Abstract Rationale Macrophages are one of the most prevalent cells in the lung at homeostasis. Pulmonary macrophages are a heterogenous, highly plastic population of immune cells which can be classified by location, developmental origin and activation; however, less is known about specific macrophage subsets and their underlying mechanisms in promoting pulmonary fibrotic disease. Evaluating macrophage heterogeneity and comprehensively characterising macrophage subsets, based on gene expression profiles, is possible with single-cell RNA sequencing (scRNA-seq). Methods Analysis was performed on available human scRNA-seq data from whole lung dissociates (IPF Cell Atlas GSE13683; control n = 27, IPF n = 32). Data was subjected to quality control, genome alignment, SCT transformation, integration, dimensionality reduction and cell clustering. Using Seurat v5.3.0 FindAllMarkers and previously described markers, cell annotation identified macrophage and monocyte cell clusters. Expression profiles of these were obtained per cluster following aggregation by DESeq2. Results Analysis of 77,228 cells showed 10 distinct cell clusters of which five were alveolar macrophages (AM) expressing markers CD206, MARCO, PPARG, CD169, CD11c and/or CHI3L1 (log2FC ≥ 0.25 padj0.05). In control cells, one subset of AMs expressed high levels of SPP1 and MMP7 (log2FC 4, padj0.05) and relatively low levels of RBP4, IL17RB and GPD1. Differentially expressed genes (DEGs) in this subset compared to others suggest involvement in ATP synthesis coupled electron transport and oxidative phosphorylation (log2FC 0.25, padj0.05). A second AM cluster expressed relatively low levels of SPP1 and MMP7, however had high expression of RBP4, IL17RB and GPD1 (log2FC 1.2, padj0.05). DEGs for this cluster suggest involvement in processes such as intracellular protein transport. Interestingly, the average proportion of cells per donor in the SPP1-high cluster was significantly increased in IPF patients (p 0.001). Conclusion To identify and better characterise pulmonary macrophage subsets at single-cell resolution, this project utilises scRNA-seq. Of the 10 cell clusters identified, we observed two distinct AM subsets with contrasting co-expression of SPP1 and MMP7 in control patients. Future work will transcriptionally define other identified clusters in control lungs and compare how cluster expression is altered during IPF to further elucidate the role of distinct macrophage subsets in disease pathogenesis. This abstract is funded by: None
Forde et al. (Fri,) studied this question.