Adult circulating monocytes contribute to the AMF pool in irradiated mice, but not in homeostasis

Previous work supporting the fact that AMFs belong to the MPS system have studied AMF pool replenishment after whole-body irradiation, illustrating that the AMF pool can derive from an adult hematopoietic progenitor. However, it has not been formally shown that circulating monocytes constitute the main precursor of the AMF pool after irradiation, or only participate minimally to the AMF pool. To test this, congenic mice coexpressing CD45.1 and CD45.2 were lethally irradiated and reconstituted with a 1:1 mixture of BM cells isolated from CD45.1⁺ C57BL/6 WT mice and from CD45.2⁺ Ccr2^−/− mice. These B6 (CD45.1) WT + B6 (CD45.2) Ccr2^−/− → B6 (CD45.1–CD45.2) competitive chimeras were analyzed 8 wk after BM transfer. As CCR2 is critical for egress of monocytes from the BM (Serbina and Pamer, 2006), competitive chimerism allowed us to address the relative contribution of circulating monocytes to AMF replenishment after whole body irradiation. As expected, Ly-6C^hi blood monocytes were primarily composed of CD45.1⁺CCR2⁺ donor cells, whereas neutrophils and B cells were comprised of almost equal percentages of CD45.1⁺ and CD45.2⁺ donor cells (Fig. 1 D, group summarized in Fig. 1 E). 8 wk after reconstitution, AMFs still contained between 5 and 10% of host-derived, radio-resistant CD45.1⁺CD45.2⁺ cells, illustrating the relative radioresistance of these cells (Fig. 1 E). The donor-derived AMFs showed CD45 chimerism identical to those of Ly-6C^hi blood monocytes, demonstrating that upon severe depletion, AMFs derive from circulating monocytes.

We next questioned whether circulating monocytes participate to the AMF pool in the adult life when AMFs are not experimentally depleted by irradiation. We therefore performed parabiosis experiments between CD45.1⁺ WT mice and CD45.2⁺ Ccr2^−/− mice. After 8 wk of parabiosis, we analyzed the contribution of CD45.1⁺ WT cells within the CD45.2⁺ Ccr2^−/− mice. There was an almost even distribution of long-lived recirculating cells, and donor CD45.1⁺ WT B lymphocytes represented 40% of circulating blood B lymphocytes of the parabiont CD45.2⁺ Ccr2^−/− partner (Fig. 1 F). However, short-lived circulating cells like neutrophils exchanged ~20% of the cells among the parabiont partner. Such uneven distribution between parabionts likely results from the fact that neutrophils do not spend enough time in the circulation to equilibrate between the parabionts (Liu et al., 2007). Due to a defect in monocyte egress from the BM in the CD45.2⁺ Ccr2^−/− mice, donor CD45.1⁺ WT monocyte constituted ~80% of the monocytes in these mice. However, in our experiments AMFs achieved a much lower chimerism in parabionts with <5% of AMFs originating from the parabiotic partner. This indicates that circulating adult hematopoietic precursors only minimally participate to the AMF pool. These data are congruent with a recent report from the Merad group (Hashimoto et al., 2013).

We next sought to find evidence for the lack of monocyte contribution to the AMF pool in unmanipulated mice. We reasoned that if monocytes were precursors to AMFs, then studying BrdU incorporation kinetics should allow us to study progenitor–product relationships (Kamath et al., 2002). We exposed C57BL/6 mice continuously to BrdU in the drinking water for 4 wk after an initial i.p. injection of BrdU. As shown in Fig. 1 G, whereas almost all monocytes were rapidly BrdU labeled within 2 d, AMFs slowly accumulated 30% of BrdU incorporation after 4 wk of continuous BrdU exposure. The slow accumulation of BrdU could come from BrdU⁺ circulating hematopoietic progenitors or from local proliferation of AMFs. We therefore evaluated the expression of the cell cycle protein Ki-67, which labels dividing and recently divided daughter cells. As shown in Fig. 1 H, a small population (<5%) of Ki-67⁺ dividing cells could be found among AMFs, suggesting a slow local proliferation of AMFs.

