Hepatocytes release ceramide-enriched pro-inflammatory extracellular vesicles in an IRE1α-dependent manner
2016; Elsevier BV; Volume: 57; Issue: 2 Linguagem: Inglês
10.1194/jlr.m063412
ISSN1539-7262
AutoresEiji Kakazu, Amy S. Mauer, Meng Yin, Harmeet Malhi,
Tópico(s)Phagocytosis and Immune Regulation
ResumoNonalcoholic steatohepatitis (NASH) is a lipotoxic disease wherein activation of endoplasmic reticulum (ER) stress response and macrophage-mediated hepatic inflammation are key pathogenic features. However, the lipid mediators linking these two observations remain elusive. We postulated that ER stress-regulated release of pro-inflammatory extracellular vesicles (EVs) from lipotoxic hepatocytes may be this link. EVs were isolated from cell culture supernatants of hepatocytes treated with palmitate (PA) to induce lipotoxic ER stress, characterized by immunofluorescence, Western blotting, electron microscopy, and nanoparticle tracking analysis. Sphingolipids were measured by tandem mass spectrometry. EVs were employed in macrophage chemotaxis assays. PA induced significant EV release. Because PA activates ER stress, we used KO hepatocytes to demonstrate that PA-induced EV release was mediated by inositol requiring enzyme 1α (IRE1α)/X-box binding protein-1. PA-induced EVs were enriched in C16:0 ceramide in an IRE1α-dependent manner, and activated macrophage chemotaxis via formation of sphingosine-1-phosphate (S1P) from C16:0 ceramide. This chemotaxis was blocked by sphingosine kinase inhibitors and S1P receptor inhibitors. Lastly, elevated circulating EVs in experimental and human NASH demonstrated increased C16:0 ceramide. PA induces C16:0 ceramide-enriched EV release in an IRE1α-dependent manner. The ceramide metabolite, S1P, activates macrophage chemotaxis, a potential mechanism for the recruitment of macrophages to the liver under lipotoxic conditions. Nonalcoholic steatohepatitis (NASH) is a lipotoxic disease wherein activation of endoplasmic reticulum (ER) stress response and macrophage-mediated hepatic inflammation are key pathogenic features. However, the lipid mediators linking these two observations remain elusive. We postulated that ER stress-regulated release of pro-inflammatory extracellular vesicles (EVs) from lipotoxic hepatocytes may be this link. EVs were isolated from cell culture supernatants of hepatocytes treated with palmitate (PA) to induce lipotoxic ER stress, characterized by immunofluorescence, Western blotting, electron microscopy, and nanoparticle tracking analysis. Sphingolipids were measured by tandem mass spectrometry. EVs were employed in macrophage chemotaxis assays. PA induced significant EV release. Because PA activates ER stress, we used KO hepatocytes to demonstrate that PA-induced EV release was mediated by inositol requiring enzyme 1α (IRE1α)/X-box binding protein-1. PA-induced EVs were enriched in C16:0 ceramide in an IRE1α-dependent manner, and activated macrophage chemotaxis via formation of sphingosine-1-phosphate (S1P) from C16:0 ceramide. This chemotaxis was blocked by sphingosine kinase inhibitors and S1P receptor inhibitors. Lastly, elevated circulating EVs in experimental and human NASH demonstrated increased C16:0 ceramide. PA induces C16:0 ceramide-enriched EV release in an IRE1α-dependent manner. The ceramide metabolite, S1P, activates macrophage chemotaxis, a potential mechanism for the recruitment of macrophages to the liver under lipotoxic conditions. Nonalcoholic fatty liver disease (NAFLD, defined by the presence of isolated hepatocellular steatosis) and nonalcoholic steatohepatitis (NASH, defined by the presence of hepatocellular steatosis plus liver inflammation and fibrosis) constitute the spectrum of obesity-associated liver diseases, the most prevalent chronic