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METABOL I SM

1State Key Laboratory of Pharmaceutical Biotechnology, University of Hong Kong,Hong Kong 999077, China. 2Department of Pharmacology and Pharmacy, Universityof Hong Kong, Hong Kong 999077, China. 3Department of Medicine, Li Ka Shing Fac-ulty of Medicine, University of Hong Kong, Hong Kong 999077, China. 4Department ofEndocrinology, Second Hospital of Shanxi Medical University, Taiyuan 030001, China.5Division of Vascular Surgery, Department of Cardiothoracic and Vascular Surgery, Uni-versity Medical Center Mainz, Mainz 55131, Germany.*These authors contributed equally to this work.†Corresponding author. Email: cwhwoo@hku.hk (C.W.W.); amxu@hku.hk (A.X.)

Chen et al., Sci. Signal. 10, eaal3336 (2017) 8 August 2017

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TRIF-dependent Toll-like receptor signaling suppressesScd1 transcription in hepatocytes and preventsdiet-induced hepatic steatosisJing Chen,1,2* Jin Li,1,3,4* Jensen H. C. Yiu,1,2 Jenny K. W. Lam,2 Chi-Ming Wong,1,3

Bernhard Dorweiler,5 Aimin Xu,1,2,3† Connie W. Woo1,2†

Nonalcoholic fatty liver disease (NAFLD) includes a spectrum of diseases that ranges in severity from hepaticsteatosis to steatohepatitis, the latter of which is a major predisposing factor for liver cirrhosis and cancer. Toll-likereceptor (TLR) signaling, which is critical for innate immunity, is generally believed to aggravate disease progres-sion by inducing inflammation. Unexpectedly, we found that deficiency in TIR domain–containing adaptor-inducinginterferon-b (TRIF), a cytosolic adaptor that transduces some TLR signals, worsened hepatic steatosis induced by ahigh-fat diet (HFD) and that such exacerbation was independent of myeloid cells. The aggravated steatosis in Trif −/−

mice was due to the increased hepatocyte transcription of the gene encoding stearoyl–coenzyme A (CoA) desaturase1 (SCD1), the rate-limiting enzyme for lipogenesis. Activation of the TRIF pathway by polyinosinic:polycytidylic acid[poly(I:C)] suppressed the increase in SCD1 abundance induced by palmitic acid or an HFD and subsequently preventedlipid accumulation in hepatocytes. Interferon regulatory factor 3 (IRF3), a transcriptional regulator downstream of TRIF,acted as a transcriptional suppressor by directly binding to the Scd1 promoter. These results suggest an unconventionalmetabolic function for TLR/TRIF signaling that should be taken into consideration when seeking to pharmacologicallyinhibit this pathway.

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INTRODUCTIONNonalcoholic fatty liver disease (NAFLD) is a spectrum of diseasesthat ranges from hepatic steatosis to steatohepatitis. The major char-acteristic of NAFLD is excessive hepatic lipid accumulation, and withthe presence of inflammation, steatohepatitis predisposes patients toliver cancer and cirrhosis. In recent years, the increased prevalence ofobesity and diabetes has led to the concern in the NAFLD epidemic (1).Toll-like receptor (TLR) pathway is a key part of the innate immuneresponse that is associated with various metabolic diseases (2). It isgenerally believed that activation of the TLR pathway in immune cellsis the source of local and systemic inflammatory molecules that aggra-vate the disease progression from simple steatosis to steatohepatitis(2–6). Moreover, TLR pathway can regulate various proteins involvedin metabolism in immune cells such as macrophages and dendriticcells during activation and differentiation (7–9). Different TLRs aredetected in metabolic cells including hepatocytes, but whether TLRpathway also has metabolic functions in these cells remains to beinvestigated.

The signals of most TLRs are mediated by two key cytosolic adap-tors: TIR domain–containing adaptor-inducing interferon-b (TRIF) andmyeloid differentiation primary response 88 (MyD88). MyD88 trans-duces signaling mediated by all TLRs except TLR3, and TRIF is a cyto-solic adaptor for only TLR3 and TLR4. Unlike TLR3, which can directlybind to TRIF, TLR4 requires another adaptor, TRIF-related adaptor mol-ecule (TRAM), to activate the TRIF-dependent signal. Many studies havedemonstrated the pathological role of TLR4 and MyD88 in meta-

bolic diseases; however, the effects of TLR3 and TRIF are inconclusive(5, 10–13). Activation of TRIF by low-dose lipopolysaccharides (LPS)prevents chemical endoplasmic reticulum (ER) stressor–induced he-patic steatosis (12). TRIF can also restore protein translation and delayapoptosis by activating eukaryotic initiation factor 2B (14). Interferonregulatory factor 3 (IRF3), a downstream transcription factor of TRIFsignaling, can decrease the abundance of retinoid X receptor–a (RXR-a),which controls the transcription of various metabolism-related genesupon challenge with vesicular stomatitis virus (VSV), LPS (a TLR4 ligand),or polyinosinic:polycytidylic acid [poly(I:C)] (a TLR3 ligand) in macro-phages (15). These studies imply that TLR/TRIF pathway may play adistinct role in metabolic pathways and diseases (16).

Here, we investigated the function of TRIF pathway in the devel-opment of diet-induced hepatic steatosis. Our results showed that Trif −/−

mice under high-fat diet (HFD) exhibited more severe hepatic steatosiscompared with wild-type mice, and the TRIF deficiency–induced aggra-vation was independent of myeloid cells. Activation of TRIF inhibitedlipid accumulation in hepatocytes by suppressing the expression of thegene encoding the rate-limiting enzyme of lipogenesis, namely, stearoyl–coenzyme A (CoA) desaturase 1 (SCD1), and such inhibition was fa-cilitated by IRF3, which acted as a transcriptional suppressor of Scd1.

