Haruyuki Fujita

Bortezomib suppresses function and survival of plasmacytoid dendritic cells by targeting intracellular trafficking of Toll-like receptors and endoplasmic reticulum homeostasis

Dendritic cells (DCs) play a pivotal role in the pathogenesis of inflammatory disorders, so suppressing the activity of DCs is instrumental in treating such diseases. In the present study, we show that a proteasome inhibitor, bortezomib, suppresses the survival and immunostimulatory function of human plasmacytoid DCs (pDCs) by targeting 2 critical points, intracellular trafficking of nucleic acid-sensingToll-like receptors (TLRs) and endoplasmic reticulum (ER) homeostasis. Among the immune cells in blood, pDCs were the most susceptible to the killing effect of bortezomib. This correlates with a decrease in the spliced form of a transcription factor XBP1, which rescues cells from apoptosis by maintaining ER homeostasis. Bortezomib suppressed the production of interferon-α and interleukin-6 by pDCs activated with a TLR9-stimulating CpG DNA and a TLR7-stimulating influenza virus, which appears to be partially independent of apoptosis. Bortezomib inhibited translocation of TLR9 from the ER to endolysosomes but not of an ER membrane protein, Unc93B1, that delivers TLR9 to endolysosomes. Thus, bortezomib suppresses the activity of pDCs by inhibiting intracellular trafficking of TLRs through disrupting the coordinated translocation of TLRs and Unc93B1 and by disturbing ER homeostasis. This study suggests that proteasome inhibitors may alleviate inflammatory disorders such as lupus and psoriasis that involve pDCs.

Efficient delivery of immunostimulatory DNA to mouse and human immune cells through the construction of polypod-like structured DNA.

UNLABELLED Investigation of mouse macrophage-like RAW264.7 cells showed that the immunostimulatory activity of CpG DNA is increased by formation of polypod-like structured DNA (polypodna), an assembly consisting of three or more oligodeoxynucleotides. To apply CpG polypodna to immunotherapy, its activity was examined in murine dendritic DC2.4 cells, splenic macrophages, and bone marrow-derived dendritic cells (BMDCs). In all cell types, increasing the pod number increased the cellular uptake of DNA and cytokine release. No significant release of cytokines was observed in macrophages lacking Toll-like receptor 9. Similar results were obtained after intradermal injection of polypodna. The polypodna preparations produced significantly higher amounts of interferon α in human peripheral blood mononuclear cells (PBMCs) compared with single-stranded DNA. The conditioned medium of hexapodna-treated human PBMCs effectively inhibited the activity of a hepatitis C virus subgenomic replicon reporter system. These results indicate that polypodna preparations are useful as an immunostimulator. FROM THE CLINICAL EDITOR This study demonstrates the utility of polypoid-like structured DNA (polypodna) preparations as potent immunostimulators in a murine model.

The tyrosine kinase inhibitor dasatinib suppresses cytokine production by plasmacytoid dendritic cells by targeting endosomal transport of CpG DNA

Plasmacytoid dendritic cells (pDCs) produce a vast amount of interferon (IFN)‐α in response to nucleic acids from viruses and damaged self‐cells through Toll‐like receptor (TLR)7 and TLR9. Pharmaceutical agents that suppress IFN‐α production by pDCs are instrumental in elucidating the mechanisms behind IFN‐α production, and in developing novel therapies for inflammatory disorders that involve pDCs. Here, we show that a tyrosine kinase inhibitor for chronic myeloid leukemia with multiple targets, dasatinib, strongly suppresses production of IFN‐α and proinflammatory cytokines by human pDCs stimulated with multimeric CpG oligodeoxynucleotides (CpG‐A) without reducing viability. In contrast, other tyrosine kinase inhibitors, imatinib, and nilotinib, did not suppress the cytokine production at clinically relevant concentrations. Inhibitors of SRC family kinases (SFKs), which are prominent targets of dasatinib, also suppressed the cytokine production. Notably, however, dasatinib, but not SFK inhibitors, abrogated prolonged localization of CpG‐A in early endosomes, which is a critical step for pDCs to produce a large amount of IFN‐α. This study suggests that dasatinib suppresses IFN‐α production by pDCs by inhibiting SFK‐dependent pathways and SFK‐independent endosomal retention of CpG DNA. Kinases controlling the distinctive endosomal trafficking in pDCs may be exploited as targets to develop novel therapies for pDC‐related inflammatory disorders.

Human CD1c+ Myeloid Dendritic Cells Acquire a High Level of Retinoic Acid–Producing Capacity in Response to Vitamin D3

All-trans-retinoic acid (RA) plays a critical role in maintaining immune homeostasis. Mouse intestinal CD103+ dendritic cells (DCs) produce a high level of RA by highly expressing retinal dehydrogenase (RALDH)2, an enzyme that converts retinal to RA, and induce gut-homing T cells. However, it has not been identified which subset of human DCs produce a high level of RA. In this study, we show that CD1c+ blood myeloid DCs (mDCs) but not CD141high mDCs or plasmacytoid DCs exhibited a high level of RALDH2 mRNA and aldehyde dehydrogenase (ALDH) activity in an RA- and p38-dependent manner when stimulated with 1α,25-dihydroxyvitamin D3 (VD3) in the presence of GM-CSF. The ALDH activity was abrogated by TLR ligands or TNF. CD103− rather than CD103+ human mesenteric lymph node mDCs gained ALDH activity in response to VD3. Furthermore, unlike in humans, mouse conventional DCs in the spleen and mesenteric lymph nodes gained ALDH activity in response to GM-CSF alone. RALDH2high CD1c+ mDCs stimulated naive CD4+ T cells to express gut-homing molecules and to produce Th2 cytokines in an RA-dependent manner. This study suggests that CD1c+ mDCs are a major human DC subset that produces RA in response to VD3 in the steady state. The “vitamin D – CD1c+ mDC – RA” axis may constitute an important immune component for maintaining tissue homeostasis in humans.

