Dietary fish oil exacerbates concanavalin A induced hepatitis through

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The Journal of Toxicological Sciences (J. Toxicol. Sci.) Vol.39, No.2, 179-190, 2014

Original Article

Dietary fish oil exacerbates concanavalin A induced hepatitis through promoting hepatocyte apoptosis and altering immune cell populations Sheng Xia1,2, Mutian Han1, Xiaoping Li1, Lu Cheng1, Yetao Qiang2, Shuiyun Wu2, Miaomiao Zhang1, Huaxi Xu1,2, Xia Liu1 and Qixiang Shao1,2 Department of Immunology, School of Medical Science and Laboratory Medicine, Jiangsu University, 301 Xuefu Road, Zhenjiang, Jiangsu, 212013, China 2Institute of Clinic Laboratory Diagnosis, School of Medical Science and Laboratory Medicine, Jiangsu University, 301 Xuefu Road, Zhenjiang, Jiangsu, 212013, China 1

(Received December 1, 2013; Accepted January 7, 2014)

ABSTRACT — The development of hepatitis is associated with the infiltration and activation of immune cells in liver. N-3 polyunsaturated fatty acids (n-3 PUFAs) rich fish oil (FO) is used to prevent and treat inflammatory diseases. But, the effects of dietary FO on autoimmune hepatitis remain largely unknown. In this study, Concanavalin A (Con A) induced hepatitis was used to evaluate the actions of dietary FO. Unexpectedly, 2-week FO treatment had not shown any protection, on the contrary, exacerbated liver injury in this hepatitis model. The levels of alanine aminotransferase (ALT) and lactate dehydrogenase (LDH) statistically increased from 10,501 ± 2,154 and 30,394 ± 2,420 in low fat diet (LFD)/Con A group to 17,579 ± 693 and 49,439 ± 4,628 in FO/Con A group. Simultaneously, FO diet induced more necrotic liver tissues and apoptotic hepatocytes, and up-regulated the hepatic expression of TNF-α and IFN-γ after Con A challenge. Interestingly, FO promoted severe liver injury was accompanied by decreasing the percentage of CD4+ T cell, NK1.1+ cells and CD8+ T cells in CD45+ liver non-parenchymal hepatic cells (NPCs) through inducing apoptosis. Further experiments declared 2-week FO diet intake firstly increased the proportion of CD11b+Gr-1hi neutrophils in liver, but then dramatically expanded CD11b+Gr-1int inflammatory monocytes population after Con A administration. Collectively, our study indicated that high FO intake not only aggravated liver injury, but also altered the population of immune cells in liver. Thus, these results indicated that when dietary FO was used to benefit health in autoimmune diseases, its potential risks of side effect also need paying close attention. Key words: Hepatitis, Concanavalin A, Lymphocyte, Myeloid cell, Fatty acids, Fish oil INTRODUCTION Liver is one of major metabolic organs in mammals. Unfortunately, many factors can induce different types of liver diseases, which affect nearly 10% of total people population in the world (Dam-Larsen et al., 2004). So, it will be greatly helpful to alleviate this health problem by exploring its etiologies and effective treatment. Generally, the development of liver diseases, such as viral hepatitis, primary biliary cirrhosis and autoimmune hepatitis, is thought depending on the balance of inflammation and/or immune response (Crispe, 2009). As an unique immunological organ, many distinctive kinds of liver res-

ident immune cells can be found in liver, including CD8+ T, natural killer (NK) and natural killer T (NKT), stellate cells, kupffer cells and dendritic cells (Crispe, 2009; Seki et al., 2000). If antigens were presented by antigen presenting cells, immune response would be initiated and then mediated pathological effects in liver. For example, activated NK/NKT cells and CD8+ T cells can kill hepatocytes through Fas/FasL pathway, perforins/ granzyme B and TNF-α secretion (Ando et al., 1997; Santodomingo-Garzon and Swain, 2011). So, both acute and chronic hepatitis are characterized by the modification in function and phenotype of these immune cells, and the imbalance of immune response in liver (Ferri et al.,

