Toll-Like Receptor-7 Signaling Promotes Nonalcoholic Steatohepatitis by Inhibiting Regulatory T Cells in Mice
2018; Elsevier BV; Volume: 188; Issue: 11 Linguagem: Inglês
10.1016/j.ajpath.2018.07.011
ISSN1525-2191
AutoresYoon Seok Roh, Jong‐Won Kim, Surim Park, Chang-Ho Shon, Sokho Kim, Seong Kug Eo, Jung Kee Kwon, Chae Woong Lim, Bumseok Kim,
Tópico(s)Diabetes and associated disorders
ResumoToll-like receptor 7 (TLR7) signaling regulates the production of type 1 interferons (IFNs) and proinflammatory cytokines, such as tumor necrosis factor (TNF)-α, implicated in the control of regulatory T (Treg) cell activity. However, the mechanistic interplay between TLR7 signaling and Treg cells in nonalcoholic steatohepatitis (NASH) has not been elucidated. Our aim was to clarify the role of TLR7 signaling in the pathogenesis of NASH. Steatohepatitis was induced in wild-type (WT), TLR7-deficient, IFN-α/β receptor 1–deficient, and Treg cell–depleted mice. TLR7-deficient and IFN-α/β receptor 1–deficient mice were more protective to steatohepatitis than WT mice. Of interest, both TNF-α and type 1 IFN promoted apoptosis of Treg cells involved in the prevention of NASH. Indeed, Treg cell–depleted mice had aggravated steatohepatitis compared with WT mice. Finally, treatment with immunoregulatory sequence 661, an antagonist of TLR7, efficiently ameliorated NASH in vivo. These results demonstrate that TLR7 signaling can induce TNF-α production in Kupffer cells and type I IFN production in dendritic cells. These cytokines subsequently induce hepatocyte death and inhibit Treg cells activities, leading to the progression of NASH. Thus, manipulating the TLR7-Treg cell axis might be used as a novel therapeutic strategy to treat NASH. Toll-like receptor 7 (TLR7) signaling regulates the production of type 1 interferons (IFNs) and proinflammatory cytokines, such as tumor necrosis factor (TNF)-α, implicated in the control of regulatory T (Treg) cell activity. However, the mechanistic interplay between TLR7 signaling and Treg cells in nonalcoholic steatohepatitis (NASH) has not been elucidated. Our aim was to clarify the role of TLR7 signaling in the pathogenesis of NASH. Steatohepatitis was induced in wild-type (WT), TLR7-deficient, IFN-α/β receptor 1–deficient, and Treg cell–depleted mice. TLR7-deficient and IFN-α/β receptor 1–deficient mice were more protective to steatohepatitis than WT mice. Of interest, both TNF-α and type 1 IFN promoted apoptosis of Treg cells involved in the prevention of NASH. Indeed, Treg cell–depleted mice had aggravated steatohepatitis compared with WT mice. Finally, treatment with immunoregulatory sequence 661, an antagonist of TLR7, efficiently ameliorated NASH in vivo. These results demonstrate that TLR7 signaling can induce TNF-α production in Kupffer cells and type I IFN production in dendritic cells. These cytokines subsequently induce hepatocyte death and inhibit Treg cells activities, leading to the progression of NASH. Thus, manipulating the TLR7-Treg cell axis might be used as a novel therapeutic strategy to treat NASH. Nonalcoholic fatty liver disease (NAFLD) is a major form of chronic liver diseases in adults and children.1Roberts E.A. Nonalcoholic steatohepatitis in children.Curr Gastroenterol Rep. 2003; 5: 253-259Crossref PubMed Scopus (76) Google Scholar Nonalcoholic steatohepatitis (NASH) is characterized by simple steatosis, hepatitis, and progressive fibrosis. It can ultimately lead to end-stage liver disease.2Powell E.E. Cooksley W.G. Hanson R. Searle J. Halliday J.W. Powell L.W. The natural history of nonalcoholic steatohepatitis: a follow-up study of forty-two patients for up to 21 years.Hepatology. 