To further evaluate whether circulating monocytes contribute to the AMF pool of unmanipulated mice, we transferred 2 × 10⁶ CD45.1⁺ Ly-6C^hi CD11b^hi monocytes into adult Ccr2^−/− CD45.2⁺ mice, but we could not recover any transferred monocyte developing into SiglecF^hiCD11c^hi AMFs 7 d after the transfer, although we readily recovered transferred monocytes as mature intestinal MFs 7 d after transfer (not depicted; Tamoutounour et al., 2012; Bain et al., 2013).

Alveolar MFs appear in the alveolar space during the first week of life

Microglia and Langerhans cells (LCs) derive from embryonic precursors that seed the brain and the skin before birth and maintain themselves locally throughout life (Chorro et al., 2009; Ginhoux et al., 2010; Hoeffel et al., 2012). As AMFs also did not derive from circulating monocytes and showed signs of low-grade proliferation, we next addressed the ontogeny of AMF before and after birth. Little is known about the ontogeny of the lung immune system in relation to lung development, which proceeds via a pseudoglandular stage (E9.5–E16.5), a canalicular stage (E16.5–17.5), and a saccular stage (E18.5 to postnatal day [PND] 5). During the latter stage, canaliculi develop numerous sacs that are the precursors to the alveoli. As shown in Fig. 2 A, we first measured the kinetics of CD45⁺ leukocyte accumulation in the lung. The percentage of CD45⁺ cells started expanding around E18, coinciding with the start of the saccular stage (Fig. 2 C). We next addressed the ontogeny of mononuclear cells (Fig. 2 B). At day E12, the major population of mononuclear cells in the CD11b⁺ F4/80⁺ gate consisted of F4/80^hi CD11b^int cells, which had an intensity of staining resembling adult mature AMFs. However, these cells were completely negative for SiglecF and CD11c. They were most reminiscent of the phenotype of primitive MFs that arise at E9 and E12 from the yolk sac, and are also CD11b^int F4/80^hi (unpublished data). Progressively, the mononuclear gate became replete with a second F4/80^int CD11b^hi population, and there was a gradual increase in the cells expressing intermediate levels of CD11c. However, before birth, the mononuclear gate was completely devoid of SiglecF^hiCD11c^hi AMFs. Around the date of birth (DOB), the mononuclear gate was in transition, and it was no longer possible to clearly discern F4/80^hi from F4/80^int cells. There was a marked increase in CD11c^int cells. Mature SiglecF^hiCD11c^hi AMFs only appeared between PND1 and PND3, when a predominant population of F4/80^hi cells was again apparent in the mononuclear gate. Interestingly, this time point coincided with the first appearance of large mononuclear cells in the alveolar space (Fig. 2 D). Mature SiglecF-expressing AMFs were first found in the lung septa at PND1, and accumulated in the alveolar lumen at PND3 (Fig. 2 E). When we studied lungs before birth, the developing airspaces were completely devoid of mononuclear cells (Fig. 2 D and E). Therefore, bona fide SiglecF^hiCD11c^hi AMFs only appear in the alveolar space during the first days of life.

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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.

Fetal MFs, fetal monocytes, preAMFs, and mature AMFs appear in consecutive waves during lung development

