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, 183 (5), 3109-17

Requirement for DNA CpG Content in TLR9-dependent Dendritic Cell Activation Induced by DNA-containing Immune Complexes

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Requirement for DNA CpG Content in TLR9-dependent Dendritic Cell Activation Induced by DNA-containing Immune Complexes

Kei Yasuda et al. J Immunol.

Abstract

Although TLR9 was originally thought to specifically recognize microbial DNA, it is now evident that mammalian DNA can be an effective TLR9 ligand. However, the DNA sequence required for TLR9 activation is controversial, as studies have shown conflicting results depending on the nature of the DNA backbone, the route of DNA uptake, and the cell type being studied. In systemic lupus erythematosus, a major route whereby DNA gains access to intracellular TLR9, and thereby activates dendritic cells (DCs), is through uptake as a DNA-containing immune complex. In this report, we used defined dsDNA fragments with a natural (phosphodiester) backbone and show that unmethylated CpG dinucleotides within dsDNA are required for murine DC TLR9 activation induced by a DNA-containing immune complex. The strongest activation is seen with dsDNA fragments containing optimal CpG motifs (purine-purine-CpG-pyrimidine-pyrimidine) that are common in microbial DNA but rare in mammalian DNA. Importantly, however, activation can also be induced by CpG-rich DNA fragments that lack these optimal CpG motifs and that we show are plentiful in CpG islands within mammalian DNA. No activation is induced by DNA fragments lacking CpG dinucleotides, although this CpG-free DNA can induce DC activation if internalized by liposomal transfection instead of as an immune complex. Overall, the data suggest that the release of CpG-rich DNA from mammalian DNA may contribute to the pathogenesis of autoimmune diseases such as systemic lupus erythematosus and psoriasis in which activation of TLR9 in DCs by self DNA has been implicated in disease pathogenesis.

