Development of a Spontaneous Liver Disease Resembling Autoimmune Hepatitis in Mice Lacking Tyro3, Axl and Mer Receptor Tyrosine Kinases

Autoimmune hepatitis (AIH) is a severe type of chronic liver disease. The lack of appropriate animal models has resulted in a limited understanding regarding the etiology of AIH. Here, we demonstrated that mice deficient in Tyro3, Axl and Mer (TAM) receptor tyrosine kinases (RTKs) developed persistent inflammatory liver damage resembling AIH. Tyro3−/−Axl−/−Mer−/− triple mutant (TAM−/−) mice exhibited chronic hepatitis, manifested by progressive appearance of interface hepatitis, immune cell infiltrations and elevated inflammatory cytokine levels in the liver. Accordingly, increased levels of transaminases were observed. Moreover, characteristic autoantibodies and high levels of plasma immunoglobulin G for AIH were detected as TAM−/− mice aged. Finally, we provided evidence that the liver damage in TAM−/− mice mainly result from bone marrow-derived cells and could be rescued by transplantation of WT bone marrow cells. Results suggest that TAM RTKs play an important role in maintaining immune tolerance of the liver.


Introduction
Although the liver is an immunoprivileged site, autoimmune liver diseases, such as autoimmune hepatitis (AIH), develop globally in diverse ethnic groups [1]. AIH is a progressive inflammatory liver disorder that is characterized histologically by interface hepatitis and serologically by high levels of transaminases and the presence of non-organ specific autoantibodies. Two AIH types have been defined according to the autoantibody profile: type 1 (AIH-1) is positive for anti-nuclear antibody (ANA) and/or smooth muscle antibody (SMA), and type 2 (AIH-2) is positive for anti-liver kidney microsomal type 1 (anti-LKM1) antibody [2]. Children and adults of any age may develop AIH [3,4]. AIH, which is often diagnosed late in its progression, usually results in severe consequences for the patients. Unfortunately, the etiology of the disease and the mechanisms leading to liver damage in AIH are poorly understood. One reason for this limited understanding regarding the etiology of AIH is the absence of reliable animal models. Several mouse models of AIH have been described [5,6,7,8]. However, none of these models show persistent autoimmune liver damage [9]. Studies on immune reactivity against the liver have indicated that self-reactive lymphocytes alone are not sufficient for disease induction without additional inflammatory signals, namely ''liver tolerance'' [10]. However, upregulation of costimulatory factors in the liver during pathogeninduced inflammation can break this tolerance [11]. Activation of toll-like receptors (TLRs) is engaged to break the immuno-tolerant status of the liver and convert autoreactivity into overt AIH [12].
TLRs belong to a family of pattern recognition receptors that recognize pathogen-associated molecular patterns (PAMPs) from microbes to induce production of pro-inflammatory cytokines for the initial host defense against pathogens [13]. Endogenous components derived from dying host cells, termed damageassociated molecular patterns (DAMPs), can also activate TLRs [14]. Various TLRs are expressed and regulate innate immune responses in the liver [15,16]. TLR signaling must be tightly controlled for the homeostasis of immune responses, because unrestrained TLR activation generates a chronic inflammatory milieu that can lead to the development of autoimmune diseases [17,18]. Several mechanisms for the negative regulation of TLR signaling have been identified [19,20]. TAM subfamily of receptor tyrosine kinases (RTKs) contains three members: Tyro3, Axl and Mer [21]. Two highly similar vitamin K-dependent proteins, product of growth arrest-specific gene 6 (Gas6) and Protein S (ProS, a blood anticoagulant cofactor), are the common ligands of TAM RTKs [22]. Gene knockout studies have provided directly insights into the physiologic functions of TAM RTKs. TAM RTK triple mutant (TAM 2/2 ) mice displayed multiple defects in the immune, neuro, reproductive and hematopoietic systems [23,24,25,26,27]. One of the most prominent functions of TAM RTKs is the negative regulation of innate immune response via inhibition of TLR signaling [28,29].
In the present study, we demonstrate that TAM RTKs are required for the immune tolerance of the liver, and loss of these receptors results in progressive inflammatory liver damage resembling AIH. The finding fits the previous reports that TAM RTKs inhibit inflammatory response.

