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.

PAT in the ER for Transmembrane Protein Folding.

Integral membrane proteins (IMPs) have crucial roles in many cellular processes. A novel intramembrane chaperone complex, recently elucidated by Chitwood and Hedge, provides mechanistic insight of IMP biogenesis and folding, illustrating how IMPs with multiple transmembrane domains (TMDs) are assembled within the endoplasmic reticulum (ER) membrane.

Immune checkpoints are suppressed during pregnancy following influenza A virus infection.

Influenza A virus (IAV) infection is a major health risk during pregnancy. Whilst vaccination and antiviral agents are widely employed and reduce IAV-induced symptoms, they are not sufficient to control IAV infections in pregnancy, especially during pandemics. Respiratory viruses like IAV, exploit immune alterations that occur during pregnancy, including the upregulation of immune checkpoint proteins (ICPs) like PDL1, PD1 and CTLA4. We hypothesize that blocking expression of PDL1 on innate immune cells will improve maternal immunity following IAV infection. We utilized murine models of IAV infection during pregnancy with and without treatment with the immune checkpoint inhibitor (ICI), a-PDL1. Pregnant and non-pregnant mice were infected with mouse adapted IAV (A/PR/8) and assessed at 3 days post infection (3dpi). Lung cells were analyzed using flow cytometry. Lung mRNA expression of inflammatory and antiviral markers and histology was measured. Protein concentrations of inflammatory and antiviral markers, as well as viral titers was measured from lung bronchiolar lavage fluid (BALF). Lung function was also assessed. Following IAV infection, immune cells from pregnant mice had significant increases in the ICPs, PDL1, PD1 and CTLA4. a-PDL1 treatment effectively suppressed these ICPs and increased the activation marker, CD86. A-PDL1 treatment also reduced lung inflammatory cell infiltration and viral titres, increased antiviral responses, and improved lung function. Overall, IAV infection in pregnancy activates key inhibitory ICPs, leading to worsened disease outcomes. a-PDL1 treatment during IAV infection in pregnancy is an effective method to reduce ICP expression and improve overall immune cell responses.

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.

Inflammation-induced loss of CFTR-expressing airway ionocytes in non-eosinophilic asthma.

BACKGROUND AND OBJECTIVE: Severe asthma is a heterogeneous disease with subtype classification according to dominant airway infiltrates, including eosinophilic (Type 2 high), or non-eosinophilic asthma. Non-eosinophilic asthma is further divided into paucigranulocytic or neutrophilic asthma characterized by elevated neutrophils, and mixed Type 1 and Type 17 cytokines in the airways. Severe non-eosinophilic asthma has few effective treatments and many patients do not qualify for biologic therapies. The cystic fibrosis transmembrane conductance regulator (CFTR) is dysregulated in multiple respiratory diseases including cystic fibrosis and chronic obstructive pulmonary disease and has proven a valuable therapeutic target. We hypothesized that the CFTR may also play a role in non-eosinophilic asthma. METHODS: Patient-derived human bronchial epithelial cells (hBECs) were isolated and differentiated at the air-liquid interface. Single cell RNA-sequencing (scRNAseq) was used to identify epithelial cell subtypes and transcriptional activity. Ion transport was investigated with Ussing chambers and immunofluorescent quantification of ionocyte abundance in human airway epithelial cells and murine models of asthma. RESULTS: We identified that hBECs from patients with non-eosinophilic asthma had reduced CFTR function, and did not differentiate into CFTR-expressing ionocytes compared to those from eosinophilic asthma or healthy donors. Similarly, ionocytes were also diminished in the airways of a murine model of neutrophilic-dominant but not eosinophilic asthma. Treatment of hBECs from healthy donors with a neutrophilic asthma-like inflammatory cytokine mixture led to a reduction in ionocytes. CONCLUSION: Inflammation-induced loss of CFTR-expressing ionocytes in airway cells from non-eosinophilic asthma may represent a key feature of disease pathogenesis and a novel drug target.

IL-33 Expression Is Lower in Current Smokers at both Transcriptomic and Protein Levels.

