SAP expression on human NK cells contributes to HBV persistence - PubMed (original) (raw)

TGF-β1 down-regulation of NKG2D/DAP10 and 2B4/SAP expression on human NK cells contributes to HBV persistence

Cheng Sun et al. PLoS Pathog. 2012.

Abstract

The 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.

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Conflict of interest statement

The authors have declared that no competing interests exist.

Figures

Figure 1. NKG2D and 2B4 expression is decreased on circulating NK cells during the IT phase of HBV infection.

Immunofluorescent staining of human cells was performed to investigate the expression of NKG2D and 2B4 on NK cells, as gated by FACS analysis. Horizontal bars denote the means. (A) NKG2D and 2B4 expression on total CD3+CD56− NK cells within the lymphocyte gate from a representative healthy control or CHB patient. (B and C) Differential NKG2D and 2B4 expression on total NK cells within the lymphocyte gate in HC and samples from patients in the IT, IA and IN phases. (D and E) Absolute counts of NKG2D+ and 2B4+NK cells in HC, IT, IA and IN. The expression of NKG2D and 2B4 was lower, both by percentage and absolute count, on IT NK cells than on NK cells from both healthy controls and other chronic patients. (F) Inverse correlation between serum 2B4 levels and NKG2D levels on NK cells in HBV patients. Pearson's correlation coefficient: r = 0.7695, P<0.0001. * p<0.05 and *** p<0.001.

Figure 2. Deficient NK cell function in IT patients.

(A) Deficient NK cell cytotoxicity from IT patients. NK cell cytotoxicity was assessed in primary NK cells from IT, IA, and IN patients and healthy controls using a 51Cr-release assay with K562 target cells. (B) Results show the mean ± SEM values in 19 patients and 5 controls. Values for IT patients were less than controls (P = 0.0384). (C) Positive correlation between NK cell cytotoxicity and the percentage of NK cells expressing NKG2D and 2B4. (D) Fresh PBMCs were stimulated with/without IL-12 as described in the Materials and Methods. After 16 h, IFN-γ production was determined using flow cytometry by gating on CD3−CD56+ NK cells. A representative dot plot displaying intracellular IFNγ staining in NK cells with the subsets indicated is shown. (E) Cumulative data are shown. (F, G) Fresh PBMCs from patients before treatment (black) or after treatment (white) were stimulated with IL-12 for 16 h. IFNγ (F) and CD107a (G) production was determined using flow cytometry by gating on CD3−CD56+ NK cells. (H) Induction of ex vivo NK cell cytotoxic activity after in vivo administration of antiviral treatment with nucleoside analogues until ALT reached normal levels. NK cytotoxicity was determined ex vivo by measuring the lysis of 51Cr-labelled target cells before treatment (red) or after treatment (green). Cumulative data are shown.

Figure 3. Reduced expression of DAP10 and SAP in NK cells from IT patients.

(A) The mRNA expression levels of DAP10, SAP and EAT-2 in NK cells were investigated in cells from HBV patients and healthy controls. Confocal immunofluorescence images of DAP10 (green) (B) or SAP (green) (E) staining in primary NK cells in HC, IT, IA and IN patients, showing the down-regulation of DAP10 and SAP in NK cells from IT patients compared with healthy controls and other chronic patients in cells also co-stained with DAPI. (C and F) Graph showing the percentages of NK cells expressing DAP10 or SAP. (D) Absolute counts of DAP10+ NK cells in HC, IT, IA and IN. (G) Differential MFI of SAP+ NK cells from HC and IT-, IA- and IN-phase patients. The results are representative of 1000 cells. Scale bars in the immunofluorescence images represent 100 µm. The data are represented as the means ± SEM. *** P<0.001. Original magnification: 20× (B, E).

Figure 4. Deficient NK92 cell function from siR-DAP and siR-SAP.

(A, B) Real-time PCR analysis of the mRNA expression levels of DAP10, SAP on NK92 cells were investigated 48 h after transfection with either an siRNA negative control (siR.NC), DAP10 siRNA (siR-DAP) or SAP siRNA (siR-SAP). (C) NK92 cell cytotoxicity was assessed 20 h after transfection with siR.NC, siR-DAP or siR-SAP using a 51Cr-release assay with K562 target cells. The data are shown as the mean ± SE, siR-DAP had a mean of 10.1%±0.3% and siR-SAP had a mean of 3.4%±1.2% K562 lysis at a 8∶1 E∶ T ratio compared with 19.5%±3.7% for the controls. Values for siR-DAP and siR-SAP were lower than for the negative control (P = 0.027 and P = 0.002, respectively).

Figure 5. Ca2+ flux down-regulation is induced by synergy between NKG2D and 2B4 in NK cells from IT patients.

Freshly isolated, resting NK cells from the peripheral blood of HC (A), IA (B) and IT (C) patients were loaded with Fluo-4 and Fura Red, and preincubated with mAbs specific for NKG2D (blue), 2B4 (purple) or both (red) on ice for 30 min. Cells were washed, resuspended in cold HBSS with 1% FBS, and prewarmed at 37°C. Fluorescence was measured by Zeiss 510 confocal microscopy. Sixty seconds after the beginning of each scan, secondary F(ab')2 goat anti–mouse IgG or KCL (green) was added to each chamber. Traces of the Fluo-4/Fura Red ratios of the representative NK cells are shown. Fluo-4/Fura Red ratios are plotted as a function of time. Green lines show activation with the isotype control (KCL). Blue and purple lines show activation by the single receptors. Red lines show activation by the combination of both receptors. The experiment shown is representative of five independent experiments. The Ca2+ mobilisation induced by NKG2D and 2B4 synergy was measured in >100 NK cells from representative healthy controls or patients at 200 (D) and 500 s (E).

