Akiko Miyazato

IL-18 contributes to host resistance against infection with Cryptococcus neoformans in mice with defective IL-12 synthesis through induction of IFN-γ production by NK cells.

The aim of this study was to examine the contribution of IL-18 in host defense against infection caused by Cryptococcus neoformans in mice with defective IL-12 production. Experiments were conducted in mice with a targeted disruption of the gene for IL-12p40 subunit (IL-12p40−/− mice). In these mice, host resistance was impaired, as shown by increased number of organisms in both lungs and brains, compared with control mice. Serum IFN-γ was still detected in these mice at a considerable level (20–30% of that in control mice). The host resistance was moderately impaired in IL-12p40−/− mice compared with IFN-γ−/− mice. Neutralizing anti-IFN-γ mAb further increased the lung burdens of organisms. In addition, treatment with neutralizing anti-IL-18 Ab almost completely abrogated the production of IFN-γ and also impaired the host resistance. Host resistance in IL-12p40−/− IL-18−/− mice was more profoundly impaired than in IL-12p40−/− mice. Administration of IL-12 as well as IL-18 increased the serum levels of IFN-γ and significantly restored the reduced host resistance. Spleen cells obtained from infected IL-12p40−/− mice did not produce any IFN-γ upon restimulation with the same organisms, while those from infected and IL-12-treated mice produced IFN-γ. In contrast, IL-18 did not show such effect. Finally, depletion of NK cells by anti-asialo GM1 Ab mostly abrogated the residual production of IFN-γ in IL-12p40−/− mice. Our results indicate that IL-18 contributes to host resistance to cryptococcal infection through the induction of IFN-γ production by NK cells, but not through the development of Th1 cells, under the condition in which IL-12 synthesis is deficient.

Toll-Like Receptor 9-Dependent Activation of Myeloid Dendritic Cells by Deoxynucleic Acids from Candida albicans

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 β-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 pmr1Δ 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-κB 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.

Deoxynucleic Acids from Cryptococcus neoformans Activate Myeloid Dendritic Cells via a TLR9-Dependent Pathway

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-κB 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.

Dectin‐1 Is Not Required for the Host Defense to Cryptococcus neoformans

Dectin‐1 is known as a sole receptor for β‐glucan, a major cell wall component of fungal microorganisms. In the current study, we examined the role of this molecule in the host defense to Cryptococcus neoformans, an opportunistic fungal pathogen in AIDS patients. There was no significant difference in the clinical course and cytokine production between dectin‐1 gene‐deficient and control mice. These results indicate that dectin‐1 is not likely essential for the development of host protective responses to C. neoformans.

Toll-like receptor 4-dependent activation of myeloid dendritic cells by leukocidin of Staphylococcus aureus

Leukocidin (Luk), an exotoxin of Staphylococcus aureus consisting of LukF and LukS, is a hetero-oligomeric pore-forming cytolytic toxin toward human and rabbit polymorphonuclear leukocytes. However, it is uncertain how Luk affects the host immune response. In the present study, we investigated whether Luk has the ability to stimulate mouse bone marrow-derived myeloid dendritic cells (BM-DCs). LukF activated BM-DCs to generate IL-12p40 mRNA, induce intracellular expression and extracellular secretion of this cytokine and express CD40 on their surface, whereas LukS showed a much lower or marginal ability in the activation of BM-DCs than its counterpart component. Similarly, TNF-alpha was secreted by BM-DCs upon stimulation with these components. Combined addition of these components did not lead to a further increase in IL-12p40 secretion. IL-12p40 production caused by LukF was completely abrogated in BM-DCs from TLR4-deficient mice similarly to the response to lipopolysaccharide (LPS). Polymixin B did not affect the LukF-induced IL-12p40 production, although the same treatment completely inhibited the LPS-induced response. Boiling significantly inhibited the response caused by LukF, but not by LPS. Finally, in a luciferase reporter assay, LukF induced the activation of NF-kappaB in HEK293T cells transfected with TLR4, MD2 and CD14, whereas LukS did not show such activity. These results demonstrate that LukF caused the activation of BM-DCs by triggering a TLR4-dependent signaling pathway and suggests that Luk may affect the host inflammatory response as well as show a cytolytic effect on leukocytes.

Chemokine synthesis and cellular inflammatory changes in lungs of mice bearing p40tax of human T-lymphotropic virus type 1.

