Novel flow cytometry approach to identify bronchial epithelial cells from healthy human airways

Sampling various compartments within the lower airways to examine human bronchial epithelial cells (HBEC) is essential for understanding numerous lung diseases. Conventional methods to identify HBEC in bronchoalveolar lavage (BAL) and wash (BW) have throughput limitations in terms of efficiency and ensuring adequate cell numbers for quantification. Flow cytometry can provide high-throughput quantification of cell number and function in BAL and BW samples, while requiring low cell numbers. To date, a flow cytometric method to identify HBEC recovered from lower human airway samples is unavailable. In this study we present a flow cytometric method identifying HBEC as CD45 negative, EpCAM/pan-cytokeratin (pan-CK) double-positive population after excluding debris, doublets and dead cells from the analysis. For validation, the HBEC panel was applied to primary HBEC resulting in 98.6% of live cells. In healthy volunteers, HBEC recovered from BAL (2.3% of live cells), BW (32.5%) and bronchial brushing samples (88.9%) correlated significantly (p = 0.0001) with the manual microscopy counts with an overall Pearson correlation of 0.96 across the three sample types. We therefore have developed, validated, and applied a flow cytometric method that will be useful to interrogate the role of the respiratory epithelium in multiple lung diseases

Effect of di-butyl phthalate exposure in the human airway and systemic immunology : a double-blind, crossover study

Phthalates are used as softeners in commercial products. They leak into the environment and become wide-spread contaminants. Epidemiological studies suggest an association between phthalate inhalation and development/worsening of airway diseases, but a firm link has not been established. Asthma is a complex disorder associated with inflammation resulting in airway hyper-responsiveness (AHR). Increasing interest is focused on abnormal immune responses as an underlying mechanism contributing to increasing the risk of asthma. This study is the first to investigate airway and systemic effects in humans due to inhalation of a known concentration of a single phthalate. Di-butyl phthalate (DBP), exists in high concentrations in indoor air and has shown inflammatory potential. We hypothesize that DBP inhalation, prior to allergen inhalation will: a) enhance airway inflammation and responsiveness to allergen, and b) alter the activation state and functionality of immune cells in the airway and peripheral blood. In this novel double-blind, order-randomized, crossover study, 16 participants were exposed by inhalation to controlled levels of DBP or clean air (CA) for 3h, followed immediately by an allergen (dust-mite, grass or birch) inhalation. To assess lung function and airway inflammation, spirometry and exhaled nitric oxide was measured before, 3h and 20h post-DBP/CA exposure and allergen inhalation. Blood, bronchoalveolar wash and lavage (BAL) were collected for quantification and measurement of the activation pattern of immune cells and inflammatory mediator release. DBP inhalation followed by an allergen inhalation, significantly augmented the airflow decline (FEV1) in response to an inhaled allergen, compared to CA. Moreover, DBP enhanced the recruitment of BAL macrophages, specifically the M2 phenotype with increased expression of CD206 to the lungs. Meanwhile, the percent of T helper cells increased, while T regulatory and non-classical monocytes decreased, in peripheral blood. Only minor effects were observed for systemic inflammatory mediators. Moreover, significant effect modifications were observed for sex, AHR status and type of allergen inhaled. The results suggest significant effects of a common commercial chemical, DBP, on clinically relevant airway and systemic endpoints in the context of allergen exposure in sensitized individuals. Future research should aim to validate and connect these findings within relevant policy and public health contexts.Medicine, Faculty ofExperimental Medicine, Division ofMedicine, Department ofGraduat

Peroxisome proliferator-activated receptor gamma gene variants modify human airway and systemic responses to indoor dibutyl phthalate exposure

Abstract Background Single nucleotide polymorphisms (SNPs) of peroxisome proliferator-activated receptor gamma (PPAR-γ; gene: PPARG) and oxidative stress genes are associated with asthma risk. However, whether such variants modulate responses to dibutyl phthalate (DBP), a common plasticizer associated with increased asthma development, remains unknown. The purpose of this study is to investigate how SNPs in PPARG and oxidative stress genes, as represented by two separate genetic risk scores, modify the impact of DBP exposure on lung function and the airway and systemic response after an inhaled allergen challenge. Methods We conducted a double-blinded human crossover study with sixteen allergen-sensitized participants exposed for three hours to DBP and control air on distinct occasions separated by a 4-week washout. Each exposure was followed by an allergen inhalation challenge; subsequently, lung function was measured, and blood and bronchoalveolar lavage (BAL) were collected and analyzed for cell counts and allergen-specific immunoglobulin E (IgE). Genetic risk scores for PPAR-γ (P-GRS; weighted sum of PPARG SNPs rs10865710, rs709158, and rs3856806) and oxidative stress (OS-GRS; unweighted sum of 16 SNPs across multiple genes) were developed, and their ability to modify DBP effects were assessed using linear mixed-effects models. Results P-GRS and OS-GRS modified DBP effects on allergen-specific IgE in blood at 20 h (interaction effect [95% CI]: 1.43 [1.13 to 1.80], p = 0.005) and 3 h (0.99 [0.98 to 1], p = 0.03), respectively. P-GRS also modified DBP effects on Th2 cells in blood at 3 h (− 25.2 [− 47.7 to − 2.70], p = 0.03) and 20 h (− 39.1 [− 57.9 to − 20.3], p = 0.0005), and Th2 cells in BAL at 24 h (− 4.99 [− 8.97 to − 1.01], p = 0.02). An increasing P-GRS associated with reduced DBP effect on Th2 cells. Neither GRS significantly modified DBP effects on lung function parameters. Conclusions PPAR-γ variants modulated several airway and systemic immune responses to the ubiquitous chemical plasticizer DBP. Our results suggest that PPAR-γ variants may play a greater role than those in oxidative stress-related genes in airway allergic responses to DBP.Medicine, Faculty ofNon UBCMedical Genetics, Department ofMedicine, Department ofReviewedFacultyResearcherPostdoctoralGraduat