Journal of Nutritional Biochemistry 25 (2014) 219–226
Role of immunodeficient animal models in the development of fructoseinduced NAFLD☆,☆☆
Jashdeep Bhattacharjeea,1, Jerald Mahesh Kumarb,1, Shailendra Arindkara, Barun Dasa, Upadhyay Pramoda,Ramesh C. Juyala, Subeer S. Majumdara, Perumal Nagarajana,⁎
aExperimental Animal Facility, National Institute of Immunology, New Delhi - 100 067, IndiabCSIR - Centre for Cellular and Molecular Biology, Hyderabad, India
Received 4 May 2013; received in revised form 11 October 2013; accepted 18 October 2013
Keywords: NAFLD; T cells; B cells; NK cells; Fructose; Mouse models
1. Introduction
Non-alcoholic fatty liver disease (NAFLD) is associated withobesity, insulin resistance and Type 2 diabetes. NAFLD represents awide spectrum of diseases ranging from fatty liver (hepatic steatosis),steatosis with inflammation and necrosis, to cirrhosis. The animalmodel to study NAFLD/non-alcoholic steatohepatitis (NASH) isparticularly useful to decide the unappreciated events involved inthe pathology of the disease. The studies from the established animalmodels have provided some clues for the pathogenesis of steatosis
and steatohepatitis; nevertheless, therapeutic options are still limitedas the mechanisms involved in the development of NAFLD are not yetfully understood [1]. Therefore, a better understanding of thebiochemical and pathological changes that led to early stages ofNAFLD is essential to develop therapeutic strategies [2].
Recent studies in humans show that the diet rich in carbohydrates,particularly fructose may be a significant cause of NAFLD [3]. Fructoserich diet has been postulated to be a key reason in the development ofNAFLD [4]. In line with these findings, it has also been shown inanimal studies that increased consumption of fructose may result inhepatic steatosis accompanied by insulin resistance, elevated plasmatriglyceride levels, and oxidative stress in the liver [5,6].
Understanding the mechanisms that led to progress from simplesteatosis to NASH is essential for designing realistic treatmentstrategies for patients who develop the progressive disease [7]. Inaddition, oxidative stress, cytokine production and other pro-inflammatory mediators have been implicated for delivering a secondhit during the switch from simple steatosis to NASH [8]. Altogether,hepatic immune responses also play key roles in the pathogenesis ofNASH and other progressive diseases [9].
Lymphocytes are part of the adaptive immune response and assuch, are crucial for normal immune functions. T or B cell deficienciesare known to result in severe immunodeficiency. T cells are keyregulators of adipose inflammation, and thus the adaptive immune
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system is also crucially important [10]. In mouse models, modulationof T cell function ameliorated not only adipose inflammation but alsosystemic insulin resistance induced by obesity. On the other hand,natural killer (NK) cells are an important part of the innate immuneresponse. Studies on NK cell deficiency have been carried out in micemodels for the development of atherosclerosis and obesity [11] butnot NAFLD. Recently, increasing attention has been given todetermine the role of T, B cells and NK cells in the development ofNAFLD. Although there are a few reports on involvement of B cells, Tcells and NK on the development of obesity, inflammation anddiabetes mellitus, only few addressed their role in the development ofNAFLD. The present study is to understand the role of T cells, B cellsand NK cells in the development of NAFLD.
2. Experimental procedures
2.1. Animals
Experimental procedures for this study were duly approved by theInstitutional Animal Ethics Committee. Six-week-oldmice (n=5) thatwere T cell-deficient [NU/J (nude)], T and B cell deficient [B6.129S7-Rag1tm1Mom/J (Rag-1)], NK cell deficient [C57BL/6J-Lystbg/j (beige)], Bcells defect [CBA/CaHN-Btkxid/J (xid)] and wild type (C57BL/6J (B6))(Jackson Laboratories, Bar Harbour, USA)were used for this study. Theanimals were housed in a pathogen-free environment under standardlight (14 h light, 10 hdark cycle), temperature (23±1°C) and humidity(50±5%) conditions. All mice received standard chow diet containing4.8% fat, 21% crude protein, and 4.5% crude fibre accountingmetabolizable energy 3395 kcal/kg with all micronutrients incorpo-rated. (Nutrilab, India) (The details of the ingredients in the chow areattached in Supplementary Table 1). Animals from each genotypewere divided into control (n=5) and treatment group (n=5)receiving plain water and 30% (w/v) fructose (Himedia, India),respectively, for 12 weeks. The daily intake of chow and liquid intakewasmeasured bymonitoring theweight and volume of the remainingfood and liquid. Solid food intakewas corrected for any visible spillageandwasmeasured every day using a balancewith a precision of 0.01 g.
