A novel quinoline with airway relaxant effects and anti-inflammatory properties.

BACKGROUND: In chronic pulmonary diseases characterized by inflammation and airway obstruction, such as asthma and COPD, there are unmet needs for improved treatment. Quinolines is a group of small heterocyclic compounds that have a broad range of pharmacological properties. Here, we investigated the airway relaxant and anti-inflammatory properties of a novel quinoline (RCD405). METHODS: The airway relaxant effect of RCD405 was examined in isolated airways from humans, dogs, rats and mice. Murine models of ovalbumin (OVA)-induced allergic asthma and LPS-induced airway inflammation were used to study the effects in vivo. RCD405 (10 mg/kg) or, for comparisons in selected studies, budesonide (3 mg/kg), were administered intratracheally 1 h prior to each challenge. Airway responsiveness was determined using methacholine provocation. Immune cell recruitment to bronchi was measured using flow cytometry and histological analyses were applied to investigate cell influx and goblet cell hyperplasia of the airways. Furthermore, production of cytokines and chemokines was measured using a multiplex immunoassay. The expression levels of asthma-related genes in murine lung tissue were determined by PCR. The involvement of NF-κB and metabolic activity was measured in the human monocytic cell line THP-1. RESULTS: RCD405 demonstrated a relaxant effect on carbachol precontracted airways in all four species investigated (potency ranking: human = rat > dog = mouse). The OVA-specific IgE and airway hyperresponsiveness (AHR) were significantly reduced by intratracheal treatment with RCD405, while no significant changes were observed for budesonide. In addition, administration of RCD405 to mice significantly decreased the expression of proinflammatory cytokines and chemokines as well as recruitment of immune cells to the lungs in both OVA- and LPS-induced airway inflammation, with a similar effect as for budesonide (in the OVA-model). However, the effect on gene expression of Il-4, IL-5 and Il-13 was more pronounced for RCD405 as compared to budesonide. Finally, in vitro, RCD405 reduced the LPS-induced NF-κB activation and by itself reduced cellular metabolism. CONCLUSIONS: RCD405 has airway relaxant effects, and it reduces AHR as well as airway inflammation in the models used, suggesting that it could be a clinically relevant compound to treat inflammatory airway diseases. Possible targets of this compound are complexes of mitochondrial oxidative phosphorylation, resulting in decreased metabolic activity of targeted cells as well as through pathways associated to NF-κB. However, further studies are needed to elucidate the mode of action.

The effect of mitochondria-targeted slow hydrogen sulfide releasing donor AP39-treatment on airway inflammation.

Mitochondrial dysfunction has been shown to contribute to the pathophysiology of airway diseases. Therefore, mitochondria are targeted in the development of new therapeutic approaches. Hydrogen sulfide (H2S) has been shown to be involved in the pathophysiological processes of airway inflammation. We aimed to evaluate the effect of mitochondria-targeted slow H2S releasing donor AP39 [(10-oxo-10-(4-(3-thioxo-3H-1,2-dithiol5yl)phenoxy)decyl)triphenylphosphoniumbromide)] on lipopolysaccharide (LPS)-induced airway inflammation in mice. LPS was applied to female Balb/c mice by intranasal (i.n.) route to induce airway inflammation and the subgroups of mice were treated with i.n. AP39 (250-1000 nmol/kg). 48 h after LPS administration airway reactivity was evaluated in vivo, then bronchoalveolar lavage (BAL) fluid and lungs were collected. LPS application led to bronchial hyperreactivity and neutrophil infiltration into the lung tissues along with increased TNF-α, IL-1ß and IL-6 levels in BAL fluid. LPS also induced an increase in the rate of glycolysis, glycogenolysis and Krebs-cycle. AP39 treatment prevented the LPS-induced bronchial hyperreactivity and reversed the increase in TNF-α and IL-6 levels in BAL fluid. The increase in neutrophil numbers in BAL fluid was also prevented by AP39 treatment at the highest dose. Our results indicate that AP39 can prevent bronchial hyperreactivity and decrease airway inflammation. Targeting H2S to the mitochondria may be a new therapeutic approach in airway inflammation.

Novel acute hypersensitivity pneumonitis model induced by airway mycosis and high dose lipopolysaccharide.

