Increased Airway Hyperresponsiveness Research Articles

Cryptotanshinone attenuates allergic airway inflammation through negative regulation of NF-κB and p38 MAPK

This study is to determine the role and mechanism of cryptotanshinone (CTS) in allergic airway inflammation. Asthma induced by OVA was established in BALB/c mice. We found increased airway hyperresponsiveness (AHR), increased inflammatory cell infiltration, elevated levels of TNF-α, interleukin-1β (IL-1β), IL-4, IL-5, IL-6 and IL-13, decreased interferon gamma (IFN-γ) in lung tissue, increased content of total immunoglobulin E (IgE), OVA specific IgE, Eotaxin, ICAM-1, VCAM-1, nuclear factor-kappaB (NF-κB) and phosphorylation of p38 MAPK in lung tissue. However, the administration of CTS significantly decreased AHR in asthmatic mice, reduced inflammation around the bronchioles and inflammatory cells around airway, regulated cytokine production, reduced the total IgE and OVA-specific IgE levels, and inhibited NF-κB activation and p38 MAPK phosphorylation. In vitro experiments in 16 HBE cells revealed that CTS attenuated CAM-1 and IL-6 expression. These results indicate that CTS alleviates allergic airway inflammation by modulating p38 MAPK phosphorylation and NF-κB activation.

Efficacy of Optimized Treatment Protocol Using LAU-7b Formulation against Ovalbumin (OVA) and House Dust Mite (HDM) -Induced Allergic Asthma in Atopic Hyperresponsive A/J Mice.

To assess the efficacy of the novel clinical formulation of fenretinide (LAU-7b) for the treatment of allergic asthma. To study the association between LAU-7b treatment in allergic asthma and the modulation of very long chain ceramides (VLCC). We used two allergens (OVA and HDM) to induce asthma in mouse models and we established a treatment protocol with LAU-7b. The severity of allergic asthma reaction was quantified by measuring the airway resistance, quantifying lung inflammatory cell infiltration (Haematoxylin and eosin stain) and mucus production (Periodic acid Schiff satin). IgE levels were measured by ELISA. Immunophenotyping of T cells was done using Fluorescence-activated cell sorting (FACS) analysis. The analysis of the specific species of lipids and markers of oxidation was performed using mass spectrometry. Our data demonstrate that 10mg/kg of LAU-7b was able to protect OVA- and HDM-challenged mice against increase in airway hyperresponsiveness, influx of inflammatory cells into the airways, and mucus production without affecting IgE levels. Treatment with LAU-7b significantly increased percentage of regulatory T cells and CD4+ IL-10-producing T cells and significantly decreased percentage of CD4+ IL-4-producing T cells. Our data also demonstrate a strong association between the improvement in the lung physiology and histology parameters and the drug-induced normalization of the aberrant distribution of ceramides in allergic mice. 9days of 10mg/kg of LAU-7b daily treatment protects the mice against allergen-induced asthma and restores VLCC levels in the lungs and plasma.

Assessment of psychiatric co-morbidities in patient of bronchial asthma attending a tertiary medical centre (Multicentric study).

Aims and Objectives:Asthma is a chronic inflammatory condition, which is associated with increase in airway hyper responsiveness that leads to recurrent episodes of wheezing, breathlessness, chest tightness and coughing. Asthma is a very common respiratory illness, in which some of the disease related factors may increases the vulnerability to psychiatric disorders. This study was done to determine the prevalence of psychiatric co-morbidity in patients of bronchial asthma.Methodology:It is an observational study conducted in 110 follow-up patients of bronchial asthma attending respiratory medicine OPD at tertiary care centre in central India. Psychiatric co-morbidities are assessed by pre-designed short-structured questionnaire using Mini international neuropsychiatric interview.Result:Among 110 patients of bronchial asthma 28% had psychiatric co-morbidity mainly depressive episode (59%). A significant association is found between lower socioeconomic status (P = 0.01), duration of of active illness (more than 1 year) (P = 0.001), and age of patient above 60 years (P = 0.001) with psychiatric co-morbidity of asthma patient.Conclusion:Our study shows there is increased prevalence of psychiatric co-morbidities in patients of bronchial asthma, higher than the national average. The predominant psychiatric disorder seen is depressive disorder, so treatment of asthma should be a multidisciplinary approach including medical treatment of asthma and psychiatric evaluation to prevent psychiatric co-morbidity or its early management. This will greatly reduce the morbidity, visits to hospital, expenditure on treatment and thereby having better outcomes in our patients of asthma.

