Dysregulation of Kupffer Cells/Macrophages and Natural Killer T Cells in Steatohepatitis in LXRα Knockout Male Mice

Dysregulation of Kupffer Cells/Macrophages and Natural Killer T Cells in Steatohepatitis in LXRα... Abstract Liver X receptor (LXR) α expression is mainly localized to metabolic tissues, such as the liver, whereas LXRβ is ubiquitously expressed. LXRα is activated by oxysterols and plays an important role in the regulation of lipid metabolism in metabolic tissues. In macrophages, LXRs stimulate reverse cholesterol transport and regulate immune responses. Although a high-cholesterol diet induces severe steatohepatitis in LXRα-knockout (KO) mice, the underlying mechanisms linking lipid metabolism and immune responses remain largely unknown. In this study, we investigated the role of LXRα in the pathogenesis of steatohepatitis by assessing the effects of a high-fat and high-cholesterol diet (HFCD) on hepatic immune cell proportion and function as well as lipid metabolism in wild-type (WT) and LXRα-KO mice. HFCD feeding induced severe steatohepatitis in LXRα-KO mice compared with WT mice. These mice had higher cholesterol levels in the plasma and the liver and dysregulated expression of LXR target and proinflammatory genes in both whole liver samples and isolated hepatic mononuclear cells. Flow cytometry showed an increase in CD68+CD11b+ Kupffer cells/macrophages and a decrease in invariant natural killer T cells in the liver of HFCD-fed LXRα-KO mice. These mice were more susceptible to lipopolysaccharide-induced liver injury and resistant to inflammatory responses against α-galactosylceramide or concanavalin-A treatment. The findings provide evidence for activation of bone marrow–derived Kupffer cells/macrophages and dysfunction of invariant natural killer T cells in LXRα-KO mouse liver. These findings indicate that LXRα regulates hepatic immune function along with lipid metabolism and protects against the pathogenesis of nonalcoholic steatohepatitis. Lipid metabolism is influenced by dietary and endogenous lipids and is regulated by several regulatory mechanisms, including nuclear receptor function. Lipid dysregulation is involved in the pathogenesis of metabolic diseases, including fatty liver, diabetes, and atherosclerosis (1–3). Impaired regulation of cholesterol metabolism and hepatic cholesterol accumulation result in inflammation and play a role in the development of nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), liver fibrosis, liver cirrhosis, and hepatic carcinoma (4–6). A number of immune cells, including Kupffer cells, natural killer (NK) cells, and natural killer T (NKT) cells, are present in the liver and participate in the induction and progression of hepatic inflammation (7). Kupffer cells/macrophages are classified into two subsets, radio-resistant resident Kupffer cells and radio-sensitive bone marrow–derived Kupffer cells/macrophages (8, 9). Whereas resident Kupffer cells express F4/80 and CD68 and have high phagocytic activity and reactive oxygen species production, bone marrow–derived Kupffer cells/macrophages express F4/80 and CD11b and produce proinflammatory cytokines efficiently (8). Mice fed a high-cholesterol diet or a high-fat and high-cholesterol diet (HFCD) have increased numbers of CD11b+ Kupffer cells/macrophages in the liver and are highly susceptible to acute hepatic inflammation induced by cytosine-phosphate-guanine oligonucleotide or α-galactosylceramide (α-GalCer) administration (10, 11). A high-cholesterol diet also induces free cholesterol accumulation in hepatic stellate cells, which are activated by inflammation, and exacerbates hepatic fibrosis induced by bile duct ligation or carbon tetrachloride treatment (12). Thus, dysregulation of lipid metabolism, specifically cholesterol accumulation, activates hepatic immune cells and induces hepatic inflammatory diseases including NASH. The possible pathophysiological roles of hepatic immune cells, such as the two subsets of Kupffer cells/macrophages, in NAFLD/NASH induced by dietary cholesterol overload remain largely unknown. The nuclear receptors liver X receptor (LXR) α and LXRβ are ligand-dependent transcription factors that are activated by oxysterols and regulate lipid metabolism and immunity (13, 14). LXRα is mainly expressed in metabolic tissues, such as liver, adipose tissue, and intestine, whereas LXRβ is ubiquitously expressed. LXR activation stimulates conversion of cholesterol to bile acids by inducing expression of cholesterol 7α-hydroxylase in the rodent liver and also promotes cholesterol efflux into bile by inducing adenosine triphosphate–binding cassette (ABC) transporter G5 and ABCG8. In macrophages, LXRs stimulate reverse cholesterol transport by inducing expression of ABCA1 and apolipoprotein E (ApoE). A high-cholesterol diet induces more severe hepatic cholesterol accumulation and NASH in LXRα-knockout (KO) mice than in LXRβ-KO mice (15, 16). Hepatic LXRα is necessary for effective biliary and fecal cholesterol excretion and reverse cholesterol transport (17). Because the liver plays important roles in both lipid metabolism and innate immunity as a gateway for dietary signals, LXRα has been suggested to have a key function as the hepatic cholesterol sensor. In this study, we investigated the role of LXRα in hepatic immune responses in NAFLD mice induced by HFCD and found that LXRα regulates not only hepatic lipid metabolism, but also immune cell function, specifically in CD11b+ Kupffer cells/macrophages and NKT cells. Materials and Methods Animal studies Wild-type (WT) C57BL/6J mice were obtained from Nihon CLEA (Tokyo, Japan), and Nr1h3–/– (Lxrα–/–, LXRα-KO) mice were kindly provided by Dr. David J. Mangelsdorf (University of Texas Southwestern Medical Center, Dallas, TX) (15). Mice were maintained under controlled temperature (23°C ± 1°C) and humidity (45% to 56%) with free access to water and chow. Male mice between 7 and 8 weeks of age were fed a control normal diet (ND) (CE-2; Nihon CLEA) or an HFCD (CE-2 supplemented with 1.25 g/100 g cholesterol and 12.5 g/100 g cocoa butter) as reported previously with minor modification (18). After the 4-week feeding period, mice were subjected to sample collection or intravenous treatment with Escherichia coli–derived lipopolysaccharide (LPS; Sigma-Aldrich, St. Louis, MO; 2.5 mg/kg of body weight), α-GalCer (Funakoshi Co., Tokyo, Japan; 0.1 mg/kg of body weight), or concanavalin-A (Con-A; Vector Laboratories, Burlingame, CA; 12.5 mg/kg of body weight). Mice were euthanized with carbon dioxide. The experimental protocol adhered to the Nihon University Rules Concerning Animal Care and Use and was approved by the Nihon University Animal Care and Use Committee. Plasma analysis Plasma cholesterol, triglyceride, glucose, nonesterified fatty acid (NEFA), aspartate aminotransferase (AST)/alanine aminotransferase (ALT), and total/direct bilirubin levels were measured with Cholesterol E-Test Wako, Triglyceride E-Test Wako, Glucose CII-Test Wako, NEFA C-Test Wako, GOT•GPT CII-Test Wako, and Bilirubin BII-Test Wako (Wako Pure Chemical Industries, Osaka, Japan), respectively. Plasma tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin-12p70 (IL-12p70), chemokine (C-C motif) ligand 2 (CCL2), and IL-4 levels were measured with enzyme-linked immunosorbent assay kits (R&D Systems, Minneapolis, MN). Liver histology Liver sections were fixed with 10% neutral buffered formalin (Muto Pure Chemicals, Tokyo, Japan) for 24 hours and embedded with paraffin. The embedded tissues were cut into 5-μm sections, deparaffinized, stained with hematoxylin and eosin (Sakura Finetek Japan, Tokyo, Japan), washed with ethanol and xylene, and mounted with the Histofine Mousestain Kit (Nichirei Corporation, Tokyo, Japan). Oil red O staining was performed on liver frozen sections by Medical & Biological Laboratories Co. (Nagoya, Japan). We used the NASH Clinical Research Network scoring system (19) to assess steatosis, ballooning, and inflammation for a diagnosis of NASH in mice as reported previously (20). For immunohistochemistry, sections were treated with anti-F4/80 antibody (Thermo Fisher Scientific, Waltham, MA; Table 1), the Histofine Simple Stain Mouse MAX-PO (Rat) (Nichirei Corporation), and ImmPACT DAB Substrate solution (Vector Laboratories), and counterstained with hematoxylin. The F4/80-positive area was quantified with ImageJ software (National Institutes of Health, Bethesda, MD). Table 1. Antibodies Used in This Study Peptide/Protein Target  Antigen Sequence (if Known)  Name of Antibody  Manufacturer, Catalog No.  Species Raised in; Monoclonal or Polyclonal  RRID  Dilution  CD16/32    Anti-CD16/CD32 purified  Thermo Fisher Scientific, 14-0161, Clone no. 93  Rat; monoclonal  AB_467133  1:30  F4/80    Anti-F4/80 FITC  Thermo Fisher Scientific, 11-4801, Clone no. BMB  Rat; monoclonal  AB_2637191  1:10  F4/80    F4/80 monoclonal antibody  Thermo Fisher Scientific, 14-4801, Clone no. BMB  Rat; monoclonal  AB_467559  1:10  CD11b    Anti-mouse CD11b PE-Cy5  Thermo Fisher Scientific, 15-0112, Clone no. M1/70  Rat; monoclonal  AB_468714  1:30  CD68    Rat anti-mouse CD68: biotin  Bio-Rad Laboratories, MCA1957B, Clone no. FA-11  Rat; monoclonal  AB_323443  1:8  βTCR    Anti-mouse TCR β FITC  Thermo Fisher Scientific, 11-5961, Clone no. H57-597  Armenian hamster; monoclonal  AB_465323  1:10  βTCR    Anti-mouse TCR β PE-Cy5  Thermo Fisher Scientific, 15-5961, Clone no. H57-597  Armenian hamster; monoclonal  AB_468816  1:30  CD69    Anti-mouse CD69 biotin  Thermo Fisher Scientific, 13-0691, Clone no. H1.2F3  Armenian hamster; monoclonal  AB_466495  1:30  NK1.1    Anti-mouse NK1.1 FITC  Thermo Fisher Scientific, 11-5941, Clone no. PK136  Mouse; monoclonal  AB_465318  1:30  NK1.1    Anti-mouse NK1.1 PE  Thermo Fisher Scientific, 12-5941, Clone no. PK136  Mouse; monoclonal  AB_466050  1:30  IgG1    PE rat anti-mouse IgG1  BD Biosciences,550083, Clone no. A85-1  Rat; monoclonal  AB_393553  1:2  Peptide/Protein Target  Antigen Sequence (if Known)  Name of Antibody  Manufacturer, Catalog No.  