Measurements Of Airway Responsiveness Research Articles

Nebulized Mycobacterium vaccae protects against asthma by attenuating the imbalance of IRF4/IRF8 expression in dendritic cells

Objective: To assess the effects of nebulized inhaled Mycobacterium vaccae on allergic airway inflammation, airway hyperresponsiveness, and Th1/Th2 cell imbalance in mice with ovalbumin (OVA)-induced asthma. Methods: Mice received OVA sensitization and challenge for establishment of the asthmatic model. For intervention, mice received Mycobacterium vaccae nebulization once every other day from the first day of sensitization to the day before challenge. After challenge, pulmonary histological analysis and airway responsiveness measurement were performed. In addition, Th1/Th2 cytokines and OVA-specific IgE levels in bronchoalveolar lavage fluid were measured by ELISA. Th1/Th2 subset ratios and the expression of interferon-regulatory factor 4 (IRF4), IRF8 and Toll-like receptor 4 (TLR4) in dendritic cells were evaluated by flow cytometry. Results: Severe inflammatory infiltration and airway hyperresponsiveness were observed in OVA-induced asthmatic mice. Asthmatic mice showed higher Th2 cytokine concentration and increased percentage of Th2 cells, along with lower Th1 cytokine concentration and reduced percentage of Th1 cells compared with the normal control. Moreover, an imbalance of IRF4+ and IRF8+ in dendritic cells was found in asthmatic mice. Nebulized inhaled Mycobacterium vaccae reduced airway hyperresponsiveness and inflammation in OVA-induced asthmatic mice. In addition, nebulized inhaled Mycobacterium vaccae enhanced TLR4 and IRF8 expression, and alleviated the imbalance of Th1/Th2 as well as IRF4+ and IRF8+ in dendritic cells. Conclusions: Nebulized inhaled Mycobacterium vaccae protects against asthma by alleviating the imbalance of Th1/Th2 and IRF4/ IRF8 in OVA-induced asthmatic mice.

The Evaluation of Acute Lung Injury Induced by Nitrogen Mustard in Mouse Precision Cut Lung Slices

Nitrogen Mustard (NM; mechlorethamine hydrochloride) is a cytotoxic alkylating agent known to cause progressive pulmonary injury. Its toxic properties elicit extensive tissue remodeling within the respiratory tract, contributing to the development of chronic lung disease. Precision-Cut Lung Slices (PCLS) represent an ex vivo alternative to in vivo studies, minimizing animal numbers while increasing experimental replicates, which is advantageous for mechanistic toxicology studies and evaluating potential therapeutics. PCLS are three-dimensional excerpts of the lung that include all resident cell types; they remain viable in culture for up to 14 days, allowing for evaluation of functional activity, structural alterations and cytotoxicity. In the present studies, PCLS were used to assess NM-induced lung injury and alterations in pulmonary mechanics. C57BL/6 mice were euthanized, the lungs filled with agarose, isolated after congealment, and sliced (300 mM in thickness); slices were cultured in Dulbecco's Modified Eagle's Medium (DMEM) and Ham's F-12 Nutrient Mixture (DMEM/F-12). Twenty-four hours later, PCLS were treated with NM (1, 10, and 100 mM) for one hour, washed, refed with medium and cultured for 3 days. NM caused a dose-related increase in cytotoxicity and a decrease in mitochondrial activity as measured by lactate dehydrogenase (LDH) and WST-1 assays, respectively. Analysis of histologic sections revealed that NM caused damage to the airway epithelium; this was associated with decreased mRNA expression of collagen1A1 and collagen3A1, markers of tissue repair. To determine if these changes were linked to altered pulmonary function, we measured airway responsiveness using video microscopy. In these studies, changes in airway area after stimulation with increasing concentrations of methacholine were assessed in cross-sectional airways. Consistent with structural alterations, NM caused a ~30% decrease in maximal airway contraction after methacholine, relative to control, indicating a loss in airway smooth muscle function. These data demonstrate that PCLS can be used effectively to mechanistically assess the progression of injury induced by NM. Further understanding of the progression of damage and repair processes following exposure to mustards is essential in order to develop therapeutics for treating pulmonary toxicity.

Effect of San'ao decoction with scorpio and bombyx batryticatus on CVA mice model via airway inflammation and regulation of TRPA1/TRPV1/TRPV5 channels