Materials and Methods
Animals TAM RTK mutant mice were kindly provided by Dr. Qingxian Lu (Salk Institute for Biological Studies, La Jolla, CA), and were progenies of the original colony with a genetic background of 50% 129/SV 6 50% C57BL/6. Wild-type (WT) control mice were the littermates of the mutant mice. The mice were inbred in pathogenfree conditions, and had free access to food and water. Animal study protocol was reviewed and approved by the Institutional Animal Care and Use Committee of Peking Union Medical College Hospital, China [the permit number: SCXK (Jing) 2007-0001]. Female mice were used in this study. All efforts were made to minimize suffering.

Transaminase activity assay
Peripheral blood was collected from the tail vena of mice. Activities of serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) were measured using ALT and AST enzymatic assay kits (Rongsheng Co., Shanghai, China) following the supplier9s protocol.

Histological analysis and immunohistochemistry
For histological analysis, the livers were removed from mice and embedded in paraffin. A series of sections (4 mm thick) were cut, and stained with hematoxylin and eosin (H&E).
Immunohistochemistry was performed based on previous description [30]. Briefly, the frozen liver sections (8 mm thick) were incubated with the primary antibodies F4/80, ED1 or ED2 for 60 min at room temperature. The sections were then incubated with the appropriate biotinylated secondary antibodies (Zhongshan Biotechnology Co., Beijing, China) for 60 min, followed by incubation with streptavidin-peroxidase complex for 30 min. Peroxidase binding sites were demonstrated by diaminobenzidine method.

Immunofluorescence staining
Indirect IF staining was used to detect serum autoantibodies. Hepa1-6 cells cultured on Lab-Tek chamber slides (Nunc, Naperville, IL) were fixed with cold methanol at -20uC for 3 min. After blocking with 10% normal goat serum in PBS at room temperature for 1 h, the cells were incubated with sera obtained from TAM 2/2 and WT mice at 37uC for 1 h, followed by incubation with fluorescent isothiocyanate (FITC)-conjugated goat anti-mouse immunoglobulin G (IgG) (Zhongshan) for 30 min.
Direct IF staining was performed to examine lymphocytes in the liver. The frozen liver sections were incubated with FITCconjugated antibodies against B220, CD4 or CD8 for 90 min at room temperature. After rinsing with PBS, the sections were mounted with Canada balsam (Sigma, St. Louis, MO) for observation under a fluorescence microscope (IX71, Olympus).
Isolation and analysis of lymphocytes from the liver Lymphocytes were isolated from the liver based on previous description [31]. Briefly, mice were anesthetized with 10 ml 16 PBS (pH 7.0) from the portal vein. The liver was cut into , 1 mm 3 pieces using a surgical scissor, then were treated with 0.5 mg/ml collagenase type 1 (Sigma) at room temperature for 20 min with gentle pipetting. The suspensions were filtered through 80-mm copper meshes. The cells were separated in 70% percoll solution (Sigma) according to manufacturer9s instructions. After washing twice with PBS, the cells were labeled with FITCconjugated antibodies against CD4, CD8, or B220, and subsequently analyzed using FACSCanto flow cytometer (BD Biosciences, San Jose, CA).

Autoantibody detection
The liver was lysed by freezing and grinding. Cytokine concentration in the supernatant was measured using ELISA kits (eBioscience, San Diego, CA). Serum autoantibodies against actin and total plasma IgG were determined using Quanta Lite TM ELISA (INOVA Diagnostic, Inc, San Diego, CA), in accordance with manufacturer9s instructions. The titers of serum autoantibodies against SMA were detected using microscope slides (Beijing EIAab Science Co. Beijing, China) according to manufacturer9s instructions.

