Ayumi Saeki

Involvement of suppressor of cytokine signalling-1-mediated degradation of MyD88-adaptor-like protein in the suppression of Toll-like receptor 2-mediated signalling by the murine C-type lectin SIGNR1-mediated signalling

Dendritic cells recognize pathogens through pattern recognition receptors such as Toll‐like receptors and phagocytose and digest them by phagocytic receptors for antigen presentation. This study was designed to clarify the cross‐talk between recognition and phagocytosis of microbes in dendritic cells. The murine dendritic cell line XS106 cells were stimulated with the murine C‐type lectin SIGNR1 ligand lipoarabinomannan and the Toll‐like receptor 2 ligand FSL‐1. The co‐stimulation significantly suppressed FSL‐1‐mediated activation of NF‐κB as well as production of TNF‐α, IL‐6 and IL‐12p40 in a dose‐dependent manner. The suppression was significantly but not completely recovered by knock‐down of SIGNR1. SIGNR1 was associated with Toll‐like receptor 2 in XS106 cells. The co‐stimulation upregulated the expression of suppressor of cytokine signalling‐1 in XS106 cells, the knock‐down of which almost completely recovered the suppression of the FSL‐1‐mediated cytokine production by lipoarabinomannan. In addition, it was found that the MyD88‐adaptor‐like protein in XS106 cells was degraded by co‐stimulation with FSL‐1 and lipoarabinomannan in the absence, but not the presence, of the proteasome inhibitor MG132 and the degradation was inhibited by knock‐down of suppressor of cytokine signalling‐1. This study suggests that Toll‐like receptor 2‐mediated signalling is negatively regulated by SIGNR1‐mediated signalling in dendritic cells, possibly through suppressor of cytokine signalling‐1‐mediated degradation of the MyD88‐adaptor‐like protein.

Chlamydophila pneumoniae in human immortal Jurkat cells and primary lymphocytes uncontrolled by interferon-γ

Lymphocytes are a potential host cell for Chlamydophila pneumoniae, although why the bacteria must hide in lymphocytes remains unknown. Meanwhile, interferon (IFN)-γ is a crucial factor for eliminating chlamydiae from infected cells through indoleamine 2,3-dioxygenase (IDO) expression, resulting in depletion of tryptophan. We therefore assessed if lymphocytes could work as a shelter for the bacteria to escape IFN-γ. C. pneumoniae grew normally in human lymphoid Jurkat cells, even in the presence of IFN-γ or under stimulation with phorbol myristate acetate plus ionomycin. Although Jurkat cells expressed IFN-γ receptor CD119, their lack of IDO expression was confirmed by RT-PCR and western blotting. Also, C. pneumoniae survived in enriched human peripheral blood lymphocytes, even in the presence of IFN-γ. Furthermore, C. pneumoniae in spleen cells obtained from IFN-γ knockout mice with C57BL/6 background was maintained in a similar way to wild-type mice, supporting a minimal role of IFN-γ-related response for eliminating C. pneumoniae from lymphocytes. Thus, we concluded that IFN-γ did not remove C. pneumoniae from lymphocytes, possibly providing a shelter for C. pneumoniae to escape from the innate immune response, which has direct clinical significance.

