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. 2008 May;76(5):2123-9.
doi: 10.1128/IAI.00047-08. Epub 2008 Mar 10.

Toll-like receptor 9-dependent immune activation by unmethylated CpG motifs in Aspergillus fumigatus DNA

Zaida G Ramirez-Ortiz  1 , Charles A Specht, Jennifer P Wang, Chrono K Lee, Daniella C Bartholomeu, Ricardo T Gazzinelli, Stuart M Levitz
Affiliations

Toll-like receptor 9-dependent immune activation by unmethylated CpG motifs in Aspergillus fumigatus DNA

Zaida G Ramirez-Ortiz et al. Infect Immun. 2008 May.

Abstract

Phagocytic defenses are critical for effective host defenses against the opportunistic fungal pathogen Aspergillus fumigatus. Previous studies found that following challenge with A. fumigatus, Toll-like receptor 9 (TLR9) knockout mice survived longer than wild-type mice. However, the mechanism responsible was not defined. Here we demonstrate that A. fumigatus contains unmethylated CpG sequences, the natural ligands for TLR9. A. fumigatus DNA and synthetic CpG-rich oligodeoxynucleotides (ODNs) containing sequences found in the A. fumigatus genome potently stimulated the production of proinflammatory cytokines in mouse bone marrow-derived dendritic cells (BMDCs) and human plasmacytoid dendritic cells. The response was decreased when the fungal DNA was treated with a CpG methylase or with CpG-specific endonucleases. A role for TLR9 was demonstrated as cytokine production was abolished in BMDCs from TLR9-deficient mice. Moreover, transfection of HEK293 cells with human TLR9 conferred responsiveness to synthetic CpG-rich ODNs containing sequences found in A. fumigatus DNA. Taken together, these data demonstrate that TLR9 detects A. fumigatus DNA, resulting in the secretion of proinflammatory cytokines, which may contribute to the immune response to the pathogen.

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Figures

FIG. 1.
FIG. 1.
A. fumigatus DNA contains unmethylated CpG sequences. Genomic DNA was isolated from A. fumigatus and left untreated or treated with the indicated enzymes. “+” indicates treatment; “−” indicates absence of treatment. The digest was then analyzed by 1% agarose gel electrophoresis with ethidium bromide staining. The left lane shows a DNA ladder with numbers indicating the size of the DNA, in kb. The figure is representative of 12 individual experiments, each with similar results.
FIG. 2.
FIG. 2.
Stimulation of murine BMDCs by A. fumigatus DNA. BMDCs were stimulated by transfecting 2.5 μg/ml of A. fumigatus DNA or E. coli DNA that was left untreated (DNA) or treated with the CpG methyltransferase M.SssI (Meth DNA), MspI, or HpaII. CpG 1826 at 3 μg/ml served as a positive control, while unstimulated cells and GpC 2137 were negative controls. After 24 h, supernatants were collected and TNF-α (A) and IL-12p70 (B) concentrations were determined by ELISA. Data are means ± SE of five individual experiments. P was <0.001 according to one-way ANOVA with a Bonferroni posttest for TNF-α and IL-12p70 stimulated by untreated A. fumigatus or E. coli DNA compared with any treatment.
FIG. 3.
FIG. 3.
A. fumigatus DNA stimulates TNF-α secretion in a TLR9-dependent manner. BMDCs from WT and TLR9−/− mice were transfected with the indicated concentrations of A. fumigatus DNA that was left untreated (Af DNA) or treated with the CpG methyltransferase M.SssI (Met Af DNA) and incubated at 37°C. After 24 h, supernatants were collected and TNF-α concentrations were determined by ELISA. The concentrations of TNF-α released from unstimulated WT and TLR9−/− BMDCs were 225 ± 40 pg/ml and 134 ± 18 pg/ml, respectively. Data are means ± SE of a representative (out of four) experiment performed in triplicate. P was <0.001 according to one-way ANOVA with a Bonferroni posttest for TNF-α stimulated by Af DNA in WT BMDCs compared with any other group at DNA concentrations of 3 and 10 μg/ml. DC, dendritic cells; KO, knockout.
FIG. 4.
FIG. 4.
Stimulation of human pDCs by A. fumigatus DNA. Human pDCs were isolated from PBMCs by positive selection using CD304+ magnetic beads. (A) pDCs were stimulated with 10 μg of A. fumigatus DNA which was either left untreated or methylated with the CpG methyltransferase M.SssI. Unstimulated and CpG 2336 (10 μg/ml)-stimulated pDCs served as negative and positive controls, respectively. After 18 to 24 h, supernatants were collected and analyzed by ELISA for IFN-α. Results are shown as means ± SE of seven individual donors. P was <0.001 comparing untreated and methylated DNA by one-way ANOVA with a Bonferroni posttest. (B) As in panel A, except a dose-response curve was performed comparing untreated and methylated A. fumigatus DNA. Data are means ± SE of a representative (out of three) experiment performed in triplicate. P was <0.001 comparing untreated and methylated DNA at concentrations of 1, 3, and 10 μg/ml by two-way ANOVA with a Bonferroni posttest.
FIG. 5.
FIG. 5.
Stimulation of murine BMDCs by ODNs containing CpG-rich motifs present in the A. fumigatus genome. BMDCs from WT (top panel) and TLR9−/− (bottom panel) mice were stimulated with the indicated mouse-like CpG motifs (see Table S1 in the supplemental material). CpG 1826 served as a positive control, while unstimulated (Unstim) cells and GpC 2137 were negative controls. After 24 h, supernatants were collected and TNF-α concentrations were determined by ELISA. Data are means ± SE of a representative (out of three) experiment performed in triplicate.
FIG. 6.
FIG. 6.
Stimulation of hTLR9-transfected HEK293 cells by ODNs containing CpG-rich motifs present in the A. fumigatus genome. HEK293 cells stably coexpressing hTLR9 and the NF-κB-driven luciferase reporter construct were stimulated with the indicated human-like CpG motifs (see Table S1 in the supplemental material). CpG 2007 served as a positive control, while GpC 2137 was a negative control. After 18 h, luciferase activity was determined in the cell lysates. Results are expressed as n-fold induction over unstimulated cells. Data are means ± SE of a representative (out of four) experiment performed in triplicate.
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