Regulatory T cells suppress excessive lipid accumulation in alcoholic liver disease.

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.


Introduction
Alcohol is consumed worldwide and has long been identified as a leading cause of global morbidity and mortality [1].As the primary organ for alcohol metabolism, liver also suffer the principal damage from excessive alcohol consumption [2]. The spectrum of alcohol induced liver disease(ALD)，ranging from steatosis to steatohepatitis (ASH), progressive fibrosis, cirrhosis, and the development of hepatocellular cancer (HCC) [3].
Fatty liver is the earliest and most common pathological feature in ALD. Although fatty liver had been typically considered as a benign and reversible disease, recent evidence has indicated fat accumulation renders the liver more vulnerable to inflammatory mediators and toxic agents, leading to progress to the advanced stage of ALD [4,5].
Some studies suggested that in active drinkers, liver steatosis was associated with more rapid progression of fibrosis [6]. 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 [7]. Therefore, elucidating the mechanism of the pathogenesis by which chronic alcohol consumption leads to excessive triglyceride accumulation and liver damage is important and may provide new insights into prevention and therapy of ALD.
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 [16].
In this study, we investigated the role of Tregs in the development of alcoholic fatty liver in mice. We identified a reversible impact of Tregs on liver lipid metabolism in mice model of alcoholic fatty liver, which relies on IL-10-mediated immunosuppressive effects on proinflammatory macrophages activation in hepatic microenvironment at least in part.

Animal Experiments
Adult male wild-type C57BL/6 mice at 6-8 weeks old were purchased from the Vital River Laboratories (Beijing, China). 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 (vol/vol) 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 dextranmaltose 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 spleen of C57BL/6 mice at 6-8 weeks old with a MACS Treg isolation kit (MiltenyiBiotec, Auburn, CA).The purity of the cell was ＞95%, as assessed by flow cytometry. These mice were divided into three groups randomly and fed with Lieber-DeCarli liquid diet with 5% ethanol (vol/vol).
One group was adoptively transferred with 1.5×10 6 CD4 + CD25 + Treg and another one with CD4 + CD25 -T (Teff) cells at 1-week intervals from forth weeks of alcoholic feeding via the tail vein injection, respectively. 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℃ for future analysis. Livers were snap-frozen in liquid nitrogen for proteins and RNA extraction, or fixed in 10% neutral-buffered formalin for histopathological analysis. Liver samples for cell quantification were prepared as previously described [17]. 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 that was multiplied by 20 to assume the total cell number of different tissues. The absolute number of Treg cells was subsequently determined by the percentage of Tregs and the total cell number.

Cells isolation, quantitation, and Treg staining
Anti-mouse fluorescent antibodies against CD25, or CD4 were purchased from eBioscience (San Diego, CA, USA). 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, San Diego, CA).

Flow cytometric analysis
Stained cells were analyzed by the BD FACSCantoII (BD Bioscience) instrument.
Lymphocytes were firstly gated according to forward scatter and side scatter; then the Tregs (CD4+CD25+Foxp3+) were gated and the percentage of Tregs was determined.
Homogenates were centrifuged with Tris-HCl buffer at 12,000×g for 15 min at 4°C; then 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.

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 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-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 (reduced glutathione) to oxidized glutathione (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 milligrams of protein (U/mg protein). The activity of CAT was expressed as (U/g protein).

Assay of hepatic lipid peroxidation
Hepatic lipid peroxidation was evaluated via the thiobarbituric acid (TBA)-reactive substances method and was expressed as MDA (malonaldehyde) level, which was assayed with a commercial kit according to the manufacturer's instructions. (Nanjing Jiancheng Bioengineering Institute, Nanjing, China). Briefly, after mixing by guest, on April 28, 2019 www.jlr.org Downloaded from trichloroacetic acid with the homogenate and centrifuging, the supernatant was obtained, and TBA 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 nmol/mg protein.

Hepatic cytokine measurement
Liver samples for cytokine quantification were prepared as previously described [18].

