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of February 15, 2018. This information is current as after Chronic Ethanol Exposure Signaling in Rat Kupffer Cells and in Mice Suppress TLR4/MyD88-Independent Adiponectin and Heme Oxygenase-1 Brian T. Pratt, Thierry Roger and Laura E. Nagy Palash Mandal, Sanjoy Roychowdhury, Pil-Hoon Park, ol.1002060 http://www.jimmunol.org/content/early/2010/09/22/jimmun published online 22 September 2010 J Immunol Material Supplementary 0.DC1 http://www.jimmunol.org/content/suppl/2010/09/20/jimmunol.100206 average * 4 weeks from acceptance to publication Fast Publication! Every submission reviewed by practicing scientists No Triage! from submission to initial decision Rapid Reviews! 30 days* Submit online. ? The JI Why Subscription http://jimmunol.org/subscription is online at: The Journal of Immunology Information about subscribing to Permissions http://www.aai.org/About/Publications/JI/copyright.html Submit copyright permission requests at: Email Alerts http://jimmunol.org/alerts Receive free email-alerts when new articles cite this article. Sign up at: Print ISSN: 0022-1767 Online ISSN: 1550-6606. All rights reserved. 1451 Rockville Pike, Suite 650, Rockville, MD 20852 The American Association of Immunologists, Inc., is published twice each month by The Journal of Immunology by guest on February 15, 2018 http://www.jimmunol.org/ Downloaded from by guest on February 15, 2018 http://www.jimmunol.org/ Downloaded from

Adiponectin and Heme Oxygenase-1 Suppress TLR4/MyD88

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Page 1: Adiponectin and Heme Oxygenase-1 Suppress TLR4/MyD88

of February 15, 2018.This information is current as

after Chronic Ethanol ExposureSignaling in Rat Kupffer Cells and in MiceSuppress TLR4/MyD88-Independent Adiponectin and Heme Oxygenase-1

Brian T. Pratt, Thierry Roger and Laura E. NagyPalash Mandal, Sanjoy Roychowdhury, Pil-Hoon Park,

ol.1002060http://www.jimmunol.org/content/early/2010/09/22/jimmun

published online 22 September 2010J Immunol 

MaterialSupplementary

0.DC1http://www.jimmunol.org/content/suppl/2010/09/20/jimmunol.100206

        average*  

4 weeks from acceptance to publicationFast Publication! •    

Every submission reviewed by practicing scientistsNo Triage! •    

from submission to initial decisionRapid Reviews! 30 days* •    

Submit online. ?The JIWhy

Subscriptionhttp://jimmunol.org/subscription

is online at: The Journal of ImmunologyInformation about subscribing to

Permissionshttp://www.aai.org/About/Publications/JI/copyright.htmlSubmit copyright permission requests at:

Email Alertshttp://jimmunol.org/alertsReceive free email-alerts when new articles cite this article. Sign up at:

Print ISSN: 0022-1767 Online ISSN: 1550-6606. All rights reserved.1451 Rockville Pike, Suite 650, Rockville, MD 20852The American Association of Immunologists, Inc.,

is published twice each month byThe Journal of Immunology

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The Journal of Immunology

Adiponectin and Heme Oxygenase-1 SuppressTLR4/MyD88-Independent Signaling in Rat Kupffer Cellsand in Mice after Chronic Ethanol Exposure

Palash Mandal,* Sanjoy Roychowdhury,* Pil-Hoon Park,† Brian T. Pratt,*

Thierry Roger,‡,x and Laura E. Nagy*,{

Alcoholic liver disease is mediated via activation of TLR4 signaling; MyD88-dependent and -independent signals are important

contributors to injury in mouse models. Adiponectin, an anti-inflammatory adipokine, suppresses TLR4/MyD88-dependent

responses via induction of heme oxygenase-1 (HO-1). Here we investigated the interactions between chronic ethanol, adiponectin,

and HO-1 in regulation of TLR4/MyD88-independent signaling in macrophages and an in vivo mouse model. After chronic eth-

anol feeding, LPS-stimulated expression of IFN-b and CXCL10 mRNA was increased in primary cultures of Kupffer cells

compared with pair-fed control mice. Treatment of Kupffer cells with globular adiponectin (gAcrp) normalized this response.

LPS-stimulated IFN-b/CXCL10 mRNA and CXCL10 protein was also reduced in RAW 264.7 macrophages treated with gAcrp or

full-length adiponectin. gAcrp and full-length adiponectin acted via adiponectin receptors 1 and 2, respectively. gAcrp decreased

TLR4 expression in both Kupffer cells and RAW 264.7 macrophages. Small interfering RNA knockdown of HO-1 or inhibition of

HO-1 activity with zinc protoporphyrin blocked these effects of gAcrp. C57BL/6 mice were exposed to chronic ethanol feeding,

with or without treatment with cobalt protoporphyrin, to induce HO-1. After chronic ethanol feeding, mice were sensitized to

in vivo challenge with LPS, expressing increased IFN-b/CXCL10 mRNA and CXCL10 protein in liver compared with control

mice. Pretreatment with cobalt protoporphyrin 24 h before LPS challenge normalized this effect of ethanol. Adiponectin and

induction of HO-1 potently suppressed TLR4-dependent/MyD88-independent cytokine expression in primary Kupffer cells from

rats and in mouse liver after chronic ethanol exposure. These data suggest that induction of HO-1 may be a useful therapeutic

strategy in alcoholic liver disease. The Journal of Immunology, 2010, 185: 000–000.

