OMIP-100: A flow cytometry panel to investigate human neutrophil subsets.

This 14-color, 13-antibody optimized multicolor immunofluorescence panel (OMIP) was designed for deep profiling of neutrophil subsets in various types of human samples to contextualize neutrophil plasticity in a range of healthy and diseased states. Markers present in the OMIP allow the profiling of neutrophil subsets associated with ontogeny, migration, phagocytosis capacity, granule release, and immune modulation. For panel design, we ensured that the commonly available fluorophores FITC/AF488, PE, and APC were assigned to the intracellular subset marker Olfactomedin 4, the maturity and activation marker CD10, and whole blood subset marker CD177, respectively. These markers can be easily replaced without affecting the core identification of neutrophils, enabling antibodies to new neutrophil antigens of interest or for fluorescent substrates to assess different neutrophil functions to be easily explored. Panel optimization was performed on whole blood and purified neutrophils. We demonstrate applications on clinical samples (whole blood and saliva) and experimental endpoints (purified neutrophils stimulated through an in vitro transmigration assay). We hope that providing a uniform platform to analyze neutrophil plasticity in various sample types will facilitate the future understanding of neutrophil subsets in health and disease.

Pseudomonas aeruginosa modulates neutrophil granule exocytosis in an in vitro model of airway infection.

A population of neutrophils recruited into cystic fibrosis (CF) airways is associated with proteolytic lung damage, exhibiting high expression of primary granule exocytosis marker CD63 and reduced phagocytic receptor CD16. Causative factors for this population are unknown, limiting intervention. Here we present a laboratory model to characterize responses of differentiated airway epithelium and neutrophils following respiratory infection. Pediatric primary airway epithelial cells were cultured at the air-liquid interface, challenged individually or in combination with rhinovirus (RV) and Pseudomonas aeruginosa, then apically washed with medical saline to sample epithelial infection milieus. Cytokine multiplex analysis revealed epithelial antiviral signals, including IP-10 and RANTES, increased with exclusive RV infection but were diminished if P. aeruginosa was also present. Proinflammatory signals interleukin-1α and ß were dominant in P. aeruginosa infection milieus. Infection washes were also applied to a published model of neutrophil transmigration into the airways. Neutrophils migrating into bacterial and viral-bacterial co-infection milieus exhibited the in vivo CF phenotype of increased CD63 expression and reduced CD16 expression, while neutrophils migrating into milieus of RV-infected or uninfected cultures did not. Individually, bacterial products lipopolysaccharide and N-formylmethionyl-leucyl-phenylalanine and isolated cytokine signals only partially activated this phenotype, suggesting that additional soluble factors in the infection microenvironment trigger primary granule release. Findings identify P. aeruginosa as a trigger of acute airway inflammation and neutrophil primary granule exocytosis, underscoring potential roles of airway microbes in prompting this neutrophil subset. Further studies are required to characterize microbes implicated in primary granule release, and identify potential therapeutic targets.

ACE2 expression is elevated in airway epithelial cells from older and male healthy individuals but reduced in asthma.

BACKGROUND AND OBJECTIVE: COVID-19 is complicated by acute lung injury, and death in some individuals. It is caused by SARS-CoV-2 that requires the ACE2 receptor and serine proteases to enter AEC. We determined what factors are associated with ACE2 expression particularly in patients with asthma and COPD. METHODS: We obtained lower AEC from 145 people from two independent cohorts, aged 2-89 years, Newcastle (n = 115) and Perth (n = 30), Australia. The Newcastle cohort was enriched with people with asthma (n = 37) and COPD (n = 38). Gene expression for ACE2 and other genes potentially associated with SARS-CoV-2 cell entry was assessed by qPCR, and protein expression was confirmed with immunohistochemistry on endobronchial biopsies and cultured AEC. RESULTS: Increased gene expression of ACE2 was associated with older age (P = 0.03) and male sex (P = 0.03), but not with pack-years smoked. When we compared gene expression between adults with asthma, COPD and healthy controls, mean ACE2 expression was lower in asthma patients (P = 0.01). Gene expression of furin, a protease that facilitates viral endocytosis, was also lower in patients with asthma (P = 0.02), while ADAM-17, a disintegrin that cleaves ACE2 from the surface, was increased (P = 0.02). ACE2 protein expression was also reduced in endobronchial biopsies from asthma patients. CONCLUSION: Increased ACE2 expression occurs in older people and males. Asthma patients have reduced expression. Altered ACE2 expression in the lower airway may be an important factor in virus tropism and may in part explain susceptibility factors and why asthma patients are not over-represented in those with COVID-19 complications.