Liver Triglyceride Determination
Liver Triglyceride levels were determined as previously described [19]. 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 1250×g for 10 min, the supernatants were harvested and the intrahepatic Triglyceride levels quantified by commercial Triglyceride detecting 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-

Histopathological and immunohistochemistry staining
Sections of formalin-fixed livers were stained with hematoxylin/eosin and analyzed by microscopy. Frozen sections were prepared from liver tissue frozen in OCT media, and stained with Oil-Red. Immunohistochemistry was performed using antibodies against

Dual immunofluorescence staining
Sections were prepared from formalin-fixed, paraffin-embedded liver samples. After

In vitro Experiments
Usage of peripheral blood from healthy volunteers was approved by the Ethics

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 means ± standard error of the mean (SEM), and P-value < 0.05 was considered significant.

Alcohol consumption induced steatosis and decline of Treg in the liver
Previous study showed a decrease of Treg in peripheral blood in AH [16], however, the dynamic involvement of Treg during the development of alcoholic fatty liver remains unclear. Here, we found C57BL/6 mice fed with Leiber-Decali alcohol diet developed steatosis as revealed by aberrant TG accumulation when compared to those with pairfed diet (Fig 1C-D). Interestingly, a significant decrease of hepatic Tregs number was observed in mice after 6 weeks' alcohol feeding, (Fig 1.A- interplay between the Treg and steatosis.

Adoptive transfer of Tregs protect against alcoholic fatty liver
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 alcoholfed mice were introduced hepatic lipid deposition, micro and macro-steatosis (data not shown). However, adoptive transfer of Tregs relieved such phenotype as examined by H-E staining (data not shown) and oil-red staining for hepatic TG level (Fig 2B-C), while adoptive transfer of Treg negative lymphocytes (Teff) did not give rise to such effect. Alcoholic TG accumulation was accompanied with impaired liver injury and function as demonstrated by increased ALT level in alcohol diet mice (Fig.2D), and Treg transfer ameliorated ALT levels (Fig. 2D). Moreover, there is no significant change in the hepatic cholesterol level before and after Treg transfer (supplement Fig.1) Collectively, these data suggest decrease of Tregs accounts for TG accumulation and liver injury in mice with chronic alcoholic consumption.

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. Interesting, we found that SREBP-1c, the key transcriptional factor in fatty acid synthesis, was up-regulated 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 by guest, on April 28, 2019 www.jlr.org Downloaded from also decreased significantly when examined at transcription level after Treg transfer ( Fig 3C). AMPK was a master regulator for energy homeostasis and act as central actor for stimulating lipolysis and inhibition of lipogenesis [22,23]. AMPK phosphorylation inhibited SREBP-1c cleavage and nuclear translocation, a vital step for its transactivation of lipogenic genes [23] In our experiment, chronic alcohol consumption inhibited AMPK activation as determined by dampened phosphorylation, however, Treg replenishment substantially antagonized such effect (Fig.3D). These data suggested Treg decrease induced by alcoholic consumption resulted in lipid biogenesis, which accounted at least in part for lipid accumulation.

Adoptive transfer of Tregs increases fatty acid β-oxidation in alcoholic fatty liver
To further identify whether alcoholic lipid accumulation was also ascribed to impaired lipid consumption, we also explored lipid oxidation in the livers of alcoholic fed mice.
We found PPARα, master regulator of fatty acid oxidation [24], was down-regulated at both mRNA level and protein level (Fig 4A-B). While activation of PPARα included nuclear translocation, which was critical for transactivation of lipolytic targets [25,26], 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 the Acox and Cpt, the direct targets of PPARα forβ-oxidation, were down-regulated upon alcoholic feeding but recovered substantially after Treg transfer (Fig.4D).
However, the expression of long-chain acyl CoA dehydrogenase (LCAD), and medium- Our results showed that the activities of SOD, CAT, and GSH-Px decreased significantly in the liver from alcohol-fed mice, as compared to Pair-fed controls ( Fig 5A). However, such effect was inhibited significantly in the liver of Tregtransferred 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 cytochrome P4502E1 (CYP2E1), a critical enzyme for alcohol-induced reactive oxygen species (ROS) generation in hepatic microsomes [28], were 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.

