MyD88-dependent pathway accelerates the liver damage of Concanavalin A-induced hepatitis

Biochemical and Biophysical Research Communications 399 (2010) 744–749

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MyD88-dependent pathway accelerates the liver damage of Concanavalin A-induced hepatitis Keisuke Ojiro, Hirotoshi Ebinuma, Nobuhiro Nakamoto, Kanji Wakabayashi, Yohei Mikami, Yuichi Ono, Chu Po-Sung, Shingo Usui, Rumiko Umeda, Hiromasa Takaishi, Yoshiyuki Yamagishi, Hidetsugu Saito, Takanori Kanai ⇑, Toshifumi Hibi ⇑⇑ Department of Gastroenterology and Hepatology, Keio University School of Medicine, Tokyo 160-8582, Japan

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Article history: Received 2 August 2010 Available online 7 August 2010 Keywords: Concanavalin A Hepatitis Macrophage MyD88 Toll-like receptors

a b s t r a c t We have explored the pathological role of the MyD88 signaling pathway via Toll-like receptors (TLRs) that mediate the recognition of pathogen-associated molecular patterns (PAMPs) in a murine model of autoimmune hepatitis induced by administering Concanavalin A (ConA). We first found that various TLRs and MyD88 molecules were expressed in liver of Con A-treated and untreated wild-type (WT) mice including liver macrophages. Flowcytometric analysis revealed that liver CD11b+CD11c and CD11b+CD11c+ antigen-presenting cells express TLR2, although NK and NKT cells did not. When WT and MyD88/ mice were intravenously administered with Con A, the severity of hepatitis was significantly lower in Con A-injected MyD88/ mice than in WT mice in terms of the histopathology, the levels of serum transaminase and pro-inflammatory cytokines (TNF-a, IFN-c, and IL-6), and upregulation of CD80/CD86 and TNF-a on/in liver macrophages. The results provide evidence of a possible contribution of the TLRs-MyD88 signaling pathway in activating TLR-expressing liver macrophages in the autoimmune hepatitis model, and thus indicate that the strategy of blockade of pathological pathogens via the intestinal lumen may be feasible for the treatment of the disease. Ó 2010 Elsevier Inc. All rights reserved.

1. Introduction The liver receives large amounts of blood that is abundant in commensal bacterial products from the intestines, presumably supplied via the portal vein [1–3]. Nevertheless, the liver is a site of immune tolerance, although various immune compartments, such as NK, NKT, T, macrophages (Kupffer cells), and dendritic cells (DCs), are reported to physiologically reside in the liver [1,2]. These previous findings suggest that a set of immune cells in the liver contributes to its tolerance to commensal bacterial products. Nevertheless, various autoimmune diseases occur in the liver, such as autoimmune hepatitis (AIH) [4], primary biliary cirrhosis (PBC), and primary sclerosing cholangitis (PSC), when the immunological tolerance is disrupted, presumably by environmental agents. As such, innate immune cells, such as macrophages (Kupffer cells), DC cells, NK, and NKT cells, are suspected to play a crucial role in these diseases, but the precise mechanisms involved in activating these cells, as well as T and B cells, are unclear. Abbreviations: Con A, Concanavalin A; NK, natural killer; DC, dendritic cell; PAMP, pathogen-associated molecular pattern; TLR, Toll-like receptor; WT, wildtype. ⇑ Corresponding author. Fax: +81 3 3341 3631. ⇑⇑ Corresponding author. Fax: +81 3 3341 3631. E-mail addresses: [email protected] (T. Kanai), [email protected] (T. Hibi). 0006-291X/$ - see front matter Ó 2010 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2010.08.012

To clarify this issue, we here investigate the role of innate immune responses via MyD88 molecules [3,5], which are adaptor molecules for the main pathway of Toll-like receptors (TLR.), using a murine Concanavalin A (Con A)-induced autoimmune hepatitis model [6,7]

