Inflammation and Ectopic Fat Deposition in the Aging Murine Liver Is Influenced by CCR2
2019; Elsevier BV; Volume: 190; Issue: 2 Linguagem: Inglês
10.1016/j.ajpath.2019.10.016
ISSN1525-2191
AutoresElizabeth C. Stahl, Evan R. Delgado, Frances Alencastro, Samuel T. LoPresti, Patrick D. Wilkinson, Nairita Roy, Martin J. Haschak, Clint D. Skillen, Satdarshan P. Monga, Andrew W. Duncan, Bryan N. Brown,
Tópico(s)Adipose Tissue and Metabolism
ResumoAging is associated with inflammation and metabolic syndrome, which manifests in the liver as nonalcoholic fatty liver disease (NAFLD). NAFLD can range in severity from steatosis to fibrotic steatohepatitis and is a major cause of hepatic morbidity. However, the pathogenesis of NAFLD in naturally aged animals is unclear. Herein, we performed a comprehensive study of lipid content and inflammatory signature of livers in 19-month–old aged female mice. These animals exhibited increased body and liver weight, hepatic triglycerides, and inflammatory gene expression compared with 3-month–old young controls. The aged mice also had a significant increase in F4/80+ hepatic macrophages, which coexpressed CD11b, suggesting a circulating monocyte origin. A global knockout of the receptor for monocyte chemoattractant protein (CCR2) prevented excess steatosis and inflammation in aging livers but did not reduce the number of CD11b+ macrophages, suggesting changes in macrophage accumulation precede or are independent from chemokine (C-C motif) ligand–CCR2 signaling in the development of age-related NAFLD. RNA sequencing further elucidated complex changes in inflammatory and metabolic gene expression in the aging liver. In conclusion, we report a previously unknown accumulation of CD11b+ macrophages in aged livers with robust inflammatory and metabolic transcriptomic changes. A better understanding of the hallmarks of aging in the liver will be crucial in the development of preventive measures and treatments for end-stage liver disease in elderly patients. Aging is associated with inflammation and metabolic syndrome, which manifests in the liver as nonalcoholic fatty liver disease (NAFLD). NAFLD can range in severity from steatosis to fibrotic steatohepatitis and is a major cause of hepatic morbidity. However, the pathogenesis of NAFLD in naturally aged animals is unclear. Herein, we performed a comprehensive study of lipid content and inflammatory signature of livers in 19-month–old aged female mice. These animals exhibited increased body and liver weight, hepatic triglycerides, and inflammatory gene expression compared with 3-month–old young controls. The aged mice also had a significant increase in F4/80+ hepatic macrophages, which coexpressed CD11b, suggesting a circulating monocyte origin. A global knockout of the receptor for monocyte chemoattractant protein (CCR2) prevented excess steatosis and inflammation in aging livers but did not reduce the number of CD11b+ macrophages, suggesting changes in macrophage accumulation precede or are independent from chemokine (C-C motif) ligand–CCR2 signaling in the development of age-related NAFLD. RNA sequencing further elucidated complex changes in inflammatory and metabolic gene expression in the aging liver. In conclusion, we report a previously unknown accumulation of CD11b+ macrophages in aged livers with robust inflammatory and metabolic transcriptomic changes. A better understanding of the hallmarks of aging in the liver will be crucial in the development of preventive measures and treatments for end-stage liver disease in elderly patients. Nonalcoholic steatohepatitis (NASH) is the leading cause of chronic liver disease in the world and is currently the second leading indication for liver transplantation in the United States.1Mikolasevic I. Filipec-Kanizaj T. Mijic M. Jakopcic I. Milic S. Hrstic I. Sobocan N. Stimac D. Burra P. Nonalcoholic fatty liver disease and liver transplantation: where do we stand?.World J Gastroenterol. 2018; 24: 1491-1506Crossref PubMed Scopus (72) Google Scholar, 2Wong R.J. Aguilar M. Cheung R. Perumpail R.B. Harrison S.A. Younossi Z.M. 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Targeting hepatic macrophages to treat liver diseases.J Hepatol. 2017; 66: 1300-1312Abstract Full Text Full Text PDF PubMed Scopus (502) Google Scholar The production of CCL2 is regulated by the canonical NF-κB signaling pathway and is inversely correlated to liver X receptor and retinoid X receptor signaling in both macrophages and hepatocytes.21Chinenov Y. Gupte R. Rogatsky I. Nuclear receptors in inflammation control: repression by GR and beyond.Mol Cell Endocrinol. 