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Regulatory T cells suppress excessive lipid accumulation in alcoholic liver disease

Open AccessPublished:January 21, 2019DOI:https://doi.org/10.1194/jlr.M083568
      Sensitization of hepatic immune cells from chronic alcohol consumption gives rise to inflammatory accumulation, which is considered a leading cause of liver damage. Regulatory T cells (Tregs) are an immunosuppressive cell subset that plays an important role in a variety of liver diseases; however, data about pathological involvement of Tregs in liver steatosis of alcoholic liver disease (ALD) is insufficient. In mouse models of ALD, we found that increased lipid accumulation by chronic alcohol intake was accompanied by oxidative stress, inflammatory accumulation, and Treg decline in the liver. Adoptive transfer of Tregs relieved lipid metabolic disorder, oxidative stress, inflammation, and, consequently, ameliorated the alcoholic fatty liver. Macrophages are a dominant source of inflammation in ALD. Aberrant macrophage activation and cytokine production were activated during chronic alcohol consumption, but were significantly inhibited after Treg transfer. In vitro, macrophages were co-activated by alcohol and lipopolysaccharide to mimic a condition for alcoholic liver microenvironment. Tregs suppressed monocyte chemoattractant protein-1 and TNF-α production from these macrophages. However, such effects of Tregs were remarkably neutralized when interleukin (IL)-10 was blocked. Altogether, our data uncover a novel role of Tregs in restoring liver lipid metabolism in ALD, which partially relies on IL-10-mediated suppression of hepatic pro-inflammatory macrophages.
      Alcohol is consumed worldwide and has long been identified as a leading cause of global morbidity and mortality (
      • Rehm J.
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      Global burden of alcoholic liver disease.
      ). As the primary organ for alcohol metabolism, liver also suffers the principal damage from excessive alcohol consumption (
      • Nagy L.E.
      The role of innate immunity in alcoholic liver disease.
      ). The spectrum of alcoholic liver disease (ALD) ranges from steatosis to steatohepatitis, progressive fibrosis, cirrhosis, and the development of hepatocellular cancer (
      • Gao B.
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      Alcoholic liver disease: pathogenesis and new therapeutic targets.
      ).
      Fatty liver is the earliest and most common pathological feature in ALD. Although fatty liver had typically been considered as a benign and reversible disease, recent evidence has indicated that fat accumulation renders the liver more vulnerable to inflammatory mediators and toxic agents, leading to progression to the advanced stage of ALD (
      • Purohit V.
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      Molecular mechanisms of alcoholic fatty liver.
      ,
      • Nishiyama Y.
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      HIF-1alpha induction suppresses excessive lipid accumulation in alcoholic fatty liver in mice.
      ). Some studies suggested that, in active drinkers, liver steatosis was associated with more rapid progression of fibrosis (

      European Association for the Study of Liver. 2012. EASL clinical practical guidelines: management of alcoholic liver disease. J. Hepatol. 57: 399–420.

      ). Other studies also suggested that cirrhosis might occur after a median of 10.5 years in 10% of patients with a histological diagnosis of simple steatosis without evidence of fibrosis or alcoholic steatohepatitis (
      • Teli M.R.
      • Day C.P.
      • Burt A.D.
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      Determinants of progression to cirrhosis or fibrosis in pure alcoholic fatty liver.
      ). Therefore, elucidating the mechanism of the pathogenesis by which chronic alcohol consumption leads to excessive triglyceride (TG) accumulation and liver damage is important and may provide new insights into prevention and therapy of ALD.
      Regulatory T cells (Tregs) are generally characterized by the coexpression of CD4, interleukin (IL)-2 receptor α-subunit (CD25), and the transcription factor, forhead box P3 (Foxp3), the key factor in Treg development and function (
      • Bacchetta R.
      • Gambineri E.
      • Roncarolo M.G.
      Role of regulatory T cells and FOXP3 in human diseases.
      ). In the liver, the deficiency of Tregs is shown to exacerbate inflammatory injury in nonalcoholic fatty liver disease (NAFLD) (
      • Ma X.
      • Hua J.
      • Mohamood A.R.
      • Hamad A.R.
      • Ravi R.
      • Li Z.
      A high-fat diet and regulatory T cells influence susceptibility to endotoxin-induced liver injury.
      ), autoimmune hepatitis (
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      • Mieli-Vergani G.
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      Impairment of CD4(+)CD25(+) regulatory T-cells in autoimmune liver disease.
      ), primary biliary cholangitis (
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      • Yang G.X.
      • Moritoki Y.
      • Chuang Y.H.
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      • et al.
      Liver-targeted and peripheral blood alterations of regulatory T cells in primary biliary cirrhosis.
      ), and acute rejection of transplant grafts (
      • Demirkiran A.
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      • Kwekkeboom J.
      • Kusters J.G.
      • Metselaar H.J.
      • Tilanus H.W.
      • van der Laan L.J.
      Low circulating regulatory T-cell levels after acute rejection in liver transplantation.
      ). However, restoring or expanding the Treg pool leads to increased immune tolerance after liver transplantation (
      • Cabello-Kindelan C.
      • Mackey S.
      • Bayer A.L.
      Adoptive T regulatory cell therapy for tolerance induction.
      ), chronic hepatitis virus infection (
      • Stoop J.N.
      • van der Molen R.G.
      • Baan C.C.
      • van der Laan L.J.
      • Kuipers E.J.
      • Kusters J.G.
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      Regulatory T cells contribute to the impaired immune response in patients with chronic hepatitis B virus infection.
      ), and hepatocellular carcinoma (
      • Unitt E.
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      • Marshall A.
      • Davies S.
      • Gibbs P.
      • Morris L.S.
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      Compromised lymphocytes infiltrate hepatocellular carcinoma: the role of T-regulatory cells.
      ). A recent study indicated a significant decrease of peripheral blood Tregs in patients with alcoholic hepatitis, implying the significance of Tregs in the pathogenesis of ALD (
      • Almeida J.
      • Polvorosa M.A.
      • Gonzalez-Quintela A.
      • Marcos M.
      • Pastor I.
      • Hernandez Cerceno M.L.
      • Orfan A.
      • Laso F.J.
      Decreased peripheral blood CD4+/CD25+ regulatory T cells in patients with alcoholic hepatitis.
      ). In this study, we investigated the role of Tregs in the development of alcoholic fatty liver (AFL) in mice. We identified a reversible impact of Tregs on liver lipid metabolism in a mouse model of AFL, which relies on IL-10-mediated immunosuppressive effects on pro-inflammatory macrophage activation in the hepatic microenvironment, at least in part.

      MATERIALS AND METHODS

      Animal experiments

      Adult male wild-type C57BL/6 mice (6–8 weeks old) were purchased from the Vital River Laboratories (Beijing, China). The mice were randomized into either ethanol-fed or pair-fed groups. The ethanol-fed group was habituated to a Lieber-DeCarli liquid diet with 5% ethanol (v/v) over a period of 1 week, and then maintained on the 5% diet for 5 weeks. Isocaloric amounts of a non-alcohol-containing diet (in which dextran-maltose was used to replace the calories from ethanol) were dispensed to pair-fed animals. Consumption was recorded daily, and weights were recorded weekly throughout the study. All animal studies were carried out according to the guidelines of the Chinese Council on Animal Care and approved by the Tongji Hospital of Tongji Medical School Committees on Animal Experimentation.

      Cell purification and adoptive transfer

      Mouse CD4+CD25+ Tregs were isolated from the spleens of C57BL/6 mice at 6–8 weeks of age with a MACS Treg isolation kit (Miltenyi Biotec, Auburn, CA).The purity of the cells was >95%, as assessed by flow cytometry. These mice were divided randomly into three groups and fed with Lieber-DeCarli liquid diet with 5% ethanol (v/v). One group was adoptively transferred with 1.5 × 106 CD4+CD25+ Tregs and another group with CD4+CD25− T (Teff) cells, respectively, at 1 week intervals from the fourth week of alcoholic feeding via the tail vein injection. Food intake was recorded and the average volume per mouse was calculated daily. The volume was calculated to adjust the amount of liquid diet given to pair-fed mice and Treg- or Teff-transferred groups so that mice in each group consumed equal amounts of diet. Serum was collected and stored at −80°C for future analysis. Livers were snap-frozen in liquid nitrogen for protein and RNA extraction or fixed in 10% neutral-buffered formalin for histopathological analysis.