In vivo anti- and pro-tumour activities of the TLR2 ligand FSL-1

TLR ligands as Th1 inducers have been investigated as potential anti-tumour agents. However, few attempts have been made to investigate the anti-tumour activity of TLR ligands as Th2 inducers. This study, therefore, was carried out to determine whether the TLR2 ligand FSL-1 as a Th2 inducers affects the growth of a QRsP tumour, a fibrosarcoma derived from the C57BL/6 (TLR2(+/+)) mouse in vivo. Tumour volumes in TLR2(+/+) mice immunized with both FSL-1 and tumour-associated antigens were significantly smaller than those in control mice. Immunization with both FSL-1 and tumour-associated antigens increased the survival rate of TLR2(+/+) mice. However, surprisingly, immunization with FSL-1 alone significantly enhanced the growth of tumour. Both anti- and pro-tumour activities of FSL-1 were not observed in TLR2(-/-) mice. Immunization of both FSL-1 and tumour-associated antigens induced tumour-associated antigen-specific cytolytic T cells, antibody-dependent cell-mediated cytotoxicity of natural killer cells by production of the tumour-specific antibodies, tumour lysis by complement activation and reduction of the number of regulatory T cells in the draining lymph node. Immunization with FSL-1 alone increased the number of regulatory T cells in the draining lymph node, and in vivo administration of anti-CD25 antibody into mice abrogated the pro-tumour activity of FSL-1, suggesting that regulatory T cells are involved in the pro-tumour activity. This study demonstrated that FSL-1 exhibited TLR2-mediated anti- and pro-tumour activities when immunized with and without tumour-associated antigens, respectively.

Physicochemical fabrication of antibacterial calcium phosphate submicrospheres with dispersed silver nanoparticles via coprecipitation and photoreduction under laser irradiation.

We achieved rapid, surfactant-free, and one-pot fabrication of antibacterial calcium phosphate (CaP) submicrospheres containing silver nanoparticles by combining physical laser and chemical coprecipitation processes. In this physicochemical process, weak pulsed laser irradiation (20min) was performed on a labile CaP reaction mixture supplemented with silver ions as a light-absorbing agent. The silver content in the submicrospheres was controlled for a wide range (Ag/P elemental ratio varied from 0.60 to 62.0) by tuning the initial concentration of silver ions (from 5 to 20mM) in the CaP reaction mixture. At the silver concentration of 5mM, we obtained unique nanocomposite particles: CaP submicrospheres (average diameter of approximately 500nm) containing metallic silver nanoparticles dispersed throughout, as a result of CaP and silver coprecipitation with simultaneous photoreduction of silver ions and spheroidization of the coprecipitates. These CaP submicrospheres containing silver nanoparticles (ca. 0.3mg silver per 1mg submicrospheres) exhibited antibacterial activity against major pathogenic oral bacteria, i.e., Streptococcus mutans, Aggregatibacter actinomycetemcomitans, and Porphyromonas gingivalis. Moreover, the CaP submicrospheres dissolved and neutralized the acidic environment generated by Streptococcus mutans, demonstrating their potential as acid-neutralizing and remineralizing agents. The present process and resulting antibacterial CaP-based submicrospheres are expected to be useful in dental healthcare and infection control. STATEMENT OF SIGNIFICANCE Nano- and microsized spheres of calcium phosphate (CaP) containing silver nanoparticles have great potential in dental applications. Conventional fabrication processes were time-consuming or weak regarding the size/shape control of the spheres. In this study, we achieved a simple (one-pot), rapid (20-min irradiation), and surfactant-free fabrication of CaP submicrospheres containing silver nanoparticles by pulsed laser irradiation to a mixture of calcium, phosphate, and silver ion solutions. The resulting CaP submicrospheres contained metallic silver nanoparticles dispersed throughout in a sequence of reactions: CaP and silver coprecipitation, laser-induced melting and spheroidization of the coprecipitates, and photoreduction of silver ions. These submicrospheres showed antibacterial activity against oral bacteria and acid-neutralizing property in the bacterial suspension, and hence are worth considering for dental applications.

Differences in recognition of wild-type and lipoprotein-deficient strains of oral Streptococci in vitro and in vivo

