Unsaturated fatty acids prevent activation of NLRP3 inflammasome in human monocytes/macrophages

• DOI: 10.1194/jlr.m037861
• Publication Date: 2013-09-05T00:13:11Z
• Authors: Laurent L'homme; Nathalie Esser; Laura Riva; André Scheen; Nicolas Paquot; Jacques Piette; Sylvie Legrand-Poels
• Keywords: 0301 basic medicine; obesity; interleukin-1beta; Inflammasomes; Macrophages; Interleukin-1beta; Palmitates; QD415-436; Biochemistry; Monocytes; Cell Line; 3. Good health; 03 medical and health sciences; inflammation; stearate; NLR Family, Pyrin Domain-Containing 3 Protein; Stearates; Fatty Acids, Unsaturated; Humans; palmitate; Carrier Proteins; innate immunity; Protein Processing, Post-Translational

The NLRP3 inflammasome is involved in many obesity-associated diseases, such as type 2 diabetes, atherosclerosis, and gouty arthritis, through its ability to induce interleukin (IL)-1β release. molecular link between obesity activation still unclear, but free fatty acids have been proposed one triggering event. Here we reported opposite effects of saturated (SFAs) compared with unsaturated (UFAs) on human monocytes/macrophages. Palmitate stearate, both SFAs, triggered IL-1β secretion a caspase-1/ASC/NLRP3-dependent pathway. Unlike the UFAs oleate linoleate did not lead secretion. In addition, they totally prevented release induced by SFAs and, less efficiency, broad range inducers, including nigericin, alum, monosodium urate. affect transcriptional effect suggesting specific activation. These results provide new anti-inflammatory mechanism preventing therefore, processing. By this way, might play protective role NLRP3-associated diseases. 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Free (FFAs), usually elevated plasma people, event Recently, acid (SFA) palmitate demonstrated activate (14Wen Gris Lei Y. Jha Huang M.T. Brickey W.J. Fatty acid-induced NLRP3-ASC interferes signaling.Nat. 12: 408-415Crossref (1272) present study, tested four most important FFAs (15Hodson Skeaff Fielding B.A. composition humans use biomarker dietary intake.Prog. 2008; 47: 348-380Crossref (923) two (UFAs), linoleate. We stearate physiological inducer, acting same efficiency caspase-1/ASC/NLRP3 UFAs, revealed unexpected property inducers urate (MSU). palmitic (#P0500), stearic (#S4751), oleic (#O1008), linoleic (#L1376) purchased Sigma. 100 mM stock solution sodium salt was prepared dissolving 0.1 M NaOH 30°C, 37°C, 65°C, 75°C acid, respectively. Stock solutions aliquoted stored −20°C than year. 5% free, endotoxin BSA (Sigma, #A8806) RPMI 1640. FFA mixed together obtain 2.5 working FFA:BSA molar ratio 3.4:1. After pH adjustment, filtered 0.2 µm pore size membrane filter, aliquoted, months. THP-1 monocytic line (ATCC) (authentication 2011 DSMZ, Germany) THP1-XBlue™ (InvivoGen) cultured 0.5 1.0 × 106 cells/ml 1640 glutamine supplemented 10%1 heat-inactivated FBS, U/ml penicillin, streptomycin maintained 37°C under CO2 atmosphere. Medium reagents Lonza. differentiated ng/ml PMA (Sigma) 24 h, washed PBS, kept night rest fresh medium before stimulation. Primary isolated peripheral mononuclear (PBMCs) using CD14 MicroBeads (Miltenyi Biotec) according manufacturer's instructions. PBMCs obtained single-step density gradient centrifugation Ficoll-Paque PLUS (GE Healthcare) buffy coats (Red Cross, Belgium). Buffy healthy donors after informed consent, experiments approved ethical committee University Liege. monocyte purity evaluated flow cytometry fluorescence-activated sorting (FACS) Canto II (BD Biosciences) FITC-coupled anti-human antibody Pharmingen), it greater 92%. Freshly resuspended 1640, plated, rest. next day, primary primed 200 lipopolysaccharide (LPS) 3 h treatment. Monocyte-derived (MDMs) culturing freshly 20% FBS M-CSF