RESULTSTRIF deficiency caused aggravation of HFD-induced hepaticsteatosis in mice, which was independent of myeloid cellsSix-week-old male wild-type and Trif −/− mice in C57BL/6J backgroundfed a normal chow diet (NCD) or a 60% fat kcal HFD for 10 weeks didnot show differences in body weight or fat mass (fig. S1, A and B). He-matoxylin and eosin (H&E) and Oil Red O staining showed more lipidvacuoles in the livers of Trif −/− than wild-type mice on an HFD (Fig. 1,A and B). Quantification of extracted hepatic lipids revealed that bothtriglyceride and total cholesterol were significantly higher in the Trif −/−

mice compared with the wild-type control (Fig. 1C). Furthermore, Trif −/−

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mice had significantly greater serum aspartate transaminase (AST)and alanine transaminase (ALT) activities and fasting blood glucose(Fig. 1D and fig. S1C). On an NCD, Trif −/− mice displayed AST andALT activities, hepatic triglyceride and cholesterol content, and fastingblood glucose similar to those in wild-type mice (Fig. 1, A to D, andfig S1C). However, these NCD-fed Trif −/− mice had lower glucose tol-erance, an impairment that was further aggravated by HFD feeding(fig. S1D). The effects of TLR pathway on metabolic dysfunction aremediated by immune cells such as macrophages (5, 17). We used bone

Chen et al., Sci. Signal. 10, eaal3336 (2017) 8 August 2017

marrow transplantation to find out whether the observed phenotypeswere mediated by myeloid cells (5). Irradiated wild-type and Trif −/−

mice received bone marrow isolated from either wild-type or Trif −/−

donors before being fed an HFD. Successful transplantation was con-firmed by DNA sequencing to detect the presence or absence of gua-nine at codon 708 of Trif in the bone marrow of recipients at the endof HFD feeding. Similar to previous data, mice undergoing bone mar-row transplantation had lower body weights and fat mass without sig-nificant differences between the two genotypes (fig. S1, E to F) (18). The

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Trif −/−mice that received either wild-typeor Trif −/− bone marrow had increased he-patic triglyceride and cholesterol content,degree of steatosis, serum AST and ALTactivities, and fasting blood glucose com-pared with the corresponding wild-typerecipients (Fig. 1, E to H, and fig. S1G).

Inflammation aggravates metabolic dys-function. Nevertheless, in spite of the wors-ened hepatic steatosis, hepatic inflammationwas decreased in a nonstatistically signif-icant manner in Trif −/− mice comparedwith the wild-type littermates (fig. S2A).However, serum insulin, triglyceride andcholesterol, and low-density lipoproteinand high-density lipoprotein did not differbetween HFD-fed wild-type and Trif −/−

mice (fig. S2, B to D). Together, these re-sults suggest that deficiency in TRIF innonmyeloid cells worsened the degreeof HFD-induced hepatic steatosis.

Increased hepatic lipid content inTrif −/− mice was due to augmentedScd1 expression in hepatocytesActivation of the TRIF pathway inhibitsthe ER stress–induced increase in the ex-pression of Chop [which encodes CCAAT/enhancer binding protein (C/EBP) homolo-gous protein] (12), and CHOP can dimerizeand inhibit C/EBPs, resulting in the disrup-tion of lipid regulation (19). We speculatedthat the increased hepatic steatosis in Trif −/−

mice might be due to an increase in Chopexpression. However, HFD-fed Trif −/−

and wild-type mice did not show increasedCHOP abundance at the mRNA or proteinlevels (Fig. 2A and fig. S3, A to B), and he-patic apoptosis was also absent (fig. S3C).Because hepatic triglyceride content wasincreased in both whole-body knockoutand bone marrow transplantation models,we next examined the expression of sev-eral genes involved in lipogenesis in liver(Fig. 2A). Trif −/−mice fed an NCD did notshow any significant changes in lipogenicgene expression (fig. S4). However, uponHFD feeding, Scd1 expression and SCD1protein abundance were significantly in-creased in Trif −/− mice compared with

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Fig. 1. The aggravation of diet-induced hepatic steatosis in Trif −/− mice was mediated by nonmyeloid cells.(A to D) Six-week-old wild-type (WT) and Trif −/− mice were fed an NCD or HFD (60% fat kcal) for 10 weeks. (E to H)Irradiated eight-week-old WT and Trif −/− mice received bone marrow (BM) transplantation and were fed an HFD for10 weeks as in (A) to (D). Livers were subjected to H&E (A and E) and Oil Red O staining (B and F). Scale bars, 100 mm(A and E) and 20 mm (B and F). Hepatic total triglyceride (TG) and total cholesterol (TC) content (C and G) and serumAST and ALT activities were measured (D and H). For (A) to (D), n = 8 to 10 mice per genotype and diet; for (E) to (H),n = 4 to 6 mice per genotype and treatment. *P < 0.05 and **P < 0.01. Representative images are shown.

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wild-type mice (Fig. 2, A and B). Furthermore, palmitic acid, a long-chain saturated fatty acid, stimulated Scd1 expression and SCD1 proteinin hepatocytes (Fig. 2, C and D), and the TLR3 agonist poly(I:C) alle-viated such induction in wild-type but not Trif −/− hepatocytes (Fig. 2, Cand D). TLR3/TRIF signaling results in the activation of IRF3 by phos-phorylation (20, 21), and poly(I:C) induced IRF3 phosphorylation atSer396 in hepatocytes (fig. S5A). The inhibition of the palmitic acid–induced increase in SCD1 abundance by poly(I:C), which was TRIF-

Chen et al., Sci. Signal. 10, eaal3336 (2017) 8 August 2017

dependent, also correlated with lower tri-glyceride and cholesterol content of he-patocytes as shown by Oil Red O staining(Fig. 2E) and quantification of hepatic lip-ids (Fig. 2F). The ability of poly(I:C) to in-hibit the palmitic acid–induced increasein Scd1 expression in hepatocytes wasblocked by knockdown of TLR3 with smallinterfering RNA (siRNA) (fig. S5, B to D)but not by treating the cells with LPS to ac-tivate TLR4, which can stimulate TRIF (fig.S5D). This result might be due to the differ-ent extents of TRIF activation elicited byTLR3 and TLR4 in hepatocytes. Comparedwith other cell types in the liver, hepatocytesproduce less cytokines and downstreamsignaling molecules upon LPS stimula-tion, implying that hepatocyte TLR4 is lesssensitive (22). These results show that acti-vation of TLR3/TRIF pathway by poly(I:C)could inhibit SCD1 in hepatocytes at bothmRNA and protein levels and prevent lipidaccumulation.

