Cheng Sun1, Binqing Fu1,2, Yufeng Gao3, Xiaofeng Liao1, Rui Sun1,2, Zhigang Tian1,2*, Haiming Wei1,2* 1 Institute of Immunology, School of Life Sciences, University of Science and Technology of China, Hefei, China, 2 Hefei National Laboratory for Physical Sciences at Microscale, University of Science and Technology of China, Hefei, China, 3 Department of Liver Diseases of the Second Affiliated Hospital of Anhui Medical University, Hefei, China http://www.plospathogens.org/article/info%3Adoi%2F10.1371%2Fjournal.ppat.1002594
Abstract TopThe mechanism underlying persistent hepatitis B virus (HBV) infection remains unclear. We investigated the role of innate immune responses to persistent HBV infection in 154 HBV-infected patients and 95 healthy controls. The expression of NKG2D- and 2B4-activating receptors on NK cells was significantly decreased, and moreover, the expression of DAP10 and SAP, the intracellular adaptor proteins of NKG2D and 2B4 (respectively), were lower, which then impaired NK cell-mediated cytotoxic capacity and interferon-γ production. Higher concentrations of transforming growth factor-beta 1 (TGF-β1) were found in sera from persistently infected HBV patients. TGF-β1 down-regulated the expression of NKG2D and 2B4 on NK cells in our in vitro study, leading to an impairment of their effector functions. Anti-TGF-β1 antibodies could restore the expression of NKG2D and 2B4 on NK cells in vitro. Furthermore, TGF-β1 induced cell-cycle arrest in NK cells by up-regulating the expression of p15 and p21 in NK cells from immunotolerant (IT) patients. We conclude that TGF-β1 may reduce the expression of NKG2D/DAP10 and 2B4/SAP, and those IT patients who are deficient in these double-activating signals have impaired NK cell function, which is correlated with persistent HBV infection.
Author Summary TopNK cells have been viewed as the most important effectors of the initial antiviral innate immune response. Their activation depends on the integration of signals from “co-activation” receptors, and the cytotoxic effects of NK cells on target cells are tempered by a need for combined signals from multiple activating receptors, such as NKG2D and 2B4. In this study, we showed that NKG2D and 2B4 expression levels were decreased on NK cells from patients in the IT phase of HBV infection. We further demonstrated that lower levels of intracellular adaptor proteins (DAP10 and SAP) were associated with lower surface expression of NKG2D and 2B4. As a result, the synergistically co-activated signalling pathway initiated by NKG2D and 2B4 did not operate properly in IT-phase patients. We demonstrated that high levels of soluble TGF-β1 were associated with the reduction of NKG2D and 2B4 in patients. In addition, we showed that TGF-β1 causes the cell-cycle arrest of NK cells by up-regulating the levels of p15 and p21 in NK cells from IT patients. Collectively, these findings may contribute to our understanding of the immune tolerance mechanism and aid in the development of novel therapeutic methods to clear HBV infection during the initial phase.