      Cell isolation, quantitation, and Treg staining

      Mouse livers were weighed and dissected, pressed through a 200 gauge stainless steel mesh, suspended in RPMI 1640 medium (Gibco, Shanghai, China) containing 5% heat-inactivated FBS (Gibco), and centrifuged at 30 g for 3 min. The suspension was transferred and centrifuged at 500 g for 10 min. The pellet was resuspended in 40% Percoll (Sigma-Aldrich, Shanghai, China) solution in RPMI 1640 medium, layered onto 70% Percoll solution, and centrifuged for 20 min at 800 g. The mononuclear cells were collected from the interphase.
      Liver samples for cell quantification were prepared as previously described (
      • Zou Y.
      • Chen T.
      • Han M.
      • Wang H.
      • Yan W.
      • Song G.
      • Wu Z.
      • Wang X.
      • Zhu C.
      • Luo X.
      • et al.
      Increased killing of liver NK cells by Fas/Fas ligand and NKG2D/NKG2D ligand contributes to hepatocyte necrosis in virus-induced liver failure.
      ). The isolated cells from liver were suspended in 2 ml of FACS buffer, and then the number of cells in 100 μl was determined by FACS and multiplied by 20 to assume the total cell number of different tissues. The absolute number of Tregs was subsequently determined by the percentage of Tregs and the total cell number.
      Anti-mouse fluorescent antibodies against CD25 or CD4 were purchased from eBioscience (San Diego, CA). For intracellular Foxp3 staining, HMNCs were first stained with surface antibodies recognizing CD4 or CD25 and then washed, permeabilized, and stained with antibodies against Foxp3 (eBioscience).

      Flow cytometric analysis

      Stained cells were analyzed by the BD FACSCanto II (BD Bioscience) instrument. Lymphocytes were first gated according to forward scatter and side scatter; then the Tregs (CD4+CD25+Foxp3+) were gated and the percentage of Tregs was determined.

      Microsomal preparations and CYP2E1 assays

      Liver tissue was homogenized in cold Tris-HCl buffer (0.01 mM, pH 7.4). Homogenates were centrifuged with Tris-HCl buffer at 12,000 g for 15 min at 4°C; then, the supernatant was centrifuged in Tris-HCl buffer at 100,000 g for 60 min at 4°C; the pellets were resuspended in Tris-HCl buffer to a final concentration of 1 g/ml. Cytochrome P450 2E1 (CYP2E1) was detected by ELISA kits (Elabscience Biotechnology, Wuhan, China) according to the manufacturer's instructions.

      Assay of hepatic antioxidative level

      Liver tissue was homogenized in cold saline to prepare for the assay of activity of antioxidant enzymes. The activities of three enzymes, T-SOD, GPx, and catalase (CAT), were determined using commercial kits (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). 1) T-SOD activity was assayed using the xanthine/xanthine oxidase method based on the production of superoxide anions. 2) GSH peroxidase (GSH-Px) activity was determined by the velocity method using a GSH-Px kit (Nanjing Jiancheng Bioengineering Institute). The reaction was initiated by the addition of H2O2. A series of enzymatic reactions was activated by GSH-Px in the homogenate, which subsequently led to the conversion of GSH to GSSG. The change in absorbance during the conversion of GSH to GSSG was recorded spectrophotometrically at 412 nm. 3) CAT activity was measured by analyzing the rate at which it caused the decomposition of H2O2 at 240 nm, the substrate of the enzyme contained in various tissue samples. Activities of T-SOD and GSH-Px were expressed as units per milligram of protein (U/mg protein). The activity of CAT was expressed as units per gram of protein (U/g protein).

      Assay of hepatic lipid peroxidation

      Hepatic lipid peroxidation was evaluated via the thiobarbituric acid-reactive substances method and was expressed as malondialdehyde (MDA) level, which was assayed with a commercial kit according to the manufacturer's instructions (Nanjing Jiancheng Bioengineering Institute). Briefly, after mixing trichloroacetic acid with the homogenate and centrifuging, the supernatant was obtained and thiobarbituric acid was added. The resulting reaction developed a red color, which was measured at 532 nm with a spectrophotometer. Other procedures were carried out following the manufacturer's protocols. MDA level was expressed as nanomoles per milligram of protein.

      Hepatic cytokine measurement

      Liver samples for cytokine quantification were prepared as previously described (
      • Wolf D.
      • Schümann J.
      • Koerber K.
      • Kiemer A.K.
      • Vollmar A.M.
      • Sass G.
      • Papadopoulos T.
      • Bang R.
      • Klein S.D.
      • Brune B.
      • et al.
      Low-molecular-weight hyaluronic acid induces nuclear factor-kappaB-dependent resistance against tumor necrosis factor alpha-mediated liver injury in mice.
      ). Briefly, liver tissue samples were cut, weighed, and disintegrated in ice-cold PBS buffer (pH 7.4) containing protease inhibitors (1 μg/ml aprotinin, 10 μg/ml leupeptin, and 1 μg/ml pepstatin) and DNase (0.05 mg/ml). After incubation on ice for 30 min, samples were centrifuged twice at 20,000 g for 15 min at 4°C. The resulting supernatants were harvested and the intrahepatic cytokines quantified by commercial TNF-α and monocyte chemoattractant protein-1 (MCP-1) ELISA kits (Dakewe Biotech Co., Shenzhen, China) according to the manufacturer's instructions.

      Liver TG determination

      Liver TG levels were determined as previously described (
      • Peng J.H.
      • Cui T.
      • Huang F.
      • Chen L.
      • Zhao Y.
      • Xu L.
      • Xu L.L.
      • Feng Q.
      • Hu Y.Y.
      Puerarin ameliorates experimental alcoholic liver injury by inhibition of endotoxin gut leakage, Kupffer cell activation, and endotoxin receptors expression.
      ). Briefly, liver tissue was disintegrated and homogenized mechanically in cold PBS buffer (pH 7.4) at a mass-volume concentration of 10%. After centrifugation at 1,250 g for 10 min, the supernatants were harvested and the intrahepatic TG levels quantified by commercial TG detection kits (Dongou Bioengineering Co. Ltd., Zhejiang, China) according to the manufacturer's instructions.

      RNA analysis

      Total RNA was extracted and dissolved in RNA-free water and quantified using UV-clear microplates. Subsequently, single-stranded cDNA was synthesized from 2 μg total RNA by using ReverTra Ace qPCR RT kit (Toyobo Life Science Department, Shanghai, China). Real-time PCR was performed using Real-time PCR Master Mix (Toyobo Life Science Department, Shanghai, China). Primer sequences are shown in Table 1.
      TABLE 1.Primer sequences for real-time quantitative PCR gene validation
      GenePrimer Sequences
      ACC1-FATGGGCGGAATGGTCTCTTTC
      ACC1-RTGGGGACCTTGTCTTCATCAT
      SERBPlc-FTTGTGGAGCTCAAAGACCTG
      SERBPlc-RTGCAAGAAGCGGATGTAGTC
      PPARa-FAAACATCGAGTGTCGAATATGTGG
      PPARa-RAGCCGAATAGTTCGCCGAAAG
      CD68-FCCCACAGGCAGCACAGTGGAC
      CD68-RTCCACAGCAGAAGCTTTGGCCC
      MCP-1-FTGTGCTGACCCCAAGAAGGA
      MCP-l-RTGAGGTGGTTGTGGAAAAGG
      βactin-FTCTTTTCCAGCCTTCCTTCTTG
      βactin-RGAGGTCTTTACGGATGTCAACG
      Fas-FGTGATAGCCGGTATGTCGGG
      Fas-RTAGAGCCCAGCCTTCCATCT
      SREBP1-FTCTGGAGACATCGCAAACAAG
      SREBP1-RTGGTAGACAACAGCCGCATC
      Cpt1a-FAGCGACTCTTCAATACTTCCCG
      Cpt1a-RCAAAATAGGTCTGCCGACACTTC
      Acox-FTCAGATGTCACAGGTAAACAGTCG
      Acox-RGTCTTGTTCAGTGGGGACTTCTT
      LCAD-FTACAAGGAGTAAGAACGAACGCC
      LCAD-RCGACGATCACGAGATCACTTAAC
      MCAD-FTAAGACCAAAGCAGAGAAGAAGG
      MCAD-RAACACATTTTCCTTAGGCACTCT
      Acox, acyl-coA oxidase; CPT1, Carnitinepalmitoyltransferase1; Fas, fatty acid synthase; F, forward; R, reverse.

