Elsevier

Journal of Hepatology

Volume 54, Issue 2, February 2011, Pages 209-218
Journal of Hepatology

Research Article
Viral load reduction improves activation and function of natural killer cells in patients with chronic hepatitis B

https://doi.org/10.1016/j.jhep.2010.07.009Get rights and content

Background & Aims

Natural killer (NK) cells play a major role in anti-viral immunity as first line defense and regulation of virus-specific T cell responses. This study aimed to investigate phenotype and function of NK cells in patients with chronic hepatitis B virus (HBV) infection and to study the effect of anti-viral therapy.

Methods

Peripheral blood NK cells from 40 chronic HBV patients were compared to NK cells of 25 healthy controls. The effect of entecavir-induced viral load reduction on NK cell phenotype and function was investigated in 15 chronic HBV patients.

Results

NK cell numbers and subset distribution did not differ between HBV patients and normal subjects. In chronic HBV patients, the cytotoxic capacity was retained, but NK cell activation and subsequent IFNγ and TNFα production, especially of the CD56dim subset, were strongly hampered. This functional dichotomy was paralleled by an altered activation state, elevated expression of NKG2A, and downregulated expression of CD16 and NKp30, which correlated with serum HBV-DNA load. Anti-viral therapy partially restored NK cell phenotype, as shown by NKG2A downregulation. Moreover, viral replication inhibition improved IFNγ production as a result of an increased ability of CD56dim NK cells to become activated de novo. This improved NK cell activation and function which correlated with therapy-induced reduction in serum ALT levels, but not HBV-DNA load.

Conclusions

The specific defect in CD56dim NK cell activation and the reduced capacity to produce anti-viral and Th1-skewing cytokines may play a role in HBV persistence. Restoration of this NK cell cytokine-producing capacity, as achieved by viral load reduction, could therefore contribute to definite clearance of the virus.

Introduction

Chronic hepatitis B is the result of an inadequate immune response towards the virus [6], [11]. Treatment strategies should thus be geared towards achieving effective anti-HBV immunity in all patients. The development of an effective anti-HBV therapy, however, requires insight into the mechanisms of HBV persistence. It is known that the lack of a coordinated and efficient T cell response against HBV underlies the viral persistence [3], [11] but the mechanism behind this failure has not yet been elucidated.

Natural killer (NK) cells are innate immune cells that are crucial in the defense against viral infections [4], [33]. Human NK cells can be divided into two subsets based on cell-surface density expression of CD56 [12], [39]. Despite the absence of antigen-specific receptors at their surface, NK cells can selectively eliminate virus-infected cells. A dynamic and precisely coordinated balance between activating and inhibitory receptors governs NK cell activation programs [25], [29]. Also, a pro-inflammatory cytokine milieu can skew NK cells to become functionally active [4], [14].

NK cells mediate their anti-viral effects through at least three different mechanisms [37]: (1) release of cytolytic granules for lysis of infected cells, (2) induction of target cell apoptosis through crosslinking of cell surface death receptors, and (3) release of cytokines. Although not absolute, cytotoxicity is mainly exerted by the CD56dim subset, whereas the CD56bright subset specializes in the release of cytokines [13]. In addition to the direct anti-viral effect, NK cell-derived cytokines, notably IFNγ, are important for skewing T cell responses towards Th1 and subsequent cytotoxicity [27].

Only limited information has been published on the role of NK cells in HBV infection. In acute HBV, an early rise in circulating NK cells has been documented, suggesting their contribution to the initial viral containment [18], [21], [38]. In the context of persistent HBV infection, NK cell studies mainly focused on NK cell-induced tissue injury [10], [15]. However, very little is known about the quality of the anti-viral functions of NK cells during chronic HBV, and the effect of anti-viral therapy on these cells has not been reported. By analysing the function of peripheral blood NK cells of chronic HBV patients prior to and during anti-viral therapy, we provide insight into the anti-viral function of NK cells during chronic HBV infection and into the effect of viral load reduction on NK cell phenotype and function.

The data show that, in chronic HBV, the cytotoxic capacity is retained, but NK cell activation and IFNγ production are strongly hampered, which may contribute to viral persistence. Anti-viral therapy resulted in a partially enhanced activation state of NK cells in vivo, as well as in improved production of IFNγ, as a result of an increased susceptibility for de novo activation ex vivo. These findings may contribute to the development of novel therapeutic approaches to clear the viral infection.

Section snippets

Patients and healthy subjects

Peripheral heparinized blood samples were obtained from 40 patients with chronic hepatitis B for cross-sectional analysis and from 15 other patients for longitudinal analysis during anti-viral therapy (Table 1). Blood samples of the latter group were obtained at baseline (t = 0) and after 6 months (t = 6) of anti-viral therapy. All patients were negative for antibodies against hepatitis C, hepatitis D, and human immunodeficiency virus. Patients were treated with the nucleoside analogue entecavir (0.5

Altered frequencies of activated CD56dim NK cells in chronic HBV patients

In patients as well as controls, the peripheral blood lymphocyte population contained about 14% NK cells, with equal distribution in both subsets (Fig. 1A). We determined the in vivo expression of the early activation marker CD69 and the late activation marker HLA-DR on NK cells. In chronic HBV, CD56dim NK cells less frequently expressed CD69 (Fig. 1B and C) but more HLA-DR than controls (Fig. 1D). Within the CD56bright NK cells, the HLA-DR expressing cells were not increased, but, like the CD56

Discussion

Natural killer (NK) cells play a major role in anti-viral immunity as first line defense and regulation of virus-specific T cell responses. Originally, CD56bright NK cells were regarded as the prime cytokine producing subset, whereas CD56dim NK cells were held responsible for cytotoxicity [13]. Since hepatocytes, the major target of HBV, are known to be resistant to perforin/granzyme-mediated killing [24], the production of anti-viral cytokines is of major importance for combating viruses that

Conflict of interest

The authors who have taken part in this study declared a relationship with the manufacturers of the drugs involved. This study was supported by an unrestricted Educational grant from Bristol Myers Squibb.

Acknowledgements

The authors thank A. Boonstra (Dept. of Gastroenterology and Hepatology Erasmus MC University Medical Centre, Rotterdam, The Netherlands) for useful discussions and critically reading the manuscript.

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