Research Article

Toxic lipids stored by Kupffer cells correlates with theirpro-inflammatory phenotype at an early stage of steatohepatitis

Anne Leroux1,2, Gladys Ferrere1,2, Vanessa Godie1,2, Frédéric Cailleux1,2, Marie-Laure Renoud1,2,Françoise Gaudin1,2,3, Sylvie Naveau1,2,4, Sophie Prévot2,5, Samira Makhzami6,

Gabriel Perlemuter1,2,4, Anne-Marie Cassard-Doulcier1,2,⇑

1INSERM U996, Clamart F-92140, France; 2Univ Paris-Sud, Faculté de médecine Paris-Sud, Kremlin-Bicêtre F-94270, France;3Institut Paris-Sud d’Innovation Thérapeutique (IPSIT), IFR141, Faculté de Pharmacie, Univ Paris-Sud 11, Châtenay-Malabry F-92296, France;

4AP-HP, Hôpital Antoine Béclère, Service d’hépato-gastroentérologie, Clamart F-92140, France; 5AP-HP, Hôpital Antoine Béclère, Serviced’anatomie pathologique, Clamart F-92140, France; 6INRA-Plateforme Iso Cell Express, Jouy-en-Josas, F-78350, France

In memory of Professor Dominique Emilie.

Background & Aims: Non-alcoholic steatohepatitis (NASH) is Results: Although the number of KCs was not modified in stea-

characterized by steatosis associated with liver inflammation.Steatosis causes recruitment of lymphocytes into the liver andthis is worsened by lipopolysaccharides (LPS). As macrophagesmay be involved in the lymphocyte homing, we studied the roleof lipids in determining the phenotype of Kupffer cells (KCs) atthe stage of steatosis.Methods: Steatosis was induced in mice by a high fat diet. Theturnover and the recruitment of KCs were analyzed in vivo byflow cytometry. KCs phenotype was assessed by optical and elec-tron microscopy, cell culture and lymphocyte recruitment byin vitro chemotaxis. Lipidomic analysis was carried out bymass-spectrometry and gene expression analysis by TaqManlow density array.

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Keywords: NASH; Kupffer cells; Inflammation; Lipidomic; Obesity.Received 7 October 2011; received in revised form 27 January 2012; accepted 10February 2012; available online 14 March 2012⇑Corresponding author. Address: INSERM U996, 32 rue des Carnets, Clamart F-92140, France. Tel.: +33 1 41 28 80 37; fax: +33 1 46 32 79 93.E-mail address: cassard.doulcier@u-psud.fr (A.-M. Cassard-Doulcier).Abbreviations: NASH, non-alcoholic steatohepatitis; LPS, lipopolysaccharide; KC,Kupffer cell; NAFLD, non-alcoholic fatty liver disease; TNFa, tumor necrosis fac-tor-a; IFNc, interferon-c; IL, interkeukin; ND, normal diet; HFD, high fat diet; TG,triacylglycerol; NPC, non-parenchymateous cells; TOFA, 5-(tetradecyloxy)-2-fur-oic acid; TLDA, TaqMan low density array; OGTT, oral glucose tolerance test;HOMA, homeostasis model assessment of insulin-resistance; ALT, alanine ami-notransferase; CCL, C-C motif chemokine ligand; ATM, adipose tissue macro-phages; FA, fatty acid; FAS, fatty acid synthase; ACC1, acetyl-CoA carboxylase 1;ChREBP, carbohydrate-responsive element binding protein; PPAR, peroxisomeproliferator-activated receptor; FABP, fatty acid-binding protein; FATP5, fatty acidtransport protein 5; DGAT, diacylglycerol acyltransferase; SCD1, stearoyl-CoA d-esaturase 1; CPT1A, carnitine parmitoyltransferase 1A; HMG-CoA, b-hydroxy-b-methylglutaryl coenzyme A; ACAT1, acyl-CoA cholesterol acyltransferase 1; CE,cholesterol esters; ABCA6, ATP binding cassette transporter A6; MARCO, macro-phage receptor with collagenous structure; LDL, low-density lipoprotein; TLR, tolllike receptors; MTP, microsomal triglyceride transfer protein; VLDL, very low-density lipoprotein; LXRa, liver X receptors alpha; MSR1 (SR-AI), macrophagescavenger receptor; FC, free cholesterol; DAG, diacylglycerol; CXCL10, C-X-C m-otif chemokine 10; SREBP1c, sterol regulatory element binding proteins isoform1c; SR-PSOX, scavenger receptor binding phosphatidylserine and oxidizedlipoprotein.

totic livers compared to normal livers, their phenotypes were dif-ferent. Electron microscopy demonstrated that the KCs from fattylivers were enlarged and loaded with lipid droplets. Lipid synthe-sis and trafficking were dysregulated in fat-laden KCs and toxiclipids accumulated. Fat-laden KCs recruited more CD4+ T and Blymphocytes in response to LPS stimulation than did controlKCs and produced high levels of pro-inflammatory cytokines/che-mokines, which could be reversed by inhibition of lipogenesis.Conclusions: Lipid accumulation in fat-laden KCs is due to a dys-regulation of lipid metabolism and trafficking. Fat-laden KCs are‘‘primed’’ to recruit lymphocytes and exhibit a pro-inflammatoryphenotype, which is reversible with inhibition of lipogenesis.� 2012 European Association for the Study of the Liver. Publishedby Elsevier B.V. All rights reserved.

Introduction

The prevalence of non-alcoholic fatty liver disease (NAFLD) isincreasing worldwide and is linked to the expansion of obesityand type 2 diabetes [1]. NAFLD ranges from pure steatosis tonon-alcoholic steatohepatitis (NASH), fibrosis, cirrhosis, andhepatocellular carcinoma [2]. Obesity with or without associatedinsulin resistance is largely involved in excessive fat accumula-tion in the liver [3]. Although weight loss and correction of insu-lin resistance are relevant therapeutic targets to improve NASH[4], there is currently no treatment for advanced NAFLD [3]. Inthis regard, understanding the mechanisms responsible for theinflammatory processes leading to NASH remains a significantchallenge.

Recruitment of inflammatory cells into the liver and their sub-sequent activation are key steps in the progression of liver dis-ease. NAFLD is associated with alterated hepatic lymphocytesubsets [5] including reduced numbers of hepatic NKT lympho-cytes and T regulatory lymphocytes. Correcting these NKT or Tregulatory lymphocyte deficiencies by adoptive transfer in

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Research Article

murine models of NASH reduces liver damage [6,7]. In addition tothe redistribution of hepatic lymphocyte subsets, we have shownthat liver inflammation in obese mice results not only from stea-tosis but also from lymphocyte hyper-responsiveness to chemo-tactic agents [8].