Endogenous mRNA released by apo-ptotic or necrotic cells and gut-derivedviromes can activate TLR3 under non-infectious conditions (23, 24). We observedan increase in circulating RNAs in HFD-fed mice (Fig. 2G), and these circulatingRNAs consisted of ribosomal RNAs (fig.S6A). As a proof of concept, we appliedfreshly isolated RNAs complexed withLipofectamine and palmitic acid to pri-mary mouse hepatocytes and found thatthe cells took up these RNAs (fig. S6B). Sim-ilar to poly(I:C), these isolated RNAs par-tially inhibited the palmitic acid–inducedincrease in SCD1 at the mRNA and pro-tein levels and triglyceride accumulationin wild-type but not Trif −/− hepatocytes(Fig. 2, H to I).

Reconstitution of TRIF by adenovirusgene delivery inhibited the increasein SCD1 and hepatic steatosis inTrif −/− miceTo verify the specific role of hepatic TRIFin lipogenesis, we reconstituted TRIF ex-pression in Trif −/− mice using adenovirusgene delivery (Ad-TRIF). When wild-typeand Trif −/− hepatocytes were infected with

the control virus [Ad–green fluorescent protein (GFP)], we observedthat poly(I:C) failed to suppress the palmitic acid–induced increasein SCD1 and lipid accumulation in Trif −/− cells. Infection with Ad-TRIFrestored poly(I:C)–mediated suppression in Trif −/− hepatocytes (Fig. 3, Ato C). Moreover, the reconstitution of TRIF in vivo reversed the increasein SCD1 at the mRNA and protein levels and reduced hepatic triglycerideaccumulation in Trif −/− mice to amounts comparable to those of wild-type mice infected with Ad-GFP (Fig. 3, D to G).

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Fig. 2. Activation of TRIF by poly(I:C) inhibited palmitic acid–induced hepatic SCD1 expression and lipid ac-cumulation. (A and B) Six-week-old WT and Trif −/− mice were fed an NCD or HFD for 10 weeks. (A) Hepatic Chop,Acc1, Fasn, Scd1, and Dgat2 expression was examined by real-time polymerase chain reaction (PCR) and normalizedto Actb. (B) Western immunoblotting was performed to detect hepatic SCD1 and b-actin proteins. (C to F) Primaryhepatocytes from WT and Trif −/− mice were treated with or without palmitic acid (PA; 250 mM) (ctrl, control) andpoly(I:C) (2.5 mg/ml) for (C) 6 hours or (D to F) 8 hours. Scd1mRNA (C) and SCD1 protein abundance (D) were measured.Oil Red O staining (E) and quantification of hepatic TG and TC content (F) were performed. Scale bar, 40 mm. Repre-sentative images are shown. (G) The concentrations of serum RNA from WT mice fed an NCD or HFD for 10 weeks weremeasured. (H and I) WT and Trif −/− hepatocytes were treated with PA and RNA (2.4 mg/ml) isolated from adipose tissueand complexed with Lipofectamine as in (D). (H) SCD1 protein abundance and (I) intracellular TG content weremeasured. For (A), (B), and (G), n = 4 to 7 mice per genotype or diet; for (C) to (F) and (H) and (I), n = 3 independentexperiments. *P < 0.05 and **P < 0.01.

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Silencing SCD1 decreased intracellular triglyceridecontent in Trif −/− hepatocytesScd1−/− mice are resistant to HFD-induced hepatic steatosis (25).To determine whether attenuation of SCD1 prevented TRIF-mediatedaggravation of hepatic steatosis, we treated wild-type and Trif −/− he-patocytes that had been infected with lentivirus encoding SCD1 siRNA(Lv-SCD1-siRNA) or scrambled RNA (Lv-Scr-RNA) with palmitic acid(Fig. 4, A to C, and fig. S7, A and B). The palmitic acid–induced increasein Scd1 expression was successfully inhibited by Lv-SCD1-siRNA in bothwild-type and Trif −/− hepatocytes (Fig. 4A). Similar to the above finding,poly(I:C) inhibited the palmitic acid–induced increase in Scd1 expressionand triglyceride accumulation in infected wild-type but not Trif −/−

cells (Fig. 4, A to C). Conversely, infection with Lv-SCD1-siRNA abol-ished the palmitic acid–induced increase in intracellular triglyceridecontent in hepatocytes from both wild-type and Trif −/− mice (Fig. 4,B and C). Scd1 expression and hepatic triglyceride accumulation weredecreased in HFD-fed Trif −/− mice infected with Lv-SCD1-siRNA com-pared to those infected with Lv-Scr-RNA (Fig. 4, D to F). Together, thesefindings suggest that silencing SCD1 alone was sufficient to reversehepatic lipid accumulation in Trif −/− mice.

Chen et al., Sci. Signal. 10, eaal3336 (2017) 8 August 2017

IRF3 acted as a transcriptional suppressor of Scd1Because TRIF inhibited Scd1 expression, we next tested whether thiseffect occurred at the promoter region of the Scd1 gene. First, we de-livered a bioluminescent reporter plasmid containing the promoter re-gion of Scd1 (26) into both wild-type and Trif −/− mice fed an HFD (27).Trif −/− mice displayed a threefold more intense chemiluminescent signalin the liver area compared with wild-type mice (Fig. 5A). Activation ofTLR pathway can induce transcriptional activity of several IRFs (28).We speculated that IRF3, which is selectively responsive to TRIF, wasinvolved in poly(I:C)–induced transcriptional suppression of Scd1 (28).In HeLa cells, SCD1 abundance was reduced by overexpression of aphosphomimetic IRF3(5D) [in which the five Ser/Thr sites in the regionbetween amino acid residues 395 and 407 (ISNSHPLSLTSDQ) at the Cterminus of IRF3 were substituted with Asp (21)] but not wild-type IRF3(Fig. 5B). Bioinformatics analysis identified two putative IRF3 bindingsites (GAAANN) at the −248 and −593 base pair of the 5′ flanking regionof Scd1 gene. Two different lengths of the upstream segments of Scd1 gene(−816 + 229 and −469 + 229) covering the trans- and/or cis-regulatoryelements were inserted into pcDNA3.1/V5-His/lacZ reporter vector.Palmitic acid or poly(I:C) treatment did not alter reporter activity in

