Increased Muc5AC and Decreased Ciliated Cells in Severe Asthma Partially Restored by Inhibition of IL-4Rα Receptor.

BACKGROUND: The role of IL-13 on the airway epithelium in severe asthma leading to airway remodeling remains poorly understood. OBJECTIVE: To study IL-13 induced airway remodeling on goblet cells and cilia in the airway epithelium in severe asthma and the impact of an anti-IL4Rα antibody, dupilumab, in vitro. METHODS: Quantitative CT (qCT) lungs and endobronchial biopsies and brushings were obtained in 51 participants (22 severe, 11 non-severe asthma and 18 healthy participants) in the Severe Asthma Research Program (SARPIII) and measured for mucin and cilia related proteins. Epithelial cells were differentiated in air-liquid interphase (ALI) with IL-13 +/-dupilumab and assessed for mucin, cilia, cilia beat frequency (CBF) and epithelial integrity (transepithelial electrical resistance, TEER). RESULTS: Increased Muc5AC (Δ+263.2±92.7 lums/EpiArea) and decreased ciliated cells (Δ-0.07±0.03 Foxj1+cells/EpiArea) were observed in biopsies from severe asthma when compared to healthy (p<0.01 and p=0.047 respectively). RNAseq of epithelial cell brushes confirmed a Muc5AC increase with a decrease in a 5-gene cilia-related mean in severe asthma compared to healthy (all p<0.05). IL-13 (5 ng/mL) differentiated ALI cultures of healthy and asthmatic (severe and non-severe participants) increased Muc5AC, decreased cilia (α-acytl-tubulin) in healthy (Δ+6.5±1.5%, Δ-14.1±2.7%; all p<0.001 respectively) and asthma (Δ+4.4±2.5%, Δ-13.1±2.7%; p=0.084, p<0.001 respectively); decreased epithelial integrity (TEER) in healthy (-140.9±21.3 [ohms], p<0.001) while decreasing CBF in asthma (Δ-4.4±1.7 [Hz], p<0.01). When dupilumab was added to ALI with IL-13, there was no significant decrease in Mu5AC but there was restoration of cilia in healthy and asthma participants (absolute increase of 67.5% and 32.5% cilia, all p<0.05 respectively) while CBF increased (Δ+3.6±1.1 [Hz], p<0.001) and TEER decreased (only in asthma Δ-37.8±16.2 [ohms] p<0.05). CONCLUSIONS: IL-13 drives features of airway remodeling in severe asthma which are partially reversed by inhibiting IL-4Rα receptor in vitro.

Discovery and Validation of a Volatile Signature of Eosinophilic Airway Inflammation in Asthma.

RATIONALE: Volatile organic compounds (VOCs) in asthmatic breath may be associated with sputum eosinophilia. We developed a volatile biomarker-signature to predict sputum eosinophilia in asthma. METHODS: VOCs emitted into the space above sputum samples (headspace) from severe asthmatics (n=36) were collected onto sorbent tubes and analysed using thermal desorption gas chromatography-mass spectrometry (TD-GC-MS). Elastic net regression identified stable VOCs associated with sputum eosinophilia ≥3% and generated a volatile biomarker signature. This VOC signature was validated in breath samples from: (I) acute asthmatics according to blood eosinophilia ≥0.3x109cells/L or sputum eosinophilia of ≥ 3% in the UK EMBER consortium (n=65) and U-BIOPRED-IMI consortium (n=42). Breath samples were collected onto sorbent tubes (EMBER) or Tedlar bags (U-BIOPRED) and analysed by gas-chromatography-mass spectrometry (GC×GC-MS -EMBER or GC-MS -U-BIOPRED). MAIN RESULTS: The in vitro headspace identified 19 VOCs associated with sputum eosinophilia and the derived VOC signature yielded good diagnostic accuracy for sputum eosinophilia ≥ 3% in headspace (AUROC (95% CI) 0.90(0.80-0.99), p<0.0001), correlated inversely with sputum eosinophil % (rs= -0.71, p<0.0001) and outperformed FeNO (AUROC (95% CI) 0.61(0.35-0.86). Analysis of exhaled breath in replication cohorts yielded a VOC signature AUROC (95% CI) for acute asthma exacerbations of 0.89(0.76-1.0) (EMBER cohort) with sputum eosinophilia and 0.90(0.75-1.0) in U-BIOPRED - again outperforming FeNO in U-BIOPRED 0.62 (0.33-0.90). CONCLUSIONS: We have discovered and provided early-stage clinical validation of a volatile biomarker signature associated with eosinophilic airway inflammation. Further work is needed to translate our discovery using point of care clinical sensors.

