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

Hepatitis C virus (HCV) relies on host lipids and lipid droplets for replication and morphogenesis. The accumulation of lipid droplets in infected hepatocytes manifests as hepatosteatosis, a common pathology observed in chronic hepatitis C patients. One way by which HCV promotes the accumulation of intracellular lipids is through enhancing de novo lipogenesis by activating the sterol regulatory element-binding proteins (SREBPs). In general, activation of SREBPs occurs during cholesterol depletion. Interestingly, during HCV infection, the activation of SREBPs occurs under normal cholesterol levels, but the underlying mechanisms are still elusive. Our previous study has demonstrated the activation of the inflammasome complex in HCV-infected human hepatoma cells. In this study, we elucidate the potential link between chronic hepatitis C-associated inflammation and alteration of lipid homeostasis in infected cells. Our results reveal that the HCV-activated NLRP3 inflammasome is required for the up-regulation of lipogenic genes such as 3-hydroxy-3-methylglutaryl-coenzyme A synthase, fatty acid synthase, and stearoyl-CoA desaturase. Using pharmacological inhibitors and siRNA against the inflammasome components (NLRP3, apoptosis-associated speck-like protein containing a CARD, and caspase-1), we further show that the activation of the NLRP3 inflammasome plays a critical role in lipid droplet formation. NLRP3 inflammasome activation in HCV-infected cells enables caspase-1-mediated degradation of insulin-induced gene proteins. This subsequently leads to the transport of the SREBP cleavage-activating protein·SREBP complex from the endoplasmic reticulum to the Golgi, followed by proteolytic activation 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 inflammasome to activate SREBPs and host lipid metabolism, leading to liver disease pathogenesis associated with chronic HCV.

Chronic liver disease resulting from HCV infection represents a major global health problem. HCV infection often leads to chronic hepatitis in up to 60 -80% of infected adults and progresses to liver fibrosis, cirrhosis, and hepatocellular carcinoma (HCC) 2 (1). The HCV genome is a 9.6-kb, positive-sense, single-stranded RNA molecule containing a 5Ј UTR, a single open reading frame, and a 3Ј UTR (2). The 5Ј UTR contains an internal ribosome entry site that directs cap-independent translation of a polyprotein precursor of ϳ3000 amino acids that is cleaved by viral proteases and host cell signal peptidases into mature structural proteins (core, E1, E2, and p7) and nonstructural (NS) proteins (NS2, NS3, NS4A, NS4B, NS5A, and NS5B) (2).
The majority of HCV-infected individuals develop a persistent infection that promotes chronic inflammation, which is considered to be the primary catalyst for progressive liver disease and development of HCC. Our recent work highlights a mechanism of chronic inflammation through activation of the NLRP3 inflammasome in HCV-infected hepatoma cells (3). In addition, previous studies have shown activation of the NLRP3 inflammasome in hepatic macrophages and monocytes (4 -7). Activation of the inflammasome is a major mechanism of inflammation, leading to the production of proinflammatory IL-1␤ and IL-18 cytokines via caspase-1 activation (8). Most inflammasomes consist of a member of the NOD-like receptor (NLR) family of cytosolic receptors that either directly interact with caspase-1 or are coupled indirectly to it by the adaptor protein apoptosis-associated speck-like protein containing a CARD (ASC) and procaspase-1 (8). Activated caspase-1 processes pro-IL-1␤ and IL-18 into their mature forms. In chronic HCV 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 recognized to have inflammasome/cytokine-independent functions (11- 15). Recent studies have demonstrated that inflammasomeindependent NLRP3 augments TGF-␤1 signaling in the kidney epithelium and cardiac fibroblasts (12,13). NLRP3 is also known to interact with ubiquitin ligase-associated protein SGT1, heat shock protein 90 (HSP90), and thioredoxin-interacting protein (16,17). Typically, caspase-1 mediates the maturation of IL-1␤ and IL-18 in immune and non-immune cells (18). However, studies have shown that several proteins associated with the glycolytic pathway are cleaved by caspase-1, which is suggestive of a broader role of caspase-1 in addition to maturation of IL-1␤ and IL-18 (19). Activation of caspase-1 leads to pyroptosis of the cells infected with intracellular bacteria (20). In contrast, the ability of caspase-1 to prevent hepatocyte death during redox stress by up-regulating beclin 1 expression signifies its protective function in non-immune cells (11). Caspase-1 has also been shown to regulate the expression of NF-B target genes through caspase-7-mediated cleavage of PARP1 (21). In addition, recent studies have implicated caspase-1 in cell survival by facilitating membrane biogenesis and cellular repair via regulation of lipid metabolism (22).
