Background
Liver failure (LF) has a very high mortality rate due to the loss of functional liver mass below a critical level [
1]. The loss of liver functions, such as detoxification, metabolic and regulatory activities, may cause severe complications, including hepatic coma, systemic hemodynamic dysfunction and multi-organ failure [
1]-[
3]. Hepatitis B related LF is the most common severe disease requiring immediate hospitalization in China [
4]. Although the pathologic mechanisms underlying hepatitis B related LF are not fully understood, evidence suggests that the immune response is involved in the pathogenesis of liver injury [
2].
Natural killer (NK) cells are a fundamental component of the innate immune system, and they play an important role in the first-line defense against viral infections and tumor transformation without prior sensitization [
5]-[
7]. Hepatic NK cells which represent 20%-30% of liver lymphocytes, are located in the liver sinusoids and are adherent to the endothelium [
8]-[
10]. Increasing evidence suggests that NK cells play a pivotal role in the pathogenesis of liver injury, thus contributing to LF. Hepatic NK cells can directly induce hepatocyte injury through the surface expression of death ligands (TRAIL/TRAIL receptor, Fas/Fas ligand and NKG2D/NKG2D ligand) and the release of perforin [
11]-[
14]. The production of IFN-γ and TNF-γ, a hallmark of NK cell activation, is another important mechanism contributing to liver injury, which occurs through the induction of hepatocyte apoptosis and activation/recruitment of other immune effector cells [
12],[
15],[
16]. However, few studies have investigated the phenotypes and functions of NK cells involved in hepatitis B related LF, and the precise mechanism underlying NK cell regulation is not fully understood.
Fibroblasts are ubiquitous cells that provide more than a source of scaffolding on which other cells function and migrate. Fibroblasts play an important role in initiating inflammation via leukocyte recruitment to the site of tissue injury [
17]. Moreover, recent research has reported that fibroblasts isolated from different tumors can modulate T or NK cell functions [
18],[
19]. Following hepatic injury, the liver stroma undergoes extensive remodeling by liver myofibroblasts (LMFs) that are principally derived from activated hepatic stellate cells (HSCs) [
20],[
21]. LMFs can release cytokines and chemokines, such as IL-6, IL-12, HGF, VEGF and CXCL8, to promote the recruitment and positioning of lymphocytes in the inflamed liver as well as affect immune responses [
22]. In a murine study, it was shown that activated HSCs attenuated intrahepatic T cell activation [
23],[
24]. However, few studies have focused on the effect of LMFs from hepatitis B related LF patients on NK cells.
In the present study, we found that the percentage of peripheral NK cells was down-regulated with dysfunction in hepatitis B related LF patients. Our study also consistently showed that LMFs inhibited the IL-2-induced up-regulation of NK cell triggering receptors, cytokine production and cytotoxicity via prostaglandin (PG) E2 production in vitro using a cell-cell direct interaction model. Our research may provide novel insight into the pathogenesis of hepatitis B related LF.
Methods
Patients and specimens
Liver tissues and peripheral blood were all obtained from patients in the medical center of Sun Yat-sen University as described in our previous report [
25]. Blood were from 20 patients with hepatitis B induced liver failure (Additional file
1: Table S1) and 20 healthy individuals as controls; diseased liver tissues were from 4 patients undergoing transplantation for hepatitis B induced LF (Additional file
1: Table S1); healthy livers were from 3 patients undergoing surgery for hepatic hemangioma; normal skin fibroblasts (NFs) were from 2 patients undergoing circumcision. All the samples were anonymously coded in accordance with the local ethical guidelines, as stipulated by the Declaration of Helsinki. Written informed consent was obtained from the patients, and the protocol was approved by the Review Board of Sun Yat-sen University.
Isolation and culture of fibroblasts
NFs and LMFs were isolated as described previously [
25]. Briefly, 50 grams of liver tissue or 20 grams of foreskin sample was diced and digested using type-I collagenase (100 U/mL; GIBCO, USA) and hyaluronidase (125 U/mL; Sigma-Aldrich, St. Louis, MO) followed by mechanical homogenization in a Stomacher 60 Circulator (Seward, NY, USA). The cell suspensions derived from liver specimens were cultured in DMEM medium plus 10% FBS (GIBCO, USA). Fibroblasts that had been passaged for up to 3-8 passage doublings were used for subsequent experiments to minimize clonal selection and culture stress, which can occur during extended tissue culture.