1990; 11: 74-80Crossref PubMed Scopus (1326) Google Scholar NASH has been observed in a subset of patients with NAFLD. However, the exact mechanisms by which a simple steatosis leads to NASH remain poorly understood. Recent studies have suggested that overgrowth of intestinal bacteria and increased intestinal permeability might play roles in the development of NASH.3Miele L. Valenza V. La Torre G. Montalto M. Cammarota G. Ricci R. Masciana R. Forgione A. Gabrieli M.L. Perotti G. Vecchio F.M. Rapaccini G. Gasbarrini G. Day C.P. Grieco A. Increased intestinal permeability and tight junction alterations in nonalcoholic fatty liver disease.Hepatology. 2009; 49: 1877-1887Crossref PubMed Scopus (1000) Google Scholar, 4Son G. Kremer M. Hines I.N. Contribution of gut bacteria to liver pathobiology.Gastroenterol Res Pract. 2010; 2010 (453563)Crossref PubMed Scopus (108) Google Scholar, 5Brun P. Castagliuolo I. Di Leo V. Buda A. Pinzani M. Palu G. Martines D. Increased intestinal permeability in obese mice: new evidence in the pathogenesis of nonalcoholic steatohepatitis.Am J Physiol Gastrointest Liver Physiol. 2007; 292: G518-G525Crossref PubMed Scopus (668) Google Scholar In chronic liver diseases, including NASH, intestinal permeability is increased because of bacterial overgrowth or altered compositions of bacterial microflora.6Wu W.C. Zhao W. Li S. Small intestinal bacteria overgrowth decreases small intestinal motility in the NASH rats.World J Gastroenterol. 2008; 14: 313-317Crossref PubMed Scopus (62) Google Scholar Systemic inflammation related to NASH also injures epithelial tight junctions,3Miele L. Valenza V. La Torre G. Montalto M. Cammarota G. Ricci R. Masciana R. Forgione A. Gabrieli M.L. Perotti G. Vecchio F.M. Rapaccini G. Gasbarrini G. Day C.P. Grieco A. Increased intestinal permeability and tight junction alterations in nonalcoholic fatty liver disease.Hepatology. 2009; 49: 1877-1887Crossref PubMed Scopus (1000) Google Scholar resulting in deregulation of intestinal barrier functions. Indeed, plasma levels of lipopolysaccharides (LPSs) are elevated in patients with chronic liver diseases, including NASH.7Farhadi A. Gundlapalli S. Shaikh M. Frantzides C. Harrell L. Kwasny M.M. Keshavarzian A. Susceptibility to gut leakiness: a possible mechanism for endotoxaemia in non-alcoholic steatohepatitis.Liver Int. 2008; 28: 1026-1033Crossref PubMed Scopus (177) Google Scholar These findings suggest that hepatic immune cells might be exposed to high levels of Toll-like receptor (TLR) ligands derived from gut bacterial products, which might trigger liver injury and progress to NASH. Several reports have detailed the importance of TLR4 and intestine-derived LPS in animal models of NASH.8Csak T. Velayudham A. Hritz I. Petrasek J. Levin I. Lippai D. Catalano D. Mandrekar P. Dolganiuc A. Kurt-Jones E. Szabo G. Deficiency in myeloid differentiation factor-2 and toll-like receptor 4 expression attenuates nonalcoholic steatohepatitis and fibrosis in mice.Am J Physiol Gastrointest Liver Physiol. 2011; 300: G433-G441Crossref PubMed Scopus (182) Google Scholar, 9Rivera C.A. Adegboyega P. van Rooijen N. Tagalicud A. Allman M. Wallace M. Toll-like receptor-4 signaling and Kupffer cells play pivotal roles in the pathogenesis of non-alcoholic steatohepatitis.J Hepatol. 2007; 47: 571-579Abstract Full Text Full Text PDF PubMed Scopus (504) Google Scholar Recently, it has been revealed that TLR7, initially considered a receptor of single-stranded RNA virus, can also recognize RNA from bacteria.10Eberle F. Sirin M. Binder M. Dalpke A.H. Bacterial RNA is recognized by different sets of immunoreceptors.Eur J Immunol. 2009; 39: 2537-2547Crossref PubMed Scopus (58) Google Scholar, 11Mancuso G. Gambuzza M. Midiri A. Biondo C. Papasergi S. Akira S. Teti G. Beninati C. Bacterial recognition by TLR7 in the lysosomes of conventional dendritic cells.Nat Immunol. 