premium grade six days, followed additional 18 stimulation LPS 20 interferon-γ acquire M1-polarization. differentiation, monocytes/macrophages treated FFAs, (Sigma), imject alum (Pierce), MSU (InvivoGen), nigericin (Enzo Life Sciences), muramyl dipeptide (MDP) doxorubicin anti-mycin tunicamycin monensin ATP flagellin poly(dA:dT) FSL-1 (InvivoGen). All compounds used times concentrations indicated figure legends. Flagellin transfected DOTAP liposomal transfection reagent (Roche). Caspase inhibitor Z-VAD-FMK (Promega) Z-YVAD-FMK (BioVision), added 10 µM simultaneously stimuli. stably expressing shRNA lentiviral transduction carried out GIGA-Viral Vectors Platform (GIGA, Liege, summary, promoter sequences amplified PCR commercial plasmids encoding #TRCN0000427726) nontarget sequence #SHC002) cloned pHRSin-CSGW plasmid GFP [a kind gift Dr. Els Veroyen (University Lyon)]. New cotransfected pSPAX2 (Addgene, #12260) VSV-G-encoding (16Emi N. Friedmann Yee J.K. Pseudotype formation leukemia virus G vesicular stomatitis virus.J. Virol. 1991; 65: 1202-1207Crossref Lenti-X 293T (Clontech, #632180). Viral supernatants collected, filtered, concentrated 100× ultracentrifugation. Finally, transduced vectors GFP-positive sorted FACS. Cells HiPerFect (Qiagen) instructions suspension lines, minor modifications. (0.2 105) plated 24-well plate µl medium. Transfection without serum adding 400 ng esiRNA final volume 6 added. differentiation performed previously described. esiRNAs Sigma: esiPYCARD (#EHU066851) ASC, esiNLRP3 (#EHU071121) esiEGFP (#EHUEGFP) negative control. washed, scraped lysis buffer [50 Tris-HCl 7.5, 150 NaCl, 1 EDTA, 1% Igepal, 0.25% deoxycholate, PMSF, complete protease cocktail (Roche Applied Science)], centrifuged remove remnant pellets. Supernatants −20°C. For phosphorylation experiments, lysed total phospho (62.5 6.8, 10% glycerol, 2% SDS, 3% β-mercaptoethanol, 0.03% bromophenol blue, DTT, orthovanadate, 25 β-glycerophosphate, 15 fluoride, cocktail), sonicated, Culture also collected Proteins (40 µg) supernatant (20 µl) subjected SDS-PAGE. electrophoresis, proteins transferred gel PVDF Healthcare). antibodies follows: anti-ASC (Adipogen, #AG-25B-0006), anti-cleaved (Cell Signaling, #2021), anti-IL-1β propeptide (R & D Systems, #MAB6964), #AF-201-NA), anti-NLRP3 #HPA012878), anti-phospho-IκBα #9246), anti-phospho-p65 #3031). secondary conjugated HRP anti-rabbit #7074), anti-goat (Dako, #P0160), anti-mouse #P0447). detection ECL plus Western blotting substrate (Pierce) digital imaging system ImageQuant LAS 4000 Quantification achieved TL software (version 7.0, GE Mature quantified ELISA Quantikine Systems) recommendations. Total RNAs extracted RNeasy Mini Kit protocol, DNase treatment quantification spectrophotometer Nanodrop 1000 (Thermo scientific), 500 RNA reverse-transcribed moloney reverse transcriptase (Invitrogen). Obtained cDNA submitted qRT-PCR LightCycler 480 Science). Relative gene calculated 2-delta delta CT method (17Livak Schmittgen T.D. Analysis relative data real-time quantitative 2(-Delta Delta C(T)) method.Methods. 2001; 25: 402-408Crossref (123392) HPRT1 housekeeping gene. Results representative three independent triplicate. following primers used: IL-1β-Fw: 5′-CCTTGGGCCTCAAGGAAAA-3′ IL-1β-Rv: 5′-CTCCAGCTGTAGAGTGGGCTTA-3′ ASC-Fw: 5′-CGCGAGGGTCACAAACGT-3′ ASC-Rv: 5′-TGCTCATCCGTCAGGACCTT-3′ Caspase-1-Fw: 5′-CCGCAAGGTTCGATTTTCA-3′ Caspase-1-Rv: 5′-ACTCTTTCAGTGGTGGGCATCT-3′ NLRP3-Fw: 5′-CTGTAACATTCGGAGATTGTGGTT-3′ NLRP3-Rv: 5′-GACCAAGGAGATGTCGAAGCA-3′ HPRT1-Fw: 5′-TGACACTGGCAAAACAATGCA-3′ HPRT1-Rv: 5′-GCTTGCGACCTTGACCATCT-3′. fluorescence-quenching (7-methoxycoumarin-4-yl)acetyl- L-tyrosyl- L-valyl- L-alanyl- L-aspartyl- L-prolyl- Nε- (2,4-dinitrophenyl)- L-lysine amide (MOCAc-YVADAPK(Dnp)-NH2) (PeptaNova, #3183-v) DMSO, then light month. 