Immunofluorescence
LMFs were cultured in 48-well flat-bottom plates. When the cells were approximately 50% confluent, they were fixed with 100% carbinol (15 minutes), rinsed and pre-wetted with phosphate-buffered saline (PBS) prior to the addition of mouse anti-human monoclonal antibodies (mAbs)-vimentin, fibronectin and α-smooth muscle actin (α-SMA), rabbit anti-human mAb-fibroblast surface protein (FSP) and immunoglobulin (IgG) controls in Tris-buffered saline (pH 7.4) for 60 minutes. The mAbs were all purchased from Abcam (Cambridge, MA, USA). The cells were washed and incubated for 30 minutes in isotype-relevant donkey anti-mouse or anti-rabbit fluorescein-isothiocyanate-conjugated secondary antibodies (IgG-AF555 or IgG-AF488, respectively; Molecular Probes, Carlsbad, CA), and the nuclei were counterstained with 40-6-diamidino-2-phenylindole (Sigma Aldrich, St. Louis, MO, USA). The images were viewed and assessed using a fluorescence microscope (LEICA DMI 4000B; Germany) at 488 nm and analyzed with Leica Application suite software (version 4.0).
Immunohistochemistry
Paraffin-embedded samples were cut into 5 μm sections and processed for immunohistochemistry according to Kuang et al. [
26]. Following incubation with anti-CD56Ab and α-SMA (Abcam, Cambridge, MA), the sections were stained using the Envision System with diaminobenzidine (DakoCytomation, Glostrup, Denmark).
Isolation of NK cells
To obtain purified NK cells, peripheral blood mononuclear cells from healthy donors were first isolated via density gradient centrifugation, according to Pradier et al. [
27], and magnetic active cell sorting (Miltenyi Biotec GmbH, Bergisch Gladbach, Germany) was subsequently used to obtain NK cells. Only the populations displaying more than 90% CD56
+CD3
- NK cells were selected.
NK cell/fibroblast co-culture
NK cells from healthy donors were cultured in RPMI-1640 plus 10% FBS in 48-well flat-bottom microtiter plates (5 × 104 cells per well) in the absence or presence of fibroblasts (NK:fibroblast ratio, 2.5:1), and 100 IU/mL rhIL-2 (R&D systems, Oxford, United Kingdom) was added when indicated. At the indicated time intervals, NK cells were harvested, counted and analyzed. When indicated, 0.5 mM 1-methyl-tryptophan (Sigma, St. Louis, MO, USA) and/or 5 μm NS398 (Cayman Chemicals, Ann Arbor, MI, USA) were added at the onset of the co-cultures.
Flow cytometry analysis
Mouse anti-human mAbs against CD73, CD56, CD69, NKp44, NKp30, NKp46, NKG2D, DNAM-1 and granzyme B were purchased from BD Biosciences (San Jose, CA, USA), mouse anti-human mAbs against CD105, CD90, CD13, CD44, CD31, CD34, CD45 and CD3 were purchased from eBioscience (San Diego, CA, USA) and mAbs against TNF-α, IFN-γ and perforin were obtained from Beckman Coulter (Fullerton, CA, USA) and Biolegend (San Diego, CA, USA).
The cells were collected, washed, and resuspended in PBS supplemented with 1% heat-inactivated FBS. Thereafter, the cells were directly stained extracellularly with 2 μg mAb in a total staining volume of 100 μL per 106 cells. The cells were stimulated at 37°C for 4 hours with Leukocyte Activation Cocktail (BD Pharmingen, San Jose, CA, USA) and fixed and permeabilized prior to intracellular staining using Pharmingen's staining protocol. The data were acquired on a Gallios instrument (Beckman Coulter, Brea, CA, USA) and analyzed with FlowJo software.
Cytotoxicity assays
The NK cells were tested for cytolytic activity via killing assays. Briefly, the purified NK cells derived from healthy donors were cultured with rhIL-2 in the presence or absence of fibroblasts. K562 cells were used as target cells (a generous gift from Dr. Dongjun Lin, Leukemia Research Institute of Sun Yat-sen University) and cocultured with NK cells for 2 hours at 37°C in 96-well V-bottom plates (2 × 10
4 K562 cells per well). Thereafter, the target cells were stained with 5 μL annexin V (AV)-FITC and propidium iodide (PI), according to Kurschus et al. [
28], and incubated in the dark for 15 minutes at room temperature. The cells were analyzed using multicolor flow cytometry (FACS Vantage-SE, BD Immunocytometry Systems, San Diego, CA, USA). Apoptotic and dead cells were identified by annexin V and PI staining. The lytic potential of the NK cells was tested by plating cells at different effector-to-target cell (E/T) ratios.