BACKGROUND: Inhalation of fungal spores is a strong risk factor for severe asthma and experimentally leads to development of airway mycosis and asthma-like disease in mice. However, in addition to fungal spores, humans are simultaneously exposed to other inflammatory agents such as lipopolysaccharide (LPS), with uncertain relevance to disease expression. To determine how high dose inhalation of LPS influences the expression of allergic airway disease induced by the allergenic mold Aspergillus niger (A. niger). METHODS: C57BL/6J mice were intranasally challenged with the viable spores of A. niger with and without 1 µg of LPS over two weeks. Changes in airway hyperreactivity, airway and lung inflammatory cell recruitment, antigen-specific immunoglobulins, and histopathology were determined. RESULTS: In comparison to mice challenged only with A. niger, addition of LPS (1 µg) to A. niger abrogated airway hyperresponsiveness and strongly attenuated airway eosinophilia, PAS+ goblet cells and TH2 responses while enhancing TH1 and TH17 cell recruitment to lung. Addition of LPS resulted in more severe, diffuse lung inflammation with scattered, loosely-formed parenchymal granulomas, but failed to alter fungus-induced IgE and IgG antibodies. CONCLUSIONS: In contrast to the strongly allergic lung phenotype induced by fungal spores alone, addition of a relatively high dose of LPS abrogates asthma-like features, replacing them with a phenotype more consistent with acute hypersensitivity pneumonitis (HP). These findings extend the already established link between airway mycosis and asthma to HP and describe a robust model for further dissecting the pathophysiology of HP.

Metformin prevents airway hyperreactivity in rats with dietary obesity.

Increased insulin is associated with obesity-related airway hyperreactivity and asthma. We tested whether the use of metformin, an antidiabetic drug used to reduce insulin resistance, can reduce circulating insulin, thereby preventing airway hyperreactivity in rats with dietary obesity. Male and female rats were fed a high- or low-fat diet for 5 wk. Some male rats were simultaneously treated with metformin (100 mg/kg orally). In separate experiments, after 5 wk of a high-fat diet, some rats were switched to a low-fat diet, whereas others continued a high-fat diet for an additional 5 wk. Bronchoconstriction and bradycardia in response to bilateral electrical vagus nerve stimulation or to inhaled methacholine were measured in anesthetized and vagotomized rats. Body weight, body fat, caloric intake, fasting glucose, and insulin were measured. Vagally induced bronchoconstriction was potentiated only in male rats on a high-fat diet. Males gained more body weight, body fat, and had increased levels of fasting insulin compared with females. Metformin prevented development of vagally induced airway hyperreactivity in male rats on high-fat diet, in addition to inhibiting weight gain, fat gain, and increased insulin. In contrast, switching rats to a low-fat diet for 5 wk reduced body weight and body fat, but it did not reverse fasting glucose, fasting insulin, or potentiation of vagally induced airway hyperreactivity. These data suggest that medications that target insulin may be effective treatment for obesity-related asthma.

Small airway dysfunction in patients with cough variant asthma: a retrospective cohort study.

BACKGROUND: Cough variant asthma (CVA) is one of the special populations of asthma. The aim of the study was to compare small airways, the degree of bronchial hyperresponsiveness (BHR) and airway inflammatory subtypes between CVA and classic asthma (CA), and investigate the relationship between these markers to determine the accuracy as indicators of CVA. METHODS: A total of 825 asthmatic patients participated in the study and 614 were included. 614 patients underwent spirometry and a bronchial challenge with methacholine and 459 patients performed induction sputum cell test. RESULTS: The number of CVA patients showed less small airway dysfunction than those of CA patients (p < 0.005). The degree of small airways dysfunction was higher in the CA group compared with the CVA group (p < 0.001). Small airways dysfunction was severer in the eosinophilic airway inflammatory subtype compared with other subtypes (p < 0.05).The area under curve of MMEF, FEF50 and FEF75 (% predicted) was 0.615, 0.621, 0.606, respectively. 0.17mcg of PD20 and 4.7% of sputum eosinophils was the best diagnostic value for CVA with an AUC of 0.582 and 0.575 (p = 0.001 and p = 0.005, respectively). CONCLUSIONS: The eosinophilic airway inflammatory subtype may be increased small airway dysfunction. The value of small airways, BHR and induction sputum cells in CVA prediction, which reflected significant, but not enough to be clinically useful.

Platelets Independently Recruit into Asthmatic Lungs and Models of Allergic Inflammation via CCR3.

