type 2 immunity is protective in metabolic disease but...

SC I ENCE TRANS LAT IONAL MED I C I N E | R E S EARCH ART I C L E

F I BROS I S

1Laboratory of Parasitic Diseases, National Institute of Allergy and Infectious Diseases,National Institutes of Health, Bethesda, MD 20892, USA. 2Centre de Recherche du CentreHospitalier de l’Université de Montréal (CRCHUM), Montréal, Québec, Canada. 3Départe-ment de microbiologie, infectiologie et immunologie, Université de Montréal, Montréal,Québec, Canada. 4Tissue Fibrosis and Repair Group, Institute of Cellular Medicine, New-castle University, Newcastle upon Tyne, UK. 5Département de pathologie et biologiecellulaire, Université de Montréal, Montréal, Québec, Canada. 6Département de méde-cine, Université de Montréal, Montréal, Québec, Canada. 7Genentech Inc., South SanFrancisco, CA 94080, USA.*Corresponding author. Email: [email protected]

Hart et al., Sci. Transl. Med. 9, eaal3694 (2017) 28 June 2017

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Type 2 immunity is protective in metabolic disease butexacerbates NAFLD collaboratively with TGF-bKevin M. Hart,1 Thomas Fabre,2,3 Joshua C. Sciurba,1 Richard L. Gieseck III,1 Lee A. Borthwick,4

Kevin M. Vannella,1 Thomas H. Acciani,1 Rafael de Queiroz Prado,1 Robert W. Thompson,1

Sandra White,1 Genevieve Soucy,2,5 Marc Bilodeau,2,6 Thirumalai R. Ramalingam,1

Joseph R. Arron,7 Naglaa H. Shoukry,2,6 Thomas A. Wynn1*

Nonalcoholic fatty liver disease (NAFLD) is now the most common progressive liver disease in developed countriesand is the second leading indication for liver transplantation due to the extensive fibrosis it causes. NAFLD progres-sion is thought to be tied to chronic low-level type 1 inflammation originating in the adipose tissue during obesity;however, the specific immunological mechanisms regulating the progression of NAFLD-associated fibrosis in theliver are unclear. To investigate the immunopathogenesis of NAFLD more completely, we investigated adipose dys-function, nonalcoholic steatohepatitis (NASH), and fibrosis in mice that develop polarized type 1 or type 2 immuneresponses. Unexpectedly, obese interleukin-10 (IL-10)/IL-4–deficient mice (type 1–polarized) were highly resistant toNASH. This protection was associated with an increased hepatic interferon-g (IFN-g) signature. Conversely, IFN-g–deficient mice progressed rapidly to NASH with evidence of fibrosis dependent on transforming growth factor–b(TGF-b) and IL-13 signaling. Unlike increasing type 1 inflammation and the marked loss of eosinophils seen inexpanding adipose tissue, progression of NASH was associated with increasing eosinophilic type 2 liver inflamma-tion in mice and human patient biopsies. Finally, simultaneous inhibition of TGF-b and IL-13 signaling attenuated thefibrotic machinery more completely than TGF-b alone in NAFLD-associated fibrosis. Thus, although type 2 immunitymaintains healthy metabolic signaling in adipose tissues, it exacerbates the progression of NAFLD collaborativelywith TGF-b in the liver.

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INTRODUCTIONTreating the individual sequelae of the metabolic syndrome associatedwith obesity and type 2 diabetes represents oneway to alleviate the over-all burden of disease. For example, nonalcoholic fatty liver disease(NAFLD), the hepatic manifestation of metabolic syndrome, is nowthe most common form of chronic liver disease, is growing in preva-lence, and will soon be the most common cause of liver transplantationin the United States (1–3). This is highlighted by a recent estimate thatNAFLD affects 64 million people in the United States, with annual di-rect medical costs projected at $103 billion (4). Although a great deal ofresearch has focused on obesity, metabolic alterations in disease, adi-pose tissue dysregulation, and the pathophysiology of NAFLD, the mo-lecular and immunological mechanisms that facilitate the progressionof NAFLD to nonalcoholic steatohepatitis (NASH) and cirrhosis re-mainmuch less clear (5). Because severe cirrhosis is the ultimate reasonpatients require liver transplant and the presence of fibrosis has beenidentified as the best predictor of clinical outcome and mortality (6, 7),a better understanding of the mechanisms that drive fibrogenesis inNAFLD could reveal novel treatment strategies and have amajor impacton morbidity and mortality. However, progress in this area has beenstymied by the absence of preclinical models that reliably reproduce in-flammatory and fibrotic components seen in human disease (8, 9).

It is clear that the initiation and progression of NAFLD along itsspectrum of severity are intricately associated with the inflammatoryprocesses that are driven by chronic obesity. The initiation of dyslipide-mia and lipogenesis in the liver is tied to insulin resistance andunderlying adipose tissue inflammation.How the dysregulated immuneresponse in the adipose tissue affects whole organism metabolism dur-ing homeostasis and obesity has been the topic of intense study. Theemerging model suggests that homeostatic type 2 immunity in adiposetissues driven by interleukin-5 (IL-5), IL-4, and IL-13 productionfrom innate type 2 lymphocytes, resident eosinophils, regulatoryT cells, and alternatively activated macrophages maintains healthymetabolic signaling (10–15). During obesity, this set point is skewedtoward type 1 inflammation with increasing production of IL-1b,tumor necrosis factor–a (TNF-a), IL-6, IL-12, and interferon-g(IFN-g), resulting in loss of local and then systemic insulin sensitivityand establishment of the chronic low-level type 1 inflammation thatis the hallmark of the metabolic syndrome. Thus, prevailing modelshold that the inflammation contributing to NASH, cirrhosis, andeventual liver failure is an extension of dysregulated adipose tissueinflammation (16).

Here, we investigated whether type 1 inflammatory response inobese mice directly contributes to the development of NAFLD. Usinga variety of transgenic mice that develop highly polarized type 1 andtype 2 immune responses, we examined whether the progression ofhigh-fat diet (HFD)–induced NASH and fibrosis is influenced bychanges in type 2 immunity. Surprisingly, our studies reveal a dis-connect in howobesity and the loss of type 2 effector function in adiposetissues affects the progression of inflammation, NASH, and fibrosis inthe liver. In contrast to its protective role in metabolism, adipose tissuehomeostasis, and obesity, we show that the type 2 cytokine IL-13 collab-orates with transforming growth factor–b (TGF-b) to drive liver fibro-genesis in HFD-induced obesity.

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RESULTSNASH and fibrosisdevelop late in micefed an HFDNASH and the accompanyingdevelopment of hepatic fibro-sis are complications linkedto chronic obesity. To establisha timeline for the developmentof NASH and to determine thetimepoint atwhich obesemicedevelop NASH with fibrosis,we subjected animals to HFDfor 15 and 40 weeks and thenexamined them for the hall-mark features of steatohepati-tis. Mice on HFD for 15 weeksgained appreciable weightand developed hepatomegaly(Fig. 1A). Despite being stea-totic, there were no detectableinflammation and hepatocytedamage at this early time point,as assessed by liver leukocytenumbers (Fig. 1B) and serumaminotransferase level (Fig. 1C).Oil Red O staining was modestat this timepoint comparedwith40 weeks (Fig. 1D). We also didnotdetect staining forballooninghepatocytes (Fig. 1E) or fibro-sis by picrosirius red stainingof liver sections (Fig. 1F, top).Nevertheless, after 40 weeks,mice on HFD displayed in-creases in leukocyte infiltrationof the liver (Fig. 1B), with his-tological evidence of markedlobular inflammation (Fig. 1E,inset). Additionally, increasesin aminotransferases in serum(Fig. 1C) suggested that chron-ic HFD led to substantial hep-atocellular damage. This wascharacterized by hepatocellularmacrosteatosis (Fig. 1D) andballooning degeneration,identified through immuno-histochemical and immuno-fluorescence staining forubiquitin and hepatic loss ofkeratin 8/18 staining (Fig. 1Eand fig. S1A). There was alsosignificant pericellular zone 3accumulation of collagen inthe livers of mice fed an HFD(Fig. 1F, bottom) and increasedexpression (fig. S1B) and im-munofluorescence staining for

