Abstract Rationale Chronic beryllium disease(CBD) is a granulomatous lung disease resulting from beryllium exposure in those sensitized to beryllium(Be). The key drivers of Be-specific immune response in CBD compared to the disease precursor, Be-sensitization(BeS) are not well-defined, and could help define targets for disease therapy or diagnostics. We performed cellular indexing of transcriptomes and epitopes by sequencing (CITE-seq) on bronchoalveolar (BAL) cells from CBD, BeS and controls stimulated with Be over time to identify molecular changes associated with CBD and BeS at the cellular level. Methods BAL cells from CBD, BeS and control (n = 6 each) were cultured with BeSO4 for 0, 1, 4 and 24 hours. CITE-seq was performed using 22 antibody tags on 5-10,000 cells/sample. Protein epitopes and transcriptome data were used to cluster cells and identify broad cell types. Macrophage and non-macrophage populations were further subclustered to identify more granular sub-populations. We identified cell type-specific gene expression changes by disease/control status at each time point, adjusting for age and sex using linear mixed effects models on normalized pseudobulk counts. Ingenuity Pathway Analysis(IPA) was used to identify pathway enrichment. Results Multiple alveolar macrophages populations were identified, including monocyte-derived recruited and metallophilic macrophages and two specialized populations exhibiting high expression of 1) cytokines and chemokines and 2) NF-ĸB genes (Figure 1A), with changes in abundance of specialized and recruited macrophage populations noted by disease and time. Clusters of T helper cells, cytotoxic T lymphocytes (CTLs), NK/NKT cells, dendritic cells (pDC and mDC), and mast cells were identified, with changes in CTLs noted (Figure 1B). The most significant differential gene expression was observed in macrophages at 24 hours in CBD vs. BeS. Known and novel pathway enrichment was found in CBD in all macrophage populations. Comparing CBD to BeS, NF-ĸB macrophages showed activation of TNFR2-mediated non-canonical NF-ĸB pathway, mTOR and senescence pathways, whereas metallophillic macrophages showed enrichment in multiple development-related pathways (several Hedgehog signaling-related pathways, NOTCH4 signaling, degradation of β-catenin), regulation of apoptosis, noncanonical NF-ĸB signaling, and cellular response to hypoxia. Other groups exhibited activation of known and novel pathways. Conclusions Macrophages exhibited disease- and time-dependent transcriptional differences in CBD, BeS and controls using CITE-seq. Differences in gene expression were also apparent, and associated with different pathways in distinct macrophage subpopulations, including known and novel pathways to CBD. These pathways may serve as therapeutic targets, and we are exploring their functional potential in cell models. This abstract is funded by: R01ES033678
Maier et al. (Fri,) studied this question.