Ethnopharmacological relevanceCough variant asthma (CVA) is characterized with its long-lasting cough symptom on clinic. The mechanism of CVA is related to chronic persistent airway inflammation, airway hyperresponsiveness, etc. The traditional Chinese prescription has achieved good curative effect on CVA treatment through reducing cough counts, decreasing airway hyperresponsiveness and alleviating airway inflammation. The mechanism is associated with reducing IL4, IL-13, NGF and CGRP levels, as well as down-regulating TRPA1/TRPV1/TRPV5 channels in both lung and brain tissues. Aim of the studyThe Chinese prescription, San'ao decoction with scorpio and bombyx batryticatus (SSB), is well known in treating cough in asthmatic patients. In this study, the anti-tussive and anti-asthmatic role of SSB, as well as its mechanism on CVA mice model were explored and evaluated via alleviating airway inflammation and regulation of TRP channels. Materials and methodsThe major chemical components in SSB were detected and analyzed by UPLC-QTOF-MS under an optimized chromatographic and MS condition. 60 BALB/c mice were randomly divided into six groups: normal group, model group, dexamethasone group (0.1178 mg/kg/d), SSB high dose group (9.74 g/kg/d), SSB middle dose group (4.87 g/kg/d) and SSB low dose group (2.435 g/kg/d). The cough variant asthma mice model was established by ovalbumin sensitization and challenge. The protective role of SSB on CVA mice model was studied through inducing cough counts by capsaicin, assessing inflammatory cells in peripheral blood and bronchoalveolar lavage fluid (BALF), measuring airway responsiveness, detecting histopathological changes in lung tissues, analyzing cytokines and neuropeptides levels in BALF, as well as examining the mRNA and protein expressions of TRPA1, TRPV1 and TRPV5 in both lung and brain tissues. Results17 signal peaks of the chemical components in SSB were identified by using UPLC-QTOF-MS. SSB (especially the high dose and middle dose), showed significantly effects on mice model by reducing mice cough counts (P ＜ 0.01), decreasing eosinophil (EOS) counts in blood (P ＜ 0.01) and inflammatory cell numbers in BALF (P ＜ 0.01), decreasing airway hyperresponsiveness (P ＜ 0.05), reducing the levels of IL-4 (P ＜ 0.05), IL-13 (P ＜ 0.01), NGF (P ＜ 0.01) and CGRP (P ＜ 0.01) in BALF, as well as down regulating the mRNA and protein expressions of TRPA1, TRPV1 and TRPV5 in both lung and brain tissues (P ＜ 0.01). ConclusionsSSB showed anti-tussive and anti-asthmatic effects on cough variant asthma mice model by reducing cough counts, improving lung function, alleviating lung injury and airway inflammation. The mechanism of SSB might be associated with the regulation of cytokines and neuropeptides in BALF, as well as the regulation of TRPA1, TRPV1, TRPV5 channels in both lung and brain tissues.

Divergent Roles of miR-3162-3p in Pulmonary Inflammation in Normal and Asthmatic Mice as well as Antagonism of miR-3162-3p in Asthma Treatment

MicroRNA (miRNA) mimics or antagomirs hold great promise for asthma treatment compared with glucocorticoids as mainstay therapy for asthma. But the role of miRNA in regulating asthmatic inflammation is largely unclear. We previously reported that miR-3162-3p in the peripheral blood of children with asthma was obviously upregulated compared to that in healthy children. This study aimed to elucidate the role of miR-3162-3p in pulmonary inflammation in normal and asthmatic mice as well as preliminarily explore the potential of miR-3162-3p antagomir in asthma treatment. A noninvasive whole-body plethysmograph measured airway responsiveness. Both qRT-PCR and Western blot were used to detect the expression of miRNA, mRNA, or protein. Cells in bronchoalveolar lavage fluid were counted by platelet counting and Wright’s staining. Inflammatory infiltration and mucus secretion were identified by hematoxylin and eosin and periodic acid-Schiff staining, respectively. Cytokines in the lungs were detected by ELISA. The miR-3162-3p mimic intraperitoneally administered to normal mice decreased β-catenin levels in the lungs without obviously altering the lung histology and cytokine levels. Antagonizing miR-3162-3p in ovalbumin-induced asthmatic mice effectively alleviated the typical features of asthma, such as airway hyper­responsiveness, airway inflammation, and Th1/Th2 cytokine imbalance, and concomitantly rescued the total and active β-catenin expression. Collectively, we discovered divergent roles of miR-3162-3p in lung inflammation between normal and asthmatic mice. The anti-inflammatory effects of the miR-3162-3p antagomir were comparable to those of glucocorticoid treatment. Our study helped in understanding the contribution of miRNAs to the pathogenesis of asthma.

Edaravone attenuates experimental asthma in mice through induction of HO-1 and the Keap1/Nrf2 pathway.