2.2. Glucose tolerance (GTT) and insulin tolerance (ITT) tests
Mice fasted for 6 h were injected intraperitoneally with 2 g/kgbody weight of dextrose, and blood glucose was measured at timepoints of 0, 30, 60 and 120 min post injection using a glucose monitor(Accu check India). For ITT, insulin was injected intraperitoneally at 1U/kg body weight, and blood glucose was measured at time points of0, 30, 60 and 120 minute post injection. GTT and ITT were performedon 11th and 12th week respectively before euthanizing the animals.
2.3. Biochemical analysis
At the end of the experimental period the animals were fasted for6 h and euthanized under anaesthesia. The levels of aspartateaminotransferase (AST), alanine aminotransferase (ALT), triglycer-ides (TGY), and cholesterol (CHO) in blood serum were quantified byan automated analyzer (Screen Master 3000, Goa, India).
2.4. Histological analysis
Liver tissues were isolated after euthanasia, paraffin embeddedsections of 4 micron thickness were made and stained withhaematoxylin and eosin. Frozen sections of formalin-fixed liverwere stained with Oil-Red O using standard techniques. Representa-tive photomicrographs were captured at 40× magnification using asystem incorporated in a microscope. NAFLD activity score wascalculated essentially as described by Kleiner et al. [12]. Briefly;
steatosis was graded on a four-point scale: grade 0, steatosis involvingb5% of hepatocytes; grade 1, steatosis involving up to 33%; grade 2,steatosis involving 33–66% and grade 3, steatosis involving N66%.Lobular inflammation was graded on a four-point scale: grade 0, nofoci; grade 1, less than two foci per 200× field; grade 2, two to fourfoci per 200× field; grade 3, more than four foci per 200× field.Hepatocyte ballooningwas graded from 0 to 2: 0, none; 1, few ballooncells; 2, many/prominent balloon cells. The stage of a fibrosis wasquantified in a four-point scale: Stage 0, no fibrosis; Stage 1,perisinusoidal or periportal; Stage 2, perisinusoidal and portal/periportal; Stage 3, bringing to stage cirrhosis. Portal tract inflamma-tion was graded from 0 to 3 (0, none; 1, mild; 2, moderate; 3, severe).NAFLD activity score (NAS) was quantified by summing scores ofsteatosis (0–3), inflammation (0–3), liver cell injury ballooning (0–3).NASH was defined in the cases of NAS of N5.The rest of the liversamples was frozen immediately at −80°C until analysis.
2.5. Flow cytometry analysis
For estimating levels of CD3, B220 and NK cells in blood, 40 μl ofblood was collected from retro-orbital plexus from mice in 40 μl CPDbuffer. For each staining parameter, 20 μl of blood–CPD mixture wastaken in a tube and 50 μl of antibody solution CD3-FITC, NK1.1-APCCy7; B220-PE (1:200) (BD Pharmingen, San Diego, CA, USA) wasadded to it and incubated on ice for 30 min. For RBC lysis, 1 ml of 1×BD FACS Lysing Solution (BD Biosciences, USA) was added to a tubeand incubated at room temperature for 10 min; 4 ml of phosphate-buffered saline (PBS) was then added to both the tubes andcentrifuged at 300×g at 4°C. The cell pellet was finally resuspendedin 200 μl PBS. BD FACS AriaIII (BD Biosciences, San Jose, CA, USA) wasused for flow cytometry. The data were analysed using FlowJo(TreeStar, Ashland, OR, USA).