Whole cells of wild-type strains of Streptococcus gordonii and Streptococcus mutans induced Toll-like receptor 2 (TLR2)-mediated nuclear factor-κB (NF-κB) activation, whereas those of lipoprotein (LP)-deficient strains did not. All strains upregulated the proliferation of TLR2(+/+) splenocytes more strongly than TLR2(-/-) splenocytes. However, significant differences were not observed between the cytokine-inducing activities of wild-type and LP-deficient strains toward TLR2(+/+) and TLR2(-/-) splenocytes. Muramyl dipeptide as well as whole cells not only induced nucleotide-binding oligomerization domain 2 (NOD2)-mediated activation of NF-κB but also enhanced the proliferation of TLR2(-/-) as well as TLR2(+/+) splenocytes. Wild-type strains of these streptococci were more resistant to clearance from blood and organs (liver and spleen) in TLR2(+/+) but not TLR2(-/-) mice and induced production of larger amounts of blood TNF-α than the LP-deficient strains. Wild-type strains of both species adhered to human vascular endothelial cells more strongly than did the LP-deficient strains. Thus, this study suggested that LP plays an important role in the recognition of these streptococci by the host in vivo as well as in vitro and that these streptococci possess some components recognized by NOD2 and/or TLR2 that are involved in the mitogenic activity toward splenocytes.

Activation of nucleotide‐binding domain‐like receptor containing protein 3 inflammasome in dendritic cells and macrophages by Streptococcus sanguinis

Streptococcus sanguinis is frequently isolated from the blood of patients with infective endocarditis and contributes to the pathology of this disease through induction of interleukin (IL)‐1β responsible for the development of the disease. However, the mechanism of IL‐1β induction remains unknown. In this study, S. sanguinis activated a murine dendritic cell (DC) to induce IL‐1β and this activity was attenuated by silencing the mRNAs of nucleotide‐binding domain‐like receptor containing protein 3 (NLRP3) and caspase‐1.

Mycoplasma removal from cell culture using antimicrobial photodynamic therapy.

OBJECTIVE The objective of this research was to determine the effectiveness of antimicrobial photodynamic therapy (aPDT) in the removal of mycoplasmas from contaminated cells. BACKGROUND DATA Mycoplasmas often contaminate cell cultures. The cell-contaminating mycoplasmas are removed by antibiotics, but the use of antibiotics usually induces antibiotic-resistant bacteria. aPDT is expected to be a possible alternative to antibiotic treatments for suppressing infections. MATERIALS AND METHODS Mycoplasma salivarium (Ms)-infected human embryonic kidney (HEK) 293 cells were irradiated using a red light-emitting diode (LED) in the presence of methylene blue (MB) as a photosensitizer. The Ms viable count was determined using culture on agar plates or using a mycoplasma detection kit. RESULTS aPDT performed using red LED irradiation was effective in decreasing live Ms in the presence of MB without damaging the HEK293 cells. aPDT removed live Ms from the infected cells after washing the cells with sterilized phosphate-buffered saline (PBS) to decrease the initial number of live Ms before aPDT. CONCLUSIONS This study suggests that aPDT could remove mycoplasmas from contaminated cells.

Differences in interleukin-1β release-inducing activity of Candida albicans toward dendritic cells and macrophages

OBJECTIVE The purpose of this study is to elucidate differences in the mechanism of the IL-1β release-inducing activity of Candida albicans toward dendritic cells and macrophages because IL-1β is one of the proinflammatory cytokines which is crucial in host defense against candidiasis. DESIGN Two C. albicans strains were used in this study. One strain is uridine-auxotrophic (CAI4) that needs uridine to grow and form hyphae, and another is a strain without any specific auxotrophy (pACT1-GFP), which forms hyphae naturally by culturing with serum components. Murine macrophage and dendritic cell lines were primed with LPS and then stimulated with C. albicans CAI4 or pACT1-GFP. RESULTS Both strains of C. albicans induced IL-1β release from dendritic cells, and C. albicans pACT1-GFP induced IL-1β release but CAI4 induced little amounts in macrophages. These differences were suggested to be due to the difference in the amount of extracellular ATP released in the cell culture supernatants induced by C. albicans CAI4 or pACT1-GFP. For induction of IL-1β release from both macrophages and dendritic cells by C. albicans, direct contacts of the microbes with cells were required. In addition, macrophages required morphological change of C. albicans from yeast to hyphae for induction of IL-1β release, whereas dendritic cells did not require it. Dead C. albicans could induce IL-1β release from dendritic cells, but could not from macrophages. CONCLUSIONS There are different mechanisms by which C. albicans induces IL-1β release from dendritic cells and macrophages.