Yuko Matsunaga

Role of Mast Cells and Basophils in IgE Responses and in Allergic Airway Hyperresponsiveness

We established a diphtheria toxin (DT)-based conditional deletion system using Il4 enhancer elements previously shown to be specific for IL-4 production in mast cells (MCs) or basophils (Mas-TRECK and Bas-TRECK mice). DT treatment of Bas-TRECK mice resulted in specific deletion of basophils, whereas both MCs and basophils were deleted in Mas-TRECK mice. DT-treated Mas-TRECK mice had impaired passive cutaneous anaphylaxis, IgE-mediated passive systemic anaphylaxis, and IgE-mediated chronic allergic inflammation, whereas DT-treated Bas-TRECK mice had impaired IgE-mediated chronic allergic inflammation. Using these mice, we also sought to tease out the role of MCs and basophils in airway hyperresponsiveness (AHR). Although MC deletion resulted in a slight increase in basal Ag-specific IgE levels and significant increases in basal IgE levels, we found that this deletion markedly impaired the AHR effector phase and was accompanied by decreased histamine levels. By contrast, basophil deletion had no effect on the AHR effector phase or on IgE production induced by systemic OVA immunization. Our results, using these newly established Mas-TRECK and Bas-TRECK models, demonstrated an indispensable role for MCs as effector cells in AHR.

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.

Leukotriene B4 receptor BLT2 negatively regulates allergic airway eosinophilia

Leukotriene B4 (LTB4) has been implicated in the pathogenesis of allergic diseases. BLT2, a low‐affinity LTB4 receptor, is activated by LTB4 and 12(S)‐hydroxyheptadeca‐5Z,8E,10E ‐trienoic acid (12‐HHT). Although the high‐affinity LTB4 receptor BLT1 has been shown to exert proinflammatory roles, the role of BLT2 in allergic inflammation has not been clarified. To study the function of BLT2 in development of asthma, we used mice model of ovalbumin (OVA)‐induced allergic airway disease. The 12‐HHT levels were elevated in bronchoalveolar lavage (BAL) fluids of OVA‐sensitized/challenged wild‐type mice. BLT2‐deficient mice exhibited enhanced eosinophilia in BAL fluids after OVA exposure. Interleukin (IL)‐13 levels in BAL fluids and IL‐13‐producing CD4+ T cells in the lungs were elevated in BLT2‐deficient mice compared to wild‐type mice, whereas the levels of IL‐4, IL‐5, and interferon (IFN)‐γ in BAL fluids and serum OVA‐specific IgE were comparable. Transfection of BLT2‐specific small interfering RNA enhanced IL‐13 production in CD4+ T cells in vitro. Expression of BLT2 mRNA in CD4+ T cells was significantly reduced in patients with asthma compared to healthy control subjects. These findings indicate that BLT2 has a protective role in allergic airway inflammation and that diminished BLT2 expression in CD4+ T cells may contribute to the pathophysiology of asthma.—Matsunaga, Y., Fukuyama, S., Okuno, T., Sasaki, F., Matsunobu, T., Asai, Y., Matsumoto, K., Saeki, K., Oike, M., Sadamura, Y., Machida, K., Nakanishi, Y., Kubo, M., Yokomizo, T., Inoue, H., Leukotriene B4 receptor BLT2 negatively regulates allergic airway eosinophilia. FASEBJ. 27, 3306‐3314 (2013). www.fasebj.org

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.

Cigarette smoke impairs phagocytosis of apoptotic neutrophils by alveolar macrophages via inhibition of the histone deacetylase/Rac/CD9 pathways

Efferocytosis, which is the homeostatic phagocytosis of apoptotic cells, prevents the release of toxic intracellular contents and subsequent tissue damage. Impairment of efferocytosis was reported in alveolar macrophages (AMs) of patients with chronic obstructive pulmonary disease (COPD), a common disease caused by smoking. In COPD, histone deacetylase (HDAC) activity is reduced in AMs. We investigated whether the reduction of HDAC activity is associated with the impairment of efferocytosis. Murine AMs were collected by bronchoalveolar lavage and their ability to efferocytose apoptotic human polymorphonuclear leukocytes was assessed. Pre-treatment of AMs with cigarette smoke extract (CSE) or trichostatin A (TSA), an HDAC inhibitor, suppressed efferocytosis and CSE reduced HDAC activity. TSA inhibited the activity of Rac, a key mediator of efferocytosis. These TSA-induced impairments were restored by treatment of AMs with aminophylline, a potent activator of HDAC. To further elucidate the underlying mechanism, we explored a role of CD9 in TSA-induced impairment of efferocytosis. CD9 is a transmembrane protein of the tetraspanin family that facilitates the uptake of several pathogens and other material. TSA profoundly down-regulated the expression of CD9 on AMs. The expression of CD9 was partly down-regulated by the Rac inhibitor. Pretreatment with an anti-CD9 mAb or CD9 small interfering RNA inhibited efferocytosis, which was attributable to the reduced binding of AMs to apoptotic cells. These results suggest that smoking impairs efferocytosis via inhibition of HDAC/Rac/CD9 pathways. Aminophylline/theophylline is effective in restoring the impairment of efferocytosis and might have benefit for the treatment of patients with COPD.

