Toll-like receptor 9-dependent activation of myeloid dendritic cells by Deoxynucleic acids from Candida albicans - PubMed (original) (raw)

. 2009 Jul;77(7):3056-64.

doi: 10.1128/IAI.00840-08. Epub 2009 May 11.

Kiwamu Nakamura, Natsuo Yamamoto, Héctor M Mora-Montes, Misuzu Tanaka, Yuzuru Abe, Daiki Tanno, Ken Inden, Xiao Gang, Keiko Ishii, Kiyoshi Takeda, Shizuo Akira, Shinobu Saijo, Yoichiro Iwakura, Yoshiyuki Adachi, Naohito Ohno, Kotaro Mitsutake, Neil A R Gow, Mitsuo Kaku, Kazuyoshi Kawakami

Affiliations

Toll-like receptor 9-dependent activation of myeloid dendritic cells by Deoxynucleic acids from Candida albicans

Akiko Miyazato et al. Infect Immun. 2009 Jul.

Abstract

The innate immune system of humans recognizes the human pathogenic fungus Candida albicans via sugar polymers present in the cell wall, such as mannan and beta-glucan. Here, we examined whether nucleic acids from C. albicans activate dendritic cells. C. albicans DNA induced interleukin-12p40 (IL-12p40) production and CD40 expression by murine bone marrow-derived myeloid dendritic cells (BM-DCs) in a dose-dependent manner. BM-DCs that lacked Toll-like receptor 4 (TLR4), TLR2, and dectin-1, which are pattern recognition receptors for fungal cell wall components, produced IL-12p40 at levels comparable to the levels produced by BM-DCs from wild-type mice, and DNA from a C. albicans pmr1Delta null mutant, which has a gross defect in mannosylation, retained the ability to activate BM-DCs. This stimulatory effect disappeared completely after DNase treatment. In contrast, RNase treatment increased production of the cytokine. A similar reduction in cytokine production was observed when BM-DCs from TLR9(-/-) and MyD88(-/-) mice were used. In a luciferase reporter assay, NF-kappaB activation was detected in TLR9-expressing HEK293T cells stimulated with C. albicans DNA. Confocal microscopic analysis showed similar localization of C. albicans DNA and CpG-oligodeoxynucleotide (CpG-ODN) in BM-DCs. Treatment of C. albicans DNA with methylase did not affect its ability to induce IL-12p40 synthesis, whereas the same treatment completely eliminated the ability of CpG-ODN to induce IL-12p40 synthesis. Finally, impaired clearance of this fungal pathogen was not found in the kidneys of TLR9(-/-) mice. These results suggested that C. albicans DNA activated BM-DCs through a TLR9-mediated signaling pathway using a mechanism independent of the unmethylated CpG motif.

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Figures

FIG. 1.

FIG. 1.

C. albicans DNA and S. cerevisiae DNA activate BM-DCs. BM-DCs were cultured under various conditions for 24 h. IL-12p40 levels in the culture supernatants were determined by ELISA (a, b, c, and e), and surface expression of CD40 on BM-DCs was determined by flow cytometry (d). The data are means ± SD of triplicate cultures. Similar results were obtained in three independent experiments. (−) indicates C. albicans DNA (CA-DNA) stimulation without polymyxin B (b) or DNase or RNase (c) treatment. *, P < 0.05 for a comparison of a preparation treated with polymyxin B, DNase, or RNase and a preparation that was not treated. The amount of DNA from C. albicans THK519 and S. cerevisiae DNA used was 10 μg/ml, unless indicated otherwise (a and e). The concentration of LPS used was 1 μg/ml, and the concentration of DNase used was 10 μg/ml. PMB, polymyxin B (10 μg/ml); CpG, CpG1826 (1 μg/ml).

FIG. 2.

FIG. 2.

Role of C. albicans cell wall components in the activation of BM-DCs. BM-DCs from TLR4−/− and wild-type mice (a), TLR2−/− and wild-type mice (b), and dectin-1−/− and wild-type mice (c) were cultured with DNA from C. albicans THK519 (10 μg/ml), CpG1826 (1 μg/ml), LPS (1 μg/ml), OX-CA (3 μg/ml), or PG (1 μg/ml) for 24 h, and IL-12p40 levels in the culture supernatants were determined by ELISA. The data are means ± SD of triplicate cultures. Similar results were obtained in three independent experiments. CA-DNA, C. albicans DNA; CpG, CpG1826. *, P < 0.05 for a comparison of TLR4−/−, TLR2−/−, or dectin-1−/− mice and wild-type mice.