      Western blot analysis

      Total cell protein and the nucleoprotein fractions were extracted by protein extraction kits (KeyGen Biotech, Nanjing, China) according to the manufacturer's instructions. Protein concentrations were determined using the Enhanced BCA protein assay kit (Beyotime Biotechnology, Nanjing, China). Samples of cell lysates were separated by 10% SDS-PAGE and then transferred onto nitrocellulose membranes. After being placed in blocking buffer, the membranes were incubated with the following primary antibodies: rabbit polyclonal anti-PPARα (1:300 dilution; Santa Cruz Biotechnology, Inc., Dallas, TX), rabbit monoclonal anti-SREBP-1 (1:300 dilution; Santa Cruz Biotechnology, Inc.), mouse monoclonal anti-pAMPK (1:400 dilution; Cell Signaling Technology, Boston, MA), and rabbit monoclonal anti-CD68 (1:2,000 dilution; Abcam Inc., Cambridge, MA). Then, goat anti-rabbit- or goat anti-mouse-HRP-labeled secondary antibodies were used at a 1:80,000 dilution. The protein bands were visualized with an ECL kit. The protein band intensities were subsequently analyzed by BandScan 4.3 software (Glyko, Novato, CA). β-Actin or Lamb protein was used as the internal control.

      Biochemical analysis

      Serum alanine aminotransferase (ALT), aspartate aminotransferase, and TGs were determined with a Roche cobas 8000 (F. Hoffmann-La Roche Ltd., Basel, Switzerland) instrument.

      Histopathological and immunohistochemistry staining

      Sections of formalin-fixed livers were stained with H&E and analyzed by microscopy. Frozen sections were prepared from liver tissue frozen in OCT medium and stained with Oil Red O. Immunohistochemistry was performed using antibodies against PPARα (Santa Cruz Biotechnology, Inc.), CYP2E1 (Biosynthesis Biotechnology, Beijing, China), SREBP-1 (Santa Cruz Biotechnology, Inc.), and pAMPK (Cell Signaling Technology).

      Dual immunofluorescence staining

      Sections were prepared from formalin-fixed paraffin-embedded liver samples. After slice dewaxing, antigen retrieval, and normal serum block, sections were stained with two primary antibodies, which were chosen from the following for different fluorescent matching: rabbit polyclonal for MCP-1, rat monoclonal for F4/80, FITC-conjugated rat monoclonal for F4/80, mouse monoclonal for CD68, mouse monoclonal for TNF-α (Abcam Inc.). Cy3-conjugated goat anti-mouse IgG (Boster Bio-Engineering, Wuhan, China), FITC-conjugated goat anti-rabbit IgG (Boster Bio-Engineering), and DyLight®594-conjugated rabbit anti-rat IgG (H&L) were labeled as secondary antibodies (ImmunoReagents, Raleigh, NC), respectively. Nuclei were counterstained with DAPI (Beyotime Biotechnology).

      In vitro experiments

      Usage of peripheral blood from healthy volunteers was approved by the Ethics Committee of Tongji Hospital, Huazhong University of Science and Technology and was conducted according to the principles of the Declaration of Helsinki. Written informed consent was obtained from the subjects.
      Isolation of human peripheral blood mononuclear cells obtained from healthy volunteers was performed using Ficoll-Hypaque density gradient centrifugation following the manufacturer's protocol (Hao Yang, Tianjin, China). CD4+CD25+ (>93% purity) and CD4+CD25− (>97% purity) T cells were isolated from total peripheral blood mononuclear cells with a MACS Treg isolation kit (Miltenyi Biotec). Monocytes (>85% purity) were isolated by using anti-CD14 microbeads or by depleting nonmonocytes with a MACS isolation kit (Miltenyi Biotec).
      Monocytes and T cells (2:1 ratio) were cocultured in RPMI medium 1640 supplemented with 1% penicillin/streptomycin, 1% glutamine, and 10% heat-inactivated FCS. Monocytes (5 × 105 per milliliter) were cultured in 200 μl cultures without T cells (no T), with CD4+CD25+ T cells (CD25+), or with CD4+CD25− T cells (CD25−) for 40 h in the presence of 50 ng/ml anti-CD3 mAb (BioLegend, San Diego, CA), after which the different cultures were stimulated for 24 h with lipopolysaccharide (LPS) (50 ng/ml; Sigma) and ethanol (25 mM). The 25 mM in vitro ethanol concentration approximates a 0.1 g/dl blood alcohol level, which is achieved in vivo after a single moderate drink and is a little above the legal limit for blood alcohol concentration (
      • Mandrekar P.
      • Bala S.
      • Catalano D.
      • Kodys K.
      • Szabo G.
      The opposite effects of acute and chronic alcohol on lipopolysaccharide-induced inflammation are linked to IRAK-M in human monocytes.
      ). Cell viability was not affected by ethanol or LPS treatment.
      For alcohol exposure, cells exposed to 25 mM alcohol were placed in a Billups-Rothenburg chamber (Billups-Rothenburg, San Diego, CA) with twice the alcohol concentration in the bottom of the chamber to saturate the chamber and maintain the alcohol concentration in the wells, as described earlier by other research groups (
      • Mandrekar P.
      • Bala S.
      • Catalano D.
      • Kodys K.
      • Szabo G.
      The opposite effects of acute and chronic alcohol on lipopolysaccharide-induced inflammation are linked to IRAK-M in human monocytes.
      ,
      • Zhang Z.
      • Bagby G.J.
      • Stoltz D.
      • Oliver P.
      • Schwarzenberger P.O.
      • Kolls J.K.
      Prolonged ethanol treatment enhances lipopolysaccharide/phorbol myristate acetate-induced tumor necrosis factor-alpha production in human monocytic cells.
      ).
      The concentrations of TNF-α and MCP-1 in the supernatant were measured by ELISA kits (Neobioscience, Beijing, China). For neutralization experiments, neutralizing antibodies against IL-10 (5 μg/ml; BioLegend), TGF-β (5 μg/ml; BioLegend), and the isotype control (BioLegend) were added at the start of the coculture.

      Statistical analysis

      GraphPad Prism 5 was used for statistical analysis. Comparisons between the two groups were evaluated using the Student's t-test, and those between multiple groups were analyzed by one-way ANOVA followed by a Tukey post hoc test. All data are represented as mean ± SEM, and P < 0.05 was considered significant.

      RESULTS

      Alcohol consumption induced steatosis and decline of Tregs in the liver

      A previous study showed a decrease of Tregs in peripheral blood in alcoholic hepatitis (
      • Almeida J.
      • Polvorosa M.A.
      • Gonzalez-Quintela A.
      • Marcos M.
      • Pastor I.
      • Hernandez Cerceno M.L.
      • Orfan A.
      • Laso F.J.
      Decreased peripheral blood CD4+/CD25+ regulatory T cells in patients with alcoholic hepatitis.
      ); however, the dynamic involvement of Tregs during the development of AFL remains unclear. Here, we found that C57BL/6 mice fed with Leiber-Decali alcohol diet developed steatosis, as revealed by aberrant TG accumulation when compared with those with the pair-fed diet (Fig. 1C, D). Interestingly, a significant decrease of hepatic Treg number was observed in mice after 6 weeks of alcohol feeding (Fig. 1A, B), suggesting a potential interplay between the Tregs and steatosis.
      Figure thumbnail gr1
      Fig. 1.Decreased hepatic Tregs are correlated with steatosis in AFL in mice. C57BL/6 mice were fed a 5% alcohol-containing Leiber-DeCarli diet or isocaloric pair-fed diet for 6 weeks. A: Percentage of hepatic Tregs (CD4+CD25+FoxP3+) in the sixth week after ethanol (EtOH) or pair feeding. Representative dot plots of gated Tregs (CD4+CD25+FoxP3+) from each animal of one experiment. The number of cells per gram of liver tissue (B) and TGs (milligrams per gram of liver tissue) (C) were analyzed (**P < 0.01, compared with ethanol-fed group; n = 10). D: Oil red O staining of livers of pair- or ethanol-fed mice (magnification, ×200). Serum ALT was measured (E) (**P < 0.01, compared with ethanol-fed group; n = 10). The values shown in the graphs are mean ± SEM.

