A18-07 Single-Cell RNA Sequencing (scRNA-seq) of Asthmatic Individuals Exposed to Traffic-Related Air Pollution and an Inhaled Corticosteroid

Abstract Rationale Exposure to environmental pollutants such as traffic-related air pollution (TRAP) contributes to the exacerbation of asthma symptoms. Inhaled corticosteroids (ICS) are prescribed as anti-inflammatory medications to control asthma symptoms. However, recent studies suggest that TRAP exposure may reduce ICS effectiveness. Using single-cell RNA-sequencing (scRNA-seq) technology, we sought to identify immune cell populations and biological mechanisms that may contribute. Methods Thirteen research participants (aged 19-38; 3 males and 9 females) with physician-diagnosed asthma completed this double-blinded, crossover, controlled human exposure study. Participants inhaled 1.6mg of the ICS budesonide or a placebo. Participants were subsequently exposed to filtered air (FA) or diesel exhaust (DE), standardized 300µg/m3 of fine particulate matter (PM2.5), for 2 hours. Bronchoscopies were conducted to obtain bronchoalveolar lavage (BAL) samples 6 hours post-inhalation. Each participant completed four study arms (FA+Placebo, FA+ICS, DE+Placebo, DE+ICS) in a randomized order, with 4-week washouts. BAL cells from 4 participants with complete visits underwent scRNA-seq on a NextSeq 2000 platform. Approximately 40,000 cells were captured per sample at 10,000 reads/cell using 10X Genomics GEM-X Flex Gene Expression kit. Data analysis was conducted using the R package Seurat and annotated with the Human Lung Cell Atlas. Analysis on pseudobulk profiles was conducted using linear mixed-effects models to identify differentially expressed genes (DEGs). Results We captured 221,349 single cells from 16 distinct samples, identifying alveolar macrophages (AMs), T lymphocytes, B lymphocytes, monocytes, dendritic, mast, and basal cells. Within AMs, the largest cell population, we assessed DE exposure effects, with and without ICS treatment. Without ICS, there were 973 significant DEGs (Benjamini-Hochberg-corrected p-value 0.05 for DE+Placebo vs FA+Placebo) where 379 were downregulated and 594 were upregulated. With ICS use, the number of DEGs increased to 1,841 where 953 were downregulated and 888 were upregulated (DE+ICS vs FA+ICS). In addition, we assessed the effect of ICS inhalation with and without DE exposure. Without DE exposure, we found 78 DEGs where 18 were downregulated and 60 were upregulated (FA+ICS vs FA+Placebo). However, with DE exposure this increased to 3,323 where 1,760 were downregulated and 1,563 were upregulated (DE+ICS vs DE+Placebo). Interestingly, DEGs were associated with reduced interferon-γ and interferon-α response activity following DE exposure (DE+Placebo vs FA+Placebo) or ICS usage (FA+ICS vs FA+Placebo). Conclusion ScRNA-seq data of BAL from TRAP-exposed asthmatic individuals showed altered gene profiles in AMs. DEGs associated with DE exposure or ICS inhalation reflected reduced activity associated with immune responses that may impair biological function. This abstract is funded by: CIHR, BC Lung Foundation