FIG. 3.

FIG. 3.

Role of manosyl residues in the activation of BM-DCs. BM-DCs were cultured with C. albicans DNA extracted from wild-type strain CAI4 or the _C. albicans pmr1_Δ null mutant at concentrations of 1, 3, and 10 μg/ml for 24 h, and IL-12p40 levels in the culture supernatants were determined by ELISA (a). C. albicans DNA was pretreated with or without DNase (b). (−) indicates C. albicans DNA stimulation without DNase treatment. *, P < 0.05 for a comparison of a preparation treated with DNase and a preparation that was not treated. The data are means ± SD of triplicate cultures. Similar results were obtained in three independent experiments.

FIG. 4.

FIG. 4.

Inhibitory effect of _C. albicans pmr1_Δ DNA on THK DNA activity. BM-DCs were incubated with 3 μg/ml DNA from THK519 alone or with various concentrations of _pmr1_Δ null mutant DNA (50, 25, and 12.5 μg/ml) or with _pmr1_Δ DNA alone (50, 25, and 12.5 μg/ml) for 24 h. IL-12p40 levels in the culture supernatants were measured by ELISA. The data are means ± SD of triplicate cultures. Similar results were obtained in three independent experiments. *, P < 0.05 for a comparison of a preparation incubated with THK519 alone and a preparation incubated with _pmr1_Δ null mutant DNA.

FIG. 5.

FIG. 5.

Indispensable role of TLR9-MyD88 signaling pathway in BM-DC activation. BM-DCs from TLR9−/− and wild-type mice (a) and MyD88−/− and wild-type mice (b) were cultured with C. albicans ATCC 18804 DNA (10 μg/ml), CpG (1 μg/ml), or LPS (1 μg/ml) for 24 h, and IL-12p40 levels in the culture supernatants were measured by ELISA. The data are means ± SD of triplicate cultures. Similar results were obtained in three independent experiments. CA-DNA, C. albicans DNA; CpG, CpG1826. *, P < 0.05 for a comparison of TLR9−/− or MyD88−/− mice and wild-type mice.

FIG. 6.

FIG. 6.

NF-κB activation via TLR9 by C. albicans DNA. HEK293T cells transfected with a TLR9 gene or control vector were cultured with CpG1826 (300 nM) or DNA from C. albicans THK519 (100 μg/ml) for 6 h. The luciferase activity in each sample was determined as described in Materials and Methods. Data are expressed as relative values based on the value for an unstimulated sample in each group and are means ± SD of triplicate cultures. Similar results were obtained in three independent experiments. CA-DNA, C. albicans DNA; CpG, CpG1826.

FIG. 7.

FIG. 7.

Trafficking of C. albicans DNA in BM-DCs. BM-DCs were simultaneously incubated with 10 μg/ml Alexa 647-conjugated DNA from C. albicans THK519 (green) and 3 μM CpG-rhodamine (red) (a). BM-DCs were incubated with 3 μM CpG-rhodamine (red) for 30 min. After fixation, TLR9 was stained intracellularly by direct immunofluorescence with FITC-conjugated anti-TLR9 antibody (green) (b). Cells were analyzed using a confocal microscope. The data are representative of three or four independent experiments. CA-DNA, C. albicans DNA; CpG, CpG1826.

FIG. 8.

FIG. 8.

Effect of methylation of C. albicans DNA. BM-DCs were cultured with ds CpG1826 (4.5 μM) or DNA from C. albicans THK519 (10 μg/ml) pretreated or not pretreated with methylase. IL-12p40 levels in the cultures supernatants were measured by ELISA. The data are means ± SD of triplicate cultures. Similar results were obtained in three independent experiments. (−) indicates C. albicans DNA stimulation without methylase treatment. CA-DNA, C. albicans DNA; ds-CpG, ds CpG1826. *, P < 0.05 for a comparison of a preparation treated with methylase and a preparation that was not treated with methylase.

FIG. 9.

FIG. 9.

Fungal growth in TLR9−/− and wild-type mice with systemic C. albicans infections. Wild-type or TLR9−/− mice were inoculated intravenously with 105 C. albicans yeast cells in 0.1 ml. Fungal growth in the kidneys was assessed 5 days after infection. Fungal growth in the kidneys (means ± standard errors) is expressed as the number of CFU per organ. WT, wild-type mice; TLR9KO, TLR9−/− mice; N.S., difference not significant.

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