Doctor of Philosophy, Kyoto University (2018)
Master of Science, Kyoto University (2015)
Bachelor of Science, Osaka University of Pharmaceutical Sciences (2013)
Objective: Granulomatosis with polyangiitis (GPA) is a multi-organ vasculitic syndrome typically associated with neutrophil extracellular trap (NET) formation and aggressive tissue inflammation. Manifestations in head and neck (H&N) GPA include septal perforations, saddle-nose deformities, bony erosions of the orbital and sinus walls, middle ear damage and epiglottitis, indicative of bone, cartilage, and connective tissue destruction. Whether H&N-centric lesions engage disease pathways distinctive from the ischemic tissue damage in the lungs, kidneys, skin, and peripheral nerves is unknown. We have compared inflammatory responses triggered by neutrophilic NETs in patients with H&N GPA and systemic GPA (sGPA). Methods: Neutrophils and monocytes were isolated from the peripheral blood of patients with H&N GPA, sGPA, and age/gender matched healthy individuals. Neutrophil NETosis was induced. NETs were isolated and cocultured with monocytes. Gene induction was quantified by RT-PCR, protein upregulation by flow cytometry. Tissue invasiveness of monocytes was measured in a 3D collagen matrix system. Expression of MMP-9 in tissue-residing macrophages was assessed by immunohistochemistry in tissue biopsies. Results: Neutrophils from H&N GPA patients showed more intense NETosis with higher frequencies of netting neutrophils (P < 0.001) and release of higher amounts of NETs (P < 0.001). Isolated NETs from H&N GPA functioned as an inducer of danger-associated molecular patterns in monocytes; specifically, alarmin S100A9. NET-induced upregulation of monocyte S100A9 required recognition of DNA. S100A9 release resulted in the induction of metalloproteinases, including MMP-9, and enabled monocytes to invade into extracellular matrix. Anti-MMP-9 treatment attenuated the tissue invasiveness of monocytes primed with NETs from H&N GPA patients. MMP-9-producing macrophages dominated the tissue infiltrates in naso-sinal biopsies from H&N GPA patients. Conclusion: Distinct disease patterns in GPA are associated with differences in NET formation and NET content. H&N GPA patients with midline cartilaginous and bony lesions are highly efficient in generating NETs. H&N GPA neutrophils trigger the induction of the alarmin S100A9, followed by production of MMP-9, endowing monocytes with tissue-invasive capabilities.
View details for DOI 10.3389/fimmu.2019.02617
View details for PubMedID 31798577
View details for PubMedCentralID PMC6874157
Despite the importance of the interaction between DNA and cells for its biological activity, little is known about exactly how DNA interacts with cells. To elucidate the relationship between the structural properties of DNA and its cellular uptake, a single-stranded circular DNA of 1801 bases was designed and folded into a series of rectangular DNA (RecDNA) nanostructures with different rigidities using DNA origami technology. Interactions between these structures and cells were evaluated using mouse macrophage-like RAW264.7 cells. RecDNA with 50 staple DNAs, including four that were Alexa Fluor 488-labeled, was designed. RecDNA with fewer staples, down to four staples (all Alexa Fluor 488-labeled), was also prepared. Electrophoresis and atomic force microscopy showed that all DNA nanostructures were successfully obtained with a sufficiently high yield. Flow cytometry analysis showed that folding of the single-stranded circular DNA into RecDNA significantly increased its cellular uptake. In addition, there was a positive correlation between uptake and the number of staples. These results indicate that highly folded DNA nanostructures interact more efficiently with RAW264.7 cells than loosely folded structures do. Based on these results, it was concluded that the interaction of DNA with cells can be controlled by folding using DNA origami technology.
