Three Unique Interstitial Macrophages in the Murine Lung at Steady State

Abstract

The current paradigm in macrophage biology is that some tissues mainly contain macrophages from embryonic origin, such as microglia in the brain, whereas other tissues contain postnatal-derived macrophages, such as the gut. However, in the lung and in other organs, such as the skin, there are both embryonic and postnatal-derived macrophages. In this study, we demonstrate in the steady-state lung that the mononuclear phagocyte system is comprised of three newly identified interstitial macrophages (IMs), alveolar macrophages, dendritic cells, and few extravascular monocytes. We focused on similarities and differences between the three IM subtypes, specifically, their phenotype, location, transcriptional signature, phagocytic capacity, turnover, and lack of survival dependency on fractalkine receptor, CX₃CR1. Pulmonary IMs were located in the bronchial interstitium but not the alveolar interstitium. At the transcriptional level, all three IMs displayed a macrophage signature and phenotype. All IMs expressed MER proto-oncogene, tyrosine kinase, CD64, CD11b, and CX₃CR1, and were further distinguished by differences in cell surface protein expression of CD206, Lyve-1, CD11c, CCR2, and MHC class II, along with the absence of Ly6C, Ly6G, and Siglec F. Most intriguingly, in addition to the lung, similar phenotypic populations of IMs were observed in other nonlymphoid organs, perhaps highlighting conserved functions throughout the body. These findings promote future research to track four distinct pulmonary macrophages and decipher the division of labor that exists between them.

Keywords: dendritic cells; interstitial macrophages; monocytes; mononuclear phagocytes; pulmonary.

Figure 1.

Analysis of pulmonary mononuclear phagocytes. (A) FACS gating strategy used to identify pulmonary interstitial macrophages (IMs) among other myeloid cells in naive mice. Myeloid cells were gated on CD11c versus CD11b (Figure E1A). Total myeloid cells were then plotted as MerTK versus CD64. Top row, the MerTK⁺CD64⁺ macrophage gate was plotted as CD11c versus CD11b or MHCII to illustrate the alveolar macrophages (AMs) and three IMs. Top middle row, MerTK⁻CD64⁻ macrophage–deficient gate was plotted with CD11c and MHCII to illustrate the dendritic cells (DCs). CD11c⁺MHCII⁺ DCs were plotted as CD11c versus CD11b to identify CD11b^lo (Batf3⁺) and CD11b^hi (Irf4⁺) DCs. Bottom middle row, macrophage/DC–deficient gate was plotted as side scatter (SSC) versus F4/80 to identify neutrophils (Neuts), eosinophils (Eos), and monocytes (Mono). Bottom row, monocytes were plotted as Ly6C versus MHCII to illustrate MHCII⁺ monocytes. (B) Frequency of individual myeloid subtypes as a proportion of the total lung extravascular myeloid cells and total number per lung. (C) Frequency of IM subtypes as a proportion of the total IM pool. Data represent four independent experiments (n = 4). (D) Pulmonary mononuclear phagocytes were plotted as SSC versus CD64 to illustrate the granularity of pulmonary mononuclear phagocytes. MHC, major histocompatibility complex.

Figure 2.

IMs are transcriptionally distinct from other pulmonary mononuclear phagocytes. Defined pulmonary myeloid cells, AMs, IM1, IM2, and IM3, from naive mice were sorted in triplicates for RNA sequencing analysis. (A) Principal component analysis plot illustrates IMs clustering distal from AMs and separate from each other. (B and C) Heat maps demonstrate RNA transcript expression from AM, IM1, IM2, and IM3. Genes of interest were separated into (B) macrophage- and monocyte-lineage–associated genes and (C) functional molecules: M1/M2 polarization markers; inflammatory mediators; chemokine ligands; and receptors. Rows are displayed as signal intensity relative to minimum and maximum values, with the exception of macrophage signature genes, where expression intensity is scaled globally across all genes of interest. P values and transcript per million (TPM) values are shown in Tables E2 and E3. Comp, component. Mac, macrophage.

Figure 3.