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g. 1. NASH and fibrosis develop late in mice fed an HFD. Mice were maintained on a normal diet (filled symbols; ND) or a% kcal from fat diet (open symbols; HFD) for 15 (circles) or 40 (squares) weeks. (A) Body weights and liver weight and liverdex (% of body weight) were collected (n = 10 to 20). Characteristics of NASH were measured including isolated liver leukocyteunts (n = 9 to 12) (B), serum ALT and aspartate aminotransferase (AST) (n = 9 to 10) (C), and steatosis by Oil Red O staining (D). Scalers, 100 mm. (E) Liver sections were stained with keratin 8/18 and ubiquitin (Ub.) to assess hepatocyte ballooning and hematoxylind eosin (H&E) for hepatic inflammation (inset showing example of lobular inflammation). Scale bars, 40 mm. (F) Picrosirius redaining was performed to assess fibrotic collagen deposition (scale bars, 200 mm) and viewed under polarized light to enhancesualization (inset scale bars, 200 mm). Adipose inflammation was assessed from Giemsa-stained sections from 40-week–treatedimals (scale bars, 150 mm) (G), crown-like structures marked with arrows, expression of tnf (H) (n = 4 to 5), and flow cytometryalysis of Siglec-F+ adipose eosinophils among CD45 leukocytes (I) (n = 5 to 12). All data points represent a single mouse, andpresentative or pooled data from two or more independent experiments are shown (two-tailed t tests, n = 2 to 10; *P < 0.05,P < 0.01, ***P < 0.005, ****P < 0.0001).

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smooth muscle actin (fig. S1C), which are defining features of NASH.This timeline is in agreement with previously published work, whichreports NASH phenotypes present in wild-type (WT) mice on HFDby 34 weeks (17). As predicted, the NASH phenotype was accompaniedby increases in adipose inflammationmeasured by histological immuneinfiltration (Fig. 1G), presence of crown-like structures (Fig. 1G, arrow-heads), and increased tnf expression (Fig. 1H). Furthermore, theincreased inflammation in adipose tissues was accompanied by a lossof eosinophils as has been previously reported (Fig. 1I) (15). Theincreased inflammation in adipose tissue resulted in increased TGF-bexpression and induction of a number of collagens andmetalloprotein-ases; however, we did not detect development of substantial fibrosis bypicrosirius red staining of adipose tissue at either time point (fig. S2).These studies demonstrate that obese mice are susceptible to thesequelae of NAFLD but only when chronically exposed to HFD. Thesedata also support the model that progression of NAFLD in obesity isassociated with a loss of type 2 homeostatic set points and expansionof inflammation in adipose tissues.

Type 1 immunity drives metabolic disease but regulatesprogression of NAFLDTo determine whether the dysregulated proinflammatory immune re-sponse in adipose tissues of obese mice contributes to the developmentand progression of NAFLD, we investigated whether primary drivers oftype 2 immunity protect against NAFLD. We used IL-4−/− and doubleIL-10−/−/IL-4−/−mice, whichwere previously shown to develop increas-ingly polarized type 1 inflammation after sterile or infectious challenge(18, 19). Our goal with the IL-10−/−/IL-4−/− mice was to assess extremetype 1 inflammatory skewing rather than the specific contributionof IL-10to NAFLD, which remains unclear despite being previously studied(20–25). The single IL-10−/−mice, in contrast to the IL-10−/−/IL-4−/−

mice, develop more of a mixed T helper cell 1 (TH1)/TH2 responsewhen challenged with various inflammatory stimuli, which may, inpart, explain the contradictory results regarding IL-10 in NAFLD(18, 19). We observed consistently low and unchanging levels ofIL-10 with disease in mice and in human microarray data (fig. S3).Thus, we hypothesized that the IL-4−/− and IL-10−/−/IL-4−/− geneti-cally deficient mice would display a progressive worsening of metabolichomeostasis, obesity, adipose tissue dysregulation, and increased pro-gression toward NASH and fibrosis because of their highly polarizedtype 1 inflammatory response.

When challenged with HFD for 15 weeks, IL-4−/− mice devel-oped similar obesity as WT mice measured by body composition(Fig. 2A) and serum leptin levels (Fig. 2B). However, the IL-4−/− micedisplayed slightly worse hepatomegaly (Fig. 2C), despite having similarliver triglycerides (Fig. 2D). Unexpectedly, IL-10−/−/IL-4−/− mice weresignificantly protected from hepatomegaly, increased serum amino-transferases, and their liver triglyceride content, and steatosis wasmarkedly reduced relative to WT and IL-4−/− mice (Fig. 2, C to E).This was surprising because the IL-10−/−/IL-4−/− mice were similarlysusceptible to obesity compared to WT mice as measured by bodycomposition and serum leptin levels (Fig. 2, A and B). This protectionfrom the features of NAFLD despite normal susceptibility to obesitywas sustained as long as 40 weeks on HFD in the IL-10−/−/IL-4−/−

mice (fig. S4). Perigonadal adipose tissues were also analyzed for ev-idence of dysregulated proinflammatory mediator expression. Allthree groups showed marked increases in tnf and ccl2, as well as read-ily observable increases in histological adipose immune infiltrationand abundant presence of crown-like structures when fed an HFD


(Fig. 2, F and G). The switch toward type 1 inflammation in adiposetissues was also accompanied by a substantial reduction in the numberof adipose tissue eosinophils (Fig. 2H). Compared to WT mice, bothgenetically deficient animals displayed similar or increased inflamma-tion in the adipose tissue, but the IL-10−/−/IL-4−/− mice were resistantto steatosis and NASH when placed on HFD as measured by hepatictriglyceride levels, serum aminotransferase levels, and histological eval-uations. Together, these findings suggest that there are disconnectsbetween the mechanisms driving inflammation and metabolic dysreg-ulation in adipose tissues and the mechanisms driving developmentof NAFLD.

Obese IFN-g−/− mice develop accelerated NAFLDwith fibrosisTo elucidate themechanism for the reduced development of NAFLD inIL-10−/−/IL-4−/−mice, we performedRNA sequencing (RNA-seq) anal-ysis onwhole liver tissue isolated fromWT, IL-4−/−, and IL-10−/−/IL-4−/−

mice on a normal diet orHFD for 15weeks. Ingenuity PathwayAnalysisof significantly altered genes between the genotypes on a normal diet orHFD [analysis of variance (ANOVA)] identified interferon-g (ifng) asthe most activated pathway among predicted upstream regulatorypathways at baseline in the IL-10−/−/IL-4−/− livers (P = 2.65 × 10−31).This pathway was down-regulated slightly in WT animals on HFD(z score = −1.362). In IL-4−/− mice, the IFN-g pathway was down-regulated whether the mice were on a normal diet (z score = −3.805)or HFD (z score = −2.64). In contrast, the IFN-g pathway was thehighest predicted up-regulated pathway in IL-10−/−/IL-4−/− mice on anormal diet (z score = 7.678) and was only slightly reduced when themice were fed the HFD (z score = −1.069) (Fig. 3A). The IFN-g signa-ture in IL-10−/−/IL-4−/− mice was further validated when we searchedfor genes that, according to their annotation, are positively linkedwith IFN-g activity (Fig. 3A, heat map). These data suggest thatelevated baseline hepatic levels of IFN-g that are highly activatedin IL-10−/−/IL-4−/−, but not in IL-4−/− mice, may be protective againstNAFLD.

