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Identification of an atypical monocyte and committed progenitor involved in fibrosis

Abstract

Monocytes and macrophages comprise a variety of subsets with diverse functions1,2,3,4,5. It is thought that these cells play a crucial role in homeostasis of peripheral organs, key immunological processes and development of various diseases. Among these diseases, fibrosis is a life-threatening disease of unknown aetiology. Its pathogenesis is poorly understood, and there are few effective therapies. The development of fibrosis is associated with activation of monocytes and macrophages6,7,8. However, the specific subtypes of monocytes and macrophages that are involved in fibrosis have not yet been identified. Here we show that Ceacam1+Msr1+Ly6CF4/80Mac1+ monocytes, which we term segregated-nucleus-containing atypical monocytes (SatM), share granulocyte characteristics, are regulated by CCAAT/enhancer binding protein β (C/EBPβ), and are critical for fibrosis. Cebpb deficiency results in a complete lack of SatM. Furthermore, the development of bleomycin-induced fibrosis, but not inflammation, was prevented in chimaeric mice with Cebpb−/− haematopoietic cells. Adoptive transfer of SatM into Cebpb−/− mice resulted in fibrosis. Notably, SatM are derived from Ly6CFcεRI+ granulocyte/macrophage progenitors, and a newly identified SatM progenitor downstream of Ly6CFcεRI+ granulocyte/macrophage progenitors, but not from macrophage/dendritic-cell progenitors. Our results show that SatM are critical for fibrosis and that C/EBPβ licenses differentiation of SatM from their committed progenitor.

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Figure 1: Cebpb−/− chimaeric mice are resistant to fibrosis.
Figure 2: Critical role of C/EBPβ in the differentiation of SatM.
Figure 3: Identification of SatM progenitor cells.
Figure 4: C/EBPβ licenses SatM differentiation from committed progenitor cells.

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Acknowledgements

We thank S. Saeki, S. Watanabe, R. Takenaka and M. Miyamoto for assistance with experiments, and T. Matsuki, T. Kawasaki, H. Kanemaru, H. Tanaka and K. Kuniyoshi for helpful discussions. We thank T. Kitamura for providing PlatE cells. We also thank E. Kamada for secretarial assistance, and C. Funamoto, N. Kitagaki, A. Wataki, K. Yokoyama and R. Kawaguchi for technical assistance. This work was supported by the Government of Japan and the Japan Society for the Promotion of Science (JSPS) through the funding program for World-Leading Innovative R&D on Science and Technology (FIRST Program), by Japan Science and Technology Agency (JST) thorough funding for a Grant-in-Aid for Young Scientists (A)(16H06234), Specially Promoted Research (15H05704) and the US National Institutes of Health (P01-AI070167) and ‘Visionary Research Fund’ from Takeda Science Foundation. A part of this work was supported by the Nanotechnology Platform (project number 12024046) of the Ministry of Education, Culture, Sports, Science and Technology (MEXT), Japan.

Author information

Affiliations

Authors

Contributions

T.S. designed and performed the experiments and wrote the manuscript. K.F., I.E., F.Y. and A.K. helped with experiments. K.N. performed the experiments. M.A. and Y.M. performed bioinformatics analysis. F.S. and Y.Y. performed the MRI analysis. R.K. performed the electron microscopy analysis. S.A. wrote the manuscript and supervised the project.

Corresponding author

Correspondence to Shizuo Akira.

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Competing interests

S.A. has research support from Chugai Pharmaceutical Co., Ltd. The terms of this arrangement have been reviewed and approved by the Osaka University in accordance with its policy on objectivity in research.

Additional information

Reviewer Information Nature thanks I. Amit, D. Brenner, F. Geissmann and the other anonymous reviewer(s) for their contribution to the peer review of this work.