Toll-like Receptor 4 Deficiency Aggravates Airway Hyperresponsiveness and Inflammation by Impairing Neutrophil Apoptosis in a Toluene Diisocyanate-Induced Murine Asthma Model.

PurposeAccumulating evidence has suggested that toll-like receptor 4 (TLR4) is critically involved in the pathogenesis of asthma. The aim of this study was to investigate the role of TLR4 in toluene diisocyanate (TDI)-induced allergic airway inflammation.MethodsTLR4−/− and wild-type (WT) C57BL/10J mice were sensitized and challenged with TDI to generate a TDI-induced asthma model. B-cell lymphoma 2 (Bcl-2) inhibitors, ABT-199 (4 mg/kg) and ABT-737 (4 mg/kg), were intranasally given to TDI-exposed TLR4−/− mice after each challenge.ResultsTDI exposure led to increased airway hyperresponsiveness (AHR), granulocyte flux, bronchial epithelial shedding and extensive submucosal collagen deposition, which were unexpectedly aggravated by TLR4 deficiency. Following TDI challenge, TLR4−/− mice exhibited down-regulated interleukin-17A and increased colony-stimulating factor 3 in bronchoalveolar lavage fluid (BALF), while WT mice did not. In addition, TLR4 deficiency robustly suppressed the expression of NOD-like receptor family pyrin domain containing 3 and NLR family CARD domain containing 4, decreased caspase-1 activity in TDI-exposed mice, but had no effect on the level of high mobility group box 1 in BALF. Flow cytometry revealed that TDI hampered both neutrophil and eosinophil apoptosis, of which neutrophil apoptosis was further inhibited in TDI-exposed TLR4−/− mice, with marked up-regulation of Bcl-2. Moreover, inhibition of Bcl-2 with either ABT-199 or ABT-737 significantly alleviated neutrophil recruitment by promoting apoptosis.ConclusionsThese data indicated that TLR4 deficiency promoted neutrophil infiltration by impairing its apoptosis via up-regulation of Bcl-2, thereby resulting in deteriorated AHR and airway inflammation, which suggests that TLR4 could be a negative regulator of TDI-induced neutrophilic inflammation.

