© 2015 Nature America, Inc. All rights reserved. work. Correspondence work. should Correspondence be addressed to B.P. ( University Hadassah Medical School, Jerusalem, Israel. Rijeka, Croatia. 1 factor necrosis (TNF) andtumor IL-1 as such cytokines, proinflammatory of tion involved become cells and factors more as time over aggravated is that response immune of T in cells VAT mal cells lead to the and activation of accumulation proinflammatory nutrients and oxygen to access their limits of adipocytes hypertrophy induced tissue adipose peripheral the from it reach that of metabolites and the composition signals the endocrinological macrophages. of importantly and accumulation of immune activation most cells, proinflammatory formation, the prevent thereby and IL-13, and (IL-5) 5 interleukin (T T cell 2 helper type secrete cells These cells ( type 2 innate lymphoid cells (ILC2 cells) and invariant natural killer T eosinophils, including subsets, cell immune various individuals, lean (VAT) tissue adipose visceral the in originating inflammation systemic chronic is resistance insulin obesity-induced of cause lying mortality and morbidity of cause global major a insulin resistance, which can ultimately lead to diabetes mellitus type 2, health challenges of our time. An important complication in obesity is worldwide With an estimated half a billion obese and 1 billion overweight people insulin resistance in response to adipocyte obesity-induced stress. macrophages in VAT and greatly ameliorated insulin sensitivity. Thus NK cells are key regulators of macrophage polarization and and promoted insulin resistance. Deficiency of NK cells, NCR1 or IFN- and proliferation interferon- Obesity drove the upregulation of ligands of the NK receptor cell–activating NCR1 on adipocytes; this stimulated NK cell natural tissue-resident killer (NK) cells represented a crucial link between adipose obesity-induced stress and VAT inflammation. signals immunological that trigger their accumulation remain unknown. We found that a distinct phenotypically population of tissue (VAT). VAT inflammation is associated with the accumulation of macrophages in proinflammatory adipose tissue, but the An important cause of insulin obesity-induced resistance is chronic systemic inflammation originating in visceral adipose Jens C Brüning Theurich Sebastian Felix M Wensveen to inflammation and insulin resistance NK cells link adipose obesity-induced stress nature immunology nature Received 16 December 2014; accepted 9 February 2015; published online 2 March 2015; Department Department of Histology and Embryology, Faculty of Medicine, University of Rijeka, Rijeka, Croatia. Obesity deregulates homeostasis in VAT by inducing changes in in changes VATinducing by in homeostasis deregulates Obesity H 1 1 CD4 i NKT NKT cells) 12 1 , , , overt metabolic dysregulation is one of the most pressing 1 5 + 3 . Obesity-induced inflammation of VATinflammation . Obesity-induced an drives influx T cells, CD8 T cells, . The resulting stress responses of adipocytes and stro and adipocytes of responses stress resulting The . 3 Max Max Planck Institute for Metabolism Research Cologne, Cologne, Germany. β , , which in time reduces systemic insulin sensitivity 6– 3 9 , , Ofer Mandelboim , , are important in VATmaintaining homeostasis. 14– 1 , , Vedrana Jelencˇić

3 1 , Ariella , Glasner Ariella 6 aDV + . This results in chronic systemic produc systemic chronic in results This . T cells and B cells, generating a T generating and B T cells, cells g A NCE ONLINE PUBLIC ONLINE NCE (IFN- g ) ) production, which in turn triggered the of differentiation macrophages proinflammatory H 2)-type cytokines, such as such cytokines, 2)-type 10 [email protected] 4 , & Bojan Polić 1 4 1 1 , , Davor Mendrila . In addition, obesity- addition, In . , 5 6 Department Department of Surgery, University Hospital Rijeka, Rijeka, Croatia. 2 , , Sonja Valentić . A primary under primary A . A TION

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, , , Davor Štimac 6 , , Marko Šestan important in the defense against influenza infection influenza against defense the in important is that cells NK on receptor activating potent particularly a is mice) feron- such on as NK to stimulates cells inter target cells produce cytokines ’) self (‘induced receptors cell NK for activating ligands of stress tion upregula or ’) self ‘missing so-called (the molecules I class complex while ensuring self-tolerancecytopathology of signs different detect to calibrated are that receptors are They and a with armed inhibitory of number activating cells. ‘stressed’ otherwise or transformed infected, for tissues survey infection. microbial overt of absence the in especially initiated, is the how transition unclear M2-to-M1 still is it macrophages, of number the in increase resist insulin ance obesity-induced of development the for pivotal be IL-1 of source dominant the be to considered are mediators pro proinflammatory to duce able features are subjects obese VAT (M1)-like of the in cells many proinflammatory Although develop and increase number rapidly in macrophages feeding, high-fat After phenotype. (M2)-like anti-inflammatory an have macrophages adipose tissue–resident conditions, lean Under tissue. adipose in macrophages of that unknown. largely is signal system immune the a activates into translated is stress tissue adipose how However, Natural killer (NK) cells are important sentinels of the body that that body the of sentinels important are cells (NK) killer Natural activation the is VAT in inflammation event early important An g 1 8 doi:10.1038/ni.312 4 prevented the accumulation of proinflammatory γ . Whereas much is known about the factors that stimulate the the stimulate that factors the about known is much . Whereas The The Lautenberg Center for General and Tumor Immunology, The Hebrew (IFN- 2 γ Department Department of Internal Medicine, University Hospital Rijeka, ) or mediate a cytolytic response cytolytic a mediate or ) 2 , , F Thomas Wunderlich 1 , 6 , , Tamara Turk Wensveen 1 0 9 . Downregulation of major histocompatibility 6 These These authors contributed equally to this 15 , 1 6 , adipose tissue macrophages macrophages tissue adipose , 3

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© 2015 Nature America, Inc. All rights reserved. after 4 weeks of high-fat feeding, NK cells cells NK feeding, high-fat VAT of weeks mouse 4 after from isolated leukocytes the produce IFN- not did cells B and macrophages of >95% IFN- all represented together cells T cells NK and tissue, lean the in that VAT in showed leukocytes of tion in than (NCD; diet chow weeks a normal 4 receiving for mice (HFD) diet high-fat a fed mice VAT of the in IFN- (DIO). obesity induced IFN- measured we ance, IFN- whether unclear is process this in role exact its but ance, IFN- IFN- cell NK increases diet High-fat RESULTS and resistance. formation insulin to leads macrophage M1 drives that signal immunological an into translated is stress adipose obesity-induced which by nisms mecha and primary the of one tolerance delineates study Our glucose sensitivity. insulin ameliorated in resulting reduced, was strongly polarization macrophage M1 obesity-induced prevented, was activation cell NK When differentiation. macrophage M1 induced prolifera their promoted IFN- and tion. cells NK VAT-resident which activated adipocytes, on ligands NCR1 of adipose upregulation caused stress Obesity-induced macro resistance. stress, insulin and adipose activation phage obesity-induced among connections the independent experiments. Panel are representative of two ( (Mann-Whitney and Student’s * fed an mice with NCD or an HFD ( ( one. minus fluorescence FMO, cells. VAT-resident for CD3 Gated NK cells. ( in ( mice lean ( of in cells the gate. the percentage represents adjacent to the outlined area in the panel lower ( 4 of after weeks NCD or HFDionomycin feeding PMA and ester the with phorbol stimulation IFN- ( units. RU, relative fed mice an NCD or an HFD for 4 ( weeks of expression transcript showing PCR analysis quantitative by NK in cells VAT.production ( 1 Figure resistance insulin obesity-associated of development the adipose M1 of that macrophages to tissue–resident similar phenotype a develop macrophages polarization macrophage IFN- NK tissues, cell–derived state inflammatory an to a homeostatic from switch to unclear. still is diabetes 2 type of development the in cells 1 (ref. type mellitus diabetes of development the s e l c i rt A  data from three experiments. n e d c P

