Induction of the nuclear receptor PPAR-γ by ...

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Induction of the nuclear receptor PPAR-G by the cytokine GM-CSF is critical for the differentiation of fetal monocytes into alveolar macrophages © 2014 Nature America, Inc. All rights reserved.

Christoph Schneider1,5, Samuel P Nobs1, Michael Kurrer2, Hubert Rehrauer3, Christoph Thiele4 & Manfred Kopf1 Tissue-resident macrophages constitute heterogeneous populations with unique functions and distinct gene-expression signatures. While it has been established that they originate mostly from embryonic progenitor cells, the signals that induce a characteristic tissue-specific differentiation program remain unknown. We found that the nuclear receptor PPAR-G determined the perinatal differentiation and identity of alveolar macrophages (AMs). In contrast, PPAR-G was dispensable for the development of macrophages located in the peritoneum, liver, brain, heart, kidneys, intestine and fat. Transcriptome analysis of the precursors of AMs from newborn mice showed that PPAR-G conferred a unique signature, including several transcription factors and genes associated with the differentiation and function of AMs. Expression of PPAR-G in fetal lung monocytes was dependent on the cytokine GM-CSF. Therefore, GM-CSF has a lung-specific role in the perinatal development of AMs through the induction of PPAR-G in fetal monocytes. Tissue-resident macrophages constitute a heterogeneous group of professional phagocytes with distinct functions in organ homeostasis. The long-held dogma that bone marrow–derived blood monocytes constantly replenish tissue macrophages is now under revision, as several reports over the past 4 years have demonstrated that most of the tissue-resident macrophages are maintained independently of the adult blood monocyte system through longevity and local self-renewal, with probably a few exceptions, such as intestinal macrophages that are continuously renewed by circulating bone marrow– derived monocytes1–3. Fate-mapping experiments have shown that most tissue macrophages are of embryonic origin4,5, which has been further elucidated for microglia6, Langerhans cells7, alveolar macrophages (AMs)8 and cardiac macrophages9. Despite sharing fetal monocytes and/or fetal macrophages as common embryonic progenitors, tissue-resident macrophages show distinct transcriptional signatures10, which suggests that the local environment ‘imprints’ identity and ‘instructs’ a specific transcriptional program that allows them to exert organ-specific functions. However, such organ-specific signals and transcription factors remain largely unknown. The transcription factor PPAR-G is a member of a ligand-activated family of nuclear receptors. Initially described as a master regulator of adipocyte differentiation that controls the expression of key genes encoding molecules involved in lipid transport and metabolism11, PPAR-G has since been found to have high expression in macrophages of atherosclerotic lesions, indicative of a role for PPAR-G in the development of this inflammatory cardiovascular disease12,13. Stimulation of PPAR-G by synthetic ligands suppresses inflammatory

gene expression in macrophages14,15. In alternatively activated (M2) macrophages, which are involved in tissue repair and immunity to parasites, PPAR-G contributes to the polarization of macrophages by modulating gene expression in a manner dependent on interleukin 4 (IL-4) and the transcription factor STAT6 (ref. 16). Published reports have described expression of PPAR-G in macrophages of mucosaassociated tissues, lymph nodes, lung16 and human AMs17, results that have been extended by another report showing the highest PPAR-G expression in mouse macrophages of the lungs and lower expression in the spleen and Ly6Clo ‘patrolling’ blood monocytes under steadystate conditions18. Such studies indicate a role for PPAR-G in certain myeloid populations and particularly in AMs. Accordingly, mice with loxP-flanked alleles encoding PPAR-G (Ppargfl/fl) and with expression of Cre recombinase under control of the gene encoding the lysozyme LysM (Lyz2-Cre; called ‘Lysm-Cre’ here) (Lysm-CrePpargfl/fl mice) are reported to develop pulmonary alveolar proteinosis indicative of impaired function of AMs that, however, becomes manifest only in aging mice (>4 months of age)19. Other studies have found a moderately altered transcriptome but normal numbers of AMs in LysmCrePpargfl/fl mice18. Together these data do not support the hypothesis of a major function for PPAR-G in AMs. Whether PPAR-G has a role during the development of AMs has so far not been addressed. Given advances in the ontogeny and development of tissue macrophages, we characterized the role of PPAR-G in myeloid populations of adult mice as well as during the perinatal development of AMs. By crossing Ppargfl/fl mice with mice expressing Cre under control of the gene encoding the common DC marker CD11c (Itgax) (called

1Institute

of Molecular Health Sciences, Department of Biology, Swiss Federal Institute of Technology Zurich, Zurich, Switzerland. 2Pathology Institute, Zurich, Switzerland. 3Functional Genomics Center, Zurich, Switzerland. 4LIMES Institute, University of Bonn, Bonn, Germany. 5Present address: Howard Hughes Medical Institute, University of California San Francisco, San Francisco, California, USA. Correspondence should be addressed to M.Ko. ([email protected]). Received 6 May; accepted 5 September; published online 28 September 2014; doi:10.1038/ni.3005

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‘Cd11c-Cre’ here) or mice expressing ‘codon-improved’ Cre under control of the hematopoietic compartment–specific promoter of the gene encoding the guanine nucleotide–exchange factor Vav1 (called ‘Vav1-Cre’ here), we found that PPAR-G was the main transcription factor that regulated the perinatal development and signature gene expression of AMs. Although dendritic cells (DCs) and macrophages in many tissues developed normally in the absence of PPAR-G, we found a profound defect in the perinatal development of AMs. Differentiation was arrested at an immature AM stage (called ‘preAM’ here) in Cd11c-CrePpargfl/fl mice and was abrogated at the level of the lung fetal monocyte progenitor cell in Vav1-CrePpargfl/fl mice, similar to its differentiation in mice deficient in the cytokine GMCSF. The defect in AM development was rather moderate in LysmCrePpargfl/fl mice due to inefficient deletion of Pparg. Furthermore, our data suggested that GM-CSF ‘licensed’ the development of AMs by inducing PPAR-G expression selectively in lung fetal monocytes. Thus, we have established the link between a signal provided by the tissue and a cell-intrinsic transcription factor that mediates tissue macrophage–specific differentiation. RESULTS AM differentiation requires PPAR-G To investigate the role of PPAR-G in myeloid cells, we generated mice lacking PPAR-G in a cell type–specific manner by crossing Ppargfl/fl mice20 with mice expressing Cre in macrophages (Lysm-Cre mice) or DCs (Cd11c-Cre mice). Using a combination of antibodies (i.e., antibodies to the macrophage marker F4/80, the common myeloid marker CD11b and CD11c), we characterized macrophage and DC populations in various organs of naive mice by flow cytometry. The frequency and expression patterns of CD11c+, F4/80+ and CD11b+ populations were unaltered in Lysm-CrePpargfl/fl and Cd11c-CrePpargfl/fl mice relative to that in their Ppargfl/fl control littermates without expression of Cre (called simply ‘Ppargfl/fl’ here) in the heart, kidneys, lamina propria and white adipose tissue (Supplementary Fig. 1a,b and data not shown), which indicated normal development and recruitment of macrophages and DCs in these tissues in the absence of PPAR-G. As reported before18, there were fewer splenic macrophages in the absence of PPAR-G (data not shown). Strikingly, mature AMs, characterized as CD11c+CD11bloF4/80+ cells with high autofluorescence, were almost completely absent from the bronchoalveolar lavage (BAL) fluid and lungs of Cd11c-CrePpargfl/fl mice (Fig. 1a,b). Instead, the BAL fluid and lungs of Cd11c-CrePpargfl/fl mice contained a population of CD11chiCD11bhi cells that were not present in Ppargfl/fl mice and that shared several features with mature AMs, including high autofluorescence21 and expression of F4/80 (‘AM-like cells’) (Fig. 1c). Thus, according to the classical markers that define AMs, the population present in Cd11c-CrePpargfl/fl mice seemed to be AM-like cells that differed from Ppargfl/fl AMs only in high expression of CD11b. Moreover, the total number of AM-like cells together with the few mature AMs in BAL fluid and lungs of Cd11c-CrePpargfl/fl mice was lower than that of AMs in Ppargfl/fl mice (Fig. 1b). Analysis of Lysm-CrePpargfl/fl mice showed only a minor difference in the number of mature AMs in the lungs and slightly but significantly lower number of mature AMs in BAL fluid relative to that in Ppargfl/fl mice (Fig. 1b). Microscopic analysis revealed that most of the cells in BAL fluid from Cd11c-CrePpargfl/fl mice were enlarged and/or displayed intracellular accumulation of lipids, as indicated by staining with Oil Red O (Fig. 1d), reminiscent of foam-cell macrophages found in atherosclerotic lesions22. Consistent with the slightly lower number of mature AMs, only a few BAL fluid cells from Lysm-CrePpargfl/fl mice contained 2

lipid droplets (Fig. 1d). Furthermore, foam macrophages were also present in lung sections from Cd11c-CrePpargfl/fl mice (Fig. 1e). We next sorted CD11chiCD11bhiSiglec-F+ AM-like cells from BAL fluid of Cd11c-CrePpargfl/fl mice and found that almost every AM-like cell was considerably enlarged and contained visible lipid droplets, as shown by diffuse staining with Oil Red O, indicative of lipid accumulation (Supplementary Fig. 2a). Consistent with that, Cd11c-CrePpargfl/fl mice developed pulmonary alveolar proteinosis, indicated by increased amounts of total protein and surfactant protein D in their BAL fluid, which were undetectable in Lysm-CrePpargfl/fl and Ppargfl/fl mice (Fig. 1f,g). Macrophages contribute to the maintenance of tissue integrity by the phagocytosis and processing of opsonized material such as apoptotic cells. Dead or dying cells are usually quickly removed to prevent necrosis and inflammation23. Whereas BAL fluid from Ppargfl/fl or Lysm-CrePpargfl/fl mice contained only few dead cells, BAL fluid from Cd11c-CrePpargfl/fl mice showed considerable enrichment for dead cells (Fig. 1h and Supplementary Fig. 2b). Neutrophils were present in increased numbers in the BAL fluid of Cd11c-CrePpargfl/fl mice (Fig. 1i), indicative of low-grade inflammation possibly induced by the accumulation of dead cells. Together the results presented above indicated that Cd11cCrePpargfl/fl mice had a considerable defect in the development of typical CD11c+CD11blo AMs. Their BAL fluid and lungs contained a population of functionally immature AM-like cells with high expression of CD11b. Notably, this defect was rather mild in Lysm-CrePpargfl/fl mice. To elucidate the difference in the phenotypes of Cd11c-CrePpargfl/fl mice and Lysm-CrePpargfl/fl mice, we analyzed the degree of Cre-mediated recombination in the Pparg locus. Comparison of F4/80+CD11c+Siglec-F+ autofluorescencepositive cells sorted from the lungs of Lysm-CrePpargfl/fl and Cd11cCrePpargfl/fl mice showed that the latter had five-to tenfold more recombination of the Pparg alleles (Fig. 1j) and, accordingly, much less Pparg mRNA (Fig. 1k). These results suggested that insufficient Lysm-Cre–mediated deletion of Pparg underlay the ‘weaker’ phenotype of Lysm-CrePpargfl/fl mice compared with the substantial defects observed in Cd11c-CrePpargfl/fl mice. PPAR-G-dependent development of AMs from fetal monocytes Primitive hematopoiesis in the yolk sac and definitive hematopoiesis from hematopoietic stem cells contribute differently to the development of tissue macrophages in the embryo24. We therefore characterized AM development in the lungs before and after birth. At embryonic day 17.5 (E17.5), CD45+ cells in the lungs consisted of mainly two myeloid populations, including F4/80hiCD11bintCD11clo fetal macrophages and F4/80intCD11bhiCD11cloLy6Chi fetal monocytes (Fig. 2a). Over the course of the following days until day 2 after birth, fetal monocytes increased in number and downregulated CD11b expression concomitantly with upregulation of CD11c expression and subsequently also expression of Siglec-F (Fig. 2a,b). By going through a CD11cintCD11bintSiglec-Fint ‘pre-AM’ stage around day 2 after birth, they eventually differentiated into mature CD11chiCD11bloSiglec-Fhi AMs. The number of fetal macrophages remained relatively constant during this process (Fig. 2c). Kinetic analysis of perinatal AM development in the lungs of Cd11c-CrePpargfl/fl mice showed that fetal monocytes were fully able to upregulate CD11c expression and to differentiate into CD11cintCD11bintSiglec-Fint pre-AMs shortly after birth (Fig. 2a,b). However, further development was arrested at this stage, as indicated by the failure to downregulate CD11b expression and upregulate Siglec-F expression (Fig. 2a,b). Consistent with that, the timing of the developmental defect was paralleled by the induction of Cre activity ADVANCE ONLINE PUBLICATION