2009; 10: 587-594Crossref PubMed Scopus (262) Google Scholar Furthermore, host-derived denatured RNA from apoptotic cells can also be recognized by TLR7 as an endogenous ligand.12Guiducci C. Tripodo C. Gong M. Sangaletti S. Colombo M.P. Coffman R.L. Barrat F.J. Autoimmune skin inflammation is dependent on plasmacytoid dendritic cell activation by nucleic acids via TLR7 and TLR9.J Exp Med. 2010; 207: 2931-2942Crossref PubMed Scopus (160) Google Scholar, 13Barrat F.J. Meeker T. Gregorio J. Chan J.H. Uematsu S. Akira S. Chang B. Duramad O. Coffman R.L. Nucleic acids of mammalian origin can act as endogenous ligands for Toll-like receptors and may promote systemic lupus erythematosus.J Exp Med. 2005; 202: 1131-1139Crossref PubMed Scopus (759) Google Scholar As a consequence, hepatic immune cells might be exposed to effective levels of TLR7 ligands after liver injury, thus playing a role in the pathogenesis of NASH. Indeed, TLR4- or TLR9-deficient mice have exhibited reduced liver injury and NASH induced by a methionine-choline–deficient (MCD) or choline-deficient amino acid–defined diet,9Rivera C.A. Adegboyega P. van Rooijen N. Tagalicud A. Allman M. Wallace M. Toll-like receptor-4 signaling and Kupffer cells play pivotal roles in the pathogenesis of non-alcoholic steatohepatitis.J Hepatol. 2007; 47: 571-579Abstract Full Text Full Text PDF PubMed Scopus (504) Google Scholar, 14Miura K. Kodama Y. Inokuchi S. Schnabl B. Aoyama T. Ohnishi H. Olefsky J.M. Brenner D.A. Seki E. Toll-like receptor 9 promotes steatohepatitis by induction of interleukin-1beta in mice.Gastroenterology. 2010; 139: 323-334.e7Abstract Full Text Full Text PDF PubMed Scopus (557) Google Scholar indicating that TLR ligands originated from bacteria (bacterial LPS or DNA) or host (host DNA) might play a crucial role in NASH pathogenesis.15Seki E. De Minicis S. Osterreicher C.H. Kluwe J. Osawa Y. Brenner D.A. Schwabe R.F. TLR4 enhances TGF-beta signaling and hepatic fibrosis.Nat Med. 2007; 13: 1324-1332Crossref PubMed Scopus (1499) Google Scholar, 16Watanabe A. Hashmi A. Gomes D.A. Town T. Badou A. Flavell R.A. Mehal W.Z. Apoptotic hepatocyte DNA inhibits hepatic stellate cell chemotaxis via toll-like receptor 9.Hepatology. 2007; 46: 1509-1518Crossref PubMed Scopus (201) Google Scholar, 17Gabele E. Muhlbauer M. Dorn C. Weiss T.S. Froh M. Schnabl B. Wiest R. Scholmerich J. Obermeier F. Hellerbrand C. Role of TLR9 in hepatic stellate cells and experimental liver fibrosis.Biochem Biophys Res Commun. 2008; 376: 271-276Crossref PubMed Scopus (109) Google Scholar Recently, it has been reported that TLR7 affects lipid accumulation in hepatocytes by controlling autophagy and lipid peroxidation.18Kim S. Park S. Kim B. Kwon J. Toll-like receptor 7 affects the pathogenesis of non-alcoholic fatty liver disease.Sci Rep. 2016; 6: 27849Crossref PubMed Scopus (33) Google Scholar However, it is currently unclear what mechanisms regulate steatohepatitis by TLR7 signaling in vivo. Therefore, the objective of this study was to clarify the role of TLR7 in the pathogenesis of NASH. We fed the MCD diet to mice to induce experimental NASH because of the reproducibility of this well-established model, which allows the assessment of inflammatory pathway.19McCuskey R.S. Ito Y. Robertson G.R. McCuskey M.K. Perry M. Farrell G.C. Hepatic microvascular dysfunction during evolution of dietary steatohepatitis in mice.Hepatology. 2004; 40: 386-393Crossref PubMed Scopus (169) Google Scholar, 20Rinella M.E. Green R.M. The methionine-choline deficient dietary model of steatohepatitis does not exhibit insulin resistance.J Hepatol. 