      Adoptive transfer of Tregs protects against AFL

      To investigate the role of Tregs in ALD, Tregs were isolated from pair-fed mice and adoptively transferred to the alcohol-fed mice (Fig. 2A). As expected, chronic alcohol-fed mice were introduced hepatic lipid deposition, as demonstrated by micro-steatosis and macro-steatosis in hepatic H&E staining (data not shown). However, adoptive transfer of Tregs relieved this phenotype, as examined by H&E staining (data not shown) and Oil Red O staining for hepatic TG level (Fig. 2B, C), while adoptive transfer of Treg-negative lymphocytes (Teff) did not give rise to such an effect. Alcoholic TG accumulation was accompanied by liver injury and impaired liver function, as demonstrated by increased ALT level in alcohol diet mice (Fig. 2D), and Treg transfer ameliorated ALT levels (Fig. 2D). Moreover, there was no significant change in the hepatic cholesterol level before and after Treg transfer (supplemental Fig. S1). Collectively, these data suggest that decrease of Tregs accounts for TG accumulation and liver injury in mice with chronic alcoholic consumption.
      Figure thumbnail gr2
      Fig. 2.Adoptive transfer of Tregs prevents steatosis and liver injury in AFL in mice. C57BL/6 mice were fed a 5% alcohol-containing Leiber-DeCarli diet or isocaloric pair-fed diet for 6 weeks. A: Schematic view of the experimental procedure in the mouse model. B: Liver sections were stained with Oil Red O (magnification, ×200). Liver TGs (milligrams per gram of liver tissue) (C) and serum ALT (D) were measured (*P < 0.05, **P < 0.01; n = 8). Values shown in the graphs are mean ± SEM. EtOH, ethanol.

      Adoptive transfer of Tregs inhibits alcohol-induced hepatic lipogenesis

      TG accumulation occurs when lipid biogenesis exceeds degradation. To obtain the metabolic role of Tregs on alcohol-induced lipid accumulation, we first examined the effects of Treg transfer on lipogenesis in the liver. Interestingly, we found that SREBP-1c, the key transcriptional factor in fatty acid synthesis, was upregulated upon alcohol feeding at both mRNA and protein levels, but declined after Treg transfer (Fig. 3A, B). Consistently, Acc and Fas, direct targets of SREBP-1c involved in lipogenesis, were also decreased significantly when examined at transcription level after Treg transfer (Fig. 3C). AMP-dependent protein kinase (AMPK) was a master regulator for energy homeostasis and acted as the central factor for stimulating lipolysis and inhibition of lipogenesis (
      • Long Y.C.
      • Zierath J.R.
      AMP-activated protein kinase signaling in metabolic regulation.
      ,
      • Li Y.
      • Xu S.
      • Mihaylova M.M.
      • Zheng B.
      • Hou X.
      • Jiang B.
      • Park O.
      • Luo Z.
      • Lefai E.
      • Shyy J.Y.
      • et al.
      AMPK phosphorylates and inhibits SREBP activity to attenuate hepatic steatosis and atherosclerosis in diet-induced insulin-resistant mice.
      ). AMPK phosphorylation inhibited SREBP-1c cleavage and nuclear translocation, a vital step for SREBP-1c dependent transactivation of lipogenic genes by SREBP-1c (
      • Li Y.
      • Xu S.
      • Mihaylova M.M.
      • Zheng B.
      • Hou X.
      • Jiang B.
      • Park O.
      • Luo Z.
      • Lefai E.
      • Shyy J.Y.
      • et al.
      AMPK phosphorylates and inhibits SREBP activity to attenuate hepatic steatosis and atherosclerosis in diet-induced insulin-resistant mice.
      ). In our experiment, chronic alcohol consumption inhibited AMPK activation, as determined by dampened phosphorylation; however, Treg replenishment substantially antagonized such an effect (Fig. 3D). These data suggested that the Treg decrease induced by alcohol consumption resulted in lipid biogenesis, which accounted, at least in part, for lipid accumulation.
      Figure thumbnail gr3
      Fig. 3.Adoptive transfer of Tregs prevents hepatic lipid synthesis in AFL in mice. C57BL/6 mice were fed a 5% alcohol-containing Leiber-DeCarli diet or isocaloric pair-fed diet for 6 weeks, and livers were subjected to analysis of SREBP-1C mRNA (A) (*P < 0.05, **P < 0.01; n = 8) by real-time PCR. B: Nuclear SREBP-1 levels by Western blotting are shown in representative gels (upper panel) and graphs of density units (lower panel) (*P < 0.05, **P <0.01; n = 6). C: SREBP-1C target gene expression by real-time PCR for ACC1 mRNA and FAS mRNA (*P < 0.05; n = 8). D: The pAMPK(Thr172) and AMPK levels by Western blotting are shown in representative gels (upper panel) and graphs of density units (lower panel) (*P < 0.05, **P < 0.01; n = 6). The values shown in the graphs are mean ± SEM. EtOH, ethanol.

      Adoptive transfer of Tregs increases fatty acid β-oxidation in AFL

      To further identify whether alcoholic lipid accumulation was also ascribed to impaired lipid consumption, we also explored lipid oxidation in the livers of alcohol-fed mice. We found that PPARα, master regulator of fatty acid oxidation (
      • Bensinger S.J.
      • Tontonoz P.
      Integration of metabolism and inflammation by lipid-activated nuclear receptors.
      ), was downregulated at both the mRNA level and the protein level (Fig. 4A, B). While activation of PPARα included nuclear translocation, which was critical for transactivation of lipolytic targets (
      • Rosen E.D.
      • Spiegelman B.M.
      Molecular regulation of adipogenesis.
      ,
      • Kliewer S.A.
      • Umesono K.
      • Noonan D.J.
      • Heyman R.A.
      • Evans R.M.
      Convergence of 9-cis retinoic acid and peroxisome proliferator signalling pathways through heterodimer formation of their receptors.
      ), we also detected attenuated expression and activation of PPARα in alcohol-fed mice (Fig. 4C). Expectedly, Treg transfer restored PPARα expression and nuclear translocation to a comparable extent. In agreement with the above results, transcription of Acox and carnitine palmitoyltransferase (Cpt), the direct targets of PPARα for β-oxidation, were downregulated upon alcohol feeding, but recovered substantially after Treg transfer (Fig. 4D). However, the expression of long-chain acyl-CoA dehydrogenase (LCAD) and medium-chain acyl-CoA dehydrogenase (MCAD) was not affected in the ALD model with Treg transfer (Fig. 4D). As mentioned above, AMPK activation is essential for stimulating lipolytic gene expression. The cross-talk between AMPK phosphorylation and Treg number further supported lipolytic disorder in the ALD model. Taken together, our results showed that inhibition of lipolytic gene expression by chronic alcohol consumption was largely due to reduced Treg numbers, and such an effect could be reversed substantially by replenishing the Treg population.
      Figure thumbnail gr4
      Fig. 4.Adoptive transfer of Tregs improves the hepatic fatty acid β-oxidation in AFL in mice. C57BL/6 mice were fed a 5% alcohol-containing Leiber-DeCarli diet or isocaloric pair-fed diet for 6 weeks, and livers were subjected to analysis of PPARα mRNA (A) (*P < 0.05, **P < 0.01; n = 8) by real-time PCR. B: Nuclear PPARα levels by Western blotting are shown in representative gels (upper panel) and graphs of density units (lower panel). (*P < 0.05; n = 6). C: Representative sections of immunohistochemistry stained PPARα in livers with brown staining in hepatic nuclei as positive (magnification, ×400). D: PPARα target gene expression by real-time PCR for MCAD mRNA, LCAD mRNA, ACOX mRNA, and CPT1 mRNA (*P < 0.05; n = 8). The values shown in the graphs are mean ± SEM. EtOH, ethanol; ns, nonsignificant.