2. Materials and methods 2.1. Mice Eight-to 12-week-old wild-type (WT) C57BL/6J mice were purchased from Japan Clea (Tokyo, Japan). Age-matched MyD88/ mice with C57BL/6 background were kindly provided by Dr. Shizuo Akira (Osaka University) [8]. All animals were maintained under specific pathogen-free conditions in the Animal Care Facility of Keio University School of Medicine. All experiments were approved by the regional animal study committees and were done according to Institutional guidelines and Home Office regulations. 2.2. Con A-induced hepatitis model Concanavalin A (Con A, type IV) was purchased from SigmaAldrich (St. Louis, MO, USA). Intravenous injections of Con A

K. Ojiro et al. / Biochemical and Biophysical Research Communications 399 (2010) 744–749

(20 mg/kg) were performed into the tail vein of animals 10 h before examination under anesthesia. 2.3. Preparation of liver mononuclear cells Liver mononuclear cells were separated from the liver as described previously. Briefly, livers were perfused through the portal vein with phosphate-buffered saline, then minced and passed through 100 lm nylon mesh. The filtrate was centrifuged at 50g for 1 min and supernatant was washed once. Cells were suspended in a Histopaque solution (Sigma–Aldrich) and overlaid on a HBSS solution. After centrifugation at 2500 rpm for 20 min, the cells were collected from the upper face of the Histopaque. 2.4. Measurement of liver injury Serum alanine aminotransferase (ALT) levels were measured using a LDH-UV kinetic method (SRL Inc., Tokyo, Japan). Livers were fixed in 10% formalin, embedded in paraffin. Sections were stained with hematoxylin and eosin (H&E) and examined.

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TLR9, 50 -GGCGTTCTGAGATAAACACGACC-30 , 50 -TGTCCCTAGTCA GGGCTGTACTCAG-30 ; MyD88, 50 -CCGGAACAATCTGGCACTCC-30 , 50 -TCATCTTCCCCTCT GCCCTA-30 . After PCR amplification, the products were detected by 2.0% agarose gel electrophoresis and stained with ethidium bromide. To measure the quantity of cytokine, real-time PCR was performed using TaqMan Universal Master Mix and StepOne Plus systems (Applied Biosystems). The level of target gene expression was normalized against glyceraldehyde-3-phosphate dehydrogenase expression in each sample. 2.8. Statistical analysis The results are expressed as mean ± standard error of mean (SEM). Groups of data were compared by Mann–Whitney U test. Differences were considered to be statistically significant when p < 0.05. 3. Results

2.5. Flowcytometry

3.1. TLR2 and TLR4 are expressed on liver macrophages

After blocking with anti-FcR (CD16/32, BD Pharmingen) for 20 min, the cells were incubated with the specific fluorescence-labeled mAbs at 4 °C for 30 min. The following mAbs were used: anti-mouse anti-CD3e (FITC), anti-NK1.1 (APC), and anti-CD11b (APC), anti-CD11c (FITC), 7-AAD (PerCP), anti-TLR2 (PE), antiTLR4 (PE) (eBioscience, BD Pharmingen). Background fluorescence was assessed by the staining of irrelevant anti-rat isotype (BD Pharmingen). The stained cells were analyzed by flowcytometry (FACS CantoII, Becton Dickinson Co.) and the data were analyzed using FlowJo software (Tree Star Inc.).[9]

To investigate the role of the innate immune system in a murine model of Con A-induced autoimmune hepatitis [7], we first examined whether mRNAs of TLR1–9 and their adaptor molecule, MyD88, are expressed in the murine liver. RT-PCR analysis revealed that not only whole liver and spleen tissue samples but also their single cell suspended mononuclear cells expressed all members of TLR1–9, especially TLR2 and TLR4, and MyD88 (Fig. 1A). To next determine which cell compartments expressed these molecules, we isolated mononuclear cells from normal liver and stained CD11b/CD11c with TLR2 or TLR4. Almost all CD11b+CD11c macrophages and CD11b+/CD11c+ DC cells and a very small proportion of CD3+NK1.1 T cells, CD3NK1.1+ NK cells, and CD3+NK1.1+ NKT cells expressed TLR2 (Fig. 1B). In contrast, a small but substantial proportion of liver macrophages, but not other compartments, expressed TLR4 (Fig. 1B).