2013; 380: 55-64Crossref PubMed Scopus (47) Google Scholar This study investigates hepatic steatosis in aging mice, and identifies phenotypical and functional changes in hepatic macrophages, toward a better understanding of mechanisms underlying the development of fatty liver disease and inflammation in the natural aging process. The Institutional Animal Care and Use Committee of the University of Pittsburgh (Pittsburgh, PA) approved all mouse experiments. C57BL/6 wild-type (WT) mice were obtained at the ages of 2 and 18 months from the National Institute on Aging (NIA) Rodent Colony (Charles River Laboratories, Wilmington, MA), where they received the NIH 31 diet (Ziegler Feed, Gardners, PA) and were used for experiments 1 month after arrival. CCR2 knockout (KO) mice (B6.129S4-CCr2 ) or WT controls were obtained from The Jackson Laboratory (Bar Harbor, ME) as retired breeders (7 to 8 months of age), where they received JL Rat and Mouse Auto 6F (LabDiet, St. Louis, MO) and were aged in house at the University of Pittsburgh for an additional 11 to 12 months, where they received Prolab Isopro RMH 3000 (Lab Diet). Female mice were included exclusively in this study, as sex-specific differences in the age of onset of NAFLD/NASH have been noted in the literature.22Ballestri S. Nascimbeni F. Baldelli E. Marrazzo A. Romagnoli D. Lonardo A. NAFLD as a sexual dimorphic disease: role of gender and reproductive status in the development and progression of nonalcoholic fatty liver disease and inherent cardiovascular risk.Adv Ther. 2017; 34: 1291-1326Crossref PubMed Scopus (289) Google Scholar Females are more likely to develop NAFLD/NASH with aging and after menopause, whereas males are less well associated with age as a risk factor for developing NAFLD/NASH.22Ballestri S. Nascimbeni F. Baldelli E. Marrazzo A. Romagnoli D. Lonardo A. NAFLD as a sexual dimorphic disease: role of gender and reproductive status in the development and progression of nonalcoholic fatty liver disease and inherent cardiovascular risk.Adv Ther. 2017; 34: 1291-1326Crossref PubMed Scopus (289) Google Scholar Mice were maintained on a standard 12-hour light and 12-hour dark system at the University of Pittsburgh specific pathogen-free animal facility. Mice were housed in Optimice cages (AnimalCare Systems, Centennial, CO) with Sani-Chip Coarse bedding (P.J. Murphy, Montville, NJ) and provided ad libitum access to water. Mice were provided huts and running wheels for enrichment. Mice were weighed and sacrificed by carbon dioxide inhalation, followed by cervical dislocation, in accordance with University of Pittsburgh Institutional Animal Care and Use Committee standards. Livers were excised and rinsed in phosphate-buffered saline (PBS). Liver mass was recorded. Small pieces of liver were dissected and i) fixed in 2% paraformaldehyde (Acros Organics; Fisher Scientific, Pittsburgh, PA) for 2 hours, followed by 60% sucrose overnight, then embedded in OCT compound (Fisher Scientific); ii) fixed in 10% neutral-buffered formalin (Fisher Scientific) and embedded into paraffin blocks by the McGowan Institute for Regenerative Medicine histology core; or iii) snap frozen in liquid nitrogen and stored at −80°C for later analysis of RNA, protein, or lipids. Primary hepatocytes were isolated using a two-step collagenase perfusion.23Overturf K. Al-Dhalimy M. Tanguay R. Brantly M. Ou C.N. Finegold M. Grompe M. Hepatocytes corrected by gene therapy are selected in vivo in a murine model of hereditary tyrosinaemia type I.Nat Genet. 1996; 12: 266-273Crossref PubMed Scopus (493) Google Scholar Briefly, after general anesthesia induction, a catheter was inserted into the portal vein or inferior vena cava and 0.3 mg/mL collagenase II (Worthington, Lakewood, NJ) was circulated through the liver. Digested livers were placed in Dulbecco's modified Eagle's medium with 10% fetal bovine serum (Corning, Tewksburg, MA), passed through 100- and 70-μm cell strainers, and washed twice using low-speed centrifugation (50 × g for 3 minutes) to remove nonparenchymal cells (NPCs). Hepatocyte viability, determined by trypan blue staining, was typically >80%, with purity >90%. Hepatocytes were stored in RNA-later (Fisher Scientific) or radioimmunoprecipitation assay (RIPA) buffer (1% IgePAL