      Adoptive transfer of Tregs decreases alcohol-induced oxidative stress in AFL

      Superoxide ( O2) is believed to be a key initiator of oxidative stress, as it forms more potent oxidants via catalytic pathways in alcohol exposure and plays a central role in alcohol-induced liver injury (
      • Beier J.I.
      • McClain C.J.
      Mechanisms and cell signaling in alcoholic liver disease.
      ). To further test the effects of Tregs on hepatic oxidative stress induced by chronic alcohol consumption, we detected the activities of superoxide dismutase (SOD), CAT, and GSH-Px, the main enzymes for clearing O2. Our results showed that the activities of SOD, CAT, and GSH-Px decreased significantly in the livers of alcohol-fed mice, as compared with pair-fed controls (Fig. 5A). However, such an effect was inhibited significantly in the livers of Treg-transferred mice (Fig. 5A). In accordance with these findings, hepatic content of MDA, a major hepatotoxicity product found to be increased in the settings of alcoholic oxidative stress in alcohol-fed mice, was decreased in Treg-transferred mice (Fig. 5A). On the other hand, the expression of CYP2E1, a critical enzyme for alcohol-induced reactive oxygen species (ROS) generation in hepatic microsomes (
      • Leung T.M.
      • Nieto N.
      CYP2E1 and oxidant stress in alcoholic and non-alcoholic fatty liver disease.
      ), was induced significantly in alcohol-fed mice. However, Treg-transfer decreased CYP2E1 expression to basal level (Fig. 5B, C). These results indicated that Tregs protected against alcohol-induced oxidative stress via rectifying the expression of enzymes for both clearing the ROS and ROS generation.
      Figure thumbnail gr5
      Fig. 5.Adoptive transfer of Tregs prevents chronic alcohol-induced oxidative stress in AFL in mice. C57BL/6 mice were fed a 5% alcohol-containing Leiber-DeCarli diet or isocaloric pair-fed diet for 6 weeks, and livers were subjected to analysis of the hepatic antioxidant activities of CAT, GSH-PX, SOD, and MDA (*P < 0.05; n = 7) levels were analyzed by chemical chromatometry (A). CYP2E1 was detected in liver microsomal fractions by ELISA (*P < 0.05,**P < 0.01; n = 8) (B) and immunohistochemistry (C). C: Representative sections of CYP2E1 immunohistochemistry staining (magnification, ×100). The values shown in the graphs are mean ± SEM. EtOH, ethanol.

      Adoptive transfer of Tregs decreases hepatic MCP-1 and TNF-α production and macrophage activation in AFL

      It has been accepted that alcohol metabolism generates a number of metabolites, such as acetate, ROS, and acetaldehyde, which could initiate and aggravate inflammation (
      • Wang H.J.
      • Gao B.
      • Zakhari S.
      • Nagy L.E.
      Inflammation in alcoholic liver disease.
      ,
      • Louvet A.
      • Mathurin P.
      Alcoholic liver disease: mechanisms of injury and targeted treatment.
      ). Many inflammatory cytokines and chemokines are involved in the development of ALF disease (
      • Neuman M.G.
      • French S.W.
      • French B.A.
      • Seitz H.K.
      • Cohen L.B.
      • Mueller S.
      • Osna A.N.
      • Kharbanda K.K.
      • Seth D.
      • Bautista A.
      • et al.
      Alcoholic and non-alcoholic steatohepatitis.
      ). Of these cytokines and chemokines, TNF-α and MCP-1 are described to regulate lipid metabolism, especially suppressed AMPK phosphorylation and, thus, activation (
      • Steinberg G.R.
      • Kemp B.E.
      Adiponectin: starving for attention.
      ,
      • Mizutani K.
      • Sud S.
      • McGrego N.A.
      • Martinovski G.
      • Rice B.T.
      • Craig M.J.
      • Varsos Z.S.
      • Roca H.
      • Pienta K.J.
      The chemokine CCL2 increases prostate tumor growth and bone metastasis through macrophage and osteoclast recruitment.
      ,
      • Mandrekar P.
      • Ambade A.
      • Lim A.
      • Szabo G.
      • Catalano D.
      An essential role for monocyte chemoattractant protein-1 in alcoholic liver injury: regulation of proinflammatory cytokines and hepatic steatosis in mice.
      ). Indeed, chronic alcohol consumption led to upregulation of CCL2 and MCP-1 expression at both mRNA and protein levels (Fig. 6A, B). Resident macrophages or Kupffer cells account for about 70% of resident immune cells and are considered as the major sources of inflammatory factors (
      • Ju C.
      • Tacke F.
      Hepatic macrophages in homeostasis and liver diseases: from pathogenesis to novel therapeutic strategies.
      ). Adipose tissue from obese people contains an increased number of CD68+ macrophages with a pro-inflammatory phenotype (
      • Westerbacka J.
      • Corner A.
      • Kolak M.
      • Makkonen J.
      • Turpeinen U.
      • Hamsten A.
      • Fisher R.M.
      • Yki-Jarvinen H.
      Insulin regulation of MCP-1 in human adipose tissue of obese and lean women.
      ). In our experiments, we detected an increase of CD68 expression in the liver (Fig. 6C), suggesting the infiltration of macrophages. Furthermore, the population of F4/80+CD68+, F4/80+TNF-α+, and F4/80+MCP-1+ macrophages was increased after alcohol feeding, supporting our hypothesis that chronic alcohol consumption underpins an inflammatory microenvironment in the liver. As expected, adoptive transfer of Tregs restricted pro-inflammatory phenotype, as manifested by decreasing TNF-α and MCP-1 expression (Fig. 6A, B), dampening macrophage infiltration (Fig. 6C) and macrophage activation that linked to an inflammatory phenotype (Fig. 6D-F) in alcohol fed-mice.
      Figure thumbnail gr6
      Fig. 6.Adoptive transfer of Tregs prevents hepatic MCP-1 and TNF-α overexpression in AFL in mice. C57BL/6 mice were fed a 5% alcohol-containing Leiber-DeCarli diet or isocaloric pair-fed diet for 6 weeks. A: Livers were subjected to analysis of TNF-α mRNA expression was by real-time PCR, and liver tissue TNF-α levels were estimated by ELISA (*P < 0.05, **P < 0.01; n = 8). B: MCP-1 mRNA by real-time PCR and liver tissue MCP-1 by ELISA (*P < 0.05, **P < 0.01; n = 8). C: CD68 mRNA was estimated by real-time PCR (*P < 0.05, **P < 0.01; n = 8). Liver tissue CD68 levels by Western blotting are shown in representative gels (upper panel) and graphs of density units (lower panel). D: Representative sections of TNF-α immunofluorescence staining (magnification, ×400; green, F4/80; red, TNF-α; yellow, F4/80 and TNF-α double positive). E: Representative sections of MCP-1 immunofluorescence staining (magnification, ×400; red, F4/80; green, MCP-1; yellow, F4/80 and MCP-1 double positive). F: Representative sections of CD68 immunofluorescence staining (magnification, ×400; green, F4/80; red, CD68; yellow, F4/80 and CD68 double positive). EtOH, ethanol.