Downregulation of otulin induces inflammasome activation in neutrophilic asthma.

BACKGROUND: Neutrophilic asthma (NA) is a severe asthma phenotype associated with steroid resistance and IL-1ß overproduction; however, the exact mechanism remains unclear. Moreover, the dysfunction of TNF-α signaling pathway, a regulator of IL-1ß production, was associated with the deficiency of ovarian tumor protease deubiquitinase with linear linkage specificity (otulin) in autoimmune patients. OBJECTIVE: We hypothesized that otulin downregulation in macrophages (Mφ) could trigger Mφ activation via the nucleotide-binding domain, leucine-rich repeat, and pyrin domain-containing protein 3 (NLRP3) inflammasome signaling pathway. METHODS: We assessed the expressions of otulin in blood monocyte subsets from NA patients and in alveolar Mφ from NA mice. Additionally, we evaluated the functional consequences of otulin deficiency in bone marrow-derived Mφ. The effects of inhibiting receptor-interacting protein kinase (RIPK)-1 and RIPK-3 on neutrophils and group 3 innate lymphoid cells (ILC3s) were assessed in vitro and in vivo. RESULTS: When comparing nonclassical monocytes, a significant downregulation of otulin in the intracellular components was observed in NA patients compared to healthy controls (P = .005). Moreover, isolated alveolar Mφ from the NA mice exhibited lower otulin expression compared to those from control mice. After otulin knockdown in bone marrow-derived Mφ, we observed spontaneous IL-1ß production depending on NLRP3 inflammasome. Moreover, the infiltrated neutrophils and ILC3s were significantly decreased by combined treatment of RIPK-1 and RIPK-3 inhibitors through blocking IL-1ß release in NA. CONCLUSIONS: IL-1ß overproduction caused by a deficiency of otulin, an upstream triggering factor, could be a promising diagnostic and therapeutic target for NA.

Anti-IL-4R versus anti-IL-5/5R after anti-IL-5/5R failure in asthma: An emulated target trial.

BACKGROUND: Switching biologics is now common practice in severe eosinophilic asthma. After insufficient response to anti-IL-5 or 5 receptor (anti-IL-5/5R), the optimal switch between an anti-IL-4R mAb (interclass) or another anti-IL-5/5R drug (intraclass) remains unknown. OBJECTIVE: We sought to compare the effectiveness of these 2 strategies in asthma control in patients with severe eosinophilic asthma and insufficient response to an anti-IL-5/5R mAb. METHODS: We emulated a target randomized trial using observational data from the Recherche sur les AsthMes SEvèreS (RAMSES) cohort. Eligible patients were switched to an anti-IL-4R mAb or another anti-IL-5/5R drug after insufficient response to an anti-IL-5/5R mAb. The primary outcome was the change in Asthma Control Test score at 6 months. RESULTS: Among the 2046 patients in the cohort, 151 were included in the study: 103 switched to an anti-IL-4R mAb and 48 to another anti-IL-5/5R. At 6 months, the difference in Asthma Control Test score improvement was not statistically significant (mean difference groups, 0.82 [-0.47 to 2.10], P = .213). The interclass group exhibited greater cumulative reduction in oral corticosteroid dose (Pinter-intra, -1.05 g [-1.76 to -0.34], P = .041). The interclass group had a better effect, although not significantly, on reducing exacerbations (Δinter-intra, -0.37 [-0.77 to 0.02], P = .124) and increasing lung function (FEV1) (126.8 mL [-12.7 to 266.4], P = .124). CONCLUSIONS: After anti-IL-5/5R mAb insufficient response, switching to dupilumab demonstrated similar improvement in Asthma Control Test scores compared with intraclass switching. However, it appeared more effective in reducing oral corticosteroid use. Larger studies are warranted to confirm these results.

