Endoplasmic reticulum stress enhances the expression of TLR3-induced TSLP by airway epithelium.

Thymic stromal lymphopoietin (TSLP) is an epithelial-derived pleiotropic cytokine that regulates T-helper 2 (Th2) immune responses in the lung and plays a major role in severe uncontrolled asthma. Emerging evidence suggests a role for endoplasmic reticulum (ER) stress in the pathogenesis of asthma. In this study, we determined if ER stress and the unfolded protein response (UPR) signaling are involved in TSLP induction in the airway epithelium. For this, we treated human bronchial epithelial basal cells and differentiated primary bronchial epithelial cells with ER stress inducers and the TSLP mRNA and protein expression was determined. A series of siRNA gene knockdown experiments were conducted to determine the ER stress-induced TSLP signaling pathways. cDNA collected from asthmatic bronchial biopsies was used to determine the gene correlation between ER stress and TSLP. Our results show that ER stress signaling induces TSLP mRNA expression via the PERK-C/EBP homologous protein (CHOP) signaling pathway. AP-1 transcription factor is important in regulating this ER stress-induced TSLP mRNA induction, though ER stress alone cannot induce TSLP protein production. However, ER stress significantly enhances TLR3-induced TSLP protein secretion in the airway epithelium. TSLP and ER stress (PERK) mRNA expression positively correlates in bronchial biopsies from participants with asthma, particularly in neutrophilic asthma. In conclusion, these results suggest that ER stress primes TSLP that is then enhanced further upon TLR3 activation, which may induce severe asthma exacerbations. Targeting ER stress using pharmacological interventions may provide novel therapeutics for severe uncontrolled asthma.NEW & NOTEWORTHY TSLP is an epithelial-derived cytokine and a key regulator in the pathogenesis of severe uncontrolled asthma. We demonstrate a novel mechanism by which endoplasmic reticulum stress signaling upregulates airway epithelial TSLP mRNA expression via the PERK-CHOP signaling pathway and enhances TLR3-mediated TSLP protein secretion.

Faecal microbial transfer and complex carbohydrates mediate protection against COPD.

OBJECTIVE: Chronic obstructive pulmonary disease (COPD) is a major cause of global illness and death, most commonly caused by cigarette smoke. The mechanisms of pathogenesis remain poorly understood, limiting the development of effective therapies. The gastrointestinal microbiome has been implicated in chronic lung diseases via the gut-lung axis, but its role is unclear. DESIGN: Using an in vivo mouse model of cigarette smoke (CS)-induced COPD and faecal microbial transfer (FMT), we characterised the faecal microbiota using metagenomics, proteomics and metabolomics. Findings were correlated with airway and systemic inflammation, lung and gut histopathology and lung function. Complex carbohydrates were assessed in mice using a high resistant starch diet, and in 16 patients with COPD using a randomised, double-blind, placebo-controlled pilot study of inulin supplementation. RESULTS: FMT alleviated hallmark features of COPD (inflammation, alveolar destruction, impaired lung function), gastrointestinal pathology and systemic immune changes. Protective effects were additive to smoking cessation, and transfer of CS-associated microbiota after antibiotic-induced microbiome depletion was sufficient to increase lung inflammation while suppressing colonic immunity in the absence of CS exposure. Disease features correlated with the relative abundance of Muribaculaceae, Desulfovibrionaceae and Lachnospiraceae family members. Proteomics and metabolomics identified downregulation of glucose and starch metabolism in CS-associated microbiota, and supplementation of mice or human patients with complex carbohydrates improved disease outcomes. CONCLUSION: The gut microbiome contributes to COPD pathogenesis and can be targeted therapeutically.

Clinical Subtypes of Neutrophilic Asthma: A Cluster Analysis From Australasian Severe Asthma Network.