Correspondence: Sheng Xia (E-mail: [email protected]) Vol. 39 No. 2

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2010; Liberal et al., 2012). Concanavalin A (Con A) induced hepatitis is an immune cells mediated disease, many kinds of immune cells, including CD4+ T cells, NK1.1+ NK/NKT cells, CD8+ T cells, Kupffer cells and neutrophils contribute to Con A-induced hepatitis (Bonder et al., 2004; Nakashima et al., 2008; Tiegs et al., 1992). So, it mimics a number of human liver diseases in many respects, and is widely used as experimental animal hepatitis model to explore pathological mechanisms or evaluate treatment effects. Many studies showed that regulatory immune cells or molecules negatively regulated immune cells activation in liver, and alleviated Con A-induced liver injury (Erhardt et al., 2007; Lee et al., 2012; Tomiyama et al., 2011). These results suggested that maintaining immune homeostasis in liver is very important for hepatitis treatment. In recent decades, many studies showed that different diet lipids play different roles in immune cell functions (Galli and Calder, 2009). For example, as essential fatty acids, n-3 polyunsaturated fatty acids (n-3 PUFAs) or n-3 PUFAs rich fish oil have shown anti-inflammatory effects through multiple mechanisms, including modifying lipid rafts of immune cells, suppressing antigen presenting cell (APC cell) phagocytosis and T-cell functions (Calder, 2007). So, fish oil or n-3 PUFAs itself, especially eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), were used as drugs or dietary intervention in chronic inflammatory diseases, such as cardiovascular diseases and autoimmune diseases (James et al., 2010; Mozaffarian et al., 2005). Indeed, fish oil showed its protection through reducing inflammatory in hepatocytes in non-alcoholic steatohepatitis, hepatic ischemia/reperfusion injury, and chemical mediated liver injury (Atakisi et al., 2013; Kim et al., 2013; López-Vicario et al., 2014; Schmocker et al., 2007; Zuniga et al., 2011). But, some studies showed conflicting results that dietary intake of fish oil not only had no protection, but also even aggravated liver injury (de Castro et al., 2012; de Meijer et al., 2013; Vollmar et al., 2002). So, up to now, the exact properties of dietary fish oil in inflammatory diseases are still not well explored, especially in autoimmune hepatitis. In this study, we hypothesized that n-3 PUFAs rich fish oil ameliorate liver injury in hepatitis, and evaluate the treatment effects of dietary fish on Con A induced autoimmune hepatitis model. Unfortunately, the data in this study showed fish oil exacerbated the development of hepatitis and indicated its harmful aftereffects in liver.

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MATERIALS AND METHODS Diets and Animals 6 to 8 weeks old, male C57BL/6 mice were purchased from the laboratory animal facility of Yangzhou University (Yangzhou, China) and maintained in the facility under controlled conditions (22°C, 12-hr day/night rhythm). After being housed and fed with standard lowfat diet (LFD, Teklad 2914) for one week, the mice were assigned to feed with low-fat diet (LFD, Teklad 2914) or high fish oil diet (FO diet, 60% kcal, Research Diets, Inc. New Brunswick, NJ, USA. D01112604) for 2 weeks, and then used for further experiments. This fish oil diet contained 40 mg EPA (eicosapentaenoic acid, C20:5n3), 15 mg DPA (docosapentaenoic acid, C22:5n3) and 26 mg DHA (docosahexanoic acid, C22:6n3) per gram diet. To prevent peroxidation, fish oil diet was provided in clean cups and changed daily. All experimental procedures for using laboratory animals were approved by the Scientific Investigation Board of Jiangsu University in China. Con A induced hepatitis model Con A induced hepatitis was prepared as previously described with modification (Lafdil et al., 2009). Con A (type IV) (Sigma-Aldrich, Shanghai, China) was dissolved in PBS at 1 mg/ml and administered intravenously at 20 mg/kg to induce hepatitis. Control mice were injected with PBS. After being treated with Con A for 18 hr, blood and tissues were collected for further experiments. Detection of plasma ALT, AST and LDH The mice were anesthetized with sodium pentobarbital and blood samples were collected from the eyes. Liver injury was quantified by automated measurement of plasma activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and lactate dehydrogenase (LDH) 18 hr after Con A administration using AU680 system (Beckman Coulter, S. Kraemer Boulevard Brea, CA, USA). Isolation of RNA and qPCR analysis for cytokine mRNAs Liver tissues were homogenized and total RNA was extracted using Trizol (Invitrogen, Carlsbad, CA, USA). One microgram total RNA was used to generate cDNA by using RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, Waltham, MA,USA). qPCR analysis was performed using SYBR Premix ExTaq TM (TaKaRa, Dalian, Liaoning, China) with CFX Connect™ RealTime PCR Detection System (185-5200, BIO-RAD), and