The innate immune system is involved in many stages ofan organism’s response to injury or stress. Componentsof the innate immune response, including NK and NKT

cells (1), Kupffer cells (2), and the complement system (3, 4), areinvolved in the hepatic response to chronic alcohol exposure.Kupffer cells, the resident macrophages in the liver, are particu-larly critical to the onset of ethanol-induced liver injury. Ablationof Kupffer cells prevents the development of fatty liver and in-flammation in rats chronically exposed to ethanol via intragastricfeeding (5). Increased exposure of Kupffer cells to LPS duringchronic ethanol consumption, associated with impaired barrier

function of the intestine (6), results in activation of TLR4 andincreased production of inflammatory mediators (7). Mice defi-cient in TLR4 or TNF-aR1 expression are protected from chronicethanol-induced liver injury (7).TLR4 signaling is mediated via MyD88-dependent and -inde-

pendent pathways (8). Although the rapid LPS-stimulated expres-sion of TNF-a is characteristic of MyD88-dependent signaling,MyD88-independent signals are more slowly activated and resultin increased expression of type 1 IFN and IFN-dependent genes(8). Chronic ethanol exposure sensitizes Kupffer cells to TLR4-MyD88–dependent responses, such as rapid activation of MAPKsand NF-kB, as well as TNF-a expression (2). Treatment of Kup-ffer cells with adiponectin, an abundant 30-kDa adipokine withpotent anti-inflammatory properties (9), normalizes these effectsof chronic ethanol on LPS-induced MAPK and NF-kB activation,as well as TNF-a production in primary cultures of Kupffer cells(10). Treatment of mice with supraphysiologic concentrations ofadiponectin during chronic ethanol exposure prevents the devel-opment of liver injury, decreasing both steatosis and TNF-a ex-pression in the liver (11).Despite the efficacy of adiponectin in decreasing LPS-stimulated

responses in Kupffer cells and preventing liver injury in mice,the development of adiponectin for therapeutic interventions inpatients with alcoholic liver disease (ALD) is likely of limitedutility, because of the high concentration of adiponectin in thecirculation, as well as the complex oligomeric structure of adipo-nectin. Therefore, recent studies have focused on the downstreammolecular targets of adiponectin to identify anti-inflammatorytargets that are more amenable to pharmacologic intervention.We recently identified an IL-10 and heme oxygenase-1 (HO-1)-

*Department of Pathobiology and {Department of Gastroenterology, ClevelandClinic, Cleveland, OH 44195; †College of Pharmacy, Yeungnam University, Gyeong-san, Gyeongsangbuk-do 712-749, South Korea; ‡Centre Hospitalier UniversitaireVaudois; and ‡University of Lausanne, Lausanne, Switzerland

Received for publication June 21, 2010. Accepted for publication August 16, 2010.

This work was supported by National Institutes of Health Grants RO1AA011975,R56AA011975, and P20AA17069 (to L.E.N.) and Swiss National Science Founda-tion Grants 3100-118266.07 and 310000-114073 (to T.R.).

Address correspondence and reprint requests to Dr. Laura E. Nagy at the currentaddress: Cleveland Clinic Foundation, Lerner Research Institute/NE-40, 9500 EuclidAvenue, Cleveland, OH 44195. E-mail address: [email protected]

The online version of this article contains supplemental material.

Abbreviations used in this paper: adipoR, adiponectin receptor; ALD, alcoholic liverdisease; CoPP, cobalt protoporphyrin; CORM2, CO-releasing molecule-2; Ct, com-parative threshold; EtOH, ethanol; flAcrp, full-length adiponectin; gAcrp, globularadiponectin; HO-1, heme oxygenase-1; MD2, lymphocyte Ag 96; overexp, overex-pression; QRT-PCR, quantitative real-time PCR; siRNA, small interfering RNA; TRIF,Toll/IL-1R domain-containing adapter-inducing IFN-b; ZnPP, zinc protoporphyrin.

Copyright� 2010 by The American Association of Immunologists, Inc. 0022-1767/10/$16.00

www.jimmunol.org/cgi/doi/10.4049/jimmunol.1002060

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dependent pathway in Kupffer cells that mediates the anti-inflammatory effects of globular adiponectin (gAcrp) on LPS-stimulated TNF-a expression (12). Similarly, induction of HO-1expression in mice by treatment with cobalt protoporphyrin (CoPP)normalizes TNF-a expression in response to in vivo LPS challengeafter chronic ethanol exposure (12).A critical role for TLR4 has been identified in the progression of

multiple forms of liver disease, including ALD, non-ALD, and viralhepatitis (13). More recently, data have suggested that the MyD88-independent/Toll/IL-1R domain-containing adapter-inducing IFN-b(TRIF)-dependent pathway of TLR4 signaling is also a key con-tributor to chronic ethanol-induced liver injury (14, 15) and acutehepatitis (16) in mice. Little is known about the impact of chronicethanol on the regulation of MyD88-independent signaling. Herewe report that chronic ethanol exposure sensitizes Kupffer cells toLPS-stimulated expression of IFN-b and CXCL10 mRNA, twocritical TLR4–MyD88-independent responses. This sensitizationwas also observed in an in vivo mouse model of LPS challenge afterchronic ethanol exposure. Treatment of Kupffer cells with adipo-nectin decreased these MyD88-independent responses. Importantly,induction of HO-1 was sufficient to alleviate ethanol-induced sen-sitization of MyD88-independent responses in both Kupffer cellsand mice.

Materials and MethodsAnimals

Adult male Wistar rats weighing 140–150 g were purchased from HarlanSprague Dawley (Indianapolis, IN). Female C57BL/6 mice were purchasedfrom The Jackson Laboratory (Bar Harbor, ME). Lieber–DeCarli ethanoldiet (regular, no. 710260) was purchased from Dyets (Bethlehem, PA).

Materials

Recombinant human gAcrp expressed in Escherichia coli was purchasedfrom Peprotech (Rocky Hill, NJ). Recombinant human full-length Acrpexpressed in human embryonic kidney 293 cells were purchased fromBiovendor Research and Diagnostic Products (Candler, NC). Full-lengthTLR4-LUC (containing 22715 to +52 of the TLR4 promoter in a lucif-erase reporter vector) was previously described (17). HO-1 overexpressionplasmid (18) was from Prof. M.P. Soares, Instituto Gulbenkian de Ciencia,Oeiras, Portugal. Cell culture reagents were from Life Technologies-BRL(Grand Island, NY). Abs were from the following sources: total ERK1/2(Upstate Biotechnology, Lake Placid, NY) and phospho-STAT1 (CellSignaling Technology, Danvers, MA), HO-1 monoclonal and HO-1 poly-clonal Abs (Assay Designs, Ann Arbor, MI), Hsc70 (Alpha Diagnostic,San Antonio, TX). Anti-rabbit and anti-mouse IgG-peroxidase Abs werepurchased from Boehringer Mannheim (Indianapolis, IN). LPS fromE. coli serotype 026:B6 (tissue culture-tested, L-2654) was purchasedfrom Sigma (St. Louis, MO); all experiments were conducted with a singlelot of LPS (lot no. 064K4077). Adiponectin preparations contained lessthan 0.2 ng LPS/mg of protein. Mouse RAW 264.7 cell nucleofection kitwas purchased from Lonza (Cologne, Germany). PE-conjugated TLR4-lymphocyte Ag 96 (MD2) Ab and rat anti-mouse IgG1 mAbs andCD32/CD16 Fcg were purchased from eBioscience (San Diego, CA). Co(III) protoporphyrin IX chloride (CoPP) was purchased from FrontierScientific (Logan, UT). Endotoxin-free plasmid preparation kits were fromQiagen (Valencia, CA).