Species Raised in; Monoclonal or Polyclonal  RRID  Dilution  CD16/32    Anti-CD16/CD32 purified  Thermo Fisher Scientific, 14-0161, Clone no. 93  Rat; monoclonal  AB_467133  1:30  F4/80    Anti-F4/80 FITC  Thermo Fisher Scientific, 11-4801, Clone no. BMB  Rat; monoclonal  AB_2637191  1:10  F4/80    F4/80 monoclonal antibody  Thermo Fisher Scientific, 14-4801, Clone no. BMB  Rat; monoclonal  AB_467559  1:10  CD11b    Anti-mouse CD11b PE-Cy5  Thermo Fisher Scientific, 15-0112, Clone no. M1/70  Rat; monoclonal  AB_468714  1:30  CD68    Rat anti-mouse CD68: biotin  Bio-Rad Laboratories, MCA1957B, Clone no. FA-11  Rat; monoclonal  AB_323443  1:8  βTCR    Anti-mouse TCR β FITC  Thermo Fisher Scientific, 11-5961, Clone no. H57-597  Armenian hamster; monoclonal  AB_465323  1:10  βTCR    Anti-mouse TCR β PE-Cy5  Thermo Fisher Scientific, 15-5961, Clone no. H57-597  Armenian hamster; monoclonal  AB_468816  1:30  CD69    Anti-mouse CD69 biotin  Thermo Fisher Scientific, 13-0691, Clone no. H1.2F3  Armenian hamster; monoclonal  AB_466495  1:30  NK1.1    Anti-mouse NK1.1 FITC  Thermo Fisher Scientific, 11-5941, Clone no. PK136  Mouse; monoclonal  AB_465318  1:30  NK1.1    Anti-mouse NK1.1 PE  Thermo Fisher Scientific, 12-5941, Clone no. PK136  Mouse; monoclonal  AB_466050  1:30  IgG1    PE rat anti-mouse IgG1  BD Biosciences,550083, Clone no. A85-1  Rat; monoclonal  AB_393553  1:2  Abbreviations: IgG, immunoglobulin G; RRID, Research Resource Identifier. View Large Measurement of lipid contents in liver samples Liver tissues (0.1 g) were homogenized in cold phosphate-buffered saline on ice. Lipids were extracted with 3 mL of chloroform/methanol (1:2 by volume), next with 3.8 mL of chloroform/methanol (1:2 by volume), and then twice with 3 mL of chloroform. Collected samples were dried up and dissolved in isopropanol. Liver cholesterol, triglyceride, and NEFA levels were measured with Cholesterol E-Test Wako, Triglyceride E-Test Wako, and NEFA C-Test Wako (Wako Pure Chemical Industries), respectively. Cholesterol content in hepatic mononuclear cells (MNCs) was also measured with Cholesterol E-Test Wako (Wako Pure Chemical Industries). Isolation of mouse hepatic MNCs Hepatic MNCs were isolated with collagenase digestion and Percoll gradient centrifugation as previously described (11). For detection of NK and NKT cells, hepatic MNCs were isolated without collagenase digestion. Reverse transcription and quantitative real-time polymerase chain reaction Complementary DNAs (cDNAs) were synthesized from total RNAs from whole liver or hepatic MNCs using the ImProm-II reverse transcription system (Promega Corporation, Madison, WI), as described previously (21). Quantitative real-time polymerase chain reaction (PCR) was used with the ABI PRISM 7000 sequence detection system (Thermo Fisher Scientific) using Power SYBR Green PCR Master Mix (Thermo Fisher Scientific). Briefly, after incubation at 95°C for 10 minutes, cDNAs were denatured at 95°C for 15 seconds and annealed and extended with primers at 60°C for 1 minute for each PCR cycle. Intron-spanning primer sequences were as follows: Ccl2, 5′-ATG CAG GTC CCT GTC ATG CTT-3′ and 5′-AGC TCT CCA GCC TAC TCA TTG-3′; Adgre1 (the gene encoding F4/80), 5′-GCA TCA TGG CAT ACC TGT TC-3′ and 5′-GAG CTA AGG TCA GTC TTC CT-3′; Itgam (the gene encoding CD11b), 5′-TCC TGT ACC ACT CAT TGT GG-3′ and 5′-GGG CAG CTT CAT TCA TCA TG-3′; Cd68, 5′-CTG CTG TGG AAA TGC AAG CA-3′ and 5′-TGG TCA CGG TTG CAA GAG AA-3′; Abcg5, 5′-TCA GGA CCC CAA GGT CAT GAT-3′ and 5′-AGG CTG GTG GAT GGT GAC AAT-3′; Abcg8, 5′-GAC AGC TTC ACA GCC CAC AA-3′ and 5′-GCC TGA AGA TGT CAG AGC GA-3′; Il4, 5′-CTT CCA AGG TGC TTC GCA TA-3′ and 5′-GAC TCA TTC ATG GTG CAG CT-3′; and Apoe, 5′-TGC TGT TGG TCA CAT TGC TGA-3′ and 5′-CTT GTG TGA CTT GGG AGC TCT G-3′. Primer sequences for Nr1h3 (the gene encoding LXRα), Nr1h2 (the gene encoding LXRβ), Tnf, Il12b, Ifng, Abca1, Ppib (the gene encoding cyclophilin B), and Gapdh (the gene encoding glyceraldehyde-3-phosphate dehydrogenase) were reported previously (22–25). Each primer was used at a final concentration of 0.2 μM. PCR products were quantified with standard curves that were linear over a range of 0.02 to 200 ng/mL for corresponding cDNAs inserted into pcDNA3.1 plasmids (Thermo Fisher Scientific) as reported previously (26). The calculated RNA values were normalized with the Ppib or Gapdh messenger RNA (mRNA) levels. Flow cytometric analysis For analysis of Kupffer cells/macrophages, hepatic MNCs were isolated with collagenase digestion, incubated with anti-CD16/32 Fc blocker (Thermo Fisher Scientific) for 15 minutes at 4°C, and stained with fluorescein isothiocyanate (FITC)–conjugated anti-F4/80 (Thermo Fisher Scientific), biotin-conjugated anti-CD68 (Bio-Rad Laboratories, Hercules, CA), phycoerythrin (PE)-Cy5-conjugated anti-CD11b, and PE-streptavidin (Thermo Fisher Scientific). For analysis of NK cells and NKT cells, hepatic MNCs were isolated without collagenase digestion, incubated with anti-CD16/32 Fc blocker, and stained with FITC-anti-βTCR, PE-anti-NK1.1, biotin-anti-CD69, and PE-Cy5-streptavidin (Thermo Fisher Scientific). For analysis of invariant natural killer T (iNKT) cells, hepatic MNCs were isolated without collagenase digestion, incubated with anti-CD16/32 Fc blocker, and stained with FITC-anti-NK1.1 (Thermo Fisher Scientific), PE-CD1d-IgG1 dimer (BD Biosciences, San Jose, CA) complexed with α-GalCer, and PE-Cy5-anti-NK1.1 (Thermo Fisher Scientific). Flow cytometric analysis was performed with the Cytomics FC500 (Beckman Coulter, Indianapolis, IN). The antibodies used are listed in Table 1. Independent flow cytometric analysis was performed at least 3 times. Statistical analysis Data are presented as means ± standard deviation. We performed two-tailed unpaired Student t test to assess significant differences between the two groups and one-way analysis of variance (ANOVA) with the Tukey post hoc test or two-way ANOVA for multiple comparisons. Results HFCD feeding induces NASH in LXRα-KO mice Under high-cholesterol diet conditions, LXRα-KO mice exhibit severe cholesterol accumulation in the liver (15, 16). NAFLD and NASH are associated with lifestyle factors such as a “Western diet” high in fat and cholesterol (1, 27). We used HFCD as a model for a Western diet and examined the effect of HFCD on lipid metabolism and liver damage in LXRα-KO mice. Peet et al. (15) demonstrate that high-cholesterol diet feeding significantly increases liver mass and plasma AST and ALT levels in LXRα-KO mice within 30 days. Because we also observed that HFCD feeding for 3 to 4 weeks increased liver weight and plasma AST and ALT levels in LXRα-KO mice in preliminary experiments, we compared the effects of 4-week feeding of an HFCD on the development of NAFLD and NASH in LXRα-KO mice vs WT mice. Similar to high-cholesterol diet feeding (15), HFCD feeding for 4 weeks increased liver weight without changing body weight in LXRα-KO mice, but not in WT mice [Fig. 1(a)]. There was no difference in body weight or liver weight between WT and LXRα-KO mice fed an ND. Whereas plasma cholesterol levels were similar in WT and LXRα-KO mice fed an ND, HFCD feeding increased plasma cholesterol levels in LXRα-KO mice more effectively than WT mice [Fig. 1(b)]. Plasma triglyceride levels were lower in LXRα-KO mice than in WT mice and were decreased by HFCD feeding compared with ND feeding in both mice [Fig. 1(b)]. Plasma glucose and NEFA levels were not altered in all groups [Fig. 1(b)]. Plasma AST and ALT levels were strongly elevated in HFCD-fed LXRα-KO mice [Fig. 1(c)]. Figure 1. View largeDownload slide Body and liver weights and plasma biochemical profiles in WT and LXRα-KO mice fed an ND or HFCD. After WT and LXRα-KO mice were fed an ND or HFCD for 4 weeks, (a) body weight and liver weight per 20 g body weight, (b) plasma cholesterol (CHO), triglyceride (TG), glucose, and NEFA levels, (c) and plasma AST and ALT levels were measured (n = 8 for each group). **P < 0.01; ***P < 0.001 (one-way ANOVA followed by Tukey multiple comparisons). Figure 1. View largeDownload slide Body and liver weights and plasma biochemical profiles in WT and LXRα-KO mice fed an ND or HFCD. After WT and LXRα-KO mice were fed an ND or HFCD for 4 weeks, (a) body weight and liver weight per 20 g body weight, (b) plasma cholesterol (CHO), triglyceride (TG), glucose, and NEFA levels, (c) and plasma AST and ALT levels were measured (n = 8 for each group). **P < 0.01; ***P < 0.001 (one-way ANOVA followed by Tukey multiple comparisons). We next evaluated liver histology and hepatic lipid contents. Hematoxylin and eosin staining showed that the liver of HFCD-fed LXRα-KO mice had lipid accumulation, hepatocyte ballooning, and inflammatory cell infiltration [Fig. 2(a)]. Oil red O staining showed lipid accumulation in both WT and LXRα-KO mice fed an HFCD, and lipid droplets in LXRα-KO mice were larger than in WT mice [Fig. 2(a)]. LXRα-KO had higher scores for steatosis, ballooning, and inflammation compared with WT mice, and a total score in these mice was 7 to 8 [Fig. 2(b)], supporting a diagnosis of NASH (19). Hepatic cholesterol, triglyceride, and NEFA levels were elevated in HFCD-fed LXRα-KO mice [Fig. 2(c)]. Cholesterol levels were also increased in hepatic MNCs [Fig. 2(d)]. These findings indicate that HFCD feeding induces NASH in LXRα-KO mice. Figure 2. View largeDownload slide Increased lipid accumulation in the liver of HFCD-fed LXRα-KO mice. (a) Hematoxylin and eosin (HE) staining and oil red O staining of liver samples. White and black arrowheads indicate immune cell infiltration and hepatocyte ballooning, respectively. White scale bar, 100 μm; black scale bar, 50 μm. (b) NAFLD/NASH score. Steatosis, ballooning, and inflammation scores were evaluated in HFCD-fed WT and LXRα-KO mice (n = 4 for each group). **P < 0.01; ***P < 0.001 compared with WT mice (two-tailed unpaired Student t test). (c) Hepatic cholesterol, triglyceride (TG), and NEFA levels. (d) Cholesterol content in hepatic MNCs. WT and LXRα-KO mice were fed an ND or HFCD for 4 weeks (n = 3 for each group). **P < 0.01; ***P < 0.001 (one-way ANOVA followed by Tukey multiple comparisons). Figure 2. View largeDownload slide Increased lipid accumulation in the liver of HFCD-fed LXRα-KO mice. (a) Hematoxylin and eosin (HE) staining and oil red O staining of liver samples. White and black arrowheads indicate immune cell infiltration and hepatocyte ballooning, respectively. White scale bar, 100 μm; black scale bar, 50 μm. (b) NAFLD/NASH score. Steatosis, ballooning, and inflammation scores were evaluated in HFCD-fed WT and LXRα-KO mice (n = 4 for each group). **P < 0.01; ***P < 0.001 compared with WT mice (two-tailed unpaired Student t test). (c) Hepatic cholesterol, triglyceride (TG), and NEFA levels. (d) Cholesterol content in hepatic MNCs. WT and LXRα-KO mice were fed an ND or HFCD for 4 weeks (n = 3 for each group). **P < 0.01; ***P < 0.001 (one-way ANOVA followed by Tukey multiple comparisons). HFCD feeding increases expression of genes encoding macrophage activation makers and inflammatory cytokines LXRα is a transcription factor that induces expression of genes involved in cholesterol metabolism, such as Abcg5 and Abcg8, and represses expression of inflammatory cytokine genes, such as Ccl2 (the gene encoding CCL2, also called monocyte chemotactic protein 1) (28, 29). We examined expression of immune cell genes to assess whether NASH in LXRα-KO mice is accompanied by increased inflammatory gene expression. In the whole liver of HFCD-fed LXRα-KO mice, expression of Ccl2 and genes of the macrophage activation markers, Adgre1 (the gene encoding F4/80), Itgam (the gene encoding CD11b), and Cd68, was strongly elevated [Fig. 3(a)]. HFCD feeding induced expression of the LXR target genes Abcg5 and Abcg8 in an LXRα-dependent manner [Fig. 3(b)]. The expression of the gene encoding LXRα (Nr1h3) was not changed by HFCD in WT mice. There was no difference in expression of the gene encoding LXRβ (Nr1h2) in WT and LXRα-KO mice fed an ND or HFCD. Figure 3. View largeDownload slide Expression of (a) genes involved in inflammation and (b) LXRs and their target genes in the whole liver of LXRα-KO mice fed an ND or HFCD. WT and LXRα-KO mice were fed an ND or HFCD for 4 weeks, and gene expression analysis was performed in the whole liver samples (ND-fed WT mice, n = 3; HFCD-fed WT mice, n = 4; ND-fed LXRα-KO mice, n = 3; HFCD-fed LXRα-KO mice, n = 4). Adgre1, Itgam, Nr1h3, and Nr1h2 encode F4/80, CD11b, LXRα, and LXRβ, respectively. *P < 0.05; **P < 0.01; ***P < 0.001 (one-way ANOVA followed by Tukey multiple comparisons). Figure 3. View largeDownload slide Expression of (a) genes involved in inflammation and (b) LXRs and their target genes in the whole liver of LXRα-KO mice fed an ND or HFCD. WT and LXRα-KO mice were fed an ND or HFCD for 4 weeks, and gene expression analysis was performed in the whole liver samples (ND-fed WT mice, n = 3; HFCD-fed WT mice, n = 4; ND-fed LXRα-KO mice, n = 3; HFCD-fed LXRα-KO mice, n = 4). Adgre1, Itgam, Nr1h3, and Nr1h2 encode F4/80, CD11b, LXRα, and LXRβ, respectively. *P < 0.05; **P < 0.01; ***P < 0.001 (one-way ANOVA followed by Tukey multiple comparisons). Next, we examined gene expression in isolated hepatic MNCs from WT and LXRα-KO mice. Similar to the findings