Satoshi Ano

Transcription Factors GATA-3 and RORγt Are Important for Determining the Phenotype of Allergic Airway Inflammation in a Murine Model of Asthma

In refractory asthma, neutrophils, rather than eosinophils, often predominate in the airways. Neutrophilic airway inflammation appears to be resistant to steroids and may be related to the Th17, rather than the Th2, cytokine milieu. However, the role of GATA-3 and RORγt, transcription factors for Th2 and Th17 cell differentiation, respectively, in the pathogenesis of steroid-insensitive asthma remains unclear. To examine the effect of GATA-3– and RORγt-overexpression backgrounds on airway inflammation and steroid sensitivity, we generated two strains of transgenic mice overexpressing GATA-3 or RORγt. Mice were sensitized and challenged with OVA. Some OVA-sensitized/challenged mice were treated with dexamethasone, anti–IL-17 Ab, CXCR2 antagonist, or anti–IL-6R Ab to demonstrate their therapeutic effects on airway inflammation. Although Ag-specific airway inflammation and hyperresponsiveness were induced in each mouse, the phenotype of inflammation showed a distinct difference that was dependent upon the genotype. GATA-3–overexpressing mice exhibited steroid-sensitive eosinophilic inflammation with goblet cell hyperplasia and mucus hyperproduction under Th2-biased conditions, and RORγt-overexpressing mice developed steroid-insensitive neutrophilic inflammation under Th17-biased conditions. The levels of keratinocyte-derived chemokine, MIP-2, IL-6, and other neutrophil chemotaxis-related mediators were significantly elevated in OVA-exposed RORγt-overexpressing mice compared with wild-type mice. Interestingly, airway hyperresponsiveness accompanied by neutrophilic airway inflammation in RORγt-overexpressing mice was effectively suppressed by anti–IL-17 Ab, CXCR2 antagonist, or anti–IL-6R Ab administration. In conclusion, our results suggest that the expression levels of GATA-3 and RORγt may be important for determining the phenotype of asthmatic airway inflammation. Furthermore, blockade of the Th17-signaling pathway may be a treatment option for steroid-insensitive asthma.

Role of Nrf2 in Host Defense against Influenza Virus in Cigarette Smoke-Exposed Mice

ABSTRACT Influenza virus is a common respiratory tract viral infection. Although influenza can be fatal in patients with chronic pulmonary diseases such as chronic obstructive pulmonary disease, its pathogenesis is not fully understood. The Nrf2-mediated antioxidant system is essential to protect the lungs from oxidative injury and inflammation. In the present study, we investigated the role of Nrf2 in protection against influenza virus-induced pulmonary inflammation after cigarette smoke exposure with both in vitro and in vivo approaches. For in vitro analyses, peritoneal macrophages isolated from wild-type and Nrf2-deficient mice were treated with poly(I:C) and/or cigarette smoke extract. For in vivo analysis, these mice were infected with influenza A virus with or without exposure to cigarette smoke. In Nrf2-deficient macrophages, NF-κB activation and the induction of its target inflammatory genes were enhanced after costimulation with cigarette smoke extract and poly(I:C) compared with wild-type macrophages. The induction of antioxidant genes was observed for the lungs of wild-type mice but not those of Nrf2-deficient mice after cigarette smoke exposure. Cigarette smoke-exposed Nrf2-deficient mice showed higher rates of mortality than did wild-type mice after influenza virus infection, with enhanced peribronchial inflammation, lung permeability damage, and mucus hypersecretion. Lung oxidant levels and NF-κB-mediated inflammatory gene expression in the lungs were also enhanced in Nrf2-deficient mice. Our data indicate that the antioxidant pathway controlled by Nrf2 is pivotal for protection against the development of influenza virus-induced pulmonary inflammation and injury under oxidative conditions.

Th17-Associated Cytokines as a Therapeutic Target for Steroid-Insensitive Asthma

Steroid-insensitive asthma is an infrequent but problematic airway disease that presents with persistent symptoms, airflow limitation, or recurrent exacerbations even when treated with steroid-based therapies. Because of unsatisfactory results obtained from currently available therapies for steroid-insensitive asthma, a better understanding of its pathogenesis and the development of new targeted molecular therapies are warranted. Recent studies indicated that levels of interleukin (IL)-17 are increased and both eosinophils and neutrophils infiltrate the airways of severe asthmatics. IL-17 is a proinflammatory cytokine mainly secreted from helper T (Th) 17 cells and is important for the induction of neutrophil recruitment and migration at sites of inflammation. This review focuses on the pathogenetic role of Th17 cells and their associated cytokines in steroid-insensitive asthma and discusses the prospects of novel therapeutic options targeting the Th17 signaling pathway.