Culture of RAW 264.7 macrophages and luciferase assays

The murine RAW 264.7 macrophage-like cell line was routinely culturedin DMEMwith 10% FBS and penicillin-streptomycin at 37˚C and 5% CO2.For luciferase reporter assays, RAW 264.7 macrophages were cotrans-fected with a luciferase reporter plasmid containing the full-length TLR4promoter (17) and pRenilla-thymidine kinase (Promega, Madison, WI), anexpression vector for Renilla luciferase under the control of the thymidinekinase promoter, as a control for transfection efficiency. After 24 h, me-dium was removed and cells stimulated or not with adiponectin, LPS, orboth, as described in the figure legends, in DMEM/FBS. Cells were thenlysed and luciferase activities measured using the Dual Luciferase assaysystem (Promega).

Chronic ethanol feeding and Kupffer cell isolation

All procedures involving animals were approved by the Institutional AnimalCare and Use Committee at the Cleveland Clinic. Chronic ethanol feeding torats and mice, as well as the isolation and culture of Kupffer cells, wereperformed as previously described (19, 20). In brief, rats were allowed freeaccess to the Lieber–Decarli high-fat complete liquid diet for 2 d. Ratswere then randomly assigned to pair- or ethanol-fed groups. Ethanol-fedrats were allowed free access to a liquid diet containing 17% of the calories(3.3% v/v) from ethanol for 2 d and then a liquid diet containing 35% ofthe calories (6.6% v/v) from ethanol for 4 wk (19). Control rats were pair-fed a liquid diet in which maltose dextrins were substituted isocaloricallyfor ethanol over the entire feeding period. Kupffer cells were isolated andcultured, as previously described (19, 21). In brief, isolated Kupffer cellswere suspended in Connaught Medical Research Laboratories media with10% FBS, L-glutamine, and antibiotic-antimycotic (Connaught MedicalResearch Laboratories–FBS) at a concentration of 2 3 106 cells/ml. Cellsuspensions were immediately plated onto 96-well (0.2 3 106 cells/wellfor analysis of mRNA) or 24-well (1.5 3 106 cells/well for flow cytometryand Western blot analysis) culture plates. One hour after plating the iso-lated Kupffer cells, nonadherent cells were removed by aspiration, andfresh media with or without gAcrp or CoPP were added. After 18 h inculture, cells were treated with or without 100 ng/ml LPS, as indicated inthe figure legends.

Mouse ethanol feeding

Ten- to 12-wk-old female C57BL/6 mice were randomly assigned toethanol- or pair-fed groups. Ethanol-fed mice were allowed free access toa complete Lieber-DeCarli high-fat diet with increasing concentrations ofethanol (5.5% of calories [1% v/v] for 2 d, 11% of calories [2% v/v] for 2 d,22% of calories [4% v/v] for 7 d, 27% of calories [5% v/v] for 7 d, andfinally, 32% of calories [6% v/v] for 7 d) (22). Control mice were pair-feddiets that isocalorically substituted maltose dextrins for ethanol. HO-1expression was induced using the following protocols. At the end of thefeeding protocol, mice were treated with 5 mg/kg CoPP or vehicle (saline)via i.p. injection. Twenty-four hours later, mice were injected i.p. with 0.7mg LPS/g body weight or an equivalent volume of sterile, endotoxin-freesaline (0.09%). Mice were anesthetized, blood was collected into lithiumtubes, and plasma was isolated and stored at 280˚C to terminate the ex-periment. Livers were perfused and excised as previously described (22).Portions of each liver were either fixed in formalin or frozen in optimalcutting temperature compound (Sakura Finetek U.S.A., Torrance, CA) forhistology, preserved in RNA later (Ambion, Austin, TX), and stored at220˚C for RNA isolation, or flash frozen in liquid nitrogen and stored at280˚C until further analysis.

Transfection of RAW 264.7 macrophages

For transfection with DNA plasmids, RAW 264.7 macrophages were grownin six-well plates to 60–70% confluency and then transiently transfectedwith control and expression vectors using SuperFect transfection reagent(for DNA-only transfections) according to the manufacturer’s instructions.Transfected cells were subcultured and seeded at 10.2 3 104/cm2 in 96-well plates.

Nucleofection in RAW 264.7 macrophages

For small interfering RNA (siRNA) knockdown experiments, RAW 264.7cells were transfected using the Amaxa Nucleofector apparatus (Lonza).Transfected cells were seeded at 10.2 3 104/cm2 in 96-well plates and cul-tured for 24 h before experimental manipulation. A nucleofection protocolwas used to transfect RAW264.7macrophages with siRNA. In brief, 23 106

cells were resuspended in 105ml Cell Line Nucleofector Solution V (AmaxaBiosystems, Cologne, Germany) andwere nucleofectedwith 100 nM specificor scrambled siRNA (siRNA sequences are shown later) in the Nucleofectordevice using the D-032 program, according to the instructions of the man-ufacturer. After nucleofection, 500 ml prewarmed DMEM was added to thetransfection mix and cells were transferred to 12-well plates containing 1.5ml prewarmed DMEM per well. After nucleofection, gene expression wasanalyzed at different times, as indicated in the figure legends. Validated Si-lencer Select siRNA predesigned sequences were purchased from Ambion/Applied Biosystems: mouse adiponectin receptor (adipoR)1 sequencessense: 59-GGCUGAAAGACA ACGA CUAtt-39 and antisense: 59-UAG-UCGUUGUCUUUCAGCCag-39, mouse adipoR2 sequences sense: 59-GGCCCAUCAUGCUAUGGAAtt-39 and antisense: 59-UUCCAUAG CAU-GAU GGGCCtg-39, nonspecific siRNA scrambled duplex sense: 59-GCGCGC UUU GUA GGA UUC G-39 and antisense: 59-CGA AUC CUA CAAAGC GCG C-39. Efficiency of knockdown was determined by Western blotanalysis and quantitative real-time PCR (QRT-PCR).

