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Published on: December 24, 2016
Toll-like receptor 9-dependent activation of bone marrow-derived dendritic cells by URA5 DNA from Cryptococcus
Misuzu Tanaka1, Keiko Ishii, Yuri Nakamura
1Department of Medical Microbiology, Mycology and Immunology, Tohoku University Graduate School of Medicine, Sendai, Japan.
Abstract:
Cryptococcus neoformans is an opportunistic fungal pathogen that causes meningoencephalitis in immunocompromised patients. Recently, we reported that Toll-like receptor 9 (TLR9) is involved in host defense against C. neoformans: specifically, it detects the pathogen's DNA. In the present study, we aimed to elucidate the mechanisms underlying TLR9-mediated activation of innate immune responses by using the URA5 gene, which encodes a virulent component of this fungal pathogen. A PCR-amplified 345-bp URA5 gene fragment induced interleukin-12 p40 (IL-12p40) production by bone marrow-derived dendritic cells (BM-DCs) in a TLR9-dependent manner. Similar activity was detected in the 5' 129-bp DNA fragment of URA5 and in a synthesized oligodeoxynucleotide (ODN) with the same sequence. Shorter ODN fragments, which contained GTCGGT or GACGAT but had only 24 or 21 bases, induced IL-12p40 production and CD40 expression by BM-DCs, but this activity vanished when the CG sequence was replaced by GC or when a phosphorothioate modification was introduced. IL-12p40 production caused by active ODN was strikingly enhanced by treatment with DOTAP, a cationic lipid that increases the uptake of DNA by BM-DCs, though DOTAP failed to induce IL-12p40 production by inactive ODN and did not affect the activity of an ODN-containing canonical CpG motif. There was no apparent difference in intracellular trafficking between active and inactive ODNs. Finally, an extremely high dose of inactive ODN suppressed IL-12p40 production by BM-DCs that had been stimulated with active ODN. These results suggest that the C. neoformans URA5 gene activates BM-DCs through a TLR9-mediated signaling pathway, using a mechanism possibly independent of the canonical CpG motif.
Insights
The Cryptococcus neoformans URA5 gene activates immune cells via Toll-like receptor 9 (TLR9). This mechanism may not rely on the typical CpG motif, offering new insights into fungal pathogen recognition.
Area of Science:
- Immunology
- Mycology
- Molecular Biology
Background:
- Cryptococcus neoformans is an opportunistic fungal pathogen causing severe infections in immunocompromised individuals.
- Toll-like receptor 9 (TLR9) has been identified as a key component in the host's defense against C. neoformans, recognizing the fungal DNA.
- Understanding the precise mechanisms of TLR9-mediated immune activation is crucial for developing targeted therapies.
Purpose of the Study:
- To elucidate the mechanisms of TLR9-mediated innate immune response activation by the C. neoformans URA5 gene.
- To investigate the role of specific DNA sequences within the URA5 gene in activating bone marrow-derived dendritic cells (BM-DCs).
- To explore the influence of sequence modifications and delivery agents on TLR9-dependent immune activation.
Main Methods:
- Utilized PCR-amplified DNA fragments of the C. neoformans URA5 gene and synthesized oligodeoxynucleotides (ODNs).
- Assessed the induction of interleukin-12 p40 (IL-12p40) production and CD40 expression in BM-DCs.
- Investigated the effects of sequence modifications (e.g., CG to GC, phosphorothioate backbone) and DOTAP treatment on ODN activity.
- Analyzed intracellular trafficking of ODNs and the suppressive effects of inactive ODNs.
Main Results:
- A 345-bp URA5 gene fragment and a 129-bp 5' fragment induced IL-12p40 production in a TLR9-dependent manner.
- Shorter ODNs containing specific GTCGGT or GACGAT sequences induced IL-12p40 and CD40 expression, which was abolished by sequence modification or phosphorothioate treatment.
- DOTAP enhanced IL-12p40 production by active ODNs but not inactive ones, and inactive ODNs suppressed responses to active ODNs at high doses.
Conclusions:
- The C. neoformans URA5 gene activates BM-DCs through a TLR9-mediated signaling pathway.
- The activation mechanism appears to be independent of the canonical CpG motif, suggesting novel recognition patterns.
- These findings provide insights into the innate immune recognition of fungal DNA and potential therapeutic targets.
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