Kiwamu Nakamura

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.

Lower expression of Th1‐related cytokines and inducible nitric oxide synthase in mice with streptozotocin‐induced diabetes mellitus infected with Mycobacterium tuberculosis

Diabetes mellitus is an important predisposing factor for tuberculosis. The aim of this study was to investigate the mechanism underlying this association using a murine model. Mice with streptozotocin‐induced diabetes mellitus were prone to Mycobacterium tuberculosis infection, as indicated by increased numbers of live bacteria in lung, liver and spleen. In diabetic mice, the levels of IL‐12 and IFN‐γ in the lung, liver and spleen were lower than those in control animals on day 14 postinfection, while the opposite was true for IL‐4 levels in the lung and liver. The expression pattern of inducible nitric oxide synthase (iNOS), in the two mice types was as for IL‐12 and IFN‐γ. In addition, peritoneal exudate cells obtained from diabetic mice produced lower amounts of IL‐12 and NO than those from control mice, when stimulated in vitro with M. bovis BCG. Spleen cells from diabetic mice infected with M. tuberculosis produced a significantly lower amount of IFN‐γ upon restimulation with purified protein derivatives (PPD) than those from infected nondiabetic mice. Interestingly, addition of high glucose levels (33 mM) to the cultures of PPD‐restimulated spleen cells reduced the synthesis of IFN‐γ only in diabetic mice, and not in nondiabetic mice. Finally, control of blood glucose levels by insulin therapy resulted in improvement of the impaired host protection and Th1‐related cytokine synthesis. Our results suggest that the reduced production of Th1‐related cytokines and NO account for the hampered host defense against M. tuberculosis infection under diabetic conditions.

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.

CpG oligodeoxynucleotides promote the host protective response against infection with Cryptococcus neoformans through induction of interferon-gamma production by CD4+ T cells.

In the present study, we elucidated the effect of synthetic CpG‐containing oligodeoxynucleotides (ODN) on pulmonary and disseminated infection caused by Cryptococcus neoformans. CDF‐1 mice were inoculated intratracheally with a highly virulent strain of this pathogen, which resulted in massive bacterial growth in the lung, dissemination to the brain and death. Administration of CpG‐ODN promoted the clearance of C. neoformans in the lungs, decreased their dissemination to brain and prolonged the survival of infected mice. These effects correlated well with the enhanced production of interleukin (IL)‐12 and interferon (IFN)‐γ and attenuated secretion of IL‐4 in bronchoalveolar lavage fluids (BALF) and promoted development of Th1 cells, as indicated by the increased production of IFN‐γ by paratracheal lymph node cells upon restimulation with cryptococcal antigens. The IFN‐γ synthesis in BALF was inhibited by depletion of CD8+ and CD4+ T cells on days 7 and 14 after infection, respectively, but not by depletion of NK and γδ T cells. Consistent with these data, intracellular expression of IFN‐γ was detected predominantly in CD8+ and CD4+ T cells in the lung on days 7 and 14, respectively. The protective effect of CpG‐ODN, as shown by the prolonged survival, was completely and partially inhibited by depletion of CD4+ or CD8+ T cells, respectively, but not by depletion of other cells. Finally, TNF‐α was markedly induced by CpG‐ODN, and the protective effect of this agent was strongly inhibited by neutralizing anti‐TNF‐α MoAb. Our results indicate that CpG‐ODN alters the Th1–Th2 cytokine balance and promotes host resistance against infection with C. neoformans.

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.

Activation of myeloid dendritic cells by deoxynucleic acids from Cordyceps sinensis via a Toll-like receptor 9-dependent pathway

The mechanism by which host cells recognize Cordyceps sinensis, a Chinese herbal medicine that is known to exhibit immunomodulating activity, remains poorly understood. In this study, we investigated whether the DNA of this fungus could activate mouse bone marrow-derived dendritic cells (BM-DCs). Upon stimulation with C. sinensis DNA, BM-DCs released IL-12p40 and TNF-alpha and expressed CD40. Cytokine production and CD40 expression were attenuated by chloroquin and bafilomycin A. Activation of BM-DCs by C. sinensis DNA was almost completely abrogated in TLR9KO mice. According to a luciferase reporter assay, C. sinensis DNA activated NF-kappaB in HEK293T cells transfected with the TLR9 gene. Finally, a confocal microscopic analysis showed that C. sinensis DNA was co-localized with CpG-ODN and partly with TLR9 and LAMP-1, a late endosomal marker, in BM-DCs. Our results demonstrated that C. sinensis DNA caused activation of BM-DCs in a TLR9-dependent manner.

Early production of tumor necrosis factor-α by Gr-1+ cells and its role in the host defense to pneumococcal infection in lungs

In this study, we elucidated the role of tumor necrosis factor (TNF)-alpha in the host defense to pulmonary infection with Streptococcus pneumoniae and defined the cellular source of this cytokine at an early stage of infection. Administration of anti-TNF-alpha monoclonal antibody (mAb) resulted in the reduced accumulation of neutrophils in bronchoalveolar lavage fluids (BALFs) and severe exacerbation of this infection. In a flow cytometric analysis, the intracellular expression of TNF-alpha was detected in Gr-1(bright+) and Gr-1(dull+) cells during the time intervals postinfection, and F4/80(+) cells expressed intracellular TNF-alpha before Gr-1(dull+) cells appeared. The Gr-1(bright+) and Gr-1(dull+) cells sorted from BALF cells at 24 h were identified as neutrophils and macrophage-like cells, respectively, and the Gr-1(dull+) cells expressing CD11c, partially CD11b and a marginal level of F4/80 secreted TNF-alpha in in vitro cultures. Finally, deletion of Gr-1(+) cells by administration of the specific mAb significantly reduced the concentrations of this cytokine in BALF at 6 and 12 h postinfection, but not the expression of TNF-alpha in F4/80(+) cells. Thus, these results demonstrated that neutrophils, F4/80(+) macrophages and Gr-1(dull+) CD11c(+) macrophage-like cells played an important role in the production of TNF-alpha in lungs at an early stage of infection with S. pneumoniae.