liver diseases in the Western world (1.Rinella M.E. Nonalcoholic fatty liver disease: a systematic review.JAMA. 2015; 313: 2263-2273Crossref PubMed Scopus (1480) Google Scholar). NASH patients are at risk for progressive liver fibrosis, which can culminate in cirrhosis with its attendant risks of hepatocellular carcinoma and liver failure. Besides weight loss, there are no effective regulatory agency-approved pharmacologic therapies for NASH (2.Lassailly G. Caiazzo R. Buob D. Pigeyre M. Verkindt H. Labreuche J. Raverdy V. Leteurtre E. Dharancy S. Louvet A. et al.Bariatric surgery reduces features of nonalcoholic steatohepatitis in morbidly obese patients.Gastroenterology. 2015; 149: 379-388Abstract Full Text Full Text PDF PubMed Scopus (480) Google Scholar). Therefore, understanding NASH pathogenesis to facilitate rational drug development is an urgent unmet need. Circulating and hepatic saturated free fatty acid levels, especially palmitate (PA), are elevated in the obese insulin-resistant state and in NAFLD (3.Nehra V. Angulo P. Buchman A.L. Lindor K.D. Nutritional and metabolic considerations in the etiology of nonalcoholic steatohepatitis.Dig. Dis. Sci. 2001; 46: 2347-2352Crossref PubMed Scopus (139) Google Scholar). PA, both directly and indirectly, as a precursor for other toxic lipid moieties such as lysophosphatidyl choline, can activate the apoptotic machinery in hepatocytes (4.Malhi H. Bronk S.F. Werneburg N.W. Gores G.J. Free fatty acids induce JNK-dependent hepatocyte lipoapoptosis.J. Biol. Chem. 2006; 281: 12093-12101Abstract Full Text Full Text PDF PubMed Scopus (562) Google Scholar, 5.Kakisaka K. Cazanave S.C. Fingas C.D. Guicciardi M.E. Bronk S.F. Werneburg N.W. Mott J.L. Gores G.J. Mechanisms of lysophosphatidylcholine-induced hepatocyte lipoapoptosis.Am. J. Physiol. Gastrointest. Liver Physiol. 2012; 302: G77-G84Crossref PubMed Scopus (143) Google Scholar). PA is also a precursor for ceramide biosynthesis via the de novo pathway. Ceramides are bioactive lipids that are the backbone for sphingolipid synthesis and important structural components of eukaryotic cell membranes (6.Holland W.L. Brozinick J.T. Wang L.P. Hawkins E.D. Sargent K.M. Liu Y. Narra K. Hoehn K.L. Knotts T.A. Siesky A. et al.Inhibition of ceramide synthesis ameliorates glucocorticoid-, saturated-fat-, and obesity-induced insulin resistance.Cell Metab. 2007; 5: 167-179Abstract Full Text Full Text PDF PubMed Scopus (906) Google Scholar). They also play important roles in signaling. In the context of obesity-associated disorders, ceramides are known to accumulate in adipose tissues, and the inhibition of ceramide accumulation ameliorates diabetes and atherosclerosis in lipotoxic disorders (6.Holland W.L. Brozinick J.T. Wang L.P. Hawkins E.D. Sargent K.M. Liu Y. Narra K. Hoehn K.L. Knotts T.A. Siesky A. et al.Inhibition of ceramide synthesis ameliorates glucocorticoid-, saturated-fat-, and obesity-induced insulin resistance.Cell Metab. 2007; 5: 167-179Abstract Full Text Full Text PDF PubMed Scopus (906) Google Scholar, 7.Park T.S. Rosebury W. Kindt E.K. Kowala M.C. Panek R.L. Serine palmitoyltransferase inhibitor myriocin induces the regression of atherosclerotic plaques in hyperlipidemic ApoE-deficient mice.Pharmacol. Res. 2008; 58: 45-51Crossref PubMed Scopus (86) Google Scholar). Recent studies have implicated hepatic C16:0 ceramide accumulation in the pathogenesis of insulin resistance and NASH (8.Turpin S.M. Nicholls H.T. Willmes D.M. Mourier A. Brodesser S. Wunderlich C.M. Mauer J. Xu E. Hammerschmidt P. Bronneke H.S. et al.Obesity-induced CerS6-dependent C16:0 ceramide production promotes weight gain and glucose intolerance.Cell Metab. 