The liver contains an array of potential antigen-presentingcells, including Kupffer cells (KCs), which could participate inthe redistribution of hepatic lymphocyte subsets during NAFLD.KCs constitute 20–25% of the non-parenchymal cells in the liver.KCs are the primary source of hepatic pro-inflammatory cyto-kines, such as tumor necrosis factor-a (TNFa) [9]. In addition totheir essential role as phagocytes, KCs participate in liver T celltolerance [10]. Consequently, modified KC phenotypes could beinvolved in an altered immune response by disrupting T cell tol-erance in the liver. Macrophage phenotypes are based on thepresence of specific receptors and on the cytokines and chemo-kines they produce, classifying them into two subsets definedas M1 and M2 [11]. M1 macrophages are induced by pro-inflam-matory mediators, such as lipopolysaccharide (LPS) or interferon-c (IFNc), and produce pro-inflammatory cytokines and chemo-kines. Conversely, M2 macrophages are stimulated by interleukin(IL) 4 and IL13 and this stimulation induces low levels of pro-inflammatory cytokines and high levels of IL10. In a recent studythat addressed the role of lipid metabolism in macrophage acti-vation, lipogenesis was found to be a pre-requisite for macro-phage activation [12]. We therefore aimed at studying theinvolvement of lipids in determining KC phenotypes at the stepof steatosis.

Materials and methods

Animals and diets

C57BL/6J and ob/ob mice were purchased from Janvier (France) and maintainedunder a 12-h light/dark schedule, with food and water ad libitum and treated inaccordance with the Guide for the Care and Use of Laboratory Animals (NationalResearch Council, 1996). Mice were fed either a normal diet (ND) or a high-fatdiet (HFD) (Safe, France) as described in Supplementary Materials.

Lipidomic analysis

Hepatic triacylglycerols (TGs) were extracted with a chloroform–methanol mix-ture and separated by thin layer chromatography. TGs were then extracted fromthe silica plate with acetone and measured with a colorimetric diagnostic kit (Tri-glycerides FS; Diasys). For lipidomic analysis, the liver and KCs were homoge-nized in 1 ml of methanol/water (2:1, v/v) containing 5 mM EGTA. Lipids wereextracted and molecular species were quantified by gas liquid chromatographyas previously described [13].

Isolation and culture of KCs

Livers were homogenized and non-parenchymal cells (NPC) and KCs were recov-ered as described in Supplementary Materials. NPC were resuspended in PBS 2%FCS prior to staining with mAbs and flow cytometry analysis. KCs (106 cells) wereplated in RPMI, 10% FCS (PAA, Austria) and 1% penicillin/streptomycin for 24 hand then stimulated with 100 ng/ml LPS from Escherichia coli serotype O55:B5endotoxin-free (Alexis, Switzerland) or not for 24 h. For TOFA (5-(tetradecyl-oxy)-2-furoic acid) (Merrel National Laboratories, USA) exposure, KCs were trea-ted with 200 lM TOFA for 24 h. KC culture supernatants were stored frozen orused in an in vitro chemotaxis assay. Cell viability was evaluated by trypan blueand was higher than 90%. There was no contamination by hepatocytes. The puritywas assessed by F4/80, CD31, and CD11c labeling and ranged from 85% to 89%with some contamination by endothelial cells (4.8%) or dendritic cells (2.4%) (datanot shown).

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Liver monocyte recruitment

Aggregates of PKH26-PCL (0.1 mM; Sigma, MO) or saline were intravenouslyinjected into mice fed with HFD or ND at the age of 4 weeks. Mice fed the HFDand ND diets were sacrificed 4, 8, 12, and 16 weeks after the PKH26-PCL or salineinjection. Macrophages were stained with a rat anti-F4/80 mAb (AbD Serotec, UK)and analyzed using a FACSCalibur cytometer (Becton Dickinson, USA).

Lymphocyte recruitment assay

Lymphocyte chemotaxis was evaluated using a Transwell� system (5 lm pores,Corning Costar, MA). Splenocytes from lean mice were purified by centrifugationthrough a Lympholyte� density gradient (Cedarlane, Canada) at 800 g for 20 min.Lymphocytes were stained with anti-CD3, anti-CD4, anti-CD8, and anti-CD19mAbs. KC supernatants were placed in the lower chamber and 1.5 � 106 lympho-cytes in 150 ll were placed into the upper chamber (same culture medium inboth chambers). Uncultured medium in the lower chamber with 1.5 � 106 cellsin the upper chamber were used as input control. After 4 h of incubation, cellsin the lower chamber were analyzed by flow cytometry. To quantify the lympho-cytes subsets infiltrating the liver, anti-CD45 or antibodies described above wereused. NPC were stained and we counted the number of labeled lymphocytes per gof liver.

RNA and TaqMan low-density array analysis (TLDA)

RNAs were extracted using the RNeasy Lipid Tissue kit (Qiagen, CA) including aDNase treatment. TLDA was performed following the manufacturer’s instructionsand described in Supplementary Materials.

Statistical analysis

All analyses were performed using the StatView (version 5.0 statistical softwaresystem (Abacus Concepts, CA).

Results

HFD-induced steatosis does not modify recruitment of KCs

To determine whether KCs were involved in the early steps ofNASH, C57BL/6 J mice were fed the HFD for 16 weeks. This dietinduced a mean weight gain of 26% (±4%) and obesity-relatedmetabolic disorders (Table 1). Insulinemia, blood glucose levels,oral glucose tolerance test (OGTT) results and calculated homeo-stasis model assessment of insulin-resistance (HOMA) clearlydemonstrated that the HFD mice were insulin-resistant. Theplasma levels of TGs, leptin, resistin, and CCL2 were significantlyhigher in HFD mice compared to those of ND mice. The HFDmouse liver exhibited clear signs of steatosis and had TG levelsthat were fourfold higher than the liver TG levels of ND mice(Fig. 1A and B). Alanine aminotransferase (ALT) levels were notsignificantly modified by the HFD diet (Table 1). There was noliver inflammation in HFD mice, as shown by the absence ofinflammatory infiltrates at histological examination and by thenumbers of liver CD45 lymphocytes on flow cytometry analysis,which were not higher than those of ND mice (Fig. 1 A and C).Therefore, HFD mice were at an early step of NAFLD, with a fattyliver but no significant inflammation.

