thehepatitiscvirus-inducednlrp3inflammasome … · 2018-12-11 · to liver disease pathogenesis...

The Hepatitis C Virus-induced NLRP3 InflammasomeActivates the Sterol Regulatory Element-binding Protein(SREBP) and Regulates Lipid Metabolism*

Received for publication, September 23, 2015, and in revised form, December 18, 2015 Published, JBC Papers in Press, December 23, 2015, DOI 10.1074/jbc.M115.694059

Steven McRae‡, Jawed Iqbal‡, Mehuli Sarkar-Dutta‡, Samantha Lane‡, Abhiram Nagaraj‡, Naushad Ali§,and Gulam Waris‡1

From the ‡Department of Microbiology and Immunology, H. M. Bligh Cancer Research Laboratories, Rosalind Franklin University ofMedicine and Science, Chicago Medical School, North Chicago, Illinois 60064 and the §Department of Medicine, Section ofDigestive Diseases and Nutrition, University of Oklahoma, Oklahoma City, Oklahoma 73104

Hepatitis C virus (HCV) relies on host lipids and lipid dropletsfor replication and morphogenesis. The accumulation of lipiddroplets in infected hepatocytes manifests as hepatosteatosis, acommon pathology observed in chronic hepatitis C patients.One way by which HCV promotes the accumulation of intracel-lular lipids is through enhancing de novo lipogenesis by activat-ing the sterol regulatory element-binding proteins (SREBPs). Ingeneral, activation of SREBPs occurs during cholesterol deple-tion. Interestingly, during HCV infection, the activation ofSREBPs occurs under normal cholesterol levels, but the under-lying mechanisms are still elusive. Our previous study has dem-onstrated the activation of the inflammasome complex in HCV-infected human hepatoma cells. In this study, we elucidate thepotential link between chronic hepatitis C-associated inflam-mation and alteration of lipid homeostasis in infected cells. Ourresults reveal that the HCV-activated NLRP3 inflammasome isrequired for the up-regulation of lipogenic genes such as 3-hy-droxy-3-methylglutaryl-coenzyme A synthase, fatty acid syn-thase, and stearoyl-CoA desaturase. Using pharmacologicalinhibitors and siRNA against the inflammasome components(NLRP3, apoptosis-associated speck-like protein containing aCARD, and caspase-1), we further show that the activation of theNLRP3 inflammasome plays a critical role in lipid droplet for-mation. NLRP3 inflammasome activation in HCV-infected cellsenables caspase-1-mediated degradation of insulin-inducedgene proteins. This subsequently leads to the transport of theSREBP cleavage-activating protein�SREBP complex from theendoplasmic reticulum to the Golgi, followed by proteolyticactivation of SREBPs by S1P and S2P in the Golgi. Typically,inflammasome activation leads to viral clearance. Paradoxically,here we demonstrate how HCV exploits the NLRP3 inflam-masome to activate SREBPs and host lipid metabolism, leadingto liver disease pathogenesis associated with chronic HCV.

Chronic liver disease resulting from HCV infection repre-sents a major global health problem. HCV infection often leadsto chronic hepatitis in up to 60 – 80% of infected adults andprogresses to liver fibrosis, cirrhosis, and hepatocellular carci-noma (HCC)2 (1). The HCV genome is a 9.6-kb, positive-sense,single-stranded RNA molecule containing a 5� UTR, a singleopen reading frame, and a 3� UTR (2). The 5� UTR contains aninternal ribosome entry site that directs cap-independenttranslation of a polyprotein precursor of �3000 amino acidsthat is cleaved by viral proteases and host cell signal peptidasesinto mature structural proteins (core, E1, E2, and p7) and non-structural (NS) proteins (NS2, NS3, NS4A, NS4B, NS5A, andNS5B) (2).

The majority of HCV-infected individuals develop a persis-tent infection that promotes chronic inflammation, which isconsidered to be the primary catalyst for progressive liver dis-ease and development of HCC. Our recent work highlights amechanism of chronic inflammation through activation of theNLRP3 inflammasome in HCV-infected hepatoma cells (3). Inaddition, previous studies have shown activation of the NLRP3inflammasome in hepatic macrophages and monocytes (4 –7).Activation of the inflammasome is a major mechanism ofinflammation, leading to the production of proinflammatoryIL-1� and IL-18 cytokines via caspase-1 activation (8). Mostinflammasomes consist of a member of the NOD-like receptor(NLR) family of cytosolic receptors that either directly interactwith caspase-1 or are coupled indirectly to it by the adaptorprotein apoptosis-associated speck-like protein containing aCARD (ASC) and procaspase-1 (8). Activated caspase-1 pro-cesses pro-IL-1� and IL-18 into their mature forms. In chronicHCV infection, induction of proinflammatory molecules,including IL-1�, plays a central role in the pathogenesis of HCV(9, 10).

In addition to their role in IL-1� and IL-18 regulation,NLRP3, ASC, and caspase-1 are increasingly being recognizedto have inflammasome/cytokine-independent functions (11–

* This work was supported by National Institutes of Health Grant1R56AI089772– 01A1 from NIAID, the Rosalind Franklin University of Med-icine and Science, and the Bligh Cancer Research Fund (to G. W.). Theauthors declare that they have no conflicts of interest with the contents ofthis article. The content is solely the responsibility of the authors and doesnot necessarily represent the official views of the National Institutes ofHealth.

1 To whom correspondence should be addressed. Tel.: 847-578-8839; Fax:847-578-3349; [email protected].

2 The abbreviations used are: HCC, hepatocellular carcinoma; NS, nonstruc-tural; NLR, NOD-like receptor; HCV, hepatitis C virus; LD, lipid droplet;SREBP, sterol regulatory element-binding protein; ER, endoplasmic reticu-lum; SCAP, SREBP cleavage-activating protein; Insig, insulin-induced gene;Z-YVAD-fmk,benzyloxycarbonyl-YVAD-fluoromethylketone;ASC,apopto-sis-associated speck-like protein containing a CARD; FAS, fatty acid syn-thase; SCD, stearoyl-CoA desaturase; HMGCS, 3-hydroxy-3-methylglutaryl-coenzyme A synthase; PDI, protein disulfide isomerase.

crossmarkTHE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 291, NO. 7, pp. 3254 –3267, February 12, 2016

© 2016 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

3254 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 291 • NUMBER 7 • FEBRUARY 12, 2016

This article has been withdrawn by Steven McRae, Jawed Iqbal, MehuliSarkar-Dutta, Naushad Ali, and Gulam Waris. Samantha Lane and Abhiram

Nagaraj could not be reached. The ASC immunoblot from Fig. 1A was reused as HCV core in Fig. 1G. Additionally, these images were reused in Burdette et al. (2010) J. Gen. Virol. 91, 681-690, Burdette et al. (2012) J. Gen. Virol. 93, 235-246, Presser et al. (2013) PLOS One 8, e56367, and

Iqbal et al. (2014) PLOS One 9, e87464. The NLRP3 immunoblot in Fig. 1D was reused from Fig. 9 of Iqbal et al. (2013) J. Biol. Chem. 288, 36994-

37009, representing different experimental conditions. The actin immunoblot from Fig. 1D was also reused in Fig. 1C of Burdette et al. (2012) J. Gen. Virol. 93, 235-246, representing different experimental

conditions. The actin immunoblot from Fig. 1F was reused in Fig. 1G as actin. The Journal also determined that this immunoblot was reused in the

following publications representing different experimental conditions: Waris et al. (2003) J. Biol. Chem. 278, 40778-40787, Waris et al. (2005) J. Virol. 79, 1569-1580, Waris and Siddiqui (2005) J. Virol. 79, 9725-9734,

Waris et al. (2007) J. Virol. 81, 8122-8130, Nasimuzzaman et al. (2007) J. Virol. 81, 10249-10257, Burdette et al. (2010) J. Gen. Virol. 91, 681-690, Burdette et al. (2012) J. Gen. Virol. 93, 235-246, and Presser et al. (2013)

PLOS One 8, e56367. Gulam Waris does not agree that the actin immunoblot was reused in other publications. The SCD immunoblot from

Fig. 1F was reused in Fig.6A as actin. The mature SREBP-2 immunoblotfrom Fig. 3B was reused as SCD in Fig. 1G. Insig 2 of Fig. 7A was reused in

Fig. 7B as Insig 1. The actin immunoblot from Fig. 7A was reused in lanes 3 and 4 of the actin immunoblot in Fig. 7B. Columns f and g were duplicated

in Fig. 2A. Column a from Fig. 4C was duplicated in a of Fig. 5D. The withdrawing authors sincerely apologize to the readers.