A unique feature of HCV is its absolute reliance on host lipids in the various stages of the viral life cycle (23). To favor its proliferation, HCV alters cellular lipid metabolism by stimulating lipogenesis, impairing mitochondrial ␤-oxidation and cellular lipid export, and promoting a lipid-rich intracellular environment (23,24). This alteration of lipid homeostasis results in the intracellular accumulation of cellular lipid storage organelles, termed "lipid droplets" (LDs), that play crucial roles in the HCV life cycle, hepatic steatosis, and HCC (24 -26).
Sterol regulatory element-binding proteins (SREBPs) are the master regulators of lipid homeostasis that activate the transcription of genes encoding enzymes involved in the biosynthesis of cholesterol, triglycerides, phospholipids, and fatty acids (27). Previously, we have shown the activation of SREBPs in HCV-infected human hepatoma cells (28). However, the underlying mechanism by which HCV activates SREBPs is not clearly understood. To be active, SREBPs must be cleaved to produce the active/mature forms. There are three SREBP isoforms, designated SREBP-1a, SREBP-1c, and SREBP-2 (27). SREBP-1a activates all SREBP target genes, whereas SREBP-2 and SREBP-1c activate genes involved in cholesterol and fatty acid synthesis, respectively (27). SREBPs are synthesized as endoplasmic reticulum (ER)-membrane-bound precursors and exist in complex with SREBP cleavage-activating protein (SCAP) (27). SCAP is both an escort for SREBPs and a sensor of sterol. Retention of the SCAP-SREBP complex in the ER is mediated by the binding of SCAP to insulin-induced gene (Insig) proteins (29). Insig-1 and Insig-2 are membrane-bound proteins that reside in the ER and play a central role in the regulation of SREBP activation (30). When cells are depleted of cholesterol, SCAP transports SREBPs from the ER to the Golgi, where site 1 proteases (S1Ps) and site 2 proteases (S2Ps) act specifically and sequentially to release the active forms of SREBPs, which actively translocate into the nucleus and bind to the sterol response elements of the target genes.
In this study, we investigated the mechanism of increased lipid biosynthesis in cells infected with HCV. Our studies show that HCV-induced NLRP3 inflammasome activates SREBPs and stimulates lipogenic gene expression and formation of LDs. Our results demonstrate that the proteolytic activation of SREBPs in HCV-infected cells is mediated by interaction of the NLRP3 inflammasome with SCAP in the ER. We also demonstrate that caspase-1 activity is critical for SREBP activation. Collectively, these observations provide insights into the novel role of the NLRP3 inflammasome in lipid homeostasis during chronic HCV infection.
Cell Culture-The human hepatoma cell line Huh-7.5 was obtained from Dr. C. Rice (31). Huh-7.5 cells were cultured at 37°C in a humidified atmosphere containing 5% CO 2 with DMEM supplemented with 10% fetal calf serum, 100 units of penicillin/ml, and 100 g of streptomycin sulfate/ml.
HCV Cell Culture Infection System-Fifteen micrograms of in vitro transcribed J6/JFH-1 RNA was delivered into Huh-7.5 cells by electroporation as described previously (3,28,32). Cells were passaged every 3-5 days. The presence of HCV in these cells and the corresponding supernatants was determined as described previously (33). The cell-free virus was propagated in Huh7.5 cell culture as described previously (32)(33)(34). The expression of HCV protein in HCV-infected cells was analyzed by Western blotting. The HCV cell culture supernatant was collected at appropriate time points and used to infect naïve Huh7.5 cells at a multiplicity of infection of 1 for 5-6 h at 37°C and 5% CO 2 (32,33). The viral titer in the cell culture supernatant was expressed as focus forming units per milliliter, which was determined by the average number of HCV-NS5A-positive foci detected at the highest dilutions, as described previously (33). The cell culture supernatant collected from Huh7.5 cells expressing JFH-1/GND (replication-defective virus) was used as a negative control.
Immunoprecipitation and Western Blotting Analysis-Cellular lysates from mock-and HCV-infected cells were prepared by incubation in radioimmune precipitation assay buffer (50 mM Tris (pH 7.5), 150 mM NaCl, 1% Nonidet P-40, 0.5% sodium deoxycholate, 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 of cellular lysates were immunoprecipitated with the indicated antibodies overnight at 4°C. The immune complexes were incubated with protein A-Sepharose (Invitrogen) for 1 h at 4°C, washed three to four times with radioimmune precipitation assay buffer, and boiled for 5 min in SDS-containing sample buffer. The samples were then subjected to SDS-PAGE. Gels were electroblotted onto a nitrocellulose membrane (Thermo Scientific) in 25 mM Tris, 192 mM glycine, and 20% methanol. Membranes were incubated overnight in blocking buffer (20 mM Tris/HCl (pH 7.5), 150 mM NaCl, and 5% nonfat dry milk) and probed with primary antibody of interest for 1 h at room temperature. The membranes were then washed three times for 10 min in Tris-buffered saline with 1% Tween 20 (TBS-T), followed by incubation with secondary antibody for 45 min at room temperature. After an additional washing cycle with TBS-T, the immunoblots were visualized using the LICOR Odyssey system.