2.6. Apoptosis by TUNEL assay
The numbers of apoptotic cells in liver sections were determinedby terminal deoxynucleotide transferase-mediated deoxyuridinetriphosphate nick-end labelling (TUNEL) assay with a commercialkit (Promega, Madison, WI, USA). Tissue sections were prepared bydeparaffinization in xylene followed by gradual rehydration withdecreasing concentrations of ethanol. The TUNEL assay was thenperformed according to the manufacturer's instructions, and thetissue section for apoptotic cells were detected using confocalfluorescence microscope.
2.7. Immunodetection of p53 in liver
The level of expression of p53 was determined by immunofluo-rescence. Briefly, frozen tissue sections (16 μm) of liver sample werefixed with acetone for 20 min followed by permeabilization with 0.5%(v/v) Triton×100 in PBS for 10 min at room temperature. Afterblocking with 5% horse serum in PBS for 1 h, the sections wereincubated in primary antibody [mouse monoclonal antibodies to p53(Santa Cruz Biotechnology, USA] for 1 h and then incubated with FITCconjugated secondary antibody for 1 h at room temperature. Toreduce auto fluorescence, the sections were treated with coppersulphate (10 mM) in the ammonium acetate buffer (50 mM, pH5.5)for 30 min. The sections were counterstained with DAPI-(4′, 6-diamidino-2-phenylindole) for 5 min and mounted in vector shield(Vector Laboratories). After mounting the slides, the sections wereviewed for immunofluorescence under a confocal laser scanningimmunofluorescence microscope. Image analysis was done usingLSM510 META software (Carl Zeiss), and images were assembledusing Adobe Photoshop 7.0 (Adobe Systems, Inc., San Jose, CA, USA).
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2.8. Immunodetection of tumor necrosis factor alpha (TNFα) in liver
To deduct the inflammatory response of treated groups, the levelof expression of TNFα (ab1793, Abcam) was checked by using animmunofluorescence assay. Briefly, paraffin sections (7 μm) of liversamples were treated with xylene for 30 min to remove the paraffinwax, then followed by two treatments with 90% and 100% alcoholand subsequently the liver samples fixed with acetone for 20 min.Then the samples were treated with 0.5% (v/v) TritonX-100 in PBS for10 min at room temperature by permeabilization. After blocking with5% horse serum in PBS for 1 h, the sections were incubated in primaryantibody for 1 h and then incubated in FITC conjugated secondaryantibody for 1 h at room temperature. To reduce auto fluorescence,the sections were treated with CuSO4 (10 mM) in ammoniumacetate buffer (50 mM CH3COONH4, pH5.5) for 30 min. The sectionswere counterstained with propidium iodide (PI) for 5 min andmounted in a vector shield (Vector laboratories). The normal andtreated liver sections treated as above, but without primary antibody,served as secondary controls. Confocal laser scanning immunofluo-rescence microscopy was carried out using a Zeiss LSM 510 METAconfocal microscope. Image analysis was done using LSM510 METAsoftware (Carl Zeiss), and images were assembled using adobePhotoshop 7.0.
2.9. qRT-PCR
Total RNA was extracted from the liver of mice by TRI Reagent(Sigma-Aldrich, St. Louis, MO, USA). cDNA synthesis was performedby ProtoScript M-MuLV First Strand cDNA Synthesis Kit (NEB,Ipswich, MA, USA) using 1 μg total RNA. Quantitative real-time PCRwas performed with MESA GREEN Master Mixes Plus (Eurogentac,Seraing, Liège, Belgium) on the Master cycler RealPlex4 platform(Eppendorf, Germany). The primer pairs used are listed in Supple-mentary Table 2. The PCR conditions used were as follows: initialdenaturation 95°C for 5 min, followed by 40 cycles of 95°C for 15 s(denaturation), 60°C for 60 s (annealing and extension).The expres-sion of genes was normalized to housekeeping gene, (18S rRNA) andrelative expressionwas calculated. Target gene levels are presented asa ratio of levels in all fructose treated groups vs. B6 untreated. Forinflammatory genes, the relative expression of genes was calculatedwith respect to the individual control strains. Relative expression wascalculated using ΔΔCt method.
