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Identification and Therapeutic Targeting of a Central DNA-Based Mechanism Through Which Movement Augments Inflammation
Shailesh Agarwal, MD, Shawn Loder, BS, David Cholok, BS, Michael Chung, MD, Arminder Kaura, BS, John Li, MD, Kavitha Ranganathan, MD, Christopher Breuler, BS, Joseph Habbouche, BS, John Butts, BS, Hsiao Hsieh Hsung, DDS, Shuli Li, PhD, Yuji Mishina, PhD, Benjamin Levi, MD.
University of Michigan, Ann Arbor, MI, USA.

PURPOSE: Musculoskeletal trauma and surgery poses a unique challenge to surgeons: early mobilization facilitates rehabilitation but also promotes local inflammation and pathologic wound healing. However the mechanism by which extremity movement promotes local inflammation remains poorly characterized. Recently, discrete structural webs composed of DNA and histones, known as neutrophil extracellular traps (NETs), have been identified as key components of the inflammatory cascade elicited by neutrophils. Here we demonstrate that physically or chemically disrupted NETs are responsible for augmenting local inflammation by directly inducing NET formation (NETosis) yielding a therapeutic target to prevent inflammation caused by early movement.
METHODS: Mice received dorsal hindlimb tendon transection +/- cast immobilization with subsequent treatments to destabilize NETs (+/- DNase I), inhibit NETosis (+/- Cl-Amidine), and/or inhibit NET-induced NETosis (+/- ODN-2088). MicroCT imaging was performed to evaluate In vitro experiments were performed to confirm that NETs produced by neutrophils in response to the inducing agent PMA are able to induce neutrophils to form NETs (NET-induced NETosis). Furthermore, in vitro experiments were performed to confirm that Cl-Amidine but not ODN-2088 inhibits PMA-induced NETosis.
RESULTS: In vitro experiments confirmed that NETs are capable of inducing secondary NET formation (NET-induced NETosis; Fig. 1A). Both Cl-Amidine and the toll-like receptor 7/8/9 inhibitor ODN-2088 reduced the number of NETs formed through NET-induced NETosis (Cl-Amidine: 46.6 v. 9.1, p<0.05; ODN-2088: 46.6 v. 11.0, p<0.05) (Fig. 1A). Cast-immobilization of mice after tendon transection eliminated ectopic cartilage and heterotopic ossification (Fig. 1B). Flow cytometry showed that cast-immobilization significantly reduced the normalized presence of neutrophils (1.0 v. 0.08, p<0.05) and macrophages (1.0 v. 0.13, p<005) 48 hours after injury (Fig. 1C). However, treatment of cast-immobilized mice with DNase to destabilize NETs led to a rebound increase in inflammation (neutrophils: 1.0 v. 6.39, p<0.05; macrophages: 1.0 v. 3.0, p<0.05) and caused ectopic cartilage and heterotopic ossification (Fig. 1D). Furthermore, Cl-Amidine and ODN-2088 both reduced early neutrophil presence in mice with the mobile hindlimb (Cl-Amidine: 1.0 v. 0.4, p<0.05; ODN-2088: 1.0 v. 0.27, p<0.05) and in DNase-treated mice with cast-immobilization (Cl-Amidine: 1.0 v. 0.27, p<0.05; ODN-2088: 1.0 v. 0.28, p<0.05).
CONCLUSION: These results elucidate a central mechanism by which movement induces inflammation - NETs produced during the early inflammation are disrupted and further induce NETosis. These experiments identify a class of toll-like receptors such as ODN-2088, which are capable of reducing inflammation caused by movement by targeting NET-induced NETosis. These findings have immense value in preventing wound healing pathology associated with unchecked inflammation during movement, while allowing the initial inflammatory response to injury to proceed unimpaired.


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