A Specialized Population of Monocyte-Derived Tracheal Macrophages Promote Airway Epithelial Regeneration

Immune cells are a critical part of the airway stem cell niche and are responsible for maintenance and immune surveillance of mucosal tissues. To understand the role of the immune microenvironment in regulating airway basal stem cell response to injury, we profiled the immune compartment of the murine trachea before and after airway epithelial injury. Sterile polidocanol detergent was administered intraorally to damage the luminal tracheal epithelium, which induces a significant infiltration of immune cells within 24 hours of injury. Basal cells then proliferate and differentiate to regenerate the tracheal epithelium within 7-14 days. Characterization of immune cell dynamics was accomplished using single cell RNA sequencing and a 15-color immunophenotyping flow cytometry panel. These studies identified tracheal macrophages as the immune cell type that undergoes the greatest expansion following epithelial injury and revealed significant phenotypic differences between tracheal macrophages and classically-defined pulmonary macrophages in the distal lung parenchyma. Tracheal macrophages are CD11b+/SiglecF- and resemble interstitial macrophages found in the distal lung. However, tracheal macrophages, under normal homeostatic conditions, display increased levels of cell surface markers normally associated with alveolar macrophages involved in phagocytosis or antigen presentation, including CD206, CD80, CD11c, and MHC II. Notably, within 3 days of epithelial injury, a significant proportion of these specialized macrophages express activation markers consistent with an M2-like pro-regenerative phenotype. To characterize the origin of these cells, transgenic Flk-Switch mice were used to discriminate macrophages derived from hematopoietic stem cells (HSCs) in the adult bone marrow versus tissue-resident macrophages derived from other sources. A significant increase in Flk2-mediated GFP expression indicates injury-responsive tracheal macrophages are HSC-derived and recruited to the trachea from outside the site of injury. In this population of mobilized cells, robust expression of monocyte-specific cell surface markers and the minimal expression of proliferation markers confirm that tracheal macrophages are largely monocyte-derived following epithelial injury, including at least 90% of M2-like activated macrophages. Lastly, we demonstrate that Ccr2-knockout mice lacking functional monocytes and macrophages fail to repair the tracheal epithelium within the normal 7-14 day time period. Ccr2-knockout mice have a significant reduction in the total number of macrophages, and in the number of M2-like macrophages following epithelial injury. Taken together, these studies indicate that tracheal macrophages are phenotypically distinct from distal lung macrophages at homeostasis, most closely resembling monocyte-derived interstitial macrophages, with some features of phagocytic or pathogen-experienced alveolar macrophages. Following epithelial injury, HSC-derived monocytes are recruited to the trachea from circulation to become M2-like activated macrophages. These M2-like activated macrophages likely promote basal stem cell activation to efficiently repair the tracheal epithelium.

No relevant conflicts of interest to declare.