Rationale: IL-33 is a proinflammatory cytokine thought to play a role in the pathogenesis of asthma and chronic obstructive pulmonary disease (COPD). A recent clinical trial using an anti-IL-33 antibody showed a reduction in exacerbation and improved lung function in ex-smokers but not current smokers with COPD. Objectives: This study aimed to understand the effects of smoking status on IL-33. Methods: We investigated the association of smoking status with the level of gene expression of IL-33 in the airways in eight independent transcriptomic studies of lung airways. Additionally, we performed Western blot analysis and immunohistochemistry for IL-33 in lung tissue to assess protein levels. Measurements and Main Results: Across the bulk RNA-sequencing datasets, IL-33 gene expression and its signaling pathway were significantly lower in current versus former or never-smokers and increased upon smoking cessation (P < 0.05). Single-cell sequencing showed that IL-33 is predominantly expressed in resting basal epithelial cells and decreases during the differentiation process triggered by smoke exposure. We also found a higher transitioning of this cellular subpopulation into a more differentiated cell type during chronic smoking, potentially driving the reduction of IL-33. Protein analysis demonstrated lower IL-33 levels in lung tissue from current versus former smokers with COPD and a lower proportion of IL-33-positive basal cells in current versus ex-smoking controls. Conclusions: We provide strong evidence that cigarette smoke leads to an overall reduction in IL-33 expression in transcriptomic and protein level, and this may be due to the decrease in resting basal cells. Together, these findings may explain the clinical observation that a recent antibody-based anti-IL-33 treatment is more effective in former than current smokers with COPD.

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.

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.

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.

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.

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.

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.

Increased SARS-CoV-2 Infection, Protease, and Inflammatory Responses in Chronic Obstructive Pulmonary Disease Primary Bronchial Epithelial Cells Defined with Single-Cell RNA Sequencing.

Rationale: Patients with chronic obstructive pulmonary disease (COPD) develop more severe coronavirus disease (COVID-19); however, it is unclear whether they are more susceptible to severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection and what mechanisms are responsible for severe disease. Objectives: To determine whether SARS-CoV-2 inoculated primary bronchial epithelial cells (pBECs) from patients with COPD support greater infection and elucidate the effects and mechanisms involved. Methods: We performed single-cell RNA sequencing analysis on differentiated pBECs from healthy subjects and patients with COPD 7 days after SARS-CoV-2 inoculation. We correlated changes with viral titers, proinflammatory responses, and IFN production. Measurements and Main Results: Single-cell RNA sequencing revealed that COPD pBECs had 24-fold greater infection than healthy cells, which was supported by plaque assays. Club/goblet and basal cells were the predominant populations infected and expressed mRNAs involved in viral replication. Proteases involved in SARS-CoV-2 entry/infection (TMPRSS2 and CTSB) were increased, and protease inhibitors (serpins) were downregulated more so in COPD. Inflammatory cytokines linked to COPD exacerbations and severe COVID-19 were increased, whereas IFN responses were blunted. Coexpression analysis revealed a prominent population of club/goblet cells with high type 1/2 IFN responses that were important drivers of immune responses to infection in both healthy and COPD pBECs. Therapeutic inhibition of proteases and inflammatory imbalances reduced viral titers and cytokine responses, particularly in COPD pBECs. Conclusions: COPD pBECs are more susceptible to SARS-CoV-2 infection because of increases in coreceptor expression and protease imbalances and have greater inflammatory responses. A prominent cluster of IFN-responsive club/goblet cells emerges during infection, which may be important drivers of immunity. Therapeutic interventions suppress SARS-CoV-2 replication and consequent inflammation.

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.

Pathogenesis, clinical features of asthma COPD overlap, and therapeutic modalities.

Both asthma and COPD are heterogeneous diseases identified by characteristic symptoms and functional abnormalities, with airway obstruction common in both diseases. Asthma COPD overlap (ACO) does not define a single disease but is a descriptive term for clinical use that includes several overlapping clinical phenotypes of chronic airways disease with different underlying mechanisms. This literature review was initiated to describe published studies, identify gaps in knowledge, and propose future research goals regarding the disease pathology of ACO, especially the airway remodeling changes and inflammation aspects. Airway remodeling occurs in asthma and COPD, but there are differences in the structures affected and the prime anatomic site at which they occur. Reticular basement membrane thickening and cellular infiltration with eosinophils and T-helper (CD4+) lymphocytes are prominent features of asthma. Epithelial squamous metaplasia, airway wall fibrosis, emphysema, bronchoalveolar lavage (BAL) neutrophilia, and (CD8+) T-cytotoxic lymphocyte infiltrations in the airway wall are features of COPD. There is no universally accepted definition of ACO, nor are there clearly defined pathological characteristics to differentiate from asthma and COPD. Understanding etiological concepts within the purview of inflammation and airway remodeling changes in ACO would allow better management of these patients.

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.

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.

Endoplasmic reticulum-unfolded protein response signalling is altered in severe eosinophilic and neutrophilic asthma.

