Hyperresponsiveness In Mice Research Articles

Eosinophils prevent diet-induced airway hyperresponsiveness in mice on a high-fat diet.

Eosinophils contribute to metabolic homeostasis and airway hyperresponsiveness, but their specific role in obesity-related airway hyperresponsiveness remains unclear. To address this, we used transgenic mice that overexpress interleukin-5 (IL-5) in peripheral T cells (+IL-5T) and wild-type controls. On a normal diet, +IL-5T and wild-type mice have similar body weight, body fat, and airway nerve-mediated reflex bronchoconstriction in response to inhaled serotonin. Feeding wild-type mice a 61.6% high-fat diet resulted in significantly increased body weight, body fat, fasting glucose, fasting insulin, and reflex bronchoconstriction induced by serotonin, which was blocked by vagotomy. In contrast, +IL-5T mice on a high-fat diet gained less body weight and fat than wild-type mice on the same diet and did not exhibit potentiation in fasting glucose, fasting insulin, or reflex bronchoconstriction induced by serotonin. Compared with wild-type mice, +IL-5T mice on normal diet had significantly more adipose tissue eosinophils, and this was further increased by high-fat diet. High-fat diet did not increase adipose tissue eosinophils in wild-type mice. Our findings suggest that adipose tissue eosinophils may play a role in regulating body fat, thereby reducing insulin, which is a mediator of obesity-related airway hyperresponsiveness. Thus, our data indicate adipose tissue eosinophils may be an important avenue for research in obesity-related asthma.NEW & NOTEWORTHY This study investigates how eosinophils influence systemic metabolism and airway function in obesity. Known for their immune functions, eosinophils also mitigate obesity-related hyperinsulinemia, reducing airway hyperresponsiveness in obese mice models. The findings suggest potential therapeutic strategies targeting the intricate interplay among neurons, eosinophils, and the endocrine system to alleviate asthma in obesity. This research provides novel insights into the critical neuro-immune-endocrine interactions essential for managing obesity-related asthma.

Unveiling the potent effect of vitamin D: harnessing Nrf2/HO-1 signaling pathways as molecular targets to alleviate urban particulate matter-induced asthma inflammation

BackgroundAsthma is the most common allergic disease characterized by an inflammatory response in the airways. Mechanismly, urban particulate matter (PM) is the most widely air pollutant associated with increased asthma morbidity and airway inflammation. Current research found that vitamin D is an essential vitamin with anti-inflammatory, antioxidant and other medical efficacy. Inadequate or deficient vitamin D often leads to the pathogenesis and stability of asthma. NGF exacerbates airway inflammation in asthma by promoting smooth muscle cell proliferation and inducing the Th2 immune response. Activation of the Nrf2/HO-1 signaling pathway can exert a protective effect on the inflammatory response in bronchial asthma. However, the specific mechanism of this pathway in PM-involved asthmatic airway smooth muscle cells remains unclear.MethodsMice were sensitized and challenged with Ovalbumin (OVA) to establish an asthma model. They were then exposed to either PM, vitamin D or a combination of both, and inflammatory responses were observed. Including, acetylcholine stimulation at different concentrations measured airway hyperresponsiveness in mice. Bronchoalveolar lavage fluid (BALF) and serum were collected for TNF-α, IL-1β, IL-6, and Nerve growth factor (NGF) analysis. Additionally, lung tissues underwent histopathological examination to observe alveolar structure and inflammatory cell infiltration. Specific ELISA kits were utilized to determine the levels of the inflammatory factors TNF-α, IL-1β, IL-6, and Nerve growth factor (NGF). Nrf2/HO-1 signaling pathways were examined by western blot analysis. Meanwhile, we constructed a cell system with low HO-1 expression by lentiviral transfection of airway smooth muscle cells. The changes of Nrf2, HO-1, and NGF were observed after the treatment of OVA, PM, and Vit D were given.ResultsThe in vivo results showed that vitamin D significantly alleviated pathological changes in lung tissue of PM-exposed mice models. Mechanismly, vitamin D decreased substantial inflammatory cell infiltration in lung tissue, as well as the number of inflammatory cells in BALF. Furthermore, vitamin D reduced the heightened inflammatory factors including of TNF-α, IL-1β, IL-6, and NGF caused by PM exposure, and triggered the activity of nucleus Nrf2 and HO-1 in PM-exposed asthmatic mice. Notably, knockdown HO-1 weakens the Vitamin D- mediated inhibition to pollution toxicity in asthma. Importantly, in vitro experiments on OVA-stimulated mice airway smooth muscle cells, the results showed that OVA and PM, respectively, reduced Nrf2/HO-1 and increased NGF’s expression, while vitamin D reversed the process. And in the HO-1 knockdown cell line of Lenti-si-HO-1 ASMCs, OVA and PM reduced Nrf2’s expression, while HO-1 and NGF’s expression were unchanged.ConclusionsThe above results demastrate that vitamin D downregulated the inflammatory response and the expression of NGF by regulating the Nrf2/HO-1 signaling pathways in airway smooth muscle cells, thereby showing potent anti-inflammatory activity in asthma.

