Takafumi Matsumoto

Effects of a Janus kinase inhibitor, pyridone 6, on airway responses in a murine model of asthma

Th2 cytokines and their downstream Janus kinase (JAK)-signal transducer and activation of transcription (STAT) pathways play a critical role in allergic asthma. We studied the effects of a pan-JAK inhibitor, pyridone 6 (P6), on asthmatic responses in a mouse model and investigated the mechanism for its biological effects. Mice were sensitized and challenged by ovalbumin (OVA). P6 treatment during the challenge phase suppressed eosinophilia in bronchoalveolar lavage (BAL) fluids but did not affect airway hyperresponsiveness (AHR). To improve the efficacy of the JAK inhibitor, P6 was encapsulated in polylactic-coglycolic acid nanoparticles (P6-PLGA). P6-PLGA treatment just before OVA challenge suppressed both airway eosinophilia and AHR. Although the IL-13 levels in BAL fluids and the OVA-specific IgE levels in serum after the challenge phase treatment with P6-PLGA were similar to those after a sham treatment, the eotaxin levels in BAL fluids and lung mCLCA3/Gob-5 expression were decreased in P6-PLGA-treated mice. Interestingly, the local IL-13 levels and serum OVA-specific IgE were decreased, while IL-17-producing T cells were increased by P6-PLGA treatment during the sensitization plus challenge phases. In vitro, P6 strongly suppressed the differentiation of Th2 from naive CD4 T cells, but it partly enhanced Th17 differentiation. P6 potently suppressed IL-13-mediated STAT6 activation and mCLCA3/Gob-5 expression in mouse tracheal epithelial cells. These findings suggest that the JAK inhibitor P6 suppresses asthmatic responses by inhibiting Th2 inflammation and that application of PLGA nanoparticles improves the therapeutic potency of P6.

Frequency of Foxp3+CD4+CD25+ T cells is associated with the phenotypes of allergic asthma

Background and objective:  A forkhead/winged‐helix family transcriptional repressor, Foxp3, is highly expressed on CD4+CD25+ T regulatory cells. The role of Foxp3+CD4+CD25+ T regulatory cells in asthma remains to be elucidated. Using mouse models and peripheral blood mononuclear cells (PBMC) from subjects with allergic asthma, we aimed to explore whether Foxp3+CD4+CD25+ T regulatory cells associate with asthma phenotypes.

Demethylallosamidin, a chitinase inhibitor, suppresses airway inflammation and hyperresponsiveness.

Acidic mammalian chitinase is upregulated in response to allergen exposure in the lung. We investigated the effects of chitinase inhibitors, allosamidin (Allo) and demethylallosamidin (Dma), on asthmatic responses. Mice were subjected to IL-13 instillation into the airways or to ovalbumin sensitization plus exposure with or without treatment of Allo or Dma. Airway hyperresponsiveness (AHR) and inflammation were evaluated. Allo and Dma attenuated airway eosinophilia and the upregulation of eotaxin after IL-13 instillation, while Dma, but not Allo, suppressed AHR in IL-13-induced asthma. Allo or Dma suppressed the elevated chitinase activity in BAL fluids after IL-13 to similar levels. The bronchoprotective PGE(2) levels in BAL fluids were elevated after IL-13 instillation. Allo, but not Dma, suppressed the overproduction of PGE(2) and the expression of COX-2 and PGE synthase-1 induced by IL-13. In ovalbumin-induced asthma, Dma suppressed AHR more strongly than Allo. These findings suggest that Dma attenuates asthmatic responses induced by IL-13 without affecting PGE(2) synthesis. Dma may have potential as therapeutic agents for asthma.

T cell treatment with small interfering RNA for suppressor of cytokine signaling 3 modulates allergic airway responses in a murine model of asthma