      Treg-mediated inhibitory effect on monocyte/macrophage activation upon costimulation of LPS and alcohol is partially dependent on IL-10

      Alcohol-induced sensitization of liver macrophages to portal gut-derived endotoxin/ LPS is considered a hallmark of ALD (
      • Yamashina S.
      • Takei Y.
      • Ikejima K.
      • Enomoto N.
      • Kitamura T.
      • Sato N.
      Ethanol-induced sensitization to endotoxin in Kupffer cells is dependent upon oxidative stress.
      ). The portal circulation of endotoxins such as LPS and alcohol acts as a stimulus to resident macrophages and infiltrated monocytes and thus differentiates macrophages to a pro-inflammatory phenotype (
      • Szabo G.
      Gut-liver axis in alcoholic liver disease.
      ).
      To verify the hypothesis that adoptive transfer of Tregs inhibits macrophage activation, we analyzed the inhibitory effects of Tregs on human CD14+ macrophages with the costimulation of LPS and alcohol for 48 h in vitro. Upon LPS and alcohol costimulation, the expression of MCP-1 and TNF-α were increased significantly (Fig. 7A, B). However, induced expression of MCP-1 and TNF-α declined considerably in macrophages when cocultured with Tregs. Inhibitory molecules like IL-10 and TGF-β are typical effectors of Tregs, so IL-10 and/or TGF-β might be involved in the Treg-mediated suppression of pro-inflammatory cytokine expression in macrophages upon costimulation with LPS and alcohol. Interestingly, blockage of IL-10 but not TGF-β reversed such an effect, as indicated by MCP-1 and TNF-α expression in CD14+ macrophages recovered when neutralizing IL-10 activity (Fig. 7C, D).
      Figure thumbnail gr7
      Fig. 7.Tregs suppress MCP-1 and TNF-α expression in monocytes/macrophages with LPS and ethanol costimulation in vitro. Monocytes/macrophages (M) were cultured with CD4+CD25+ T cells (Treg), or with CD4+CD25- T cells (Teff) in the presence of anti-CD3 mAb (50 ng/ml). After 40 h of culture, LPS (50 ng/ml) and ethanol (25 mM) were added, and 24 h later supernatant MCP-1 (A) and TNF-α (B) were measured by ELISA. Some experiments are in the presence of neutralizing mAbs to IL-10 and TGFβ or isotype control mAbs. After 40 h of culture, LPS (50 ng/ml) and ethanol (25 mM) were added and 24 h later supernatant MCP-1 (C) and TNF-α (D) were measured by ELISA. Data are shown as mean ± SEM of triplicate wells and are representative of at least three independent experiments (*P < 0.05; **P < 0.01 vs. monocytes/macrophages in the presence of LPS and ethanol; n = 9).