2014; 20: 678-686Abstract Full Text Full Text PDF PubMed Scopus (423) Google Scholar, 9.Raichur S. Wang S.T. Chan P.W. Li Y. Ching J. Chaurasia B. Dogra S. Ohman M.K. Takeda K. Sugii S. et al.CerS2 haploinsufficiency inhibits beta-oxidation and confers susceptibility to diet-induced steatohepatitis and insulin resistance.Cell Metab. 2014; 20 (–. [Erratum. 2014. Cell Metab. 20: 919.]): 687-695Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar). However, the exact pathways by which C16:0 ceramides mediate liver injury and inflammation remain incompletely defined. Extracellular vesicles (EVs) are cell-derived membrane-defined circulating nano-particles that are shed both basally and under stress conditions (10.Raposo G. Stoorvogel W. Extracellular vesicles: exosomes, microvesicles, and friends.J. Cell Biol. 2013; 200: 373-383Crossref PubMed Scopus (5145) Google Scholar). EVs are heterogeneous and composed of particles shed by different mechanisms and of variable sizes. Depending on these characteristics, they can be further classified into exosomes, microparticles, oncosomes, etc. Until advancements permit distinction between these particle types, the all-encompassing term, EV, is most appropriate. EVs are a route for cell-derived cargo to be shed from cells, and a mechanism for delivery of specific cargoes from donor cells to recipient cells, thus acting as carriers of a stress-stimulated message. Recently, studies have demonstrated an increase in circulating EV release under in vitro lipotoxic conditions, circulating EVs in mouse models of NASH and ischemia/reperfusion injury, and in patients with chronic hepatitis C and NASH (11.Povero D. Eguchi A. Niesman I.R. Andronikou N. de Mollerat du Jeu X. Mulya A. Berk M. Lazic M. Thapaliya S. Parola M. et al.Lipid-induced toxicity stimulates hepatocytes to release angiogenic microparticles that require Vanin-1 for uptake by endothelial cells.Sci. Signal. 2013; 6: ra88Crossref PubMed Scopus (132) Google Scholar, 12.Kornek M. Lynch M. Mehta S.H. Lai M. Exley M. Afdhal N.H. Schuppan D. Circulating microparticles as disease-specific biomarkers of severity of inflammation in patients with hepatitis C or nonalcoholic steatohepatitis.Gastroenterology. 2012; 143: 448-458Abstract Full Text Full Text PDF PubMed Scopus (140) Google Scholar, 13.Nojima H. Freeman C.M. Schuster R.M. Japtok L. Kleuser B. Edwards M.J. Gulbins E. Lentsch A.B. Hepatocyte exosomes mediate liver repair and regeneration via sphingosine-1-phosphate.J. Hepatol. August 5, 2015; (Epub ahead of print. doi:10.1161/01.cir.0000437738.63853.7a)PubMed Google Scholar). Furthermore, it has been reported that ceramides are needed for the formation of EVs via the multivesicular body (MVB) endosomal trafficking pathway (14.Trajkovic K. Hsu C. Chiantia S. Rajendran L. Wenzel D. Wieland F. Schwille P. Brugger B. Simons M. Ceramide triggers budding of exosome vesicles into multivesicular endosomes.Science. 2008; 319: 1244-1247Crossref PubMed Scopus (2286) Google Scholar). However, whether PA-induced ceramide biosynthesis drives an EV release response is not known. Hepatocytes are enriched in the endoplasmic reticulum (ER), a membrane-bound organelle that serves as the subcellular site for lipid and sterol synthesis, as well as the folding factory for secreted proteins (15.Malhi H. Kaufman R.J. Endoplasmic reticulum stress in liver disease.J. Hepatol. 2011; 54: 795-809Abstract Full Text Full Text PDF PubMed Scopus (840) Google Scholar). Perturbations in ER function result in ER stress. ER stress is observed in NAFLD and PA-induced lipotoxicity (15.Malhi H. Kaufman R.J. Endoplasmic reticulum stress in liver disease.J. Hepatol. 