2004; 40: 47-51Abstract Full Text Full Text PDF PubMed Scopus (335) Google Scholar, 21Diehl A.M. Lessons from animal models of NASH.Hepatol Res. 2005; 33: 138-144Crossref PubMed Scopus (131) Google Scholar Our data indicate that TLR7 signaling mediated TNF-α and type 1 interferon (IFN) production in Kupffer cells (KCs) and dendritic cells (DCs), respectively, subsequently suppressing regulatory T (Treg) cells and leading to steatohepatitis. In the current study, TLR7 knockout (KO) mice, type 1 IFN receptor (IFNAR1) KO mice, depletion of regulatory T cells (DEREG) mice, and C57BL/6 littermate controls (eight to nine weeks of age, 25 to 30 g of weight) were used. TLR7-deficient mice were kindly provided by Dr. Shizou Akira (Osaka University, Suita, Japan). IFNAR1 KO mice were purchased from B&K Universal Limited (Hull, UK) and backcrossed with C57BL/6 mice for at least 10 generations. DEREG mice were provided by Dr. Tim Sparwasser (Technische Universität München, Munich, Germany).22Lahl K. Loddenkemper C. Drouin C. Freyer J. Arnason J. Eberl G. Hamann A. Wagner H. Huehn J. Sparwasser T. Selective depletion of Foxp3+ regulatory T cells induces a scurfy-like disease.J Exp Med. 2007; 204: 57-63Crossref PubMed Scopus (727) Google Scholar CD11c-DTR transgenic mice were purchased from the Jackson Laboratory (Bar Harbor, ME). These mice received humane care according to US National Institutes of Health recommendations outlined in the Guide for the Care and Use of Laboratory Animals.23Committee for the Update of the Guide for the Care and Use of Laboratory AnimalsNational Research CouncilGuide for the Care and Use of Laboratory Animals: Eighth Edition. National Academies Press, Washington, DC2011Crossref Google Scholar Experimental and animal management procedures were undertaken in accordance with requirements from the Animal Care and Ethics Committees of Chonbuk National University. Mice were used to prepare two different NASH models: acute and chronic. For the acute model, TLR7 KO, IFNAR1 KO, and WT mice (n = 6 mice per group) were fed an MCD or methionine-choline–sufficient (MCS) diet (Dyets Inc., Bethlehem, PA) for 17 days. For the chronic model, TLR7 KO, IFNAR1 KO, DEREG, and WT mice (n = 10 mice per group) were fed an MCD diet for six weeks, an established protocol to prepare NASH model.24Rangnekar A.S. Lammert F. Igolnikov A. Green R.M. Quantitative trait loci analysis of mice administered the methionine-choline deficient dietary model of experimental steatohepatitis.Liver Int. 2006; 26: 1000-1005Crossref PubMed Scopus (30) Google Scholar After establishing the short-term NASH model, mice were intraperitoneally injected with a single dose of R848 (2.5 mg/kg, Invivogen, San Diego, CA) or normal saline. At 6 hours after injection, mice were sacrificed to obtain serum samples and liver tissues. Pentoxifylline (100 mg/kg, Sigma-Aldrich Co., St. Louis, MO), an inhibitor of TNF-α production, was intraperitoneally injected at 1 hour and 24 hours before R848 treatment. For histologic review of hematoxylin and eosin–stained liver sections by light microscopy (BX-51, Olympus Corp., Tokyo, Japan), liver tissues were collected, fixed in 10% neutral buffered formalin solution for 48 hours, routinely processed, and then embedded in paraffin. Tissue sections (4 μm in thickness) were prepared using a microtome (HM-340E, Thermo Fisher Scientific Inc., Waltham, MA) and placed on glass slides. Hematoxylin and eosin staining was performed according to standard techniques. The NAFLD score was determined by two pathologists independently (Y.S.R. and B.K.) as described previously.25Kleiner D.E. Brunt E.M. Van Natta M. Behling C. Contos M.J. Cummings O.W. Ferrell L.D. Liu Y.C. Torbenson M.S. Unalp-Arida A. Yeh M. McCullough A.J. Sanyal A.J. Design and validation of a histological scoring system for nonalcoholic fatty liver disease.Hepatology. 