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* Department of Infection Control and Laboratory Diagnostics, Internal Medicine, Tohoku University Graduate School of Medicine,
Microbiology and Immunology, Department of Medical Technology, Tohoku University School of Health Sciences,
Comprehensive Research and Education Center for Planning of Drug Development and Clinical Evaluation Project, Tohoku University, and
Infection-Control Research Center, Tohoku University Hospital, Sendai;
¶ Department of Medicine and Therapeutics, Control and Prevention of Infectious Diseases, Faculty of Medicine, University of the Ryukyus, Nishihara;
|| Department of Microbiology and Immunology, Graduate School of Medicine and
# Department of Host Defense, Research Institute for Microbial Disease, Osaka University, Osaka;
** Department of Microbiology and Immunology, Center for Experimental Medicine, Institute of Medical Science, University of Tokyo;
Laboratory for Immunopharmacology of Microbial Products, Tokyo University of Pharmacy and Life Science, Tokyo; and
* Department of Immunology, Inflammatory Program, Chiba University School of Medicine, Chiba, Japan
The mechanism of host cell recognition of Cryptococcus neoformans, an opportunistic fungal pathogen in immunocompromised patients, remains poorly understood. In the present study, we asked whether the DNA of this yeast activates mouse bone marrow-derived myeloid dendritic cells (BM-DCs). BM-DCs released IL-12p40 and expressed CD40 upon stimulation with cryptococcal DNA, and the response was abolished by treatment with DNase, but not with RNase. IL-12p40 production and CD40 expression were attenuated by chloroquine, bafilomycin A, and inhibitory oligodeoxynucleotides (ODN) that suppressed the responses caused by CpG-ODN. Activation of BM-DCs by cryptococcal DNA was almost completely abrogated in TLR9 gene-disrupted (TLR9–/–) mice and MyD88–/– mice, similar to that by CpG-ODN. In addition, upon stimulation with whole yeast cells of acapsular C. neoformans, TLR9–/– BM-DCs produced a lower amount of IL-12p40 than those from wild-type mice, and TLR9–/– mice were more susceptible to pulmonary infection with this fungal pathogen than wild-type mice, as shown by increased number of live colonies in lungs. Treatment of cryptococcal DNA with methylase resulted in reduced IL-12p40 synthesis by BM-DCs. Furthermore, using a luciferase reporter assay, cryptococcal DNA activated NF-
B in HEK293 cells transfected with the TLR9 gene. Finally, confocal microscopy showed colocalization of fluorescence-labeled cryptococcal DNA with CpG-ODN and the findings merged in part with the distribution of TLR9 in BM-DCs. Our results demonstrate that cryptococcal DNA causes activation of BM-DCs in a TLR9-dependent manner and suggest that the CpG motif-containing DNA may contribute to the development of inflammatory responses after infection with C. neoformans.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by a Grant-in-Aid for Science Research (C) (18590413) from the Ministry of Education, Culture, Sports, Science and Technology of Japan, the Ministry of Health and Welfare of Japan, and Tohoku University 21st Center of Excellence Program "Comprehensive Research and Education Center for Planning of Drug Development and Clinical Evaluation Project."
2 K.N. and A.M. contributed equally to this work.
3 Current address: Department of Laboratory Medicine, The 6th Affiliated Hospital, Sun Yat-sen University School of Medicine, Guangzhou, China.
4 Address correspondence and reprint requests to Dr. Kazuyoshi Kawakami, Microbiology and Immunology, Department of Medical Technology, Tohoku University School of Health Sciences, 2-1 Seiryo-cho, Aoba-ku, Sendai, Miyagi 980-8575, Japan. E-mail address: kawakami{at}mail.tains.tohoku.ac.jp
5 Abbreviations used in this paper: Cn, Cryptococcus neoformans; DC, dendritic cell; BM-DC, bone marrow-derived DC; ODN, oligodeoxynucleotide; WT, wild type; OX-CA, NaClO-oxidized Candida albicans.
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