Single-cell RNA sequencing reveals unique monocyte-derived interstitial macrophage subsets during lipopolysaccharide-induced acute lung inflammation

Interstitial macrophages (IMs) reside in the lung tissue surrounding key structures including airways, vessels, and alveoli. Recent work has described IM heterogeneity during homeostasis, however, there are limited data on IMs during inflammation. We sought to characterize 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 distin-guished by surface and transcriptional expression of folate receptor I3 (Folr2/FRI3). These subsets inhabited distinct niches within the lung interstitium. Within FRI3+ IMs we identified a subpopulation marked by coexpression 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 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 FRI3- resident IMs but retained expression in several origin-specific genes, such as IL-1I3. FRI3+ IMs were of near-pure resident origin. Taken together our data show that during LPS-induced inflammation, there are distinct populations of IMs that likely have unique functions. FRB+ IMs comprise a stable, resident population, whereas FRI3- IMs represent a mixed population of resident and recruited IMs.