2005; 41: 1313-1321Crossref PubMed Scopus (7163) Google Scholar Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) assay was performed on paraffin-embedded sections using an ApopTaq Peroxidase in situ apoptosis detection kit (Chemicon, Temecula, CA) according to the manufacturer's instructions. Positive reactions were visualized with 3,3′-diaminobenzidine substrate. Next, nuclear counterstaining was performed using methyl green dye. TUNEL-labeled cells were quantified by the percentage of positive area in high-power field. A total of 10 high-power fields of liver tissues were analyzed for each animal. Data are expressed as percentages of TUNEL-positive areas. Total liver section images were analyzed using a light microscope (BX-51, Olympus Corp.) and digital image software (analySIS TS Auto version 5.1, Olympus Corp.). Serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were determined using AM101-K spectrophotometric assay kits (ASAN Pharmaceutical, Hwasung, Korea). Triglyceride and total cholesterol contents in the liver were determined using an AM202-K spectrophotometric assay kit (ASAN Pharmaceutical, Hwasung, Korea). Total RNA was isolated from liver tissue using Easy-Spin Total RNA extraction kit (iNtRon Biotech, Seoul, Korea). After incubation with RNase-free DNase I (Promega, Madison, WI), reverse transcription was performed using a random primer and MultiScribe MuLV Reverse Transcriptase (Applied Biosystems, Foster City, CA) according to the manufacturer's instructions. cDNA was subjected to real-time PCR on a CFX96 Real-Time PCR Detection System (Bio-Rad Laboratories, Hercules, CA) using SYBR Green I as a dye for binding double-strand DNA. All PCR primers were obtained from Bioneer (Daejeon, Korea). After real-time quantitative RT-PCR reaction was completed, specificity was verified by melting curve analysis. Quantification was performed by comparing Ct values of samples after normalization with glyceraldehyde-3-phosphate dehydrogenase as internal control. Sequences of primers were summarized in Table 1.Table 1Sequences of Primers Used for Real-Time PCRGeneForwardReverseTnfa5′-TCTACTCCCAGGTTCTCTTCAAGG-3′5′-GCAAATCGGCTGACGGTGTG-3′Foxp35′-GGAGAGGCAGAGGACACTCAATG-3′5′-TCAGGTTGTGGCGGATGGC-3′Ifna45′-CCTGCTGGCTGTGAGGACATAC-3′5′-TCTTGCCAGCAAGTTGGTTGAG-3′Lpl5′-TCTGTACGGCACAGTGG-3′5′-CCTCTCGATGACGAAGC-3′Fabp15′-TGGACCCAAAGTGGTCCGCA-3′5′-AGTTCAGTCACGGACTTTAT-3′Pparg5′-CGGAAGCCCTTTGGTGACTTTATG-3′5′-GCAGCAGGTTGTCTTGGATGTC-3′Adipoq5′-TGACGGCAGCACTGGCAAG-3′5′-GATACTGGTCGTAGGTGAAGAGAAC-3′Gapdh5′-ACGGCAAATTCAACGGCACAG-3′5′-AGACTCCACGACATACTCAGCAC-3′ Open table in a new tab Liver tissue was directly lyzed with an extraction buffer (T-PER, Thermo Fisher Scientific Inc.) for 30 minutes on ice. After centrifugation at 13,000 × g for 15 minutes at 4°C, protein concentration in the supernatant was measured using Bradford's reagent (Thermo Fisher Scientific Inc.). Protein (30 μg) was resolved by sodium dodecyl sulfate–polyacrylamide gel electrophoresis on a gradient gel and then transferred onto polyvinylidene difluoride membranes. Blocking was performed using blocking buffer [5% nonfat dairy milk in Tris-buffered saline (20 mmol/L Tris, 150 mmol/L NaCl, pH 7.4) containing 0.05% Tween-20] for 1 hour at room temperature. Primary antibodies were diluted 1:1000 in a blocking buffer and incubated at 4°C overnight. The following antibodies were used: anti-Bcl-2, anti-Bax, and β-actin (mouse or rabbit antibody, Cell Signaling Technology, Danvers, MA). To detect antigen antibody complexes, anti-rabbit or anti-mouse horseradish peroxidase–conjugated secondary antibodies (Santa Cruz Biotechnology Inc., Santa Cruz, TX) were diluted 1:3000 in blocking buffer and incubated at room temperature for 45 minutes. Immune complexes were visualized using chemiluminescent substrate (Millipore, Burlington, MA) and