ELISA
Supernatants were generated by seeding 5 × 104 cells per well into 48-well plates in 500 μL of RPMI-1640/1% bovine serum albumin (BSA) containing 2 mmol/L L-glutamine, 60 μg/mL benzylpenicillin and 100 μg/mL streptomycin (all purchased from Sigma Aldrich, St. Louis, MO, USA). The conditioned culture supernatants were collected and analyzed for the presence of PGE2 by specific ELISA kits according to the manufacturer’s instructions (R&D systems, Abingdon, UK).
Western blotting
The fibroblasts were cultured for 5 days in RPMI-1640 supplemented with 10% FBS and 100 U/mL rhIL-2 in 24-well cell culture cluster flat-bottom plates (2.5 × 105 cells per well) in the absence or presence of NK cells (NK:fibroblast ratio, 2.5:1). Equal amounts of cellular protein were separated by 10% SDS-PAGE, immunoblotted with Abs against COX-2 (Abcam, Cambridge, MA, USA) and β-actin (Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and visualized with an ECL kit.
Statistical analysis
Multiple comparisons were made between the different groups via the Mann-Whitney U test. All of the calculations were performed using Prism software (release 5.00, GraphPad Software, San Diego, CA, USA). A value of P <0.05 was considered statistically significant.
Discussion
In the present study, we provide evidence that LMFs isolated from hepatitis B related LF patients interfere substantially with the activation of NK cells in vitro. We show that LMFs attenuated rhIL-2-induced NK cell triggering receptors, cytotoxicity and cytokine production. This suppressive function, which is largely dependent on PGE2 release, appears to be specifically induced by LMFs; NFs had a minimal effect on NK cells and released low levels of PGE2.
In the current study, we observed that NKp30 were expressed at lower levels on peripheral NK cells in hepatitis B related LF patients compared to healthy controls (P = 0.0201). However, Zou Y et al. reported up-regulated NKp30 expression on peripheral NK cells in patients with HBV-related acute-on-chronic LF [
12]. The controversy regarding NKp30 expression on NK cells of LF patients may derive from the use of different controls and the complexity of the disease. Firstly, the controls in our study were all from healthy donors, but in the report by Zou Y et al., all the controls were from chronic hepatitis B patients. Secondly, all the samples in our study were from patients with late stage LF, which means that the patients were preparing for liver transplantation. However, in the report by Zou Y et al., the conditions of the patients appeared improved, and they may have recovered as a result of conservative treatments. We speculate that at the early stage of liver injury, NK cells may be activated and accelerate inflammation, but at the late stage of liver disease, the function of NK cells is suppressed. This notion is also supported by evidence that the cytolytic activities of NK cells increase during HBV-related spontaneous “hepatic flares” (the early stage of liver injury) [
14],[
30] and that NK cell functions are suppressed during advanced liver injury [
31].
We studied differentiated LMFs isolated directly from different diseased human livers. There were no consistent differences between the LMFs isolated from patients with hepatitis B related LF, and all the LMFs expressed similar markers and could secrete similar levels of PGE2 (data not shown). Interestingly, the HSCs from healthy livers could also be activated into LMFs through in vitro culture, and no differences were found in their levels of PGE2 secretion (Additional file
3: Figure S2), suggesting their stability for further research. Consistent with our results, other investigators have recently reported that LMF preparations from diseased livers of different etiologies secreted similar patterns of proinflammatory cytokines and chemokines [
22].
The present investigation provides evidence that LMFs can regulate NK cell activation in vitro. Hepatic NK cells are comparable to in vitro IL-2-activated NK cells derived from the spleen or blood and are thus considered to be naturally activated by the hepatic microenvironment [
32]. Therefore, we used IL-2-stimulated NK cells isolated from the peripheral blood of healthy donors for our cultures. With respect to the mechanisms of inhibition, we showed that the NK cell activation receptors, including NKp30, DNAM-1, NKp44 and NKG2D, were partially inhibited when exposed to LMFs. Indeed, the surface expression of CD69 and NKp46 was not inhibited upon LMF co-culture in the study. We believe that the effect of LMFs on NK cells did not involve a general blockade of NK cell activation. Consistent with our findings, investigators have reported that the surface expression of CD69 and NKp46 that are either up-regulated (NKp46) or induced ex-novo (CD69) after exposure to IL-2 was not inhibited by the interaction with tumor-associated fibroblasts [
19]. Elucidating the molecular mechanisms of action by LMFs on NK cells is an interesting and ongoing research project in our laboratory.