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© 2015 Nature America, Inc. All rights reserved. synthase (iNOS) and low expression of the costimulatory molecule molecule costimulatory the of expression low oxide and (iNOS) nitric synthase inducible and CD11c integrin of expression high had VAT-resident M1 macrophages, to classical Similar M1 macrophages NK1.1 of numbers the on effect considerable a have not did but tenfold than protocol depletion antibody NK1.1- a low-dose we used types, cell on other is expressed receptor NK1.1 the Because antibodies. NK1.1 by an HFD using with feeding during macrophages M1-like of accumulation the on depletion cell ( T mice and HFD-fed VAT or the NCD- in either mice, of detectable hardly were NCD-fed with compared mice HFD-fed in of CD4 percentage note a higher ( animals NCD-fed (CD11b activated more a acquired HFD an fed VAT mice in weeks, cells NK 4 After feeding. high-fat of stages early the in polarization rophage mac M1-like in are involved cells NK whether we investigated Next HFD activates NK cells and drives M1 macrophage polarization VAT-resident population. cell NK IFN- an of expansion the with associated primarily was and HFD IFN- IFN- of induction early the suggests that T cells are probably not the main source of IFN- ( feeding of weeks 8 after ( each. in CD3 for gated were Cells weeks. 4 for feeding HFD 2 Figure nature immunology nature IL-4R and arginase CD86, are plasma glucose concentrations and area under the curve (AUC). ( (AUC). curve the under area and concentrations glucose plasma are kg g (1 glucose of injection i.p. by accomplished ( d 5 every antibodies control isotype ( day other every once injection i.p. via antibodies ( d 5 every (CD19 ( feeding HFD or NCD of weeks 4 after ionomycin representative of four ( four of cells representative unstimulated to relative expression Protein experiments. * given. independent is two VAT. from and data liver of pooled shows samples tissue in Quantification p-Akt) Ser473; control. at loading a as used was (phosphorylated Akt phosphorylated quantify to used was Immunoblotting (+). insulin ( concentrations. glucose blood of measurement and upeetr Fg 2a Fig. Supplementary HF NC a D D γ was increased in was VATincreased early (at 4 with anduring feeding weeks) − CD27 10 10 10 10 CD11b g

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© 2015 Nature America, Inc. All rights reserved. promoted the induction of glucose intolerance in response to an an to response in reports published with HFD. Consistent intolerance glucose of induction the promoted intolerance. glucose and resistance insulin obesity-induced of generation the for responsible tissue primary the ( operation it but was significantly less than controls, that in HFD-fed mice that underwent NCD-fed sham than intolerance glucose greater develop ( frame time resistance this in insulin hyperinsulinemia or develop not did mice VATectomized HFD-fed ( sham operation that underwent animals HFD-fed with compared VATectomized mice in tissue adipose ous subcutane of mass the in increase large a in resulted feeding HFD operation. sham undergone had that mice with compared mice fed after weeks Twelve NCD- in responses feeding. VATectomy, HFD glucose-tolerance normal found we of initiation the before weeks 2 (VATectomy) pads fat adipose visceral of excision surgical to mice the of development in mice, the we resistance obese insulin subjected To VAT,show that for is required or organs tissues, other than rather resistance insulin HFD-induced for required VAT are cells NK resistance. insulin obesity-induced of development the VAT and in macrophages M1 of accumulation the in important are cells NK together, that show data All these directly. metabolism VAT than depletion rather cell inflammation NK by affected not ( was contrast, in gain, weight ( values these normalized cells NK of depletion whereas animals, NCD-fed ( Data post-test). ( PCR quantitative by determined was expression depleting or PBS received and weeks 12 for HFD-fed or NCD- were ( AUC and concentrations glucose IRT. and plasma GTT to are Shown subjected were mice weeks, 12 After 5 d. every antibodies ( AUC and concentrations glucose plasma GTT.to are Shown subjected were mice weeks, 10 After 2 weeks. every liposomes control or clodronate-loaded received and HFD-fed ( and AUC) and concentrations glucose plasma are ( to subjected were they later weeks 12 and feeding, HFD or NCD (VATectomy). began mice removed surgery, fat Two after weeks 3 Figure s e l c i rt A  d a upeetr Fg 2j Fig. Supplementary

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* * b Quantitative PCR analysis of VAT tissue homogenates from these these from homogenates VATtissue of ( analysis PCR mice Quantitative SCID HFD-fed in gain weight obesity-induced of amount the alter not did cells NK of Depletion ( depleted not were cells NK M1 of number macrophagethe cells comparedin with that in reduction HFD-fed SCID a mice in which with corresponded which mice, SCID in HFD-fed sensitivity and insulin tolerance improved glucose cells are not required for this step. NK cell also significantly depletion mice ( and CD11b of induction the KLRG1 by on these cells, was in still observed the VAT measured of HFD-fed SCID as activation, cell NK ( condition this of development the for sufficient is system immune innate the that indicating diet, same the on mice wild-type in seen that to similar resistance insulin developed and tolerance glucose impaired showed mice SCID HFD, resistance insulin develop and HFD an to their use in this particular context, but they do gain weight in response defects in hepatic lipogenesis cells B and T lack therefore they and recombination, for V(D)J DNA-PKcs,is essential subunit which ciency (SCID). SCID mice are deficient for theimmunodefi DNA-PKcombined severe proteinwith mice kinase to HFD an fed we cells, T of in absence the tissue in adipose of M1 macrophages accumulation model. our in intolerance glucose HFD-induced for ( tered adminis been had liposomes control which to mice with compared liposomes of clodronate-loaded by administration the was disturbed function macrophage which in mice in tolerance glucose improved α P -NK1.1 < 0.05, ** < 0.05, To investigate whether VAT the promote to To whether are sufficient investigate cells NK HFD +VATectomy HFD +sham NCD +sham Insulin (A580) Supplementary Fig. Supplementary 3d Fig. 3 Fig. 0.2 0.4 0.6 e F4/80 (RU) 0 10 20 30 40 50 aDV a 0 , c * * P b ). Thus, macrophages in adipose tissue were important important were tissue adipose in macrophages Thus, ). ) Mice either underwent sham operation or had abdominal abdominal had or operation sham underwent either ) Mice < 0.01, *** < 0.01, NCD +isotype A NCE ONLINE PUBLIC ONLINE NCE c CD206 (RU) Glucose (mM) 10 15 20 25 100 200 300 400 0 5 0 2 0 GTT P < 0.001 (ANOVA with Bonferroni’s Bonferroni’s with (ANOVA < 0.001 3 ** 5 5 HFD +isotype 5 ), ), which suggests that adaptive immune Fig. 3 Fig. , , which might represent a limitation for Time (min) Supplementary Fig. 3c Fig. Supplementary 7 0 3 HFD +clodronate HFD +control NCD +clodronate NCD +control ** 4 CD11c (RU) 10 15 . As a note, SCID mice also have have also mice SCID note, a As . n 0 5 d A = 3–5). ( = 3–5). 5 and and TION *** α 100 upeetr Fg 3f Fig. Supplementary * -NK1.1 or isotype control control isotype or -NK1.1