To study the number of KCs and their recruitment to the liverduring steatosis development, we injected mice intravenouslywith aggregates of PKH26 [14]. After 24 h, more than 95% ofKCs were PKH26+ and all monocytes were PKH26� (data notshown). Mice were then maintained on HFD or ND for 4, 8, 12,or 16 weeks. Recruitment of new liver macrophages was assessedby the number of PKH26�/F4/80+ cells found in the liver. The

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Table 1. Characteristics of ND and HFD mice.

ND HFD

Mice weight (g) 29.36 ± 0.30 39.73 ± 1.4***Liver weight (g) 1.52 ± 0.038 1.48 ± 0.049ALT (IU/L) 173.31 ± 43.03 155.67 ± 39.73Epididymal adipose tissue weight (g)

0.41 ± 0.03 2.19 ± 0.21**

Plasma TG 0.51 ± 0.014 0.86 ± 0.079*Blood glucose (mg/dl) 119 ± 11.61 172.55 ± 5.45*Insulin (pg/ml) 162.06 ± 17.31 654.23 ± 80.41***HOMA 1.23 ± 0.20 7.14 ± 0.96***OGTT (arbitrary unit) 30,162 ± 312 37,055 ± 1182*Leptin (pg/ml) 273.72 ± 64.52 7085.52 ± 828.11***Resistin (pg/ml) 833.05 ± 57.35 1391.21 ± 95.94***IL6 (pg/ml) 20.9 ± 8.89 22.99 ± 5.66CCL2 (pg/ml) 24.72 ± 7.31 55.91 ± 5.98*

Mice were fed either ND or HFD for 16 weeks. Data represent the mean ± SEM ofgroups of at least eight mice. ⁄p <0.05, ⁄⁄p <0.001, and ⁄⁄⁄p <0.0001.Statistical analysis was performed using the Mann–Whitney test.

A

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Fig. 1. No modification of KCs recruitment in the liver of HFD mice at the stepof pure steatosis. (A) Histological examination of liver tissue from micemaintained for 16 weeks on the ND or the HFD: macro-vesicular and micro-vesicular steatosis. (B) Hepatic triglyceride content and (C) quantification by flowcytometry of CD45+ lymphocytes from ND and HFD mice after 16 weeks on theirrespective diets. Data represent the mean ± SEM of 14 animals. (D) Mice wereinjected iv with PKH26 aggregates before starting the diet. PKH26+ and PKH26�KCs in the livers of mice were quantified by flow cytometry after 4, 8, 12, and16 weeks of ND or HFD. The percentage represents the recruited KCs (PKH26–) vs.the non-recruited KCs (PKH26+). Data represent the mean ± SEM of four animals.(E) Quantification of KCs by flow cytometry after 16 weeks of ND or HFD. Datarepresent the mean ± SEM of 14 animals. (Mann–Whitney, ⁄⁄⁄p <0.0001).

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percentage of recruited KCs was similar for ND and HFD micesuggesting that accumulation of fat in the liver does not modifymonocyte recruitment (Fig. 1D). This was confirmed by quantify-ing total liver KCs by flow cytometry or immunohistochemistry,for which there were no significant differences between HFDand ND mice (Fig. 1E and Supplementary Fig. 1).

HFD-induced steatosis led to lipid accumulation in KCs

As the number of Kupffer cells in a steatotic liver was unchangedcompared to a normal liver, we assessed their phenotype. Themorphology of KCs from HFD mice was different from that ofKCs from ND mice. Indeed, observation by optic microscopyand Red Oil labeling showed that neutral lipids had accumulatedin the cytoplasm of KCs and these cells were larger than those ofND mice (Fig. 2A). These observations were confirmed by electronmicroscopy which demonstrated the accumulation of numerousand enlarged lipid droplets in the cytoplasm of KCs from HFD(Fig. 2B). This lipid droplets accumulation is also observed in adi-pose tissue macrophages (ATMs) and in KCs from another modelof NASH, ob/ob mice (Supplementary Figs. 2A and 3A).

Dysregulation of genes expression involved in lipid metabolism infat-laden KCs

To study the mechanisms involved in lipid accumulation of KCs,we determined by TLDA the gene expression levels of enzymesinvolved in lipid metabolism and transport (Table 2). The de novofatty acid (FA) synthesis pathways were altered in the KCs of HFDmice. Fatty acid synthase (Fas) gene expression was significantlyhigher, although acetyl-CoA carboxylase 1 (Acc1) gene expressionwas unaltered. This increase could be relayed by elevated expres-sion levels of the carbohydrate-responsive element binding pro-tein 1 (Chrebp1) and peroxisome proliferator-activated receptorc (Pparc) genes, which were also upregulated. The FA transport-ers gene expression such as FA binding proteins 2 gene (Fabp2)

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and fatty acid transport protein 5 (Fatp5) was also increased.The TG synthesis pathway was also activated as shown by a3.98-fold increase in diacylglycerol acyltransferase 2 (Dgat2) geneexpression. The stearoyl-CoA desaturase 1 (Scd1) gene was upreg-ulated, suggesting that the storage of unsaturated lipids wasincreased. Upregulation of the Ppara gene and one of its targetscarnitine parmitoyltransferase 1A (Cpt1A) suggested an increasein b-oxidation.

We found that the cholesterol metabolism was dysregulatedas the b-hydroxy-b-methylglutaryl coenzyme A (Hmg-CoA) geneexpression was downregulated. The expression level of theacyl-CoA cholesterol acyltransferase 1 (Acat1) gene, whichencodes one of the enzymes required for the cholesterol esters(CE) synthesis, was not significantly modified. The expression ofthe ATP-binding cassette transporter A6 (Abac6) gene, whichbelongs to a family of transporters involved in the cholesterolefflux [15], was higher in KCs from HFD mice. Among the scaven-ger receptors involved in the uptake of modified LDLs (oxidizedor acetylated), several were significantly upregulated, such asmacrophage receptor with collagenous structure (Marco) andCd163. As some toll like receptors (TLRs) could bind fatty acidsand/or are known to be involved in NASH such as TLR4, TLR2,and TLR9 [16], we analyzed their expression. TLR2 and TLR4 werenot modified but we found a large increase of TLR6 and TLR9expression which were, at least for TLR9, previously involved inNASH development [17]. Expression of the microsomal TG

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Fig. 2. Morphological analysis of KCs. (A) KCs, from mice sacrificed after16 weeks of diet, stained with Red Oil and examined by light microscopy. (B)Electron microscopy of KCs from ND and HFD mice.

