Platelet-instructed SPP1+ macrophages drive myofibroblast activation in fibrosis in a CXCL4-dependent manner

Konrad Hoeft; Gideon J.L. Schaefer; Hyojin Kim; David Schumacher; Tore Bleckwehl; Qingqing Long; Barbara Mara Klinkhammer; Fabian Peisker; Lars Koch; James Nagai; Maurice Halder; Susanne Ziegler; Elisa Liehn; Christoph Kuppe; Jennifer Kranz; Sylvia Menzel; Ivan Costa; Adam Wahida; Peter Boor; Rebekka K. Schneider; Sikander Hayat; Rafael Kramann

doi:10.1016/j.celrep.2023.112131

Platelet-instructed SPP1⁺ macrophages drive myofibroblast activation in fibrosis in a CXCL4-dependent manner

Konrad Hoeft, Gideon J.L. Schaefer, Hyojin Kim, David Schumacher, Tore Bleckwehl, Qingqing Long, Barbara Mara Klinkhammer, Fabian Peisker, Lars Koch, James Nagai, Maurice Halder, Susanne Ziegler, Elisa Liehn, Christoph Kuppe, Jennifer Kranz, Sylvia Menzel, Ivan Costa, Adam Wahida, Peter Boor, Rebekka K. SchneiderSikander Hayat, Rafael Kramann^*

^*Corresponding author for this work

Research output: Contribution to journal › Article › Academic › peer-review

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Abstract

Fibrosis represents the common end stage of chronic organ injury independent of the initial insult, destroying tissue architecture and driving organ failure. Here we discover a population of profibrotic macrophages marked by expression of Spp1, Fn1, and Arg1 (termed Spp1 macrophages), which expands after organ injury. Using an unbiased approach, we identify the chemokine (C-X-C motif) ligand 4 (CXCL4) to be among the top upregulated genes during profibrotic Spp1 macrophage differentiation. In vitro and in vivo studies show that loss of Cxcl4 abrogates profibrotic Spp1 macrophage differentiation and ameliorates fibrosis after both heart and kidney injury. Moreover, we find that platelets, the most abundant source of CXCL4 in vivo, drive profibrotic Spp1 macrophage differentiation. Single nuclear RNA sequencing with ligand-receptor interaction analysis reveals that macrophages orchestrate fibroblast activation via Spp1, Fn1, and Sema3 crosstalk. Finally, we confirm that Spp1 macrophages expand in both human chronic kidney disease and heart failure.

Original language	English
Article number	112131
Journal	Cell Reports
Volume	42
Issue number	2
DOIs	https://doi.org/10.1016/j.celrep.2023.112131
Publication status	Published - 28 Feb 2023

Bibliographical note

Funding Information:
This work was supported by RWTH Aachen University Clinician Scientist grants to K.H. and D.S. as well as an RWTH Aachen University START grant (139/21) to K.H. G.S. received funding from the German Society of Internal Medicine (DGIM, Peter-Scriba MD grant) and German Society for Cardiology (DGK, Otto-Hess MD grant). A.W. was funded by the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (grant agreement No. GA 884754). This work was mainly funded by grants of the German Research Foundation (DFG: SFBTRR219 322900939, CRU344-4288578857858, CRU5011-445703531), by grants of the European Research Council (ERC-StG 677448 and ERC-CoG 101043403), the ERA-CVD MENDAGE consortium (BMBF 01KL1907), and the NWO VIDI 09150172010072 (all to R.K.). R.K. and R.K.S. received support of the BMBF Consortia FibroMap and CureFib. R.K.S. further received funding from ERC Starting grant deFIBER; ERC-StG 757339. P.B. is supported by the German Research Foundation (DFG, Project IDs: 322900939, 454024652, 432698239, and 445703531, the latter also to B.M.K.), the European Research Council (ERC, Consolidator Grant No 101001791). Individual images for the design of experimental timelines were taken from the Smart servier medical art server (https://smart.servier.com). The graphical abstract was created using biorender. K.H. G.S. and R.K. designed the study and interpreted the data. K.H. H.K. and S.H. designed the data analysis plan. K.H. G.S. and R.K. wrote the manuscript and organized the figures. R.K.S. A.W. I.C. and S.H. provided conceptual support, advised on data interpretation, and edited the manuscript. D.S. G.S. K.H. and E.L. performed myocardial infarction surgeries and mice echocardiography. G.S. performed echocardiographic data analysis. K.H. and G.S. performed all other mouse experiments with assistance from S.M. S.Z. established the Gli1+ fibroblast cell line. G.S. and K.H. performed all other cell culture experiments with assistance from Q.L. and L.K. G.S. B.M.K. and P.B. performed staining, imaging, and quantification of picrosirius red stains. G.S. performed ISH and IF stainings and data analysis with assistance from Q.L. L.K. and M.H. K.H. G.S. and F.P. performed single nuclei isolation and single nuclear RNA library preparation. K.H. H.K. S.H. and G.S. carried out single-cell and high-throughput data analysis. T.B. performed Symphony Reference Mapping Analysis with assistance from J.N. C.K. and J.K. collected patient nephrectomy tissues. C.K. performed TMA embedding. K.H. and R.K. initiated the study. All authors read and approved the final manuscript. The authors declare no competing interests.