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driven by the Cd11c promoter in pre-AMs around day 1 after birth, as indicated by results obtained with the progeny of Cd11c-Cre mice crossed with fate-reporter mice with a loxP-flanked transcriptional stop element and sequence encoding red fluorescent protein (RFP) knocked into the ubiquitous Rosa26 locus (Cd11c-CreRosa26-RFP mice; this construct reports Cre activity driven by the Cd11c promoter) (Fig. 2d). In contrast, kinetic analysis of reporter-gene expression during AM development in the progeny of Lysm-Cre mice crossed with Rosa26RFP mice (Lysm-CreRosa26-RFP mice) revealed that Lysm-Cre– mediated recombination already started in fetal monocyte at E17.5–E19.5, but it was less efficient in these cells (30–50% RFP+ cells) than in neutrophils (70–80% RFP+ cells) (Fig. 2d). Moreover, mature AMs at day 5 after birth were targeted at a significantly lower frequency in LysmCreRosa26-RFP mice (60%) than in Cd11c-CreRosa26-RFP mice (80%), consistent with the lower abundance of AMs with recombined Pparg alleles in adult Lysm-CrePpargfl/fl mice (Fig. 1j,k). Overall, these results suggested that PPAR-G was essential for the terminal AM differentiation of fetal monocyte precursor cells. NATURE IMMUNOLOGY

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Figure 1 Impaired development and function of AMs in Cd11c-CrePpargfl/fl mice. (a) Flow cytometry of AMs in BAL fluid and lungs of Ppargfl/fl, Lysm-CrePpargfl/fl and Cd11cCrePpargfl/fl mice, gated on CD45 + cells. Arrow (top right) indicates accumulating dead cells and debris typically found in BAL fluid of Cd11c-CrePpargfl/fl mice. Numbers adjacent to outlined areas indicate percent CD11c +CD11blo cells (red) and CD11c+CD11bhi cells (blue). (b) Total CD11c+CD11blo mature AMs (mAM) and CD11c+CD11bhi AM-like cells (AM-like), gated as in a. P values, compared with mature AMs. (c) Expression of CD11b and F4/80 and autofluorescence (AF) of cells as in a, gated on CD11c hiCD45+eFluor780− (viable) cells. (d) Microscopy of cytospins of BAL fluid from mice as in a, stained with Oil Red O. Original magnification, ×63; scale bar, 50 Mm. (e) Lung sections from mice as in a, stained by the Verhoeff-Van Gieson protocol. Arrows indicate foam cells. Scale bar, 250 Mm. (f,g) Surfactant protein D (SP-D) (f) and total protein concentration (g) in BAL fluid from mice as in a. A405 (f), absorbance at 405 nm. (h,i) Absolute number of dead (eF780 +) cells (h) and CD11b+CD11c−Ly-6CintGr-1+ neutrophils (i) in BAL fluid from mice as in a, assessed by flow cytometry. (j) Quantitative real-time PCR analysis of the recombination of Ppargfl/fl alleles in genomic DNA from AMs obtained from mice as in a and sorted by flow cytometry; results are presented relative to those of a Pparg control allele. (k) Quantitative real-time PCR analysis of Pparg mRNA in sorted AMs; results are presented relative to those of the control gene G6pdx (which encodes glucose-6-phosphate dehydrogenase). NS, not significant; *P < 0.01 and **P < 0.0001 (Student’s t-test). Data are from one experiment representative of three independent experiments (a–i; mean and s.d. of three to six mice per group) or one experiment (j,k; mean and s.d. of three mice per group).

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Irradiation-induced replenishment of AMs also requires PPAR-G Studies of irradiation chimeras have shown that most tissue macrophages, including AMs, are replaced by bone marrow–derived blood monocytes1,2,4,5,8. To investigate whether PPAR-G is also required for replenishment of AMs after irradiation-induced depletion, we generated chimeras by adoptive transfer of a mixture of bone marrow (BM) cells from Cd11c-CrePpargfl/fl (CD45.2+) mice and wild-type (CD45.1+) mice at a ratio 4:1 (or Ppargfl/fl (CD45.2+) BM and wildtype (CD45.1+) BM as a control) into lethally irradiated wild-type mice (Supplementary Fig. 3a). AMs in lungs and BAL fluid of the recipient mice were almost exclusively reconstituted by BM from wild-type mice (Fig. 3a,b), while the remaining PPAR-G-deficient AM-like counterparts were arrested in a CD11chiCD11bhiSiglec-Flo state (Fig. 3c and Supplementary Fig. 3b–d). Notably, the frequency of arrested PPAR-G-deficient AMs was much lower (five- to sevenfold) than that of PPAR-G-sufficient mature AMs, despite a fourfold excess of transferred PPAR-G-deficient BM cells relative to that of wild-type BM cells (Fig. 3b); this indicated that the absence of PPAR-G resulted 3

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in not only arrested AM development but also reduced AM development. Furthermore, we observed no difference in the number of dead AMs (Supplementary Fig. 3e). Consistent with impaired AM development in the absence of PPAR-G, we found that the expression of many AM signature genes, as well as those encoding chemokines and proinflammatory cytokines, was deregulated in PPAR-G-deficient arrested AMs compared with their expression in wild-type AMs in the same recipient mice (Fig. 3d,e). Furthermore, PPAR-G-deficient AMs contained lipid droplets, which we did not observe in their wildtype counterparts (Fig. 3f). However, lipid enrichment and foamcell character was less pronounced in PPAR-G-deficient AMs from the mixed-BM chimeras than in AMs isolated from non-irradiated Cd11c-CrePpargfl/fl mice (Fig. 1), probably because in the mixed-BM chimeras, 80–90% of AMs were derived from wild-type BM and completely prevented alveolar proteinosis and the associated influx of neutrophils (Fig. 3g,h). Consistent with results obtained with Cd11c-CrePpargfl/fl mice (data not shown), results obtained with the mixed-BM chimeras also confirmed the contribution of PPAR-G to 4

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Figure 2 AMs develop perinatally from fetal monocytes through a pre-AM intermediate that requires PPAR-G for terminal differentiation. (a) Expression of Siglec-F and CD11b (second and bottom CD11c CD11c CD11c CD11c rows) by gated subpopulations (outlined E17.5 E19.5 DAB1 DAB5 + areas with arrows) of CD45 cells isolated 100 100 100 100 * from the lungs of time-mated Ppargfl/fl 80 80 80 80 fl/fl and Cd11c-CrePparg mice before 60 60 60 60 birth (at E17.5 or E19.5) and at 2 d 40 40 40 40 (DAB2), 11 d (DAB11) or 8 weeks 20 20 20 20 after birth (above plots) and sorted by 0 0 0 0 expression of F4/80 and CD11c (top row and third row). Numbers adjacent to outlined areas indicate percent cells in each throughout. aAM, arrested AMs. (b) Total fetal monocytes (mo), pre-AMs, mature AMs and arrested AMs among cells as in a. (c) Total fetal macrophages (M&) among cells as in a, gated as F4/80hiCD11cloCD11bintSiglecF−Ly-6C− and autofluorescence-positive. (d) Cre activity (bottom) in myeloid cells in the lungs of Lysm-CreRosa26-RFP, Cd11c-CreRosa26-RFP and Vav1-CreRosa26-RFP mice at various times before birth (at E17.5 or E19.5) and at 1 d (DAB1) or 5 d (DAB5) after birth, among CD45 + cells gated by expression of F4/80 and CD11c (outlined areas, top; horizontal axis, bottom), with fetal macrophages further gated as F4/80 hiCD11cloCD11bintSiglecF−Ly-6C− and autofluorescence-positive, and neutrophils further gated as F4/80 −CD11bhiSiglec-F−; Cre activity is presented as frequency of RFP+ cells. *P < 0.0001 (Student’s t-test). Data are pooled from two independent experiments (a–c; mean and s.e.m. of four to seven mice per group) or two experiments (d; mean and s.d. of four to nine mice per group). F4


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the development of spleen macrophages, as their reconstitution from Cd11c-CrePpargfl/fl bone marrow was impaired (Fig. 3i). However, DC subsets in the lungs (i.e., CD103+ and CD11b+ DCs) and spleen (i.e., SIRPA+ and CD8+ DCs), as well as blood monocytes (Ly6C− and Ly6C+), were equally well reconstituted from Cd11c-CrePpargfl/fl BM cells and wild-type BM cells (Fig. 3j,k). These results demonstrated a cell-autonomous defect in development of AMs in the absence of PPAR-G. They further suggested that PPAR-G promoted the differentiation of precursor cells into AMs and ‘licensed’ the terminal differentiation of AMs, including upregulation of Siglec-F expression and downregulation of CD11b expression. To verify the results reported above in an experimental setting without ablation of AMs by irradiation, we transferred wild-type (CD45.1+) lung fetal monocytes into newborn Ppargfl/fl or Cd11c-CrePpargfl/fl mice. Strikingly, we observed competition for AM development between cells of donor origin and recipient mouse–derived cells only in recipients that lacked PPAR-G (Fig. 3l). When transferred into Cd11c-CrePpargfl/fl recipients, donor wild-type fetal monocytes efficiently competed with their ADVANCE ONLINE PUBLICATION

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ARTICLES Pparg-deficient counterparts and were able to entirely fill up the ‘empty space’ of mature AMs, while recipient mouse–derived Pparg-deficient cells remained in a CD11bhiSiglec-Flo stage. In contrast, donor wild-type fetal monocytes were efficiently ‘competed out’ by endogenous Ppargfl/fl precursors of AMs. These results suggested that AM development was limited by space and/or extrinsic factors and that successful transfer of pre-AMs or mature AMs into recipient mice with intact AMs would probably be doomed to fail.