2011; 54: 795-809Abstract Full Text Full Text PDF PubMed Scopus (840) Google Scholar, 16.Malhi H. Kropp E.M. Clavo V.F. Kobrossi C.R. Han J. Mauer A.S. Yong J. Kaufman R.J. C/EBP homologous protein-induced macrophage apoptosis protects mice from steatohepatitis.J. Biol. Chem. 2013; 288: 18624-18642Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Indeed, PA can directly activate all three ER stress sensors: i) inositol requiring enzyme 1α (IRE1α); ii) activating transcription factor 6α (ATF6α); and iii) protein kinase-like ER kinase (PERK). Because PA-driven de novo ceramide synthesis occurs at the ER, we asked whether PA-induced lipotoxic ER stress would lead to an EV response, and whether any of the three ER stress sensors would mediate PA-induced EV release. Although a full spectrum of inflammatory cells exists in the injured liver, macrophages have received the most attention in NASH, as they are recruited to the liver during lipotoxicity (17.Morinaga H. Mayoral R. Heinrichsdorff J. Osborn O. Franck N. Hah N. Walenta E. Bandyopadhyay G. Pessentheiner A.R. Chi T.J. et al.Characterization of distinct subpopulations of hepatic macrophages in HFD/obese mice.Diabetes. 2015; 64: 1120-1130Crossref PubMed Scopus (115) Google Scholar). Furthermore, inhibition of macrophage activation, recruitment, or accumulation in the liver ameliorates steatohepatitis (16.Malhi H. Kropp E.M. Clavo V.F. Kobrossi C.R. Han J. Mauer A.S. Yong J. Kaufman R.J. C/EBP homologous protein-induced macrophage apoptosis protects mice from steatohepatitis.J. Biol. Chem. 2013; 288: 18624-18642Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Herein, we report that PA-induced ER stress leads to EV release. Furthermore, ceramides are enriched in PA-induced EVs, and this phenomenon occurs in an IRE1α-dependent manner. The ceramide-enriched vesicles also contain sphingosine-1-phosphate (S1P), a known inflammatory mediator and activator of macrophages. We speculate that inhibition of this pathway and/or S1P signaling may be salubrious in NASH. Previously described immortalized mouse hepatocyte (IMH) cell lines derived from IRE1α KO and WT mice (18.Zhang K. Wang S. Malhotra J. Hassler J.R. Back S.H. Wang G. Chang L. Xu W. Miao H. Leonardi R. et al.The unfolded protein response transducer IRE1alpha prevents ER stress-induced hepatic steatosis.EMBO J. 2011; 30: 1357-1375Crossref PubMed Scopus (255) Google Scholar), ATF6α-WT and KO IMH cell lines, and eIF2α S51A phosphorylation resistant (AA) and WT (SS) IMH cell lines were a kind gift of Dr. Randal Kaufman. The IMH cell lines were cultured in DMEM (Life Technologies) supplemented with 10% FBS, penicillin, and streptomycin. Huh7 cells were cultured in DMEM supplemented with 10% FBS, penicillin, and streptomycin. Primary mouse hepatocytes were isolated by collagenase perfusion followed by Percoll purification, cultured as previously described by us (19.Faubion W.A. Guicciardi M.E. Miyoshi H. Bronk S.F. Roberts P.J. Svingen P.A. Kaufmann S.H. Gores G.J. Toxic bile salts induce rodent hepatocyte apoptosis via direct activation of Fas.J. Clin. Invest. 1999; 103: 137-145Crossref PubMed Scopus (465) Google Scholar), and used in experiments if viability exceeded 95%. For EV isolation, cells were cultured in growth medium supplemented with 5% EV-depleted FBS [prepared by overnight centrifugation at 100,000 g at 4°C according to standard protocols (20.Thery C. Amigorena S. Raposo G. Clayton A. Isolation and characterization of exosomes from cell culture supernatants and biological fluids.Curr. Protoc. Cell Biol. 2006; Chapter 3: 3.22Google Scholar)] and 1% BSA, with the addition of either PA or oleate (OA) as previously described by us (4.Malhi H. Bronk S.F. Werneburg N.W. Gores G.J. Free fatty acids induce JNK-dependent hepatocyte lipoapoptosis.J. Biol. Chem. 2006; 281: 12093-12101Abstract Full Text Full Text PDF PubMed Scopus (562) Google Scholar), thapsigargin (Tg), or vehicle, respectively. Bone