Laser-scanning Confocal Microscopy-Mock-and HCV-infected cells on coverslips were washed with PBS, fixed with 4% paraformaldehyde for 10 min at room temperature, permeabilized for 5 min with 0.2% Triton X-100, and blocked for 45 min with 5% bovine serum albumin in PBS. The cells were then incubated with primary antibody against the specific protein for 1 h at room temperature or overnight at 4°C, followed by incubation with Alexa Fluor-labeled secondary antibodies (Invitrogen) for 1 h. After washing with PBS, cells were mounted with anti-fade reagent containing DAPI (Invitrogen) and observed under a laser-scanning confocal microscope (Fluoview FV10i).
Immunohistochemistry-Liver biopsies from normal and HCV-associated cirrhosis and HCC (no history of hepatitis B virus, HIV infection, and fatty liver) were obtained from the Liver Tissue Cell Distribution System (University of Minnesota, Minneapolis, MN). Immunohistochemistry was performed according to the protocol of the manufacturer using the Leica BOND-III TM polymer refined detection system (DS 9800) at the Stephenson Cancer Center Pathology core laboratory (University of Oklahoma Health Sciences Center, Institutional Review Board (IRB) Number 3405). The tissue sections from normal and HCV-associated cirrhosis and HCC were deparaffinized and rehydrated in an automated multistainer (Leica ST5020). The tissue section slides were subjected to antigen retrieval at 100°C for 20 min in a retrieval solution, fol-lowed by incubation in blocking solution for 1 h. The sections were stained with primary antibody for 1 h, followed by the secondary antibody (poly-HRP IgG). The detection was performed using 3,3Ј-diaminobenzidine tetrachloride, and counterstaining was done with hematoxylin. For double-staining, the Leica BOND-III TM polymer refined detection system (DS 9800) and Leica BOND-III TM refined red detection system (DS 9390) were used sequentially. For Western blotting analysis, frozen liver tissues were thawed in radioimmune precipitation assay buffer and crushed gently, followed by sonication and incubation on ice for 30 min. Samples were centrifuged at 4°C, and the supernatant was collected.
Silencing of Target Gene Expression-Mock-and HCV-infected cells on day 2 were transfected with siRNA targeted against control (sicontrol), siNLRP3, siASC, and sicaspase-1 according to the protocols of the manufacturers (Santa Cruz Biotechnology and Qiagen). Each siRNA consisted of pools of three to five target-specific, 19-to 25-nt siRNA designed to knock down target gene expression. For sicontrol and sicaspase-1 transfections, two solutions were prepared. For solution A, 60 pmol of siRNA duplex was mixed with 100 l of siRNA transfection medium. For solution B, 6 l of transfection reagent was added to 100 l of siRNA transfection medium. Solutions A and B were allowed to incubate at room temperature for 20 min. After 20 min, solutions A and B were combined and allowed to incubate for another 20 min at room temperature. The combined solutions were then added to the cells in 6-well plates and incubated for 5 h at 37°C and 5% CO 2 . Then the transfection solution was replaced with 2 ml of complete DMEM.
siASC was transfected according to the protocol of the manufacturer (Qiagen). 256 ng of the siRNA duplex was diluted in 100 l of serum-free medium along with 20 l of HiPerFect transfection reagent. The solution was allowed to incubate at room temperature for 10 min. The transfection solution was then added to the cells, and the cells were harvested at different time points.
Quantitative RT-PCR-Total cellular RNA was extracted from mock-and HCV-infected cells using TRIzol (Invitrogen) and treated with RQ1 RNase-free DNase prior to cDNA synthesis. The cDNA was reverse-transcribed from 1 g of total RNA using a reverse transcription kit (Life Technologies). Quantitative RT-PCR was carried out using SYBR Green Master Mix (Life Technologies) and specific primers as described previously (3,28,31). Amplification reactions were performed under 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 transcript levels were calculated using the ⌬⌬Ct method as specified by the manufacturer.
Cell Viability Assay-Mock-infected cells (Huh7.5), HCVinfected cells, and HCV-infected cells transfected with various siRNA or treated 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 of the manufacturer using the CellTitre-Glo luminescent cell viability assay kit (Promega). The percent viability was calculated considering 100% viability for mock cells. The values represent the mean ϩ S.D. of three independent experiments performed in duplicate.
