 7. Saenz, S. A., M. C. Siracusa, J. G. Perrigoue, S. P. Spencer, J. F. Urban, Jr., 28. Okumura, T. 2011. Role of lipid droplet proteins in liver steatosis. J. Physiol. J. E. Tocker, A. L. Budelsky, M. A. Kleinschek, R. A. Kastelein, T. Kambayashi, Biochem. 67: 629–636. http://www.jimmunol.org/ et al. 2010. IL25 elicits a multipotent progenitor cell population that promotes T 29. Xu, L., L. Zhou, and P. Li. 2012. CIDE proteins and lipid metabolism. Arte- (H)2 cytokine responses. Nature 464: 1362–1366. rioscler. Thromb. Vasc. Biol. 32: 1094–1098. 8. Fort, M. M., J. Cheung, D. Yen, J. Li, S. M. Zurawski, S. Lo, S. Menon, 30. Zhou, L., L. Xu, J. Ye, D. Li, W. Wang, X. Li, L. Wu, H. Wang, F. Guan, and T. Clifford, B. Hunte, R. Lesley, et al. 2001. IL-25 induces IL-4, IL-5, and IL-13 P. Li. 2012. Cidea promotes hepatic steatosis by sensing dietary fatty acids. Immunity and Th2-associated pathologies in vivo. 15: 985–995. Hepatology 56: 95–107. 9. Zhao, A., J. F. Urban, Jr., R. Sun, J. Stiltz, M. Morimoto, L. Notari, 31. McManaman, J. L., E. S. Bales, D. J. Orlicky, M. Jackman, P. S. MacLean, K. B. Madden, Z. Yang, V. Grinchuk, T. R. Ramalingam, et al. 2010. Critical role S. Cain, A. E. Crunk, A. Mansur, C. E. Graham, T. A. Bowman, and of IL-25 in nematode infection-induced alterations in intestinal function. J. A. S. Greenberg. 2013. Perilipin-2-null mice are protected against diet-induced Immunol. 185: 6921–6929. obesity, adipose inflammation, and fatty liver disease. J. Lipid Res. 54: 1346– 10. Zaph, C., Y. Du, S. A. Saenz, M. G. Nair, J. G. Perrigoue, B. C. Taylor, 1359. A. E. Troy, D. E. Kobuley, R. A. Kastelein, D. J. Cua, et al. 2008. Commensal- 32. Dixon, L. J., M. Barnes, H. Tang, M. T. Pritchard, and L. E. Nagy. 2013. Kupffer

dependent expression of IL-25 regulates the IL-23-IL-17 axis in the intestine. J. cells in the liver. Comprehensive Physiol. 3: 785–797. by guest on September 28, 2021 Exp. Med. 205: 2191–2198. 33. Gordon, S. 2003. Alternative activation of macrophages. Nat. Rev. Immunol. 3: 11. Ikeda, K., H. Nakajima, K. Suzuki, S. Kagami, K. Hirose, A. Suto, Y. Saito, and 23–35. I. Iwamoto. 2003. Mast cells produce interleukin-25 upon Fc epsilon RI- 34. Lanthier, N., O. Molendi-Coste, P. D. Cani, N. van Rooijen, Y. Horsmans, and mediated activation. Blood 101: 3594–3596. I. A. Leclercq. 2011. Kupffer cell depletion prevents but has no therapeutic effect 12. Kang, C. M., A. S. Jang, M. H. Ahn, J. A. Shin, J. H. Kim, Y. S. Choi, on metabolic and inflammatory changes induced by a high-fat diet. FASEB J. 25: T. Y. Rhim, and C. S. Park. 2005. Interleukin-25 and interleukin-13 production 4301–4311. by alveolar macrophages in response to particles. Am. J. Respir. Cell Mol. Biol. 35. Wan, J., M. Benkdane, F. Teixeira-Clerc, S. Bonnafous, A. Louvet, F. Lafdil, 33: 290–296. F. Pecker, A. Tran, P. Gual, A. Mallat, et al. 2014. M2 Kupffer cells promote M1 13. Owyang, A. M., C. Zaph, E. H. Wilson, K. J. Guild, T. McClanahan, Kupffer cell apoptosis: a protective mechanism against alcoholic and nonalco- H. R. Miller, D. J. Cua, M. Goldschmidt, C. A. Hunter, R. A. Kastelein, and holic fatty liver disease. Hepatology 59: 130–142. D. Artis. 2006. Interleukin 25 regulates type 2 cytokine-dependent immunity and 36. Chu, D., M. Du, X. Hu, Q. Wu, and J. Shen. 2011. Paeoniflorin attenuates limits chronic inflammation in the gastrointestinal tract. J. Exp. Med. 203: 843– schistosomiasis japonica-associated liver fibrosis through inhibiting alternative 849. activation of macrophages. Parasitology 138: 1259–1271. 