Airway wall extracellular matrix changes induced by bronchial thermoplasty in severe asthma.

BACKGROUND: Airway remodeling is a prominent feature of asthma, which involves increased airway smooth muscle mass and altered extracellular matrix composition. Bronchial thermoplasty (BT), a bronchoscopic treatment for severe asthma, targets airway remodeling. OBJECTIVE: We sought to investigate the effect of BT on extracellular matrix composition and its association with clinical outcomes. METHODS: This is a substudy of the TASMA trial. Thirty patients with severe asthma were BT-treated, of whom 13 patients were treated for 6 months with standard therapy (control group) before BT. Demographic data, clinical data including pulmonary function, and bronchial biopsies were collected. Biopsies at BT-treated and nontreated locations were analyzed by histological and immunohistochemical staining. Associations between histology and clinical outcomes were explored. RESULTS: Six months after treatment, it was found that the reticular basement membrane thickness was reduced from 7.28 µm to 5.74 µm (21% relative reduction) and the percentage area of tissue positive for collagen increased from 26.3% to 29.8% (13% relative increase). Collagen structure analysis revealed a reduction in the curvature frequency of fibers. The percentage area positive for fibulin-1 and fibronectin increased by 2.5% and 5.9%, respectively (relative increase of 124% and 15%). No changes were found for elastin. The changes in collagen and fibulin-1 negatively associated with changes in FEV1 reversibility. CONCLUSIONS: Besides reduction of airway smooth muscle mass, BT has an impact on reticular basement membrane thickness and the extracellular matrix arrangement characterized by an increase in tissue area occupied by collagen with a less dense fiber organization. Both collagen and fibulin-1 are negatively associated with the change in FEV1 reversibility.

A novel syndrome of silent rhinovirus-associated bronchoalveolitis in children with recurrent wheeze.

BACKGROUND: Rhinovirus (RV) infections trigger wheeze episodes in children. Thus, understanding of the lung inflammatory response to RV in children with wheeze is important. OBJECTIVES: This study sought to examine the associations of RV on bronchoalveolar lavage (BAL) granulocyte patterns and biomarkers of inflammation with age in children with treatment-refractory, recurrent wheeze (n = 616). METHODS: Children underwent BAL to examine viral nucleic acid sequences, bacterial cultures, granulocyte counts, and phlebotomy for both general and type-2 inflammatory markers. RESULTS: Despite the absence of cold symptoms, RV was the most common pathogen detected (30%), and when present, was accompanied by BAL granulocytosis in 75% of children. Compared to children with no BAL pathogens (n = 341), those with RV alone (n = 127) had greater (P < .05) isolated neutrophilia (43% vs 16%), mixed eosinophils and neutrophils (26% vs 11%), and less pauci-granulocytic (27% vs 61%) BAL. Children with RV alone furthermore had biomarkers of active infection with higher total blood neutrophils and serum C-reactive protein, but no differences in blood eosinophils or total IgE. With advancing age, the log odds of BAL RV alone were lower, 0.82 (5th-95th percentile CI: 0.76-0.88; P < .001), but higher, 1.58 (5th-95th percentile CI: 1.01-2.51; P = .04), with high-dose daily corticosteroid treatment. CONCLUSIONS: Children with severe recurrent wheeze often (22%) have a silent syndrome of lung RV infection with granulocytic bronchoalveolitis and elevated systemic markers of inflammation. The syndrome is less prevalent by school age and is not informed by markers of type-2 inflammation. The investigators speculate that dysregulated mucosal innate antiviral immunity is a responsible mechanism.

