Tuning The Immune Response To Improve Musculoskeletal Extremity Trauma Wound Healing Through Macrophage Directed Tgfb1 Modification
Michael Sorkin, MD1, Charles Hwang, BS1, Serra Ucer, PhD1, John Li, MD1, David Cholok, BS2, Michael T. Chung, MD3, Shailesh Agarwal, MD1, Shawn Loder, MD4, Chase Pagani, none5, Kaetlin Vasquez, MS1, Shuli Li, MD, PhD1, Aaron James, MD, PhD6, Yuji Mishina, PhD1, Benjamin Levi, MD1.
1University of Michigan, Ann Arbor, MI, USA, 2Boston University, Boston, MA, USA, 3Stanford University, Stanford, CA, USA, 4University of Pittsburgh, Pittsburgh, PA, USA, 5Colgate University, Hamilton, NY, USA, 6Johns Hopkins University, Baltimore, MD, USA.
PURPOSE: Musculoskeletal extremity injury is the most common trauma seen in civilians and combat casualties. Despite its high frequency, little is known about how to tune the immune response to maximize wound healing and prevent complications. Here, we assess the circulatory monocyte/macrophage recruitment to abnormal extremity wound healing as seen with heterotopic ossification (HO) and evaluate the role of macrophage derived Tgfβ1 in the pathogenesis and therapeutic targeting. We hypothesize that macrophage phenotype and cytokine secretion can be tuned to improve traumatic extremity wound healing.
METHODS: A proven musculoskeletal extremity trauma model of HO (30% total body surface area dorsal burn and Achilles tenotomy) was used. Inflammation at the injury site was followed with bioluminescent in-vivo imaging of myeloperoxidase activity and recruited inflammatory cell subpopulations were quantified using flow cytometry. Circulatory monocyte depletion was performed with intravenous injection of clodronate. Early HO was assessed histologically at 3 weeks. In-vitro cultured bone marrow derived macrophages were polarized to M1 and M2 phenotypes and expression of Tgfβ1 was analyzed on RNA and protein levels. In-vivo, TGFβ1 in macrophages and downstream signaling in HO progenitor cells was visualized with immunostaining of HO anlagen in human and mouse samples. Furthermore, a CD47 receptor activating peptide was used to attenuate macrophage Tgfβ1 activity.
RESULTS: Extremity trauma resulted in a profound inflammatory response at the injury site with initial recruitment of circulatory inflammatory Ly6Chi monocytes peaking 3 days post injury comprising over 30% of cells. These were replaced by regenerative Ly6Clow macrophages by day 14 which remained the prevalent macrophage population indicating a role in HO (Fig A). Chemical ablation of circulatory monocytes with clodronate reduced recruitment of Ly6Clow monocytes and F480+/MHCII+ macrophages (Fig B) and attenuated HO (Fig C). Expression of profibrotic Tgf-β1, a cytokine commonly secreted by regenerative macrophages, was highly expressed at the injury site and co-localized with recruited macrophages in human early HO anlagen (Fig D). These findings were confirmed in-vitro where Tgfβ1 expression was significantly increased in M2 polarized macrophages compared to M1. Interestingly, treatment with CD47 receptor activating peptide decreased macrophage Tgfβ1 expression in macrophages in vitro (Fig E) and systemic administration showed attenuation of early HO anlagen in vivo.
CONCLUSION: Our data demonstrate that recruited circulatory macrophages play an instrumental role in the inflammatory response and aberrant musculoskeletal wound healing through contribution from distinct macrophage sub-populations. Furthermore, we identify macrophage secreted TGF-β1 as a key cytokine driving HO. Systemic administration of therapeutics targeting macrophage phenotype and TGF-β1 secretion have a high potential to reduce aberrant musculoskeletal wound healing. These results uncover a novel role of macrophages in musculoskeletal wound healing and allow for new cell specific HO prevention. This macrophage targeted therapy can also be translated to additional wound healing disorders.
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