Potential conflict of interest: Nothing to report.
The immunopathogenic process from hepatitis B virus (HBV) infection to liver fibrosis is incompletely understood because it lacks an animal model. In this study we observed the development of liver fibrosis in HBV transgenic (HBV-tg) mice and found the roles of natural killer T (NKT) cells in HBV-related liver fibrosis. We found liver fibrosis spontaneously developed in HBV-tg mice with the elevated transcription of col1a1, matrix metalloproteinase (MMP)2, and tissue inhibitor of metalloproteinase (TIMP)1. Mice were then injected with repetitive hepatotoxin carbon tetrachloride (CCl4) to induce prominent liver fibrosis. After chronic CCl4 treatment, the serum alanine aminotransferase (ALT) was higher, the liver regenerative nodules became more and bigger, and the fibrosis area was remarkably increased in HBV-tg mice than in C57BL/6 mice. Moreover, the increase in col1a1 and MMP2 transcription was greater, with a sustaining high level of TIMP1 and a greater activation of hepatic stellate cells (HSCs) in the livers of CCl4-treated HBV-tg mice. Our data also showed that there were more liver mononuclear cells (MNCs) in HBV-tg mice after CCl4 injection, and Rag1−/− mice adoptive transferred lymphocytes from HBV-tg mice displayed increased collagen deposition. Further study demonstrated the number of liver NKT cells increased after CCl4 treatment and NKT cells were overactivated in HBV-tg mice in the long term. It was further confirmed that NKT cells were critical for HSCs activation by depletion of NKT cells of HBV-tg mice and adoptive transfer of purified NKT cells from HBV-tg mice into recipient Rag1−/− mice. The inflammatory cytokines IL-4 and IL-13 produced by NKT cells played a pivotal role in HSCs activation in an in vitro coculture experiment. Conclusion: These data suggest that NKT cells from HBV-tg mice induce the HSC activation in liver fibrogenesis. (HEPATOLOGY 2011;.)
Liver fibrosis is considered as an outcome of chronic liver injury during a long-term wound-healing response,1, 2 which causes increasing amounts of extracellular matrix (ECM) deposition in the liver and eventually leads to liver fibrosis and later cirrhosis.1, 2 The hepatic stellate cell (HSC) is the main ECM-producing cell in liver fibrosis,1-6 and upon activation, HSCs differentiate from quiescent vitamin A-storing cell into proliferative myofibroblasts.1-4, 6 Activated HSCs express many ECM proteins including collagen, α-smooth muscle actin (α-SMA), transforming growth factor-β1 (TGFβ), matrix metalloproteinase (MMP), and tissue inhibitors of metalloproteinases (TIMP), which all contribute to liver fibrosis.2
Clinical studies suggest that chronic infection with hepatitis B virus (HBV) has a high risk for the development of liver fibrosis and later cirrhosis in human patients.7-10 Most studies on the relationship between HBV infection and liver fibrosis were based on clinical data, the results of which demonstrated that the pathologic mechanisms are relatively variable and complex. For example, the human gene polymorphisms such as glutathione and angiotensinogen were associated with HBV-related liver cirrhosis11, 12 and the HBV gene mutation was another factor in the severity of the disease.13 Recently, one study revealed that the HBV x gene-transfected hepatocyte cell lines could activate human HSCs, suggesting a direct interaction between HBV infection and activation of HSCs.14 An in vivo study demonstrated that infection with HBV in the severe combined immunodeficiency, urokinase-type plasminogen activator-transgenic mouse (uPA-SCID) xenografted with human hepatocytes could induce liver fibrosis.15 Immune dysfunction is always considered the cause of acute and chronic liver injury, and the immune cells, including T cell,16 B cell,17 Kupffer cell,18, 19 NK cell,20, 21 and natural killer T (NKT) cell22 affected the immunopathogenesis of liver fibrosis, but the host's immune mechanisms underlying HBV-related fibrosis are still unknown because of the lack of a practical animal model.2
NKT cells are abundant in the liver. They recognize lipid antigens presented by CD1d and had different roles in liver diseases. NKT cells produce a wide range of cytokines promptly after activation.23 It is well accepted that Th1 cytokines suppress fibrosis, whereas Th2 cytokines promote fibrosis.24 In wildtype (WT) mice, it was reported that NKT cells can suppress the activation of HSC.22 But in different animal models and in human patients the conclusions were controversial.25, 26
Although the acceleration of HBV infection to liver fibrosis have been extensively observed in clinical settings, the immune response during this process is not clear, especially in the condition of the HBV carriers with no obvious symptoms. In this study, by using HBV transgenic mice (HBV-tg) that mimic human HBV healthy carriers,27 we found liver fibrosis spontaneously occurred in old age of HBV-tg mice, and, importantly, HBV-tg mice were much more sensitive to the hepatotoxin CCl4-induced liver injury and liver fibrosis with the accompanied overactivation of HSCs. Further study demonstrated that hepatic NKT cells from HBV-tg mice could directly activate HSCs and thereafter induce liver fibrosis in the experiments of cellular depletion and adoptive transfer, and IL-4 and IL-13 secreted by NKT cells were considered a crucial step for the activation of HSCs.