The kinetic analysis of lung mononuclear cells and AMF ontology demonstrated that the phenotype of lung mononuclear cells was highly dynamic, and composed of cells resembling fetal monocytes, fetal MFs, and immature AMFs, with significant overlap in expression of marker sets. We therefore studied the phenotype of mononuclear cells in greater detail, again using the basic mononuclear gate and three populations CD11c^hiSiglecF^hi, CD11c^intSiglecF^lo, and CD11c^loSiglecF^lo as the starting gate, just like in adult mice (Fig. 3 A). As illustrated in Fig. 3 B for the E17 time point, most mononuclear cells were SiglecF^loCD11c^lo cells that could be readily subdivided into CD11b^intF4/80^hi and CD11b^hiF4/80^int cells. These cells also differentially expressed the monocytic marker Ly-6C (Fig. 3 B). Ly-6C^lo F4/80^hi cells highly expressed CD64 (Fig. 3 C), a core MF marker (Gautier et al., 2012b; Tamoutounour et al., 2012), showed a typical MF morphology on SEM (Fig. 3 D), and adhered to plastic in vitro. On the contrary, Ly-6C^hiF4/80^int did not express CD64 and had the typical monocytic FSC^loSSC^lo profile (not depicted) and displayed a nonadherent monocytic morphology on scanning electron microscopy (SEM; Fig. 3 D), thus constituting fetal monocytes. Given their kinetic appearance and resemblance to fetal liver monocytes (not depicted), these were likely derived from the liver, the major hematopoietic organ at this time period of development. From E18 onwards, but culminating around DOB (Fig. 3, A–C, second column), a predominant population of SiglecF^loCD11c^int cells appeared. These cells were mainly CD11b^hiF4/80^int and were Ly-6C^int and expressed intermediate levels of CD64. On SEM, these cells were larger and more granular like AMFs, but did not adhere well to plastic (Fig. 3 D, preAMF). Very few MHCII⁺ DCs were found in this gate (data not shown). At DOB, the SiglecF^loCD11c^lo cells still contained some Ly-6C^loF4/80^hi fetal MFs, but Ly-6C^hiF4/80^int fetal monocytes were the predominant population. Within the SiglecF^loCD11c^lo cells, some monocytes had already lost Ly-6C, a common feature of monocytes differentiating into mature MFs or DCs (Tamoutounour et al., 2012; Plantinga et al., 2013). Between PND1 and PND3, SiglecF^hiCD11c^hi cells appeared very suddenly, and these were Ly-6C^loF4/80^hi cells, like adult AMFs. The SiglecF^loCD11c^int cells contained more MHCII DCs (not depicted), but still contained many mononuclear cells that were losing Ly-6C. At PND3 (Fig. 3, A–C, third column), the SiglecF^loCD11c^lo fraction hardly contained any F4/80^hi fetal MFs anymore. At PND14 (Fig. 3, A–C, fourth column), the ensemble of lung mononuclear cell types resembled the adult state, and the CD11c^int population had lower mean fluorescence expression of SiglecF. In Fig. 3 E, the relative distribution of all these cell populations is plotted against time. The appearance of mature AMFs at PND3 was preceded by a surge in SiglecF^loCD11c^intCD64^int cells that looked like immature AMFs on SEM. We will therefore call this population preAMFs.

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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.

Fetal monocytes give rise to self-maintaining AMFs through a preAMF intermediate step

To verify whether fetal monocytes or fetal MFs are the direct precursors to AMFs, we designed a competitive transfer experiment (Fig. 4). CD45.1 fetal MFs and CD45.2 monocytes were isolated from E17 lungs and transferred together in a 1:1 ratio i.n.) into CD45.1⁺ CD45.2⁺ mice on their DOB, i.e., when the AMF niche is still empty. 7 d after transfer, the fate of the donor cells was evaluated. Whereas at day 7, most of the AMFs were of host CD45.1⁺CD45.2⁺ phenotype in these nonirradiated mice (Fig. 4 A), the fate of donor cells could clearly be defined. As shown in Fig. 4 (A and B), transferred AMFs were significantly more derived from fetal monocyte–derived MFs than from fetal MFs. Similar results were found when the congenic donor pair was switched over (i.e. when fetal monocytes were CD45.1 and fetal MFs were CD45.2) or when the recipient mice were analyzed at 6 weeks after transfer (unpublished data) instead of 7 days after transfer (Fig. 4, A and B). A similar setup also allowed us to follow the differentiation of developing AMFs from adoptively transferred fetal monocytes. Therefore, we i.n. transferred fetal CD45.1⁺ monocytes into CD45.2⁺ mice on DOB and checked the phenotypic changes of the cells in the following days and weeks. Strikingly, transferred CD45.1 cells could be found up to 11 wk after transfer (Fig. 4 C). Fetal monocytes gradually increased expression of CD11c, SiglecF, F4/80, and CD64 levels, while down-regulating first Ly-6C and then CD11b, going through a Ly-6C^intCD11b^hiF4/80^intLy-6C^intCD64^int intermediate stage, essentially validating these cells as preAMFs. The kinetic of the differentiation of the transferred monocytes fits a developmental scheme that would go from fetal monocytes over preAMFs to mature AMFs within 6 to 7 d, which was also seen in Fig. 3 E. We could still trace back AMF originating from transferred monocytes 3 mo after transfer. Moreover, within one transfer experiment, the percentage of donor-derived cells among mature AMFs only dropped very slowly during adult life (Fig. 4 D), suggesting that transferred monocytes gave rise to a stable self-maintaining population.