Research Article

transfer protein (Mtp) gene, which encodes a key molecule forVLDL assembly and export, was markedly increased. The higherexpression levels of liver X receptor a (Lxra) gene, and the Pparagene, which are involved in the upregulation of cholesterol effluxfrom macrophages, suggested that cholesterol trafficking wasstimulated [18]. These differences of expression were alsoobserved except for two membrane receptor, Msr1 and TLR9, inanother advanced model of NASH, ob/ob mice. As gene expressionof several enzymes involved in lipid synthesis and trafficking wasdysregulated, we assessed the lipid species that accumulated inthe total liver and specifically in KCs.

Toxic lipids are concentrated in fat-laden KCs

Lipidomic analysis showed that TG accumulation was signifi-cantly higher in total liver from HFD mice compared to that ofND mice (Table 3). In contrast, the total amounts of TGs in NDand HFD KCs were not significantly different. However, the levelsof some TG species were significantly higher in KCs from HFDmice (Table 3).

The levels of free cholesterol (FC) and CE were higher in boththe total liver and KCs from HFD mice compared to ND mice(Table 3). The same species of CE were higher in the total liverand in KCs.

Diacylglycerols (DAGs) are toxic lipids since they activate pro-inflammatory and nutrient-sensing pathways [19,20]. Theamount of DAGs was much higher in HFD KCs than in ND KCs(Table 3). In contrast, DAG content was significantly lower in totalliver extracts from HFD mice compared to that of ND mice.

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Sphingolipids, including ceramides, are involved in many bio-logical processes including inflammation [21]. Total sphingomye-lin levels were similar in livers and KCs from ND and HFD mice(data not shown). Conversely, the total ceramide level was signif-icantly higher for the KCs of HFD mice than for the KCs of NDmice (Table 3). The species of ceramides were also substantiallyredistributed. In contrast, the ceramide levels in total liversextracts of HFD and ND mice were not significantly different.Overall, these results demonstrate that lipid homeostasis in theKC from HFD mice was profoundly dysregulated with higheramounts of toxic lipids in fat-laden KCs.

Lipogenesis induces a pro-inflammatory phenotype of fat-laden KCs

In response to LPS challenge, KCs from HFD mice secreted morepro-inflammatory cytokines and chemokines than KCs from NDmice (Fig. 3A). In particular, KCs from HFD mice secreted moreIFNc, TNFa, IL10, CCL2, and CCL5 under basal conditions and afterLPS challenge than did the KCs from ND mice (Fig. 3B). Secretionof IL6, IL1b, and CXCL10 was higher in KCs from HFD mice than inKCs from ND mice only after LPS challenge.

We then inhibited the acetyl-CoA carboxylase (ACC), first stepof lipogenesis, by TOFA in short term experiments. The inhibitionof lipogenesis decreased the production of these pro-inflamma-tory cytokines/chemokines (Fig. 3C and D). Moreover, weobserved a similar inhibition of the pro-inflammatory phenotypeunder lipogenesis inhibiton in KCs from another model of NASH,ob/ob mice and also in ATMs (Supplementary Figs. 2 and 3). Thisresult demonstrates that lipid accumulation in KCs and ATM cor-related with the pro-inflammatory phenotype of KCs and isreversible with inhibition of lipogenesis.

Pro-inflammatory fat-laden KCs induced a higher recruitment oflymphocytes

HFD mice challenged by LPS showed a higher recruitment ofCD45+ lymphocytes into the liver compared to ND mice(Fig. 4A). To further analyze the involvement of KCs fromHFD mice in recruiting lymphocytes to the liver, we testedthe ability of KCs to recruit lymphocytes in vitro using a chemo-taxis assay. KCs from HFD-fed mice or their lean controls wereincubated with or without LPS for 24 h and the resulting cul-ture supernatants were loaded into the lower chamber of atranswell system. Freshly isolated splenocytes from C57BL/6Jmice, labeled with antibodies to lymphocyte markers, wereloaded in the upper chamber. Uncultured medium in the lowerchamber with 1.5 � 106 cells in the upper chamber were usedas input control. Lymphocyte migration into the lower chamberwas quantified by flow cytometry. The culture supernatantfrom KC of HFD-fed mice recruited significantly more CD4+ Tand B cells than the control supernatant (Fig. 4C). In vivo, LPSchallenge showed that the recruitment of lymphocytes subsetsinto the liver was also increased (Fig. 4B). Overall, these resultsdemonstrate that KCs from HFD mice are ‘‘primed’’ to recruitlymphocytes.

Discussion

We have previously shown that liver inflammation in obese miceresults not only from lymphocyte hyper-responsiveness to

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Table 2. Quantification of gene expression in KCs from ND, HFD and ob/ob mice.

Gene symbol 2-ΔCT ND 2-ΔCT HFD Fold HFD vs. ND

p value HFD vs. ND

2-ΔCT ob/ob Fold ob/ob vs. ND

p value ob/ob vs. ND

Lipid metabolismSrebp1c 2.00 ± 0.09 2.00 ± 0.08 0.99 n.s. 2.00 ± 0.00 0.91 n.s.

Chrebp 0.13 ± 0.09 0.58 ± 0.14 1.71 * 0.40 ± 0.25 1.98 n.s.Pparα <0.01 ± 0.00 0.61 ± 0.12 3.10 *** 1.00 ± 0.32 5.09 *Pparγ 1.25 ± 0.11 2.11 ± 0.24 1.67 * 2.60 ± 0.40 2.18 *Lxrα 4.13 ± 0.33 15.60 ± 1.29 3.82 *** 15.60 ± 2.29 3.80 **Fas 2.07 ± 0.15 2.63 ± 0.30 1.37 * 4.40 ± 1.03 2.24 *Acc1 1.81 ± 0.10 1.74 ± 0.10 0.93 n.s. 2.00 ± 0.00 1.04 n.s.Scd1 24.88 ± 3.93 87.79 ± 15.78 3.54 ** 252.00 ± 68.30 10.17 **Dgat1 2.31 ± 0.12 2.05 ± 0.05 0.89 * 2.20 ± 0.20 0.99 n.s.Dgat2 1.31 ± 0.20 5.00 ± 0.87 3.98 ** 6.00 ± 1.34 4.85 *Cpt1a 6.69 ± 0.50 8.16 ± 0.36 1.24 * 8.60 ± 0.81 1.28 0.09Hmg-CoA 4.80 ± 0.24 3.59 ± 0.17 0.78 *** 3.75 ± 0.48 0.79 0.08Acat1 11.40 ± 1.34 9.42 ± 0.58 0.83 n.s. 10.80 ± 1.32 0.93 n.s.