Real-time RT-PCR
Total RNA was isolated from the liver and cells using TRIzol reagent (Invitrogen, Carlsbad, CA) according to the manufactur-er9s instructions. RNA (1 mg) was reverse-transcribed into cDNA using Moloney murine leukemia virus reverse transcriptase and random hexamer primers (Promega, Madison, WI). PCR was performed using Power SYBR Green PCR master mix kit (Applied Biosystems, Foster City, CA) in an ABI PRISM 7300 real-time cycler (Applied Biosystems). The relative mRNA levels of target genes were normalized to b-actin. The primers for PCR are listed in Table 1.

Isolation of mouse parenchymal and nonparenchymal liver cells
Primary parenchymal hepatocytes were isolated as described previously [32]. Briefly, the livers from 10-week-old mice were perfused and digested using 0.05% collagenase IV (Sigma) for 10 min. The liver cell suspension was separated by natural sedimentation once and centrifugation twice at 30 g for 5 min. The parenchymal hepatocytes were recovered from the cell pellets. The fraction of cells in supernatant contained mainly nonparenchymal liver cells including Kupffer cells and sinusoidal endothelial cells, which were separated via density centrifugation on Percoll gradients at 800 g for 15 min based on procedures described previously [33].

Bone marrow transplantation
Bone marrow cells were collected from donor mice at age 2 months. WT and TAM 2/2 recipients at 6 months age were lethally irradiated with a single dose of 8.5 Gy 60 Co and injected with bone marrow cells (5610 6 cells each mouse) from donors through tail vein. At 6 months after transplantation, the engrafted mice were analyzed.

Statistical analyses
Data are presented as mean 6 SEM for n given samples. Student's t-test was used to determine statistical significance for all comparisons. Calculations were performed with SPSS version 11.0 statistical software (SPSS Inc., Chicago, IL). P,0.05 was considered significant.

Persistent liver damage in TAM 2/2 mice
Based on histological analysis, a phenotype of chronic hepatitis was found in TAM RTK triple mutant (Tyro3 2/2 Axl 2/2 Mer 2/2 , TAM 2/2 ) mice. Severe portal inflammation with piecemeal necrosis and cellular infiltrations into parenchymal regions were frequently observed as TAM 2/2 mice aged ( Figure 1A). Accordingly, increased ALT and AST activities were detected in the sera of TAM 2/2 mice ( Figure 1B). Although a great variance in ATL and AST levels was observed in different mice, all 14 female TAM 2/2 mice at age 12 months exhibited significant high serum ALT and AST activities compared to age-matched WT controls ( Figure 1B). Mean values of ALT and AST activities reached 781 and 653 U/ liter serum respectively, which were approximately 15-fold that of WT controls. The liver damage occurred progressively as mice aged. At age 2 months, we detected only basal ALT and AST activities in WT and TAM 2/2 mice. However, ALT and AST levels were significantly increased at age 6 months and sharply elevated at 12 months in TAM 2/2 mice ( Figure 1C).