BACKGROUND: Clinical heterogeneity may exist within asthma subtypes defined by inflammatory markers. However, the heterogeneity of neutrophilic asthma (NA) remains largely unexplored. OBJECTIVE: To explore potential clusters and the stability of NA. METHODS: Participants with NA from the Australasian Severe Asthma Network underwent a multidimensional assessment. They were then asked to participate in a 12-month longitudinal cohort study. We explored potential clusters using a hierarchical cluster analysis and validated the differential future risk of asthma exacerbations in the identified clusters. A decision tree analysis was developed to predict cluster assignments. Finally, the stability of prespecified clusters was examined within 1 month. RESULTS: Three clusters were identified in 149 patients with NA. Cluster 1 (n = 99; 66.4%) was characterized by female-predominant nonsmokers with well-controlled NA, cluster 2 (n = 16; 10.7%) by individuals with comorbid anxiety/depressive symptoms with poorly controlled NA, and cluster 3 by older male smokers with late-onset NA. Cluster 2 had a greater proportion of participants with severe exacerbations (P = .005), hospitalization (P = .010), and unscheduled visits (P = .013) and a higher number of emergency room visits (P = .039) than that of the other two clusters. The decision tree assigned 92.6% of participants correctly. Most participants (87.5%; n = 7) in cluster 2 had a stable NA phenotype, whereas participants of clusters 1 and 3 had variable phenotypes. CONCLUSIONS: We identified three clinical clusters of NA, in which cluster 2 represents an uncontrolled and stable NA subtype with an elevated risk of exacerbations. These findings have clinical implications for the management of NA.

Ventilation Heterogeneity Is a Treatable Trait in Severe Asthma.

BACKGROUND: Ventilation heterogeneity (VH) is a feature of asthma and indicates small airway disease. Nuclear imaging methods assess VH, which can facilitate clinical diagnosis and further our understanding of disease aetiology. OBJECTIVE: We sought to assess VH in severe eosinophilic asthma (SEA) using ventilation/perfusion single-photon emission computed tomography (V/P SPECT), and to assess its use as an objective test of the effect of biologic treatment for ventilation defects in SEA. METHODS: Adults (≥18 y) with severe asthma were recruited to participate in a cross-sectional observational study. Participants underwent a clinical assessment and V/P SPECT CT using Technegas as the ventilation agent. Measures were repeated for a nested before-after treatment study in people with SEA commencing biologics. RESULTS: A total of 62 participants with severe asthma were recruited. From this, 38 participants with SEA were included in the before-after study. The VH was associated with clinical variables such as lung function impairment and significantly improved after monoclonal antibody treatment in the severe asthma group. The changes in VH correlated with change in post bronchodilator forced expiratory volume in 1 second (FEV1) %predicted (r = -0.503; P = .001) and post bronchodilator FEV1/FVC (forced vital capacity) (r = -0.415; P = .01). CONCLUSIONS: The VH is clinically significant, measurable, and treatable, which establishes VH as a treatable trait in severe asthma.

Airway inflammatory changes in the lungs of patients with asthma-COPD overlap (ACO): a bronchoscopy endobronchial biopsy study.

BACKGROUND: Although asthma and chronic obstructive pulmonary disease (COPD) are two distinct chronic airway inflammatory diseases, they often co-exist in a patient and the condition is referred to as asthma-COPD overlap (ACO). Lack of evidence regarding the inflammatory cells in ACO airways has led to their poor prognosis and treatment. The objective of this endobronchial biopsy (EBB) study was to enumerate inflammatory cellular changes in the airway wall of ACO compared with asthma, COPD current smokers (CS) and ex-smokers (ES), normal lung function smokers (NLFS), and non-smoker controls (HC). METHODS: EBB tissues from 74 patients were immunohistochemically stained for macrophages, mast cells, eosinophils, neutrophils, CD8+ T-cells and CD4+ T-cells. The microscopic images of stained tissues were evaluated in the epithelium, reticular basement membrane (RBM) cells/mm RBM length, and lamina propria (LP) cells/mm2 up to a depth of 120 µM using the image analysis software Image-Pro Plus 7.0. The observer was blinded to the images and disease diagnosis. Statistical analysis was performed using GraphPad Prism v9. RESULTS: The tissue macrophages in ACO were substantially higher in the epithelium and RBM than in HC (P < 0.001 for both), COPD-ES (P < 0.001 for both), and -CS (P < 0.05 and < 0.0001, respectively). The ACO LP macrophages were significantly higher in number than COPD-CS (P < 0.05). The mast cell numbers in ACO were lower than in NLFS (P < 0.05) in the epithelium, lower than COPD (P < 0.05) and NLFS (P < 0.001) in RBM; and lower than  HC (P < 0.05) in LP. We noted lower eosinophils in ACO LP than HC (P < 0.05) and the lowest neutrophils in both ACO and asthma. Furthermore, CD8+ T-cell numbers increased in the ACO RBM than HC (P < 0.05), COPD-ES (P < 0.05), and NLFS (P < 0.01); however, they were similar in number in epithelium and LP across groups. CD4+ T-cells remained lower in number across all regions and groups. CONCLUSION: These results suggest that the ACO airway tissue inflammatory cellular profile differed from the contributing diseases of asthma and COPD with a predominance of macrophages.