Lipid transporter Fabp2 0.75 ± 0.31 2.37 ± 0.47 2.94 * 4.40 ± 1.17 5.53 *Fabp4 193.44 ± 26.25 142.37 ± 17.52 0.74 n.s. 355.00 ± 50.70 1.83 *Fatp5 1.13 ± 0.27 6.37 ± 1.36 5.43 * 7.80 ± 2.18 6.58 *Mtp 0.44 ± 0.20 4.95 ± 1.03 7.81 ** 7.40 ± 2.06 11.69 *Abca6 0.31 ± 0.12 1.83 ± 0.34 4.38 ** 2.60 ± 0.75 6.55 *

Scavenger receptor Cd36 14.75 ± 0.99 14.47 ± 0.49 0.99 n.s. 15.00 ± 1.08 1.05 n.s.Cd163 3.69 ± 0.62 12.32 ± 1.13 3.44 *** 14.40 ± 1.33 3.97 **Marco 5.44 ± 1.03 36.68 ± 6.65 6.73 ** 31.60 ± 8.15 5.78 **Msr1 (Sr-ai) 62.75 ± 3.99 52.63 ± 3.23 0.84 n.s. 102.00 ± 6.89 1.62 *Msr2 0.19 ± 0.10 <0.01 ± 0.00 0.58 *** 0.00 ± 0.00 0.35 **Sr-psox 44.47 ± 3.01 28.74 ± 1.09 0.65 *** 35.00 ± 3.29 0.80 0.10

CytokineTgfß1 40.90 ± 2.42 33.20 ± 1.43 0.82 * 33.00 ± 3.74 0.81 0.08

Il6 0.50 ± 0.13 0.83 ± 0.25 1.63 n.s. 1.00 ± 0.32 1.60 n.s.

Tnfα 5.00 ± 0.51 5.89 ± 0.64 1.18 n.s. 7.00 ± 0.84 1.45 0.07

Il1b 10.90 ± 1.33 18.90 ± 2.25 1.72 * 23.40 ± 4.06 2.15 *

Il10 0.63 ± 0.13 2.32 ± 0.50 4.19 * 2.80 ± 0.80 5.15 ***

TLR

Tlr2 3.81 ± 0.23 3.58 ± 0.18 0.96 n.s. 3.40 ± 0.51 0.91 *

Tlr4 15.20 ± 0.89 15.60 ± 0.71 1.01 n.s. 15.00 ± 1.79 0.97 n.s.

Tlr5 <0.01 ± 0.00 <0.01 ± 0.00 1.29 n.s. <0.01 ± 0.00 1.51 n.s.

Tlr6 0.06 ± 0.06 0.58 ± 0.12 9.25 *** 0.60 ± 0.25 9.61 0.07

Tlr9 1.19 ± 0.10 1.89 ± 0.19 1.57 ** 0.80 ± 0.20 0.67 *

mRNAs from KCs of ND, HFD and ob/ob mice were isolated and subjected to TLDA as described in Materials and methods. Results are expressed relative to the expressionlevels in ND mice and represent the mean ± SEM and were multiplied by an arbitrary factor: 104; n = 16 for ND mice, n = 19 for HFD mice and n = 4 for ob/ob mice; ⁄p <0.05,⁄⁄p <0.001, and ⁄⁄⁄p <0.0001. Statistical analysis was performed using the Student’s t test between ND and HFD mice and the Mann–Withney test between ND and ob/obmice.

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chemotactic agents, but also from steatosis [8]. KCs are the pri-mary source of pro-inflammatory cytokines and chemokines inthe liver. Consequently, modified KC phenotypes could be

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involved in an altered immune response by disrupting T cell tol-erance. In this study, we investigated the involvement of lipids indetermining KC phenotypes at the step of steatosis.

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Table 3. Fat-laden KCs produced higher levels of toxic lipids.

Lipid molecular species Liver KCsND HFD Fc ND HFD Fc

Cholesterol 36.18 ± 3.83 40.69 ± 1.00* 1.12 4.58 ± 1.45 13.63 ± 4.04* 2.98Cholesterol ester C16 2.09 ± 0.26 6.23 ± 1.25* 2.98 0.10 ± 0.02 2.61 ± 1.55* 26.10

C18 3.19 ± 0.40 7.88 ± 1.23* 2.47 0.17 ± 0.04 3.02 ± 1.59* 17.76C20:4 u.n. u.n. u.n. 0.17 ± 0.09*

total 5.28 ± 0.64 14.11 ± 2.44* 2.67 0.27 ± 0.06 5.80 ± 3.22* 21.48Diacylglycerol C33 (14/16) 0.15 ± 0.07 0.38 ± 0.10 2.53 u.n. 0.14 ± 0.06

C35 (16/16) 2.44 ± 0.27 2.45 ± 0.20 1.00 0.15 ± 0.03 1.02 ± 0.51* 6.80C37 (16/18) 11.69 ± 1.11 9.80 ± 0.77* 0.84 0.67 ± 0.13 3.89 ± 1.98* 5.81C39 (18/18) 16.08 ± 1.26 11.43 ± 0.76 0.71 1.36 ± 0.23 5.13 ± 2.22 3.77C41 (18/20) 1.89 ± 0.30 1.05 ± 0.24* 0.56 0.06 ± 0.02 0.66 ± 0.37 11.00total 32.25 ± 2.67 25.13 ± 1.24* 0.78 2.24 ± 0.41 10.84 ± 5.12* 4.84

Triacylglycerol C49 (14/16/16) 2.66 ± 0.78 10.50 ± 2.94* 3.95 1.46 ± 0.25 8.98 ± 4.07* 6.15C51 (16/16/16) 5.66 ± 0.93 42.92 ± 13.88* 7.58 1.50 ± 0.37 44.37 ± 23.89* 29.58C53 (16/16/18) 43.70 ± 7.25 168.40 ± 56.44* 3.85 8.25 ± 2.04 135.75 ± 56.57* 16.45C55 (16/18/18) 105.97 ± 16.78 292.61 ± 83.54 2.76 18.79 ± 4.83 219.81 ± 82.80* 11.70C57 (18/18/18) 36.52 ± 6.11 61.88 ± 19.82 1.69 7.00 ± 2.51 67.33 ± 33.03 9.62total 194.52 ± 31.00 576.30 ± 175.31* 2.96 36.99 ± 9.84 476.44 ± 197.97 12.88

Ceramide C16:0-D18:1 0.66 ± 0.10 0.91 ± 0.30 1.38 0.12 ± 0.03 0.54 ± 0.19* 4.50C18:0-D18:1 u.n. u.n. 0.04 ± 0.04 u.n.

