RESEARCH ARTICLE
Free Fatty Acids Differentially Downregulate Chemokines in Liver Sinusoidal Endothelial Cells: Insights into Non-Alcoholic Fatty Liver Disease Rachel H. McMahan1, Cara E. Porsche1, Michael G. Edwards2, Hugo R. Rosen1,3,4*
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1 Department of Medicine, Division of Gastroenterology and Hepatology, University of Colorado Denver, Aurora, Colorado, United States of America, 2 Department of Medicine, Division of Pulmonary Sciences and Critical Care Medicine, University of Colorado Denver, Aurora, Colorado, United States of America, 3 Department of Microbiology and Immunology, University of Colorado Denver, Aurora, Colorado, United States of America, 4 Denver Veteran’s Affairs Medical Center, Denver, Colorado, United States of America *
[email protected]
OPEN ACCESS Citation: McMahan RH, Porsche CE, Edwards MG, Rosen HR (2016) Free Fatty Acids Differentially Downregulate Chemokines in Liver Sinusoidal Endothelial Cells: Insights into Non-Alcoholic Fatty Liver Disease. PLoS ONE 11(7): e0159217. doi:10.1371/journal.pone.0159217 Editor: Hervé Guillou, INRA, FRANCE Received: April 7, 2016 Accepted: June 28, 2016 Published: July 25, 2016 Copyright: © 2016 McMahan et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: Microarray data is available in the Gene Expression Omnibus (GEO) repository (NCBI) under the accession # GSE67651. All other data is within the paper and its Supporting Information files. Funding: RHM was funded by NIH/NIDDK K01DKO96025. HRR was funded by a VA Merit Review Grant. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist.
Abstract Non-alcoholic fatty liver disease is a prevalent problem throughout the western world. Liver sinusoidal endothelial cells (LSEC) have been shown to play important roles in liver injury and repair, but their role in the underlying pathogenetic mechanisms of non-alcoholic fatty liver disease remains undefined. Here, we evaluated the effects of steatosis on LSEC gene expression in a murine model of non-alcoholic fatty liver disease and an immortalized LSEC line. Using microarray we identified distinct gene expression profiles following exposure to free fatty acids. Gene pathway analysis showed a number of differentially expressed genes including those involved in lipid metabolism and signaling and inflammation. Interestingly, in contrast to hepatocytes, fatty acids led to decreased expression of pro-inflammatory chemokines including CCL2 (MCP-1), CXCL10 and CXCL16 in both primary and LSEC cell lines. Chemokine downregulation translated into a significant inhibition of monocyte migration and LSECs isolated from steatotic livers demonstrated a similar shift towards an antiinflammatory phenotype. Overall, these pathways may represent a compensatory mechanism to reverse the liver damage associated with non-alcoholic fatty liver disease.
Introduction Non-alcoholic fatty liver disease (NAFLD) is the fastest-growing liver disease in the western world. NAFLD represents a disease spectrum that is histologically defined and can range from simple hepatic steatosis to non-alcoholic steatohepatitis (NASH). NASH includes steatosis along with liver inflammation, hepatocyte injury and often fibrosis [1]. The mechanisms that lead to the different pathological outcomes are not well defined but hepatic lipid accumulation, primarily as triacylglycerol (TAG), is a key pathogenic feature of NAFLD [2]. Hepatic TAG synthesis results in part from increased uptake of hepatic fatty acids. Circulating free fatty acids
PLOS ONE | DOI:10.1371/journal.pone.0159217 July 25, 2016
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(FFA) make up the majority of FFA encountered by the liver [3] although they can also originate from de novo lipogenesis (DNL) in hepatocytes [4]. In the context of NAFLD elevated plasma FFA are observed and are at least one source for TAG synthesis in hepatocytes [5]. In addition, elevated levels of the saturated palmitic acid (PA) and the mono-unsaturated oleic acid (OA) are found in NAFLD patients and make up a majority of the FFA in TAG [3,6]. In vitro studies of the cellular and metabolic effects of FFA on hepatic cells have focused primarily on hepatocyte function with some studies showing lipotoxic effects on hepatocytes [7–9] and others suggesting that the effects vary depending on the composition of the FFA [10]. Liver sinusoidal endothelial cells (LSEC) play an important role in the regulation of the transport of macromolecules between the blood and liver parenchyma including lipids and lipoproteins. LSEC lack a basement membrane and have pores or fenestrae which allow for regulation of macromolecule transport [11]. In addition, LSEC are also important regulators of lymphocyte adhesion and migration across the sinusoidal endothelium into the parenchyma via expression of adhesion molecules and chemokines [12]. It has been demonstrated both in tissue culture and in murine hepatotoxicity fibrosis models that LSEC can produce a number of inflammatory mediators including pro-inflammaotry cytokines (TNFα, IL-6 and IL-1) and chemokines (CCL2 (MCP-1), CCL3 (MIP1α), CCL4 (Mip1β), CCL5 (Rantes), CXCL1 (KC), CXCL2 (MIP2α))[13,14]. Taken together this suggests that LSEC may participate in the inflammatory response associated with NAFLD. The aim of current study was to evaluate the effects of FFA on LSEC phenotype including cell survival, lipid metabolism and inflammatory mediators in both primary LSEC and immortalized LSEC lines. Here we show that, in contrast to hepatocytes, FFAs inhibit LPS-induced pro-inflammatory chemokine production in LSEC and inhibit inflammatory cell recruitment. This data suggests that LSEC could potentially play a protective role when the liver is presented with an overabundance of FFA as seen in NAFLD.