Upper Respiratory Tract OC43 Infection Model for Investigating Airway Immune-Modifying Therapies.

Respiratory virus infections initiate and transmit from the upper respiratory tract (URT). Coronaviruses, including OC43, are a major cause of respiratory infection and disease. Failure to mount an effective antiviral immune response in the nasal mucosa increases the risk of severe disease and person-to-person transmission, highlighting the need for URT infection models to support the development of nasal treatments that improve coronavirus antiviral immunity. We aimed to determine if OC43 productively infected the mouse URT and would therefore be a suitable model to assess the efficacy and mechanism of action of nasal-targeting immune-modifying treatments. We administered OC43 via intranasal inoculation to wild-type Balb/c mice and assessed virus airway tropism (by comparing total respiratory tract vs. URT-only virus exposure) and characterized infection-induced immunity by quantifying specific antiviral cytokines and performing gene array assessment of immune genes. We then assessed the effect of immune-modulating therapies, including an immune-stimulating TLR2/6 agonist (INNA-X) and the immune-suppressing corticosteroid fluticasone propionate (FP). OC43 replicated in nasal respiratory epithelial cells, with peak viral RNA observed 2 days after infection. Prophylactic treatment with INNA-X accelerated expression of virus-induced IFN-λ and IFN-stimulated genes. In contrast, intranasal FP treatment increased nasal viral load by 2.4 fold and inhibited virus-induced IFN and IFN-stimulated gene expression. Prior INNA-X treatment reduced the immune-suppressive effect of FP. We demonstrate that the mouse nasal epithelium is permissive to OC43 infection and strengthen the evidence that TLR2 activation is a ß-coronavirus innate immune determinant and therapeutic target.

Comparison of commercially available differentiation media on cell morphology, function, and anti-viral responses in conditionally reprogrammed human bronchial epithelial cells.

Primary air liquid interface (ALI) cultures of bronchial epithelial cells are used extensively to model airway responses. A recent advance is the development of conditional reprogramming that enhances proliferative capability. Several different media and protocols are utilized, yet even subtle differences may influence cellular responses. We compared the morphology and functional responses, including innate immune responses to rhinovirus infection in conditionally reprogrammed primary bronchial epithelial cells (pBECs) differentiated using two commonly used culture media. pBECs collected from healthy donors (n = 5) were CR using g-irradiated 3T3 fibroblasts and Rho Kinase inhibitor. CRpBECs were differentiated at ALI in either PneumaCult (PN-ALI) or bronchial epithelial growth medium (BEGM)-based differentiation media (BEBM:DMEM, 50:50, Lonza)-(AB-ALI) for 28 days. Transepithelial electrical resistance (TEER), immunofluorescence, histology, cilia activity, ion channel function, and expression of cell markers were analyzed. Viral RNA was assessed by RT-qPCR and anti-viral proteins quantified by LEGENDplex following Rhinovirus-A1b infection. CRpBECs differentiated in PneumaCult were smaller and had a lower TEER and cilia beat frequency compared to BEGM media. PneumaCult media cultures exhibited increased FOXJ1 expression, more ciliated cells with a larger active area, increased intracellular mucins, and increased calcium-activated chloride channel current. However, there were no significant changes in viral RNA or host antiviral responses. There are distinct structural and functional differences in pBECs cultured in the two commonly used ALI differentiation media. Such factors need to be taken into consideration when designing CRpBECs ALI experiments for specific research questions.