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15). Recent studies have demonstrated that inflammasome-independent NLRP3 augments TGF-�1 signaling in the kidneyepithelium and cardiac fibroblasts (12, 13). NLRP3 is alsoknown to interact with ubiquitin ligase-associated proteinSGT1, heat shock protein 90 (HSP90), and thioredoxin-inter-acting protein (16, 17). Typically, caspase-1 mediates the mat-uration of IL-1� and IL-18 in immune and non-immune cells(18). However, studies have shown that several proteins associ-ated with the glycolytic pathway are cleaved by caspase-1,which is suggestive of a broader role of caspase-1 in addition tomaturation of IL-1� and IL-18 (19). Activation of caspase-1leads to pyroptosis of the cells infected with intracellular bac-teria (20). In contrast, the ability of caspase-1 to prevent hepa-tocyte death during redox stress by up-regulating beclin 1expression signifies its protective function in non-immune cells(11). Caspase-1 has also been shown to regulate the expressionof NF-�B target genes through caspase-7-mediated cleavage ofPARP1 (21). In addition, recent studies have implicatedcaspase-1 in cell survival by facilitating membrane biogenesisand cellular repair via regulation of lipid metabolism (22).

A unique feature of HCV is its absolute reliance on host lipidsin the various stages of the viral life cycle (23). To favor itsproliferation, HCV alters cellular lipid metabolism by stimulat-ing lipogenesis, impairing mitochondrial �-oxidation and cel-lular lipid export, and promoting a lipid-rich intracellular envi-ronment (23, 24). This alteration of lipid homeostasis results inthe intracellular accumulation of cellular lipid storage organ-elles, termed “lipid droplets” (LDs), that play crucial roles in theHCV life cycle, hepatic steatosis, and HCC (24 –26).

Sterol regulatory element-binding proteins (SREBPs) are themaster regulators of lipid homeostasis that activate the tran-scription of genes encoding enzymes involved in the biosynthe-sis of cholesterol, triglycerides, phospholipids, and fatty acids(27). Previously, we have shown the activation of SREBPs inHCV-infected human hepatoma cells (28). However, theunderlying mechanism by which HCV activates SREBPs is notclearly understood. To be active, SREBPs must be cleaved toproduce the active/mature forms. There are three SREBP iso-forms, designated SREBP-1a, SREBP-1c, and SREBP-2 (27).SREBP-1a activates all SREBP target genes, whereas SREBP-2and SREBP-1c activate genes involved in cholesterol and fattyacid synthesis, respectively (27). SREBPs are synthesized asendoplasmic reticulum (ER)-membrane-bound precursors andexist in complex with SREBP cleavage-activating protein(SCAP) (27). SCAP is both an escort for SREBPs and a sensor ofsterol. Retention of the SCAP-SREBP complex in the ER ismediated by the binding of SCAP to insulin-induced gene(Insig) proteins (29). Insig-1 and Insig-2 are membrane-boundproteins that reside in the ER and play a central role in theregulation of SREBP activation (30). When cells are depleted ofcholesterol, SCAP transports SREBPs from the ER to the Golgi,where site 1 proteases (S1Ps) and site 2 proteases (S2Ps) actspecifically and sequentially to release the active forms ofSREBPs, which actively translocate into the nucleus and bind tothe sterol response elements of the target genes.

In this study, we investigated the mechanism of increasedlipid biosynthesis in cells infected with HCV. Our studies showthat HCV-induced NLRP3 inflammasome activates SREBPs

and stimulates lipogenic gene expression and formation of LDs.Our results demonstrate that the proteolytic activation ofSREBPs in HCV-infected cells is mediated by interaction of theNLRP3 inflammasome with SCAP in the ER. We also demon-strate that caspase-1 activity is critical for SREBP activation.Collectively, these observations provide insights into the novelrole of the NLRP3 inflammasome in lipid homeostasis duringchronic HCV infection.

Experimental Procedures

Plasmids and Reagents—The infectious HCV J6/JFH-1cDNA (genotype 2a) and the replication-defective HCV JFH-1/GND constructs were obtained from Dr. C. Rice (RockefellerUniversity, NY). Recombinant IL-1� was purchased from R&DSystems (Minneapolis, MN). The wild-type human pFLAG-NLRP3 expression vector was obtained from Dr. J. Tschopp(University of Lausanne, Switzerland). MG132, ALLM (N-Acetyl-Leu-Leu-Met-CHO), and inhibitors of caspase-1 (Z-YVAD-fmk), and caspase-3 (DEVD) were from EMD Millipore(Massachusetts, MA). BODIPY (4,4-difluoro-4-bora-3a,4a-diaza-s-indacene) was purchased from Invitrogen.

Antibodies—The following antibodies were used accordingto the protocols of the manufacturer: HCV NS3 (Virogen,Watertown, MA); actin and �-tubulin (Sigma); NLRP3 forWestern blotting (Abcam, Cambridge, MA) and for immuno-fluorescence (ProSci, Atlanta, GA); ASC (MBL, Woods Hole,MA); caspase-1 (Invitrogen); SREBP-1, SREBP-2, Insig-1,Insig-2, and SCAP (Santa Cruz Biotechnology, Dallas, TX); andalbumin (Thermo Scientific, Rockford, IL). The rabbit poly-clonal antibody against HCV NS5A was a gift from Dr. CraigCameron (Pennsylvania State University). The organelle local-ization immunofluorescence antibody sampler kit was fromCell Signaling Technology (Danvers, MA).

Cell Culture—The human hepatoma cell line Huh-7.5 wasobtained from Dr. C. Rice (31). Huh-7.5 cells were cultured at37 °C in a humidified atmosphere containing 5% CO2 withDMEM supplemented with 10% fetal calf serum, 100 units ofpenicillin/ml, and 100 �g of streptomycin sulfate/ml.

HCV Cell Culture Infection System—Fifteen micrograms ofin vitro transcribed J6/JFH-1 RNA was delivered into Huh-7.5cells by electroporation as described previously (3, 28, 32). Cellswere passaged every 3–5 days. The presence of HCV in thesecells and the corresponding supernatants was determined asdescribed previously (33). The cell-free virus was propagated inHuh7.5 cell culture as described previously (32–34). Theexpression of HCV protein in HCV-infected cells was analyzedby Western blotting. The HCV cell culture supernatant wascollected at appropriate time points and used to infect naïveHuh7.5 cells at a multiplicity of infection of 1 for 5– 6 h at 37 °Cand 5% CO2 (32, 33). The viral titer in the cell culture superna-tant was expressed as focus forming units per milliliter, whichwas determined by the average number of HCV-NS5A-positivefoci detected at the highest dilutions, as described previously(33). The cell culture supernatant collected from Huh7.5 cellsexpressing JFH-1/GND (replication-defective virus) was usedas a negative control.