IMs display distinct cell surface protein expression. Left, FACS plot demonstrates how to identify IM1, IM2, and IM3 from CD45⁺MerTK⁺CD64⁺CD11b⁺ IMs. First, plotting CD206 versus CD11c separates IM3 from IM1 and IM2. CD206^int/hiCD11c^lo cells were then plotted by MHCII to identify IM1 and IM2. Top and middle rows, histograms display overlays of all three IMs (IM1, red; IM2, blue; and IM3, green) for the given protein, relative to isotype antibody control (gray). Bottom row, reporter mice display histograms for the expression of CX₃CR1, Csfr1, and Zbtb46 in all three IMs relative to a control mouse (gray).

Figure 4.

Phagocytic capacity of IMs in vivo and ex vivo. In vivo, carboxylated latex beads, Escherichia coli, or zymosan bioparticles were delivered via the intranasal route. Particle uptake was assessed after 3 hours. Data are representative of two experiments with n = 3 per group. Ex vivo, Percoll-enriched pulmonary macrophages were given carboxylated beads or bioparticles in culture. Particle uptake was assessed after 1 hour. Data are representative of three experiments with n = 3 per group. ****P < 0.0005, ***P < 0.001, **P < 0.005, *P < 0.05.

Figure 5.

IMs are slowly replaced by circulating bone marrow (BM)-derived cells. (A) Wild-type mice were irradiated using lead strips to protect the lungs before delivery of congenic donor BM cells. Lung mononuclear phagocytes were analyzed for donor origin 5 weeks after BM reconstitution. Data are representative of two experiments with n = 4. BL, blood. (B) Parabiotic mice: 15 weeks after surgical joining of congenic wild-type (WT) mice (CD45.1 and CD45.2), the contribution of circulating partner-derived cells to the lung mononuclear phagocytes was analyzed. Data are represented as the ratio of partner-derived lung mononuclear phagocytes to partner-derived blood Ly6C⁺ monocytes within the same mouse. Data are representative of two experiments with n = 4. ****P < 0.0005, ***P < 0.001, **P < 0.005, *P < 0.05. mo, monocytes.

Figure 6.

IMs are located in the bronchial interstitium. (A) FACS analysis of gated pulmonary myeloid cells from naive CX₃CR1^GFP mice plotted as MerTK versus CX₃CR1. (B and C) Frozen sections from CX₃CR1^GFP reporter mice (green) were stained with antibody against MerTK (red) and 4′,6-diamidino-2-phenylindole (DAPI; blue). (B) Pleural and alveolar space images (20× magnification) were analyzed for the presence of double-positive CX₃CR1⁺ MerTK⁺ IMs. (C) Bronchial interstitium was also stained for Lyve-1 (cyan) to identify lymphatic vessels (LV) and Lyve-1⁺ IMs (40× magnification). Arrows identify CX₃CR1⁺ MerTK⁺ IMs; white arrows point to Lyve-1⁺ IMs and gray arrows point to Lyve-1⁻ IMs. Images are representative of the indicated regions of lung tissue surveyed over five experiments. Scale bar, 10 μm.

Figure 7.

Three IMs are also present in other organs. (A) RNA-seq data were analyzed for the expression of genes previously associated with dermal or cardiac IMs. Heat map shows the relative transcript expression by AMs compared with IM1, IM2, and IM3. Rows are displayed as signal intensity relative to minimum and maximum, values with minimum and maximum TPM counts displayed to the left of each row. (B and C) The presence of IMs was analyzed in naive WT or CX₃CR1^GFP mice in the lung, skin, colon, and heart. (B) CD45⁺CD11b⁺Ly6C⁻ cells were plotted as MerTK versus CD64 to identify MerTK⁺CD64⁺ macrophages (upper panel). Gated macrophages were then plotted as CD206 versus MHCII (lower panel). Data are representative of four independent experiments with n = 3. (C) Histograms display overlays of all three IMs for the expression of CD11c, CX₃CR1 (reported), and CCR2 from the indicated organs. Gray lines illustrate isotype control antibody or WT (GFP⁻) controls.