Under co-culture conditions, NFs, which don’t release PGE2, also inhibit the expression of NKp30 and DNAM-1 on NK cells (albeit to a lesser extent). This was consistent with previous observations and suggested that fibroblasts, which are rather numerous in organisms, may represent a cell population that is underrated for its involvement in immunomodulation [
33]. In a recent study, we observed that NFs could also release different soluble factors [
34]. We accept that it is highly likely that other soluble factors may also be responsible for fibroblast-induced immunosuppression on NK cells. However, we argue that PGE2 is likely to represent a driver of inhibition on NK cells.
In the current study, we observed that LMFs produced more PGE2 when they were in contact with NK cells, which in turn led to dysfunctional NK cells. These data suggest a negative feedback mechanism involving the LMF-PGE2-NK cell axis. The downregulation of NK cell function during liver injury may facilitate liver regeneration and prevent further hepatocyte injury [
10]. However, accumulating evidence supports an antifibrotic role for NK cells via the inhibition of HSCs by apoptosis and IFN-γ production [
35]-[
37]. Therefore, the dysfunction of NK cells during liver injury may facilitate liver fibrosis or cirrhosis [
38]. Simultaneously, decreased NK cell activity in cirrhotic patients has been related to an increased incidence and invasiveness of hepatocellular carcinoma [
38],[
39]. The tumor cells may escape from NK cell immune surveillance and directly and indirectly facilitate tumor progression and metastasis [
40]. Therefore, we believe that LMFs are double-edged swords.
The precise mechanisms underlying the deterioration of liver function during hepatitis B related LF remain unclear. However, the impairment of cellular immunity is believed to be a contributing factor. In general, activated NK cells accelerate liver injury by producing proinflammatory cytokines, such as granzyme B and IFN-γ, and by killing hepatocytes [
41]. Moreover, the function of NK cells can be further enhanced by other cytokines, such as IFN-γ, IL-8, IL-12 and IL-15 [
14],[
30]. The induction of PGE2 by LMFs may represent a mechanism of limiting liver damage, such that few patients with fibrosis develop fulminant LF [
42]. Consistent with our results, a recent study reports that liver fibrosis may be protective in the context of acute liver injury [
43]. We hypothesize that NK cells may be inhibited and preferentially skewed toward suppression activity during hepatitis B related LF, depending on the balance between the levels of PGE2 and cytokines mentioned above. Further investigation is therefore warranted to determine the hypothesis.
Our study has limitations. First, most of our experiments were done in vitro, which may not stand for the in vivo state of hepatitis B related LF patients. Nevertheless, our study is of hypothesis-generating value for the design of future in vivo studies. Second, the 20 patients from whom serum was studied and the 4 liver tissues of subjects with hepatitis B related LF are not representative for all phases of LF and these numbers are small with respect to the different conditions of these LF patients. In China, hepatitis B is involved in more than 80% of LF cases due to a high incidence of HBV infection, and most LF patients died except for liver transplantation [
44]. However it is very difficult to obtain sufficient quantities of liver tissue from these kind of patients for the shortage of donor organs. Nonetheless, since there were no consistent differences that characterized LMFs isolated from different hepatitis B related LF patients in our study, we believe that this phenomenon is not peculiar to a few patients and as seems more likely applicable to most LF patients. Despite these shortcomings, we believe our analysis shows the role of LMFs in the regulation of NK cell functions during the process of hepatitis B related LF. Larger studies are necessary to fully address this important question.
In conclusion, LMFs not only regulate remodeling against liver injury by producing the extracellular matrix but also release PGE2, which can regulate immune responses. The notion that NK cells can trigger a paradoxical suppressive loop by enhancing the release of PGE2 by LMFs in vitro reveals an interesting area of investigation for the field of immune subversion in hepatitis B related LF patients. Indeed, this observation suggests that LMFs not only suppress NK cells but may also interact with them, thereby eliciting reciprocal cross-talk to protect hepatocytes from further injury. Further studies should be performed to extend our knowledge on this matter in view of the attempts to exploit NK cell-based cellular therapies [
45].
Authors’ contributions
MZ and FW (the principle investigators) performed cell isolations, cell-coculture studies, immunofluorescence, flow cytometry analysis, created figures and contributed to the design of the study and the writing of the manuscript. YC performed cell isolations, immunohistochemistry, flow cytometry and Western blotting studies. QT and QZ performed flow cytometry, ELISA and assisted in liver myofibroblasts evaluation and in manuscript preparation. YZ and LP acquired specimens and contributed to the design of the study. SL and ZG (the corresponding authors) designed and coordinated the study, contributed to experimental setup, data analysis and interpretation, and drafted and edited the manuscript. All authors read and approved the final manuscript.
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Competing interests
The authors declare that they have no competing interests.