Preparation of Nuclear Extracts—Nuclear lysates were pre-pared from mock and HCV-infected cells. Cells were lysed in

HCV-activated NLRP3 Inflammasome Regulates Lipid Metabolism

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hypotonic buffer (20 mM HEPES (pH 7.9), 10 mM KCl, 0.1 mM

Na3VO4, 1 mM EDTA, 10% glycerol, 1 mM PMSF, 3 mg/mlaprotinin, 1 mg/ml pepstatin, 20 mM NaF, and 1 mM DTT with0.2% Nonidet P-40) on ice for 15 min. After centrifugation at4 °C (13,000 rpm) for 1 min, the nuclear pellet was resuspendedin high-salt buffer (hypotonic buffer with 20% glycerol and 420mM NaCl) at 4 °C by rocking for 30 min after centrifugation.The supernatant was collected and stored at �80 °C in aliquots.

Immunoprecipitation and Western Blotting Analysis—Cellu-lar lysates from mock- and HCV-infected cells were preparedby incubation in radioimmune precipitation assay buffer (50mM Tris (pH 7.5), 150 mM NaCl, 1% Nonidet P-40, 0.5% sodiumdeoxycholate, 0.1% SDS, 1 mM sodium orthovanadate, 1 mM

sodium formate, and 10 �l/ml protease inhibitor mixture(Thermo Scientific) for 30 min on ice. Equal concentrations ofcellular lysates were immunoprecipitated with the indicatedantibodies overnight at 4 °C. The immune complexes wereincubated with protein A-Sepharose (Invitrogen) for 1 h at 4 °C,washed three to four times with radioimmune precipitationassay buffer, and boiled for 5 min in SDS-containing samplebuffer. The samples were then subjected to SDS-PAGE. Gelswere electroblotted onto a nitrocellulose membrane (ThermoScientific) in 25 mM Tris, 192 mM glycine, and 20% methanol.Membranes were incubated overnight in blocking buffer (20mM Tris/HCl (pH 7.5), 150 mM NaCl, and 5% nonfat dry milk)and probed with primary antibody of interest for 1 h at roomtemperature. The membranes were then washed three timesfor 10 min in Tris-buffered saline with 1% Tween 20 (TBS-T),followed by incubation with secondary antibody for 45 min atroom temperature. After an additional washing cycle withTBS-T, the immunoblots were visualized using the LICOROdyssey system.

Laser-scanning Confocal Microscopy—Mock- and HCV-in-fected cells on coverslips were washed with PBS, fixed with 4%paraformaldehyde for 10 min at room temperature, permeabi-lized for 5 min with 0.2% Triton X-100, and blocked for 45 minwith 5% bovine serum albumin in PBS. The cells were thenincubated with primary antibody against the specific proteinfor 1 h at room temperature or overnight at 4 °C, followed byincubation with Alexa Fluor-labeled secondary antibodies(Invitrogen) for 1 h. After washing with PBS, cells weremounted with anti-fade reagent containing DAPI (Invitrogen)and observed under a laser-scanning confocal microscope (Flu-oview FV10i).

Immunohistochemistry—Liver biopsies from normal andHCV-associated cirrhosis and HCC (no history of hepatitis Bvirus, HIV infection, and fatty liver) were obtained from theLiver Tissue Cell Distribution System (University of Minne-sota, Minneapolis, MN). Immunohistochemistry was per-formed according to the protocol of the manufacturer using theLeica BOND-IIITM polymer refined detection system (DS9800) at the Stephenson Cancer Center Pathology core labora-tory (University of Oklahoma Health Sciences Center, Institu-tional Review Board (IRB) Number 3405). The tissue sectionsfrom normal and HCV-associated cirrhosis and HCC weredeparaffinized and rehydrated in an automated multistainer(Leica ST5020). The tissue section slides were subjected to anti-gen retrieval at 100 °C for 20 min in a retrieval solution, fol-

lowed by incubation in blocking solution for 1 h. The sectionswere stained with primary antibody for 1 h, followed by thesecondary antibody (poly-HRP IgG). The detection was per-formed using 3,3�-diaminobenzidine tetrachloride, and coun-terstaining was done with hematoxylin. For double-staining,the Leica BOND-IIITM polymer refined detection system (DS9800) and Leica BOND-IIITM refined red detection system (DS9390) were used sequentially. For Western blotting analysis,frozen liver tissues were thawed in radioimmune precipitationassay buffer and crushed gently, followed by sonication andincubation on ice for 30 min. Samples were centrifuged at 4 °C,and the supernatant was collected.

Silencing of Target Gene Expression—Mock- and HCV-in-fected cells on day 2 were transfected with siRNA targetedagainst control (sicontrol), siNLRP3, siASC, and sicaspase-1according to the protocols of the manufacturers (Santa CruzBiotechnology and Qiagen). Each siRNA consisted of pools ofthree to five target-specific, 19- to 25-nt siRNA designedto knock down target gene expression. For sicontrol andsicaspase-1 transfections, two solutions were prepared. Forsolution A, 60 pmol of siRNA duplex was mixed with 100 �l ofsiRNA transfection medium. For solution B, 6 �l of transfectionreagent was added to 100 �l of siRNA transfection medium.Solutions A and B were allowed to incubate at room tempera-ture for 20 min. After 20 min, solutions A and B were combinedand allowed to incubate for another 20 min at room tempera-ture. The combined solutions were then added to the cells in6-well plates and incubated for 5 h at 37 °C and 5% CO2. Thenthe transfection solution was replaced with 2 ml of completeDMEM.

siASC was transfected according to the protocol of the man-ufacturer (Qiagen). 256 ng of the siRNA duplex was diluted in100 �l of serum-free medium along with 20 �l of HiPerFecttransfection reagent. The solution was allowed to incubate atroom temperature for 10 min. The transfection solution wasthen added to the cells, and the cells were harvested at differenttime points.

Quantitative RT-PCR—Total cellular RNA was extractedfrom mock- and HCV-infected cells using TRIzol (Invitrogen)and treated with RQ1 RNase-free DNase prior to cDNA syn-thesis. The cDNA was reverse-transcribed from 1 �g of totalRNA using a reverse transcription kit (Life Technologies).Quantitative RT-PCR was carried out using SYBR Green Mas-ter Mix (Life Technologies) and specific primers as describedpreviously (3, 28, 31). Amplification reactions were performedunder the following conditions: 2 min at 50 °C, 10 min at 95 °C,40 cycles for 10 s at 95 °C, and 1 min at 60 °C. Relative transcriptlevels were calculated using the ��Ct method as specified bythe manufacturer.

Cell Viability Assay—Mock-infected cells (Huh7.5), HCV-infected cells, and HCV-infected cells transfected with varioussiRNA or treated with caspase-1 and caspase-3 inhibitors wereplaced in a 96-well plate. The cells were lysed, and ATP wasquantitated according to the instructions of the manufacturerusing the CellTitre-Glo luminescent cell viability assay kit (Pro-mega). The percent viability was calculated considering 100%viability for mock cells. The values represent the mean � S.D. ofthree independent experiments performed in duplicate.




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Statistical Analysis—Error bars show mean � S.D. of datafrom three individual experiments. Two-tailed unpaired t testswere used to compare experimental conditions with those ofthe respective controls. In all tests, p � 0.05 was consideredstatistically significant.