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Events (% of max)

a

PPAR-G confers AM signature and identity The block in AM differentiation in the absence of PPAR-G and the associated functional impairment suggested a major role for PPAR-G as an AM-specific differentiation factor. A published study has characterized the gene-expression profiles of tissue macrophages isolated from the lungs, spleen, brain and peritoneal cavity and has identified a distinct gene-expression signature for each tissue macrophage population10. Many signature genes encode molecules that are probably

Figure 3 Cell-autonomous requirement for PPAR-G during the terminal differentiation of AMs. (a) Flow cytometry of CD11c+Siglec-F+ autofluorescencehigh AMs in the lungs of chimeras generated by the reconstitution of lethally irradiated wild-type mice with a mixture of wild-type (CD45.1 +) BM plus Ppargfl/fl (CD45.2+) BM (WT:Ppargfl/fl) or a mixture of wild-type (CD45.1+) BM plus Cd11c-CrePpargfl/fl (CD45.2+) BM (WT:Cd11c-CrePpargfl/fl). Numbers adjacent to outlined areas indicate percent CD45.1 + cells (top left) and CD45.2+ cells (bottom right). (b) Frequency of CD45.2+ (Ppargfl/fl or Cd11c-CrePpargfl/fl) cells among CD11c+Siglec-F+ autofluorescence-high AMs in the BAL fluid and lungs of chimeras as in a, presented relative to the frequency of CD45.2+CD19+ B cells in the same chimera. (c) Expression of CD11b and Siglec-F on CD45.1+ (wild-type) and CD45.2+ (Ppargfl/fl or Cd11c-CrePpargfl/fl) AMs in the lungs of chimeras as in a. (d,e) Quantitative real-time RT-PCR analysis of selected AM signature genes (d) or cytokines and chemokines (e) in Cd11c-CrePpargfl/fl (CD45.2+) arrested AMs, presented relative to their expression in wild-type (CD45.1 +) AMs sorted from the same chimera. (f) Microscopy of wild-type (CD45.1+) mature AMs and PPAR-G-deficient (CD45.2+) arrested AMs sorted from the same chimera and stained with Oil Red O; arrows indicate lipid droplets. Original magnification, ×63; scale bar, 25 Mm. (g) Total protein concentration in BAL fluid from chimeras as in a, measured by BCA (bicinchoninic acid) assay. (h) Absolute number of neutrophils in the BAL fluid and lungs of chimeras as in a. (i–k) Frequency of CD45.2+ (Ppargfl/fl or Cd11c-CrePpargfl/fl) cells among F4/80+CD64+MHCIIloCD11cint spleen macrophages (i) or among DC subsets (j) in the lung (gated on CD11c+Siglec-F−) and the spleen (gated on CD11chiautofluorescencelo), or among peripheral blood leukocytes (k), presented as in b. (l) Flow cytometry (middle) of cells obtained from the lungs of neonatal CD45.2 + Cd11c-CrePpargfl/fl or Ppargfl/fl recipient mice 8 weeks after transfer of fetal monocytes sorted by flow cytometry from lungs of CD45.1 + mouse embryos at E18.5 (+ fetal mo) or after no transfer of cells (untreated (UT)), assessing the frequency of donor-derived AMs (red) and recipient-derived AMs (blue) in the lungs (top row) and their expression of Siglec-F and CD11b (bottom row). Right, total donor- or recipient-derived AMs in BAL fluid and lungs of recipient mice as at left. Left, experimental protocol. *P < 0.001 and **P < 0.0001 (Student’s t-test). Data are from one experiment representative of two independent experiments (a–c,i–k; mean and s.d. of four chimeras per group) or are from one experiment (d–h,l; mean and s.d. of three to four chimeras per group (d–h) or three mice per group (l)).

NATURE IMMUNOLOGY


5

ARTICLES

fl/ fl

fl/ fl

fl/ fl

Adult

Signature-up

Signature-down

4 2 0

–1

lia ro g ic

M

P

lM ea

ng Lu

R

AM

lia ro g ic

M

Sp le en Pe R P rit on ea lM

AM ng

–2

–2

Lu

Lu

Adult

Pe rit on

DAB2 DAB11 Adult

DAB11

en

0

DAB2

le

0

Slc11a1 Neurl3 Rcsd1 Msi2 Abcc3 Abca9 Tspan13 Ckb Ninj1 Etv1 CD86 B4galt6 Ptgs1 Mafb Nsmaf C1qb C1qc C1qa Tnfrsf14 Cmklr1 Prkcb Apoe Acer3 Ifitm3 Lrrc25 Lpcat2 Itga9

Sp

1

Sp

DAB11

2

1

–2

Pp a C rg fl/fl d1 1 Pp c-C a re C rg fl/fl Pp d1 ar g 1c Pp -C ar re C g fl/fl Pp d1 ar g 1c -C re Pp ar g

Pp a C rg fl/fl d1 1 Pp c-C ar re C g fl/fl Pp d1 ar g 1 Pp c-C ar re P g f C l d1 /fl pa rg 1c -C re Pp ar g DAB2

–1

fl/ fl

fl/ fl

fl/ fl

fl/ fl

fl/ fl

Adult

Signature TFs

2

ng

–4

DAB2 DAB11 Adult

DAB11

lia

–2

DAB2

e

ro g

W

W

tr1

Adult

co a4 Ra ra

DAB11

N

af f M

o4 r Ls

Lm

a1 m Li

hd

C

Ba z

1a


0

5

DAB2

b

ic

Expression (fold)

M

3

0

le en Pe R P rit on ea lM

–3

Adult

d

Afap1 C530008M17Rik Agpat9 Spp1 Ptpn12 Naaa Card11 Flt1 A430107O13Rik Gpnmb Fabp1 II12rb2 Cidec Cd69 Clec7a Olr1 Siglec5 Atp10a Ucp3 Itgal Itgax Cdh1 Rab11fip1 Mtmr7 Cyp4f18 Mmp8 Trim29 Mcam 1600029D21Rik Anxa2 Nrg4 Acaa1b Htr2c Plp2 Slc9a7 L1cam

Atp13a3 Trem1 Epcam Cpne5 S100a11 Lipf Gpr120 Kazald1 Gal Scgb1a1 Tmem216 Tmem138 Mamdc2 Fam189a2 Ch25h II1rn Kynu Plp2 Bub1b Ly75 Prr5l Sulf2 Nceh1 Tmem154 Kcnn3 S100a11 Ctsk Bcar3 Mcoln3 Tlr2 Cd2 Chi3/3 Lmo4 Lepr Atp6v0d2 Sema3e

AM

Atad2 Hat1 Lrrfip1 Trerf1 Wwtr1


Figure 4 PPAR-G confers the AM M DC signature and identity. (a) Expression Height CD11b+ CD103+ of mRNA encoding selectively expressed transcription factors in Ppargfl/fl AMs and Cd11c-CrePpargfl/fl Liver CD11b+ immature arrested AMs sorted from Lung CD11b+ 11-day-old mice (DAB11) and 7- to + 8-week-old mice (Adult) and in Lung CD103 fl/fl 1 –0.188 –0.319 0.492 –0.07 –0.14 0.836 Pparg DC pre-AMs from Ppargfl/fl and Cd11cSpleen –0.206 Liver CD103+ CrePpargfl/fl mice at day 2 after birth –0.291 Microglia Spleen CD4+ Lung 0.264 (DAB2). (b) Expression of select M hi 0.017 Peritoneal F4/80 genes in a in Cd11c-CrePpargfl/fl cells Spleen CD8+ –0.077 Peritoneal F4/80lo (mouse ages as in a), relative to that Lung Cd11c-CrePpargfl/fl in Ppargfl/fl cells. (c,d) Expression of Spleen CD8+ Cd11c-CrePpargfl/fl mRNA of ‘signature-up’ genes (c) Spleen CD4+ and ‘signature-down’ genes (d) in Ppargfl/fl Lung cells as in a. (e) Expression of all genes DC CD103+ M Peritoneal F4/80lo Liver in the groups of signature transcription Liver Peritoneal F4/80hi factors (left), ‘signature-up’ genes CD11b+ Lung (middle) and signature-down-genes Microglia (right) (previously associated with various Spleen 10 fl/fl tissue macrophage subsets ) in Cd11c-CrePparg cells relative to their expression in Ppargfl/fl cells (results presented as average ‘fold’ values). (f) Correlation matrix showing the relatedness between Ppargfl/fl adult mature AMs, Cd11c-CrePpargfl/fl arrested AMs and various tissue macrophage and DC populations on the basis of gene-expression data available from the Immunological Genome Project; expression values underwent ‘gene-wise’ normalization before correlation computation. Numbers in plot indicate correlation coefficient value. (g) Hierarchical clustering based on all available gene probes; red, cell populations from our transcriptome analysis. Data are from one experiment (with cells from two individual mice pooled for each microarray sample).

6

Lung

Liver

Liver

Lung

+

Spleen CD4

Spleen CD8+

0 0. 5 1.0 1.5 2 2. 5 3 3. 5

fl/fl

lo

Cd11c-CrePparg

Peritoneal F4/80

3

17 0. 03 –0 .1 –0 .2 –0 3 .3 –0 6 .5

0.

g

0.

Peritoneal F4/80

57 44 0.

Microglia

Lung

0.

0.

7

Pparg

Spleen

hi

f

fl/fl


Vdr


Maff Ncoa4 Rara Rbl1 Runx2


Lsr

DAB11


Slc9a4 Rufy4 Plscr1 Uck2 Perp Dna2 Phlda1 Plxnc1 Vstm2a Slc6a4 Aldoc Car4 A430084P05Rik Krt19 Prkch Gm5068 Akap5 Mfsd7c Zfp125 Prkar2b Tc2n Clmn Dnahc11 Gcnt2 Net1 Mak Ear2 Slc39a2 Ear1 Ear10 Sftpc Matn2 Krt79 Cd200r4 Ccdc80 Cldn1

Baz1a Chd5 Creb5 Ctnnb1 Egr2 Fosl2 Hmgn2 Lima1 Lmo4

DAB2

fl/ fl

fl/ fl

fl/ fl


fl/ fl

fl/ fl

c

Pp a C rg fl/fl d1 1 Pp c-C a re C rg fl/fl Pp d1 ar g 1c Pp -C ar re P g f C l d1 /fl pa rg 1c -C re Pp ar g

fl/ fl

a

fl/ fl

and mature AMs from age-matched Ppargfl/fl mice and focused our analysis on AM signature genes described before10. Among the 21 transcription factors whose expression is described to be selectively upregulated in lung AMs, we found that the expression of 8 was downregulated more than twofold in arrested immature AMs sorted from Cd11c-CrePpargfl/fl mice relative to their expression in AMs from

involved in the unique development and function of a particular type of tissue macrophage. To address whether deletion of PPAR-G changed the AM-specific transcriptional signature, we compared the transcriptome of the arrested PPAR-G-deficient pre-AMs in neonatal mice (day 2 after birth), prepubescent mice (day 11 after birth) and adult mice of the Cd11c-CrePpargfl/fl genotype with pre-AMs


NATURE IMMUNOLOGY

ARTICLES

r1 Em

ax Itg

ec 5 gl

Itg

am



–4

0 –2 –4 –6

cg Ab 1 ca 1 C D 3 Sc 6 ar b1 O Sl lr1 c2 7 Sl a1 c2 7a Sl 4 c2 7a An 6 gp tI4 C id ec Pl in Fa 2 bp Fa 1 bp 4