Because the IFN-g pathway was such a strong predictor ofresistance or susceptibility to NAFLD in the IL-4 and IL-10/IL-4genetically deficient mice, we investigated whether IFN-g plays an im-portant regulatory role in the progression of NAFLD. IFN-g−/− miceon HFD for 15 weeks had similar increases in body weight and fatmass as WT mice (Fig. 3B). However, the IFN-g−/− mice displayedmarkedly increased hepatomegaly and significantly increased steatosiscompared to WT animals when assessed by liver triglyceride contentand Oil Red O staining (Fig. 3, B and C). In additional studies, wefound increased NAFLD in IL-12−/− and IL-10−/−/IL-12−/− mice com-pared to WT mice but no differences in NAFLD between IL-12−/− andIL-10−/−/IL-12−/− mice, suggesting that increased presence of type1 regulatory mechanisms driven by IL-12 and IFN-g is ultimately re-sponsible for the differences we observed in the IL-10−/−/IL-4−/− micerather than a specific effect of IL-10 (fig. S5). We also observed areasof NASH-like fibrotic deposition in the IFN-g−/− mice by picrosiriusred staining, which were undetectable in WT livers at the early 15-weektime point (Fig. 3C, bottom). Additionally, the IFN-g−/− mice hadevidence of increased liver damage as measured by serum alanineaminotransferase (ALT) (Fig. 3D). The early detection of fibrosis inIFN-g−/− mice was also accompanied by increased expression ofcollagen3a1 (col3a1), alpha smooth muscle actin, and the extracellularmatrix–associated protein periostin, which is produced by activatedmyofibroblasts in response to TGF-b1 and IL-13 stimulation (Fig. 3D)

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(26, 27). Given that progression to fibrosis was accelerated in obeseIFN-g−/−mice, we also examined whether TGF-b1 activity was altered.Expression of Tgfb1 was significantly increased in the livers of IFN-g−/−

mice, as were tissue inhibitor of matrix metalloproteinases–1 (TIMP-1)and matrix metalloproteinase–2 (MMP-2), which are both regulated byactivated TGF-b1 (Fig. 3E) (28, 29). Furthermore, we observed local-ization of nuclear phosphorylated Smad3 in a greater frequency andvariety of cell types present in the livers of HFD-challenged IFN-g−/−

mice (Fig. 3F).


Next, we analyzed adipose tissues for markers of inflammation anddetected increased tnf and ccl2 expression in the obese IFN-g−/− miceand a substantial decrease in the number of tissue eosinophils (fig.S6A). Nevertheless, despite these changes, we failed to detect any sig-nificant increase in gross histological inflammation in the adipose tis-sues of IFN-g−/−, which contrasts with the findings obtained in theadipose tissues of obese IL-4−/− mice (fig. S6B). The worseningNASH-associated fibrosis is likely explained, in part, by the increasedTGF-b1 signature in the livers of IFN-g−/−mice relative toWT or IL-4−/−

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Fig. 2. Type 1 immunity drives metabolic disease but protects against NAFLD. WT (circles), IL-4−/− (squares), and IL-10/IL-4−/− (triangles) mice were maintained ona normal diet (filled symbols) or HFD (open symbols) for 15 weeks. Obesity was measured by magnetic resonance imaging (MRI) body composition (A) (n = 4 to 6), andserum leptin levels were assessed by enzyme-linked immunosorbent assay (n = 3 to 7) (B). NAFLD progression was assessed by liver weight and indices (n = 8 to 15)(C), hepatic triglyceride measurement (n = 8 to 11), serum aminotransferase levels (n = 4 to 17) (D), and Giemsa-stained liver sections (E). KO, knockout. Adiposeinflammation was assessed through histological analysis of H&E-stained adipose sections (scale bars, 150 mm) (F), adipose tissue expression of tnf and ccl2 (n = 3 to11) (G), and flow cytometry analysis of adipose eosinophils (n = 4 to 11) (H). All data points represent a single mouse, and representative or pooled data from two ormore independent experiments are shown (two-tailed t tests, n = 2 to 10; *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.0001).

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mice, as well as the increased expression of TGF-b1 in adipose tissues(fig. S6C).

NASH-driven fibrosis is partly TGF-b–dependentAlthough TGF-b activity has been documented in the livers ofNAFLD/NASH patients and in animal models of NAFLD/NASHand proposed as a therapeutic target, detailed experimental manipu-lation of the pathway to determine its importance in NASH-associatedfibrosis and immune activation has not been performed (30–33). Thus,to determine the role for TGF-b signaling in chronic HFD–inducedNASH and to validate its potential as a therapeutic target in NASH,we placed WT mice on HFD for 40 weeks, and TGF-b activity was


inhibited therapeutically during the final 4 weeks of the study. Comparedto animals receiving control antibody (Ab) injections, the animalstreated with a pan TGF-b blocking Ab did not display significant dif-ferences in body composition, hepatomegaly, or steatosis as measuredby liver triglycerides (Fig. 4, A and B). When we analyzed histologicalfibrosis using picrosirius red staining, there was a modest yet signifi-cant decrease in characteristic NASH fibrosis quantified as fibroticfraction in the mice receiving anti–TGF-b therapy after just 4 weeks(Fig. 4B). This was also associated with a decrease in expression ofpro-col3a1 and pro-col1a1. However, TGF-b Ab blockade did not af-fect periostin or pro-col6a1 mRNA expression (Fig. 4C). Periostin hasbeen proposed as a biomarker of type 2 inflammation in asthma (34),

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Fig. 3. Obese IFN-g−/− mice develop accelerated NAFLD with fibrosis. (A) Activation scores of the IFN-g pathway were assessed by Ingenuity Pathway Analysis ofsignificantly differentially expressed genes as determined by RNA-seq from whole liver tissue from WT, IL-4−/−, and IL-10/IL-4−/− mice on HFD for 15 weeks. Expression ofgenes annotated as being involved in positive regulation of IFN-g is shown in the heat map. (B) WT (circles) and IFN-g−/− (squares) mice were maintained for 15 weekson a normal diet (filled symbols) or HFD (open symbols) and assessed for obesity by MRI body composition measurement (n = 3 to 5). NAFLD progression was alsoassessed by liver weight (n = 9 to 11), liver index, hepatic triglyceride measurement (n = 3 to 6) (B), steatosis by Oil Red O staining (C) (left, normal diet; right, HFD) (scalebars, 100 mm), picrosirius red staining of collagen (scale bars, 125 mm) (C) (bottom) (visualized under polarized light; inset scale bars, 125 mm), and serum ALT levels (n =5) (D). (D) Expression of fibrotic markers col3a1 (n = 9 to 11), postn (n = 9 to 11), and acta2 (SD in shaded regions) (n = 3 to 5) was assessed by quantitative polymerasechain reaction (qPCR). (E) Expression of tgfb1 and TGF-b–regulated genes timp1 and mmp2 were assessed (n = 9 to 11). (F) Immunofluorescence staining for phospho-Smad3 (green), keratin 8/18 (gray), and 4′,6-diamidino-2-phenylindole (DAPI) (blue) (arrows indicate overlapping Smad3 and DAPI staining). All data points represent asingle mouse, and representative or pooled data from two or more independent experiments are shown (two-tailed t tests, n = 2 to 10; *P < 0.05, **P < 0.01, ***P <0.005, ****P < 0.0001).

