Cellular Origin and Residence Time in the Airspace Alters Macrophage Activation and Differentiation in Acute Lung Injury

Rationale Resident pulmonary macrophages are seeded during embryonic development and live for up to a year in mice. These macrophages’ unique phenotype allows them to protect the lung from environmental exposures without eliciting an inflammatory response. These cells are programmed to the appropriate phenotype by their interactions with the epithelial lining. In response to lung injury this resident population can be depleted and restoration occurs via recruitment from both lung progenitors and blood monocytes. Epithelial programming is critical to establishing appropriate tissue phenotype in these recruited cells. Both resident and recruited macrophages play a role in intratracheal bleomycin mediated acute lung injury (ITB) and adopt phenotypes dependent on their origin. We propose that epithelial programming of macrophages that arise in the lung biases these cells to alternative activation post ITB. Methods C57BL/6 mice were adoptively transferred with green fluorescent protein (GFP+) positive bone marrow cells. Resultant chimeras had GFP+ bone marrow derived cells and GFP‐resident cells. Chimeras were allowed 6 weeks for bone marrow repopulation then were either sham instilled or instilled with saline or bleomycin (3U/kg). Cells collected from the airspace by bronchoalveolar lavage, with (BALM) or without (BAL) massage, were phenotypically assessed by flow cytometry and IHC. Results BAL cells collected from sham mice were mature pulmonary macrophages (F4/80+ Cd11c+ Cd11b−). 8 days post sham, 11% of macrophages collected by BAL were GFP+, which increased to 20% in BALM. One week later BALM GFP+ cells increased to 37%. GFPminus; cells in the BAL did not change in number with time. Saline administration did not alter number or phenotype at 8 days. At 15 days, BAL macrophages were not altered but massage collected more GFPminus; and GFP+ macrophages. Histological assessment of tissue after lavage identified GFP+ cells near the blood vessels in sham and saline treated mice. ITB increased the number of GFP+ macrophages in the airspace that were predominately Ly6C+ at 8 days. GFPminus; macrophage number was not altered by ITB. GFPminus; macrophages were a mix of mature (Cd11c+ Cd11b‐Cd206+) and immature (Cd11cminus; Cd11b+ Cd206minus;). Massage increased the number of Cd11cminus; but not Cd11c+ GFPminus; macrophages. At 15 days, GFP+ macrophages are found in both BAL and BALM. Those in BAL express high levels of Cd206 while in BALM cells are either high Cd206 or Ly6C. At 15 days the number of GFPminus; CD11cminus; macrophages is increased compared to 8 days, and these express neither Cd206 nor Ly6C. Cd11c+ GFPminus; macrophages express high levels of Cd206. Those cells identified as GFP+ in tissue by IHC are found in areas of consolidation and septal destruction. IHC staining of Cd11b identifies a similar pattern of positive cells. Conclusions Resident pulmonary macrophages are turned over at baseline without change in number or phenotype. Repopulation of the resident cells occurs in the face of acute lung injury via a tissue intermediate. Vascular recruited macrophages respond to injury by adopting a M1 phenotype during inflammation and M2 phenotype during resolution. Resident macrophages adopt a M2 phenotype as a result of epithelial interaction that is accentuated by injury. Immature resident cells are undifferentiated or polarized. This work implicates the pulmonary epithelium as critical to alternative activation during acute lung injury. Support or Funding Information HL086621, ES005022, GM108463