Fine particulate matter induces airway inflammation by disturbing the balance between Th1/Th2 and regulation of GATA3 and Runx3 expression in BALB/c mice.

The present study aimed to examine the effects of 2.5 µm particulate matter (PM2.5) on airway inflammation and to investigate the possible underlying mechanism. Specifically, the focus was on the imbalance of T helper (Th)1/Th2 cells and the dysregulated expression of transcription factors, including trans­acting T cell­specific transcription factor 3 (GATA3), runt­related transcription factor 3 (Runx3) and T­box transcription factor TBX21 (T­bet). In this study, ambient PM2.5 was collected and analyzed, male BALB/c mice were sensitized and treated with PBS, ovalbumin (OVA), PM2.5 or OVA + PM2.5. The effects of PM2.5 alone or PM2.5 + OVA on immunopathological changes, the expression of transcription factors GATA3, Runx3 and T­bet, and the imbalance of Th1/Th2 were investigated. It was found that PM2.5 + OVA co­exposure significantly enhanced inflammatory cell infiltration, increased higher tracheal secretions in lung tissue and upregulated respiratory resistance response to acetylcholine compared with PM2.5 or OVA single exposure and control groups. In addition, higher protein and mRNA expression levels of Th2 inflammatory mediators interleukin (IL)­4, IL­5 and IL­13 in bronchoalveolar lavage fluid were observed in PM2.5 + OVA treated mice, whereas the expression levels of GATA3 and STAT6 were exhibited in mice exposed to OVA + PM2.5 compared with the OVA and PM2.5 groups. By contrast, PM2.5 exposure decreased the protein and mRNA expression levels of Th1 cytokine interferon­Î³ and transcription factors Runx3 and T­bet, especially among asthmatic mice, different from OVA group, PM2.5 exposure only failed to influence the expression of T­bet. To conclude, PM2.5 exposure evoked the allergic airway inflammation response, especially in the asthmatic mouse model and led to Th1/Th2 imbalance. These effects worked mainly by upregulating GATA3 and downregulating Runx3. These data suggested that Runx3 may play an important role in PM2.5­aggravated asthma in BALB/c mice.

The Proximal Airway Is a Reservoir for Adaptive Immunologic Memory in Idiopathic Subglottic Stenosis.

OBJECTIVES/HYPOTHESIS: Characterization of the localized adaptive immune response in the airway scar of patients with idiopathic subglottic stenosis (iSGS). STUDY DESIGN: Basic Science. METHODS: Utilizing 36 patients with subglottic stenosis (25 idiopathic subglottic stenosis [iSGS], 10 iatrogenic post-intubation stenosis [iLTS], and one granulomatosis with polyangiitis [GPA]) we applied immunohistochemical and immunologic techniques coupled with RNA sequencing. RESULTS: iSGS, iLTS, and GPA demonstrate a significant immune infiltrate in the subglottic scar consisting of adaptive cell subsets (T cells along with dendritic cells). Interrogation of T cell subtypes showed significantly more CD69+ CD103+ CD8+ tissue resident memory T cells (TRM ) in the iSGS airway scar than iLTS specimens (iSGS vs. iLTS; 50% vs. 28%, P = .0065). Additionally, subglottic CD8+ clones possessed T-cell receptor (TCR) sequences with known antigen specificity for viral and intracellular pathogens. CONCLUSIONS: The human subglottis is significantly enriched for CD8+ tissue resident memory T cells in iSGS, which possess TCR sequences proven to recognize viral and intracellular pathogens. These results inform our understanding of iSGS, provide a direction for future discovery, and demonstrate immunologic function in the human proximal airway. Laryngoscope, 131:610-617, 2021.

A Novel Presentation of the Acute Airway: Anti-IgLON5 Disease.

Anti-IgLON5 disease is a newly discovered novel sleep disorder at the crossroads of neurology and immunology. In addition to the underlying sleep disorder, anti-IgLON5 manifests with progressive aerodigestive symptoms such as dysphagia, stridor, and vocal cord paresis in 90% cases and may present to the otolaryngologist. Herein we present a case of a patient with anti-IgLON5 disease who presented to the hospital with an acute airway including marked stridor and respiratory failure requiring intubation and subsequently a tracheostomy. Laryngoscope, 131:E724-E726, 2021.