Asthma is a chronic disease that threatens public health worldwide. Multiple studies have shown that oxidative stress plays an important role in the pathogenesis of asthma. Edaravone (Eda), a free radical scavenger, has been found to have a protective effect against lung injury due to its ability to eliminate reactive oxygen species. The present study aimed to investigate the effect of Eda on asthma and the mechanism underlying its actions. An experimental asthma model was induced in mice, before they were treated with different doses of Eda. Measurements of airway responsiveness to methacholine (Mch), cell counts and cytokine levels in bronchoalveolar lavage fluid (BALF) and of the oxidative products and antioxidant enzymes in lung tissue were taken in these asthma model mice and compared with control mice. Protein levels of kelch-like ECH-associated protein-1 (Keap1)/nuclear factor erythroid 2-related factor 2 (Nrf2) and hemeoxygenase-1 (HO-1) were determined in the lung tissue of normal mice and Nrf2 and HO-1-deficient mice subject to the asthma model to investigate the mechanisms underlying Eda action. The results indicated that Eda effectively reduced airway responsiveness to Mch. The total number of cells and the numbers of eosinophils, lymphocytes and neutrophils in BALF of asthma model mice were also significantly reduced by Eda treatment when compared with normal saline treatment. Eda treatment significantly alleviated perivascular edema, peribronchial inflammation and macrophage infiltration in the alveolar space and decreased the levels of inflammatory cytokines released in BALF compared with control. Eda also significantly reduced the levels of oxidative stress markers in BALF and restored the levels of antioxidative enzyme, superoxide dismutase, when compared with control. The Keap1/Nrf2 ratio was significantly decreased with Eda compared with control due to an increase in Nrf2 and a decrease in Keap1 expression. HO-1 expression was increased by Eda. The airway responsiveness of Nrf2-/- mice or HO-1-/- mice to Mch was significantly higher compared with normal mice treated with Eda. Taken together, the results of the present study show that Eda exerts anti-inflammatory and antioxidative effects, which suggests a potential use for Eda in reduction of asthma severity. The activated Keap1/Nrf2 pathway and HO-1 may be involved in the anti-asthmatic effect of Eda.

Endomorphin-2- and Neurotensin- Based Chimeric Peptide Attenuates Airway Inflammation in Mouse Model of Nonallergic Asthma.

We examined anti-inflammatory potency of hybrid peptide-PK20, composed of neurotensin (NT) and endomorphin-2 (EM-2) pharmacophores in a murine model of non-atopic asthma induced by skin sensitization with 2,4-dinitrofluorobenzene and intratracheal challenge of cognate hapten. Mice received intraperitoneally PK20, equimolar mixture of its structural elements (MIX), dexamethasone (DEX), or NaCl. Twenty-four hours following hapten challenge, the measurements of airway responsiveness to methacholine were taken. Bronchoalveolar lavage (BALF) and lungs were collected for further analyses. Treatment with PK20, similarly to dexamethasone, reduced infiltration of inflammatory cells, concentration of mouse mast cell protease, IL-1β, IL-12p40, IL-17A, CXCL1, RANTES in lungs and IL-1α, IL-2, IL-13, and TNF-α in BALF. Simple mixture of NT and EM-2 moieties was less potent. PK20, DEX, and MIX significantly decreased malondialdehyde level and secretory phospholipase 2 activity in lungs. Intensity of NF-κB immunoreactivity was diminished only after PK20 and DEX treatments. Neither PK20 nor mixture of its pharmacophores were as effective as DEX in alleviating airway hyperresponsiveness. PK20 effectively inhibited hapten-induced inflammation and mediator and signaling pathways in a manner seen with dexamethasone. Improved anti-inflammatory potency of the hybrid over the mixture of its moieties shows its preponderance and might pose a promising tool in modulating inflammation in asthma.

Androgens augment pulmonary responses to ozone in mice.

Ozone causes airway hyperresponsiveness, a defining feature of asthma, and is an asthma trigger. In mice, ozone‐induced airway hyperresponsiveness is greater in males than in females, suggesting a role for sex hormones in the response to ozone. To examine the role of androgens in these sex differences, we castrated 4‐week‐old mice. Controls underwent sham surgery. At 8 weeks of age, mice were exposed to ozone (2ppm, 3 h) or room air. Twenty‐four hours later, mice were anesthetized and measurements of airway responsiveness to inhaled aerosolized methacholine were made. Mice were then euthanized and bronchoalveolar lavage was performed. Castration attenuated ozone‐induced airway hyperresponsiveness and reduced bronchoalveolar lavage cells. In intact males, flutamide, an androgen receptor inhibitor, had similar effects to castration. Bronchoalveolar lavage concentrations of several cytokines were reduced by either castration or flutamide treatment, but only IL‐1α was reduced by both castration and flutamide. Furthermore, an anti‐IL‐1α antibody reduced bronchoalveolar lavage neutrophils in intact males, although it did not alter ozone‐induced airway hyperresponsiveness. Our data indicate that androgens augment pulmonary responses to ozone and that IL‐1α may contribute to the effects of androgens on ozone‐induced cellular inflammation but not airway hyperresponsiveness.

Emodin Alleviates the Airway Inflammation of Cough Variant Asthma in Mice by Regulating the Notch Pathway.