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and we have similarly identified pro-col6a1 as a marker of IL-13–dependent inflammation and fibrosis (35). Thus, we hypothesized thattype 2 inflammation was likely contributing to the persistent expres-sion of these profibrotic biomarkers in the anti–TGF-b–treated mice.Therefore, to investigate whether TGF-b blockade was having anyimpact on immune homeostasis, we analyzed the cytokine responsein restimulated CD4+ T cells isolated from the liver. Production ofIFN-g was largely unaffected by the chronic HFD or Ab treatment. How-ever, production of IL-4 by T cells was markedly increased in mice treatedwith anti–TGF-bmonoclonal antibody (mAb) (Fig. 4D). We also noted amodest increase in IL-13 production in the control Ab-treated mice fedan HFD (Fig. 4E). We also observed increased expression of chi3l3, aninducible marker of type 2 immunity, in the HFD group, which was fur-ther augmented by anti–TGF-b therapy (Fig. 4E). Collectively, these datarevealed the presence of low-level type 2 cytokine-associated inflamma-tion in the livers of mice with progressive NAFLD, which was furtherexacerbated by TGF-b blockade. Therefore, emergence of compensatoryprofibrotic type 2 cytokine response could impair the efficacy of TGF-bpathway inhibitors in NAFLD-associated fibrosis.


Accelerated NASH-driven fibrogenesis in obese IFN-g−/− miceis characterized by eosinophilic inflammation duringTGF-b blockadeWe repeated the TGF-b blockade studies in both WT and IFN-g−/−

mice on HFD for 20 weeks to test whether the highly susceptibleIFN-g−/− mice would serve as a more rapid model of NASH and tofurther investigate the potential involvement of type 2 immunity inthe progression of NAFLD. Once again, IFN-g−/− mice developedsevere hepatomegaly by 20 weeks, which contrasted with WT micethat displayed little evidence of advanced NAFLD at this time point.Despite variability in histological fibrosis at this time point assessedby picrosirius red staining (Fig. 5A), we detected significant increasesin pro-col3a1 and pro-col6a1 mRNA expression in the IFN-g−/−

mice and modest increases in periostin expression in mice treatedwith anti–TGF-b mAbs, confirming that the machinery driving fi-brosis was substantially increased (Fig. 5B). Because IFN-g is knownto cross-regulate type 2 inflammation, we hypothesized that the in-creases in periostin and pro-col6a1 expression could be due to fur-ther dysregulation of cytokine responses in the livers of these mice,

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Fig. 4. NASH-driven fibrosis is partly TGF-b–dependent. (A) WT mice were maintained for 40 weeks on a normal diet (filled symbols) or HFD (open symbols) andreceived 4 weeks of semiweekly control (Cont.) immunoglobulin (Ig) (squares) or anti–TGF-b (triangles) (250 mg each). Mice were assessed for obesity by MRI bodycomposition measurement (n = 5 to 9) and hepatomegaly by liver weight and liver index scores (n = 5 to 12). (B) Hepatic triglycerides were measured, and fibroticfraction was calculated by measuring picrosirius red–stained liver sections for characteristic NASH-associated fibrosis (n = 4 to 12). (C) Expression of four fibrosis-associated genes was measured (n = 5 to 12). Phorbol 12-myristate 13-acetate (PMA)/ionomycin–restimulated hepatic CD4 T cell production of IFN-g, IL-4 (D), andIL-13 (left) (E) was assessed by intracellular cytokine staining (n = 5 to 10). (E) Expression of the type 2 inflammation marker chi3l3 was quantified from whole liver tissue(n = 4 to 11) (right). Freq., frequency. All data points represent a single mouse, and representative or pooled data from two or more independent experiments areshown (two-tailed t tests, n = 2 to 10; *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.0001).

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which was validated by increases in IL-13 mRNA expression observedin both WT and IFN-g−/− mice after TGF-b blockade (Fig. 5C).

To determine the functional impact of the enhanced type 2 responseon the liver microenvironment, we examined ccl11 expression andquantified tissue eosinophils, which have been shown to promote in-flammation and fibrosis. Both were increased in the livers of IFN-g−/−

mice and further exacerbated by TGF-b blockade (Fig. 5, C and D), butwe were surprised to find a similar but less marked increase in eosino-phils in WT mice as well, despite the fact that ccl11 expression was not


increased in this group. This increase in eosinophils with TGF-b block-adewas recapitulated in 40-week–treatedWTmice (fig. S7A). The pres-ence of tissue eosinophilswas confirmed through histological analysis ofGiemsa-stained liver sections (Fig. 5E) and immunofluorescence imag-ing of siglec-F+ leukocytes in liver tissue (Fig. 5F). Few, if any, eosino-phils were observed in WT or IFN-g−/− mice fed a normal diet, withoccasional eosinophils found in microgranulomas. In contrast, eosino-phils were scattered throughout the liver sinusoids in the HFD mice.They were also localized in areas of marked lobular inflammation

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Fig. 5. Accelerated NASH-driven fibrogenesis in obese IFN-g−/− mice is characterized by severe eosinophilic inflammation during TGF-b blockade. (A) WT(circles) and IFN-g−/− (squares) mice were maintained for 20 weeks on normal diet (filled symbols) or HFD (open symbols) and received 4 weeks of biweekly controlIg or anti–TGF-b (250 mg each). Liver weight and liver index were determined (n = 9 to 14), and fibrotic fraction was calculated from picrosirius red–stained liver sections(n = 3 to 12). (B) Expression of interstitial collagen genes. Periostin, IL-13, and CCL11 expression were quantified from whole liver tissue (n = 6 to 14). (C) Hepaticeosinophils were measured by flow cytometry analysis of Siglec-F+ cells among CD45 leukocytes (n = 7 to 9) (D). Eosinophils were observed in hepatic tissue byGiemsa-stained liver sections (scale bars, 50 mm) (E) and by immunofluorescence staining of tissue sections for Siglec-F+ cells (scale bars, 125 mm) (F). WT mice weremaintained for 40 weeks on normal diet (filled symbols) or HFD (open symbols) and received 4 weeks of biweekly control Ig (circles), anti–TGF-b (squares), or combinedanti–TGF-b and IL-13 mAbs (triangles) (250 mg each). (G) Expression of col3a1 and col6a1 from whole liver tissue were assessed by qPCR (n = 3 to 6). All data pointsrepresent a single mouse, and representative or pooled data from two or more independent experiments are shown (two-tailed t tests, n = 2 to 10; *P < 0.05, **P < 0.01,***P < 0.005, ****P < 0.0001).

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and macrosteatosis where fibrosis is observed. To further validate theseobservations and to confirmwhether increases in the presence of type 2inflammation are associated with the worsening NASH phenotype, wealso assessed livers from WT animals on a chronic 40-week HFD forccl11 expression and presence of eosinophils (fig. S7B) and detected sig-nificant increases in both measures.

Because IFN-g negatively regulates both TGF-b and type 2 immu-nity, we wanted to verify that the marked protection observed in thelivers of IL-10−/−/IL-4−/− mice was not accompanied by any inductionof a profibrotic type 2 response. Confirming the evidence of hepatic pro-tection in thesemice, there was almost no induction of tgfb1 or evidenceof increased TGF-b or IL-13 activity measured by timp1 and postn ex-pression or accumulation of eosinophils at 40 weeks on HFD. In con-trast, all of these measures were increased in WT mice (fig. S8). Thisdemonstrates that the increased IFN-g signaling in the livers of theIL-10−/−/IL-4−/− mice is associated with protection and that activationof TGF-b or type 2 inflammation requires an additional stimulus suchas steatosis or hepatic damage.We also examined the livers for the pres-ence of tissue neutrophils and expression of neutrophil elastase, whichwe would have predicted to increase, based onmodels of type 1 inflam-mation serving as themajor driver ofNASHpathogenesis. Althoughweobserved modest increases in expression of neutrophil elastase, partic-ularly during TGF-b blockade, we found no significant changes in thefrequencies of tissue neutrophils associated with HFD alone, TGF-bblockade, the absence of IFN-g, or chronic HFD (fig. S7, C and D).As such, these observations are in stark contrast to the shifts in immunebalance and loss of resident eosinophils seen in obese adipose tissue,indicating a significant divergence in the pathogenic mechanisms driv-ing disease in each organ.