Inflammatory and antiviral responses to influenza A virus infection are dysregulated in pregnant mice with allergic airway disease.

Influenza A virus (IAV) infections are increased during pregnancy especially with asthma as a comorbidity, leading to asthma exacerbations, secondary bacterial infections, intensive care unit admissions, and mortality. We aimed to define the processes involved in increased susceptibility and severity of IAV infections during pregnancy, especially with asthma. We sensitized mice to house dust mite (HDM), induced pregnancy, and challenged with HDM to induce allergic airway disease (AAD). At midpregnancy, we induced IAV infection. We assessed viral titers, airway inflammation, lung antiviral responses, mucus hypersecretion, and airway hyperresponsiveness (AHR). During early IAV infection, pregnant mice with AAD had increased mRNA expression of the inflammatory markers Il13 and IL17 and reduced mRNA expression of the neutrophil chemoattractant marker Kc. These mice had increased mucous hyperplasia and increased AHR. miR155, miR574, miR223, and miR1187 were also reduced during early infection, as was mRNA expression of the antiviral ß-defensins, Bd1, Bd2, and Spd and IFNs, Ifnα, Ifnß, and Ifnλ. During late infection, Il17 was still increased as was eosinophil infiltration in the lungs. mRNA expression of Kc was reduced, as was neutrophil infiltration and mRNA expression of the antiviral markers Ifnß, Ifnλ, and Ifnγ and Ip10, Tlr3, Tlr9, Pkr, and Mx1. Mucous hyperplasia was still significantly increased as was AHR. Early phase IAV infection in pregnancy with asthma heightens underlying inflammatory asthmatic phenotype and reduces antiviral responses.NEW & NOTEWORTHY Influenza A virus (IAV) infection during pregnancy with asthma is a major health concern leading to increased morbidity for both mother and baby. Using murine models, we show that IAV infection in pregnancy with allergic airway disease is associated with impaired global antiviral and antimicrobial responses, increased lung inflammation, mucus hypersecretion, and airway hyperresponsiveness (AHR). Targeting specific ß-defensins or microRNAs (miRNAs) may prove useful in future treatments for IAV infection during pregnancy.

The Complex Association between COPD and COVID-19.

Chronic obstructive pulmonary disease (COPD) is significant cause of morbidity and mortality worldwide. There is mounting evidence suggesting that COPD patients are at increased risk of severe COVID-19 outcomes; however, it remains unclear whether they are more susceptible to acquiring SARS-CoV-2 infection. In this comprehensive review, we aim to provide an up-to-date perspective of the intricate relationship between COPD and COVID-19. We conducted a thorough review of the literature to examine the evidence regarding the susceptibility of COPD patients to COVID-19 infection and the severity of their disease outcomes. While most studies have found that pre-existing COPD is associated with worse COVID-19 outcomes, some have yielded conflicting results. We also discuss confounding factors such as cigarette smoking, inhaled corticosteroids, and socioeconomic and genetic factors that may influence this association. Furthermore, we review acute COVID-19 management, treatment, rehabilitation, and recovery in COPD patients and how public health measures impact their care. In conclusion, while the association between COPD and COVID-19 is complex and requires further investigation, this review highlights the need for careful management of COPD patients during the pandemic to minimize the risk of severe COVID-19 outcomes.

Mechanical forces suppress antiviral innate immune responses from asthmatic airway epithelial cells following rhinovirus infection.