Neutrophil Extracellular Traps Increase Airway Mucus Viscoelasticity and Slow Mucus Particle Transit.

Mucus obstruction is a key feature of many inflammatory airway diseases. Neutrophil extracellular traps (NETs) are released upon neutrophil stimulation and consist of extracellular chromatin networks studded with cytotoxic proteins. When released in the airways, these NETs can become part of the airway mucus. We hypothesized that the extracellular DNA and/or oxidative stress (e.g., by the release of reactive oxygen species and myeloperoxidase during NETs formation in the airways) would increase mucus viscoelasticity. We collected human airway mucus from endotracheal tubes of healthy patients admitted for elective surgery and coincubated these samples with NETs from phorbol 12-myristate 13-acetate-stimulated neutrophils. Unstimulated neutrophils served as controls, and blocking experiments were performed with dornase alfa for extracellular DNA and the free radical scavenger dimethylthiourea for oxidation. Compared with controls, the coincubation of mucus with NETs resulted in 1) significantly increased mucus viscoelasticity (macrorheology) and 2) significantly decreased mesh pore size of the mucus and decreased movement of muco-inert nanoparticles through the mucus (microrheology), but 3) NETs did not cause visible changes in the microstructure of the mucus by scanning EM. Incubation with either dornase alfa or dimethylthiourea attenuated the observed changes in macrorheology and microrheology. This suggests that the release of NETs may contribute to airway mucus obstruction by increasing mucus viscoelasticity and that this effect is not solely due to the release of DNA but may in part be due to oxidative stress.

The Airway Epithelium-A Central Player in Asthma Pathogenesis.

Asthma is a chronic inflammatory airway disease characterized by variable airflow obstruction in response to a wide range of exogenous stimuli. The airway epithelium is the first line of defense and plays an important role in initiating host defense and controlling immune responses. Indeed, increasing evidence indicates a range of abnormalities in various aspects of epithelial barrier function in asthma. A central part of this impairment is a disruption of the airway epithelial layer, allowing inhaled substances to pass more easily into the submucosa where they may interact with immune cells. Furthermore, many of the identified susceptibility genes for asthma are expressed in the airway epithelium. This review focuses on the biology of the airway epithelium in health and its pathobiology in asthma. We will specifically discuss external triggers such as allergens, viruses and alarmins and the effect of type 2 inflammatory responses on airway epithelial function in asthma. We will also discuss epigenetic mechanisms responding to external stimuli on the level of transcriptional and posttranscriptional regulation of gene expression, as well the airway epithelium as a potential treatment target in asthma.

Inflammatory patterns in fixed airflow obstruction are dependent on the presence of asthma.

RATIONALE: Fixed airflow obstruction (FAO) can complicate asthma. Inflammation is a proposed underlying mechanism. OBJECTIVE: Our aim in this cross-sectional investigation was to evaluate the blood leucocyte pattern and level of exhaled nitric oxide in asthmatics and non-asthmatics with or without FAO. METHODS: A total of 11,579 individuals aged ≥20 years from the US National Health and Nutrition Examination Survey were included. They were grouped as: controls without asthma and FAO (n = 9,935), asthmatics without FAO (n = 674), asthmatics with FAO (n = 180) and non-asthmatics with FAO (n = 790). FAO was defined as post-bronchodilator FEV1/FVC < lower limit of normal. Exhaled nitric oxide ≥ 25ppb, blood eosinophil levels ≥300 cells/µL, and blood neutrophil levels ≥5100 cells/µL were defined as elevated. Stratified analyses for smoking and smoking history were performed. RESULTS: Elevated blood eosinophil levels were more common in all groups compared to the controls, with the highest prevalence in the group with asthma and fixed airflow obstruction (p<0.01). In a multiple logistic regression model adjusted for potential confounders including smoking, the asthma groups had significantly higher odds ratios for elevated B-Eos levels compared to the control group (odds ratio 1.4, (confidence interval: 1.1-1.7) for the asthma group without fixed airflow obstruction and 2.5 (1.4-4.2) for the asthma group with fixed airflow obstruction). The group with fixed airflow obstruction without asthma had higher odds ratio for elevated blood neutrophil levels compared to the controls: 1.4 (1.1-1.8). Smoking and a history of smoking were associated to elevated B-Neu levels. CONCLUSION: Fixed airflow obstruction in asthma was associated with elevated blood eosinophil levels, whereas fixed airflow obstruction without asthma was associated with elevated blood neutrophil levels.