Finally, because of the increases in IL-13 production, up-regulationof IL-13–responsive genes, and increased presence of eosinophils, whichboth respond and contribute to IL-13 signaling, we investigated thecombined rolesof IL-13andTGF-b in theprogressionofNASH-associatedfibrosis. Returning to the 40-week model of NASH, we treated weight-matched animals therapeutically during the final 4 weeks with control,anti–TGF-b, or combined anti–TGF-b and anti–IL-13. Although thetreatment regimen was too short to detect meaningful changes in dis-ease, it was clear that the Abs were blocking fibrogenesis because weobserved significant decreases in expression of both col3a1 and col6a1with the dual blockade (Fig. 5G). This confirmed our hypothesis thatcol6a1 expression was being driven by IL-13 signaling because it wasunaffected by the single TGF-b blockade. These observations demon-strate that TGF-b and IL-13 cooperatively drive fibrogenic pathways inthe murine NASH liver.

AMLN diet–induced NASH recapitulates IFN-g regulationand type 2 inflammation even with modestadipose inflammationTo determine the broad applicability of our findings in NASH, werepeated our analyses with WT and IFN-g−/− knockout animals af-ter 15 weeks on the amylin liver NASH model (AMLN) diet (40%fat, 20% fructose, and 2% cholesterol) (32, 36). This more severemodel of NASH recapitulated our main findings, with IFN-g−/−

mice displaying similar increases in body weight (Fig. 6A) but havingsignificantly increased hepatomegaly (Fig. 6A), expression of fibroticmarkers (Fig. 6B), evidence of fibrosis (Fig. 6C), and hepatic damage(Fig. 6D). Furthermore, we foundmarked increases in induction of type 2inflammation in both WT and IFN-g−/− knockout animals measuredby frequencies of IL-13 producing CD4 T cells and tissue expression


of periostin (Fig. 6E), with significant increases in both measures inIFN-g−/− mice. This was associated with increases in eosinophilsand not neutrophils in the livers of IFN-g−/− mice (Fig. 6F). As sug-gested by the reduced weights of mice on the AMLN diet, reduction ineosinophils and increases in expression of TNF-a in the adipose tissuewere less marked than what was observed with mice on HFD (Fig.6G), suggesting that the induction of type 2 inflammation and fibrosiscan occur somewhat independently from immune alterations in theadipose tissue.

Human NASH is characterized by type 2eosinophilic inflammationTo confirm whether our NASH models were accurately recapitulatinghuman disease and to determine whether our findings would be rele-vant to patients and potential therapeutics, we performed an analysisof NASH biopsies and publicly available microarrays from NASHpatients. First, to determine whether there is a component of type2 inflammation present in NASH patients, we assessed cytokine pro-duction from isolated and expanded intrahepatic lymphocytes(IHLs) after anti-CD3/CD28 restimulation. Compared to lympho-cytes isolated from healthy controls, NASH-associated lymphocytesproduced increased amounts of IL-4, IL-5, and IL-13 (Fig. 7A). Fur-thermore, when stratified by pathologist-assigned METAVIR fibro-sis scores, we observed increased type 2 cytokine production withworsening fibrosis, with significant increases observed in IL-5 andIL-4 between lower (F0-2) and higher (F3-4) fibrosis scores (Fig.7B). Second, we determined that eosinophils were present in NASHpatient livers by histological analysis and observed eosinophils pres-ent in similar patterns as seen in the murine livers in every NASHbiopsy analyzed (n = 26) (Fig. 7C and fig. S10).

To further support eosinophils as an indicator of type 2 inflamma-tion and potential contributor in progressive NASH, we performed ananalysis of publicly available microarray data from healthy, steatotic,and NASH livers for genes with significant differential expression(37). Of the transcripts with significantly altered expression by steatosisor NASH, we identified 37 genes as either annotated or implicated ineosinophil function (38). Hierarchical clustering by samples and prin-cipal components analysis using this gene set separates the NASH fromthe healthy control and steatotic biopsies (Fig. 7D). Finally, we assessedpotential mechanisms of eosinophil accumulation in the humanmicro-array data. Althoughwe did not detect significant changes in expressionof the eotaxin genes in NASH microarrays, the eosinophil-associatedchemoattractant genes ccl5, ccl23, and ptgds were all significantly up-regulated in NASH patients (fig. S9) (36, 38–43). These data indicatea previously unappreciated role for aspects of the type 2 response as acomponent of the inflammatory milieu contributing to NASH in pa-tient livers and suggest that chronic HFD, AMLN diet, and the inflam-matory pathway modifications we explore here reliably replicate thisaspect of progression of NAFLD.

DISCUSSIONResearch on the inflammation associated with NAFLD progression toNASH and cirrhosis has almost universally focused on components ofthe proinflammatory type 1 response including IL-1b, TNF-a, IL-6, IL-12,and Toll-like receptor (TLR) activation (16, 44). Here, we observed type 2inflammatory components in both mouse and human NASH, suggest-ing a previously unappreciated role for type 2 inflammation in the pro-gression of NASH to fibrosis, as IL-13 is a well-known contributor to

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progressive end-stage fibrosis in diseases of diverse etiology. The datapresented here illustrate a much more mixed inflammatory milieu inthe fatty liver than currentmodels suggest, which could have significantimplications for therapeutics that are based on altering the character ofthe inflammatory response in patients with NASH. Although eosino-phils and other type 2–associated inflammatory cells play a protectiverole in adipose tissues, the findings here demonstrate that dysregulatedtype 2 inflammation contributes to the progression of steatosis and fi-brosis in the liver (fig. S11).

We initiated these studies to better understand the protective andpathogenic roles of type 1 and type 2 inflammation in the pathogenesis


of NAFLD, as recent studies have suggested that type 2 immunity playsa protective role in metabolic syndrome. Although we confirmed animportant counterregulatory role for type 2 cytokines in adipose tissueinflammation (11, 12, 14, 15), our studies in IL-10−/−/IL-4−/− and IFN-g–deficient animals revealed an unexpected protective role for somecomponents of type 1 inflammation in NAFLD progression. Mecha-nistically, we show that targets of this IFN-g regulation include thecytokines TGF-b and type 2–associated IL-13, which were dysregulatedin the absence of IFN-g. TGF-b has also previously been observed inNAFLD, and this pathway has been proposed as a possible therapeutictarget for NASH-associated fibrosis (33, 45).

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Fig. 6. AMLN diet–induced NASH recapitulates IFN-g regulation and type 2 inflammation despite modest adipose inflammation. (A) WT (circles) and IFN-g−/−

(squares) mice were maintained for 15 weeks on normal diet (filled symbols) or AMLN diet (open symbols) and assessed for obesity by body weight (n = 5 to 8). NAFLDprogression was assessed by liver weight and liver index, (A), expression of fibrotic markers col1a1, col3a1, and acta2 as assessed by qPCR (n = 5 to 7) (B), picrosiriusred– and FAST green-stained liver sections from WT (top) and IFN-g−/− (bottom) mice on a normal diet (left) and HFD (right) (C), and serum alkaline phosphatase andALT levels (n = 4 to 8) (D). Type 2 response was measured by intracellular staining of hepatic CD4 T cells for IL-13 after PMA/ionomycin stimulation (n = 4 to 8), postnexpression (n = 5 to 8) (E), and flow cytometry analysis of hepatic eosinophils and neutrophils (n = 5 to 8) (F). (G) Adipose inflammation was measured by flowcytometry analysis of eosinophils and tnf expression (n = 5 to 8). All data points represent a single mouse, and representative or pooled data from two or moreindependent experiments are shown (two-tailed t tests, n = 2 to 10; *P < 0.05, **P < 0.01, ***P < 0.005, ****P < 0.0001).