Statistical Analysis-Error bars show mean Ϯ S.D. of data from three individual experiments. Two-tailed unpaired t tests were used to compare experimental conditions with those of the respective controls. In all tests, p Ͻ 0.05 was considered statistically significant.

Results
The NLRP3 Inflammasome Induces Lipogenesis in HCV-infected Cells-We have demonstrated previously that HCV stimulates lipogenesis by activating SREBPs (28). However, the underlying mechanism(s) by which HCV activates SREBPs is not clearly understood. Recently, studies have shown the role of the inflammasome complex in cell survival by facilitating membrane biogenesis and cellular repair via regulation of lipid metabolism (22). To determine whether HCV induces lipogenesis through the activation of the NLRP3 inflammasome, HCV-infected Huh7.5 cells were transfected with siRNA against each component of the inflammasome complex (i.e. siNLRP3, siASC, and sicaspase-1) or scrambled siRNA (sicontrol). Equal amounts of cellular lysates were subjected to immunoblot analysis. A marked reduction in the expression of NLRP3, ASC, and caspase-1 protein levels suggests efficient knockdown of these proteins by their corresponding siRNA. The specificity of siRNA activity was indicated by the fact that control siRNA did not inhibit the expression of these proteins (Fig. 1, A-C). HCV-infected cells transfected with siNLRP3 specifically down-regulated the expression of NLRP3 but not the other component (ASC) of the inflammasome complex (Fig. 1A,  lane 4). This is also true for HCV-infected cells transfected with siASC (Fig. 1B, lane 4). In addition, HCV-infected cells transfected with independent siNLRP3 duplexes show similar inhibition in the expression of NLRP3 (Fig. 1D). The cell viability assay was performed in the above siRNA-transfected cells. We did not observe any significant change in ATP levels under various conditions (Fig.  1E).
To determine the role of the NLRP3 inflammasome in lipogenic gene expression, lysates from mock-and HCV-infected Huh7.5 cells silenced with siNLRP3, siASC, and sicaspase-1 were subjected to immunoblot analysis. The results show increased expression of fatty acid synthase (FAS) and stearoyl-CoA desaturase (SCD) in HCV-infected cells that were reduced significantly in cells transfected with siNLRP3, siASC, and sicaspase-1 compared with sicontrol (Fig. 1F, lanes 3-6). Furthermore, we also analyzed the expression of FAS and SCD in the presence of inhibitors of caspase-1 and caspase-3 (negative control). Our results showed significantly reduced expression of FAS and SCD in HCV-infected cells treated with caspase-1 inhibitor compared with caspase-3 inhibitor (Fig. 1F, lanes 7  and 8). In addition, we also observed a significant reduction in the expression of SCD in HCV-infected cells transfected with siNLRP3#2, suggesting that siNLRP3#1 and #2 produce similar phenotypes and not likely to be the off-target effects of these siRNA (Fig. 1G). The effect of silencing of NLRP3 on SCD expression was rescued by siRNA-resistant ectopic expression of NLRP3 (pFLAG-NLRP3del) (Fig. 1H, lane 4).
The HCV-activated NLRP3 Inflammasome Induces LDs Formation-To determine the role of the HCV-induced NLRP3inflammasome in LDs formation, mock-and HCV-infected Huh7.5 cells transfected with siNLRP3, siASC, sicaspase-1, and sicontrol were stained with the neutral lipid-specific green fluorescent dye BODIPY 493/503. The results show increased staining of LDs in HCV-infected cells compared with mockinfected cells ( Fig. 2A, a and b). In contrast, LDs were reduced significantly 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 cells treated with caspase-1 inhibitor but not with caspase-3 inhibitor ( Fig. 2A, g and h). Furthermore, treatment of Huh7.5 cells with 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 of inflammatory cytokine production. These results suggest that the activation of the NLRP3 inflammasome stimulates formation of LDs in HCV-infected cells.
To determine the sequence of NLRP3 inflammasome activation and LD formation in HCV-infected cells, we analyzed the activation of caspase-1 and staining of LDs at various time points. Our results suggest that HCV induces activation of the NLRP3 inflammasome, which is followed by lipogenesis and LD accumulation in HCV-infected cells (data not shown).
