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Airway mucus is a hydrogel with unique biophysical properties due to its primary water composition and a small proportion of large anionic glycoproteins or mucins. The predominant mucins in human mucus, MUC5AC and MUC5B, are secreted by specialized cells within the airway epithelium both in normal conditions and in response to various stimuli. Their relative proportions are correlated with specific inflammatory responses and disease mechanisms. The dysregulation of mucin expression is implicated in numerous respiratory diseases, including asthma, COPD, and cystic fibrosis, where the pathogenic role of mucus has been extensively described yet often overlooked. In airway diseases, excessive mucus production or impaired mucus clearance leads to mucus plugging, with secondary airway occlusion that contribute to airflow obstruction, asthma severity and poor control. Eosinophils and Charcot Leyden crystals in sputum contribute to the mucus burden and tenacity. Mucin may also contribute to eosinophil survival. Other mechanisms, including eosinophil-independent IL-13 release, mast-cell activation and non-type-2 (T2) cytokines, are also likely to participate in mucus pathobiology. An accurate assessment of mucus and its clinical and functional consequences require a thorough approach that includes evaluation of cellular predominance in sputum, airway cytokines and other inflammatory markers, mucus characteristics and composition and structural and functional impact measured by advanced lung imaging. This review, illustrated with clinical scenarios, provides an overview of current methods to assess mucus and its relevance to the choice of biologics to treat patients with severe asthma.

The role of interleukin-18 (IL-18) and inflammasomes in chronic inflammatory airway diseases, such as asthma and chronic obstructive pulmonary disease (COPD), has garnered significant attention in recent years. This review aims to provide an overview of the current understanding of IL-18 biology, the associated signaling pathways, and the involvement of inflammasome complexes in airway diseases. We explore the multifaceted role of IL-18 in asthma pathophysiology, including its interactions with other cytokines and contributions to both T2 and non-T2 inflammation. Importantly, emerging evidence highlights IL-18 as a critical player in severe asthma, contributing to chronic airway inflammation, airway hyperresponsiveness (AHR), and mucus impaction. Furthermore, we discuss the emerging evidence of IL-18’s involvement in autoimmunity and highlight potential therapeutic targets within the IL-18 and inflammasome pathways in severe asthma patients with evidence of infections and airway autoimmune responses. By synthesizing recent advancements and ongoing research, this review underscores the importance of IL-18 as a potential novel therapeutic target in the treatment of severe asthma and other related conditions.

Abstract Background Respiratory conditions, such as asthma, are infrequently associated with auto-inflammatory diseases. We describe five patients with uncontrolled respiratory symptoms that were seen at St. Joesph’s Healthcare in Hamilton for severe asthma management diagnosed with rare autoinflammatory conditions using genetic molecular analysis. Case presentation Five patients are included in this case series. Gene mutations associated with familial Mediterranean fever, Yao syndrome, Cryopyrin-associated periodic syndrome, and Majeed syndrome were considered to explain partly the patient’s clinical manifestation after comprehensive clinical, biochemical, hematological investigations ruled out other disorders such as parasitosis, Allergic Bronchopulmonary Fungosis, Eosinophilic Granulomatosis with Poly Angitis, IgG4 disease, and Hypereosinophilia syndrome. Conclusions Complex patients initially presenting with respiratory conditions in addition to unexplained autoinflammatory conditions are a diagnostic challenge. Genetic molecular testing provides healthcare practitioners with useful information that may diagnose underlying auto-inflammatory diseases in undifferentiated patients. Role of inflammasome-activation in asthma and eosinophilia needs further investigation.

Abstract Eosinophils are granulocytes that play a significant role in the pathogenesis of asthma and other airway diseases. Directing patient treatment based on the level of eosinophilia has been shown to be extremely effective in reducing exacerbations and therefore has tremendous potential as a routine clinical test. Herein, we describe the in vitro selection and optimization of DNA aptamers that bind to eosinophil peroxidase (EPX), a protein biomarker unique to eosinophils. Fifteen rounds of magnetic bead aptamer selection were performed prior to high throughput DNA sequencing. The top 10 aptamer candidates were assessed for EPX binding using a mobility shift assay. This process identified a lead aptamer candidate termed EAP1-05 with low nanomolar affinity and high specificity for EPX over other common sputum proteins. This aptamer sequence was further optimized through truncation and used to develop an easy-to-use colourimetric pull-down assay that can detect EPX over a concentration range from 1 – 100 nM in processed sputum. Forty-six clinical samples were processed using a new sputum dispersal method, appropriate for a rapid assessment assay, that avoids centrifugation and lengthy processing times. The assay showed 89% sensitivity and 96% specificity to detect eosinophilia (compared to gold standard sputum cytometry), with results being produced in under an hour. This assay could allow for an easy assessment of eosinophil activity in the airway to guide anti-inflammatory therapy for several airway diseases.