Bronchoconstriction is the main physiological event in asthma, which leads to worsened clinical symptoms and generates mechanical stress within the airways. Virus infection is the primary cause of exacerbations in people with asthma, however, the impact that bronchoconstriction itself on host antiviral responses and viral replication is currently not well understood. Here we demonstrate how mechanical forces generated during bronchoconstriction may suppress antiviral responses at the airway epithelium without any difference in viral replication. Primary bronchial epithelial cells from donors with asthma were differentiated at the air-liquid interface. Differentiated cells were apically compressed (30 cmH2O) for 10 min every hour for 4 days to mimic bronchoconstriction. Two asthma disease models were developed with the application of compression, either before ("poor asthma control model," n = 7) or following ("exacerbation model," n = 4) rhinovirus (RV) infection. Samples were collected at 0, 24, 48, 72, and 96 h postinfection (hpi). Viral RNA, interferon (IFN)-ß, IFN-λ, and host defense antiviral peptide gene expressions were measured along with IFN-ß, IFN-λ, TGF-ß2, interleukin-6 (IL-6), and IL-8 protein expression. Apical compression significantly suppressed RV-induced IFN-ß protein from 48 hpi and IFN-λ from 72 hpi in the poor asthma control model. There was a nonsignificant reduction of both IFN-ß and IFN-λ proteins from 48 hpi in the exacerbation model. Despite reductions in antiviral proteins, there was no significant change in viral replication in either model. Compressive stress mimicking bronchoconstriction inhibits antiviral innate immune responses from asthmatic airway epithelial cells when applied before RV infection.NEW & NOTEWORTHY Bronchoconstriction is the main physiological event in asthma, which leads to worsened clinical symptoms and generates mechanical stress within the airways. Virus infection is the primary cause of exacerbations in people with asthma, however, the impact of bronchoconstriction on host antiviral responses and viral replication is unknown. We developed two disease models, in vitro, and found suppressed IFN response from cells following the application of compression and RV-A1 infection. This explains why people with asthma have deficient IFN response.

Severe asthma ILC2s demonstrate enhanced proliferation that is modified by biologics.

BACKGROUND AND OBJECTIVE: Type 2 (T2) innate lymphoid cells (ILC2s) contribute to airway inflammation and disease in asthma. We hypothesize that ILC2s isolated from people with severe allergic and eosinophilic asthma would exhibit an enhanced T2 inflammatory activity that would be altered following treatment with mepolizumab and omalizumab. We compare peripheral blood (PB) isolated ILC2's proliferative capacity, IL-5 and IL-13 secretion and phenotype between healthy without asthma (HC), non-asthma allergic (NAA), mild asthma (MA) and severe allergic and eosinophilic asthma (SA) subjects. We then determined the impact of 6 months treatment with either mepolizumab or omalizumab on ILC2s physiology of SA subjects. METHODS: ILC2s were sorted and cultured in the presence of IL-2, IL-25, IL-33 and thymic stromal lymphopoietin (TSLP) for 14 days. ILC2s proliferation, phenotypes and functions were assessed using flowcytometry. The ILC2s response was then reassessed following clinically successful treatment of SA subjects with mepolizumab and omalizumab. RESULTS: SA ILC2s demonstrated increased proliferative capacity, TSLP receptor (TSLPR), GATA3 and NFATc1 protein expressions and increased IL-5 and IL-13 release. ILC2s were also capable of releasing IL-6 in response to stimulation. Mepolizumab treatment reduced ILC2s proliferative capacity and expression of TSLPR, GATA3 and NFATc1. Both mepolizumab and omalizumab were associated with reduced ILC2s release of IL-5 and IL-13, only mepolizumab reduced IL-6. CONCLUSION: ILC2s from severe allergic and eosinophilic asthma demonstrated an active phenotype typified by increased proliferation, TSLPR, GATA3 and NFATc1 expression and increased IL-5, IL-13 and IL-6 release. Mepolizumab reduced markers of ILC2s activation.

Type 2 Immunity and Its Impact on COVID-19 Infection in the Airways.