The NLRP3 Inflammasome Activates SREBPs in HCV-infected Cells-SREBPs are known to regulate cholesterol and fatty acid biosynthesis pathways (27,29). To determine whether the master inducers of lipid metabolism, SREBP-1 and SREBP-2, are regulated by the NLRP3 inflammasome complex in HCVinfected cells, total cellular lysates from mock-and HCV-infected cells transfected with siNLRP3, siASC, sicaspase-1, and sicontrol were analyzed by Western blotting. We observed proteolytic cleavage of SREBP-1 and SREBP-2 in HCV-infected cells compared with mock-infected cells (Fig. 3, A and B, lanes 1  and 2) that were reduced in cells silenced with siNLRP3, siASC, and sicaspase-1 but not in sicontrol cells (Fig. 3, A and B, lanes  3-6). To determine the role of caspase-1 activity on SREBP-1 and SREBP-2 proteolytic activation, mock-and HCV-infected cells were incubated with inhibitors of caspase-1 and caspase-3. Our results show significantly reduced activation of SREBP-1 and SREBP-2 in the presence of caspase-1 inhibitor but not caspase-3 inhibitor (Fig. 3, C and D, lanes 3 and 4). These results suggest the role of the NLRP3 inflammasome-mediated caspase-1 in HCV-induced proteolytic cleavage of SREBP-1 and SREBP-2 into their mature forms. To further demonstrate the activation and nuclear translocation of the mature forms of SREBPs in HCV-infected cells, cytoplasmic and nuclear lysates were subjected to Western blotting. The results show the induction of precursor SREBP-1 in HCV-infected cytoplasmic FIGURE 1. The HCV-activated NLRP3 inflammasome induces lipogenic gene expression. Mock-(Huh7.5) and HCV-infected cells (infected with HCV at a multiplicity of infection of 1 for 2 days) were transfected with sicontrol, siNLRP3, siASC, and sicaspase-1. 72 h post-transfection, cellular lysates were subjected to Western blotting using the respective antibodies. A, equal amounts of cellular lysates from mock-, HCV-, and HCV-infected cells transfected with siNLRP3 were immunoblotted with anti-NLRP3 and anti-ASC antibodies. B, cellular lysates from HCV-infected cells transfected with siASC were immunoblotted with anti-ASC and anti-NLRP3 antibodies. C, HCV-infected cells were transfected with sicontrol and sicaspase-1. D, HCV-infected cells were transfected with sicontrol and two different individual siNLRP3 duplexes (siNLRP3#1 and siNLRP3#2) that were present in the siRNA pool used above (Santa Cruz Biotechnology). Actin represents HCV infection. E, mock-(Huh7.5), HCV-, and HCV-infected cells transfected with sicontrol, siNLRP3, siASC, and sicaspase-1 at various time points or treated 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 of the manufacturer using a CellTitre-Glo luminescence cell viability assay kit (Promega). The percent viability was calculated considering 100% viability for mock-infected cells compared with HCV-infected cells transfected with various siRNA or treated with caspase-1/-3 inhibitors. The values represent mean ϩ 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 of cellular lysates from mock-and HCV-infected cells transfected with sicontrol, siNLRP3#1, and siNLRP3#2 were subjected to Western blotting using anti-SCD antibodies. 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 a deletion of the 3Ј UTR of NLRP3 (pFLAG-NLRP3del) along with siNLRP3 were transfected in HCV-infected cells. The pFLAG-NLRP3del expression plasmid was generated using a site-directed mutagenesis kit according to the protocols of the manufacturer (Stratagene). Cellular lysates were subjected to Western blotting using the respective antibodies. The siNLRP3 target sequence was 5Ј-CACGCTAATGATCGACTTCAA-3Ј (Qiagen). I, total cellular RNA was extracted from mock-and HCV-infected cells transfected with the above siRNA and subjected to quantitative RT-PCR using FAS-, HMGCS-, and SCD-specific primers and a SYBR Green probe. The values represent mean ϩ S.D. of three independent experiments performed in triplicate. *, p Ͻ 0.05 compared with mock-infected Huh7.5 cells; **, p Ͻ 0.05 compared with sicontrol-transfected cells; ***, p Ͻ 0.05 compared with HCV-infected cells treated with the caspase-3 inhibitor (DEVD).
lysates and the presence of a significant amount of mature SREBP-1 in the nuclear lysates (Fig. 3C, lanes 2 and 4). In contrast, we did not detect any mature SREBP-1 in nuclear lysates of mock-infected cells (Fig. 3C, lane 3).