Introduction: The ideal contrast agents for ventilation SPECT and MRI are Technegas and 129Xe gas, respectively. Despite increasing interest in the clinical utility of ventilation imaging, these modalities have not been directly compared. Therefore, our objective was to compare the ventilation defect percent (VDP) assessed by Technegas SPECT and hyperpolarized 129Xe MRI in patients scheduled to undergo lung cancer resection with and without pre-existing obstructive lung disease.Methods: Forty-one adults scheduled to undergo lung cancer resection performed same-day Technegas SPECT, hyperpolarized 129Xe MRI, spirometry, and diffusing capacity of the lung for carbon monoxide (DLCO). Ventilation abnormalities were quantified as the VDP using two different methods: adaptive thresholding (VDPT) and k-means clustering (VDPK). Correlation and agreement between VDP quantified by Technegas SPECT and 129Xe MRI were determined by Spearman correlation and Bland-Altman analysis, respectively.Results: VDP measured by Technegas SPECT and 129Xe MRI were correlated (VDPT: r = 0.48, p = 0.001; VDPK: r = 0.63, p < 0.0001). A 2.0% and 1.6% bias towards higher Technegas SPECT VDP was measured using the adaptive threshold method (VDPT: 23.0% ± 14.0% vs. 21.0% ± 5.2%, p = 0.81) and k-means method (VDPK: 9.4% ± 9.4% vs. 7.8% ± 10.0%, p = 0.02), respectively. For both modalities, higher VDP was correlated with lower FEV1/FVC (SPECT VDPT: r = −0.38, p = 0.01; MRI VDPK: r = −0.46, p = 0.002) and DLCO (SPECT VDPT: r = −0.61, p < 0.0001; MRI VDPK: r = −0.68, p < 0.0001). Subgroup analysis revealed that VDP measured by both modalities was significantly higher for participants with COPD (n = 13) than those with asthma (n = 6; SPECT VDPT: p = 0.007, MRI VDPK: p = 0.006) and those with no history of obstructive lung disease (n = 21; SPECT VDPT: p = 0.0003, MRI VDPK: p = 0.0003).Discussion: The burden of ventilation defects quantified by Technegas SPECT and 129Xe MRI VDP was correlated and greater in participants with COPD when compared to those without. Our observations indicate that, despite substantial differences between the imaging modalities, quantitative assessment of ventilation defects by Technegas SPECT and 129Xe MRI is comparable.

Abstract Rationale Patients with asthma who have neutrophilic bronchitis may have an underlying cause leading to increased susceptibility to airway infections. Methods Retrospective review of patients with asthma who had a previous history of recurrent exacerbations that had been associated with airway or sinus infections referred to a tertiary asthma center between 2005 and 2020. Demographics, clinical features, and airway inflammation type determined by sputum cytometry were compared between CFTR carriers and non-carriers. Multiple linear regression was used to identify clinical predictors of CFTR carrier status. Response to nebulized hypertonic saline was assessed by comparing the number of infective exacerbations before and after its initiation. Results 75 patients underwent CFTR mutation testing. Of these, 13 (17%) were CFTR carriers. The most common mutation was $$\Delta$$ Δ F508. CFTR carriers were older (adjusted odds ratio 1.06 (CI 95% 1.01, 1.13)) and had more frequent flares requiring hospitalization (4.19 (1.34, 24.74)). Neutrophilic airway inflammation was the most common inflammatory subtype in CFTR carriers, though 8/13 also had eosinophilic bronchitis. Nebulized hypertonic saline was well tolerated by most and reduced the frequency of infective exacerbations. Conclusions The prevalence of CFTR heterozygosity in this cohort with recurrent neutrophilic bronchitis is higher than in the general population. Respiratory disease in CFTR carriers is associated with older age and may cause significant morbidity. Airway neutrophilia is the most common inflammatory subtype, but > 50% had eosinophilic bronchitis requiring treatment. Hypertonic saline appears to be well tolerated and effective in reducing the number of infective exacerbations.

Abstract Background Excess pulmonary iron has been implicated in the pathogenesis of lung disease, including asthma and COPD. An association between higher iron content in sputum macrophages and infective exacerbations of COPD has previously been demonstrated. Objectives To assess the mechanisms of pulmonary macrophage iron sequestration, test the effect of macrophage iron-loading on cellular immune function, and prospectively determine if sputum hemosiderin index can predict infectious exacerbations of COPD. Methods Intra- and extracellular iron was measured in cell-line-derived and in freshly isolated sputum macrophages under various experimental conditions including treatment with exogenous IL-6 and hepcidin. Bacterial uptake and killing were compared in the presence or absence of iron-loading. A prospective cohort of COPD patients with defined sputum hemosiderin indices were monitored to determine the annual rate of severe infectious exacerbations. Results Gene expression studies suggest that airway macrophages have the requisite apparatus of the hepcidin-ferroportin axis. IL-6 and hepcidin play roles in pulmonary iron sequestration, though IL-6 appears to exert its effect via a hepcidin-independent mechanism. Iron-loaded macrophages had reduced uptake of COPD-relevant organisms and were associated with higher growth rates. Infectious exacerbations were predicted by sputum hemosiderin index (β = 0.035, p = 0.035). Conclusions We demonstrate in-vitro and population-level evidence that excess iron in pulmonary macrophages may contribute to recurrent airway infection in COPD. Specifically, IL-6-dependent iron sequestration by sputum macrophages may result in immune cell dysfunction and ultimately lead to increased frequency of infective exacerbation.

IntroductionChronic obstructive pulmonary disease (COPD) is a common, preventable lung disease which affects more than 300 million people worldwide. People with COPD have elevated levels of inflammatory biomarkers, which are linked to physiological alterations in the respiratory system and extrapulmonary manifestations. Pulmonary rehabilitation (PR) is one of the strategies used in the management of individuals with COPD irrespective of severity, however its effect on systemic inflammation is poorly understood. We report the protocol of a systematic review on the effects of PR on systemic inflammation in patients with COPD.Materials and methodsUsing the search terms "chronic obstructive pulmonary disease", "pulmonary rehabilitation", and "inflammatory biomarkers" and their synonyms, five databases (AMED, CINAHL, Ovid MEDLINE, MEDLINE (Pubmed), EMBASE) will be searched from their inception to identify primary literature evaluating the effects of PR on systemic inflammation. Two reviewers will independently screen titles, abstracts, and full texts for eligibility using the Covidence web-based software. Eligible studies must be published in a peer-reviewed journal and include: (1) participants with COPD undergoing PR with an exercise component of at least 4 weeks in length and (2) a measure of systemic inflammation (e.g., bloodwork or sputum sample) as an outcome of interest. We will use the Cochrane Risk of Bias Tools (ROB2 and ROBINS-I) and will rate the quality of the evidence using the Grading of Recommendations, Assessment, Development and Evaluations (GRADE) tool. This protocol has followed the Preferred Reporting Items for Systematic Reviews and Meta-Analyses Protocols (PRISMA-P) guidelines and is registered with the International Prospective Register of Systematic Reviews (PROSPERO).ConclusionThe results of this systematic review will summarize the status of the evidence highlighting the effect of PR on systemic inflammation. A manuscript will be drafted and submitted to a peer-reviewed journal and shared at conferences.