Platelet activation and pulmonary recruitment occur in patients with asthma and in animal models of allergic asthma, in which leukocyte infiltration, airway remodeling, and hyperresponsiveness are suppressed by experimental platelet depletion. These observations suggest the importance of platelets to various characteristics of allergic disease, but the mechanisms of platelet migration and location are not understood. The aim of this study was to assess the mechanism of platelet recruitment to extravascular compartments of lungs from patients with asthma and after allergen challenge in mice sensitized to house dust mite (HDM) extract (contains the DerP1 [Dermatophagoides pteronyssinus extract peptidase 1] allergen); in addition, we assessed the role of chemokines in this process. Lung sections were immunohistochemically stained for CD42b+ platelets. Intravital microscopy in allergic mice was used to visualize platelets tagged with an anti-mouse CD49b-PE (phycoerythrin) antibody. Platelet-endothelial interactions were measured in response to HDM (DerP1) exposure in the presence of antagonists to CCR3, CCR4, and CXCR4. Extravascular CD42b+ platelets were detected in the epithelium and submucosa in bronchial biopsy specimens taken from subjects with steroid-naive mild asthma. Platelets were significantly raised in the lung parenchyma from patients with fatal asthma compared with postmortem control-lung tissue. Furthermore, in DerP1-sensitized mice, subsequent HDM exposure induced endothelial rolling, endothelial adhesion, and recruitment of platelets into airway walls, compared with sham-sensitized mice, via a CCR3-dependent mechanism in the absence of aggregation or interactions with leukocytes. Localization of singular, nonaggregated platelets occurs in lungs of patients with asthma. In allergic mice, platelet recruitment occurs via recognized vascular adhesive and migratory events, independently of leukocytes via a CCR3-dependent mechanism.

Diagnostic comparison of methacholine and mannitol bronchial challenge tests for identifying bronchial hyperresponsiveness in asthma: a systematic review and meta-analysis.

OBJECTIVE: Bronchial hyperresponsiveness (BHR) is a representative feature of asthma. Although methacholine and mannitol are commonly used for bronchial challenge tests, the optimal roles of the two agents for assessing BHR remain unclear. We compared the diagnostic performance of methacholine and mannitol in bronchial challenge tests. METHODS: A systematic literature search was performed using MEDLINE, EMBASE, and the Cochrane Central Register. The sensitivity, specificity, diagnostic odds ratio (DOR), and a hierarchical summary of the receiver-operating characteristic curve (HSROC) of the two agents for detecting BHR in asthma were pooled using meta-analysis. A meta-regression analysis was used to identify potential sources of heterogeneity within the selected studies. RESULTS: We identified six studies comprising 565 patients. The pooled sensitivity, specificity, and DOR of methacholine were 0.61 (95%CI, 0.44-0.76), 0.93 (95%CI, 0.70-0.99), and 23.47 (95% CI, 2.51-219.89), respectively. The pooled sensitivity, specificity, and diagnostic odds ratio of mannitol were 0.50 (95%CI, 0.28-0.73), 0.97 (95% CI, 0.94-0.99), and 35.22 (95% CI, 8.82-140.62), respectively. The area under the HSROC for mannitol was higher than that for methacholine (0.97 vs. 0.81, p < 0.01). Considerable between-study heterogeneity was present for sensitivity and specificity in studies of both index tests. Univariate meta-regression analysis revealed that age and sex of the study participants were probable sources of heterogeneity for specificity in studies of methacholine. CONCLUSION: Although mannitol showed better diagnostic performance than methacholine for identifying BHR in asthma, substantial between-study heterogeneity necessitates caution when interpreting the data.

A mouse allergic asthma model induced by shrimp tropomyosin.

Allergic asthma remains an important worldwide health issue. Animal models are valuable for understanding the pathophysiological mechanisms of asthma and the development of effective therapeutics. This study aims to develop an alternative murine model induced by shrimp tropomyosin (ST) instead of ovalbumin (OVA). To investigate responses to short-term exposure to antigens, mice were sensitized with intraperitoneal injections of ST or ST plus aluminum adjuvant on days 0, 7, 14 followed by an intranasal challenge with ST for seven consecutive days. We reveal that sensitization with ST alone or ST plus aluminum induces significant levels of serum total IgE and ST-specific IgE in mice. Challenge results show that ST causes severe eosinophilic airway inflammation. Histology analysis of the lung tissues demonstrates airway inflammation and mucus hypersecretion within the bronchi in mice exposed to ST. Analysis of the cell composition in bronchoalveolar lavage fluid (BALF) shows a significant increase in eosinophil count in ST alone and ST plus aluminum groups. We also detect increased CD4+ T lymphocytes in lung tissues and production of helper T cell type 2-associated cytokines (IL-4 and IL-5) in BALF. In addition, airway hyperresponsiveness to methacholine in ST alone and ST plus aluminum groups is much higher than that in control groups. For the chronic model, mice were sensitized by ST or ST plus aluminum adjuvant for 3weeks and challenged with ST for 6weeks. We find severe structural changes in animals upon prolonged exposure to ST, including goblet cell hyperplasia, collagen deposition, and smooth muscle thickening. In conclusion, ST-induced asthma is a simple murine model for studying pathogenesis of asthma and evaluating new therapeutic drugs.

