Rationale: Epidemiologic studies on patients with asthma and in vitro data suggest a protective role of type 2 (T2) inflammation in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Objectives: Using a large, multisite cohort, we studied clinical outcomes after SARS-CoV-2 infection in multiple asthma endotypes and examined the effects of T2-directed biologics in infected patients with asthma. Methods: The National COVID Cohort Collaborative Data Enclave was used to identify and stratify patients with asthma by endotype to include those with non-T2 and T2 asthma, as well as exposure to T2-directed biologic therapy. We evaluated the risk of hospitalization, invasive mechanical ventilation, and 90-day mortality by endotype and exposure to biologics. Results: For this study, 402,376 patients met the inclusion criteria, of whom 138,142 (34%) were characterized as having non-T2 asthma and 264,234 (66%) as having T2 asthma, a group further divided into 104,823 (26%) atopic, 84,440 (21%) eosinophilic, and 74,971 (19%) T2-high asthmatic endotypes. Compared with patients with non-T2 asthma, those with atopic and T2-high asthma experienced decreased odds of hospitalization and 90-day mortality. Conversely, patients with eosinophilic asthma experienced higher odds of hospitalization, intubation, and 90-day mortality. Exposure to T2-directed biologic therapies did not alter outcomes after propensity score matching. In contrast, maximum eosinophil count and recent systemic corticosteroid use were directly correlated with increased odds of all outcomes. Conclusions: Coronavirus disease (COVID-19) outcomes differ depending on asthma endotype, with patients with atopic asthma experiencing lower odds and those with eosinophilic asthma experiencing higher odds of deleterious outcomes. T2-directed biologic treatment did not alter these outcomes, but recent systemic corticosteroid use predisposes all patients with asthma to adverse outcomes.

ABSTRACTAirway mucociliary clearance (MCC) is required for host defense and often diminished in chronic lung diseases. Effective clearance depends upon coordinated actions of the airway epithelium and a mobile mucus layer. Dysregulation of the primary secreted airway mucin proteins, MUC5B and MUC5AC, is associated with a reduction in the rate of MCC; however, how other secreted proteins impact the integrity of the mucus layer and MCC remains unclear. We previously identified the geneBpifb1/Lplunc1as a regulator of airway MUC5B levels using genetic approaches. Here, we show that BPIFB1 is required for normal mucociliary clearancein vivousingBpifb1knockout (KO) mice. Reduced MCC inBpifb1KO mice occurred in the absence of defects in sodium or chloride ion transport or reduced ciliary beat frequency. BPIFB1 loss resulted in airway mucus flakes with significantly increased complex viscosity, a key biophysical property of mucus known to impact MCC. Finally, we detected colocalization of BPIFB1 and MUC5B in secretory granules in mice and in the protein mesh of secreted mucus in human airway cultures. Collectively, our findings demonstrate that BPIFB1 is an important component of the mucociliary apparatus in mice and a key component of the mucus protein network.

Background Mucus hyperconcentration in cystic fibrosis (CF) lung disease is marked by increases in both mucin and DNA concentration. Additionally, it has been shown that half of the mucins present in bronchial alveolar lavage fluid (BALF) from preschool-aged CF patients are present in as non-swellable mucus flakes. This motivates us to examine the utility of mucus flakes, as well as mucin and DNA concentrations in BALF as markers of infection and inflammation in CF airway disease. Methods In this study, we examined the mucin and DNA concentration, as well as mucus flake abundance, composition, and biophysical properties in BALF from three groups; healthy adult controls, and two CF cohorts, one preschool aged and the other school aged. BALFs were characterized via refractometry, PicoGreen, immunofluorescence microscopy, particle tracking microrheology, and fluorescence image tiling. Results Mucin and DNA BALF concentrations increased progressively from healthy young adult controls to preschool-aged people and school-aged people with CF. Notably, mucin concentrations were increased in bronchoalveolar lavage fluid (BALF) from preschool-aged patients with CF prior to decreased pulmonary function. Infrequent small mucus flakes were identified in normal subjects. A progressive increase in the abundance of mucus flakes in preschool and school-aged CF patients was observed. Compositionally, MUC5B dominated flakes from normal subjects, whereas an increase in MUC5AC was observed in people with CF, reflected in a reduced flaked MUC5B/MUC5AC mucin ratio. Conclusion These findings suggest mucus composition and flake properties are useful markers of inflammatory and infection-based changes in CF airways.