Introduction TopHepatitis B virus (HBV) infects more than 350 million people worldwide, accounting for over 1 million deaths annually due to immune-mediated chronic liver damage [1]–[3]. The course of HBV infection is complicated. Three phases of chronic HBV (CHB) infection are now widely accepted: 1) an immune tolerant (IT) phase, characterised by an HBV DNA concentration >200,000 IU/mL, normal alanine aminotransferase (ALT) levels, normal liver biopsy or only minimal inflammation and perinatal infection of infants born to HBsAg/HBeAg-positive mothers; 2) an immune active (IA) phase, which is also referred to as the “chronic hepatitis B phase” or the “immune clearance phase”, characterised by an HBV DNA concentration >20,000 IU/mL, elevated ALT levels and active hepatic inflammation on biopsy; and 3) an inactive (IN) phase, characterised by HBV DNA levels <2000 IU/mL, normal ALT levels and minimal or absent hepatic inflammation [4]. During the IA phase, the immune system of the host recognises the virus as foreign and initiates the immune clearance response, which results in hepatocyte damage. After one or more episodes of reactivation to the IA phase, patients begin the IN phase [5]–[7]. Patients in the IT phase have normal ALT levels and elevated levels of HBV DNA, commonly well above 1 million IU/mL; this phase can last anywhere from a few to >30 years [8]. The IT phase has been suggested to occur most frequently in patients who were infected via perinatal transmission from HBeAg-positive mothers [9]. In China, approximately 2 million infants are infected with HBV via perinatal transmission from HBsAg/HBeAg-positive mothers annually, and many of them are at high risk of developing chronic liver inflammation resulting in cirrhosis and hepatocellular carcinoma (HCC) in later life [10]–[13]. The risk of developing HCC with HBV infection is higher in East Asian countries than in Western countries, possibly due to the frequency of earlier viral infection [14]. During the IT phase of CHB infection, the virus evolves strategies to evade immune clearance in the majority of patients. However, the tolerance mechanism of the IT phase has not been widely studied. Due to their normal ALT levels, there is no available treatment to reduce the very high HBV DNA levels or alleviate psychological pressure in IT-phase patients. The development of an anti-HBV therapy for such patients will require insight into the mechanisms of HBV persistence.
The innate immune system provides the first line of defence in antiviral responses and activates adaptive immune responses. NK cells have been viewed as the most important effectors of the initial antiviral innate immune system [15]–[16]. Previous investigations have demonstrated that NK cells may be particularly important in patients with CHB. NK cells are highly enriched in the liver, and the site of HBV replication, and they are partially functionally tolerant in CHB [13], [17]. The substantial quantity of NK cells in the liver suggests that they act as “watcher cells”, surveying the liver for indications of cellular stress and implying that HBV has to evade NK cell-mediated immune responses to establish a persistent viral infection. The evidence shows that the cytotoxic capacity of NK cells is retained. Moreover, the activation of NK cells and the secretion of IFN-γ are strongly inhibited during CHB infection [18]. Blockage of IL-10 with or without TGF-β1 can restore the capacity of NK cells to produce the antiviral cytokine IFN-γ in CHB patients [19]. NKG2D, the activating receptor of NK cells, is constitutively expressed on human NK cells and CD8+ T cells [20]. The importance of the NKG2D pathway is highlighted by evidence that tumours and viruses have developed distinct escape mechanisms to avoid NKG2D-mediated recognition [21]–[26]. The signalling lymphocyte activation molecule (SLAM)-related receptor 2B4 is predominantly expressed on human NK cells and CD8+ T cells. The immunoregulatory role of 2B4 as an activating or inhibitory receptor depends on three factors: 1) surface expression, because costimulatory qualities are associated with low expression and inhibitory qualities are associated with high expression; 2) the coexpression of additional inhibitory molecules; and 3) the presence of the intracellular adaptor protein SLAM-associated protein (SAP) [18], [27]–[30]. NKG2D and 2B4 are the main triggering receptors of NK cells [31]. Many studies have provided evidence for a functional dichotomy in patients with chronic HBV that may contribute to virus persistence [32]. NK cell-mediated cytotoxicity is efficiently initiated by the NKG2D activation signal on NK cells. NKG2D recognises pressure-induced antigen signals on a target cell, whereas 2B4 receives the costimulatory signal. Therefore, these two molecules play a key role in NK cell activation and function. The tolerance mechanism of HBV persistence and the contribution of the NKG2D/DAP10 and 2B4/SAP pathways to the control of persistent HBV infection are unclear. Here, we show that NKG2D/DAP10 and 2B4/SAP are down-regulated on circulating NK cells and are associated with the impaired functionally of NK cells in IT-phase patients. Moreover, this defect is mediated by TGF-β1, which causes NK cell-cycle arrest by inducing high expression of p15 and p21. These findings may contribute to our understanding of immune tolerance mechanisms and may aid in the development of novel therapeutic methods to clear the viral infection during the initial phase.