Materials and Methods Cell Lines and Animals The immortalized murine LSEC cell line TSEC [15] was provided by Dr. Vijay Shah (Mayo Clinic, Rochester, MN) and was cultured in Dulbecco’s modified Eagle medium (DMEM) with 10% fetal bovine serum (FBS), 1% Pen/Strep (Invitrogen) and 1% Endothelial Cell Growth Supplement (Sciencell). Human immortalized LSEC (TMNK-1) [16] were kindly provided by Dr. Alejandro Soto-Gutierrez (University of Pittsburgh, PA) and were cultured in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin (Invitrogen). The murine (AML12) and human (HepG2) hepatocyte cell lines were cultured according to the vendors instructions (ATCC, Manassas, VA). Eight-week-old male C57Bl/6 mice and sixteen-week-old male mice fed a 60% fat diet (380050 DIO) or a 10% fat diet (380056 NCD) for 12 weeks were purchased from Jackson Laboratories (Bar Harbor, ME). All animal experiments were performed humanely as approved by the Animal Care and Use Committee at the University of Colorado Denver under protocol number B-94114(02)1D. Animals were sacrificed using CO2 asphyxiation followed by cervical dislocation and the appropriate organs were harvested following sacrifice.
Cell Culture AML12 and TSEC cell lines (5x105 and 1.25x105 respectively) were plated in 24 well plates with culture media (mentioned previously). After cells had adhered, media was replaced with media containing 1% FFA-free bovine serum albumin (BSA) (Sigma) and variable concentrations of the long chain FFAs palmitic acid (PA, Sigma), oleic acid (OA, Sigma) or both.
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100mM stock solutions of PA and OA were made up in DMSO and fatty acid were pre-incubated with 2.5mM FFA-free BSA prior to adding to culture. For LPS stimulation 100ng/ml LPS (Sigma) was added to the wells for 6 hours. For signaling experiments the MEK1/2 inhibitor PD-0325901 (EMD Calbiochem) was added to LSEC culture 1 hour prior to treatment with FFA. Stock solutions of PD-0325901 were made up at 100mM in DMSO and 1 and 0.3μM were added to the wells. RNA was isolated for quantitative PCR and CCL2 levels in the supernatants were determined using the mouse CCL2 DuoSet ELISA according to the standard protocol (R&D).
Microarray Analysis For microarray analysis triplicate wells of the murine LSEC line, TSEC, were treated with PA and OA (0.33mM/0.33mM) for 18 hours. RNA was harvested using RNeasy Mini kit (Qiagen) and RNA quality verified using the Eukaryote Total RNA Nano assay on an Agilent 2100 Bioanalyzer (Agilent Technologies). Gene expression profiling was performed using Mouse GeneChip ST 2.0 arrays (Affymetrix). Partek1 Genomics Suite™ ver.6.6 (www.partek.com) was used to process and perform the statistical analysis on these array data, using GC-RMA to compute log2 expression values. A gene was considered present if it had an expression value > 4.5 in at least one sample (26,084 out of 41,353 transcripts total). A P-value