BackgroundSerum periostin associates with type-2 inflammation in asthmatic airways, but also reflects whole body periostin levels originating from multiple sources. Less is known about sputum periostin as a biomarker in asthma as detection levels are low using currently available periostin assays. We aimed to investigate detection of sputum periostin using ELISA assays targeting different periostin epitopes and relate levels to clinical characteristics.MethodsTwo ELISA systems were developed using antibodies detecting whole periostin or cleavage products, the molecular weight and amino acid sequences of which were confirmed. The ELISA assays were applied to sputum from 80 patients with mild-to-moderate and severe asthma enrolled in the European, multi-center study BIOAIR. Results were related to clinical characteristics.ResultsSputum was found to contain smaller periostin fragments, possibly due to proteolytic cleavage at a C-terminal site. Comparing ELISA methodology using antibodies against cleaved versus whole periostin revealed detectable levels in 90% versus 44% of sputum samples respectively. Sputum periostin showed associations with blood and sputum eosinophils. Furthermore, sputum, but not serum, periostin correlated with reduced lung function and sputum IL-13 and was reduced by oral corticosteroid treatment.ConclusionsWe present an ELISA method for improved analysis of sputum periostin by detecting cleavage products of the periostin protein. Using this assay, sputum periostin was detectable and associated with more disease-relevant parameters in asthma than serum periostin. Sputum periostin is worth considering as a phenotype-specific biomarker in asthma as its proximity to the airways may eliminate some of the confounding factors known to affect serum periostin.

Abstract Background Pulmonary fibrosis is thought to be driven by recurrent alveolar epithelial injury which leads to the differentiation of fibroblasts into α-smooth muscle actin (α-SMA)-expressing myofibroblasts and subsequent deposition of extracellular matrix (ECM). Transforming growth factor beta-1 (TGF-β1) plays a key role in fibroblast differentiation, which we have recently shown involves human antigen R (HuR). HuR is an RNA binding protein that also increases the translation of hypoxia inducible factor (HIF-1α) mRNA, a transcription factor critical for inducing a metabolic shift from oxidative phosphorylation towards glycolysis. This metabolic shift may cause fibroblast differentiation. We hypothesized that under hypoxic conditions, HuR controls myofibroblast differentiation and glycolytic reprogramming in human lung fibroblasts (HLFs). Methods Primary HLFs were cultured in the presence (or absence) of TGF-β1 (5 ng/ml) under hypoxic (1% O2) or normoxic (21% O2) conditions. Evaluation included mRNA and protein expression of glycolytic and myofibroblast/ECM markers by qRT-PCR and western blot. Metabolic profiling was done by proton nuclear magnetic resonance (1H- NMR). Separate experiments were conducted to evaluate the effect of HuR on metabolic reprogramming using siRNA-mediated knock-down. Results Hypoxia alone had no significant effect on fibroblast differentiation or metabolic reprogramming. While hypoxia- together with TGFβ1- increased mRNA levels of differentiation and glycolysis genes, such as ACTA2, LDHA, and HK2, protein levels of α-SMA and collagen 1 were significantly reduced. Hypoxia induced cytoplasmic translocation of HuR. Knockdown of HuR reduced features of fibroblast differentiation in response to TGF-β1 with and without hypoxia, including α-SMA and the ECM marker collagen I, but had no effect on lactate secretion. Conclusions Hypoxia reduced myofibroblasts differentiation and lactate secretion in conjunction with TGF-β. HuR is an important protein in the regulation of myofibroblast differentiation but does not control glycolysis in HLFs in response to hypoxia. More research is needed to understand the functional implications of HuR in IPF pathogenesis.

Abstract Rationale On Wright-stained sputum cytospins, eosinophil differential of ≥ 1.2% is considered abnormal, and ≥ 2.3% identifies an eosinophilic endotype. We hypothesized that failure to consider free eosinophil granules (FEG), and the re-emergence (unmasking) of eosinophilia due to various reasons underestimate the prevalence of the eosinophilic endotype. Methods This is a retrospective analysis of our Institutional Review Board-approved clinical sputum database. Of the 24,176 examinations of sputa from patients with various airway diseases, 17,693 were viable cell counts from 9570 patients (6604 on a single occasion, 2967 from multiple occasions). The prevalence of intact eosinophil % at 1.2 and 2.3% thresholds was first examined. Then, additional evidence of eosinophilia was assessed by semi-quantitative enumeration of FEGs. In those patients whose sputa were examined on multiple occasions (at the time of an exacerbation or after corticosteroid dose was reduced), re-emergence (unmasking) of eosinophilia was assessed . Results Using the threshold of eosinophilia ≥ 1.2%, 6289/17693 (35.6%) of sputa were classified as eosinophilic. This increased to 7850/17693 (44.4%) when the presence of FEGs was considered. Using the threshold of eosinophilia ≥ 2.3%, 4647/17693 (26.3%) of sputa were classified as eosinophilic. This increased to 5435/17693 (30.7%) when the presence of FEG were considered. Extrapolating from the prevalence of re-emergence observed in the 2967 patients who had sputa examined on multiple occasions to the whole sample, we estimated that eosinophilia at 1.2% threshold would be observed in at least 60% of the samples, and a clinically relevant eosinophilia at 2.3% threshold would be observed in at least 48.5% of the samples. Conclusions Using a large sputum cytometry clinical database (17,693 viable cell counts), we demonstrate that a single time point intact cell count underestimates the prevalence of eosinophilia in a variety of airway diseases. The prevalence of eosinophilia increases from 35.6 to 60% (40% underestimation) at the 1.2% threshold, and from 26.3 to 48.5% (45% underestimation) at the 2.3% clinically relevant threshold, when free granules and a second examination are considered. This has important implications to identify the eosinophilic and Th2 high endotype both for clinical trials of anti-eosinophil therapies, and to select patients who may respond well to glucocorticosteroids and anti-IL5 therapies.

