Alveolar macrophages develop from fetal monocytes that differentiate into long-lived cells in the first week of life via GM-CSF

Abstract

Tissue-resident macrophages can develop from circulating adult monocytes or from primitive yolk sac-derived macrophages. The precise ontogeny of alveolar macrophages (AMFs) is unknown. By performing BrdU labeling and parabiosis experiments in adult mice, we found that circulating monocytes contributed minimally to the steady-state AMF pool. Mature AMFs were undetectable before birth and only fully colonized the alveolar space by 3 d after birth. Before birth, F4/80(hi)CD11b(lo) primitive macrophages and Ly6C(hi)CD11b(hi) fetal monocytes sequentially colonized the developing lung around E12.5 and E16.5, respectively. The first signs of AMF differentiation appeared around the saccular stage of lung development (E18.5). Adoptive transfer identified fetal monocytes, and not primitive macrophages, as the main precursors of AMFs. Fetal monocytes transferred to the lung of neonatal mice acquired an AMF phenotype via defined developmental stages over the course of one week, and persisted for at least three months. Early AMF commitment from fetal monocytes was absent in GM-CSF-deficient mice, whereas short-term perinatal intrapulmonary GM-CSF therapy rescued AMF development for weeks, although the resulting AMFs displayed an immature phenotype. This demonstrates that tissue-resident macrophages can also develop from fetal monocytes that adopt a stable phenotype shortly after birth in response to instructive cytokines, and then self-maintain throughout life.

Figure 1.

AMFs self-maintain locally with minimal contribution from circulating hematopoietic precursors. BAL (A) and total lung (B) were harvested from adult mice and stained for CD11b, F4/80, SiglecF, CD64, Ly-6C, and MHCII and evaluated for autofluorescence, FSC, and SSC profile (C). (D) CD45.1⁺CD45.2⁺ mice were lethally irradiated and reconstituted with equal amounts of CD45.1⁺ WT and CD45.2⁺CCR2^−/− BM cells. Radio-resistant cells, distinguished as CD45.1⁺CD45.2⁺ cells, were gated out, and the remaining BM-derived cells of the indicated populations were analyzed for their ratio of CD45.1⁺ WT and CD45.2⁺CCR2^−/− cells. (E) Summary data from five independent chimeric mice. (F) Parabiotic mice were generated by suturing together CD45.1⁺ WT and CD45.2⁺CCR2^−/− mice. The percentage of cells of CD45.1⁺ WT donor origin was determined in B cells, neutrophils, and monocytes in the blood and AMFs in the lungs of the CD45.2⁺CCR2^−/− parabionts. (G) Adult C57BL/6 mice received BrdU continuously for 28 d. Mice were sacrificed at regular intervals, and incorporation of BrdU in lung AMFs and monocytes was evaluated. (H) Adult C57BL/6 mice were sacrificed and Ki-67 expression (left) was determined in AMFs. The isotype staining is shown (right). Data represent at least three (A– E) or two (F– H) independent experiments involving at least six (E) and four (F and G) independent mice.

Figure 2.

Alveolar MFs appear in the alveolar space during the first week of life. Lungs were harvested at various time points before and after (PND) the DOB. (A and C) Flow cytometry staining for CD45⁺ cells. (B) CD11b⁺F4/80⁺ myeloid cells were gated and analyzed for CD11c and SiglecF expression. (D) Paraffin lung sections stained with hematoxylin. (E) Cryosection of lungs stained with DAPI (blue) and SiglecF (red). Data in A–E represent at least two independent experiments involving at least three independent mice per time point.

Figure 3.

Fetal MFs, fetal monocytes, preAMFs, and mature AMFs appear in consecutive waves during lung development. Lungs were harvested at various time points before and after (PND3, PND14) the date of birth (DOB). CD11b⁺F4/80⁺ myeloid cells were subdivided into CD11c^loSigle-F^lo, CD11c^intSigle-F^lo, and CD11c^hiSiglecF^hi cells (A) and analyzed for Ly-6C, F4/80 (B), and CD64 expression (C). (D) Fetal monocytes (isolated at E17), fetal MFs (isolated at E17), preAMFs (isolated at the DOB), and mature AMFs (isolated from adult mice) were sorted, put in culture in vitro overnight in complete medium, and subjected to electromagnetic microscopy to assess their capacity to adhere to cell culture plastic and general morphology. (E) Percentage of fetal MFs, fetal monocytes, preAMFs, and mature AMFs among CD45⁺ cells in the lungs at the indicated time points. Data in A–E represent at least two independent experiments involving at least three independent mice per time point.

Figure 4.