Callicarpa japonica Thunb. ameliorates allergic airway inflammation by suppressing NF-κB activation and upregulating HO-1 expression.

ETHNOPHARMACOLOGICAL RELEVANCE: Callicarpa japonica Thunb., as an herbal medicine has been used for the treatment of inflammatory diseases in China and Korea. MATERIALS AND METHODS: Ultra performance liquid chromatography-photodiode array-quadrupole time-of-flight mass spectrometer (UPLC-PDA-QTof MS) was used to detect the major phenylethanoid glycosides in the C. japonica extract. BALB/c mice were intraperitoneally sensitized by ovalbumin (OVA) (on days 0 and 7) and challenged by OVA aerosol (on days 11-13) to induce airway inflammatory response. The mice were also administered with C. japonica Thunb. (CJT) (20 and 40 mg/kg Per oral) on days 9-13. CJT pretreatment was conducted in lipopolysaccharide (LPS)-stimulated RAW264.7 or phorbol 12-myristate 13-acetate (PMA)-stimulated A549 cells. RESULTS: CJT administration significantly reduced the secretion of Th2 cytokines, TNF-α, IL-6, immunoglobulin E (IgE) and histamine, and the recruitment of eosinophils in an OVA-exposed mice. In histological analyses, the amelioration of inflammatory cell influx and mucus secretion were observed with CJT. The OVA-induced airway hyperresponsiveness (AHR), iNOS expression and NF-κB activation were effectively suppressed by CJT administration. In addition, CJT led to the upregulation of HO-1 expression. In an in vitro study, CJT pretreatment suppressed the LPS-induced TNF-α secretion in RAW264.7 cells and attenuated the PMA-induced IL-6, IL-8 and MCP-1 secretion in A549 cells. These effects were accompanied by downregulated NF-κB phosphorylation and by upregulated HO-1 expression. CONCLUSION: These results suggested that CJT has protective activity against OVA-induced airway inflammation via downregulation of NF-κB activation and upregulation of HO-1, suggesting that CJT has preventive potential for the development of allergic asthma.

Asthma onset after exposure to fluorinated hydrocarbons in the presence of combustion.

Fluorinated hydrocarbons, which can thermally degrade into toxic hydrofluoric acid, are widely used as, for example, cooling agents in refrigerators and air conditioning systems and as medical aerosol propellants. Hydrofluoric acid is a known causative agent of irritant-induced asthma. We report on two patients with asthma initiation shortly after exposure to fluorinated hydrocarbon-based cooling agents while welding or smoking cigarettes in a confined space. Both cases developed respiratory symptoms and headache and later demonstrated nonspecific bronchial hyperresponsiveness. In follow-up, asthma was persistent and responded poorly to asthma medication. Exposure to the fluorinated hydrocarbons themselves is unlikely to have caused asthma due to their low toxicity. Instead, exposure to small amounts of hydrofluoric acid via the thermal degradation of the fluorinated hydrocarbons was considered the most likely cause of asthma onset. This is supported by the typical clinical picture of irritant-induced asthma and acute symptoms resembling hydrofluoric acid poisoning. When fluorinated hydrocarbons are used in the presence of combustion, thermal degradation may lead to the formation of hydrofluoric acid. In confined spaces, this exposure may induce asthma via irritation. Welding, smoking, and other sources of combustion in confined spaces may be a risk in workplaces and other places in which fluorinated hydrocarbons are used.

LPS aggravates lung inflammation induced by RSV by promoting the ERK-MMP-12 signaling pathway in mice.