Aspergillus Infections and Progression of Structural Lung Disease in Children with Cystic Fibrosis.

Rationale: Recent data show that Aspergillus species are prevalent respiratory infections in children with cystic fibrosis (CF). The biological significance of these infections is unknown.Objectives: We aimed to evaluate longitudinal associations between Aspergillus infections and lung disease in young children with CF.Methods: Longitudinal data on 330 children participating in the Australian Respiratory Early Surveillance Team for Cystic Fibrosis surveillance program between 2000 and 2018 who underwent annual chest computed tomography (CT) imaging and BAL were used to determine the association between Aspergillus infections and the progression of structural lung disease. Results were adjusted for the effects of other common infections, associated variables, and repeated visits. Secondary outcomes included inflammatory markers in BAL, respiratory symptoms, and admissions for exacerbations.Measurements and Main Results:Haemophilus influenzae, Staphylococcus aureus, Pseudomonas aeruginosa, and Aspergillus infections were all associated with worse CT scores in the same year (Poverall < 0.05). Only P. aeruginosa and Aspergillus were associated with progression in CT scores in the year after an infection and worse CT scores at the end of the observation period. P. aeruginosa was most significantly associated with development of bronchiectasis (difference, 0.9; 95% confidence interval, 0.3-1.6; P = 0.003) and Aspergillus with trapped air (difference, 3.2; 95% confidence interval, 1.0-5.4; P = 0.004). Aspergillus infections were also associated with markers of neutrophilic inflammation (P < 0.001) and respiratory admissions risk (P = 0.008).Conclusions: Lower respiratory Aspergillus infections are associated with the progression of structural lung disease in young children with CF. This study highlights the need to further evaluate early Aspergillus species infections and the feasibility, risk, and benefit of eradication regimens.

Elastase Exocytosis by Airway Neutrophils Is Associated with Early Lung Damage in Children with Cystic Fibrosis.

RATIONALE: Neutrophils are recruited to the airways of individuals with cystic fibrosis (CF). In adolescents and adults with CF, airway neutrophils actively exocytose the primary granule protease elastase (NE), whose extracellular activity correlates with lung damage. During childhood, free extracellular NE activity is measurable only in a subset of patients, and the exocytic function of airway neutrophils is unknown. OBJECTIVES: To measure NE exocytosis by airway neutrophils in relation to free extracellular NE activity and lung damage in children with CF. METHODS: We measured lung damage using chest computed tomography coupled with the Perth-Rotterdam Annotated Grid Morphometric Analysis for Cystic Fibrosis scoring system. Concomitantly, we phenotyped blood and BAL fluid leukocytes by flow and image cytometry, and measured free extracellular NE activity using spectrophotometric and Förster resonance energy transfer assays. Children with airway inflammation linked to aerodigestive disorder were enrolled as control subjects. MEASUREMENTS AND MAIN RESULTS: Children with CF but not disease control children harbored BAL fluid neutrophils with high exocytosis of primary granules, before the detection of bronchiectasis. This measure of NE exocytosis correlated with lung damage (R = 0.55; P = 0.0008), whereas the molecular measure of free extracellular NE activity did not. This discrepancy may be caused by the inhibition of extracellular NE by BAL fluid antiproteases and its binding to leukocytes. CONCLUSIONS: NE exocytosis by airway neutrophils occurs in all children with CF, and its cellular measure correlates with early lung damage. These findings implicate live airway neutrophils in early CF pathogenesis, which should instruct biomarker development and antiinflammatory therapy in children with CF.