0 –2

2

Ab

2


4

c

4 Cd11c-CrePpargfl/fl vs WT

DAB2 DAB11 Adult

2 0 –2 –4

Ac s Ac I1 o Eh x1 h H ad sd h 17 Ac b4 aa Ac 1a aa 1b Ph Ec os h1 ph Ag o1 pa t G 9 pd So 1 at So 1 a C t2 h2 5h

b

6

Si



a

I

Ppargfl/fl mice (Fig. 4a,b). Beyond the 21 transcription factors, the AM signature was composed of another 110 genes whose expression was selectively higher in AMs than in other tissue macrophages (‘signature-up’ genes) and 27 genes whose expression was selectively lower in AMs than in other tissue macrophages (‘signature-down’ genes). Notably, expression of 48 of the 110 ‘signature-up’ genes was least twofold lower in the arrested AMs from adult Cd11c-CrePpargfl/fl mice than in mature AMs from Ppargfl/fl mice, and only 4 genes were further upregulated (twofold or more) in arrested PPAR-G-deficient cells relative to their expression in PPAR-G-sufficient cells (Fig. 4c). In contrast, 24 of the 27 ‘signature-down’ genes were upregulated (twofold or more) in arrested AMs from adult Cd11c-CrePpargfl/fl mice relative to their expression in mature AMs from Ppargfl/fl mice (Fig. 4d). We largely confirmed those transcriptional changes in arrested AMs from prepubescent Cd11c-CrePpargfl/fl mice (at day 11 after birth) before establishment of pathology (i.e., proteinosis and inflammation) (Fig. 4a–d). Moreover, comparison of the expression profiles of sorted pre-AMs from Ppargfl/fl and Cd11c-CrePpargfl/fl mice at day 2 after birth, the stage at which further development was blocked in the absence of PPAR-G, revealed a set of deregulated AM signature genes almost identical to that in 11-day-old and adult Cd11c-CrePpargfl/fl mice (Fig. 4a–d). These results suggested that the alteration in the expression of AM signature genes identified was due to the absence PPAR-G during differentiation and was not due to secondary environmental effects such as proteinosis and inflammation. The absence of PPAR-G resulted specifically in downregulation of the expression of genes encoding AM signature transcription factors and other transcripts, while the expression of ‘signature-up’ genes of other tissue macrophage subsets was increased (Fig. 4e), which suggested a considerable loss of AM identity. In contrast, the expression of ‘signature-down’ transcripts from AMs was selectively upregulated in cells from Cd11c-CrePpargfl/fl mice, but that of ‘signature-down’ transcripts from other tissue macrophage subsets was not (Fig. 4e). NATURE IMMUNOLOGY


hb

Ld

1

am

Pg

kI

Pf

k2

H

a1

c2

Sl

H


if1

a

Cd11c-CrePparg

CE (fold)

fl/fl

vs WT

I/f


Pp ar g

fI/

fI

C d Pp 11c ar -C gf

re

Figure 5 Diminished lipid catabolism– d e f associated gene expression and enhanced fI/fI Pparg 4 cholesterol esterification in PPAR-G-deficient Cd11c-Cre St1 1 2 1 2 3 US St2 fI/fI AMs. (a) Expression of genes encoding the Pparg 0.3 TAG CE surface markers CD11b (Itgam), Siglec-F DAG (Siglec5), CD11c (Itgax) and F4/80 (Emr1) 0.2 Ch fFA 2 in Ppargfl/fl AMs and Cd11c-CrePpargfl/fl immature arrested AMs sorted from 0.1 11-day-old and adult mice and of pre-AMs PE from Ppargfl/fl and Cd11c-CrePpargfl/fl mice 0 0 PC at day 2 after birth, presented as expression in Cd11c-CrePpargfl/fl cells relative to that in Ppargfl/fl cells. (b,c) Expression of genes encoding molecules involved in lipid import and storage (b) and lipid metabolism (c) in cells as in a (presented as in a). (d) Incorporation of fatty acids into intracellular lipids in AMs sorted from BAL fluid of Ppargfl/fl and Cd11c-CrePpargfl/fl mice (numbers above lanes indicate individual wells of the same pool of sorted cells), followed by in vitro stimulation with alkyne-oleate and alkyne-palmitate or medium (unstimulated (US)), assessed by thin-layer chromatography and fluorescence imaging of the extracted lipids and presented with standard St1 (cholesteryl ester (CE) (red arrow) and cholesterol (Ch)), standard St2 (triacylglycerol (TAG), diacylglycerol (DAG), phosphatidylethanolamine (PE) and phosphatidylcholine (PC)). (e) Signal intensity of the cholesteryl ester bands of the stimulated samples in d. (f) Expression of genes encoding molecules involved in glycolysis in cells as in a. Data are from one experiment (a–c,f; same experiment as in Fig. 4) or are from one experiment with cells pooled from at least 20 mice per group (d,e; error bars (e), s.d.).

To better understand to what extent AM identity was changed by the transcriptional alterations in arrested immature PPAR-G-deficient AMs, we assessed the degree of relatedness with different tissue macrophages and DC gene-expression profiles using publicly accessible data sets (from the Immunological Genome Project). Correlation analysis revealed that the strongest relatedness of our Ppargfl/fl mature AMs and PPAR-G-deficient arrested AMs was with published wild-type AMs (Fig. 4f,g). However, for PPAR-G-deficient arrested AMs, this correlation coefficient was smaller (Fig. 4f). This demonstrated a greater distance between PPAR-G-deficient arrested AMs and published wild-type AMs than between our Ppargfl/fl mature AMs and published wild-type AMs. Furthermore, there was a slightly smaller distance between PPAR-G-deficient arrested AMs and published peritoneal macrophages than between Ppargfl/fl mature AMs and published peritoneal macrophages (Fig. 4f). This was in line with the upregulation of several genes specific for peritoneal macrophages in arrested PPAR-G-deficient AMs (Supplementary Fig. 4a,b). Overall, these results demonstrated that PPAR-G conferred the AM signature and identity. PPAR-G controls the AM-specific gene-expression program To better understand how the markedly altered transcriptional pattern in the absence of PPAR-G influences AM development, we focused our analysis on genes encoding molecules involved in the phenotype and function of AMs. The expression of Itgam (which encodes CD11b) was more than 50-fold higher in PPAR-G-deficient arrested cells than in PPAR-G-sufficient cells, and the expression of Siglec5 (which encodes Siglec-F) was ninefold lower in PPAR-G-deficient arrested cells than in PPAR-G-sufficient cells, while the expression of Itgax (which encodes CD11c) and Emr1 (which encodes F4/80) was unaltered (Fig. 5a); this confirmed the flow cytometry data reported above (Fig. 1a,c). However, we observed that many of the genes that were downregulated twofold or more encoded molecules that mapped to metabolic pathways in lipid and energy metabolism (data 7

ARTICLES Msr1) was higher in PPAR-G-deficient cells than in Ppargfl/fl cells, the expression of other genes encoding molecules involved in lipid transport and intracellular storage (Fabp1, Fabp4, Cd36, Olr1 and Cidec) was much lower in PPAR-G-deficient cells than in Ppargfl/fl

WT

preAM 14.9

4 2

mAM+aAM

0 E17.5 E19.5 DAB2 mAM aAM

89.4

87.9

pre-AM 9.62

32.4

3.07

81 CD11b 14.7

22

F4/80

30.1

84.9

Csf2 (fold)

10–3 10–4

n

rt

10

–3

10–4

ai

ea

ey

ND Br

H

ve r Li

dn

Ki

en

ng

le

Lu

Sp

ND

10–2

DAB2

DAB11

+fetal mo 7.44

+fetal M 0.14

j

WT UT

100

101

10–1

100

10–2 –3

10

10–4 Csf2–/– 6x PBS

Csf2–/– 6x rGM-CSF

4 2

0

0

f

Fetal M Fetal Mo

0.4 0.2 0

Siglec-F

i

Fetal Mo Fetal M CD45–

10–1 10

–2

100 10

WT Csf2–/–

**

–1

10–2 10–3

10–3 Cd11cCre Ppargfl/fl UT

*

0.6

o

10–1

10–2

ND

E19.5

Siglec-F

h

Csf2 Il4

–1

10–5

51.5

6

0.8 E17.5

96.2

66.5

2

lm

23.6

58.7

4

CD11c

31.1

CD11b

g 10

2

d

0

2.19

6

Lung

4

CD11c

Siglec-F

11.2

Csf2ra (fold)

Csf2

6 Fetal M

0 CD11c

–/–

Expression (fold)

k

WT Cd11c-CrePpargfl/fl

8

L N sr co W a4 W t C r1 id e Kr c t1 C 9 1q c

B

az 1 Li a m a Lm 1 o4

U M UT rG -CST M F -C S PB F S rG


l

Pparg mRNA (fold)

F4/80

Figure 6 GM-CSF drives the development Csf2–/– Cd11c-CrePpargfl/fl 13.1 0.16 7.86 20.4 of AMs from fetal monocytes by the 100 Csf2–/– + rGM-CSF * induction of PPAR-G. (a) Expression of –1 4 ** 10 Siglec-F and CD11b (second and 2 10–2 bottom rows) by CD45+ cells isolated 0 CD11c –2 at various times before and after birth –3 10 5.45 3.22 0 89.5 –4 (as in Fig. 2a) from the lungs of time-mated –6 10–4 wild-type and Csf2−/− mice and sorted by –8 expression of F4/80 and CD11c (top and third row; gating and arrows as in Fig. 2a). CD11b mAM or Fetal (b) Total fetal monocytes, pre-AMs, aAM M mature AMs and arrested AMs in mice as in a. (c) Total fetal macrophages in mice as in a, gated as F4/80hiCD11cloCD11bintSiglec-F−Ly-6C− and autofluorescence-positive cells. Samples in a–c were assessed in parallel with the analysis of Cd11c-CrePpargfl/fl mice (values from Fig. 2b,c are presented here for the wild-type groups). (d) Flow cytometry of cells from neonatal Csf2rb−/− (CD45.2+) recipient mice 4 weeks after transfer of fetal monocytes and fetal macrophages sorted by flow cytometry from the lungs of CD45.1+ embryos at E17.5, assessing the presence of donor-derived AMs. (e) Total AMs in the BAL fluid and lungs of mice as in d. (f) Pulmonary proteinosis in BAL fluid from mice as in d. (g) Quantitative real-time RT-PCR analysis of Csf2 mRNA and Il4 mRNA in the lungs, spleen, liver, kidneys, heart and brain of wild-type embryos at E17.5; results are presented relative to those of the control gene Tubb1 (encoding tubulin B-1). ND, not detected. (h) Quantitative real-time RT-PCR analysis of Csf2, Csf2ra and Csf2rb mRNA in fetal monocytes, fetal macrophages and CD45− cells sorted from lungs of wildtype fetuses at E17.5 (presented as in g). (i) Quantitative real-time RT-PCR analysis of Pparg mRNA in fetal monocytes and fetal macrophages sorted from lungs of wild-type and Csf2−/− embryos at E17.5; results are presented relative to those of the control gene Eef1a1 (encoding eukaryotic translation elongation factor-1A 1). (j) Flow cytometry of AMs in the lungs of neonatal Csf2−/− mice treated with PBS (6× PBS) or recombinant GM-CSF (6× rGM-CSF) every other day from the day of birth until day 10 after birth, and in untreated (UT) wild-type and Cd11c-CrePpargfl/fl mice, assessed on day 12. (k) Quantitative real-time RT-PCR analysis of Pparg mRNA in mature wild-type AMs or in arrested AMs sorted by flow cytometry from Cd11c-CrePpargfl/fl mice or from Csf2−/− mice treated with recombinant GM-CSF (left), and in fetal macrophages from Csf2−/− mice treated with recombinant GM-CSF or PBS (right) (presented as in i). (l) Quantitative real-time RT-PCR analysis of mRNA from selected AM signature genes in arrested AMs from Cd11c-CrePpargfl/fl and from Csf2−/− mice treated with recombinant GM-CSF, presented (as log2 values) relative to that in wild-type mice. *P < 0.01 and **P < 0.0001 (Student’s t-test). Data are pooled from two independent experiments (a–c; mean and s.e.m. of four to seven mice per group), are from one experiment representative of two independent experiments (d–f,j–l; mean and s.d. of three (d–f) or four (j–l) mice per group) or three independent experiments (i; mean and s.d. of four mice per group) or are from one experiment (g,h; mean and s.d. of four (g) or three (h) mice per group). Siglec-F