Funding Information:
This work was supported by RWTH Aachen University Clinician Scientist grants to K.H. and D.S., as well as an RWTH Aachen University START grant ( 139/21 ) to K.H. G.S. received funding from the German Society of Internal Medicine (DGIM, Peter-Scriba MD grant) and German Society for Cardiology (DGK, Otto-Hess MD grant). A.W. was funded by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant agreement No. GA 884754 ). This work was mainly funded by grants of the German Research Foundation (DFG: SFBTRR219 322900939 , CRU344-4288578857858, CRU5011-445703531 ), by grants of the European Research Council ( ERC-StG 677448 and ERC-CoG 101043403 ), the ERA-CVD MENDAGE consortium ( BMBF 01KL1907 ), and the NWO VIDI 09150172010072 (all to R.K.). R.K. and R.K.S. received support of the BMBF Consortia FibroMap and CureFib. R.K.S. further received funding from ERC Starting grant deFIBER; ERC-StG 757339 . P.B. is supported by the German Research Foundation (DFG, Project IDs: 322900939 , 454024652 , 432698239 , and 445703531 , the latter also to B.M.K.), the European Research Council (ERC, Consolidator Grant No 101001791 ). Individual images for the design of experimental timelines were taken from the Smart servier medical art server ( https://smart.servier.com ). The graphical abstract was created using biorender.

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© 2023 The Author(s)

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Platelet-instructed SPP1+ macrophages drive myofibroblast activation in fibrosis in a CXCL4-dependent mannerFinal published version, 13.4 MBLicence: CC BY

Cite this

Hoeft, K., Schaefer, G. J. L., Kim, H., Schumacher, D., Bleckwehl, T., Long, Q., Klinkhammer, B. M., Peisker, F., Koch, L., Nagai, J., Halder, M., Ziegler, S., Liehn, E., Kuppe, C., Kranz, J., Menzel, S., Costa, I., Wahida, A., Boor, P., ... Kramann, R. (2023). Platelet-instructed SPP1⁺ macrophages drive myofibroblast activation in fibrosis in a CXCL4-dependent manner. Cell Reports, 42(2), Article 112131. https://doi.org/10.1016/j.celrep.2023.112131

@article{4c4204406ea24d20b749d3b4b1c143ac,

title = "Platelet-instructed SPP1+ macrophages drive myofibroblast activation in fibrosis in a CXCL4-dependent manner",

abstract = "Fibrosis represents the common end stage of chronic organ injury independent of the initial insult, destroying tissue architecture and driving organ failure. Here we discover a population of profibrotic macrophages marked by expression of Spp1, Fn1, and Arg1 (termed Spp1 macrophages), which expands after organ injury. Using an unbiased approach, we identify the chemokine (C-X-C motif) ligand 4 (CXCL4) to be among the top upregulated genes during profibrotic Spp1 macrophage differentiation. In vitro and in vivo studies show that loss of Cxcl4 abrogates profibrotic Spp1 macrophage differentiation and ameliorates fibrosis after both heart and kidney injury. Moreover, we find that platelets, the most abundant source of CXCL4 in vivo, drive profibrotic Spp1 macrophage differentiation. Single nuclear RNA sequencing with ligand-receptor interaction analysis reveals that macrophages orchestrate fibroblast activation via Spp1, Fn1, and Sema3 crosstalk. Finally, we confirm that Spp1 macrophages expand in both human chronic kidney disease and heart failure.",

