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, a substantial number of U.S. military personnel report deployment-related lung disease (DRLD) symptoms, including those consistent with an asthma-like airways disease. DRLD is thought to be caused by prolonged inhalation of toxic desert particulate matter, which can persist in the postdeployment setting such as exposure to common household allergens. The goal of this study was to define the transcriptomic responses of lung leukocytes of mice exposed to Afghanistan desert particulate matter (APM) and house dust mite (HDM). C57BL/6 mice (n = 15/group) were exposed to filtered air or aerosolized APM for 12 days, followed by intranasal PBS or HDM allergen challenges for 24 h. Bronchoalveolar lavage (BAL) cells were collected for single-cell RNA sequencing (scRNAseq), and assessment of inflammation and airway hyper-responsiveness. 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 the expression of genes involved in allergic asthma, including Alox15. We validated Alox15 expression in monocytes via immunostaining of lung tissue. APM pre-exposure, followed by the HDM challenge, led to significantly increased total respiratory system resistance compared with filtered air controls. Using this mouse model to mimic DRLD, we demonstrated that inhalation of airborne PM during deployment may prime airways to be more responsive to allergen exposure after returning home, which may be linked to dysregulated immune responses such as induction of a unique lung monocyte population.

Keywords: ALOX15; allergic asthma; particulate matter; single-cell RNA sequencing.

Figure 1.

Overview of in vivo mouse APM and allergen exposures. C57BL/6 mice were exposed to aerosolized Afghanistan PM (APM) at a concentration of 5 mg/m³ or filtered air (FA, control) for 6 h per day on days 1–12 days. On days 22 and 29, mice were sensitized with PBS (control) or house dust mite (HDM, 10 μg/mouse) then challenged with PBS or HDM on days 36–38. Mice were euthanized on day 39.

Figure 2.

scRNAseq revealed unique cell populations induced by different treatments. tSNE plots of mouse BAL cells were constructed in Seurat. Unsupervised clustering of mouse BAL cells revealed 17 distinct clusters (A). Clusters were named according to cell marker genes (B). Proportions of different cell types vary across treatments, revealing a unique cluster of monocytes is induced by only one treatment—HDM and APM (C and D).

Figure 3.

Combination of APM and HDM treatment induces expression of allergic asthma-associated genes in the monocyte cluster. Heatmap of top genes expressed in the monocyte cluster, induced by APM + HDM treatment (A). Yellow indicates upregulation and purple indicates downregulation, presented as average log fold change. In (B), a signaling pathway of genes involved in allergic asthma shows how Alox15 and other genes in the monocyte cluster contribute to allergic asthma. Alox15 is expression is concentrated in the monocyte cluster (C). A tSNE plot colored by Alox15 expression demonstrates that Alox15 gene expression is concentrated highly in cells of the monocyte cluster, which is induced only by combination APM + HDM treatment. Alox15 expressing cells are shown on the spectrum of yellow to red, gray indicates no expression.

Figure 4.

Presence of Alox15 positive monocytes in lung tissue from APM + HDM exposed mice. Lung tissue was harvest after BAL collection and stained with F4/80, a monocyte/macrophage marker (conjugated to APC 647, pseudocolored in green), and an Alox15 antibody (with secondary conjugated to AF555, red). Nuclei were stained with DAPI (blue). Representative images of lung tissue from one mouse per group show that only APM + HDM treated mice (bottom) had cells that stain positive for both markers (white arrows, overlay) (A). Alox15 positive cells that were not monocytes were present in FA + HDM treated lungs (yellow arrows). A close-up of an Alox15 positive monocyte from the APM + HDM treated group shows double staining (B). Slides were imaged using a Zeiss LSM 700 Confocal Microscope at 20× (A) and 63× (B).

Figure 5.

Positive Alox15 expression in lung tissue from APM + HDM exposed mice. Lung tissue was harvest after BAL collection and stained with Alox15 antibody then counterstained with Mayer’s hematoxylin. Representative images of tissue show that APM + HDM treated mice (bottom right) had more eosinophils (red arrows) and monocytes (black arrows) positive for Alox15 than in other groups.

Figure 6A.

APM pre-exposure enhances inflammation and AHR after the HDM challenge. AHR was assessed 24 h after the last challenge (n = 8/group). BAL was collected 24 h after the last challenge from mice after airway physiology assessment. APM pre-exposure followed by allergen (APM vs APM + HDM) challenge significantly increased total cell count (A), absolute eosinophil numbers (B), % eosinophils (C), Eotaxin-2 protein (D), and KC protein (E). Data are presented with bars indicating the median. APM + HDM exposure increased AHR compared with filtered air + PBS control AHR in a dose-dependent manner (G). APM pre-exposure followed by allergen challenge significantly increased AHR at the highest Mch concentration (H). Data presented as means + SEM (G) or bars to represent median (H). p-Values represent a one-way ANOVA and p-value of less than .05 was considered significant. An outlier (determined via the outlier test) was removed from the FA + HDM group at the 50 mg/ml dose. A two-way ANOVA was performed to compare filtered air versus APM versus HDM versus APM + HDM: HDM = 0.0003, APM = 0.54, Interaction = 0.52 (A); HDM = 0.0001, APM = 0.39, Interaction = 0.1 (B); HDM = 0.0002, APM = 0.61, Interaction = 0.61 (C); HDM = 0.0001, APM = 0.86, Interaction = 0.91 (D); HDM = 0.003, APM = 0.22, Interaction = 0.35 (E); HDM = 0.0001, APM = 0.69, Interaction = 0.67 (E); HDM = 0.0027, APM = 0.058, Interaction = 0.20 (H).

Figure 6B