Adoptive transfer of Tregs decreases hepatic MCP-1 and TNF-α production and macrophage activation in alcoholic fatty liver
It has been accepted that alcohol metabolism generated a number of metabolites such as acetate, reactive oxygen species and acetaldehyde, which could initiate and aggravate inflammation [29,30]. Many inflammatory cytokines and chemokines were involved in the development of alcoholic fatty liver disease [31]. Of these cytokines and chemokines, TNF-α and MCP-1 were described to regulate lipid metabolism, especially suppressed AMPK phosphorylation and thus activation [32,33,34]. Indeed, chronic alcoholic consumption led to up-regulation of CCL2 and MCP-1 expression at both mRNA and protein levels ( Fig. 6A-B). Resident macrophages or Kupffer cells accounted for about 70% of resident immune cells and were considered as the major sources of inflammatory factors [35]. Adipose tissue from obese people contains an increased number of CD68+ macrophages with a pro-inflammatory phenotype [36]. In our experiments, we detected an increase of CD68 expression in the liver (Fig. 6C), suggesting the infiltration macrophages. Furthermore, the population of F4/80+CD68+, F4/80+TNF-α+ and F4/80+MCP-1+ macrophage was increased after alcohol feeding, by guest, on April 28, 2019 www.jlr.org Downloaded from supporting our hypothesis that chronic alcohol consumption underpinned 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.

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/lipopolysaccharide (LPS) is considered a hallmark of ALD [37]. The portal circulation of endotoxin such as LPS and alcohol acts as stimulus to resident macrophages and infiltrated monocytes and thus differentiated macrophage to a proinflammatory phenotype [38].
To verify the hypothesis that adoptive transfer of Tregs inhibited macrophages activation, we analyzed the inhibitory effects of Tregs on human CD14+ macrophages with the co-stimulation of LPS and alcohol for 48h in vitro. Upon LPS and the alcohol co-stimulation, the expression of MCP-1 and TNF-α were increased significantly ( Fig.7A-B). However, induced expression of MCP-1and TNF-α declined considerably in macrophages when co-cultured with Tregs. Inhibitory molecules like IL-10, TGF-β are typical effectors of Tregs, so IL-10 and/or TGF-β might be involved in the Tregmediated suppression of pro-inflammatory cytokine expression in macrophages upon by guest, on April 28, 2019 www.jlr.org Downloaded from co-stimulation with LPS and alcohol. Interestingly, Blockage of IL-10 but not TGF-β reversed such effect as indicated by MCP-1 and TNF-α expression in CD14+ macrophages recovered when neutralizing IL-10 activity (Fig. 7C-D).

Discussion
Alcoholic fatty liver disease caused by chronic alcohol consumption manifests with altered lipid metabolic events including increased lipogenesis and decreased βoxidation and results in aberrant lipid accumulation, has long been proposed [39,40,41].
In addition to alcohol induced lipogenesis [39], ethanol consumption inhibits lipid oxidation directly by suppression of DNA-binding ability and transactivation of PPARα, a master receptor that controls transcription of a serial of genes involved in free fatty acid transport and oxidation, through alcohol metabolite acetaldehyde [40,42]. Alcohol consumption also inhibit PPAR-α indirectly via up-regulation of oxidative stress and adenosine [43,44]. Additionally, transcription of PPAR-α is suppressed in response to TNF-α or LPS stimulation [45,46] Among the transcription factors for lipid metabolism, AMPK, is a serine-threonine kinase considered as key regulator as phosphorylation of