96-well ViewPlate (Perkin Elmer, #6005182) 105 cells/well, PMA, stimulated described per well. treatment, concentration. 90 min incubation, fluorescence measured EnSpire 2300 Multilabel Reader Elmer) excitation wavelength 328 nm emission 393 area scan mode. PMA-differentiated (107 cells/dish) seeded 60 cm2 culture dishes. 4 PBS oligoASC [20 HEPES-KOH KCl, 1.5 MgCl2, EGTA, 320 sucrose, cocktail, disuccinimidyl suberate (DSS; Pierce)] room temperature agitation. microcentrifuge tube. shearing 20× 21 gauge needle, agitation min. Crosslinking stopped addition Tris, samples ice. To nuclei, 5 filter. Crosslinked precipitated full speed 4°C, pellets immunoblot analysis. describe above. QUANTI-Blue™ detect alkaline phosphatase activity. Absorbance 630 Reader. statistical analyses GraphPad Prism Windows (GraphPad Software, Inc.) presented means ± SEM. Student t-test simple comparison (two groups) ANOVA test Bonferroni post-test multiple comparisons (more groups). Significance symbol. poorly soluble aqueous solution, them bound albumin physiology (18Richieri G.V. Kleinfeld A.M. Unbound serum.J. 1995; 36: 229-240Abstract vitro often substituted did. Among (C16:0) pathway (Fig. 1A, bottom), showed that (C18:0) able similar when vehicle alone (BSA) top). On other hand, (C18:1) (C18:2) had no secretion, showing UFAs. Similar findings observed MDMs 1B) 1C), predominant obese. LPS-priming 1A) 1C) linked synthesis. constitutive activity (19Kahlenberg Dubyak G.R. Differing states versus models IL-1beta processing release.J. Leukoc. Biol. 76: 676-684Crossref (49) resulting background related content. explore signaling SFA-induced release, macrophage-like PMA. This classical (20Menu Mayor Zhou Ichijo Mori ER stress activates UPR-independent pathway.Cell Dis. 3: e261Crossref (249) 21Zhou mitochondria activation.Nature. 469: 221-225Crossref (3447) highly responsive allowed us large amounts blot focus process. monocytes/macrophages, increased dose-dependent manner 2A). 17 kDa Fig. 2A known be proteolytic cleavage 3Schroder investigate involvement, along pan-caspase (Z-VAD) caspase-1-specific (Z-YVAD). Inhibition caspases completely abolished while inhibition repressed almost 2B). result suggests key even if contribution caspase excluded. It composed domains protein-protein interactions: pyrin domain (PYD) domain, essential homotypic interaction When knocked down decrease 2C). can participate types complexes: pyroptosome inflammasome. dimerization allowing examine extracts crosslinked DSS dimers. Compared high concentration, well-characterized inducer neither nor led 2D). indicates involvement due formation. complex formed appears PYD protein, pyroptosome, leads recruitment. extensively studied, wide variety knockdown strong 2E), confirming inflammasome-dependent possess double bounds confer particular properties. previously, cannot SFAs. determine impact LPS-primed combination Interestingly, presence equal 3A). decreased 3B, C). affinity long-chain differs each (22Spector A.A. John Fletcher Binding bovine albumin.J. 1969; 10: 56-67Abstract Therefore, more do could mediate inhibitory decreasing unbound fraction, active exclude option, normalized concentration evaluate palmitate-induced 3D). With BSA, inhibiting persisted obviously dose dependent. Moreover, effect. 3E). efficient small concentrations, UFA:SFA 1:2 provided good inhibition. 1:1 all subsequent experiments. Processing regulatory events identify step targeted assessed process Cotreatment reduced intracellular 4A) 4B) Considering probably operates hallmark ASC. Unfortunately, amount endogenous lack make difficult observe over-expressing tagged protein. overcome problem, approach used. explained inflammasom