Restoration of TLR3-activated myeloid dendritic cell activity leads to improved natural killer cell function in chronic hepatitis B virus infection

Abstract

There is increasing evidence that the function of NK cells in patients with chronic hepatitis B (CHB) infection is impaired. The underlying mechanism for the impaired NK cell function is still unknown. Since myeloid dendritic cells (mDC) are potent inducers of NK cells, we investigated the functional interaction of mDC and NK cells in CHB and the influence of antiviral therapy. Blood BDCA1(+) mDC and NK cells were isolated from 16 healthy controls or 39 CHB patients at baseline and during 6 months of antiviral therapy. After activation of mDC with poly(I · C) and gamma interferon (IFN-γ), mDC were cocultured with NK cells. Phenotype and function were analyzed in detail by flow cytometry and enzyme-linked immunosorbent assay. Our findings demonstrate that on poly(I · C)/IFN-γ-stimulated mDC from CHB patients, the expression of costimulatory molecules was enhanced, while cytokine production was reduced. In cocultures of poly(I · C)/IFN-γ-stimulated mDC and NK cells obtained from CHB patients, reduced mDC-induced NK cell activation (i.e., CD69 expression) and IFN-γ production compared to those in healthy individuals was observed. Antiviral therapy normalized mDC activity, since decreased expression of CD80 and CD86 on DC and of HLA-E on NK cells was observed, while poly(I · C)/IFN-γ-induced cytokine production by mDC was enhanced. In parallel, successful antiviral therapy resulted in improved mDC-induced NK cell activation and IFN-γ production. These data demonstrate that CHB patients display a diminished functional interaction between poly(I · C)/IFN-γ activated mDC and NK cells due to impaired mDC function, which can be partially restored by antiviral therapy. Enhancing this reciprocal interaction could reinforce the innate and thus the adaptive T cell response, and this may be an important step in achieving effective antiviral immunity.

Fig 1

Diminished mDC-induced activation and function of NK cells in CHB. Purified fresh NK cells were cocultured with stimulated BDCA1⁺ mDC as described in Materials and Methods. Cells were isolated from 11 healthy controls (HC) and 22 CHB patients (HBV) (HBV DNA, log₁₀ 8.1 ± 0.4 IU/ml; ALT, 77 ± 7 IU/ml [mean ± SEM]). After 48 h, the early activation marker CD69 on NK cells was determined by fluorescence-activated cell sorter (FACS) analysis, and supernatants of these cultures were evaluated for IFN-γ content by ELISA. Data are expressed as means ± SEM and represent CD69-expressing NK cells within the total NK cell population (A) or IFN-γ levels in supernatant (B). *, P < 0.05; **, P < 0.01.

Fig 2

Expression of markers of maturation is enhanced on stimulated mDC of CHB patients. Isolated BDCA1⁺ mDC were stimulated with poly(I · C) and IFN-γ for 18 h. Cells were isolated from 11 healthy controls (HC) and 22 CHB patients (HBV). Expression of CD80, CD86, MHC class I molecules (HLA-A, HLA-B, and HLA-C), MIC-A, and HLA-E was determined without stimulation (no stim) and after 18 h of stimulation with poly(I · C) and IFN-γ (stim). Expression levels of maturation markers CD80 and CD86 (A), expression levels and percentages of cells positive for ligands MHC-I, MIC-A, and HLA-E (B), and representative dot plots and histograms (C) as determined by flow cytometry are presented as means ± SEM. *, P < 0.05; **, P < 0.01; ***, P < 0.0001.

Fig 3

Expression of CD48 and 2B4 on mDC and NK cells of CHB patients. NK cells and mDC were purified from 5 healthy controls (HC) and 17 CHB patients (HBV) (HBV-DNA, log₁₀ 6.3 ± 0.5 IU/ml; ALT, 96 ± 17 IU/ml), and mDC were stimulated with poly(I · C) and IFN-γ for 18 h. Expression levels of CD48 and 2B4 were determined on mDC without stimulation (unstim) and after 18 h of stimulation with poly(I · C) and IFN-γ (stim) and on NK cells. Expression levels of CD48 and 2B4 on mDC (A) and NK cells (B), determined by FACS, are presented as means ± SEM. *, P < 0.05; ***, P < 0.001.

Fig 4

Impaired cytokine production upon stimulation of mDC of CHB patients. Purified mDC isolated from 5 healthy controls (HC) and 17 CHB patients (HBV) were stimulated with poly(I · C) and IFN-γ for 18 h. Supernatants of these cultures were evaluated by ELISA or FACS (labeled beads). Data represent mean concentrations (pg/ml) ± SEM of IL-6, IL-12p40, IL-12p70, and IL-18. **, P < 0.01; ***, P < 0.001.

Fig 5

Antiviral therapy improves NK cell function as a result of improved mDC-NK cell interaction. Isolated NK cells were cocultured for 48 h with isolated BDCA1⁺ mDC after stimulation with poly(I · C) and IFN-γ for 18 h. Cells were isolated from 22 CHB patients (HBV) at baseline (t = 0) and after 6 months of treatment with entecavir 0.5 mg o.i.d. (t = 6). The frequency of fresh (A) or thawed (B) CD69-positive NK cells after 48 h of coculture with simultaneously isolated mDC was determined at t = 0 and t = 6 by FACS analysis. Fresh NK cells were isolated simultaneously with mDC at t = 0 and t = 6. Thawed NK cells were isolated 2 weeks prior to baseline and frozen at −80°C before use. Supernatants of these cultures were evaluated for IFN-γ content by ELISA. Data represent CD69-expressing NK cells within the total NK cell population or mean levels of IFN-γ ± SEM. *, P < 0.05.

Fig 6

Diminished activation and decreased expression of HLA-E on mDC upon antiviral therapy. Purified fresh NK cells and mDC were isolated from 11 healthy controls (HC) and 22 CHB patients at baseline (t = 0) and after 6 months of treatment (t = 6). Expression of CD80 and CD86 on mDC upon coculture with simultaneously isolated NK cells was determined by FACS analysis. Expression of HLA-E at t = 0 and t = 6 was determined on mDC without stimulation (no stim) and after 18 h of stimulation with poly(I · C) and IFN-γ (stim). (A and B) Mean (±SEM) levels of expression of CD80 and CD86 (A) and HLA-E (B) on mDC. *, P < 0.05. (C) Spearman's rank correlation coefficient between the change in HBV DNA (log₁₀ IU/ml) and the change in mean fluorescence intensity (MFI) of HLA-E expression on mDC after stimulation at t = 0 and t = 6. Change in HBV DNA was defined as the absolute difference between levels at baseline and at 6 months. Change in MFI was defined as the percent difference between levels at baseline and at 6 months. Spearman's rank correlation coefficient between the MFI of HLA-E expression on mDC after stimulation and levels of ALT (IU/ml) at t = 6 was determined.

Fig 7

Antiviral therapy ameliorates cytokine production upon stimulation Purified mDC isolated from 17 CHB patients (HBV) were stimulated with poly(I · C) and IFN-γ for 18 h at baseline (t = 0) and after 6 months of treatment (t = 6). Supernatants of these cultures were evaluated by immunoassay. Data represent the mean concentrations (±SEM) of IL-6, IL-12p40, IL-12p70, and IL-18. *, P < 0.05; **, P < 0.01; ***, P < 0.001.