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Induction of the nuclear receptor PPAR-γ by the cytokine GM-CSF is critical for the differentiation of fetal monocytes into alveolar macrophages

Abstract

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-γ determined the perinatal differentiation and identity of alveolar macrophages (AMs). In contrast, PPAR-γ 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-γ conferred a unique signature, including several transcription factors and genes associated with the differentiation and function of AMs. Expression of PPAR-γ 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-γ in fetal monocytes.

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Figure 1: Impaired development and function of AMs in Cd11c-CrePpargfl/fl mice.
Figure 2: AMs develop perinatally from fetal monocytes through a pre-AM intermediate that requires PPAR-γ for terminal differentiation.
Figure 3: Cell-autonomous requirement for PPAR-γ during the terminal differentiation of AMs.
Figure 4: PPAR-γ confers the AM signature and identity.
Figure 5: Diminished lipid catabolism–associated gene expression and enhanced cholesterol esterification in PPAR-γ-deficient AMs.
Figure 6: GM-CSF drives the development of AMs from fetal monocytes by the induction of PPAR-γ.
Figure 7: Prenatal PPAR-γ is essential for the development of AMs.

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Acknowledgements

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).

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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.

Corresponding author

Correspondence to Manfred Kopf.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 PPAR-γ is dispensable for the development of tissue macrophages in the heart, kidneys, lamina propria and white adipose tissue.

Plots show the expression of F4/80 and CD11b (a) or F4/80 and CD11c (b) among CD45+ cells in the indicated organs (LP, lamina propria). Bar graphs display the percentage of cells gated as shown in flow cytometry plots. Data are from one experiment representative of two independent experiments (mean and s.d. of three mice per group).

Supplementary Figure 2 Cd11c-CrePpargfl/fl mice with functionally impaired AMs accumulate apoptotic cells in the bronchoalveolar space.

(a) CD11chiCD11bloautofluorescencehiSiglec-F+ Ppargfl/fl AM and CD11chiCD11bhiautofluorescencehiSiglec-F+ arrested immature AM from Cd11cCrePpargfl/fl mice were sorted by flow cytometry followed by cytospin and Oil Red O staining. Micrographs were taken at 20× magnification. Scale bar = 50 μm. (b) BAL of Ppargfl/fl, LysmCrePpargfl/fl and Cd11cCrePpargfl/fl mice was analyzed by flow cytometry for the presence of dead eFluor780+ cells. Representative pictures and plots of three to four mice per group are shown.

Supplementary Figure 3 Cell-autonomous requirement for PPAR-γ during AM development.

Mixed BM chimeras (1:4 mixture of CD45.1+WT:CD45.2+Cd11cCrePpargfl/fl or CD45.1+WT:CD45.2+Ppargfl/fl) were analyzed as in Fig. 3. (a) Reconstitution ratio in peripheral blood leukocyte populations shown as CD45.2+ fold over CD45.1+ cells. (b-d) Histograms show the levels of CD11b and Siglec-F in CD45.1+WT and CD45.2+Ppargfl/fl or CD45.2+Cd11cCrePpargfl/fl AM in the BAL of the same mouse (b) and the degree of CD11c expression and autofluorescence in the BAL (c) and lung (d). (e) Bar graphs display the frequencies of eF780+ apoptotic AM among CD45.2+Ppargfl/fl and CD45.2+Cd11cCrePpargfl/fl cells and their CD45.1+ WT counterparts. Data are from one experiment representative of two independent experiments (mean and s.d. of four chimeras per group, dot plots from one mouse representative of the group).

Supplementary Figure 4 PPAR-γ is required for the maintenance of AM identity.

Transcriptomes of Ppargfl/fl AM and arrested immature Cd11cCrePpargfl/fl AM sorted from adult mouse lungs by flow cytometry were analysed by microarray. (a,b) Heat maps representing mRNA levels in Ppargfl/fl and Cd11cCrePpargfl/fl AM of peritoneal macrophage signature-up-genes (a) and transcription factors (b). (c,d) Heat maps representing mRNA levels in Ppargfl/fl and Cd11cCrePpargfl/fl AM of microglia signature-up-genes (c) and transcription factors (d). The list of signature transcripts was obtained from Reference.

Supplementary Figure 5 PPAR-γ is required for the induction of an AM-specific gene-expression profile and maintenance of AM identity.

Transcriptomes of Ppargfl/fl AM and immature arrested Cd11cCrePpargfl/fl AM sorted from 11 days old and adult mice and of pre-AM from DAB2 were analysed by microarray. Bar graphs show relative expression levels plotted as log2-fold change in Cd11cCrePpargfl/fl cells compared to Ppargfl/fl. Effects of Pparg-deficiency on genes involved in phagocytosis of apoptotic cells (a), cytokines and modulators of inflammation (b), chemokines and chemokine receptors (c) and tissue remodeling factors (d).

Supplementary Figure 6 PPAR-γ is dispensable for the development and maintenance of most tissue macrophages.

(a) Fetal monocytes were sorted from lungs of the indicated strains on E17.5 and recombination of the Ppargfl/fl alleles was assessed by quantitative real-time PCR on genomic DNA. (b,c) E17.5 WT and Vav1CrePpargfl/fl fetuses were analyzed for the presence of fetal monocytes in the blood (b) and the liver (c). (d) Adult WT and Vav1CrePpargfl/fl mice were analyzed for the presence of macrophages in the indicated organs. Numbers represent the frequencies among total cells (blood), CD11b+CD19 (peritoneum), eF780CD45+ (brain, liver, perigonadal white adipose tissue (WAT)), eF780CD45+CD64+ (kidney), eF780CD45+CD64+autofluorescence+ (heart) or eF780CD45+CD11b+ cells (small intestine lamina propria (LP)). (e) Macrophages were sorted as gated in (d), from the indicated organs of adult WT and Vav1CrePpargfl/fl mice and recombination of the Ppargfl/fl alleles was assessed by quantitative real-time PCR on genomic DNA. Subsets of blood monocytes and peritoneal Mø subsets were pooled, respectively. NS, not significant (Student's t-test). Data are from one experiment (a; mean and s.d. of three to five mice per group), from one experiment representative of two independent experiments (b,c; dot plots of one mouse per group representative of three mice per group), from one experiment representative of two independent experiments (d; dot plots of one mouse per group representative of five mice per group) or from one experiment (e; mean and s.d. of four mice per group).

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–6 and Supplementary Table 1 (PDF 1854 kb)

Enrichment analysis in pathway maps for differentially expressed genes.

Enrichment analysis was performed using MetaCore. List of 100 pathways enriched in differentially expressed genes are displayed for upregulated and downregulated genes (log2 ratio ≥1, P<0.05). (XLS 125 kb)

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Schneider, C., Nobs, S., Kurrer, M. et al. Induction of the nuclear receptor PPAR-γ by the cytokine GM-CSF is critical for the differentiation of fetal monocytes into alveolar macrophages. Nat Immunol 15, 1026–1037 (2014). https://doi.org/10.1038/ni.3005

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