Immune cell infiltrations in the liver
Histological analysis revealed an inflammatory phenotype characterized by immune cell infiltrations in the liver parenchymal regions of TAM 2/2 mice (Figure 2A). This phenotype was more severe as mice aged. No apparent cell infiltration was observed in the liver of 2-month-old mice (M2). However, evident immune cell infiltrations were found in the liver of TAM 2/2 mice at age 6 months (M6). The colonies of infiltrated cells grew larger as mice aged. At age 12 months (M12), infiltrated cell colonies were confluent, and most clusters of hepatocytes were disrupted. In contrast, no cellular infiltration was found in WT controls at any age from 1 to 12 months (data not shown).
The infiltrated cells contain macrophages and lymphocytes. The lymphocytes were confirmed by immunofluorescence staining, which consisted predominantly of CD4 + and CD8 + T cells, and relative few B cells ( Figure 2B, upper panels). By contrast, much less lymphocytes were observed in the liver of age-matched WT mice ( Figure 2B, lower panels). The three types of lymphocytes in the liver of TAM 2/2 mice were quantitatively analyzed using flow cytometry ( Figure 2C). The percentage of CD4 + cells was about 3fold more than CD8 + cells. The macrophages in the parenchyma of TAM 2/2 liver were determined by immunohistochemistry for F4/80. Abundant macrophages were detected in the TAM 2/2 liver, which consisted of ED1 + circulating macrophages and ED2 + resident macrophages ( Figure 2D, upper panels). In controls, only few F4/80 + and ED2 + cells were observed in the liver parenchymal regions of WT mice ( Figure 2D, lower panels).
Increased plasma IgG level and generation of autoantibodies in TAM 2/2 mice Given that TAM 2/2 mice exhibited phenotypes with several autoimmune diseases [25], we speculated that they may develop chronic AIH. Therefore, we examined characteristic serologic hallmarks of AIH, circulating autoantibodies and IgG level. Immunofluorescence staining on mouse hepatoma (Hepa1-6) cells showed the presence of ANA in the sera from TAM 2/2 mice at age 12 months ( Figure 3A). All sera obtained from 10 TAM 2/2 female mice positively stained nuclei of Hepa1-6 cells at 1:800 times dilution ( Figure 3B). The positive staining was even detected using 1,600 times diluted sera from three of these mice. In contrast, no ANA in the sera from WT mice was detected at .1:20 dilution. The anti-actin autoantibody component of SMA was detected by ELISA in 7 of the 10 sera from TAM 2/2 mice ( Figure 3C). Titration analysis showed that SMA titers were more than 1:20 in 8 of the 10 sera from TAM 2/2 mice ( Figure 3D). Plasma total IgG level in TAM 2/2 mice at age 12 months was about 4-fold greater than that in WT mice ( Figure 3E). These data indicate that TAM 2/2 mice generate characteristic autoantibodies and high level of IgG.

Expression of TAM RTKs and their ligands in the liver
To understand the function of TAM RTKs in the liver, we examined the expression of TAM RTKs and their ligands (ProS and Gas6). All TAM RTKs and their ligands were detected in the liver of WT mice ( Figure 4A). In contrast, TAM RTKs were absent in the liver of TAM 2/2 mice, confirming that TAM alleles were null. To clarify the cell type-specific expression of TAM Figure 4B, Tyro3 was only detected in KCs. However, Axl was abundantly expressed at a similar level in the three types of the liver cells. Mer was detected in KCs and SECs, but not in PCs. Gas6 and Protein S were prominently expressed in PCs, and relative weakly in KCs and SECs ( Figure 4B). Immunohistochemistry confirmed that Tyro3 and Mer were localized in spindleshaped sinusoidal cells, whereas Axl was evidently detected in some PCs ( Figure 4C). Gas6 and Protein S were observed in all types of the liver cells based on immunohistochemical staining ( Figure 4C).

Upregulation of inflammatory cytokines in the liver of TAM 2/2 mice
Given that TLR-mediated inflammatory cytokine production is involved in the development of autoimmune liver diseases [12] and that TAM RTKs are inhibitors of inflammatory responses [28], we examined the expression of inflammatory cytokines in the liver of TAM 2/2 mice. We found that, the inflammatory cytokines including interleukin (IL)-1b, IL-6, tumor necrosis factor alpha (TNF-a) and type 1 interferons (IFN-a and IFN-b) were dramatically upregulated in the liver of TAM 2/2 mice ( Figure  5A). These cytokines can be induced by the activation of nuclear factor (NF)-kB and interferon regulatory factor 3 (IRF3), leading to the induction of numerous inflammatory cytokines. Accordingly, NF-kB and IRF3 are activated in the liver of TAM 2/2 mice as evident phosphorylation of NF-kB and IRF3 was observed ( Figure  5B).