Cytokine profile in the sputum of subjects with post-tuberculosis airflow obstruction and in those with tobacco related chronic obstructive pulmonary disease.

BACKGROUND: Previous studies have shown that tuberculosis (TB) is a risk factor for chronic airflow limitation. Chronic obstructive pulmonary disease (COPD) is recognized as the result of chronic inflammation, usually related to noxious particles. Post-TB airflow obstruction and tobacco-related COPD have the same functional pathway characterized by persistent airflow limitation. We sought to compare the profile of 29 cytokines in the sputum of subjects with post-TB airflow obstruction and those with COPD related to tobacco. RESULTS: The forced expiratory volume in the first second (FEV1) and forced expiratory volume/forced vital capacity (FEV/FVC) ratio were lower in the COPD patients with the history of smoking compared to the post-TB airflow obstruction subgroup. The stages of the disease were more advanced in COPD / tobacco patients. Among the cytokines, IL-1α, IL-1ß, MIP-1ß, sCD40L and VEGF levels were higher in COPD patients, compared to the controls with p values ​​of 0.003, 0.0001, 0.03, 0.0001 and 0.02 respectively. When the two COPD subgroups were compared, IL-1α, IL-6, TNF-α and IL-8 levels were higher in the COPD patients with the history of tobacco compared to the COPD patients with the history of TB with p-values ​​of 0.031, 0.05, 0.021 and 0.016, respectively. CONCLUSION: COPD related to tobacco is more severe than post-TB airflow obstruction. The pathogenesis of post-TB airflow obstruction appears to involve the cytokines IL-1RA, IL-1α, IL-1ß, IL-17, GRO and sCD40L, while COPD related to tobacco involves more cytokines.

Immunopathology of Airway Surface Liquid Dehydration Disease.

The primary purpose of pulmonary ventilation is to supply oxygen (O2) for sustained aerobic respiration in multicellular organisms. However, a plethora of abiotic insults and airborne pathogens present in the environment are occasionally introduced into the airspaces during inhalation, which could be detrimental to the structural integrity and functioning of the respiratory system. Multiple layers of host defense act in concert to eliminate unwanted constituents from the airspaces. In particular, the mucociliary escalator provides an effective mechanism for the continuous removal of inhaled insults including pathogens. Defects in the functioning of the mucociliary escalator compromise the mucociliary clearance (MCC) of inhaled pathogens, which favors microbial lung infection. Defective MCC is often associated with airway mucoobstruction, increased occurrence of respiratory infections, and progressive decrease in lung function in mucoobstructive lung diseases including cystic fibrosis (CF). In this disease, a mutation in the cystic fibrosis transmembrane conductance regulator (CFTR) gene results in dehydration of the airway surface liquid (ASL) layer. Several mice models of Cftr mutation have been developed; however, none of these models recapitulate human CF-like mucoobstructive lung disease. As an alternative, the Scnn1b transgenic (Scnn1b-Tg+) mouse model overexpressing a transgene encoding sodium channel nonvoltage-gated 1, beta subunit (Scnn1b) in airway club cells is available. The Scnn1b-Tg+ mouse model exhibits airway surface liquid (ASL) dehydration, impaired MCC, increased mucus production, and early spontaneous pulmonary bacterial infections. High morbidity and mortality among mucoobstructive disease patients, high economic and health burden, and lack of scientific understanding of the progression of mucoobstruction warrants in-depth investigation of the cause of mucoobstruction in mucoobstructive disease models. In this review, we will summarize published literature on the Scnn1b-Tg+ mouse and analyze various unanswered questions on the initiation and progression of mucobstruction and bacterial infections.

Assessment of serum allergen-specific IgE levels in horses with seasonal allergic dermatitis and recurrent airway obstruction in Spain.