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cells transfected with the plasmid contain-ing the sequence between −469 and +229of the Scd1 gene promoter (Fig. 5C). How-ever, palmitic acid treatment stimulated re-porter activity in cells transfected with theplasmid containing the region between−816 and +229, an effect that was blockedby poly(I:C) (Fig. 5C). In this region, theIRF3 binding motif is located between−599 and −593 and is highly conservedacross different species (table S1). Mutationof this sequence did not affect the palmiticacid–mediated induction but abolishedthe poly(I:C)–mediated repression of Scd1promoter activity (Fig. 5C). Chromatin im-munoprecipitation analysis further demon-strated that in poly(I:C)–treated cells, IRF3directly bound to the wild-type version ofthe Scd1 promoter but not to the mutantversion of the promoter (Fig. 5D). In ad-dition, poly(I:C) did not suppress palmiticacid–induced Scd1 promoter activity inHepG2 human liver cells expressing themutant promoter (Fig. 6A). Overexpressionof IRF(5D) inhibited the palmitic acid–induced increase in Scd1 promoter activity,SCD1 protein abundance, and intracellu-lar triglyceride content in HepG2 cells(Fig. 6, B to D). Moreover, the suppressiveeffect of poly(I:C) was abolished after silenc-ing IRF3 (Fig. 6, E and F). These data sug-gest that IRF3 is a transcriptional suppressorof Scd1 in both rodents and humans.

Poly(I:C) prevented thedevelopment of HFD-inducedhepatic steatosis in miceBecause poly(I:C) inhibited palmiticacid–induced Scd1 expression and lipid

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mice. (A to C) WT and Trif −/− hepatocytes infected with adenovirus encodingGFP (Ad-GFP) or TRIF (Ad-TRIF) were treated with or without PA (250 mM) andpoly(I:C) (2.5 mg/ml) for 8 hours. GFP was detected in cells transfected withAd-GFP and Ad-TRIF (A). Scale bar, 40 mm. SCD1 protein (B) and intracellularTG content (C) were determined. (D to G) Six-week-old WT and Trif −/− mice werefed an HFD for 4 weeks, injected once with 109 viral particles (vp) per mouse ofAd-GFP or Ad-TRIF, and continued on the HFD for two more weeks. Livers wereanalyzed for GFP fluorescence (D), Scd1mRNA expression (E), SCD1 protein abun-dance (F), and TG content (G). Scale bar, 20 mm. For (A) to (C), n = 3 independentexperiments; for (D) to (G), n = 4 to 6 mice per genotype and/or infection con-dition. *P < 0.05 and **P < 0.01. Representative images are shown.

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accumulation in hepatocytes, we next tested whether administration ofpoly(I:C) prevented HFD-induced hepatic steatosis in mice. Withoutaffecting body weight and fat mass (fig. S8, A and B), administrationof poly(I:C) significantly decreased Scd1 expression, SCD1 protein abun-dance, hepatic triglyceride and cholesterol accumulation, and fastingblood glucose in wild-type mice, protective effects of poly(I:C) that weremarkedly diminished in Trif −/− mice (Fig. 7, A to C, and fig. S8C). Thisdosage of poly(I:C) did not aggravate hepatic inflammation (as assessedby analysis of the expression of inflammatory cytokine–encodingmRNAs) or serum ALT activities in mice of both genotypes but loweredAST activity in Trif −/−mice (Fig. 7D and fig. S8, D to F). These resultssuggest a possible pharmacological option for treating hepatic steatosisby selective activation of TRIF in hepatocytes.

DISCUSSIONHere, we show that the increased Scd1 expression in hepatocytes inTRIF-deficient mice was attributed to the worsened hepatic steatosisunder HFD. Activation of TRIF pathway stimulated phosphorylationand transcriptional activity of IRF3, which acted as a suppressor onthe promoter region of Scd1 gene. Treatment with poly(I:C) resultingin phosphorylation of IRF3 suppressed both HFD- and palmitic acid–

Chen et al., Sci. Signal. 10, eaal3336 (2017) 8 August 2017

induced SCD1 expression and lipid accu-mulation in liver and hepatocytes (Fig. 7E).

NAFLDis a commonlyunderdiagnosedmedicalproblembecause, inmost situations,it is asymptomatic. Currently, there is no de-finitive treatment for NAFLD. Preventingthe progression to nonalcoholic steato-hepatitis (NASH) or fibrosis using anti-inflammatory and antioxidant therapiesis the goal of treatment for susceptive pa-tients with obesity or diabetes. TLRs playa key role in the progression of simple he-patic steatosis to NASH by driving the he-patic inflammation, and their antagonistshave been considered as potential treat-ments (2, 5, 29, 30). Hepatocyte-specificdeficiency of TLR4 improves glucose tol-erance and insulin sensitivity and amelio-rates hepatic steatosis and adipose tissueinflammation in HFD-induced obesity(10). Here, we showed that suppressionof SCD1 in hepatocytes was mediated byTLR3/TRIF but not TLR4/TRIF (fig. S5D).The hepatocyte-specific ablation of TLR4wouldyield adouble benefit by suppressinginflammation through the TLR4/MyD88pathway and inhibiting lipogenesis throughthe intact TLR3/TRIF pathway. The TRIFpathway can be activated throughTLR3byendogenousRNAandgut-derived viromeduring HFD-induced obesity (23, 24).High-fat intake can weaken the gut barri-er, resulting in penetration of the intestinalbacteria andviruses and their products intothe bloodstream, providing a panel of TLRligands (24, 31, 32). Adipose tissue expan-sion during obesity is associated with ne-

crosis and apoptosis of adipocytes, and cellular RNAreleased bynecrotictissue can activate TLR3 (23, 33, 34). We found that circulating RNAswere increased in HFD-fed mice (Fig. 2G), which may be derived fromthe turnover of adipocytes. CirculatingRNAs are encapsulated inmicro-vesicles or exosomes (35). We showed that Lipofectamine-complexedRNAsuppressedpalmitic acid–induced SCD1expression and triglycerideaccumulation in a TRIF-dependentmanner (Fig. 2, H and I). The activa-tionofTLR3/TRIFduringHFD-induced obesitymay intrinsically provideaprotectivemechanismagainst hepatic lipid accumulation.The absenceofsuch a pathway aggravatedHFD-induced hepatic steatosis (Fig. 1, A toC).