      DISCUSSION

      It has long been proposed that AFL disease caused by chronic alcohol consumption manifests with altered lipid metabolic events, including increased lipogenesis and decreased β-oxidation, and results in aberrant lipid accumulation (
      • You M.
      • Fischer M.
      • Deeg M.A.
      • Crabb D.W.
      Ethanol induces fatty acid synthesis pathways by activation of sterol regulatory element-binding protein (SREBP).
      ,
      • Galli A.
      • Pinaire J.
      • Fischer M.
      • Dorris R.
      • Crabb D.W.
      The transcriptional and DNA binding activity of peroxisome proliferator-activated receptor alpha is inhibited by ethanol metabolism. A novel mechanism for the development of ethanol-induced fatty liver.
      ,
      • Kim M.J.
      • Sim M.O.
      • Lee H.I.
      • Ham J.R.
      • Seo K.I.
      • Lee M.K.
      Dietary umbelliferone attenuates alcohol-induced fatty liver via regulation of PPARalpha and SREBP-1c in rats.
      ). In addition to alcohol-induced lipogenesis (
      • You M.
      • Fischer M.
      • Deeg M.A.
      • Crabb D.W.
      Ethanol induces fatty acid synthesis pathways by activation of sterol regulatory element-binding protein (SREBP).
      ), ethanol consumption inhibits lipid oxidation directly by suppression of DNA-binding ability and transactivation of PPAR-α, a master receptor that controls transcription of a series of genes involved in free fatty acid transport and oxidation through the alcohol metabolite, acetaldehyde (
      • Galli A.
      • Pinaire J.
      • Fischer M.
      • Dorris R.
      • Crabb D.W.
      The transcriptional and DNA binding activity of peroxisome proliferator-activated receptor alpha is inhibited by ethanol metabolism. A novel mechanism for the development of ethanol-induced fatty liver.
      ,
      • Navidshad B.
      • Royan M.
      Peroxisome proliferator-activated receptor alpha (PPARalpha), a key regulator of lipid metabolism in avians.
      ). Alcohol consumption also inhibits PPAR-α indirectly via upregulation of oxidative stress and adenosine (
      • Lu Y.
      • Zhuge J.
      • Wang X.
      • Bai J.
      • Cederbaum A.I.
      Cytochrome P450 2E1 contributes to ethanol-induced fatty liver in mice.
      ,
      • Peng Z.
      • Borea P.A.
      • Varani K.
      • Wilder T.
      • Yee H.
      • Chiriboga L.
      • Blackburn M.R.
      • Azzena G.
      • Resta G.
      • Cronstein B.N.
      Adenosine signaling contributes to ethanol-induced fatty liver in mice.
      ). Additionally, transcription of PPAR-α is suppressed in response to TNF-α or LPS stimulation (
      • Beier K.
      • Volkl A.
      • Fahimi H.D.
      TNF-alpha downregulates the peroxisome proliferator activated receptor-alpha and the mRNAs encoding peroxisomal proteins in rat liver.
      ,
      • Drosatos K.
      • Khan R.S.
      • Trent C.M.
      • Jiang H.
      • Son N.H.
      • Blaner W.S.
      • Homma S.
      • Schulze P.C.
      • Goldberg I.J.
      Peroxisome proliferator-activated receptor-gamma activation prevents sepsis-related cardiac dysfunction and mortality in mice.
      ). Moreover, AMPK, a serine-threonine kinase, is considered to be a key regulator for lipid metabolism. The phosphorylation of AMPK inactivates acetyl-CoA carboxylase (ACC), a rate-limiting enzyme for fatty acid synthesis. As for SREBP-1c, a master regulator of lipogenic process by transactivation of genes involved in fatty acid and TG synthesis (
      • Steinberg G.R.
      • Kemp B.E.
      Adiponectin: starving for attention.
      ,
      • Viollet B.
      • Guigas B.
      • Leclerc J.
      • Hebrard S.
      • Lantier L.
      • Mounier R.
      • Andreelli F.
      • Foretz M.
      AMP-activated protein kinase in the regulation of hepatic energy metabolism: from physiology to therapeutic perspectives.
      ), is also deactivated by the phosphorylation of AMPK. On the other hand, phosphorylated AMPK directly mediates PPAR-α activation in response to high AMP/ATP ratio (
      • Bronner M.
      • Hertz R.
      • Bar-Tana J.
      Kinase-independent transcriptional co-activation of peroxisome proliferator-activated receptor alpha by AMP-activated protein kinase.
      ,
      • Yoon M.J.
      • Lee G.Y.
      • Chung J.J.
      • Ahn Y.H.
      • Hong S.H.
      • Kim J.B.
      Adiponectin increases fatty acid oxidation in skeletal muscle cells by sequential activation of AMP-activated protein kinase, p38 mitogen-activated protein kinase, and peroxisome proliferator-activated receptor alpha.
      ). AMPK's activation is inhibited during alcohol consumption because of low AMP level (
      • You M.
      • Matsumoto M.
      • Pacold C.M.
      • Cho W.K.
      • Crabb D.W.
      The role of AMP-activated protein kinase in the action of ethanol in the liver.
      ). AMPK's activity is inhibited in settings of chronic inflammation in obesity and diabetes (
      • O'Neill L.A.
      • Hardie D.G.
      Metabolism of inflammation limited by AMPK and pseudo-starvation.
      ,
      • Steinberg G.R.
      • Michell B.J.
      • van Denderen B.J.
      • Watt M.J.
      • Carey A.L.
      • Fam B.C.
      • Andrikopoulos S.
      • Proietto J.
      • Gorgun C.Z.
      • Carling D.
      • et al.
      Tumor necrosis factor alpha-induced skeletal muscle insulin resistance involves suppression of AMP-kinase signaling.
      ). Therefore, targeted activation of AMPK becomes a therapeutic strategy to treat metabolic liver disease (
      • Viollet B.
      • Foretz M.
      • Guigas B.
      • Horman S.
      • Dentin R.
      • Bertrand L.
      • Hue L.
      • Andreelli F.
      Activation of AMP-activated protein kinase in the liver: a new strategy for the management of metabolic hepatic disorders.
      ).
      Mechanistic investigation of the pathogenesis of AFL elicits a “two hits” hypothesis: 1) that the duration of alcohol intake gives rise to steatosis as the first hit; and 2) that steatotic liver is vulnerable to secondary insults by ROS, gut-derived endotoxins, and adipocytokines such as TNF-α and MCP-1 as the second hit (
      • Rasineni K.
      • Casey C.A.
      Molecular mechanism of alcoholic fatty liver.
      ). Another concept is that intermediate metabolites released by adipose tissue may elicit lipotoxicity (
      • Zhong W.
      • Zhao Y.
      • Tang Y.
      • Wei X.
      • Shi X.
      • Sun W.
      • Sun X.
      • Yin X.
      • Sun X.
      • Kim S.
      • et al.
      Chronic alcohol exposure stimulates adipose tissue lipolysis in mice: role of reverse triglyceride transport in the pathogenesis of alcoholic steatosis.
      ,
      • Wei X.
      • Shi X.
      • Zhong W.
      • Zhao Y.
      • Tang Y.
      • Sun W.
      • Yin X.
      • Bogdanov B.
      • Kim S.
      • McClain C.
      • et al.
      Chronic alcohol exposure disturbs lipid homeostasis at the adipose tissue-liver axis in mice: analysis of triacylglycerols using high-resolution mass spectrometry in combination with in vivo metabolite deuterium labeling.
      ). TNF-α and MCP-1, typical inflammatory indicators, were reported to be vital for hepatic steatosis and early liver injury, as manifested by fatty acid oxidation inhibition, decrease of lipoprotein lipase activity, and increased hepatic lipogenesis (
      • Hardardóttir I.
      • Doerrler W.
      • Feingold K.R.
      • Grünfeld C.
      Cytokines stimulate lipolysis and decrease lipoprotein lipase activity in cultured fat cells by a prostaglandin independent mechanism.
      ,
      • Clément S.
      • Juge-Aubry C.
      • Sgroi A.
      • Conzelmann S.
      • Pazienza V.
      • Pittet-Cuenod B.
      • Meier C.A.
      • Negro F.
      Monocyte chemoattractant protein-1 secreted by adipose tissue induces direct lipid accumulation in hepatocytes.
      ,
      • Feingold K.R.
      • Grunfeld C.
      Tumor necrosis factor-alpha stimulates hepatic lipogenesis in the rat in vivo.
      ,
      • Nachiappan V.
      • Curtiss D.
      • Corkey B.E.
      • Kilpatrick L.
      Cytokines inhibit fatty acid oxidation in isolated rat hepatocytes: synergy among TNF, IL-6, and IL-1.
      ). Genetic ablation of either MCP-1 or TNFR2 facilitates fatty acid oxidation, oxidative stress inhibition, and alcoholic liver injury (
      • Mandrekar P.
      • Ambade A.
      • Lim A.
      • Szabo G.
      • Catalano D.
      An essential role for monocyte chemoattractant protein-1 in alcoholic liver injury: regulation of proinflammatory cytokines and hepatic steatosis in mice.
      ,
      • Yin M.
      • Wheeler M.D.
      • Kono H.
      • Bradford B.U.
      • Gallucci R.M.
      • Luster M.I.
      • Thurman R.G.
      Essential role of tumor necrosis factor alpha in alcohol-induced liver injury in mice.
      ).
      Chronic ethanol consumption increases gut permeability and induces gut-derived LPSs in the liver, in turn activating innate immune cells, including hepatic macrophages and infiltrated monocytes (
      • Nagy L.E.
      The role of innate immunity in alcoholic liver disease.
      ). Sensitization of alcohol in portal circulation activates hepatic macrophages/monocytes by endotoxins/LPSs (
      • Nagy L.E.
      The role of innate immunity in alcoholic liver disease.
      ), which provoke robust cytokine production and liver inflammation (
      • Thakur V.
      • McMullen M.R.
      • Pritchard M.T.
      • Nagy L.E.
      Regulation of macrophage activation in alcoholic liver disease.
      ). Increase of CYP2E1-mediated ROS generation during alcohol metabolism, which can react with and damage intracellular molecules such as lipids, proteins, or DNAs, has also been proposed (
      • Galicia-Moreno M.
      • Gutierrez-Reyes G.
      The role of oxidative stress in the development of alcoholic liver disease.
      ). Oxidative stress and ROS generation due to alcohol metabolism not only increase lipid accumulation in hepatocytes but also sensitize liver to subsequent insults by cytokines. Antioxidant therapy is not recommended because attempted use of antioxidants failed to bring benefits to patients of ALD (
      • Frazier T.H.
      • Stocker A.M.
      • Kershner N.A.
      • Marsano L.S.
      • McClain C.J.
      Treatment of alcoholic liver disease.
      ). All of these cues linking liver damage to alcohol-induced inflammatory insults imply the crucial importance of anti-inflammation against ALD.
      Tregs are a population of a subset of immune-suppressive T cells in the circulation and peripheral organs that maintain immune homeostasis and regulate immune response to various pathogenic antigens (
      • Liston A.
      • Gray D.H.
      Homeostatic control of regulatory T cell diversity.