observed in whole liver samples, expression of Adgre1, Itgam, and Cd68, but not Ccl2, was increased in hepatic MNCs isolated from HFCD-fed LXRα-KO mice [Fig. 4(a)]. Expression of Ccl2 in the liver of HFCD-fed LXRα-KO mice is not derived from MNCs, but likely from other cells, such as hepatocytes and hepatic stellate cells. Hepatic MNCs from HFCD-fed LXRα-KO mice showed elevated expression of inflammatory cytokine genes, Tnf, Il12b, and Ifng, and decreased expression of Il4 [Fig. 4(a)]. HFCD feeding increased Abcg5 and Abcg8 expression in hepatic MNCs from WT mice, but this effect was not observed in hepatic MNCs from LXRα-KO mice [Fig. 4(b)]. Interestingly, HFCD feeding did not change expression of Abca1 or Apoe in hepatic MNCs from WT mice, but increased these mRNA levels in hepatic MNCs from LXRα-KO mice [Fig. 4(b)]. Expression of Nr1h3 and Nr1h2 was not changed in hepatic MNCs after HFCD feeding. Thus, deletion of LXRα induces inflammatory gene expression in hepatic MNCs in mice fed an HFCD, consistent with the NASH phenotype observed in LXRα-KO mice. In hepatic MNCs, LXRα is necessary for induction of LXR target genes involved in cholesterol metabolism in a gene-selective manner. Figure 4. View largeDownload slide Expression of (a) genes involved in inflammation and (b) LXRs and their target genes in hepatic MNCs from LXRα-KO mice fed an ND or HFCD. WT and LXRα-KO mice were fed an ND or HFCD for 4 weeks, and gene expression analysis was performed in isolated hepatic MNCs (ND-fed WT mice, n = 4; HFCD-fed WT mice, n = 5; ND-fed LXRα-KO mice, n = 4; HFCD-fed LXRα-KO mice, n = 5). Adgre1, Itgam, Nr1h3, and Nr1h2 encode F4/80, CD11b, LXRα, and LXRβ, respectively. *P < 0.05; **P < 0.01; ***P < 0.001 (one-way ANOVA followed by Tukey multiple comparisons). Figure 4. View largeDownload slide Expression of (a) genes involved in inflammation and (b) LXRs and their target genes in hepatic MNCs from LXRα-KO mice fed an ND or HFCD. WT and LXRα-KO mice were fed an ND or HFCD for 4 weeks, and gene expression analysis was performed in isolated hepatic MNCs (ND-fed WT mice, n = 4; HFCD-fed WT mice, n = 5; ND-fed LXRα-KO mice, n = 4; HFCD-fed LXRα-KO mice, n = 5). Adgre1, Itgam, Nr1h3, and Nr1h2 encode F4/80, CD11b, LXRα, and LXRβ, respectively. *P < 0.05; **P < 0.01; ***P < 0.001 (one-way ANOVA followed by Tukey multiple comparisons). F4/80+CD11b+ Kupffer cells/macrophages are markedly increased in the liver of HFCD-fed LXRα mice We previously reported that F4/80+CD11b+ Kupffer cells/macrophages are elevated in the liver of mice fed a high-cholesterol diet and HFCD (10). To investigate whether NASH in HFCD-fed LXRα-KO mice is accompanied by an increase of Kupffer cells/macrophages, we performed flow cytometric analysis on hepatic MNCs. Consistent with the histological finding of inflammatory cell infiltration [Fig. 2(a)], total MNC number was elevated and F4/80-positive area was increased in the liver of HFCD-fed LXRα-KO mice [Fig. 5(a) and 5(b)]. The flow cytometric analysis showed that the proportion of F4/80+CD68+CD11b+ cells, but not F4/80+CD68+CD11b– cells, was increased in the liver of HFCD-fed LXRα-KO mice [Fig. 5(c)]. There was no increase in the total MNC number or the proportion of F4/80+CD68+CD11b+ cells in ND-fed or HFCD-fed WT mice or ND-fed LXRα-KO mice [Fig. 5(a)–5(c)]. Thus, the expanded population of MNCs in the liver of HFCD-fed LXRα-KO mice are bone marrow–derived Kupffer cells/macrophages (F4/80+CD11b+). Figure 5. View largeDownload slide Elevation of F4/80+CD68+CD11b+ Kupffer cells/macrophages in HFCD-fed LXRα-KO mice. WT and LXRα-KO mice were fed an ND or HFCD for 4 weeks, and hepatic MNCs were isolated with collagenase digestion and Percoll solution. (a) Total number of hepatic MNCs (n = 5 for each group). (b) Immunostaining of liver with anti-F4/80 antibody. Scale bar, 50 μm. Relative F4/80-positive area was quantified (n = 4 for each group). (c) Hepatic MNCs were stained with FITC-anti-F4/80, PE-Cy5-anti-CD11b, biotin-anti-CD68, and PE-streptavidin and analyzed with flow cytometry. The percentages of F4/80+CD68+CD11b+ Kupffer cells/macrophages are indicated in the right panel (n = 5 for each group). ***P < 0.001 (one-way ANOVA followed by Tukey multiple comparisons). Figure 5. View largeDownload slide Elevation of F4/80+CD68+CD11b+ Kupffer cells/macrophages in HFCD-fed LXRα-KO mice. WT and LXRα-KO mice were fed an ND or HFCD for 4 weeks, and hepatic MNCs were isolated with collagenase digestion and Percoll solution. (a) Total number of hepatic MNCs (n = 5 for each group). (b) Immunostaining of liver with anti-F4/80 antibody. Scale bar, 50 μm. Relative F4/80-positive area was quantified (n = 4 for each group). (c) Hepatic MNCs were stained with FITC-anti-F4/80, PE-Cy5-anti-CD11b, biotin-anti-CD68, and PE-streptavidin and analyzed with flow cytometry. The percentages of F4/80+CD68+CD11b+ Kupffer cells/macrophages are indicated in the right panel (n = 5 for each group). ***P < 0.001 (one-way ANOVA followed by Tukey multiple comparisons). Expanded F4/80+CD68+CD11b+ Kupffer cells/macrophages mediate more severe LPS-induced liver inflammation Bone marrow–derived F4/80+CD11b+ Kupffer cells/macrophages are involved in hepatic inflammation through the production of proinflammatory cytokines (8). To investigate proinflammatory responses in mice, we administered LPS to mice fed an HFCD for 4 weeks. LPS treatment increased immune cells in the periportal area of the liver of WT mice and induced more drastic accumulation of immune cells in LXRα-KO mice [Fig. 6(a)]. We measured plasma levels of TNF-α, IFN-γ, IL-12p70, and CCL2 and found that TNF-α, IFN-γ, and CCL2 levels were slightly increased in LXRα-KO mice prior to LPS administration when compared with WT mice and that LPS treatment induced higher levels of TNF-α, INF-γ, IL-12p70, and CCL2 in LXRα-KO mice than in WT mice [Fig. 6(b)]. Plasma AST and ALT levels were also increased after LPS treatment in LXRα-KO mice, whereas these changes were minimal in WT mice [Fig. 6(c)]. Figure 6. View largeDownload slide HFCD feeding exacerbates LPS-induced acute hepatic inflammation in LXRα-KO mice. WT and LXRα-KO mice were fed an ND or HFCD for 4 weeks and subjected to intravenous LPS injection. (a) Hematoxylin and eosin staining of liver samples 12 hours after LPS injection. Arrowheads indicate infiltration of MNCs in periportal areas. Scale bar, 100 μm. Plasma samples were collected at 0, 1, 3, and 6 hours after LPS injection and subjected to measurement of (b) proinflammatory cytokines and (c) aminotransferases (n = 8 for each group). Insets show magnified views of the indicated data points. There were significant differences between WT mice and LXRα-KO mice for all panels of (b) and (c) (P < 0.001; two-way ANOVA). *P < 0.05; **P < 0.01; ***P < 0.001 compared with WT mice at the same time point (two-tailed unpaired Student t test). Figure 6. View largeDownload slide HFCD feeding exacerbates LPS-induced acute hepatic inflammation in LXRα-KO mice. WT and LXRα-KO mice were fed an ND or HFCD for 4 weeks and subjected to intravenous LPS injection. (a) Hematoxylin and eosin staining of liver samples 12 hours after LPS injection. Arrowheads indicate infiltration of MNCs in periportal areas. Scale bar, 100 μm. Plasma samples were collected at 0, 1, 3, and 6 hours after LPS injection and subjected to measurement of (b) proinflammatory cytokines and (c) aminotransferases (n = 8 for each group). Insets show magnified views of the indicated data points. There were significant differences between WT mice and LXRα-KO mice for all panels of (b) and (c) (P < 0.001; two-way ANOVA). *P < 0.05; **P < 0.01; ***P < 0.001 compared with WT mice at the same time point (two-tailed unpaired Student t test). iNKT cells are decreased and functionally impaired in the liver of HFCD-fed LXRα-KO mice Because NKT cells are present in substantial numbers in the mouse liver (30), we examined the characteristics of NKT cells in the liver of mice. Interestingly, NKT cells (βTCR+NK1.1+ cells) were decreased in the liver of HFCD-fed LXRα-KO mice but not WT mice or ND-fed LXRα-KO mice, whereas the proportion of a related immune cell–type, NK cells (βTCR–NK1.1+ cells), did not differ in all mouse groups [Fig. 7(a)]. CD1d-α-GalCer dimer-positive iNKT cells were also decreased in HFCD-fed LXRα-KO mice, though HFCD feeding did not change the proportion of these cells [Fig. 7(b)]. We examined expression of the activation antigen CD69 in NK cells and NKT cells (31). In the liver of LXRα-KO mice, CD69-positive cells were increased in the NK cell population, but there was no change in the proportion of CD69-positive NKT cells [Fig. 7(c)]. Figure 7. View largeDownload slide Decreased iNKT cell population in the liver of HFCD-fed LXRα-KO mice. WT and LXRα-KO mice were fed an ND or HFCD for 4 weeks, and hepatic MNCs were isolated without collagenase digestion. (a) Cells were stained with FITC-anti-βTCR, PE-anti-NK1.1, biotin-anti-CD69, and PE-Cy5-streptavidin and analyzed with flow cytometry. The percentages of βTCR–NK1.1+ cells (NK cells) and βTCR+NK1.1+ cells (NKT cells) are indicated in the right panels (n = 4). (b) Cells were stained with FITC-anti-NK1.1, PE-anti-CD1d dimer complexed with α-GalCer, and PE-Cy5-anti-NK1.1 and analyzed with flow cytometry. The percentages of βTCR+CD1d dimer+ cells (iNKT cells) are indicated in the right panel (n = 4). (c) Expression of CD69 on the membranes of βTCR–NK1.1+ cells (NK cells) and βTCR+NK1.1+ cells (NKT cells) (n = 4). *P < 0.05; **P < 0.01; ***P < 0.001 (one-way ANOVA followed by Tukey’s multiple comparisons). Figure 7. View largeDownload slide Decreased iNKT cell population in the liver of HFCD-fed LXRα-KO mice. WT and LXRα-KO mice were fed an ND or HFCD for 4 weeks, and hepatic MNCs were isolated without collagenase digestion. (a) Cells were stained with FITC-anti-βTCR, PE-anti-NK1.1, biotin-anti-CD69, and PE-Cy5-streptavidin and analyzed with flow cytometry. The percentages of βTCR–NK1.1+ cells (NK cells) and βTCR+NK1.1+ cells (NKT cells) are indicated in the right panels (n = 4). (b) Cells were stained with FITC-anti-NK1.1, PE-anti-CD1d dimer complexed with α-GalCer, and PE-Cy5-anti-NK1.1 and analyzed with flow cytometry. The percentages of βTCR+CD1d dimer+ cells (iNKT cells) are indicated in the right panel (n = 4). (c) Expression of CD69 on the membranes of βTCR–NK1.1+ cells (NK cells) and βTCR+NK1.1+ cells (NKT cells) (n = 4). *P < 0.05; **P < 0.01; ***P < 0.001 (one-way ANOVA followed by Tukey’s multiple comparisons). Next, we examined whether the diminished number of NKT cells is associated with impaired function of these cells. We administered the iNKT cell-specific activator α-GalCer to WT and LXRα-KO mice after HFCD feeding for 4 weeks. Before α-GalCer treatment, HFCD-fed LXRα-KO mice had slightly higher levels of IFN-γ and TNF-α and lower levels of IL-4 compared with HFCD-fed WT mice [Fig. 8(a)]. α-GalCer treatment increased plasma levels of IL-4, IFN-γ, and TNF-α in WT mice, and this effect was markedly diminished in LXRα-KO mice [Fig. 8(a)]. Because NKT cells are involved in Con-A-induced hepatitis (32, 33), we also examined the activity of NKT cells by evaluating Con-A-induced acute hepatic injury. Necrotic hepatocytes were widely distributed in the liver of HFCD-fed WT mice, but were not observed in LXRα-KO mice [Fig. 8(b)]. Con-A treatment increased plasma levels of AST, ALT, total bilirubin, and direct bilirubin in WT mice, but these liver injury markers were increased at lower levels in LXRα-KO mice [Fig. 8(c)]. Therefore, hepatic NKT cells are decreased and functionally impaired in the liver of HFCD-fed LXRα-KO mice. Figure 8. View largeDownload slide iNKT cells