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Single Cell RNA Sequencing Reveals a Unique Monocyte Population in Bronchoalveolar Lavage Cells of Mice Challenged with Afghanistan Particulate Matter and Allergen

ABSTRACT: Upon returning from deployment to Afghanistan, increasing numbers of United States service personnel are reporting symptoms consistent with an asthma-like airways disease, thought to be caused by prolonged inhalation of toxic desert particulate matter. Deployment Related Lung Disease (DRLD) symptoms persist in the post-deployment setting, where soldiers are then exposed to common household allergens. The goal of this study was to define the transcriptomic responses of leukocytes found in the airway lumen and airspace in a post-deployment setting, mimicked by inhalation of Afghanistan desert particulate matter (APM) and subsequent challenge with house dust mite (HDM) in mice. C57BL/6 mice (n=15/group) were exposed to filtered air or aerosolized APM for 6 hours per day for 12 consecutive days using a whole body exposure system, followed by intranasal PBS or HDM allergen challenges. One day after the last HDM challenge, mice were euthanized to collect bronchoalveolar lavage (BAL) cells for single cell RNA sequencing (scRNAseq) using the 10x Genomics Platform and assessment of inflammation and airway hyperresponsiveness (AHR). Unsupervised clustering of BAL cell scRNAseq data revealed a unique monocyte population induced only by both APM and allergen treatments. This population of monocytes is characterized by expression of genes involved in allergic asthma, including ALOX15, which is involved in airway obstruction and promoting the inflammatory response. We validated ALOX15 expression in monocytes via immunohistochemistry and immunofluorescence staining of lung tissue. APM pre-exposure, followed by HDM challenge lead to significantly increased total respiratory system resistance compared to filtered air controls. Using this post-deployment mimic in mice, we demonstrated that inhalation of desert PM while on deployment to Afghanistan may prime airways to be more responsive to allergen exposure after returning home. DRLD symptoms in post deployment setting may involve induction of a unique lung monocyte population that has the ability to regulate AHR and inflammation, possibly through increased expression of ALOX15.

ORGANISM(S): Mus musculus

PROVIDER: GSE155391 | GEO | 2022/01/06

REPOSITORIES: GEO

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Project description:Background: Allergen inhalation challenge in mild asthmatics induces airflow obstruction, airway hyperresponsiveness and inflammation, providing a model for hypothesis-generating experiments to understand regulation of these responses. We have sought to evaluate the peripheral whole blood transcriptome, post-challenge compared to pre-challenge, and to determine the effect of globin mRNA reduction methodology. Methods: Asthmatic subjects (20-60 years of age, with stable, mild allergic asthma, n=17) underwent allergen inhalation challenges. All subjects had an early asthmatic response of ≥ 20% fall in FEV1; most had a late phase response of ≥ 15% fall in FEV1. Blood was collected immediately prior to, and two hours after allergen challenge. Transcriptome analysis was performed using Affymetrix GeneChip® Human Gene 1.0 ST arrays, with and without globin mRNA reduction (PAX-GR and PAX-NGR, respectively) in 4 subjects. Replication studies for expression of nine genes contributing to the top canonical pathway, Nrf2-mediated oxidative stress response pathway, were performed with 5 independent subjects with microarray data, also with the same 4 subjects had microarray results and 8 other independent subjects with QPCR. Data were analyzed using paired t-test and Partek® Genomics Suite™. Results: The number of differentially expressed probe sets from PAX-NGR samples was twice that of PAX-GR samples. Paired analysis of each subjects' pre- and post-sample additionally demonstrated twice the power to detect differentially expressed probe sets. The Nrf2-mediated oxidative stress response pathway was identified as the top canonical pathway in the PAX-NGR samples. ATP-binding cassette sub-family C member 1 (ABCC1) gene was significantly reduced two hours after allergen challenge in the 5 subjects’ microarray dataset and the 2 QPCR replication datasets with P < 0.05. Conclusions: Globin mRNA reduction does not provide benefits to detect differentially expressed genes during allergen inhalation challenge. Allergen inhalation challenge is associated with decreased peripheral blood cell transcript level of ABCC1 gene. 26 array were analyzed. We first identified differentially expreed genes between post- and pre-challenge samples from 4 asthmatic subjects by using globin reduction samples and non-globin reduction samples (16 arrays). Then we replicated our findings using 10 arrays from 5 asthmatic subjects challenged with allergen. Includes 4 normal blood samples.

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Single Cell RNA Sequencing Reveals a Unique Monocyte Population in Bronchoalveolar Lavage Cells of Mice Challenged with Afghanistan Particulate Matter and Allergen

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