Targeted Inhibition of Select Extracellular Signal-regulated Kinases 1 and 2 Functions Mitigates Pathological Features of Asthma in Mice.

ERK1/2 (extracellular signal-regulated kinases 1 and 2) regulate the activity of various transcription factors that contribute to asthma pathogenesis. Although an attractive drug target, broadly inhibiting ERK1/2 is challenging because of unwanted cellular toxicities. We have identified small molecule inhibitors with a benzenesulfonate scaffold that selectively inhibit ERK1/2-mediated activation of AP-1 (activator protein-1). Herein, we describe the findings of targeting ERK1/2-mediated substrate-specific signaling with the small molecule inhibitor SF-3-030 in a murine model of house dust mite (HDM)-induced asthma. In 8- to 10-week-old BALB/c mice, allergic asthma was established by repeated intranasal HDM (25 μg/mouse) instillation for 3 weeks (5 days/week). A subgroup of mice was prophylactically dosed with 10 mg/kg SF-3-030/DMSO intranasally 30 minutes before the HDM challenge. Following the dosing schedule, mice were evaluated for alterations in airway mechanics, inflammation, and markers of airway remodeling. SF-3-030 treatment significantly attenuated HDM-induced elevation of distinct inflammatory cell types and cytokine concentrations in BAL and IgE concentrations in the lungs. Histopathological analysis of lung tissue sections revealed diminished HDM-induced pleocellular peribronchial inflammation, mucus cell metaplasia, collagen accumulation, thickening of airway smooth muscle mass, and expression of markers of cell proliferation (Ki-67 and cyclin D1) in mice treated with SF-3-030. Furthermore, SF-3-030 treatment attenuated HDM-induced airway hyperresponsiveness in mice. Finally, mechanistic studies using transcriptome and proteome analyses suggest inhibition of HDM-induced genes involved in inflammation, cell proliferation, and tissue remodeling by SF-3-030. These preclinical findings demonstrate that function-selective inhibition of ERK1/2 signaling mitigates multiple features of asthma in a murine model.

Azithromycin inhibits mucin secretion, mucous metaplasia, airway inflammation, and airways hyperresponsiveness in mice exposed to house dust mite extract.

Excessive production, secretion, and retention of abnormal mucus is a pathological feature of many obstructive airways diseases including asthma. Azithromycin is an antibiotic that also possesses immunomodulatory and mucoregulatory activities, which may contribute to the clinical effectiveness of azithromycin in asthma. The current study investigated these nonantibiotic activities of azithromycin in mice exposed daily to intranasal house dust mite (HDM) extract for 10 days. HDM-exposed mice exhibited airways hyperresponsiveness to aerosolized methacholine, a pronounced mixed eosinophilic and neutrophilic inflammatory response, increased airway smooth muscle (ASM) thickness, and elevated levels of epithelial mucin staining. Azithromycin (50 mg/kg sc, 2 h before each HDM exposure) attenuated HDM-induced airways hyperresponsiveness to methacholine, airways inflammation (bronchoalveolar lavage eosinophil and neutrophils numbers, and IL-4, IL-5, IL-6, IL-10, IL-12, IL-13, and RANTES levels), and epithelial mucin staining (mucous metaplasia) by at least 50% (compared with HDM-exposed mice, P < 0.05). Isolated tracheal segments of HDM-exposed mice secreted Muc5ac and Muc5b (above baseline levels) in response to exogenous ATP. Moreover, ATP-induced secretion of mucins was attenuated in segments obtained from azithromycin-treated, HDM-exposed mice (P < 0.05). In additional ex vivo studies, ATP-induced secretion of Muc5ac (but not muc5b) from HDM-exposed tracheal segments was inhibited by in vitro exposure to azithromycin. In vitro azithromycin also inhibited ATP-induced secretion of Muc5ac and Muc5b in tracheal segments from IL-13-exposed mice. In summary, azithromycin inhibited ATP-induced mucin secretion and airways inflammation in HDM-exposed mice, both of which are likely to contribute to suppression of airways hyperresponsiveness.