are functionally impaired in LXRα-KO mice. WT and LXRα-KO mice were fed an ND or HFCD for 4 weeks and subjected to (a) intravenous α-GalCer injection and (b, c) Con-A injection. (a) Plasma IL-4, IFN-γ, and TNF-α levels collected after α-GalCer injection at the indicated time point (HFCD-fed WT mice, n = 6; HFCD-fed LXRα-KO mice, n = 6). Insets show magnified views of the indicated data points. There was a significant difference between WT mice and LXRα-KO mice (P < 0.001; two-way ANOVA). *P < 0.05; **P < 0.01; ***P < 0.001 compared with WT mice at the same time point (two-tailed unpaired Student t test). (b) Hematoxylin and eosin staining of liver samples 12 and 24 hours after Con-A injection. Arrowheads indicate necrotic hepatocytes. Scale bar, 100 μm. (c) Plasma levels of aminotransferases and (d) total bilirubin (T-Bil) and direct bilirubin (D-Bil) before (–) or 12 and 24 hours after Con-A injection. *P < 0.05; **P < 0.01; ***P < 0.001 compared with the same genotype mice before Con-A injection; #P < 0.05; ##P < 0.01; ###P < 0.001 compared with WT mice at the same time condition (one-way ANOVA followed by Tukey multiple comparisons). Figure 8. View largeDownload slide iNKT cells are functionally impaired in LXRα-KO mice. WT and LXRα-KO mice were fed an ND or HFCD for 4 weeks and subjected to (a) intravenous α-GalCer injection and (b, c) Con-A injection. (a) Plasma IL-4, IFN-γ, and TNF-α levels collected after α-GalCer injection at the indicated time point (HFCD-fed WT mice, n = 6; HFCD-fed LXRα-KO mice, n = 6). Insets show magnified views of the indicated data points. There was a significant difference between WT mice and LXRα-KO mice (P < 0.001; two-way ANOVA). *P < 0.05; **P < 0.01; ***P < 0.001 compared with WT mice at the same time point (two-tailed unpaired Student t test). (b) Hematoxylin and eosin staining of liver samples 12 and 24 hours after Con-A injection. Arrowheads indicate necrotic hepatocytes. Scale bar, 100 μm. (c) Plasma levels of aminotransferases and (d) total bilirubin (T-Bil) and direct bilirubin (D-Bil) before (–) or 12 and 24 hours after Con-A injection. *P < 0.05; **P < 0.01; ***P < 0.001 compared with the same genotype mice before Con-A injection; #P < 0.05; ##P < 0.01; ###P < 0.001 compared with WT mice at the same time condition (one-way ANOVA followed by Tukey multiple comparisons). Discussion In this study, we demonstrate that LXRα regulates not only lipid metabolism, but also immune responses in the liver of mice. HFCD feeding induced apparent NASH in LXRα-KO mice, but only mild hepatic steatosis in WT mice (Figs. 1 and 2). Plasma and liver cholesterol levels were elevated in HFCD-fed WT mice with more profound elevation in HFCD-fed LXRα-KO mice (Fig. 1). A similar pattern of elevated plasma and liver cholesterol levels was reported in LXRα-KO mice fed a high-cholesterol diet (15). LXRα-KO mice fed a high-cholesterol diet have decreased triglyceride levels in both plasma and liver (15). Although plasma triglyceride levels were decreased in HFCD-fed LXRα-KO mice, liver triglyceride levels were increased (Fig. 1). The dietary fat component of the HFCD may affect hepatic triglyceride and fatty liver accumulation in LXRα-KO mice through a lipogenesis-independent mechanism, because LXRα activation induces gene expression involved in lipogenesis (34). HFCD-fed LXRα-KO mice demonstrated hepatic inflammation (Figs. 2, 4, and 5), which has been suggested to contribute to the accumulation of triglycerides and fatty acids by decreasing fatty acid oxidation and/or lipoprotein secretion. Gene expression analysis revealed that HFCD feeding increased expression of Abcg5 and Abcg8 in the liver and hepatic MNCs in WT mice but not LXRα-KO mice (Figs. 3 and 4), consistent with the previous report (28). Interestingly, mRNA levels of Abca1 and Apoe in hepatic MNCs were elevated in HFCD-fed LXRα-KO mice compared with HFCD-fed WT mice and ND-fed LXRα-KO mice (Fig. 4). LXR ligand treatment increases expression of these genes in macrophages in a manner dependent on both LXRα and LXRβ (35, 36). Synthetic LXR ligand treatment can induce Abca1 expression and stimulate cholesterol efflux in macrophages from LXRα-KO mice (37, 38). HFCD feeding did not change expression of Nr1h2 (the gene encoding LXRβ) in hepatic MNCs from WT mice or LXRα-KO mice (Fig. 4), but increased cholesterol levels in hepatic MNCs from LXRα-KO mice (Fig. 2). These findings suggest that LXRβ is activated and induces expression of Abca1 and Apoe in HFCD-fed LXRα-KO mice, but that activity is insufficient to compensate for the loss of LXRα. We cannot exclude the possibility of an LXR-independent mechanism of regulation of Apoe and Abca1 expression. Further studies using hepatic MNCs from LXRβ-KO mice and/or LXRα/β double KO mice are needed to elucidate the role of LXRβ in NASH pathogenesis. Expression of Adgre1, Itgam, and Cd68, which encode F4/80, CD11b, and CD68, respectively, was increased in both whole liver and hepatic MNCs of HFCD-fed LXRα-KO mice (Figs. 3 and 4). In agreement with the gene expression results, flow cytometry revealed an increase of the F4/80+CD68+CD11b+ cell population (Fig. 5). By contrast, Ccl2 mRNA levels were elevated in whole liver but not in hepatic MNCs in HFCD-fed LXRα-KO mice (Figs. 3 and 4). Ccl2 expression is induced in hepatocytes, hepatic stellate cells, and sinusoidal endothelial cells after inflammatory stimulation (39–41), and LXRα regulates metabolism and immunity in these cells (42–44). Increased Ccl2 mRNA expression in the liver of HFCD-fed LXRα-KO mice may be derived from hepatocytes, hepatic stellate cells, and/or sinusoidal endothelial cells and may be involved in recruitment of myeloid cells expressing the CCL2 receptor (40). The elevated number of F4/80+CD68+CD11b+ cells was associated with increased susceptibility to LPS-induced liver inflammation. HFCD-fed LXRα-KO mice had increased proinflammatory cytokines in the plasma (Fig. 6), in agreement with the role of CD68+CD11b+ Kupffer cells/macrophages in cytokine production after LPS stimulation (8). This type of Kupffer cell/macrophage also accumulates in the liver of fibroblast growth factor 5–deficient mice fed a high-fat diet, a model of severe NASH (20). Because LXRα is involved in transrepression of proinflammatory gene expression (14, 45), LXRα is suggested to control recruitment and activation of CD68+CD11b+ Kupffer cells/macrophages through regulation of gene expression in Kupffer cells/macrophages and other hepatic cells, such as hepatocytes, hepatic stellate cells, and sinusoidal endothelial cells. On the other hand, cholesterol overload enhances activation of Kupffer cells/macrophages by inflammatory stimuli (10). Because cholesterol accumulation induces inflammasome activation in macrophages (4), LXRα may regulate Kupffer cell/macrophage activation through control of cellular cholesterol balance. Conversely, iNKT cells were significantly decreased in the liver of HFCD-fed LXRα-KO mice (Fig. 7). The function of iNKT cells in LXRα-KO mice in producing IL-4 and IFN-γ after α-GalCer stimulation was also suppressed, and Con-A-induced liver injury was attenuated in LXRα-KO mice (Fig. 8). These findings identify decreased number and function of iNKT cells in the liver of LXRα-KO mice. Kupffer cell activation results in decreased hepatic NKT cell number in mice with hepatic steatosis (46, 47). A decrease of NKT cells was also observed in human livers with severe steatosis (46). Thus, decreased iNKT cells in the liver of LXRα-KO mice may be a consequence of Kupffer cell activation. On the other hand, NKT cell activation enhances the pathogenesis of atherosclerosis and hepatic steatosis with increased proinflammatory cytokine levels (48–50). However, a causative relationship between activation of Kupffer cells/macrophages and dysfunction of NKT cells still remains unclear. Coculture experiments may be useful to elucidate the interaction of these cells. Recently, we reported that fibroblast growth factor 5–deficient mice fed a high-fat diet develop severe NASH and have increased F4/80+CD68+CD11b+ Kupffer cells/macrophages and decreased NKT cells in the liver (20). Depletion of F4/80+CD68+CD11b+ Kupffer cells/macrophages by irradiation in these mice inhibits liver inflammation without attenuating steatosis. Kupffer cells/macrophages and NKT cells may play distinct roles in NASH pathogenesis. Depletion experiments and/or transplantation of Kupffer cells/macrophages and NKT cells can provide clues to the NASH pathogenesis in LXRα-KO mice. Con-A-induced hepatitis is mediated by NKT cells (32, 33). LXRα-KO mice were resistant to Con-A-induced liver injury compared with WT mice, although they had more lipid accumulation with HFCD feeding (Fig. 8). Recently, it was reported that NKT cells play a role in the healing response after liver injury by regulating the inflammatory reaction (51). Decreased iNKT function may enhance liver damage in HFCD-fed LXRα-KO mice. Although Con-A is widely used in a murine model of autoimmune hepatitis, Con-A is not a causative agent of human hepatitis and the role of NKT cells in human autoimmune hepatitis remains elusive (52). Further studies are needed to elucidate potential roles of LXRα in hepatic iNKT cell function and pathogenesis of autoimmune hepatitis. In conclusion, LXRα controls hepatic immune cell populations and function along with lipid metabolism. LXRα is suggested to play a role in the gateway function of the liver as a lipid sensor and regulator of metabolism and immunity. Abbreviations: Abbreviations: ABC adenosine triphosphate–binding cassette ALT alanine aminotransferase ANOVA analysis of variance ApoE apolipoprotein E AST aspartate aminotransferase CCL2 chemokine (C-C motif) ligand 2 cDNA complementary DNA Con-A concanavalin-A FITC fluorescein isothiocyanate HFCD high-fat and high-cholesterol diet IFN-γ interferon-γ iNKT invariant natural killer T IL-12p70 interleukin-12p70 KO knockout LPS lipopolysaccharide LXR liver X receptor MNC mononuclear cell mRNA messenger RNA NAFLD nonalcoholic fatty liver disease NASH nonalcoholic steatohepatitis ND normal diet NEFA nonesterified fatty acid NK natural killer NKT natural killer T PCR polymerase chain reaction PE phycoerythrin TNF-α tumor necrosis factor-α WT wild-type α-GalCer α-galactosylceramide Acknowledgments We thank Dr. Shigeyuki Uno, Dr. Makoto Ayaori, and members of the Makishima and Seki Laboratories for technical assistance and helpful comments, Dr. David J. Mangelsdorf of the Howard Hughes Medical Institute and University of Texas Southwestern Medical Center at Dallas for providing LXRα-KO mice, and Dr. Andrew I. Shulman for editorial assistance. Financial Support: This work was supported by Japan Society for the Promotion of Science KAKENHI Grants JP 25860248 and JP 16K19061 (to K.E.-U.) and a Nihon University School of Medicine Toki Fund Research Grant (to K.E.-U.). Disclosure Summary: The authors have nothing to disclose. References 1. Yasutake K, Kohjima M, Kotoh K, Nakashima M, Nakamuta M, Enjoji M. Dietary habits and behaviors associated with nonalcoholic fatty liver disease. World J Gastroenterol . 2014; 20( 7): 1756– 1767. Google Scholar CrossRef Search ADS PubMed  2. Hosseini Z, Whiting SJ, Vatanparast H. Current evidence on the association of the metabolic syndrome and dietary patterns in a global perspective. Nutr Res Rev . 2016; 29( 2): 152– 162. Google Scholar CrossRef Search ADS PubMed  3. Wagner M, Zollner G, Trauner M. 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Dysregulation of Kupffer Cells/Macrophages and Natural Killer T Cells in Steatohepatitis in LXRα Knockout Male Mice

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Copyright © 2018 Endocrine Society
