Emi Kanno

Structures of antifungal diarylheptenones, gingerenones A, B, C and isogingerenone B, isolated from the rhizomes of Zingiber officinale

Abstract Four new diarylheptenones, 1,7-bis(4-hydroxy-3-methoxyphenyl)hept-4-en-3-one (gingerenone A), 7-(3,5-dimethoxy-4-hydroxyphenyl)-1-(4-hydroxy-3-methoxyphenyl) hept-4-en-3-one(gingerenone B), 1-(3,5-dimethoxy-4-hydroxyphenyl)-7-(4-hydroxy-3-methoxyphenyl)hept-4-en-3-one (isogingerenone B) and 1-(4-hydroxy-3-methoxyphenyl)-7-(4-hydroxyphenyl)hept-4-en-3-one (gingerenone C) have been isolated from the rhizomes of Zingiber officinale , and their structures established by spectral methods and some chemical transformations. The results indicate the similarity in the metabolic fates of the curcuminoid constituents to those of the arylalkanoids (dehydrogingerdione → gingerol → shogaol) of zingiberaceous plants. Gingerenone A exhibited a moderate anticoccidium activity in vitro and a strong antifungal effect to Pyricularia oryzae .

Defect of CARD9 Leads to Impaired Accumulation of Gamma Interferon-Producing Memory Phenotype T Cells in Lungs and Increased Susceptibility to Pulmonary Infection with Cryptococcus neoformans

ABSTRACT Caspase recruitment domain-containing protein 9 (CARD9) is an adaptor molecule signal that is critical for NF-κB activation and is triggered through C-type lectin receptors (CLRs), which are pattern recognition receptors that recognize carbohydrate structures. Previous studies have reported that Cryptococcus neoformans, a fungal pathogen that causes meningoencephalitis in AIDS patients, is recognized through some CLRs, such as mannose receptors or DC-SIGN. However, the role of CARD9 in the host defense against cryptococcal infection remains to be elucidated. In the present study, we analyzed the role of CARD9 in the host defense against pulmonary infection with C. neoformans. CARD9 gene-disrupted (knockout [KO]) mice were highly susceptible to this infection, as shown by the reduced fungal clearance in the infected lungs of CARD9 KO mice, compared to that in wild-type (WT) mice. Gamma interferon (IFN-γ) production was strongly reduced in CARD9 KO mice during the innate-immunity phase of infection. Reduced IFN-γ synthesis was due to impaired accumulation of NK and memory phenotype T cells, which are major sources of IFN-γ innate-immunity-phase production; a reduction in the accumulation of these cells was correlated with reduced CCL4, CCL5, CXCL9, and CXCL10 synthesis. However, differentiation of Th17 cells, but not of Th1 cells, was impaired at the adaptive-immunity phase in CARD9 KO mice compared to WT mice, although there was no significant difference in the infection susceptibility between interleukin 17A (IL-17A) KO and WT mice. These results suggest that CARD9 KO mice are susceptible to C. neoformans infection probably due to the reduced accumulation of IFN-γ-expressing NK and memory phenotype T cells at the early stage of infection.

Dectin-2 Deficiency Promotes Th2 Response and Mucin Production in the Lungs after Pulmonary Infection with Cryptococcus neoformans

ABSTRACT Dectin-2 is a C-type lectin receptor that recognizes high mannose polysaccharides. Cryptococcus neoformans, a yeast-form fungal pathogen, is rich in polysaccharides in its cell wall and capsule. In the present study, we analyzed the role of Dectin-2 in the host defense against C. neoformans infection. In Dectin-2 gene-disrupted (knockout) (Dectin-2KO) mice, the clearance of this fungus and the inflammatory response, as shown by histological analysis and accumulation of leukocytes in infected lungs, were comparable to those in wild-type (WT) mice. The production of type 2 helper T (Th2) cytokines in lungs was higher in Dectin-2KO mice than in WT mice after infection, whereas there was no difference in the levels of production of Th1, Th17, and proinflammatory cytokines between these mice. Mucin production was significantly increased in Dectin-2KO mice, and this increase was reversed by administration of anti-interleukin 4 (IL-4) monoclonal antibody (MAb). The levels of expression of β1-defensin, cathelicidin, surfactant protein A (Sp-A), and Sp-D in infected lungs were comparable between these mice. In in vitro experiments, IL-12p40 and tumor necrosis factor alpha (TNF-α) production and expression of CD86 and major histocompatibility complex (MHC) class II by bone marrow-derived dendritic cells and alveolar macrophages were completely abrogated in Dectin-2KO mice. Finally, the disrupted lysates of C. neoformans, but not of whole yeast cells, activated Dectin-2-triggered signaling in an assay with nuclear factor of activated T cells (NFAT)-green fluorescent protein (GFP) reporter cells expressing this receptor. These results suggest that Dectin-2 may oppose the Th2 response and IL-4-dependent mucin production in the lungs after infection with C. neoformans, and it may not be required for the production of Th1, Th17, and proinflammatory cytokines or for clearance of this fungal pathogen.