Discussion TopThere are over 350 million persistent HBV carriers worldwide, and approximately 90% of children become chronic carriers after HBV infection [1], [3]. Immune tolerance is a serious problem in CHB carriers, who are at high risk of developing cirrhosis and HCC later in life [4], [10]. HBV persistence is thought to result from inefficiencies of innate and adaptive immune responses. NK cells are a major component of innate immunity. Accumulating evidence has suggested a role for NK cells in the fight to control persistent virus infection [15], [50]–[52]. However, the tolerance mechanisms of HBV persistence have not been well explored. For the first time (to our knowledge), we demonstrated that NKG2D/DAP10 and 2B4/SAP were down-regulated on circulating NK cells. Consequently, these NK cells were functionally impaired in IT-phase patients. The loss of these molecules was mediated by TGF-β1, which resulted in cell-cycle arrest due to the induction of p15 and p21. Our results indicated that NKG2D and 2B4 expression were decreased on circulating NK cells from IT-phase patients but not CHB patients in other phases or healthy controls. Furthermore, DAP10 and SAP, the intracellular adaptor proteins of NKG2D and 2B4 in humans, were also significantly reduced in NK cells from IT patients. It has been reported that NK cell cytotoxicity towards target cells is tempered by a request for combined signals from multiple activating receptors, such as NKG2D and 2B4 [16], [35]–[36]. To evaluate the functional consequences of the observed reduction in the proportion of NKG2D and 2B4, Ca2+ mobilisation triggered by the double-activating signals was analysed. Our data revealed that the down-regulation of Ca2+ flux was induced by synergism between NKG2D and 2B4 in NK cells from IT patients but occurred at normal levels in IA patients and healthy controls. In addition, NK cell cytotoxicity and IFN-γ production were decreased in IT patients compared to healthy controls and IA patients. Anti-TGF-β1 Abs could partially restore Ca2+ flux in NK cells from healthy controls incubated with sera from IT patients. Moreover, anti-TGF-β1 also restored NKG2D and 2B4 surface expression on NK cells incubated with sera from IT patients. p21 and p15 were elevated in IT patients and induced the arrest of the NK cell cycle. Taken together, these results suggest that TGF-β1 reduces NKG2D/DAP10 and 2B4/SAP expression on NK cells during persistent HBV infection and suppresses innate antiviral immunity by blocking the cell cycle, which would eventually provide an additional HBV strategy to avoid NK cell-mediated recognition.
Our results suggest that NK cells may be functionally impaired in IT patients. This conclusion is supported by at least four important findings. First, the percentage of NKG2D and 2B4 levels was lower on NK cells from patients in the IT phase compared to patients in other phases and healthy controls, which indicates an activation defect in circulating NK cells. Second, lower levels of intracellular adaptor proteins were associated with lower surface expression levels of NKG2D and 2B4, which implies that the signalling pathways leading to NK-cell activation might be impeded. Specific silencing of DAP10 or SAP led to deficient NK-cell cytotoxicity. Third, patients in the IT phase had a specific defect in NK cell cytotoxic activity. Moreover, there was a significant reduction in the production of IFN-γ by NK cells from patients in the IT phase compared to healthy controls and IA- and IN-phase patients. Fourth, NKG2D and 2B4 receptor synergy down-regulated the mobilisation of Ca2+ in primary NK cells from IT patients. These observations indicate that NK cells are not completely functional during the IT phase, which may contribute to the persistence of HBV infections. In our study, IT-phase HBV patients were characterised by lower levels of NKG2D and 2B4 compared with healthy individuals. To our knowledge, this is the first report of reduced expression of NKG2D/DAP10 and 2B4/SAP in IT patients, thus indicating that the function of NK cells was impaired due to deficiencies in the double-activating signals. In this context, abundant data in cancer patients has shown that impaired NK function can be attributed to the down-modulation of activating receptors, such as NKG2D, which can be inhibited via TGF-β1 [42]–[44]. Notably, NKG2D-dependent NK cell functions are also modulated during chronic HCV infection [26]. These findings provide further evidence for our observation that NKG2D was down-regulated during the IT phase of HBV infection. The increased expression of 2B4 on