marrow-derived macrophages (BMDMϕs) were isolated from the long leg bones of C57Bl/6J mice, as previously described by us (16.Malhi H. Kropp E.M. Clavo V.F. Kobrossi C.R. Han J. Mauer A.S. Yong J. Kaufman R.J. C/EBP homologous protein-induced macrophage apoptosis protects mice from steatohepatitis.J. Biol. Chem. 2013; 288: 18624-18642Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Briefly, dissected long bones were flushed with serum-free RPMI and cells were differentiated in 20% L929 cell conditioned medium containing RPMI and used on day 7 after differentiation. Cells were grown to 90% confluency on 150 mm tissue culture plastic dishes. Before treating, cells were washed twice with PBS and then the medium was changed to assay medium supplemented with PA, OA, or Tg (4.Malhi H. Bronk S.F. Werneburg N.W. Gores G.J. Free fatty acids induce JNK-dependent hepatocyte lipoapoptosis.J. Biol. Chem. 2006; 281: 12093-12101Abstract Full Text Full Text PDF PubMed Scopus (562) Google Scholar). After 14–16 h, supernatant was recovered and sequential low-speed centrifugation was performed to deplete cells and cellular debris at 2,000 g for 20 min followed by 20,000 g for 30 min (20.Thery C. Amigorena S. Raposo G. Clayton A. Isolation and characterization of exosomes from cell culture supernatants and biological fluids.Curr. Protoc. Cell Biol. 2006; Chapter 3: 3.22Google Scholar). The supernatant was further ultracentrifuged at 100,000 g for 90 min to pellet EVs, which were washed once by resuspending in PBS followed by ultracentrifugation at 100,000 g for 90 min. The final EV pellet was resuspended in PBS and either used for downstream experiments or stored at −80°C. For each experimental condition, isolated EVs were normalized to cell number and expressed relative to the vehicle-treated condition, unless indicated otherwise. Circulating vesicles were isolated from platelet-poor plasma by differential ultracentrifugation, as previously described (20.Thery C. Amigorena S. Raposo G. Clayton A. Isolation and characterization of exosomes from cell culture supernatants and biological fluids.Curr. Protoc. Cell Biol. 2006; Chapter 3: 3.22Google Scholar). For human plasma samples, 900 ¼l each were used. For mouse plasma samples, 100 ¼l each were used. Briefly, plasma was diluted with an equal volume of PBS and centrifuged at 2,000 g for 30 min at 4°C. The clear supernatant was transferred to new tubes and centrifuged at 12,000 g for 30 min at 4°C. EVs were isolated from the second spin supernatant by ultracentrifugation at 110,000 g for 120 min. Isolated vesicles were characterized by nanoparticle tracking analysis and stored frozen until further analyses. The Nanosight NTA NS300 (Malvern Instruments, UK) equipped with a fast video capture and nanoparticle tracking analysis (NTA) software was used to characterize EV particle size and concentration (21.van der Pol E. Coumans F.A. Grootemaat A.E. Gardiner C. Sargent I.L. Harrison P. Sturk A. van Leeuwen T.G. Nieuwland R. Particle size distribution of exosomes and microvesicles determined by transmission electron microscopy, flow cytometry, nanoparticle tracking analysis, and resistive pulse sensing.J. Thromb. Haemost. 2014; 12: 1182-1192Crossref PubMed Scopus (560) Google Scholar). The instrument was calibrated according to the manufacturer's protocol. EV samples were diluted in PBS to perform measurements in the linear dynamic range of the instrument (2E+08 to 8E+08 particles/ml). Each sample was perfused through the sample chamber at a constant rate of 25 ¼l/min using a syringe pump. The light scatter and Brownian motion of each sample of nanoparticles was recorded at least three times, 30 s each at constant room temperature (22.5°C); particle tracks were analyzed by NanoSight