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relative mRNA levels of target genes were normalized to reference Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) levels. Primers used in qPCR analysis are listed below: Murine TNF-α: forward 5’-GAACTGGCAGAAGAGGCACT-3’, reverse 5’-GGTCTGGGCCATAGAACTGA-3’. Murine IL-6: forward 5’-GAGGAGACTTCACAGAGGATAC-3’, reverse 5’-GACTCTGGCTTTGTCTTTCTTG-3’ Murine IFN-γ: forward 5’-GCGTCATTGAATCACACCTG-3’, reverse 5’-TGAGCTCATTGAATGCTTGG-3’ Murine MCP-1: forward 5’-ATGCAGGTCCCTGTCATG-3’ reverse 5’-GCTTGAGGTGGTTGTGGA -3’ Murine CCR2: forward 5’-GTGATTGACAAGCACTTAGACC-3’ reverse 5’-TTTACAACCCAACCGAGA-3’ Murine GAPDH: forward 5’-GGCATTGCTCTCAATGACAA-3’ reverse 5’-TGTGAGGGAGATGCTCAGTG -3’ Hoechst 33342-staining and apoptotic hepatocytes quantification 4% paraformaldehyde fixed liver samples were embedded using paraffin and then sliced. Semithin sections were deparaffinized and stained with Hoechst 33342 (10 μg/ml) for 5 min at room temperature. Hoechst 33342 stained apoptotic cell shows nuclear condensation and DNA fragmentation, and can be examined under fluorescence microscope. To quantify apoptotic hepatocytes, stained liver tissues were examined under fluorescence microscope, and randomly being accounted 200 nuclei by 3 independent operators. Isolation of nonparenchymal hepatic cells Liver nonparenchymal hepatic cells (NPCs) were prepared as described previously with modification (Xia et al., 2008). Briefly, liver tissues were pressed through a 200-gauge steel mesh and single cell suspensions were collected. After being washed with PBS, cells were resuspended in 40% Percoll (Pharmacia) solution, then carefully added on 70% Percoll solution, and further purified NPCs by density centrifugation at 1,200 g for 20 min at 4°C. The buffy layer over the 70% Percoll was aspirated, and cells were washed and resuspended with PBS or 10% FCS RPMI-1640 for further experiments.

Antibodies and flow cytometery analysis All fluorescence conjugated CD4, CD8, NK1.1, CD45, CD11b, Gr-1, CD69 and respective isotype controls were purchased from eBioscience (San Diego, CA, USA). After being incubated with rat sera to block FcR on cell membrane, cells were incubated with fluorescence-conjugated mAbs in labeling solution in 96-well plate at 4°C for 20 min. Then cells were washed 3 times, and examined by flow cytometer (FACSCalibur, BD Biosciences, Shanghai, China). Fluorescence-conjugated isotype-matched irrelevant mAbs were used as control. For immune cell apoptosis detection, single liver NPCs were incubated with fluorescence-conjugated CD4, CD8, NK1.1 and annexin-V in apopotosis labeling buffer. After being washed for three times, cells were further labeled with 7-AAD, and analyzed with flow cytometer (FACSCalibur, BD Biosciences, Shanghai, China). Flow data were analyzed with CELLQuest (BD Bioscience) or FlowJo 8.7 software (Tree Star, Inc., Ashland, OR, USA). H&E staining Portions of the liver were excised and fixed in 4% paraformaldehyde and embedded in paraffin. 4-μm thickness sections were sliced, deparaffinized and stained with routine hematoxylin and eosin (H&E). The liver injury was examined under microscope (ZEISS, Munich, Germany) and necrosis grade was assessed with a four-point score for severity: 0, no necrosis; 1, 1-20% necrosis; 2, 20-40% necrosis; and 3, > 40% necrosis (Liu et al., 2007). Statistics All data in this study were expressed as the mean ± S.E.M., and analyzed by Student’s t test or ANOVA, as appropriate. p < 0.05 was considered significant. RESULTS FO diet feeding exacerbated Con A-induced liver injury To explore the exact role of fish oil on Con A-induced hepatitis, FO feeding C57BL/6 male mice were challenged with Con A. Compared with PBS injection in LFD and FO feeding mice, the results in Fig. 1A showed that Con A usage greatly increased the levels of ALT, AST and LDH in serum. These data indicated that Con A injection induced pathological injury in liver in both diets mice. But, between Con A challenged LFD and FO feeding mice, the levels of ALT were significantly increased from (10,501 ± 2,154, n = 6) in LFD/Con A group to (17,579 ± 693, n = 6) in FO/Con A group. Similarly, the levels of LDH were changed from (30,394 ± 2,420, n = 6) in LFD Vol. 39 No. 2

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Fig. 1.