The prevalence of NAFLD in diabetic and obese individuals is high,and their risk of developing into NASH and fibrosis is greater than thatof the general public (36). Because there is no confirmative diagnostictool to identify the initiation of NASH, intervention at the early stage ofbenign hepatic steatosis would be beneficial in these patients. SeveralScd1-deficient mouse models do not develop diet-induced hepaticsteatosis (25, 37), and inhibition of SCD1 using antisense oligonucleotidecan also prevent HFD-induced hepatic insulin resistance and obesity(38, 39). Pharmacological approaches to inhibiting SCD1 for treatingmetabolic diseases have been explored but have been unsuccessful dueto the potential nonspecific actions on other peripheral tissues such asvascular wall and pancreas (40). Here, we found that Scd1 expression

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Fig. 4. Silencing SCD1 abolished lipidaccumulation in Trif−/− hepatocytes.(A to C) Isolated WT and Trif−/− hepato-cytes infected with lentivirus encodingeither SCD1 siRNA (Lv-SCD1-siRNA) orscrambled RNA (Lv-Scr-RNA) were treatedwith or without PA (250 mM) and poly(I:C)(2.5 mg/ml) for 8 hours. Scd1 mRNA ex-pression (A) and TG content (B) weremeasured, and Oil Red O staining (C)

was performed. Scale bar, 40 mm. (D to F) Six-week-old Trif −/− mice were fed an HFD for 10 days, injected with eitherLv-SCD1-siRNA or Lv-Scr-RNA, and continued on the HFD for another 3 weeks. Hepatic Scd1mRNA (D), SCD1 protein(E), and TG content (F) were determined. A group of age-matched Trif −/− mice fed an NCD served as controls. n = 4independent experiments for (A) to (C); n = 6 mice per genotype, diet, and/or injection condition for (D) to (F).*P < 0.05 and **P < 0.01. Representative images are shown.

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was inhibited by intraperitoneal injection of poly(I:C), which is a mimeticof double-stranded RNA (dsRNA). A cell- and pathway- specific syn-thetic ligand mimicking our observed poly(I:C) effect would be an idealSCD1 inhibitor. Hepatocyte-specific delivery of siRNA has been explored,and, for example, siRNA conjugated to triantennary N-acetylgalactosamineis effectively taken up by the liver due to binding to a liver-specific asialo-glycoprotein receptor (41). Specific delivery of a dsRNA mimetic to he-patocytes appears to be feasible. Moreover, a stereochemically alteredmimetic of LPS, CRX-527, can activate the TRIF pathway without sti-mulating the production of MyD88-mediated proinflammatory mole-

Chen et al., Sci. Signal. 10, eaal3336 (2017) 8 August 2017

cules (42). The architecture of the TIR domain signalosome revealsthat MyD88- and TRIF-dependent pathways cannot be simulta-neously induced upon TLR4 activation because of the shared bind-ing site on a single TLR4 dimer (43). Because deficiency of TLR4 yieldsa protective anti-inflammatory effect in metabolic diseases, a TLR4 an-tagonist with intact TRIF-dependent effects could ameliorate NAFLDand prevent progression to NASH. The detailed understanding of howthe known ligands stereochemically interact with different TLRs andtrigger the downstream pathway would help to precisely design small-molecule drugs.

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Fig. 5. Suppressive action on the Scd1 promoter by TRIF was mediated by IRF3. (A) Six-week-old WT and Trif −/− micefed an HFD for 10 weeks were hydrodynamically injected with the luciferase-expressing plasmid pGL3/–1537+155mSCD1.Luciferase activity was analyzed 1 hour after administration of D-luciferin. (B) HeLa cells expressing IRF3(WT) or IRF3(5D) wereimmunoblotted for IRF3 and SCD1 with b-tubulin as loading control. (C) b-Galactosidase activity was measured in HeLacells expressing WT pcDNA3.1/–469+229mSCD1 and WT or mutated pcDNA3.1/–816+229mSCD1 that were treatedwith or without PA (250 mM) and poly(I:C) (2.5 mg/ml) for 8 hours. (D) HeLa cells transfected with WT or mutated

pcDNA3.1/–816+229mSCD1 were treated with PA (250 mM) and poly(I:C) (2.5 mg/ml) for 2 hours. Chromatin immunoprecipitation with IRF3 antibody and PCR amplificationusing specific primers against the Scd1 promoter region were performed. IgG, immunoglobulin G. For (A), n = 4 mice per genotype; for (B) to (D), n = 3 to 4 independentexperiments. *P < 0.05 and **P < 0.01. Representative images are shown.

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ig. 6. IRF3-mediated SCD1 suppression was validated in human hepatic cells. (A) b-Galactosidase activity was measured in HepG2ells transfected with WT or mutated pcDNA3.1/–816+229mSCD1 followed by the treatment of PA and poly(I:C). (B) HepG2 cells expressingT pcDNA3.1/–816+229mSCD1 and vector or IRF3(5D) plasmids were treated with or without PA (250 mM) and poly(I:C) (2.5 mg/ml) for 8 hours

and reporter activity was measured. (C and D) HepG2 cells were treated as in (B), and SCD1 protein with b-tubulin as loading control (C) and TG

ontent (D) were measured. (E and F) HepG2 cells transfected with scrambled RNA or IRF3 siRNA were treated with or without PA and poly(I:C) for 8 hours. IRF3 and SCD1 proteinbundance (E) and TG content (F) were measured. n = 3 to 4 independent experiments for (A) to (F). *P < 0.05 and **P < 0.01. Representative images are shown.

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Several key metabolic transcription factors can independently reg-ulate Scd1 expression including sterol regulatory element–binding proteintranscription factor 1c (SREBP-1c), peroxisome proliferator–activatedreceptor a (PPARa), and liver X receptor (26, 44). We found that IRF3directly bound to the Scd1 promoter and suppressed its transcription.The transcription-suppressing activity of IRF3 can occur by differentmechanisms (15, 45). For example, poly(I:C) can suppress the transcrip-tion of Rxra through IRF3, which stimulates a transcriptional sup-pressor, Hes1 (Hes family BHLH transcription factor 1), leading to therecruitment of transcriptional repression machinery to the Rxra pro-moter (15). Activated IRF3 also interacts with Smad3 and disrupts func-tional Smad3 transcription complexes by competing with co-regulators(45). PPARa and SREBP-1c binding sites are in close proximity of theIRF3 binding motif on the Scd1 promoter. The binding of IRF3 couldphysically obstruct the attachment of other transcription activatorson the promoter region, resulting in inhibition of transcription. Globalknockout of IRF3 promotes HFD-induced hepatic insulin resistanceand steatosis, suggesting that the protective effect of IRF3 is mediatedby suppressing inflammation because IRF3 can prevent nuclear factorkB (NFkB) activation by interacting with inhibitor of NFkB kinase b(46). These studies suggest that activation of IRF3 in liver results inboth metabolic and anti-inflammatory effects. Moreover, our find-ing of the attenuation of SCD1 by TLR3/TRIF/IRF3 pathway mayalso be considered as an immune response. Hepatitis C virus (HCV),a single-stranded RNA virus able to generate dsRNA, can hijack hostlipogenesis for its own viral assembly (47). Host SCD1 activity can fa-cilitate HCV viral replication, and supplementation of the productsof SCD1 such as oleate and palmitoleate restores HCV replication inScd1-knockdown hepatocytes (48). TRIF/IRF3-induced suppression