Chronic eosinophilic Leukemia with the fip1l1-pdgfr± fusion gene in a patient with a history of combination chemotherapy

Hypereosinophilic syndrome (HES) was diagnosed in December 2000 in a 43-year-old man on the basis of persistent eosinophilia (11.7 x 109/L) and a normal karyotype of the bone marrow cells. He had developed intra-abdominal non-Hodgkin’s lymphoma and in 1992 had received 3 courses of combination chemotherapy with doxorubicin (Adriamycin), cyclophosphamide, vincristine, methotrexate, bleomycin, and prednisolone. The patient was orally given prednisolone (10 mg/day) and cyclophosphamide (50 mg/day) as HES treatment without a subsequent improvement of the eosinophilia. In May 2003, anemia (hemoglobin, 7.9 g/dL) and thrombocytopenia (65 x 109/L) manifested with progressive eosinophilia (21.0 ×109/L) and a small number of blasts. The patient became febrile and was admitted in July 2003. Cytogenetic reexamination of the bone marrow cells disclosed the deletion of 4q12, indicating the presence of a fusion of the Fip1-like 1 (FIP1L1) gene to the plateletderived growth factor receptor α (PDGFRα) gene and consequently the clonal nature of his hematopoietic cells. DNA sequence analysis demonstrated that the breakpoints of the FIP1L1 and PDGFRα genes were present in exon 9 and exon 12, respectively. Treatment with imatinib mesylate (300 mg/day) promptly brought about complete remission. Although a number of similar eosinophilic cases have been reported, our patient may be the first such patient with a history of chemotherapy.

Experiment on atomic hydrogen reflection by use of a permeation probe

Experiments on reflection behavior of atomic hydrogen with lower energies were conducted by use of a palladium membrane as a probe for detecting atomic hydrogen. The palladium membrane was exposed to a deuterium rf-plasma, and reflectors were located between them. Hydrogen atoms easily permeated through palladium, which acted as a window for hydrogen atoms. By changing the arrangement of the reflectors of nickel, graphite, glass, tungsten and polytetrafluoroethylene, the corresponding permeation flux was observed, and from which the relative value of the incident atoms to the membrane was estimated. The incident atoms reflected at least once by nickel were nearly the same as those which were reflected more than twice. This indicated that an atomic reflection was very significant. The other reflector materials also showed high atomic reflection. These results may mean that atomic hydrogen is transported for a long distance in openings of structures in fusion devices.

Influence of hydrogen surface coverage on atomic particle reflection

Atomic reflection of deuterium on metal samples of nickel, palladium and type 304 stainless steel (SS-304) was observed by use of a permeation probe. The surface densities of deuterium on the sample materials, which were continuously irradiated by atomic deuterium particles, were measured by the nuclear reaction analysis (NRA). As a result, for each sample, the amount of reflected deuterium decreased with increasing the sample temperature and temperature dependence of the amount was very similar to that of the surface density of deuterium. This would be explained by the fact that atomic hydrogen is reflected as being atoms with a significant probability on hydrogen-covered surface but not reflected on hydrogen-free surface.

Experiments on potential energy diagram for hydrogen isotopes on nickel surface

Activation energies for some processes concerned with hydrogen recombination were studied experimentally. One side of a nickel membrane was exposed to a deuterium plasma and the deuterium permeation flux J at a steady state was monitored. At the same time, the surface density S of deuterium on the plasma-exposed side was observed by nuclear reaction analysis. The results showed that both the data of S -2 and J/S fell on straight lines in Arrhenius diagrams. Considering the particle balance equations under the experimental conditions, two values of the activation energy for the rate constant were obtained: one for two atoms recombining and subsequently leaving from the surface of 0.40 eV and the other for one atom migrating from the surface to the bulk of 0.57 eV. From these values, the energy term in the conventional form of the recombination coefficient was found to be 0.08 eV.

In-situ depth-profiling of deuterium in nickel exposed to RF plasma

Depth-profiling of deuterium near the surface of nickel membranes was performed by using the D(3He, p)4He nuclear reaction, and the permeation rate of deuterium was measured at the same time. When the surface was exposed to RF plasma, remarkable PDP (plasma driven permeation) was observed. The depth profile consists of a high and sharp peak and a flat plateau, which correspond to the surface and the bulk concentration of deuterium, respectively. The permeation rate was proportional to the square of the bulk concentration under our experimental conditions. As an application of the depth-profiling, values of recombination coefficient of the downstream side were determined in case of GDP (gas driven permeation). It was shown that depth-profiling near the surface exposed to plasma was a useful method for understanding hydrogen recycling for fusion reactors.

Durable hematological response and improvement of nephrotic syndrome on thalidomide therapy in a patient with refractory light chain deposition disease

Light chain deposition disease (LCDD) is a rare disease for which an optimal treatment is not yet available. Here, we report the clinical course of a 32-year-old woman with LCDD who was successfully treated with thalidomide. She presented with nephrotic syndrome. Based on the renal biopsy findings and the presence of monoclonal immunoglobulin light chains in her serum and urine, LCDD was diagnosed. Prednisolone and cytotoxic chemotherapy used for multiple myeloma proved ineffective. We initiated administration of thalidomide (100 mg daily) and dexamethasone (20 mg for 4 days per month). After 8 months of treatment, she achieved complete hematological remission, defined as the disappearance of monoclonal protein and a normalized free light chain ratio, which led to improvement of her renal insufficiency. She has shown sustained hematological and organ response for 31 months with thalidomide therapy. Thus, thalidomide therapy seems to be a promising approach to the treatment of LCDD.