Quantitative sputum cytometry facilitates in assessing the nature of bronchitis associated with exacerbations of chronic obstructive pulmonary disease (COPD). This is not assessed in most clinical trials that evaluate the effectiveness of strategies to prevent or to treat exacerbations. While up to a quarter of exacerbations may be associated with raised eosinophil numbers, the vast majority of exacerbations are associated with neutrophilic bronchitis that may indicate airway infections. While eosinophilia may be a predictor of response to corticosteroids (oral and inhaled), the limited efficacy of anti–interleukin 5 therapies would suggest that eosinophils may not directly contribute to those exacerbations. However, they may contribute to airspace enlargement in patients with COPD through various mechanisms involving the interleukin 13 and matrix metalloprotease pathways. The absence of eosinophils may facilitate in limiting the unnecessary use of corticosteroids. The presence of neutrophiia could prompt an investigation for the specific pathogens in the airway. Additionally, sputum measurements may also provide insight into the mechanisms of susceptibility to airway infections. Iron within sputum macrophages, identified by hemosiderin staining (and by more direct quantification) may impair macrophage functions while the low levels of immunoglobulins in sputum may also contribute to airway infections. The assessment of sputum at the time of exacerbations thus would facilitate in customizing treatment and treat current exacerbations and reduce future risk of exacerbations.

Cannabis (marijuana) is the most commonly used illicit product in the world and is the second most smoked plant after tobacco. There has been a rapid increase in the number of countries legalizing cannabis for both recreational and medicinal purposes. Smoking cannabis in the form of a joint is the most common mode of cannabis consumption. Combustion of cannabis smoke generates many of the same chemicals as tobacco smoke. Although the impact of tobacco smoke on respiratory health is well-known, the consequence of cannabis smoke on the respiratory system and, in particular, the inflammatory response is unclear. Besides the combustion products present in cannabis smoke, cannabis also contains cannabinoids including Δ9-tetrahydrocannabinol (Δ9-THC) and cannabidiol (CBD). These compounds are hydrophobic and not present in aqueous solutions. In order to understand the impact of cannabis smoke on pathological mechanisms associated with adverse respiratory outcomes, the development of in vitro surrogates of cannabis smoke exposure is needed. Therefore, we developed a standardized protocol for the generation of cannabis smoke extract (CaSE) to investigate its effect on cellular mechanisms in vitro. First, we determined the concentration of Δ9-THC, one of the major cannabinoids, by ELISA and found that addition of methanol to the cell culture media during generation of the aqueous smoke extract significantly increased the amount of Δ9-THC. We also observed by LC-MS/MS that CaSE preparation with methanol contains CBD. Using a functional assay in cells for CB1 receptors, the major target of cannabinoids, we found that this CaSE contains Δ9-THC which activates CB1 receptors. Finally, this standardized preparation of CaSE induces an inflammatory response in human lung fibroblasts. This study provides an optimized protocol for aqueous CaSE preparation containing biologically active cannabinoids that can be used for in vitro experimentation of cannabis smoke and its potential impact on various indices of pulmonary health.

Patient-specific localisation of ventilation defects using hyperpolarised gas magnetic resonance imaging (MRI) introduces the possibility of regionally targeted bronchial thermoplasty (BT) for the treatment of severe asthma. We aimed to demonstrate that BT guided by MRI to ventilation defects reduces the number of radiofrequency activations while resulting in improved asthma quality-of-life and control scores that are non-inferior to standard BT. In a 1-year pilot randomised controlled trial, 14 patients with severe asthma who were clinically eligible to receive BT underwent hyperpolarised gas MRI to characterise ventilation defects and were randomised to MRI-guided or standard BT. End-points were improved Asthma Quality of Life Questionnaire (AQLQ) and Asthma Control Questionnaire (ACQ) scores, the proportion of AQLQ and ACQ responders and the number of radiofrequency activations and bronchoscopy sessions. Participants who underwent MRI-guided BT received 53% fewer radiofrequency activations than those who had standard BT (p=0.003). At 12 months, the mean improvement from baseline was similar between the MRI-guided group (n=5) and the standard group (n=7) for AQLQ score (MRI-guided: 1.8, 95% CI 0.1–3.5, p=0.04; standard: 0.7, 95% CI −0.9–2.3, p=0.30) (p=0.25) and ACQ-5 score (MRI-guided: −1.4, 95% CI −2.6– −0.2, p=0.03; standard: −0.7, 95% CI −1.3–0.0, p=0.04) (p=0.17). A similar proportion of participants in both groups achieved a clinically relevant improvement in AQLQ score (MRI-guided: 80%; standard: 71%) and ACQ-5 score (MRI-guided: 80%; standard: 57%). Hyperpolarised gas MRI-guided BT reduced the number of radiofrequency activations, and resulted in asthma quality of life and control improvements at 12 months that were non-inferior to standard BT.

Abstract Omalizumab, a recombinant humanized monoclonal antibody targeting the IgE molecule, is the first biologic approved for moderate-to-severe allergic asthmatics, who remain uncontrolled despite high dose inhaled corticosteroid and bronchodilators. Steroid-sparing effect of omalizumab has not been demonstrated in asthmatics with persistent airway eosinophilia in a randomised controlled trial till date. From this double-blind, placebo-controlled, multi-centred, randomized parallel group design, we report that omalizumab is possibly inadequate to control sputum eosinophilia, and therefore may not have a steroid-sparing effect, especially in those maintained on oral corticosteroids daily. This needs to be confirmed or refuted in a larger trial, which may be a challenge with respect to recruitment, since there are currently three additional biologics available to prescribe. Trial registration Clinicaltrials.gov, NCT02049294, Registered 30th January 2014, https://clinicaltrials.gov/ct2/show/NCT02049294