Airway and Esophageal Eosinophils in Children With Severe Uncontrolled Asthma

To examine the frequency of and correlation between esophageal and airway eosinophils in children with severe uncontrolled asthma (SUA).Researchers in the study included children with SUA according to international European Respiratory Society and American Thoracic Society guidelines who were referred for refractory respiratory symptoms to the Aerodigestive Center at Hassenfeld’s Children’s Hospital at New York University Langone Health from April 2015 to June 2017. All children had either poor symptom control despite use of high-dose inhaled corticosteroids with long-acting β-agonists medication or worsening of asthma control during attempts to taper those medications.This was a retrospective review of prospectively collected data on 24 children aged 2 to 16 years old (n = 24, 21 boys). All children had skin prick tests to food and/or aeroallergens. All subjects underwent triple endoscopy (sleep laryngoscopy, bronchoscopy with bronchoalveolar lavage [BAL] and endobronchial biopsy [EBB], and esophagogastroduodenoscopy with esophageal biopsy). Elevated BAL eosinophils were defined as >1% and neutrophils as >3.5%. Eosinophils in EBB were considered elevated if ≥1 per high-power field. Presence of 1 or more eosinophils per high-power field on esophagogastroduodenoscopy with esophageal biopsy was considered elevated. Airway eosinophils were defined as elevated BAL and/or elevated EBB eosinophils. Aerodigestive eosinophils were defined as the presence of elevated airway eosinophils and elevated esophageal eosinophils together.There was no difference at enrollment in gastrointestinal symptoms, previous diagnosis of reflux and allergy, and use of inhaled corticosteroids, long-acting β-agonists, and proton pump inhibitors between children with and without elevated esophageal eosinophils, airway eosinophils, and aerodigestive eosinophils. All patients with eosinophilic esophagitis (>15 eosinophils per high-power field) (n = 3) and 5 of 8 patients with reflux esophagitis (>5 eosinophils per high-power field) had elevated aerodigestive eosinophils, but only eosinophilic esophagitis correlated with gastrointestinal symptoms and food allergies. Elevated airway eosinophils were present in 71% of patients, and elevated esophageal eosinophils were present in 46% of patients. The number of eosinophils correlated between the BAL and EBB, which then correlated with esophageal eosinophils. Laryngomalacia, tracheal stenosis, or tracheomalacia were present in 4 patients. Over half had bacterial bronchitis on the basis of positive BAL culture results. All patients had BAL neutrophilia, which did not correlate with EBB or BAL eosinophils or the BAL culture.Children with SUA frequently have elevated airway and esophageal eosinophils, and there is a correlation between the airway and esophageal eosinophils.Reflux esophagitis has been strongly associated with increased airway hyperreactivity, and this study suggests that reflux esophagitis should be suspected even in asymptomatic severe asthmatics. Limitations include the small sample size and the low cutoff in defining elevated esophageal eosinophils, which could reduce the significance of this measure. Also, this study does not provide any specific implications for pathogenesis or how this would modify treatment of SUA. Collecting a larger sample size and potentially studying other eosinophil or inflammatory biomarkers would help to better characterize the role, if any, of aerodigestive eosinophils in SUA.

Immune Sensing of Aeroallergen-Associated Double-Stranded RNA Triggers an IFN Response and Modulates Type 2 Lung Inflammation

The innate immune sensing of allergens or allergen-associated components regulate the development of type 2 inflammatory responses. However, the underlying molecular basis by which allergens or allergen-associated components are detected by innate immune receptors remains elusive. In this study, we report that the most common aeroallergen, house dust mite (HDM), harbors a dsRNA species (HDM-dsRNA) that can activate TLR3-mediated IFN responses and counteract the development of an uncontrolled type 2 immune response. We demonstrate that the mouse strains defective in the dsRNA-sensing pathways show aggravated type 2 inflammation defined by severe eosinophilia, elevated level of type 2 cytokines, and mucus overproduction in a model of allergic lung inflammation. The inability to sense HDM-dsRNA resulted in significant increases in airway hyperreactivity. We further show that the administration of the purified HDM-dsRNA at a low dose is sufficient to induce an immune response to prevent the onset of a severe type 2 lung inflammation. Collectively, these results unveil a new role for the HDM-dsRNA/TLR3-signaling axis in the modulation of a type 2 lung inflammation in mice.

IL-1RA regulates immunopathogenesis during fungal-associated allergic airway inflammation.

Severe asthma with fungal sensitization (SAFS) defines a subset of human asthmatics with allergy to 1 or more fungal species and difficult-to-control asthma. We have previously reported that human asthmatics sensitized to fungi have worse lung function and a higher degree of atopy, which was associated with higher IL-1 receptor antagonist (IL-1RA) levels in bronchoalveolar lavage fluid. IL-1RA further demonstrated a significant negative association with bronchial hyperresponsiveness to methacholine. Here, we show that IL-1α and IL-1β are elevated in both bronchoalveolar lavage fluid and sputum from human asthmatics sensitized to fungi, implicating an association with IL-1α, IL-1β, or IL-1RA in fungal asthma severity. In an experimental model of fungal-associated allergic airway inflammation, we demonstrate that IL-1R1 signaling promotes type 1 (IFN-γ, CXCL9, CXCL10) and type 17 (IL-17A, IL-22) responses that were associated with neutrophilic inflammation and increased airway hyperreactivity. Each of these were exacerbated in the absence of IL-1RA. Administration of human recombinant IL-1RA (Kineret/anakinra) during fungal-associated allergic airway inflammation improved airway hyperreactivity and lowered type 1 and type 17 responses. Taken together, these data suggest that IL-1R1 signaling contributes to fungal asthma severity via immunopathogenic type 1 and type 17 responses and can be targeted for improving allergic asthma severity.