Allergic conditions are prevalent equine diseases that can be diagnosed by clinical examination alone, but definitive diagnosis is more likely with laboratory testing. The ELISA Allercept© test was used to analyse the serum samples of 73 horses with allergic diseases. Sixty-one horses (83.5%) had allergen-specific IgE levels ≥ 150 ELISA Units (EU), the cut-off defined by the assay. Fifty-four horses had allergic dermatitis (AD) with high IgE levels to Tyrophagus putrescentiae (51.9%), Rumex crispus (48.1%), Tabanus (46.3%) and Dermatophagoides farinae/ D. pteronyssinus (40.7%). Seven horses with recurrent airway obstruction (RAO) had a high prevalence of T. putrescentiae (85.7%), followed by that of Acarus siro (57.1%) and D. farinae/D. pteronyssinus (57.1%). Horses affected with RAO had more positive reactions to mites (2.22 ± 0.84) than did horses with AD (1.51 ± 0.61, P < 0.05). A strong correlation of serum allergen-specific IgE level was found between Culex tarsalis and Stomoxys (r = 0.943) and between Dactylis glomerata and both Secale cereale (r = 0.79) and R. crispus (r = 0.696). These results indicate that among horses with allergic diseases in Spain, ELISA tests demonstrated a high prevalence of serum allergen-specific IgE in response to mites. Our study emphasises the importance of laboratory testing and updating allergy panels to improve the likelihood of a definitive diagnosis and the identification of allergens that should be included in allergic disease treatment.

CT and Functional MRI to Evaluate Airway Mucus in Severe Asthma.

BACKGROUND: Intraluminal contributor(s) to airflow obstruction in severe asthma are patient-specific and must be evaluated to personalize treatment. The occurrence and functional consequence of airway mucus in the presence or absence of airway eosinophils remain undetermined. OBJECTIVE: The objective of this study was to understand the functional consequence of airway mucus in the presence or absence of eosinophils and to identify biomarkers of mucus-related airflow obstruction. METHODS: Mucus plugs were quantified on CT scans, and their contribution to ventilation heterogeneity (using MRI ventilation defect percent [VDP]) was evaluated in 27 patients with severe asthma. Patients were dichotomized based on sputum eosinophilia such that the relationship between mucus, eosinophilia, and ventilation heterogeneity could be investigated. Fractional exhaled nitric oxide (Feno) and related cytokines in sputum were measured. RESULTS: Mucus plugging was present in 100% of asthma patients with sputum eosinophils and 36% of those without sputum eosinophils (P = .0006) and was correlated with MRI VDP prebronchodilator (r = 0.68; P = .0001) and postbronchodilator (r = 0.72; P < .0001). In a multivariable regression, both mucus and eosinophils contributed to the prediction of postbronchodilator MRI VDP (R2 = 0.75; P < .0001). Patients with asthma in whom the mucus score was high had raised Feno (P = .03) and IL-4 (P = .02) values. Mucus plugging correlated with Feno (r = 0.63; P = .005). CONCLUSIONS: Both airway eosinophils and mucus can contribute to ventilation heterogeneity in patients with severe asthma. Patients in whom mucus is the dominant cause of airway obstruction have evidence of an upregulated IL-4/IL-13 pathway that could be identified according to increased Feno level.

Systemic levels of anti-PAD4 autoantibodies correlate with airway obstruction in cystic fibrosis.

Cystic fibrosis (CF) airway disease is characterized by the long-term presence of neutrophil granulocytes. Formation of neutrophil extracellular traps (NETs) and/or autoantibodies directed against extracellular components of NETs are possible contributors to neutrophil-mediated lung damage in CF. The goal of this study was to measure their levels in CF adults compared to healthy controls and subjects with rheumatologic diseases known to develop NET-related autoantibodies and pathologies, rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE). Sera were analyzed from the following number of subjects: 37 CF, 23 healthy controls (HC), 20 RA, and 21 SLE. CF had elevated serum myeloperoxidase (MPO) concentrations (347.5±56.1 ng/ml, mean+/-S.E.M., p = .0132) compared to HC (144.5±14.6 ng/ml) but not of neutrophil elastase (NE) complexed with alpha-1-antitrypsin, cell-free DNA or NE-DNA complexes. The peptidylarginine deiminase 4 (PAD4) enzyme is required for NET formation and associated DNA release in neutrophils. Serum levels of anti-PAD4 antibodies (Ab) were elevated in CF (p = .0147) compared to HC and showed an inverse correlation with a measure of lung function, FEV1% predicted (r = -0.5020, p = .015), as did MPO levels (r = -0.4801, p = .0026). Anti-PAD4 Ab levels in CF sera associated with lung infection by P. aeruginosa, but not that by S. aureus, age, sex, CF-related diabetes or the presence of musculoskeletal pain. Serum levels of anti-citrullinated protein Abs (ACPAs) and anti-nucleosome Abs were not elevated in CF compared to HC (p = .7498, p = .0678). In summary, adult CF subjects develop an autoimmune response against NET components that correlates with worsening lung disease.