author = "Konrad Hoeft and Schaefer, {Gideon J.L.} and Hyojin Kim and David Schumacher and Tore Bleckwehl and Qingqing Long and Klinkhammer, {Barbara Mara} and Fabian Peisker and Lars Koch and James Nagai and Maurice Halder and Susanne Ziegler and Elisa Liehn and Christoph Kuppe and Jennifer Kranz and Sylvia Menzel and Ivan Costa and Adam Wahida and Peter Boor and Schneider, {Rebekka K.} and Sikander Hayat and Rafael Kramann",

note = "Funding Information: This work was supported by RWTH Aachen University Clinician Scientist grants to K.H. and D.S. as well as an RWTH Aachen University START grant (139/21) to K.H. G.S. received funding from the German Society of Internal Medicine (DGIM, Peter-Scriba MD grant) and German Society for Cardiology (DGK, Otto-Hess MD grant). A.W. was funded by the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (grant agreement No. GA 884754). This work was mainly funded by grants of the German Research Foundation (DFG: SFBTRR219 322900939, CRU344-4288578857858, CRU5011-445703531), by grants of the European Research Council (ERC-StG 677448 and ERC-CoG 101043403), the ERA-CVD MENDAGE consortium (BMBF 01KL1907), and the NWO VIDI 09150172010072 (all to R.K.). R.K. and R.K.S. received support of the BMBF Consortia FibroMap and CureFib. R.K.S. further received funding from ERC Starting grant deFIBER; ERC-StG 757339. P.B. is supported by the German Research Foundation (DFG, Project IDs: 322900939, 454024652, 432698239, and 445703531, the latter also to B.M.K.), the European Research Council (ERC, Consolidator Grant No 101001791). Individual images for the design of experimental timelines were taken from the Smart servier medical art server (https://smart.servier.com). The graphical abstract was created using biorender. K.H. G.S. and R.K. designed the study and interpreted the data. K.H. H.K. and S.H. designed the data analysis plan. K.H. G.S. and R.K. wrote the manuscript and organized the figures. R.K.S. A.W. I.C. and S.H. provided conceptual support, advised on data interpretation, and edited the manuscript. D.S. G.S. K.H. and E.L. performed myocardial infarction surgeries and mice echocardiography. G.S. performed echocardiographic data analysis. K.H. and G.S. performed all other mouse experiments with assistance from S.M. S.Z. established the Gli1+ fibroblast cell line. G.S. and K.H. performed all other cell culture experiments with assistance from Q.L. and L.K. G.S. B.M.K. and P.B. performed staining, imaging, and quantification of picrosirius red stains. G.S. performed ISH and IF stainings and data analysis with assistance from Q.L. L.K. and M.H. K.H. G.S. and F.P. performed single nuclei isolation and single nuclear RNA library preparation. K.H. H.K. S.H. and G.S. carried out single-cell and high-throughput data analysis. T.B. performed Symphony Reference Mapping Analysis with assistance from J.N. C.K. and J.K. collected patient nephrectomy tissues. C.K. performed TMA embedding. K.H. and R.K. initiated the study. All authors read and approved the final manuscript. The authors declare no competing interests. Funding Information: This work was supported by RWTH Aachen University Clinician Scientist grants to K.H. and D.S., as well as an RWTH Aachen University START grant ( 139/21 ) to K.H. G.S. received funding from the German Society of Internal Medicine (DGIM, Peter-Scriba MD grant) and German Society for Cardiology (DGK, Otto-Hess MD grant). A.W. was funded by the European Research Council (ERC) under the European Union{\textquoteright}s Horizon 2020 research and innovation program (grant agreement No. GA 884754 ). This work was mainly funded by grants of the German Research Foundation (DFG: SFBTRR219 322900939 , CRU344-4288578857858, CRU5011-445703531 ), by grants of the European Research Council ( ERC-StG 677448 and ERC-CoG 101043403 ), the ERA-CVD MENDAGE consortium ( BMBF 01KL1907 ), and the NWO VIDI 09150172010072 (all to R.K.). R.K. and R.K.S. received support of the BMBF Consortia FibroMap and CureFib. R.K.S. further received funding from ERC Starting grant deFIBER; ERC-StG 757339 . P.B. is supported by the German Research Foundation (DFG, Project IDs: 322900939 , 454024652 , 432698239 , and 445703531 , the latter also to B.M.K.), the European Research Council (ERC, Consolidator Grant No 101001791 ). Individual images for the design of experimental timelines were taken from the Smart servier medical art server ( https://smart.servier.com ). The graphical abstract was created using biorender. Publisher Copyright: {\textcopyright} 2023 The Author(s)",