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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.

GM-CSF drives the development of preAMFs around birth

Primary pulmonary alveolar proteinosis (PAP) is a disease associated with the accumulation of surfactant in the lungs that has been linked to nonfunctional GM-CSF signaling in patients (Suzuki et al., 2008) and could be reproduced in genetically modified mice lacking GM-CSF (Csf2^−/−) or GM-CSF-R (Csf2r^−/−) expression (Huffman et al., 1996; Reed et al., 1999; Reed et al., 2000; Zsengellér et al., 1998). Although the PAP syndrome was originally proposed to derive from the fact that AMFs were not functional in these mice (REFs; Golde, 1976, 1979; Harris, 1979; Gonzalez-Rothi and Harris, 1986; Nugent and Pesanti, 1983; LeVine et al., 1999; Yoshida et al., 2001; Paine et al., 2001; Thomassen et al., 2007), Shibata et al. (2001) later proposed that a MF developmental defect rather than a functional defect may lie at the basis of the disease. However, it is not known how absence of GM-CSF signaling impacts on AMF ontogeny before and shortly after birth. We found that lung epithelial cells show an increased expression of GM-CSF mRNA that is paralleled by high GM-CSF protein levels in lung tissues around DOB, yet quickly wanes thereafter (Fig. 5, A and B). Because fetal MFs, fetal monocytes, preAMFs, and mature AMFs all express the GM-CSF-R (CD116, Fig. 5 D), we decided to investigate the development of AMFs in Csf2^−/− mice. On their DOB, Csf2^−/− mice completely lacked preAMFs (Fig. 5 C). However, they contained fetal MFs and monocytes in ratios similar to WT mice (see Fig. 3 for comparison). Note that the small CD11c^int population that was found in Csf2^−/− mice on DOB was Ly-6C^int, but had a SiglecF^loCD64^loFSC^loSSC^lo profile. Therefore, the wave of preAMFs that is found in WT mice around birth is absent in Csf2^−/− mice. Importantly, development of preAMFs or mature AMFs remained absent in Csf2^−/−mice throughout life (Fig. 5 E). Liver and splenic CD11b^intF4/80^hi-resident MFs, however, could be readily identified in Csf2^−/− mice (Fig. 5, F and G). In contrast, we had great difficulty identifying MFs in the BAL of Csf2^−/− mice. Proteinosis had already developed by ~4 wk of age and yielded autofluorescent debris in the BAL fluid. The presence of many dead cells (unpublished data) also rendered the analysis of the cellular composition of the BAL difficult. However, others have described AMFs with functional defects in adult Csf2^−/− mice. To characterize these cells, we first performed a Percoll gradient to remove debris and dead cells form the BAL fluid and then used a more general MF gating strategy using F4/80 and CD64 (Fig. 5 H). In doing so, we could indeed identify some rare F4/80⁺CD64⁺ cells in the BAL of Csf2^−/− mice. These cells did not express a bona fide AMF profile, but were found to be SiglecF^loCD11b^hiCD11c^intLy-6C^hi-to-int. Moreover we could only identify such cells after 4 wk of age, i.e., once the first signs of PAP developed.

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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.

We sincerely thank Saskia Lippens and Anneke Kremer of the VIB Bio-Imaging Core Facility for most valuable help with scanning EM pictures.

This work was supported by a European Research Council consolidator grant to B.N. Lambrecht, by a University of Ghent Multidisciplinary Research Partnerships grant (GROUP-ID consortium) to B.N. Lambrecht, by a Marie Curie re-integration grant (CIG), a Belspo Return Grant, a Post-Doc FWO grant and a Odysseus Grant to M. Guilliams, and by a FWO project grant to H. Hammad. I. De Kleer is the recipient of a European Respiratory Society/Marie Curie Joint Research Fellowship number MC 1231-2009 and of a Marie Curie Inter-European Fellowship (FP7-PEOPLE-2009-IEF, grant agreement number 253370). This work was also supported by Centre National de la Recherche Scientifique, Institut National de la Santé et de la Recherche Médicale, and European Communities (MUGEN Network of Excellence and MASTERSWITCH Integrating Project) for B. Malissen and S. Henri.

The authors have no conflicting financial interests.