Chen et al., Sci. Signal. 10, eaal3336 (2017) 8 August 2017

of SCD1 may limit viral infection by counteracting the virus-hijackedmetabolic pathway. Of the HCV genotypes, infection with HCVgenotype 3 (GT3) is highly associated with hepatic steatosis that ismore prone to develop into fibrosis (49). The degree of steatosis is pro-portional to the virus load (50). A dsRNA mimetic targeted to the liverwould be an ideal adjuvant treatment for this particular HCV genotype.By activating the TRIF pathway, it would inhibit hepatic lipogenesiswhile maintaining interferon production, yielding a dual benefit againstHCV GT3 infection. Activation of TRIF signaling can counterbalancethe ER stress–induced protein translation suppression and delay apo-ptosis, and such mechanism is essential to maintain proper secretoryfunctions of both immune and metabolic cells in the early stages ofmetabolic stress (12, 14).

Activation of TLR pathways by microbial and endogenous stimuliin metabolic disorders (2, 3, 51) is detrimental because of the release ofinflammatory molecules from immune cells (2, 17, 22). TLRs are presentin various nonimmune cell types and have unique functions (16, 52).Here, we showed that activation of the TRIF-dependent TLR pathwaysuppressed Scd1 expression and prevented hepatic lipid accumulationand that the absence of this pathway exacerbated hepatic steatosis un-der metabolic stress. It is important to take this metabolic function ofTLRs into consideration when using antagonists to ameliorate meta-bolic inflammation in chronic diseases.

MATERIAL AND METHODSAnimalsC57BL/6J wild-type mice and Trif −/−male mice (stock no. 005037) werepurchased from the Jackson Laboratory. All animal experimental

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Fig. 7. Poly(I:C) ameliorated HFD-induced lip-id accumulation. (A to D) Six-week-old WT andTrif −/− mice were injected with phosphate-buffered saline (PBS) or poly(I:C) (5 mg/g) intra-peritoneally three times a week while beingfed an HFD for 4 weeks. Hepatic Scd1 expression(A), SCD1 protein abundance (B), TG and TC con-tent (C), and serum AST and ALT activities (D)were measured. n = 8 to 10 mice per genotypeand treatment condition for (A) to (D). *P < 0.05and **P < 0.01. (E) Working model. IRF3 in re-sponse to TLR/TRIF signaling serves as a tran-scriptional suppressor of Scd1 in hepatocytes,resulting in the suppression of lipogenesis.

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procedures were approved by the Committee on the Use of Live Ani-mals in Teaching and Research of the University of Hong Kong. Forthe HFD model, mice were co-housed and fed a 60% kcal fat diet pur-chased from Research Diets Inc. for 10 weeks. In one set of experiment,poly(I:C) (5 mg/g) (P9582, Sigma-Aldrich) was injected intraperitoneallyinto mice three times a week starting on the day when the HFD wasintroduced until euthanasia after 4 weeks of HFD. For experiments usinglentivirus, mice were fed the HFD for 10 days before lentiviral infection(3.6 × 105 transduction units of Lv-SCD1-siRNA or Lv-Scr-RNA permouse) and continued on the HFD for another 3 weeks because theeffectiveness of lentivirus lasts for about 3 weeks. For experiments usingadenovirus, mice were fed the HFD for 4 weeks, and the wild-type andhalf of the Trif −/− mice received Ad-GFP (5 × 109 vp per mouse),whereas the other half of Trif −/− mice received Ad-TRIF (5 × 109 vpper mouse). The HFD was continued for two more weeks.

Bone marrow transplantationMice received total body irradiation of 9 gray in a Lucite ionizationchamber. Bone marrow cells were isolated from the femurs of wild-typeand Trif −/− mice and resuspended in serum-free RPMI medium. A totalof 5 × 106 cells were injected into each of the irradiated recipient micethrough the lateral tail vein on the day of irradiation. The mice were kepton antibiotic-supplemented water for 2 weeks after irradiation (5). Toverify that irradiation was successful, two mice were not injected withbone marrow and died 8 days after initial irradiation. HFD feeding wasinitiated after the recovery. Successful transplantation was further con-firmed by genotyping the bone marrow collected after euthanasia. Theirradiated mice were co-housed based on the genotypes of the recipientsdue to the large number of mice and cage size limitations, and the cagesof mice with different background genotypes were swapped periodicallyto achieve co-housed conditions of four groups of mice (WT/WT-BM,Trif −/−/WT-BM, WT/Trif −/−-BM, and Trif −/−/Trif −/−-BM).

Isolation of primary hepatocytes and cell culturePrimary hepatocytes were isolated from six- to eight-week-old wild-type or Trif −/− mice as previously mentioned with modification (53).Briefly, liver was perfused with PBS through the portal vein with anoutlet at the inferior vena cava, followed by digestion with type I col-lagenase (~40 collagen digestion units/ml, Sigma-Aldrich, C2674). Di-gested liver was minced and filtered, and cell suspension was collectedand further separated using Percoll (Sigma-Aldrich). Hepatocytes werecollected after centrifugation at 100g for 5 min and cultured in Dulbecco’smodified Eagle’s medium (DMEM) containing 10% fetal bovine serum(FBS) on collagen-coated plate (BD BioCoat Collagen). Cells were in-cubated overnight at 37°C under 5% CO2 and 95% relative humidity.HeLa and HepG2 cells (American Type Culture Collection) were rou-tinely maintained in DMEM with 10% FBS. Mouse TLR3 siRNA andhuman IRF3 siRNA were purchased from Santa Cruz BiotechnologyInc. and Qiagen Inc., respectively.