Systematic Characterization of the Group 2 House Dust Mite Allergen in Dermatophagoides microceras.

BackgroundAllergic asthma, a chronic airway inflammatory disease, is a critical public health problem. Indoor house dust mites (HDMs) could cause allergic asthma. The prevalence of sensitization to Dermatophagoides microceras (Der m) was approximately 80% and is related to the immunoglobulin E crossing-reactivity of mites belonging to the same genus, Dermatophagoides pteronyssinus (Der p) and Dermatophagoides farina (Der f). However, studies on Der m are scant.MethodsWe used integrated OMICs approaches to identify and characterize the group 2 mite allergen-like protein in Der m (Der m 2). We established a Der m 2-induced allergic asthma mouse model and treated the mice with a fungal immunomodulatory protein (FIP-fve) isolated from Flammulina veluptipes to evaluate the allergenicity of Der m 2 and the immunomodulatory effects of FIP-fve.ResultsBy performing de novo draft genome assembly and comparative genome analysis, we identified the putative 144-amino acid Der m 2 in silico and further confirmed its existence through liquid chromatography–tandem mass spectrometry. Der m 2 is a lipopolysaccharides (LPS)-binding protein. Thus, we examined the LPS-binding activity of recombinant Der m 2 by performing molecular docking analysis, co-immunoprecipitation (Co-IP), and a pull-down assay. Der m 2 elicited the production of pro-inflammatory cytokines, interleukin (IL)-6, and IL-8 in BEAS-2B cells, a human bronchial epithelial cell line, and induced airway hyperresponsiveness in mice. Furthermore, in mice sensitized with Der m 2, the administration of FIP-fve in either the earlier stage or the late stage, FIP-fve alleviated allergic asthma by moderating airway inflammation and remodeling.Conclusions Der m 2 induced inflammatory responses in cell and mouse models. FIP-fve alleviated inflammation in Der m 2-induced asthma in mice by exerting an immunomodulatory effect.

Measurement of Airway Hyperresponsiveness in Mice.

Asthma has been the most prevalent chronic respiratory disease (Mensah et al. J Allergy Clin Immunol 142:744-748, 2018). To explore pathogenic mechanism or new treatments of asthma, mice have been utilized to model the disease. Eosinophilic airway inflammation, allergen specific-IgE, and airway hyperresponsiveness have been characteristic features of allergic asthma (Drake et al. Pulm Ther 5:103-115, 2019). In mouse models, airway hyperresponsiveness to inhaled broncho-constrictor agents such as methacholine chloride (MCh) has been a key disease marker (Alessandrini et al. Front Immunol 11:575936, 2020). A variety of systems to assess airway reactivity in mice are currently available. Here, three distinct systems are described as these have been used in many publications. In the first system, an invasive system in which mice are anesthetized and intubated followed by mechanical ventilation, lung resistance (R), dynamic compliance (C), and other respiratory parameters with MCh challenge are measured. In the second system, a noninvasive system equipped with a chamber in which mice can move freely and spontaneously breathe, changes in airways with MCh challenge are measured as enhanced pause (Penh) values. In the third system, in vitro airway smooth muscle (ASM) reactivity is monitored in an extracted mouse tracheal duct with a cholinergic agonist challenge or electrical stimulation. Each of these systems has unique features, benefits, or disadvantages.