CA-630 [octylphenoxy poly(ethyleneoxy)ethanol], 0.5% sodium deoxycholate, and 0.1% SDS; Sigma-Aldrich, St. Louis, MO), with Pierce protease and phosphatase inhibitor mini tablet (ThermoScientific, Waltham, MA) at −20°C for RNA/protein analysis at a later time. NPCs were used for flow cytometry or were further purified to collect hepatic macrophages. Briefly, NPCs were blocked with Fc receptor (1:100; rat anti-mouse CD16/32; BD Biosciences, San Jose, CA), stained with F4/80-phosphatidylethanolamine (F4/80-PE) [1:20; rat anti-mouse (BM8); eBioscience, ThermoFisher Scientific, San Diego, CA], and incubated with anti-PE microbeads (1:10; Miltenyi Biotec, Auburn, CA). Cells were applied to LS columns on the QuadroMACS separator (Miltenyi Biotec) and washed three times to remove nontagged NPCs. F4/80+ cells were eluted and counted. Viability of collected F4/80+ cells was typically >85%, with purity >95%. Serum was collected from experimental animals; and the levels of circulating proteins, including alanine transaminase (ALT), aspartate transaminase (AST), albumin, and total bilirubin, or serum cholesterol and triglycerides were determined by the University of Pittsburgh Medical Center Clinical Laboratory. Frozen liver pieces (approximately 100 mg) were shipped overnight on dry ice to the Lipids and Lipoproteins Subcore at the Vanderbilt Mouse Metabolic Phenotyping Center Analytical Resources Core (Nashville, TN). Lipids were extracted from liver tissue by thin-layer chromatography, and total cholesterol, triglycerides, and fatty acids were quantified by gas chromatography. Frozen liver pieces (approximately 100 mg) were homogenized in 75% ethanol (DeconLabs, King of Prussia, PA) and incubated at 50°C for 2 hours. Digested lysates were centrifuged at 6000 × g for 10 minutes, and the supernatant was collected. Bile acids were quantified using the Mouse Total Bile Acids Kit (Crystal Chem, Elk Grove Village, IL). Glucose levels were measured from blood using EmbracePRO glucose monitor (Omnis Health, Nashville, TN) after 6 hours of fasting. Mice received i.p. injection of 10% d-glucose solution (1g/kg; Fisher Scientific). Levels of glucose in the blood were measured at 15, 30, 60, and 120 minutes after injection and recorded. Paraffin blocks were divided into sections (4 μm thick) and affixed to glass slides. Sections were deparaffinized using xylene and graded ethanol (100% to 95%) washes and then placed in tap water. Hematoxylin and eosin, Masson trichrome, and Picrosirius red staining was performed in house, according to standard protocols (Sigma-Aldrich). Staining was performed by the McGowan Institute for Regenerative Medicine histology core using ApopTag Peroxidase In Situ Apoptosis Detection Kit (EMD Millipore, Burlington MA). OCT blocks were divided into sections (7 μm thick) and affixed to glass slides. Sections were brought to room temperature for 10 minutes, then stained with Oil Red O (Sigma-Aldrich), or blocked with 5% donkey serum (Fisher Scientific), 1% bovine serum albumin (Sigma-Aldrich), and 0.01% Triton X–Tween 20 (Fisher Scientific) for 1 hour for immunostaining. Primary antibodies, rabbit anti-mouse CD68 (ab125212; 1:250; Abcam, Cambridge, MA), rat anti-mouse CD11b (550282; 1:100; BD Biosciences, San Jose, CA), or rabbit anti-p21 (sc-471; Santa Cruz Biotechnology Inc., Dallas, TX), were applied to tissue sections overnight at 4°C. Slides were washed three times in PBS, then secondary antibodies were applied for 1 hour at 1:250 dilution (donkey anti-rabbit 647 or donkey anti-rat 594; ThermoFisher Scientific). Slides were washed three times in PBS, then blocked in 5% rat serum (ThermoFisher Scientific) for 1 hour at room temperature. F4/80–FITC (ab105155; 1:50; Abcam) was applied for 1 hour at room temperature. Slides were washed three times in PBS, counterstained with DAPI (BioLegend, San Diego, CA), mounted, and coverslipped. Light microscopy was performed on Eclipse50i microscope (Nikon Instruments Inc., Melville, NY). Fluorescence microscopy was performed on EclipseTiU (Nikon Instruments Inc.). Staining was quantitated in ImageJ software version 1.50i (NIH, Bethesda, MD; http://imagej.nih.gov/ij) or NIS Elements Basic Research software version 4.13 (Nikon Instruments Inc.). RNA-later solution was removed from cell pellets. RNA was isolated using the RNeasy mini kit (Qiagen, Gaithersburg, MD). Briefly, cells were lysed with the RLT buffer and 70% ethanol, then applied to