Inhibition of Scleraxis Signaling Provides a Target to Reduce Mesenchymal Cell Inflammation During Wound Healing
Arminder Kaura, BS, Michael Chung, MD, David Cholok, BS, Shawn Loder, BS, Christopher Breuler, BS, John Butts, BS, Joseph Habbouche, BS, John Li, MD, Shuli Li, PhD, Shailesh Agarwal, MD, Benjamin Levi, MD.
University of Michigan, Ann Arbor, MI, USA.
PURPOSE: Inflammation following trauma is a critical step during both normal and pathologic wound healing. Recently, we have identified Scleraxis, a transcription factor with conserved topoisomerase homology, as a mediator of pathologic wound healing. Here we demonstrate that ciprofloxacin, a known topoisomerase inhibitor, reduces exhibits anti-inflammatory properties during injury through a reduction in Scleraxis signaling.
METHODS: Mesenchymal cells were isolated from mice with genetic loss of Scleraxis (Prx-cre/Scxfl/fl) and wild type mice. Separately, wild type cells were treated with ciprofloxacin (10 mg/kg) or vehicle control. In vitro assays were performed to quantify proliferation and Scleraxis signaling. Furthermore, mutant or wild type mice underwent hindlimb tendon transection and subsequent flow cytometry and histologic analysis during the first week after injury. This was similarly performed for mice treated with ciprofloxacin or vehicle control.
RESULTS: Flow cytometry demonstrated that genetic loss of scleraxis among mesenchymal cells significantly reduced the presence of macrophages (F4/80+) and neutrophils (Cd11b+Ly6G+) at the injury site within 48 hours after injury. The presence of PDGFRa+ mesenchymal cells was also significantly reduced based on both flow cytometry and histologic analysis. These findings were confirmed with ciprofloxacin treatment. Furthermore, genetic loss of Scleraxis and ciprofloxacin both corresponded with a significant reduction in mesenchymal cell proliferation (Fig. 1A). Ciprofloxacin treatment led to reduced chondrogenic differentiation and aggrecan expression (Fig. 1A). Genetic loss of Scleraxis reduced ectopic cartilage formation when compared with wild type controls (Fig. 1B).
CONCLUSION: These findings indicate that Scleraxis is a potent target to prevent mesenchymal cell proliferation and inflammation. Ciprofloxacin, an FDA-approved drug, has therapeutic efficacy as an anti-inflammatory agent with translational potential to prevent pathologic wound healing.
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