SQSTM1/p62/A170 regulates the severity of Legionella pneumophila pneumonia by modulating inflammasome activity

Sequestosome1/A170/p62 (SQSTM1) is a scaffold multifunctional protein involved in several cellular events, such as signal transduction, cell survival, cell death, and inflammation. SQSTM1 expression by macrophages is induced in response to environmental stresses; however, its role in macrophage‐mediated host responses to environmental stimuli, such as infectious pathogens, remains unclear. In this study, we investigated the role of SQSTM1 in host responses to Legionella pneumophila, an intra‐cellular pathogen that infects macrophages, in both an SQSTM1‐deficient (SQSTM1−/−) mouse model and macrophages from these mice. Compared with wild‐type (WT) macrophages, the production and secretion of the proinflammatory cytokine IL‐1β was significantly enhanced in SQSTM1−/− macrophages after infection with L. pneumophila. Inflammasome activity, indicated by the level of IL‐18 and caspase‐1 activity, was also elevated in SQSTM1−/− macrophages after infection with L. pneumophila. SQSTM1 may interact with nucleotide‐binding oligomerization domain‐like receptor family, caspase recruitment domain‐containing 4 and nucleotide‐binding oligomerization domain like receptor family, pyrin domain containing 3 proteins to inhibit their self‐dimerization. Acute pulmonary inflammation induced by L. pneumophila and silica was enhanced in SQSTM1−/− mice with an increase in IL‐1β levels in the bronchoalveolar lavage fluids. These findings suggest that SQSTM1 is a negative regulator of acute pulmonary inflammation, possibly by regulating inflammasome activity and subsequent proinflammatory cytokine production.

Role of Th1/Th17 Balance Regulated by T-bet in a Mouse Model of Mycobacterium avium Complex Disease

Th1 immune responses are thought to be important in protection against intracellular pathogens. T-bet is a critical regulator for Th1 cell differentiation and Th1 cytokine production. The aim of this study was to determine the role of T-bet in host defense against Mycobacterium avium complex (MAC) infection. Wild-type mice, T-bet–deficient mice, and T-bet–overexpressing mice were infected with MAC via intratracheal inoculation. Macrophages and dendritic cells obtained from these mice were incubated with MAC. T-bet–deficient mice were highly susceptible to MAC, compared with wild-type mice and T-bet–overexpressing mice. Neutrophilic pulmonary inflammation was also enhanced in T-bet–deficient mice, but attenuated in T-bet–overexpressing mice, following MAC infection. Cytokine expression shifted toward Th1 in the lung and spleen of T-bet–overexpressing mice, but toward Th17 in T-bet–deficient mice. IFN-γ supplementation to T-bet–deficient mice reduced systemic MAC growth but did not reduce pulmonary inflammation. In contrast, neutralization of IL-17 in T-bet–deficient mice reduced pulmonary inflammation but did not affect mycobacterial growth in any organs tested. T-bet–deficient T cells tended to differentiate toward Th17 cells in vitro following exposure to MAC. Treatment with NO donor suppressed MAC-induced Th17 cell differentiation of T-bet–deficient T cells. This study identified that the fine balance between Th1 and Th17 responses is essential in defining the outcome of MAC disease. T-bet functions as a regulator for Th1/Th17 balance and is a critical determinant for host resistance to MAC infection by controlling cytokine and NO levels.

Carbocisteine reduces virus-induced pulmonary inflammation in mice exposed to cigarette smoke.

Carbocisteine (S-CMC) inhibits viral infection and prevents acute exacerbation of chronic obstructive pulmonary disease. We recently demonstrated the protective effects of NF-E2-related factor (Nrf) 2 against influenza virus (FluV)-induced pulmonary inflammation in mice exposed to cigarette smoke (CS). In our current study, we investigated the effects of S-CMC on Nrf2 activation in cultured macrophages, and in mice infected with influenza after exposure to CS. Nuclear translocation of Nrf2 and the expression of Nrf2-targeted antioxidant genes, such as heavy and light subunits of γ glutamyl cysteine synthetase and heme oxigenase-1, were enhanced in a dose-dependent manner after treatment with S-CMC in peritoneal and alveolar macrophages of wild-type mice, but not in those of Nrf2-deficient mice. Nuclear translocation of Nrf2 in macrophages was inhibited by the phosphatidylinositol 3-kinase inhibitor, LY294002. Phosphorylated Akt, Nrf2, and heme oxigenase-1 were induced in the alveolar macrophages of the lungs in wild-type mice after S-CMC administration. The extent of oxidative stress, inflammatory cell infiltration, pulmonary edema, and goblet cell hyperplasia was suppressed by S-CMC administration in the lungs of wild-type mice after exposure to both CS and FluV. Our findings suggest that S-CMC reduces pulmonary inflammation and mucus overproduction in mice exposed to CS after infection with FluV via the activation of Nrf2.