2 ADIPONECTIN SUPPRESSES MyD88-INDEPENDENT SIGNALING

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RNA isolation and QRT-PCR

Total RNA was isolated, reverse transcribed, and QRT-PCR amplificationwas performed. The relative amount of target mRNAwas determined usingthe comparative threshold (Ct) method by normalizing target mRNA Ctvalues to those of 18S or b-actin. RNA was isolated from Kupffer cellsand RAW 264.7 macrophages using the RNeasy Micro Kit (Qiagen), withon-column DNA digestion using the RNase-free DNase set (Qiagen)according to the manufacturer’s instructions. Total RNA (200–300 ng) wasreverse transcribed using the RETROscript kit (Ambion/Applied Bio-systems) with random decamers as primers. RT-PCR amplification wasperformed in a Mx3000p (Stratagene, La Jolla, CA) using SYBR GreenPCR Core Reagents (Applied Biosystems, Warrington, U.K.). All primersused for RT-PCR analysis were synthesized by Integrated DNA Technol-ogies (Coralville, IA). Primer sequences are shown in Supplemental Ta-ble 1. Statistical analysis of RT-PCR data were performed using DCtvalues.

Immunohistochemistry of HO-1

Formalin-fixed paraffin-embedded liver sections (4 mm) were deparaffi-nized in SafeClear II xylene substitute (three times 3 min each; Protocol,Kalamazoo, MI) and hydrated consecutively in 100% (two times), 70%,and 30% ethanol, followed by two washes in PBS to detect HO-1 protein in

mouse liver. Sections were then blocked with 2% BSA (diluted in PBS)containing 0.1% Triton X-100 for 1 h, followed by overnight incubationwith the primary Ab (1:300 dilution) at 4˚C. All sections were then washedin PBS (three times 5 min each), incubated with the fluorochrome-conjugated secondary Ab (Alexa Fluor 488-labeled goat anti-rabbit IgG,1:300) for 2 h in the dark at room temperature, washed again in PBS, andmounted with VECTASHIELD containing DAPI and antifade reagent(Vector Laboratories, Burlingame, CA). Fluorescent images were acquiredusing a Leica confocal microscope (Cologne, Germany). No specificimmunostaining was seen in sections incubated with PBS rather than theprimary Ab.

Western blot analysis

After treatment of RAW 264.7 macrophages with gAcrp or CoPP for 18h, cells were washed by cold PBS and lysed in radio immunoprecipitationassay lysis buffer containing 100 mM sodium orthovanadate. Total cellularextracts (20 mg) were separated in 10% SDS polyacrylamide gel andtransferred onto polyvinylidene difluoride membranes. Membranes werefirst probed with HO-1 Ab, followed by secondary Ab conjugated withHRP, and then visualized by ECL detection reagents (Amersham Bio-sciences). The membranes were then stripped and reprobed with eithertotal ERK1/2 or Hsc70 Ab for loading control. Western blot analysiswas performed using ECL for signal detection. Signal intensities were

FIGURE 1. Effect of adiponectin on LPS-stimulated IFN-b and CXCL10 mRNA expression in macrophages. A and B, Kupffer cells isolated from pair-

and ethanol (EtOH)-fed rats were cultured with 1 mg/ml gAcrp for 18 h. Cells were then stimulated with 100 ng/ml LPS for 4 h. The expression of IFN-b

and CXCL10 mRNAwas normalized to 18S mRNA. Values are expressed relative to Kupffer cells from pair-fed rats not treated with gAcrp. n = 4; pp ,0.05 ethanol-fed compared with pair-fed; +p , 0.05 compared with LPS-stimulated cells not treated with gAcrp. C–F, RAW 264.7 cells were cultured for

18 h in the absence or presence of increasing doses of either gAcrp (0–1 mg/ml) or full-length adiponectin (flAcrp; 0–10 mg/ml). Cells were then stimulated

with 100 ng/ml LPS for 4 h, and expression of IFN-b and CXCL10 mRNAwas normalized to 18S mRNA. n = 4; +p, 0.05 compared with LPS-stimulated

cells not treated with adiponectin. G and H, RAW 264.7 cells were cultured for 18 h in the absence or presence of 10 mg/ml gAcrp or full-length adi-

ponectin. Cells were then stimulated with 100 ng/ml LPS for 8 h, and the concentration of CXCL10 protein accumulated in the media was measured by

ELISA. n = 6; +p , 0.05 compared with LPS-stimulated cells not treated with adiponectin.

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quantified by densitometry using Image J software (National Institutes ofHealth, Bethesda, MD).

Flow cytometry analysis

After 18-h culture with or without gAcrp, RAW 264.7 cells were gentlyscraped and adjusted to 1 million/ml with culture media. Cells were.90%viable as determined by trypan blue exclusion. The cells were centrifugedat 100 3 g for 10 min. The pellet was washed with PBS and resuspendedin 100 ml PBS + 0.1% sodium azide and then blocked with 1.0 mG anti-mouse CD32/CD16 Fcg Block Abs for 15 min at 4˚C. Cells were thenstained with 0.5 mg fluorochrome conjugated TLR4-MD2 (PE-conjugatedTLR4-MD2) or isotype control (PE-conjugated IgG1) diluted in Dulbec-co’s PBS containing 0.1% sodium azide for 30 min. Cells were washedtwice with PBS and resuspended in 0.5 ml wash buffer (final concentration∼106 cells in 0.5 ml) and kept on ice until flow cytometric measurementswere performed on a FACScan flow cytometer (Becton Dickinson Im-munocytometry Systems, Mountain View, CA). Data were acquired andprocessed using FlowJo software (Becton Dickinson).