2.6. Intracellular cytokine staining After stimulation for 6 h with or without lipopolysaccharide (LPS, from Escherichia coli B5, Sigma) and brefeldin A (10 lg/ml), the cells were stained using anti-TNF-a (PE, BD Pharmingen), anti-IFN-c (PE, BD Pharmingen), and Cytofix/Cytoperm kit (BD Pharmingen). 2.7. Rt-PCR Total RNA was extracted from liver homogenates using RNeasy Mini Kit (QIAGEN, Valencia, CA, USA). Complementary DNA was synthesized from 1 lg of total RNA by reverse transcription. To determine the expression of TLRs in the liver, polymerase chain reaction was performed using AmpliTaq Gold Fast PCR MasterMix (Applied Biosystems) and the following primers [10]: Tlr1, 50 -AGAGACTTCCGAAACAGCTATGT-30 , 50 -GACAGAGCCTGT AAGCATATTCG-30 ; Tlr2, 50 -AGATTTCAGCTAGGCGCTGTA-30 , 50 -TACCCAGCTCGCTCA CTACGT-30 ; TLR3, 50 -AACAGAAGACGTGCTTGGAC-30 , 50 -CGCAACGCAAGGA TTTTATT-30 ; TLR4, 50 -GTTCTTGTATCTAGACTCGAAGTTGGG-30 , 50 -GCTGTCCA ATAGGGAAGCTTTCTAGAG-30 ; TLR5, 50 -TGACTTAAGGAATTCGCTGCAT-30 , 50 -AGAAGATAAAGCC GTGCGAAA-30 ; TLR6, 50 -TTGTCCTATGCCTCGGAACT-30 , 50 -CCAGGAAAGTCAGCT TCGTC-30 ; TLR7, 50 -AAGGCTATGCTACTTATACGTGC-30 , 50 -TGAGTTTGTCCAGAAGCCGTAAT-30 ; TLR8, 50 -CCGTGTTGAGGGAACACTAA-30 , 50 -CATTTGGGTGCTGTT GTTTG-30 ;

3.2. Liver macrophages markedly produced TNF-a in Con A-induced hepatitis Given the evidence that several proportions of immune compartments, especially macrophages, in liver expressed TLRs, we next examined whether their expression is affected by Con A treatment, which induces autoimmune liver damage. However, RT-PCR analysis using whole Con A-treated and untreated liver samples revealed that no dramatic changes of TLR expression after Con A treatment: all TLRs, especially TLR2 and TLR4, were constitutively expressed in both Con A-treated and untreated liver tissue (Fig. 2A). Although previous studies have shown that intravenous administration of Con A causes acute autoimmune hepatitis [7] and that TNF-a plays a crucial role in this model [11], it remains unknown which immune compartments produce TNF-a. We therefore examined which cell compartments in liver produced TNF-a before and after Con A treatment by intracellular cytokine staining and flowcytometric analysis. The major producer of TNF-a after Con A treatment was found to be macrophages rather than DCs, since a large proportion of macrophages expressed TNF-a (Fig. 2B, upper), although a small proportion of liver DCs, but not CD11bCD11c other immune compartments, also expressed TNF-a. In contrast, neither macrophages nor DCs or other immune compartments in liver produced IFN-c before or after Con A administration (Fig. 2B, lower). Given the evidence that liver macrophages are the main compartment of innate immunity in liver and that they express various TLRs, especially TLR2 and 4 (Figs. 1

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Fig. 1. Various TLRs are preferentially expressed on liver macrophages but not on NK and NKT cells. (A) Expression of various TLRs and MyD88 mRNAs in liver and spleen of WT mice. To comfirm that TLRs and MyD88 were expressed in the tissues and single suspended mononuclear cells from liver and spleen of WT mice, total RNA was extracted from whole tissue or mononuclear cells. The RNA was reverse-transcribed, then PCR was performed. Data are representative of three independent experiments. L, liver; Sp, spleen. (B) Expression of TLR-2 and TLR-4 proteins in various immune cells in liver of WT mice. Liver mononuclear cells isolated from WT mice were stained with specific mAbs and analyzed by flowcytometry. CD3 + NK1.1- T, CD3-NK1.1 + NK, CD3 + NK1.1 + NKT, CD11b+/CD11c + DC and CD11b + CD11c- macrophages were gated, and the expression of TLR2 and TLR4 was examined. Data are representative of five independent experiments. Mac, macrophages.