Results

The NLRP3 Inflammasome Induces Lipogenesis in HCV-infectedCells—We have demonstrated previously that HCV stimulateslipogenesis by activating SREBPs (28). However, the underlyingmechanism(s) by which HCV activates SREBPs is not clearlyunderstood. Recently, studies have shown the role of the inflam-masome complex in cell survival by facilitating membrane biogen-esis and cellular repair via regulation of lipid metabolism (22). Todetermine whether HCV induces lipogenesis through the activa-tion of the NLRP3 inflammasome, HCV-infected Huh7.5 cellswere transfected with siRNA against each component of theinflammasome complex (i.e. siNLRP3, siASC, and sicaspase-1) orscrambled siRNA (sicontrol). Equal amounts of cellular lysateswere subjected to immunoblot analysis. A marked reduction in theexpression of NLRP3, ASC, and caspase-1 protein levels suggestsefficient knockdown of these proteins by their correspondingsiRNA. The specificity of siRNA activity was indicated by the factthat control siRNA did not inhibit the expression of these proteins(Fig. 1, A–C). HCV-infected cells transfected with siNLRP3specifically down-regulated the expression of NLRP3 but not theother component (ASC) of the inflammasome complex (Fig. 1A,lane 4). This is also true for HCV-infected cells transfected withsiASC (Fig. 1B, lane 4). In addition, HCV-infected cells transfectedwith independent siNLRP3 duplexes show similar inhibition in theexpression of NLRP3 (Fig. 1D). The cell viability assay was per-formed in the above siRNA-transfected cells. We did not observeany significant change in ATP levels under various conditions (Fig.1E).

To determine the role of the NLRP3 inflammasome in lipo-genic gene expression, lysates from mock- and HCV-infectedHuh7.5 cells silenced with siNLRP3, siASC, and sicaspase-1were subjected to immunoblot analysis. The results showincreased expression of fatty acid synthase (FAS) and stearoyl-CoA desaturase (SCD) in HCV-infected cells that were reducedsignificantly in cells transfected with siNLRP3, siASC, andsicaspase-1 compared with sicontrol (Fig. 1F, lanes 3– 6). Fur-thermore, we also analyzed the expression of FAS and SCD inthe presence of inhibitors of caspase-1 and caspase-3 (negativecontrol). Our results showed significantly reduced expressionof FAS and SCD in HCV-infected cells treated with caspase-1inhibitor compared with caspase-3 inhibitor (Fig. 1F, lanes 7and 8). In addition, we also observed a significant reduction inthe expression of SCD in HCV-infected cells transfected withsiNLRP3#2, suggesting that siNLRP3#1 and #2 produce similarphenotypes and not likely to be the off-target effects of thesesiRNA (Fig. 1G). The effect of silencing of NLRP3 on SCDexpression was rescued by siRNA-resistant ectopic expressionof NLRP3 (pFLAG-NLRP3del) (Fig. 1H, lane 4).

To determine whether NLRP3 inflammasome-mediatedinduction of lipogenic genes was due to increased expressionof their mRNA, total cellular RNA from the above siRNA-transfected cells were subjected to quantitative RT-PCR using

3-hydroxy-3-methylglutaryl-coenzyme A synthase (HMGCS)-,FAS-, and SCD-specific primers. We observed significantlyhigher expression of FAS mRNA (�12-fold), HMGCS mRNA(�9-fold), and SCD mRNA (�7-fold) in HCV-infected cellsthat was decreased in cells transfected with siNLRP3, siASC,and sicaspase-1 compared with sicontrol (Fig. 1I). In addition,treatment with caspase-1 inhibitor (Z-YVAD-fmk) but notwith caspase-3 inhibitor (DEVD) also blocked HCV-inducedlipogenic gene expression (Fig. 1I). Taken together, theseresults suggest that HCV-infected cells induce lipogenic genesthrough activation of the NLRP3 inflammasome and are depen-dent on caspase-1 activity.

The HCV-activated NLRP3 Inflammasome Induces LDsFormation—To determine the role of the HCV-induced NLRP3-inflammasome in LDs formation, mock- and HCV-infectedHuh7.5 cells transfected with siNLRP3, siASC, sicaspase-1, andsicontrol were stained with the neutral lipid-specific green flu-orescent dye BODIPY 493/503. The results show increasedstaining of LDs in HCV-infected cells compared with mock-infected cells (Fig. 2A, a and b). In contrast, LDs were reducedsignificantly in HCV-infected cells transfected with siNLRP3,siASC, and sicaspase-1 compared with sicontrol (Fig. 2A, c–f).To determine the effect of caspase-1 activity on LD formation,HCV-infected cells were incubated with caspase-1 inhibitor.The increased LDs in HCV-infected cells were reduced in cellstreated with caspase-1 inhibitor but not with caspase-3 inhibi-tor (Fig. 2A, g and h). Furthermore, treatment of Huh7.5 cellswith recombinant IL-1� did not result in accumulation of LDs(Fig. 2A, i), suggesting that this event is not mediated by IL-1�signaling and is probably a consequence of events upstream ofinflammatory cytokine production. These results suggest thatthe activation of the NLRP3 inflammasome stimulates forma-tion of LDs in HCV-infected cells.

To determine the sequence of NLRP3 inflammasome activa-tion and LD formation in HCV-infected cells, we analyzed theactivation of caspase-1 and staining of LDs at various timepoints. Our results suggest that HCV induces activation of theNLRP3 inflammasome, which is followed by lipogenesis and LDaccumulation in HCV-infected cells (data not shown).

The NLRP3 Inflammasome Activates SREBPs in HCV-in-fected Cells—SREBPs are known to regulate cholesterol andfatty acid biosynthesis pathways (27, 29). To determine whetherthe master inducers of lipid metabolism, SREBP-1 and SREBP-2, are regulated by the NLRP3 inflammasome complex in HCV-infected cells, total cellular lysates from mock- and HCV-in-fected cells transfected with siNLRP3, siASC, sicaspase-1, andsicontrol were analyzed by Western blotting. We observed pro-teolytic cleavage of SREBP-1 and SREBP-2 in HCV-infectedcells compared with mock-infected cells (Fig. 3, A and B, lanes 1and 2) that were reduced in cells silenced with siNLRP3, siASC,and sicaspase-1 but not in sicontrol cells (Fig. 3, A and B, lanes3– 6). To determine the role of caspase-1 activity on SREBP-1and SREBP-2 proteolytic activation, mock- and HCV-infectedcells were incubated with inhibitors of caspase-1 and caspase-3.Our results show significantly reduced activation of SREBP-1and SREBP-2 in the presence of caspase-1 inhibitor but notcaspase-3 inhibitor (Fig. 3, C and D, lanes 3 and 4). These resultssuggest the role of the NLRP3 inflammasome-mediated




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caspase-1 in HCV-induced proteolytic cleavage of SREBP-1and SREBP-2 into their mature forms. To further demonstratethe activation and nuclear translocation of the mature forms of

SREBPs in HCV-infected cells, cytoplasmic and nuclear lysateswere subjected to Western blotting. The results show theinduction of precursor SREBP-1 in HCV-infected cytoplasmic