View details for DOI 10.1039/c9nr08695a
View details for PubMedID 31799532
We previously demonstrated that the immunostimulatory activity of CpG DNA is increased by the formation of polypod-like structures. The present study was designed to elucidate the mechanism underlying this increase.Tripodna (three pods) and hexapodna (six pods) were prepared. The cellular uptake of Alexa Fluor 488-labeled DNA samples was examined in several cell lines by measuring the MFI of cells. TNF-α release from RAW264.7 cells was measured after addition of polypodna containing CpG motifs. Dissociation of double stranded DNA was evaluated using FRET.Tripodna and hexapodna were efficiently taken up by macrophage-like RAW264.7 cells and dendritic DC2.4 cells, but not by fibroblast or endothelial cell lines. The uptake by RAW264.7 cells was highest for hexapodna, followed by tripodna, dsDNA, and ssDNA. The release of TNF-α from RAW264.7 cells was also highest for hexapodna. The ratio of TNF-α release to cellular uptake was highest for ssDNA, and lowest for dsDNA. Tripodna and hexapodna were more easily dissociated into single strands after cellular uptake than was dsDNA.The efficient cellular uptake and prompt dissociation into single strands can be directly related to the high immunostimulatory activity of polypod-like structured DNAs containing CpG motifs.
View details for DOI 10.1007/s11095-017-2243-y
View details for PubMedID 28791533
We used human Toll-like receptor 9 (hTLR9)-expressing HEK-Blue hTLR9 cells, which release secreted embryonic alkaline phosphatase (SEAP) upon response to CpG DNA, to evaluate the immunological properties of nucleic acid drug candidates. Our preliminary studies showed that phosphodiester CpG DNA hardly induced any SEAP secretion in HEK-Blue hTLR9 cells. In the current study, therefore, we developed HEK-Blue hTLR9 cells transduced with human macrophage scavenger receptor-1 (hMSR1), a cell-surface DNA receptor, and determined whether HEK-Blue hTLR9/hMSR1 cells respond to phosphorothioate (PS) CpG DNA and phosphodiester (PO) CpG DNA. We selected PS CpG2006, a single-stranded PO CpG DNA (ssCpG), and a tetrapod-like structured DNA (tetrapodna) containing ssCpG (tetraCpG) as model TLR9 ligands. Alexa Fluor 488-labeled ligands were used for flow cytometry. Unlike the mock-transfected HEK-Blue hTLR9 cells, the HEK-Blue hTLR9/hMSR1 cells efficiently took up all three CpG DNAs. SEAP release was almost proportional to the uptake. Treatment of HEK-Blue hTLR9/hMSR1 cells with an anti-hMSR1 antibody significantly reduced the uptake of ssCpG and tetraCpG. Collectively, reconstruction of TLR9-mediated responses to CpG DNA in HEK-Blue hTLR9 cells can be used to evaluate the toxicity of nucleic acid drug candidates with diverse physicochemical properties.
View details for DOI 10.1038/s41598-017-13890-3
View details for PubMedID 29057947
View details for PubMedCentralID PMC5651873
Success of tumor photothermal immunotherapy requires a system that induces heat stress in cancer cells and enhances strong anti-tumor immune responses. Here, we designed a composite-type immunostimulatory DNA hydrogel consisting of a hexapod-like structured DNA (hexapodna) with CpG sequences and gold nanoparticles. Mixing of the properly designed hexapodna and oligodeoxynucleotide-modified gold nanoparticles resulted in the formation of composite-type gold nanoparticle-DNA hydrogels. Laser irradiation of the hydrogel resulted in the release of hexapodna, which efficiently stimulated immune cells to release proinflammatory cytokines. Then, EG7-OVA tumor-bearing mice received an intratumoral injection of a gold nanoparticle-DNA hydrogel, followed by laser irradiation at 780 nm. This treatment increased the local temperature and the mRNA expression of heat shock protein 70 in the tumor tissue, increased tumor-associated antigen-specific IgG levels in the serum, and induced tumor-associated antigen-specific interferon-γ production from splenocytes. Moreover, the treatment significantly retarded the tumor growth and extended the survival of the tumor-bearing mice.