and 2A), and produce a large amount of TNF-a after Con A treatment (Fig. 2B), we hypothesized that the innate immune signaling pathway through TLRs-MyD88 molecules critically contributes to the pathogenesis of autoimmune Con A-induced hepatitis for the following experiments. However, it should be addressed whether MyD88-mediated cytokines, such as IL-1b, IL-18, and IL-33. are also involved in the pathogenesis of Con A-induced hepatitis in addition to TLR signaling pathway. To this end, we assessed the mRNA expression of IL-1b, IL-18, and IL-33, in PBS- and Con Aadministered liver, and of interest found that the expression of IL-1b and IL-33 in Con A-administered liver was significantly increased as compared to that in PBS-administered liver, whereas that of IL-18 in Con A-administered liver was significantly decreased as compared to that in PBS-administered liver (Fig. 2C), suggesting that some cytokine signaling pathways mediated by MyD88 molecules are also involved in the pathogenesis of Con Ainduced hepatitis in addition to TLRs-MyD88 signaling pathway. 3.3. Severity of Con A-induced hepatitis in MyD88/ mice was significantly reduced compared to wild-type mice Although receptors for Con A in any species are presently unknown, to test our hypothesis that TLRs-MyD88 signaling pathway in liver macrophages is critically involved in the pathogenesis of Con A-induced liver damage, we assessed Con A-induced hepatitis

using MyD88/ mice, in which the TLRs signaling pathway is mostly defective. To this end, we administered 20 mg/ml Con A intravenously into WT and MyD88/ mice, and sacrificed 10 h after the injection. As expected, serum alanine aminotransferase (ALT) in MyD88/ mice after Con A administration was significantly lower than that in WT mice after Con A administration (p < 0.05) (Fig. 3A), while that in PBS-injected control MyD88/ and WT mice was at baseline level. Furthermore, the liver histology of Con A-injected MyD88/ mice revealed milder degenerative changes, whereas Con A-injected WT mice showed marked damage with hepatocyte necrosis and inflammation (Fig. 3B). PBS-treated MyD88/ and WT mice both showed no signs of necrosis or inflammation (Fig. 3B). Consistent with these findings, quantitative RT-PCR analysis revealed that pro-inflammatory TNF-a, IL-6 and IFN-c levels in the liver of Con A-injected MyD88/ mice were significantly reduced as compared to those of Con A-injected WT mice, whereas the level of IL-4 was not significantly different between Con A-injected WT and MyD88/ mice (Fig. 3C). 3.4. Macrophages of MyD88/ mice could not be activated after Con A administration The finding that MyD88/ mice developed milder Con A-induced hepatitis than the paired WT mice suggested that the severity of Con A-induced hepatitis is affected by the innate immunity

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Fig. 2. TLRs were constitutively expressed in the livers of both Con A-treated and untreated mice, and liver acrophages in Con A-injected WT mice increased production of TNF-a. (A) Expression of various TLRs and MyD88 mRNAs in liver of PBS- and Con A-treated mice. Total RNA was extracted from whole liver tissues of WT mice administered with PBS or Con A. Data are representative of three independent experiments. (B) Expression of TNF-a and IFN-c proteins in macrophages and DCs of PBS- and Con Aadministered liver. The mononuclear cells separated from livers of Con A-treated and untreated WT mice were incubated for 6 h with LPS, subjected to intracellular staining with cytokine-conjugated fluorescent mAbs, and analyzed by flowcytometry. Data are representative of three independent experiments. (C) Expression of MyD88-mediated cytokines in liver of PBS- and Con A-treated mice. Total RNA was extracted from whole liver tissues of WT mice administered with PBS or Con A. Data are indicated as the mean ± SEM in each group. *p < 0.05.