Preterm birth: Born too soon for the developing airway epithelium?

Birth prior to term interrupts the normal development of the respiratory system and consequently results in poor respiratory outcomes that persist throughout childhood. The mechanisms underpinning these poor respiratory outcomes are not well understood, but intrinsic abnormalities within the airway epithelium may be a contributing factor. Current evidence suggests that the airway epithelium is both structurally and functionally abnormal after preterm birth, with reports of epithelial thickening and goblet cell hyperplasia in addition to increased inflammation and apoptosis in the neonatal intensive care unit. However, studies focusing on the airway epithelium are limited and many questions remain unanswered; including whether abnormalities are a direct result of interrupted development, a consequence of exposure to inflammatory stimuli in the perinatal period or a combination of the two. In addition, the difficulty of accessing airway tissue has resulted in the majority of evidence being collected in the pre-surfactant era which may not reflect contemporary preterm birth. This review examines the consequences of preterm birth on the airway epithelium and explores the clinical relevance of currently available models whilst highlighting the need to develop a clinically relevant in vitro model to help further our understanding of the airway epithelium in preterm birth.

Visualisation of Multiple Tight Junctional Complexes in Human Airway Epithelial Cells.

BACKGROUND: Apically located tight junctions in airway epithelium perform a fundamental role in controlling macromolecule migration through paracellular spaces. Alterations in their expression may lead to disruptions in barrier integrity, which subsequently facilitates entry of potential bacterial and other pathogens into the host. Furthermore, there is emerging evidence that the barrier integrity of the airway in certain airway inflammatory diseases may be altered. However, there is little consensus on the way this is assessed and measured and the type of cells used to achieve this. METHODS: Here, we assessed four fixation methods including; (i) 4% (v/v) paraformaldehyde; (ii) 100% methanol; (iii) acetone or; (iv) 1:1 methanol: acetone. Pre-extraction with Triton X-100 was also performed and assessed on cells prior to fixation with either methanol or paraformaldehyde. Cells were also permeabilized with 0.1% (v/v) Saponin in 1× TBS following fixation and subsequently stained for tight junction proteins. Confocal microscopy was then used to visualise, compare and evaluate staining intensity of the tight junctional complexes in order to determine a standardised workflow of reproducible staining. RESULTS: Positive staining was observed following methanol fixation for claudin-1 and ZO-1 tight junction proteins but no staining was detected for occludin in 16HBE14o- cells. Combinatorial fixation with methanol and acetone also produced consistent positive staining for both occludin and ZO-1 tight junction proteins in these cells. When assessed using primary cells cultured at air-liquid interface, similar positive staining for claudin-1 and ZO-1 was observed following methanol fixation, while similar positive staining for occludin and ZO-1 was observed following the same combinatorial fixation with methanol and acetone. CONCLUSIONS: The present study demonstrates the importance of a personalised approach to optimise staining for the visualisation of different tight junction proteins. Of significance, the workflow, once optimised, can readily be translated into primary airway epithelial cell air-liquid interface cultures where it can be used to assess barrier integrity in chronic lung diseases.

Alpha-1 Antitrypsin Mitigates the Inhibition of Airway Epithelial Cell Repair by Neutrophil Elastase.