Fig. 1. Histological analysis — histological analyses of liver sections from five strains of mice: rpanel-Oil Red O staining; down panel-hematoxylin and eosin staining) are shown with an oristeatosis in the livers of fructose treated B6 and xid mice.
2.10. Statistical analysis
Statistical analysis was performed using Graph Pad Prism (Version4.03; Graph Pad Software, La Jolla, CA, USA) on untransformed data.Results are presented as mean±S.E. Food, water intake levels andclinical biochemistry were analyzed by two-way analysis of variance(ANOVA), with pair-wise comparisons being made by the Student–Newman–Keuls method. Histopathology scores were evaluated byone-way ANOVA, using strain as the fixed factor.
3. Results
To detect any physiological changes in mice in response totreatment with fructose, we analyzed body weight, food and waterintake and biochemical parameters of all strain of mice usedthroughout this study. There was an increase in body weight in B6,nude and xid mice (Supplementary Graph1). There was a significantdecrease in food intake in fructose-treated groups in all genotypes, butfluid intakewas decreased significantly in fructose-treated B6, xid andnude mice compared to their respective control (Table1 and S-Figure1A and 1B). In contrast, the mean serum AST, were significantlyhigher in fructose treated xidmice andB6mice as comparedwith theircontrol. Therewere significant increases in ALT, cholesterol (CHO) andtriglyceride (TGY) level in xid mice treated with fructose (Table 1 andS-Figure 2A and 2B). While examining the GTT and ITT, we observedthat only fructose treated B6 and xid mice developed both glucosetolerance and insulin resistance (S-Figure 2C and 2D).
Liver histopathology. We next studied the morphological changeswithin the liver following fructose treatment. There was no morpho-logical change in the liver of nude, Rag-1 and beige mice. However, B6and xid mice demonstrated micro vesicular and macro vesicular fattydegeneration in their liver. The steatosis appeared more in thecentrilobular region of the liver parenchyma. Frozen samples fromfructose-fed B6 and xid mice stained for Oil-Red O showed red-coloured micro- and macrovesicular fatty droplets and is remarkablymore than other strains (Fig. 1), the analysis of the histological scoringsystem for NAFLD activity revealed that only B6 and xid mice were toachieve the NAS of N5 (Fig. 2).
3.1. FACS analysis
Detailed FACS analysis on isolated peripheral blood mononuclearcell (PBMCs) revealed that the percentage of NK cellswas decreased in
epresentative photomicrographs of hematoxylin-and-eosin and Oil-Red O staining (topginal magnification of ×40. H and E stain revealed markedly micro and macrovesicular
Table 1Comparative table showing feed, water intake, energy intake, AST, ALT, CHO, TGY, NAS score
Rag-1 control Rag-1 treated Nude control Nude treated Beige control Beige treated B6 control B6 treated xid control xid treated
The values are compared between control and treated groups among different strains and represented as mean±S.E. (n=5). Asterisk statistically significances. Statistical significancefor each genotype is compared with its own baseline and marked with *Pb.05 and **Pb.001.
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nude, beige and B6mice, but there was no change in the percentage ofNK cells in Rag-1 and xid mice with respect to their control groups.However, the percentage of B220 cells decreased in fructose-fed beigeand xidmicewith respect to their control, but no changewas observedin other strains. The percentage of CD3 cells remains unchanged incontrol and treated groups in all the strains (Fig. 3).
3.2. TUNEL assay and expression of p53 and TNF-α
TUNEL-positive hepatocytes were readily observed and found tobe in a greater number in xid than in B6 mice (Fig. 4). There was apositive correlation between p53 expression and the stage of liversteatosis. Over expression of p53 protein was observed in the nucleusof hepatocytes of fructose-fed B6 and xid mice indicating apoptosis inhepatocytes (Fig. 5). We could not find TUNEL-positive cells andexpression of p53 in Rag-1, nude and beige strains. There wasoverexpression of TNFα in the periportal and sinusoidal spaces infructose-treated, B6 and xid mice. In fructose-treated Rag-1 mice,there wasmild over expression in the cytoplasm of hepatocytes in theentire liver, there was no over expression of TNFα in treated beigeand nude mice (Fig. 6).