spin columns, washed, and eluted, per the manufacturer's directions. RNA was quantified using a NanoDrop Lite Spectrophotometer (ThermoFisher Scientific). cDNA was generated using the RNA-to-cDNA kit (Applied Biosystems, Foster City, CA). Quantitative RT-PCR was performed with 10 μg of cDNA, TaqMan Gene Expression Master Mix (ThermoFisher Scientific), and TaqMan primers (ThermoFisher Scientific) on the Quant Studio 3 machine (Applied Biosystems). The primers used were Ccl2 (Mm00441242_m1), Tnfa (Mm00443258_m1), Il1b (Mm00434228_m1), Il6 (Mm00446190_m1), and β-actin (Mm00607939_s1). Fold change was calculated using the 2−Δ(ΔCt) method compared with young or young wild-type control samples, unless otherwise stated. Hepatocyte protein lysates were quantified using a BCA Assay (ThermoFisher Scientific). CCL2 was measured with the murine JE/MCP-1 (CCL2) enzyme-linked immunosorbent assay kit (Peprotech, Rocky Hill, NJ), according to the manufacturer's directions. NPCs were washed in fluorescence-activated cell sorting buffer [5% fetal bovine serum and 1% bovine serum albumin in Hank's Buffered Saline Solution (Sigma-Aldrich)] and stained with fixable near-infrared viability dye (1:1000; FisherScientific). Cells were blocked with Fc blocker (1:100; CD16/32; BD Biosciences), then surface stained with flow antibodies [1:20; F4/80-PE (BM8; eBioscience, ThermoFisher Scientific), CD11b-APC (1:20; M1/70; eBioscience, ThermoFisher Scientific), CD146-PerCypCy5.5 (1:20; ME-9F1; BD Biosciences), and CD45–FITC (1:20; 30-F11; BD Biosciences)] for 20 minutes at 4°C. Cells were fixed with Fixation Buffer (BioLegend) and stored overnight in fluorescence-activated cell sorting buffer. The following morning, cells were permeabilized with Intracellular Staining Permeabilization Wash Buffer (BioLegend) and stained with CD68-BV421 (1:20; FA-11; BioLegend). Cells were washed and analyzed on the MACSQuant Flow Cytometer (Miltenyi Biotec) and analyzed with FlowJo Software version 9.9.5 (FlowJo LLC, Ashland OR). Bone marrow was harvested from 2-month–old female WT mice (NIA) as follows. First, the harvested tibia and femur bones were rinsed in a sterile dish containing macrophage complete medium consisting of Dulbecco's modified Eagle's medium (Gibco, Grand Island, NY), 10% fetal bovine serum (Invitrogen, Carlsbad, CA), 10% L929 supernatant, 0.1% β-mercaptoethanol (Gibco), 100 U/mL penicillin, 100 μg/mL streptomyocin, 10 mm nonessential amino acids (Gibco), and 10 mmol/L HEPES buffer. Then, the ends of each bone were cut and the marrow cavity was flushed with the medium using a 30-gauge needle. The cells were plated at 106 cell/mL, and allowed to differentiate into macrophages for 7 days at 37°C, 5% CO2, with complete media changes every 48 hours. Isolated bone marrow macrophages or F4/80+ hepatic macrophages were cultured at 100,000 cells in a tissue culture–treated 96-well plate (Corning; 31.6 mm2 growth volume or 3165 cells/mm2) in RPMI 1640 media (Sigma-Aldrich) supplemented with 10% fetal bovine serum (Corning), 1% 1 mol/L HEPES (Sigma-Aldrich), and 1% penicillin-streptomycin (Fisher Scientific). After allowing hepatic macrophages to adhere to the cell culture plates for 3 hours, medium was replaced with fresh warmed medium (M0 group) or fresh medium supplemented with recombinant murine interferon-γ (20 ng/mL; Peprotech) and lipopolysaccharides from Escherichia coli O55:B5 (100 ng/mL; Sigma-Aldrich; M1 group), or supplemented with IL-4 (20 ng/mL; Peprotech; M2 group) for 12 hours. After 12 hours, the supernatants were collected for nitric oxide production assay using the Griess test. Briefly, supernatant was mixed with equal amounts of 1% sulfanilamide in 5% phosphoric acid and 0.1% N-(1-napthyl) ethylenediamine dihydrochloride (Sigma-Aldrich) and absorbance was read at 540 nm on Synergy HTX plate reader (BioTek, Winooski VT), compared with a standard curve prepared from 0.1 mol/L sodium nitrite (Sigma-Aldrich). Cells were fixed with 2% paraformaldehyde, then blocked with 5% donkey serum (ThermoFisher Scientific), 1% bovine serum albumin (Sigma-Aldrich), and 0.01% Triton X–Tween 20 (Fisher Scientific) for 1 hour for immunostaining. Primary antibodies, rabbit anti-mouse inducible nitric oxide synthase (1:100; ab3523; Abcam) and rabbit anti-mouse liver arginase (1:400; ab91279; Abcam), were applied to cells overnight at 4°C. Cells were washed three times in PBS, then secondary antibodies were applied for 1 