Chronic ethanol consumption increases gut permeability and induces gut-derived LPS
in the liver, in turn activating innate immune cells, including hepatic macrophages and infiltrated monocytes [2]. Sensitization of alcohol in portal circulation activates hepatic macrophages/monocytes by endotoxins/LPS [2], which provoke robust cytokines production and liver inflammation [62]. Increase of CYP2E-1 mediated ROS generation during alcohol metabolism, which can react with and damage intracellular molecules such as lipids, proteins, or DNAs, has also been proposed [63]. Oxidative stress and ROS generation due to alcohol metabolism not only increase lipids accumulation in hepatocytes but also sensitize liver to subsequent insults by cytokines. Antioxidant therapy is not recommended as attempts using antioxidants fails to bring benefits in patient of ALD [64]. All of these cues linking liver damage to alcoholic-induced inflammatory insults imply anti-inflammation against alcoholic liver disease.
Treg is a population of subset of immune-suppressive T cells in circulation and peripheral organs, maintaining immune homeostasis and regulating immune response to various pathogenic antigens [65]. Previous studies demonstrated that adoptive transfer of Tregs ameliorated a variety of immuno-inflammatory diseases such as atherosclerosis [53], cerebral ischemia [66], angiotensin II induced cardiac damage [67], angiotensin II induced hypertension and vascular injury [68], and aldosterone-induced vascular injury [69]. Here, we have described a novel finding that adoptive transfer of hepatic Tregs was not increased significantly at 12 hours after Tregs transfer (Supplement Figure 2). By two weeks post Treg transfer, peripheral Tregs were significantly decreased to a level little higher than the normal (supplement Figure 3), however, the number of hepatic Tregs was increased close to normal level ( Supplement   Fig 2). These results indicated that transferred Tregs could gradually enter the liver from periphery.
Indeed, inflammatory stress is induced by lipid accumulation in multiple organ in mice [70], 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 non-immune 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 [71]. In our study, Treg transfer inhibited the increase of hepatic inflammatory cytokines in alcohol fed mice significantly，while did not change the levels of them in normal mice. (Supplement Figure4).
The modulation of monocytes by Tregs is shown to be dependent on both soluble factors as well as cell-contact [72], though cell contact of Treg to T cells confers cytolysis of target cells but not monocytes [73.74]. Mechanism for suppression of Treg includes release of inhibitory cytokines TGF-β and IL-10, cytolysis of targets via granzymes, metabolic disruption via Il-2 consumption or ATP degradation via ectonucleotidases CD39 and CD73 [75]. It couldn't be excluded that other mechanism besides IL-10 conferred suppressive function of Treg on macrophage in the liver. A number of data inflammatory have revealed cytokines in ALD and NAFLD but remains controversial. It has been proposed that IL-10 deficiency ameliorates both ALD and NAFLD by elevating of IL6-STAT3 signaling as demonstrated concomitant either IL6 or STAT3 depletion correct metabolic abnormality [76]. However, data from IL-6 deficiency mice shows a comparable liver steatosis score despite of less sphingomyelin and ceramide in with a methionine and choline-deficient diet (MCD) [77].
Neutralization of with IL-6 signaling enhanced steatosis but improved liver damage MCD-induced NASH [78]. 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 macrophage [79].
Macrophage in ALD has been recognized as pivotal to pathogenesis of ALD by gutderived endotoxins/LPS which favors M1 macrophage. Depletion of macrophage prevents chronic ethanol-induced fat accumulation, inflammation, and necrosis [80].
The ratio of M1/M2 macrophage seems to be determinant to alcoholic liver injury [81,82]. Recent finding reveals M2 macrophage selectively triggers M1 macrophage apoptosis via IL-10 secretion in ALD [82]. These data suggest distinct role of IL-10 in pathogenesis of ALD in that whole mount depletion of IL-10 induced hyper-activation of IL6-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 proinflammatory macrophage activation and thus an inflammatory microenvironment, increased TG accumulation, reduced lipid oxidation and enhanced ROS generation, and by guest, on April 28, 2019 www.jlr.org Downloaded from 24 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.

Disclosure
The authors have no conflict of interest.

Financial Support
This study was supported by National Natural Science Foundation of China NSFC