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0013-7227
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10.1210/en.2017-03141
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Abstract

Abstract Liver X receptor (LXR) α expression is mainly localized to metabolic tissues, such as the liver, whereas LXRβ is ubiquitously expressed. LXRα is activated by oxysterols and plays an important role in the regulation of lipid metabolism in metabolic tissues. In macrophages, LXRs stimulate reverse cholesterol transport and regulate immune responses. Although a high-cholesterol diet induces severe steatohepatitis in LXRα-knockout (KO) mice, the underlying mechanisms linking lipid metabolism and immune responses remain largely unknown. In this study, we investigated the role of LXRα in the pathogenesis of steatohepatitis by assessing the effects of a high-fat and high-cholesterol diet (HFCD) on hepatic immune cell proportion and function as well as lipid metabolism in wild-type (WT) and LXRα-KO mice. HFCD feeding induced severe steatohepatitis in LXRα-KO mice compared with WT mice. These mice had higher cholesterol levels in the plasma and the liver and dysregulated expression of LXR target and proinflammatory genes in both whole liver samples and isolated hepatic mononuclear cells. Flow cytometry showed an increase in CD68+CD11b+ Kupffer cells/macrophages and a decrease in invariant natural killer T cells in the liver of HFCD-fed LXRα-KO mice. These mice were more susceptible to lipopolysaccharide-induced liver injury and resistant to inflammatory responses against α-galactosylceramide or concanavalin-A treatment. The findings provide evidence for activation of bone marrow–derived Kupffer cells/macrophages and dysfunction of invariant natural killer T cells in LXRα-KO mouse liver. These findings indicate that LXRα regulates hepatic immune function along with lipid metabolism and protects against the pathogenesis of nonalcoholic steatohepatitis. Lipid metabolism is influenced by dietary and endogenous lipids and is regulated by several regulatory mechanisms, including nuclear receptor function. Lipid dysregulation is involved in the pathogenesis of metabolic diseases, including fatty liver, diabetes, and atherosclerosis (1–3). Impaired regulation of cholesterol metabolism and hepatic cholesterol accumulation result in inflammation and play a role in the development of nonalcoholic fatty liver disease (NAFLD), nonalcoholic steatohepatitis (NASH), liver fibrosis, liver cirrhosis, and hepatic carcinoma (4–6). A number of immune cells, including Kupffer cells, natural killer (NK) cells, and natural killer T (NKT) cells, are present in the liver and participate in the induction and progression of hepatic inflammation (7). Kupffer cells/macrophages are classified into two subsets, radio-resistant resident Kupffer cells and radio-sensitive bone marrow–derived Kupffer cells/macrophages (8, 9). Whereas resident Kupffer cells express F4/80 and CD68 and have high phagocytic activity and reactive oxygen species production, bone marrow–derived Kupffer cells/macrophages express F4/80 and CD11b and produce proinflammatory cytokines efficiently (8). Mice fed a high-cholesterol diet or a high-fat and high-cholesterol diet (HFCD) have increased numbers of CD11b+ Kupffer cells/macrophages in the liver and are highly susceptible to acute hepatic inflammation induced by cytosine-phosphate-guanine oligonucleotide or α-galactosylceramide (α-GalCer) administration (10, 11). A high-cholesterol diet also induces free cholesterol accumulation in hepatic stellate cells, which are activated by inflammation, and exacerbates hepatic fibrosis induced by bile duct ligation or carbon tetrachloride treatment (12). Thus, dysregulation of lipid metabolism, specifically cholesterol accumulation, activates hepatic immune cells and induces hepatic inflammatory diseases including NASH. The possible pathophysiological roles of hepatic immune cells, such as the two subsets of Kupffer cells/macrophages, in NAFLD/NASH induced by dietary cholesterol overload remain largely unknown. The nuclear receptors liver X receptor (LXR) α and LXRβ are ligand-dependent transcription factors that are activated by oxysterols and regulate lipid metabolism and immunity (13, 14). LXRα is mainly expressed in metabolic tissues, such as liver, adipose tissue, and intestine, whereas LXRβ is ubiquitously expressed. LXR activation stimulates conversion of cholesterol to bile acids by inducing expression of cholesterol 7α-hydroxylase in the rodent liver and also promotes cholesterol efflux into bile by inducing adenosine triphosphate–binding cassette (ABC) transporter G5 and ABCG8. In macrophages, LXRs stimulate reverse cholesterol transport by inducing expression of ABCA1 and apolipoprotein E (ApoE). A high-cholesterol diet induces more severe hepatic cholesterol accumulation and NASH in LXRα-knockout (KO) mice than in LXRβ-KO mice (15, 16). Hepatic LXRα is necessary for effective biliary and fecal cholesterol excretion and reverse cholesterol transport (17). Because the liver plays important roles in both lipid metabolism and innate immunity as a gateway for dietary signals, LXRα has been suggested to have a key function as the hepatic cholesterol sensor. In this study, we investigated the role of LXRα in hepatic immune responses in NAFLD mice induced by HFCD and found that LXRα regulates not only hepatic lipid metabolism, but also immune cell function, specifically in CD11b+ Kupffer cells/macrophages and NKT cells. Materials and Methods Animal studies Wild-type (WT) C57BL/6J mice were obtained from Nihon CLEA (Tokyo, Japan), and Nr1h3–/– (Lxrα–/–, LXRα-KO) mice were kindly provided by Dr. David J. Mangelsdorf (University of Texas Southwestern Medical Center, Dallas, TX) (15). Mice were maintained under controlled temperature (23°C ± 1°C) and humidity (45% to 56%) with free access to water and chow. Male mice between 7 and 8 weeks of age were fed a control normal diet (ND) (CE-2; Nihon CLEA) or an HFCD (CE-2 supplemented with 1.25 g/100 g cholesterol and 12.5 g/100 g cocoa butter) as reported previously with minor modification (18). After the 4-week feeding period, mice were subjected to sample collection or intravenous treatment with Escherichia coli–derived lipopolysaccharide (LPS; Sigma-Aldrich, St. Louis, MO; 2.5 mg/kg of body weight), α-GalCer (Funakoshi Co., Tokyo, Japan; 0.1 mg/kg of body weight), or concanavalin-A (Con-A; Vector Laboratories, Burlingame, CA; 12.5 mg/kg of body weight). Mice were euthanized with carbon dioxide. The experimental protocol adhered to the Nihon University Rules Concerning Animal Care and Use and was approved by the Nihon University Animal Care and Use Committee. Plasma analysis Plasma cholesterol, triglyceride, glucose, nonesterified fatty acid (NEFA), aspartate aminotransferase (AST)/alanine aminotransferase (ALT), and total/direct bilirubin levels were measured with Cholesterol E-Test Wako, Triglyceride E-Test Wako, Glucose CII-Test Wako, NEFA C-Test Wako, GOT•GPT CII-Test Wako, and Bilirubin BII-Test Wako (Wako Pure Chemical Industries, Osaka, Japan), respectively. Plasma tumor necrosis factor-α (TNF-α), interferon-γ (IFN-γ), interleukin-12p70 (IL-12p70), chemokine (C-C motif) ligand 2 (CCL2), and IL-4 levels were measured with enzyme-linked immunosorbent assay kits (R&D Systems, Minneapolis, MN). Liver histology Liver sections were fixed with 10% neutral buffered formalin (Muto Pure Chemicals, Tokyo, Japan) for 24 hours and embedded with paraffin. The embedded tissues were cut into 5-μm sections, deparaffinized, stained with hematoxylin and eosin (Sakura Finetek Japan, Tokyo, Japan), washed with ethanol and xylene, and mounted with the Histofine Mousestain Kit (Nichirei Corporation, Tokyo, Japan). Oil red O staining was performed on liver frozen sections by Medical & Biological Laboratories Co. (Nagoya, Japan). We used the NASH Clinical Research Network scoring system (19) to assess steatosis, ballooning, and inflammation for a diagnosis of NASH in mice as reported previously (20). For immunohistochemistry, sections were treated with anti-F4/80 antibody (Thermo Fisher Scientific, Waltham, MA; Table 1), the Histofine Simple Stain Mouse MAX-PO (Rat) (Nichirei Corporation), and ImmPACT DAB Substrate solution (Vector Laboratories), and counterstained with hematoxylin. The F4/80-positive area was quantified with ImageJ software (National Institutes of Health, Bethesda, MD). Table 1. Antibodies Used in This Study Peptide/Protein Target  Antigen Sequence (if Known)  Name of Antibody  Manufacturer, Catalog No.  Species Raised in; Monoclonal or Polyclonal  RRID  Dilution  CD16/32    Anti-CD16/CD32 purified  Thermo Fisher Scientific, 14-0161, Clone no. 93  Rat; monoclonal  AB_467133  1:30  F4/80    Anti-F4/80 FITC  Thermo Fisher Scientific, 11-4801, Clone no. BMB  Rat; monoclonal  AB_2637191  1:10  F4/80    F4/80 monoclonal antibody  Thermo Fisher Scientific, 14-4801, Clone no. BMB  Rat; monoclonal  AB_467559  1:10  CD11b    Anti-mouse CD11b PE-Cy5  Thermo Fisher Scientific, 15-0112, Clone no. M1/70  Rat; monoclonal  AB_468714  1:30  CD68    Rat anti-mouse CD68: biotin  Bio-Rad Laboratories, MCA1957B, Clone no. FA-11  Rat; monoclonal  AB_323443  1:8  βTCR    Anti-mouse TCR β FITC  Thermo Fisher Scientific, 11-5961, Clone no. H57-597  Armenian hamster; monoclonal  AB_465323  1:10  βTCR    Anti-mouse TCR β PE-Cy5  Thermo Fisher Scientific, 15-5961, Clone no. H57-597  Armenian hamster; monoclonal  AB_468816  1:30  CD69    Anti-mouse CD69 biotin  Thermo Fisher Scientific, 13-0691, Clone no. H1.2F3  Armenian hamster; monoclonal  AB_466495  1:30  NK1.1    Anti-mouse NK1.1 FITC  Thermo Fisher Scientific, 11-5941, Clone no. PK136  Mouse; monoclonal  AB_465318  1:30  NK1.1    Anti-mouse NK1.1 PE  Thermo Fisher Scientific, 12-5941, Clone no. PK136  Mouse; monoclonal  AB_466050  1:30  IgG1    PE rat anti-mouse IgG1  BD Biosciences,550083, Clone no. A85-1  Rat; monoclonal  AB_393553  1:2  Peptide/Protein Target  Antigen Sequence (if Known)  Name of Antibody  Manufacturer, Catalog No.  