47.9

0.4

4.01

3.56

DAB11

8

8

ta

31.6

c Cells (104)

63.5

51.7

Csf2rb (fold)

Siglec-F

44.4

BAL

10

AMs (105)

F4/80 CD11c

Fetal M Fetal Mo

12.7

67.9

e

WT –/– Csf2

lM

62

6 Fetal mo AM

ta

2.24

13.1

41.9

b

Fe

7.75

DAB11

Pparg mRNA (fold)

23.5

DAB2

BAL total protein (mg/ml)

E19.5

Fe

E17.5

AMs (104)

a

Cells (105)

not shown). We found markedly altered gene expression in a network of genes encoding molecules involved in lipid uptake, transport, storage and processing. While the expression of genes encoding some fatty acid receptors and transporters (Slc27a1, Slc27a6 and


NATURE IMMUNOLOGY

ARTICLES b

8 weeks E18.5

DAB2

12.0

1.9 67.0

–4

CD11b

CD11b

CD11b

79.7

F4/80

92.6

0.1

1

30 20 10

+

nt

C F4/ D 8 11 0 b hi

C F4 D /8 11 0 bi

+

0 +

C+ L N y-6 eu C – tro ph ils

-6 Ly

2

0

0

CD115+

Cells (%) of live

Cells (×105)

NS

2

40 Kidney

C F4/ D 80 11 b+

2

11

AB

8 weeks

4

0

AB

8.

5

D

E1

7.

M aA

AM

5

0

2

lo

2

6

hi

4

Peritoneum

8 4

Vav1-CrePpargfl/fl

3 Brain

F M 4/8 H 0 C II – F M 4/8 H 0 C II +

6

Ppargfl/fl 10

91.3

90.6

91.4

6 Blood

Pparg Vav1-CrePpargfl/fl

D

8 weeks

1.32

82.5

Cells (%) of live

1

m

M aA

AM

M

DAB11

m

aA

pr AB eA 2 M m AM

5 8.

11.7

0.1

9.9

fl/fl

E1

Cells (×10 )

5

**

2

0

D

1.85 13.4

0.31

CD11b 2.47

e

Fetal M 8

Cells (104)

3

0

5

0.6

CD11b

Cells (×105)

BAL

2

7.

1.8

95.3

19.5

15.1

78.6

d

6

E1

CD11b

84.9

20.3

AM

Pparg Vav1-CrePpargfl/fl

E1

93.8

4.5

fl/fl

**

33.2

fl

fl

fl/

fl

fl/

fl

fl/

fl/

Ly s C m-C Ppa d1 r 1c reP g p Va -Cr arg v1 eP - C pa re rg Pp ar g

Vav1-Cre Ppargfl/fl

Lung

4

CD11c

89.8

49.2

Fetal mo

8 Cells (105)

CD11b

55.6

70.4

10

63.4

16.9

19.8

5.34

NATURE IMMUNOLOGY


C M D6 H 4 hi C I + C I M D6 H 4 int C II +

+

Cells (%) of live

Cells per g tissue (×105)

F C 4/8 D 0 64 h i

+

+

F M 4/8 H 0 C II – F M 4/8 H 0 C II +

cells (Fig. 5b and Supplementary Fig. 5a). In addition, the expression of genes encoding molecules involved in lipid metabolism was much lower in PPAR-G-deficient cells than in Ppargfl/fl cells (Fig. 5c). In particular, the expression of genes encoding enzymes crucial for fatty acid B-oxidation (Acsl1, Acox1, Gpd1, Ehhadh, Hsd17b4, Acaa1a, Acaa1b, Ech1) was much lower (Fig. 5c), consistent with the intracellular accumulation of lipids. Experimentally, we observed less uptake of fatty acids in AMs from Cd11c-CrePpargfl/fl mice than in those from Ppargfl/fl mice, whereas incorporation into major phospholipids in Cd11c-CrePpargfl/fl mice was only slightly affected relative to that in Ppargfl/fl mice (Fig. 5d). Notably, we observed significantly more cholesterol esterification in PPAR-G-deficient arrested cells than in Ppargfl/fl arrested cells after treatment with fatty acids in vitro (Fig. 5d,e), consistent with higher expression of Soat2, which encodes a cholesterol acyltransferase, and of Ch25h, which encodes cholesterol

F C 4/8 D 0h 11 i b+

Cells (×104)

Heart Figure 7 Prenatal PPAR-G is essential for the development of AMs. 8 WAT 20 SI LP 3 Liver 10 (a) Quantitative real-time RT-PCR analysis of Pparg mRNA in 8 6 15 fetal monocytes sorted from lungs of Ppargfl/fl, Lysm-CrePpargfl/fl, Cd11c2 fl/fl fl/fl CrePparg and Vav1-CrePparg mice at E17.5 (presented as in Fig. 6g). 6 4 10 (b) Expression of Siglec-F, CD11b and Ly6C by CD45+ cells obtained from the 4 1 lungs of time-mated Ppargfl/f and Vav1-CrePpargfl/fl mice at various times before 2 5 2 and after birth (above plots) and sorted by expression of F4/80 and CD11c (gating and arrows as in Fig. 2a). (c) Total fetal monocytes, pre-AM, mature 0 0 0 0 AMs and arrested AMs in the lungs and BAL fluid of mice as in b. hi lo int − − (d) Fetal macrophages gated as F4/80 CD11c CD11b Siglec-F Ly6C and autofluorescence-positive cells in the lungs of mice as in b. (e) Total number or frequency (among live cells) of myeloid cells in various organs (above plots) of adult Ppargfl/fl and Vav1-CrePpargfl/fl mice (gated as in Supplementary Fig. 6d). WAT, perigonadal white adipose tissue; SI LP, small intestine lamina propria. *P < 0.05 and **P < 0.01 (Student’s t-test). Data are from one experiment (a; mean and s.d. of three to five mice per group), from two independent experiments (b–d; mean and s.d. of four to nine mice per group) or one experiment representative of two independent experiments (e; mean and s.d. of five mice per group). Cells (×104)


F4/80 29.8

97.9

CD11c

58.4

Siglec-F

41.7

52.8

Ly-6C

60.9

10–5

c

CD11c 36.6

Siglec-F

11.6

Ly6C

10–1 10–3 10

CD11c 58.3

0

Siglec-F

Pparg mRNA (fold)

10

CD11c

Ppargfl/fl

*

21.9

BAL

18.3

14.7

10.0

F4/80

F4/80

NS 101

52.6

Lung

F4/80

21.6

DAB11

Siglec-F

E17.5

Siglec-F

a

25 hydroxylase, and lower expression of Angptl4, which encodes a known inhibitor of foam-cell formation, in PPAR-G-deficient cells than in Ppargfl/fl cells (Fig. 5c). The diminished catabolic lipid metabolism was paralleled by higher expression of genes encoding molecules involved in glycolysis (Fig. 5f). Such metabolic changes have been associated with an enhanced inflammatory phenotype25. Consistent with that, the expression of several cytokines and modulators of inflammation (Supplementary Fig. 5b), chemokines and chemokine receptors (Supplementary Fig. 5c), and various factors involved in tissue remodeling (Supplementary Fig. 5d) was higher in PPAR-G-deficient cells than in PPAR-G-sufficient cells; this might have been involved in the low-grade inflammation we observed in the lungs of adult Cd11cCrePpargfl/fl mice (Fig. 1). In summary, our transcriptome analysis of pre-AMs and arrested immature AMs from Cd11c-CrePpargfl/fl mice 9

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showed that they failed to induce a functionally mature AM signature gene-expression profile and did not develop the ability to degrade lipids or perform fatty acid B-oxidation but displayed enhanced cholesterol esterification. GM-CSF induces PPAR-G expression in fetal monocytes GM-CSF has a crucial role in AM development2,8,26. Consistent with that, we found that the development of AMs in GM-CSFdeficient (Csf2−/−) mice was already abrogated at the stage of the early fetal monocyte–derived AM precursor cell at E17.5, which failed to upregulate CD11c expression or expand in number (Fig. 6a,b). In contrast, fetal macrophages remained unaffected in Csf2−/−mice (Fig. 6c). To unequivocally demonstrate that the monocytes identified in the embryonic lungs represented GM-CSF-dependent true precursors of AMs, we sorted fetal monocytes and fetal macrophages from lungs of wild-type (CD45.1+) embryos (at E17.5) and infused them intranasally into newborn mice deficient in the B-chain of the receptor for GM-CSF (Csf2rb−/− (CD45.2+) mice), which lack AMs, similar to Csf2−/− mice. The transfer of sorted F4/80intCD11bhi fetal monocytes from mice at E17.5 into Csf2rb−/− neonatal mice completely restored AM development and prevented pulmonary proteinosis, while the transfer of F4/80hiCD11bint fetal macrophages failed to reconstitute AM development (Fig. 6d–f). Consistent with a prenatal role for GM-CSF in early AM differentiation, we found higher expression of GM-CSF in the lungs than in other organs in fetuses at E17.5, whereas we did not detect IL-4 (Fig. 6g), which is known to drive PPAR-G expression and the polarization of M2 macrophages. Sorting of lung cells from mice at E17.5 into distinct populations and expression analysis showed that transcripts encoding GM-CSF were 10- to 40-fold greater in abundance in CD45− cells (epithelial and lung stromal cells) and fetal macrophages than in fetal monocytes (Fig. 6h). In contrast, fetal monocytes had high expression of the A-chain (Csf2ra) and B-chain (Csf2rb) of the receptor for GM-CSF (Fig. 6h), which suggested that lung epithelial cells and fetal macrophages induced the differentiation of lung fetal monocytes in a paracrine manner. Pparg mRNA expression was 20-fold higher in fetal monocytes than in fetal macrophages in mice at E17.5 (at the onset of AM differentiation), and it was 10-fold lower in fetal monocytes of Csf2−/−mice than in those of wild-type mice (Fig. 6i). Overall, these results demonstrated that GM-CSF was required for the induction of PPAR-G expression in lung fetal monocytes that are precursors of AMs. To address whether supplementation with GM-CSF can restore PPAR-G expression and neonatal AM development in Csf2−/− mice, we administered recombinant GM-CSF intranasally every other day during the first 10 d after birth and analyzed lungs at 12 d after birth. This treatment resulted in the development of CD11chiCD11bintSiglec-Fint AM-like cells, similar to the arrested AMs found in Cd11c-CrePpargfl/fl mice (Fig. 6j). Thus, GM-CSF therapy restored development of preAMs but failed to induce terminal AM differentiation in Csf2−/− mice. The treatment of neonatal Csf2−/− mice with GM-CSF resulted in higher expression of PPAR-G in AM-like cells than that in fetal macrophages, but expression of mRNA encoding PPAR-G was still significantly lower than that in mature AMs from wild-type mice (Fig. 6k). Furthermore, AM-like cells showed reduced expression of various AM signature genes, including those encoding transcription factors (Fig. 6l), which supported the proposal that PPAR-G controlled perinatal AM differentiation in response to stimulation with GM-CSF. We note that the transfer of immature AMs from Csf2−/− mice treated with recombinant GM-CSF into a GM-CSF-proficient host results in complete AM maturation8, which suggests that the amount and/or bioavailability of recombinant 10

