Hepatic cholesterol crystals and crown-like structures distinguish NASH from simple steatosis

• DOI: 10.1194/jlr.m034876
• Publication Date: 2013-02-18T20:15:04Z
• Authors: George N. Ioannou; W. Geoffrey Haigh; David Thorning; Christopher Savard
• Keywords: Steatosis; Lipid droplet; Oil Red O; Steatohepatitis

We sought to determine whether hepatic cholesterol crystals are present in patients or mice with nonalcoholic fatty liver disease/nonalcoholic steatohepatitis (NASH), and their presence distribution correlates the of NASH as compared simple steatosis. identified, by filipin staining, free within hepatocyte lipid droplets C57BL/6J that developed following a high-fat high-cholesterol diet. Under polarized light these exhibited strong birefringence suggesting some was form crystals. Activated Kupffer cells aggregated around dead hepatocytes included strongly birefringent crystals, forming "crown-like structures" similar those recently described inflamed visceral adipose tissue. These appeared process turning it into activated lipid-laden "foam cells" numerous small cholesterol-containing droplets. In contrast, steatosis did not exhibit crown-like structures transform foam cells. Our results suggest crystallization aggregation activation such represent an important, novel mechanism for progression NASH. Nonalcoholic disease (NAFLD), defined increased deposition absence viral hepatitis excessive alcohol consumption, is extremely common affecting 15–46% adults United States (1Browning J.D. Szczepaniak L.S. Dobbins R. Nuremberg P. Horton Cohen J.C. Grundy S.M. Hobbs H.H. Prevalence urban population States: impact ethnicity.Hepatology. 2004; 40: 1387-1395Crossref PubMed Scopus (2927) Google Scholar, 2Bellentani S. Saccoccio G. Masutti F. Croce Brandi Sasso Cristanini Tiribelli C. risk factors Northern Italy.Ann. Intern. Med. 2000; 132: 112-117Crossref (1065) 3Williams C.D. Stengel J. Asike M.I. Torres D.M. Shaw Contreras M. Landt C.L. Harrison S.A. among largely middle-aged utilizing ultrasound biopsy: prospective study.Gastroenterology. 2011; 140: 124-131Abstract Full Text PDF (1586) Scholar). However, majority NAFLD have "simple steatosis" substantial inflammation fibrosis. Such generally benign clinical course very low probability developing progressive dysfunction cirrhosis (4Matteoni C.A. Younossi Z.M. Gramlich T. Boparai N. Liu Y.C. McCullough A.J. disease: spectrum pathological severity.Gastroenterology. 1999; 116: 1413-1419Abstract (2798) 10–30% develop more aggressive condition known (NASH) (3Williams Scholar), characterized varying degrees inflammation, balloon hepatocytes, fibrosis addition Steatohepatitis can progress cirrhosis, failure, hepatocellular carcinoma variable proportion 5Bugianesi E. Leone Vanni Marchesini Brunello Carucci Musso A. De Paolis Capussotti L. Salizzoni et al.Expanding natural history steatohepatitis: from cryptogenic carcinoma.Gastroenterology. 2002; 123: 134-140Abstract (1262) It clearly established central obesity insulin resistance important development responsible remain unclear. Determining would: i) clarify pathogenesis steatohepatitis; ii) help distinguish subgroup who likely cirrhosis; iii) potentially point targeted treatments. Recent reports our group others dietary critical animal models (6Matsuzawa Takamura Kurita Misu H. Ota Ando Yokoyama Honda Zen Y. Nakanuma al.Lipid-induced oxidative stress causes fed atherogenic diet.Hepatology. 2007; 46: 1392-1403Crossref (410) 7Zheng Hoos Cook Tetzloff Davis Jr, van Heek Hwa J.J. Ezetimibe improves high fat diet-induced non-alcoholic mice.Eur. Pharmacol. 2008; 584: 118-124Crossref (142) 8Subramanian Goodspeed Wang Kim Zeng Ioannou G.N. Haigh W.G. Yeh M.M. Kowdley K.V. O舗Brien K.D. al.Dietary exacerbates obese LDL receptor-deficient mice.J. Lipid Res. 52: 1626-1635Abstract (174) 9Van Rooyen Larter C.Z. Kuver Lee S.P. Teoh N.C. Farrell G.C. Hepatic accumulates obese, diabetic steatohepatitis.Gastroenterology. 141: 1393-1403Abstract (238) Human studies also support hypothesis plays role steatohepatitis. large nationally representative epidemiological study, we reported consumption independently associated (10Ioannou Morrow O.B. Connole M.L. Association between nutrient composition incidence cancer population.Hepatology. 2009; 50: 175-184Crossref (130) Finally, inhibition intestinal absorption administration ezetimibe (11Yoneda Fujita K. Nozaki Endo Takahashi Hosono Suzuki Mawatari Kirikoshi Inamori al.Efficacy treatment open-label, pilot study.Hepatol. 