Kodak X-OMAT film (Eastman Kodak, Rochester, NY) according to the manufacturer's instructions. Hepatic thiobarbituric acid–reactive substances (TBARSs) as oxidative stress markers were measured using a Lab Assay TBARS kit (Wako Pure Chemical Industries, Osaka, Japan) according to the manufacturer's instructions. Plasma concentration of mouse TNF-α was measured by enzyme-link immunosorbent assay using a Quantikine kit (R&D Systems, Minneapolis, MN) according to the manufacturer's instructions. To isolate hepatocytes and hepatic nonparenchymal cells (NPCs), livers of MCS or MCD-fed WT mice, TLR7 KO mice, and IFNAR1 KO mice were perfused (1 mL per minute) and digested with collagenase 1 (Worthington Biochemical Corporation, Lakewood, NJ) after cannulation of portal vein. Liver cell suspension was centrifuged at 50 × g for 3 minutes. After centrifugation, the pellet representing hepatocytes was resuspended, filtered, and washed several times using Dulbecco's modified Eagle's medium (PAA Laboratories, Piscataway, NJ) supplemented with 5% fetal bovine serum (Thermo Fisher Scientific Inc.), 100 nmol/L dexamethasone (Sigma-Aldrich), 1% insulin-transferrin-selenium-X supplement (Thermo Fisher Scientific Inc.), 100 IU/mL of penicillin, and 100 μg/mL of streptomycin. Viability of hepatocytes was assessed using trypan blue (Sigma-Aldrich). After centrifuging digested liver cell suspension at 50 × g for 3 minutes, the supernatant containing hepatic NPCs was collected, washed with phosphate-buffered saline, and resuspended in 40% Percoll in RPMI 1640 media. The cell suspension was gently overlaid onto 70% Percoll and centrifuged at 750 × g for 20 minutes with off-brake setting. NPCs were collected from the interface, washed twice with phosphate-buffered saline, and resuspended in RPMI 1640 media. Murine normal hepatocyte cell line TIB-73 was obtained from the ATCC (Manassas, VA). Murine monocyte/macrophage cell line RAW 264.7 was obtained from the ATCC. These cells were cultured at 37°C under 5% CO2 in Dulbecco's modified Eagle's medium (PAA Laboratories) supplemented with 10% fetal bovine serum, 4 mmol/L l-glutamine (PAA Laboratories), 100 IU/mL of penicillin, and 100 μg/mL of streptomycin. For co-culture experiments, primary hepatocytes and NPCs isolated from WT mice, TLR7 KO mice, and IFNAR1 KO mice were co-cultured in collagen-coated 12-well Transwell plate (Sigma-Aldrich) with or without 0.4 mmol/L palmitic acid for 24 hours. Hepatocyte death was evaluated using a cytotoxicity detection kit (Sigma-Aldrich) based on the measurement of activity of lactate dehydrogenase released from the cytosol into the culture medium following the manufacturer's instruction. Absorbance of sample was measured at wavelength of 490 nm using an EMax spectrophotometer (Molecular Devices, Sunnyvale, CA). Cell Counting Kit-8 (Dojindo Molecular Technologies Inc., Rockville, MD) was used to detect cellular activity of dehydrogenase, a well-known hallmark of viability. A total of 2.5 × 104 primary hepatocytes per well were plated onto a 48-well collagen-coated plate in duplicates and incubated overnight. These cells were then treated with 0, 20, or 100 μmol/L H2O2 for 5 minutes. After that, 20 ng/mL of TNF-α was added into each well. After incubating for 16 hours at 37°C, samples were assayed according to the manufacturer's instructions. The following endotoxin-free oligodeoxyribonucleotides (ODNs, Bioneer) were used for in vitro or in vivo studies: immunoregulatory sequence (IRS) 661, 5′-TGCTTGCAAGCTTGCAAGCA-3′, and control ODNs, 5′-TCCTGCAGGTTAAGT-3′. Various groups of mice were treated with saline, IRS 661, or control ODN at dose of 50 μg on alternate days during the MCD diet. Hepatic NPCs obtained from livers were stained with fluorescein