QuestionCystic fibrosis (CF) is characterised by the accumulation of viscous adherent mucus in the lungs. While several hypotheses invoke a direct relationship with cystic fibrosis transmembrane conductance regulator (CFTR) dysfunction (i.e.acidic airway surface liquid (ASL) pH, low bicarbonate (HCO3−) concentration, airway dehydration), the dominant biochemical alteration of CF mucus remains unknown.Materials/methodsWe characterised a novel cell line (CFTR-KO Calu3 cells) and the responses of human bronchial epithelial (HBE) cells from subjects with G551D or F508del mutations to ivacaftor and elexacaftor-tezacaftor-ivacaftor. A spectrum of assays such as short-circuit currents, quantitative PCR, ASL pH, Western blotting, light scattering/refractometry (size-exclusion chromatography with inline multi-angle light scattering), scanning electron microscopy, percentage solids and particle tracking were performed to determine the impact of CFTR function on mucus properties.ResultsLoss of CFTR function in Calu3 cells resulted in ASL pH acidification and mucus hyperconcentration (dehydration). Modulation of CFTR in CF HBE cells did not affect ASL pH or mucin mRNA expression, but decreased mucus concentration, relaxed mucus network ultrastructure and improved mucus transport. In contrast with modulator-treated cells, a large fraction of airway mucins remained attached to naïve CF cells following short apical washes, as revealed by the use of reducing agents to remove residual mucus from the cell surfaces. Extended hydration, but not buffers alkalised with sodium hydroxide or HCO3−, normalised mucus recovery to modulator-treated cell levels.ConclusionThese results indicate that airway dehydration, not acidic pH and/or low [HCO3−], is responsible for abnormal mucus properties in CF airways and CFTR modulation predominantly restores normal mucin entanglement.

Cystic fibrosis (CF) is characterized by abnormal transepithelial ion transport. However, a description of CF lung disease pathophysiology unifying superficial epithelial and submucosal gland (SMG) dysfunctions has remained elusive. We hypothesized that biophysical abnormalities associated with CF mucus hyperconcentration provide a unifying mechanism. Studies of the anion secretion-inhibited pig airway model of CF revealed elevated SMG mucus concentrations, osmotic pressures, and SMG mucus accumulation. Human airway studies revealed hyperconcentrated CF SMG mucus with raised osmotic pressures and cohesive forces predicted to limit SMG mucus secretion/release. Using proline-rich protein 4 (PRR4) as a biomarker of SMG secretion, CF sputum proteomics analyses revealed markedly lower PRR4 levels compared to healthy and bronchiectasis controls, consistent with a failure of CF SMGs to secrete mucus onto airway surfaces. Raised mucus osmotic/cohesive forces, reflecting mucus hyperconcentration, provide a unifying mechanism that describes disease-initiating mucus accumulation on airway surfaces and in SMGs of the CF lung.

Significance Gaining insights into severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) high transmissibility and the role played by inflammatory mediators in viral proliferation are critical to investigating new therapeutic targets against COVID-19. Electron microscopy reveals important SARS-CoV-2 features, including the combination of large, rapidly released viral clusters and the massive shedding of epithelial cells packed with virions. Interleukin-13 (IL-13), a Th2 cytokine up-regulated in allergic asthma and associated with less severe COVID-19, protects against SARS-CoV-2 viral and cell shedding. Using gene expression analyses and biochemical assays, IL-13 is shown to affect viral entry, replication, and cell-to-cell transmission. Given the broad spectrum of COVID-19 clinical symptoms, it is important to elucidate intrinsic factors that modulate viral load and spreading mechanisms.

In the United States, millions of adults use electronic cigarettes (e-cigs), and a majority of these users are former or current cigarette smokers. It is unclear, whether prior smoking status affects biological responses induced by e-cigs. In this study, differentiated human nasal epithelial cells (hNECs) from nonsmokers and smokers at air-liquid interface were acutely exposed to the e-cig generated aerosols of humectants, propylene glycol (PG), and glycerol (GLY). Mucin levels were examined in the apical washes, and cytokine levels were assessed in the basolateral supernatants 24 h postexposure. The aerosol from the GLY exposure increased mucin 5, subtype AC (MUC5AC) levels in the apical wash of hNECs from nonsmokers, but not smokers. However, the aerosol from GLY induced pro-inflammatory responses in hNECs from smokers. We also exposed hNECs from nonsmokers and smokers to e-cig generated aerosol from PG:GLY with freebase nicotine or nicotine salt. The PG:GLY with freebase nicotine exposure increased MUC5AC and mucin 5, subtype B (MUC5B) levels in hNECs from nonsmokers, but the nicotine salt exposure did not. The PG:GLY with nicotine salt exposure increased pro-inflammatory cytokines in hNECs from smokers, which was not seen with the freebase nicotine exposure. Taken together, these data indicate that the e-cig generated aerosols from the humectants, mostly GLY, and the type of nicotine used cause differential effects in airway epithelial cells from nonsmokers and smokers. As e-cig use is increasing, it is important to understand that the biological effects of e-cig use are likely dependent on prior cigarette smoke exposure.