BackgroundThis study investigated the effects and underlying mechanisms of emodin on cough variant asthma (CVA) in mice.Material/MethodsThe bronchial asthma mouse model was successfully established by use of ovalbumin (OVA) sensitization and challenge. The BALB/c mice were divided into 6 groups: a control group, an OVA model without or with emodin (15, 30, 60 mg/kg) group, and a dexamethasone (0.5 mg/g) group. The effect of the treatment was determined by measuring airway responsiveness. The levels of immunoglobulin molecules, as well as inflammatory cytokines in bronchoalveolar lavage fluid (BALF) and serum, were determined by ELISA. The lung tissues were stained by hematoxylin-eosin (HE). The expressions of Notch receptors (Notch 1–3) and Delta-like (DLL) 4 in the lung tissues were detected by RT-PCR and Western blot analysis.ResultsCompared with the model group, emodin treatment significantly increased the levels of immunoglobulin E (IgE) and IgG1/IgG2a in BALF and serum (p<0.05). HE results indicated that emodin inhibited the infiltration of inflammatory cells and that emodin reduced the levels of inflammatory cytokines, interleukin (IL)-5, IL-17, and interferon (IFN)-γ in BALF and serum (p<0.05). Furthermore, the expressions of Notch 1, 2, 3, and DLL4 in lung tissue were inhibited by emodin treatment.ConclusionsThe results demonstrated that emodin alleviated inflammation in CVA mice, which might be associated with suppression of the Notch pathway. Emodin might be a promising therapeutic agent for allergic asthma.

Diagnostic Value of Astograph Airway Responsiveness Measurement in Chest Suffocation Variant Asthma

Objective To investigate the diagnostic value of Astograph methacholine provocation test in patients with chest tightness variant asthma(CTVA). Methods From January 2011 to February 2017, 156 patients with CTVA in outpatient or inpatient department of respiratory medicine of Kailuan General Hospital affiliated to North China University of Science and Technology were selected as case group (chest tightness variant asthma group). The control group were 361 non-asthmatic patients including interstitial lung disease(23 cases), coronary disease (157 cases), hypertensive cardiopathy(22 cases), myocardiosis (16 cases), congenital heart disease (3 cases), rheumatic valvular heart disease (6 cases), central airway disease (3 cases), thyromegaly (10 cases), mediastinal tumor (5 cases), thoracic or spinal deformity (8 cases), phrenoparalysis (2 cases) and vegetative nerve functional disturbance (106 cases). All participants received pulmonay ventilation test, average daily and nightly variation rate of PEF(Peak expiratory flow) or PEF weekly variability, Astograph methacholine provocation test (forced expirataory volume in one second≥70% expectation), and other relevant examinations.The diagnostic value of Astograph methacholine provocation test on CTVA was assessed by analyzing the sensitivity, specificity, positive predictive value, negative predictive value, and Yunden index of Astograph methacholine airway. Results Compared with the control group((1.18±0.44)%), theforced expiratory flow from 75% of Forced vital capcacity (FEF75)index of CTVA group((1.29±0.50)%) had significant difference(, t=2.96, P=0.006). The sensitivity, specificity, positive predictive value, negative predictive value, Yunden index, and diagnostic accuracy of Astograph methacholine provocation test on CTVA were 0.814, 0.695, 0.536, 0.305, 0.509 and 0.731, respectively. Conclusion The sensitivity, negative predictive value, Yunden index and diagnostic accuracy of Astograph methacholine provocation test on CTVA were higher, whereas the specificity and positive predictive value were relatively lower, suggesting that Astograph methacholine provocation test had a reliable diagnostic value on CTVA, with lower false negative and higher false positive. Key words: Astograph method; Chest tightness variant asthma; Diagnosis

Absence of Angiotensin ll type 2 (AT2) Receptors Increases Airway Inflammation and Responsiveness in an Allergic Mouse Model of Asthma

Angiotensin ll (Ang ll) is produced locally in the lungs and may potentiate airway responsiveness in asthmatic patients. Ang ll induces its effects by activating two GPCR subtypes: Ang II receptor type‐1 and type‐2 (AT1 and AT2). This lab has previously found that AT2 receptor agonist plays a significant role in reducing airway inflammation and reactivity (FASEB J April 2018 32: 829.6). For the current study, we characterized the role of AT2 receptors in airway hyperreactivity and inflammation using AT2 receptor knockout (AT2KO; Hein L, Barsh et. al. Nature. 1995;377:744–747) and corresponding wild‐type (WT) mice that were divided into two experimental groups each: control (CON) and allergen sensitized‐challenged (SEN). Mice were sensitized (i.p.) on days 1, 6 with 0.2μg ovalbumin (OVA) followed by 5% OVA aerosol challenges on days 11–13. Whole body plethysmography (measuring airway responsiveness as enhanced pause, Penh) and bronchoalveolar lavage (BAL) studies were performed. Methacholine (MCh; 48mg/ml) produced significantly higher airway responsiveness in AT2KO SEN mice compared to WT SEN (324.7±66% in AT2KO SEN vs 166±56.3%, p&lt;0.05). Total cell count analysis showed increased number of cells in AT2KO SEN (3.3±0.2 × 106 vs. 1.4±5 × 106 in WT SEN, p&lt;0.001). Differential BAL cell analysis showed increased eosinophils (73±8% in AT2KO SEN vs. 50.11±2.84% in WT SEN, p&lt;0.05). Macrophages were lower in AT2KO SEN than WT SEN (15±4% vs. 40±3%; p&lt;0.001). These data indicate that absence of AT2 receptors increases airway inflammation and responsiveness in our model of asthma.Support or Funding InformationInstitutional Research Grant Long Island UniversityThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Kininogen deficiency or depletion reduces enhanced pause independent of pulmonary inflammation in a house dust mite-induced murine asthma model.