Abstract Background Sputum induction with hypertonic saline in obstructive airway diseases is generally safe. However, saline induces bronchoconstriction in some patients despite pre-medication with Salbutamol. Our objectives were to investigate the predictors of failure of Salbutamol to protect against saline-induced-bronchoconstriction in patients with asthma and COPD and to evaluate implications for asthma therapy. Methods Retrospective survey on a database of 3565 patients with obstructive airway diseases who had sputum induced with hypertonic saline. The effect of baseline FEV1, bronchitis and concomitant medication on saline-induced-bronchoconstriction (≥ 15% drop in FEV1) were examined by logistic regression analysis. A subgroup had this re-examined 8–12 weeks after decreasing long-acting-beta-2-agonist dose or after adding Montelukast, which included an assessment of mast cell activity in sputum. Results 222 (6.2%) patients had saline-induced-bronchoconstriction despite pre-treatment with inhaled Salbutamol. Baseline airflow obstruction (FEV1% predicted

Abstract Background Chronic obstructive pulmonary disease (COPD) is a heterogeneous disease of the lungs that is currently the fourth leading cause of death worldwide. Genetic factors account for only a small amount of COPD risk, but epigenetic mechanisms, including DNA methylation, have the potential to mediate the interactions between an individual’s genetics and environmental exposure. DNA methylation is highly cell type-specific, and individual cell type studies of DNA methylation in COPD are sparse. Fibroblasts are present within the airway and parenchyma of the lung and contribute to the aberrant deposition of extracellular matrix in COPD. No assessment or comparison of genome-wide DNA methylation profiles in the airway and parenchymal fibroblasts from individuals with and without COPD has been undertaken. These data provide valuable insight into the molecular mechanisms contributing to COPD and the differing pathologies of small airways disease and emphysema in COPD. Methods Genome-wide DNA methylation was evaluated at over 485,000 CpG sites using the Illumina Infinium HumanMethylation450 BeadChip array in the airway (non-COPD n = 8, COPD n = 7) and parenchymal fibroblasts (non-COPD n = 17, COPD n = 29) isolated from individuals with and without COPD. Targeted gene expression was assessed by qPCR in matched RNA samples. Results Differentially methylated DNA regions were identified between cells isolated from individuals with and without COPD in both airway and parenchymal fibroblasts. Only in parenchymal fibroblasts was differential DNA methylation associated with differential gene expression. A second analysis of differential DNA methylation variability identified 359 individual differentially variable CpG sites in parenchymal fibroblasts. No differentially variable CpG sites were identified in the airway fibroblasts. Five differentially variable-methylated CpG sites, associated with three genes, were subsequently assessed for gene expression differences. Two genes (OAT and GRIK2) displayed significantly increased gene expression in cells isolated from individuals with COPD. Conclusions Differential and variable DNA methylation was associated with COPD status in the parenchymal fibroblasts but not airway fibroblasts. Aberrant DNA methylation was associated with altered gene expression imparting biological function to DNA methylation changes. Changes in DNA methylation are therefore implicated in the molecular mechanisms underlying COPD pathogenesis and may represent novel therapeutic targets.

Abstract Background Asthma and obesity, two growing epidemics worldwide, may share an underlying causal relationship. Airway hyperresponsiveness (AHR), a defining component of asthma, has been documented in both ‘obese’ animal models and non-asthmatic obese individuals. However, there is a paucity of evidence that obesity-derived factors directly affect human airway smooth muscles (ASM). Methods Experiments were designed with primary ASM and adipocytes isolated from the same human tissue explants (n = 6). The modulatory effects of human adipocytes extracted from subcutaneous (extrathoracic) and visceral (intrathoracic) depots, on ASM biology was examined with respect to proliferation, migration, contractility and pro-inflammatory cytokine synthesis. Results Adipocyte-conditioned media as well as myocyte-adipocyte co-cultures failed to show any significant changes in the proliferative or migrational properties of the ASM. Adipocyte-conditioned media also had no effect on the contractility or relaxation of bovine tracheal muscle strips. In contrast, there was a moderate yet significant increase of IL-6 and eotaxin release by ASM incubated with adipocyte-conditioned media (P = 0.0035 and P = 0.0067, vs. control, respectively), thereby further consolidating the altered inflammatory state reported for both diseases. Conclusion We report, for the first time, that adipocytes from either subcutaneous or visceral depots can trigger an inflammatory state in the ASM, with negligible modulatory effects on hyperplasia, hypertrophy or contractile properties.

Patients with COPD may be at an increased risk for severe illness from COVID-19 because of ACE2 upregulation, the entry receptor for SARS-CoV-2. Chronic exposure to cigarette smoke, the main risk factor for COPD, increases pulmonary ACE2. How ACE2 expression is controlled is not known but may involve HuR, an RNA binding protein that increases protein expression by stabilizing mRNA. We hypothesized that HuR would increase ACE2 protein expression. We analyzed scRNA-seq data to profile ELAVL1 expression in distinct respiratory cell populations in COVID-19 and COPD patients. HuR expression and cellular localization was evaluated in COPD lung tissue by multiplex immunohistochemistry and in human lung cells by imaging flow cytometry. The regulation of ACE2 expression was evaluated using siRNA-mediated knockdown of HuR. There is a significant positive correlation between ELAVL1 and ACE2 in COPD cells. HuR cytoplasmic localization is higher in smoker and COPD lung tissue; there were also higher levels of cleaved HuR (CP-1). HuR binds to ACE2 mRNA but knockdown of HuR does not change ACE2 protein levels in primary human lung fibroblasts (HLFs). Our work is the first to investigate the association between ACE2 and HuR. Further investigation is needed to understand the mechanistic underpinning behind the regulation of ACE2 expression.

Survivors of severe SARS-CoV-2 infections frequently suffer from a range of post-infection sequelae. Whether survivors of mild or asymptomatic infections can expect any long-term health consequences is not yet known. Herein we investigated lasting changes to soluble inflammatory factors and cellular immune phenotype and function in individuals who had recovered from mild SARS-CoV-2 infections (n = 22), compared to those that had recovered from other mild respiratory infections (n = 11). Individuals who had experienced mild SARS-CoV-2 infections had elevated levels of C-reactive protein 1–3 months after symptom onset, and changes in phenotype and function of circulating T-cells that were not apparent in individuals 6–9 months post-symptom onset. Markers of monocyte activation, and expression of adherence and chemokine receptors indicative of altered migratory capacity, were also higher at 1–3 months post-infection in individuals who had mild SARS-CoV-2, but these were no longer elevated by 6–9 months post-infection. Perhaps most surprisingly, significantly more T-cells could be activated by polyclonal stimulation in individuals who had recently experienced a mild SARS-CoV-2, infection compared to individuals with other recent respiratory infections. These data are indicative of prolonged immune activation and systemic inflammation that persists for at least three months after mild or asymptomatic SARS-CoV-2 infections.

