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. Author manuscript; available in PMC: 2021 Mar 1.
Published in final edited form as: J Endocrinol. 2019 Dec;243(3):199–209. doi: 10.1530/JOE-19-0198

Mice Lacking Adenosine 2A Receptor Reveal Increased Severity of MCD-induced NASH

Jing Zhou 1,*, Honggui Li 1,*, Yuli Cai 1,2, Linqiang Ma 1,3,4, Destiny Matthews 1, Bangchao Lu 1,5, Bilian Zhu 1,6, Yanming Chen 6, Xiaoxian Qian 7, Xiaoqiu Xiao 4, Qifu Li 3, Shaodong Guo 1, Yuqing Huo 8,†, Liang Zhao 1,9, Yanan Tian 10, Qingsheng Li 11, Chaodong Wu 1,†
PMCID: PMC7050433  NIHMSID: NIHMS1539527  PMID: 31505462

Abstract

Adenosine 2A receptor (A2AR) exerts a protective role in obesity-related non-alcoholic fatty liver disease. Here, we examined whether A2AR protects against non-alcoholic steatohepatitis (NASH). In C57BL/6J mice, feeding a methionine- and choline-deficient diet (MCD) resulted in significant weight loss, overt hepatic steatosis, and massive aggregation of macrophages in the liver compared with mice fed a chow diet. MCD feeding also significantly increased the numbers of A2AR-positive macrophages/Kupffer cells in liver sections although decreasing A2AR amount in liver lysates compared with chow diet feeding. Next, MCD-induced NASH phenotype was examined in A2AR-disrupted mice and control mice. Upon MCD feeding, A2AR-disruptd mice and control mice displayed comparable decreases in body weight and fat mass. However, MCD-fed A2AR-disrupted mice revealed greater liver weight and increased severity of hepatic steatosis compared with MCD-fed control mice. Moreover, A2AR-disupted mice displayed increased severity of MCD-induced liver inflammation, indicated by massive aggregation of macrophages and increased phosphorylation states of Jun-N terminal kinase (JNK) p46 and nuclear factor kappa B (NFκB) p65 and mRNA levels of tumor necrosis factor alpha, interleukin-1 beta, and interleukin-6. In vitro, incubation with MCD-mimicking media increased lipopolysaccharide (LPS)-induced phosphorylation states of JNK p46 and/or NFκB p65 and cytokine mRNAs in control macrophages and RAW264.7 cells, but not primary hepatocytes. Additionally, MCD-mimicking media significantly increased lipopolysaccharide-induced phosphorylation states of p38 and NFκB p65 in A2AR-deficient macrophages, but insignificantly decreased lipopolysaccharide-induced phosphorylation states of JNK p46 and NFκB p65 in A2AR-deficient hepatocytes. Collectively, these results suggest that A2AR disruption exacerbates MCD-induced NASH, which is attributable to, in large part, increased inflammatory responses in macrophages.

Keywords: Adenosine 2A receptor, non-alcoholic steatohepatitis, lipodystrophy, macrophage

INTRODUCTION

Non-alcoholic fatty liver disease (NAFLD) is characterized by excessive fat deposition in hepatocytes (steatosis) (Browning, et al. 2004; Sanyal 2005). When the liver displays overt inflammatory damage due to fat deposition and inflammatory mediators from extrahepatic tissues, simple steatosis progresses to non-alcoholic steatohepatitis (NASH) as the advanced form of NAFLD (Chalasani, et al. 2018; Cohen, et al. 2011). Epidemiological data indicate that NASH affects 1.5 to 6.45 percent of the general populations (Estes, et al. 2018; Younossi, et al. 2016). Alarmingly, the incidence of NASH in both adults and children is rising continuously due to ongoing epidemics of obesity (Estes et al. 2018; Younossi et al. 2016). NASH is one of the most common causes of liver cirrhosis and hepatocellular carcinoma (Bugianesi, et al. 2002; Marrero, et al. 2002; Powell, et al. 2005; Starley, et al. 2010). To date, there is no effective treatment for NASH (Chalasani et al. 2018; Neuschwander-Tetri 2010).