Bone marrow-derived cells are critical in the liver damage
To determine the contribution of infiltrated cells and resident liver cells to the liver damage in TAM 2/2 mice, we performed bone marrow transplantation assays. At 6 months after transplan-tation, WT bone marrow cells transferred into irradiated TAM 2/2 (WTRTAM 2/2 ) mice resulted in dramatically down-regulation of inflammatory cytokine expression in the liver ( Figure 6A). The liver damage was reduced in the engrafted mice, as serum ALT and AST activities in TAM 2/2 recipients were significantly decreased ( Figure  6B). By contrast, the transplantation of TAM 2/2 bone marrows into WT (TAM 2/2 RWT) mice resulted in significant augmentation in inflammatory cytokine production in the liver and serum ALT/AST activities at 6 months after transplantation ( Figure 6C,  D). Notably, histological analysis revealed that cellular infiltrations were observed in the liver of TAM 2/2 RWT mice, but not in the liver of WTRTAM 2/2 mice (data not shown). To confirm the origin of circulating cells, the genotype of peripheral leukocytes was examined by PCR. Amplification of mutant and wild-type Axl showed that the circulating leukocytes in engrafted mice were from donor mice ( Figure 6E). The results suggest that bone marrowderived cells lacking TAM RTKs are critical in the liver damage.