HBV transgenic mice C57BL/6J-TgN (AlblHBV) 44Bri, which contains HBV genome S, pre-S, and X domains, were purchased from VITALRIVER experiment animal company (Beijing, China), who obtained the animals from Jackson Laboratory (Bar Harbor, ME). C57BL/6 mice were also purchased from VITALRIVER experiment animal company. Rag1−/− mice were purchased from Model Animal Research Center (Nanjing, China), who obtained the mice from Jackson Laboratory. Mice were housed in a specific pathogen-free facility and used according to the regulations of animal care of University of Science and Technology of China.
Models of Hepatic Injury and Fibrosis.
For chronic liver injury and fibrosis, male 7 to 10-week-old C57BL/6 and HBV-tg mice (weighing about 20-25 g) were injected (intraperitoneally, i.p., 2 times a week) with 0.5 μL per gram of body weight of pure CCl4 diluted with olive oil (Sigma). After several weeks' injections (2, 4, 10, and 14 weeks, respectively), mice were sacrificed 72 hours following the last CCl4 injection, and liver tissues and serum were collected. For acute liver injury, both mice were injected CCl4 once and then killed and analyzed at different timepoints.
Total RNA was isolated from liver tissue using Trizol Reagent (Invitrogen) according to the manufacturer's instructions. The same quantity of total RNA was reverse-transcribed to complementary DNA (cDNA) using M-MLV Transcriptase (Invitrogen) in the presence of oligo-dT primers (Shenggong, China). Quantitative PCR was performed using SYBR Green I (Takara) for 45 cycles at 15 seconds at 95° and 60 seconds at 60° with Rotor-Gene 6000 (Corbett Research) according to the manufacturer's instructions. Quantitative PCR primers were included in Supporting Information materials. Results were analyzed by ΔΔCt method as described before.28 Values were expressed as fold change in comparison with control.
Adoptive Cell Transfer for Liver Fibrosis.
Donor splenocytes were isolated from biweekly CCl4-treated C57BL/6 and HBV-tg mice. Four million splenocytes were adoptive transferred weekly (i.p.) to Rag1−/− recipient mice from the same genetic background and age for 4 weeks. The recipient Rag1−/− mice were sacrificed and liver tissues were collected 72 hours after the fourth transfer.
Coculture of HSCs and NKT Cells.
HSCs were isolated from liver of HBV-tg mice by collagenase and pronase perfusion and 8.2% Nycodenz (Sigma) gradient centrifugation.29 For isolation of hepatic NKT cells, first, HBV-tg mice were injected CCl4 (i.p. 0.5 μL of body weight) 12 hours and isolation of liver mononuclear cells (MNCs). Liver MNCs were stained for PE-NK1.1 and APC-CD3 (BD PharMingen, San Diego, CA) and sorted by flow cytometry (Becton Dickinson) for CD3+NK1.1+ NKT cells. For the coculture experiment, HSCs were previously cultured for 24 hours before constitution of NKT cells, and then cocultured (1:10) with CCl4-pretreated NKT cells for another 24 hours with or without functional purified neutralizing cytokine antibodies of IL-4, IL-13, or IFN-γ at a concentration of 5 μg/mL (eBioscience). After coculture, NKT cells were removed by washing, HSCs were visualized with phase-contrast microscopy, and collected by mild trypsinization for analyzing the transcription of α-SMA. HSCs RNA extraction were using RNAprep pure Micro Kit (Tiangen Biotech, Beijing, China).
