A specialized population of monocyte-derived tracheal macrophages promote airway epithelial regeneration through a CCR2-dependent mechanism.

Macrophages are critical for maintenance and repair of mucosal tissues. While functionally distinct subtypes of macrophage are known to have important roles in injury response and repair in the lungs, little is known about macrophages in the proximal conducting airways. Single-cell RNA sequencing and flow cytometry demonstrated murine tracheal macrophages are largely monocyte-derived and are phenotypically distinct from lung macrophages at homeostasis. Following sterile airway injury, monocyte-derived macrophages are recruited to the trachea and activate a pro-regenerative phenotype associated with wound healing. Animals lacking the chemokine receptor CCR2 have reduced numbers of circulating monocytes and tracheal macrophages, deficient pro-regenerative macrophage activation and defective epithelial repair. Together, these studies indicate that recruitment and activation of monocyte-derived tracheal macrophages is CCR2-dependent and is required for normal airway epithelial regeneration.

Airway-Associated Macrophages in Homeostasis and Repair.

There is an increasing appreciation for the heterogeneity of myeloid lineages in the lung, but relatively little is known about populations specifically associated with the conducting airways. We use single-cell RNA sequencing, flow cytometry, and immunofluorescence to characterize myeloid cells of the mouse trachea during homeostasis and epithelial injury/repair. We identify submucosal macrophages, similar to lung interstitial macrophages, and intraepithelial macrophages. Following injury, there are early increases in neutrophils and submucosal macrophages, including M2-like macrophages. Intraepithelial macrophages are lost after injury and later restored by CCR2+ monocytes. We show that repair of the tracheal epithelium is impaired in Ccr2-deficient mice. Mast cells and group 2 innate lymphoid cells are sources of interleukin-13 (IL-13) that polarize macrophages and directly influence basal cell behaviors. Their proximity to the airway epithelium establishes these myeloid populations as potential therapeutic targets for airway disease.

The Cellular and Physiological Basis for Lung Repair and Regeneration: Past, Present, and Future.

The respiratory system, which includes the trachea, airways, and distal alveoli, is a complex multi-cellular organ that intimately links with the cardiovascular system to accomplish gas exchange. In this review and as members of the NIH/NHLBI-supported Progenitor Cell Translational Consortium, we discuss key aspects of lung repair and regeneration. We focus on the cellular compositions within functional niches, cell-cell signaling in homeostatic health, the responses to injury, and new methods to study lung repair and regeneration. We also provide future directions for an improved understanding of the cell biology of the respiratory system, as well as new therapeutic avenues.

Isolation, Maintenance and Differentiation of Primary Tracheal Basal Cells from Adult Rhesus Macaque.

In this report, we describe methodologies for the isolation and culture of primary rhesus macaque tracheal basal cells, their cryopreservation, long term storage and differentiation. These are comparable to state-of-the-art protocols that have been developed for mouse and human airway basal cells. This method is based on the use of proprietary media, providing an easily reproducible and applicable protocol for usage in biosafety level 2 (BSL2) settings. Tracheas from rhesus macaques were isolated after animal euthanasia and subjected to enzymatic digestion overnight. Cells of the epithelial layer were scraped off of the trachea for cell culture. Twenty-four hours after plating basal cells had attached and nonadherent cells were removed. First passages of basal cells can be frozen for early passage storage in liquid nitrogen or propagated and differentiated on an air-liquid interface and in a tracheosphere assay up to passage seven. This protocol provides a platform for the analysis of basal cells from a close evolutionary relative to humans.