FO diet feeding exacerbates Con A induced liver injury. (A) ALT, AST and LDH levels in serum were detected in different mice after Con A injection for 18 hr. N = 5 mice each group. (B) Morphology of livers after being treated with Con A for 18 hr. (C-D) Representative images (C) and quantified necrosis grades (D) of hematoxylin-eosin staining of liver tissues from lives in (B). In the upper images, 500 μm bar was used; the lower images use 200 μm bar for marking. 5 mice per group. Data are representative of more than 3 independent experiments. Data were shown as mean ± S.E.M. *p < 0.05, LFD/Con A treated mice versus FO/Con A treated mice.

group to (49,439 ± 4,628, n = 6 ) in FO group with statistic significance after Con A challenge. Although there was no significance in the levels of AST, these data unexpectedly showed FO feeding hadn’t showed any protection functions, in contrast, aggravates Con A-induced hepatitis. This was further confirmed by morphological and histological changes of livers in Figs. 1B and 1C. Compared with smooth surface with red and brown color of livers in normal mice, the livers of Con A treated mice showed slight blood stasis, small granules and edema, which can be seen by naked eyes. FO feeding aggravated these pathological changes in livers (Fig. 2B). After H&E staining, necrotic liver tissue emerged lighter staining and can be easily detected under microscope. As Figs. 1C and 1D showed, more necrotic lesions, which showed severe focal confluent in liver tissue, can be founded in FO/Con A group than in LFD/Con A mice. Vol. 39 No. 2

Some studies indicated that hepatocytes apoptosis are responsible for the liver damage in Con A induced hepatitis model (Torisu et al., 2008). So, we further explored the effects of FO feeding on the Con A induced hepatocytes apoptosis. By using Hoechest 33342 staining to detect apoptotic hepatocytes, the results in Fig. 2 showed that Con A treatment induced high numbers of hepatocytes into apoptosis. Moreover, compared with LFD mice, FO feeding induced more apoptotic hepatocytes (8.2 ± 2.1 versus 4.9 ± 1.3, n = 5) in ConA-induced hepatitis (Fig. 2), and indicated severe liver damage in FO group, which in keeping with data in Fig. 1. Thus, these results suggest that FO feeding aggravates Con A-induced liver damages.


Fig. 2.

Con A induced hepatocellular apoptosis is aggravated by FO diet feeding. Representative images of liver sections stained by Hoechst 33342(A), and the percentage of apoptotic hepatocytes were quantified in (B). Bar: 200 μm. Data were shown as mean ± S.E.M. *p < 0.05, LFD/Con A group versus FO/Con A group. ***p < 0.001. LFD or FO groups versus Con A treated mice respectively.

FO feeding increased the levels of inflammatory cytokines in liver after Con A treatment As Con A treatment promoted inflammation in liver, and increased the concentration of TNF-α, IL-6 and IFN-γ in serum and liver tissue (Cao et al., 1998; Kusters et al., 1996), we next detected the levels of these inflammatory cytokines in liver tissues by using qPCR analysis. Similar with previous studies reported (Cao et al., 1998; Kusters et al., 1996), Con A challenge greatly increased the levels of TNF-α, IL-6 and IFN-γ in both LFD and FO feeding groups (Fig. 3). In accord with previous serum enzyme data in Fig. 1A, more TNF-α and IFN-γ can be detected in liver in FO/Con A mice than LFD/Con A mice. But, there was no statistical significance in the level of IL-6 (Fig. 3). Collectively, these data demonstrate that FO diet feeding augment inflammatory cytokines expression in Con A-induced hepatitis model. FO diet lowered lymphocytes populations through inducing apoptosis in hepatitis Lymphocytes, including NK/NKT cells and CD4/CD8 T cells, are responsible for the liver damage in Con A-induced hepatitis (Kaneko et al., 2000; Tiegs et al., 1992; Toyabe et al., 1997). To investigate the effects of FO feed-