isothiocyanate anti-CD4 antibody and allophycocyanin anti-CD25 antibody (e-Biosciences, San Diego, CA) on ice for 30 minutes followed by fixation with permeabilization concentrate buffer (e-Biosciences) at 4°C for 6 hours. After fixation, cells were washed twice with permeabilization buffer and stained with phosphatidylethanolamine anti-Foxp3 antibody in permeabilization buffer at room temperature for 30 minutes. For detection of early and late apoptosis, the liver NPCs were stained with anti-CD4 antibody and anti-CD25 antibody and washed with phosphate-buffered saline. Then, cells were suspended in binding buffer and supplemented with annexin V (AV) and propidium iodide (PI) for 15 minutes at room temperature without light exposure. The cells with fluorescence AV+/PI− were considered to be early apoptotic cells, and those with fluorescence AV+/PI+ were considered to be late apoptotic cells. Data were analyzed using CellQuest software version 3.1 (BD Biosciences, Franklin Lakes, NJ) or FlowJo software version 7.8 (FlowJo, Ashland, OR). All data are expressed as means ± SEM. Differences between the two groups were compared using a two-tailed t-test. Statistically significant difference between groups was considered at P < 0.05. All Western blotting, real-time quantitative RT-PCR, flow cytometry, and immunocytochemistry experiments were repeated at least twice with five to 10 different mice or samples per group. Only representative results are shown in the figures. To investigate the role of TLR7 in the pathogenesis of NASH, WT and TLR7-deficient mice were fed with an MCD or MCS diet (control) for 6 weeks. After feeding with an MCD diet, livers of WT mice had marked lipid accumulation with infiltration of inflammatory cells. In contrast, livers of TLR7-deficient mice had significant reduction of steatosis and inflammation compared with those of WT mice (Figure 1A). NAFLD activity scores as determined by the degree of steatosis and inflammation25Kleiner D.E. Brunt E.M. Van Natta M. Behling C. Contos M.J. Cummings O.W. Ferrell L.D. Liu Y.C. Torbenson M.S. Unalp-Arida A. Yeh M. McCullough A.J. Sanyal A.J. Design and validation of a histological scoring system for nonalcoholic fatty liver disease.Hepatology. 2005; 41: 1313-1321Crossref PubMed Scopus (7163) Google Scholar were significantly lower in TLR7-deficient mice than that in WT mice (Figure 1A). Reduced liver injury in TLR7-deficient mice was confirmed by lower serum ALT and AST levels (Figure 1B). Several lines of evidence have indicated that adiponectin and peroxisome proliferator-activated receptor gamma (PPARγ) can negatively regulate the severity of NASH.26Wu C.W. Chu E.S. Lam C.N. Cheng A.S. Lee C.W. Wong V.W. Sung J.J. Yu J. PPARgamma is essential for protection against nonalcoholic steatohepatitis.Gene Ther. 2010; 17: 790-798Crossref PubMed Scopus (68) Google Scholar, 27Gupte A.A. Liu J.Z. Ren Y. Minze L.J. Wiles J.R. Collins A.R. Lyon C.J. Pratico D. Finegold M.J. Wong S.T. Webb P. Baxter J.D. Moore D.D. Hsueh W.A. Rosiglitazone attenuates age- and diet-associated nonalcoholic steatohepatitis in male low-density lipoprotein receptor knockout mice.Hepatology. 2010; 52: 2001-2011Crossref PubMed Scopus (82) Google Scholar, 28George J. Liddle C. Nonalcoholic fatty liver disease: pathogenesis and potential for nuclear receptors as therapeutic targets.Mol Pharm. 