Macrophage-derived oncostatin M contributes to human and mouse neurogenic heterotopic ossifications
Frédéric Torossian, Bernadette Guerton, Adrienne Anginot, Kylie A. Alexander, Christophe Desterke, Sabrina Soave, Hsu-Wen Tseng, Nassim Arouche, Laetitia Boutin, Irina Kulina, Marjorie Salga, Beulah Jose, Allison R. Pettit, Denis Clay, Nathalie Rochet, Erica Vlachos, Guillaume Genet, Charlotte Debaud, Philippe Denormandie, François Genet, Natalie A. Sims, Sébastien Banzet, Jean-Pierre Levesque, Jean-Jacques Lataillade, Marie-Caroline Le Bousse-Kerdilès
Frédéric Torossian, Bernadette Guerton, Adrienne Anginot, Kylie A. Alexander, Christophe Desterke, Sabrina Soave, Hsu-Wen Tseng, Nassim Arouche, Laetitia Boutin, Irina Kulina, Marjorie Salga, Beulah Jose, Allison R. Pettit, Denis Clay, Nathalie Rochet, Erica Vlachos, Guillaume Genet, Charlotte Debaud, Philippe Denormandie, François Genet, Natalie A. Sims, Sébastien Banzet, Jean-Pierre Levesque, Jean-Jacques Lataillade, Marie-Caroline Le Bousse-Kerdilès
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Research Article Bone biology Hematology

Macrophage-derived oncostatin M contributes to human and mouse neurogenic heterotopic ossifications

Abstract

Neurogenic heterotopic ossification (NHO) is the formation of ectopic bone generally in muscles surrounding joints following spinal cord or brain injury. We investigated the mechanisms of NHO formation in 64 patients and a mouse model of spinal cord injury–induced NHO. We show that marrow from human NHOs contains hematopoietic stem cell (HSC) niches, in which mesenchymal stromal cells (MSCs) and endothelial cells provide an environment supporting HSC maintenance, proliferation, and differentiation. The transcriptomic signature of MSCs from NHOs shows a neuronal imprinting associated with a molecular network required for HSC support. We demonstrate that oncostatin M (OSM) produced by activated macrophages promotes osteoblastic differentiation and mineralization of human muscle-derived stromal cells surrounding NHOs. The key role of OSM was confirmed using an experimental model of NHO in mice defective for the OSM receptor (OSMR). Our results provide strong evidence that macrophages contribute to NHO formation through the osteogenic action of OSM on muscle cells within an inflammatory context and suggest that OSM/OSMR could be a suitable therapeutic target. Altogether, the evidence of HSCs in ectopic bones growing at the expense of soft tissue in spinal cord/brain-injured patients indicates that inflammation and muscle contribute to HSC regulation by the brain-bone-blood triad.

Authors

Frédéric Torossian, Bernadette Guerton, Adrienne Anginot, Kylie A. Alexander, Christophe Desterke, Sabrina Soave, Hsu-Wen Tseng, Nassim Arouche, Laetitia Boutin, Irina Kulina, Marjorie Salga, Beulah Jose, Allison R. Pettit, Denis Clay, Nathalie Rochet, Erica Vlachos, Guillaume Genet, Charlotte Debaud, Philippe Denormandie, François Genet, Natalie A. Sims, Sébastien Banzet, Jean-Pierre Levesque, Jean-Jacques Lataillade, Marie-Caroline Le Bousse-Kerdilès

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Figure 1

Human NHOs contain functional hematopoietic stem cells.

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Human NHOs contain functional hematopoietic stem cells. (A) Fragment of ...
(A) Fragment of human neurogenic heterotopic ossification (NHO) with hematopoietic activity resected from a patient with stroke. Osteoblasts (ob), osteocytes (oc), hematopoietic cells (he), adipocytes (ad), and chondrocytes (ch) on sections of human NHO biopsies stained with H&E. Scale bar: 100 μm. (B) Flow cytometry characterization on human mononuclear leukocytes from one hematopoietic NHO representative of the 5–14 analyzed. (C) Percentage of CD34+ cells within the CD45+ mononuclear leukocyte fraction from peripheral blood (PB) and bone marrow (BM) from healthy donors and from NHOs. Each dot represents a different donor/patient. Bars represent mean ± SEM (n = 3–14). Kruskal-Wallis test followed by Dunn’s post-hoc tests were used for the statistical analysis (*P ≤ 0.05, **P ≤ 0.01). (D) CD34+ cells from PB, BM, and NHOs were isolated using immunomagnetic cell separation and plated in colony assay with human cytokines. Images show representative colonies from erythroid (BFU-E), granulocyte/erythroid/macrophage/megakaryocyte (CFU-GEMM), granulocytic (CFU-G), and macrophage (CFU-M) progenitors. Data show relative frequencies of granulocyte/macrophage (CFU-GM), BFU-E, and CFU-GEMM progenitors after 14 days of culture (n = 3–4 patients/donors per tissue). Results are expressed as mean ± SEM. Two-way ANOVA followed by Tukey’s post-hoc tests were used (***P ≤ 0.001). (E) NHOs contain side population (SP) CD34+ cells. Lineage-negative cells from NHOs were isolated using immunomagnetic cell separation. SP cells (blue circles) were analyzed by flow cytometer with or without 50 μM verapamil for expression CD34 and CD38 markers (n = 3). (F) CD34+ cells from human NHOs reconstitute human hematopoiesis in immunodeficient mice. CD34+ cells from human NHOs were transplanted intravenously into NSG mice. After 2 months, BM from NSG mice was analyzed by flow cytometry for expression human CD45, CD34, B lymphoid CD19, and myeloid CD11b and CD15 markers (n = 6).