ing on the population of lymphocytes, liver nonparenchymal hepatic cells were further isolated after Con A administration, and percentages of CD4 +, NK1.1 + and CD8 + lymphocytes in CD45+ cells were analyzed. The results in Figs. 4A and 4B showed the percentage of CD4+, NK1.1+ and CD8+ lymphocytes in liver CD45+ cells were 7.1 ± 0.9, 6.0 ± 1.2 and 3.5 ± 1.5 in LFD mice. After FO feeding, the percentage of these cells changed to 6.3 ± 1.2 (CD4+), 8.2 ± 0.5 (NK1.1+) and 5.2 ± 0.6 (CD8+) respectively without statistic significance. These data indicated that 2-week FO feeding had not changed the constitution of these lymphocytes in liver. Similar with previous studies, Con A treatment induced more NK1.1+ cells in liver (Figs. 4A and 4B), and its percentage raised from 6.0 ± 1.2 to 15.8 ± 1.8, nearly 3 times increased. Furthermore, although there was no statistic change in CD4+ T cells, the percentage of CD8+ T in liver also increased 2 folds by Con A administration. But, when we detected the percentage of these lymphocytes in FO feeding mice after Con A challenge, the results unexpectedly showed all percentages of CD4+, NK1.1+ and CD8+ cells dramatically decreased 3 or 4 folds in FO diet group than in LFD hepatitis group (Figs. 4A and 4B). These data suggest 2-week FO diet feeding decreases lymphocyte populations in Con Vol. 39 No. 2

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Fig. 3.

FO diet increases expression of inflammatory cytokines in livers in hepatitis models. TNF-α(A), IL-6(B) and IFN-γ(C) mRNA levels in the liver were quantified by qPCR after hepatitis induction. mRNAs of different target genes were normalized to GAPDH. Representative of three independent experiments. Data were shown as mean ± S.E.M. **p < 0.01, ***p < 0.001.

Fig. 4.

Effects of FO diet on the percentage and activation of lymphocytes in liver. The percentage of CD4+, NK1.1+ and CD8+ lymphocytes in liver NPCs were analyzed using flow cytometer (A), and quantified its percentages in (B). The percentage of CD69+ cells respectively in CD4+, NK1.1+ and CD8+ lymphocytes in liver NPCs were analyzed as (A), and representative images were shown in (C), and quantified its percentages in (D). Cells were gated on CD45+ cells first, and then for further CD4, NK1.1, CD8 and CD69 analysis. Numbers in the image mean the percentage of CD45+ cells. Data were presented as mean ± S.E.M., and from one representative experiment of three independent experiments. *p < 0.05, **p < 0.01.

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Fig. 5.

FO diet feeding promote more lymphocytes into apoptosis in hepatitis. Representative graphs (A) and quantitative analysis (B) of apoptotic lymphocytes in LFD and FO cohorts after being treated with Con A. Cells were firstly gated on CD4, CD8 and NK1.1 respectively, and then analyzed early apoptotic cells (annexin-V+7-AAD-) and late stage apoptotic cells (annexin-V+7-AAD+) in these cells. Experiments were repeated at least three times.

A-induced autoimmune hepatitis. As the Con A-induced liver damage largely depended on the activation of lymphocytes (Tiegs et al., 1992; Toyabe et al., 1997), we further detected the activation of these lymphocytes in different groups by using lymphocyte activation marker CD69. The data in Figs. 4C and 4D show that Con A administration activated 87.9% CD4+ T cells, 51.0% NK1.1+ cells and 94.5% CD8+ T cells in liver in LFD mice. Further experiments data showed there was no difference in the activation of CD4+ T cells, NK1.1+ cells and CD8+ T cells between LFD and FO diet groups after Con A challeng (Fig. 4). These results suggest that FO feeding has no effects on lymphocytes activation in Con A-induced hepatitis. Moreover, we analyzed the effects of dietary FO on lymphocytes apoptosis after Con A treatment. After being labeled with annexin-V and 7-AAD, cells were analyzed by flow cytometer. The results in Fig. 5 showed both early

and late stage apoptotic cells were significantly increasing in three CD4+, CD8+ and NK1.1+ lymphocytes. These data indicate that after being treated with FO, lymphocytes are easy to be induced into apoptosis by Con A. FO intake promoted more CD11b+Gr-1+ myeloid cells in liver Myeloid cells, including neutrophil and macrophages, also contributed to Con A induced or other acute hepatitis (Bonder et al., 2004; Nakashima et al., 2008), we further analyzed the populations of these myeloid cells in liver. After nonparenchymal hepatic cells being isolated, CD11b and Gr-1 were used as markers to identify CD11b+Gr-1hi neutrophil and CD11b+Gr-1int monocyte in liver. The data in Fig. 6A showed that Con A administration greatly recruited more CD11b+Gr-1hi neutrophil in LFD mice, similar with previous reported (Bonder et al., 2004). The percentage of neutrophil cells in CD45+ Vol. 39 No. 2

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Fig. 6.