Wound healing in skin promoted by inoculation with Pseudomonas aeruginosaPAO1: The critical role of tumor necrosis factor‐α secreted from infiltrating neutrophils

Wound healing is promoted by the presence of replicating microorganisms adhering to the wounded tissue, but the precise mechanism is not fully understood. In the present study, using a rat model with full‐thickness dermal wounds, we examined the effect of Pseudomonas aeruginosa inoculation on wound healing and the role of neutrophils infiltrating the wound site. Within 3 days, inoculation with this bacterium had accelerated re‐epithelialization, epidermal cell proliferation, and neo‐vascularization, as well as the local infiltration of neutrophils, which reached a peak at 24 hours. Tumor necrosis factor (TNF)‐α was detected in the wound tissues on the mRNA and protein levels within 24 hours. Flow cytometry and immunohistochemical analyses detected higher levels of TNF‐α in the infiltrating neutrophils in rats inoculated with P. aeruginosa than in uninoculated rats. Neutropenic rats treated with anti‐neutrophil mAb or cyclophosphamide exhibited significant attenuation in re‐epithelialization, epidermal cell proliferation, neo‐vascularization, and TNF‐α synthesis compared with control; administration of TNF‐α reversed these attenuations. These wound‐healing responses were decelerated in rats treated with anti‐TNF‐α mAb, as was the infiltration of neutrophils. These results indicate that inoculation with P. aeruginosa promotes wound healing by inducing the infiltration of neutrophils, which play a critical role as a major source of TNF‐α.

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.

Biofilm formation on rat skin wounds by Pseudomonas aeruginosa carrying the green fluorescent protein gene

Please cite this paper as: Biofilm formation on rat skin wounds by Pseudomonas aeruginosa carrying the green fluorescent protein gene. Experimental Dermatology 2010; 19:154–156.

Involvement of high mobility group box 1 and the therapeutic effect of recombinant thrombomodulin in a mouse model of severe acute respiratory distress syndrome

Acute respiratory distress syndrome (ARDS) is accompanied by severe lung inflammation induced by various diseases. Despite the severity of the symptoms, therapeutic strategies have been ineffective. High mobility group box 1 (HMGB1), which was identified originally as a DNA binding protein, has been proposed as a mediator of acute lung injury. In addition to its anti‐coagulant activity, recombinant thrombomodulin (rTM) possesses an ability to suppress the inflammatory response through neutralizing HMGB1. T regulatory (Treg) cells in the lungs are reported to modify innate immune responses during resolution of acute lung injury. In the present study, we investigated the therapeutic effect of rTM, and the contribution of Treg cells to this effect, in a mouse model of severe ARDS. C57BL/6 mice received sequential intratracheal administration of α‐galactosylceramide (α‐GalCer) and lipopolysaccharide (LPS), which resulted in the development of severe ARDS. HMGB1 levels in the lungs increased to a higher level in ARDS mice compared to those in mice treated with LPS alone. HMGB1 was expressed in the infiltrating neutrophils and macrophages in lungs. Treg cells were reduced significantly in the lungs of ARDS mice compared to those in mice treated with LPS alone. rTM administration prolonged the survival time and ameliorated the development of ARDS, which was associated with increased Treg cells and synthesis of interleukin (IL)‐10 and transforming growth factor (TGF)‐β in the lungs. These results suggest that HMGB1 is involved in the development of severe ARDS and rTM shows therapeutic effects through promoting the accumulation of Treg cells at the inflammatory sites.