Analysis of liver transaminase activity, liver histology, and immunohistochemistry for α-SMA, liver mononuclear cell (MNC) isolation and flow cytometric analysis, cell depletion, NKT cell preparation, and adoptive transfer to Rag1−/− mice in vivo CD1d block methods are included in the Supporting Information Materials online.
Student t test was chosen to compare values between two groups. Analysis of variance (ANOVA) was used to compare values from multiple groups. Data are expressed as means ± standard deviation (SD). P < 0.05 was considered statistically significant.
Spontaneous Occurring Liver Injury and Liver Fibrosis in HBV-tg Mice.
It was found that HBV-tg mice had an elevated level of serum ALT at the age of 2, 3, 4, and 6 months than that of normal C57BL/6 mice, showing that ALT levels were much higher in HBV-tg mice compared with C57BL/6 mice (all below 40 IU/L) (Fig. 1A), indicating that liver injury, as an important early disease indicator of chronic liver fibrosis,1, 2 spontaneously occurred in HBV-tg mice. We then examined liver transcription by real-time qPCR of three fibrosis-related genes to see if HBV-tg mice developed liver fibrosis at 6 months of age. As shown in Fig. 1B, col1a1, MMP2, and TIMP1 mRNA were all apparently up-regulated in 6-month-old HBV-tg mice. α-SMA is a marker extensively recognized for HSC activation. In Fig. 1C, the transcription of α-SMA was also increased in HBV-tg mice than in C57BL/6 mice. From Sirius Red staining (Fig. 1D), we found the obvious staining of fibrosis in 6-month-old HBV-tg mice, but not in C57BL/6 mice. These results indicate that, similar to human chronic hepatitis B patients, HBV-tg mice also develop liver fibrosis.
HBV-tg Mice Are Oversensitive to CCl4-Induced Liver Fibrosis.
We then investigated liver injury in C57BL/6 mice and HBV-tg mice after CCl4 treatment (0.5 μL/g of body weight). At first, the kinetics of liver injury was analyzed in C57BL/6 and HBV-tg mice 12, 24, 48, and 72 hours after a single CCl4 injection, as an acute liver injury model (Supporting Information Fig. 1). Following one CCl4 injection, serum ALT was elevated in both groups of mice, but the increase was much higher in HBV-tg mice at 12 and 24 hours after CCl4 treatment (Supporting Information Fig. 1A). For example, at 24 hours after single CCl4 injection the serum ATL value was 1,895 ± 361 IU/L in C57BL/6 mice but was 6,684 ± 946 IU/L in HBV-tg mice (Supporting Information Fig. 1A). The histopathological changes of liver were visualized in liver sections by hematoxylin and eosin (H&E) staining. Hepatic necrosis and inflammation were much more severe in HBV-tg mice than C57BL/6 mice (Supporting Information Fig. 1B).
Second, C57BL/6 and HBV-tg mice were treated with several CCl4 injections (0.5 μL/g of body weight, twice a week) for a longer time, as a sustained chronic liver injury model. Mice were killed at 72 hours following the last CCl4 injection and the results demonstrated that although the ALT value sustained a much lower level than the acute liver injury, which was previously described as a feature of chronic inflammatory fibrosis,30 the ALT level was significantly higher in HBV-tg mice than C57BL/6 mice after twice-a-week CCl4 treatment for 10 and 14 weeks (Fig. 2A). The H&E staining of liver tissue of HBV-tg mice showed more liver inflammation after CCl4 injections for 10 weeks (Supporting Information Fig. 2A). Furthermore, there was more inflammation and more distorted hepatic architectural formation in HBV-tg mice than that of C57BL/6 mice at 14 weeks of CCl4 injections (Fig. 2B).