Because NAFLD is highly prevalent in obese populations (Estes et al. 2018; Younossi et al. 2016), obesity-associated inflammation is accepted as a critical factor that initiates or exacerbates NAFLD. As supported by evidence from both human and animal studies, inflammation can impair hepatic insulin resistance and dysregulate hepatic fat metabolism, which in turn brings about pathological increases in hepatocyte fat deposition (Kang, et al. 2008; Menghini, et al. 2009; Odegaard, et al. 2008). In addition, obesity-associated adipose tissue dysfunction has also been implicated to play a critical role in development of NAFLD. Indeed, this role of dysfunctional adipose tissue is highlighted by the “second hit” hypothesis. In support of this, adipocyte-specific overexpression of monocyte chemoattractant protein-1 (MCP1), an inflammatory molecule up-regulated in adipose tissue of obese mice and human subjects, mediates the effect of adipose tissue inflammation to bring about an increase in hepatic triglyceride content (Kamei, et al. 2006). These results and many others suggest that dysfunctional adipose tissue contributes to hepatic steatosis by increasing the delivery of fatty acid flux to the liver (Tilg and Moschen 2010) and by impairing liver insulin signaling through adipose tissue-driven inflammation (Kelley, et al. 2003; Schaffler, et al. 2005). During obesity, however, inflammation exists in both the liver and adipose tissue (Guo, et al. 2016; Xu, et al. 2014) and complicates with fat deposition, e.g., adiposity and hepatic steatosis. This makes it difficult to separate the effects of inflammation from those of fat deposition. Furthermore, increased adiposity, when displaying fat composition characterized by an elevation of palmitoleate, can promote hepatic steatosis, but decrease liver inflammation (Huo, et al. 2012). Given this, there is a critical need to better understand the role of inflammation in regulating NAFLD/NASH in the absence of obesity.

Adenosine 2A receptor (A2AR) is one of the four adenosine receptors that belong to the superfamily of G-protein-coupled receptors and displays powerful anti-inflammatory effects in immune cells such as macrophages and neutrophils (Gessi, et al. 2000; Haskó, et al. 2008). Previous studies have validated a critical role for A2AR in the pathophysiology of NAFLD/NASH. Specifically, A2AR activation is shown to ameliorate NASH phenotype in both rats and mice (Alchera, et al. 2017; Imarisio, et al. 2012). In contrast, using A2AR-disrupted mice, Cai et al. provide complementary evidence to support a protective role for A2AR in NAFLD (Cai, et al. 2018); although the study by Csoka et al. suggests a contradictory role for A2AR in obesity and NAFLD (Csóka, et al. 2017). Of importance, the protective role for A2AR is largely attributed to the effect of A2AR on suppressing inflammation derived from lipotoxicity (Alchera et al. 2017; Imarisio et al. 2012). However, it is not clear in the liver how the A2AR is altered by inflammation in the absence of obesity as it relates to development and progression of NAFLD/NASH. In the present study, we examined the expression of A2AR in livers of mice fed a methionine- and choline-deficient diet (MCD). We also examined the effect of A2AR disruption on MCD-induced NASH phenotype, and examined the effects of MCD-mimicking media on the proinflammatory responses in both hepatocytes and macrophages.

RESEARCH DESIGN AND METHODS

Animal experiments

A2AR-disrupted (A2AR−/− or A2AR+/−) mice, in which A2AR was disrupted in all cells, and their wild-type (WT) littermates (A2AR+/+ mice) were generated as previously described (Cai et al. 2018). Additional C57BL/6J mice were obtained from the Jackson Laboratory (Bar Harbor, ME). All mice were maintained on a 12:12-h light-dark cycle (lights on at 06:00). Study 1: male C57BL/6J mice, at 9 – 10 weeks of age (when body weight was greater than 20 g), were fed an MCD for 5 weeks or maintained on a chow diet (CD) to examine A2AR abundance in relation to diet-induced NASH. Study 2: both male and female A2AR-disrupted mice and A2AR+/+ mice, at 9 – 10 weeks of age, were fed an MCD for 5 weeks to induce NASH (Luo, et al. 2018; Rinella, et al. 2008). All diets are products of Research Diets, Inc (New Brunswick, NJ). At one day prior to harvest, all mice were subjected to EchoMRI™ analyzer (EchoMRI LLC, Houston, TX 77079, USA) to measure body composition. After the feeding period, all mice were fasted for 4 hr before sacrifice for collection of blood and tissue samples as previously described (Cai et al. 2018; Guo, et al. 2010; Huo, et al. 2010). The levels of plasma alanine aminotransferase (ATL) measured using an assay kit (BioVision, Inc. Milpitas, CA). These protocols were approved by the Institutional Animal Care and Use Committee of Texas A&M University.