More CD11b+Gr-1+ inflammatory myeloid cells are recruited into liver by FO diet feeding. The percentage of CD11b+Gr-1hi granulocytes (up circle) and CD11b+Gr-1int monocytes (down circle) in liver NPCs were analyzed using flow cytometer (A), and quantified the percentages of granulocytes (left) and monocytes (right) in (B). (C) Q-PCR analysis for mRNA levels of MCP-1 and CCR2 in liver tissues. Data are normalized to the level of the corresponding GAPDH and shown as fold change. Data were shown as mean ± S.E.M., and from one representative experiment of more than three independent experiments. * p < 0.05, **p < 0.01, ***p < 0.001.

nonparenchymal hepatic cells changed from 1.7 ± 0.9 to 10.6 ± 3.6, nearly increased 5 folds. Meanwhile, the percentage of CD11b+Gr-1int monocyte only increased from 7.8 ± 1.7 to 12.3 ± 2.1 without statistic significance (Figs. 6A and 6B). Interestingly, 2-week FO feeding also significantly promoted more CD11b+Gr-1hi neutrophil in liver, its percentage increased up to 7.1 ± 1.2 in CD45+ liver nonparenchymal hepatic cells. In the meantime, the percentage of CD11b+Gr-1int monocyte only showed lightly increasing, from 7.8 ± 1.7 to 11.5 ± 2.2, similar with Con A treatment in LFD mice. But, if FO diet feeding mice were challenged with Con A, not CD11b+Gr-1 hi neutrophils, but CD11b+Gr-1int monocytes were dramatically increased. The percentage of CD11b+Gr-1int monocytes in liver reached as high as 30% of total CD45+ liver nonparenchymal hepatic cells, while the percentage of Vol. 39 No. 2

CD11b+Gr-1hi neutrophil was 12.1 ± 2.3, which is close to the number in Con A treated LFD mice (Figs. 6A and 6B). As MCP-1 and its specific receptor CCR2 worked as one potent monocyte chemoattractant axis (Lu et al., 1998; Mulle et al., 1998), we further analyzed its expression in liver tissue using qPCR analysis. The data in Fig. 6C show that Con A treatment significantly increased the level of MCP-1 in liver in both LFD and FO diet gropes, but there were no statistic significance in Con A treated LFD mice and FO diet mice. Interestingly, high CCR2 expression can be detected in Con A treated FO diet mice (Fig. 6C). Collectively, these results suggest that FO diet promoted more myeloid cells (CD11b+Gr-1hi neutrophils and CD11b+Gr-1int monocytes) in liver in Con A treated mice.


DISCUSSION This study explored the effects of dietary FO intake on Con A-induced liver injury. The data presented here showed that 2-week FO feeding boosted up the severity of Con A-induced hepatitis. Compared with LFD mice, FO feeding enhanced the liver tissue damage grading, increased the levels of ALT and LDH in serum and inflammatory cytokines secretion. By using flow cytometer analysis, the FO diet aggravated inflammation was characterized with lower percentage of activated lymphocytes (CD4+ T cells, NK1.1+ cells and CD8+ T cells), but more inflammatory CD11b+Gr-1+ myeloid cells in liver after Con A administration. These finding demonstrated that fish oil consumption would promote liver inflammation in some conditions. Since being discovered in the food of Eskimos, fish oil and its main components n-3 PUFAs have attracted great interest in food science and clinic. Based on its functions in anti-inflammation, n-3 PUFAs rich fish oil has been added in food or use as drug directly to prevent or treat chronic inflammatory diseases (Rizos et al., 2012; Yan et al., 2013). Unfortunately, many studies stated contradictory conclusions (de Meijer et al., 2013; Woodworth et al., 2010), and indicated the exact role of n-3 PUFAs in inflammation might depend on many factors. The data from Schmöcker C showed that endogenously synthesized n-3 PUFAs in fat-1 mice alleviate D-galactosamine/lipopolysaccharide (D-GalN/LPS) induced hepatitis through suppressing inflammatory cytokines secretion (Schmocker et al., 2007). Atakisi O and his colleague also declared that diethylnitrosamine induced toxicity in liver would be protected if n-3 PUFAs was used via subcutaneous route (Atakisi et al., 2013). In contrast to these studies, our experiments showed that n-3 PUFAs rich fish oil aggravated Con A-induced hepatitis when it being consumed in diet. This difference might depend on the difference of different liver injury models and the origins of n-3 PUFAs in experiments. The result from de Meijer VE and colleague showed if n-3 PUFAs used in diet, it exaggerated paracetamol-induced liver injury in mice. While, if it used through intravenously, there were no significant differences in hepatic injury between soybean-based and fish oil-based lipid, followed by paracetamol intoxication (de Meijer et al., 2013). Furthermore, some studies showed that the dose of n-3 PUFAs also decided its role in inflammation. Woodworth H.L. observed dietary fish oil induced exacerbation in colitis was dose dependent, and mice consuming 6.00% DFO expressed the highest degree of inflammation and dysplasia than other lower DFO diets (Woodworth et