MiR-511-3p protects against cockroach allergen-induced lung inflammation by antagonizing CCL2.

miR-511-3p, encoded by CD206/Mrc1, was demonstrated to reduce allergic inflammation and promote alternative (M2) macrophage polarization. Here, we sought to elucidate the fundamental mechanism by which miR-511-3p attenuates allergic inflammation and promotes macrophage polarization. Compared with WT mice, the allergen-challenged Mrc1-/- mice showed increased airway hyperresponsiveness (AHR) and inflammation. However, this increased AHR and inflammation were significantly attenuated when these mice were pretransduced with adeno-associated virus-miR-511-3p (AAV-miR-511-3p). Gene expression profiling of macrophages identified Ccl2 as one of the major genes that was highly expressed in M2 macrophages but antagonized by miR-511-3p. The interaction between miR-511-3p and Ccl2 was confirmed by in silico analysis and mRNA-miR pulldown assay. Further evidence for the inhibition of Ccl2 by miR-511-3p was given by reduced levels of Ccl2 in supernatants of miR-511-3p-transduced macrophages and in bronchoalveolar lavage fluids of AAV-miR-511-3p-infected Mrc1-/- mice. Mechanistically, we demonstrated that Ccl2 promotes M1 macrophage polarization by activating RhoA signaling through Ccr2. The interaction between Ccr2 and RhoA was also supported by coimmunoprecipitation assay. Importantly, inhibition of RhoA signaling suppressed cockroach allergen-induced AHR and lung inflammation. These findings suggest a potentially novel mechanism by which miR-511-3p regulates allergic inflammation and macrophage polarization by targeting Ccl2 and its downstream Ccr2/RhoA axis.

Inhibitions of HMGB1 and TLR4 alleviate DINP-induced asthma in mice.

We studied the effects of high mobility group box chromosomal protein 1 (HMGB1) and toll-like receptor (TLR4) in diisonoyl phthalate (DINP)-induced asthma. Mice with DINP-induced asthma were treated with a TLR4-signaling inhibitor or anti-HMGB1 antibody, and various markers of asthma were measured 24 h later. DINP increased airway hyperresponsiveness, numbers of cells in BALF, numbers of inflammatory cells (leukocytes, lymphocytes, monocytes, eosinophils, neutrophils, basophils) in blood, mucus production, pulmonary fibrosis, Th2 type cytokine levels in BALF, and lung cell apoptosis. On the other hand, administrations of TLR4-signaling inhibitors (TAK-242) or anti-HMGB1 antibodies to a mouse model of DINP-induced asthma reduced biological markers of asthma. These results show TLR4 and HMGB1 both contribute to DINP-induced asthma, and that the inhibitions of TLR4 or HMGB1 offer potential means of treating asthma induced by phthalates like DINP.

Comment on "An extracellular matrix fragment drives epithelial remodeling and airway hyperresponsiveness".

Increased airway hyperresponsiveness and epithelial remodeling in asthmatic LTA4H-KO mice may be mediated by CysLTs rather than elevated tripeptide PGP.

The Club Cell Marker SCGB1A1 Downstream of FOXA2 is Reduced in Asthma.

