Both cell-to-cell contact and cytokines were involved in the crosstalk between NK cells and macrophages.

<p>Freshly isolated murine NK cells were co-cultured with poly I:C-stimulated primary macrophages separated by a transwell for 24 h. Then NK cells were stained with antibodies for cell-surface CD69 and NKG2D, and for intracellular IFN-γ. The numbers of CD69- or NKG2D-positive as well as IFN-γ-secreting NK cells were determined by flow cytometry. Data are expressed as the percentage of positively-stained cells. The data are representatives from at least three separate experiments.</p

Protein kinase R (PKR) over-expression enhanced type I interferon (IFN) production and HBx-siRNA-mediated hepatitis B virus (HBV) inhibition.

<p>HepG2.2.15 cells were transfected with 2 µg of full-length PKR plasmid or the control plasmid. 24 h later, the cells were transfected with HBx-siRNA (150 nM). (A) Over-expression of PKR at the transcriptional level. (B, C) Levels of IFN-α and IFN-β were detected by real-time PCR at 24 h. (D, E) mRNA expression levels for HBx and HBs/p were detected by real-time RT-PCR at 24 h. (F) HepG2.2.15 cells were transfected with 1 µg of hPKR-K296R or the control plasmid. Then cells were collected and 6-His was assayed by FACS (top). When transfected with hPKR-K296R for 24 h, the cells were transfected with HBx-siRNA4 (150 nM). Another 24 h later, levels of IFN-α and IFN-β were detected. The levels of all examined parameters were expressed as percentages of the corresponding parameter in LF-treated control-vector transfected cells (bottom). Data are expressed as the mean ± SD from at least three independent experiments. *<i>p<</i>0.05 versus corresponding control-vector transfected cells.</p

Poly I:C-treated macrophages enhanced NKG2D expression and NK cell function.

<p>Freshly isolated splenic NK cells were co-cultured with RAW264.7 cells that were treated with 0 or 100 µg/ml of poly I:C for 24 h. NK cells were then isolated and NKG2D expression was determined by RT-PCR and flow cytometry (A). Data are expressed as the fold change in mRNA expression normalized to untreated cells or as the percentage of positively-stained cells. * p<0.05 versus untreated cells using the paired Student’s test. The isolated NK cells from co-culture with macrophages were co-incubated with saturating amounts of anti-NKG2D mAb or isotype control, washed and used as effector cells against YAC-1 targets (B). The expression of CD69 (C), cytotoxic effector molecules FasL, TRAIL, and perforin (D), and seceretion of IFN-γ (E) were determined by flow cytometry, RT-PCR, and ELISA separately. Data are expressed as the mean ± SD from at least three separate experiments. *p<0.05, **p<0.01 compared with poly I:C-untreated RAW264.7 group; ^# p<0.05, ^## p<0.01 compared with isotype control group.</p

Working model of innate recognition-mediated interaction between NK cells and macrophages during anti-tumor immune response.

<p>As a response to stimulation from TLR3 ligand poly I:C, macrophages up-express surface RAE-1 (NKG2D ligand) and produce soluble IL-12, IL-15, and IFN-β. RAE-1 on surface of macrophages and their cytokines will strongly activate NK cells to produce IFN-γ and up-expression of NKG2D. IFN-γ produced by NK cells will stimulate macrophages in feedback. NK cells after activation will directly attack tumor cells by recognizing tumor’s RAE-1 and indirectly arrest tumor growth by secreting IFN-γ. At the same time, poly I:C-induced up-regulation of NKG2A ligand Qa-1 protects macrophages themselves from cytolysis by NK cells in order to keep the positive feedback loop in the anti-tumor immune response.</p

HBx-siRNA inhibits hepatitis B virus (HBV) expression in HepG2.2.15 cells.

<p>(A) Scramble siRNA or HBx-siRNA (siRNA1, siRNA2, siRNA3, siRNA4) (100 nM) was transfected into HepG2.2.15 cells using lipofectamine 2000 (LF). LF-treated group was used as a control. The cells were harvested, and RNA was extracted at 24 h after transfection. Expression of HBx mRNA were detected by real-time PCR (top). The level of each sample is expressed as the percentage of the RNA level in scramble siRNA-treated cells. The levels of HBx protein were detected by western blot at 48 h after transfection (bottom). (B) HepG2.2.15 cells were transfected for 48 h, and then cells were fixed, immunostained for HBcAg and visualized under a microscope. Representative fields from each cell group are shown (original magnification ×100). (C, D) The supernatants were harvested after 48 h, and the dose-dependent reductions in HBsAg (C) and HBeAg (D) were detected with ELISA. Data showing the silencing effect are representative of three independent experiments. Data are expressed as the mean ± SD from at least three separate experiments. *<i>p</i><0.05 versus the LF-treated group.</p

Up-regulation of NKG2D ligands on poly I:C-treated murine macrophages.