al., 2010). Con A is one of T lymphocytes mitogen, and induces these cells into activation in vitro and in vivo (Tiegs et al., 1992). So, Con A-induced hepatitis in the mouse is mainly mediated by activated CD4+ T cells and NK1.1+(NK/ NKT) cells in liver, and has been used to mimic autoimmune liver diseases in human (Takeda et al., 2000; Tiegs et al., 1992). In this study, we confirmed that Con A administration can dramatically recruit more CD4+ T cells, NK1.1+ cells and CD8+ T cells into liver, and induced most of these cells into activation in LFD mice (Figs. 4A and 4B). Thus, Con A challenge induced hepatocytes into apoptosis and promoted hepatitis in these mice. It worthwhile to note that if mice pretreated with FO diet for 2 weeks, these mice were all susceptible to liver necrosis than LFD treated mice when it were treated with Con A. The high severity of liver injury and high concentration of ALT and LDH indicated more activated lymphocytes, for example NK1.1+(NK/NKT) cells, CD4+ T cells and CD8+ T cells, should be detected in liver in these mice. Unexpectedly, the results in Fig. 4 showed lower percentage of CD4+ T cells, NK1.1+ cells and CD8+ T cells in CD45+ nonparenchymal hepatic cells in FO/Con A group, but the expressions of activation marker CD69 in these cells were similar with LFO/Con A mice. So, combined with aggravating liver injury, these data indicated more lymphocytes might be in over-activation, and then were induced into apoptosis or activation-induced cell death (AICD). AICD is one major mechanism to induce activated lymphocytes into apoptotic death and maintains immune homeostasis. Except repeated antigen stimulation, the levels of survival factors or proapoptotic signals of lymphocytes, for example IFN-γ and TNF-α, also contribute to AICD of activated lymphocytes (Liu and Janeway, 1990; Murray and Crispe, 2004). In this study, we found FO/ Con A mice produced higher IFN-γ and TNF-α. As one effect cytokine in cell-mediated immunity, IFN-γ also worked as a negative regulator of lymphocytes through regulating Fas/FasL pathway and caspases production (Badovinac et al., 2000; Liu and Janeway, 1990; Refaeli et al., 2002). TNF-α is another pleiotropic inflammatory cytokine, and also has similar negative regulatory functions on lymphocytes as IFN-γ, which induces mature T cells into apoptosis. If it is blocked with neutralizing Abs, activated CD8+ T cells will accumulate into liver (Murray and Crispe, 2004; Zheng et al., 1995). Indeed, the data in Fig. 5 declared more lymphocytes were induced into apoptosis in FO groupe after being activated by Con A. So, the decreased number of activated lymphocytes (CD4+ T cells, NK1.1+ cells and CD8+ T cells) in liver in FO/Con A mice may be partially depend on AICD Vol. 39 No. 2