It is well established that the mature forms of SREBPs translocate into the nucleus and bind to the SRE of the target genes (27). To determine whether the translocation of mature forms of SREBP-1 and SREBP-2 into the nucleus is regulated by the NLRP3 inflammasome, mock-and HCV-infected cells, as described in Fig. 2, were subjected to immunofluorescence. The results show significant nuclear translocation of mature SREBP-1 and SREBP-2 in HCV-infected cells compared with mockinfected cells (Fig. 4, A and B). In contrast, we observed reduced translocation of mature SREBP-1 and SREBP-2 in HCV-infected cells transfected with siNLRP3, siASC, and sicaspase-1 or incubated with caspase-1 inhibitor but not with sicontrol or treated with caspase-3 inhibitor (Fig. 4, A and B). Taken together, these results suggest that the NLRP3 inflammasome in HCV-infected cells plays a critical role in the activation and nuclear translocation of SREBPs. To demonstrate that SREBP-1 is transported to the Golgi during HCV infection, mock-and HCV-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 the Golgi in HCV-infected cells (Fig. 4C, yellow spots) compared with mock cells. In addition, we also observed a significant migration of the mature form of SREBP-1 into the nucleus of the HCV-infected cells (Fig. 4C).
The NLRP3 Inflammasome Colocalizes with SCAP in HCV-infected Cells-SCAP transports SREBPs from the ER to the Golgi. To determine whether the NLRP3 inflammasome interacts with SCAP and facilitates the transport of SREBPs from the ER to the Golgi, we performed confocal microscopy. The results show significant colocalization of NLRP3, ASC, and caspase-1 with SCAP in HCV-infected cells compared with mock-infected cells (Fig. 5,  A-C, b, yellow dots). These results suggest the interaction of the NLRP3 inflammasome with SCAP. To determine whether the NLRP3 inflammasome associates with the ER, colocalization of 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, and caspase-1 primarily with the ER (Fig. 5, D-F). However, we did not observe any colocalization of NLRP3 with endosome and lysosome markers except weak colocalization with the Golgi marker (Fig. 5, G-I). Collectively, these results clearly suggest the association of the NLRP3 inflammasome with the ER in HCV-infected cells.  -1 and SREBP-2. A and B, mock-infected cells, HCV-infected cells, 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 was used 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, and their secondary antibodies as described above. Arrows represent colocalization of SREBP-1 with the Golgi (yellow dots).
Association of the NLRP3 Inflammasome with SCAP-The association of NLRP3, ASC, and caspase-1 with SCAP was also confirmed by a protein-protein interaction approach. Cellular lysates from mock-and HCV-infected cells were immunoprecipitated with anti-SCAP, followed by Western blotting using anti-NLRP3, anti-caspase-1, and anti-SCAP antibodies. The results showed that SCAP was pulled down with NLRP3 and caspase-1 in HCV-infected cells compared with mock-infected cells (Fig. 6A, lanes 3 and 4). However, immunoprecipitation of HCV-infected lysates with an isotype control antibody did not pull down NLRP3 and caspase-1 (Fig. 6A, lane 5). We could not show the expression of ASC during immunoprecipitation with SCAP because the banding pattern of the IgG light chain overlapped with ASC (26 kDa). The interaction of the NLRP3 inflammasome with SCAP was further confirmed by reciprocal co-immunoprecipitation using anti-NLRP3, anti-ASC, and anti-caspase-1 antibodies. We observed that NLRP3, ASC, and caspase-1 were pulled down with SCAP in HCV-infected cells compared with mock-infected cells but not with an isotype control antibody (Fig. 6B, lanes 3-10). Collectively, these results suggest that the NLRP3 inflammasome interacts with SCAP in HCV-infected cells.
The NLRP3 Inflammasome Induces Degradation of Insig Proteins in HCV-infected Cells-Because Insigs are ER-resident proteins and play an important role in the activation of SREBP-1 and SREBP-2, we examined the status of Insig-1 and Insig-2 proteins in HCV-infected cells. Mock-and HCV-infected cellular lysates were subjected to Western blotting using anti-Insig-1 and anti-Insig-2 antibodies. The results showed reduced expression of Insig-1 and Insig-2 expression in HCVinfected cells compared with mock-infected cells (Fig. 7A, lane  2). However, we did not observe any change in the expression of SCAP. Previously, it has been demonstrated that the dissociation of Insig from the ER retention complex leads to protea-some-mediated degradation of Insig (30). Our results clearly showed the degradation of Insig-1 and Insig-2 in HCV-infected cells, which was blocked by proteasome inhibitor but not by calpain inhibitor (negative control) (Fig. 7B, lanes 3 and 4), suggesting that Insig-1 and Insig-2 play critical roles in SREBP activation in HCV-infected cells.
To determine whether the interaction of the NLRP3 inflammasome/caspase-1 with SREBP activation machinery (SREBP-SCAP-Insig complex) mediates the degradation of Insig-1 and Insig-2, mock-and HCV-infected cells were silenced with sicaspase-1. The cellular lysates were analyzed by Western blotting. The results show degradation of Insig-1 in HCV-infected cells that was blocked in cells silenced with sicaspase-1 but not in sicontrol cells (Fig. 7C, lanes 3 and 4). In addition, we observed significant silencing (68%) of caspase-1 expression in HCV-infected cells (Fig. 7C, lane 4). These results suggest a role of the caspase-1⅐inflammasome complex in HCV-mediated degradation of Insig proteins.