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© 2015 Nature America, Inc. All rights reserved. NK cells in spleen were donor cell derived; however, very few GFP few very however, derived; cell donor were spleen in of cells NK (~6%) fraction considerable A transfer. after d 1 quantified was (GFP donor of frequency the and mice, NCD-fed wild-type GFP splenic of injections i.v. 2 received for mice HFD an weeks, or NCD an with fed being After expansion. local or cells peripheral of influx an to VAT due in was cells NK of number 4a Fig. ~4%; fat, and under lean ~13%) conditions ( tissue, subcutaneous adipose brown ~8%; liver, ~3%: (spleen, tissues other amounts in VAT (~13% of relative leukocytes) to the amounts in most immunity eral periph of independent responses immune local in aid that subsets Many tissues in humans and mice contain residentspecialized NK cell VATin cells NK of proliferation local the drives HFD resistance. insulin and inflammation tion, and sufficient to promote obesity-induced M1 macrophage accumula but only in of the presence NK Thus, NK cells. are cells required both in mice increased HFD-fed compared with that in NCD-fed controls, as IL-1 such cytokines proinflammatory effects of NK cells on M1 macrophage accumulation, the expression of the with Consistent were present. NK cells when only mice SCID fed HFD- in increased notably was CD11c marker polarization M1 the ( cytometry flow by confirmed was mice NCD-fed in that with compared mice SCID F4/80 of number increased The significance. cal ( HFD the to VATin induced was CD206 response marker in polarization M2 the and F4/80 marker macrophage the of expression that showed mice IFN- the in and pool cell NK total the within cells donor of frequency the and restimulated were leukocytes Total8 d. another for HFD-fed or NCD- were and cells 5 × 10 received mice Next, 1 week. for HFD-fed ( VATand cytometry flow by spleen in determined were numbers cell NK donor relative GFP × 10 1.4 administered 2 weeks, for HFD an or NCD an fed ( VAT and cytometry spleen in flow by determined were numbers cell NK donor relative and absolute × 10 1.4 received then and ( HFD. an to 4 Figure nature immunology nature cells (~1% of total NK in could be cells) the detected VAT of NCD- or plots were gated for CD3 for gated were plots ( feeding 8 d of last the during CD3 for gated were plots Representative (CD3 cells NK donor of number total The mice. the of pads fat the into injected were cells NK ** a P Donor cells < 0.01, *** < 0.01, + 10 10 10 10 10 10 NK cells and HFD-fed for another 8 d. Absolute and and Absolute 8 d. another for HFD-fed and cells NK 0 1 2 3 4 5 6 , b

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VAT the last 8 d of the feeding period. Similar numbers of numbers BrdU Similar 8 last the d period. of feeding the for day second every (BrdU) 5-bromodeoxyuridine of injections i.p. administered and weeks 2 for HFD an or NCD an with mice fed we proliferation, cell NK local to due was HFD the to VATresponse in cells. NK of numbers greater supported that environment local a generated subjects obese ( recipients fed GFP donor more significantly had recipients HFD-fed but recipients, NCD-fed and HFD-fed both in lean and animals. obese Donor GFP GFP Next, tions. condi HFD under cells NK resident to equivalent functionally were ( mice the total NK cell population in the VAT of either NCD-fed or HFD-fed cells did not contribute more to the IFN- faster VAT cells. proliferate tissue-resident than the reach do that cells but feeding, HFD of in influx the ( VATthat indicates peripheral ~1.2% is to not promoted as a result increased had this d 8 after cells, NK of number total of the ~0.5% only constituted cells donor transfer after donor GFP more fourfold to threefold had mice trans recipient after d HFD-fed VATfer. 8 in quantified was cells NK donor of number the and transfer, cell before for 2 weeks or an HFD an NCD fed mice VAT obese in cells ( NK donor fewer relatively in resulted this sue, tis lean than cells NK more contained VAT HFD the an fed mice of Because mice. recipient NCD-fed and HFD-fed in VATsimilar was ( mice HFD-fed Fig. 4 Fig. To determine whether the increase in the number of NK cells in in cells NK of number the in increase the whether Todetermine + 6 + BrdU NK cells (× 10 ) c NK cells than did NCD-fed recipients ( recipients NCD-fed did than cells NK Fig. 4 Fig. 0 2 4 6 8 a Donor cells (%) d ). Next, splenic NK cells were transferred intravenously into into intravenously transferred were cells NK splenic Next, ). 0.2 0.4 c and and Spleen 0 ), two ( two ), NS

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α e Obese VAT Obese induces 0 -Ncr1 and and a HFD Ctr ) Paraffin-embedded ) Paraffin-embedded *** 0.1 + a n *** NK cells were detected in the VAT of HFD-fed mice mice HFD-fed VAT of the in detected were cells NK , ( + h = 5). ( = 5). immunoglobulin b µ Supplementary Fig. 4c Fig. Supplementary γ NCR1 represent the percentages of cells in gates. * gates. in cells of percentages the represent g ml 1 ), three ( three ), HFD NCR1- production by production µ − - m. Irrelevant fusion proteins were used as negative controls ( controls negative as used were proteins fusion Irrelevant m. NK1.1 5 –1 ) c + ) Human VAT and Sc fat sections were stained with hNKp46-immunoglobulin fusion proteins and visualized with DAB ( DAB with visualized and proteins fusion hNKp46-immunoglobulin with stained were VAT ) Human sections fat Sc and ) were found in the spleens of HFD-fed mice mice HFD-fed of spleens the in found were )