GM-CSF was insufficient and that stable levels of GM-CSF are required for optimal induction of PPAR-G expression and thus AM signature gene expression and terminal differentiation. Together our data showed that GM-CSF was essential for the induction of PPAR-G expression in fetal monocytes at E17.5, the onset of AM differentiation. Prenatal PPAR-G activity is essential for AM development The GM-CSF-dependent induction of PPAR-G expression in fetal monocytes indicated a prenatal function for PPAR-G in these precursors of AMs during early AM development. Notably, we were unable to address such a role in Cd11c-CrePpargfl/fl mice, as Cd11c reached high expression only around the day of birth (Fig. 2a,d). Moreover, analysis of reporter-gene expression in the lungs of fetal Lysm-CreRosa26RFP mice revealed that Lysm-Cre–mediated recombination at E17.5– E19.5 targeted only a small fraction of fetal monocytes (Fig. 2d). Given the disadvantage of fetal monocyte–derived progenitors of AMs lacking PPAR-G in competition with their PPAR-G-competent counterparts in the development of true AMs, we speculated that inefficient deletion of Pparg in neonatal mice might have been the underlying reason for the almost normal AM differentiation in LysmCrePpargfl/fl mice. To find mice with efficient Cre-mediated deletion of loxP-flanked alleles in myeloid cells during late embryogenesis, we assessed Vav1-Cre mice27. Analysis of the lungs of Vav1-CreRosa26RFP embryos revealed potent Cre-mediated recombination in about 90% of fetal monocytes at E17.5 and in almost 100% of most hematopoietic cell types at E19.5 (Fig. 2d). To achieve more Cremediated recombination of Ppargfl/fl in fetal monocytes, we crossed Ppargfl/fl mice with Vav1-Cre mice. Accordingly, in contrast to results obtained for Lysm-CrePpargfl/fl mice and Cd11c-CrePpargfl/fl mice, in fetal monocytes from Vav1-CrePpargfl/fl mice at E17.5, both the Ppargfl alleles (Supplementary Fig. 6a) and Ppargfl/fl mRNA (Fig. 7a) were efficiently deleted, which resulted in abrogated AM development even before birth, as indicated by impaired expansion of the fetal monocyte progenitor cell population, concomitant with ‘paused’ expression of the surface marker Ly6C (at E17.5) and of CD11b (at E18.5) and no upregulation of Siglec-F expression (at day 2 after birth) (Fig. 7b,c). Notably, we observed a similar block in Csf2−/− mice at E17.5 (Fig. 6a), when GM-CSF induced Pparg expression in fetal monocytes. Thereafter, only a few progenitors made it to the CD11cintCD11bintSiglec-Fint stage of arrested preAMs observed in Cd11c-CrePpargfl/fl mice (Fig. 7b,c). We speculate that these ‘escapees’ might have shut down PPAR-G protein activity a few days later, consistent with the observation that ‘only’ 90% of the fetal monocytes in Vav1-CreRosa26-RFP mice showed reporter activity at E17.5 (Fig. 2d). Notably, fetal macrophages in the lungs and fetal monocytes in the blood and liver remained unaffected (Fig. 7d and Supplementary Fig. 6b,c), which demonstrated a specific local requirement for PPAR-G in regulating the differentiation of fetal monocytes into AMs in the lungs. Moreover, the number of macrophages in peritoneum, brain, kidneys, heart, liver, intestines and white adipose tissue was similar in Ppargfl/fl mice and Vav1-CrePpargfl/fl mice (Fig. 7e and Supplementary Fig. 6d), despite efficient deletion of the Pparg alleles in these macrophage populations (Supplementary Fig. 6e). Together our data have provided evidence of a crucial role for PPAR-G as a key transcription factor in the ‘imprinting’ of an AMspecific differentiation program during perinatal AM development. Prenatal, GM-CSF-dependent expression of PPAR-G was essential for the local differentiation of AMs from fetal monocytes in the lungs. Moreover, PPAR-G deficiency resulted in a low number of arrested ADVANCE ONLINE PUBLICATION

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immature AMs that had not fully acquired AM identity and thus AM function, and upregulated genes described as being specific for other tissue macrophages. DISCUSSION The majority of tissue-resident macrophages have been shown to originate from common embryonic progenitors that are generated during early hematopoiesis in the yolk sac and fetal liver6–9,24. AM have been proposed to derive from yolk-sac macrophages in a manner independent of transcription factor c-Myb24. However, a published report has concluded that AMs develop mainly from fetal monocytes8. In that report, the transfer of a 50:50 mixture of purified congenically marked fetal monocytes and fetal macrophages into the lungs of newborn mice showed that the majority of mature AMs developed from fetal monocytes8. A disadvantage, however, was that the vast majority (>95%) of mature AMs were derived from progenitor cells of the recipient mice8. We circumvented that problem by using newborn Csf2rb−/− mice as recipients; these mice fail to develop any endogenous AMs. Adoptive transfer of fetal lung monocytes isolated at E17.5 resulted in efficient population expansion and development of mature and functionally competent AMs that were all derived from the donor progenitor cells. Notably, transfer of fetal lung macrophages isolated at E17.5 failed to restore AM development. These data unequivocally demonstrated that AMs were derived from fetal lung monocytes and not fetal lung macrophages and confirmed the previously published conclusion8. However, the identification and definition of fetal monocytes and fetal macrophages was based entirely on a F4/80intCD11bhi and F4/ 80hiCD11bint phenotype, respectively, as proposed by others24. The current concept of tissue macrophage development proposes that the tissue niche provides instructive signals for the differentiation of progenitor cells into specialized effector cells with a distinct geneexpression profile10. While the cell-intrinsic signals in the progenitor cells remain poorly defined, the tissue environmental signals for the development of resident macrophages are beginning to emerge. Neuron- and keratinocyte-derived IL-34 drives the development of brain microglia and skin Langerhans cells28,29, while lung stromal cells induce perinatal AM development by GM-CSF production8. Our results demonstrated that GM-CSF ‘instructed’ AM development through the induction of a PPAR-G-regulated transcriptional program selectively in fetal monocytes. Prenatal deletion of Pparg in hematopoietic cells of Vav1-CrePpargfl/fl mice resulted in blockade of AM development at the fetal monocyte progenitor stage at E18.5, similar to results obtained for GM-CSF-deficient mice, except for a few cells that progressed to the CD11cintCD11bintSiglec-Fint pre-AM stage before arrest. Notably, Cd11c-CrePpargfl/fl mice effectively deleted Ppargfl concomitant with upregulation of CD11c expression shortly after birth, with the consequence that AM development proceeded to the pre-AM stage characterized as CD11cintCD11bintSiglec-Fint. Together these results suggested that PPAR-G was already required for the population expansion and differentiation of the AM progenitor before birth, as well as for terminal AM differentiation after birth. The presence of arrested immature pre-AMs in Cd11c-CrePpargfl/fl mice allowed us to characterize PPAR-G-regulated genes in AMs, which revealed selective downregulation of various AM signature genes10, indicative of a loss of AM identity. Specifically, we established PPAR-G-dependent regulation of genes encoding molecules involved in intracellular lipid metabolism during AM differentiation, in particular fatty acid B-oxidation. Immature AMs in Cd11c-CrePpargfl/fl mice looked like large foam macrophages and accumulated intracellular lipid droplets. Moreover, they showed a pronounced atypical accumulation of NATURE IMMUNOLOGY


cholesterol esters when cultured with labeled fatty acids. These defects were most probably the underlying reason for the development of pulmonary alveolar proteinosis in the Cd11c-CrePpargfl/fl mice. Lipid accumulation and foam macrophage formation are hallmarks of atherosclerotic lesions22 and have been associated mostly with diminished cholesterol efflux due to decreased levels of the cholesterol transporters ABCG1 and ABCA1 (ref. 30). Our results have identified an array of PPAR-G-regulated genes encoding molecules involved in the intracellular binding, storage and metabolism of lipids, which together prevent foam-cell formation. Notably, several genes with higher expression in arrested immature pre-AMs than in PPAR-Gsufficient mature AMs, such as Mmp12, Mmp14 and Pla2g7, also have higher expression in atherosclerosis and encode molecules linked to the propagation of inflammation31,32. Therefore, our data might provide insight into the mechanisms of protection from lipotoxicity and maintenance of macrophage function in a lipid-rich environment beyond AMs. The defect in lipid metabolism was paralleled by higher expression of genes encoding molecules involved in glycolysis. A metabolic switch from fatty acid oxidation to glycolysis has been described for many cells of the immune system with an activated and inflammatory phenotype25. Furthermore, the high ‘efferocytic’ activity of AMs26 imposes an excess metabolic load and requires efficient degradation pathways33, which might explain the accumulation of apoptotic cells and cellular debris in the lungs of Cd11c-CrePpargfl/fl mice. Notably, the immature arrested CD11bhi AMs found predominantly in Cd11c-CrePpargfl/fl mice are reminiscent of a minor population of CD11b+ lung macrophages that are not derived from the adult blood monocyte system34. However, whether there is a developmental link between the two populations needs to be investigated further. In contrast to Vav1-CrePpargfl/fl mice and Cd11c-CrePpargfl/fl mice, Lysm-CrePpargfl/fl mice showed only a slightly lower abundance of mature AMs and mild pulmonary alveolar proteinosis, due to inefficient deletion of the Ppargfl alleles in fetal monocytes and supersession of PPAR-G-deficient progenitors of AMs by PPAR-G-sufficient ‘sisters’ that failed to delete the Ppargfl alleles. Indeed, the proposal of an advantage of PPAR-G-sufficient cells over PPAR-G-deficient cells in a competitive situation during AM development was further supported by our results obtained with mixed-BM chimeras and the transfer of wild-type progenitor cells into Cd11c-CrePpargfl/fl mice. Overall, our findings indicated that the role described for PPAR-G in AMs has been greatly underestimated by the use of Lysm-CrePpargfl/fl mice18,19,35,36. Notably, our transcriptional profile of arrested AMs from Cd11c-CrePpargfl/fl mice was fundamentally different from published transcriptome data of AMs from Lysm-CrePpargfl/fl mice18 and revealed a vast number of PPAR-G-dependent AM signature genes not observed in the former study18, consistent with our results showing a mostly wild-type phenotype and Pparg expression for AMs from Lysm-CrePpargfl/fl mice. Thus, our data warrant caution in drawing conclusions on the role of a particular gene product in the development of fetal monocyte–derived tissue macrophages in studies using Lysm-Cre for deletion of the gene. Notably, we found that PPAR-G was dispensable for the development and homeostasis of macrophages in many organs, which demonstrated a specific role in the development of AMs. Although a few transcription factors have been identified as having a discrete role in a particular tissue macrophage subset, such as Spi-C and LXRA in spleen macrophages37,38 or Bach2 in AMs39, their tissue-specific induction is poorly understood. Exemplified by AM development in the lungs, our results have shown how a tissue-specific extrinsic signal (i.e., GM-CSF) secreted by lung epithelial cells and fetal macrophages acted on lung fetal monocytes to induce AM 11

ARTICLES development in paracrine manner by the induction of a specific transcriptional program controlled by PPAR-G. Notably, similar function for a local environmental factor in regulating organ-specific macrophage differentiation by the induction of a specific transcription factor has been described for retinoic acid–induced transcription factor GATA-6 in the development of peritoneal macrophages 40. Together our results have illustrated how differentiation of the heterogeneous subsets of tissue macrophages of a common origin can be regulated in situ by environment-specific factors, which induce discrete intrinsically active transcriptional regulators that eventually define the character of a member of these highly specialized professional phagocytes. METHODS Methods and any associated references are available in the online version of the paper.