Histological and immunohistochemical analyses

The sections of paraffin-embedded liver blocks were stained with H&E and/or stained for F4/80 expression with rabbit anti-F4/80 antibodies (1:100) (AbD Serotec, Raleigh, NC). Also, co-staining of F4/80 (rat anti-mouse, MCA497, Bio-Rad, Hercules, CA) and A2AR (7F6-G5-A2, Cat# sc-32261, Santa Cruz Biotechnology, Inc., Dallas, TX, USA) in liver sections was evaluated by double immunofluorescent labeling according to the manufacturer’s instructions (Vector Laboratories, Inc. Burlingame, CA) as previously described (Pei, et al. 2018). Following staining, images were obtained using Leica TCS SPE Confocal Microscope System (Buffalo Grove, IL). The sections of frozen livers were stained with Oil-Red-O as previously described (Guo et al. 2016).

Cell culture and treatment

Primary mouse hepatocytes were isolated from male A2AR−/− mice and A2AR+/+ mice, at 11 – 12 weeks of age, using a collagenase digestion method as previously described (Cai et al. 2018; Luo et al. 2018). After attachment, hepatocytes were further incubated in M199 supplemented with 10% fetal bovine serum (FBS) and 100 U/mL penicillin and 100 μg/mL streptomycin for 24 hr. Thereafter, hepatocytes were incubated in MCD-mimicking media (US Biological Life Sciences, Salem, MA 01970, USA) for an additional 24 hr. Prior to harvest, hepatocytes were treated with or without lipopolysaccharide (LPS, 100 ng/mL) for 30 min. Cell lysates were prepared for Western blot analysis. Also, bone marrow cells were isolated from male A2AR−/− mice and A2AR+/+ mice, at 11 – 12 weeks of age, and differentiated into macrophages (BMDM) as previously described (Xu et al. 2014). After differentiation, BMDM were incubated with MCD-mimicking media for 24 hr and treated with or without LPS (100 ng/mL) for the last 30 min. Cell lysates were measured for proinflammatory signaling using Western blot analysis. For a confirmatory study, RAW264.7 cells were treated with MCD-mimicking media and/or control media, and assayed for proinflammatory signaling and cytokine expression same as BMDM.

Western blot analysis

Frozen liver tissues and cultured cells were prepared in a lysis buffer containing 50 mM HEPES (pH 7.4), 10 mM EDTA, 50 mM sodium pyrophosphate, 0.1 M sodium fluoride, 10 mM sodium orthovanadate, 2 mM phenylmethylsulfonyl fluoride, 10 μg/ml aprotinin, 10 μg/ml leupeptin, 2 mM benzamidine, and 1% Triton X-100. After protein electrophoresis and transfer, immunoblots were performed using rabbit anti-serum as primary antibody at a 1:1,000 dilution. This dilution was used for each of the primary antibodies used for the present study. After washing, the blot was incubated with a 1:10,000 dilution of goat anti-rabbit horseradish peroxidase-conjugated secondary antibody and followed by a chemiluminescent kit (Immobilon™ Western; EMD Millipore, Billerica, MA, USA) as described (Qi, et al. 2017). GAPDH was used as a loading control. The maximum intensity of each band was quantified using ImageJ software. Protein amount of αSMA and/or A2AR was normalized to GAPDH and adjusted relative to the average of CD-fed mice. Similarly, ratios of Pp46/p46 and/or Pp65/65 were normalized to GAPDH and adjusted relative to the average of CD-fed WT mice, MCD-fed A2AR+/+ mice, or control medium-treated primary hepatocytes or BMDM from A2AR+/+ mice, which was arbitrarily set as 1 (AU). Antibodies against Pp46, p46, Pp65, p65, αSMA, and A2AR were products of Cell Signaling (Danvers, MA, USA).

RNA isolation, reverse transcription, and real-time PCR

Total RNA was isolated from liver tissues. Reverse transcription was performed using the GoScript™ Reverse Transcription System (Promega) and real-time PCR analysis was performed using SYBR Green (LightCycler® 480 system; Roche) (Guo, et al. 2013; Guo, et al. 2012). The mRNA levels were analyzed for tumor necrosis factor alpha (Tnfa), interleukin 1 beta (Ilb), Il6, fatty acid synthase (Fas), carnitine palmitoyltransferase 1a (Cpt1a), and sterol regulatory element-binding protein 1c (Srebp1c). A total of 0.1 μg RNA was used for the determination. Results were normalized to 18s ribosomal RNA and plotted as relative expression to the average of MCD-fed A2AR+/+ mice, which was set as 1. Primer sequences are available upon request.

Statistical Methods

Numeric data are presented as means ± SEM (standard error). Statistical significance was assessed by unpaired, two-way ANOVA (for comparisons including three or more groups) and/or two-tailed Student’s t tests (for variables only involving two groups). Differences were considered significant at the two-tailed P < 0.05. Tukey’s range test was used for post-hoc test.

