Novel mechanisms of Collagenase Santyl Ointment (CSO) in wound macrophage polarization and resolution of wound inflammation

Abstract

Collagenases are useful in enzymatic wound debridement. Clostridial collagenase, marketed as Collagenase Santyl Ointment (CSO), is FDA approved for such use. Building on the scientific premise that collagenases as well as collagen degradation products may regulate immune cell function, we sought to investigate the potential role of CSO in wound inflammation. We tested the hypothesis that in addition to enacting debridement, CSO contributes to the resolution of persistent wound inflammation. Wound macrophages were isolated from PVA sponges loaded with CSO or petrolatum and implanted in mice. Significant increase in pro-reparative and decrease in pro-inflammatory polarization was noted in macrophages of acute as well as diabetic wounds. Wound macrophages from CSO-treated group displayed increased production of anti-inflammatory cytokines IL-10 and TGF-β, and decreased levels of pro-inflammatory cytokines TNF-α and IL-1β. The active ingredient of CSO, CS-API, induced the expression of mϕheal /M(IL-4) polarization markers ex vivo. CS-API treatment attenuated transactivation of NF-κB and significantly induced STAT6 phosphorylation. A significant role of a novel PGE2-EP4 pathway in CS-API induced STAT6 activation and the mϕheal /M(IL-4) polarization was identified. Taken together, findings of this work reposition CSO as a potential agent that may be effective in resolving wound inflammation, including diabetic wounds.

A Modified Collagen Dressing Induces Transition of Inflammatory to Reparative Phenotype of Wound Macrophages

“…Collagen and its degradation products have been recognized to induce signal transduction which in turn modulates several physiological functions like cell adhesion and migration, hemostasis and immune function 28,29 . Interestingly, a clostridial collagenase ointment resolves inflammation through a PGE 2 -EP 4 -STAT6 signaling pathway 30 . Collagen degraded to its peptide components is readily phagocytosed by macrophages at the wound-site 31,32 .…”

“…MCG promoted an anti-inflammatory proangiogenic mϕ heal -like macrophage phenotype via miR-21-cJun/JNK mediated signaling pathway. Results of this study were obtained from the study of macrophages that were activated in vivo employing a standardized PVA sponge implantation model 19,30,53,57 . The reported observations provide a valuable paradigm that is ready to be tested on macrophages directly isolated from the chronic wound patients.…”

“…The animal studies were approved by, and all methods were performed in accordance with the relevant guidelines and regulations set by The Ohio State University’s Institutional Animal Care and Use Committee and Indiana University’s Institutional Animal Care and Use Committee. Circular sterile PVA sponges (8 mm diameter) were subcutaneously implanted on the back of 8-12-week old C57BL/6 mice under anesthesia induced by isoflurane inhalation as previously described 19,30,53,57 . PVA sponges were soaked in MCG stock solution (2.5 g/ml) for overnight (16 h).…”

“…Markers used to determine monocyte and/or macrophage subsets comprised: FITC-F4/80 (Serotec) and PE-CD16/32, PE-CD11c, PE-CD40, PE-CD284, PE-CD206, PE-CD23(eBioscience). The cells were stained with surface markers and recorded using BD Accuri flow cytometry (BD Biosciences) and analyzed as previously described 17,30,58,59 .…”

“…For the assay, wound cells or wound mϕ (isolated from subcutaneously implanted PVA sponges) or THP-1 cells were seeded in 24-well plates cells (equally seeded for control and treatment). Cytokine levels were measured from commercially available ELISA kits as previously described 12,18,19,30,53,57 and are expressed as per million cells.…”