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= 6). ( = 6). < 0.01, *** < 0.01, m). RU, relative units. ( units. relative RU, m). Sc Fat + Scfat immunoglobulin fusion proteins ligands are currently unknown, but they can be detected using NCR1- 1 in type mice diabetes promotes cells beta on NCR1 pancreatic receptor NK cell–activating that cells NK activate and proteins attract cell-surface of expression the induce tissues Stressed ligands NCR1 via cells NK activate adipocytes Stressed proliferation. local to due VAT the in primarily cells probably NK is of number the in increase n = 5). ( = 5). f − g shows pooled data from two experiments. two from data pooled shows CD11b 30 8.4 ) ) IFN- n = 3). ( = 3). NCR1-immunoglobulin 10 15 20 25 30 b P 5 n (GMI) ) Total hepatocytes and purified mature adipocytes isolated isolated adipocytes mature purified and ) Total hepatocytes 1 < 0.001 (Student’s (Student’s < 0.001 γ 20 40 60 80 = 5). ( = 5). + aDV production by VAT NK cells measured by flow cytometry upon upon cytometry VAT by flow by measured production cells NK 0 F4/80 e ) NK cells were stimulated with the cell-membrane fraction fraction cell-membrane the with stimulated were ) cells NK + VAT HF NCD * A VAT FMO NCD HF ** NCE ONLINE PUBLIC ONLINE NCE D h + ) Macrophages were left unstimulated (“M unstimulated left were ) Macrophages GR1 D 50 10 d ) Fraction of CD27 of ) Fraction −/Dim 2 f 5 2

. . Unfortunately, of the identities exact NCR1 + c IFN-γ (%) immunoglobulin immunoglobulin Sc fatNKp46 cells ( cells 10 Sc fatCtr- 2 4 6 8 0 3 t 8 -test). Data ( Data -test). . Induced expression of ligands for the the for of ligands expression . Induced WT * 2 n

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α + 0.10 -NCR1 IFN-γ cells (%) investigated whether + 10 15 α ** NCR1 0 5 -IFN-

( RU 100 µ γ 20 40 60 80 g ml * + 0 1 cells ( cells ** Φ –1 only”) or only”) n ) = 6). = 6). VAT Sc fa n * HF NCD 5 *** = 5). t D © 2015 Nature America, Inc. All rights reserved. mice showed increased IFN- showed increased mice ( mice HFD-fed from membrane adipocyte of type either with cultured when production (ref. stimuli activating other to normally respond but NCR1 lack which IFN- increase ( mice VAT NCD-fed of the from membranes with compared IFN- production in increase significant a induced mice from HFD-fed VAT of membranes the Adipocyte mice. HFD-fed or NCD-fed from fat isolated membrane of from fraction VATadipocytes or subcutaneous direct make adipocytes whole c and cells NK of buoyancies ferent ( phenotype of induction IFN- concentration-dependent the in resulted antibodies activation. ( ligands NKp46 expressed fat, subcutaneous VAT,not but that demonstrated teins pro fusion NKp46-immunoglobulin with stained tissues human of fat from mouse ( subcutaneous isolated or adipocytes hepatocytes in not observed was upregulation NCR1-l expression after 4 weeks of high-fat feeding ( VAT from adipocytes that induced showed analysis cytometric Flow on ( NK receptor cells ing stress activat another NKG2D, for ligands of not but NCR1 for ligands of ysis, adipocytes in VAT of HFD-fed mice showed increased expression ( molecules these for negative were epididymis, the adjacent in as such VAT, tissues, whereas mouse lean in adipocytes on detected was islets in Langerhans cells atic beta immunohistological analysis. Similar to what was in observed pancre VAT in obese is of induced ligands NCR1 expression the by of means ( ( cytometry flow by ( ( cytometry flow by VATin determined was ( NCR1. for deficient α depleting or PBS received Animals 5 weeks. for HFD-fed ( DIO. after resistance insulin 6 Figure nature immunology nature ( animals NCD-fed from VAT to cells NK relative ulation three experiments. * experiments. three ( given is animals NCD-fed to relative controls PBS-injected in induction p-Akt control. VAT. and a loading as liver of used was samples Calnexin tissue in p-Akt) Ser473; U kg 1.0 or (−) PBS either with intraperitoneally injected ( n b f Fig. o -NK1.1 antibodies every 5 d. 5 d. every antibodies -NK1.1 , a = 4–5). ( = 4–5). VAT-resident of determined was cells NK phenotype ) The g cultivation cultivation of these cells complicated, we cultured NK cells with the We next investigated the effect of NCR1 stimulation on NK cell cell NK on stimulation NCR1 of effect the investigated Wenext 3 ) The indicated groups of mice were NCD- or HFD-fed for 12 weeks and ( and weeks 12 for HFD-fed or NCD- were mice of groups indicated ) The NK cells (× 10 )

100 2 γ 5 20 40 60 80 1 and differentiation toward a mature CD27 mature a toward differentiation and Fig. 5 Fig. a 0 and and

and HFD + HFD + HFD NC NCR1 deficiency delays the development of development the delays deficiency NCR1 d * D ) Expression of of ) Expression In vitro In Supplementary Fig. 5c Fig. Supplementary Supplementary Fig. 5a e Fig. 5 Fig. ), whereas membranes from su from membranes whereas ), Ncr α γ * -NK1. production. NK cells isolated from from isolated cells NK production. * n Fig. 5 Fig. 1 = 2–3). Data ( Data = 2–3). n gfp/gf = 4–5). Cells of NK-depleted mice were not analyzed. ( analyzed. not were mice NK-depleted of Cells = 4–5). d P 1 a stimulation of NK cells with agonistic NCR1 NCR1 agonistic with cells NK of stimulation and and < 0.05, ** < 0.05, ) The total number of NK cells cells NK of number total ) The p Fig. c b ).

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0

c f ) subjected to GTT and IRT ( and GTT to ) subjected ) The total number of M1 macrophages was determined by flow cytometry cytometry flow by determined was macrophages M1 of number total ) The HFD + HFD + HFD NCD IFN- mice ( in seen NK cell (antibody)-depleted that to similar reduction a controls, wild-type did than HFD the of HFD-fed IFN- produce to cells NK stimulate mice HFD-fed from adipocytes that IFN- to antibodies neutralizing ( of addition the by blocked was macrophages, on expression CD11c as well as effect, this and etry, NK cells induced the expression of iNOS, as measured by flow cytom NK or cells from with stimulated supernatant with NCR1-stimulated cocultured Macrophages differentiation. macrophage M1 induced rl ( trols con NCD-fed to compared weight pad fat increased had and obese were mice cell–depleted NK and NCR1-deficient HFD shown). not the (data of weeks 12 after tolerance glucose not did reduced NKG2D, demonstrate receptor activating cell NK ( the lack mice which mice, cell–deficient NK in seen that to degree a similar to resistance insulin reduced and tolerance glucose amelio rated deficiency NCR1 that found and weeks) (12 feeding HFD process. this in role dominant a play not does stimulation NCR1 to 6a Fig. M1 of number the in VAT( in feeding macrophages of HFD 6 weeks after reduction no showed receptor, TNF the of fed ( controls mice and mice NK compared cell–depleted to that HFD- in wild-type ( feeding HFD of weeks 6 of end the at controls wild-type bers or activation were not observed in polarization macrophage M1 HFD-induced affects Next NCR1 deficiency we whether investigated resistance insulin of development delays deficiency NCR1 differentiation. macrophage M1 ** Fig. Fig. 5 ** We then investigated whether NCR1 stimulation of NK cells cells NK of stimulation NCR1 whether investigated then We We subjected Wesubjected * a + + HFD – γ Ncr α γ c and TNF was greatly reduced in the VAT of HFD-fed -NK1. via the induction of NCR1 ligands, and in turn IFN- turn in and ligands, NCR1 of induction the via h upeetr Fg 6b Fig. Supplementary , ), which indicates that TNF produced by NK cells in response in response by NK cells produced TNF that indicates ), which f 1 and e ) independent experiments. Panel Panel experiments. ) independent gfp/gf Tnf (RU) + + Ncr1 1 200 150 100 p 50 – Supplementary Fig. 5d Supplementary 0 Fig. 6d Fig. α gfp/gfp HFD -NK1. ** HFD + HFD + HFD NCD Ncr1 ** + + + * 1 mice had fewer M1 macrophages after 6 weeks weeks 6 after macrophages M1 fewer had mice , e Ncr α gfp/gfp ). ). -NK1. 1 – Tnfrsf1a f gfp/gf n Ncr Glucose (mM) = 4–5). ( = 4–5). HFD + 1 10 15 20 and NK cell–depleted mice to prolonged prolonged to mice cell–depleted NK and + + 5 p 1 gfp/gf GTT in vivo in 0 , p −/− + c 25 Time (min) ), which indicated that NK cells cells NK that indicated which ), g mice, which lack the p55 the subunit lack which mice, 50 Liver VAT ) Mice were fasted overnight and and overnight fasted were ) Mice * , . Changes in VAT in Changes . num cell NK e HFD + HFD + HFD NCD Ncr1 ). ). Together, data indicate these 75