RESULTS

MCD feeding induces lipoatrophy and severe NASH in mice

Feeding a high-fat diet (HFD) to C57BL/6J mice induces hepatic steatosis and inflammation that are associated with obesity and increased adiposity (Cai et al. 2018; Luo et al. 2018). In this type of mouse model, dysfunctional adipose tissue is thought to also contribute to NAFLD phenotype. Considering the existence of steatohepatitis in human subjects without obesity, we sought to feed male C57BL/6J mice an MCD and examined hepatic steatosis and inflammation in the presence of lipoatrophy. Compared with CD-fed male mice, MCD-fed male C57BL/6J mice revealed significant decreases in food intake and body weight (Figure 1A,B). Additionally, MCD-fed mice displayed nearly no fat mass (Figure 1C). When NASH phenotype was analyzed, the levels of plasma alanine aminotransferase (ALT) in MCD-fed mice were significantly increased compared with those in CD-fed mice (Figure 1D). In contrast, MICD-fed mice revealed significantly decreased liver weight relative to CD-fed mice (Figure 1E). Consistent with NASH phenotype, liver sections of MCD-fed mice displayed overt hepatic steatosis and revealed mass aggregation of F4/80+ macrophages (Figure 1F). In addition, liver lysates of MCD-fed mice showed significant increases in the phosphorylation states of JNK1 p46 and the amount of αSMA, a marker of liver fibrosis (Figure 1G). These results validate MCD-fed mice as a model of NASH, which reveals hepatic steatosis and severe inflammation in the presence of lipoatrophy.

Figure 1. MCD feeding induces severe hepatic steatosis and inflammation while causing lipoatrophy.

Figure 1.

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Male C57BL/6J mice, at 9 – 10 weeks of age, were fed a methionine- and choline-deficient (MCD) for 5 weeks or maintained on a chow diet (CD). (A, B) Body weight was measured during the feeding period (A). Also, foods consumed by the mice were recorded during the feeding period and used to calculate food intake (B). (C) At one day prior to the end of feeding period, body composition of the mice was analyzed using an EchoMRI analyzer. (D) Plasma levels of alanine aminotransferase (ALT). (E) Liver weight was measured after harvest of mice. (F) Liver sections were stained with H&E or for F4/80 expression. (G) Liver lysates were examined for the phosphorylation states of JNK p46 and the amount of αSMA. Bar graphs, quantification of blots. For A - E and G, numeric data are means ± SEM (standard error), n = 8 – 10. AU, arbitrary unit. *, P < 0.05 and **, P < 0.01 MCD vs. CD (in D, E, and G) for the same time point (in A and B) or for the same type of mass (in C).

MCD feeding induces NASH and alters hepatic A2AR expression in mice

We showed before that hepatic A2AR expression was increased in mice with obesity-associated NAFLD, and validated the increase in A2AR expression as a defensive response (Cai et al. 2018). Here we examined hepatic A2AR expression in MCD-fed mice. Unlike the changes in HFD-fed mice, the amount of A2AR in liver lysates of MCD-fed C57BL/6 mice was decreased compared with that in CD-fed mice (Figure 2A). Next, we used immunofluorescent staining to examine the expression pattern of A2AR in liver macrophages/Kupffer cels; given that liver sections of MCD-fed C57BL/6J mice displayed marked aggregation of F4/80+ cells. Compared with those of CD-fed mice, liver sections of MCD-fed mice contained much more A2AR-postive cells (Figure 2B), most of which were F4/80+ cells (macrophages/Kupffer cells). Together, these results suggest that during MCD-induced NASH the liver revealed increased A2AR expression in macrophages although A2AR expression was decreased in liver lysates.

Figure 2. MCD feeding increases A2AR expression in liver macrophages/Kupffer cells although decreasing liver A2AR abundance.

Figure 2.

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Male C57BL/6J mic, at 9 – 10 weeks of age, were fed an MCD for 5 weeks or maintained on a CD. (A) Liver lysates were examined for A2AR amount using Western blot analysis. Bar graph, quantification of blots. Data are means ± SEM. n = 6 – 10. AU, arbitrary unit. **, P < 0.01 MCD vs. CD. (B) Liver sections were examined for the expression of F4/80 and/or A2AR using immunofluorescent staining. The scale bar is 75 μm for 10× images.