Type 2 immune responses are characterized by elevated type 2 cytokines and blood eosinophilia. Emerging evidence suggests that people with chronic type 2 inflammatory lung diseases are not particularly susceptible to SARS-CoV-2 infection. Intriguingly, recent in vitro, ex vivo research demonstrates type 2 cytokines, particularly IL-13, reduce the risk of SARS-CoV-2 infection in the airway epithelium. IL-13 treatment in airway epithelial cells followed by SARS-CoV-2 diminished viral entry, replication, spread, and cell death. IL-13 reduces the expression of the angiotensin-converting enzyme 2 (ACE2) receptor in the airway epithelium and transmembrane serine protease 2 (TMPRSS2), particularly in ciliated cells. It also alters the cellular composition toward a secretory-cell-rich phenotype reducing total ciliated cells and, thus, reducing viral tropism. IL-13 enhances Muc5ac mucin and glycocalyx secretion in the periciliary layer, which acts as a physical barrier to restrict virus attachment. Moreover, type 2 airway immune cells, such as M2 alveolar macrophages, CD4+ tissue-resident memory T cells, and innate lymphoid 2 cells, may also rescue type 2 airways from SARS-CoV-2-induced adverse effects. In this review, we discuss recent findings that demonstrate how type 2 immunity alters immune responses against SARS-CoV-2 and its consequences on COVID-19 pathogenesis.

Regional variation in prevalence of difficult-to-treat asthma and oral corticosteroid use for patients in Australia: heat map analysis.

BACKGROUND: In Australia, the regional prevalence of difficult-to-treat asthma is unknown. We aimed to describe regional variation in difficult-to-treat asthma prevalence and oral corticosteroid (OCS) use. METHODS: In this retrospective, observational, longitudinal study using data from March 2018-February 2019 in the NostraData longitudinal database, prescriptions dispensed for obstructive airway disease were processed through a high-level algorithm to identify patients with asthma. Difficult-to-treat asthma was defined by ≥2 high-dosage inhaled corticosteroids plus long-acting beta-agonist prescriptions over 6 months. Patients who additionally received OCS prescriptions sufficient to treat ≥2 exacerbations over 6 months were classified as having uncontrolled difficult-to-treat asthma. Patient-level data were analyzed across 340 geographic areas in Australia to determine regional prevalence of difficult-to-treat asthma, uncontrolled difficult-to-treat asthma, and OCS use. RESULTS: Of 1 851 129 people defined as having asthma, 440 800 (24%) were classified as having difficult-to-treat disease. Of those difficult-to-treat asthma patients, 96 338 (22%) were considered to have uncontrolled disease. Between 29% and 48% of patients had difficult-to-treat asthma in 49 geographic areas, most frequently located in Western Australia. Between 26% and 67% of patients had uncontrolled difficult-to-treat asthma in 29 geographic areas (mostly in Eastern Australia). Overall, a wide variability of asthma severity and control was observed among regions. CONCLUSIONS: Despite global and national guidelines, regional differences in the prevalence of difficult-to-treat asthma and uncontrolled difficult-to-treat asthma and OCS use exist in Australia. Understanding these regional variations should inform policy and target management in the areas with the greatest unmet need.

The establishment of COPD organoids to study host-pathogen interaction reveals enhanced viral fitness of SARS-CoV-2 in bronchi.

Chronic obstructive pulmonary disease (COPD) is characterised by airflow limitation and infective exacerbations, however, in-vitro model systems for the study of host-pathogen interaction at the individual level are lacking. Here, we describe the establishment of nasopharyngeal and bronchial organoids from healthy individuals and COPD that recapitulate disease at the individual level. In contrast to healthy organoids, goblet cell hyperplasia and reduced ciliary beat frequency were observed in COPD organoids, hallmark features of the disease. Single-cell transcriptomics uncovered evidence for altered cellular differentiation trajectories in COPD organoids. SARS-CoV-2 infection of COPD organoids revealed more productive replication in bronchi, the key site of infection in severe COVID-19. Viral and bacterial exposure of organoids induced greater pro-inflammatory responses in COPD organoids. In summary, we present an organoid model that recapitulates the in vivo physiological lung microenvironment at the individual level and is amenable to the study of host-pathogen interaction and emerging infectious disease.