2008; 5: 49-59Crossref PubMed Scopus (68) Google Scholar To understand how TLR7 signaling might regulate lipid accumulation, expression levels of PPARγ and PPARγ-dependent target genes (LFABP and LPL)26Wu C.W. Chu E.S. Lam C.N. Cheng A.S. Lee C.W. Wong V.W. Sung J.J. Yu J. PPARgamma is essential for protection against nonalcoholic steatohepatitis.Gene Ther. 2010; 17: 790-798Crossref PubMed Scopus (68) Google Scholar, 29Liu L. Yu S. Khan R.S. Ables G.P. Bharadwaj K.G. Hu Y. Huggins L.A. Eriksson J.W. Buckett L.K. Turnbull A.V. Ginsberg H.N. Blaner W.S. Huang L.S. Goldberg I.J. DGAT1 deficiency decreases PPAR expression and does not lead to lipotoxicity in cardiac and skeletal muscle.J Lipid Res. 2011; 52: 732-744Crossref PubMed Scopus (61) Google Scholar were examined in livers of WT and TLR7-deficient mice fed with an MCD diet. Expression levels of PPARγ, LFABP, and LPL in TLR7-deficient mice fed with an MCD diet were significantly higher than those in WT mice fed with the same diet (Figure 1C). Moreover, expression level of adiponectin, an anti-inflammatory adipokine, was up-regulated in livers of TLR7-deficient mice fed with an MCD diet compared with that in WT mice fed with the same diet (Figure 1C). These results suggest that TLR7 signaling is involved in the progression of NASH. To demonstrate the mechanistic effects of the TLR7–type I IFN pathway on steatotic hepatocyte death, ex vivo experiments were conducted using a hepatocytes-KCs coculture system. Viabilities of WT and TLR7-deficient hepatocytes were comparable after treatment with palmitic acid (Supplemental Figure S1A). To mimic in vivo condition, hepatocytes were co-cultured with KCs. Hepatocytes co-cultured with KCs from livers of TLR7-deficient mice had significantly less cell death compared with those from livers of WT mice (Figure 2A). In contrast, these effects were dampened in the presence of TNF-α (Figure 2B). Furthermore, R848-induced cell death was significantly inhibited by pentoxifylline treatment, suggesting that TLR7-mediated hepatocyte death is dependent on KC-derived TNF-α (Figure 2C). Consistently, the expression of TNF-α was significantly decreased in TLR7-deficient mice fed with an MCD diet. The expression level of TNF-α was markedly down-regulated in co-cultured TLR7 KO KCs compared with that in WT KCs (Figure 2D), suggesting that KCs might be the major source for inducing TNF-α–mediated hepatocyte death. R848 treatment dramatically up-regulated hepatic mRNA expression of TNF-α in MCD-fed mice compared with that in MCS-fed mice (Supplemental Figure S1, B and C). The serum ALT level was also significantly increased by R848 treatment in both diet groups (Figure 2E), which was corroborated by the results of hepatocyte apoptosis using TUNEL results (Supplemental Figure S2, A and B). These results indicate that TNF-α might be a key mediator in the pathogenesis of lipid-mediated hepatocyte death. Oxidative stress is known to play an important role in the pathogenesis of NASH.9Rivera C.A. Adegboyega P. van Rooijen N. Tagalicud A. Allman M. Wallace M. Toll-like receptor-4 signaling and Kupffer cells play pivotal roles in the pathogenesis of non-alcoholic steatohepatitis.J Hepatol. 2007; 47: 571-579Abstract Full Text Full Text PDF PubMed Scopus (504) Google Scholar, 14Miura K. Kodama Y. Inokuchi S. Schnabl B. Aoyama T. Ohnishi H. Olefsky J.M. Brenner D.A. Seki E. Toll-like receptor 9 promotes steatohepatitis by induction of interleukin-1beta in mice.Gastroenterology. 2010; 139: 323-334.e7Abstract Full Text Full Text PDF PubMed Scopus (557) Google Scholar, 30Rinella M.E. Elias M.S. Smolak R.R. Fu T. Borensztajn J. Green R.M. Mechanisms of hepatic steatosis in mice fed a lipogenic methionine choline-deficient diet.J Lipid Res. 2008; 49: 1068-1076Crossref PubMed Scopus (321) Google Scholar Therefore, lipid peroxidation was assessed by quantifying levels of TBARSs, a major indicator of oxidative stress.31Armstrong D. Browne R. The analysis of free radicals, lipid peroxides, antioxidant enzymes and compounds related to oxidative stress as applied to the clinical chemistry laboratory.Adv Exp Med Biol. 1994; 36
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