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FIGURE 1. The HCV-activated NLRP3 inflammasome induces lipogenic gene expression. Mock- (Huh7.5) and HCV-infected cells (infected with HCV at amultiplicity of infection of 1 for 2 days) were transfected with sicontrol, siNLRP3, siASC, and sicaspase-1. 72 h post-transfection, cellular lysates were subjectedto Western blotting using the respective antibodies. A, equal amounts of cellular lysates from mock-, HCV-, and HCV-infected cells transfected with siNLRP3were immunoblotted with anti-NLRP3 and anti-ASC antibodies. B, cellular lysates from HCV-infected cells transfected with siASC were immunoblotted withanti-ASC and anti-NLRP3 antibodies. C, HCV-infected cells were transfected with sicontrol and sicaspase-1. D, HCV-infected cells were transfected with sicontroland two different individual siNLRP3 duplexes (siNLRP3#1 and siNLRP3#2) that were present in the siRNA pool used above (Santa Cruz Biotechnology). Actinrepresents HCV infection. E, mock- (Huh7.5), HCV-, and HCV-infected cells transfected with sicontrol, siNLRP3, siASC, and sicaspase-1 at various time points ortreated with caspase-1 and caspase-3 inhibitors were placed in a 96-well plate. The cells were lysed, and ATP was quantitated according to the instructions ofthe manufacturer using a CellTitre-Glo luminescence cell viability assay kit (Promega). The percent viability was calculated considering 100% viability formock-infected cells compared with HCV-infected cells transfected with various siRNA or treated with caspase-1/-3 inhibitors. The values represent mean � S.D.of three independent experiments performed in duplicate. F, equal amounts of cellular lysates from mock- and HCV-infected cells transfected with siNLRP3,siASC, and sicaspase-1 were subjected to Western blotting using anti-FAS and anti-SCD antibodies. Lane 1, mock cells; lane 2, HCV-infected cells; lanes 3– 6,HCV-infected cells transfected with sicontrol, siNLRP3, siASC, and sicaspase-1, respectively; lanes 7 and 8, HCV-infected cells treated with inhibitors of caspase-1(50 �M Z-YVAD-fmk for 2 h) and caspase-3 (100 �M DEVD for 2 h); right panel (lanes 9 and 10), basal level expression of FAS in mock cells. G, equal amounts ofcellular lysates from mock- and HCV-infected cells transfected with sicontrol, siNLRP3#1, and siNLRP3#2 were subjected to Western blotting using anti-SCDantibodies. Lane 1, mock cells; lane 2, HCV-infected cells; lanes 3–5, HCV-infected cells transfected with sicontrol, siNLRP3#1 and siNLRP3#2, respectively. H,rescue of NLRP3 gene silencing. The wild-type NLRP3-expressing plasmid (pFLAG-NLRP3wt) and the plasmid expressing siRNA-resistant mRNA containing adeletion of the 3� UTR of NLRP3 (pFLAG-NLRP3del) along with siNLRP3 were transfected in HCV-infected cells. The pFLAG-NLRP3del expression plasmid wasgenerated using a site-directed mutagenesis kit according to the protocols of the manufacturer (Stratagene). Cellular lysates were subjected to Westernblotting using the respective antibodies. The siNLRP3 target sequence was 5�-CACGCTAATGATCGACTTCAA-3� (Qiagen). I, total cellular RNA was extracted frommock- and HCV-infected cells transfected with the above siRNA and subjected to quantitative RT-PCR using FAS-, HMGCS-, and SCD-specific primers and a SYBRGreen probe. The values represent mean � S.D. of three independent experiments performed in triplicate. *, p � 0.05 compared with mock-infected Huh7.5cells; **, p � 0.05 compared with sicontrol-transfected cells; ***, p � 0.05 compared with HCV-infected cells treated with the caspase-3 inhibitor (DEVD).

FIGURE 2. The NLRP3 inflammasome induces LD formation in HCV-infected cells. A, mock- (Huh7.5), HCV-, and HCV-infected cells were transfected withsicontrol, siNLRP3, siASC, or sicaspase-1; incubated with 50 �M caspase-1 inhibitor (Z-YVAD-fmk), 100 �M caspase-3 inhibitor, or negative control (DEVD) for 2 hat 37 °C; and incubated with recombinant IL-1� (20 ng/ml for 24 h). These cells were fixed with 4% paraformaldehyde, permeabilized with 0.2% Triton X-100,and incubated with 20 �M BODIPY 493/503 (Invitrogen) for 30 min, followed by incubation with anti-HCV NS5A antibodies (red fluorescence). Cells werevisualized under a laser-scanning confocal microscope (Fluoview FV1Oi). DAPI was used as a nuclear stain. B, LDs were quantified manually in 15 individual cellimages under various conditions using ImageJ software.




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lysates and the presence of a significant amount of matureSREBP-1 in the nuclear lysates (Fig. 3C, lanes 2 and 4). In con-trast, we did not detect any mature SREBP-1 in nuclear lysatesof mock-infected cells (Fig. 3C, lane 3).

It is well established that the mature forms of SREBPs trans-locate into the nucleus and bind to the SRE of the target genes(27). To determine whether the translocation of mature formsof SREBP-1 and SREBP-2 into the nucleus is regulated by theNLRP3 inflammasome, mock- and HCV-infected cells, asdescribed in Fig. 2, were subjected to immunofluorescence. Theresults show significant nuclear translocation of mature SREBP-1 and SREBP-2 in HCV-infected cells compared with mock-infected cells (Fig. 4, A and B). In contrast, we observed reducedtranslocation of mature SREBP-1 and SREBP-2 in HCV-in-fected cells transfected with siNLRP3, siASC, and sicaspase-1or incubated with caspase-1 inhibitor but not with sicontrol ortreated with caspase-3 inhibitor (Fig. 4, A and B). Takentogether, these results suggest that the NLRP3 inflammasome

in HCV-infected cells plays a critical role in the activation andnuclear translocation of SREBPs. To demonstrate that SREBP-1is transported to the Golgi during HCV infection, mock- andHCV-infected cells were stained with anti-SREBP-1 and anti-RCAS1 (a Golgi marker) and subjected to confocal microscopy.The results show a significant association of SREBP-1 with theGolgi in HCV-infected cells (Fig. 4C, yellow spots) comparedwith mock cells. In addition, we also observed a significantmigration of the mature form of SREBP-1 into the nucleus ofthe HCV-infected cells (Fig. 4C).

The NLRP3 Inflammasome Colocalizes with SCAP in HCV-in-fected Cells—SCAP transports SREBPs from the ER to the Golgi.To determine whether the NLRP3 inflammasome interacts withSCAP and facilitates the transport of SREBPs from the ER to theGolgi, we performed confocal microscopy. The results show sig-nificant colocalization of NLRP3, ASC, and caspase-1 with SCAPin HCV-infected cells compared with mock-infected cells (Fig. 5,A–C, b, yellow dots). These results suggest the interaction of the

FIGURE 3. The NLRP3-inflammasome induces proteolytic activation of SREBP-1 and SREBP-2 in HCV-infected cells. A and B, cellular lysates from mock-or HCV-infected cells and cells transfected with siNLRP3, siASC, and sicaspase-1 were subjected to Western blotting using anti-SREBP-1 and anti-SREBP-2antibodies. Lane 1, mock-infected cells; lane 2, HCV-infected cells; lanes 3– 6, HCV-infected cells transfected with sicontrol, siNLRP3, siASC, and sicaspase-1,respectively. C and D, mock- and HCV-infected cells were treated with caspase-1 inhibitor (50 �M Z-YVAD-fmk) and caspase-3 inhibitor (100 �M DEVD) for 2 hat 37 °C. Cellular lysates were subjected to immunoblotting using anti-SREBP-1 and anti-SREBP-2 antibodies. Lane 1, mock-infected cells; lane 2, HCV-infectedcells; lanes 3 and 4, HCV-infected cells treated with inhibitors of caspase-1 and caspase-3 as described above. Actin and tubulin represent protein loadingcontrols. E, cytoplasmic and nuclear extracts from mock- and HCV-infected cells were subjected to Western blotting using anti-SREBP-1 antibody. Lanes 1 and3, cytoplasmic and nuclear extracts from mock-infected cells; lanes 2 and 4, cytoplasmic and nuclear extracts from HCV-infected cells. Tubulin and TATA-binding protein (TBP) represented protein controls. NS3 represented HCV infection.