INTRODUCTION: The significance of endoplasmic reticulum (ER) stress in asthma is unclear. Here, we demonstrate that ER stress and the unfolded protein response (UPR) are related to disease severity and inflammatory phenotype. METHODS: Induced sputum (n=47), bronchial lavage (n=23) and endobronchial biopsies (n=40) were collected from participants with asthma with varying disease severity, inflammatory phenotypes and from healthy controls. Markers for ER stress and UPR were assessed. These markers were also assessed in established eosinophilic and neutrophilic murine models of asthma. RESULTS: Our results demonstrate increased ER stress and UPR pathways in asthma and these are related to clinical severity and inflammatory phenotypes. Genes associated with ER protein chaperone (BiP, CANX, CALR), ER-associated protein degradation (EDEM1, DERL1) and ER stress-induced apoptosis (DDIT3, PPP1R15A) were dysregulated in participants with asthma and are associated with impaired lung function (forced expiratory volume in 1 s) and active eosinophilic and neutrophilic inflammation. ER stress genes also displayed a significant correlation with classic Th2 (interleukin-4, IL-4/13) genes, Th17 (IL-17F/CXCL1) genes, proinflammatory (IL-1b, tumour necrosis factor α, IL-8) genes and inflammasome activation (NLRP3) in sputum from asthmatic participants. Mice with allergic airway disease (AAD) and severe steroid insensitive AAD also showed increased ER stress signalling in their lungs. CONCLUSION: Heightened ER stress is associated with severe eosinophilic and neutrophilic inflammation in asthma and may play a crucial role in the pathogenesis of asthma.

Adverse roles of mast cell chymase-1 in COPD.

BACKGROUND: COPD is the third leading cause of death worldwide. Cigarette smoke (CS)-induced chronic inflammation inducing airway remodelling, emphysema and impaired lung function is the primary cause. Effective therapies are urgently needed. Human chymase (hCMA)1 and its orthologue mCMA1/mouse mast cell protease (mMCP)5 are exocytosed from activated mast cells and have adverse roles in numerous disorders, but their role in COPD is unknown. METHODS: We evaluated hCMA1 levels in lung tissues of COPD patients. We used mmcp5-deficient (-/-) mice to evaluate this protease's role and potential for therapeutic targeting in CS-induced experimental COPD. In addition, we used ex vivo/in vitro studies to define mechanisms. RESULTS: The levels of hCMA1 mRNA and CMA1+ mast cells were increased in lung tissues from severe compared to early/mild COPD patients, non-COPD smokers and healthy controls. Degranulated mast cell numbers and mMCP5 protein were increased in lung tissues of wild-type mice with experimental COPD. mmcp5 -/- mice were protected against CS-induced inflammation and macrophage accumulation, airway remodelling, emphysema and impaired lung function in experimental COPD. CS extract challenge of co-cultures of mast cells from wild-type, but not mmcp5 -/- mice with wild-type lung macrophages increased in tumour necrosis factor (TNF)-α release. It also caused the release of CMA1 from human mast cells, and recombinant hCMA-1 induced TNF-α release from human macrophages. Treatment with CMA1 inhibitor potently suppressed these hallmark features of experimental COPD. CONCLUSION: CMA1/mMCP5 promotes the pathogenesis of COPD, in part, by inducing TNF-α expression and release from lung macrophages. Inhibiting hCMA1 may be a novel treatment for COPD.

Pathways linked to unresolved inflammation and airway remodelling characterize the transcriptome in two independent severe asthma cohorts.

BACKGROUND AND OBJECTIVE: Severe asthma (SA) is a heterogeneous disease. Transcriptomic analysis contributes to the understanding of pathogenesis necessary for developing new therapies. We sought to identify and validate mechanistic pathways of SA across two independent cohorts. METHODS: Transcriptomic profiles from U-BIOPRED and Australian NOVocastrian Asthma cohorts were examined and grouped into SA, mild/moderate asthma (MMA) and healthy controls (HCs). Differentially expressed genes (DEGs), canonical pathways and gene sets were identified as central to SA mechanisms if they were significant across both cohorts in either endobronchial biopsies or induced sputum. RESULTS: Thirty-six DEGs and four pathways were shared across cohorts linking to tissue remodelling/repair in biopsies of SA patients, including SUMOylation, NRF2 pathway and oxidative stress pathways. MMA presented a similar profile to HCs. Induced sputum demonstrated IL18R1 as a shared DEG in SA compared with healthy subjects. We identified enrichment of gene sets related to corticosteroid treatment; immune-related mechanisms; activation of CD4+ T cells, mast cells and IL18R1; and airway remodelling in SA. CONCLUSION: Our results identified differentially expressed pathways that highlight the role of CD4+ T cells, mast cells and pathways linked to ongoing airway remodelling, such as IL18R1, SUMOylation and NRF2 pathways, as likely active mechanisms in the pathogenesis of SA.