Isolation of serum RNA and RNA for treatmentof hepatocytesSerum RNA and adipose tissue RNA for in vitro experiment were iso-lated using a commercially available kit (Zymo Research). RNA con-centrations were measured by NanoDrop 2000 (Thermo Fisher).

Histochemical stainingLivers were collected and fixed in 10% buffered formalin followed byembedding in paraffin. Paraffin-embedded sections (5 mm thick) were

Chen et al., Sci. Signal. 10, eaal3336 (2017) 8 August 2017

prepared and subjected to H&E staining. Another portion of the liverwas embedded in frozen Tissue-Tek OCT compound, and 7-mm frozensections were subjected to Oil Red O staining or GFP detection.

Lipid extraction and measurementLipids were extracted with a chloroform/methanol/water ratio of 4:3:2,and dried lipid pellets were resuspended in 100% ethanol. Hepatic andcellular triglyceride and total cholesterol were assessed by using com-mercially available kits (Stanbio Diagnostics). Triglyceride and choles-terol amounts were normalized to liver wet weight or cellular protein.

Fasting blood glucose and liver function testMice were fasted for 5 hours for blood glucose measurement using aglucometer (Accu-Chek Performa, Roche Diagnostics). Serum AST andALT activities were measured using commercially available kits (StanbioDiagnostics).

Real-time PCRTotal RNA from hepatocytes or liver tissues were isolated using TRIzolreagent (Invitrogen). RNA was reverse-transcribed into complementaryDNA (cDNA) using oligo(dT) and ImProm-II Reverse TranscriptionSystem (Promega). Real-time PCR was conducted using the SYBR GreenPCR reagent (Roche) and StepOnePlus System (Applied Biosystems).The mRNA expression of target genes was normalized to Actb. Thesequences of the primers are listed in table S2.

Western immunoblottingImmunoblots were conducted as described previously (12). Briefly, cul-tured cells were lysed with Laemmli sample buffer (Bio-Rad), and liverswere homogenized in a lysis buffer containing 20 mM tris (pH 7.4),150 mM NaCl, 1% Triton X-100, and phosphatase and protease in-hibitor cocktails. Protein samples were separated by electrophoresis ona 10% SDS polyacrylamide gel and transferred to polyvinylidene di-fluoride membranes. The membranes were probed with the indicatedprimary antibodies against SCD1, IRF3, or phospho-IRF3(Ser396) (CellSignaling), and the protein bands were detected with horseradishperoxidase–conjugated secondary antibodies (Jackson ImmunoResearch)and WesternBright ECL reagent (Advansta). The membranes were re-probed with antibody against b-actin (Sigma-Aldrich) or b-tubulin (CellSignaling) to detect differences in protein loading. Densitometry anal-ysis of the gels was carried out using ImageJ software from the Na-tional Institutes of Health.

Adenovirus constructionAdenovirus encoding TRIF (Ad-TRIF) was constructed by WelgenInc. Briefly, mouse Trif cDNA (GE Dharmacon) was subcloned intopEntCMV-Ef1aGFP vector predigested with Pme 1. The ligation mix-ture was transformed into Escherichia coli, and the positive clones werescreened with Eco R5 and sequenced. The pEntCMV-mTRIF-Ef1aGFPwas treated with LR Clonase II enzyme (Invitrogen) and ligated to apAd-REP plasmid that contains the remaining adenovirus genome.The recombination products were transformed into E. coli cells. Afterovernight incubation, positive clones were selected, and cosmid DNAwas purified. The purified cosmid DNA was digested with Pac 1 andthen transfected into human embryonic kidney (HEK) 293 cells. Theadenovirus plaques were seen 7 days after transfection. The low titervirus was further amplified and purified. The purified high titer virus(1012 vp/ml) was diluted into 109 vp/ml to infect HEK293 cells followedby sequencing to validate the absence of contamination.

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Lentivirus constructionVector carrying scrambled or SCD1 siRNA was purchased from Ap-plied Biological Materials Inc. Lentiviruses were produced by transfect-ing HEK293TN cells with the purchased vectors expressing GFP andthree helper plasmids (pVSV-G, pPACKH1-GAG, and pPACKH1-REV). The medium were harvested 48 and 72 hours after initial trans-fection and filtered through a 0.22-mm pore size filter. The lentiviruswas precipitated by adding 5% polyethylene glycol–8000 and 0.15 MNaCl to the medium followed by incubation overnight at 4°C alongwith mixing every 20 min. The virus was pelleted by centrifugation at7000g for 15 min (54). Titer was determined by performing flow cytom-etry on transduced HeLa cells.

Plasmid construction and transfectionThe plasmid pGL3/–1537+155mSCD1 was provided by J. Ntambi(University of Wisconsin). The promoter regions (−816 + 229) and(−469 + 229) were constructed by PCR using 5′-CGGGGTACC-TGATTCCTTAGTCCCTTTTCTTGGA-3′ and 5′-CGCGG-ATCCTGGTGTAGGCGAGTGGCGGAACTGC-3′ and 5′-CGGGGTACCCCTCACTTCTTTCGATGCGATTTCC-3′ and 5′-CGCGGATCCTGGTGTAGGCGAGTGGCGGAACTGC-3′, re-spectively. The PCR product was subcloned into pcDNA3.1/lacZ(Invitrogen) at Kpn I and Bam HI restriction sites. The IRF3 bind-ing site at −593 was mutated using GENSTART site-directed mutagenesissystem (Invitrogen) and the primers 5′-CTGAAGGGATACACTAT-TACCCCTCCGGGTCAGAGCCCTGGG-3′ and 5′-CCCAGGGCTCT-GACCCGGAGGGGTAATAGTGTATCCCTTCAG-3′. b-Galactosidaseactivity was determined by a colorimetrical method using o-nitrophenyl-b-D-galactopyranoside (Sigma-Aldrich) as substrate. The plasmids encod-ing wild-type and mutated IRF3 including pEGFPC1-IRF3(WT) andpEGFPC1-IRF3(5D) were provided by J. Hiscott (University of Florida).All the plasmids were verified by sequencing service provided by BeijingGenomics Institute.