Human SCGB1A1 protein has been shown to be significantly reduced in BAL, sputum, and serum from humans with asthma as compared with healthy individuals. However, the mechanism of this reduction and its functional impact have not been entirely elucidated. By mining online datasets, we found that the mRNA of SCGB1A1 was significantly repressed in brushed human airway epithelial cells from individuals with asthma, and this repression appeared to be associated with reduced expression of FOXA2. Consistently, both Scgb1A1 and FoxA2 were downregulated in an ovalbumin-induced mouse model of asthma. Furthermore, compared with wild-type mice, Scgb1a1 knockout mice had increased airway hyperreactivity and inflammation when they were exposed to ovalbumin, confirming the antiinflammatory role of Scgb1a1 in protection against asthma phenotypes. To search for potential asthma-related stimuli of SCGB1A1 repression, we tested T-helper cell type 2 cytokines. Both IL-4 and IL-13 repressed epithelial expression of SCGB1A1 and FOXA2. Importantly, infection of epithelial cells with human rhinovirus similarly reduced expression of these two genes, which suggests that FOXA2 may be the common regulator of SCGB1A1. To establish the causal role of reduced FOXA2 in SCGB1A1 repression, we demonstrated that FOXA2 was required for SCGB1A1 expression at baseline. FOXA2 overexpression was sufficient to drive promoter activity and expression of SCGB1A1 and was also able to restore the repressed SCGB1A1 expression in IL-13-treated or rhinovirus-infected cells. Taken together, these findings suggest that low levels of epithelial SCGB1A1 in asthma are caused by reduced FOXA2 expression.

Modulation of ILC2 trafficking and effector functions in ILC2-driven airway hyperreactivity

Abstract Background Asthma is a heterogeneous disease of the airways characterized by increased airway hyperreactivity (AHR) to specific and unspecific stimuli. Group 2 innate lymphoid cells (ILC2s) are type 2 secreting cells that are sufficient to induce eosinophilic lung inflammation and AHR independent of adaptive immunity in murine models. Identifying pathways that modulate ILC2 homeostasis and function are therefore an important step towards treating ILC2-dependent asthma. Objective We examined the expression of adhesion molecule lymphocyte-associated antigen 1 (LFA-1) and ligand intercellular adhesion molecule (ICAM)-1 on ILC2s and their effect on the development of lung inflammation and AHR in murine models. Methods Genetically modified mice constitutively lacking LFA-1 or ICAM-1 were assessed for the development of lung inflammation and AHR. We further generated mixed bone marrow chimeras and performed transcriptomic analysis to characterize their role on ILC2 homeostasis and function. Results LFA-1 is expressed on murine naïve and IL-33-activated ILC2s, while ICAM-1 is surprisingly expressed – and upregulated – upon activation. Compared to WT mice, both LFA-1−/− and ICAM-1−/− mice develop less lung inflammation and AHR in response to intranasal IL-33 challenge. Furthermore, experiments with chimeric mice revealed that LFA-1−/− ILC2s significantly traffic less to the lungs, unlike ICAM-1−/− ILC2s. Strikingly, ICAM-1−/− ILC2s however show a defect in proliferation and cytokine production, ultimately affecting lung inflammation. Conclusions The LFA-1-ICAM-1 interaction is involved in lung ILC2 trafficking, homeostasis and function. Such findings could lead to the development of new therapeutic targets.

Blockade of the NLRP3/Caspase-1 Axis Ameliorates Airway Neutrophilic Inflammation in a Toluene Diisocyanate-Induced Murine Asthma Model.