Neutrophil elastase (NE) activity is associated with many destructive lung diseases and is a predictor for structural lung damage in early cystic fibrosis (CF), which suggests normal maintenance of airway epithelium is prevented by uninhibited NE. However, limited data exist on how the NE activity in airways of very young children with CF affects function of the epithelia. The aim of this study was to determine if NE activity could inhibit epithelial homeostasis and repair and whether any functional effect was reversible by antiprotease alpha-1 antitrypsin (α1AT) treatment. Viability, inflammation, apoptosis, and proliferation were assessed in healthy non-CF and CF pediatric primary airway epithelial cells (pAECnon-CF and pAECCF, respectively) during exposure to physiologically relevant NE. The effect of NE activity on pAECCF wound repair was also assessed. We report that viability after 48 hours was significantly decreased by 100 nM NE in pAECnon-CF and pAECCF owing to rapid cellular detachment that was accompanied by inflammatory cytokine release. Furthermore, both phenotypes initiated an apoptotic response to 100 nM NE, whereas ≥ 50 nM NE activity significantly inhibited the proliferative capacity of cultures. Similar concentrations of NE also significantly inhibited wound repair of pAECCF, but this effect was reversed by the addition of α1AT. Collectively, our results demonstrate free NE activity is deleterious for epithelial homeostasis and support the hypothesis that proteases in the airway contribute directly to CF structural lung disease. Our results also highlight the need to investigate antiprotease therapies in early CF disease in more detail.

Effect of human rhinovirus infection on airway epithelium tight junction protein disassembly and transepithelial permeability.

RATIONALE: No studies have assessed the effects of human rhinovirus (HRV) infection on epithelial tight junctions (TJs) and resultant barrier function. AIM OF THE STUDY: To correlate viral infection with TJ disassembly, epithelial barrier integrity, and function. MATERIALS AND METHODS: Human airway epithelial cells were infected with HRV minor serotype 1B (HRV-1B) at various 50% tissue culture infectivity doses (TCID50) over 72 hours. HRV replication was assessed by quantitative-polymerase chain reaction (qPCR) while cell viability and apoptosis were assessed by proliferation and apoptotic assays, respectively. Protein expression of claudin-1, occludin, and zonula occludens protein-1 (ZO-1) was assessed using In-Cell™ Western assays. Transepithelial permeability assays were performed to assess effects on barrier functionality. RT2 Profiler focused qPCR arrays and pathway analysis evaluating associations between human TJ and antiviral response were performed to identify potential interactions and pathways between genes of interests. RESULTS: HRV-1B infection affected viability that was both time and TCID50 dependent. Significant increases in apoptosis and viral replication post-infection correlated with viral titer. Viral infection significantly decreased claudin-1 protein expression at the lower TCID50, while a significant decrease in all three TJ protein expressions occurred at higher TCID50. Decrease in protein expression was concomitant with significant increases in epithelial permeability of fluorescein isothiocynate labeled-dextran 4 and 20 kDa. Analysis of focused qPCR arrays demonstrated a significant decrease in ZO-1 gene expression. Furthermore, network analysis between human TJ and antiviral response genes revealed possible interactions and regulation of TJ genes via interleukin (IL)-15 in response to HRV-1B infection. CONCLUSION: HRV-1B infection directly alters human airway epithelial TJ expression leading to increased epithelial permeability potentially via an antiviral response of IL-15.

Airway epithelial repair in health and disease: Orchestrator or simply a player?

Epithelial cells represent the most important surface of contact in the body and form the first line of defence of the body to external environment. Consequently, epithelia have numerous roles in order to maintain a homeostatic defence barrier. Although the epithelium has been extensively studied over several decades, it remains the focus of new research, indicating a lack of understanding that continues to exist around these cells in specific disease settings. Importantly, evidence is emerging that airway epithelial cells in particular have varied complex functions rather than simple passive roles. One area of current interest is its role following injury. In particular, the epithelial-specific cellular mechanisms regulating their migration during wound repair remain poorly understood and remain an area that requires much needed investigation. A better understanding of the physiological, cellular and molecular wound repair mechanisms could assist in elucidating pathological processes that contribute to airway epithelial pathology. This review attempts to highlight migration-specific and cell-extracellular matrix (ECM) aspects of repair used by epithelial cells under normal and disease settings, in the context of human airways.