IL-17A promotes neutrophilic inflammation and disturbs acute wound healing in skin

In the wound healing process, neutrophils are the first inflammatory cells to move to the wound tissues. They sterilize wounds by killing microbes, and they stimulate other immune cells to protect the host from infection. In contrast, neutrophil‐derived proteases cause damage to host tissues, so neutrophils play dual opposite roles in wound healing. Interleukin‐17A (IL‐17A) is a proinflammatory cytokine that promotes the recruitment of these cells. The role of this cytokine in the wound healing process is not fully clarified. In the present study, therefore, we examined how defect in IL‐17A production affected the wound healing in skin. IL‐17A‐knockout (KO) mice showed promoted wound closure, myofibroblast differentiation and collagen deposition and decreased the neutrophil accumulation compared with wild‐type (WT) mice. In contrast, the administration of recombinant IL‐17A led to delayed wound closure, low collagen deposition and accelerated neutrophilic accumulation. In addition, the treatment of IL‐17A‐administered mice with a neutrophil elastase inhibitor improved the wound repair to the same level as that of WT mice. These results indicated that IL‐17A hampered the wound healing process and suggested that neutrophilic inflammation caused by IL‐17A may be associated with impaired wound healing in skin.

Reconsideration of iodine in wound irrigation: the effects on Pseudomonas aeruginosa biofilm formation

OBJECTIVE Chronic skin wounds are usually colonised with bacteria and subsequent infection may develop. Topical antiseptics are commonly used to control bacterial colonisation. The topical antiseptic, 1% polyvinylpyrrolidone-iodine (PVP-I), that is used on chronic open skin wounds remains controversial in the clinical setting because of its cytotoxicity. Here, we tested 1% PVP-I solution against saline to determine if it reduces bacterial count on the wound surface and within the tissue that may lead to wound reduction. METHOD Open wounds that were created on the backs of Sprague Dawley rats were inoculated with Pseudomonas aeruginosa at the wound surface. Wounds were kept covered except for wound irrigation, two days post-wounding, wounds were irrigated daily using a 10ml syringe and spray tip. RESULTS Our results indicate that 1% PVP-I irrigation resulted in a reduced bacterial count on the wound surface and within the tissue compared with saline irrigation. The 1% PVP-I irrigation promoted wound re-epithelialisation compared with saline irrigation, but it did not reach significance. CONCLUSION These results indicated that irrigation with 1% PVP-I was an effective way to reduce bacterial count on the wound surface, and allow the wound to progress to healing.

Cryptococcus neoformans Infection in Mice Lacking Type I Interferon Signaling Leads to Increased Fungal Clearance and IL-4-Dependent Mucin Production in the Lungs

Type I interferons (IFNs) are secreted by many cell types upon stimulation via pattern recognition receptors and bind to IFN-α/β receptor (IFNAR), which is composed of IFNAR1 and IFNAR2. Although type I IFNs are well known as anti-viral cytokines, limited information is available on their role during fungal infection. In the present study, we addressed this issue by examining the effect of IFNAR1 defects on the host defense response to Cryptococcus neoformans. In IFNAR1KO mice, the number of live colonies was lower and the host immune response mediated not only by Th1 but also by Th2 and Th17-related cytokines was more accelerated in the infected lungs than in WT mice. In addition, mucin production by bronchoepithelial cells and expression of MUC5AC, a major core protein of mucin in the lungs, were significantly higher in IFNAR1KO mice than in WT mice. This increase in mucin and MUC5AC production was significantly inhibited by treatment with neutralizing anti-IL-4 mAb. In contrast, administration of recombinant IFN-αA/D significantly suppressed the production of IL-4, but not of IFN-γ and IL-17A, in the lungs of WT mice after cryptococcal infection. These results indicate that defects of IFNAR1 led to improved clearance of infection with C. neoformans and enhanced synthesis of IFN-γ and the IL-4-dependent production of mucin. They also suggest that type I IFNs may be involved in the negative regulation of early host defense to this infection.