14. Sarra, M., M. L. Cupi, R. Bernardini, G. Ronchetti, I. Monteleone, M. Ranalli, 37. Liu, Y., C. R. Gardner, J. D. Laskin, and D. L. Laskin. 2013. Classical and al- E. Franze`, A. Rizzo, A. Colantoni, F. Caprioli, et al. 2013. IL-25 prevents and ternative activation of rat hepatic sinusoidal endothelial cells by inflammatory cures fulminant hepatitis in mice through a myeloid-derived suppressor cell- stimuli. Exp. Mol. Pathol. 94: 160–167. dependent mechanism. Hepatology 58: 1436–1450. 38. Finkelman, F. D., T. Shea-Donohue, S. C. Morris, L. Gildea, R. Strait, 15. Ahmadian, M., J. M. Suh, N. Hah, C. Liddle, A. R. Atkins, M. Downes, and K. B. Madden, L. Schopf, and J. F. Urban, Jr. 2004. Interleukin-4- and R. M. Evans. 2013. PPARg signaling and metabolism: the good, the bad and the interleukin-13-mediated host protection against intestinal nematode parasites. future. Nat. Med. 19: 557–566. Immunol. Rev. 201: 139–155. 16. Kleiner, D. E., E. M. Brunt, M. Van Natta, C. Behling, M. J. Contos, 39. Akaiwa, M., B. Yu, R. Umeshita-Suyama, N. Terada, H. Suto, T. Koga, O. W. Cummings, L. D. Ferrell, Y. C. Liu, M. S. Torbenson, A. Unalp-Arida, K. Arima, S. Matsushita, H. Saito, H. Ogawa, et al. 2001. Localization of human et al; Nonalcoholic Steatohepatitis Clinical Research Network. 2005. Design and interleukin 13 receptor in non-haematopoietic cells. Cytokine 13: 75–84. validation of a histological scoring system for nonalcoholic fatty liver disease. 40. Gabay, C., B. Porter, D. Guenette, B. Billir, and W. P. Arend. 1999. Interleukin-4 Hepatology 41: 1313–1321. (IL-4) and IL-13 enhance the effect of IL-1beta on production of IL-1 receptor 17. Yang, Z., V. Grinchuk, A. Smith, B. Qin, J. A. Bohl, R. Sun, L. Notari, Z. Zhang, antagonist by human primary hepatocytes and hepatoma HepG2 cells: differ- H. Sesaki, J. F. Urban, Jr., et al. 2013. Parasitic nematode-induced modulation of ential effect on C-reactive protein production. Blood 93: 1299–1307. body weight and associated metabolic dysfunction in mouse models of obesity. 41. Stanya, K. J., D. Jacobi, S. Liu, P. Bhargava, L. Dai, M. R. Gangl, K. Inouye, Infect. Immun. 81: 1905–1914. J. L. Barlow, Y. Ji, J. P. Mizgerd, et al. 2013. Direct control of hepatic glucose 18. Chalasani, N., Z. Younossi, J. E. Lavine, A. M. Diehl, E. M. Brunt, K. Cusi, production by interleukin-13 in mice. J. Clin. Invest. 123: 261–271. M. Charlton, and A. J. Sanyal, American Gastroenterological Association, 42. Neill, D. R., S. H. Wong, A. Bellosi, R. J. Flynn, M. Daly, T. K. A. Langford, American Association for the Study of Liver Diseases, American College of C. Bucks, C. M. Kane, P. G. Fallon, R. Pannell, et al. 2010. Nuocytes represent Gastroenterologyh. 2012. The diagnosis and management of non-alcoholic fatty a new innate effector leukocyte that mediates type-2 immunity. Nature 464: liver disease: practice guideline by the American Gastroenterological Associa- 1367–1370. tion, American Association for the Study of Liver Diseases, and American 43. Fina, D., E. Franze`, L. Rovedatti, G. R. Corazza, L. Biancone, P. P. Sileri, College of Gastroenterology. Gastroenterology 142: 1592–1609. G. Sica, T. T. MacDonald, F. Pallone, A. Di Sabatino, and G. Monteleone. 2011. 19. Zhao, A., Z. Yang, R. Sun, V. Grinchuk, S. Netzel-Arnett, I. E. Anglin, Interleukin-25 production is differently regulated by TNF-a and TGF-b1inthe K. H. Driesbaugh, L. Notari, J. A. Bohl, K. B. Madden, et al. 2013. SerpinB2 is human gut. Mucosal Immunol. 4: 239–244.