Multiple studies have addressed the vital role of Nod-like receptor protein 3(NLRP3)/caspase-1/IL-1β signaling in asthma. Yet, the role of NLRP3/caspase-1 in toluene diisocyanate (TDI)-induced asthma is still obscure. The aim of this study is to investigate the role of the NLRP3/caspase-1 axis in TDI-induced asthma. Using an established murine model of TDI-induced asthma as described previously, we gave the asthmatic mice a highly selective NLRP3 inhibitor, MCC950, as well as the specific caspase-1 inhibitors VX-765 and Ac-YVAD-CHO for therapeutic purposes. Airway resistance was measured and bronchoalveolar lavage fluid was analyzed. Lungs were examined by histology, immunohistochemistry, Western blotting, and flow cytometry. TDI exposure elevated the expression of NLRP3 and caspase-1 that was coupled with increased airway hyperresponsiveness (AHR), neutrophil-dominated cell infiltration, pronounced goblet cell metaplasia, extensive collagen deposition, and increased TH2/TH17 responses. Both VX-765 and Ac-YVAD-CHO effectively inhibited the activation of caspase-1 in TDI-asthmatic mice that was accompanied by dramatic attenuation of AHR, airway inflammation, and airway remodeling, in addition to a decreased TH2 response and lower levels of IL-18 and IL-1β. MCC950 blocked the activation of NLRP3 and downregulated protein expression of caspase-1, IL-1β, and IL-18 in TDI-exposed mice. Furthermore, MCC950 remarkably alleviated AHR, airway inflammation, airway remodeling, and significantly suppressed TH2/TH17 responses. These findings suggested that blockade of the NLRP3/caspase-1 axis effectively prevents the progression of TDI-induced asthma and could be used as therapeutic targets for asthmatics.

Cystathionine γ-lyase deficiency enhances airway reactivity and viral-induced disease in mice exposed to side-stream tobacco smoke.

BACKGROUND:Environmental tobacco smoke (ETS) is a known risk factor for severe respiratory syncytial virus (RSV) infections, yet the mechanisms of ETS/RSV co-morbidity are largely unknown. Cystathionine γ-lyase regulates important physiological functions of the respiratory tract.METHODS:We used mice genetically deficient in the cystathionine γ-lyase enzyme (CSE), the major H2S-generating enzyme in the lung to determine the contribution of H2S to airway disease in response to side-stream tobacco smoke (TS), and to TS /RSV co-exposure.RESULTS:Following a two-week period of exposure to TS, CSE-deficient mice (KO) showed a dramatic increase in airway hyperresponsiveness (AHR) to methacholine challenge, and greater airway cellular inflammation, compared to wild type (WT) mice. TS-exposed CSE KO mice that were subsequently infected with RSV exhibited more severe clinical disease, airway obstruction and AHR, enhanced viral replication and lung inflammation, compared to TS-exposed RSV-infected WT mice. TS-exposed RSV-infected CSE KO mice had also a significant increase in the number of neutrophils in bronchoalveolar lavage (BAL) fluid and increased levels of inflammatory cytokines and chemokines.CONCLUSION:This study demonstrates the critical contribution of the H2S-generating pathway to airway reactivity and disease following exposure to ETS alone or in combination with RSV infection.

Human albumin augmented airway inflammation induced by PM2.5 in NC/Nga mice.

The synergic allergic inflammatory effects of particulate matter (PM) 2.5 and human albumin were investigated in NC/Nga mice, which are hypersensitive to mite allergens. PM2.5 or PM2.5 plus human albumin with aluminum oxide was injected twice intraperitoneally for sensitization. After 7 days, PM2.5 or PM2.5 plus human albumin was administered five times intranasally to mice for further sensitization. Subsequently, PM2.5 was administered as a challenge on the 11th day. On the 12th day, mice were examined for airway hyperresponsiveness (AHR), bronchoalveolar lavage fluid (BALF) cell count, mRNA expression of Th1 , Th2 cytokines, chemokines, and mucus proteins (MUC5AC and MUC5B) in the lung tissue and histopathology. Although PM2.5 or human albumin alone did not induce allergic airway inflammation, simultaneous inoculation of PM2.5 and human albumin-induced airway inflammation showing increase in AHR, total BALF cell numbers, mRNA levels of IL-13, eotaxin 1, eotaxin 2, and MUC5AC, and anti-IG against human serum albumin. Inflammation was observed around the bronchus in PM2.5 plus human albumin-induced lungs. These results demonstrate that PM2.5 can induce allergic airway inflammation through the synergistic action with human albumin in NC/Nga mice.

