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 distinguished by surface and transcriptional expression of folate receptor ß (Folr2/FRß). These subsets inhabited distinct niches within the lung interstitium. Within FRß+ 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 FRß- resident IMs but retained expression in several origin-specific genes, such as IL-1ß. FRß+ 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. FRΒ+ IMs comprise a stable, resident population, whereas FRß- ΙΜs represent a mixed population of resident and recruited IMs.

Polyamine import and accumulation causes immunomodulation in macrophages engulfing apoptotic cells.

Phagocytosis of apoptotic cells, termed efferocytosis, is critical for tissue homeostasis and drives anti-inflammatory programming in engulfing macrophages. Here, we assess metabolites in naive and inflammatory macrophages following engulfment of multiple cellular and non-cellular targets. Efferocytosis leads to increases in the arginine-derived polyamines, spermidine and spermine, in vitro and in vivo. Surprisingly, polyamine accumulation after efferocytosis does not arise from retention of apoptotic cell metabolites or de novo synthesis but from enhanced polyamine import that is dependent on Rac1, actin, and PI3 kinase. Blocking polyamine import prevents efferocytosis from suppressing macrophage interleukin (IL)-1ß or IL-6. This identifies efferocytosis as a trigger for polyamine import and accumulation, and imported polyamines as mediators of efferocytosis-induced immune reprogramming.

Airspace Macrophages and Monocytes Exist in Transcriptionally Distinct Subsets in Healthy Adults.

Rationale: Macrophages are the most abundant immune cell in the alveoli and small airways and are traditionally viewed as a homogeneous population during health. Whether distinct subsets of airspace macrophages are present in healthy humans is unknown. Single-cell RNA sequencing allows for examination of transcriptional heterogeneity between cells and between individuals. Understanding the conserved repertoire of airspace macrophages during health is essential to understanding cellular programing during disease.Objectives: We sought to determine the transcriptional heterogeneity of human cells obtained from BAL of healthy adults.Methods: Ten subjects underwent bronchoscopy with BAL. Cells from lavage were subjected to single-cell RNA sequencing. Unique cell populations and putative functions were identified. Transcriptional profiles were compared across individuals.Measurements and Main Results: We identify two novel subgroups of resident airspace macrophages-defined by proinflammatory and metallothionein gene expression profiles. We define subsets of monocyte-like cells and compare them with peripheral blood mononuclear cells. Finally, we compare global macrophage and monocyte programing between males and females.Conclusions: Healthy human airspaces contain multiple populations of myeloid cells that are highly conserved between individuals and between sexes. Resident macrophages make up the largest population and include novel subsets defined by inflammatory and metal-binding profiles. Monocyte-like cells within the airspaces are transcriptionally aligned with circulating blood cells and include a rare population defined by expression of cell-matrix interaction genes. This study is the first to delineate the conserved heterogeneity of airspace immune cells during health and identifies two previously unrecognized macrophage subsets.