Species Raised in; Monoclonal or Polyclonal  RRID  Dilution  CD16/32    Anti-CD16/CD32 purified  Thermo Fisher Scientific, 14-0161, Clone no. 93  Rat; monoclonal  AB_467133  1:30  F4/80    Anti-F4/80 FITC  Thermo Fisher Scientific, 11-4801, Clone no. BMB  Rat; monoclonal  AB_2637191  1:10  F4/80    F4/80 monoclonal antibody  Thermo Fisher Scientific, 14-4801, Clone no. BMB  Rat; monoclonal  AB_467559  1:10  CD11b    Anti-mouse CD11b PE-Cy5  Thermo Fisher Scientific, 15-0112, Clone no. M1/70  Rat; monoclonal  AB_468714  1:30  CD68    Rat anti-mouse CD68: biotin  Bio-Rad Laboratories, MCA1957B, Clone no. FA-11  Rat; monoclonal  AB_323443  1:8  βTCR    Anti-mouse TCR β FITC  Thermo Fisher Scientific, 11-5961, Clone no. H57-597  Armenian hamster; monoclonal  AB_465323  1:10  βTCR    Anti-mouse TCR β PE-Cy5  Thermo Fisher Scientific, 15-5961, Clone no. H57-597  Armenian hamster; monoclonal  AB_468816  1:30  CD69    Anti-mouse CD69 biotin  Thermo Fisher Scientific, 13-0691, Clone no. H1.2F3  Armenian hamster; monoclonal  AB_466495  1:30  NK1.1    Anti-mouse NK1.1 FITC  Thermo Fisher Scientific, 11-5941, Clone no. PK136  Mouse; monoclonal  AB_465318  1:30  NK1.1    Anti-mouse NK1.1 PE  Thermo Fisher Scientific, 12-5941, Clone no. PK136  Mouse; monoclonal  AB_466050  1:30  IgG1    PE rat anti-mouse IgG1  BD Biosciences,550083, Clone no. A85-1  Rat; monoclonal  AB_393553  1:2  Abbreviations: IgG, immunoglobulin G; RRID, Research Resource Identifier. View Large Measurement of lipid contents in liver samples Liver tissues (0.1 g) were homogenized in cold phosphate-buffered saline on ice. Lipids were extracted with 3 mL of chloroform/methanol (1:2 by volume), next with 3.8 mL of chloroform/methanol (1:2 by volume), and then twice with 3 mL of chloroform. Collected samples were dried up and dissolved in isopropanol. Liver cholesterol, triglyceride, and NEFA levels were measured with Cholesterol E-Test Wako, Triglyceride E-Test Wako, and NEFA C-Test Wako (Wako Pure Chemical Industries), respectively. Cholesterol content in hepatic mononuclear cells (MNCs) was also measured with Cholesterol E-Test Wako (Wako Pure Chemical Industries). Isolation of mouse hepatic MNCs Hepatic MNCs were isolated with collagenase digestion and Percoll gradient centrifugation as previously described (11). For detection of NK and NKT cells, hepatic MNCs were isolated without collagenase digestion. Reverse transcription and quantitative real-time polymerase chain reaction Complementary DNAs (cDNAs) were synthesized from total RNAs from whole liver or hepatic MNCs using the ImProm-II reverse transcription system (Promega Corporation, Madison, WI), as described previously (21). Quantitative real-time polymerase chain reaction (PCR) was used with the ABI PRISM 7000 sequence detection system (Thermo Fisher Scientific) using Power SYBR Green PCR Master Mix (Thermo Fisher Scientific). Briefly, after incubation at 95°C for 10 minutes, cDNAs were denatured at 95°C for 15 seconds and annealed and extended with primers at 60°C for 1 minute for each PCR cycle. Intron-spanning primer sequences were as follows: Ccl2, 5′-ATG CAG GTC CCT GTC ATG CTT-3′ and 5′-AGC TCT CCA GCC TAC TCA TTG-3′; Adgre1 (the gene encoding F4/80), 5′-GCA TCA TGG CAT ACC TGT TC-3′ and 5′-GAG CTA AGG TCA GTC TTC CT-3′; Itgam (the gene encoding CD11b), 5′-TCC TGT ACC ACT CAT TGT GG-3′ and 5′-GGG CAG CTT CAT TCA TCA TG-3′; Cd68, 5′-CTG CTG TGG AAA TGC AAG CA-3′ and 5′-TGG TCA CGG TTG CAA GAG AA-3′; Abcg5, 5′-TCA GGA CCC CAA GGT CAT GAT-3′ and 5′-AGG CTG GTG GAT GGT GAC AAT-3′; Abcg8, 5′-GAC AGC TTC ACA GCC CAC AA-3′ and 5′-GCC TGA AGA TGT CAG AGC GA-3′; Il4, 5′-CTT CCA AGG TGC TTC GCA TA-3′ and 5′-GAC TCA TTC ATG GTG CAG CT-3′; and Apoe, 5′-TGC TGT TGG TCA CAT TGC TGA-3′ and 5′-CTT GTG TGA CTT GGG AGC TCT G-3′. Primer sequences for Nr1h3 (the gene encoding LXRα), Nr1h2 (the gene encoding LXRβ), Tnf, Il12b, Ifng, Abca1, Ppib (the gene encoding cyclophilin B), and Gapdh (the gene encoding glyceraldehyde-3-phosphate dehydrogenase) were reported previously (22–25). Each primer was used at a final concentration of 0.2 μM. PCR products were quantified with standard curves that were linear over a range of 0.02 to 200 ng/mL for corresponding cDNAs inserted into pcDNA3.1 plasmids (Thermo Fisher Scientific) as reported previously (26). The calculated RNA values were normalized with the Ppib or Gapdh messenger RNA (mRNA) levels. Flow cytometric analysis For analysis of Kupffer cells/macrophages, hepatic MNCs were isolated with collagenase digestion, incubated with anti-CD16/32 Fc blocker (Thermo Fisher Scientific) for 15 minutes at 4°C, and stained with fluorescein isothiocyanate (FITC)–conjugated anti-F4/80 (Thermo Fisher Scientific), biotin-conjugated anti-CD68 (Bio-Rad Laboratories, Hercules, CA), phycoerythrin (PE)-Cy5-conjugated anti-CD11b, and PE-streptavidin (Thermo Fisher Scientific). For analysis of NK cells and NKT cells, hepatic MNCs were isolated without collagenase digestion, incubated with anti-CD16/32 Fc blocker, and stained with FITC-anti-βTCR, PE-anti-NK1.1, biotin-anti-CD69, and PE-Cy5-streptavidin (Thermo Fisher Scientific). For analysis of invariant natural killer T (iNKT) cells, hepatic MNCs were isolated without collagenase digestion, incubated with anti-CD16/32 Fc blocker, and stained with FITC-anti-NK1.1 (Thermo Fisher Scientific), PE-CD1d-IgG1 dimer (BD Biosciences, San Jose, CA) complexed with α-GalCer, and PE-Cy5-anti-NK1.1 (Thermo Fisher Scientific). Flow cytometric analysis was performed with the Cytomics FC500 (Beckman Coulter, Indianapolis, IN). The antibodies used are listed in Table 1. Independent flow cytometric analysis was performed at least 3 times. Statistical analysis Data are presented as means ± standard deviation. We performed two-tailed unpaired Student t test to assess significant differences between the two groups and one-way analysis of variance (ANOVA) with the Tukey post hoc test or two-way ANOVA for multiple comparisons. Results HFCD feeding induces NASH in LXRα-KO mice Under high-cholesterol diet conditions, LXRα-KO mice exhibit severe cholesterol accumulation in the liver (15, 16). NAFLD and NASH are associated with lifestyle factors such as a “Western diet” high in fat and cholesterol (1, 27). We used HFCD as a model for a Western diet and examined the effect of HFCD on lipid metabolism and liver damage in LXRα-KO mice. Peet et al. (15) demonstrate that high-cholesterol diet feeding significantly increases liver mass and plasma AST and ALT levels in LXRα-KO mice within 30 days. Because we also observed that HFCD feeding for 3 to 4 weeks increased liver weight and plasma AST and ALT levels in LXRα-KO mice in preliminary experiments, we compared the effects of 4-week feeding of an HFCD on the development of NAFLD and NASH in LXRα-KO mice vs WT mice. Similar to high-cholesterol diet feeding (15), HFCD feeding for 4 weeks increased liver weight without changing body weight in LXRα-KO mice, but not in WT mice [Fig. 1(a)]. There was no difference in body weight or liver weight between WT and LXRα-KO mice fed an ND. Whereas plasma cholesterol levels were similar in WT and LXRα-KO mice fed an ND, HFCD feeding increased plasma cholesterol levels in LXRα-KO mice more effectively than WT mice [Fig. 1(b)]. Plasma triglyceride levels were lower in LXRα-KO mice than in WT mice and were decreased by HFCD feeding compared with ND feeding in both mice [Fig. 1(b)]. Plasma glucose and NEFA levels were not altered in all groups [Fig. 1(b)]. Plasma AST and ALT levels were strongly elevated in HFCD-fed LXRα-KO mice [Fig. 1(c)]. Figure 1. View largeDownload slide Body and liver weights and plasma biochemical profiles in WT and LXRα-KO mice fed an ND or HFCD. After WT and LXRα-KO mice were fed an ND or HFCD for 4 weeks, (a) body weight and liver weight per 20 g body weight, (b) plasma cholesterol (CHO), triglyceride (TG), glucose, and NEFA levels, (c) and plasma AST and ALT levels were measured (n = 8 for each group). **P < 0.01; ***P < 0.001 (one-way ANOVA followed by Tukey multiple comparisons). Figure 1. View largeDownload slide Body and liver weights and plasma biochemical profiles in WT and LXRα-KO mice fed an ND or HFCD. After WT and LXRα-KO mice were fed an ND or HFCD for 4 weeks, (a) body weight and liver weight per 20 g body weight, (b) plasma cholesterol (CHO), triglyceride (TG), glucose, and NEFA levels, (c) and plasma AST and ALT levels were measured (n = 8 for each group). **P < 0.01; ***P < 0.001 (one-way ANOVA followed by Tukey multiple comparisons). We next evaluated liver histology and hepatic lipid contents. Hematoxylin and eosin staining showed that the liver of HFCD-fed LXRα-KO mice had lipid accumulation, hepatocyte ballooning, and inflammatory cell infiltration [Fig. 2(a)]. Oil red O staining showed lipid accumulation in both WT and LXRα-KO mice fed an HFCD, and lipid droplets in LXRα-KO mice were larger than in WT mice [Fig. 2(a)]. LXRα-KO had higher scores for steatosis, ballooning, and inflammation compared with WT mice, and a total score in these mice was 7 to 8 [Fig. 2(b)], supporting a diagnosis of NASH (19). Hepatic cholesterol, triglyceride, and NEFA levels were elevated in HFCD-fed LXRα-KO mice [Fig. 2(c)]. Cholesterol levels were also increased in hepatic MNCs [Fig. 2(d)]. These findings indicate that HFCD feeding induces NASH in LXRα-KO mice. Figure 2. View largeDownload slide Increased lipid accumulation in the liver of HFCD-fed LXRα-KO mice. (a) Hematoxylin and eosin (HE) staining and oil red O staining of liver samples. White and black arrowheads indicate immune cell infiltration and hepatocyte ballooning, respectively. White scale bar, 100 μm; black scale bar, 50 μm. (b) NAFLD/NASH score. Steatosis, ballooning, and inflammation scores were evaluated in HFCD-fed WT and LXRα-KO mice (n = 4 for each group). **P < 0.01; ***P < 0.001 compared with WT mice (two-tailed unpaired Student t test). (c) Hepatic cholesterol, triglyceride (TG), and NEFA levels. (d) Cholesterol content in hepatic MNCs. WT and LXRα-KO mice were fed an ND or HFCD for 4 weeks (n = 3 for each group). **P < 0.01; ***P < 0.001 (one-way ANOVA followed by Tukey multiple comparisons). Figure 2. View largeDownload slide Increased lipid accumulation in the liver of HFCD-fed LXRα-KO mice. (a) Hematoxylin and eosin (HE) staining and oil red O staining of liver samples. White and black arrowheads indicate immune cell infiltration and hepatocyte ballooning, respectively. White scale bar, 100 μm; black scale bar, 50 μm. (b) NAFLD/NASH score. Steatosis, ballooning, and inflammation scores were evaluated in HFCD-fed WT and LXRα-KO mice (n = 4 for each group). **P < 0.01; ***P < 0.001 compared with WT mice (two-tailed unpaired Student t test). (c) Hepatic cholesterol, triglyceride (TG), and NEFA levels. (d) Cholesterol content in hepatic MNCs. WT and LXRα-KO mice were fed an ND or HFCD for 4 weeks (n = 3 for each group). **P < 0.01; ***P < 0.001 (one-way ANOVA followed by Tukey multiple comparisons). HFCD feeding increases expression of genes encoding macrophage activation makers and inflammatory cytokines LXRα is a transcription factor that induces expression of genes involved in cholesterol metabolism, such as Abcg5 and Abcg8, and represses expression of inflammatory cytokine genes, such as Ccl2 (the gene encoding CCL2, also called monocyte chemotactic protein 1) (28, 29). We examined expression of immune cell genes to assess whether NASH in LXRα-KO mice is accompanied by increased inflammatory gene expression. In the whole liver of HFCD-fed LXRα-KO mice, expression of Ccl2 and genes of the macrophage activation markers, Adgre1 (the gene encoding F4/80), Itgam (the gene encoding CD11b), and Cd68, was strongly elevated [Fig. 3(a)]. HFCD feeding induced expression of the LXR target genes Abcg5 and Abcg8 in an LXRα-dependent manner [Fig. 3(b)]. The expression of the gene encoding LXRα (Nr1h3) was not changed by HFCD in WT mice. There was no difference in expression of the gene encoding LXRβ (Nr1h2) in WT and LXRα-KO mice fed an ND or HFCD. Figure 3. View largeDownload slide Expression of (a) genes involved in inflammation and (b) LXRs and their target genes in the whole liver of LXRα-KO mice fed an ND or HFCD. WT and LXRα-KO mice were fed an ND or HFCD for 4 weeks, and gene expression analysis was performed in the whole liver samples (ND-fed WT mice, n = 3; HFCD-fed WT mice, n = 4; ND-fed LXRα-KO mice, n = 3; HFCD-fed LXRα-KO mice, n = 4). Adgre1, Itgam, Nr1h3, and Nr1h2 encode F4/80, CD11b, LXRα, and LXRβ, respectively. *P < 0.05; **P < 0.01; ***P < 0.001 (one-way ANOVA followed by Tukey multiple comparisons). Figure 3. View largeDownload slide Expression of (a) genes involved in inflammation and (b) LXRs and their target genes in the whole liver of LXRα-KO mice fed an ND or HFCD. WT and LXRα-KO mice were fed an ND or HFCD for 4 weeks, and gene expression analysis was performed in the whole liver samples (ND-fed WT mice, n = 3; HFCD-fed WT mice, n = 4; ND-fed LXRα-KO mice, n = 3; HFCD-fed LXRα-KO mice, n = 4). Adgre1, Itgam, Nr1h3, and Nr1h2 encode F4/80, CD11b, LXRα, and LXRβ, respectively. *P < 0.05; **P < 0.01; ***P < 0.001 (one-way ANOVA followed by Tukey multiple comparisons). Next, we examined gene expression in isolated hepatic MNCs