virus-specific CD8+ T cells, both in the peripheral blood and in the liver, is believed to mediate inhibitory signalling in the absence of SAP during CHB infection [53], but this has not been evaluated in NK cells in the presence of HBV infection. Interestingly, the absence of 2B4 resulted in diminished LCMV-specific CD8+ T cell responses and prolonged viral persistence in mice persistently infected with LCMV. Additionally, long-lasting viral persistence was regulated by 2B4-deficient NK cells acting early in infection. These observations illustrate the value of NK cell self-tolerance to activated CD8+ T cells in early infection, similar to the IT phase of HBV infection; these results also demonstrate how NK cells can regulate a persistent infection that appears to be dependent on T cell responses [54]. We also analysed the frequency of NKp30, NKp44, NKp46, CD16, CD27 and CD226 expression in circulating NK cells from healthy controls and CHB patients. We observed that the frequency of NKp30 was slightly decreased in patients in the IT phase relative to the healthy controls. No statistical differences existed on other receptors between patients and healthy controls. It has been previously shown that TGF-β1 can down-regulate NKp30 and NKG2D in vitro, suggesting that slightly decrease of NKp30 in IT-phase patients may related to the high levels of TGF-β1 [39]. Evidence from previous studies suggested that SAP deficiency could lead to the inhibition of NK cytotoxicity in humans [28], [55]–[56]. It has also been shown that in the absence of SAP, lymphoma development would normally be eliminated by apoptosis in patients with X-linked lymphoproliferative disease [57]. However, the analysis of potential apoptosis caused by reducing expression of SAP indicated that the deficiency of SAP would not lead to the significant apoptosis of NK92 cells (Data not show). Here, we show that the expression of SAP is deficient in NK cells from patients in the IT phase, suggesting that 2B4 receptors may become inhibitory, rather than activating, during the IT phase of HBV infection due to the absence of SAP. 2B4 molecules with inhibitory functions are responsible for the inability of NK cells to kill virus-infected cells. This feature may further compromise NK cell function in IT patients in the absence of SAP, which implies a positive regulatory role for SAP during NK cell activation. Furthermore, TGF-β down-modulates NKG2D expression on NK cells, leading to the impairment of their cytolytic activity and ability to produce IFN-γ during HCV infection [26]. Therefore, we suggest that there may be a functional impairment in NK cells in patients in the IT phase of HBV infection.
T and B cells each possess a single antigen receptor, which regulates their development and activation. Signals initiated through antigen receptors need co-stimulatory molecules to augment the signals. In contrast, NK cells do not possess one dominant receptor but instead depend on synergistic co-activation by NK cell receptors to initiate effector functions. In our studies, we have observed that Ca2+ mobilisation cannot be induced by NKG2D and 2B4 synergy in NK cells from IT patients, providing an explanation for the dominance of the double-activating signals in controlling NK cell activation. Strikingly, the deficiencies of the double-activating signals may be significant with respect to NK-cell tolerance. TGF-β1 inhibits NK cell activity and cytotoxicity by down-regulating NKG2D, as originally proposed in various cancer fields [42]–[44]. It has also been observed that TGF-β can impair NK cell cytolytic activity and IFN-γ production during HCV infection [26]. However, the role of TGF-β1 in blocking NK-cell function during the IT phase of HBV infection has not yet been well defined. Our data demonstrate that TGF-β1 can down-regulate NKG2D and 2B4 surface expression and restrain the Ca2+ mobilisation triggered by NKG2D and 2B4 receptor synergy. Moreover, anti-TGF-β1 Abs can restore NKG2D and 2B4 expression and also partially restore Ca2+ flux. HCV infection can induce TGF-β1, which can regulate HCV RNA replication [58]. HCV-specific Th17 cells are suppressed by virus-induced TGF-β [59]–[60]. Furthermore, the HBV X protein significantly up-regulates the expression of TGF-β1 and TGF-βRII in the LX-2 human hepatic stellate cell line [61]. The HBV-encoded pX oncoprotein amplifies TGF-β signalling [62], and HBV X antigen promotes TGF-β1 activity [63]. These findings suggest that during the IT phase of HBV infection, there may be a similar mechanism by which the virus evades host-protective immune responses.