Abstract Airway remodelling is an important feature of asthma pathogenesis. A key structural change inherent in airway remodelling is increased airway smooth muscle mass. There is emerging evidence to suggest that the migration of airway smooth muscle cells may contribute to cellular hyperplasia, and thus increased airway smooth muscle mass. The precise source of these cells remains unknown. Increased airway smooth muscle mass may be collectively due to airway infiltration of myofibroblasts, neighbouring airway smooth muscle cells in the bundle, or circulating hemopoietic progenitor cells. However, the relative contribution of each cell type is not well understood. In addition, although many studies have identified pro and anti-migratory agents of airway smooth muscle cells, whether these agents can impact airway remodelling in the context of human asthma, remains to be elucidated. As such, further research is required to determine the exact mechanism behind airway smooth muscle cell migration within the airways, how much this contributes to airway smooth muscle mass in asthma, and whether attenuating this migration may provide a therapeutic avenue for asthma. In this review article, we will discuss the current evidence with respect to the regulation of airway smooth muscle cell migration in asthma.

Abstract Background Prednisone dependence in asthma is usually described based on clinical and spirometric characteristics. It is generally believed that these patients have frequent exacerbations and lose lung function rapidly because of uncontrolled airway eosinophilia. Objectives The objectives of this study are to report the effect on asthma exacerbations and the change in lung function over time in prednisone-dependent asthma when severe asthma is managed using a protocol that aims to maintain normal sputum cell counts. Methods A retrospective survey of patients prospectively assessed in a university tertiary care asthma clinic. Results 52 patients (30 males, mean age 51 years, 64% non-atopic) were followed for a median period of 5.4 years (min–max: 0.2–35.2). Monitoring with the aim of keeping sputum eosinophils below 3% resulted in higher doses of corticosteroids (median daily dose of prednisone was 10 mg and for inhaled corticosteroids was 1500 μg of fluticasone equivalent) than at baseline and this was associated with predictable adverse effects. Despite the disease severity, 10 patients (19%) did not require LABA for symptom control. Most importantly, over the period of follow-up, there were only 0.3 eosinophilic exacerbations/patient/year. Overall, there was an increase in FEV1 over the period of follow-up (mean +84.6 ml/year) rather than an expected decline. Conclusions Monitoring of eosinophils in sputum enables to maintain symptom control and preserve FEV1 in patients with severe prednisone-dependent asthma.

Abstract Background Infective exacerbations of COPD are common and are accompanied by neutrophilic bronchitis in sputum. Increased respiratory iron content has been associated with respiratory tract infection, though it is unclear if this represents a predisposing factor for infection or the sequelae of inflammation. Iron overload, as assessed in the airways, may be an important biomarker for recurrent infective exacerbations of COPD. The purpose of our study was to determine if hemosiderin in sputum macrophages is related to infective exacerbations of COPD. Methods We undertook a retrospective observational study of 54 consecutive patients who presented with an exacerbation of COPD and had sputum examined including assessment for hemosiderin in alveolar macrophages. The relation between infective exacerbations in the previous two years and the percent of hemosiderin-positive macrophages was analyzed with linear regression. To account for the non-parametric distribution of infective exacerbations, negative binomial regression modelling was used to account for other covariates. Results The percent of hemosiderin positive alveolar macrophages (hemosiderin index), analyzed parametrically and non-parametrically, demonstrated a significant correlation with increasing numbers of infective exacerbations in the previous two years. In a multivariate regression analysis, hemosiderin index was an independent predictor of infective exacerbations. COPD patients with raised hemosiderin index (≥20%) had higher levels of sputum IL-6 compared to patients with lower levels (

Nibu Parameswaran Nair, Leanne Chalmers, Gregory M Peterson, Bonnie J Bereznicki, Ronald L Castelino, Luke R Bereznicki Division of Pharmacy, School of Medicine, Faculty of Health, University of Tasmania, Hobart, TAS, Australia Abstract: Adverse drug reactions (ADRs) represent a major burden on society, resulting in significant morbidity, mortality, and health care costs. Older patients living in the community are particularly susceptible to ADRs, and are at an increased risk of ADR-related hospitalization. This review summarizes the available evidence on ADR-related hospital admission in older patients living in the community, with a particular focus on risk factors for ADRs leading to hospital admission and the need for a prediction tool for risk of ADR-related hospitalization in these individuals. The reported proportion of hospital admissions due to ADRs has ranged from 6% to 12% of all admissions in older patients. The main risk factors or predictors for ADR-related admissions were advanced age, polypharmacy, comorbidity, and potentially inappropriate medications. There is a clear need to design intervention strategies to prevent ADR-related hospitalization in older patients. To ensure the cost-effectiveness of such strategies, it would be necessary to target them to those older individuals who are at highest risk of ADR-related hospitalization. Currently, there are no validated tools to assess the risk of ADRs in primary care. There is a clear need to investigate the utility of tools to identify high-risk patients to target appropriate interventions toward prevention of ADR-related hospital admissions. Keywords: adverse drug reactions, hospital admission, prediction, older patients, primary care, risk factors

The authors illustrate the merits of identifying the components of diseases (eg, bronchitis and airway hyper-responsiveness) that contribute to exacerbations in the management of a patient with severe asthma. Quantitative cell counts in sputum identified a neutrophilic – as opposed to eosinophilic – bronchitis that enabled a stepwise weaning of prednisone. Molecular microbiology and extended culture methods identified anaerobes and other airway microbiome that helped to guide the use of antibiotics. Further control of asthma was achieved by performing bronchial thermoplasty.