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which induced by higher IFN-γ and TNF-α production in liver. Previous observation had shown the recruitment and activation of myeloid cells, such as neutrophils and macrophages, can be found in the liver parenchyma, and these cells worked as effectors to damage hepatocyte in Con A-induced hepatitis (Bonder et al., 2004; Nakamura et al., 2001; Schumann et al., 2000). Neutrophil-depleted mice failed to produce inflammatory IFN-γ, and hardly caused liver injury after Con A administration (Hatada et al., 2005). This neutrophil infiltration and its associated hepatocyte death were depended on purinergic P2Y2 receptors on bone marrow-derived cells (Ayata et al., 2012). Thus, these data indicated the accessory role of neutrophil in Con A-induced hepatitis. Indeed, as previous studies showed, in this study, Con A injection significantly increased the number of CD11b+Gr-1hi neutrophils in LFD. Interestingly, FO diet also promoted more CD11b+Gr-1hi neutrophils in liver, similar with LFD/Con A mice, but not showed obevious liver injury. This might be dependent on the different activation status in hepatic neutrophils in these mice. But, if FO feeding mice were challenged with Con A, FO/Con A mice showed severe liver damage than FO control and LFD/ Con A mice (Figs. 6A and 6B). These suggested the activation of more FO induced- neutrophils in liver contributed to this serious consequence in FO/Con A mice. Interestingly, not only the percentage of CD11b+Gr-1hi neutrophils, but also the percentage of CD11b +Gr-1 int monocytes was increased in FO/Con A mice. It is worthwhile to note that Con A challenge dramatically increased the percentage of CD11b+Gr-1int monocytes in liver (Figs. 6A and 6B). Except its contribution in the percentage decreasing of lymphocytes in liver, more recruited monocytes might play roles in hepatic inflammation in Con A-induced hepatitis. Recently, two major monocyte subsets have been classified in mice based on its properties of migration and differentiation (Geissmann et al., 2003; Ingersoll et al., 2010). One subset is ‘classical’ inflammatory monocytes (CD11b+, Gr-1+, CCR2+), another subset is ‘non-classical’ monocytes (CD11b+, Gr-1-, CCR2-). Inflammatory monocytes are considered as precursors for kupffer cells in inflammatory liver, and emerge from the bone marrow in response to MCP-1 and other chemokines (Engel et al., 2008). To the contrary, Gr-1+ monocytes work as precursors of resident tissue macrophages in steady state (Tacke et al., 2007). Although we had not use CCR2 to label CD11b+ Gr-1int monocytes in liver, high hepatic level of CCR2 in FO/Con A mice suggested these monocytes were in inflammatory condition (Fig. 6). The results from Karlmark K.R. and colleague showed inflamVol. 39 No. 2

matory Gr-1+ monocyte subset can be recruit into injured liver via MCP-1/CCR2 axis, then differentiated into proinflammatory iNOS-producing CD11b+F4/80+ intrahepatic macrophage, and promoted liver fibrogenesis (Karlmark et al., 2009). So, more CD11b +Gr-1int monocytes were found in FO/Con A mice suggest these cells not only contribute to severe inflammation, but also can be different into more kupffer cells, and might play vital roles in liver fibrogenesis in Con A-induced liver damage. In summary, our study explored the effects of high fish oil intake on T cell mediated hepatitis, and found it can aggravated liver pathology through modifying immune cell populations. Thus, these results indicated that when n-3 PUFAs were used to benefit health in autoimmune diseases, its potential risks of side effect also need paying more close attention. ACKNOWLEDGMENT This work was supported by grants from the National Natural Science Foundation of China (81172834,31200676), Jiangsu University Faculty Initiation Funding (08JDG044) and the project-sponsored by SRF for ROCS, SEM. REFERENCES Ando, K., Hiroishi, K., Kaneko, T., Moriyama, T., Muto, Y., Kayagaki, N., Yagita, H., Okumura, K. and Imawari, M. (1997): Perforin, Fas/Fas ligand, and TNF-alpha pathways as specific and bystander killing mechanisms of hepatitis C virus-specific human CTL. J. Immunol., 158, 5283-5291. Atakisi, O., Atakisi, E., Ozcan, A., Karapehlivan, M. and Kart, A. (2013): Protective effect of omega-3 fatty acids on diethylnitrosamine toxicity in rats. Eur. Rev. Med. Pharmacol. Sci., 17, 467471. Ayata, C.K., Ganal, S.C., Hockenjos, B., Willim, K., Vieira, R.P., Grimm, M., Robaye, B., Boeynaems, J.M., Di Virgilio, F., Pellegatti, P., Diefenbach, A., Idzko, M. and Hasselblattm, P. (2012): Purinergic P2Y(2) receptors promote neutrophil infiltration and hepatocyte death in mice with acute liver injury. Gastroenterology, 143, 1620-1629 e1624. Badovinac, V.P., Tvinnereim, A.R. and Harty, J.T. (2000): Regulation of antigen-specific CD8+ T cell homeostasis by perforin and interferon-gamma. Science, 290, 1354-1358. Bonder, C.S., Ajuebor, M.N., Zbytnuik, L.D., Kubes, P. and Swain, M.G. (2004). Essential role for neutrophil recruitment to the liver in concanavalin A-induced hepatitis. J. Immunol., 172, 45-53. Calder, P.C. (2007): Immunomodulation by omega-3 fatty acids. Prostaglandins Leukot Essent Fatty Acids, 77, 327-335. Cao, Q., Batey, R., Pang, G., Russell, A. and Clancy, R. (1998): IL-6, IFN-gamma and TNF-alpha production by liver-associated T cells and acute liver injury in rats administered concanavalin A. Immunol. Cell Biol., 76, 542-549. Crispe, I.N. (2009): The liver as a lymphoid organ. Annu. Rev. Immunol., 27, 147-163.

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