3.3. Gene's expression analysis by q RT-PCR
The relative expression of genes involved in lipogenesis (SREBP-1c), insulin signalling pathway, monocyte attracting chemokines-1
Fig. 2. NAFLD activity score — NAFLD activity score of fructose treated groups was assesseinflammation is greater in B6 and xid mice than nude, beige and Rag-1and NAS activity score
(MCP-1) and stearoyl-CoA Desaturase-1 (SCD-1) was over expressedin fructose-fed B6 and xid mice as compared to control B6 mice fedwith plain water. However, no noticeable fold changes were observedin other strains for these genes (Fig. 7 and Supplementary table-3).Among inflammatory genes studied we could find increase in foldof expression of IL6, TNFα and C-reactive protein in nude and Rag-1mice when compared with their respective control.
4. Discussion
The present study addresses the question whether the absence offunctional T, B and NK cells will affect NAFLD development inimmunodeficient mice. High-fructose consumption clearly increasesvisceral fat in healthy adults and in animal models. In rodent models,a high-fructose diet induces a “metabolic syndrome” that includesdevelopment of insulin resistance, hypertriglyceridemia, abdominalobesity, hypertension and hepatic steatosis. Based on the publishedreport on fructose induced NAFLD in mouse models, we hypothesizedthat fructose may have some role in immune cells in the developmentof NAFLD. There are published reports [13–15] on the role of T, B andNK cells in the development of obesity and diabetes, but littleattention has been focused on NAFLD. Elisavet Kodela et al., [16]studied the role of lymphocytes in the pathogenesis of obesity and themetabolic syndrome in Rag-1 mice with high-fat diet and concludedRag-1 mice exhibited increased insulin sensitivity, as assessed by theinsulin tolerance test, and importantly, this strain showed no
d by histopathology of H&E stained livers. The score range for steatosis and portalis greater than five in B6 and xid mice.
Fig. 3. Flow cytometry analyses — the figures shown are FACS dot plots of one representative mouse indicating the percentage of CD3, B220 and NK cells blood of fructose treatedstrains with their respective controls. Numbers in quadrants indicate percent cells in each throughout.
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histological signs of liver steatosis in contrast to thewild-typemice. Inour study, Rag-1 mice did not develop insulin resistance or glucoseintolerance even after 12 weeks of fructose treatment. Studies on Bcell deficient mice (B null) conclude that B cells and IgG are keypathogenic effectors in the development of obesity associated insulinresistance and glucose intolerance, and they showed B cells alsoexacerbate metabolic disease through production of IgG [13]. In our
Fig. 4. Apoptosis by TUNEL assay — shows enhanced apop
study, the mice having the disorder in B-cell maturation with a lowlevel IgM and IgG production developed glucose intolerance andinsulin resistance after treatment with high fructose. The character-istic features of NAFLD have been seen in this mouse strain. Thereason for this contradictory observation needs further evaluation byusing other mutant B deficient mouse strains. Schiller et al., [17]demonstrated that NK cells were antiatherogenic with high-fat diet.
totic cells in B6 and xid (arrow denotes apoptosis).
Fig. 5. Immunodetection of p53 expression — representative photomicrographs of fructose treated B6 and xid mice liver for Immunodetection of p53 expression. Intensity of greenfluorescence indicates the expression level of p53 at 100×). Treated xid and B6 mice liver showing enhanced expression of p53 (original magnification ×100) FITC, DAPI, MERGE[FITC+DAPI].