Guidelines for the management of severe asthma do not emphasize the measurement of the inflammatory component of airway disease to indicate appropriate treatments or to monitor response to treatment. Inflammation is a central component of asthma and contributes to symptoms, physiological, and structural abnormalities. It can be assessed by a number of endotyping strategies based on “omics” technology such as proteomics, transcriptomics, and metabolomics. It can also be assessed using simple cellular responses by quantitative cytometry in sputum. Bronchitis may be eosinophilic, neutrophilic, mixed-granulocytic, or paucigranulocytic (eosinophils and neutrophils not elevated). Eosinophilic bronchitis is usually a Type 2 (T2)-driven process and therefore a sputum eosinophilia of greater than 3% usually indicates a response to treatment with corticosteroids or novel therapies directed against T2 cytokines such as IL-4, IL-5, and IL-13. Neutrophilic bronchitis represents a non-T2-driven disease, which is generally a predictor of response to antibiotics and may be a predictor to therapies targeted at pathways that lead to neutrophil recruitment such as TNF, IL-1, IL-6, IL-8, IL-23, and IL-17. Paucigranulocytic disease may not warrant anti-inflammatory therapy. These patients, whose symptoms may be driven largely by airway hyper-responsiveness may benefit from smooth muscle-directed therapies such as bronchial thermoplasty or mast-cell directed therapies. This review will briefly summarize the current knowledge regarding “omics-based signatures” and cellular endotyping of severe asthma and give an overview of segmentation of asthma therapeutics guided by the endotype.

Heightened inflammation, including expression of COX-2, is associated with chronic obstructive pulmonary disease (COPD) pathogenesis. The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor that is reduced in COPD-derived lung fibroblasts. The AhR also suppresses COX-2 in response to cigarette smoke, the main risk factor for COPD, by destabilizing the Cox-2 transcript by mechanisms that may involve the regulation of microRNA (miRNA). Whether reduced AhR expression is responsible for heightened COX-2 in COPD is not known. Here, we investigated the expression of COX-2 as well as the expression of miR-146a, a miRNA known to regulate COX-2 levels, in primary lung fibroblasts derived from non-smokers (Normal) and smokers (At Risk) with and without COPD. To confirm the involvement of the AhR, AhR knock-down via siRNA in Normal lung fibroblasts and MLE-12 cells was employed as were A549-AhRko cells. Basal expression of COX-2 protein was higher in COPD lung fibroblasts compared to Normal or Smoker fibroblasts but there was no difference in Cox-2 mRNA. Knockdown of AhR in lung structural cells increased COX-2 protein by stabilizing the Cox-2 transcript. There was less induction of miR-146a in COPD-derived lung fibroblasts but this was not due to the AhR. Instead, we found that RelB, an NF-κB protein, was required for transcriptional induction of both Cox-2 and miR-146a. Therefore, we conclude that the AhR controls COX-2 protein via mRNA stability by a mechanism independent of miR-146a. Low levels of the AhR may therefore contribute to the heightened inflammation common in COPD patients.

Airway inflammation is a central feature of many airway diseases such as asthma, chronic bronchitis, bronchiectasis and chronic cough; therefore, it is only logical that it is measured to optimize its treatment. However, most treatment recommendations, including the use of anti-inflammatory therapies such as corticosteroids, are based on assessments of only airflow and symptoms. Over the past 10 years, methods have been developed to assess airway inflammation relatively noninvasively. Quantitative cell counts in sputum and the fraction of exhaled nitric oxide are the most validated tests. Judicious use of currently available drugs, such as corticosteroids, bronchodilators and antibiotics, and other anti-inflammatory therapies guided by sputum eosinophil and neutrophil counts, have been demonstrated to decrease exacerbations of asthma and chronic obstructive pulmonary disease, ameliorate cough, improve quality of life in patients with these diseases and is cost effective compared with treatment strategies based on guidelines that do not incorporate these measurements. Thus, it is unfortunate that this is not used more widely in the management of airway diseases, particularly in patients with severe asthma and chronic obstructive pulmonary disease who experience frequent exacerbations.

Many common diseases affecting the airways are characterized by airway inflammation. The measurement of this inflammation has a significant role in the management of these diseases. Quantitative sputum cell counts provide a measurement of the type and severity of inflammation present. Sputum cell counts are used in routine clinical practice in some centres but their use is not widespread. The present study used a standardized questionnaire to determine both patients’ and physicians’ attitudes toward the use of sputum cell counts. The use of sputum cell counts was well accepted by patients and physicians. Ninety per cent of patients were satisfied with the test. Sixty per cent of family physicians were satisfied with the test and 80% were in favour of it being funded by the government. The authors recommend more widespread use of sputum cell counts to guide the management of airway diseases.

Chronic obstructive pulmonary disease (COPD) is characterized by fixed airflow limitation and progressive decline of lung function and punctuated by occasional exacerbations. The disease pathogenesis may involve activation of the bone marrow stimulating mobilization and lung-homing of progenitor cells. We investigated the hypothesis that lower circulating numbers of vascular endothelial progenitor cells (VEPCs) are a consequence of increased lung-sequestration in COPD. Nonatopic, current or ex-smokers with diagnosed COPD and nonatopic, nonsmoking normal controls were enrolled. Blood and induced sputum extracted primitive hemopoietic progenitors (HPCs) and VEPC were enumerated by flow cytometry. Migration and adhesive responses to fibronectin were assessed. In sputum, VEPC numbers were significantly greater in COPD compared to normal controls. In blood, VEPCs were significantly lower in COPD versus normal controls. There were no differences in HPC levels between the two groups in either compartment. Functionally, there was a greater migrational responsiveness of progenitors from COPD subjects to stromal cell-derived factor-1alpha (SDF-1α) compared to normal controls. This was associated with greater numbers of CXCR4+ progenitors in sputum from COPD. Increased migrational responsiveness of progenitor cells may promote lung-homing of VEPC in COPD which may disrupt maintenance and repair of the airways and contribute to COPD disease pathogenesis.