governed by liver macrophages. Therefore, we next investigated the differences in quantity and functional activity of macrophages between MyD88/ and WT mice in Con A-induced hepatitis. First, we demonstrated that there was no difference between PBS-treated MyD88/ and WT mice, and that the proportion of CD11bCD11c macrophages separated from the livers of Con A-injected MyD88/ and WT mice significantly increased compared with the paired controls (Fig. 4A and B). However, the increased ratio of liver macrophages was comparable between MyD88/ and WT mice diminished in number in MyD88/ mice compared with WT mice (Fig. 4A and B). Second, activation markers CD80 and CD86 of liver macrophages from MyD88/ mice were markedly diminished as compared to those from Con A-injected WT mice in terms of the percentages of positive fraction and the MFI, whereas the baselines of PBS-treated mice were comparable (Fig. 4C). 4. Discussion In the present study, we demonstrated that (1) various TLRs are expressed in liver, especially in liver macrophages, irrespective of physiological and inflammatory conditions, (2) liver macrophages are major producers of TNF-a in the development of Con A-induced liver injury, and (3) MyD88/ mice, in which the TLRs signaling pathway is mostly defective, develop less severe Con A-induced hepatitis with less elevation of serum ALT, less severe liver pathology, and less production of pro-inflammatory cytokines such as TNF-a.

Collectively, these results suggest that the TLRs-MyD88 signaling pathway in liver macrophages is critically involved in the pathogenesis of Con A-induced liver damage. Initially, it was believed that the Con A-induced liver injury model established by Tiegs et al. was mediated by CD4+ T cells due to the amelioration of the disease in T cell-deficient nude mice and glucocorticosteroid-treated mice [7], and thus it was categorized as a T cell-mediated autoimmune hepatitis model. Subsequently, however, Takeda et al. discovered that CD1d/ mice, in which the function of NKT cells is impaired, are resistant to this model [12], concluding that the model is mediated by CD3+CD4+NK1.1+NKT T cells rather than conventional CD3+CD4+ NK1.1 T cells. Consistent with this, it is now known that liver lymphocytes contain an unusually high frequency of NKT cells in mice [13] and also in human [14]. However, it remains unknown how NKT cells are activated only in the liver of Con A-administered mice, since no receptor for Con A has yet been identified. It is also known that NKT cells are activated in other models of liver injury, such as LPS-induced liver injury [15], suggesting that liver APCs including macrophages (Kupffer cells) and DCs are critically involved in the pathogenesis of Con A-induced liver injury. Surprisingly, however, given that MyD88/ mice are often used to examine the role of the overall innate immune system in various murine autoimmune models, it has not been so far determined whether these mice are resistant to Con A-induced liver injury. Therefore, the present study is the first to demonstrate that

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Fig. 3. Severity of Con A hepatitis was significantly reduced in the MyD88/ mice compared with in WT mice. WT and MyD88/ mice were injected with 20 mg/kg Con A and sacrificed 10 h after injection. (A) Serum ALT level was significantly diminished in Con A-injected MyD88/ mice compared with Con A-injected WT mice. (B) The severity of liver damage in Con A-injected MyD88/ mice was markedly reduced as compared to that in Con A-injected WT mice. (C) Expression of pro-inflammatory cytokines in Con A-injected MyD88/ mice was significantly less than that in Con A-injected WT mice. RT-PCR was performed at least three times on independent samples. Data are indicated as the mean ± SEM in each group. *p < 0.05.

MyD88/ mice develop less severe Con A-induced hepatitis than the control mice. Importantly, we showed that liver macrophages in Con A-administered WT mice are activated in terms of TNF-a production and CD80/CD86 upregulation, whereas such activation was impaired in Con A-administered MyD88/ mice. Classically, Con A is one of representative T cell mitogens, but it is known that APCs are needed to stimulate T cells in in vitro Con A stimulation, since very highly purified CD4+ T cells are unable to be activated by Con A. Thus, we speculate that APCs including liver macrophages are the direct target of Con A in liver, although we have presented no experimental evidence herein. To solve this issue, we stimulated highly purified CD11b+ liver macrophages with various concentrations of Con A in vitro, but did not detect their activation (data not shown). This may indicate that the mechanisms of liver macrophage activation in Con A-injected mice are greatly complicated with the involvement of hepatocytes, Ito cells, and others, or more systemic mechanisms for the activation of liver macrophages. Nevertheless, the crosstalk between activated liver macrophages and other effector compartments, such as NKT cells and T cells, would be essential for the establishment of Con A-induced liver injury, since T cell-deficient nude mice [7] and RAG-2/ mice (data not shown) do not develop this model. How do commensal bacteria-derived pathogen-associated molecular patterns (PAMPs) or living bacteria themselves contribute to the activation of liver macrophages in the development of Con A-induced liver injury? Since the liver is supplied with blood from the arterial system and the portal vein returning from the intestine, it is reason-