As is known, chronic inflammation with HBV infection is tightly linked to liver fibrosis in human patients.9, 10 We then observed liver fibrosis in company with chronic liver injury. The liver appearance showed many more regenerative nodules (Fig. 3A), and Sirius Red staining showed more fibrous septa at week 10 and week 14 after chronic CCl4 treatment in HBV-tg mice than that of C57BL/6 mice (Supporting Information Fig. 2B; Fig. 3B), suggesting that HBV-tg mice are more sensitive to CCl4-induced liver fibrosis.
The mRNA expression of fibrosis-related genes were also examined in CCl4-induced liver fibrosis of HBV-tg mice by real-time qPCR (Fig. 4). Interestingly, we found that three fibrosis-related genes were significantly up-regulated in HBV-tg mice even without CCl4 injection (oil-treated control): col1a1 was higher in oil-treated HBV-tg mice at 10 and 14 weeks, MMP2 was higher in oil-treated HBV-tg mice at 4, 10, and 14 weeks, and TIMP1 was higher in HBV-tg mice at all timepoints. Moreover, CCl4 injection induced more overexpression of col1a1 at 2, 4, 10, and 14 weeks' treatment and MMP2 at 10 and 14 weeks' treatment in HBV-tg mice, but CCl4 injection did not have any impact on the increase of TIMP1 in HBV-tg mice, as TIMP1 expression was much higher in control HBV-tg mice (e.g., spontaneous occurring, as shown in Fig. 1B) than that of C57BL/6 mice.
Because HSCs are the main collagen-producing cells in liver fibrosis,1-6 we analyzed HSCs in CCl4-treated HBV-tg mice by detecting α-SMA. As shown by immunohistochemistry analysis in Supporting Information Fig. 2C and Fig. 5, injection of CCl4 twice a week for 10 or 14 weeks induced a greater deposition of α-SMA in the livers of HBV-tg mice. At the mRNA level, the transcription of α-SMA was significantly increased in HBV-tg mice at most timepoints (Supporting Information Fig. 3).
NKT Cells Activate HSCs and Aggravate Liver Fibrosis in HBV-tg Mice.
We evaluated the number of liver MNCs in the mice in response to CCl4 treatment, and as shown in Fig. 6A, there were more liver MNCs in HBV-tg mice after acute CCl4 injection at 24 hours and chronic CCl4 administration at 3 weeks, whereas there were no changes in the corresponding C57BL/6 mice. We then explored the roles of the immune response in liver fibrosis by the adoptive transfer experiment. Splenocytes were isolated from CCl4-treated C57BL/6 mice and HBV-tg mice, and then adoptively transferred to Rag1−/− mice once a week for 4 weeks. Interestingly, Rag1−/− mice receiving lymphocytes from HBV-tg mice showed increased collagen deposition by Sirius Red staining, whereas there was no change in Rag1−/− mice receiving splenocytes from C57BL/6 mice (Fig. 6B), which was consistent with the α-SMA transcription (Fig. 6C). This result indicates that immune cells from CCl4-treated HBV-tg mice are able to induce liver fibrosis.
We then wanted to know which cell population in liver MNCs exerted a function on the activation of HSCs. We found the numbers of both NK and NKT cells increased in HBV-tg mice after CCl4 treatment for at hours (e.g., acute fibrosis) or 3 weeks (e.g., chronic fibrosis) (Fig. 7A). The percentage and the number of CD69-positive NKT cells were more in 6-month-old HBV-tg mice than that of C57BL/6 mice, but the percentage of CD69-positive NK cells decreased in HBV-tg mice (Fig. 7B). In order to find the role of NK and NKT cells in CCl4-induced HSCs activation and liver fibrosis of HBV-tg mice, we depleted NK cells alone using AsGM1 antibody or NK and NKT cells together by using anti-NK1.1 antibody (PK136) before CCl4 injection. Interestingly, in HBV-tg mice, depletion of NK cells before CCl4 administration had no effect on HSCs activation but depletion of NKT and NK cells together decreased HSCs activation by examining transcription of α-SMA (Fig. 7C), indicating NKT cells possibly play a critical role in the HSC activation. On the contrary in C57BL/6 mice, depletion of NK or NKT cells, both increased the transcription of α-SMA (Fig. 7C), which was similarly documented previously.20-22 These results suggest that NKT cells play a critical role in HSCs overactivation and liver fibrosis only in HBV-tg mice, but both NK and NKT cells are antifibrotic in C57BL/6 mice.