ELISA assays

Cell culture supernatants from RAW 264.7 macrophages were collectedafter treatment with adiponectin and LPS, and CXCL10 concentrationmeasured by ELISA according to the manufacturer’s instructions (R&D,Minneapolis, MN). The concentration of CXCL10 in mouse plasma andliver lysates was also assayed by ELISA.

Statistical analysis

Because of the limited number of Kupffer cells available from each animal,data from several feeding trials are presented in this study. All values arereported as means 6 SEM. Data were analyzed by general linear modelsprocedure (SAS Institute, Carey, NC). Data were log transformed, ifneeded, to obtain a normal distribution. Follow-up comparisons were madeby least square means testing.

ResultsAdiponectin alleviates the sensitization of Kupffer cells toTLR4–MyD88-independent cytokine expression after chronicethanol

Chronic ethanol exposure sensitizes Kupffer cells to activation ofTLR4/MyD88-dependent signaling by LPS, resulting in enhanced

activation of MAPKs and NF-kB, and increased expression ofTNF-a (2). Treatment of Kupffer cells with gAcrp normalizesthese MyD88-dependent responses (10). Because recent studieshave identified MyD88-independent/TRIF-dependent pathwaysas critical for the development of ethanol-induced liver injury inmice (14, 15), we investigated the interactions between chronicethanol and gAcrp on the sensitivity of MyD88-independent sig-nals in Kupffer cells. Expression of IFN-b and CXCL10 mRNA,considered a molecular signature for TRIF signaling, was in-creased in response to a 4-h challenge with LPS in Kupffer cellsfrom both pair- and ethanol-fed rats. However, expression was 2-fold greater in Kupffer cells from ethanol-fed rats (Fig. 1A, 1B).Treatment of Kupffer cells with gAcrp completely prevented LPS-stimulated IFN-b and CXCL10 mRNA expression (Fig. 1A, 1B).

adipoRs and suppression of MyD88-independent responses inRAW 264.7 macrophages

In RAW 264.7 macrophages, LPS increased expression of IFN-band CXCL10 mRNA expression; both messages reached a maxi-mal response at 4 h (Supplemental Fig. 1). LPS-induced increasesin IFN-b and CXCL10 were dose-dependently decreased bygAcrp at concentrations from 0.1–1 mg/ml (Fig. 1C, 1D). Treat-ment of RAW 264.7 macrophages with 1–10 mg/ml full-lengthadiponectin also decreased LPS-induced IFN-b and CXCL10mRNA expression, but only at greater concentrations (Fig. 1E,1F). A similar suppression of CXCL10 expression was reported inhuman macrophages in response to 10 mg/ml full-length adipo-nectin (23). Pretreatment of RAW 264.7 macrophages with gAcrpand full-length adiponectin also suppressed LPS-stimulated ac-cumulation of CXCL10 protein in the cell culture media after 8 h(Fig. 1G, 1H).Adiponectin signals primarily via two receptors, adipoR1 and

adipoR2 (24). RAW 264.7 macrophages were transfected or notwith siRNA against adipoR1, adipoR2, or scrambled siRNA toidentify the adipoR used by gAcrp and full-length adiponectin in

FIGURE 2. adipoR1 and adipoR2 mediated the in-

hibitory effects of gAcrp and full-length adiponectin

(flAcrp), respectively, on LPS-induced IFN-b and

CXCL10 mRNA expression in RAW 264.7 macro-

phages. RAW 264.7 cells were transfected or not with

100 nM adipoR1 siRNA, adipoR2 siRNA, or scram-

bled siRNA, and then cultured with or without 1 mg/ml

gAcrp or 10 mg/ml flAcrp for 18 h. Cells were then

stimulated with LPS for 4 h. IFN-b and CXCL10

mRNA was normalized to 18S mRNA. n = 4. Values

with different superscripts (a, b, c) are significantly

different from each other; p , 0.05.

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macrophages. Transfection with specific siRNAs effectively de-creased expression of adipoR1 and adipoR2 (Supplemental Fig.2A, 2B). In cells not transfected with siRNA or transfected witha scrambled siRNA control, both gAcrp and full-length adipo-nectin suppressed LPS-stimulated IFN-b and CXCL10 mRNAexpression (Fig. 2). siRNA knockdown of adipoR1 prevented theeffects of gAcrp, but had no effect on the response to full-lengthadiponectin. In contrast, siRNA knockdown of adipoR2 preventedonly the response to full-length adiponectin (Fig. 2). Therefore,gAcrp and full-length adiponectin suppressed TLR4-stimulatedIFN-b and CXCL10 expression via adipoR1 and adipoR2, re-spectively, in line with the observation that gAcrp has a relativelyhigher affinity for adipoR1, and full-length adiponectin hasa higher affinity for adipoR2 (24).

gAcrp decreases expression of TLR4 in Kupffer cells and RAW264.7 macrophages

Because adiponectin suppressed both TLR4–MyD88-dependent(10) and MyD88-independent/TRIF-dependent responses, we hy-pothesized that the anti-inflammatory effects of gAcrp may bemediated via decreased expression of TLR4 or CD14, componentsof the LPS-receptor complex required for LPS sensing by innateimmune cells. TLR4 and CD14 mRNA expression was increasedin Kupffer cells after ethanol feeding (Fig. 3A, 3B) (25). Treatmentof Kupffer cells with increasing concentrations of gAcrp dose-dependently decreased TLR4 mRNA (Fig. 3A), with no effect

on CD14 mRNA (Fig. 3B). Kupffer cells from ethanol-fed ratswere more sensitive to the effects of gAcrp on TLR4 mRNA(Fig. 3A).Using a luciferase reporter construct driven by the full-length

TLR4 promoter, we assessed the effect of gAcrp on the tran-scription of TLR4 in RAW 264.7 macrophages. Treatment of RAW264.7 macrophages with gAcrp decreased TLR4 promoter activityby ∼45–55% (Fig. 3C). gAcrp decreased the expression of TLR4mRNA by 60% (Fig. 3D) and had no effect on the mRNA stabilityof TLR4 (Supplemental Fig. 3). This decrease in TLR4 mRNAwas associated with decreased surface expression of TLR4/MD2complex, measured by flow cytometry (66 6 14%; n = 4; p ,0.03) (Fig. 3E).