Extended data figures and tables

Extended Data Figure 1 Characterization of various macrophage/monocytes.

a, Indicated cell types were sorted and RNA was collected from them. Each cell type was subjected to microarray analysis. Principal component analysis by using gene expression data was performed. b, c, Indicated cell types in bone marrow were sorted, and each cell type was adoptively transferred into the wild-type mice, which were administered bleomycin. Images of Azan staining (b) and quantity of hydroxyproline are shown (c). Similar results were obtained in three independent experiments (ac). Scale bars, 100 μm. *P < 0.05, **P < 0.01.

Extended Data Figure 2 Cebpb−/− chimaeras are resistant to the development of fibrosis.

a, Quantity of hydroxyproline 14 days after bleomycin administration was determined. b, Indicated cytokines in the BAL 3 days after bleomycin administration were determined by ELISA. c, d, Images of H&E staining in the lung tissue 4 days (c) and 13 days (d) after intratracheal injection with bleomycin. Scale bars, 50 μm. e, MRI imaging, H&E, Azan and Sirius red staining of lung. f, MRI images of T2 weighted (T2W), T1 weighted (T1W) and water suppression (WS) was shown (left panels). Azan staining of fibrotic lungs and corresponding schematic images of fibrotic region (right panels). g, Indicated cell types in bone marrow were sorted, and adoptively transferred into the wild-type mice with bleomycin administration. Quantity of hydroxyproline was shown. Similar results were obtained in three independent experiments (ag). *P < 0.05, **P < 0.01.

Extended Data Figure 3 Cebpd is dispensable for fibrosis and SatM differentiation.

a, The survival rate of chimaeras (at least n = 5) inoculated with bleomycin (3 μg per g body weight). Similar results were obtained in three independent experiments. b, The proportions of SatM in bone marrow (upper left panel), spleen (bottom left panel), blood (upper right panel) and lung (bottom right panel) were shown. Similar results were obtained in five independent experiments.

Extended Data Figure 4 Modified high-fat diet liver fibrosis was repressed in Cebpb−/− chimaeras.

a, Liver was collected from wild-type and Cebpb−/− chimaeric mice fed on CDA-HFD, and indicated mRNA were determined by qPCR. b, Images of Azan staining of liver tissue. Scale bar, 50 μm. c, Quantity of hydroxyproline was determined. Similar results were obtained in three independent experiments (ag). *P < 0.05, **P < 0.01.

Extended Data Figure 5 Wound healing progressed normally in Cebpb−/− chimaeras.

a, b, Chimaeric mice with wild-type and Cebpb−/− cells (at least n = 5) were injured by biopsy (diameter (Φ) = 6 mm). The repair rate of the wound area was monitored. c, H&E analysis of skin was performed. Similar results were obtained in three independent experiments.

Extended Data Figure 6 FACS analysis of various immune cells in Cebpb−/− chimaeras.

a, FACS analysis of spleen. The proportions of neutrophils (upper left), eosinophils (middle left), natural killer cells (bottom left), B cells, T cells (upper right), plasmacytoid dendritic cells (pDC) and conventional dendritic cells (cDC) were shown. b, FACS analysis of SatM. The proportions of these cells in blood (left panel), spleen (centre panel) and lung (right panel) were shown. Similar results were obtained in five independent experiments (a, b).

Extended Data Figure 7 Initiation factor of fibrosis produced from activated SatM.

a, Sorted SatM were cultured with Toll-like receptor ligands for 48 h, and concentrations of indicated cytokines were determined by ELISA. b, Fibroblasts were cultured with TNF-α for indicated time, and expression level of Spp1 mRNA was determined by qPCR. Similar results were obtained in three independent experiments (a, b). *P < 0.05, **P < 0.01.