Prevalence of asthma with airflow limitation, COPD, and COPD with variable airflow limitation in older subjects in a general Japanese population: The Hisayama Study

BACKGROUND Elucidating the prevalence of asthma and chronic obstructive pulmonary disease (COPD) is important for designing a public health strategy. Recent studies have discriminated a phenotype of COPD with variable airflow limitation (COPD-VAL) associated with asthma-COPD overlap syndrome. Its prevalence remains uncertain. The age and occupational distributions in the town of Hisayama and in Japan are nearly identical. Each diseases prevalence was estimated for the towns residents. METHODS In 2008, town residents (≥ 40 years) were solicited to participate in a health checkup. Individuals with abnormal spirometry (forced expiratory volume in 1s/forced vital capacity [FEV1/FVC]<70% and/or %FVC<80%) were recommended for further evaluations. Two pulmonologists in a blinded fashion reviewed their medical records, including bronchodilator reversibility. Individuals with airflow limitation were classified as having asthma, COPD, COPD-VAL, or other diseases. The prevalence of each disease was then estimated. RESULTS A total of 2100 residents (43.4% of residents in the age group) completed spirometry. In 455 residents with abnormal spirometry, 190 residents had further evaluations, and the medical records of 174 residents were reviewed. The prevalence of asthma with airflow limitation, COPD, and COPD-VAL, were 2.0%, 8.4%, and 0.9%, respectively. The prevalence of COPD and COPD-VAL were higher in men and smokers than in women and never-smokers. The prevalence of COPD, but not COPD-VAL or asthma, increased with age. CONCLUSION The prevalence of asthma with airflow limitation, COPD, and COPD-VAL were estimated in a population of residents (≥ 40 years) in Hisayama.

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.

IL-6 Induced by Double-Stranded RNA Augments Allergic Inflammation via Suppression of Foxp3+ T-Cell/IL-10 Axis

Activation of innate immunity against viruses in the respiratory tracts affects the development of asthma. Most respiratory viruses generate double-stranded (ds)RNA during their replication. We recently showed that a low-dose administration of polyinosinic polycytidylic acid (poly IC), a mimetic of viral dsRNA, during allergen sensitization augments airway eosinophilia and hyperresponsiveness in mice via enhanced production of IL-13 from T cells. However, a phenotype of asthma under severer load of dsRNA remains unknown. d-galactosamine (d-GalN) is known as a strong sensitizer of poly IC. Mice were treated with poly IC plus d-GalN during allergen sensitization. A sublethal dose of poly IC/d-GalN augmented airway eosinophilia and CD4(+) T-cell accumulation in the lungs but not airway hyperresponsiveness. The augmented inflammation was associated with decreased IL-10 in the bronchoalveolar lavage fluid and decreased Foxp3(+) regulatory T cells in the lungs. Serum IL-6 was prominently higher in the mice treated with poly IC/d-GalN than in that with poly IC alone or d-GalN alone. Poly IC/d-GalN did not affect IL-17-producing T cells in the lungs. Poly IC/d-GalN failed to augment airway eosinophilia after anti-IL-10 receptor monoclonal antibody treatment during allergen challenge. Finally, anti-IL-6 receptor monoclonal antibody treatment before poly IC/d-GalN completely prevented the decrease of IL-10 and Foxp3(+) regulatory T cells and the augmentation of airway inflammation. These results indicate that enhanced production of IL-6 by poly IC/d-GalN induces the augmentation of allergic inflammation via suppression of Foxp3(+) regulatory T-cell/IL-10 axis. IL-6 may be a target for preventing asthma augmentation related to severe virus infection.

Mast cells contribute to double-stranded RNA-induced augmentation of airway eosinophilia in a murine model of asthma

BackgroundClinical studies showed the contribution of viral infection to the development of asthma. Although mast cells have multiple roles in the pathogenesis of allergic asthma, their role of in the virus-associated pathogenesis of asthma remains unknown. Most respiratory viruses generate double-stranded (ds) RNA during their replication. dsRNA provokes innate immune responses. We recently showed that an administration of polyinocinic polycytidilic acid (poly IC), a mimetic of viral dsRNA, during allergen sensitization augments airway eosinophilia and hyperresponsiveness in mice via enhanced production of IL-13.MethodsThe effect of poly IC on allergen-induced airway eosinophilia was investigated for mast cell-conserved Kit+/+ mice and -deficient KitW/KitW-v mice. The outcome of mast cell reconstitution was further investigated.ResultsAirway eosinophilia and IL-13 production were augmented by poly IC in Kit+/+ mice but not in KitW/KitW-v mice. When KitW/KitW-v mice were reconstituted with bone marrow-derived mast cells (BMMCs), the augmentation was restored. The augmentation was not induced in the mice systemically deficient for TIR domain-containing adaptor-inducing IFN-β (TRIF) or interferon regulatory factor (IRF)-3, both mediate dsRNA-triggered innate immune responses. The augmentation was, however, restored in KitW/KitW-v mice reconstituted with TRIF-deficient or IRF-3-deficient BMMCs. Although leukotriene B4 and prostaglandin D2 are major lipid mediators released from activated mast cells, no their contribution was shown to the dsRNA-induced augmentation of airway eosinophilia.ConclusionsWe conclude that mast cells contribute to dsRNA-induced augmentation of allergic airway inflammation without requiring direct activation of mast cells with dsRNA or involvement of leukotriene B4 or prostaglandin D2.