C18:0-D18:2 u.n. u.n. 0.01 ± 0.01 0.02 ± 0.02 2.00

C20:0-D18:2 0.30 ± 0.09 0.48 ± 0.14 1.60 0.01 ± 0.01 0.02 ± 0.02 2.00C22:0-D18:1 0.84 ± 0.16 1.80 ± 0.31* 2.14 0.03 ± 0.01 0.65 ± 0.42* 21.67C24:0-D18:1 0.36 ± 0.06 0.57 ± 0.11 1.58 0.01 ± 0.01 0.22 ± 0.13* 22.00C24:1-D18:1 0.67 ± 0.11 0.64 ± 0.16 0.96 0.04 ± 0.03 0.39 ± 0.27 9.75total 2.83 ± 0.40 4.40 ± 0.78 1.57 0.26 ± 0.10 1.81 ± 1.01* 6.00

Livers and KCs were recovered from mice after they had received ND or HFD for 16 weeks. Cholesterol, cholesterol ester, diacylglycerol, triacylglycerol, and ceramidecontents were quantified by gas liquid chromatography. Data represent the mean (expressed as nmol of lipid/mg of tissue or as nmol of lipid/106 KCs) ± SEM of n = 12animals for liver analysis; n = 4 for ND KCs; n = 9 for HFD KCs; ⁄p <0.05. FC represents the fold change between the respective values determined for HFD and ND mice.Statistical analysis was performed using the Student’s t test for liver samples and the Mann–Whitney test for KCs samples. u.n., undetected.

Research Article

KCs from HFD mice with pure steatosis were enlarged andshowed typical lipids droplets in their cytoplasm. It has beenreported that KCs stored lipids in rabbit models of hypercholes-terolemia and NASH [22,23]. The HFD enriched with 37% of fat,used in our study, induces fat-laden KCs after 16 weeks of dietconversely to a HFD with 21% of fat during 21 days [24]. Whileseveral publications have suggested that there are higher num-bers of KCs in the fatty liver, this has not been clearly established.However, semi-quantitative analysis of KCs by immunohisto-chemistry in a choline-deficient diet inducing hepatosteatosisshowed that the amount of KCs in the liver did not change [25].Our experiments showed that the number of KCs and theirrecruitment were unchanged by lipid accumulation in the liver.

Macrophages are a heterogeneous population of cells, whichhave diverse functions and phenotypes based on their locationand activation state. The inflammatory environment determinesthe macrophage phenotype generating different subsets. TheM1 subset produces pro-inflammatory cytokines and chemokinesand the M2 subset produces low levels of pro-inflammatory cyto-

146 Journal of Hepatology 201

kines and high levels of IL10 [11,26]. The M1/M2 classificationwas often determined by gene expression analysis. As we couldmeasure the production of cytokines and chemokines, we directlyassessed the inflammatory status of KCs. We have shown that fat-laden KCs produced higher level of pro-inflammatory cytokinesand chemokines in basal conditions, and were more sensitive toLPS. Recently, it has been shown that adipose tissue macrophageshad a similar phenotype during the obesity development [18].However, we also detected high levels of IL10 in fat-laden KCsin addition to a typical M1 phenotype. Such an apparent contra-diction may be due to KC heterogeneity; indeed, some regulatorymacrophage subsets expressed high levels of IL10 without theother typical M2-associated cytokines/chemokines [27]. In vivo,LPS challenge induced a significant increase in the recruitmentof CD4+ and CD8+ lymphocytes and a tendency to recruit moreCD19+ lymphocytes. Pro-inflammatory fat-laden KCs recruitedmore CD4+ T and B lymphocytes under an LPS challenge, showingthat KCs efficiently participated in lymphocyte recruitment intothe liver.

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pg/m

l

0100200300400500600

0250050007500

10,00012,500

050

100150200250

0500

1000150020002500

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10,00015,00020,00025,00030,000

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NDHFD

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IFNγ IL6 TNFα IL1β IL10 CXCL10 CCL5 CCL2

LPS +-+- +-+- +-+- +-+- +-+- +-+- +-+- +-+-

TOFA +-+- +-+- +-+- +-+- +-+- +-+- +-+- +-+-

*

**

** *

*

**

*** * * *

**

A B

C D

Fig. 3. KCs from HFD mice produced pro-inflammatory cytokines and chemokines dampered by inhibition of lipogenesis. KCs were cultured from ND or HFD mice. (Aand B) Cytokines and chemokines levels produced by KCs challenged or not with LPS (100 ng/ml). Data represent the mean ± SEM of 14 animals. (C) Cytokines andchemokines levels in the supernatant of KCs challenged or not with TOFA (200 lM). Data represent the mean ± SEM of four mice. Statistical analysis was performed usingthe Mann–Whitney and Kruskal–Wallis tests; ⁄p <0.05, ⁄⁄p <0.001 and ⁄⁄⁄p <0.0001.

JOURNAL OF HEPATOLOGY

Induction of lipogenesis is a key step in the differentiation ofmonocytes into macrophages and suppression of FA synthesisprevents phagocytosis development and function [12]. A FASdeficiency in macrophages decreases foam cell formation anddiet-induced atherosclerosis [28]. On the other hand, TG synthe-sis also helps protect liver cells from lipotoxicity by bufferingthe accumulation of FA [20]. RT-qPCR and lipidomic analysisshowed that lipid homeostasis was dysregulated. DAGs, whichare toxic lipids [19,20], were greatly increased in fat-ladenKCs. Inhibition of TG synthesis in the liver causes DAG accumu-lation, which exacerbates liver damage. Conversely, specificoverexpression of DGAT in macrophages increases their capacityfor TG storage and protects them against inflammatory activa-tion [29]. Here, the accumulation of DAG in KCs from HFD micedid not result from a defect in TG synthesis. The upregulation ofScd1 suggested that the level of unsaturated FAs had increased,which could serve as a protective function against lipotoxicitythrough promotion of TG accumulation [20]. The inhibition ofthe lipogenesis via the inhibition of the ACC by TOFA in fat-laden KCs was able to reverse the pro-inflammatory phenotype.Therefore, upregulation of lipogenesis directly participates inthe pro-inflammatory phenotype of fat-laden KCs. The analysisof ATMs suggests that the dysregulation of lipid metabolismprofoundly disturbs the phenotype of monocytes/macrophagessubsets.