year = "2023",

month = feb,

day = "28",

doi = "10.1016/j.celrep.2023.112131",

language = "English",

volume = "42",

journal = "Cell Reports",

issn = "2211-1247",

publisher = "Cell Press",

number = "2",

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Hoeft, K, Schaefer, GJL, Kim, H, Schumacher, D, Bleckwehl, T, Long, Q, Klinkhammer, BM, Peisker, F, Koch, L, Nagai, J, Halder, M, Ziegler, S, Liehn, E, Kuppe, C, Kranz, J, Menzel, S, Costa, I, Wahida, A, Boor, P, Schneider, RK, Hayat, S & Kramann, R 2023, 'Platelet-instructed SPP1⁺ macrophages drive myofibroblast activation in fibrosis in a CXCL4-dependent manner', Cell Reports, vol. 42, no. 2, 112131. https://doi.org/10.1016/j.celrep.2023.112131

TY - JOUR

T1 - Platelet-instructed SPP1+ macrophages drive myofibroblast activation in fibrosis in a CXCL4-dependent manner

AU - Hoeft, Konrad

AU - Schaefer, Gideon J.L.

AU - Kim, Hyojin

AU - Schumacher, David

AU - Bleckwehl, Tore

AU - Long, Qingqing

AU - Klinkhammer, Barbara Mara

AU - Peisker, Fabian

AU - Koch, Lars

AU - Nagai, James

AU - Halder, Maurice

AU - Ziegler, Susanne

AU - Liehn, Elisa

AU - Kuppe, Christoph

AU - Kranz, Jennifer

AU - Menzel, Sylvia

AU - Costa, Ivan

AU - Wahida, Adam

AU - Boor, Peter

AU - Schneider, Rebekka K.

