Although the antibody-based recognition of cell-surface markers has been widely used for the identification of immune cells, overlap in the expression of markers by different cell types and the inconsistent use of antibody panels have resulted in a lack of clearly defined signatures for myeloid cell subsets. We developed a 10-fluorochrome flow cytometry panel for the identification and quantitation of myeloid cells in the lungs, including pulmonary monocytes, myeloid dendritic cells, alveolar and interstitial macrophages, and neutrophils. After the initial sorting of viable CD45+ leukocytes, we detected three leukocyte subpopulations based on CD68 expression: CD68−, CD68low, and CD68hi. Further characterization of the CD68hi population revealed CD45+/CD68hi/F4/80+/CD11b−/CD11c+/Gr1− alveolar macrophages and CD45+/CD68hi/F4/80−/CD11c+/Gr1−/CD103+/major histocompatibility complex (MHC) class IIhi dendritic cells. The CD68low population contained primarily CD45+/CD68low/F4/80+/CD11b+/CD11c+/Gr1−/CD14low interstitial macrophages and CD45+/CD68low/F4/80+/CD11b+/CD11c−/Gr1low/CD14hi monocytes, whereas the CD68− population contained neutrophils (CD45+/CD68−/F4/80−/CD11b+/Gr1hi). The validity of cellular signatures was confirmed by a morphological analysis of FACS-sorted cells, functional studies, and the depletion of specific macrophage subpopulations using liposomal clodronate. We believe our approach provides an accurate and reproducible method for the isolation, quantification, and characterization of myeloid cell subsets in the lungs, which may be useful for studying the roles of myeloid cells during various pathological processes.

Clear identification of specific cell populations by flow cytometry is important to understand functional roles. A well-defined flow cytometry panel for myeloid cells in human bronchoalveolar lavage (BAL) and lung tissue is currently lacking. The objective of this study was to develop a flow cytometry–based panel for human BAL and lung tissue. We obtained and performed flow cytometry/sorting on human BAL cells and lung tissue. Confocal images were obtained from lung tissue using antibodies for cluster of differentiation (CD)206, CD169, and E cadherin. We defined a multicolor flow panel for human BAL and lung tissue that identifies major leukocyte populations. These include macrophage (CD206+) subsets and other CD206− leukocytes. The CD206− cells include: (1) three monocyte (CD14+) subsets, (2) CD11c+ dendritic cells (CD14−, CD11c+, HLA-DR+), (3) plasmacytoid dendritic cells (CD14−, CD11c−, HLA-DR+, CD123+), and (4) other granulocytes (neutrophils, mast cells, eosinophils, and basophils). Using this panel on human lung tissue, we defined two populations of pulmonary macrophages: CD169+ and CD169− macrophages. In lung tissue, CD169− macrophages were a prominent cell type. Using confocal microscopy, CD169+ macrophages were located in the alveolar space/airway, defining them as alveolar macrophages. In contrast, CD169− macrophages were associated with airway/alveolar epithelium, consistent with interstitial-associated macrophages. We defined a flow cytometry panel in human BAL and lung tissue that allows identification of multiple immune cell types and delineates alveolar from interstitial-associated macrophages. This study has important implications for defining myeloid cells in human lung samples.

Monocyte-derived alveolar macrophages drive lung injury and fibrosis in murine models and are associated with pulmonary fibrosis in humans. Monocyte-derived alveolar macrophages have been suggested to develop a phenotype that promotes lung repair as injury resolves. We compared single-cell and cytokine profiling of the alveolar space in a cohort of 35 patients with post-acute sequelae of COVID-19 who had persistent respiratory symptoms and abnormalities on a computed tomography scan of the chest that subsequently improved or progressed. The abundance of monocyte-derived alveolar macrophages, their gene expression programs, and the level of the monocyte chemokine CCL2 in bronchoalveolar lavage fluid positively associated with the severity of radiographic fibrosis. Monocyte-derived alveolar macrophages from patients with resolving or progressive fibrosis expressed the same set of profibrotic genes. Our findings argue against a distinct reparative phenotype in monocyte-derived alveolar macrophages, highlighting their utility as a biomarker of failed lung repair and a potential target for therapy.

Despite IL-9 functioning as a pleiotropic cytokine in mucosal environments, the IL-9–responsive cell repertoire is still not well defined. Here, we found that IL-9 mediates proallergic activities in the lungs by targeting lung macrophages. IL-9 inhibits alveolar macrophage expansion and promotes recruitment of monocytes that develop into CD11c ⁺ and CD11c ⁻ interstitial macrophage populations. Interstitial macrophages were required for IL-9–dependent allergic responses. Mechanistically, IL-9 affected the function of lung macrophages by inducing Arg1 activity. Compared with Arg1-deficient lung macrophages, Arg1-expressing macrophages expressed greater amounts of CCL5. Adoptive transfer of Arg1 ⁺ lung macrophages but not Arg1 ⁻ lung macrophages promoted allergic inflammation that Il9r ^−/− mice were protected against. In parallel, the elevated expression of IL-9, IL-9R, Arg1, and CCL5 was correlated with disease in patients with asthma. Thus, our study uncovers an IL-9/macrophage/Arg1 axis as a potential therapeutic target for allergic airway inflammation.

The chemokine, CXCL10, and its cognate receptor, CXCR3, are important mediators of the pathobiology of lung fibrosis. Macrophages are a known source of CXCL10, but their specific source in the lung is poorly defined due to incomplete characterization of macrophage subpopulations. We recently developed a novel flow cytometric approach that discriminates resident alveolar macrophages from recruited exudative macrophages (ExMacs) after infectious lung injury. We hypothesized that ExMacs are present after noninfectious lung injury with bleomycin, and are a source of CXCL10. We found that ExMacs are recruited to the lung after injury, peaking at Day 7, then maintained through Day 28. ExMac recruitment was significantly reduced, but not abolished, in CCR2 null mice. ExMacs, but not alveolar macrophages, produce CXCL10, both constitutively and after stimulation with hyaluronan (HA) fragments. Interestingly, ExMac stimulation with LPS resulted in complete suppression of CXCL10. In contrast, ExMacs produced TNF-α and CXCL2/MIP-2 (Macrophage Inflammatory Protein-2) after stimulation with both HA and LPS. ExMacs were present in CXCR3 null mice after bleomycin, but produced minimal CXCL10. This impairment was overcome by administration of exogenous IFN-γ or IFN-γ with HA. Collectively, these data suggest that ExMacs are recruited and maintained in the lung after noninfectious lung injury, are a source of a variety of cytokines, but importantly, are essential for the production of antifibrotic CXCL10. Understanding the contribution of ExMacs to the pathobiology of lung injury and repair could lead to new treatment options for fibrosing lung diseases.