d 100 shows pooled data from from data pooled shows Fig. Fig. 6

gfp/gfp p-Akt induction (% of NCD) 125 Ncr α HFD NCD 100 120 -NK1. 20 40 60 80 0 1 gfp/gf

c Glucose (% initial) 1 mice compared with VAT 100 ). ). The expression of 50 75 ** 25 p * i. 6 Fig. ticles e l c i rt A * IRT Supplementary Supplementary 0

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© 2015 Nature America, Inc. All rights reserved. rnfr f CD45.1 of Transfer intolerance relative to that of wild-type mice ( IFN- in feeding HFD ( not did cells NK NCR1-deficient of transfer whereas polarization, M1 restored mice ( transfer cell VATNK in without macrophages M1 of accumulation ( cytometry flow by of confirmed was weeks 2 efficiency Transfer during feeding. d HFD 5 every once pads fat mouse the into directly (CD45.1 CD45.2 HFD-fed supplemented we activation, NCR1 to VATresponse in in macrophages M1 of lation IFN- cell–derived NK that prove Tofurther IFN- via macrophages M1 promote cells NK to in an response NCR1. requires HFD resistance insulin of systemic ( p-GSK3 p-p70, for made were observations Similar ( mice NCD-fed in those to similar amounts in induction HFD-fed in VAT, and cells NK liver capacity to induce Akt in phosphorylation response to insulin in both after injection. Whereas HFD-fed wild-type mice showed an impaired VAT and liver of homogenates min 30 tissue whole in measured was phorylation of signaling mediators downstream of the phos insulin and receptor overnight, fasted being after insulin or PBS with injected wild-type, HFD-fed resistance, insulin systemic mediating in NCR1 of involvement the address To VATmetabolism. not and VATinflammation mediate ( one to relative levels glucose plasma are Shown NCR1-immunoglobulin. or control-immunoglobulin PBS, with treatment of weeks 10 after groups indicated ( cytometry kg 5 mg or PBS with intraperitoneally injected 0, point (time HFD the of start the to relative levels IRT ( and GTT to subjected were mice Next, 8 weeks. additional an for week per once and 2 weeks first the 4 d in every CD19 ( × 10 (4 cells NK or wild-type either or Ifng 2 weeks. for HFD-fed or NCD- and ( activation. NCR1 to response in formation macrophage IFN- 7 Figure s e l c i rt A  impaired significantly pad fat the into injection via week every n Supplementary Fig. 6d Supplementary Fig. 7a Supplementary Fig. 7 Fig. a

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0 b HFD/NCR1-immunoglobulin HF NCD ) Representative flow cytometry plots of macrophages. Gated for for Gated macrophages. of plots cytometry flow ) Representative +

– 38. nlee ( unaltered remained gain weight diet-induced whereas feeding, high-fat longed pro to response in intolerance glucose prevent to able was feeding HFD weeks) (10 prolonged during administration immunoglobulin ( phages macro M2 to M1 of ratio the reversed and mice, NCR1-deficient in observed that to similar degree a to accumulation macrophage M1 prevented feeding, HFD of weeks 2 VAT in after cells NK of lation 7f Fig. (ref. ligands NCR1 blocked specifically that proteins fusion per week twice with NCR1-immunoglobulin injected vivo in the way in which they are presented, NCR1 antibodies cannot be used to ing NCR1 antibodies can on stimulate this also protein, depending many block Because activation. NCR1 that prevents protocol peutic by be prevented could a intolerance and thera glucose accumulation intolerance glucose feeding. HFD during of induction the VATand the in polarization IFN- ( transfer after VAT the in IFN- of polarization rophage CD45.1 donor injected cells at 2 weeks after transfer ( CD69 the acquire not did sue tis this in detected be could that cells NK donor VAT, the the and IFN- HFD-fed HFD an with feeding weeks) ( (10 prolonged during amounts lin insu plasma and resistance insulin increased and tolerance ­glucose CD27 D Fig. Fig. 7c 0

Finally, we investigated M1 proinflammatory whether macrophage CD11b + Ncr1

γ + from tissue-resident NK cells is required for M1 macrophage macrophage M1 for required is cells NK tissue-resident from CD27 – HFD ( * * , gfp/gf h d Fig. 7e Fig. Supplementary Fig. 7e Fig. Supplementary CD11b + g ). NCR1-l blocking prevented the activation and accumu and activation the prevented blocking NCR1-l ). ). ). When CD45.1 ) Absolute number of M1 macrophages. ( macrophages. M1 of number ) Absolute 17. + p aDV

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TION Glucose (% initial) 125 c * dim 100 200 300 gfp/gfp ). Shown are plasma glucose glucose plasma are Shown ). 0 ). Thus, we concluded that that concluded we Thus, ). γ GT phenotype of resident NK NK resident of phenotype 2 0 –deficient mice at 2 weeks weeks 2 at mice –deficient

T Glucose (% initial) NK cells (4 × 10 (4 cells NK 100 nature immunology nature 2 25 50 75 IR – 0 2 5 5 * k and and T 2 0 h Time (min) ). Moreover,). NCR1- ) GTT results for for results ) GTT 7 0 ). In conclusion, conclusion, In ). * 5 5 Supplementary Supplementary ). ). Intravenously Time (min) HFD + HFD + HFD 5 immunoglobulin HFD/NCR1- immunoglobulin HFD/control- NCD 7 0 100 * Ncr1 WT