To further demonstrate the role of NKT cells in HBV-related liver fibrosis, we adoptively transferred the purified liver NKT cells from C57BL/6 or HBV-tg mice to Rag1−/− mice and then treated the cellular-adoptively transferred Rag1−/− mice with CCl4. It was noted that the α-SMA expression was increased (Fig. 7D), along with more inflammatory cells in the liver (Supporting Information Fig. 4), if the transferred NKT cells were derived from HBV-tg mice but not from C57BL/6 mice, indicating NKT cells from HBV-tg mice might exert a function to activate HSCs in liver fibrosis. These results raise the possibility that more inflammation exists in HBV-tg mice-derived NKT cell-transferred Rag1−/− mice, which may initiate the activation of the stellate cells. Because CD1d expression by antigen-presenting cells is required for CD1d-restricted NKT cell activation, we blocked CD1d-NKT cell recognition by injecting anti-CD1d antibody before CCl4 injection. We observed that the HSC activation was reduced in CD1d antibody-pretreated HBV-tg mice (Fig. 7E). We also found that the transcription levels of TIMP1, one of the representative fibrotic genes, correlated with the change of α-SMA in NKT cell-depleted HBV-tg mice (Supporting Information Fig. 5A), HBV-tg mice-derived liver NKT cell-transferred Rag1−/− mice (Supporting Information Fig. 5B), and anti-CD1d mAb-treated HBV-tg mice (Supporting Information Fig. 5C). Taken together, these data suggest that NKT cells from HBV-tg mice aggravate the HSC activation to cause liver fibrosis.
Increased Production of IL-4, IL-13, and IFN-γ by NKT Cells from CCl4-Treated HBV-tg Mice.
NKT cells are well known for their strong and rapid production of cytokines. We observed that the transcriptional expression of IL-4, IL-13, and IFN-γ were significantly higher in the livers of HBV-tg mice after CCl4 injection (Fig. 8A). Moreover, the absolute number of NKT cells increased much more in HBV-tg mice after CCl4 injection at 6, 12, and 24 hours, respectively (Fig. 8B), along with significantly more increase in the number of IL-4-, IL-13-, or IFN-γ-secreting NKT cells in HBV-tg mice after CCl4 treatment than that of C57BL/6 mice (Fig. 8C). In the HSC and NKT cell coculture experiments, we found that neutralizing antibodies against IL-4 and IL-13 could attenuate the activation of HSC, but not the one against IFN-γ (Fig. 8D), which was further confirmed by microscopy that the cocultured HSCs with NKT cells showed a more fibroblast-like appearance, which became less when neutralizing with anti-IL-4 or anti-IL-13 mAbs but not anti-IFN-γ mAb (Supporting Information Fig. 6).
Chronic infection with HBV has been recognized to exacerbate liver fibrosis in patients.7-10 Mouse models for liver fibrosis have been successfully established in normal mice31-33 but there was no animal model to mimic liver fibrosis occurring in long-term HBV-infected patients. In this work, to our knowledge, we are the first to observe spontaneously occurring or CCl4-induced liver fibrosis in HBV-tg mice, and thus explored the possible immunologic mechanisms. The oversensitive liver fibrosis induced by CCl4 in HBV-tg mice may help us to investigate the precise mechanisms of liver fibrosis during chronic HBV infection.