Discussion
TAM RTKs are potent negative regulators of innate immunity and required for the optimal phagocytic clearance of apoptotic cells [29,34,35]. Mice lacking function of TAM RTKs display a profound dysregulation of the immune responses [25]. Here, we demonstrate that TAM 2/2 mice develop progressively immune liver damage. This is the first report showing the liver inflammatory disease in the TAM 2/2 mice.
The lack of a reliable animal model impedes the understanding the etiology of AIH. Several attempts have been made to develop animal models that can reflect the persistent liver damage occurring in human AIH. However, most models have only been successful in causing a transient form of liver damage [36,37]. Our present study shows that TAM 2/2 mice develop progressively liver inflammation and a persistent liver damage. Moreover, we demonstrated that TAM 2/2 mice generate increased plasma IgG level, non-organ specific autoantibodies including ANA and SMA. These phenotypes are the characteristics of chronic AIH [37]. Notably, ANA and SMA in TAM 2/2 mice could be secondary to an antigen driven response by the accumulation of apoptotic and necrotic cells in different tissues, because TAM receptor mutation impairs phagocytic clearance of apoptotic cells by macrophages [35].
The liver is constantly exposed to the microbial products from the enteric microflora through the portal circulation to the liver in physiological condition [38]. However, no obvious inflammation occurs under what would be pathologic condition in other tissues, due to '' liver tolerance '' [10]. The liver tolerance may arise from different mechanisms [39]. Immune damage of the liver requires the priming of liver-specific T cells, which then migrate into the liver to induce autoimmunity [40,41]. Our present study showed that the immune cells including macrophages and T lymphocytes evidently infiltrate into the liver of TAM 2/2 mice. Notably, more CD4 + vs CD8 + T cells were found, which correspond to previous observation in AIH [42]. A recent study indicated that activated liver-specific T cells alone were not sufficient to trigger the liver damage, and TLR3-driven inflammatory cytokine release was Figure 1. Liver damage in TAM 2/2 mice. (A) Representative images of H&E stained paraffin-embedded liver sections of 12-month-old WT and TAM 2/2 female mice. Scale bar = 10 mm. (B) Serum ALT and AST levels in 12-month-old mice. Each circle represents unit of ALT or AST in sera of an individual mouse. (C) Serum ALT and AST levels in mice at the indicated ages. Note that the livers were progressively damaged as TAM 2/2 mice aged. (D) Serum ALT and AST levels in 12-month-old mice mutant singly, doubly, and triply for TAM RTKs. T, A, and M represent Tyro3, Axl and Mer, respectively. Data are mean 6 SEM, n$10. *P,0.05, **P,0.01. doi:10.1371/journal.pone.0066604.g001 Immunofluorescence was performed using FITC-conjugated antibodies against lymphocyte markers. Note that the infiltrated lymphocytes predominantly consist of CD4 + and CD8 + T cells. (C) Quantitatively analysis of lymphocytes. The cells were labeled with FITC-conjugated antibodies against CD4, CD8 and B220, and subsequently subject to flow cytometry. (D) Identification of macrophages. Immunohistochemistry was used for the identification of total macrophages (F4/80 + ), circulating macrophages (ED1 + ) and resident macrophages (ED2 + ). Note that the macrophages consist of resident and infiltrated cells in the liver of TAM 2/2 mice, whereas only resident macrophages were observed in WT controls. Frozen liver sections of 10-month-old mice were used for the immune staining. Images are representatives of at least 5 mice. Scale bar = 20 mm. doi:10.1371/journal.pone.0066604.g002 required to induce autoimmune liver disease [43]. The immune cell infiltrations and elevated inflammatory factors in the liver of TAM 2/2 mice should be responsible for the liver damage. This speculation is also supported by the previous reports that IFNa,IFN-b and TNF-a are involved in the pathogenesis of autoimmune diseases and that elevated TNF-a plays an essential role in the progression of liver injury [44,45,46]. Moreover, we observed augmentations in the expression of cell adhesion proteins including VCAM-1, ICAM-1 and E-selectin in the liver of TAM 2/2 mice (data not shown), which should be responsible for the immune cell infiltrations.
The induction of inflammatory cytokines could result from activation of NF-kB and IRF3. We showed that NF-kB and IRF3 are activated in the liver of TAM 2/2 mice. Upon pathogen stimulation, wild-type Kupffer cells and TAM 2/2 peritoneal macrophages produce high levels of inflammatory cytokines [33,47,48]. Hepatic stellate cells (HSCs) express cell adhesion molecules (VCAM-1, ICAM-1 and E-selectin) in response to invading pathogens [49]. In addition, hepatic dendritic cells (DCs) and NK cells synthesize inflammatory factors and chemokines [50,51]. Notably, these hepatic cells are less responsive to pathogen stimulation compared to those sentinel cells such as circulating DCs and macrophages [15]. Therefore, the upregulation of these inflammatory cytokines in the liver of TAM 2/2 mice should be mainly attributable to the infiltrated immune cells. This speculation is supported by the literatures showing that TAM receptors inhibit peripheral DC and macrophage activation [25]. TAM RTKs were prominently expressed in non-parenchymal cells except that Axl was also detected in parenchymal hepatocytes. Recent findings revealed that TAM RTKs are potent inhibitors of TLR-triggered innate immune response in different types of cells [28,52,53]. Further, all hepatic cells express ProS and Gas6. It should be worthwhile to investigate whether ProS/Gas6- TAM signaling inhibits innate immune responses in the liver, thus contribute to the maintenance of the liver tolerance.
Although some resident liver cells express TAM RTKs, loss of these receptors in the resident liver cells is not sufficient for the liver damage. However, bone marrow-derived cells play a critical role in the liver damage. The inflammatory cytokine expression and the liver damage in WTRTAM 2/2 mice were significantly reduced compared to TAM 2/2 mice, and cellular infiltrations were not observed in the liver of these engrafted mice. By contrast, we observed the immune cell infiltrations, upregulation of inflammatory cytokines in the liver of TAM 2/2 RWT mice and the liver damage. These results suggest that the infiltrated cells lacking TAM RTKs are responsible for the cellular infiltrations, elevated inflammatory cytokines and liver damage. We speculate that loss of TAM RTKs in the immune cells promotes cellular infiltrations into the liver, and the infiltrated cells produced the elevated inflammatory cytokines, thus results in the liver damage.
In conclusion, the present study reveals that TAM RTKs play a critical role in maintaining the liver tolerance. Disruption of TAM function converts spontaneous inflammatory responses leading to chronic inflammatory liver damage. Data provide insights into the mechanisms underlying the liver tolerance.