HO-1 is necessary and sufficient to decrease TLR4 expressionand MyD88-independent responses in RAW 264.7macrophages

In primary Kupffer cells from rats, the anti-inflammatory effectsof gAcrp on LPS-stimulated TNF-a expression are mediatedvia a HO-1–dependent pathway (12). Therefore, we investigatedwhether HO-1 was involved in gAcrp-mediated decrease in TLR4expression and reduction in LPS-stimulated IFN-b and CXCL10.gAcrp increased the expression of HO-1 mRNA and protein inRAW 264.7 macrophages (Fig. 4A, 4B). When HO-1 was over-expressed in RAW 264.7 macrophages, at expression levelscomparable with cells treated with gAcrp (Supplemental Fig. 4),

FIGURE 3. Adiponectin suppressed TLR4 expression in Kupffer cells and RAW 264.7 macrophages. A and B, Kupffer cells from pair- and ethanol

(EtOH)-fed rats were isolated and cultured overnight with 0–1000 ng/ml gAcrp for 18 h. Expression of TLR4 and CD14 mRNA was normalized to 18S

mRNA. Values are expressed as fold increases relative to Kupffer cells from pair-fed rats not treated with gAcrp. n = 4; pp , 0.05 ethanol-fed compared

with pair-fed; +p , 0.05 compared with cells not treated with gAcrp. C, TLR4 promoter activity was measured in RAW 264.7 macrophages transiently

transfected with a full-length TLR4 promoter-luciferase reporter construct and a Renilla luciferase control plasmid. After 24 h, cells were treated with or

without 1 mg/ml gAcrp for 18 h. Values represent relative luciferase activity. n = 4; + p , 0.05. D, RAW 264.7 macrophages were treated without or with

1 mg/ml gAcrp for 18 h, and expression of TLR4 mRNA measured and normalized to 18S mRNA. n = 4; +p , 0.05 compared with cells not treated with

adiponectin. E, RAW 264.7 macrophages were cultured with or without 1 mg/ml gAcrp for 18 h, and cell surface expression of TLR4-MD2 (solid line),

relative to isotype controls (dotted line), was measured by flow cytometry. Data are representative of four experiments.

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TLR4 mRNA expression was reduced by ∼50% (Fig. 4C).Transfection with the empty vector had no effect on TLR4 mRNAexpression (Fig. 4C). Further, overexpression of HO-1 alleviatedLPS-stimulated IFN-b and CXCL10 mRNA to a similar extent astreatment with gAcrp (Fig. 4D, 4E).In macrophages, TLR4/MyD88-independent signals lead to in-

creased IFN-b expression; IFN-b then activates IFN-induciblegene transcription, primarily via activation of the transcriptionfactor STAT1 (26). LPS increased STAT1 phosphorylation at 1–2 h in RAW 264.7 macrophages (data not shown), consistent withthe time courses for IFN-b and CXCL10 mRNA expression (Sup-plementary Fig. 1). LPS-activated STAT1 phosphorylation wascompletely blocked by treatment with gAcrp or overexpression ofHO-1 (Fig. 4F).

HO-1 mediates the inhibitory effects of gAcrp on LPS-stimulated MyD88-independent pathway

RAW 264.7 macrophages were transfected with siRNA againstHO-1 or scrambled siRNA (Supplemental Fig. 4C). When HO-1expression was knocked down, the anti-inflammatory effects ofgAcrp on LPS-stimulated IFN-b and CXCL10 were attenuated(Fig. 5A, 5B). Similarly, if the activity of HO-1 was inhibited bytreating RAW 264.7 macrophages with zinc protoporphyrin, in-hibition of LPS-stimulated IFN-b and CXCL10 mRNA expressionwas reduced (Fig. 5C, 5D). Taken together, these data demonstratethat increased expression of HO-1 is necessary and sufficient toreduce TLR4 expression, as well as TRIF-dependent signaling, inresponse to gAcrp.

Induction of HO-1 decreases LPS-stimulated IFN-b andCXCL10 expression in macrophages

We next tested the ability of two different small molecules to mimicthe anti-inflammatory effects of gAcrp or overexpression of HO-1in macrophages. Treatment of cells with the heme analog, CoPP,induced the expression of HO-1 in RAW 264.7 macrophages(Supplemental Fig. 4B). When RAW 264.7 macrophages werepretreated with CoPP for 18 h, LPS-stimulated expression of IFN-b and CXCL10 mRNA was suppressed to a similar extent as incells pretreated with gAcrp (Fig. 6A, 6B). The anti-inflammatoryeffects of HO-1 are primarily mediated by its enzymatic products,CO and/or biliverdin/bilirubin (27). Pretreatment of RAW 264.7macrophages with the CO-releasing molecule-2 for 4 h beforechallenge with LPS was as effective as CoPP or gAcrp in alle-viating the effects of LPS on IFN-b and CXCL10 expression(Fig. 6A, 6B). Pretreatment of Kupffer cells with CoPP was equallyeffective at suppressing TLR4-stimulated IFN-b and CXCL10mRNA expression (Fig. 6C, 6D).

Treatment of mice with CoPP reduced chronic ethanol-inducedsensitization to TLR4-MyD88–independent responses in mouseliver

Because CoPP effectively prevented TLR4–MyD88-independentresponses in isolated Kupffer cells, we next asked whether chronicethanol feeding would sensitize mice to LPS-stimulated IFN-band CXCL10 expression, and whether treatment of mice withCoPP could alleviate this effect of chronic ethanol. C57BL/6 micewere allowed free access to an ethanol-containing liquid diet for

FIGURE 4. HO-1 expression and suppression of TLR4-mediated MyD88-independent signaling in RAW 264.7 macrophages. A and B, RAW 264.7

macrophages were treated with or without 1 mg/ml gAcrp for 18 h. HO-1 mRNA expression was normalized to 18S mRNA. B, HO-1 protein expression was

measured by Western blot and normalized to total ERK1/2. n = 4; pp , 0.05 compared with cells not treated with adiponectin. C–F, RAW 264.7 mac-

rophages were transfected with an empty vector or an HO-1 overexpression (overexp) plasmid. Twenty-four hours after transfection, cells were treated or

not with 1 mg/ml gAcrp for 18 h. C, TLR4 mRNAwas measured and normalized to 18S mRNA. n = 4; +p, 0.05 compared with nontreated cells. D and E,

Transfected cells were then stimulated with LPS for 4 h. IFN-b (D) and CXCL10 (E) mRNAwere measured normalized to 18S mRNA. n = 4; +p , 0.05

compared with LPS-stimulated cells not treated with gAcrp or HO-1 overexpression. F, Transfected cells were then stimulated with LPS for 2 h, and

Western blot analysis was performed using Abs to phosphorylated STAT1 and Hsc70 (loading control). Images are representative of four experiments.

