Macrophage TGF-β signaling is critical for wound healing with heterotopic ossification after trauma
Nicole K. Patel, Johanna H. Nunez, Michael Sorkin, Simone Marini, Chase A. Pagani, Amy L. Strong, Charles D. Hwang, Shuli Li, Karthik R. Padmanabhan, Ravi Kumar, Alec C. Bancroft, Joey A. Greenstein, Reagan Nelson, Husain A. Rasheed, Nicholas Livingston, Kaetlin Vasquez, Amanda K. Huber, Benjamin Levi
Nicole K. Patel, Johanna H. Nunez, Michael Sorkin, Simone Marini, Chase A. Pagani, Amy L. Strong, Charles D. Hwang, Shuli Li, Karthik R. Padmanabhan, Ravi Kumar, Alec C. Bancroft, Joey A. Greenstein, Reagan Nelson, Husain A. Rasheed, Nicholas Livingston, Kaetlin Vasquez, Amanda K. Huber, Benjamin Levi
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Research Article Bone biology Immunology

Macrophage TGF-β signaling is critical for wound healing with heterotopic ossification after trauma

Abstract

Transforming growth factor–β1 (TGF-β1) plays a central role in normal and aberrant wound healing, but the precise mechanism in the local environment remains elusive. Here, using a mouse model of aberrant wound healing resulting in heterotopic ossification (HO) after traumatic injury, we find autocrine TGF-β1 signaling in macrophages, and not mesenchymal stem/progenitor cells, is critical in HO formation. In-depth single-cell transcriptomic and epigenomic analyses in combination with immunostaining of cells from the injury site demonstrated increased TGF-β1 signaling in early infiltrating macrophages, with open chromatin regions in TGF-β1–stimulated genes at binding sites specific for transcription factors of activated TGF-β1 (SMAD2/3). Genetic deletion of TGF-β1 receptor type 1 (Tgfbr1; Alk5), in macrophages, resulted in increased HO, with a trend toward decreased tendinous HO. To bypass the effect seen by altering the receptor, we administered a systemic treatment with TGF-β1/3 ligand trap TGF-βRII-Fc, which resulted in decreased HO formation and a delay in macrophage infiltration to the injury site. Overall, our data support the role of the TGF-β1/ALK5 signaling pathway in HO.

Authors

Nicole K. Patel, Johanna H. Nunez, Michael Sorkin, Simone Marini, Chase A. Pagani, Amy L. Strong, Charles D. Hwang, Shuli Li, Karthik R. Padmanabhan, Ravi Kumar, Alec C. Bancroft, Joey A. Greenstein, Reagan Nelson, Husain A. Rasheed, Nicholas Livingston, Kaetlin Vasquez, Amanda K. Huber, Benjamin Levi

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Figure 8

Ligand trap does not change in MSC canonical signaling or proliferation but trends toward decreased Macs.

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Ligand trap does not change in MSC canonical signaling or proliferation ...
(A) IF tile scans of the distal hind limb where the tendons are outlined in white. The yellow box indicates the zoomed-in image location to the right of the tile scan where the 2 groups are identified above the images and the color legend is below. (B) Canonical TGF-β signaling by p-SMAD3 nuclear percentage in PDGFRα+ (MPCs) cells (n = 4/group, 3 images/n). (C–E) Further quantification from A, of the nuclei at injury site, total MPCs, and percentage of MPCs from total. (F) IF images and (G) quantification of proliferation by anti–Ki-67 in MPCs (n = 4/group, 3 images/n). (H) IF images and (I) quantification of percentage of Macs by anti-F4/80 (n = 4/group, 2–3 images/n). Error bars presented in graphs represent mean ± SEM for parametric data and represent median ± interquartile range for nonparametric data. Scale bars represent 100 μm.