Comparative effectiveness of dupilumab and omalizumab on asthma exacerbations and systemic corticosteroid prescriptions: Real-world US ADVANTAGE study.

BACKGROUND: In the United States, dupilumab is approved for moderate-to-severe eosinophilic or oral corticosteroid-dependent asthma, and omalizumab is approved for managing moderate-to-severe allergic asthma uncontrolled by inhaled corticosteroids. However, limited comparative effectiveness data exist for these biologics due to differing patient characteristics and treatment histories. OBJECTIVE: This study assessed the real-world effectiveness of dupilumab and omalizumab for asthma in patients in the United States. METHODS: In this retrospective observational study, TriNetX Dataworks electronic medical record data were used to identify patients with asthma age ≥12 years who initiated (index) dupilumab or omalizumab between November 2018 and September 2020 and who had at least 12 months of pre- and post-index clinical information. Inverse probability of treatment weighting was applied to balance potential confounding in treatment groups. Asthma exacerbation rates and systemic corticosteroid (SCS) prescriptions were compared using a doubly robust negative binomial regression model, adjusting for baseline exacerbation/SCS rates and patient characteristics with ≥10% standardized differences after inverse probability of treatment weighting. RESULTS: All inclusion and exclusion criteria were met by 2138 dupilumab patients and 1313 omalizumab patients. After weighting, the majority of baseline characteristics were balanced (standard difference <10%) between the 2 groups. Dupilumab was associated with a 44% lower asthma exacerbation rate (P < .0001) versus omalizumab. Additionally, dupilumab treatment significantly (P < .05) reduced SCS prescriptions by 28% during the follow-up period compared with omalizumab treatment. CONCLUSIONS: The US ADVANTAGE real-world study demonstrated a significant reduction in severe asthma exacerbations and SCS prescriptions for patients prescribed dupilumab compared with patients prescribed omalizumab during 12 months of follow-up.

Investigation of lactotransferrin messenger RNA expression levels as an anti-type 2 asthma biomarker.

BACKGROUND: Lactotransferrin (LTF) has an immunomodulatory function, and its expression levels are associated with asthma susceptibility. OBJECTIVES: We sought to investigate LTF messenger RNA (mRNA) expression levels in human bronchial epithelial cells (BECs) as an anti-type 2 (T2) asthma biomarker. METHODS: Association analyses between LTF mRNA expression levels in BECs and asthma-related phenotypes were performed in the Severe Asthma Research Program (SARP) cross-sectional (n = 155) and longitudinal (n = 156) cohorts using a generalized linear model. Correlation analyses of mRNA expression levels between LTF and all other genes were performed by Spearman correlation. RESULTS: Low LTF mRNA expression levels were associated with asthma susceptibility and severity (P < .025), retrospective and prospective asthma exacerbations, and low lung function (P < 8.3 × 10-3). Low LTF mRNA expression levels were associated with high airway T2 inflammation biomarkers (sputum eosinophils and fractional exhaled nitric oxide; P < 8.3 × 10-3) but were not associated with blood eosinophils or total serum IgE. LTF mRNA expression levels were negatively correlated with expression levels of TH2 or asthma-associated genes (POSTN, NOS2, and MUC5AC) and eosinophil-related genes (IL1RL1, CCL26, and IKZF2) and positively correlated with expression levels of TH1 and inflammation genes (IL12A, MUC5B, and CC16) and TH17-driven cytokines or chemokines for neutrophils (CXCL1, CXCL6, and CSF3) (P < 3.5 × 10-6). CONCLUSIONS: Low LTF mRNA expression levels in BECs are associated with asthma susceptibility, severity, and exacerbations through upregulation of airway T2 inflammation. LTF is a potential anti-T2 biomarker, and its expression levels may help determine the balance of eosinophilic and neutrophilic asthma.