High-molecular-weight kininogen is an important substrate of the kallikrein-kinin system. Activation of this system has been associated with aggravation of hallmark features in asthma. We aimed to determine the role of kininogen in enhanced pause (Penh) measurements and lung inflammation in a house dust mite (HDM)-induced murine asthma model. Normal wild-type mice and mice with a genetic deficiency of kininogen were subjected to repeated HDM exposure (sensitization on days 0, 1, and 2; challenge on days 14, 15, 18, and 19) via the airways to induce allergic lung inflammation. Alternatively, kininogen was depleted after HDM sensitization by twice-weekly injections of a specific antisense oligonucleotide (kininogen ASO) starting at day 3. In kininogen-deficient mice HDM induced in Penh was completely prevented. Remarkably, kininogen deficiency did not modify HDM-induced eosinophil/neutrophil influx, T helper 2 responses, mucus production, or lung pathology. kininogen ASO treatment started after HDM sensitization reduced plasma kininogen levels by 75% and reproduced the phenotype of kininogen deficiency: kininogen ASO administration prevented the HDM-induced increase in Penh without influencing leukocyte influx, Th2 responses, mucus production, or lung pathology. This study suggests that kininogen could contribute to HDM-induced rise in Penh independently of allergic lung inflammation. Further research is warranted to confirm these data using invasive measurements of airway responsiveness.

Application of impulse oscillometry in the measurement for childhood bronchial hyperresponsiveness

Objective To explore the effectiveness of impulse oscillometry(IOS)in airway responsiveness measurement and to find out the positive threshold of IOS for asthma diagnosis. Methods Seventy-nine children aged 6-14 years who had suspicious asthma, were recruited into the study.The positive criteria of the methacholine bron-chial provocation test was a 20% reduction in forced expiratory volume in the first second (FEV1)compared to baseline. Simultaneously measured changes in various parameters of IOS, including resonant frequency(Fres), impedance at 5 Hz(Zrs), resistances at 5 and 20 Hz(R5, R20), reactance at 5 Hz(X5), and area of reactance(AX). The results of the challenge test were divided into positive and negative groups according to the pulmonary ventilation function me-thod.The differences between the 2 groups of IOS parameters before and after the challenge test, and the correlation between the change rate of FEV1 and the change rate of IOS parameters were compared, and the positive judgment criteria of IOS parameters in the determination of respiratory responsiveness were determined. Results The positive group of bronchial provocation test had 37 patients and negative group had 42 patients.There was no significant difference in the basic values of parameters between the positive group and the negative group (all P>0.05). Changes in Zrs, R5, X5 of IOS were correlated with changes in FEV1(r=0.374, 0.310, 0.449, all P<0.05). By single factor analysis, the area under the receiver operating characteristic(ROC) carve (AUC) showed: basic value of Zrs increased by 45.85%, R5 increased by 45.72%, X5 increased by 80.74% respectively compared to the baseline showed the optimal combination of sensitivity and specificity.In multivariate Logistic regression models, when Zrs and R5 were combined to measure the airway responsiveness, the sensitivity and specificity were 73.0% and 81.0%, respectively. Conclusions IOS and spirometry can be used to determine airway responsiveness in children during methacholine bronchial challenge.Zrs≥45.85%, or R5≥45.72%, or X5≥80.74%, or Zrs and R5 of multiple regression formula can be used as the positive criteria for young children with airway heperresponsiveness, the combination of Zrs and R5 has higher sensitivity and specificity. Key words: Impulse oscillometry; Child; Airway responsiveness

Intratracheal administration of adipose derived mesenchymal stem cells alleviates chronic asthma in a mouse model