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© 2015 Nature America, Inc. All rights reserved. macrophages in this of tissue, mostly number through the local proliferationin increase an VAT in mediate neutrophils of influx an and ceramides) and palmitate acids, fatty free example, (for lites in metabo increase the feeding, of days few high-fat first Within the T of production the through homeostasis tain i resistance. insulin to leads ultimately and tion VAT in results This inflamma polarization. macrophage M2-to-M1 IFN- VAT-resident produce to activates cells NK which tissue, induces in adipose expression of ligand NCR1 upregulation the obesity Diet-induced accumulation. stress macrophage M1 tissue and adipose between links immunological primary the of one identified have we Here treatment. early for target attractive an therefore is prevention its and development, disease in event a is key The accumulation of proinflammatory M1 macrophages in obese VAT VAT perpetuate inflammation that stress factors adipose ous obesity-induced with 2 type diabetes associated pathologies life-threatening most the of many for responsible is and resistance insulin of cause preventive underlying important VAT an is more obese in ronment and earlier for 2 diabetes type with of aims patients treatment practice clinical Current DISCUSSION activation. NCR1 to response in polarization macrophage IFN- cell–derived NK nature immunology nature Prolonged HFD feeding causes VATcauses Prolonged HFD feeding to macrophages a acquire proin ance with recent insights that recognize the key role of adipocytes in of adipocytes role key the recognize that insights recent with ance obesity. diet-induced in early activated are that through cells various with acquired communication reciprocal stimuli activation activating cell multiple NK by promoted activation, consider be NCR1 can we to addition in Nevertheless, that, likely adipocytes. it stressed by sensitized be to order in stimuli additional few require cells the that implies and have stimulation IFN- produced cells readily NK and VAT CD69 of lean-tissue expression high contrast, In adipocytes. metabolically on stressed expressed differentially are cell– NK molecules additional activating whether investigate to interesting be could it Nevertheless, of induction NKG2D find on adipocytes. ligands obese stimuli to additional be by activated NCR1 engagement macrophages activated cells. NK from received is signal feed-forward a system might ensure that HFDs are able to lead to pathology only after potential proinflammatory full their achieve to macrophages allows that licensing immunological the provide to needed are cells NK HFD, an of days few VAT first the obese in within macrophages of numbers increased and inflammation to contribute factors stress various whereas Thus, macrophages. M1 IFN- into differentiate the to signal lacked they but feeding, HFD VATafter the in increased still numbers macrophage mice, NCR1-deficient in or depleted, were cells NK When stimuli. inhibitory by control cell NK ands in VAT needs to exceed a threshold in certain order to overcome tion only after 2 weeks. We that speculate the expression of NCR1 lig feeding (data not shown), NK cells reached their maximum accumula of the start HFD days after in of the number within NK cells increase of B cells and CD8 flammatory phenotype that is enhanced by the increased accumulation NKT NKT cells, ILC2 cells and eosinophils predominate in VAT and main Under lean conditions, anti-inflammatory immune cells such as as such cells immune anti-inflammatory conditions, lean Under The interaction between NK cells and adipose tissue is in accord in is tissue adipose and cells NK between interaction The by especially control, immunological to subject also are cells NK in vitro in + T cells in the tissue . This corresponds with an activated phenotype phenotype activated an with corresponds This . γ 4 was found to drive early obesity-induced M1 obesity-induced early to drive found was

5 . Moreover, resting human NK cells require require cells NK human Moreover,. resting aDV A NCE ONLINE PUBLIC ONLINE NCE 4 , 14 14 , 16 , 1 , 3 6 3 11 1 . . Although we an observed 9 4 have been characterized. characterized. been have . The inflammatory envi . inflammatory The 0 , 12 . . In recent years, numer , 41 H , 2-type cytokines 2-type 4 γ 2 and thereby drive drive thereby and and immune cells A TION 4 γ 6 after NCR1 NCR1 after . . We did not γ –mediated –mediated

11 4 , 15 4 . This . This , 37 6– , 4 9 3 ------. .

expression of IFN- of expression higher has and cells NK more considerably contains people obese in VAT that demonstrating findings published with line in are findings Our cytokines. macrophage-polarizing of production and liferation pro their promote and cells NK activate to sufficient is signal This ligands. NCR1 of expression the upregulating by cells NK resident to stress their communicate directly adipocytes that show we Here has so studied. far poorly been and immune cells adipocytes between contact cell-cell Direct receptors. a and range of inhibitory activating via receive they that cues extracellular of balance by careful a trolled and transformation oncogenic infection viral cells. NK cells are important innate sensors of cellular stress caused by immune of activation and accumulation the VAT,to in leads which This drives an increase in the amount of proinflammatory adipokines endoplasmic reticulum stress and extracellular matrix confinement due to microhypoxia stroma and their of adipocytes stress cellular adipocytes of hypertrophy acute in results HFD An locally. inflammation dampen or stimulate can adipokines addition, metabolic the regulate responses of which many organs, including the liver, adipokines, brain and as pancreas to referred generally cells endocrine important as appreciated currently are they depots, nutrient-storage static as regarded long were adipocytes Whereas disease. metabolic 3. 2. 1. reprints/index.htm R The authors declare no competing interests.financial F.M.W., with input from all coauthors. studies with NCR study. F.T.W. and J.C.B. coordinated the metabolic studies. O.M. helped design patients and obtained human samples. D.Š. the setup ofsupervised the clinical generated key research reagents. D.M. obtained informed consent from V.J., S.V., M.Š., T.T.W. and V.S. performed and analyzed experiments. A.G. F.M.W. and designed outcarried most of the experiments and analyzed data. European Commission (PCIG14-GA-2013-630827 to F.M.W.). Netherlands Organization for Research Scientific (91614029 to F.M.W.) and the Knowledge Fund (15/13 to B.P.), the University of Rijeka to (13.06.1.1.03 B.P.), the Foundation for the Study of Diabetes (New Horizons Program), the Unity through D. Jurišić-Eržen and M. forZelić help with study setup. Supported by the European We thank S. Slavić-Stupac, M. Samsa and K. Miklić for technical assistance and online version of the pape Note: Any Supplementary Information and Source Data files are available in the the pap the of in version available are references associated any and Methods M diabetes. 2 type to progression of risk the reduce to syndrome is a promising new target for the treatment of patients with metabolic therefore tissue in axis adipose The NK cell–macrophage adipocytes. stressed by cells NK of VAT activation obese in the involving tiation differen macrophage and proinflammatory obesity between link cal COMP AUTHOR CO A eprints and permissions information is available online at at online available is information permissions and eprints c

ethods In conclusion, our study an identified important new immunologi kn Xu, H. Xu, G. Danaei, G.A. Stevens, of obesity-related insulin resistance. insulin obesity-related of million 2.7 and country-years 370 with participants. studies epidemiological examination health and of analysis surveys systematic 1980: since prevalence diabetes and prevalences. obesity ow E TI et al. et N le G G FI dgm N t al. et Chronic inflammation in fat plays a crucial role in the development the in role crucial a plays fat in inflammation Chronic Lancet TRIBUTIO GFP et al. et N l 1 . A 0 ainl rgoa, n goa ted i fsig lsa glucose plasma fasting in trends global and regional, National, en . Adipocytes excrete a multitude of soluble factors, factors, soluble of multitude a excrete Adipocytes . mice. B.P. directed the research and wrote the paper with N e National, regional, and global trends in adult overweight and overweight adult in trends global and regional, National, γ