Free Feeding of CpG-Oligodeoxynucleotide Particles Prophylactically Attenuates Allergic Airway Inflammation and Hyperresponsiveness in Mice.

CpG-oligodeoxynucleotides (CpG-ODNs) constitute an attractive alternative for asthma treatment. However, very little evidence is available from studies on the oral administration of CpG-ODNs in animals. Previously, we developed acid-resistant particles (named ODNcap) as an oral delivery device for ODNs. Here, we showed that free feeding of an ODNcap-containing feed prophylactically attenuates allergic airway inflammation, hyperresponsiveness, and goblet cell hyperplasia in an ovalbumin-induced asthma model. Using transcriptomics-driven approaches, we demonstrated that injury of pulmonary vein cardiomyocytes accompanies allergen inhalation challenge, but is inhibited by ODNcap feeding. We also showed the participation of an airway antimicrobial peptide (Reg3γ) and fecal microbiota in the ODNcap-mediated effects. Collectively, our findings suggest that daily oral ingestion of ODNcap may provide preventive effects on allergic bronchopulmonary insults via regulation of mechanisms involved in the gut-lung connection.

Respiratory syncytial virus nonstructural protein 1 breaks immune tolerance in mice by downregulating Tregs through TSLP-OX40/OX40L-mTOR axis

Respiratory syncytial virus (RSV) infection in early life is associated strongly with the subsequent development and exacerbation of asthma, however, the mechanism is still ambiguous. In this study, we identified that RSV nonstructural protein (NS) 1 plays a critical role. Plasmid-mediated overexpression of NS1 induced significant airway hyperresponsiveness, eosinophilia, and mucus hyperproduction in mice. In the pNS1 group, there were markedly elevated proportions of Th2 and Th17 cells, while Th1 and Foxp3+ regulatory T cells (Tregs) significantly declined compared with the control group. Serum concentrations of interleukin (IL)-4, IL-5, IL-6, IL-17, transforming growth factor-beta, and tumor necrosis factor-alpha increased but levels of interferon-gamma and interleukin-10 declined in pNS1 group. Besides, NS1 caused a significant rise of serum thymic stromal lymphopoietin (TSLP) and OX40L levels, and a neutralizing mAb anti-OX40L was capable of promoting RSV clearance and attenuating the airway allergic inflammation caused by pNS1. Otherwise, OX40L-blocking counteracts the inhibitory effect of pNS1 on Tregs in the spleen. RSV NS1 caused elevated levels of phospho-AKT, phospho-mTOR, and phospho-S6K1, which were partially attenuated by anti-OX40L. Moreover, a specific inhibitor of mTORC1 significantly relieved the inhibition of Foxp3 expression and Tregs differentiation. Together, the data indicate that RSV NS1 protein breaks immune tolerance and induces airway inflammation and hyperresponsiveness in mice. In this process, NS1-stimulated TSLP and OX40L play a major role by inhibiting the induction of Tregs, which is at least partially mediated by modulating AKT-mTOR signaling pathways.

Particulate matter 2.5 triggers airway inflammation and bronchial hyperresponsiveness in mice by activating the SIRT2-p65 pathway.

Exposure to particulate matter 2.5 (PM2.5) potentially triggers airway inflammation by activating nuclear factor-κB (NF-κB). Sirtuin 2 (SIRT2) is a key modulator in inflammation. However, the function and specific mechanisms of SIRT2 in PM2.5-induced airway inflammation are largely understudied. Therefore, this work investigated the mechanisms of SIRT2 in regulating the phosphorylation and acetylation of p65 influenced by PM2.5-induced airway inflammation and bronchial hyperresponsiveness. Results revealed that PM2.5 exposure lowered the expression and activity of SIRT2 in bronchial tissues. Subsequently, SIRT2 impairment promoted the phosphorylation and acetylation of p65 and activated the NF-κB signaling pathway. The activation of p65 triggered airway inflammation, increment of mucus secretion by goblet cells, and acceleration of tracheal stenosis. Meanwhile, p65 phosphorylation and acetylation, airway inflammation, and bronchial hyperresponsiveness were deteriorated in SIRT2 knockout mice exposed to PM2.5. Triptolide (a specific p65 inhibitor) reversed p65 activation and ameliorated PM2.5-induced airway inflammation and bronchial hyperresponsiveness. Our findings provide novel insights into the molecular mechanisms underlying the toxicity of PM2.5 exposure. Triptolide inhibition of p65 phosphorylation and acetylation could be an effective therapeutic approach in averting PM2.5-induced airway inflammation and bronchial hyperresponsiveness.