Accession codes. GEO: microarray data, GSE60249. Note: Any Supplementary Information and Source Data files are available in the online version of the paper. ACKNOWLEDGMENTS We thank P. Chambon (Université Louis Pasteur) for Ppargfl/fl mice20; B. Becher (University of Zurich) for Csf2−/− and Csf2rb−/− mice; and C. Halin (Swiss Federal Institute of Technology Zurich) for Rosa26-stopflox-tdRFP mice43. Supported by the Swiss National Science Foundation (310030-124922/1) and Swiss Federal Institute of Technology Zurich (ETH-34 13-1). AUTHOR CONTRIBUTIONS C.S. and M.Ko. designed the experiments; C.S. performed and analyzed most of the experiments; S.P.N., M.Ku., H.R. and C.T. performed and analyzed specific experiments; and C.S. and M.Ko. wrote the manuscript. COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests. Reprints and permissions information is available online at http://www.nature.com/ reprints/index.html. 1. Yona, S. et al. Fate mapping reveals origins and dynamics of monocytes and tissue macrophages under homeostasis. Immunity 38, 79–91 (2013). 2. Hashimoto, D. et al. Tissue-resident macrophages self-maintain locally throughout adult life with minimal contribution from circulating monocytes. Immunity 38, 792–804 (2013). 3. Bain, C.C. et al. Constant replenishment from circulating monocytes maintains the macrophage pool in the intestine of adult mice. Nat. Immunol. 15, 929–937 (2014). 4. Epelman, S., Lavine, K.J. & Randolph, G.J. Origin and functions of tissue macrophages. Immunity 41, 21–35 (2014). 5. Ginhoux, F. & Jung, S. Monocytes and macrophages: developmental pathways and tissue homeostasis. Nat. Rev. Immunol. 14, 392–404 (2014). 6. Ginhoux, F. et al. Fate mapping analysis reveals that adult microglia derive from primitive macrophages. Science 330, 841–845 (2010). 7. Hoeffel, G. et al. Adult Langerhans cells derive predominantly from embryonic fetal liver monocytes with a minor contribution of yolk sac-derived macrophages. J. Exp. Med. 209, 1167–1181 (2012). 8. Guilliams, M. et al. Alveolar macrophages develop from fetal monocytes that differentiate into long-lived cells in the first week of life via GM-CSF. J. Exp. Med. 10, 1977–1992 (2013). 9. Epelman, S. et al. Embryonic and adult-derived resident cardiac macrophages are maintained through distinct mechanisms at steady state and during inflammation. Immunity 40, 91–104 (2014). 10. Gautier, E.L. et al. Gene-expression profiles and transcriptional regulatory pathways that underlie the identity and diversity of mouse tissue macrophages. Nat. Immunol. 13, 1118–1128 (2012). 11. Tontonoz, P. & Spiegelman, B.M. Fat and beyond: the diverse biology of PPARG. Annu. Rev. Biochem. 77, 289–312 (2008).

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12. Tontonoz, P., Nagy, L., Alvarez, J.G., Thomazy, V.A. & Evans, R.M. PPARG promotes monocyte/macrophage differentiation and uptake of oxidized LDL. Cell 93, 241–252 (1998). 13. Ricote, M. et al. Expression of the peroxisome proliferator-activated receptor G (PPARG) in human atherosclerosis and regulation in macrophages by colony stimulating factors and oxidized low density lipoprotein. Proc. Natl. Acad. Sci. USA 95, 7614–7619 (1998). 14. Ricote, M., Li, A.C., Willson, T.M., Kelly, C.J. & Glass, C.K. The peroxisome proliferator-activated receptor-G is a negative regulator of macrophage activation. Nature 391, 79–82 (1998). 15. Jiang, C., Ting, A.T. & Seed, B. PPAR-G agonists inhibit production of monocyte inflammatory cytokines. Nature 391, 82–86 (1998). 16. Szanto, A. et al. STAT6 transcription factor is a facilitator of the nuclear receptor PPARG-regulated gene expression in macrophages and dendritic cells. Immunity 33, 699–712 (2010). 17. Bonfield, T.L. et al. Peroxisome proliferator-activated receptor-G is deficient in alveolar macrophages from patients with alveolar proteinosis. Am. J. Respir. Cell Mol. Biol. 29, 677–682 (2003). 18. Gautier, E.L. et al. Systemic analysis of PPARG in mouse macrophage populations reveals marked diversity in expression with critical roles in resolution of inflammation and airway immunity. J. Immunol. 189, 2614–2624 (2012). 19. Bonfield, T.L. et al. Peroxisome proliferator-activated receptor-G regulates the expression of alveolar macrophage macrophage colony-stimulating factor. J. Immunol. 181, 235–242 (2008). 20. Imai, T. et al. Peroxisome proliferator-activated receptor G is required in mature white and brown adipocytes for their survival in the mouse. Proc. Natl. Acad. Sci. USA 101, 4543–4547 (2004). 21. Vermaelen, K. & Pauwels, R. Accurate and simple discrimination of mouse pulmonary dendritic cell and macrophage populations by flow cytometry: methodology and new insights. Cytometry 61, 170–177 (2004). 22. Moore, K.J. & Tabas, I. Macrophages in the pathogenesis of atherosclerosis. Cell 145, 341–355 (2011). 23. Hochreiter-Hufford, A. & Ravichandran, K.S. Clearing the dead: apoptotic cell sensing, recognition, engulfment, and digestion. Cold Spring Harb. Perspect. Biol. 5, a008748 (2013). 24. Schulz, C. et al. A lineage of myeloid cells independent of Myb and hematopoietic stem cells. Science 336, 86–90 (2012). 25. O’Neill, L.A.J. & Hardie, D.G. Metabolism of inflammation limited by AMPK and pseudo-starvation. Nature 493, 346–355 (2013). 26. Schneider, C. et al. Alveolar macrophages are essential for protection from respiratory failure and associated morbidity following influenza virus infection. PLoS Pathog. 10, e1004053 (2014). 27. de Boer, J. et al. Transgenic mice with hematopoietic and lymphoid specific expression of Cre. Eur. J. Immunol. 33, 314–325 (2003). 28. Greter, M. et al. Stroma-derived interleukin-34 controls the development and maintenance of langerhans cells and the maintenance of microglia. Immunity 37, 1050–1060 (2012). 29. Wang, Y. et al. IL-34 is a tissue-restricted ligand of CSF1R required for the development of Langerhans cells and microglia. Nat. Immunol. 13, 753–760 (2012). 30. Tall, A.R., Yvan-Charvet, L., Terasaka, N., Pagler, T. & Wang, N. HDL, ABC transporters, and cholesterol efflux: implications for the treatment of atherosclerosis. Cell Metab. 7, 365–375 (2008). 31. Johnson, J.L. & Newby, A.C. Macrophage heterogeneity in atherosclerotic plaques. Curr. Opin. Lipidol. 20, 370–378 (2009). 32. Mallat, Z., Lambeau, G. & Tedgui, A. Lipoprotein-associated and secreted phospholipases A in cardiovascular disease: roles as biological effectors and biomarkers. Circulation 122, 2183–2200 (2010). 33. Han, C.Z. & Ravichandran, K.S. Metabolic connections during apoptotic cell engulfment. Cell 147, 1442–1445 (2011). 34. Jakubzick, C. et al. Minimal differentiation of classical monocytes as they survey steady-state tissues and transport antigen to lymph nodes. Immunity 39, 599–610 (2013). 35. Malur, A. et al. Deletion of PPAR G in alveolar macrophages is associated with a Th-1 pulmonary inflammatory response. J. Immunol. 182, 5816–5822 (2009). 36. Baker, A.D. et al. PPARG regulates the expression of cholesterol metabolism genes in alveolar macrophages. Biochem. Biophys. Res. Commun. 393, 682–687 (2010). 37. Kohyama, M. et al. Role for Spi-C in the development of red pulp macrophages and splenic iron homeostasis. Nature 457, 318–321 (2009). 38. A-Gonzalez, N. et al. The nuclear receptor LXRA controls the functional specialization of splenic macrophages. Nat. Immunol. 14, 831–839 (2013). 39. Nakamura, A. et al. Transcription repressor Bach2 is required for pulmonary surfactant homeostasis and alveolar macrophage function. J. Exp. Med. 210, 2191–2204 (2013). 40. Okabe, Y. & Medzhitov, R. Tissue-specific signals control reversible program of localization and functional polarization of macrophages. Cell 157, 832–844 (2014).