2010; 613-621Crossref (112) 12Park Shima Yamaguchi Mitsuyoshi Minami Yasui Itoh Yoshikawa Fukui Hasegawa long-term therapy disease.J. Gastroenterol. 101-107Crossref (158) Scholar) has been improve steatosis; were not, however, randomized controlled. Cholesterol, naturally occurring molecule abundant most tissues, traditionally viewed inert. Recently, its crystalline shown induce stimulating NLRP3 inflammasome atherosclerosis (13Duewell Kono Rayner K.J. Sirois C.M. Vladimer Bauernfeind F.G. Abela G.S. Franchi Nunez Schnurr al.NLRP3 inflammasomes required atherogenesis crystals.Nature. 464: 1357-1361Crossref (2586) therefore hypothesized may livers, which concentrations well other lipids, be hitherto unrecognized signal leads Cholesterol before livers cholesteryl ester storage (14Di Bisceglie A.M. Ishak K.G. Rabin Hoeg J.M. Cholesteryl hepatopathology effects lovastatin.Hepatology. 1990; 11: 764-772Crossref (45) but knowledge, NAFLD/NASH. aimed NAFLD/NASH mouse model Additionally, wanted distinguishes From existing human biorepository at Veterans Affairs Puget Sound Health Care System (VAPSHCS), randomly selected 4 histological NASH, activity score (NAS) 5 greater, least 1 each three components NAS (steatosis 0–3, lobular ballooning degeneration 0–2); 3 steatosis, 2–3 no (15Kleiner D.E. Brunt E.M. Van Natta Behling Contos M.J. Cummings O.W. Ferrell L.D. Torbenson M.S. Unalp-Arida al.Design validation scoring system disease.Hepatology. 2005; 41: 1313-1321Crossref (7151) retrieved tissue had flash frozen liquid nitrogen immediately after biopsy. Fasting laboratory tests prospectively performed just prior biopsy completion questionnaires collection demographic information. Male littermate 30 weeks either (HF) (15%, w/w) diet (n = 8) (15%) (1%) (HFHC) 8). The experimental diets prepared Bioserve (Frenchtown, NJ) supplementary Table I. Mice euthanized initiation cervical dislocation isoflurane anesthesia. previously HF little (simple steatosis) (16Savard Tartaglione E.V. Geoffrey W. Subramanian Chait Quinn Synergistic interaction inducing steatohepatitis.Hepatology. 2013; 57: 81-92Crossref (192) on HFHC significantly profound perisinusoidal (steatohepatitis), reduction plasma adiponectin levels formalin-fixed paraffin-embedded sectioned stained hematoxylin eosin (H E) Masson舗s trichrome Sirius red (for collagen). Histological assessed semiquantitatively "blinded" pathologist according proposed Kleiner al. biopsies placed cryovials Just cutting, embedded optimal cutting temperature (OCT) compound dry ice. Mouse portions OCT removal. Frozen sections (10 µM thickness) allowed come room temperature, coverslipped using pure glycerol mounting medium without applying any stain, examined Nikon Eclipse microscope polarizing filter, evaluate filipin, identifies interacting 3β-hydroxy (17Rudolf Curcio Esterified highly localized Bruch舗s membrane, revealed histochemistry wholemounts choroid.J. Histochem. Cytochem. 731-739Crossref (62) follows. After fixing 15 min formalin, washed phosphate buffered saline (PBS), then treated 10% fetal bovine serum (FBS) PBS min. Filipin (Sigma Chemical Co., St. Louis, MO) dissolved volume dimethylsulfoxide diluted 0.25 mg/ml FBS/PBS added h temperature. Slides once twice. Aquamount (Lerner, Pittsburgh, PA) fluorescent excitation 340-380/emission 435-485 filter place. anti-CD68 antibodies, identify macrophages (including cells), followed secondary antibodies labeled Alexa Fluor 488 (Invitrogen, Camarillo, CA) microscope. To activated, rat anti-mouse CD11b (also macrophage-1 antigen Mac-1) identified goat anti-rat antibody linked horseradish peroxidase anti-tumor necrosis factor α (TNFα) 488. will henceforth use term "Kupffer (resident liver) describe CD68-positive cells, while acknowledging recruited circulation. At time removal, pieces fixed Trump舗s fixative. postfixation osmium tetroxide, samples epoxy resin thinly sliced (0.12 µM). solution uranyl acetate aqueous lead citrate JEOL (Tokyo, Japan) transmission electron Thicker (1 µM) tetroxide counterstained methylene blue under regular Osmium binds carbon-carbon double bonds unsaturated acids, could contained only (no staining osmium) triglycerides esters. Lipids extracted Folch method (18Folch Lees Sloane Stanley G.H. A isolation purification total lipides tissues.J. Biol. Chem. 