SOCS3 overexpression in T cells ameliorates chronic airway obstruction in a murine heterotopic tracheal transplantation model.

PURPOSE: Suppressor of cytokine signaling-3 (SOCS3) is a negative feedback inhibitor of cytokine signaling with T-cell-mediated immunosuppressive effects on obliterative bronchiolitis (OB). In this study, we aimed to investigate the impact of T-cell-specific overexpression of SOCS3 using a murine heterotopic tracheal transplantation (HTT) model. METHODS: Tracheal allografts from BALB/c mice were subcutaneously transplanted into wild-type C57BL/6J (B6; WT) mice and SOCS3 transgenic B6 (SOCS3TG) mice. Tracheal allografts were analyzed by immunohistochemistry and quantitative polymerase chain reaction assays at days 7 and 21. RESULTS: At day 21, allografts in SOCS3TG mice showed significant amelioration of airway obstruction and epithelial loss compared with allografts in WT mice. The intragraft expression of IFN-γ and CXCL10 was suppressed, while that of IL-4 was enhanced in SOCS3TG mice at day 7. The T-bet levels were lower in SOCS3TG allografts than in WT allografts at day 7. CONCLUSION: We revealed that the overexpression of SOCS3 in T cells effectively ameliorates OB development in a murine HTT model by inhibiting the Th1 phenotype in the early phase. Our results suggest that the regulation of the T-cell response, through the modulation of SOCS expression, has potential as a new therapeutic strategy for chronic lung allograft dysfunction.

Role of inflammatory cells in airway remodeling in COPD.

COPD is characterized by chronic bronchitis, chronic airway obstruction, and emphysema, leading to a progressive and irreversible decline in lung function. Inflammation is central for the development of COPD. Chronic inflammation in COPD mainly involves the infiltration of neutrophils, macrophages, lymphocytes, and other inflammatory cells into the small airways. The contribution of resident airway structural cells to the inflammatory process is also important in COPD. Airway remodeling consists of detrimental changes in structural tissues and cells including airway wall thickening, epithelial metaplasia, goblet cell hypertrophy, and smooth muscle hyperplasia. Persistent airway inflammation might contribute to airway remodeling and small airway obstruction. However, the underlying mechanisms remain unclear. In this review, we will provide an overview of recent insights into the role of major immunoinflammatory cells in COPD airway remodeling.

Effect of antigen challenge on dynamics of CD62P and CD41/61 expression on platelets in horses with recurrent airway obstruction (RAO).

Recurrent airway obstruction (RAO), also known as heaves, is an allergic respiratory condition that develops in horses following an exposure to aeroallergens in hay and straw. This is manifested by airway hyperreactivity, inflammation, bronchoconstriction, as well as a leukocyte and platelet infiltration into the airways. Platelet activation and an increase in circulating platelet-leukocyte aggregates may lead to airway remodeling. The aim of this study was to explore the effect of seven-day antigen challenge on dynamics of platelet indices and CD41/61 and CD62 P expression on platelets in horses with RAO. Ten RAO-affected horses and ten healthy horses were included in this study. All horses were exposed to 7 days hay and straw challenge. Blood samples were collected prior to the challenge (Pre-challenge) and 1, 2, 3, 7 and 14 days after the initiating the antigen challenge. Blood samples were obtained to determine the platelet count (PLT), mean platelet volume (MPV) and platelet large cell ratio (P-LCR). Expression of CD62 P and CD41/61 was detected by flow cytometry on activated platelets. Antigen challenge resulted in a significant gradual decrease of PLT in RAO horses, but not in controls. MPV and P-LCR in control and RAO-affected horses remained unchanged after antigen challenge. The expression of CD62 P and CD41/61 in RAO horses was significantly higher compared to control horses. The antigen challenge resulted in an increase expression of CD62 P and CD41/61 on the platelets of RAO-affected horses, while did not lead to significant changes in the control group. An increased expression of CD62 P and CD41/61 indicates platelet activation what may contribute to the formation of platelet aggregates in their respiratory system.