A question always exists as to the relationship between liver injury and fibrosis. Although liver injury is not the only pathway involved in liver fibrosis, for example, HSCs might be directly activated without an intermediate step of aggressive liver injury through PDGF overexpression,34 in general, the severity and persistence of liver injury determines the outcome of liver fibrosis. In our study, liver fibrosis followed chronic liver injury. In Fig. 1 we show the spontaneously developed liver fibrosis (increased transcription of α-SMA, transcription of col1a1, MMP2, and TIMP1) accompanied by liver injury (elevated serum ALT) in 6-month-old HBV-tg mice. In Figs. 2-5 it is shown that in chronic CCl4-induced liver fibrosis, HBV-tg mice also had more liver fibrosis associated with more injury.
Generally, it was realized that cytotoxic T lymphocytes (CTLs) contribute to initiate hepatocyte injury.35, 36 However, the effector mechanisms are not only by CTLs but also by other immune cells, among which the roles of innate immune cells in CTL-related or -unrelated inflammatory-mediated fibrosis is unclear and needs study. In CTL-related injury, the CTL-derived cytokines might activate other innate immune cells (such as NKT cells) to produce more inflammatory cytokines, which indirectly lead to hepatocyte injury in addition to CTL direct-killing hepatocytes.37 On the other hand, in CTL-unrelated injury, previously we and others found that innate cells (NK, NKT cells) mediated liver injury in HBV transgenic mice (a mouse model without CTL function).38, 39 In our experiments with respect to HBV-related liver fibrosis, we found NKT cells are pivotal to activate HSCs (Figs. 7, 8). The accumulating data indicate that NKT cells could be activated through TCR recognition (e.g., Vα14/Vβ8 in mice) with antigen-CD1d complex (usually glycolipid) or other killer cell receptors such as NKG2D of NKT cells with their ligands (Rae-1, Mult-1).40-42 In our case, HBV infection may “stress” the hepatocyte, leading to the expression of virus-induced self-antigen-CD1d complex or the ligands of killer cell receptors on HBV-infected hepatocytes, which may be recognized by NKT cells and cause NKT cells to be activated to produce more HSC-activating inflammatory cytokines, including IL-4 and IL-13 (Fig. 8, Supporting Information Fig. 6), which was consistent with previous observations in HBV patients.26
As reported in WT mice, the naturally activated NKT cells have a protective effect on acute liver fibrosis, although no function in long-term liver fibrosis,22 which is contrary to our conclusion from HBV-tg mice in this study. We think this discrepancy may support the common idea that the NKT cell is a double-sword cell type.23, 43-45 We think this may be due to the different subsets of NKT cells in different disease models, with a different cell-differentiating environment (such as absence or presence of HBV). For example, in WT mice, although the naturally activated NKT cells could suppress stellate cell activation after CCl4 injection, the NKT cells stimulated with α-GalCer could activate stellate cells.22 In our study, we found that blockade of CD1d in HBV-tg mice may alleviate liver fibrosis (Fig. 7E), although we do not know which antigen (possibly a glycolipid which is hard to examine) was presented by CD1d molecules. The ongoing progress in CD1d signaling biology and NKT cell differentiation will help to resolve the basic questions.
Previously, we and others reported that NK cells are antifibrotic by both direct killing and the secreting of the antifibrosis cytokine interferon-γ in CCl4-treated WT mice.20, 21 Interestingly, in this study we found that NK cells sustained an inactive status with a lower level of CD69, even though the number of NK cells increased after CCl4 treatment in HBV-tg mice (Fig. 7A,B). This suggested that the inactivation of NK cells may cause the HBV-tg mice to lose the inhibitory function on HSCs, which is at least another explanation for the overactivation of HSCs in HBV-tg mice. Considering the positive regulation of NKT cells on activation of HSCs, the losing of inhibitory function of NK cells on HSCs may possibly also play an important role in liver fibrosis in HBV-tg mice, although we do not know how the NK cells become inactive.
In conclusion, the spontaneously developed liver fibrosis and aggravated CCl4-induced liver fibrosis in HBV-tg mice suggests the HBV-tg mice as a mouse model to investigate HBV-related liver fibrosis. From our findings, NKT cells exerted a positive role in HSCs activation, which implicates the inhibition of NKT cell activation (such as CD1d) or function (such as cytokine neutralization) that may attenuate HBV-related liver fibrosis.