CD4(+) T cells, particularly T helper (Th) 2 cells, play a pivotal role in the pathophysiology of allergic asthma. Suppressor of cytokine signaling (SOCS) proteins control the balance of CD4(+) T cell differentiation. Mice that lack SOCS3 in T cells by crossing SOCS3-floxed mice with Lck-Cre-transgenic mice have reduced allergen-induced eosinophilia in the airways. Here, we studied the effects of SOCS3 silencing with small interfering (si) RNA in primary CD4(+) T cells on Th2 cell differentiation and on asthmatic responses in mice. Th2 cells were generated from ovalbumin (OVA)-specific T cell receptor-transgenic mice in vitro and transferred into recipient mice. Transfection of SOCS3-specific siRNA attenuated Th2 response in vitro. Adoptive transfer of SOCS3-siRNA T cells exhibited markedly suppressed airway hyperresponsiveness and eosinophilia after OVA challenge, with a concomitant decrease in OVA-specific CD4(+) T cell accumulation in the airways. To investigate the mechanism of this impaired CD4(+) T cell accumulation, we inactivated SOCS3 of T cells by crossing SOCS3-floxed (SOCS3(flox/flox)) mice with CD4-Cre transgenic mice. CD4-Cre × SOCS3(flox/flox) mice exhibited fewer IL-4-producing cells and more reduced eosinophil infiltration in bronchoalveolar lavage fluids than control mice in a model of OVA-induced asthma. Expression of CCR3 and CCR4 in CD4(+) T cells was decreased in CD4-Cre × SOCS3(flox/flox) mice. CCR4 expression was also decreased in CD4(+) T cells after transfer of SOCS3 siRNA-treated T cells. These findings suggest that the therapeutic modulation of SOCS3 expression in CD4(+) T cells might be effective in preventing the development of allergic asthma.

Pulmonary suppressor of cytokine signaling-1 induced by IL-13 regulates allergic asthma phenotype

RATIONALE Th2 cytokines play an important role in allergic diseases. These cytokines activate signal transduction pathways, including Janus kinase/signal transducer and activator of transcription (STAT) signaling. Although the suppressor of cytokine signaling (SOCS) family protein, a negative regulator of the Janus kinase/STAT signaling pathway, contributes to helper T cell differentiation during immune responses, the role of SOCS proteins within the structural cells of a target organ has not been clarified in allergy. OBJECTIVES To study the local function of SOCS in the development of asthma. METHODS We used mouse models of IL-13- and ovalbumin (OVA)-induced allergic airway disease. Airway smooth muscle cells were cultured from patients with asthma. MEASUREMENTS AND MAIN RESULTS The administration of IL-13 induced not only airway responses but also SOCS1 expression at the local inflammatory site. The up-regulated SOCS1 markedly suppressed IL-13-dependent STAT6 activation and eotaxin expression and subsequently down-regulated IL-13-induced airway inflammatory responses. The inactivation of SOCS1 induced airway hyperresponsiveness after IL-13 treatment even in hyporesponsive C57BL/6 background mice. In an OVA-induced model of allergic airway disease, allergen exposure up-regulated local SOCS1 expression, and the induction of SOCS1 in the airways attenuated allergen-induced airway responses. Inactivation of IL-13 inhibited SOCS1 induction in a model of allergic airway disease. Interestingly, airway smooth muscle cells from individuals with asthma had impaired up-regulation of SOCS1 after IL-13 stimulation. CONCLUSIONS SOCS1 induction by IL-13 in airway structural cells is critical to negatively control allergic airway disease.

IL-13 suppresses double-stranded RNA-induced IFN-λ production in lung cells.

Acute asthma exacerbations are frequently associated with respiratory viral infections. Although impaired production of type III IFNs (IFN-λs) is related to the severity of asthma exacerbation, the mechanisms underlying deficient IFN-λ production in asthma are poorly understood. Airway epithelial cells were stimulated in vitro with a synthetic mimetic of viral double-stranded RNA (dsRNA). IL-13, a crucial cytokine responsible for asthma pathogenesis, suppressed dsRNA-induced expression of IFN-λs, and JAK inhibitor AG490 prevented the suppression by IL-13. IL-13 per se did not affect IFN-λ production or the expressions of membrane dsRNA receptor TLR3 and of cytoplasmic receptors RIG-I and MDA5. IL-13-deficient mice exhibited more enhanced IFN-λ expression after intratracheal instillation of dsRNA than wild-type mice, whereas IFN-λ expression after dsRNA was absent in the mouse lungs of the OVA-induced asthma model. These findings suggest that IL-13 may be a putative cytokine suppressing IFN-λ production against airway viral infections in asthmatics.