Fetal monocytes give rise to self-maintaining AMFs through a preAMF intermediate step. (A and B) CD45.1⁺ or CD45.2⁺ E17 embryos were sacrificed and fetal monocytes and fetal MFs were FACS sorted and mixed together at a 50:50 ratio (A; left, CD45.1⁺ fetal monocytes and CD45.2⁺ fetal MFs; right, CD45.1⁺ fetal MFs and CD45.2⁺ fetal monocytes) and transferred into CD45.1⁺CD45.2⁺ mice on their DOB. 7 d after transfer, CD45.1⁺CD45.2⁺ recipient mice were sacrificed and the presence of CD45.1⁺ or CD45.2⁺ donor-derived AMFs was evaluated. (B) Summary of data from four independent recipient mice. (C) CD45.1⁺ E17 embryos were sacrificed, and fetal monocytes were FACS sorted and transferred into CD45.2⁺ mice on their DOB. At the indicated time points after transfer, the expression of CD11c, CD11b, Ly-6C, SiglecF, and F4/80 was evaluated in CD45.1⁺ fetal monocyte–derived cells. (D) The percentage of CD45.1⁺ fetal monocyte–derived cells among mature AMFs at the indicated time points after transfer. Data represent at least two (A– D) independent experiments, with at least three recipient mice per time point.

Figure 5.

Csf2^−/− mice lack preAMFs and mature AMFs throughout life. (A) Lung epithelial cells isolated at the indicated time points before and after birth were FACS sorted, and expression of GM-CSF mRNA was measured by RT-PCR. The expression levels were normalized by comparison with expression of the HPRT housekeeping gene. (B) GM-CSF levels in lung homogenates was measured by ELISA at the indicated time points before and after birth. (C) Csf2^−/− mice were sacrificed on their DOB. Lungs were homogenized and CD11b⁺F4/80⁺ myeloid cells (gated as indicated) were assessed for Ly-6C, CD64, CD11c, F4/80, and SiglecF expression. (D) GM-CSF-R (CD116) expression was evaluated on fetal MFs, fetal monocytes, preAMFs, and mature AMFs on the days mentioned using the gating strategy depicted in Fig. 3. (E) The presence of CD11c⁺SiglecF⁺ AMFs was evaluated in the BAL of WT and Csf2^−/− mice at the indicated time points after birth. (F and G) The presence of F4/80^hiCD11b^lo splenic MFs and liver MFs (Kupffer Cells) was evaluated in adult WT and Csf2^−/− mice. (H and I) BAL cells from adult WT and Csf2^−/− mice were subjected to a Percoll gradient to remove dead cells and protein debris. The resulting CD64⁺F4/80⁺ MFs were assessed for Ly-6C, CD11b, CD11c, and SiglecF expression (I). Data represent two (H and I) and three (A–G) independent experiments.

Figure 6.

Perinatal GM-CSF treatment of Csf2^−/− mice restores the generation of self-maintaining preAMFs. (A and B) Csf2^−/− mice were treated 1 time (1x) on the first day after birth, 3 times (3x) on the first 3 d after birth, or 5 times (5x) on the first 5 d of birth with rGM-CSF or PBS i.n. (1 rGM-CSF or PBS treatment per day). rGM-CSF–treated or PBS-treated Csf2^−/− mice were sacrificed on PND 7. (A) Lungs were homogenized and CD11b⁺F4/80⁺ myeloid cells were assessed for CD11c and SiglecF expression. (B) Expression of FSC, SSC, Ly-6C, CD64, CD11c, F4/80, and SiglecF on SiglecF^loCD11c^loCD11b^hiLy-6C^hi monocytes and SiglecF^intCD11c^hiCD11b^hiLy-6C^hi immature AMFs harvested from Csf2^−/− mice treated for 5 consecutive days with rGM-CSF. (C) 5 wk after 5 consecutive rGM-CSF treatments, Csf2^−/− mice were sacrificed and the presence of CD11c⁺SiglecF⁺ cells in the BAL was evaluated. (D) WT or Csf2^−/− mice treated with 5 consecutive treatments of rGM-CSF or PBS were sacrificed at 7 wk of age, and the development of alveolar proteinosis was evaluated by measuring the protein concentration in the BAL. Data represent two (D) and three (A– C) independent experiments, with at least three recipient mice per time point.

Figure 7.

Terminal differentiation of GM-CSF–rescued immature AMFs requires a GM-CSF–replete host. Csf2^−/− mice treated with 5 consecutive treatments of rGM-CSF were sacrificed at 7 d of age. The lungs were homogenized and CD45.2⁺ CD11c^intSiglecF^int preAMFs were FACS sorted (profile of the sorted cells before transfer is shown in A) and transferred into CD45.1⁺ WT mice on their DOB. 2 d, 9 d, and 6 wk after transfer, CD45.1⁺ recipient mice were sacrificed, and the presence of CD45.2⁺ donor-derived cells was evaluated in the lungs (B–D) and BAL (E). Their CD11b, F4/80, CD11c, and SiglecF expression profile was also assessed. Data represent two independent experiments with at least three recipient mice per time point.