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ONLINE METHODS


Mice C57BL/6 mice were from Charles River. We backcrossed Ppargtm1.2Mtz (Ppargfl/fl) mice20 (provided by P. Chambon) for six generations to C57BL/6 before crossing them to Lyz2Cretm1(Cre)Ifo (Lysm-Cre) mice41, Tg(Itgaxcre)1-1Reiz (Cd11c-Cre) mice42 or B6.Cg-Tg(Vav1Cre)A2Kio/J (Vav1-Cre) mice27 to generate mice with deficiency in PPAR-G in myeloid cells (LysmCrePpargfl/fl), CD11c+ cells (Cd11c-CrePpargfl/fl) or all hematopoietic lineages (Vav1-CrePpargfl/fl). Csf2−/− and Csf2rb−/− mice were provided by B. Becher. Gt(ROSA)26Sortm1Hjf (Rosa26-RFP) mice, which have a transcriptional stop element and an inverted sequence encoding tdRFP flanked by two inverted pairs of loxP and loxP2272 sites43, were crossed with Lysm-Cre, Cd11c-Cre and Vav1-Cre mice. All animals were housed in individually ventilated cages under specific pathogen–free conditions at BioSupport (Zurich, Switzerland) and were used for experiments at between 7 and 12 weeks of age unless otherwise stated. All animal experiments were approved by the local animal ethics committee (Kantonales Veterinaersamt Zurich) and were performed according to local guidelines (Swiss Animal Protection Ordinance (TschV) Zurich) and Swiss animal protection law (TschG). Cell suspension preparations. Mice were killed by intraperitoneal injection of pentobarbital sodium at a dose of 400 mg per kg body weight (Euthasol; VIRBAC Schweiz). Erythrocytes in peripheral blood were lysed with ACK (ammonium chloride–potassium bicarbonate) buffer. BAL fluid was isolated by canulization of the trachea with a catheter. The lungs were then flushed three times with 400 Ml PBS, and BAL fluid cells were harvested by centrifugation. Peritoneal lavage was performed with 10 ml PBS. Organs were removed after whole-body perfusion with PBS, then were minced and digested and then passed through a 70-Mm cell strainer unless otherwise stated. The lungs and fetal liver were digested for 45 min at 37 °C with 2 mg/ml of type IV collagenase (Worthington) and 0.02 mg/ml DNase I (Sigma) and were passed through a 70-Mm cell strainer. Adult liver was digested for 45 min at 37 °C with 2 mg/ml of type IV collagenase and 0.02 mg/ml DNase I and passed through a 70-Mm cell strainer. Liver cell suspensions were centrifuged at 20g for 5 min and were resuspended in 30% Percoll (GE Healthcare), before density centrifugation at 2000 r.p.m. for 20 min at 25 °C, with low acceleration and no brake. The kidneys were digested for 45 min at 37 °C with 2 mg/ml of type IV collagenase and 0.02 mg/ml DNase I and passed through a 70-Mm cell strainer, and leukocytes were purified by 30% Percoll density centrifugation as described for the liver. The heart was digested for 45 min at 37 °C in 450 U/ml type I collagenase (Sigma), 125 U/ml type XI collagenase (Sigma), 60 U/ml hyaluronidase I (Sigma) and 0.02 mg/ml DNase I. The brain was digested for 90 min at 37 °C in 100 Mg/ml collagenase D (Roche) and passed through a 70-Mm cell strainer. Cells were resuspended in 30% Percoll layered over 70% Percoll before density centrifugation at 600g for 25 min at 25 °C, with low acceleration and no brake and the interphase was collected. The spleen was passed through a 70-Mm cell strainer without prior digestion. Small intestines were washed of fecal content and then were opened longitudinally and cut into pieces 3 cm in length, then were washed in PBS and were incubated on a shaker at 300 r.p.m. for 15 min at 37 °C in Hank’s balanced-salt solution supplemented with 5% FCS, 1.35 mM EDTA and 1 mM DTT. In a second step, tissues were incubated for 30 min in the same solution without EDTA. Thereafter, intestinal tissues were digested on a shaker at 100 r.p.m. for 45 min at 37 °C in 0.4 mg/ml type IV collagenase and were filtered through a 70-Mm cell strainer. Adipose tissue was digested for 45 min at 37 °C in 2 mg/ml type II collagenase (Worthington) and 0.2 mg/ml DNase I, then was passed through a 70-Mm cell strainer, followed by twice centrifugation for 4 min at 1,000 r.p.m. for the removal of floating adipocytes. ACK buffer was used for erythrocyte lysis. Flow cytometry. A FACSCanto II or LSR Fortessa (BD) was used for multiparameter analysis, and data were analyzed with FlowJo software (TreeStar). Fluorochrome-conjugated or biotinylated monoclonal antibodies specific to mouse CD11c (N418), CD11b (M1/70), F4/80 (BM8), Ly-6C (HK1.4), SiglecF (E50-2440, BD Biosciences), CD103 (2E7), CD115 (AFS98, eBioscience), CD45 (30-F11), CD45.1 (A20), CD45.2 (104), CD4 (GK1.5), CD8A (53-6.7), MHC class II (M5/114.15.2, eBioscience), Gr-1 (RB6-8C5, eBioscience), CD206 (19.2, BD Biosciences), Siglec-F (3D6.112, AbD Serotec), CD64 (X545/7.1), SIRPa (P84, eBioscience), CD19 (6D5), Ly-6G (1A8, BD Biosciences),

doi:10.1038/ni.3005

CD68 (FA-11, AbD Serotec) were from BioLegend unless otherwise stated. Dead cells were ‘outgated’ with the live-dead marker eFluor780 (eBioscience) before analysis. Prior to all staining, FcGIII/II receptors were blocked by incubation with anti-CD16/32 (2.4G2) purified from hybridoma supernatant (Swiss Federal Institute of Technology Zurich). Oil Red O staining. Cytospins of BAL fluid cells or flow cytometry–sorted AMs (BD FACSAria IIIu) were fixed for 10 min with 4% formalin, washed two times with PBS, rinsed with 60% isopropanol and stained for 30 min with a solution of 0.3% Oil Red O (Sudan IV; Sigma O0625) in 60% isopropanol, followed by 2 min of destaining in 60% isopropanol and two further washes with PBS. Subsequently, nuclei were stained with hematoxylin. Lung histology. The lungs were removed, fixed in 4% buffered formalin and processed for Verhoeff-Van Gieson staining. Protein quantification and enzyme-linked immunosorbent assay of surfactant protein D (SP-D). Total protein concentrations in BAL fluid were measured by BCA Protein Assay according to the manufacturer’s instructions (Thermo Scientific). For enzyme-linked immunosorbent assay of SP-D, wells were coated with 0.25 Mg/ml (1:4,000 dilution) polyclonal rabbit antibody to mouse SP-D (LS-C17965; LIFESPAN Biosciences), followed by detection with 2 Mg/ml biotinylated monoclonal antibody to mouse SP-D (VIF11; Abcam). Cell sorting and transfer. Fetal monocytes (F4/80intCD11cloCD11b+ and autofluorescence-negative cells) and fetal macrophages (F4/80hi CD11cloCD11bint and autofluorescence-positive cells) were sorted from the lungs of E17.5-18.5 CD45.1+ embryos with a FACSAria IIIu (BD). For transfer experiments, neonatal recipient mice were anesthetized with isoflurane, and 3 × 104 fetal monocytes or fetal macrophages were administered intranasally in a volume of 10 Ml PBS. Mature AMs were sorted as F4/80+CD11chiSiglec-F+ and autofluorescence-high cells. In another experiment, neonatal Csf2−/− mice were treated intranasally with 50 ng recombinant GM-CSF every other day during the first 10 d after birth, and AMs and fetal macrophages were sorted at day 12. Quantitative real-time PCR. For analysis of recombination efficiency, cells were sorted from fetal and adult Pparg fl/fl, Lysm-CrePparg fl/fl, Cd11c-CrePpargfl/fl and Vav1-CrePpargfl/fl mice. Genomic DNA was isolated with a DNeasy Blood & Tissue Kit according to the manufacturer’s instructions (Qiagen). Recombined Ppargfl/fl alleles were quantified by real-time PCR with KAPA SYBR FAST (Kapa Biosystems) and an iCycler (Bio-Rad Laboratories) and results were normalized to those of a Pparg control allele. The following primers were used 20: recombined allele, 5`-AAGAGAAGAGAGGATATGGAG-3` and 5`-ATATTAATATGCTTAATA TTACAGC-3`; and control allele, 5`-CAGAAACATCTCTAGTGAAG-3` and 5`-TGACATAGTAATTTTTAGTTCCC-3`. RNA was isolated with TRIzol reagent (Invitrogen) and was reverse-transcribed with GoScript reverse transcriptase according to the manufacturer’s instructions (Promega). Quantitative real-time RT-PCR was performed with KAPA SYBR FAST (primers, Supplementary Table 1). BM chimeras. For BM chimeras, C57BL/6 CD45.2+ mice were lethally irradiated (9.5 Gy, with a cesium source) and were reconstituted with 5 × 106 to 10 × 106 cells of a 1:4 mixture of CD45.1+wild-type BM cells and CD45.2+Ppargfl/fl BM cells, or CD45.1+wild-type BM cells and CD45.2+ Cd11c-CrePpargfl/fl BM cells. Mice were analyzed at 9 weeks after reconstitution. Microarray analysis. Lungs were processed as described above and were stained with the live-dead marker eFluor780 (eBioscience) and antibodies to mouse CD45 (30-F11, BioLegend), CD11c (N418, BioLegend), CD11b (M1/70, BioLegend) and Siglec-F (E50-2440, BD Biosciences). Mature AMs were sorted (with a BD FACSAria IIIu) from 11-day-old or adult Ppargfl/fl and Cd11c-CrePpargfl/fl mice as eF780−CD45+CD11chiSiglec-F+ and autofluorescence-high cells that were CD11blo or CD11bhi, respectively, and pre-AMs (eF780−CD45+F4/80+CD11c+ and Siglec-FintCD11bint or Siglec-FloCD11bhi cells) were sorted from 2-day-old mice. RNA was prepared from sorted

NATURE IMMUNOLOGY


populations with a PureLink RNA Mini Kit (Ambion; Life Technologies), then was amplified and hybridized on an Affymetrix Mouse Gene 1.1 ST array. Heat maps were visualized with MultiExperiment Viewer software44,45. Pathway analysis was performed with MetaCore software (Thomson Reuters) (Supplementary Table 2) and pathway mapping by the KEGG (Kyoto Encyclopedia of Genes and Genomes) database. For comparison of our data with data from the Immunological Genome Project, we downloaded raw data from GEO accession code GSE15907, selected the relevant samples and computed expression values from the Affymetrix CEL files with the RMA (robust multiarray average) algorithm46. GEO data files were generated with the array type MoGene 1.0, while our own data were generated using MoGene 1.1, which has a different spatial layout but holds the same probe sets. Therefore, we ran the preprocessing separately and integrated the data on the basis of the Affymetrix probe set identifier. The combined data were normalized by quantile normalization. All data analyses were conducted on logarithmic expression values. For the heat map visualization, clustering and correlation computation, we additionally shifted the mean expression of each gene to a value of 0. Additionally, we averaged results from replicates so only one value per condition is shown. The hierarchical clustering used as distance measure 1 minus the Pearson correlation coefficient (1 − r) and Ward’s linkage rule for merging clusters. Tracing of fatty acid metabolism. AMs were sorted from pooled BAL fluid from Ppargfl/fl mice or Cd11c-CrePpargfl/fl mice and were resuspended in complete IMDM (10% FCS, 50 MM B-mercaptoethanol and penicilinstreptomycin). 1.4 × 105 cells were seeded in 96-well flat-bottomed plates and were stimulated for 1 h with 50 MM alkyne-fatty acids (alkyne-oleate and

NATURE IMMUNOLOGY

alkyne-palmitate at a ratio of 2:1) or for 3 h with 10 Mg/ml alkyne-cholesterol. Cells were washed with 5 mg/ml BSA and 150 mM ammonium acetate in PBS, then lipids were extracted and were detected by thin-layer chromatography and fluorescent imaging as described47. The signal intensity of an individual cholesteryl ester band was calculated relative to the cumulated intensity of all such bands and results were normalized to the total integrated signal intensity of an individual lane. Statistical analysis. Prism software (GraphPad) was used for statistical analysis. No randomization or exclusion of data points was used. Comparisons of two groups were calculated with unpaired two-tailed Student’s t-tests. Differences with a P value of