To elucidate the pathogenic mechanisms of human T‐lymphotropic virus type 1 (HTLV‐1)‐associated lung inflammation, we conducted a histopathological and molecular analysis study using transgenic mice bearing pX region of this virus. In these mice, accumulations of inflammatory cells consisting mainly of lymphocytes were present in peribronchiolar and perivascular areas and alveolar septa, while control littermate mice did not show such changes. In situ hybridization showed that the anatomic distribution of p40tax mRNA was similar to that of inflammatory cells, typically in peribronchiolar areas and to a lesser extent in perivascular and alveolar septa. Inflammatory cytokines, including IL‐1β, tumour necrosis factor‐alpha and interferon‐gamma, and several chemokines, such as monocyte chemotactic protein‐1 (MCP‐1), RANTES, macrophage inflammatory protein‐1α (MIP‐1α) and IP‐10, were detected in lungs of transgenic mice but not control mice. Semiquantitative analysis using reverse transcription‐polymerase chain reaction showed a significant correlation between MCP‐1 mRNA expression and p40tax mRNA, but not with other chemokines. The gene expression of the above chemokines, with the exception of MIP‐1α, correlated with the severity of histopathological changes in the lung. Considered together, our results suggested that p40tax synthesis may be involved in the development of lung lesions caused by HTLV‐1 through the induction of local production of chemokines.

Comparative molecular and microbiological diagnosis of 19 infective endocarditis cases in which causative microbes were identified by PCR-based DNA sequencing from the excised heart valves

Infective endocarditis (IE) is traditionally diagnosed by microbiological analysis of blood cultures, following which therapeutic antibiotics are chosen based on antimicrobial sensitivity tests. However, such conventional techniques do not always lead to an accurate etiological diagnosis. Recently, PCR analysis of the 16S rRNA gene has been employed to identify organisms isolated from excised heart valves. In this study, we analyzed 19 valve samples from patients with confirmed IE, as identified by Duke’s criteria. Using broad-range PCR amplification, followed by direct gene sequencing, pathological agents were identified in all samples. Although blood cultures yielded negative results in 4 cases, PCR analysis of valve samples showed positive identification of causative organisms. In 3 cases, there was a difference between blood culture and PCR in identification of pathological agents, which are likely to be misidentified by the conventional method based on the phenotypic database. Postoperative antibiotics were chosen considering the severity of lesions and the results of PCR, Gram staining, and valve cultures. All patients were cured without relapse. The broad-range PCR method was therefore beneficial for the management of IE because it enabled us to identify pathogens directly from the site of infection, even organisms that were difficult to culture or likely to be misidentified by the conventional culture method. Identification of the agents provided precise knowledge of the microbiological spectrum involved in the cases of IE.

Toll-Like Receptor 9-Dependent Activation of Bone Marrow-Derived Dendritic Cells by URA5 DNA from Cryptococcus neoformans

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 pathogens 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.

Cryptococcus neoformans inhibits nitric oxide synthesis caused by CpG-oligodeoxynucleotide-stimulated macrophages in a fashion independent of capsular polysaccharides

Cryptococcus neoformans is eradicated by macrophages via production of NO. Unmethylated CpG‐ODN protect mice from infection with this fungal pathogen by inducing IFN‐γ. The present study was designed to elucidate the effect of C. neoformans on the synthesis of NO by alveolar macrophages. For this purpose, MH‐S, an alveolar macrophage cell line, was stimulated with CpG‐ODN in the presence of IFN‐γ. A highly virulent strain of C. neoformans with thick capsule suppressed the production of NO. Capsular polysaccharides were not essential for this suppression, because there was no difference between acapsular mutant (Cap67) and its parent strain. Physical or close interaction of Cap67 with MH‐S was necessary, as shown by the loss of such effect when direct contact was interfered by nitrocellulose membrane. Similar effects were observed by disrupted as well as intact Cap67. Whereas the inhibitory effect of intact Cap67 was completely abrogated by heat treatment, disrupted Cap67 did not receive such influence. Finally, disrupted Cap67 did not show any inhibitory effect on the TLR9‐mediated activation of NF‐κB in a luciferase reporter assay with HEK293T cells, although the TLR4‐mediated activation was suppressed. These results revealed that C. neoformans suppressed the synthesis of NO by CpG‐ODN and IFN‐γ‐stimulated macrophages in a fashion independent of capsular polysaccharides, although the precise mechanism remains to be elucidated.

Toll-like receptor 2 (TLR2) and dectin-1 contribute to the production of IL-12p40 by bone marrow-derived dendritic cells infected with Penicillium marneffei

The present study was designed to elucidate the role of TLR2, TLR4 and dectin-1 in the production of IL-12p40 by bone marrow-derived dendritic cells (BM-DCs) infected with Penicillium marneffei. IL-12p40 production was almost completely abrogated in BM-DCs from TLR2 gene-knockout (KO) and MyD88KO mice, but not from TLR4-defective C3H/HeJ mice compared to those from control mice. Furthermore, BM-DCs from dectin-1KO mice faintly produced IL-12p40 upon stimulation with this fungus. Using a luciferase reporter assay, P. marneffei activated NF-kappaB in HEK293 cells transfected with the TLR2 gene, but not with the dectin-1 gene, and their co-transfection did not lead to further increase in this response. These results indicate that TLR2 and dectin-1 are essential in sensing P. marneffei for the activation of BM-DCs.