Bioluminescence imagingThe luciferase reporter plasmid (pGL3/–1537+155mSCD1) was injectedinto mice by hydrodynamic injection. Briefly, DNA (50 mg/ml) in sterilePBS was injected into mice within 10 s through the tail vein, and thetotal volume injected was maintained within 8 to 10% of body weight.Mice were allowed to recover for 24 hours before receiving an intra-peritoneal injection of D-luciferin (1 mg/g) (Promega) under anesthesia.Images were captured by the IVIS Spectrum In Vivo Imaging Sys-tem (PerkinElmer) and analyzed with the Living Image Software 4.4(PerkinElmer).

Chromatin immunoprecipitationChromatin immunoprecipitation was performed as previously de-scribed with minor modification (53). Cells were treated with 3.7%formaldehyde to cross-link DNA and protein, followed by lysis with abuffer containing 150 mM NaCl, 50 mM tris-HCl (pH 7.5), 5 mMEDTA, 0.5% (v/v) NP-40, and 1% (v/v) Triton X-100. Chromatinwas prepared and sheared by sonication with Bioruptor (Diagenode,Belgium) and subjected to immunoprecipitation with an antibodyagainst IRF3 (Cell Signaling) or rabbit IgG control and protein Amagnetic beads (Biotool). An aliquot of chromatin from each samplewas reserved for analysis of DNA input before immunoprecipitation.The specific protein-DNA complexes were eluted from the magneticbeads, and cross-links were reversed. DNA was precipitated from thesamples, including from the previously reserved aliquots, and subjected

Chen et al., Sci. Signal. 10, eaal3336 (2017) 8 August 2017

to PCR amplification and detection of the segment of Scd1 promoterregion using the primers 5′-CCAATGAGTGAGTGCAGTTGTA-3′(forward) and 5′-GCATCGAAAGAAGTGAGGAAGA-3′ (reverse).

Statistical analysisStatistical analyses were performed using SPSS version 23.00. Data werepresented as means ± SEM. One-way analysis of variance (ANOVA) wasapplied for comparisons between multiple experimental groups, followedby post hoc analysis using Tukey post hoc test for data with equal vari-ance or Games-Howell for data with unequal variance. Data with smallsample size were analyzed using the Kruskal-Wallis test, a nonparametricone-way ANOVA. An unpaired Student’s t test was applied for two-group comparison with normal distribution. P values less than 0.05were considered to indicate statistically significant differences.

SUPPLEMENTARY MATERIALSwww.sciencesignaling.org/cgi/content/full/10/491/eaal3336/DC1Fig. S1. Trif−/− mice have similar body weight and fat mass as wild-type mice but increasedfasting blood glucose after HFD feeding.Fig. S2. Hepatic inflammation, serum insulin concentrations, and serum lipids were similarbetween HFD-fed wild-type and Trif−/− mice.Fig. S3. HFD did not increase CHOP abundance or apoptosis in wild-type and Trif−/− mice.Fig. S4. Lipogenic gene expression did not increase in wild-type and Trif−/− mice fed an NCD.Fig. S5. Poly(I:C)–stimulated phosphorylation of IRF3 and suppressed palmitic acid–inducedScd1 expression in hepatocytes in a TLR3-dependent manner.Fig. S6. RNA was detected in the serum of HFD-fed mice and taken up by hepatocytes.Fig. S7. Lv-SCD1-siRNA decreased SCD1 abundance.Fig. S8. Activation of TRIF by poly(I:C) decreased fasting blood glucose concentrations inHFD-fed wild-type mice without significantly affecting body weight, fat mass, or hepaticinflammation.Table S1. IRF3 binding motifs in the Scd1 promoter in different species.Table S2. Primer sequences for specific genes.

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Acknowledgments: We would like to thank I. Tabas (Columbia University) for his valuablesuggestions on this manuscript. The pGL/–1537+155mSCD1 and the IRF3 plasmids[pEGFPC1-IRF3(WT) and pEGFPC1-IRF3(5D)] were provided by J. Ntambi (University of Wisconsin)and J. Hiscott (University of Florida), respectively. Funding: This study was supported by amatching fund offered by the Research Centre of Heart, Brain, Hormone and Healthy Aging of theUniversity of Hong Kong (200007252), the National Key Basic Research Development Program973 (2015CB553603), the Hong Kong Research Grants Council/Collaborative Research Fund(C7055-14G), and a matching grant for the State Key Laboratory of Pharmaceutical Biotechnologyfrom the University of Hong Kong. Author contributions: C.W.W. designed the experiments.J.C., J.L., and J.H.C.Y. performed the experiments. J.K.W.L assisted with the RNA labeling experiment.J.C., J.L., J.H.C.Y., C.-M.W., B.D., and C.W.W. analyzed the data. C.W.W., B.D., and A.X. wrote thepaper. Competing interests: The authors declare that they have no competing interests.

Submitted 4 November 2016Resubmitted 13 April 2017Accepted 21 July 2017Published 8 August 201710.1126/scisignal.aal3336

Citation: J. Chen, J. Li, J. H. C. Yiu, J. K. W. Lam, C.-M. Wong, B. Dorweiler, A. Xu, C. W. Woo,TRIF-dependent Toll-like receptor signaling suppresses Scd1 transcription in hepatocytesand prevents diet-induced hepatic steatosis. Sci. Signal. 10, eaal3336 (2017).

10 of 10

prevents diet-induced hepatic steatosis transcription in hepatocytes andScd1TRIF-dependent Toll-like receptor signaling suppresses

Jing Chen, Jin Li, Jensen H. C. Yiu, Jenny K. W. Lam, Chi-Ming Wong, Bernhard Dorweiler, Aimin Xu and Connie W. Woo

DOI: 10.1126/scisignal.aal3336 (491), eaal3336.10Sci. Signal. 

transcription may therefore serve to limit viral infection of hepatocytes.Scd1that the hepatitis C virus co-opts host lipogenesis to ensure its replication and that TRIF-mediated suppression of

occurs in hepatocytes exposed to palmitic acid, a saturated fatty acid that is enriched in high-fat diets. The authors noteby adipose tissue prevented the increase in SCD1 abundance and the enhanced triglyceride accumulation that normally

, which encodes a key lipogenic enzyme. Viral RNA and mimetics activate TLR3, and application of RNA generatedScd1diet-induced hepatic steatosis in mice. TRIF activation downstream of TLR3 led to the transcriptional suppression of

. found that the TLR signaling adaptor TRIF in hepatocytes, rather than myeloid cells, limitedet alHowever, Chen Viral detection by TLR pathways triggers inflammatory responses, which generally aggravate metabolic diseases.

TRIF against fatty liver

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