Collagenase Resolves Wound Inflammation Through A Pge2-ep4-stat6 Mediated Pro-healing Macrophage Polarization
AMITAVA DAS1, Soma Datta1, Eric Roche2, Scott Chaffee1, Lei Shi2, Komel Grover2, Savita Khanna1, Chandan K. Sen1, Sashwati Roy1.
1Department of Surgery, Center for Regenerative Medicine and Cell Based Therapies and Comprehensive Wound Center, The Ohio State University Wexner Medical Center, Columbus, OH, USA, 2Research & Development, Smith & Nephew, Inc., Fort Worth, TX, USA.
Background- Debridement is a necessary component of bed preparation in wound care. Clostridial collagenase, marketed as Collagenase Santyl Ointment (CSO), is FDA approved for such use. Building on the scientific premise that collagenases and collagen degradation products may regulate immune cell function, we sought to investigate the potential role of CSO in regulating wound inflammation. We tested the hypothesis that in addition to enacting debridement, CSO contributes to the resolution of persistent wound inflammation. Methods- Wound macrophages were isolated from PVA sponges previously loaded with CSO or petrolatum (control) and implanted subcutaneously on the back of male mice. Results- CSO treatment significantly increased pro-healing (mϕheal) and decreased pro-inflammatory (mϕinf) polarization in acute as well as diabetic wounds macrophages (p˂0.05; n=5). CSO-treated wound macrophages functionally displayed increased production of anti-inflammatory cytokines IL-10 and TGF-β, but decreased pro-inflammatory cytokines TNF-α and IL-1β (p˂0.05; n=5). The active ingredient of CSO, CS-API, induced the expression of mϕheal and downregulated mϕinf polarization markers ex vivo (p˂0.05; n=4). Murine RAW 264.7 macrophages were used to identify the transcriptional regulation of CS-API mediated mϕheal polarization. CS-API treatment attenuated transactivation of pro-inflammatory transcription factor NF-κB, and induced the activity of STAT6 (p<0.05; n=5), a transcription factor involved in macrophage mϕheal polarization. Inhibition of STAT6 significantly abrogated the anti-inflammatory effects of CS-API (p<0.05; n=4). PGE2 drives mϕheal macrophage polarization through EP4 receptor. CS-API treatment significantly increased PGE2 in macrophages (p<0.05; n=3). Blocking the EP4 receptor significantly attenuated the CS-API-induced phosphorylation of STAT6 and decreased the shift towards mϕheal phenotype (p<0.05; n=3). Conclusion- This work presents first evidence demonstrating that CSO, otherwise known as a debridement agent in wound clinics, is powerful in resolving wound inflammation via PGE2-EP4-STAT6 pathway.
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