Lactic Acid Bacteria and Natural Product Complex Ameliorates Ovalbumin-Induced Airway Hyperresponsiveness in Mice.

The incidence of respiratory diseases, such as asthma, has substantially increased in recent times owing to environmental changes, such as air pollution. Induction of a chronic inflammatory response begins with production of biologically active mediators from the airway epithelium, which attracts and recruits inflammatory cells into the lung airway. In our previous study, we confirmed that Lactobacillus casei HY2782 and Bifidobacterium animalis spp. lactis HY8002 could improve lung inflammation in the COPD animal model. In this study, we investigated the effect of the HY2782 complex against airway hyperresponsiveness by using an ovalbumin (OVA)-induced animal model. An orally administered HY2782 complex on OVA-induced allergic asthma in a BALB/c mouse model was used. The present results showed that the HY2782 complex suppressed total immunoglobulin E in serum and bronchoalveolar lavage fluid (BALF). The cytokine production profile in BALF and serum revealed that the HY2782 complex showed reduced levels of Th2 cytokines among immune factors released due to the elevated allergic response. Levels of inflammatory mediators in BALF, MCP-1, MIP-2, and CXCL-9 were decreased by oral administration of the HY2782 complex. Lower numbers of eosinophils and neutrophils in BALF suggested that inflammation was ameliorated by the HY2782 complex. Histological observation of lung sections also showed infiltration of fewer cells. From results, we suggested that the HY2782 complex effectively responds to improvement of the immune response and airway hypersensitivity reaction because of the anti-inflammatory effect of the Pueraria lobata root extract and antioxidant effect of HY2782.

Inhibition of TRPA1 reduces airway inflammation and hyperresponsiveness in mice with allergic rhinitis.

This study was conducted to investigate whether a transient receptor potential ankyrin 1 (TRPA1) antagonist (HC-030031) can reduce airway inflammation and hyperresponsiveness in a murine allergic rhinitis (AR) model. BALB/c mice were sensitized and challenged by ovalbumin (OVA) to induce AR. HC-030031 or vehicle was administrated to mice via intraperitoneal injection prior to OVA challenges. Nose-scratching events, histopathologic alterations of the airways, and bronchial hyperresponsiveness (BHR) were assessed. Differential cells and proinflammatory cytokines in the nasal lavage (NAL) and bronchoalveolar lavage (BAL) fluid were measured. Expressions of TRPA1 in nasal mucosa were examined by immunohistochemistry. TRPA1-expressing vagal neurons were labeled by immunofluorescent staining. HC-030031-treated AR mice had markedly reduced type-2 inflammation in nasal mucosa and ameliorated-nose-scratching events than AR mice received vehicle. HC-030031 treatment also dramatically reduced leucocyte numbers and IL-8 level in the BAL fluid, inhibited lower airway remodeling and fibrosis, and nearly abolished BHR. HC-0300031 treatment significantly inhibited the upregulated number of TRPA1 expressing nasal epithelial cells and TRPA1 expressing sensory neurons, leading to downregulation of SP in both upper and lower airways. Targeting TRPA1 may represent a promising strategy for treating AR and AR-related asthma.