1957; 226: 497-509Abstract neutral fractions solid phase extraction Bond Elut Si cartridges (Varian Corp., Walnut Creek, triglycerides, diglycerides, esters, separated quantified normal HPLC/ELSD. Free acids esterified boron trifluoride/methanol methyl esters GC, 60 m HP-Innowax capillary column (Agilent Technologies, Santa Clara, CA). Insufficient available perform analysis. Total RNA isolated RNeasy minicolumns (Qiagen, Valencia, reverse transcribed cDNA. Quantitative real-time RT-PCR ABI 7500 sequence detection (Applied Biosystems, Foster City, β-actin housekeeping gene. gene expression genes related [Nalp3, ASC (apoptosis-associated speck-like caspase recruitment domain containing protein), Caspase-1, Pannexin-1] (19Ganz Csak Nath B. Szabo Lipopolysaccharide induces activates Nalp3 liver.World 17: 4772-4778Crossref (95) because Gene All procedures undertaken approval Institutional Review Board Animal Use Committee System. subjects provided informed consent participation biorepository. investigations conformed Public Policy Humane Laboratory Animals. 4) 3) males mildly elevated ALT level bilirubin (Table 1). diabetes mellitus one diabetes.TABLE 1Characteristics versus NASHSimple SteatosisNASHPatient number1234567Steatosis (0–3)1222321Inflammation (0–3)1112122Ballooning (0–2)0001222Fibrosis (0–4)0001A1A23Age37606244414561GenderMaleMaleMaleMaleMaleMaleMaleRaceWhiteWhiteWhiteBlackWhiteWhiteWhiteDiabetesNoYesNoYesYesYesYesBody mass index (Kg/m2)30323136443536Plasma levels, fastingALT (U/L)746144788367104Bilirubin (mg/dl)0.50.20.30.30.60.60.4Insulin (µU/ml)161322405996133Glucose (mg/dl)85109102143127273104Hemoglobin 1c (%)5.27.05.47.07.17.97.6 Open table new tab higher body weight weight; triglyceride, diglyceride, ester, concentration; ALT, cholesterol, levels; lower than 2).TABLE 2Characteristics (mean ± SD) weeksHF Diet Simple Steatosis (N 8)HFHC 8)Body (g)36.3 4.939.9 3.8*Body gain (%)13 432 7*Liver (g)1.7 0.83.4 0.1*Liver weight/body (%)4.7 1.38.6 1.7*Hepatic lipids (mg/g)Triglyceride97 81235 75*Diacylglyceride1.2 0.23.0 1.3*Cholesterol ester0.8 0.6108 38*Cholesterol0.48 0.231.22 0.6*Free acid3.1 0.63.7 1.5Plasma (U/L)88 83319 111*Cholesterol (mg/dl)86 49262 66*Triglyceride (mg/dl)44 1235 14Glucose (mg/dl)239 35290 44*Insulin (ng/ml)2.78 1.773.26 2.49Adiponectin (µg/ml)5.2 1.43.2 0.8*Hepatic mRNA analysis (relative expression)Nalp31 0.51.8 1.3ASC1 0.61.5 0.9Caspase-11 0.71.5 1.5Pannexin-11 0.51.2 0.7*Indicates comparison statistical significance P < 0.05. *Indicates Light microscopy H E, red, confirmed specimens steatohepatitis, (Fig. Examination humans 2) Those prominently due both species, showed neither nor steatotic hepatocytes. (Kupffer cells) formed "crown-like" aggregates (Figs. 3, 4, Fig. I), notably droplets, "Maltese crosses" 4B, D). Moreover, surrounded intensely 3), indicating they TNFα 4E, F) activated. findings observed although slender 3A, B III). Taken together, aggregate contain accumulate become activated.Fig. 4Liver [stains green (A–D)] anti-CD11b brown (E)] anti-TNFα (F)] (A, C, F), (B, D) bright field (E). Pairs B) (C, photomicrographs same section showing (green) aggregating crystallized creates Maltese cross appearance. stain panel (E) (F) show morphology colocalizes IV [(A, B, ×200 magnification; ×800 magnification].View Large Image Figure ViewerDownload Hi-res image Download (PPT) micrographs demonstrated actually occurred remnant (marked asterisks II). many instances, directly abutted cores evident cytoplasm 5D IID, H).This created identical (20Cinti Mitchell Barbatelli Murano Ceresi Faloia Fortier Greenberg A.S. Obin Adipocyte death defines macrophage localization function humans.J. 2347-2355Abstract (1770) 21Strissel Stancheva Z. Miyoshi Perfield 2nd, J.W. DeFuria Jick death, remodeling, complications.Diabetes. 56: 2910-2918Crossref (691) While LD 5) healthy gray/brown 5), asterisks) osmium, longer triglyceride Instead, granular precipitate corresponded including exhibiting crosses, form. Furthermore, enlarged filled number much smaller 5C, E IIB, F). intense lack suggests acids. together structures. Triglycerides hydrolyzed resultant oxidized, turn material 2), unesterified Although cluster filipin. On microscopy, enlarged, (supplementary (Nalp3, ASC, Pannexin-1) all greater reach 2). induced diet, Enlarged This scavenging resulted accumulation markedly took appearance pathogenetic exposure excess