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28 d, at a final concentration of 6% ethanol (v/v), equivalent to32% of total calories, or pair-fed control diets. Mice were thentreated with a single injection of CoPP or vehicle. Twenty-fourhours after the CoPP treatment, mice were challenged with LPS orsaline. Body weights and food intake for all experimental groups

are shown in Supplemental Table 2. Expression of HO-1 in liverwas low in both pair- and ethanol-fed mice at baseline (Fig. 7A).Although challenge with LPS modestly increased expression ofHO-1 in both treatment groups, treatment with CoPP robustlyincreased expression of HO-1 in livers from both pair- and

FIGURE 5. HO-1 mediates the inhibitory

effects of gAcrp on LPS-stimulated MyD88-

independent pathway. A, RAW 264.7 cells were

transfected or not with 100 nM HO-1 siRNA or

scrambled siRNA and then cultured with or

without 1 mg/ml gAcrp for 18 h. Cells were then

stimulated with LPS for 4 h. IFN-b and CXCL10

mRNA were normalized to 18S mRNA. n = 4.

Values with different superscripts (a, b, c) are

significantly different from each other; p , 0.05.

B, RAW264.7 cells were cultured with or without

0.5 mM zinc protoporphyrin (ZnPP) in the pres-

ence and absence of 1 mg/ml gAcrp for 18 h. Cells

were then stimulated with LPS for 4 h. IFN-b and

CXCL10 mRNA was normalized to 18S mRNA.

n = 4. Values with different superscripts (a, b, c)

are significantly different from each other, p ,0.05.

FIGURE 6. Induction of HO-1 decreased LPS-

stimulated IFN-b and CXCL10 expression in RAW

264.7 macrophages and Kupffer cells. A and B, RAW

264.7 cells were cultured for 18 h in the absence or

presence of either gAcrp (1 mg/ml), CoPP (10 mM), or

CO-releasing molecule-2 (CORM2; 100 mM). Cells

were then stimulated without or with 100 ng/ml LPS

for 4 h, and expression of IFN-b and CXCL10 mRNA

normalized to 18S mRNA. n = 4; +p , 0.05 compared

with LPS-stimulated cells not treated with inducers of

HO-1. C and D, Kupffer cells isolated from pair- and

ethanol (EtOH)-fed rats were cultured with 10 mM

CoPP for 18 h. Cells were then stimulated with 100 ng/

ml LPS for 4 h. Expression of IFN-b and CXCL10

mRNA was normalized to 18S mRNA. Values are

expressed relative to Kupffer cells from pair-fed rats

not treated with gAcrp. n = 4; pp , 0.05 ethanol-fed

compared with pair-fed; +p , 0.05 compared with

LPS-stimulated cells not treated with CoPP.

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ethanol-fed mice, in a predominantly sinusoidal distribution (Fig.7A). LPS challenge increased expression of IFN-b and CXCL10mRNA in both pair- and ethanol-fed mice at 4 h; this response wasgreater after chronic ethanol feeding (Fig. 7B, 7C). Similarly, theLPS-stimulated increase in CXCL10 protein in plasma (Fig. 7D)and liver lysates (Fig. 7E) was increased after chronic ethanol.Importantly, pretreatment with CoPP reduced the sensitivity ofboth pair- and ethanol-fed mice to LPS-stimulated responses(Figs. 7B–D, 8).

DiscussionThe development of ALD is a complex process involving both theparenchymal and nonparenchymal cells in the liver. Enhancedinflammation in the liver during ethanol exposure is associatedwith liver injury; activation of TLR4 by LPS and signalingvia MyD88-dependent and -independent pathways contribute tochronic ethanol-induced liver injury (7, 14, 15). Increased acti-vation of TLR4 by LPS during chronic ethanol exposure is due toincreased exposure to gut-derived endotoxins, as well as a sensi-tization of macrophages to stimulation by LPS (2). Here we reportthat, in addition to the sensitization of MyD88-dependent signal-ing reported in the past (2), chronic ethanol also enhances TLR4signaling via MyD88-independent pathways, both in isolatedKupffer cells and in an in vivo mouse model of chronic ethanolexposure. Chronic ethanol exposure increased LPS-stimulatedIFN-b mRNA expression, as well as the IFN-inducible gene,CXCL10; expression of these cytokines is a signature for TLR4–TRIF-dependent signaling. Moreover, here we have identified anadiponectin/HO-1 pathway that attenuates TLR4 signaling viaboth MyD88-dependent and -independent pathways (summarizedin Fig. 8). Induction of HO-1 after chronic ethanol feeding to micecountered this sensitization, suppressing LPS-stimulated IFN-b/CXCL10 expression in liver within 24 h.Recent studies have explored the potential utility of adiponectin

as an anti-inflammatory agent to treat ALD (11). Importantly, wefind that adiponectin treatment normalized LPS-induced TNF-aproduction (10, 12), as well as IFN-b and CXCL10 mRNA ex-pression (Fig. 1), in primary cultures of Kupffer cells after chronicethanol exposure. Interestingly, we found that both globular andfull-length adiponectin suppressed LPS-stimulated expression ofIFN-b and CXCL10 mRNA. The response to gAcrp was mediatedby adipoR1, whereas the response to full-length adiponectin wasdependent on adipoR2 (Fig. 2). These data are consistent with thedifferential affinities of gAcrp and full-length adiponectin for

FIGURE 7. Chronic ethanol (EtOH) feeding sensitizes mice to an

in vivo challenge with LPS. Induction of HO-1 rapidly normalizes this

effect of ethanol. Female C57BL/6 mice were allowed free access to in-

creasing concentrations of ethanol as part of a complete liquid diet to

a maximum concentration of 6% v/v (32% of kcal) over 25 d or pair-fed

control diets. Mice were then injected with 5 mg/kg CoPP or vehicle

(saline). After 24 h, mice were challenged with 0.7 mg/kg LPS or saline. A,

HO-1–positive cells were detected by immunohistochemistry in paraffin-

embedded liver sections as detailed in the Materials and Methods section.