BackgroundAdipose-derived mesenchymal stem cell (ASCs) exerts immunomodulatory roles in asthma. However, the underlying mechanism remains unclear. The present study aimed to explore the effects and mechanisms of ASCs on chronic asthma using an ovalbumin (OVA)-sensitized asthmatic mouse model.MethodsMurine ASCs (mASCs) were isolated from male Balb/c mice and identified by the expression of surface markers using flow cytometry. The OVA-sensitized asthmatic mouse model was established and then animals were treated with the mASCs through intratracheal delivery. The therapy effects were assessed by measuring airway responsiveness, performing immuohistochemical analysis, and examining bronchoalveolar lavage fluid (BALF). Additionally, the expression of inflammatory cytokines and lgE was detected by CHIP and ELISA, respectively. The mRNA levels of serum indices were detected using qRT-PCR.ResultsThe mASCs grew by adherence with fibroblast-like morphology, and showed the positive expression of CD90, CD44, and CD29 as well as the negative expression of CD45 and CD34, indicating that the mASCs were successfully isolated. Administering mASCs to asthmatic model animals through intratracheal delivery reduced airway responsiveness, the number of lymphocytes (P < 0.01) and the expression of lgE (P < 0.01), IL-1β (P < 0.05), IL-4 (P < 0.001), and IL-17F (P < 0.001), as well as increased the serum levels of IL-10 and Foxp3, and the percentage of CD4 + CD25 + Foxp3+ Tregs in the spleen, and reduced the expression of IL-17 (P < 0.05) and RORγ.ConclusionsIntratracheal administration of mASCs alleviated airway inflammation, improved airway remodeling, and relieved airway hyperresponsiveness in an OVA-sensitized asthma model, which might be associated with the restoration of Th1/Th2 cell balance by mASCs.

Subcutaneous immunotherapy suppresses Th2 inflammation and induces neutralizing antibodies, but sublingual immunotherapy suppresses airway hyperresponsiveness in grass pollen mouse models for allergic asthma.

Both subcutaneous and sublingual allergen immunotherapy (SCIT and SLIT) have been shown to effectively suppress allergic manifestations upon allergen exposure, providing long-term relief from symptoms in allergic disorders including allergic asthma. Clinical studies directly comparing SCIT and SLIT report a different kinetics and magnitude of immunological changes induced during treatment. Comparative studies into the mechanisms underlying immune suppression in SCIT and SLIT are lacking. We aimed to establish an experimental model for grass pollen (GP) SCIT and SLIT that would allow a head-to-head comparison of the two treatments. BALB/c mice were sensitized with GP extract, followed by SCIT and SLIT treatments with various GP dosages. Subsequently, we challenged mice with GP and measured airway responsiveness (AHR), GP-specific immunoglobulins, ear swelling tests (EST), eosinophilic inflammation in bronchoalveolar lavage fluid (BALF), and T cell cytokine release after restimulation of lung cells (IL-5, IL-10, and IL-13). We find that SLIT treatment was able to suppress allergen-induced AHR, while allergic inflammation was not effectively suppressed even at the highest GP dose in this model. In contrast, SCIT treatment induced higher levels of GP-specific IgG1, while SLIT was superior in inducing a GP-specific IgG2a response, which was associated with increased Th1 activity in lung tissue after SLIT, but not SCIT treatment. Interestingly, SCIT was able to suppress Th2-type cytokine production in lung cell suspensions, while SLIT failed to do so. In conclusion, GP-SCIT suppresses Th2 inflammation and induced neutralizing antibodies, while GP-SLIT suppresses the clinically relevant lung function parameters in an asthma mouse model, indicating that the two application routes depend on partially divergent mechanisms of tolerance induction. Interestingly, these data mirror observations in clinical studies, underscoring the translational value of these mouse models.

Limonene‐induced Activation of A2A Adenosine Receptors Reduces Airway Inflammation and Reactivity in a Mouse Model of Asthma

Animal models of asthma have shown that limonene (terpene in citrus fruits) reduces inflammation and airway reactivity. However, the mechanism of these effects is unknown. We hypothesized that limonene activates A2A adenosine receptors (A2AAR) to produce its effects in asthma, after comparing Glide scores for limonene (−5.19) and A2AAR agonist CGS 21680 (−8.43). We investigated the effects of limonene on lung inflammation and airway responsiveness to methacholine (MCh) as well NECA (nonselective adenosine analog) in A2AAR knock‐out (A2AKO) and wild‐type (WT) mice. Mice were divided into control (CON) and allergen sensitized‐challenged (SEN) groups, and were sensitized i.p. on days 1, 6 with 20 μg ovalbumin (OVA) followed by 5% OVA aerosol challenges on days 11–13 (n=6 mice per group). Limonene was administered as an inhalation prior to allergen challenges in one group of allergic mice for both WT and KO (SEN+LIM). Whole body plethysmography (measuring airway responsiveness as enhanced pause, Penh) and bronchoalveolar lavage (BAL) studies were performed. Airway responsiveness to MCh (48 mg/ml) in WT SEN group was significantly lowered with limonene treatment (420±92.57% in SEN vs. 56.23±25% in SEN+LIM). However, limonene did not have an effect in A2AKO (553±85% in SEN vs. 588±34% in SEN+LIM). NECA‐induced increased airway responsiveness was attenuated in limonene‐treated WT SEN mice (1002±161 in SEN vs. 250±226%, p&lt;0.05) while no effect was observed in A2AKO groups (541±44% in SEN vs. 528±147% in SEN+LIM). Differential BAL cell analysis showed that limonene reduced levels of eosinophils in WT SEN (70.66±2.6% in SEN vs. 28.66±1.45% in SEN+LIM, p&lt;0.05) while no difference was observed in A2AKO (76.33±1.35% in SEN vs 79±0.69% in SEN+LIM). These data suggest that limonene‐induced reduction in airway inflammation and airway reactivity occurs via activation of A2AAR.Support or Funding InformationLong Island University start‐up funding (DSP)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effect of Omega‐3 Fatty Acid Supplementation on Resolvin (RvE1)‐mediated Suppression of Inflammation in a Mouse Model of Asthma