Inflammation and airway hyperresponsiveness after chlorine exposure are prolonged by Nrf2 deficiency in mice

Rationale Chlorine gas (Cl2) is a potent oxidant and trigger of irritant induced asthma. We explored NF‐E2–related factor 2 (Nrf2)‐dependent mechanisms in the asthmatic response to Cl2, using Nrf2‐deficient mice, buthionine sulfoximine (BSO), an inhibitor of glutathione (GSH) synthesis and sulforaphane (SFN), a phytochemical regulator of Nrf2. Methods Airway inflammation and airway hyperresponsiveness (AHR) were assessed 24 and 48 h after a 5‐min nose‐only exposure to 100 ppm Cl2 of Nrf2‐deficient and wild type Balb/C mice treated with BSO or SFN. Animals were anesthetized, paralyzed and mechanically ventilated (FlexiVent™) and challenged with aerosolized methacholine. Bronchoalveolar lavage (BAL) was performed and lung tissues were harvested for assessment of gene expression. Results Cl2 exposure induced a robust AHR and an intense neutrophilic inflammation that, although similar in Nrf2‐deficient mice and wild‐type mice at 24 h after Cl2 exposure, were significantly greater at 48 h post exposure in Nrf2‐deficient mice. Lung GSH and mRNA for Nrf2‐dependent phase II enzymes (NQO‐1 and GPX2) were significantly lower in Nrf2‐deficient than wild‐type mice after Cl2 exposure. BSO reduced GSH levels and promoted Cl2‐induced airway inflammation in wild‐type mice, but not in Nrf2‐deficient mice, whereas SFN suppressed Cl2‐induced airway inflammation in wild‐type but not in Nrf2‐deficient mice. AHR was not affected by either BSO or SFN at 48 h post Cl2 exposure. Conclusions Nrf2‐dependent phase II enzymes play a role in the resolution of airway inflammation and AHR after Cl2 exposure. Moderate deficiency of GSH affects the magnitude of acute inflammation but not AHR. Graphical abstract Nrf2 activity and its modulation in response to chlorine. (A) Chlorine gas (Cl2) induces airway inflammation and airway hyperresponsiveness (AHR) in both Nrf2‐deficient (Nrf2‐/‐) and wild‐type (Nrf2+/+) mice at 24 h after Cl2 exposure. (B) In wild type mice, Nrf2 translocates to the nucleus where it binds antioxidant response element and upregulates genes associated with glutathione synthesis and antioxidant defense. As consequence, 48 h after Cl2 exposure mice expressing Nrf2 present decrease lung inflammation and resolution of AHR compared to Nrf2‐/‐ mice. sulforaphane (SFN) stimulates Nrf2 nuclear translocation and an increase of the reduced form of glutathione leading to resolution of lung inflammation but not AHR in Nrf2+/+. The inhibition of GSH synthesis by buthionine sulfoximine (BSO) decrease the anti‐oxidant defense leading to increased inflammation without affecting AHR

Overexpression of RORγt Enhances Pulmonary Inflammation after Infection with Mycobacterium Avium.

Mycobacterium avium complex (MAC) is the most common cause of nontuberculous mycobacterial disease in humans. The role of Th17 immunity in the pathogenesis of intracellular bacteria, such as MAC, is not currently understood. Transcription factor RAR-related orphan receptor gamma t (RORγt) is known as the master regulator for Th17 cell development. Here, we investigated the role of RORγt in host responses against MAC infection. Wild-type (WT) mice and RORγt-overexpressing mice were infected with MAC via intratracheal inoculation. Systemic MAC growth was not different between WT mice and RORγt-overexpressing mice. However, neutrophilic pulmonary inflammation following MAC infection was enhanced in RORγt-overexpressing mice compared with that in WT mice. The cytokine expression shifted toward a Th17 phenotype in the lungs of RORγt-overexpressing mice following MAC infection; the levels of IL-6 and IL-17 were significantly higher in the lung of these mice than in WT mice. In addition to the increase in IL-17 single-positive T cells, T cells producing both IL-17 and interferon-γ were elevated in the lung of RORγt-overexpressing mice following MAC infection. These findings suggest that RORγt overexpression-mediated Th17 bias contributes to local inflammation rather than systemic responses, by regulating neutrophil recruitment into the sites of infection during MAC infection.