View details for DOI 10.1016/j.biomaterials.2017.09.014
View details for PubMedID 28918263
Immunoinhibitory oligodeoxynucleotides (INH-ODNs) are promising inhibitors of Toll-like receptor 9 (TLR9) activation. To efficiently deliver INH-ODNs to TLR9-positive cells, we designed a Takumi-shaped DNA (Takumi) consisting of two partially complementary ODNs as the main component of a DNA hydrogel. Polyacrylamide gel electrophoresis showed that Takumi-containing INH-ODNs (iTakumi) and iTakumi-based DNA hydrogel (iTakumiGel) were successfully generated. Their activity was examined in murine macrophage-like RAW264.7 cells and DC2.4 dendritic cells by measuring tumor necrosis factor-α and interleukin-6 release after the addition of a TLR9 ligand (CpG ODN). Cytokine release was efficiently inhibited by the iTakumiGel. Flow cytometry analysis and confocal microscopy showed that cellular uptake of INH-ODN was greatly increased by the iTakumiGel. These results indicate that a Takumi-based DNA hydrogel is useful for the delivery of INH-ODNs to immune cells to inhibit TLR9-mediated hyperinduction of proinflammatory cytokines. From the Clinical Editor: Toll-like receptor 9 activation has been reported to be associated with many autoimmune diseases. DNA inhibition using oligodeoxynucleotides is one of the potential treatments. In this article, the authors described hydrogel-based platform for the delivery of the inhibitory oligodeoxynucleotides for enhanced efficacy. The positive findings could indicate a way for the future.
View details for DOI 10.1016/j.nano.2015.08.004
View details for PubMedID 26364795
DNA dendrimers consisting of several branched DNA units connected to each other using DNA ligase were quite effective for the delivery of immunostimulatory CpG DNA to immune cells. Therapeutic application of such DNA dendrimers, however, is hampered by the use of the ligase. Here, we report that self-assembling DNA dendrimers with high immunostimulatory potency can be prepared without DNA ligases. Annealing of DNA consisting of DNA units with elongated adhesive ends resulted in the formation of DNA dendrimers. Atomic force microscopy revealed that the several preparations of DNA dendrimers resulted in dendritic structures as designed. The cellular uptake of DNA dendrimers by mouse macrophage-like RAW264.7 cells and subsequent release of tumor necrosis factor-α were dependent on the structural complexity of the dendrimers. These results indicate that the ligation-free, self-assembling DNA dendrimers are a potent system for the delivery of immunostimulatory CpG DNA to immune cells.
View details for DOI 10.1021/bm501731f
View details for PubMedID 25775113
Nanosized DNA assemblies are useful for delivering immunostimulatory cytosine-phosphate-guanine (CpG) DNA to immune cells, but little is known about the optimal structure for such delivery. In this study, we designed three different DNA nanostructures using four 55-mer oligodeoxynucleotides (ODNs), that is, tetrapod-like structured DNA (tetrapodna), tetrahedral DNA (tetrahedron), and tetragonal DNA (tetragon), and compared their potencies. Electrophoresis showed that tetrapodna was obtained with high yield and purity, whereas tetrahedron formed multimers at high ODN concentrations. Atomic force microscopy revealed that all preparations were properly constructed under optimal conditions. The thermal stability of tetrapodna was higher than those of the others. Dynamic light scattering analysis showed that all of the assemblies were about 8 nm in diameter. Upon addition to mouse macrophage-like RAW264.7 cells, tetrahedron was most efficiently taken up by the cells. Then, a CpG DNA, a ligand for toll-like receptor 9, was linked to these DNA nanostructures and added to RAW264.7 cells. CpG tetrahedron induced the largest amount of tumor necrosis factor-α, followed by CpG tetrapodna. Similar results were obtained using human peripheral blood mononuclear cells. Taken together, these results indicate that tetrapodna is the best assembly with the highest yield and high immunostimulatory activity, and tetrahedron can be another useful assembly for cellular delivery if its preparation yield is improved.
View details for DOI 10.1089/nat.2014.0524
View details for PubMedID 26222130