Neisseria species as pathobionts in bronchiectasis.

Neisseria species are frequently identified in the bronchiectasis microbiome, but they are regarded as respiratory commensals. Using a combination of human cohorts, next-generation sequencing, systems biology, and animal models, we show that bronchiectasis bacteriomes defined by the presence of Neisseria spp. associate with poor clinical outcomes, including exacerbations. Neisseria subflava cultivated from bronchiectasis patients promotes the loss of epithelial integrity and inflammation in primary epithelial cells. In vivo animal models of Neisseria subflava infection and metabolipidome analysis highlight immunoinflammatory functional gene clusters and provide evidence for pulmonary inflammation. The murine metabolipidomic data were validated with human Neisseria-dominant bronchiectasis samples and compared with disease in which Pseudomonas-, an established bronchiectasis pathogen, is dominant. Metagenomic surveillance of Neisseria across various respiratory disorders reveals broader importance, and the assessment of the home environment in bronchiectasis implies potential environmental sources of exposure. Thus, we identify Neisseria species as pathobionts in bronchiectasis, allowing for improved risk stratification in this high-risk group.

The Effects of Increasing Fruit and Vegetable Intake in Children with Asthma on the Modulation of Innate Immune Responses.

Children with asthma are at risk of acute exacerbations triggered mainly by viral infections. A diet high in fruit and vegetables (F&V), a rich source of carotenoids, may improve innate immune responses in children with asthma. Children with asthma (3−11 years) with a history of exacerbations and low F&V intake (≤3 serves/d) were randomly assigned to a high F&V diet or control (usual diet) for 6 months. Outcomes included respiratory-related adverse events and in-vitro cytokine production in peripheral blood mononuclear cells (PBMCs), treated with rhinovirus-1B (RV1B), house dust mite (HDM) and lipopolysaccharide (LPS). During the trial, there were fewer subjects with ≥2 asthma exacerbations in the high F&V diet group (n = 22) compared to the control group (n = 25) (63.6% vs. 88.0%, p = 0.049). Duration and severity of exacerbations were similar between groups. LPS-induced interferon (IFN)-γ and IFN-λ production showed a small but significant increase in the high F&V group after 3 months compared to baseline (p < 0.05). Additionally, RV1B-induced IFN-λ production in PBMCs was positively associated with the change in plasma lycopene at 6 months (rs = 0.35, p = 0.015). A high F&V diet reduced asthma-related illness and modulated in vitro PBMC cytokine production in young children with asthma. Improving diet quality by increasing F&V intake could be an effective non-pharmacological strategy for preventing asthma-related illness by enhancing children's innate immune responses.

Differential airway remodeling changes were observed in patients with asthma COPD overlap compared to patients with asthma and COPD alone.

Management of patients with asthma COPD overlap (ACO) is clinically challenging due to insufficient evidence of pathological changes in these patients. In this cross-sectional study, we evaluated airway remodeling in endobronchial biopsies from a total of 90 subjects, which included 12 ACO, 14 patients with asthma, 12 COPD exsmokers (ES), 11 current smokers (CS), 28 healthy controls (HC), and 13 normal lung function smokers (NLFS). Tissue was stained with Masson's trichrome. Epithelium, goblet cells, reticular basement membrane (RBM), cellularity, lamina propria (LP), and smooth muscle (SM) changes were measured using Image-Pro Plus v7 software. Differential airway remodeling pattern was seen in patients with ACO. A limited change was noted in the ACO epithelium compared with other pathological groups. RBM was substantially thicker in patients with ACO than in HC (P < 0.0002) and tended to be thicker than in patients with asthma and NLFS. The total RBM cells were higher in ACO than in the HC (P < 0.0001), COPD-CS (P = 0.0559), -ES (P = 0.0345), and NLFS (P < 0.0002), but did not differ from patients with asthma. Goblet cells were higher in the ACO than in the HC (P = 0.0028) and COPD-ES (P = 0.0081). The total LP cells in ACO appeared to be higher than in HC, COPD-CS, and NLFS but appeared to be lower than in patients with asthma. Finally, SM area was significantly lower in the ACO than in patients with asthma (P = 0.001), COPD-CS (=0.0290), and NLFS (P = 0.0011). This first comprehensive study suggests that patients with ACO had distinguishable tissue remodeling that appeared to be more severe than patients with asthma and COPD. This study will help in informed decision-making for better patient management in clinical practice.