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In our study, the beige mice with reduced NK activity did not developsteatosis, insulin resistance and glucose tolerance. We studied a fewgenes that are associated in progress of NAFLD and could able to findrelative over expression of SREBP1c, IRS1, MCP1 and SCD-1 genes inB6 and xid mice as compared to control B6 mice fed with plain water.As stated already by Shimano et al, overexpression of either SREBP-1cor SREBP-1a in mouse liver markedly enhances the expression of
Fig. 6. Immunodetection of TNFα expression for inflammatory response -Representative photexpression. Intensity of green fluorescence indicates the expression level of TNFα at 100× (mfructose treated B6 and xidmice. In fructose treated Rag-1mice therewasmild over expressionin treated beige and nude mice.
lipogenic genes and causes massive hepatosteatosis [18]. Secondly,IRS1 gene is overexpressed due to the fact that they are the keyregulators for the synthesis and oxidation of fatty acids in the liver ofNASH [19]. MCP-1 mRNA is markedly increased in the livers ofpatients with steatosis and NASH [20]. Since apoptosis is a key featureof NASH, which is accompanied by an increase in TUNEL positive cells,we analyzed the TUNEL positive cells in the liver of fructose-fed B6
omicrographs of fructose treated B6 and xid mice liver for Immunodetection of TNFαerge). There was over expression of TNFα in the periportal and sinusoidal spaces in
in the cytoplasm of hepatocytes in the entire liver; therewas no overexpression of TNFα
Fig. 7. Relative mRNA expression of different genes in five strains of fructose-fed mice. (A) represents relative mRNA expressions of genes and expressed in the ratios among fructose-fed groups with normal control B6 mice. (B) represents relative mRNA expressions of inflammatory genes and expressed in the ratios among fructose-fed groups with their respectivecontrol. To compare the expression of mRNA among different groups of animals, entire liver samples of five animals per groups were pooled together. From pooled tissue samples theRNA was extracted followed by cDNA synthesis and RTqPCR was setup in triplicate. The average Ct values of these triplicate samples were used for the calculation of relative mRNAexpression for different genes.
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and xid mice. The results supported the immunoassay and histologyof B6 and xid mice. p53 plays an important role in the pathogenesis offatty liver disease [21]. B6 and xid mice that had excess fataccumulation in the liver predispose cells to hepatocellular injurypresumably caused by the cellular toxicity of excess free fatty acids,oxidative stress and lipid peroxidation. All of these can potentiallyactivate the p53 pathway. Donal O'Shea [22] showed that the numberof NK cells is markedly reduced in obesity. Our findings are similar tothose observed in obese rats, which have reduced NK cell, but thesame is restored uponweight loss. In our study, we could findmarkedreduction of NK cells in PBMC of mice strains that gained weightsupporting the above studies and among the weight gained strainsonly B6 and xid developed NAFLD. On studying the expression ofinflammatory genes we could find IL6, TNFα and C-reactive proteinwere overexpressed in nude and Rag-1 mice suggesting the initiationof liver cell injury is accompanied by an enhanced secretion ofproinflammatory cytokines, but lymphocytes potentially contributeto development of steatosis. Winner et al. also studied the roles ofCD4+ and CD8+ T cells in obesity-induced insulin resistance wherethe transfer of CD4+, but not CD8+, T cells in Rag-1 null mice on highfat diet dampened its- weight gain, visceral adipose tissue mass, highglucose, and elevated TNFα and IL-6 level [23].
The results clearly support a new function of T cells and NK cells asa key peripheral, contributor to hepatic lipid deposition and thedevelopment of fructose induced NAFLD in mice. The potentiallimitations of the study are the use of few immune deficient strainsand use of fructose. Further studies on other immune-deficient strainswith high-fat diet/MCD (methionine and choline deficient) dietshould be conceded before going to translational research. Toconclude, this study will extend our current knowledge on NAFLDdevelopment. Further, it is, thus, our hope that future studies will notonly help elucidate the mechanism but will allow developing bettertherapeutic approaches to repress the development of the liver failureresultant from NAFLD.
Supplementary data to this article can be found online at http://dx.doi.org/10.1016/j.jnutbio.2013.10.010.
Acknowledgment
The authors wish to thank the director, National Institute ofImmunology for providing kind support from the funds originatedfrom Department of Biotechnology, India. The authors also wish to
thank Drs. Vineeta Bal, Ashok Mukaphadhayay NII, New Delhi andNanthakumar Thirunarayanan, USA, for their kind support and adviceto carry out this study.
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