Chitin induces steroid-resistant airway inflammation and airway hyperresponsiveness in mice

BackgroundPrevious reports have shown that pathogen-associated patterns (PAMPs) induce the production of interleukin (IL)-1β in macrophages. Moreover, studies using mouse models also suggest that chitin, which acts as a PAMP, induces adjuvant effects and eosinophilic infiltration in the lung. Thus, we investigated the effects of inhaled chitin in mouse models. MethodsWe developed mouse models of inhaled chitin particle-induced airway inflammation and steroid-resistant ovalbumin (OVA)-induced airway inflammation. Some experimental groups of mice were treated additionally with dexamethasone (DEX). Murine alveolar macrophages (AMs), which were purified from bronchoalveolar lavage (BAL) fluids, were incubated with chitin, and treated with or without DEX. ResultsThe numbers of total cells, AMs, lymphocytes, eosinophils, and neutrophils among BAL-derived cells, as well as the IL-1β levels in BAL fluids and the numbers of IL-1β-positive cells in lung, were significantly increased by chitin stimulation. Airway hyperresponsiveness (AHR) was aggravated in mice of the chitin inflammation model compared to control animals. The production of IL-1β was significantly increased in murine AMs by chitin treatment, but DEX administration did not inhibit this chitin-induced IL-1β production. Furthermore, in mouse models, DEX treatment inhibited the OVA-induced airway inflammation and AHR but not the airway inflammation and AHR induced by chitin or the combination of OVA and chitin. ConclusionsThese results suggest that inhaled chitin induces airway inflammation, AHR, and the production of IL-1β. Furthermore, our findings demonstrate for the first time that inhaled chitin induces steroid-resistant airway inflammation and AHR. Inhaled chitin may contribute to features of steroid-resistant asthma.

Glutathione ethyl ester supplementation prevents airway hyper-responsiveness in mice.

BackgroundOxidative stress plays an important role in the pathogenesis of asthma. Glutathione (GSH) is considered to be one of the most important antioxidants. Our study systematically investigated the effect of the GSH alternative, glutathione ethyl ester (GSH-EE), on airway hyper-responsiveness (AHR) in mice.MethodsSixty-three male specific pathogen-free mice were used. Asthma was induced using a single dose of ovalbumin (OVA). The normal group (n=15) received vehicle only [Al(OH)3 in saline]. Then, 48 mice were divided into two groups, including a control group who received sodium phosphate buffer (pH =7.4), and the GSH-EE group who received 0.1% GSH-EE. AHR was measured 2, 6, and 12 hours after exposure to nebulized OVA (0.01%). The animals were then sacrificed, and lung tissue and the bronchi-alveolar lavage fluid (BALF) were harvested. Factors involved in the antioxidant response to asthma were then measured in these tissues, including thiol content (from GSH and protein), γ-glutamylcysteine synthetase (γ-GCS) activity and expression, and nuclear factor-erythroid-2-related factor (Nrf2) expression.ResultsThe GSH-EE group showed a significant attenuation of AHR (P<0.01) 2 hours after OVA challenge, and significantly enhanced thiol contents by approximately 45% (P<0.05) at 2 and 6 hours after the last OVA challenge, compared to the control group. γ-GCS activity was also higher in the GSH-EE group compared to the control group at different time points (P<0.01). γ-GCSh and Nrf2 protein expression increased in the GSH-EE group and the control group compared with the normal group, but there was no statistically significant difference (P>0.05) between the GSH-EE group and the control group.ConclusionsGSH-EE supplementation can prevent AHR in asthmatic mice during the early stages. It may function by serving as a precursor for GSH biosynthesis and by protecting sulfhydryl groups from oxidation.

IL-33/ST2 signaling modulates Afghanistan particulate matter induced airway hyperresponsiveness in mice

Increased symptoms of asthma-like respiratory illnesses have been reported in soldiers returning from tours of duty in Afghanistan. Inhalation of desert particulate matter (PM) may contribute to this deployment-related lung disease (DRLD), but little is known about disease mechanisms. The IL-33 signaling pathway, including its receptor ST2, has been implicated in the pathogenesis of lung diseases including asthma, but its role in PM-mediated airway dysfunction has not been studied. The goal of this study was to investigate whether IL-33/ST2 signaling contributes to airway dysfunction in preclinical models of lung exposure to Afghanistan PM (APM). Wild-type (WT) and ST2 knockout (KO) mice on the BALB/C background were oropharyngeally instilled with a single dose of saline or 50 μg of APM in saline. Airway hyperresponsiveness (AHR) and inflammation were assessed after 24 h. In WT mice, a single APM exposure induced AHR and neutrophilic inflammation. Unlike the WT mice, ST2 KO mice that lack the receptor for IL-33 did not demonstrate AHR although airway neutrophilic inflammation was comparable to the WT mice. Oropharyngeal delivery of a soluble ST2 decoy receptor in APM-exposed WT mice significantly blocked AHR. Additional data in mouse tracheal epithelial cell and lung macrophage cultures demonstrated a role of APM-induced IL-33/ST2 signaling in suppression of regulator of G protein signaling 2 (RGS2), a gene known to protect against bronchoconstriction. We present for the first time that APM may increase AHR, one of the features of asthma, in part through the IL-33/ST2/RGS2 pathway.

