Single-cell analysis of Tgfbr2-KO pulmonary monocytes and interstitial macrophages

Lung interstitial macrophages (IMs) inhabit the lung parenchyma and are thought to contribute to lung immunoregulation and homeostasis. While recent progress has been made about the development, diversity and transcriptional regulation of lung IMs, the microenvironmental signals responsible for their tissue-specific identity remain unidentified. Here we found, in mice, that lung endothelial cell-derived Tgf 1 specifically triggered a core Tgf receptor-dependent lung IM signature in bone marrow-derived monocytes and macrophages (Macs). In vivo, myeloid-specific ablation of Tgf receptor signaling severely impaired monocyte-to-IM development, resulting in the accumulation of perivascular monocytes, decreased IM numbers and a severe loss of IM-intrinsic identity. Of note, monocyte-to-IM development was similarly impaired in the absence of endothelial-specific Tgf 1. Functionally, mice selectively lacking Tgf receptor in IMs exhibited a severe impairment of the lung immunoregulatory environment and prematurely developed lung hyperinflation, increased compliance and decreased elastance, changes classically associated with ageing. Our work identifies a novel endothelial - IM axis involving Tgf 1-Tgf r interactions that shapes IM identity and thereby sustains lung tissue integrity, thus providing foundations for IM-targeted interventions in the context of lung ageing and other chronic inflammatory disorders. Lung CD45.1-CD45.2+Ly6G-SiglecF-CD11b+SSCloCD64+ cells were FACS-sorted from lung single-cell suspensions pooled from 5 chimeric mice established by adoptively transferring CD45.1-CD45.2+ LyzM-Cre Tgfbr2 fl/fl bone marrow (Tgfbr2-KO) or CD45.1-CD45.2+ Tgfbr2 fl/fl bone marrow (control) to irradiated CD45.1+CD45.2+ IM-DTR mice. Cells from each group were then labelled with BioLegend TotalSeq anti-mouse hastags (TotalSeq-B0301 for control group and TotalSeq-B0303 for Tgfbr2-KO group) before being pooled and subjected to library preparation with Chromium Controller and Chromium Single Cell 3’ GEM, Library & Gel Bead Kit v3 (10X Genomics). The two samples were then demultiplexed, separated by barcode and UMI were counted with cellranger (10X Genomics).