ResolvinE1(RvE1), an endogenous lipid mediator derived from eicosapentaenoic acid (omega 3 fatty acids), contributes to the resolution of an allergic inflammatory response. We investigated the effects of RvE1 and Omega 3 fatty acids in airway reactivity and inflammation using an asthmatic murine model. Mice were divided into control (CON) and allergen sensitized‐challenged (SEN) groups, and were sensitized i.p. on days 1, 6 with 20μg ovalbumin (OVA) followed by 5% OVA aerosol challenges on days 11–13. RvE1 was administered intraperitoneally post‐allergen challenge while fish oil was administered via oral gavage once daily (day 1 through 13). Whole body plethysmography (measuring airway responsiveness as enhanced pause, Penh) and bronchoalveolar lavage (BAL) studies were performed. RvE1attenuated airway responsiveness to methacholine (MCh; 48mg/ml) in treated mice (150±27.88% in SEN vs. 54±7.52% in SEN+RvE1, p&lt;0.05). However, no difference was observed with either omega‐3 supplementation alone (115±19.28%in SEN+omega‐3) or additive effect of omega‐3 on RvE1 treatment (39±12.37% in SEN+RvE1+omega‐3 vs. 54±7.52% in SEN+RvE1). Differential BAL cell analysis showed that post‐treatment with RVE1 decreased eosinophils (40 ± 2.7% in SEN vs.10.2 ± 1.4% in SEN+RvE1, p&lt;0.005). Omega‐3 treated SEN showed no difference (45 ± 3%) while SEN+RvE1+omega‐3 group had similar results as RvE1 treated mice (10.2 ± 1.4% in SEN+RvE1 vs.10.6 ± 1.4% in SEN+RvE1+omega‐3), suggesting that RvE1 only attenuated the eosinophilia. Our data suggests that omega‐3 supplementation has little effect on airway inflammation and reactivity. Further, omega‐3 fatty acid, although a precursor for resolvin formation, did not have additive effects on resolvin‐mediated decreases in airway inflammation and airway reactivity.Support or Funding InformationLong Island University start‐up funds (DSP)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Angiotensin ll Type 2 (AT2) Receptors: Novel Target in Asthma

Angiotensin ll (Ang ll) is produced locally in the lungs and may potentiate airway responsiveness in asthmatic patients. Ang ll induces its effects by activating two GPCR subtypes: AT1 and AT2. This lab has previously found that AT1 antagonist losartan reduces airway inflammation and reactivity. The AT2 receptors counterbalance the effects of AT1 and are known to be anti‐inflammatory. However, it is not known if AT2 receptors have any effects in asthma. We studied the effects of AT2 agonist novokinin and antagonist PD 12339 administered in vivo in a mouse model of asthma. Mice were divided into control (non‐allergic; CON) and allergen sensitized‐challenged (SEN) groups, and were sensitized i.p. on days 1, 6 with 0.2μg ovalbumin (OVA) followed by 5% OVA aerosol challenges on days 11–13. Whole body plethysmography (measuring airway responsiveness as enhanced pause, Penh) and bronchoalveolar lavage (BAL) studies were performed. Methacholine (MCh; 48mg/ml) produced significantly higher airway responsiveness in allergic mice compared to control (563.71±40% in SEN vs. 208.79±20.84% in CON, p&lt;0.05). However, novokinin (i.p. 0.3mg/kg) treated SEN mice had significantly attenuated MCh‐indcued airway responsiveness (563.71±40% in SEN vs. 252±11% in SEN+Novokinin, p&lt;0.001). The AT2 antagonist PD 12339 (i.p.5mg/kg) significantly increased MCh airway responsiveness (563.71±40% in SEN vs. 757±30% in SEN+PD 12239, p&lt;0.05). Differential BAL cell analysis showed novokinin also decreased eosinophils (221±21 in SEN vs. 122±18 in SEN+PD 12239, p&lt;0.001), while PD 12239 had no effect on eosinophils (245± 16 cells in SEN+PD 12339). Our data suggests that activation of AT2 receptors may attenuate airway reactivity and inflammation in our model of asthma.Support or Funding InformationLong Island University start‐up funds (DSP)This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A novel method for studying airway hyperresponsiveness in allergic guinea pigs in vivo using the PreciseInhale system for delivery of dry powder aerosols