Validation of A2AR disruption in livers of MCD-fed mice

MCD feeding altered liver A2AR expression. Next, we sought to use A2AR-disrupted mice to examine whether A2AR exerts a protective role in the pathogenesis of MCD-induced NASH. Initially, we analyzed A2AR expression in liver sections of MCD-fed male A2AR−/− mice and their wild-type (WT, A2AR+/+) littermates. Upon immunofluorescent staining, liver sections of MCD-fed male A2AR+/+ mice displayed significant amount of A2AR-postive cells, many of which were F4/80+ macrophages/Kupffer cells. In contrast, liver sections of MCD-fed male A2AR−/− mice contained almost no A2AR-postive cells, but revealed significant increases in the numbers of F4/80-positive cells compared with those of MCD-fed male A2AR+/+ mice (Figure 3). These results validated A2AR deficiency and confirmed that MCD feeding induced mass macrophage aggregation.

Figure 3. Validation of liver A2AR disruption.

Figure 3.

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Male A2AR−/− mice and A2AR+/+ mice, at 9 – 10 weeks of age, were fed an MCD for 5 weeks or maintained on a CD. After sacrifice of mice, liver sections were prepared and subjected to immunofluorescent staining of A2AR and/or F4/80 expression. The scale bar is 75 μm for 10× images.

A2AR disruption does not alter the effect of MCD feeding on decreasing body weight and abdominal fat mass

A2AR disruption exacerbates HFD-induced weight gain (Cai et al. 2018). However, it is not clear whether A2AR disruption influences MCD-induced weight loss. We measured body weight and analyzed body composition of MCD-fed male and female A2AR-disrupted mice and their WT littermates. During and after MCD feeding period, all mice revealed significant decreases in body weight regardless of A2AR disruption and sex of the mice. Also, all mice started to consume fewer foods after MCD feeding for 2 weeks. However, these changes in A2AR-disrupted mice were comparable with those in WT littermates (Figure 4A,B, data from male mice only). In addition, abdominal fat mass and adiposity in A2AR-disrupted mice did not differ significantly from those in WT littermates (Figure 4C,D). Specifically, epididymal fat mass was 0.079 g ± 0.009 for MCD-A2AR−/− mice, 0.078 g ± 0.0064 for MCD-A2AR+/− mice, and 0.061 g ± 0.006 for MCD-A2AR+/+ mice (data from male mice only). These results suggest that A2AR disruption did not significantly alter MCD-induced decreases in body weight and abdominal fat mass.

Figure 4. A2AR disruption does not alter MCD-induced decreases in body weight and adiposity.

Figure 4.

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Male A2AR−/− mice, A2AR+/− mice, and A2AR+/+ mice, at 9 – 10 weeks of age, were fed an MCD for 5 weeks or maintained on a CD. (A, B) During the feeding period, body weight of the mice was measured (A). Also, foods consumed by the mice were recorded during the feeding period and used to calculate food intake (B). (C) After harvest, the mass of epididymal, mesenteric, and perinephric fats was estimated as abdominal fats. (D) Adiposity was calculated by normalizing abdominal fats with body weight. For A - D, data are means ± SEM. n = 8 – 10.

A2AR disruption exacerbates MCD-induced hepatic steatosis and inflammation

MCD feeding induces NASH while decreasing liver weight. In the present study, the levels of plasma ALT in MCD-fed male A2AR−/− mice were significantly higher than those in MCD-fed male A2AR+/+ mice (Fig. 5A). Also, liver weight of A2AR-disrupted male mice was greater than that in WT male mice (Figure 5B); although all mice revealed significant decreases in liver weight relative to CD-fed mice. Compared with male A2AR+/+ mice, male A2AR-disrupted mice also revealed a significant increase in the severity of MCD-induced hepatic steatosis, indicated by the results from liver sections stained with H&E and/or Oil-Red-O (Figure 5C). Furthermore, the severity of MCD-induced hepatic steatosis in male A2AR−/− mice was greater than in male A2AR+/− mice. Male A2AR-disrupted mice also displayed a significant increase in accumulation of liver macrophages/Kupffer cells. This increase in male A2AR−/− mice was much greater than in male A2AR+/− mice. Of note, macrophages/Kupffer cells were aggregated in large groups in livers of MCD-fed male A2AR−/− mice. When inflammatory signaling was analyzed, the phosphorylation states of JNK p46 and NFκB p65 were increased in MCD-fed male A2AR−/− or A2AR+/− mice compared with MCD-fed male A2AR+/+ mice (Figure 5D). Additionally, MCD-fed male A2AR−/− or A2AR+/− mice displayed significant increases in Tnfa, Il1b, and Il6 mRNAs (Figure 5E). Next, we analyzed the expression of lipogenic genes/enzymes and observed that hepatic Acc1, Fas, and Srebp1c mRNAs in MCD-fed male A2AR−/− mice were significantly higher than their respective levels in MCD-fed male A2AR+/+ mice whereas Cpt1a mRNAs in MCD-fed male A2AR−/− or A2AR+/− mice did not differ from those in MCD-fed male A2AR+/+ mice (Figure 5E). These results suggest that A2AR disruption exacerbates NASH phenotype in MCD-fed male mice. In female mice, we observed similar phenotype upon MCD feeding (data not shown).