HCV Activates Caspase-1 in Hepatocytes of HCV-positive Liver Tissues-In this study, we examined caspase-1 activation as a readout of NLRP3-inflammasome activation in HCV-positive liver tissues. Caspase-1 is an effector molecule of the inflammasome complex (8,18). We examined liver tissues from HCV-positive patients with cirrhosis (five cases) and HCC (four cases) to validate the expression and activation of caspase-1 in cell culture studies (3). Normal and HCV-positive patient liver  tissues were subjected to immunohistochemical staining for caspase-1. The representative results are shown in Fig. 8A. Strong caspase-1 expression was detected in HCV-positive liver tissues compared with normal tissues (Fig. 8A, b and c, brown  spots). In addition, a subpopulation of mature hepatocytes identified by staining for human albumin (red) were also positive for active caspase-1 only in HCV-positive patients liver tissues (Fig.  8B, b and c). However, similar co-staining was clearly absent in normal liver tissues (Fig. 8B, a). To further confirm the staining results, liver tissue lysates were subjected to Western blotting, and caspase-1 bands were analyzed. The results showed activation of caspase-1 in two liver tissue samples (used in Fig. 8C, b and 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 HCVpositive human liver tissues.

Discussion
In recent years, activation of the inflammasomes has been implicated in various chronic diseases and in the clearance of several viruses (35)(36)(37)(38). However, the role of the inflammasome complex in HCV pathogenesis is incompletely understood. In addition to various infections, abnormal lipid metabolism has been strongly linked to chronic inflammation in a mouse obesity model (39). Recent studies have implicated the inflammasome complex/caspase-1 in cell survival by facilitating membrane biogenesis and cellular repair via regulation of lipid metabolism (22). Consistent with this observation, our studies clearly provide a link between chronic inflammatory pathways and host lipid metabolism during HCV infection. We show that activation of the NLRP3 inflammasome in HCV-infected cells causes the activation of SREBPs and induces lipogenesis and LD formation, cellular events critical for HCV proliferation and liver disease pathogenesis associated with chronic HCV.
In this study, we show the activation of caspase-1, the effector molecule of NLRP3-inflammasome, in human hepatoma cells and in the hepatocytes of liver biopsies of chronic HCV patients. Our findings are consistent with studies from other groups demonstrating activation of the NLRP3 inflammasome in isolated hepatocytes from liver samples of patients with chronic hepatitis C (11,40). These studies clearly established the potential of HCV to activate the NLRP3 inflammasome in hepatocytes infected with HCV.
HCV has also been shown to activate the NLRP3 inflammasome in hepatic macrophages and monocytes (4 -7). However, in these reports, activation of the NLRP3 inflammasome in human hepatoma cells or primary hepatocytes by HCV was not observed. The failure to observe inflammasome activation could be due to infection with a low multiplicity of infection of 0.1 and reliance on the detection of mature forms of IL-1␤ and IL-18 in cell culture supernatants. Recent in vivo studies have shown that non-immune cells, such as hepatocytes, express and activate the inflammasome complex but do not secrete adequate/detectable amounts of IL-1␤ and IL-18 compared with immune cells, suggesting that activation of the inflammasome complex in epithelial cells is likely to be involved in cytokineindependent functions (11)(12)(13)(14)(15). Our findings suggest that, unlike in immune cells, in human hepatocytes (epithelial cells), HCV modulates the NLRP3 inflammasome differently according to its specific niche to alter lipid metabolism, leading to LD accumulation and liver disease pathogenesis (Fig. 9). NLRP3 is known to interact with several proteins to modulate various cellular functions (12)(13)(14)(15)(16)(17). Apart from cleavage/maturation of IL-1␤ and IL-18, caspase-1 has been shown to cleave several proteins, suggesting a broader role of the NLRP3 inflammasome/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 the NLRP3 inflammasome in HCV-infected cells (Fig. 1). HMGCS and FAS are critical enzymes involved in the biosynthesis of cholesterol and fatty acids, respectively (27,29). SCD is a micro- , and HCC patients (c) were stained with anti-caspase-1 antibodies (brown) for 1 h. The slides were incubated with secondary antibodies at room temperature for 1 h, followed by counterstaining with hematoxylin. Arrows represent caspase-1 staining. The boxed area in b is shown enlarged within the figure. B, double immunohistochemistry. Tissue sections from normal (a) and HCV-infected liver tissues (b and c) were stained with anti-caspase-1 (brown) and anti-albumin (red), followed by incubation with secondary antibodies with alkaline phosphatase activity (red) and peroxidase activity (brown). Arrows represent caspase-1 staining. The boxed areas are enlarged at the bottom. C, Western blotting. Equal amounts of cellular lysates from normal (lane 1) and HCV-infected liver tissues (sample 1, cirrhosis; sample 2, HCC) were subjected to Western blotting using anticaspase-1 antibodies. Tubulin represents the protein loading control. somal enzyme required for the biosynthesis of oleate and palmitoleate, which are the major monounsaturated fatty acids of membrane phospholipids, triglycerides, and cholesterol esters (41). The LD core contains triglycerides and cholesterol esters covered by a phospholipid monolayer (42). These results suggest a role of HMGCS, FAS, and SCD in NLRP3 inflammasome-mediated lipogenesis and LD formation. The promoters of the HMGCS, SCD, and FAS genes have been shown to contain functional binding sites for SREBPs (27,29,41).