Original magnification 340. Data are representative of four pair-fed mice

and six ethanol-fed mice. B and C, Expression of IFN-b and CXCL10

mRNA was measured after 4 h and normalized to 18S mRNA. D and E,

CXCL10 protein in plasma and liver lysates was measured by ELISA. n =

3–4; pp , 0.05 compared with pair-fed mice; +p , 0.05 compared with

LPS-treated mice without CoPP treatment.

FIGURE 8. Model illustrating the role of HO-1 in the anti-inflammatory

effect of gAcrp. After chronic ethanol feeding, TLR4 signaling via

MyD88-dependent (10) and -independent pathways is enhanced. Induction

of HO-1, either by treatment with gAcrp or small molecule therapeutics,

suppresses TLR4-mediated signaling via both MyD88 (10) and TRIF

pathways.

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adipoR1 and adipoR2, respectively (24). Studies investigating thespecific downstream signaling events activated by adipoR1/R2 inmacrophages are currently under investigation.Chronic ethanol feeding increased expression of TLR4mRNA in

Kupffer cells isolated from rats (Fig. 3A) and in mouse liver afterchronic ethanol exposure (28). In cellular models, changes in theexpression of TLR4 mRNA influence TLR4-dependent responses(29). Therefore, increased expression of TLR4 likely contributesto ethanol-induced increases in TLR4–MyD88-dependent and-independent signaling in the liver. Treatment of macrophageswith gAcrp or CoPP to induce HO-1 reduced expression of TLR4mRNA and protein, likely via a decrease in TLR4 transcription.We show that induction of HO-1 was sufficient to suppress

TLR4–TRIF-dependent responses in macrophages and in miceafter chronic ethanol exposure. Induction of HO-1 is also effect-ive at the prevention of steatohepatitis induced by feeding amethylcholine-deficient diet, a model of nonalcoholic steatohe-patitis (30), and protects against liver ischemia-reperfusion injury(31). However, it is not yet clear which cell(s) in the liver con-tribute to these cytoprotective effects of HO-1. In our studies,HO-1 expression is primarily sinusoidal, consistent with previousreports that HO-1 is typically expressed in Kupffer cells in theliver (32). However, in methylcholine-deficient diet-fed mice,HO-1 was observed in fatty hepatocytes (30). Similarly, whereasinduction of HO-1 dampens TLR4–TRIF-dependent responses inKupffer cells and RAW 264.7 macrophages (Fig. 1) (31), as wellas in mice after chronic ethanol exposure (Fig. 7), other studiesfind that HO-1 upregulates IFN-b production in some diseasemodels (33, 34). Taken together, these studies suggest that thereare complex cell–cell interactions involving HO-1 within the liverthat contribute to the protective effect of HO-1 and its enzymaticproducts in the liver in response to different insults.Chronic ethanol-induced sensitization to the TLR4–TRIF-

dependent pathway, associated with increased expression ofTLR4 mRNA (28), likely contributes to ethanol-induced liverinjury. TRIF-dependent signaling is critical to mediating the in-nate immune response to LPS, as the expression of TLR4 targetgenes is mediated primarily through MyD88-independent, ratherthan MyD88-dependent pathways (35). Moreover, recent studieshave identified MyD88-independent/TRIF-dependent signaling asan essential element in chronic ethanol-induced liver injury inmice (14, 15). Here we find that chronic ethanol exposure sen-sitized mice to LPS-stimulated activation of typical MyD88-independent/TRIF-dependent responses, including expression ofIFN-b mRNA and the IFN-inducible CXCL10 mRNA in liver, aswell as CXCL10 protein in plasma and liver lysates. However,the specific contributions of type 1 IFNs to the pathophysiologicprogression of ALD have not been well characterized. The pro-tective effects of CoPP treatment in suppressing signature cyto-kines in both the MyD88 (12) and TRIF pathways (Fig. 8) inKupffer cells and in mice after chronic ethanol exposure suggeststhat CoPP would likely be effective in preventing ethanol-inducedliver injury, independent of the relative contributions of MyD88-dependent and -independent pathways in the progression ofethanol-induced liver injury.The controlled and appropriate resolution of inflammation is an

essential feature of the innate immune response; failure to ter-minate an inflammatory response likely contributes to a number ofchronic inflammatory diseases, including ALD (36). Importantly,the resolution of inflammation is an active, highly coordinatedresponse, with inflammatory signals initiating the induction ofnegative regulators (37). During ethanol exposure, despite highexpression of inflammatory mediators, there appears to be ageneralized failure in the resolution of inflammation. Here we

have identified an adiponectin/HO-1–mediated anti-inflammatorypathway that, when activated, can effectively dampen the effectsof chronic ethanol on TLR4-mediated signaling, resulting in de-creased activation of MyD88-mediated signaling (10, 12), as wellas MyD88-independent signaling. Induction of HO-1, either inKupffer cells isolated from rats after chronic ethanol feeding or inan in vivo mouse model of chronic ethanol exposure, suppressedLPS-stimulated expression of IFN-b and CXCL10 mRNA ex-pression. In summary, these data suggest that strategies, such asthe induction of HO-1 identified in this study, aimed at decreasingTLR4 expression and/or TLR4-mediated signaling via both theMyD88-dependent and -independent pathways will likely be use-ful in treatment or prevention, or both, of ethanol-induced liverinjury, as well as other types of liver injury, such as nonalcoholicsteatohepatitis and ischemia-reperfusion injury (30, 31).

AcknowledgmentsWe thank M.P. Soares for the hemeoxygenase-1 overexpression plasmid.

We also thank Drs. A. Stavitsky and M.T. Pritchard for useful discussions

during the course of this study and critical reading of the manuscript.

DisclosuresThe authors have no financial conflicts of interest.

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