BACKGROUND: RSV can lead to persistent airway inflammation and airway hyperresponsiveness (AHR), and is intimately associated with childhood recurrent wheezing and asthma, but the underlying mechanisms remain unclear. Lipopolysaccharide (LPS) is also implicated in the onset and exacerbation of asthma. However, whether inhalation of LPS can boost airway inflammation induced by RSV is not clear. In this study, we utilized an LPS- and RSV-superinfected mouse model to explore underlying pathogenesis. METHODS: Mice were infected with RSV on day 0 and inoculated with LPS from day 35 to day 41, samples were collected on day 42. Inflammatory cells, lung histopathology and AHR were measured. Cytokines were detected by ELISA and ERK, JNK, p38 was determined by western blot. MMP408, PD98059, SP600125 and SB203580 were used to inhibit MMP-12, ERK, JNK and p38 respectively. RESULTS: LPS exposure superimposed on RSV-infected lungs could lead to more vigorous cellular influx, lung structures damage, augmented AHR and higher MMP-12 levels. Inhibition of MMP-12 or ERK signaling pathway in vivo both diminished LPS-driven airway inflammation and AHR. CONCLUSIONS: Exposure to LPS in RSV-infected mice is associated with enhanced increases in ERK-MMP-12 expression that translates into increased lung inflammation and AHR. These findings contribute novel information to the field investigating the onset of post-RSV bronchiolitis recurrent wheezing as a result of LPS exposure.

Osthole attenuates ovalbumin­induced lung inflammation via the inhibition of IL­33/ST2 signaling in asthmatic mice.

Asthma is a common chronic inflammatory airway disease. Recent studies have reported that interleukin (IL)­33 is a potential link between the airway epithelium and Th2­type inflammatory responses, which are closely related to the progression of asthma. The IL­33 receptor, ST2, is highly expressed in group 2 innate lymphoid cells (ILC2s), Th2 cells, mast cells, eosinophils and natural killer (NK) cells. Cnidii Fructus is a Chinese herb with a long history of use in the treatment of asthma in China. Osthole is one of the major components of Cnidii Fructus. The present study examined the anti­asthmatic effects of osthole in mice and aimed to elucidate the underlying mechanisms involving the IL­33/ST2 pathway. BALB/c mice were sensitized and challenged with ovalbumin and then treated with an intraperitoneal injection of osthole (25 and 50 mg/kg). Subsequently, the airway hyper­responsiveness (AHR) and inflammation of the lungs were evaluated. The amounts of IL­4, IL­5, IL­13, interferon (IFN)­Î³ and IL­33 in the bronchoalveolar lavage fluid (BALF) were measured by Luminex assay and their mRNA levels in the lungs were measured by reverse transcription­quantitative PCR. The histopathology of the lungs was performed with H&E, PAS and Masson's staining. The expression of ST2 in the lungs was evaluated by immunohistochemistry. The data demonstrated that osthole markedly reduced AHR and decreased the number of eosinophils and lymphocytes in BALF. It was also observed that osthole significantly inhibited the release of Th2­type cytokines (IL­4, IL­5 and IL­13) and upregulated the IFN­Î³ level in BALF. Moreover, osthole significantly attenuated the IL­33 and ST2 expression in the lungs of asthmatic mice. On the whole, osthole attenuated ovalbumin­induced lung inflammation through the inhibition of IL­33/ST2 signaling in an asthmatic mouse model. These results suggest that osthole is a promising target for the development of an asthma medication.

High-dose intranasal application of titanium dioxide nanoparticles induces the systemic uptakes and allergic airway inflammation in asthmatic mice.

BACKGROUND: Titanium dioxide nanoparticles (TiO2 NPs) have a wide range of applications in several industrial and biomedical domains. Based on the evidence, the workers exposed to inhaled nanosized TiO2 powder are more susceptible to the risks of developing respiratory diseases. Accordingly, this issue has increasingly attracted the researchers' interest in understanding the consequences of TiO2 NPs exposure. Regarding this, the present study was conducted to analyze the local effects of TiO2 NPs on allergic airway inflammation and their uptake in a mouse model of ovalbumin (OVA)-induced allergic airway inflammation. METHODS: For the purpose of the study, female BALB/c mice with or without asthma were intranasally administered with TiO2 NPs. The mice were subjected to histological assessment, lung function testing, scanning electron microscopy (SEM), inductively coupled plasma mass spectrometry (ICP-MS), and NP uptake measurement. In addition, T helper (Th) 1/Th2 cytokines were evaluated in the lung homogenate using the enzyme-linked immunosorbent assay. RESULTS: According to the results, the mice receiving OVA alone or OVA plus TiO2 NPs showed eosinophilic infiltrates and mucus overproduction in the lung tissues, compared to the controls. Furthermore, a significant elevation was observed in the circulating Th2 cytokines, including interleukin (IL)-4, IL-5, and IL-13 after NP exposure. The TiO2 NPs were taken up by alveolar macrophages at different time points. As the results of the SEM and ICP-MS indicated, TiO2 NPs were present in most of the organs in both asthmatic and non-asthmatic mice. CONCLUSION: Based on the findings of the current study, intranasally or inhalation exposure to high-dose nanosized TiO2 particles appears to exacerbate the allergic airway inflammation and lead to systemic uptake in extrapulmonary organs. These results indicate the very important need to investigate the upper limit of intranasally or inhalation exposure to nanosized TiO2 particles in occupational and environmental health policy.