Conditionally reprogrammed asthmatic bronchial epithelial cells express lower FOXJ1 at terminal differentiation and lower IFNs following RV-A1 infection.

Primary bronchial epithelial cells (pBECs) obtained from donors have limited proliferation capacity. Recently, conditional reprogramming (CR) technique has overcome this and has provided the potential for extended passaging and subsequent differentiation of cells at air-liquid interface (ALI). However, there has been no donor-specific comparison of cell morphology, baseline gene expression, barrier function, and antiviral responses compared with their "parent" pBECs, especially cells obtained from donors with asthma. We, therefore, collected and differentiated pBECs at ALI from mild donors with asthma (n = 6) for the parent group. The same cells were conditionally reprogrammed and later differentiated at ALI. Barrier function was measured during the differentiation phase. Morphology and baseline gene expression were compared at terminal differentiation. Viral replication kinetics and antiviral responses were assessed following rhinovirus (RV) infection over 96 h. Barrier function during the differentiation phase and cell structural morphology at terminal differentiation appear similar in both parent and CR groups, however, there were elongated cell structures superficial to basal cells and significantly lower FOXJ1 expression in CR group. IFN gene expression was also significantly lower in CR group compared with parent asthma group following RV infection. The CR technique is a beneficial tool to proliferate pBECs over extended passages. Considering lower FOXJ1 expression, viral replication kinetics and antiviral responses, a cautious approach should be taken while choosing CR cells for experiments. In addition, as lab-to-lab cell culture techniques vary, the most appropriate technique must be utilized to best match individual cell functions and morphologies to address specific research questions and experimental reproducibility across the labs.

Airway and parenchymal transcriptomics in a novel model of asthma and COPD overlap.

BACKGROUND: Asthma and chronic obstructive pulmonary disease (COPD) are common chronic respiratory diseases, and some patients have overlapping disease features, termed asthma-COPD overlap (ACO). Patients characterized with ACO have increased disease severity; however, the mechanisms driving this have not been widely studied. OBJECTIVES: This study sought to characterize the phenotypic and transcriptomic features of experimental ACO in mice induced by chronic house dust mite antigen and cigarette smoke exposure. METHODS: Female BALB/c mice were chronically exposed to house dust mite antigen for 11 weeks to induce experimental asthma, cigarette smoke for 8 weeks to induce experimental COPD, or both concurrently to induce experimental ACO. Lung inflammation, structural changes, and lung function were assessed. RNA-sequencing was performed on separated airway and parenchyma lung tissues to assess transcriptional changes. Validation of a novel upstream driver SPI1 in experimental ACO was assessed using the pharmacological SPI1 inhibitor, DB2313. RESULTS: Experimental ACO recapitulated features of both asthma and COPD, with mixed pulmonary eosinophilic/neutrophilic inflammation, small airway collagen deposition, and increased airway hyperresponsiveness. Transcriptomic analysis identified common and distinct dysregulated gene clusters in airway and parenchyma samples in experimental asthma, COPD, and ACO. Upstream driver analysis revealed increased expression of the transcription factor Spi1. Pharmacological inhibition of SPI1 using DB2313, reduced airway remodeling and airway hyperresponsiveness in experimental ACO. CONCLUSIONS: A new experimental model of ACO featuring chronic dual exposures to house dust mite and cigarette smoke mimics key disease features observed in patients with ACO and revealed novel disease mechanisms, including upregulation of SPI1, that are amenable to therapy.