Inhaled adenosine receptor agonists induce bronchoconstriction and inflammation in asthma and are used as bronchial challenge agents for the diagnosis of asthma and in respiratory drug development. Recently developed dry powder aerosols of adenosine have several advantages over nebulised adenosine 5′-monophosphate (AMP) as bronchial challenge agents. However, reverse translation of this bronchial challenge technique to pre-clinical drug development is limited by the difficulty of administering powder aerosols to animals. The aim of the current study was to develop methods for delivering powder aerosols of adenosine receptor agonists to sensitised guinea pigs (as a model of allergic asthma) and evaluate their effect as challenge agents for the measurement of airway responsiveness. The PreciseInhale system delivered micronised AMP and adenosine powders, with mass median aerodynamic diameters of 1.81 and 3.21 μm and deposition fractions of 31 and 48% in the lungs, respectively. Bronchoconstrictor responses in passively sensitised, anaesthetised, spontaneously breathing guinea pigs were compared to responses to nebulised and intravenously administered AMP and adenosine. AMP- and adenosine-induced bronchoconstriction following all routes of administration with the magnitude of response ranking intravenous > dry powder > nebulisation, probably reflecting differences in exposure to the adenosine agonists delivered by the different routes. In conclusion, the PreciseInhale system delivered AMP and adenosine dry powder aerosols accurately into the lungs, suggesting this method can be used to investigate drug effects on airway responsiveness.

Activation of angiotensin-converting enzyme 2 (ACE2) attenuates allergic airway inflammation in rat asthma model

Angiotensin-I converting enzyme (ACE) is positively correlated to asthma, chronic obstructive pulmonary disease (COPD), acute respiratory distress syndrome (ARDS) and is highly expressed in lungs. ACE2, the counteracting enzyme of ACE, was proven to be protective in pulmonary, cardiovascular diseases. In the present study we checked the effect of ACE2 activation in animal model of asthma. Asthma was induced in male wistar rats by sensitization and challenge with ovalbumin and then treated with ACE2 activator, diminazene aceturate (DIZE) for 2weeks. 48h after last allergen challenge, animals were anesthetized, blood, BALF, femoral bone marrow lavage were collected for leucocyte count; trachea for measuring airway responsiveness to carbachol; lungs and heart were isolated for histological studies and western blotting. In our animal model, the characteristic features of asthma such as altered airway responsiveness to carbachol, eosinophilia and neutrophilia were observed. Western blotting revealed the increased pulmonary expression of ACE1, IL-1β, IL-4, NF-κB, BCL2, p-AKT, p-p38 and decreased expression of ACE2 and IκB. DIZE treatment prevented these alterations. Intraalveolar interstitial thickening, inflammatory cell infiltration, interstitial fibrosis, oxidative stress and right ventricular hypertrophy in asthma control animals were also reversed by DIZE treatment. Activation of ACE2 by DIZE conferred protection against asthma as evident from biochemical, functional, histological and molecular parameters. To the best of our knowledge, we report for the first time that activation of ACE2 by DIZE prevents asthma progression by altering AKT, p38, NF-κB and other inflammatory markers.

Neutrophilic oxidative stress mediates organic dust-induced pulmonary inflammation and airway hyperresponsiveness.

Airway exposure to organic dust (OD) from swine confinement facilities induces airway inflammation dominated by neutrophils and airway hyperresponsiveness (AHR). One important neutrophilic innate defense mechanism is the induction of oxidative stress. Therefore, we hypothesized that neutrophils exacerbate airway dysfunction following OD exposure by increasing oxidant burden. BALB/C mice were given intranasal challenges with OD or PBS (1/day for 3 days). Mice were untreated or treated with a neutrophil-depleting antibody, anti-Ly6G, or the antioxidant dimethylthiourea (DMTU) prior to OD exposure. Twenty-four hours after the final exposure, we measured airway responsiveness in response to methacholine (MCh) and collected bronchoalveolar lavage fluid to assess pulmonary inflammation and total antioxidant capacity. Lung tissue was harvested to examine the effect of OD-induced antioxidant gene expression and the effect of anti-Ly6G or DMTU. OD exposure induced a dose-dependent increase of airway responsiveness, a neutrophilic pulmonary inflammation, and secretion of keratinocyte cytokine. Depletion of neutrophils reduced OD-induced AHR. DMTU prevented pulmonary inflammation involving macrophages and neutrophils. Neutrophil depletion and DMTU were highly effective in preventing OD-induced AHR affecting large, conducting airways and tissue elastance. OD induced an increase in total antioxidant capacity and mRNA levels of NRF-2-dependent antioxidant genes, effects that are prevented by administration of DMTU and neutrophil depletion. We conclude that an increase in oxidative stress and neutrophilia is critical in the induction of OD-induced AHR. Prevention of oxidative stress diminishes neutrophil influx and AHR, suggesting that mechanisms driving OD-induced AHR may be dependent on neutrophil-mediated oxidant pathways.