5α-dihydrotestosterone abrogates sex bias in asthma like features in the mouse.

Androgen levels inversely correlate with the incidence, susceptibility and severity of asthma. However, whether male sex hormones such as 5α-dihydrotestosterone (DHT) have beneficial effects on asthma symptoms and/or could affect asthma susceptibility have not been investigated. DHT administration to female mice, during the sensitization phase, abrogates the sex bias in bronchial hyperreactivity. This effect correlates with inhibition of leukotriene biosynthesis in the lung. DHT significantly inhibits also other asthma-like features such as airway hyperplasia and mucus production in sensitized female mice. Conversely, DHT does not affect plasma IgE levels as well as CD3+CD4+ IL-4+ cell and IgE+c-Kit+ cell infiltration within the lung but prevents pulmonary mast cell activation. The in vitro study on RBL-2H3 cells confirms that DHT inhibits mast cell degranulation. In conclusion, our data demonstrate that immunomodulatory effects of DHT on mast cell activation prevent the translation of allergen sensitization into clinical manifestation of asthma.

Multiomics of World Trade Center Particulate Matter-induced Persistent Airway Hyperreactivity. Role of Receptor for Advanced Glycation End Products.

Pulmonary disease after World Trade Center particulate matter (WTC-PM) exposure is associated with dyslipidemia and the receptor for advanced glycation end products (RAGE); however, the mechanisms are not well understood. We used a murine model and a multiomics assessment to understand the role of RAGE in the pulmonary long-term effects of a single high-intensity exposure to WTC-PM. After 1 month, WTC-PM-exposed wild-type (WT) mice had airway hyperreactivity, whereas RAGE-deficient (Ager-/-) mice were protected. PM-exposed WT mice also had histologic evidence of airspace disease, whereas Ager-/- mice remained unchanged. Inflammatory mediators such as G-CSF (granulocyte colony-stimulating factor), IP-10 (IFN-γ-induced protein 10), and KC (keratinocyte chemoattractant) were differentially expressed after WTC-PM exposure. WTC-PM induced α-SMA, DIAPH1 (protein diaphanous homolog 1), RAGE, and significant lung collagen deposition in WT compared with Ager-/- mice. Compared with WT mice with PM exposure, relative expression of phosphorylated to total CREB (cAMP response element-binding protein) and JNK (c-Jun N-terminal kinase) was significantly increased in the lung of PM-exposed Ager-/- mice, whereas Akt (protein kinase B) was decreased. Random forests of the refined lung metabolomic profile classified subjects with 92% accuracy; principal component analysis captured 86.7% of the variance in three components and demonstrated prominent subpathway involvement, including known mediators of lung disease such as vitamin B6 metabolites, sphingolipids, fatty acids, and phosphatidylcholines. Treatment with a partial RAGE antagonist, pioglitazone, yielded similar fold-change expression of metabolites (N6-carboxymethyllysine, 1-methylnicotinamide, N1+N8-acetylspermidine, and succinylcarnitine [C4-DC]) between WT and Ager-/- mice exposed to WTC-PM. RAGE can mediate WTC-PM-induced airway hyperreactivity and warrants further investigation.

Insulin Modulates the Immune Cell Phenotype in Pulmonary Allergic Inflammation and Increases Pulmonary Resistance in Diabetic Mice.