software to measure the concentration of the particles (particles per milliliter) and size (in nanometers). Ceramides and nonesterified fatty acids were measured using mass spectrometry at the Mayo Clinic Metabolomics Core Laboratory. Briefly, ceramides were extracted from EVs or cell pellets suspended in 1× PBS after the addition of internal standards and sonication. The extracts were measured against a standard curve on the Thermo TSQ Quantum Ultra mass spectrometer (Thermo Scientific, West Palm Beach, FL) coupled with a Waters Acquity UPLC system (Waters, Milford, MA), as previously described (22.Blachnio-Zabielska A.U. Persson X.M. Koutsari C. Zabielski P. Jensen M.D. A liquid chromatography/tandem mass spectrometry method for measuring the in vivo incorporation of plasma free fatty acids into intramyocellular ceramides in humans.Rapid Commun. Mass Spectrom. 2012; 26: 1134-1140Crossref PubMed Scopus (69) Google Scholar). EVs isolated from equal numbers of cells treated with vehicle or PA were used to quantify changes in EV ceramides. Cell pellet ceramides were normalized to protein content. Ceramides in plasma EVs were measured in EVs isolated from equal volumes of plasma across experimental groups. Whole mount preparations of isolated EVs were prepared for electron microscopy according to established methods (20.Thery C. Amigorena S. Raposo G. Clayton A. Isolation and characterization of exosomes from cell culture supernatants and biological fluids.Curr. Protoc. Cell Biol. 2006; Chapter 3: 3.22Google Scholar). Briefly, EVs suspended in PBS were fixed with 2% paraformaldehyde, applied to Formvar carbon-coated EM grid, fixed with 1% glutaraldehyde, contrasted with uranyl-oxalate solution, embedded in a mixture of uranyl acetate and methylcellulose, air dried, and observed under the JEOL 1400 electron microscope (JEOL USA, Peabody, MA). Treated cells were collected by scraping and lysed in RIPA buffer (50 mM Tris HCL, 1% NP-40, 0.1% SDS, 150 nM NaCl, 1 mM EDTA, and 0.5% sodium deoxycholate) with protease and phosphatase inhibitors. The protein concentrations were determined by the Bio-Rad DC protein assay (Bio-Rad, Hercules, CA). Equal amounts of protein were loaded onto Criterion 12.5% Tris-HCl gel (Bio-Rad) and electro-transferred to an Immobilion® -FL PVDF membrane (EMD Millipore). After washing, the membranes were incubated in 25 ml of blocking buffer (LI-COR Biosciences, Lincoln, NE) for 1 h at room temperature. Immunostaining was performed with the primary antibody [CD63, TSG101, and Sptlc-1 (Santa Cruz Biotechnologies); phospho-eIF2α (Invitrogen, Carlsbad, CA)], followed by incubation with IRDye 680RD and 800CW secondary antibodies (LI-COR). Immunoreactive proteins were detected with an infrared imaging system (LI-COR). Cells were cultured on cover slips in 6-well plates. After removing medium, cells were washed three times with PBS and fixed (0.1 M PIPES, 1.0 mM EGTA, 3.0 mM MgSO4, and 2.5% formaldehyde). After washing with PBS, cells were permeabilized by Triton X-100. Blocking buffer (5% goat serum, 5% glycerol, and 0.04% sodium azide) was applied to fixed and permeabilized cells for 1 h followed by primary antibody overnight at 4°C. After washing with PBS, cells were incubated with secondary antibody for 1 h, washed with PBS, and mounted with 20 ¼l Prolong AntiFade (Life Technologies) on a clean glass slide. In the case of CD63 (Santa Cruz Biotechnologies), we used poly-L-lysine-coated cover slips (Sigma) for growing cells. Fluorescence was observed with confocal microscopy (LSM 780 Zeiss). After assay medium was removed, cells were washed two times with PBS, collected in 1 ml Trizol, and the aqueous phase, after addition of 200 ¼l chloroform and centrifugation, was applied to the G-eliminator column (RNeasy Plus; Qiagen) and RNA was extracted from the flow-through according to the manufacturer's instructions. Quantity and quality of RNA was measured spectrophotometrically using a NanoDrop ND1000 (Thermo Scientific), and RNA was reverse transcribed into cDNA by the iScript cDNA synthesis kit (Bio-Rad). Quantitative real-time PCR reactions were run on the LightCycler 480 (Roche), using the LightCycler 480 SYBR Green 1 Master Mix (Roche) and previously published primers (16.Malhi H. Kropp E.M. Clavo V.F. Kobrossi C.R. Han J. Mauer A.S. Yong J. Kaufman R.J. C/EBP homologous protein-induced macrophage apoptosis protects mice from steatohepatitis.J. Biol. Chem. 2013; 288: 18624-18642Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). WT IMH cells were cultured in 6-well plates. The transient knockdown of SPT1 or X-box binding protein-1 (XBP-1) was performed using SPTLC1 siRNA (Santa Cruz Biotechnologies, sc-153804) or XBP-1 siRNA (Life Technologies, s76116), with control siRNA-A (Santa Cruz Biotechnologies, sc-37007) or Silencer Negative Control siRNA (Life Technologies) as negative controls. The siRNA transfection was performed using X-tremeGENE siRNA (Roche) for SPTLC1 or Lipofectamine RNAiMAX transfection reagent (Invitrogen) for XBP-1 according to manufacturer's instructions. S1P receptor 1 (S1P1) was silenced in BMDMϕs by electroporation, as described (23.Wiese M. Castiglione K. Hensel M. Schleicher U. Bogdan C. Jantsch J. Small interfering RNA (siRNA) delivery into murine bone marrow-derived macrophages by electroporation.J. Immunol. Methods. 2010; 353: 102-110Crossref PubMed Scopus (39) Google Scholar). Briefly, 2 × 106 cells were electroporated in a 4 mm cuvette (BTX Harvard Apparatus) with GenePulser Xcell (Bio-Rad) at 400 V, 150 ¼F, and 100 Ω. S1P1 siRNA (L-05684-00-0005) and nontargeting siRNA (D-001810-10-05) were purchased from Dharmacon. After electroporation cells were grown on tissue culture plastic in serum-free RPMI for 30 min, an equal volume of bone marrow differentiation medium was added to achieve a final concentration of 10% FBS and 20% L929 conditioned medium. Seventy-two hours after electroporation cells were used for chemotaxis assays. Gene silencing was confirmed by Western blotting or reverse-transcription PCR. WT or IRE1α KO IMH cells were cultured to 90% confluency on 150 mm dishes and treated with C16 ceramide, as described (24.Rénert A.F. Leprince P. Dieu M. Renaut J. Raes M. Bours V. Chapelle J.P. Piette J. Merville M.P. Fillet M. The proapoptotic C16-ceramide-dependent pathway requires the death-promoting factor Btf in colon adenocarcinoma cells.J. Proteome Res. 2009; 8: 4810-4822Crossref PubMed Scopus (35) Google Scholar). After washing with PBS, the medium was changed to assay medium supplemented with 10 ¼M C2 ceramide (Enzo, BML-SL100) or 10 ¼M C16 ceramide (Cayman, item number 10681), and then medium was recovered after 16 h for measuring EVs. Cells were cultured for 2–4 h under high-dose C16 ceramide (50 ¼M or 100 ¼M) conditions and the medium recovered for isolation of EVs. The lentiCRISPRv1 and lentiCRISPRv2 plasmids were a gift from Dr. Feng Zhang and obtained from Addgene (25.Sanjana N.E. Shalem O. Zhang F. Improved vectors and genome-wide libraries for CRISPR screening.Nat. Methods. 