AU - Hayat, Sikander

AU - Kramann, Rafael

N1 - Funding Information: This work was supported by RWTH Aachen University Clinician Scientist grants to K.H. and D.S. as well as an RWTH Aachen University START grant (139/21) to K.H. G.S. received funding from the German Society of Internal Medicine (DGIM, Peter-Scriba MD grant) and German Society for Cardiology (DGK, Otto-Hess MD grant). A.W. was funded by the European Research Council (ERC) under the European Union's Horizon 2020 research and innovation program (grant agreement No. GA 884754). This work was mainly funded by grants of the German Research Foundation (DFG: SFBTRR219 322900939, CRU344-4288578857858, CRU5011-445703531), by grants of the European Research Council (ERC-StG 677448 and ERC-CoG 101043403), the ERA-CVD MENDAGE consortium (BMBF 01KL1907), and the NWO VIDI 09150172010072 (all to R.K.). R.K. and R.K.S. received support of the BMBF Consortia FibroMap and CureFib. R.K.S. further received funding from ERC Starting grant deFIBER; ERC-StG 757339. P.B. is supported by the German Research Foundation (DFG, Project IDs: 322900939, 454024652, 432698239, and 445703531, the latter also to B.M.K.), the European Research Council (ERC, Consolidator Grant No 101001791). Individual images for the design of experimental timelines were taken from the Smart servier medical art server (https://smart.servier.com). The graphical abstract was created using biorender. K.H. G.S. and R.K. designed the study and interpreted the data. K.H. H.K. and S.H. designed the data analysis plan. K.H. G.S. and R.K. wrote the manuscript and organized the figures. R.K.S. A.W. I.C. and S.H. provided conceptual support, advised on data interpretation, and edited the manuscript. D.S. G.S. K.H. and E.L. performed myocardial infarction surgeries and mice echocardiography. G.S. performed echocardiographic data analysis. K.H. and G.S. performed all other mouse experiments with assistance from S.M. S.Z. established the Gli1+ fibroblast cell line. G.S. and K.H. performed all other cell culture experiments with assistance from Q.L. and L.K. G.S. B.M.K. and P.B. performed staining, imaging, and quantification of picrosirius red stains. G.S. performed ISH and IF stainings and data analysis with assistance from Q.L. L.K. and M.H. K.H. G.S. and F.P. performed single nuclei isolation and single nuclear RNA library preparation. K.H. H.K. S.H. and G.S. carried out single-cell and high-throughput data analysis. T.B. performed Symphony Reference Mapping Analysis with assistance from J.N. C.K. and J.K. collected patient nephrectomy tissues. C.K. performed TMA embedding. K.H. and R.K. initiated the study. All authors read and approved the final manuscript. The authors declare no competing interests. Funding Information: This work was supported by RWTH Aachen University Clinician Scientist grants to K.H. and D.S., as well as an RWTH Aachen University START grant ( 139/21 ) to K.H. G.S. received funding from the German Society of Internal Medicine (DGIM, Peter-Scriba MD grant) and German Society for Cardiology (DGK, Otto-Hess MD grant). A.W. was funded by the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation program (grant agreement No. GA 884754 ). This work was mainly funded by grants of the German Research Foundation (DFG: SFBTRR219 322900939 , CRU344-4288578857858, CRU5011-445703531 ), by grants of the European Research Council ( ERC-StG 677448 and ERC-CoG 101043403 ), the ERA-CVD MENDAGE consortium ( BMBF 01KL1907 ), and the NWO VIDI 09150172010072 (all to R.K.). R.K. and R.K.S. received support of the BMBF Consortia FibroMap and CureFib. R.K.S. further received funding from ERC Starting grant deFIBER; ERC-StG 757339 . P.B. is supported by the German Research Foundation (DFG, Project IDs: 322900939 , 454024652 , 432698239 , and 445703531 , the latter also to B.M.K.), the European Research Council (ERC, Consolidator Grant No 101001791 ). Individual images for the design of experimental timelines were taken from the Smart servier medical art server ( https://smart.servier.com ). The graphical abstract was created using biorender. Publisher Copyright: © 2023 The Author(s)

PY - 2023/2/28

Y1 - 2023/2/28

N2 - Fibrosis represents the common end stage of chronic organ injury independent of the initial insult, destroying tissue architecture and driving organ failure. Here we discover a population of profibrotic macrophages marked by expression of Spp1, Fn1, and Arg1 (termed Spp1 macrophages), which expands after organ injury. Using an unbiased approach, we identify the chemokine (C-X-C motif) ligand 4 (CXCL4) to be among the top upregulated genes during profibrotic Spp1 macrophage differentiation. In vitro and in vivo studies show that loss of Cxcl4 abrogates profibrotic Spp1 macrophage differentiation and ameliorates fibrosis after both heart and kidney injury. Moreover, we find that platelets, the most abundant source of CXCL4 in vivo, drive profibrotic Spp1 macrophage differentiation. Single nuclear RNA sequencing with ligand-receptor interaction analysis reveals that macrophages orchestrate fibroblast activation via Spp1, Fn1, and Sema3 crosstalk. Finally, we confirm that Spp1 macrophages expand in both human chronic kidney disease and heart failure.

AB - Fibrosis represents the common end stage of chronic organ injury independent of the initial insult, destroying tissue architecture and driving organ failure. Here we discover a population of profibrotic macrophages marked by expression of Spp1, Fn1, and Arg1 (termed Spp1 macrophages), which expands after organ injury. Using an unbiased approach, we identify the chemokine (C-X-C motif) ligand 4 (CXCL4) to be among the top upregulated genes during profibrotic Spp1 macrophage differentiation. In vitro and in vivo studies show that loss of Cxcl4 abrogates profibrotic Spp1 macrophage differentiation and ameliorates fibrosis after both heart and kidney injury. Moreover, we find that platelets, the most abundant source of CXCL4 in vivo, drive profibrotic Spp1 macrophage differentiation. Single nuclear RNA sequencing with ligand-receptor interaction analysis reveals that macrophages orchestrate fibroblast activation via Spp1, Fn1, and Sema3 crosstalk. Finally, we confirm that Spp1 macrophages expand in both human chronic kidney disease and heart failure.

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DO - 10.1016/j.celrep.2023.112131

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JF - Cell Reports

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