hour at 1:250 dilution (donkey anti-rabbit 488; ThermoFisher Scientific), counterstained with DAPI (BioLegend), and imaged. Alternatively, cells were incubated with Vybrant Phagocytosis FITC-E. coli particles (ThermoFisher Scientific) at a 1:20 dilution in media for 2 hours at 37°C, then washed, fixed with 2% paraformaldehyde, and counterstained with DAPI for analysis of phagocytosis. Urea production was measured by lysing cells in 0.001% Triton-X (ThermoFisher Scientific) and combining with arginase activation solution (10 mmol/L MnCl2 and 50 mmol/L Tris-HCl; Sigma-Aldrich) for 10 minutes at 56°C, then incubating at 37°C for 22 hours with arginase substrate solution (0.5 mol/L l-arginine; Sigma-Aldrich). Urea was measured with detection solution (513 mg/L primaquine, 100 mg/L phthalaldehyde, 2.5 mol/L sulfuric acid, 2.5 g/L boric acid, and 0.03% Brij35; Sigma-Aldrich) against a urea standard curve, and absorbance was read at 430 nm on Synergy HTX plate reader. Blood was collected from female 3-month–old and 19-month–old (NIA) mice fasted for 6 hours sterilely by inserting a catheter directly into the inferior vena cava. Blood was allowed to coagulate in a sterile tube for 15 minutes and centrifuged at 10,000 × g for 10 minutes, and the serum supernatant was collected and stored at −80°C. Serum was diluted 1:10, and levels of endotoxin were quantified using the ToxinSensor LAL Chromogenic Endotoxin Quantitation Kit (VWR, Radnor, PA), according to the manufacturer's instructions. Alternatively, blood was collected in EDTA tubes (BD Biosciences), then DNA was isolated using the DNeasy Blood and Tissue Kit (Qiagen). Quantitative RT-PCR was performed on DNA for 16S rRNA (TaqMan: Ba04930791_s1), according to methods described in Gene Expression. RNA was processed by the University of Pittsburgh Health Sciences Sequencing Core for mRNA-sequencing (Seq). Libraries were prepared with TruSeq Stranded mRNA kit (Illumina, San Diego, CA), according to manufacturer’s instructions. Sequencing was on a NextSeq 500 (Illumina) using a single mid-output flow cell and 150-base single read. Adapter sequences were trimmed during demultiplexing. This project used the Pittsburgh Health Sciences Core Research Facility Genomics Research Core for RNA sequencing experiments. Raw data were processed in CLC Genomics Workbench 11 (Qiagen). Reads were mapped to the mouse reference genome; and differentially expressed genes were determined between young and aged hepatocyte samples using filters to select genes with reads per kilobase of transcript per million ≥1, absolute fold change >1.5, and false discovery rate ≤ 0.05. Differentially expressed genes were imported into Ingenuity Pathway Analysis version 01-12 (Qiagen) to determine signaling pathways and upstream regulators. Activation (positive z score), inhibition (negative z score), and interactions of canonical pathways were examined on the basis of experimentally determined gene expression changes reported in the literature. Gene fold changes were also uploaded into the BaseSpace Correlation Engine, formerly NextBio, a searchable, online database from Illumina. Pairwise correlation scores were assigned between all gene expression signatures in the database using rank-based enrichment statistics. The most correlated gene expression studies were assigned a numerical score of 100, and the remaining results are normalized to the top-ranked study. When comparing young and aged samples (WT or KO), a two-tailed t-test was used, unless otherwise stated. One-way analysis of variance was used to compare polarization of macrophages, with Tukey multiple comparisons, unless otherwise stated. Two-way analysis of variance was used to detect differences in the proportions of CD11b and CD68 populations between young and aged mice, with Sidak multiple comparisons test. P ≤ 0.05 was considered significant. Aged virgin female mice (19 months old) obtained from the NIA animal repository showed a 1.3-fold increase in total body weight and liver weight compared with young female mice (3 months old) (Table 1). The liver/body weight ratio remained proportional between the age groups (4.7% to 5.0% total body weight). To evaluate hepatic injury and function, serum levels of ALT, AST, bilirubin, and albumin were measured in the aging female mice (Table 1). There were slight, but significant, increases in serum ALT and AST, whereas ser
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