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NLRP3 inflammasome with SCAP. To determine whether theNLRP3 inflammasome associates with the ER, colocalizationof NLRP3, ASC, and caspase-1 was performed with an ER marker,PDI. Mock- and HCV-infected cells were stained with anti-NLRP3, anti-ASC, anti-caspase-1, ER marker protein (anti-PDI),Golgi (anti-RCAS1), endosome (anti-EEA1), and lysosome (anti-LAMP1). The results with anti-PDI antibodies show significant

yellow dots, indicating an association of NLRP3, ASC, andcaspase-1 primarily with the ER (Fig. 5, D–F). However, we did notobserve any colocalization of NLRP3 with endosome and lyso-some markers except weak colocalization with the Golgi marker(Fig. 5, G–I). Collectively, these results clearly suggest the associa-tion of the NLRP3 inflammasome with the ER in HCV-infectedcells.

FIGURE 4. The HCV-activated NLRP3 inflammasome mediates nuclear translocation of SREBP-1 and SREBP-2. A and B, mock-infected cells, HCV-infectedcells, and HCV-infected cells silenced with siNLRP3, siASC, sicaspase-1, and sicontrol or treated with inhibitors of caspase-1 (Z-YVAD-fmk) and caspase-3 (DEVD)were fixed and permeabilized as described in Fig. 2. The cells were incubated with anti-SREBP-1 and anti-SREBP-2 antibodies for 1 h at room temperature,followed by incubation with secondary antibodies for SREBP-1 (goat anti-mouse Alexa Fluor 488) and SREBP-2 (donkey anti-goat Alexa Fluor 488). DAPI wasused as a nuclear stain. Arrows represent staining of SREBP-1 and SREBP-2. C, Mock and HCV-infected cells were incubated with anti-SREBP-1, anti-RCAS, andtheir secondary antibodies as described above. Arrows represent colocalization of SREBP-1 with the Golgi (yellow dots).




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Association of the NLRP3 Inflammasome with SCAP—Theassociation of NLRP3, ASC, and caspase-1 with SCAP was alsoconfirmed by a protein-protein interaction approach. Cellular

lysates from mock- and HCV-infected cells were immunopre-cipitated with anti-SCAP, followed by Western blotting usinganti-NLRP3, anti-caspase-1, and anti-SCAP antibodies. The

FIGURE 5. Colocalization of NLRP3, ASC, and caspase-1 with SCAP. A–C, mock- and HCV-infected cells were fixed, permeabilized, and incubated withanti-SCAP, anti-NLRP3, anti-ASC, and anti-caspase-1 antibodies for 1 h at room temperature, followed by incubation with secondary antibodies (SCAP,anti-goat Alexa Fluor 488; NLRP3, anti-mouse Alexa Fluor 546; ASC, anti-mouse Alexa Fluor 546; caspase-1, anti-mouse Alexa Fluor 546; NS5A, anti-rabbit AlexaFluor 633). Arrows represent colocalization of SCAP with NLRP3, ASC, and caspase-1 (b). D–F, subcellular localization of the NLRP3 inflammasome complex.Mock- and HCV-infected cells were fixed, permeabilized, and incubated with ER marker protein (anti-PDI), anti-NLRP3, anti-ASC, anti-caspase-1, and anti-HCVNS5A for 1 h at room temperature. The cells were incubated with secondary antibodies (PDI, goat anti-rabbit Alexa Fluor 488; NLRP3, anti-mouse Alexa Fluor546; ASC, anti-mouse Alexa Fluor 546; caspase-1, anti-mouse Alexa Fluor 546; NS5A, anti-rabbit Alexa Fluor 633). DAPI was used as a nuclear stain. HCV NS5Arepresented HCV infection. Arrows indicate colocalization of proteins. G–I, mock- and HCV-infected cells were incubated with antibodies against the markerproteins of endosomes (anti-EEA1), lysosomes (anti-LAMP1), Golgi (anti-RCAS1), anti-NLRP3, and anti-NS5A, followed by their secondary antibodies. DAPI wasused as a nuclear stain. Arrows indicate colocalization of proteins.




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results showed that SCAP was pulled down with NLRP3 andcaspase-1 in HCV-infected cells compared with mock-infectedcells (Fig. 6A, lanes 3 and 4). However, immunoprecipitation ofHCV-infected lysates with an isotype control antibody did notpull down NLRP3 and caspase-1 (Fig. 6A, lane 5). We could notshow the expression of ASC during immunoprecipitation withSCAP because the banding pattern of the IgG light chain over-lapped with ASC (26 kDa). The interaction of the NLRP3inflammasome with SCAP was further confirmed by reciprocalco-immunoprecipitation using anti-NLRP3, anti-ASC, andanti-caspase-1 antibodies. We observed that NLRP3, ASC, andcaspase-1 were pulled down with SCAP in HCV-infected cellscompared with mock-infected cells but not with an isotype con-trol antibody (Fig. 6B, lanes 3–10). Collectively, these resultssuggest that the NLRP3 inflammasome interacts with SCAP inHCV-infected cells.

The NLRP3 Inflammasome Induces Degradation of Insig Pro-teins in HCV-infected Cells—Because Insigs are ER-residentproteins and play an important role in the activation ofSREBP-1 and SREBP-2, we examined the status of Insig-1 andInsig-2 proteins in HCV-infected cells. Mock- and HCV-in-fected cellular lysates were subjected to Western blotting usinganti-Insig-1 and anti-Insig-2 antibodies. The results showedreduced expression of Insig-1 and Insig-2 expression in HCV-infected cells compared with mock-infected cells (Fig. 7A, lane2). However, we did not observe any change in the expression ofSCAP. Previously, it has been demonstrated that the dissocia-tion of Insig from the ER retention complex leads to protea-

some-mediated degradation of Insig (30). Our results clearlyshowed the degradation of Insig-1 and Insig-2 in HCV-infectedcells, which was blocked by proteasome inhibitor but not bycalpain inhibitor (negative control) (Fig. 7B, lanes 3 and 4), sug-gesting that Insig-1 and Insig-2 play critical roles in SREBPactivation in HCV-infected cells.

To determine whether the interaction of the NLRP3 inflam-masome/caspase-1 with SREBP activation machinery (SREBP-SCAP-Insig complex) mediates the degradation of Insig-1 andInsig-2, mock- and HCV-infected cells were silenced withsicaspase-1. The cellular lysates were analyzed by Western blot-ting. The results show degradation of Insig-1 in HCV-infectedcells that was blocked in cells silenced with sicaspase-1 but notin sicontrol cells (Fig. 7C, lanes 3 and 4). In addition, weobserved significant silencing (68%) of caspase-1 expression inHCV-infected cells (Fig. 7C, lane 4). These results suggest a roleof the caspase-1�inflammasome complex in HCV-mediateddegradation of Insig proteins.