Previously, we have shown activation of SREBPs in HCVinfected cells (28). However, the underlying mechanisms by which HCV is able to override the cholesterol-dependent physiological regulation of SREBP activation remain unclear. In another study, the investigators have shown activation of SREBP by HCV NS4B via the Akt pathway (43). The authors have shown phosphorylation of the mature form of SREBP after the cleavage steps by S1P and S2P in the Golgi. However, our results demonstrate how activation of the NLRP3 inflammasome by HCV infection induces the proteolytic cleavage/processing of SREBPs prior to translocation of their mature forms into the nucleus. There are two steps in the activation of SREBP: proteolytic cleavage of SREBP in the ER/Golgi and posttranslational modification of SREBP prior to the translocation of mature forms into the nucleus (27,29). Our results are consistent with previous studies demonstrating the role of the inflammasome complex/caspase-1 in activating SREBPs to promote lipid biogenesis and cell survival in response to bacterial poreforming toxins (22). In contrast, another study has shown activation of the NLRP3 inflammasome by SREBP-2 in endothelial cells in the context of atherosclerotic lesions in a mouse model (44).
In normal cells, SCAP, SREBPs, and Insig proteins form a complex in the ER membrane (27,29). When cells are depleted of sterols, SCAP escorts SREBPs from the ER to the Golgi for proteolytic cleavage. In addition, ER stress has also been shown to induce the proteolytic cleavage of SREBPs through downregulation of Insig-1 (45). However, the regulation of Insig proteins and proteolytic activation of SREBPs in response to HCV infection is poorly understood. Our results suggest that the interaction of the inflammasome complex with SCAP in the ER may lead to the dissociation of Insig proteins from the SCAP-SREBP-Insig complex, followed by proteasome-mediated degradation (Fig. 7). In addition, our results also showed reduced activation and nuclear translocation of SREBPs in the presence of caspase-1 inhibitor, suggesting a potential role of caspase-1 activity in the SREBP proteolytic cleavage process. However, the underlying mechanism is not known. Our findings suggest a possible role of well established S1P and S2P-dependent pathways in NLRP3 inflammasome/caspase-1-mediated SREBP proteolytic activation in HCV-infected cells (27,29). However, we cannot exclude an indirect role of caspase-1 in proteolytic activation of SREBPs. A recent study has shown that, after LPS stimulation, caspase-1 activates caspase-7, which translocates into the nucleus and cleaves PARP1 to enhance the expression of NF-B target genes (21).
In summary, our studies provide, for the first time, clear evidence of the role of HCV-mediated NLRP3 inflammasome activation in regulating host lipid homeostasis. Previously, the inflammasome complex/caspase-1 has been shown to activate SREBP to promote membrane biogenesis and host cell survival (beneficial for the host) in response to bacterial pore-forming toxins (22). These studies were conducted in CHO and HeLa cells. However, our data provide evidence that activation of the NLRP3 inflammasome in HCV-infected hepatoma cells or hepatocytes (epithelial cells) is detrimental for the cells. The stimulation of lipogenesis in hepatocytes by the NLRP3 inflam- 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 to translocation of the mature/active form (N terminus) of SREBP into the nucleus for lipogenic gene expression and LD formation. Activation of the NLRP3 inflammasome may directly or indirectly regulate liver disease pathogenesis and the HCV life cycle. masome is clearly the novel aspect of this study. Collectively, our results highlight the implications of metabolic abnormalities in liver diseases and provide a conceptual framework to develop novel strategies for combating chronic liver diseases associated with HCV infection.
Author Contributions-G. W. conceived, designed, and performed the 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 analyzed the experiments. N. A. provided technical assistance and contributed to the preparation of Fig. 8. All authors reviewed the results and approved the final version of the manuscript.