Figure 5. A2AR disruption exacerbates MCD-induced hepatic steatosis and inflammation.

Figure 5.

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Male A2AR−/− mice, A2AR+/− mice, and A2AR+/+ mice, at 9 – 10 weeks of age, were fed an MCD for 5 weeks or maintained on a CD. (A) Plasma levels of ALT. (B) Liver weight. (C) Liver sections were stained with H&E or Oil Red O or for F4/80. (D) Liver lysates were examined for the phosphorylation states of JNK p46 and NFκB p65. Bar graphs, quantification of blots. (E) Liver mRNA levels were examined using real-time PCR. For A, B, D, and E, numeric data are means ± SEM. n = 8 – 10 (A and B) or 6 – 8 (D and E). AU, arbitrary unit. *, P < 0.05 and **, P < 0.01 A2AR−/− vs. A2AR+/+ (in A and B, and in bar graphs of D) for the same gene (in E); †, P < 0.05 A2AR−/− vs. A2AR+/− (in D, bar graphs) for the same gene (in E); ‡, P < 0.05 and ‡‡, P < 0.01 A2AR+/− vs. A2AR+/+ (in B and bar graphs of D) for the same gene (in D).

A2AR-disrupted macrophages, but not hepatocytes, reveal increased proinflammatory responses upon incubation with MCD-mimicking media

A2AR deficiency exacerbated MCD-induced liver inflammation. Considering that A2AR-deficient mice lacked A2AR in both hepatocytes and macrophages, we sought to determine the direct effects of MCD-mimicking media on the proinflammatory responses of A2AR-deficient hepatocytes and/or macrophages. Upon incubation with MCD-mimicking media, primary hepatocytes from either A2AR−/− mice or A2AR+/+mice did not reveal significant changes in LPS-induced phosphorylation states of JNK1 p46 and NFκB p65 compared with hepatocytes incubated with control media (Figure 6A), suggesting that MCD-mimicking media have limited effects on altering hepatocyte proinflammatory responses regardless of the presence or absence of A2AR. In contrast, incubation with MCD-mimicking media caused a significant increase in LPS-induced phosphorylation states of NFκB p65 in A2AR+/+-BMDM (Figure 6B) relative to control media. Moreover, upon incubation with MCD-mimicking media, A2AR−/−-BMDM revealed significant increases in LPS-induced phosphorylation states of p38 and NFκB p65 compared with A2AR+/+-BMDM (Figure 6B). Since MCD increased NFκB p65 phosphorylation in WT BMDM but not A2AR−/− BMDM, we next measured cytokine expression in WT BMDM incubated with MCD-mimicking media or control media. Compared with control, MCD-mimicking media significantly increased Tnfa, Il1b, and Il6 mRNAs (Figure 6C). In a confirmatory study involving RAW264.7 cells, incubation with MCD-mimicking media caused significant increases in the phosphorylation states of JNK p46 and/or NFκB p65 relative to control media under both basal and LPS-stimulated conditions (Figure 6D). Additionally, Tnfa, Il1b, and Il6 mRNAs in MCD-mimicking media-treated RAW264.7 cells were significantly higher than their respective levels in control media-treated RAW264.7 cells (Figure 6E). These results suggest that MCD-mimicking media enhance the proinflammatory responses in macrophages, but not hepatocytes.

Figure 6. A2AR deficiency enhances the effect of MCD-mimicking media on stimulating proinflammatory responses in macrophages, but not hepatocytes.

Figure 6.