Introduction: Reports have shown that the onset of diabetes mellitus (DM) in patients previously diagnosed with asthma decreases asthmatic symptoms, whereas insulin aggravates asthma. The present study evaluated the modulatory effect of insulin on the development of allergic airway inflammation in diabetic mice. Materials and Methods: To evaluate the effects of relative insulin deficiency, an experimental model of diabetes was induced by a single dose of alloxan (50 mg/kg, i.v.). After 10 days, the mice were sensitized with ovalbumin [OVA, 20 µg and 2 mg of Al(OH)3, i.p.]. A booster immunization was performed 6 days after the first sensitization [20 µg of OVA and 2 mg of Al(OH)3, i.p.]. The OVA challenge (1 mg/mL) was performed by daily nebulization for 7 days. Diabetic animals were treated with multiple doses of neutral protamine Hagedorn (NPH) before each challenge with OVA. The following parameters were measured 24 h after the last challenge: (a) the levels of p38 MAP kinase, ERK 1/2 MAP kinases, JNK, STAT 3, and STAT 6 in lung homogenates; (b) the serum profiles of immunoglobulins IgE and IgG1; (c) the concentrations of cytokines (IL-4, IL-5, IL-10, IL-13, TNF-α, VEGF, TGF-ß, and IFN-γ) in lung homogenates; (d) cells recovered from the bronchoalveolar lavage fluid (BALF); (e) the profiles of immune cells in the bone marrow, lung, thymus, and spleen; and (f) pulmonary mechanics using invasive (FlexiVent) and non-invasive (BUXCO) methods. Results: Compared to non-diabetic OVA-challenged mice, OVA-challenged diabetic animals showed decreases in ERK 1 (2-fold), ERK 2 (7-fold), JNK (phosphor-54) (3-fold), JNK/SAPK (9-fold), STAT3 (4-fold), the levels of immunoglobulins, including IgE (1-fold) and IgG1 (3-fold), cytokines, including Th2 profile cytokines such as IL-4 (2-fold), IL-5 (2-fold), IL-13 (4-fold), TNF-α (2-fold), VEGF (2-fold), and TGF-ß (2-fold), inflammatory infiltrates (14-fold), T cells, NK cells, B cells and eosinophils in the bone marrow, lung, thymus and spleen, and airway hyperreactivity. STAT6 was absent, and no eosinophilia was observed in BALF. Insulin treatment restored all parameters. Conclusion: The data suggested that insulin modulates immune cell phenotypes and bronchial hyperresponsiveness in the development of allergic airway inflammation in diabetic mice.

HDAC8 inhibitor attenuates airway responses to antigen stimulus through synchronously suppressing galectin-3 expression and reducing macrophage-2 polarization.

BACKGROUND: This study was to investigate of the mechanism by which histone deacetylase (HDAC) 8 inhibitor ameliorated airway hyperresponsiveness (AHR) and allergic airway inflammation. METHODS: Mice were sensitized and then treated with budesonide (BUD) or PCI-34051 (PCI) prior to exposing to normal saline (NS) or ovalbumin (OVA). The raw264.7 cells were treated with interleukin (IL)-4 and PCI or shRNA alone. Repetitive measurements of enhanced pause (Penh) were executed by increasing concentrations of acetyl-ß-methacholine chloride (0 - 50 mg/ml). Cells in bronchoalveolar lavage fluid (BALF) and pathological changes of lungs were examined, respectively. The expression levels of HDAC8, Galecitn (Gal)-3, CD68, CD86, CD163, Arg1 and NOS2 in lungs were measured. Co-regulation of HDAC8 and Gal-3 proteins was observed by immunofluorescence staining and co-immunoprecipitation assay (Co-IP). RESULTS: Significant increases in Penh and IL-4 level were detected with a large inflammatory infiltrate, comprised predominantly of macrophages and eosinophils, into the BALF in OVA-exposed lungs. HDAC8, Gal-3, CD68, CD86, CD163, Arg1 and NOS2 proteins were over-expressed with the significant changes in the Arg1 and NOS2 mRNA levels in the lungs and the IL-4-treated cells. PCI intervention obviously reduced the counts of CD163+ cells. Furthermore, Gal-3 knockdown suppressed Arg1 expression in the cells. Immunofluorescence staining displayed simultaneous changes in HDAC8 and Gal-3 expression in the investigated samples. Treatment with PCI resulted in synchronous reduction of HDAC8 and Gal-3 expression in the Co-IP complexes. CONCLUSIONS: The HDAC8 inhibitor ameliorates AHR and airway inflammation in animal model of allergic asthma through reducing HDAC8-Gal-3 interaction and M2 macrophage polarization.

Lipoxin A4 reduces house dust mite and TNFα-induced hyperreactivity in the mouse trachea.