3439 Role of the Airway Microbiome in Viral Bronchiolitis Associated Respiratory Failure

OBJECTIVES/SPECIFIC AIMS: This study aims to determine if a bronchiolitis specific microbiome exists and how it evolves through disease course. Objective 1. Determine the microbiome profile of the airway in virus induced bronchiolitis-associated respiratory failure. Objective 2. Identify changes in the airway microbiome profile through the course of virus induced bronchiolitis associated respiratory failure, and the relationship between microbiome composition and clinical respiratory status. Objective 3. Determine the impact of rhinovirus infection on the lung and stool microbiome in a murine asthma model. METHODS/STUDY POPULATION: Objectives 1 &2: We are conducting a single-center prospective case-control study of patients admitted to the Komansky - Weill Cornell Pediatric Critical Care Unit. Infants less than two years of age with a diagnosis of bronchiolitis requiring intubation and mechanical ventilation are enrolled as subjects. Infants less than two years of age intubated and requiring mechanical ventilation without primary lung pathology are enrolled as controls. To evaluate our primary objective, tracheal aspirates will be collected from both subjects and controls on the day of intubation. We will perform 16s RNA sequencing on the tracheal aspirate samples and compare the resulting microbiomes. To evaluate secondary objective, we will collect tracheal aspirates of our study population on a daily basis and map the microbiome in parallel with objective measures of respiratory status including oxygen index and successful extubation. Both subjects and controls are being enrolled as a convenience sample. Objective 3: Mice, heterozygous for the sptlc2 gene (Spltc2 +/−) demonstrate reduced de-novo sphingolipids and increased airway hyperresponsiveness with methacholine challenge. Airway hyper-responsiveness is a cardinal feature of asthma. This airway hyperresponsiveness is exacerbated in the setting of rhinovirus (Figure 1). Using 16s sequencing, we will examine the lung microbiome of Sptlc2 +/− ad Sptlc2 +/+ at 1- and 7-days following rhinovirus infection. RESULTS/ANTICIPATED RESULTS: This clinical study is currently IRB approved and enrollment is ongoing. We have enrolled 12 subjects and 5 controls. Sample analysis will begin following the 2018-2019 respiratory season, with an anticipated cohort of 20 subjects and 20 controls. With regards to the murine studies, we have demonstrated that the lung microbiome of Sptlc2 +/− and Sptlc2 +/+ mice is similar at baseline (Figure 2) and remains similar following 1-day infection with rhinovirus. We do however, see a distinct change in the microbiome profile of the stool of Sptlc2 +/− mice following rhinovirus infection (Figure 3). Lung analysis at day 7 post infection is pending. DISCUSSION/SIGNIFICANCE OF IMPACT: These studies will lay the groundwork for detailing the functional role of the airway microbiome in bronchiolitis, with the objective of developing new modalities for disease treatment and prevention. In addition our murine studies allow us to view the microbiome in the context of sphingolipid deficiency, providing a potential mechanistic link to rhinovirus and ORMDL3 associated asthma.

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.

Early life inhalation exposure to mine tailings dust affects lung development

Exposure to mine tailings dust from active and abandoned mining operations may be a very significant health hazard, especially to sensitive populations living in arid and semi-arid climates like the desert southwest of the US. It is anticipated that early life exposures during sensitive times of development can lead to adult disease. However, very few studies have investigated the effects of inhalation exposure to real world dusts during lung development. Using a mouse model, we have examined the effect(s) of inhalation of real world mine tailing dusts under three separate conditions: (1) Exposure only during in utero development (exposure of the pregnant moms) (2) exposure only after birth and (3) exposures that occurred continuously during in utero development, through gestation and birth until the mice reached adulthood (28 days old). We found that the most significant changes in lung structure and function were observed in male mice when exposure occurred continuously throughout development. These changes included increased airway hyper-reactivity, increased expression of epithelial to mesenchymal (EMT) transition protein markers and increased expression of cytokines related to eosinophils. The data also indicate that in utero exposures through maternal inhalation can prime the lung of male mice for more severe responses to subsequent postnatal exposures. This may be due to epigenetic alterations in gene regulation, immune response, molecular signaling, and growth factors involved in lung development that may make the neonatal lung more susceptible to continued dust exposure.