      ). Previous studies demonstrated that adoptive transfer of Tregs ameliorated a variety of immuno-inflammatory diseases, such as atherosclerosis (
      • Viollet B.
      • Foretz M.
      • Guigas B.
      • Horman S.
      • Dentin R.
      • Bertrand L.
      • Hue L.
      • Andreelli F.
      Activation of AMP-activated protein kinase in the liver: a new strategy for the management of metabolic hepatic disorders.
      ), cerebral ischemia (
      • Li P.
      • Gan Y.
      • Sun B.L.
      • Zhang F.
      • Lu B.
      • Gao Y.
      • Liang W.
      • Thomson A.W.
      • Chen J.
      • Hu X.
      Adoptive regulatory T-cell therapy protects against cerebral ischemia.
      ), angiotensin II-induced cardiac damage (
      • Kvakan H.
      • Kleinewietfeld M.
      • Qadri F.
      • Park J.K.
      • Fischer R.
      • Schwarz I.
      • Rahn H.P.
      • Plehm R.
      • Wellner M.
      • Elitok S.
      • et al.
      Regulatory T cells ameliorate angiotensin II-induced cardiac damage.
      ), angiotensin II-induced hypertension and vascular injury (
      • Barhoumi T.
      • Kasal D.A.
      • Li M.W.
      • Shbat L.
      • Laurant P.
      • Neves M.F.
      • Paradis P.
      • Schiffrin E.L.
      T regulatory lymphocytes prevent angiotensin II-induced hypertension and vascular injury.
      ), and aldosterone-induced vascular injury (
      • Kasal D.A.
      • Barhoumi T.
      • Li M.W.
      • Yamamoto N.
      • Zdanovich E.
      • Rehman A.
      • Neves M.F.
      • Laurant P.
      • Paradis P.
      • Schiffrin E.L.
      T regulatory lymphocytes prevent aldosterone-induced vascular injury.
      ). Here, we have described a novel finding that adoptive transfer of Tregs protects against alcohol-induced lipid accumulation and liver steatosis via reversing the expression of genes associated with lipogenesis, β-oxidation, and oxidative stress, and limiting alcoholic macrophage activation. As HMG-CoA and CYP7A1 are limiting enzymes suppressed by AMPK for cholesterol synthesis and degradation into bile acid, respectively, we speculated that other factors such as cholesterol intake, hydrolysis of cholesteryl ester, reverse transport, and other degradation pathways (CYP-27A1 mediates degradation of cholesterol to bile acids in both the classic and acidic pathways) may also be involved. The observation shows that hepatic cholesterol level was not changed before or after Treg replenishment. (supplemental Fig. S3).
      Interestingly, Treg frequency in peripheral blood was increased to the peak, while hepatic Tregs were not increased significantly at 12 h after Treg transfer (supplemental Figs. S1, S2). By 2 weeks post Treg transfer, peripheral Tregs were significantly decreased to a level little higher than normal (supplemental Fig. S1); however, the number of hepatic Tregs was increased close to the normal level (supplemental Fig. S2). These results indicated that transferred Tregs could gradually enter the liver from the periphery.
      Indeed, inflammatory stress is induced by lipid accumulation in multiple organs in mice (
      • Ma K.L.
      • Zhang Y.
      • Liu J.
      • Wu Y.
      • Hu Z.B.
      • Liu L.
      • Liu B.C.
      Inflammatory stress induces lipid accumulation in multi-organs of db/db mice.
      ), suggesting the reciprocal maintenance between immune homeostasis and lipid metabolism. Moreover, it has become increasingly clear that inflammation can lead to the exacerbation of immune and nonimmune diseases. Accumulating evidence in preclinical studies suggests that Tregs can quell inflammation and reduce morbidity in these diseases by contributing to the homeostasis of the local tissue microenvironment (
      • Bluestone J.A.
      • Tang Q.
      Treg cells-the next frontier of cell therapy.
      ). In our study, Treg transfer inhibited the increase of hepatic inflammatory cytokines in alcohol-fed mice significantly, while it did not change their levels in normal mice (supplemental Fig. 4).
      The modulation of monocytes by Tregs is shown to be dependent on soluble factors as well as cell contact (
      • Tiemessen M.M.
      • Jagger A.L.
      • Evans H.G.
      • van Herwijnen M.J.
      • John S.
      • Taams L.S.
      CD4+CD25+Foxp3+ regulatory T cells induce alternative activation of human monocytes/macrophages.
      ); although cell contact of Tregs to T cells confers cytolysis of target cells but not monocytes (
      • Ren X.
      • Ye F.
      • Jiang Z.
      • Chu Y.
      • Xiong S.
      • Wang Y.
      Involvement of cellular death in TRAIL/DR5-dependent suppression induced by CD4(+)CD25(+) regulatory T cells.
      ,
      • Jagger A.L.
      • Evans H.G.
      • Walter G.J.
      • Gullick N.J.
      • Menon B.
      • Ballantine L.E.
      • Gracie A.
      • Magerus-Chatinet A.
      • Tiemessen M.M.
      • Geissmann F.
      • et al.
      FAS/FAS-L dependent killing of activated human monocytes and macrophages by CD4+CD25- responder T cells, but not CD4+CD25+ regulatory T cells.
      ). Mechanisms for suppression of Tregs include release of inhibitory cytokines TGF-β and IL-10, cytolysis of targets via granzymes, metabolic disruption via IL-2 consumption, and ATP degradation via the ectonucleotidases, CD39 and CD73 (
      • André S.
      • Tough D.F.
      • Lacroix-Desmazes S.
      • Kaveri S.V.
      • Bayry J.
      Surveillance of antigen-presenting cells by CD4+ CD25+ regulatory T cells in autoimmunity: immunopathogenesis and therapeutic implications.
      ). It could not be excluded that other mechanisms besides IL-10 conferred suppressive function of Tregs on macrophages in the liver.
      A number of data have revealed the role of inflammatory cytokines in ALD and NAFLD but reports on IL-10 remain controversial. It has been proposed that IL-10 deficiency ameliorates both ALD and NAFLD by elevating of IL-6-STAT3 signaling, while concomitant either IL-6 or STAT3 depletion correct metabolic abnormality (
      • Miller A.M.
      • Wang H.
      • Bertola A.
      • Park O.
      • Horiguchi N.
      • Ki S.H.
      • Yin S.
      • Lafdil F.
      • Gao B.
      Inflammation-associated interleukin-6/signal transducer and activator of transcription 3 activation ameliorates alcoholic and nonalcoholic fatty liver diseases in interleukin-10-deficient mice.
      ). However, data from IL-6-deficient mice show a comparable liver steatosis score despite less sphingomyelin and ceramide along with a methionine- and choline-deficient diet (
      • Mas E.
      • Danjoux M.
      • Garcia V.
      • Carpentier S.
      • Segui B.
      • Levade T.
      IL-6 deficiency attenuates murine diet-induced non-alcoholic steatohepatitis.
      ). Neutralization of IL-6 signaling enhanced steatosis but improved liver damage methionine- and choline-deficient diet-induced NASH (
      • Yamaguchi K.
      • Itoh Y.
      • Yokomizo C.
      • Nishimura T.
      • Niimi T.
      • Fujii H.
      • Okanoue T.
      • Yoshikawa T.
      Blockade of interleukin-6 signaling enhances hepatic steatosis but improves liver injury in methionine choline-deficient diet-fed mice.
      ). Activation of AMPK in response to elevated IL-10 may be achieved directly in hepatocytes because robust phosphorylation of AMPK by IL-10 stimulation has been seen in macrophages (
      • Zhu Y.P.
      • Brown J.R.
      • Sag D.
      • Zhang L.
      • Suttles J.
      Adenosine 5′-monophosphate-activated protein kinase regulates IL-10-mediated anti-inflammatory signaling pathways in macrophages.
      ).
      Macrophages in ALD have been recognized as pivotal to pathogenesis of ALD by gut-derived endotoxins/LPSs, which favor M1 macrophages. Depletion of macrophages prevents chronic ethanol-induced fat accumulation, inflammation, and necrosis (
      • Koop D.R.
      • Klopfenstein B.
      • Iimuro Y.
      • Thurman R.G.
      Gadolinium chloride blocks alcohol-dependent liver toxicity in rats treated chronically with intragastric alcohol despite the induction of CYP2E1.
      ). The ratio of M1 to M2 macrophages seems to be a determinant of alcoholic liver injury (
      • Peng H.
      • Geil Nickell C.R.
      • Chen K.Y.
      • McClain J.A.
      • Nixon K.
      Increased expression of M1 and M2 phenotypic markers in isolated microglia after four-day binge alcohol exposure in male rats.
      ,
      • Wan J.
      • Benkdane M.
      • Teixeira-Clerc F.
      • Bonnafous S.
      • Louvet A.
      • Lafdil F.
      • Pecker F.
      • Tran A.
      • Gual P.
      • Mallat A.
      • et al.
      M2 Kupffer cells promote M1 Kupffer cell apoptosis: a protective mechanism against alcoholic and nonalcoholic fatty liver disease.
      ). Recent findings reveal that M2 macrophages selectively trigger M1 macrophage apoptosis via IL-10 secretion in ALD (
      • Wan J.
      • Benkdane M.
      • Teixeira-Clerc F.
      • Bonnafous S.
      • Louvet A.
      • Lafdil F.
      • Pecker F.
      • Tran A.
      • Gual P.
      • Mallat A.
      • et al.
      M2 Kupffer cells promote M1 Kupffer cell apoptosis: a protective mechanism against alcoholic and nonalcoholic fatty liver disease.
      ). These data suggest a distinct role of IL-10 in pathogenesis of ALD in that the conventional depletion of IL-10 induced hyper-activation of IL-6-STAT3 signal in hepatocytes fostering lipid degradation; however, pathological level IL-10 favors M2 macrophage and thus a low-inflammatory microenvironment.
      In conclusion, our data described chronic alcohol consumption induced hepatic pro-inflammatory macrophage activation and thus an inflammatory microenvironment, increased TG accumulation, reduced lipid oxidation and enhanced ROS generation, and adoptive transfer of Treg restored the immune balance and reversed the alcoholic liver disorders. We provide a potent link between inflammation and steatosis in the liver and provide a therapeutic implication for ALD (Fig. 8).
      Figure thumbnail gr8
      Fig. 8.Proposed mechanism of Treg-mediated control on steatosis in ALD. Treg is an important link between immunity and lipid metabolism in the liver, and highlights the effect of liver immunity on lipid metabolism in ALD. Chronic ethanol consumption induces a decrease of Tregs in the liver. Fewer Tregs were vulnerable to the switch from anti-inflammatory macrophages to pro-inflammatory macrophages upon the costimulation of ethanol and LPS, which in turn, leads to hepatic steatosis. However, adoptive transfer of Tregs reverses aberrant lipid metabolism in the liver and ameliorates pathogenesis of ALD, providing new insights into therapeutic strategies for ALD treatment.

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