Murine Pulmonary Interstitial Macrophage Subsets after Lipopolysaccharide

We sought to characterize pulmonary interstitial macrophage (IM) origin, subsets, and transcriptomic profiles during homeostasis and lipopolysaccharide (LPS) induced acute lung inflammation. During homeostasis, we used three complementary methods: spectral flow cytometry, single-cell RNA-sequencing, and gene regulatory network enrichment to demonstrate that IMs can be divided into two core subsets distinguished by surface and transcriptional expression of folate receptor β (Folr2/FRβ). Within FRβ+ IMs we identified a subpopulation marked by co-expression of LYVE1. During acute LPS-induced inflammation, lung IM numbers expand. Lineage tracing revealed IM expansion was due to recruitment of monocyte-derived IMs. At the peak of inflammation, recruited IMs were comprised of two unique subsets defined by expression of genes associated with interferon signaling and glycolytic pathways. As recruited IMs matured, they adopted the overall transcriptional state of FRβ- resident IMs but retained expression in several origin-specific genes. FRβ+ IMs were of near-pure resident origin. Male resident IM lineage tracing mice had lungs harvested and digested at homeostasis (n=3), three days after LPS (n=4), or six days after intratracheal lipopolysaccharide. Cells underwent FACS sorting to separate 1)all CD45+ cells, 2) resident IMs, or 3)recruited (monocyte-derived) IMs. Sorted cells were analyzed by scRNAseq