HCV Activates Caspase-1 in Hepatocytes of HCV-positiveLiver Tissues—In this study, we examined caspase-1 activationas a readout of NLRP3-inflammasome activation in HCV-pos-itive liver tissues. Caspase-1 is an effector molecule of theinflammasome complex (8, 18). We examined liver tissues fromHCV-positive patients with cirrhosis (five cases) and HCC (fourcases) to validate the expression and activation of caspase-1 incell culture studies (3). Normal and HCV-positive patient liver

FIGURE 6. NLRP3, ASC, and caspase-1 interact with SCAP in HCV-infectedcells. A, Equal amounts (300 �g) of cellular lysates from mock- and HCV-infected cells were immunoprecipitated (IP) with anti-SCAP or isotype controlantibodies (normal goat IgG) and immunoblotted with anti-NLRP3, anti-caspase-1, and anti-SCAP antibodies. IgG control immunoprecipitation isonly shown for HCV-infected lysates because we did not see NLRP3 andcaspase-1 expression in mock-infected (Huh7.5) cells. WB, Western blotting.B, in reciprocal experiments, lysates (300 �g) were immunoprecipitated withanti-NLRP-3, anti-ASC, anti-caspase-1, and isotype control antibodies (normalmouse IgG) and immunoblotted with anti-SCAP antibody.

FIGURE 7. The HCV-activated NLRP3 inflammasome mediates degrada-tion of Insig proteins. A, lysates from mock- and HCV-infected cells wereimmunoblotted using anti-SCAP, anti-Insig-1, anti-Insig-2, anti-NS3, and anti-Actin antibodies. B, mock- and HCV-infected cells were incubated withMG132 (20 �M for 6 h) and ALLM (100 �M for 12 h) and subjected to Westernblotting using anti-Insig-1 and anti-Insig-2 antibodies. C, mock- and HCV-infected cells were transfected with sicontrol and sicaspase-1. Equal amountsof cellular lysates were immunoblotted using anti-Insig-1 and anti-caspase-1antibodies. Tubulin and actin were used as protein loading controls.




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tissues were subjected to immunohistochemical staining forcaspase-1. The representative results are shown in Fig. 8A.Strong caspase-1 expression was detected in HCV-positive livertissues compared with normal tissues (Fig. 8A, b and c, brownspots). In addition, a subpopulation of mature hepatocytes iden-tified by staining for human albumin (red) were also positive foractive caspase-1 only in HCV-positive patients liver tissues (Fig.8B, b and c). However, similar co-staining was clearly absent innormal liver tissues (Fig. 8B, a). To further confirm the stainingresults, liver tissue lysates were subjected to Western blotting,and caspase-1 bands were analyzed. The results showed activa-tion of caspase-1 in two liver tissue samples (used in Fig. 8C, band c) derived from HCV-positive patients compared with nor-

mal healthy individuals (Fig. 8C, lanes 2 and 3). Collectively,these results confirmed the activation of caspase-1 in HCV-positive human liver tissues.

Discussion

In recent years, activation of the inflammasomes has beenimplicated in various chronic diseases and in the clearance ofseveral viruses (35–38). However, the role of the inflammasomecomplex in HCV pathogenesis is incompletely understood. Inaddition to various infections, abnormal lipid metabolism hasbeen strongly linked to chronic inflammation in a mouse obe-sity model (39). Recent studies have implicated the inflam-masome complex/caspase-1 in cell survival by facilitatingmembrane biogenesis and cellular repair via regulation of lipidmetabolism (22). Consistent with this observation, our studiesclearly provide a link between chronic inflammatory pathwaysand host lipid metabolism during HCV infection. We show thatactivation of the NLRP3 inflammasome in HCV-infected cellscauses the activation of SREBPs and induces lipogenesis and LDformation, cellular events critical for HCV proliferation andliver disease pathogenesis associated with chronic HCV.

In this study, we show the activation of caspase-1, the effectormolecule of NLRP3-inflammasome, in human hepatoma cellsand in the hepatocytes of liver biopsies of chronic HCVpatients. Our findings are consistent with studies from othergroups demonstrating activation of the NLRP3 inflammasomein isolated hepatocytes from liver samples of patients withchronic hepatitis C (11, 40). These studies clearly establishedthe potential of HCV to activate the NLRP3 inflammasome inhepatocytes infected with HCV.

HCV has also been shown to activate the NLRP3 inflam-masome in hepatic macrophages and monocytes (4 –7). How-ever, in these reports, activation of the NLRP3 inflammasomein human hepatoma cells or primary hepatocytes by HCV wasnot observed. The failure to observe inflammasome activationcould be due to infection with a low multiplicity of infection of0.1 and reliance on the detection of mature forms of IL-1� andIL-18 in cell culture supernatants. Recent in vivo studies haveshown that non-immune cells, such as hepatocytes, express andactivate the inflammasome complex but do not secrete ade-quate/detectable amounts of IL-1� and IL-18 compared withimmune cells, suggesting that activation of the inflammasomecomplex in epithelial cells is likely to be involved in cytokine-independent functions (11–15). Our findings suggest that,unlike in immune cells, in human hepatocytes (epithelial cells),HCV modulates the NLRP3 inflammasome differently accord-ing to its specific niche to alter lipid metabolism, leading to LDaccumulation and liver disease pathogenesis (Fig. 9). NLRP3 isknown to interact with several proteins to modulate variouscellular functions (12–17). Apart from cleavage/maturation ofIL-1� and IL-18, caspase-1 has been shown to cleave severalproteins, suggesting a broader role of the NLRP3 inflam-masome/caspase-1 in addition to maturation of cytokines (19).

Our results show that the induction of lipogenic genes(HMGCS, SCD, and FAS) is mediated by activation of theNLRP3 inflammasome in HCV-infected cells (Fig. 1). HMGCSand FAS are critical enzymes involved in the biosynthesis ofcholesterol and fatty acids, respectively (27, 29). SCD is a micro-

FIGURE 8. HCV activates caspase-1 in HCV-infected liver tissues. A, immu-nohistochemical (IHC) staining of tissue section slides from normal patients(a), HCV-positive patients with cirrhosis (b), and HCC patients (c) were stainedwith anti-caspase-1 antibodies (brown) for 1 h. The slides were incubated withsecondary antibodies at room temperature for 1 h, followed by counterstain-ing with hematoxylin. Arrows represent caspase-1 staining. The boxed area inb is shown enlarged within the figure. B, double immunohistochemistry. Tis-sue sections from normal (a) and HCV-infected liver tissues (b and c) werestained with anti-caspase-1 (brown) and anti-albumin (red), followed by incu-bation with secondary antibodies with alkaline phosphatase activity (red) andperoxidase activity (brown). Arrows represent caspase-1 staining. The boxedareas are enlarged at the bottom. C, Western blotting. Equal amounts of cel-lular lysates from normal (lane 1) and HCV-infected liver tissues (sample 1,cirrhosis; sample 2, HCC) were subjected to Western blotting using anti-caspase-1 antibodies. Tubulin represents the protein loading control.




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somal enzyme required for the biosynthesis of oleate andpalmitoleate, which are the major monounsaturated fatty acidsof membrane phospholipids, triglycerides, and cholesterolesters (41). The LD core contains triglycerides and cholesterolesters covered by a phospholipid monolayer (42). These resultssuggest a role of HMGCS, FAS, and SCD in NLRP3 inflam-masome-mediated lipogenesis and LD formation. The promot-ers of the HMGCS, SCD, and FAS genes have been shown tocontain functional binding sites for SREBPs (27, 29, 41).