BACKGROUND: Proximity to major roads is reported to be associated with asthma and airway hyper-responsiveness in children. Similar studies using objective measurements in adults are not available in Canada.

BACKGROUND: Sputum cell counts have identified inflammatory subtypes of bronchitis in relatively small numbers of subjects with asthma, chronic obstructive pulmonary disease (COPD) and chronic cough in research studies. The prevalence of different subtypes of bronchitis in routine clinical practice, however, has not been reported.

BACKGROUND: Air pollution caused by motor vehicle emissions has been associated with exacerbations of obstructive airway diseases; however, the nature of the resulting bronchitis has not been quantified.

BACKGROUND: Noneosinophilic asthma has been regarded as a distinct phenotype characterized by a poor response to inhaled corticosteroids (ICS).

BACKGROUND: Quantitative cell counts in sputum provide an accurate assessment of the type and severity of bronchitis.

BACKGROUND: In a four-centre trial, the use of sputum cell counts (sputum strategy [SS]) to guide treatment had resulted in fewer and less severe exacerbations without the need for a higher corticosteroid dose, compared with the use of symptoms and spirometry (clinical strategy [CS]).

The present case series describes four patients with asthma, airway hyper-responsiveness and neutrophilic bronchitis who harboured abnormal cystic fibrosis transmembrance conductance regulator (CFTR) gene mutations. It serves both to alert clinicians to consider CFTR-related disease in both young and elderly patients with persistent neutrophilic bronchitis, and to highlight the potential utility of future genetic testing for CFTR abnormalities in patients with asthma and recurrent bronchitis or pansinusitis, and the role of nebulized hypertonic saline as a therapeutic option in these patients.

Airway inflammation is considered to be the primary component contributing to the heterogeneity and severity of airway disorders. Therapeutic efficacies of diverse novel biologics targeting the inflammatory pathways are under investigation. One such target is IL-5, a type-1 cytokine that is central to the initiation and sustenance of eosinophilic airway inflammation. Over the past decade, anti-IL5 molecules have been documented to have mixed therapeutic benefits in asthmatics. Post hoc analyses of the trials reiterate the importance of identifying the IL-5-responsive patient endotypes. In fact, the currently available anti-IL5 treatments are being considered beyond asthma management; especially in clinical complications with an underlying eosinophilic pathobiology such as hypereosinophilic syndrome (HES) and eosinophilic granulomatosis and polyangitis (EGPA). In addition, closer analyses of the available data indicate alternative mechanisms of tissue eosinophilia that remain uncurbed with the current dosage and delivery platform of the anti-IL5 molecules. Keywords: Eosinophil, IL-5, Eosinophilic asthma, Hypereosinophilic syndrome (HES), Churg-strauss syndrome, Chronic bronchitis, Eosinophilic granulamatosis and polyangitis (EGPA), Chronic obstructive pulmonary disorder (COPD), Mepolizumab, Reslizumab, Benralizumab

The aryl hydrocarbon receptor (AhR), a ligand-activated transcription factor that responds to man-made environmental toxicants, has emerged as an endogenous regulator of cyclooxygenase-2 (Cox-2) by a mechanism that is poorly understood. In this study, we first used AhR-deficient (AhR(-/-) ) primary pulmonary cells, together with pharmacological tools to inhibit new RNA synthesis, to show that the AhR is a prominent factor in the destabilization of Cox-2 mRNA. The destabilization of Cox-2 mRNA and subsequent suppression of cigarette smoke-induced COX-2 protein expression by the AhR was independent of its ability to bind the dioxin response element (DRE), thereby differentiating the DRE-driven toxicological AhR pathway from its anti-inflammatory abilities. We further describe that the AhR destabilizes Cox-2 mRNA by sequestering HuR within the nucleus. The role of HuR in AhR stabilization of Cox-2 mRNA was confirmed by knockdown of HuR, which resulted in rapid Cox-2 mRNA degradation. Finally, in the lungs of AhR(-/-) mice exposed to cigarette smoke, there was little Cox-2 mRNA despite robust COX-2 protein expression, a finding that correlates with almost exclusive cytoplasmic HuR within the lungs of AhR(-/-) mice. Therefore, we propose that the AhR plays an important role in suppressing the expression of inflammatory proteins, a function that extends beyond the ability of the AhR to respond to man-made toxicants. These findings open the possibility that a DRE-independent AhR pathway may be exploited therapeutically as an anti-inflammatory target.

Cell counts in nasal secretions are not used in routine clinical practice to decide on anti-inflammatory or antimicrobial therapy. This study investigated the reproducibility, reliability (validity), and responsiveness of cell counts in blown nasal secretions with a view to implementing this in routine clinical practice. Nasal secretions were obtained from 19 subjects with allergic rhinitis on 3 days in random order (each separated by 1–2 days) by spontaneously blowing their noses (on 2 days) and by a nasal lavage by the modified Grunberg method on the 3rd day. Total and differential cell counts were performed after dispersing the solutions with dithiothreitol as described previously. At the end of the study, subjects had 1 week of open label treatment with nasal corticosteroids if they had nasal eosinophilia or an antibiotic if they had nasal neutrophilia. If the cell counts were normal, they were not treated. The proportion of eosinophil (%) was highly reproducible (intraclass correlation coefficient [ICC], 0.93), and the total cell count (X106/g) and the proportion of neutrophil (%) were modestly reproducible in blown nasal secretions (ICC, 0.46 and 0.55, respectively). The total cell count was consistently and significantly higher in the blown nasal secretions. The proportion of eosinophils (R s = 0.4; p < 0.05) and neutrophils (R s = 0.6; p < 0.05) showed modest correlation in the two types of samples. The responsiveness index for eosinophil count was 4.0 and for neutrophil count was 1.5. Total and differential cell counts can be reliably and reproducibly obtained from spontaneously blown nasal secretions. The cell counts are responsive to treatment and can help identify allergic and infective rhinosinusitis and guide therapy and are easy to implement in routine clinical practice.