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© 2015 Nature America, Inc. All rights reserved. For non-nuclear intracellular staining, permeabilization and fixation of of fixation and permeabilization staining, intracellular non-nuclear (eBioscience). A For Brefeldin and antibodies NK1.1 or NCR1 plate-bound or of in PMA and the presence Systems) (Sigma-Aldrich) ionomycin IL-2 (R&D For IFN- of nuclear proteins was done with the set (eBioscience). FoxP3 buffer staining Cells were stained and analyzed in PBS containing 1% BSA and NaN iodide (Sigma-Aldrich) or To-Pro3 (Life Technologies) to distinguish live cells. in-house at the University of Rijeka) and analyzed in the presence of propidium cytometry. Flow The BioXcell. from IFN- of capacity neutralization purchased or chromatography-purified and Rijeka of at by were controls) the facility University produced and our isotype in-house tions (CD4 CD8 (YTS191.1.2), NK1.1 (YTS169.4.2), (PK136), IFN- for Antibodies (IC5868F). Research R&D from was arginase IFN- and (B44) BrdU (mIL4R-M1), CD124 (4E5), Ly49D (1D4B), 2B4 CD107a to (RMV-7), CD51 (2B4), Antibodies eBioscience. from (H1.2F3) CD69 and (CXNFT) NOS2 (BM8), F4/80 (CXCR3-173), CXCR3 Ly49i (YLI-90), Ly49c/i (5E6), CD103 (2E7), CD93 (AA4.1), CD48 (HM48-1), Ly49h NKG2A/C/E GR1 NCR1 (CX5), (20D5), (RB6-8C5), (29A1.4), (3D10), (DX5), CD27 (LG.7F9), CD11b (M1/70), CD11c (N418), CD86 (GL1), NKG2D T-Bet (eBio4B10), CD117 (ACK2), TRAIL (PK136), (N2B2), NK1.1 (AT8), Eomes Ly49g2 (Dan11mag),(A1), Ly49a CD49b (2F1), KLRG1 (A7R34), CD127 (5H4), CD122 (18D3), CD94 (MEL-14), CD62L (A20), CD45.1 (IM7), CD44 (L3T4), CD3 (17A2), CD8- Antibodies. analysis. cytometric flow and by To-Pro3 staining determined was cells target killed of fraction the h, 5 After Systems). (R&D ( ratios indicated the in (YAC-1), cells target CFSE-labeled with mixed were Cells cytometry. tion of NK cells in total splenocytes or VAT was leukocytes determined by flow For the BSA. 1% containing PBS in resuspended and centrifuged were Cells debris. cell remaining remove to sieve a through NH mM (155 solution hypotonic and vortexed (500 thoroughly centrifuged were subsequently Suspensions shaking. with °C 37 at h 1 for and incubated in D DMEM 1 containing (Sigma-Aldrich) mg/ml Collagenase put were tissues Cut scalpel. a with pieces small in cut and excised was tissue VAT from and leukocytes of Isolation injection. after min 90 to up determined were concentrations glucose Venous blood Lilly). (Eli insulin were determined and animals were injected with 1.0 U/kg fast-working human automated an glucometer For(LifeScan). insulin resistance tests using (IRTs), blood Saphena) glucose values (Vena blood venous in measured were tions concentra glucose blood injection, after min 120 to up and Before glucose. (GTTs), mice were fasted for 6–12 h. Next, mice were injected i.p. tests. with 1.0 g/kg resistance insulin and tolerance Glucose Committee. Ethics Faculty Medical Rijeka of University the from approval with done were experiments animal All tions. condi pathogen-free specific under Croatia, Rijeka, in facility animal local from derived animal fat All (Bregi). lines were kept as breeding colonies in the ad libitum fed were old) weeks (8–12 mice Male background. this on times ten least at cally animal modified models were generated in C57BL/6 mice or backcrossed mice were used in transfer experiments using GFP-labeled NK cells. All geneti Heterozygous School). Medical O.M. (Hebrew Hadassah University Laboratory. and 000664), 002287) (strain (strain guidelines. EU C57BL/6 and/or national Wild-type institutional, with accordance in handled Mice. METHODS ONLINE doi: Most Biosciences). (BD kit Fix/Perm the with done was cells 10.1038/ni.3120 Mice were strictly age- and sex-matched within experiments and were were and experiments within sex-matched and age- strictly were Mice γ staining, cells were first stimulated for 5 h stimulated first were cells staining, with an NCD or (Mucedola) an HFD in 50% which of were calories For flow cytometry, we used monoclonal antibodies to mouse CD4 Ncr1 Cells were pretreated with Fc block (clone 2.4G2, produced produced 2.4G2, (clone Fc block with pretreated were Cells Supplementary Fig. 1e Fig. Supplementary gfp/gfp Tnfrsf1a γ ie ae en described been have mice (XMG1.2) were from BD Biosciences. Antibody to to Antibody Biosciences. BD from were (XMG1.2) β (eBioH35-17.2), CD19 (eBio1D3), CD25 (PC61.5), −/− g γ (strain 003242) mice were from the Jackson Jackson the from were mice 003242) (strain for 5 min). The pellet was resuspended in a in resuspended was pellet The min). 5 for antibodies was determined as described as determined was antibodies 4 Cl, 10 mM KHCO mM 10 Cl, γ δ ), in the presence of 1,000 U/ml IL-2 IL-2 U/ml of 1,000 presence ), in the in vitro in TCR (eBioGL3), FoxP3 (FJK-16S), (FJK-16S), FoxP3 (eBioGL3), TCR Prkdc scid killer assay. killer in in vitro For glucose tolerance tests tests tolerance glucose For (strain 001913), 001913), (strain 2 1 3 in in vitro n wr poie by provided were and , 1 mM EDTA), 1 mM run and killer assay, frac the killer with 1,000 U/ml U/ml 1,000 with

in vivo in White adipose adipose White flow cytometry cytometry flow γ 3 (R4-6A2) . . Staining Ncr1 applica Ifng gfp/+ 5 0 −/− . - - - - -