Treatment of Experimental Asthma by Lung Restricted Inhibition of Jak1

Abstract Preclinical and clinical evidence indicates that a subset of asthma is driven by type 2 cytokines such as IL-4 and IL-13 that are dependent on Jak1 for signal transduction. Because many of the asthma-related effects of these cytokines are manifested in the lung, we reasoned that combined targeting of these cytokines by means of an inhaled Jak1 inhibitor with lung restricted exposure may confer therapeutic benefit. In order to test this, we synthesized iJak-381, a novel, inhalable small molecule specifically designed for local Jak1 inhibition in the lung and tested dry powder inhaled delivery of iJak-381 in preclinical PK and efficacy studies. iJak-381 suppressed OVA induced lung inflammation and improved allergen induced airway hyper-responsiveness in mice. In a model driven by human allergens, iJak-381 had a more potent suppressive effect than a systemic corticosteroid on neutrophilic inflammation. While iJak-381 strongly inhibited lung pathology, it did not affect systemic Jak1 activity. Our data suggests that local inhibition of Jak1 in the lung might be effective for treating asthma in the clinic.

Butyrate ameliorates allergic airway inflammation by limiting eosinophil trafficking and survival

Lung eosinophilia is a hallmark of asthma, and eosinophils are believed to play a crucial role in the pathogenesis of allergic inflammatory diseases. Short-chain fatty acids (SCFAs), such as acetate, propionate, and butyrate, are produced in high amounts in the gastrointestinal tract by commensal bacteria and can be absorbed into the bloodstream. Although there is recent evidence that SCFAs are beneficial in allergic asthma models, the effect on eosinophils has remained elusive. The role of SCFAs was investigated in human eosinophil function and a mouse model of allergic asthma. Eosinophils were purified from self-reported allergic or healthy donors. Migration, adhesion to the endothelium, and eosinophil survival were studied invitro. Ca2+ flux, apoptosis, mitochondrial membrane potential, and expression of surface markers were determined by using flow cytometry and in part by using real-time PCR. Allergic airway inflammation was assessed invivo in an ovalbumin-induced asthma model by using invasive spirometry. For the first time, we observed that SCFAs were able to attenuate human eosinophils at several functional levels, including (1) adhesion to the endothelium, (2) migration, and (3)survival. These effects were independent from GPR41 and GPR43 but were accompanied by histone acetylation and mimicked by trichostatin A, a pan-histone deacetylase inhibitor. Invivo butyrate ameliorated allergen-induced airway and lung eosinophilia, reduced type 2 cytokine levels in bronchial fluid, and improved airway hyperresponsiveness in mice. These invitro and invivo findings highlight the importance of SCFAs, especially butyrate as a promising therapeutic agent in allergic inflammatory diseases.

Caloric restriction prevents the development of airway hyperresponsiveness in mice on a high fat diet

We have previously shown that high fat diet (HFD) for 2 weeks increases airway hyperresponsiveness (AHR) to methacholine challenge in C57BL/6J mice in association with an increase in IL-1β levels in lung tissue. We hypothesize that obesity increases AHR via the IL-1β mechanism, which can be prevented by caloric restriction and IL-1β blockade. In this study, we fed C57BL/6J mice for 8 weeks with several hypercaloric diets, including HFD, HFD supplemented with fructose, high trans-fat diet (HTFD) supplemented with fructose, either ad libitum or restricting their food intake to match body weight to the mice on a chow diet (CD). We also assessed the effect of the IL-1β receptor blocker anakinra. All mice showed the same total respiratory resistance at baseline. All obese mice showed higher AHR at 30 mg/ml of methacholine compared to CD and food restricted groups, regardless of the diet. Obese mice showed significant increases in lung IL-1 β mRNA expression, but not the protein, compared to CD and food restricted mice. Anakinra abolished an increase in AHR. We conclude that obesity leads to the airway hyperresponsiveness preventable by caloric restriction and IL-1β blockade.