Like TG and DAG, the levels of FC and CE were redistributed inthe fat-laden KCs, which indicated that cholesterol homeostasishad become dysregulated. FC modify membrane fluidity andcan consequently alter phagocytosis, which is known to be dis-turbed in murine models of NASH [30]. The role of cholesterolmetabolism and especially of LDL in foam cell formation and inthe development of atherosclerosis are well described [31]. Mac-

Journal of Hepatology 201

rophages are able to synthesize cholesterol and CE, and to partic-ipate via ABC transporters in cholesterol efflux [32]. Overall, ourprofile of gene expression suggests that the metabolism and traf-ficking of cholesterol and CE were modified in fat-laden KCs andcould be deleterious, as shown by the improvement of athero-sclerotic lesions in mice deficient for CD36 [32].

The increase of some specific ceramides in the livers of obeseob/ob mice has been previously described [33]. However, in ourexperiments, the ceramide levels in obese and control livers weresimilar. Conversely, ceramide levels were significantly higher inthe fat-laden KCs of HFD mice and the relative levels of the cer-amide species had been completely redistributed. Similarly toFC, ceramides are involved in membrane fluidity and phagocyto-sis. Moreover, some ceramide metabolites can activate NKT cells[34]. Liver NKT cells are decreased in NASH and the remainingliver NKT display pro-inflammatory activation [35]. Furthermore,macrophages can present antigens to NKT lymphocytes [36]. Wehypothesize that the NKT pattern in NASH could be directly dueto ceramide dysregulation in fat-laden KC.

In conclusion, we demonstrate that steatosis does not lead toincreased recruitment of KCs into the liver, but induced anincrease of their lipid content, especially toxic lipids. This lipidaccumulation is associated with a disturbance of lipid metabo-lism encompassing lipid synthesis, oxidation, uptake, and secre-tion in KCs. Fat-laden KCs display a pro-inflammatoryphenotype, which is reversible with inhibition of lipogenesis.The pro-inflammatory phenotype of fat-laden KCs induced anin vitro recruitment of lymphocytes under LPS challenge possiblycontributing to the early homing of immune cells into the fattyliver. These observations are consistent with the fact that main-taining a M2 phenotype in KCs improves liver lesions in obesity[37].

2 vol. 57 j 141–149 147

LPS

CD4 CD8 CD19

Num

ber o

f lym

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yte/

liver

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0.6

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Num

ber o

f CD

45/

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(g) x

105

A

C

B

Fig. 4. Elevated lymphocyte recruitment by fat-laden KCs. Quantification byflow cytometry of (A) CD45+ lymphocytes, (B) CD4+ T, CD8+ T, and CD19+ Blymphocytes from ND and HFD mice challenged or not with LPS (5 mg/kg of bodyweight). Data represent the mean ± SEM of at least six mice except for ND micechallenged with LPS (n = 2). (C) KCs from ND or HFD mice were cultured eitherwith or without LPS (100 ng/ml). Splenocytes isolated from a lean mouse werelabeled with antibodies and incubated in the upper transwell chamber. CD4+ T,CD8+ T, and CD19+ B cells were quantified by flow cytometry after 4 h ofincubation. Uncultured medium in the lower chamber with 1.5 � 106 cells in theupper chamber were used as input control. The values represent the ratios of thelymphocytes that migrated in response to LPS-challenged KCs supernatants to thelymphocytes migrated in response to unstimulated KCs supernatants. Datarepresent the mean ± SEM of 14 animals (Mann–Whitney test ⁄p <0.05 and⁄⁄p <0.001).

Research Article

Conflict of interest

The authors who have taken part in this study declared that theydo not have anything to disclose regarding funding or conflict ofinterest with respect to this manuscript.

Financial support

Supported by INSERM AVENIR, Université Paris-Sud, the NationalFrench Society of Gastroenterology (SNFGE), Association Franç-aise pour l’Etude du Foie (AFEF), and National Program on diabe-tes Research (PNR-diabete). AL holds fellowships from conseilrégional d’Ile-de-France and FC holds fellowships from the Min-istère de la recherche.

Acknowledgments

The authors thank Bernadette Besson-Lescure and Nadège Brunelfrom the microassay facility of IFR65; Aurore Desquesnes fromthe ANEXPLO-Phenotypage platform of IFR150; Justine Ber-trand-Michel from the lipidomic facility Core-MetaToul of IN-SERM U1048; Alain Schmitt from the electron microscopyfacility of the Cochin institute; the biochemistry department ofthe Antoine Béclère hospital and Ghyath Maarof for transaminase

148 Journal of Hepatology 201

measurements. We also thank Carina Prip-Buus for helpfuldiscussion.

Supplementary data

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.jhep.2012.02.028.

References

[1] Angulo P. Nonalcoholic fatty liver disease. N Engl J Med2002;346:1221–1231.

[2] Matteoni CA, Younossi ZM, Gramlich T, Boparai N, Liu YC, McCullough AJ.NAFLD: a spectrum of clinical and pathological severity. Gastroenterology1999;116:1413–1419.

[3] Perlemuter G, Bigorgne A, Cassard-Doulcier AM, Naveau S. NAFLD: frompathogenesis to patient care. Nat Clin Pract Endocrinol Metab2007;3:458–469.

[4] Bugianesi E, Marzocchi R, Villanova N, Marchesini G. NAFLD/NASH: treat-ment. Best Pract Res Clin Gastroenterol 2004;18:1105–1116.

[5] Guebre-Xabier M, Yang S, Lin HZ, Schwenk R, Krzych U, Diehl AM. Alteredhepatic lymphocyte subpopulations in obesity-related murine fatty livers:potential mechanism for sensitization to liver damage. Hepatology2000;31:633–640.

[6] Elinav E, Pappo O, Sklair-Levy M, Margalit M, Shibolet O, Gomori M, et al.Adoptive transfer of regulatory NKT lymphocytes ameliorates non-alcoholicsteatohepatitis and glucose intolerance in ob/ob mice and is associated withintrahepatic CD8 trapping. J Pathol 2006;209:121–128.

[7] Ma X, Hua J, Mohamood AR, Hamad AR, Ravi R, Li Z. A high-fat diet andregulatory T cells influence susceptibility to endotoxin-induced liver injury.Hepatology 2007;46:1519–1529.

[8] Bigorgne AE, Bouchet-Delbos L, Naveau S, Dagher I, Prevot S, Durand-Gasselin I, et al. Obesity-induced lymphocyte hyperresponsiveness tochemokines: a new mechanism of Fatty liver inflammation in obese mice.Gastroenterology 2008;134:1459–1469.

[9] Crespo J, Cayon A, Fernandez-Gil P, Hernandez-Guerra M, Mayorga M,Dominguez-Diez A, et al. Gene expression of TNF alpha and TNF-receptors,p55 and p75, in nonalcoholic steatohepatitis patients. Hepatology2001;34:1158–1163.