Lipoxin A4 (LXA4) is considered a specialised pro-resolving mediator that decreases inflammation: however, pro-inflammatory effects have been described in the airways. Here, we investigated whether LXA4 could influence airway hyperreactivity induced in mouse trachea by house dust mite extract (HDM) or TNFα. Intranasal instillation of HDM caused a serotonin (5-HT) mediated airway hyperreactivity ex vivo (Emax: 78.1 ± 16.2 % versus control 12.8 ± 1.0 %) that was reduced by LXA4 installation one hour prior to HDM (Emax: 49.9 ± 11.4 %). Also, in isolated tracheal segments cultured for four days, HDM induced a hyperreactivity (Emax: 33.2 ± 3.1 % versus control 9.0 ± 0.7 %) that was decreased by LXA4 (Emax: 18.7 ± 1.5 %). One part of the HDM-induced hyperreactivity could be inhibited by the TNFα-inhibitor etanercept. TNFα-induced upregulation of 5-HT responses (Emax: 51.3 ± 1.2 % versus control 13.9 ± 0.5 %) was decreased by 10-1000 nM LXA4. In precontracted tracheal segments, LXA4 had no relaxing effect. Overall, LXA4 was able to decrease airway hyperreactivity induced by both HDM and TNFα, thus having a sub-acute anti-inflammatory effect in airway inflammation.

TLR5 Activation Exacerbates Airway Inflammation in Asthma.

INTRODUCTION: Innate immune activation through exposure to indoor and outdoor pollutants is emerging as an important determinant of asthma severity. For example, household levels of the bacterial product lipopolysaccharide (LPS) are associated with increased asthma severity. We hypothesized that activation of the innate immune receptor TLR5 by its bacterial ligand flagellin will exacerbate airway inflammation and asthma symptoms. METHODS: We determined the effect of flagellin co-exposure with ovalbumin in a murine model of allergic asthma. We evaluated the presence of flagellin activity in house dust of asthma patients. Finally, we analyzed the association of a dominant-negative polymorphism in TLR5 (rs5744168) with asthma symptoms in patients with asthma. RESULTS: We showed that bacterial flagellin can be found in the house dust of patients with asthma and that this bacterial product exacerbates allergic airway inflammation in an allergen-specific mouse model of asthma. Furthermore, a dominant-negative genetic polymorphism in TLR5, the receptor for flagellin, is associated with decreased symptoms in patients with asthma. CONCLUSION: Together, our results reveal a novel genetic protective factor (TLR5 deficiency) and a novel environmental pollutant (microbial flagellin) that influence asthma severity. (Clinical trials NCT01688986 and NCT01087307).

Comparison of asthma phenotypes in OVA-induced mice challenged via inhaled and intranasal routes.

BACKGROUND: The respiratory system is exposed to various allergens via inhaled and intranasal routes. Murine models of allergic lung disease have been developed to clarify the mechanisms underlying inflammatory responses and evaluate the efficacy of novel therapeutics. However, there have been no comparative studies on differences in allergic phenotypes following inhaled vs. intranasal allergen challenge. In this study, we compared the asthmatic features of mice challenged via different routes following allergen sensitization and investigated the underlying mechanisms. METHODS: To establish ovalbumin (OVA)-induced allergic asthma models, BALB/c mice were sensitized to 20 µg OVA with 1 mg aluminum hydroxide by the intraperitoneal route and then challenged by inhalation or intranasal administration with 5% OVA for 3 consecutive days. Cellular changes and immunoglobulin (Ig) E levels in bronchoalveolar lavage fluid (BALF) and serum, respectively, were assessed. Histological changes in the lungs were examined by hematoxylin and eosin (H&E) and periodic acid Schiff (PAS) staining. Levels of T helper (Th)2 cytokines including interleukin (IL)-4, -5, and -13 in BALF and epithelial cytokines including IL-25 and -33 in BALF and lung tissues were measured by enzyme-linked immunosorbent assay and western blotting. Airway hyperresponsiveness (AHR) was evaluated by assessing airway resistance (Rrs) and elastance (E) via an invasive method. RESULTS: OVA-sensitized and challenged mice showed typical asthma features such as airway inflammation, elevated IgE level, and AHR regardless of the challenge route. However, H&E staining showed that inflammation of pulmonary vessels, alveolar ducts, and alveoli were enhanced by inhaled as compared to intranasal OVA challenge. PAS staining showed that intranasal OVA challenge induced severe mucus production accompanied by inflammation in bronchial regions. In addition, Th2 cytokine levels in BALF and AHR in lung were increased to a greater extent by inhalation than by intranasal administration of OVA. Epithelial cytokine expression, especially IL-25, was increased in the lungs of mice in the inhaled OVA challenge group. CONCLUSION: OVA-sensitized mice exhibit different pathophysiological patterns of asthma including expression of epithelial cell-derived cytokines depending on the OVA challenge route. Thus, some heterogeneous phenotypes of human asthma can be replicated by varying the mode of delivery after OVA sensitization.