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To directly address the contribution of TGF-b toNAFLD-associatedfibrosis, we performed a series of studies in which TGF-b activitywas blocked therapeutically. Although a previous study characterizedhepatocyte-specific deletion of Tgfbr2 in choline-deficient diet-inducedNASH (45), our studies represent experimental intervention of TGF-bfocusing on two critical targets of TGF-b signaling, fibroblasts and im-mune cells, without the caveat of eliminating potentially importantTGF-b signaling during liver development. We found that TGF-bblockade alleviated profibrotic signaling pathways without having anydetectable impact on average body weight, liver weight, or steatosis.However, inWTmice, we detected no changes in col6a1 and thematrixprotein periostin, which have both been linked with IL-13–dependentinflammation and fibrosis (34, 35, 46). We also observed marked in-creases in il4 and chi3l3 expression after TGF-b blockade but little tono change in markers of type 1 inflammation, suggesting that TGF-b


signaling regulates the emergence of type 2 inflammation in the fattyliver. These observations were even more striking in the highly suscep-tible IFN-g−/−mice, in which TGF-b blockade led tomarked increase incol6a1, col3a1, postn, il13, and ccl11 expression. We also observed in-creases in the number of eosinophils in the livers of the anti–TGF-b–treated IFN-g−/−mice. Thus, although we showed that TGF-b serves asa mediator of fibrosis in NASH, these studies also uncovered an impor-tant collaborative role for type 2 immunity and IL-13 specifically in theprogression of NASH and fibrosis.

The explanation for these findings remains unclear, although wehypothesize that activation of TGF-b and IL-13 signaling in the liverlikely represents a feedback loop to repair hepatic damage induced byproinflammatory type 1 cytokines, lipid TLR ligands, dyslipidemia,and bacterial byproduct translocation during obesity-associated dys-biosis. Persistent activation or dysregulation of these repair pathways

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Fig. 7. Human NASH is characterized by type 2 eosinophilic inflammation. (A) Cytokine production from healthy (circles) (n = 2) and NASH (squares; red squares,HCV+ NASH patients) (n = 14) patient liver biopsies was measured from expanded intrahepatic lymphocytes after a CD3/CD28 restimulation for IL-4, IL-5, and IL-13.(B) Combined cytokine production stratified by METAVIR fibrosis score and representative anti–smooth muscle actin histology from F0-F2 (left) and F3-F4 (right)biopsies. (C) Eosinophils were observed in H&E-stained liver biopsies from patients (arrows). Scale bar, 25 mm. (D) Publicly available microarray data from healthy,steatotic, and NASH liver biopsies were analyzed for eosinophil-associated genes that demonstrated significantly altered expression, and hierarchical clustering bysamples (left, heat map) and principal components analysis (right) was performed on the basis of this gene set. All data points represent a single patient.

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can lead to the development of pathological fibrosis. This is supportedby our detection of increased IHL production of IL-4, IL-13, and IL-5and eosinophil accumulation in NASH patients. Eosinophils and type2 cytokines have been implicated in both tissue repair and regenera-tion in models of acute liver injury (47, 48) and also in the develop-ment of fibrosis (35, 49, 50). Thus, given the hepatic injury andhepatomegaly associated with NAFLD and our observation of eosino-phils in areas of macrosteatosis and fibrosis, we hypothesize that eo-sinophils, which are major producers of type 2 cytokines, includingIL-13 (51, 52), may initially support liver repair and regeneration tomaintain liver function and, at more chronic stages, may contribute tothe development of fibrosis. The increasing pressure to repair the liveras obesity and metabolic syndrome become chronic may also explainthe rapid loss of eosinophils from adipose tissues. Thus, worseningNAFLD could provide a feed-forward loop to further exacerbate ad-ipose tissue dysregulation. Finally, our ability to stratify human NASHpatients from normal and steatotic patients based on eosinophil-related gene expression and identification of eosinophils in 100% ofNASH patient biopsy histologic sections analyzed suggests that thepresence of eosinophils or eosinophil-derived mediators in liver biop-sies may serve as a useful indicator of advancing NASH and the pres-ence of type 2 inflammation more generally.

To directly test the contribution of type 2–associated inflammationto the activation of fibrogenic pathways in NASH, we blocked the keyprofibrotic cytokine IL-13with a neutralizingmAb in combinationwithanti–TGF-b and found that IL-13, but not TGF-b, is critically requiredfor the activation of col6a1 expression in the liver. Thus, IL-13 andTGF-b appear to activate distinct profibrotic mechanisms during theprogression of NAFLD, suggesting that dual targeting of both corepathways may be needed to ameliorate the fibrotic progression ofestablished fatty liver disease. Dual targeting may be particularly im-portant because we observed the most marked increase in type 2 im-munity when TGF-b was inhibited.

There are potential limitations to this study that may serve as futureavenues of research or offer alternative interpretations of the data.Withour use of global cytokine knockout animals, we focused on the liverand adipose tissue because these mice displayed similar susceptibilityto obesity. However, we cannot exclude the possibility that the immunealterations were not having additional systemic effects, such as alteringnutrient absorption in the intestine, modifying the microbiome, ormodulating hormonal and nervous system signaling. Additionally,the number ofNASHpatient biopsieswe assessed for IHL cytokine pro-duction (n = 14), the presence of eosinophils (n = 26), and microarrayanalysis (n = 16) was relatively small, making it uncertain whether type2 inflammation serves as a universal driver of NASH or whether it onlycharacterizes a particular subset of patients. However, given the similar-ities between the mouse and human data and the fact that the biopsies,histological analyses, and microarray data were gathered from inde-pendent research groups, there is substantial evidence supporting a crit-ical role for type 2 immunity in the progression of NAFLD. Finally,although we were able to detect eosinophils in all NASH biopsies andidentified transcriptional changes that were consistent with eosinophilicinflammation, we have not yet directly quantified eosinophils in humanNASH or correlated their numbers with disease severity, which will bean important goal of future research.

In summary, we have identified a mixed inflammatory setting spe-cific to the fatty liver containing a previously unappreciated componentof type 2 immune activation marked by IL-13 and eosinophils, whichcollaborates with TGF-b through overlapping and independent


mechanisms to drive fibrosis.We found evidence of this type 2 immuneactivation and eosinophil accumulation in both mouse and humanNASH. These studies represent a paradigm shift in inflammatory char-acterization of steatohepatitis and have important implications forpotential therapeutic interventions aimed at NASH and the metabolicsyndrome.

MATERIALS AND METHODSStudy designOur primary objective was to investigate the immunological contribu-tion to NASH progression and fibrosis. To do this, we disrupted majorregulatory cytokines or experimentally blocked relevant signaling mo-lecules in diet-induced mouse models of progressive NAFLD/NASH.No statistical methods were used to predetermine sample size. Groupsample size was chosen using records of variance in past experiments,and variance is similar between groups being statistically compared. Allexperiments were independently replicated two ormore times. Samplesor data points were excluded only in the case of a technical equipmentor human error that caused a sample to be poorly controlled. Mice orsamples were randomly assigned to experimental groups or processingorders. Group allocation was blinded for all mouse work when possible(such as administration of Abs, sample quantification and analysis, andpathology scoring). The ARRIVE (Animal Research: Reporting of InVivo Experiments) guidelines in the EQUATOR (Enhancing the Qual-ity and Transparency of Health Research) Network library werefollowed for this report. Primary data are located in table S1.