[10] You Q, Cheng L, Kedl RM, Ju C. Mechanism of T cell tolerance induction bymurine hepatic Kupffer cells. Hepatology 2008;48:978–990.

[11] Mantovani A, Sica A, Sozzani S, Allavena P, Vecchi A, Locati M. Thechemokine system in diverse forms of macrophage activation and polari-zation. Trends Immunol 2004;25:677–686.

[12] Ecker J, Liebisch G, Englmaier M, Grandl M, Robenek H, Schmitz G. Inductionof fatty acid synthesis is a key requirement for phagocytic differentiation ofhuman monocytes. Proc Natl Acad Sci U S A 2010;107:7817–7822.

[13] Besteiro S, Bertrand-Michel J, Lebrun M, Vial H, Dubremetz JF. Lipidomicanalysis of Toxoplasma gondii tachyzoites rhoptries: further insights intothe role of cholesterol. Biochem J 2008;415:87–96.

[14] Lumeng CN, Deyoung SM, Bodzin JL, Saltiel AR. Increased inflammatoryproperties of adipose tissue macrophages recruited during diet-inducedobesity. Diabetes 2007;56:16–23.

[15] Ye D, Hoekstra M, Out R, Meurs I, Kruijt JK, Hildebrand RB, et al. Hepatic cell-specific ABC transporter profiling identifies putative novel candidates forlipid homeostasis in mice. Atherosclerosis 2008;196:650–658.

[16] Mencin A, Kluwe J, Schwabe RF. TLRs as targets in chronic liver diseases. Gut2009;58:704–720.

[17] Miura K, Kodama Y, Inokuchi S, Schnabl B, Aoyama T, Ohnishi H, et al. TLR9promotes steatohepatitis by induction of interleukin-1beta in mice. Gastro-enterology 2010;139:323–334, e7.

[18] Prieur X, Roszer T, Ricote M. Lipotoxicity in macrophages: evidence fromdiseases associated with the metabolic syndrome. Biochim Biophys Acta2011;1801:327–337.

[19] Schenk S, Saberi M, Olefsky JM. Insulin sensitivity: modulation by nutrientsand inflammation. J Clin Invest 2008;118:2992–3002.

[20] Yamaguchi K, Yang L, McCall S, Huang J, Yu XX, Pandey SK, et al. Inhibitingtriglyceride synthesis improves hepatic steatosis but exacerbates liver

2 vol. 57 j 141–149

JOURNAL OF HEPATOLOGY

damage and fibrosis in obese mice with NASH. Hepatology2007;45:1366–1374.

[21] Holland WL, Summers SA. Sphingolipids, insulin resistance, and metabolicdisease: new insights from in vivo manipulation of sphingolipid metabolism.Endocr Rev 2008;29:381–402.

[22] Nenseter MS, Gudmundsen O, Roos N, Maelandsmo G, Drevon CA, Berg T.Role of liver endothelial and Kupffer cells in clearing low density lipopro-tein from blood in hypercholesterolemic rabbits. J Lipid Res 1992;33:867–877.

[23] Otogawa K, Kinoshita K, Fujii H, Sakabe M, Shiga R, Nakatani K, et al.Erythrophagocytosis by liver macrophages (Kupffer cells) promotes oxida-tive stress, inflammation, and fibrosis in a rabbit model of steatohepatitis.Am J Pathol 2007;170:967–980.

[24] Wouters K, van Gorp PJ, Bieghs V, Gijbels MJ, Duimel H, Lutjohann D, et al.Dietary cholesterol, rather than liver steatosis, leads to hepatic inflammationin hyperlipidemic mouse models of NASH. Hepatology 2008;48:474–486.

[25] Kremer M, Thomas E, Milton RJ, Perry AW, van Rooijen N, Wheeler MD, et al.Kupffer cell and interleukin-12-dependent loss of NKT cells in hepatostea-tosis. Hepatology 2010;51:130–141.

[26] Gordon S, Taylor PR. Monocyte and macrophage heterogeneity. Nat RevImmunol 2005;5:953–964.

[27] Mosser DM, Edwards JP. Exploring the full spectrum of macrophageactivation. Nat Rev Immunol 2008;8:958–969.

[28] Schneider JG, Yang Z, Chakravarthy MV, Lodhi IJ, Wei X, Turk J, et al.Macrophage fatty-acid synthase deficiency decreases diet-induced athero-sclerosis. J Biol Chem 2010;285:23398–23409.

Journal of Hepatology 201

[29] Koliwad SK, Streeper RS, Monetti M, Cornelissen I, Chan L, Terayama K, et al.DGAT1-dependent triacylglycerol storage by macrophages protects micefrom diet-induced insulin resistance and inflammation. J Clin Invest2010;120:756–767.

[30] Yang SQ, Lin HZ, Lane MD, Clemens M, Diehl AM. Obesity increasessensitivity to endotoxin liver injury: implications for the pathogenesis ofsteatohepatitis. Proc Natl Acad Sci U S A 1997;94:2557–2562.

[31] Rader DJ, Pure E. Lipoproteins, macrophage function, and atherosclerosis:beyond the foam cell? Cell Metab 2005;1:223–230.

[32] Marcel YL, Ouimet M, Wang MD. Regulation of cholesterol efflux frommacrophages. Curr Opin Lipidol 2008;19:455–461.

[33] Yetukuri L, Katajamaa M, Medina-Gomez G, Seppanen-Laakso T, Vidal-PuigA, Oresic M. Bioinformatics strategies for lipidomics analysis: characteriza-tion of obesity related hepatic steatosis. BMC Syst Biol 2007;1:12.

[34] Bendelac A, Savage PB, Teyton L. The biology of NKT cells. Annu RevImmunol 2007;25:297–336.

[35] Li Z, Soloski MJ, Diehl AM. Dietary factors alter hepatic innate immunesystem in mice with NAFLD. Hepatology 2005;42:880–885.

[36] Barral P, Polzella P, Bruckbauer A, van Rooijen N, Besra GS, Cerundolo V, et al.CD169(+) macrophages present lipid antigens to mediate early activation ofiNKT cells in lymph nodes. Nat Immunol 2010;11:303–312.

[37] Odegaard JI, Ricardo-Gonzalez RR, Red Eagle A, Vats D, Morel CR, GoforthMH, et al. Alternative M2 activation of Kupffer cells by PPARdelta amelio-rates obesity-induced insulin resistance. Cell Metab 2008;7:496–507.

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