from WT and LXRα-KO mice. Similar to the findings observed in whole liver samples, expression of Adgre1, Itgam, and Cd68, but not Ccl2, was increased in hepatic MNCs isolated from HFCD-fed LXRα-KO mice [Fig. 4(a)]. Expression of Ccl2 in the liver of HFCD-fed LXRα-KO mice is not derived from MNCs, but likely from other cells, such as hepatocytes and hepatic stellate cells. Hepatic MNCs from HFCD-fed LXRα-KO mice showed elevated expression of inflammatory cytokine genes, Tnf, Il12b, and Ifng, and decreased expression of Il4 [Fig. 4(a)]. HFCD feeding increased Abcg5 and Abcg8 expression in hepatic MNCs from WT mice, but this effect was not observed in hepatic MNCs from LXRα-KO mice [Fig. 4(b)]. Interestingly, HFCD feeding did not change expression of Abca1 or Apoe in hepatic MNCs from WT mice, but increased these mRNA levels in hepatic MNCs from LXRα-KO mice [Fig. 4(b)]. Expression of Nr1h3 and Nr1h2 was not changed in hepatic MNCs after HFCD feeding. Thus, deletion of LXRα induces inflammatory gene expression in hepatic MNCs in mice fed an HFCD, consistent with the NASH phenotype observed in LXRα-KO mice. In hepatic MNCs, LXRα is necessary for induction of LXR target genes involved in cholesterol metabolism in a gene-selective manner. Figure 4. View largeDownload slide Expression of (a) genes involved in inflammation and (b) LXRs and their target genes in hepatic MNCs from LXRα-KO mice fed an ND or HFCD. WT and LXRα-KO mice were fed an ND or HFCD for 4 weeks, and gene expression analysis was performed in isolated hepatic MNCs (ND-fed WT mice, n = 4; HFCD-fed WT mice, n = 5; ND-fed LXRα-KO mice, n = 4; HFCD-fed LXRα-KO mice, n = 5). Adgre1, Itgam, Nr1h3, and Nr1h2 encode F4/80, CD11b, LXRα, and LXRβ, respectively. *P < 0.05; **P < 0.01; ***P < 0.001 (one-way ANOVA followed by Tukey multiple comparisons). Figure 4. View largeDownload slide Expression of (a) genes involved in inflammation and (b) LXRs and their target genes in hepatic MNCs from LXRα-KO mice fed an ND or HFCD. WT and LXRα-KO mice were fed an ND or HFCD for 4 weeks, and gene expression analysis was performed in isolated hepatic MNCs (ND-fed WT mice, n = 4; HFCD-fed WT mice, n = 5; ND-fed LXRα-KO mice, n = 4; HFCD-fed LXRα-KO mice, n = 5). Adgre1, Itgam, Nr1h3, and Nr1h2 encode F4/80, CD11b, LXRα, and LXRβ, respectively. *P < 0.05; **P < 0.01; ***P < 0.001 (one-way ANOVA followed by Tukey multiple comparisons). F4/80+CD11b+ Kupffer cells/macrophages are markedly increased in the liver of HFCD-fed LXRα mice We previously reported that F4/80+CD11b+ Kupffer cells/macrophages are elevated in the liver of mice fed a high-cholesterol diet and HFCD (10). To investigate whether NASH in HFCD-fed LXRα-KO mice is accompanied by an increase of Kupffer cells/macrophages, we performed flow cytometric analysis on hepatic MNCs. Consistent with the histological finding of inflammatory cell infiltration [Fig. 2(a)], total MNC number was elevated and F4/80-positive area was increased in the liver of HFCD-fed LXRα-KO mice [Fig. 5(a) and 5(b)]. The flow cytometric analysis showed that the proportion of F4/80+CD68+CD11b+ cells, but not F4/80+CD68+CD11b– cells, was increased in the liver of HFCD-fed LXRα-KO mice [Fig. 5(c)]. There was no increase in the total MNC number or the proportion of F4/80+CD68+CD11b+ cells in ND-fed or HFCD-fed WT mice or ND-fed LXRα-KO mice [Fig. 5(a)–5(c)]. Thus, the expanded population of MNCs in the liver of HFCD-fed LXRα-KO mice are bone marrow–derived Kupffer cells/macrophages (F4/80+CD11b+). Figure 5. View largeDownload slide Elevation of F4/80+CD68+CD11b+ Kupffer cells/macrophages in HFCD-fed LXRα-KO mice. WT and LXRα-KO mice were fed an ND or HFCD for 4 weeks, and hepatic MNCs were isolated with collagenase digestion and Percoll solution. (a) Total number of hepatic MNCs (n = 5 for each group). (b) Immunostaining of liver with anti-F4/80 antibody. Scale bar, 50 μm. Relative F4/80-positive area was quantified (n = 4 for each group). (c) Hepatic MNCs were stained with FITC-anti-F4/80, PE-Cy5-anti-CD11b, biotin-anti-CD68, and PE-streptavidin and analyzed with flow cytometry. The percentages of F4/80+CD68+CD11b+ Kupffer cells/macrophages are indicated in the right panel (n = 5 for each group). ***P < 0.001 (one-way ANOVA followed by Tukey multiple comparisons). Figure 5. View largeDownload slide Elevation of F4/80+CD68+CD11b+ Kupffer cells/macrophages in HFCD-fed LXRα-KO mice. WT and LXRα-KO mice were fed an ND or HFCD for 4 weeks, and hepatic MNCs were isolated with collagenase digestion and Percoll solution. (a) Total number of hepatic MNCs (n = 5 for each group). (b) Immunostaining of liver with anti-F4/80 antibody. Scale bar, 50 μm. Relative F4/80-positive area was quantified (n = 4 for each group). (c) Hepatic MNCs were stained with FITC-anti-F4/80, PE-Cy5-anti-CD11b, biotin-anti-CD68, and PE-streptavidin and analyzed with flow cytometry. The percentages of F4/80+CD68+CD11b+ Kupffer cells/macrophages are indicated in the right panel (n = 5 for each group). ***P < 0.001 (one-way ANOVA followed by Tukey multiple comparisons). Expanded F4/80+CD68+CD11b+ Kupffer cells/macrophages mediate more severe LPS-induced liver inflammation Bone marrow–derived F4/80+CD11b+ Kupffer cells/macrophages are involved in hepatic inflammation through the production of proinflammatory cytokines (8). To investigate proinflammatory responses in mice, we administered LPS to mice fed an HFCD for 4 weeks. LPS treatment increased immune cells in the periportal area of the liver of WT mice and induced more drastic accumulation of immune cells in LXRα-KO mice [Fig. 6(a)]. We measured plasma levels of TNF-α, IFN-γ, IL-12p70, and CCL2 and found that TNF-α, IFN-γ, and CCL2 levels were slightly increased in LXRα-KO mice prior to LPS administration when compared with WT mice and that LPS treatment induced higher levels of TNF-α, INF-γ, IL-12p70, and CCL2 in LXRα-KO mice than in WT mice [Fig. 6(b)]. Plasma AST and ALT levels were also increased after LPS treatment in LXRα-KO mice, whereas these changes were minimal in WT mice [Fig. 6(c)]. Figure 6. View largeDownload slide HFCD feeding exacerbates LPS-induced acute hepatic inflammation in LXRα-KO mice. WT and LXRα-KO mice were fed an ND or HFCD for 4 weeks and subjected to intravenous LPS injection. (a) Hematoxylin and eosin staining of liver samples 12 hours after LPS injection. Arrowheads indicate infiltration of MNCs in periportal areas. Scale bar, 100 μm. Plasma samples were collected at 0, 1, 3, and 6 hours after LPS injection and subjected to measurement of (b) proinflammatory cytokines and (c) aminotransferases (n = 8 for each group). Insets show magnified views of the indicated data points. There were significant differences between WT mice and LXRα-KO mice for all panels of (b) and (c) (P < 0.001; two-way ANOVA). *P < 0.05; **P < 0.01; ***P < 0.001 compared with WT mice at the same time point (two-tailed unpaired Student t test). Figure 6. View largeDownload slide HFCD feeding exacerbates LPS-induced acute hepatic inflammation in LXRα-KO mice. WT and LXRα-KO mice were fed an ND or HFCD for 4 weeks and subjected to intravenous LPS injection. (a) Hematoxylin and eosin staining of liver samples 12 hours after LPS injection. Arrowheads indicate infiltration of MNCs in periportal areas. Scale bar, 100 μm. Plasma samples were collected at 0, 1, 3, and 6 hours after LPS injection and subjected to measurement of (b) proinflammatory cytokines and (c) aminotransferases (n = 8 for each group). Insets show magnified views of the indicated data points. There were significant differences between WT mice and LXRα-KO mice for all panels of (b) and (c) (P < 0.001; two-way ANOVA). *P < 0.05; **P < 0.01; ***P < 0.001 compared with WT mice at the same time point (two-tailed unpaired Student t test). iNKT cells are decreased and functionally impaired in the liver of HFCD-fed LXRα-KO mice Because NKT cells are present in substantial numbers in the mouse liver (30), we examined the characteristics of NKT cells in the liver of mice. Interestingly, NKT cells (βTCR+NK1.1+ cells) were decreased in the liver of HFCD-fed LXRα-KO mice but not WT mice or ND-fed LXRα-KO mice, whereas the proportion of a related immune cell–type, NK cells (βTCR–NK1.1+ cells), did not differ in all mouse groups [Fig. 7(a)]. CD1d-α-GalCer dimer-positive iNKT cells were also decreased in HFCD-fed LXRα-KO mice, though HFCD feeding did not change the proportion of these cells [Fig. 7(b)]. We examined expression of the activation antigen CD69 in NK cells and NKT cells (31). In the liver of LXRα-KO mice, CD69-positive cells were increased in the NK cell population, but there was no change in the proportion of CD69-positive NKT cells [Fig. 7(c)]. Figure 7. View largeDownload slide Decreased iNKT cell population in the liver of HFCD-fed LXRα-KO mice. WT and LXRα-KO mice were fed an ND or HFCD for 4 weeks, and hepatic MNCs were isolated without collagenase digestion. (a) Cells were stained with FITC-anti-βTCR, PE-anti-NK1.1, biotin-anti-CD69, and PE-Cy5-streptavidin and analyzed with flow cytometry. The percentages of βTCR–NK1.1+ cells (NK cells) and βTCR+NK1.1+ cells (NKT cells) are indicated in the right panels (n = 4). (b) Cells were stained with FITC-anti-NK1.1, PE-anti-CD1d dimer complexed with α-GalCer, and PE-Cy5-anti-NK1.1 and analyzed with flow cytometry. The percentages of βTCR+CD1d dimer+ cells (iNKT cells) are indicated in the right panel (n = 4). (c) Expression of CD69 on the membranes of βTCR–NK1.1+ cells (NK cells) and βTCR+NK1.1+ cells (NKT cells) (n = 4). *P < 0.05; **P < 0.01; ***P < 0.001 (one-way ANOVA followed by Tukey’s multiple comparisons). Figure 7. View largeDownload slide Decreased iNKT cell population in the liver of HFCD-fed LXRα-KO mice. WT and LXRα-KO mice were fed an ND or HFCD for 4 weeks, and hepatic MNCs were isolated without collagenase digestion. (a) Cells were stained with FITC-anti-βTCR, PE-anti-NK1.1, biotin-anti-CD69, and PE-Cy5-streptavidin and analyzed with flow cytometry. The percentages of βTCR–NK1.1+ cells (NK cells) and βTCR+NK1.1+ cells (NKT cells) are indicated in the right panels (n = 4). (b) Cells were stained with FITC-anti-NK1.1, PE-anti-CD1d dimer complexed with α-GalCer, and PE-Cy5-anti-NK1.1 and analyzed with flow cytometry. The percentages of βTCR+CD1d dimer+ cells (iNKT cells) are indicated in the right panel (n = 4). (c) Expression of CD69 on the membranes of βTCR–NK1.1+ cells (NK cells) and βTCR+NK1.1+ cells (NKT cells) (n = 4). *P < 0.05; **P < 0.01; ***P < 0.001 (one-way ANOVA followed by Tukey’s multiple comparisons). Next, we examined whether the diminished number of NKT cells is associated with impaired function of these cells. We administered the iNKT cell-specific activator α-GalCer to WT and LXRα-KO mice after HFCD feeding for 4 weeks. Before α-GalCer treatment, HFCD-fed LXRα-KO mice had slightly higher levels of IFN-γ and TNF-α and lower levels of IL-4 compared with HFCD-fed WT mice [Fig. 8(a)]. α-GalCer treatment increased plasma levels of IL-4, IFN-γ, and TNF-α in WT mice, and this effect was markedly diminished in LXRα-KO mice [Fig. 8(a)]. Because NKT cells are involved in Con-A-induced hepatitis (32, 33), we also examined the activity of NKT cells by evaluating Con-A-induced acute hepatic injury. Necrotic hepatocytes were widely distributed in the liver of HFCD-fed WT mice, but were not observed in LXRα-KO mice [Fig. 8(b)]. Con-A treatment increased plasma levels of AST, ALT, total bilirubin, and direct bilirubin in WT mice, but these liver injury markers were increased at lower levels in LXRα-KO mice [Fig. 8(c)]. Therefore, hepatic NKT cells are decreased and functionally impaired in the liver of HFCD-fed LXRα-KO