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(A,B,C) Proinflammatory signaling (A and B) and cytokine expression (C) in primary hepatocytes (A) and bone marrow-derived macrophages (BMDM) (B and C). For A - C, male A2AR−/− mice and A2AR+/+ mice, at 10 – 12 weeks of age, were subjected to isolation of primary hepatocytes and bone marrow cells. Primary hepatocytes and BMDM were treated with MCD-mimicking media for 24 hr in the presence or absence of LPS (100 ng/mL) for the last 30 min (A and B) or LPS (20 ng/mL) for the last 6 hr. (D,E) Proinflammatory signaling (D) and cytokine expression (E) in RAW264.7 cells. At 80% confluence, RAW264.7 cells were incubated with MCD-mimicking media or control media for 24 hr in the presence or absence of LPS (100 ng/mL) for the last 30 min (D) or LPS (20 ng/mL) for the last 6 hr. For A, B, and D, cell lysates were examined for proinflammatory signaling using Western blot analysis. Bar graphs, quantification of blots. For all bar graphs, data are means ± SE, n = 4 – 6. AU, arbitrary unit. *, P < 0.05 A2AR−/− vs. A2AR+/+ under the same condition (Ctrl or MCD, in A and B); †, P < 0.05 and ††, P < 0.01 MCD vs. Ctrl within the same genotype (in B) or for the same gene (in C and E) or under the same condition (PBS or LPS, in D); ‡, P < 0.05 and ‡‡, P < 0.01 LPS vs. PBS within the same media (in D).

DISCUSSION

The prevalence of NAFLD is markedly increased among obese subjects due to, in large part, adiposity-associated overflow of fats and adipokines/cytokines to the liver to trigger or exacerbate hepatic steatosis and inflammation (Estes et al. 2018; Nugent and Younossi 2007; van der Poorten, et al. 2008). Interestingly, there also is good amount of evidence suggesting the presence of NAFLD/NASH in lean subjects (Feldman, et al. 2017; Kim, et al. 2019), in which genetic factors, but not obesity-related factors, are thought to contribute to the pathogenesis of NAFLD/NASH. Similarly, the prevalence of NAFLD/NASH or even liver fibrosis is also increased among HIV-infected patients who have normal BMI or lipodystrophy (Pérez-Matute, et al. 2013; van Welzen, et al. 2019). In this case, HIV infection-related inflammation is considered as a key factor to drive the pathogenesis of NAFLD/NASH or liver fibrosis; although the precise underlying mechanisms remain to be elucidated. To date, there is lack of a perfect animal model for studying NAFLD pathophysiology in the absence of obesity or in the presence of lipodystrophy. Because feeding an MCD to C57BL/6 mice causes severe hepatic steatosis and inflammation while causing lipoatrophy, we analyzed changes in hepatic expression of A2AR in MCD-fed mice and examined the effect of A2AR disruption on MCD-induced NASH phenotype.

Similar to HFD-fed C57BL/6J mice (Cai et al. 2018), MCD-fed C57BL/6J mice displayed hepatic steatosis relative to chow diet-fed mice. However, the severity of liver inflammation in MCD-fed C57BL/6J mice was much greater than that in HFD-fed C57BL/6J mice, which is consistent with our previous report (Luo et al. 2018). In particular, liver sections of MCD-fed C57BL/6J mice contained significantly more F4/80-positive cells, many of which were aggregated. In addition, MCD-fed C57BL/6J mice contained almost no fat mass, in particular visceral fat mass. These characteristics not only validated MCD-fed C57BL/6J mice as a model for studying NASH in the presence of lipoatrophy, but also indicated that MCD-fed mice developed liver inflammation via mechanism(s) different from those for HFD-fed mice. As supporting evidence, hepatic expression of A2AR, an ant-inflammatory molecule, was differentially regulated in MCD-fed mice compared with HFD-fed mice. Specifically, A2AR abundance in liver lysates, unlike that in liver lysates of HFD-C57BL/6J fed mice (Cai et al. 2018), was decreased in MCD-fed C57BL/6J mice. Upon examining A2AR expression using immunofluorescence staining, we showed that liver sections of MCD-fed mice, indeed, contained significant more A2AR-postive cells than those of CD-fed mice. Because most A2AR-postive cells were F4/80-positive cells, we argued that MCD feeding increased A2AR expression in macrophages although it decreased whole liver A2AR abundance. The latter may be attributable to decreased A2AR expression in hepatocytes, which is opposite to that in HFD-fed mice where A2AR expression was increased in hepatocytes (Cai et al. 2018). Considering that A2AR exerts power anti-inflammatory effects, we speculated that the A2AR in macrophages could play a more important role than the A2AR in hepatocyte in terms of regulating liver inflammation in MCD-fed mice. This concept was substantiated by the differences in the responses of hepatocytes versus macrophages to culture media mimicking MCD (see below).