Essential Role of B7-H1 in Double-Stranded RNA–Induced Augmentation of an Asthma Phenotype in Mice

Clinical and epidemiological studies have shown the contribution of viral infection to the development of allergic asthma. Many RNA viruses, pathogenic for the respiratory tract, generate double-stranded (ds)RNA during their replication. Typical innate immune responses triggered by dsRNA involve the endosomal and cytoplasmic pathways. The former is mediated by Toll/IL-1R domain-containing adaptor inducing IFN-β (TRIF), and the latter by IFN-β promoter stimulator 1 (IPS-1). We explored the effect of polyinocinic polycytidilic acid, a synthetic dsRNA, on the development of an asthma phenotype in mice. Administration of dsRNA during ovalbumin sensitization augmented airway eosinophilia and airway hyperresponsiveness after an antigen challenge, which was associated with enhanced induction of IL-13-producing CD8(+) T cells. The augmentation was induced in IPS-1-deficient mice but not in TRIF-deficient mice. The interactions between dendritic cells (DCs) and T cells are regulated by B7-family costimulatory molecules, including B7-H1 (also known as PD-L1), a putative ligand for programmed death-1 (PD-1). Treatment of bone marrow-derived DCs with dsRNA enhanced B7-H1 expression in a TRIF-dependent manner. Additionally, dsRNA increased B7-H1 expression on DCs in the draining lymph nodes of ovalbumin-sensitized mice. The augmentation of the asthma phenotype was prevented by the treatment of mice with anti-B7-H1 mAb but not with anti-PD-1 mAb. The augmentation was not induced in B7-H1-deficient mice. These results suggest that dsRNA-triggered activation of the innate immune system in sensitization leads to augmentation of the asthma phenotype via IL-13 mainly from CD8(+) T cells. B7-H1 plays a crucial role in the process without requiring interaction with PD-1.

Effects of Salmeterol in Patients with Persistent Asthma Receiving Inhaled Corticosteroid plus Theophylline

Background: Patients with severe asthma require multiple therapies to improve lung function and reduce symptoms. The use of long-acting inhaled β2-agonists plus theophylline in addition to high doses of inhaled corticosteroids (ICSs) for the treatment of severe asthma has not been extensively studied. Objective: The purpose of this study was to investigate the efficacy and safety of salmeterol combined with high-dose ICSs plus theophylline in severe asthma. Methods: We undertook a randomized, placebo-controlled, crossover study to compare the effect of a single dose of inhaled salmeterol (50 µg) or a placebo in patients with severe asthma whose conditions were not being adequately controlled by therapies with high-dose ICSs plus oral theophylline with or without leukotriene receptor antagonists. Results: Twenty patients took part in the trial. Compared with the placebo, the inhalation of salmeterol significantly increased the FEV1. Even in the 9 patients treated with high-dose ICSs plus theophylline plus a leukotriene receptor antagonist, the FEV1 increased significantly more after salmeterol than after the placebo. Conclusion: Patients with severe asthma receiving high-dose ICSs plus theophylline may benefit from the addition of salmeterol.

Preparation of allosamidin and demethylallosamidin photoaffinity probes and analysis of allosamidin-binding proteins in asthmatic mice

Allosamidins, metabolites of Streptomyces with strong inhibitory activities toward family 18 chitinases, show a variety of biological activities in various organisms. We prepared photoaffinity and biotinylated probes of allosamidin and demethylallosamidin, the N-demethyl derivative that shows much stronger anti-asthmatic activity than allosamidin. Mild acid hydrolysis of allosamidins afforded mono-amine derivatives, which were amidated to prepare probes with a photoactivatable aryl azide and/or biotin moieties. The derivatives with an N-acyl group at C-2 of the D-allosamine residue at the non-reducing end of allosamidins inhibited Trichoderma chitinase as strongly as the original compounds. Since the target of allosamidins in asthma is unclear, photoaffinity probes were used to analyze allosamidin-binding proteins in bronchoalveolar lavage (BAL) fluid in IL-13-induced asthmatic mice. Ym1, a chitinase-like protein, was identified as the main allosamidin-binding protein among proteins whose expression was upregulated by IL-13 in BAL fluid. Binding of allosamidins with Ym1 was confirmed by the experiments with photoaffinity probes and recombinant Ym1.

Zno nanowalls grown with PLD and their field emission and UV detector properties

Two-dimensional ZnO nanowalls were grown on sapphire substrates with high-pressure pulsed laser deposition (PLD) without using any catalysts. The structural, field emission and ultra-violet (UV) detecting properties were investigated.