mice. Figure 8. View largeDownload slide iNKT cells are functionally impaired in LXRα-KO mice. WT and LXRα-KO mice were fed an ND or HFCD for 4 weeks and subjected to (a) intravenous α-GalCer injection and (b, c) Con-A injection. (a) Plasma IL-4, IFN-γ, and TNF-α levels collected after α-GalCer injection at the indicated time point (HFCD-fed WT mice, n = 6; HFCD-fed LXRα-KO mice, n = 6). Insets show magnified views of the indicated data points. There was a significant difference between WT mice and LXRα-KO mice (P < 0.001; two-way ANOVA). *P < 0.05; **P < 0.01; ***P < 0.001 compared with WT mice at the same time point (two-tailed unpaired Student t test). (b) Hematoxylin and eosin staining of liver samples 12 and 24 hours after Con-A injection. Arrowheads indicate necrotic hepatocytes. Scale bar, 100 μm. (c) Plasma levels of aminotransferases and (d) total bilirubin (T-Bil) and direct bilirubin (D-Bil) before (–) or 12 and 24 hours after Con-A injection. *P < 0.05; **P < 0.01; ***P < 0.001 compared with the same genotype mice before Con-A injection; #P < 0.05; ##P < 0.01; ###P < 0.001 compared with WT mice at the same time condition (one-way ANOVA followed by Tukey multiple comparisons). Figure 8. View largeDownload slide iNKT cells are functionally impaired in LXRα-KO mice. WT and LXRα-KO mice were fed an ND or HFCD for 4 weeks and subjected to (a) intravenous α-GalCer injection and (b, c) Con-A injection. (a) Plasma IL-4, IFN-γ, and TNF-α levels collected after α-GalCer injection at the indicated time point (HFCD-fed WT mice, n = 6; HFCD-fed LXRα-KO mice, n = 6). Insets show magnified views of the indicated data points. There was a significant difference between WT mice and LXRα-KO mice (P < 0.001; two-way ANOVA). *P < 0.05; **P < 0.01; ***P < 0.001 compared with WT mice at the same time point (two-tailed unpaired Student t test). (b) Hematoxylin and eosin staining of liver samples 12 and 24 hours after Con-A injection. Arrowheads indicate necrotic hepatocytes. Scale bar, 100 μm. (c) Plasma levels of aminotransferases and (d) total bilirubin (T-Bil) and direct bilirubin (D-Bil) before (–) or 12 and 24 hours after Con-A injection. *P < 0.05; **P < 0.01; ***P < 0.001 compared with the same genotype mice before Con-A injection; #P < 0.05; ##P < 0.01; ###P < 0.001 compared with WT mice at the same time condition (one-way ANOVA followed by Tukey multiple comparisons). Discussion In this study, we demonstrate that LXRα regulates not only lipid metabolism, but also immune responses in the liver of mice. HFCD feeding induced apparent NASH in LXRα-KO mice, but only mild hepatic steatosis in WT mice (Figs. 1 and 2). Plasma and liver cholesterol levels were elevated in HFCD-fed WT mice with more profound elevation in HFCD-fed LXRα-KO mice (Fig. 1). A similar pattern of elevated plasma and liver cholesterol levels was reported in LXRα-KO mice fed a high-cholesterol diet (15). LXRα-KO mice fed a high-cholesterol diet have decreased triglyceride levels in both plasma and liver (15). Although plasma triglyceride levels were decreased in HFCD-fed LXRα-KO mice, liver triglyceride levels were increased (Fig. 1). The dietary fat component of the HFCD may affect hepatic triglyceride and fatty liver accumulation in LXRα-KO mice through a lipogenesis-independent mechanism, because LXRα activation induces gene expression involved in lipogenesis (34). HFCD-fed LXRα-KO mice demonstrated hepatic inflammation (Figs. 2, 4, and 5), which has been suggested to contribute to the accumulation of triglycerides and fatty acids by decreasing fatty acid oxidation and/or lipoprotein secretion. Gene expression analysis revealed that HFCD feeding increased expression of Abcg5 and Abcg8 in the liver and hepatic MNCs in WT mice but not LXRα-KO mice (Figs. 3 and 4), consistent with the previous report (28). Interestingly, mRNA levels of Abca1 and Apoe in hepatic MNCs were elevated in HFCD-fed LXRα-KO mice compared with HFCD-fed WT mice and ND-fed LXRα-KO mice (Fig. 4). LXR ligand treatment increases expression of these genes in macrophages in a manner dependent on both LXRα and LXRβ (35, 36). Synthetic LXR ligand treatment can induce Abca1 expression and stimulate cholesterol efflux in macrophages from LXRα-KO mice (37, 38). HFCD feeding did not change expression of Nr1h2 (the gene encoding LXRβ) in hepatic MNCs from WT mice or LXRα-KO mice (Fig. 4), but increased cholesterol levels in hepatic MNCs from LXRα-KO mice (Fig. 2). These findings suggest that LXRβ is activated and induces expression of Abca1 and Apoe in HFCD-fed LXRα-KO mice, but that activity is insufficient to compensate for the loss of LXRα. We cannot exclude the possibility of an LXR-independent mechanism of regulation of Apoe and Abca1 expression. Further studies using hepatic MNCs from LXRβ-KO mice and/or LXRα/β double KO mice are needed to elucidate the role of LXRβ in NASH pathogenesis. Expression of Adgre1, Itgam, and Cd68, which encode F4/80, CD11b, and CD68, respectively, was increased in both whole liver and hepatic MNCs of HFCD-fed LXRα-KO mice (Figs. 3 and 4). In agreement with the gene expression results, flow cytometry revealed an increase of the F4/80+CD68+CD11b+ cell population (Fig. 5). By contrast, Ccl2 mRNA levels were elevated in whole liver but not in hepatic MNCs in HFCD-fed LXRα-KO mice (Figs. 3 and 4). Ccl2 expression is induced in hepatocytes, hepatic stellate cells, and sinusoidal endothelial cells after inflammatory stimulation (39–41), and LXRα regulates metabolism and immunity in these cells (42–44). Increased Ccl2 mRNA expression in the liver of HFCD-fed LXRα-KO mice may be derived from hepatocytes, hepatic stellate cells, and/or sinusoidal endothelial cells and may be involved in recruitment of myeloid cells expressing the CCL2 receptor (40). The elevated number of F4/80+CD68+CD11b+ cells was associated with increased susceptibility to LPS-induced liver inflammation. HFCD-fed LXRα-KO mice had increased proinflammatory cytokines in the plasma (Fig. 6), in agreement with the role of CD68+CD11b+ Kupffer cells/macrophages in cytokine production after LPS stimulation (8). This type of Kupffer cell/macrophage also accumulates in the liver of fibroblast growth factor 5–deficient mice fed a high-fat diet, a model of severe NASH (20). Because LXRα is involved in transrepression of proinflammatory gene expression (14, 45), LXRα is suggested to control recruitment and activation of CD68+CD11b+ Kupffer cells/macrophages through regulation of gene expression in Kupffer cells/macrophages and other hepatic cells, such as hepatocytes, hepatic stellate cells, and sinusoidal endothelial cells. On the other hand, cholesterol overload enhances activation of Kupffer cells/macrophages by inflammatory stimuli (10). Because cholesterol accumulation induces inflammasome activation in macrophages (4), LXRα may regulate Kupffer cell/macrophage activation through control of cellular cholesterol balance. Conversely, iNKT cells were significantly decreased in the liver of HFCD-fed LXRα-KO mice (Fig. 7). The function of iNKT cells in LXRα-KO mice in producing IL-4 and IFN-γ after α-GalCer stimulation was also suppressed, and Con-A-induced liver injury was attenuated in LXRα-KO mice (Fig. 8). These findings identify decreased number and function of iNKT cells in the liver of LXRα-KO mice. Kupffer cell activation results in decreased hepatic NKT cell number in mice with hepatic steatosis (46, 47). A decrease of NKT cells was also observed in human livers with severe steatosis (46). Thus, decreased iNKT cells in the liver of LXRα-KO mice may be a consequence of Kupffer cell activation. On the other hand, NKT cell activation enhances the pathogenesis of atherosclerosis and hepatic steatosis with increased proinflammatory cytokine levels (48–50). However, a causative relationship between activation of Kupffer cells/macrophages and dysfunction of NKT cells still remains unclear. Coculture experiments may be useful to elucidate the interaction of these cells. Recently, we reported that fibroblast growth factor 5–deficient mice fed a high-fat diet develop severe NASH and have increased F4/80+CD68+CD11b+ Kupffer cells/macrophages and decreased NKT cells in the liver (20). Depletion of F4/80+CD68+CD11b+ Kupffer cells/macrophages by irradiation in these mice inhibits liver inflammation without attenuating steatosis. Kupffer cells/macrophages and NKT cells may play distinct roles in NASH pathogenesis. Depletion experiments and/or transplantation of Kupffer cells/macrophages and NKT cells can provide clues to the NASH pathogenesis in LXRα-KO mice. Con-A-induced hepatitis is mediated by NKT cells (32, 33). LXRα-KO mice were resistant to Con-A-induced liver injury compared with WT mice, although they had more lipid accumulation with HFCD feeding (Fig. 8). Recently, it was reported that NKT cells play a role in the healing response after liver injury by regulating the inflammatory reaction (51). Decreased iNKT function may enhance liver damage in HFCD-fed LXRα-KO mice. Although Con-A is widely used in a murine model of autoimmune hepatitis, Con-A is not a causative agent of human hepatitis and the role of NKT cells in human autoimmune hepatitis remains elusive (52). Further studies are needed to elucidate potential roles of LXRα in hepatic iNKT cell function and pathogenesis of autoimmune hepatitis. In conclusion, LXRα controls hepatic immune cell populations and function along with lipid metabolism. LXRα is suggested to play a role in the gateway function of the liver as a lipid sensor and regulator of metabolism and immunity. Abbreviations: Abbreviations: ABC adenosine triphosphate–binding cassette ALT alanine aminotransferase ANOVA analysis of variance ApoE apolipoprotein E AST aspartate aminotransferase CCL2 chemokine (C-C motif) ligand 2 cDNA complementary DNA Con-A concanavalin-A FITC fluorescein isothiocyanate HFCD high-fat and high-cholesterol diet IFN-γ interferon-γ iNKT invariant natural killer T IL-12p70 interleukin-12p70 KO knockout LPS lipopolysaccharide LXR liver X receptor MNC mononuclear cell mRNA messenger RNA NAFLD nonalcoholic fatty liver disease NASH nonalcoholic steatohepatitis ND normal diet NEFA nonesterified fatty acid NK natural killer NKT natural killer T PCR polymerase chain reaction PE phycoerythrin TNF-α tumor necrosis factor-α WT wild-type α-GalCer α-galactosylceramide Acknowledgments We thank Dr. Shigeyuki Uno, Dr. Makoto Ayaori, and members of the Makishima and Seki Laboratories for technical assistance and helpful comments, Dr. David J. Mangelsdorf of the Howard Hughes Medical Institute and University of Texas Southwestern Medical Center at Dallas for providing LXRα-KO mice, and Dr. Andrew I. Shulman for editorial assistance. Financial Support: This work was supported by Japan Society for the Promotion of Science KAKENHI Grants JP 25860248 and JP 16K19061 (to K.E.-U.) and a Nihon University School of Medicine Toki Fund Research Grant (to K.E.-U.). Disclosure Summary: The authors have nothing to disclose. References 1. Yasutake K, Kohjima M, Kotoh K, Nakashima M, Nakamuta M, Enjoji M. Dietary habits and behaviors associated with nonalcoholic fatty liver disease. World J Gastroenterol . 2014; 20( 7): 1756– 1767. Google Scholar CrossRef Search ADS PubMed  2. Hosseini Z, Whiting SJ, Vatanparast H. Current evidence on the association of the metabolic syndrome and dietary patterns in a global perspective. Nutr Res Rev . 2016; 29( 2): 152– 162. Google Scholar CrossRef Search ADS PubMed  3. Wagner M, Zollner G, Trauner M. 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EndocrinologyOxford University Press

Published: Mar 1, 2018

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