Arhgef12 drives IL17A-induced airway contractility and airway hyperresponsiveness in mice.

Patients with severe, treatment-refractory asthma are at risk for death from acute exacerbations. The cytokine IL17A has been associated with airway inflammation in severe asthma, and novel therapeutic targets within this pathway are urgently needed. We recently showed that IL17A increases airway contractility by activating the procontractile GTPase RhoA. Here, we explore the therapeutic potential of targeting the RhoA pathway activated by IL17A by inhibiting RhoA guanine nucleotide exchange factors (RhoGEFs), intracellular activators of RhoA. We first used a ribosomal pulldown approach to profile mouse airway smooth muscle by qPCR and identified Arhgef12 as highly expressed among a panel of RhoGEFs. ARHGEF12 was also the most highly expressed RhoGEF in patients with asthma, as found by RNA sequencing. Tracheal rings from Arhgef12-KO mice and WT rings treated with a RhoGEF inhibitor had evidence of decreased contractility and RhoA activation in response to IL17A treatment. In a house dust mite model of allergic sensitization, Arhgef12-KO mice had decreased airway hyperresponsiveness without effects on airway inflammation. Taken together, our results show that Arhgef12 is necessary for IL17A-induced airway contractility and identify a therapeutic target for severe asthma.

High fat diet induces airway hyperresponsiveness in mice

The experiment was conducted to examine the effect of a high fat diet (HFD) on airway hyperresponsiveness (AHR) in mice. Twenty-three adult male C57BL/6 J mice were fed with HFD or regular chow diet for two weeks. The total respiratory resistance was measured by forced oscillation technique at baseline and after methacholine aerosol challenge at 1, 3, 10 and 30 mg/mL. Bronchoalveolar lavage (BAL) was performed. Lipid levels and lipid peroxidation in lung tissue were measured along with gene expression of multiple cytokines. Lungs were digested, and IL-1β secretion by pulmonary macrophages was determined. HFD feeding resulted in 11% higher body weight compared to chow. HFD did not affect respiratory resistance at baseline, but significantly augmented airway responses to methacholine compared to chow diet (40.5 ± 17.7% increase at 30 mg/ml methacholine, p < 0.05). HFD induced a 3.2 ± 0.6 fold increase in IL-1β gene expression (p < 0.001) and a 38 fold increase in IL-1β secretion in the lungs. There was no change in BAL and no change in any other cytokines, lipid levels or lipid peroxidation. Hence, HFD induced AHR in mice prior to the development of significant obesity which was associated with up-regulation of pulmonary IL-1β.

Allergin-1 on mast cells suppresses house dust mite-induced airway hyperresponsiveness in mice.

Although airway hyperresponsiveness (AHR) is a prominent feature of asthma, how it is regulated remains incompletely understood. Allergin-1, an inhibitory immunoglobulin-like receptor containing an immunoreceptor tyrosine-based inhibitory motif (ITIM), is expressed on human and mouse mast cells (MCs) and inhibits high-affinity receptor for IgE (FcεRI)-mediated signaling. Using MC-deficient KitW-sh/W-sh mice and Mas-TRECK mice, which carries a diphtheria toxin (DT)-induced MC deletion system based on il4 enhancer elements, we demonstrate here that MCs are involved in the induction of house dust mite (HDM)-induced AHR. Further, we show that MCs deficient in Allergin-1 exacerbated HDM-induced AHR, but had no effect on airway inflammation. In vitro analysis demonstrated that Allergin-1 inhibited anti-HDM allergen antibody-dependent HDM allergen-mediated degranulation by MCs. Thus, Allergin-1 on MCs plays an important role in the regulation of HDM-induced AHR.