Previously, we have shown activation of SREBPs in HCV-infected cells (28). However, the underlying mechanisms bywhich HCV is able to override the cholesterol-dependent phys-iological regulation of SREBP activation remain unclear. Inanother study, the investigators have shown activation ofSREBP by HCV NS4B via the Akt pathway (43). The authorshave shown phosphorylation of the mature form of SREBP afterthe cleavage steps by S1P and S2P in the Golgi. However, ourresults demonstrate how activation of the NLRP3 inflam-masomebyHCVinfectioninducestheproteolyticcleavage/pro-cessing of SREBPs prior to translocation of their mature formsinto the nucleus. There are two steps in the activation of SREBP:proteolytic cleavage of SREBP in the ER/Golgi and posttransla-tional modification of SREBP prior to the translocation ofmature forms into the nucleus (27, 29). Our results are consis-tent with previous studies demonstrating the role of the inflam-masome complex/caspase-1 in activating SREBPs to promotelipid biogenesis and cell survival in response to bacterial pore-forming toxins (22). In contrast, another study has shown acti-vation of the NLRP3 inflammasome by SREBP-2 in endothelialcells in the context of atherosclerotic lesions in a mouse model(44).

In normal cells, SCAP, SREBPs, and Insig proteins form acomplex in the ER membrane (27, 29). When cells are depleted

of sterols, SCAP escorts SREBPs from the ER to the Golgi forproteolytic cleavage. In addition, ER stress has also been shownto induce the proteolytic cleavage of SREBPs through down-regulation of Insig-1 (45). However, the regulation of Insig pro-teins and proteolytic activation of SREBPs in response to HCVinfection is poorly understood. Our results suggest that theinteraction of the inflammasome complex with SCAP in the ERmay lead to the dissociation of Insig proteins from the SCAP-SREBP-Insig complex, followed by proteasome-mediated deg-radation (Fig. 7). In addition, our results also showed reducedactivation and nuclear translocation of SREBPs in the presenceof caspase-1 inhibitor, suggesting a potential role of caspase-1activity in the SREBP proteolytic cleavage process. However,the underlying mechanism is not known. Our findings suggest apossible role of well established S1P and S2P-dependent path-ways in NLRP3 inflammasome/caspase-1-mediated SREBPproteolytic activation in HCV-infected cells (27, 29). However,we cannot exclude an indirect role of caspase-1 in proteolyticactivation of SREBPs. A recent study has shown that, after LPSstimulation, caspase-1 activates caspase-7, which translocatesinto the nucleus and cleaves PARP1 to enhance the expressionof NF-�B target genes (21).

In summary, our studies provide, for the first time, clear evi-dence of the role of HCV-mediated NLRP3 inflammasome acti-vation in regulating host lipid homeostasis. Previously, theinflammasome complex/caspase-1 has been shown to activateSREBP to promote membrane biogenesis and host cell survival(beneficial for the host) in response to bacterial pore-formingtoxins (22). These studies were conducted in CHO and HeLacells. However, our data provide evidence that activation of theNLRP3 inflammasome in HCV-infected hepatoma cells orhepatocytes (epithelial cells) is detrimental for the cells. Thestimulation of lipogenesis in hepatocytes by the NLRP3 inflam-

FIGURE 9. Model illustrating the NLRP3 inflammasome-mediated regulation of lipid metabolism. Early during HCV infection, 15–20% of acutely infectedindividuals clear the infection. In the majority of HCV-infected individuals, HCV evades the host defense system, enabling it to establish persistent infection(80 – 85%). Activation of the NLRP3 inflammasome/caspase-1 in hepatocytes during chronic/persistent infection interacts with SCAP in the ER, leading totranslocation of the mature/active form (N terminus) of SREBP into the nucleus for lipogenic gene expression and LD formation. Activation of the NLRP3inflammasome may directly or indirectly regulate liver disease pathogenesis and the HCV life cycle.




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masome is clearly the novel aspect of this study. Collectively,our results highlight the implications of metabolic abnormali-ties in liver diseases and provide a conceptual framework todevelop novel strategies for combating chronic liver diseasesassociated with HCV infection.

Author Contributions—G. W. conceived, designed, and performedthe experiments, analyzed the data, and wrote the paper. G. W.,S. M., J. I., M. S. D., S. L., and A. N. designed, performed, and ana-lyzed the experiments. N. A. provided technical assistance and con-tributed to the preparation of Fig. 8. All authors reviewed the resultsand approved the final version of the manuscript.

Acknowledgments—We thank Dr. Charles Rice (Rockefeller Univer-sity, NY) for HCV genotype 2a J6/JFH-1 infectious cDNA and theHuh-7.5 cell line and the Liver Tissue Cell Distribution System (Uni-versity of Minnesota, Minneapolis, MN) for liver biopsy specimensfrom normal and HCV-positive individuals.

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Naushad Ali and Gulam WarisSteven McRae, Jawed Iqbal, Mehuli Sarkar-Dutta, Samantha Lane, Abhiram Nagaraj,Regulatory Element-binding Protein (SREBP) and Regulates Lipid Metabolism

The Hepatitis C Virus-induced NLRP3 Inflammasome Activates the Sterol

doi: 10.1074/jbc.M115.694059 originally published online December 23, 20152016, 291:3254-3267.J. Biol. Chem.

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VOLUME 291 (2016) PAGES 3254 –3267DOI 10.1074/jbc.W118.006979

Withdrawal: The hepatitis C virus-induced NLRP3inflammasome activates the sterol regulatoryelement-binding protein (SREBP) and regulates lipidmetabolism.Steven McRae, Jawed Iqbal, Mehuli Sarkar-Dutta, Samantha Lane,Abhiram Nagaraj, Naushad Ali, and Gulam Waris

This article has been withdrawn by Steven McRae, Jawed Iqbal,Mehuli Sarkar-Dutta, Naushad Ali, and Gulam Waris. Samantha Laneand Abhiram Nagaraj could not be reached. The ASC immunoblot fromFig. 1A was reused as HCV core in Fig. 1G. Additionally, these imageswere reused in Burdette et al. (2010) J. Gen. Virol. 91, 681– 690, Burdetteet al. (2012) J. Gen. Virol. 93, 235–246, Presser et al. (2013) PLOS One 8,e56367, and Iqbal et al. (2014) PLOS One 9, e87464. The NLRP3 immu-noblot in Fig. 1D was reused from Fig. 9 of Iqbal et al. (2013) J. Biol.Chem. 288, 36994 –37009, representing different experimental condi-tions. The actin immunoblot from Fig. 1D was also reused in Fig. 1C ofBurdette et al. (2012) J. Gen. Virol. 93, 235–246, representing differentexperimental conditions. The actin immunoblot from Fig. 1F wasreused in Fig. 1G as actin. The Journal also determined that this immu-noblot was reused in the following publications representing differentexperimental conditions: Waris et al. (2003) J. Biol. Chem. 278, 40778 –40787, Waris et al. (2005) J. Virol. 79, 1569 –1580, Waris and Siddiqui(2005) J. Virol. 79, 9725–9734, Waris et al. (2007) J. Virol. 81, 8122–8130, Nasimuzzaman et al. (2007) J. Virol. 81, 10249 –10257, Burdette etal. (2010) J. Gen. Virol. 91, 681– 690, Burdette et al. (2012) J. Gen. Virol.93, 235–246, and Presser et al. (2013) PLOS One 8, e56367. GulamWaris does not agree that the actin immunoblot was reused in otherpublications. The SCD immunoblot from Fig. 1F was reused in Fig.6A asactin. The mature SREBP-2 immunoblot from Fig. 3B was reused asSCD in Fig. 1G. Insig 2 of Fig. 7A was reused in Fig. 7B as Insig 1. Theactin immunoblot from Fig. 7A was reused in lanes 3 and 4 of the actinimmunoblot in Fig. 7B. Columns f and g were duplicated in Fig. 2A.Column a from Fig. 4C was duplicated in a of Fig. 5D. The withdrawingauthors sincerely apologize to the readers.

WITHDRAWALS/RETRACTIONS

J. Biol. Chem. (2018) 293(52) 20011–20011 20011© 2018 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.

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