MiceThe National Institute of Allergy and Infectious Diseases Division ofIntramural Research Animal Care and Use Program, as part of the Na-tional Institutes of Health Intramural Research Program, approved allof the experimental procedures (protocol “LPD 16E”). The programcomplies with all applicable provisions of the Animal Welfare Act(www.aphis.usda.gov/animal_welfare/downloads/awa/awa.pdf) andother federal statutes and regulations relating to animals. Six- to 8-week-old C57BL/6 male mice or knockout strains on C57BL/6 backgroundwere obtained from within the same barrier facility at Taconic FarmsInc. and thus were exposed to equivalent bacteria for microflora colo-nization.Micewere put on either a diet of 60% calories from lard fat dietfromHarlan (TD.06414)or theAMLNdietwith40%fat, 20% fructose, and2% cholesterol from Research Diets (D09100301). For therapies, 250 mgeach of anti–TGF-b (BioXCell clone 1D11.16.8, catalog #BE0057), IL-13(Genentech clone 262A-5-1), or control Ab (BioXCell clone MOPC-21,catalog #BE0083) was injected intraperitoneally twice weekly. Mousebody composition was assessed using 1H nuclear magnetic resonancespectroscopy (3-in-1 EchoMRI, Echo Medical Systems LTD) with as-sistance of O. Gavrilova and the Mouse Metabolism Core at NationalInstitute of Diabetes and Digestive and Kidney Diseases. Mice wereterminally anesthetized with sodium pentobarbital. All animals werehoused under specific pathogen–free conditions at the National In-stitutes of Health in an American Association for the Accreditation ofLaboratory Animal Care–approved facility.

Immunofluorescence and immunohistological stainingLiver tissue was snap-frozen immediately after perfusion using aCoolRack M96-ID freezing block on dry ice. Tissue was sectionedat 8 mm using a cryostat and maintained at −80°C until needed. Slideswere removed from −80°C and immediately fixed for 15 min using

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10% neutral buffered formalin. Sections were permeabilized for 20 minusing 0.2% Triton X-100 phosphate-buffered saline (PBST). Sectionswere then blocked with 2% bovine serum albumin (BSA) PBST for30 min. Sections were washed three times with PBST for 5 min each.Primary Abs were diluted in PBST 2% BSA and incubated withsections for 2 hours at room temperature. Sections were rinsed threetimes with PBST for 5 min each. Secondary antibodies were diluted inPBST 2% BSA and incubated with sections for 1 hour at room tem-perature. Sections were rinsed once with PBST for 5 min. Sections werethen stained with 300 nM DAPI in PBST for 3 min. Sections wererinsed three times with PBST for 5 min each and then mounted forimaging using Fluoromount-G (SouthernBiotech). Primary Abs wereSiglec-F (E50-2440), phospho-Smad3 (EP823Y), ubiquitin (EPR8590-448), keratin 8/18 (TROMA-1), and smooth muscle actin. Secondary Abswere goat anti-rabbit tetramethyl rhodamine isothiocyanate (A24536),goat anti-rat Alexa 488 (A-11006), chicken anti-rabbit Alexa 488 (A-21441), and chicken anti-rat Alexa 647 (A-21472). For immuno-histological staining for ubiquitin and keratin 8/18, frozen slides werethawed and treated as above, and additional blocking of endogenousenzymes was performed (Dako enzyme block). Keratin 8/18 was vi-sualized by secondary staining with goat anti-rat horseradish peroxi-dase (HRP) (ab97057) developed with DAB substrate kit (ab64238).This was followed by ubiquitin staining visualized with Mach3 RabbitHRP (M3R531) developed with Vina Green Chromogen (BRR807A).

Fibrosis quantificationPicrosirius red–stained cross sections of three liver lobes from eachmouse were imaged under polarized light. Fibrotic area was calculatedbymeasuring total area of liver with the presence of NASH-specific col-lagen deposition quantified using a Fiji image analysis software (ImageJ)and reported as fibrotic fraction.

Hepatic triglyceride measurementRelative liver lipid was assessed by measuring glycerol content ofhydrolyzed liver tissue as a surrogate of triglyceride accumulation(53–55). Briefly, glycerol was isolated from 50 to 100 mg of liver sapon-ified by addition of ethanolic KOH (two parts EtOH and one part 30%KOH) overnight at 55°C followed by addition of 1:1 water and EtOH.OnemolarMgCl2was added to precipitate fatty acids, and sampleswerespundown.Cleared supernatantswere assayed for glycerol contentwithcolorimetric Free Glycerol Reagent (Sigma-Aldrich, F6428) relative to aglycerol standard curve (Sigma-Aldrich, G7793), and liver triglyceridecontent was calculated.

Human NASH cytokine quantificationAfter informed consent, fresh human liver biopsy specimens were ob-tained from patients undergoing diagnostic liver biopsies at the Hepa-tology Clinic of the CentreHospitalier de l’Université deMontréal. Thisstudy was approved by the institutional ethics committee (protocolSL09.228), and all experiments were performed in accordance withthe Declaration of Helsinki. IHLs were isolated by enzymatic digestionwith collagenase type 2 (100 IU/ml) (Worthington) of the liver biopsyfollowed by mechanical dissociation through a 70-mm filter. IHLs wereexpanded for 15 days in 24-well plateswith 2 × 106 autologous irradiated(3000 gray) peripheral blood mononuclear cells as feeder cells in thepresence of IL-2 (50 IU/ml) (R&D Systems) and anti-CD3 (1 mg/ml)(BD Biosciences clone UCHT1). Expanded IHLs (2 × 106/ml) were re-stimulated for 3 days with anti-CD3/CD28 (1 mg/ml). Cytokine produc-tion was quantified from supernatants collected at day 3 diluted 1:2 by


LEGENDplex analysis for Human Th cytokines (BioLegend) followingthe manufacturer’s instructions. LEGENDplex data were acquired on astandard LSR II instrument (BD Biosciences) using the FACSDIVAsoftware version 8 and analyzed using LEGENDplex software version7 (BioLegend).

Murine gene expression analysesLiver and perigonadal adipose tissues were homogenized in TRIzolReagent (Life Technologies) with Precellys 24 (Bertin Technologies).Total RNA was extracted with chloroform using a MagMAX-96 TotalRNA Isolation Kit (Qiagen) and reverse-transcribed to complementaryDNA using SuperScript II Reverse Transcriptase (Life Technologies).Gene expression was quantified using Power SYBR Green PCR MasterMix (Applied Biosystems) by reverse transcription polymerase chainreaction (PCR) on an ABI Prism 7900HT Sequence Detection System(Applied Biosystems). Gene expression is described relative to RPLP2mRNA levels in naïve liver and perigonadal adipose tissues. Genes withNational Center for Biotechnology Information GenBank accessioncodes are as follows: Rplp2 (NM_026020), 5′-ACGTCGCCTCT-TACCTGCT-3′ (forward) and 5′-GACCTTGTTGAGCCGATCAT-3′ (reverse); Il13 (NM_008355), 5′-CCTCTGACCCTTAAGGAGCT-TAT-3′ (forward) and 5′-CGTTGCACAGGGGAGTCT-3′ (reverse);ccl11 (NM_011330), 5′-GAATCACCAACAACAGATGCAC-3′ (for-ward) and 5′-ATCCTGGACCCACTTCTTCTT-3′ (reverse); tnf(NM_013693), 5′-GCCTCTTCTCATTCCTGCTTGT-3′ (forward)and 5′-GGCCATTTGGGAACTTCTCAT-3′ (reverse); ccl2(NM_009915), 5′-AGGTGTCCCAAAGAAGCTGTA-3′ (forward)and 5′-ATGTCTGGACCCATTCCTTCT-3′ (reverse); postn(NM_015784), 5′-CTGGTATCAAGGTGCTATCTGC-3′ (forward)and 5′-AATGCCCAGCGTGCCATAA-3′ (reverse); col1a1(NM_007742), 5′-AACTGGACTGTCCCAACCCC-3′ (forward) and5′-TCCCTCGACTCCTACATCTTCTG-3′ (reverse); col3a1(NM_009930), 5′-AACCTGGTTTCTTCTCACCCTTC-3′ (forward)and 5′-ACTCATAGGACTGACCAAGGTGG-3′ (reverse); col6a1(NM_009933), 5′-CGCCCTTCCCACTGACAA-3′ (forward) and 5′-GCGTTCCCTTTAAGACAGTTGAG-3′ (reverse); mmp2(NM_008610), 5′-CAAGTTCCCCGGCGATGTC-3′ (forward) and5′-TTCTGGTCAAGGTCACCTGTC-3′ (reverse); timp1 (NM_011593),5′-GCAACTCGGACCTGGTCATAA-3′ (forward) and 5′-CGGC-CCGTGATGAGAAACT-3′ (reverse); tgfb1 (NM_011577), 5′-TGGAG-CAACATGTGGAACTC-3′ (forward) and 5′-GTCAGCAG-CCGGTTACCA-3′ (reverse); chi3l3 (NM_009892), 5′-CATGAGCAA-GACTTGCGTGAC-3′ (forward) and 5′-GGTCCAAACTTCCATCCTC-CA-3′ (reverse); and elane (NM_015779), 5′-TGGAGGTCAT-TTCTGTGGTG-3′ (forward) and 5′-CTGCACTGACCGGAAATT-TAG-3′ (reverse).

