Contribution of interferon-beta to the immune activation induced by double-stranded DNA

Abstract

Introducing double-stranded DNA (dsDNA) into the cytoplasm of macrophages and dendritic cells triggers the activation of these professional antigen-presenting cells (APCs). This process is characterized by the up-regulation of costimulatory molecules and the production of various cytokines, chemokines, and antibacterial/viral factors. Current findings indicate that interferon-beta (IFN-beta) plays a key role in the stimulatory cascade triggered by dsDNA. Both immune and non-immune cells respond to intracytoplasmic dsDNA by up-regulating IFN-beta) expression, a process that reduces host susceptibility to infection. The immune activation induced by dsDNA is independent of MyD88, TRIF and DNA-PKcs, indicating that a Toll-like receptor-independent mechanism underlies the cellular activation mediated by intracytoplasmic dsDNA.

Figure 1

Mammalian and synthetic dsDNA up-regulate cytokine gene expression by murine macrophages. (a) Peritoneal macrophages were transfected with Fugene6 alone (–) or combined with 3 µg/ml of CT DNA or synthetic polynucleotides. mRNA expression was monitored by RT—PCR 3 hr later. PCR conditions for each cytokine were established such that a faint band was generated using control mRNA. (b) Quantitative analysis of RT—PCR data from three independent experiments performed as described in (a). mRNA levels were quantitated by analysing PCR image intensity using ‘NIH image’ software, and the data normalized to the endogenous β-actin reference. Values represent the mean ± SD increase in mRNA levels when compared to cells treated with Fugene6 alone. *P < 0·05; **P < 0·001 (compared with Fugene6 alone).

Figure 2

Role of IFN-β in dsDNA-induced cell activation. (a) RAW 264.7 cells were untreated (grey shadow) or transfected with 3 µg/ml dsCT DNA in Fugene6 (dark line). Cells were then incubated with 2·5 µg/ml of cytokine-neutralizing antibody for 16 hr, and CD40 expression analysed by FACS. Experiments were repeated three times with similar results. (b) Peritoneal macrophages were transfected with 3 µg/ml dsCT DNA in Fugene6. Cells were then incubated with 2·5 µg/ml of cytokine-neutralizing antibody for 24 hr, and NO levels in culture supernatants determined. (c) Peritoneal macrophages were transfected with 3 µg/ml dsCT DNA in Fugene6. Cells were then incubated with 2·5 µg/ml of cytokine-neutralizing antibody for 3 hr and mRNA expression monitored by RT—PCR. Data represent the mean ± SD response of three independent populations of macrophages, and were confirmed in two additional experiments. *P < 0·05 (compared with CT DNA transfected cells alone).

Figure 3

Effect of dsDNA on IFN-β mRNA expression by macrophages and dendritic cells. Peritoneal macrophages (a) and BMDCs (b) were transfected with 10 µg/ml dsCT DNA in Fugene6 for 1–12 hr. BMDCs (c) were stimulated with the indicated dose of dsCT DNA plus Fugene6 or poly(I:C) for 3 hr. IFN-β mRNA levels were determined by TaqMan PCR. Results represent the mean fold increase ± SD of mRNA levels from three independent experiments when compared to unstimulated cells cultured for the same period.

Figure 4

Effect of DNA transfection on IFN-β mRNA expression. Peritoneal macrophages from from C57Bl/6 or TLR9 KO mice were transfected with 3 µg/ml of ds or ss DNA in the presence or absence of Fugene6. IFN-β mRNA levels were measured 3 hr later by quantitative RT—PCR. Results represent the mean fold increase ± SD in IFN-β mRNA levels compared to cells transfected with Fugene6 alone from three independent experiments. *P < 0·05 (compared with ssDNA group).

Figure 5

Induction of IFN-β by dsDNA does not involve MyD88, TRIF or DNA—PKCs. BMDDC from WT, MyD88 KO, TRIF KO and SCID mice were transfected with 10 µg/ml dsCT DNA in Fugene6. IFN-β mRNA levels were determined 3 hr later by RT—PCR. Experiments were repeated three times with similar results.

Figure 6

Stimulatory activity of dsDNA in murine and human cell lines. Non-immune cells of murine and human origin were transfected with 3 µg/ml of dsCT DNA in Fugene6 or 3 µm CpG DNA. IFN-β and IL-6 mRNA levels were determined 3 hr later by RT—PCR. Experiments were repeated twice with similar results.

Figure 7

DsDNA reduces susceptibility to HSV-2 infection. Monolayers of MEFs were transfected with 1 µg/ml of dsCT DNA or 3 µm of CpG DNA. Cells were then incubated with 10 µg/ml of IFN-β-neutralizing or control antibody for 16 hr and challenged with HSV-2. Plaques were visualized 2 days after infection by staining with crystal violet. All studies were performed in triplicate, with the histogram showing the mean ± SD virus titre/treatment group. Experiments were repeated twice with similar results. *P < 0·001 (compared with Fugene6 treatment group).