Figures

FIGURE 1
FIGURE 1
Immune complexes containing optimal CpG motifs activate FL-DCs through TLR9 and induce different cytokine profiles from plasmacytoid and conventional DCs. A and B, Bone-marrow-derived FL-DCs from wild-type (WT) BALB/c or TLR9-deficient (TLR9−/−) mice were incubated with indicated concentrations of the DNA fragment CG50 in the presence or absence of the anti-DNA mAb PA4 (10 µg/ml). A, IFN-α and IL-6 concentrations in supernatants collected after 24 h were measured by ELISA. Data shown are the mean ± SEM of six experiments (WT) and four experiments (TLR9−/−). *, p < 0.05 versus WT;CG50; **, p< 0.01 versus WT;CG50; ##, p < 0.01 versus TLR9−/−;CG50+PA4. B, After removal of supernatants for cytokine analysis, the cells were analyzed by flow cytometry for CD40 expression, with stimulus-induced staining intensity compared with staining intensity of the nonstimulated cultures. C, FL-DCs from wild-type BALB/c mice were prepared and pDC (B220high, CD11bnegative, and CD11cpositive) and cDC (B220negative, CD11bhigh and CD11cpositive) were separated by cell sorting. The cells were stimulated with CG50 (100 ng/ml) in the presence or absence of PA4 (10 µg/ml). IFN-α and IL-6 concentrations in supernatants collected after 24 h were measured by ELISA. Data represent mean ± SEM of two experiments.
FIGURE 2
FIGURE 2
CpG content is required for TLR9-dependent DC activation induced by DNA-containing IC. A and B, Increasing concentrations of unlabeled dsDNA fragments CG50, CGSubOp and CGneg were added to plate-bound PA4. After 1 h, biotinylated CGSubOp (5 ng/ml) (A), or biotinylated CG50 (5 ng/ml) (B) was added. The binding of biotinylated CGSubOp (A), or biotinylated CG50 (B), to PA4 was detected with streptavidin-HRP and TMB substrate. Data represent mean ± SEM of three experiments for both A and B. C, Biotinylated DNA fragments (100, 300, or 1000 ng/ml) with or without the anti-biotin mAb 1D4 (3 µg/ml) were added to FL-DC from wild type BALB/c mice. IFN-α and IL-6 concentrations in supernatants collected after 24 h were measured by ELISA. Data represent mean ± SEM of four experiments. *, p < 0.05. D, Bone-marrow-derived FL-DCs from wild-type (WT) BALB/c or TLR9-deficient (TLR9−/−) mice were incubated with indicated concentrations of the DNA fragments CGSubOp and CGneg in the presence or absence of the anti-DNA mAb PA4 (10 µg/ml). IL-6 concentrations in supernatants collected after 24 h were measured by ELISA. Data shown are the mean ± SEM of five experiments (WT) and three experiments (TLR9−/−). **, p< 0.01 versus WT;DNA; #, p < 0.05 versus TLR9−/−;DNA+PA4. E, After removal of supernatants for cytokine analysis, the cells were analyzed by flow cytometry for CD40 expression, with stimulus-induced staining intensity compared with staining intensity of the nonstimulated cultures.
FIGURE 3
FIGURE 3
Methylation of CpG dinucleotides abrogates the stimulatory capacity of DNA that contains unmethylated CpG dinucleotides but lacks optimal CpG motifs. A, The 660 bp DNA fragment CG50 was treated, or not treated, with M.SssI methylase (CpG methylase) and then digested, or not digested, with the methylation sensitive restriction endonuclease, HpyCH4 IV. B, Increasing concentrations of unlabeled CGSubOp and CGSubOp treated with M.SssI methylase (Met-CGSubOp) were added to plate-bound PA4. After 1 h, biotinylated CGSubOp (5 ng/ml) was added. The binding of biotinylated CGSubOp to PA4 was detected with streptavidin-HRP and TMB substrate. Data represent mean ± SEM of three experiments. C, CGSubOp fragments were treated, or not treated, with M.SssI methylase or Alu I methylase. FL-DC from wild type BALB/c mice were then incubated with the variously treated CGSubOp fragments alone (300 ng/ml) or with CGSubOp-ICs (300 ng/ml of CGSubOp plus 10 µg/ml of PA4). IL-6 concentrations in supernatants collected after 24 h were measured by ELISA. Data represent mean ± SEM of five experiments. **, p< 0.01.
FIGURE 4
FIGURE 4
CpG-free DNA internalized into DCs by liposomal transfection is stimulatory, in contrast to CpG-free DNA internalized in an IC which is not stimulatory. A, Increasing concentrations of the unlabeled DNA fragments pCpG-mcs and CGSubOp were added to plate-bound PA4. After 1 h, biotinylated CGSubOp (5 ng/ml) was added. The binding of biotinylated CGSubOp to PA4 was detected with streptavidin-HRP and TMB substrate. Data represent mean ± SEM of two experiments. B, pCpG-mcs alone, pCpG-mcs plus PA4 (10 µg/ml) or pCpG-mcs plus DOTAP (at weight ratio of 1:2) were added to FL-DC from wild-type (WT) BALB/c or TLR9-deficient (TLR9−/−) mice. IFN-α and IL-6 concentrations in supernatants collected after 24 h were measured by ELISA. Data represent mean ± SEM of three experiments. *, p < 0.05 versus pCpG-mcs and pCpG-mcs+PA4. C, Biotinylated CGneg was labeled with streptavidin-coated fluorescent Qdots. Qdot-labeled CGneg-DOTAP complexes (CGneg 1000 ng/ml + DOTAP 2 µg/ml), Qdot-labeled CGneg ICs (CGneg 1000 ng/ml + PA4 10 µg/ml), and Qdot-labeled CGneg alone (1000 ng/ml) were added to FL-DC for 2 h at 37°C. The extent of Qdot-labeled CGneg internalization or binding to pDC and cDC was determined using flow cytometry to measure fluorescence intensity. Data represent one of three representative experiments. D, CGneg alone (1000 ng/ml), CGneg-ICs (CGneg 1000 ng/ml + PA4 10 µg/ml) and CGneg-DOTAP complexes (CGneg 1000 ng/ml + DOTAP 2 µg/ml) were added to FL-DC from wild-type BALB/c mice. IL-6 concentrations in supernatants collected after 24 h were measured by ELISA. Data represent mean ± SEM of three experiments. *, p < 0.05
FIGURE 5
FIGURE 5
Endogenous mammalian CpG-rich sequences activate DCs. Biotinylated CpG island DNA fragments (clones 11, 12, 14, 15 and 23) and the CpG-poor SUMO fragment, all at 1000 ng/ml, with or without the anti-biotin mAb 1D4 (3 µg/ml) were added to FL-DC from wild type BALB/c mice. Experiments were done in the absence or the presence of a 2 h pretreatment of the FL-DC with IFN-β (300 U/ml). IFN-α and IL-6 concentrations in supernatants collected after 24 h were measured by ELISA. Data represent the mean ± SEM of three experiments. . *, p < 0.05

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