Human primary bronchial epithelial (HBE) cells have a long history in respiratory research.With the establishment of protocols for expansion of primary airway epithelial cells with conditionally reprogrammed cell methods (1, 2), cellular redifferentiation using air–liquid interface (ALI) conditions on porous membranes, and lentiviral infection and CRISPR/Cas9-mediated transduction and gene knockdown technologies (3, 4), the use of HBE cell cultures for respiratory research has greatly expanded. Because of the worldwide coronavirus disease (COVID-19) pandemic in 2020, a shortage of clinical supplies—for example, masks and hand sanitizers—emerged as a serious issue (5). Likewise, supplies for basic research have been limited. In the case of cell culture, Transwell (3460; Corning) and Millicell (PIH P01250; Millipore) inserts have been in exceptionally short supply. Owing to their limited supply and high cost, insert reuse is a rational approach to maintain timely and cost-efficient research. The only studies reporting insert reuse focused on reuse for cell line applications (6). In this study, we investigated the reuse of Transwell andMillicell inserts for primary HBE cell culture research. All experiments were conducted using cells from three to five different donor lung specimens. Detailed protocols are found in the data supplement. Protocols to remove primary airway cells from inserts were developed first. We tested four reagents (0.25% Trypsin-EDTA [25200056; Gibco], Accutase [A6964; Sigma], 6M urea in PBS, and RIPA buffer) to dissociate/solubilize and remove well-differentiated HBE cells from Transwell inserts. Dissociation solutions were applied to the apical surface for between 5 and 20 minutes, followed by pipetting or scraping with a cell lifter. Trypsin-EDTA, 6M urea, and RIPA buffer effectively removed cells. Accutase was less effective. Following cell dissociation, an overnight incubation with 0.25% Trypsin-EDTA at 37 C completed removal of residual cells and debris from inserts. Apical and basolateral surfaces of the inserts were then washed twice with PBS and twice with pure water. After air-drying in a hood, the inserts were coated with collagen (5005; Advanced BioMatrix), UV irradiated, and stored at 4 C until reuse. The reused inserts were tested in a series of HBE cell culture studies. HBE cells from new donors were seeded and cell differentiation was monitored over 4 weeks in ALI culture (7). None of the 110 second-use (U2) Transwell inserts exhibited contamination or a loss of integrity. Preservation of cellular differentiation in U1 (first-use) versus U2 cultures was tested by three approaches. First, mRNA expression of cell type markers was measured by qRT-PCR and was similar for U1 versus U2 cultures (Figure 1A) (8). Second, MUC5B protein in apical washes was measured byWestern blotting and was found to be similar between U1 versus U2 cultures (Figure 1B). Third, hematoxylin and eosin and whole mount immunofluorescent staining for ciliated (tubulin), secretory (CCSP), and goblet (MUC5B) cell markers confirmed similar differentiation between U1 and U2 inserts (Figures 1C and 1D). Measurements of HBE cell function on U1 versus U2 inserts were made for ion transport in Ussing chambers (Figure 1E) and cilia beat frequency with video microscopy (Figure 1F) (9–11). Both assays revealed virtually identical cellular functional activities of HBE cells on U1 versus U2 inserts. Importantly, donor-to-donor variability in all parameters was reproduced in assays comparing HBE cells on U1 versus U2 inserts, suggesting that insert reuse did not affect donorspecific cellular function. We then tested whether a low number of residual cells escaped the reuse protocol and caused DNA contamination of subsequent cultures. ACE2 knockout cells (GenBankMT505392, generated by introduction of gRNA/Cas9 complexes with electroporation [3]), were cultured on U2 inserts and, after 28 days in ALI, the HBE gene–modified cells were removed from the insert and the insert reconditioned for third use (U3). Wild-type (WT) donor cells were plated onU3 inserts. After attachment, expansion, and 7-day ALI culture, DNAwas extracted from the U3 culture and the gRNA target region was amplified by PCR and sequenced. Gene-editing efficiency analyses (Inference of CRISPR Edits [ICE] analysis; Synthego) (12) demonstrated that the gene-edited U2 insert cells did not contaminate U3 culturedWT cells (Figure 1G). HBE cells exposed to inflammatorymediators exhibit changes in epithelial structure/function that maymodify the produced basement membranematrix and affect subsequent culture properties. Accordingly, U2 HBE cells were treated with IL-1b (1 ng/ml, 201-LB; R&D Systems) for 7 days. IL-1b treatment resulted in induction of MUC5B and reduction of SCGB1A1mRNA (Figure 1H). Apical washes confirmedMUC5B protein induction by IL-1b (Figure 1I) (8). During IL-1b treatment, 8 of 27 U2 inserts exhibited leakage of media onto the apical surface. U2 inserts containing IL-1b–treated versus vehicle-treated HBE cells were prepared for reuse and plated with naive HBE cells (U3). HBE cells on U3 inserts from IL-1b–treated versus vehicle-treated HBE cells exhibited similarMUC5B and SCGB1A1 gene expression, similar MUC5B protein in apical washes, and absence of media leakage, suggesting residual IL-1b effects were not transmitted to subsequent HBE cultures with insert reuse (Figures 1J and 1K). We directly investigated whether the porous membrane structure of inserts was affected by reuse using scanning EM. Significant damage to or obstruction of the pores was not observed after any of the cell dissociation/solubilization protocols (Figure 1L). Finally, multiple reuses of Transwell andMillicell inserts were tested. HBE cells were grown and differentiated on U2 to U4 (fourthuse) Transwell and U2 to U3Millicell inserts (Figure 1M). Insert reuse four times with a cumulative longevity of use for 185 days was feasible. No bacterial contamination was observed at any time in these studies. In these experiments, insert reuse was limited to inserts maintained under sterile conditions at all times to minimize bacterial and/or endotoxin contamination. Therefore, it remains unknown Author Contributions: T.K. conceived and designed the study, performed experiments, analyzed data, and wrote the initial and the revised manuscript. Y.M., L.S., T.D.R, B.R.G, and C.B.M. performed experiments and analyzed data. P.R.S and L.E.O supervised cilia beat frequency assays. C.E. supervised scanning microscopy imaging. S.H.R provided human airway epithelial cells and guidance. R.C.B supervised the study, analyzed data, and revised the manuscript. All authors reviewed and approved the final version of the manuscript.

