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SUMMARY Mononuclear phagocytes, including monocytes, macrophages and dendritic cells, contribute to tissue integrity, as well as innate and adaptive immune defense. Emerging evidence for labour division indicates that manipulation of these cells could bear therapeutic potential. However, specific ontogenies of individual populations and the overall functional organisation of the cellular network are not well-defined. Here we report a fate mapping study of the murine monocyte and macrophage compartment taking advantage of constitutive and conditional CX3CR1 promoter-driven Cre recombinase expression. We have demonstrated that major tissue resident macrophage populations, including liver Kupffer cells, lung alveolar, splenic and peritoneal macrophages, are established prior to birth and maintain themselves subsequently during adulthood independent of replenishment by blood monocytes. Furthermore, we have established that the short-lived Ly6C+ monocytes constitute obligatory steady state precursors of blood-resident Ly6C− cells and that the abundance of Ly6C+ blood monocytes dynamically controls the circulation life span of their progeny.

Rationale: The contributions of diverse cell populations in the human lung to pulmonary fibrosis pathogenesis are poorly understood. Single-cell RNA sequencing can reveal changes within individual cell populations during pulmonary fibrosis that are important for disease pathogenesis. Objectives: To determine whether single-cell RNA sequencing can reveal disease-related heterogeneity within alveolar macrophages, epithelial cells, or other cell types in lung tissue from subjects with pulmonary fibrosis compared with control subjects. Methods: We performed single-cell RNA sequencing on lung tissue obtained from eight transplant donors and eight recipients with pulmonary fibrosis and on one bronchoscopic cryobiospy sample from a patient with idiopathic pulmonary fibrosis. We validated these data using in situ RNA hybridization, immunohistochemistry, and bulk RNA-sequencing on flow-sorted cells from 22 additional subjects. Measurements and Main Results: We identified a distinct, novel population of profibrotic alveolar macrophages exclusively in patients with fibrosis. Within epithelial cells, the expression of genes involved in Wnt secretion and response was restricted to nonoverlapping cells. We identified rare cell populations including airway stem cells and senescent cells emerging during pulmonary fibrosis. We developed a web-based tool to explore these data. Conclusions: We generated a single-cell atlas of pulmonary fibrosis. Using this atlas, we demonstrated heterogeneity within alveolar macrophages and epithelial cells from subjects with pulmonary fibrosis. These results support the feasibility of discovery-based approaches using next-generation sequencing technologies to identify signaling pathways for targeting in the development of personalized therapies for patients with pulmonary fibrosis.

Misharin et al. elucidate the fate and function of monocyte-derived alveolar macrophages during the course of pulmonary fibrosis. These cells persisted throughout the life span, were enriched for the expression of profibrotic genes, and their genetic ablation ameliorated development of pulmonary fibrosis.

The lung hosts multiple populations of macrophages and dendritic cells, which play a crucial role in lung pathology. The accurate identification and enumeration of these subsets are essential for understanding their role in lung pathology. Flow cytometry is a mainstream tool for studying the immune system. However, a systematic flow cytometric approach to identify subsets of macrophages and dendritic cells (DCs) accurately and consistently in the normal mouse lung has not been described. Here we developed a panel of surface markers and an analysis strategy that accurately identify all known populations of macrophages and DCs, and their precursors in the lung during steady-state conditions and bleomycin-induced injury. Using this panel, we assessed the polarization of lung macrophages during the course of bleomycin-induced lung injury. Alveolar macrophages expressed markers of alternatively activated macrophages during both acute and fibrotic phases of bleomycin-induced lung injury, whereas markers of classically activated macrophages were expressed only during the acute phase. Taken together, these data suggest that this flow cytometric panel is very helpful in identifying macrophage and DC populations and their state of activation in normal, injured, and fibrotic lungs.Keywords: pulmonary macrophages; alveolar macrophages; interstitial macrophages; macrophage polarization; lung fibrosis Cells of the innate immune system, and especially myeloid cells such as neutrophils, eosinophils, monocytes, macrophages (alveolar and interstitial), and dendritic cells (DCs, i.e., plasmacytoid DCs, CD1031 DCs, and CD11b 1 DCs), play an important role in lung development and physiology, and contribute to important lung diseases, including pulmonary infection, cancer, asthma, chronic obstructive pulmonary disease, and pulmonary fibrosis (1-5). Alveolar and interstitial lung macrophages exhibit different origins and life spans in lungs, and have been identified as key regulators of pathological and reparative processes. Alveolar macrophages, which are considered tissue-resident macrophages, populate lung tissue during early embryogenesis and remain viable for prolonged periods, with minimal replenishment from bone marrow-derived monocytes (6). In contrast, interstitial macrophages originate from bone marrow-derived monocytes and have a shorter half-life (7,8). In recent studies, several groups of investigators suggested that these two populations of lung macrophages play opposing roles in lung injury. Alveolar macrophages appear to limit neutrophil influx into the lung during acute lung injury (9) or chronic exposure to organic dust (10), whereas interstitial macrophages promote neutrophil extravasation (11,12). An additional layer of complexity is added by the phenotypic plasticity of macrophages. Classically activated macrophages (sometimes referred to as M1-polarized) have been suggested to promote the development of acute lung injury, whereas alternatively activated macrophages (M2) may play a role in limiting or resolving lu...

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