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Macrophage-specific TGF-B is a Targetable Cytokine to Prevent Heterotopic Ossification
David Cholok, BS, Shailesh Agarwal, MD, Shawn Loder, BS, Michael Chung, MD, Ramkumar Annamalai, PhD, Joseph Habbouche, BS, Caitlin Priest, BS, Beau Carson, PhD, Christopher Breuler, BS, Kavitha Ranganathan, MD, John Li, MD, John Butts, BS, Arminder Kaura, BS, Hsiao Hsung, DDS, Shuli Li, PhD, Yuji MIshina, PhD, Benjamin Levi, MD.
University of Michigan, Ann Arbor, MI, USA.

PURPOSE: Transforming growth factor beta (TGFβ) signaling is central to both normal and pathologic wound healing. However the source of TGFβ ligand during wound healing remains unknown. Heterotopic ossification (HO) is the pathologic formation of endochondral bone in soft tissue commonly occurring after severe trauma. HO is a SMAD-signaling dependent process typically occurring in response to an acute inflammatory insult. Traditionally targeted therapeutics for treatment of HO have focused on mesenchymal cell progenitors. However, macrophages which are integral orchestrators of the initial inflammatory response and are primary agents of cytokine secretion in wound regeneration, have received little attention. Here we identify a role of macrophage production of TGFβ in HO and demonstrate a role for macrophage targeted therapies in the treatment and prevention of this debilitating condition.
METHODS: Wild type and macrophage-specific TGFβ knockout mice (LysM-cre/Tgfbfl/fl) mice underwent hindlimb Achilles' tendon transection and dorsal 30% TBSA burn. Mice were euthanized at 2, 5, and 12days, and 3 and 9 weeks after injury. A concurrent cohort of wild type animals was treated with i.v. trabectedin (0.15 mg/kg), a macrophage-depleting agent, administered twice or vehicle control to assess the effect on HO. The tenotomy site of wild-type mice harvested at 2-, 5-, and 12-days were digested to a single-cell suspension, stained with antibodies against macrophage markers (Ly6G, Ly6C, F4/80, CD11b) and TGF-β for flow cytometric analysis. MicroCT images were obtained at 9 weeks after injury among wild type and TGFβ knockout mice, and among trabectedin and control-treated mice. In vitro and in vivo studies were performed to validate a novel PLGA microparticle system with preferential uptake by macrophages.
RESULTS: Flow cytometry of tissue from wild type mice demonstrated enrichment of TGFβ in F4/80+ macrophages at all time-points with macrophages present as the dominant inflammatory population by 12-days post-injury (76%; p<0.05; Fig. 1A). LysM-cre/TGFβ mice similarly demonstrated a significant reduction in tHO volume vs. wild type controls (7.49 v. 1.25 mm3, p=.007; Fig. 1B). Macrophage depletion via trabectedin led to a 12.9-fold decrease in tHO volume when compared with control-treated mice (p<0.008; Fig 1B). Histologic analysis at 3-weeks confirmed diminished cartilage presence in both mutant and trabectedin-treated mice when compared with respective controls. Finally, the PLGA microparticle demonstrated preferential uptake by macrophages both in vitro and in the wound site in vivo (Fig. 1C).
CONCLUSION: We demonstrate that TGFβ is present in macrophages at the tenotomy site and that these macrophages comprise an increasingly large portion of the inflammatory infiltrate through 12-days post-injury. Global inhibition via trabectedin therapy is effective in preventing tHO. Importantly, genetic loss of TGFβ among macrophages was sufficient to significantly reduce HO. Our findings are strengthened by the development of a novel microparticle which exhibits preferential uptake by macrophages for drug delivery.


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