Figure 6. Soluble TGF-β1 is associated with reductions in NKG2D and 2B4.

(A) Circulating concentrations of multiple cytokines detected in serum samples taken from healthy controls and HBV patients assayed by CBA (IL-1α, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-12, p70, IL-13, TNF and IFN-γ) and sandwich ELISA (TGF-β1). Significance testing was performed using the Mann-Whitney U test. (B, D) Inverse correlations between serum TGF-β1 levels and the percentage of NK cells expressing NKG2D and 2B4. (C, E) Inverse correlation between serum TGF-β1 levels and the absolute counts of NKG2D- and 2B4-expressing NK cells. Pearson's correlation coefficients are shown.

Figure 7. Anti-TGF-β1 partially restores the surface expression of NKG2D and 2B4 on NK cells.

NK cells from healthy control peripheral blood were preincubated with TGF-β1. NKG2D (A) and 2B4 (B) expression on NK cells was monitored at 72 h by flow cytometry. Histograms correspond to NK cells from one representative donor treated with the isotype controls (green lines), medium alone (RPMI 1640 supplemented with 10% FBS in the presence of IL-15 (10 ng/ml) and IL-2 (100 U/ml)) (black lines) and TGF-β1 (1 ng/ml) (red lines). NK cells from healthy controls were cultured with healthy control sera (black lines), sera from IA-phase patients (blue lines) or sera from IT-phase patients (red lines) for 3 days. Surface expression of NKG2D (C) and 2B4 (E) were assessed at 72 h using flow cytometry. Histograms showing NKG2D (C) and 2B4 (E) surface expression on NK cells from one representative donor out of three studied are shown. Freshly isolated NK cells from healthy controls co-cultured with sera from IT-phase patients alone (red lines) or with anti-TGF-β1 Ab (blue lines) or isotype control Ab (green lines). The expression levels of NKG2D (D) and 2B4 (F) were analysed using FACS. The results are representative of three independent experiments. Cumulative data are shown (G, H).

Figure 8. Anti-TGF-β1 partially restores Ca2+ flux in NK cells from healthy controls incubated with IT patient serum.

Freshly isolated resting NK cells from healthy control peripheral blood were preincubated with sera from IT-phase patients (A), isotype control Abs (B) or anti-TGF-β1 Abs (C) and stimulated with NKG2D and/or 2B4, as shown in Figure 3. Ca2+ flux was analysed using a Zeiss 510 confocal microscope. The Ca2+ mobilisation induced by NKG2D and 2B4 synergy was measured in >100 NK cells from representative healthy controls or patients at 200 (D) and 500 s (E). The experiment shown is representative of 5 independent experiments.

Figure 9. TGF-β1 inhibits the NK cell cycle in vitro.

NK cells from healthy controls were preincubated with IT serum, IA serum, IN serum or anti-TGF-β1Ab. The proportion of cells in the G1 and S phases of the cell cycle was determined using flow cytometry, which demonstrated that TGF-β1 inhibited cell proliferation and induced G1-phase arrest. * P<0.05, ** P<0.01, and *** P<0.001.

Figure 10. p21 and p15 are elevated in IT patients and induce the arrest of the NK cell cycle.

(A) NK cells were stimulated for 30 min, 12 h and 72 h with TGF-β1. Total cell lysates were analysed using immunoblotting with antibodies against Smad-2, Smad-2P, p15 and p21. Data are representative of three independent experiments. (B) NK cells were stimulated for 72 h with IT serum or hepatitis ascites in the absence (−) or presence (+) of TGF-β1 Ab. NK cells were lysed, and the lysates were immunoblotted with Abs against Smad-2, Smad-2P, p15 or p21. Data are representative of three independent experiments. (C) Freshly isolated NK cells were obtained from the peripheral blood of healthy controls and IT-phase patients and analysed by western blotting. The results for Smad-2, Smad-2P, p15 and p21 are shown. Data are representative of at least four independent experiments.

References

1. Chisari FV, Ferrari C. Hepatitis B virus immunopathogenesis. Annu Rev Immunol. 1995;13:29–60. - PubMed
1. Lee WM. Hepatitis B virus infection. N Engl J Med. 1997;337:1733–1745. - PubMed
1. Lavanchy D. Hepatitis B virus epidemiology, disease burden, treatment, and current and emerging prevention and control measures. J Viral Hepat. 2004;11:97–107. - PubMed
1. McMahon BJ. The natural history of chronic hepatitis B virus infection. Hepatology. 2009;49:S45–55. - PubMed
1. Hadziyannis SJ, Vassilopoulos D. Hepatitis B e antigen-negative chronic hepatitis B. Hepatology. 2001;34:617–624. - PubMed

TGF-β1 down-regulation of NKG2D/DAP10 and 2B4/SAP expression on human NK cells contributes to HBV persistence - PubMed (original) (raw)