2014; 11: 783-784Crossref PubMed Scopus (2688) Google Scholar). The online tool (http://crispr.mit.edu) was used to design the single guide RNA spanning the first exon of human XBP-1 (sgRNA 5′-CACCGGCGCTGTCGCTTGCGCGCC-3′), which is conserved between total and spliced XBP-1. Annealed oligonucleotides of sgRNA were used to generate a lentivirus following subcloning into the lentiCRISPRv1 plasmid (Addgene), and transfected into HEK 293T cells along with packaging plasmids, pCMV-VSV-G and pCMV-dR8.2 dvpr (Addgene), using Lipofectamine LTX reagent (Invitrogen). Virus was harvested 48 h after transfection and passed through at 0.45 ¼M pore cellulose acetate filter (Millipore). Huh7 cells were infected with XBP-1-targeting lentivirus in the presence of 8 ¼g/ml Polybrene. Successfully infected cells were selected under 2 ¼g/ml puromycin (Invitrogen) selection pressure. XBP-1 KO was confirmed by Western blotting of nuclear extracts from cells treated with tunicamycin to induce ER stress. Modified Boyden chambers (Neuro Probe, BW200S) were used to perform chemotaxis assays using 5 ¼m pore size polycarbonate track-etch membranes (Neuro Probe, PFA5), which were coated with 0.1% gelatin (Sigma, G1393) for 30 min and then air-dried. BMDMϕs were serum starved in 2% exosome-free FBS containing RPMI for 1 h and then pretreated with the appropriate treatment conditions for 1 h. Cells were detached in treatment conditions, counted, and diluted to a concentration of 250,000 cells/ml. Chambers were assembled with chemo-attractants on the bottom in 0.2 ml volume and pretreated cells were placed on top of the membrane in 0.2 ml volume. EVs isolated from equal numbers of vehicle- or PA-treated cells were used as chemo-attractants. PF543 (5 ¼M; Selleckchem) and ABC294640 (1 ¼M; Activebiochem) were employed to inhibit sphingosine kinase (SphK)1 and SphK2, respectively. W146 (1 ¼M; Cayman Chemical) or FTY720 (0.25 ¼M; Cayman Chemical) were used to antagonize S1P receptors. Cells were allowed to migrate for 4 h, fixed in 10% neutral buffered formalin for 20 min at room temperature, rinsed once in PBS and once in water, and mounted in Prolong Gold antifade reagent with DAPI (Life Technologies). Migrated cells were counted on a confocal microscope ((LSM 780 Zeiss) using a UV laser. All animal studies and use were approved by the institutional care and animal use committee of the Mayo Clinic and were conducted in accordance with the public health policy on the humane use and care of laboratory animals. Mice were housed in standard pathogen-free facilities with 12 h day-night circadian cycles and unrestricted access to food and water. C56Bl/6J mice were purchased from Jackson Laboratory (Bar Harbor, ME). Twelve-week-old mice were fed either chow or a diet high in saturated fat, fructose, and cholesterol (FFC) for 24 weeks, as previously described (26.Charlton M. Krishnan A. Viker K. Sanderson S. Cazanave S. McConico A. Masuoko H. Gores G. Fast food diet mouse: novel small animal model of NASH with ballooning, progressive fibrosis, and high physiological fidelity to the human condition.Am. J. Physiol. Gastrointest. Liver Physiol. 2011; 301: G825-G834Crossref PubMed Scopus (272) Google Scholar). This diet recapitulates the pathologic features of human NASH. Upon completion of the feeding study, blood was collected by cardiac puncture, and platelet-poor plasma was isolated by centrifugation at 1,200 g for 20 min at room temperature, followed by 13,000 g at 4°C for 2 min. Plasma was stored at −20°C until further analyses. This study was approved by the Institutional Review Board of the Mayo Clinic and all subjects provided written informed consent. Available archived liver samples from a previously published study were utilized (27.Cazanave S.C. Mott J.L. Elmi N.A. Bronk S.F. Werneburg N.W. Akazawa Y. Kahraman A. Garrison S.P. Zambetti G.P. Charlton M.R. et al.JNK1-dependent PUMA expression contributes to hepatocyte lipoapoptosis.J. Biol. Chem. 2009; 284: 26591-26602Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar). Subjects who had undergone liver biopsy during bariatric surgery for medically complicated obesity with body mass index of >30 kg/m2 [at Mayo Clinic, Rochester, MN, wi
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