able to assume that PAMPs derived from living or killed commensal bacteria can constitutively reach the liver via the portal vein. Preliminary tests by high-accuracy RT-qPCR detected various commensal bacteria, such as Clostridium coccides, C. leptam, and Enterococcus in both Con A-treated and untreated liver (data not shown). Thus, it is conceivable that such PAMPs from the intestine are critically involved in the activation of liver macrophages. Alternatively, Con A-like specific products derived from commensal bacteria might directly activate liver macrophages in the development of liver inflammation, such as AIH, PSC, and PBC in humans. Finally, it should also be considered whether the present experimental design solely assesses the role of direct TLR signaling pathway to liver APCs in the process of development of Con A-induced liver injury, because the MyD88-dependent signaling pathway is also involved in signaling for endogenous cytokines IL-1b, IL-18, and IL-33 in addition to TLR signaling [16,17]. Since we showed that some of these cytokines were significantly upregulated in Con A-administered liver as compared to normal liver, (Fig. 2B), it is possible that such MyD88-dependent cytokine signaling is also involved in the pathogenesis of Con A-induced liver injury. Moreover, a recent study reported that TLR3/ mice were also protected from Con A-induced liver injury, although TLR3 is not mediated by MyD88 [18]. Thus, the regulation of acute hepatitis must be complex. Further studies are needed to address this issue by assessing which type of TLR is the most important in stimulating liver macrophages, and by subsequent in vivo experiments using specific TLR-null mice.

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Fig. 4. Activation, but not frequency, of liver macrophages in Con A-injected MyD88/ mice was impaired as compared to that in Con A-injected WT mice. (A) Expression of CD11b and CD11c on liver mononuclear cells in PBS- or Con A-treated WT or MyD88/ mice. Dot plot analysis shows the CD11b/CD11c expression on each fraction of immune cells from WT and MyD88/ mice with or without Con A-administration. Data are representative of three independent experiments. (B) The frequency of liver macrophages in PBS- or Con A-treated WT or MyD88/ mice. The bar graphs show the percentage of CD11b+CD11c liver macrophages of each mouse. Data are indicated as the mean ± SEM in each group. *p < 0.05. (C) Expression of CD80 and CD86 molecules on liver macrophages. Upregulation of CD80 and CD86 on liver macrophages in Con Ainjected MyD88/ mice was markedly impaired as compared to that in Con A-injected WT mice. Data are representative of three independent experiments.

In summary, the current study provides evidence of the possible contribution of commensal bacteria in activating liver APCs, especially TLRs-expressing macrophages for the autoimmune hepatitis model, and thus indicates that the strategy of blockade of pathological pathogens via the intestinal lumen may be feasible for the treatment of the disease. Acknowledgments This study was supported in part by Grants-in-Aid for Scientific Research, Scientific Research on Priority Areas, Research on Measures for Intractable Diseases, Exploratory Research and Creative Scientific Research from the Japanese Ministry of Education, Culture, Sports, Science and Technology; The Japanese Ministry of Health, Labor and Welfare; Foundation for Advancement of International Science; Ohyama Health Foundation; Yakult Bio-Science Foundation; Research Fund of Mitsukoshi Health and Welfare Foundation. References [1] Crispe I.N., Hepatic T cells and liver tolerance, Nat. Rev. Immunol. 3 (2002) 51–62. [2] I.N. Crispe, The liver as a lymphoid organ, Annu. Rev. Immunol. 27 (2009) 147– 163. [3] E. Seki, D.A. Brenner, Toll-like receptors, adaptive molecules in liver disease: update, Hepatology 48 (2008) 322–335. [4] E.L. Krawitt, Autoimmune hepatitis, New Engl., J. Med. 24 (2006) 783–801. [5] S. Akira, S.S. Uematsu, O. Takeuchi, Pathogen recognition, innate immunity, Cell 24 (2006) 783–801. [6] Z. Dong, H. Wei, R. Sun, Z. Tian, The roles of innate cells in liver injury, regeneration, Cell Mol.Immunol. 4 (2007) 241–252.

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