Activated natural killer cells accelerate liver damage in patients with chronic hepatitis B virus infection - PubMed (original) (raw)

. 2015 Jun;180(3):499-508.

doi: 10.1111/cei.12597. Epub 2015 Apr 12.

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Activated natural killer cells accelerate liver damage in patients with chronic hepatitis B virus infection

Q Zheng et al. Clin Exp Immunol. 2015 Jun.

Abstract

Emerging evidence indicates that natural killer (NK) cells may contribute to liver injury in patients with hepatitis B virus (HBV) infection. Because HBV infection progresses through various disease phases, the cytolytic profiles of peripheral and intrahepatic NK cells in HBV-infected patients remain to be defined. In this study, we comprehensively characterized intrahepatic and peripheral NK cells in a cohort of HBV-infected individuals, and investigated their impact on liver pathogenesis during chronic HBV infection. The study population included 34 immune-clearance (IC) patients, 36 immune-tolerant (IT) carriers and 10 healthy subjects. We found that the activity of peripheral NK cells from IC patients was functionally elevated compared to IT carriers and controls, and NK cell activation was indicated by an increased expression of CD69, CD107a, interferon (IFN)-γ and tumour necrosis factor (TNF)-α. Further analysis showed that the increased activity of both peripheral and hepatic NK cells was correlated positively with liver injury, which was assessed by serum alanine aminotransferase levels (ALT) and the liver histological activity index (HAI). Interestingly, the frequency of peripheral NK cells was reduced in IC patients (especially those with higher HAI scores of 3-4), but there was a concomitant increase in hepatic NK cells. The functionally activated NK cells are enriched preferentially in the livers of IC patients and skew towards cytolytic activity that accelerates liver injury in chronic hepatitis B (CHB) patients.

Keywords: chronic hepatitis B; cytokine; innate immunity; liver injury; natural killer cells.

© 2015 British Society for Immunology.

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Figures

Fig 1

Fig 1

Flow cytometric analysis of peripheral CD3− CD56+/CD16+ natural killer (NK) cells in all enrolled subjects. (a) Pooled data show percentages of peripheral NK cells in immune-clearance (IC) patients (n = 34), immune-tolerant (IT) subjects (n = 36) and healthy control (HC) donors (n = 10). (b) Summarized data show the percentages of peripheral NK cells in partial IC patients with histological activity index (HAI) inflammation scores of G1–2 (n = 10) and those with HAI scores of G3–4 (n = 14). Each dot represents one individual. The horizontal bars indicate the median percentiles (*P < 0·05).

Fig 2

Fig 2

Immunohistochemical staining of CD56+ and CD3+cells in the liver sections obtained from patients with graded inflammation scores. Samples shown here were from partial immune-clearance (IC) patients with either low (G1–2, n = 10) or high (G3–4, n = 14) histological activity index (HAI) scores. (a) Hepatic CD56+and CD3+cells were stained brown and presented in both portal and lobular areas of livers (×400 magnification). (b) Quantitative analysis of CD56+ natural killer (NK) and CD3+ T cells in portal areas of liver tissues. Each bar graph indicates the mean ± standard deviation.

Fig 3

Fig 3

Flow cytometric analysis of CD69 expression on peripheral natural killer (NK) cells. CD3− CD56+/CD16 + NK cells were gated. (a) Pooled data show the percentage of peripheral NK cells expressing the CD69 activation marker from immune-clearance (IC), immune-tolerant (IT) patients and healthy controls (HCs). Boxes show the 10th, 25th, 75th and 90th percentiles and the median value (solid line) of each group (*P < 0·05). (b) Summarized data show the percentage of peripheral CD3−CD56+/CD16+cells expressing CD69 from partial IC patients who underwent percutaneous liver biopsies with either low (G1–2) or high (G3–4) HAI scores. Each dot represents one individual. The horizontal bars indicate the median percentiles (*P < 0·05).

Fig 4

Fig 4

Peripheral natural killer (NK) cells express the level of cellular cytokine [interferon (IFN)-γ, tumour necrosis factor (TNF)-α] and degranulation (perforin and CD107a) in immune-clearance (IC), immune-tolerant (IT) patients and healthy control (HC) subjects. CD3−CD56+/CD16+ NK cells were gated. (a) Representative dot-plots depict IFN-γ, TNF-α, perforin and CD107a production by peripheral NK cells among the three groups following phorbol myristate acetate (PMA)/ionomycin or interleukin (IL)-12 stimulation. Values in the upper right quadrants represent the percentage of NK cells expressing cytokine production and degranulation. (b) Pooled data show the percentage of peripheral NK cells expressing IFN-γ, TNF-α, perforin and CD107a among the three groups upon stimulation with PMA/ionomycin or IL-12. The median value (solid line) of each group (*P < 0·05).

Fig 5

Fig 5

Flow cytometry analysis of natural killer (NK) cellular degranulation and interferon (IFN)-γ production in patients categorized by histological activity index (HAI). (a,b) Pooled data show the percentages of peripheral NK cells expressing IFN-γ (a) and CD107a (b) in two indicated groups upon stimulation with phorbol myristate acetate (PMA)/ionomycin or interleukin (IL)-12. CD3−CD56+CD16+ NK cells were gated. Each dot represents one individual. The horizontal bars indicate the median percentiles (*P < 0·05).

Fig 6

Fig 6

Immunohistochemical analysis of interferon (IFN)-γ and CD107a, negative control in the liver tissues of chronic hepatitis B virus (HBV)-infected individuals categorized by histological activity index (HAI) score. (a) Representative in-situ immunohistochemical staining of liver sections from patients with either low (G1–2) or high (G3–4) HAI scores. Positively stained areas appear brown under × 200 magnification. (b) Semiquantitative analysis (expressed as integrated optical density, IOD) of IFN-γ or CD107a levels in livers of these subjects. Each dot represents one individual. The horizontal bars indicate the median percentiles (*P < 0·05).

Fig 7

Fig 7

Correlation analysis of CD69 or CD107a expression on peripheral natural killer (NK) cells and serum ALT levels. (a) CD69 and (b) CD107a expression. Results are expressed as Pearson correlation coefficients. Each dot represents one individual.

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