The Role of the Immune Response in the Pathogenesis of Bronchiectasis.

Bronchiectasis is a prevalent respiratory condition characterised by permanent and abnormal dilation of the lung airways (bronchi). There are a large variety of causative factors that have been identified for bronchiectasis; all of these compromise the function of the immune response to fight infection. A triggering factor may lead to the establishment of chronic infection in the lower respiratory tract. The bacteria responsible for the lower respiratory tract infection are usually found as commensals in the upper respiratory tract microbiome. The consequent inflammatory response to infection is largely responsible for the pathology of this condition. Both innate and adaptive immune responses are activated. The literature has highlighted the central role of neutrophils in the pathogenesis of bronchiectasis. Proteases produced in the lung by the inflammatory response damage the airways and lead to the pathological dilation that is the pathognomonic feature of bronchiectasis. The small airways demonstrate infiltration with lymphoid follicles that may contribute to localised small airway obstruction. Despite aggressive treatment, most patients will have persistent disease. Manipulating the immune response in bronchiectasis may potentially have therapeutic potential.

Characterisation of asthma subgroups associated with circulating YKL-40 levels.

The chitinase-like protein YKL-40 mediates airway inflammation and serum levels are associated with asthma severity. However, asthma phenotypes associated with YKL-40 levels have not been precisely defined.We conducted an unsupervised cluster analysis of asthma patients treated at the Yale Center for Asthma and Airways Disease (n=156) to identify subgroups according to YKL-40 level. The resulting YKL-40 clusters were cross-validated in cohorts from the Severe Asthma Research Programme (n=167) and the New York University/Bellevue Asthma Repository (n=341). A sputum transcriptome analysis revealed molecular pathways associated with YKL-40 subgroups.Four YKL-40 clusters (C1-C4) were identified. C3 and C4 had high serum YKL-40 levels compared with C1 and C2. C3 was associated with earlier onset and longer duration of disease, severe airflow obstruction, and near-fatal asthma exacerbations. C4 had the highest serum YKL-40 levels, adult onset and less airflow obstruction, but frequent exacerbations. An airway transcriptome analysis in C3 and C4 showed activation of non-type 2 inflammatory pathways.Elevated serum YKL-40 levels were associated with two distinct clinical asthma phenotypes: one with irreversible airway obstruction and another with severe exacerbations. The YKL-40 clusters are potentially useful for identification of individuals with severe or exacerbation-prone asthma.

Cutaneous vaccination with coated microneedles prevents development of airway allergy.

Allergy cases are increasing worldwide. Currently allergies are treated after their appearance in patients. However, now there is effort to make a preventive vaccine against allergies. The rationale is to target patient populations that are already sensitized to allergens but have yet to develop severe forms of the allergic disease, or who are susceptible to allergy development but have not yet developed them. Subcutaneous injections and the sublingual route have been used as the primary mode of preventive vaccine delivery. However, injections are painful, especially considering that they have to be given repeatedly to infants or young children. The sublingual route is hard to use since infants can't be trained to hold the vaccine under their tongue. In the present study, we demonstrate a microneedle (MN)-based cutaneous preventive allergy treatment against ovalbumin (Ova)-induced airway allergy in mice. Insertion of MNs coated with Ova as a model allergen and CpG oligonucleotide as an adjuvant (MNs-CIT) into the skin significantly induced Ova specific systemic immune response. This response was similar to that induced by hypodermic-needle-based delivery of Ova using the clinically-approved subcutaneous immunotherapy (SCIT) route. MNs-CIT regulated Th2 cytokines (IL-4, IL-5 & IL-13) and anti-inflammatory cytokines (IL-10) in the bronchoalveolar fluid, and IL-2 and IFN-γ cytokines in restimulated splenocyte cultures. Absence of mucus deposition inside the bronchiole wall and low collagen around the lung bronchioles after Ova-allergen challenge further confirmed the protective role of MNs-CIT. Overall, MNs-CIT represents a novel minimally invasive cutaneous immunotherapy to prevent the progression of Ova induced airway allergy in mice.