<p>Murine macrophage RAW264.7 cells were cultured for 24 h with 0, 10, 20 or 100 µg/ml poly I:C and analyzed by RT-PCR or FACS. The data are expressed as fold induction of RAE-1, H60 or MULT-1 mRNA (A) or the percentage of positive RAE-1 staining using monoclonal pan-RAE-1 antibodies (B). Resident peritoneal macrophages from BALB/c or C57BL/6 mice were treated with either 0 or 100 µg/ml poly I:C for 24 h and NKG2D ligand transcripts and surface expression were analyzed by real-time PCR (C) or FACS (B). Data shown are expressed as the mean ± SD from at least three separate experiments. *p<0.05, **p<0.01 compared with untreated cells.</p

TLR3 is necessary for poly I:C-mediated up-regulation of NKG2D ligands by macrophages.

<p>Expression of TLR3 and TLR4 by RAW264.7 cells stimulated with 100 µg/ml poly I:C for 24 h was analyzed by RT-PCR (A). TLR3 mRNA levels of RAW264.7 cells were measured following incubation in media only or following treatment with si-TLR3 or siRNA control (si-control) for 24 h (B). The data are expressed as the folds ± SD from at least three separate experiments. *p<0.05, **p<0.01 compared with the untreated group. RAW264.7 cells first transfected with si-TLR3 or si-control were stimulated with 0 or 100 µg/ml poly I:C for 24 h and the transcripts of the NKG2D ligands RAE-1, H60, and MULT-1 were detected by RT-PCR (C). The data are expressed as the mean induction folds ± SD over untreated cells from at least three separate experiments. *p<0.05, compared with si-control group.</p

Cytokines secreted by poly I:C-treated macrophages play critical roles in NK cell activation.

<p>The secretion levels of IL-12, IL-15, IL-18, IFN-β, or IFN-γ in the supernatants of RAW264.7 cells (A) or resident peritoneal macrophages from BALB/c mice (B) that were cultured with 0 or 100 µg/ml poly I:C for 24 h were analyzed by ELISA. The data are expressed as the mean ± SD from at least three separate experiments. *p<0.05, **p<0.01 compared with the untreated RAW264.7 group. NK cells were incubated with poly I:C-pretreated RAW264.7 cells in the presence of 10 µg/ml anti-IL-15 mAb, anti-IFN-β mAb or isotype control separately for another 12 h, and the expression of NKG2D were determined by flow cytometry (C). Levels of IFN-γ in the supernatants were determined by ELISA (D). NK cell-mediated lysis of YAC-1 cells was assessed by first culturing NK cells with poly I:C-treated RAW264.7 cells (Mφ) in the presence or absence of anti-IL-15 mAb, anti-IFN-β mAb or isotype control, respectively, and then NK cells were isolated and added to target cells (E). Data are expressed as the mean ± SD from at least three separate experiments. *p<0.05 compared with the isotype control group.</p

Qa-1 contributes to the protection of macrophages from NK cell-mediated lysis.

<p>Changes in mRNA and protein levels of the NKG2A ligands Qa-1a and Qa-1b (A,B), and the NKG2D ligands RAE-1, H60, and MULT-1 (C) in YAC-1 cells, macrophages (Mφ) or RAW264.7 cells were detected by RT-PCR or FACS. Data are expressed as relative expression from at least three separate experiments. *p<0.05, **p<0.01 versus YAC-1 mRNA levels using the paired Student’s test. RAW264.7 cells first stimulated with 100 µg/ml poly I:C for 24 h and then co-incubated with saturating amounts of anti-Qa-1 mAb or isotype control or transfected with si-Qa-1 or si-control, were then used as target cells. The cytolytic activity of NK cells stimulated with poly I:C-treated macrophages was assessed at various E:T rations (D). Data are expressed as the mean ± SD from at least three separate experiments. *p<0.05, **p<0.01 compared with the isotype control or si-control group.</p

HBx-siRNA induced the expression of type I interferon (IFN) and inflammatory cytokines in HepG2.2.15 cells.

<p>(A) HepG2.2.15 cells were transfected with HBx-siRNAs; 24 h later, the mRNA levels of IFN-α, IFN-β, ISG15 and ISG56 were measured by real-time PCR. The mRNA level in each sample was expressed as a fold change of the RNA level in lipofectamine (LF)-treated cells. The levels of mRNA are presented relative to mock control. (B) Cells were transfected with HBx-siRNAs for 24 h. Levels of TNF-α, IL-6, iNOS, and IL-8 mRNA were analyzed by real-time PCR and presented relative to those of LF-treated cells. (C) HepG2.2.15 cells were incubated with phycoerythrin (PE)-labelled anti-p-Stat1 antibody after HBx-siRNA transfection for 4 h, and were then analyzed by flow cytometry. p-Stat1 expression was also detected when IFN receptor was neutralized. (D) Levels of CXCL2, CCL4, and CXCL10 mRNA were analyzed by real-time PCR and presented relative to those of LF-treated cells. Data are expressed as the mean ± SD from at least three separate experiments. *<i>p</i><0.05 versus LF-treated group.</p