sandwich ELISA with capture (D6C4, Acris Antibodies) and biotinylated biotinylated and Antibodies). Acris (D3E7, antibody Antibodies) detection Acris (D6C4, capture with ELISA by sandwich quantified were levels insulin Serum (NIH). software ImageJ with done and calnexin (Thr p-p70 (30 Extracts homogenizer. tissue a in 7.4) pH benzamidin, 0.2% aprotinin, 0.67% NaCl, X-100, Triton mM 50 1% SDS, 0.1% EDTA, HEPES, mM 10 mM Na-orthovanadate, mM NaF, (50 10 mM 100 buffer lysis Triton a using generated were extracts Tissue nitrogen. liquid in snap-frozen and and liver VATisolated min, were 30 After (Novartis). insulin human U/kg 1 with i.p. injected ELISA. and Immunoblot (NIH). software ImageJ using quantified hematoxylin was Staining brief counterstaining. and (Dako) DAB with visualized was Staining (Sigma- Aldrich). peroxidase to conjugated IgG human by to antibody detected with were incubation proteins Fusion controls. negative as used were proteins fusion immunoglobulin Irrelevant method. antigen-retrieval an as used was described as proteins fusion immunoglobulin mNKG2D- or mNCR1-immunoglobulin hNKp46-immunoglobulin, using (2 Sections paraffin. in embedded and Immunohistology. (TriStar). software FlowJo with analyzed were FACScalibur files FCS a Biosciences). on (BD Biosciences). system (BD low-pressure a FACSaria using or measured were FACSverse Adipocytes a on done were experiments stimulated for 2 d with NCR1 antibodies. NCR1 with d 2 for stimulated VAT of was fraction stromal-vascular total the polarization, macrophage of For analysis staining. iNOS to intracellular A, of and subjected then Brefeldin presence the in were 5 h of last which for 2 d, the cultured were Macrophages anti NCR1 IFN- with bodies. h 48 for activated cells NK of supernatants with cultured activated with plate-bound NCR1 antibodies. Alternatively, cells macrophages NK were with ratio 1:1 a in mixed were macrophages peritoneal assays, tion polariza macrophage For A. Brefeldin of presence the in h 5 for membranes with cocultured were cells NK density. optical on based normalized was tent 12,000 min, (5 centrifuging subsequent by obtained was fraction membrane adipocyte The vortexing. by snap-freezing followed of thawing rapid and cycles three by fractionated were Cells volume. small a in resuspended and PBS ice-cold with twice washed were Cells fraction. top the 200 min, (5 speed low at centrifuged were Suspensions shaking. with D (Sigma-Aldrich) Collagenase 1 mg/ml ing contain DMEM in °C 37 at h 1 for incubated directly and excised was tissue assays. Cocultivation (ClodronateLiposomes.com). liposomes control unloaded or 2 once with weeks 45 mice were tion, every clodronate-loaded mg/kg injected as described g/kg) (0.005 proteins fusion irrelevant or NCR1-immunoglobulin with week per twice injected were mice ligands, was analyzed using the BrdU Flow Kit (BD Biosciences). For blocking of NCR1 mice were injected i.p. with 2 mg BrdUincorporation, and BrdU killed after 4 short-term h. For BrdU incorporation analysis. before d 8 for day other every BrdU mg 0.8 with i.p. injected and weeks 2 for HFD-fed or NCD- were mice once every week in the 10 weeks thereafter. For long-term BrdU incorporation, ment, 4 × 10 IFN- the In cytometry. flow by vidin-coated beads and magnetic cell sorting (Miltenyi). Purity was confirmed were isolated. NK cells were purified with biotinylated DX5 antibodies, strepta were pretreatedmice with CD4 transfers, adoptive For diet. high-fat a or normal a either on put were animals surgery, after weeks Two surgery. after week 1 removed were abdominal fat pads were surgically removed. Animals were closed, and stitches nal fat pads were taken out of the abdomen and then replaced. For VATectomy, For abdomi incision. sham operations, a with Pfannenstiel was opened small and hair was abdominal removed cream with depilation (Veet). The abdomen In vivo experiments. 389 5 µ γ NK cells were transferred once every 4 d once every in 2 NK were the first cells and transferred weeks was neutralized with antibodies in the presence of Fc block. block. Fc of presence the in antibodies with neutralized was ), pERK1/2 (Thr pERK1/2 ), g) were analyzed with polyclonal antibodies to pAkt (Ser pAkt to antibodies polyclonal with analyzed were g) β -actin from Cell Signaling. Quantification of signal was was signal of Quantification Signaling. Cell from -actin Tissues were fixed in 4% formalin for 24 h, dehydrated dehydrated h, 24 for formalin 4% in fixed were Tissues + For VATectomy, mice were with isoflurane, anesthetized - - and CD8 For NK cell–adipocyte membrane cocultures, adipose adipose cocultures, membrane cell–adipocyte For NK Mice were fasted overnight and subsequently subsequently and overnight fasted were Mice γ 202 + –sufficient NK cell–supplementation experi cell–supplementation NK –sufficient -cell–depleting -cell–depleting antibodies. After 24 h, spleens /Tyr g 204 ), and adipocytes were isolated from from isolated were adipocytes and ), µ m) were deparaffinized and stained stained and deparaffinized were m) ), pGSK3- ), 2 2 . Boiling in citrate buffer buffer citrate in Boiling . nature immunology nature 2 α 2 . . For deple macrophage (Ser g 21/9 ). Membrane con Membrane ). ), Pan-ERK1/2, Pan-ERK1/2, ), 473 ), ), ------

© 2015 Nature America, Inc. All rights reserved. elective elective laparoscopic cholecystectomy. Patients who had inflamed gall bladders samples. Human sample. each RNA in expression was normalized by quantification of and Mm00434228_m1) Itgax assays were used to quantify the expression of Taqman machine. PCR Time Fast Real a 7500 with analysis PCR quantitative transcriptase core kit (Eurogentec). The expression of reverse mRNA a was examined with by generated was cDNA and method, Trizol the via isolated was thoroughly vortexed, and a small fraction was directly dissolved in Trizol. RNA D Collagenase (Sigma-Aldrich) andmg/ml incubated for1 1 h at containing 37 °C with shaking. DMEM Suspensions werein suspended were tissues Cut RNA. nature immunology nature (CD11c, Mm00498698_m1), Mm00498698_m1), (CD11c, White adipose tissue was excised and cut in small pieces with a scalpel. a with scalpel. pieces and in cut small excised was tissue adipose White Human tissues were obtained from patients undergoing undergoing patients from obtained were tissues Human Ifng (IFN- γ Tnf , Mm00485148_m1). The relative mRNA relative The Mm00485148_m1). , (TNF, Mm00443260_g1), (TNF, Mm00443260_g1), Emr1 Rn18S (F4/80, Mm00802529_m1), (18S, Mm03928990_g1) Il1b (IL-1 β , 50. multiple for correction post-test comparisons. Bonferroni’s with ANOVA, and Wallis we Student’sused significance, lyze statistical Statistics. it before Committee started. was Ethics Faculty Medical Rijeka of University approval the received from study the and isolated, were tissues before form consent were from excluded the study.surgery during an signed informed All patients

Polic´, B. (1998). infection. latent during replication cytomegalovirus Figures represent means and s.e.m. (depicted by error bars). To ana t al. et P < 0.05 was considered statistically significant. statistically considered was 0.05 < irrhcl n rdnat ypoye ust oto precludes control subset lymphocyte redundant and Hierarchical t -test, Mann-Whitney, -test, Kruskal- J. Exp. Med. Exp. J. doi:

188 10.1038/ni.3120 , 1047–1054 , -