Extended Data Figure 8 Characterization of SatM.

a, The FSC–SSC profiles of whole-cell and SatM in bone marrow (left and centre panels) and merged image (right panel) were shown. Similar results were obtained in three independent experiments. b, Expression of phosphorylated (p-) and unphosphorylated Erk and Akt and actin in SatM stimulated with M-CSF (50 ng ml−1). c, Flow cytometric analysis of spleen. The proportions of SatM were shown by F4/80, Mac1+, Ly6C, Msr1+ and Ceacam1+. Similar results were obtained in five independent experiments (b, c). d, The expression level of indicated cell surface molecules of SatM from bone marrow were shown. Similar results were obtained in three independent experiments. e, Cell lysates were prepared from SatM in wild-type mice, and were subjected to whole-cell proteomics analysis. Substantially expressed granule proteins were described. f, g, Sorted Siglec-F+CCR3+CD4 population, Ly6G+Mac1+ population and DX5+FcεRI+c-kitCD3CD19 population were stained with Diff-Quick after cytospin centrifugation and were photographed (f) and investigated by TEM (g). Scale bars were shown in indicated images. Similar results were obtained in three independent experiments (f, g). h, Flow cytometric analysis of spleen obtained from wild-type and ΔdblGATA mutant mice. The proportions of eosinophils (upper panel) and SatM (bottom panel) were shown. Similar results were obtained in five independent experiments.

Extended Data Figure 9 Characterization of various progenitors.

a, The proportion of GMPs (left panel) and MDPs (right panel) were shown. Similar results were obtained in three independent experiments. b, Sorted Ly6CFcεRI+ GMP population were stained with Diff-Quick after cytospin centrifugation and were photographed. Similar results were obtained in five independent experiments. c, Principal component analysis by using gene expression of indicated cells was performed. d, SatM, inflammatory monocytes, SMPs, cMoPs, MDPs and GMPs were sorted and subjected to microarray analysis. Data on transcription factor genes were partitioned by k-means clustering using the threshold k = 8. Clustered molecules are shown. e, Sorted GFP+Linc-kitC5aR+CD115+FcεRI+Ly6C populations were transferred into wild-type mice, and then analysed by FACS. Similar results were obtained in three independent experiments.

Extended Data Figure 10 Macrophages regulated by Jmjd3 and Trib1 were dispensable for the development of fibrosis.

a, The proportion of SatM in Jmjd3−/− (left panel) and Trib1−/− (right panel) were shown. Similar results were obtained in five independent experiments. b, c, Chimaeric mice with wild-type, Jmjd3−/− or Trib1−/− haematopoietic cells (at least n = 5) were intratracheally inoculated with bleomycin (3 μg per g body weight). The survival rate of mice was monitored (b). Images of Azan staining for collagen fibres in the lung tissue of wild-type, Jmjd3−/− and Trib1−/− bone marrow chimaeric mice (c). Similar results were obtained in three independent experiments.

Supplementary information

Spatio−temporal analysis of lung by MRI

The animation shows lung of WT chimeric in normal condition.The images were taken by MRI at day 0. (AVI 524 kb)

Spatio−temporal analysis of BLM induced inflammation by MRI

The animation shows inflamed area (red) in lung of WT chimeric. The images were taken by MRI after 4 days BLM administration. (AVI 607 kb)

Spatio−temporal analysis of BLM induced inflammation by MRI

The animation shows inflamed area (red) in lung of C/ebpb–/– chimeric mice. The images were taken by MRI after 4 days BLM administration. (AVI 628 kb)

Spatio−temporal analysis of BLM induced fibrosis by MRI

The animation shows fibrotic area (blue) in lung of WT chimeric mice. The images were taken by MRI after 14 days BLM administration. (AVI 605 kb)

Spatio−temporal analysis of BLM induced fibrosis by MRI

The animation shows fibrotic area (blue) in lung of C/ebpb–/– chimeric mice. The images were taken by MRI after 14 days BLM administration. (AVI 556 kb)

Three−dimensional tomographic imaging of SatM in BM by STEM

SatM was sorted from BM, and the Image was taken by STEM. (AVI 1028 kb)

Three−dimensional tomographic imaging of SMP in BM by STEM

SMP was sorted from BM, and the Image was taken by STEM. (AVI 8061 kb)

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Satoh, T., Nakagawa, K., Sugihara, F. et al. Identification of an atypical monocyte and committed progenitor involved in fibrosis. Nature 541, 96–101 (2017). https://doi.org/10.1038/nature20611

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