Cystic fibrosis (CF) is characterized by abnormal transepithelial ion transport. However, a description of CF lung disease pathophysiology unifying superficial epithelial and submucosal gland (SMG) dysfunctions has remained elusive. We hypothesized that biophysical abnormalities associated with CF mucus hyperconcentration provide a unifying mechanism. Studies of the anion secretion-inhibited pig airway CF model revealed elevated SMG mucus concentrations, osmotic pressures, and SMG mucus accumulation. Human airway studies revealed hyperconcentrated CF SMG mucus with raised osmotic pressures and cohesive forces predicted to limit SMG mucus secretion/release. Utilizing proline-rich protein 4 (PRR4) as a biomarker of SMG secretion, proteomics analyses of CF sputum revealed markedly lower PRR4 levels compared to healthy and bronchiectasis controls, consistent with a failure of CF SMGs to secrete mucus onto airway surfaces. Raised mucus osmotic/cohesive forces, reflecting mucus hyperconcentration, provide a unifying mechanism that describes disease-initiating mucus accumulation on airway surfaces and within SMGs of the CF lung. One Sentence Summary Hyperconcentrated Mucus Unifies Submucosal Gland and Superficial Airway Dysfunction in Cystic Fibrosis

Although destructive airway disease is evident in young children with cystic fibrosis (CF), little is known about the nature of the early CF lung environment triggering the disease. To elucidate early CF pulmonary pathophysiology, we performed mucus, inflammation, metabolomic, and microbiome analyses on bronchoalveolar lavage fluid (BALF) from 46 preschool children with CF enrolled in the Australian Respiratory Early Surveillance Team for Cystic Fibrosis (AREST CF) program and 16 non-CF disease controls. Total airway mucins were elevated in CF compared to non-CF BALF irrespective of infection, and higher densities of mucus flakes containing mucin 5B and mucin 5AC were observed in samples from CF patients. Total mucins and mucus flakes correlated with inflammation, hypoxia, and oxidative stress. Many CF BALFs appeared sterile by culture and molecular analyses, whereas other samples exhibiting bacterial taxa associated with the oral cavity. Children without computed tomography-defined structural lung disease exhibited elevated BALF mucus flakes and neutrophils, but little/no bacterial infection. Although CF mucus flakes appeared "permanent" because they did not dissolve in dilute BALF matrix, they could be solubilized by a previously unidentified reducing agent (P2062), but not N-acetylcysteine or deoxyribonuclease. These findings indicate that early CF lung disease is characterized by an increased mucus burden and inflammatory markers without infection or structural lung disease and suggest that mucolytic and anti-inflammatory agents should be explored as preventive therapy.