Soluble E-Cadherin Contributes to Airway Inflammation in Severe Asthma.

Impaired airway epithelial barrier and decreased expression of E-cadherin are key features of severe asthma. As a gatekeeper of the mucosa, E-cadherin can be cleaved from the cell surface and released into the apical lumen as a soluble form (sE-cadherin).This study was aimed to investigate the role of sE-cadherin in severe asthma.Induced sputum was obtained from healthy subjects and patients with asthma. Two murine models of severe asthma were established using either TDI (toluene diisocyanate) or OVA (ovalbumin)/CFA (complete Freund's adjuvants). The role of sE-cadherin in severe asthma was evaluated by intraperitoneal injection of DECMA-1, a neutralizing antibody against sE-cadherin. Mice or THP-1-derived macrophages were treated with recombinant sE-cadherin to explore the pro-inflammatory mechanism of sE-cadherin.Severe asthma patients had a significantly higher sputum sE-cadherin level than the health subjects with mild to moderate asthma, which were positively correlated with sputum HMGB1 level and glucocorticoid dosage required for daily control. Allergen exposure markedly increased sE-cadherin level in the bronchoalveolar lavage fluid in mice. Treatment of DECMA-1 significantly attenuated allergen-induced airway inflammation and hyperresponsivenes in both models of severe asthma. While exposure to recombinant sE-cadherin dramatically up-regulated VEGF expression in THP-1-derived macrophages, and increased neutophlil and eosinophil infiltration into the airway as well as the release of VEGF and IL-6 in mice, both of which can be suppressed by pharmacological inhibition of ERK signaling.Taken together, our data indicated that sE-cadherin contributed to the airway inflammation of severe asthma in an ERK-depedent pathway.

MiR-155-5p Differentially Regulates IL-13Rα1 and IL-13Rα2 Expression and Signalling, Driving Abnormal Lung Epithelial Cell Phenotype in Severe Asthma.

MiR-155-5p is known to increase in innate and adaptive immune cells in response to IL-13 and is associated with asthma severity. However, little is known about its role in airway structural cells. BECs isolated from healthy donors and severe asthma patients were stimulated with IL-13. MiR-155-5p expression and release were measured by RT-PCR in BECs and in their derived exosomes. Modulation of miR-155-5p in BECs was performed using transfection of miR-155-5p inhibitor and mimic. IL-13Rα1, IL-13Rα2, MUC5AC, IL-8 and Eotaxin-1 expression were measured by RT-PCR and western blot. BECs repair process was assessed by wound healing assay. IL-13Rα1 and IL-13Rα2 expression and downstream pathways were evaluated by western blot. Dual Luciferase assay was used to determine miR-155-5p target genes associated to IL-13 receptors signaling. BECs from severe asthma showed an increased expression and exosomal release of miR-155-5p at baseline that was amplified by IL-13 stimulation. BECs from asthmatics expressed more IL-13Rα1 and less IL-13Rα2 than healthy donors and IL-13Rα1 but not IL-13Rα2 induced miR-155-5p expression under IL-13 stimulation. MiR-155-5p overexpression favored MUC5AC, IL-8 and Eotaxin-1 through IL-13Rα1/SOCS1/STAT6 pathway to the detriment of a delayed repair process with a downregulated IL-13Rα2/MAPK14/c-Jun/c-Fos signaling. Dual Luciferase assay confirmed that miR-155-5p modulates both IL-13 receptors pathways by directly targeting SOCS1, c-Fos and MAPK14. MiR-155-5p is overexpressed in severe asthma BECs and regulates IL-13Rα1 and IL-13Rα2 expression and signaling, favoring expression of mucin and eosinophils related genes to detriment of airway repair. These results show that miR-155-5p may contribute to airway epithelial cell dysfunction in severe asthma.