Activation of A2AR by an agonist has been shown to ameliorate MCD-induced NASH phenotype in both rats and mice (Alchera et al. 2017; Imarisio et al. 2012). Considering this, we speculated that the decrease in A2AR amount in liver lysates of MCD-fed mice was indicative of reduced protection of NASH phenotype; although A2AR expression was increased in macrophages/Kupffer cells of liver sections from MCD-fed mice. In agreement with our speculation, A2AR-deficient mice revealed significant increases in the severity of MCD-induced hepatic steatosis and inflammation compared with WT control mice. Of note, the severity of hepatic steatosis in MCD-fed homozygous A2AR-deficient mice, indicated by the results of liver histology, was significantly greater than that in MCD-fed heterozygous A2AR-disrupted mice. Moreover, the degrees of liver inflammation in MCD-fed homozygous A2AR-deficient mice, indicated by the numbers of F4/80-postivie macrophages/Kupffer cells, the phosphorylation states of NF-κB p65, and the mRNA levels of Il1b and Il6, in livers from MCD-fed homozygous A2AR-deficient mice were also significantly higher than their respective levels in MCD-fed heterozygous A2AR-disrupted mice. These findings, along with the results indicative of increased severity of NASH phenotype in heterozygous A2AR-dirupted mice relative to that in WT mice, strongly suggest that A2AR exerts a gene-dose-dependent effect on protecting against MCD-induced NASH phenotype. What should be noted is that A2AR exerts a similarly gene-dose-dependent effect on protecting against NAFLD phenotype in obese mice (Cai et al. 2018), where inflammation, but not adiposity, is the characteristic shared by MCD-fed mice. Therefore, the anti-inflammatory effect of A2AR likely accounts for, to a large extent, protection of NASH in MCD-fed mice.

In MCD-fed mice, A2AR expression was increased in liver macrophages/Kupffer cells. This increase appeared to a defensive response, and was different from the A2AR in non-macrophages/Kupffer cells where decreased A2AR expression might be a consequence or adaptive response to MCD feeding. In other words, it is possible that upon MCD feeding macrophage A2AR expression was increased to counter against massive inflammation whereas the A2AR in other cells, mainly, hepatocytes, was decreased and contributed to increases in hepatocyte fat deposition and proinflammatory responses. At this point, the mechanisms by which MCD feeding differentially regulates A2AR expression in macrophages/Kupffer cells and other cells, e.g., hepatocytes, are not clear. However, MCD feeding appears to primarily account for decreasing hepatocyte A2AR expression; considering that HFD feeding increases hepatocyte A2AR expression (Cai et al. 2018). To be noted, methionine and choline are key nutrients involved in methionine cycle and methylation reactions. Given this, alterations of hepatocyte methylation reactions in response to deficiency of methionine and choline in diet likely are MCD-induced upstream events of hepatocyte A2AR expression. This view, however, is in need of validation by future study. Nonetheless, the differential expression of A2AR in different liver cells led us to examine the effect of MCD-mimicking media on the direct responses of macrophages versus hepatocytes. Indeed, we validated that MCD-mimicking media generated different effects on proinflammatory responses in the two types of cells. Notably, MCD-mimicking media increased LPS-induced phosphorylation states of NFκB p65 in WT macrophages and insignificant increases in the phosphorylation states of p38 and NFκB p65 in A2AR-deficeint macrophages. In contrast, MCD-mimicking media did not significantly alter or even tended to decrease LPS-induced phosphorylation states of JNK p46 and/or NFκB p65 in both WT and A2AR-deficient hepatocytes. Because of this, we speculated that the A2AR in macrophages directly responded to MCD feeding in a defensive manner to exert an anti-inflammatory effect. However, future study is needed to examine the extent to which A2AR disruption only in myeloid cells also protects against MCD-induced NASH phenotype.

In summary, we validated that A2AR expression was increased in liver macrophages/Kupffer cells, but was decreased in liver lysates of MCD-fed WT mice. We then demonstrated a protective role for A2AR in MCD-induced NASH phenotype. At the cellular, the A2AR in macrophages appears to be more important than that in hepatocytes in terms of suppressing the effect of MCD or MCD-mimicking media on stimulating the proinflammatory responses. Therefore, we provide additional evidence supporting the potential of targeting A2AR to suppress liver inflammation as a therapeutic strategy for the treatment of NASH.

ACKNOWLEDGEMENTS

This work was supported in whole or in part by grants from the American Diabetes Association (1-17-IBS-145 to C.W.) and the National Institutes of Health (DK095862 to C.W.). Also, C.W. is supported by the Hatch Program of the National Institutes of Food and Agriculture (NIFA). Y. Chen is supported by grants from China National Science Foundation (81770826) and Guangzhou Science and Technology Plan (2060404). X.Q. is supported by Guangdong Province Science and Technology Plan (2016A050502014).

Footnotes

CONFLICT OF INTEREST

The authors declare that there is no conflict of interest.

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