Murine RNA-seq, NanoString, and human NASHmicroarray analysisRNA isolated as described abovewas submitted to theNational Instituteof Allergy and Infectious Diseases Research Technologies Branch,where RNA-seq was performed. Illumina NextSeq 500 reads werealigned to the mouse mm10 genome with TopHat2 and mapped tothe mouse reference sequence transcriptome (2015-0804) with theexpectation-maximization algorithm in Partek Flow (build 4.0.15.0702).The nCounter Analysis System (NanoString Technologies) was usedfor gene expression analysis from RNA isolated from adipose tissue.RNA-seq data have been uploaded to the Gene Expression Omnibus(www.ncbi.nlm.nih.gov/geo/) with accession numberGSE95428. Previously

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published and publicly available human microarrays from clinicallydefined normal, steatotic, and NASH patient livers were obtained fromthe ArrayExpress database (accession E-MEXP-3291). Subsequentanalyses were performed using TM4 MeV microarray software suite.Welch’s t tests were used to generate volcano plots (P < 0.05) fromwhich list subsets were generated by using fold difference cutoffs. Ad-ditional ANOVA tests were carried out between groups. Fold changevalueswereuploaded to the IngenuityPathwayAnalysis software (Qiagen)to determine potential upstream regulators.

Data availabilityAccession codes for murine gene expression analysis are providedabove with primers. Mouse RNA-seq and human NASH microarrayaccession numbers are provided above.

Statistical analysisPrism (version 6 and 7; GraphPad) was used for statistical analysis andgraphing. Mann-Whitney or t test was used to determine significantdifferences. Data sets were compared with a two-tailed t test, and differ-ences were considered significant if P values were <0.05. Error barsrepresent SD, where shown.

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SUPPLEMENTARY MATERIALS
www.sciencetranslationalmedicine.org/cgi/content/full/9/396/eaal3694/DC1Materials and MethodsFig. S1. Chronic HFD–induced NASH is associated with hepatic ballooning and activation offibroblasts.Fig. S2. Analysis of adipose tissue fibrotic pathways and collagen deposition.Fig. S3. IL-10 in mouse and human NASH.Fig. S4. NAFLD/NASH in 40-week HFD-challenged WT, IL-4, and IL-10/IL-4 knockout mice.Fig. S5. Comparison of IL-10 versus IL-12 deletion in NAFLD progression.Fig. S6. Adipose tissue inflammation in IFN-g knockouts on HFD.Fig. S7. Presence of cellular mediators of inflammation in NAFLD/NASH.Fig. S8. IL-10/IL-4 knockout animals have long-term protection from HFD-induced NASH.Fig. S9. Collagen- and eosinophil-associated chemoattractant expression alterations in patientbiopsies.Fig. S10. Representative histological eosinophils in NASH patient biopsies.Fig. S11. Inflammatory pathways in NASH progression.Table S1. Primary data.

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Acknowledgments: We wish to thank the flow cytometry core of the Centre de Recherchedu Centre Hospitalier de l’Université de Montréal and O. Gavrilova and the MouseMetabolism Core at the National Institute of Diabetes and Digestive and Kidney Diseasesfor technical help. Funding: This work was supported by grants from the CanadianInstitutes of Health Research (CIHR) (HEO-115696) and Fonds de recherche du Québec–Santé (FRQS) AIDS and Infectious Disease Network (Réseau SIDA-MI). T.F. is supported bydoctoral fellowships from CIHR and the Canadian Network on Hepatitis C (CanHepC). N.H.S.is supported by a Chercheur Boursier salary award from FRQS. This work was alsosupported by the Intramural Research Program of the National Institute of Allergy andInfectious Diseases/NIH. Author contributions: T.A.W., K.M.H., T.F., and N.H.S. conceivedthe study. K.M.H., T.A.W., T.F., N.H.S., J.R.A., and T.R.R. designed the experiments. K.M.H., T.F.,R.L.G., L.A.B., K.M.V., J.C.S., T.H.A., R.d.Q.P., R.W.T., and S.W. performed the experiments.K.M.H. and T.F. analyzed the data. T.F. and M.B. recruited the patients. M.B. and G.S.performed the clinical assessments. K.M.H., T.F., N.H.S., and T.A.W. wrote the paper. Allauthors reviewed the manuscript for intellectual content and approved the final version.Competing interests: J.R.A. is an employee of Genentech Inc. The other authors declarethat they have no competing interests. Data and materials availability: RNA-seq datahave been uploaded to the Gene Expression Omnibus (www.ncbi.nlm.nih.gov/geo/) withaccession number GSE95428. Previously published and publicly available humanmicroarrays were obtained from the ArrayExpress database (accession E-MEXP-3291).

Submitted 10 November 2016Resubmitted 7 March 2017Accepted 17 May 2017Published 28 June 201710.1126/scitranslmed.aal3694

Citation: K. M. Hart, T. Fabre, J. C. Sciurba, R. L. Gieseck III, L. A. Borthwick, K. M. Vannella,T. H. Acciani, R. de Queiroz Prado, R. W. Thompson, S. White, G. Soucy, M. Bilodeau,T. R. Ramalingam, J. R. Arron, N. H. Shoukry, T. A. Wynn, Type 2 immunity is protective inmetabolic disease but exacerbates NAFLD collaboratively with TGF-b. Sci. Transl. Med. 9,eaal3694 (2017).

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βcollaboratively with TGF-Type 2 immunity is protective in metabolic disease but exacerbates NAFLD

Thirumalai R. Ramalingam, Joseph R. Arron, Naglaa H. Shoukry and Thomas A. WynnH. Acciani, Rafael de Queiroz Prado, Robert W. Thompson, Sandra White, Genevieve Soucy, Marc Bilodeau, Kevin M. Hart, Thomas Fabre, Joshua C. Sciurba, Richard L. Gieseck III, Lee A. Borthwick, Kevin M. Vannella, Thomas

DOI: 10.1126/scitranslmed.aal3694, eaal3694.9Sci Transl Med

homeostasis of one tissue can be detrimental to another.observed in the adipose tissue during obesity. These findings reveal that cytokine activity that is beneficial for the mice and human biopsies showed type 2 inflammation and recruitment of eosinophils, unlike the inflammationmore pronounced fibrosis. Instead, they saw that these mice were resistant to steatohepatitis. Fibrotic livers from

. expected obese mice prone to type 1 cytokine responses to experienceet alinvolves type 1 cytokines, so Hart Polarized cytokine networks can drive immunopathogenesis forward. Obesity that leads to liver fibrosis

Opposing cytokines in obesity and fibrosis

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