Study effect of Ocimum basilicum seeds on mucus production and cytokine gene expression in allergic asthma mice model

Allergic asthma is a complicated disease characterized by coughing, wheezing, airway inflammation, mucus hyper-secretion and increased airway hyperresponsiveness (AHR). Basil seed, or Ocimum basilicum (OB), has an anti-inflammatory effect that is utilized in traditional medicine. The aim of this study was to investigate the effects of OB on IL-4, IL-5, IL-13 and MUC5a gene expression in an asthma model. Eighteen female BALB/c mice were divided into three groups as follows: an untreated asthmatic group, a healthy group and an asthmatic group receiving OB. Broncho-alveolar lavage fluid (BALF) was collected and Real-Time PCR was done for IL-4, 5, 13 and mucus (MUC5a) genes, and lung histopathological sections were prepared. Asthmatic mice treated with OB had suppression of cytokines and mucus gene expression (IL-4: 2.90±1.08, IL-5: 1.11±0.37, IL-13 0.72±0.19 and mucus: 1.83±1.08) in comparison to the untreated asthmatic group (IL-4: 8.00±2.54, IL-5: 2.91±0.46, IL-13 2.00±0.22 and mucus: 12.24±1.88). In the histopathological section, mucus hyper-secretion and goblet cell hyperplasia were reduced in the OB-treated group in comparison to untreated asthmatic group. Th2 cytokines and mucus gene expression were reduced in the OB-treated group in comparison to untreated asthmatic group. Mucus hyper-secretion and goblet cell hyperplasia were regulated by OB treatment in asthmatic mice. OB showed immunomodulatory and anti-inflammatory effects in allergic asthma and could reverse airway obstruction.

Epithelial folliculin enhances airway inflammation in aspirin-exacerbated respiratory disease.

Clinical features of aspirin-exacerbated respiratory disease (AERD) are characterized by overproduction of cysteinyl leukotrienes (LT) and eosinophil activation, in which epithelial cells contribute to eosinophilic airway inflammation. Folliculin (FLCN) helps maintain the integrity of epithelial barrier, but little is known about FLCN in AERD. We investigated the role of FLCN in the pathogenic mechanisms of AERD. We recruited 178 subjects with AERD, 276 subjects with aspirin-tolerant asthma (ATA) and 71 normal healthy controls (NC) at Ajou Medical Center. Levels of FLCN and interleukin (IL)-8 in sera and supernatants were measured by ELISA. Peripheral blood eosinophils isolated from asthmatic patients were cocultured with human airway epithelial cells (HAECs) pretreated with LTE4 , dexamethasone and montelukast. The intracellular expressions of FLCN, tight (TJ) (occludins, claudin-1) and adherens (AJ) junctions (E-cadherin) were analysed by Western blotting. shRNA was used to down-regulate FLCN (shFLCN) in HAECs. Serum FLCN levels were significantly higher in AERD group than in ATA and NC groups (all P < 0.001). The cut-off value of 56.6 pg/mL was used to define the high FLCN phenotype (highFLCN). Asthmatic patients with highFLCN were associated with increased airway hyperresponsiveness to methacholine (P = 0.015). The serum FLCN level could discriminate AERD group from NC group with 82% sensitivity (AUC = 0.793, P < 0.001). When HAECs were exposed to LTE4 , FLCN release was increased significantly (P < 0.05), which were amplified along with disruption of TJ and AJ expressions when HAECs were cocultured with eosinophils and LTE4 (all P < 0.05); these effects were suppressed by dexamethasone and montelukast. FLCN knockdown reduced IL-8 release and occludin expression from shFLCN HAECs. Our findings suggest that high LT and airway eosinophilia increased FLCN release from HAECs, which enhance epithelial activation and disruption. Modulation of FLCN may be a potential target for AERD.