NK cell function can be regulated by TGF-β [49] and by CD4+ CD25+ regulatory T cells (Tregs) through a TGF-β-dependent mechanism in both humans and mice [64]–[65]. We surmised that in IT patients, Treg cells might be the source of high levels of TGF-β. In a similar mechanism, Treg cells expressing membrane-bound TGF-β directly down-regulated the expression of NKG2D/2B4 on NK cell surfaces and inhibited NK cell effector functions [64]. Previous studies have reported that cancer-expanded myeloid-derived suppressor cells can directly contact NK cells and induce anergy via membrane-bound TGF-β1 [66], though other studies have found that Treg cells are the main negative regulators of NK cell function in tumours [64]. In our study, anti-TGF-β1 did not completely restore Ca2+ flux and cell-cycle arrest, suggesting that some other inhibitory molecules such as IL-10, which is also produced by regulatory CD8+ T cells [67], may also play a role in NK impairment during the IT phase of chronic HBV infection. Moreover, recent studies have shown that the partial functional tolerance induced in NK cells by the immunosuppressive cytokine environment in CHB can be corrected in vitro by the specific blockage of IL-10 and TGF-β [19]. These findings support our results for IT phase patients and suggest that regulatory CD8+ T cells may also influence NK cells in IT-phase patients by producing IL-10. Furthermore, our results demonstrate that the level of IL-10 in IT-phase serum was higher than in sera from healthy controls and IN-phase patients but lower than in sera from IA-phase patients. However, the levels of IL-10 in the sera were significantly lower than the levels of TGF-β1, which suggests that TGF-β1 was the predominant suppressive factor in IT-phase patients.
Our results suggest that the in vivo administration of nucleoside analogues partially overcomes the defect in NK cell function in patients. In addition, we observed that NK cell function was significantly lower at the onset of inflammation in IT patients, suggesting that some other inhibitory factors may restrain NK cell function during the initial phase of liver inflammation. Some studies have found that reduced IFN-γ production by NK cells in HBV infection independent of the IT phase [32], we believe this is due to different methods of classifying patient groups. Another interesting finding of our study was the observation that p21 and p15 were obviously elevated on NK cells from IT patients. The cyclin-dependent kinase inhibitors p15 and p21Waf1/Cip1 acted as inhibitors of cell-cycle progression [46]. Relatively high levels of Smad-2P and Smad2 were observed in NK cells from IT-phase patients, implying that TGF-β1 had the potential to arrest the NK cell cycle. High rates of p15 methylation in HCCs have been shown to be associated with HCV infection. TGF-β-dependent inhibition of HCV replicons was also associated with cell-cycle arrest in a Smad-dependent manner [68]. Furthermore, the expression of p21 during liver cirrhosis is related to the persistence of infection with HBV [69], which suggests that p21 plays an important role in the progression of HBV. However, one study has shown that the HBV X protein overcomes cellular senescence by down-regulating levels of p16 and p21 via DNA methylation [70]. In that study, HepG2 cells were used, which may explain why their findings differed from our own. Some previous studies reporting persistent HBV infection have focused on genetic variants [71]–[74]. Unfortunately, this study was limited by the supply of freshly isolated NK cells from patients and healthy controls, and therefore, our analysis was restricted to the circulating compartment only. A more detailed investigation of the frequency and function of intrahepatic NK cells in IT patients should be performed.
In summary, our findings demonstrate that high levels of TGF-β1 are associated with reduced NKG2D and 2B4 expression, the functional impairment of NK cell function, and consequently, with the development of persistent HBV. Our study provides a basis for improving current therapies for IT patients by blocking TGF-β1 inhibitory pathways, which could result in additive efficacy at eliminating the virus during the initial phase of CHB infection.
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发表于 2012-3-28 21:37
