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

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.

The active constituent of CSO facilitated mφ heal polarization. The active ingredients in CSO are clostridial collagenases that are produced by Clostridium histolyticum bacteria via a proprietary fermentation process 3,8,28,29 . This collagenase material contains two distinct collagenases (col), col G (114 kDa) and col H (110 kDa), and a lesser amount of a non-specific neutral protease (a metalloproteinase, 35 kDa) (data not shown). The active constituent of CSO, henceforth called as CS-API, was used to directly treat isolated wound mφ ex vivo to examine if there is a direct effect of CS-API on mφ polarization. CS-API exists in a powder form as opposed to CSO that is in ointment form suited for wound applications and not suitable for cell culture application. Based on the cell viability data (>90% viable, data not shown), 250 ng/ml dose was selected for ex vivo wound mφ studies. CS-API directly acted on the wound mφ and caused polarization to mφ heal phenotype. Substantial induction in M(IL-4) marker genes, Arginase-1, IL-10 and CD206 in wound mφ was noted as compared to control group (Fig. 5A). Such induction was concomitant with potent down-regulation of M(LPS + IFNγ), genes, e.g. NOS2, IL-12 and CD74 (Fig. 5B). The balance of Arginase-1 and NOS2 protein expression in day 3 wound mφ and BMDM were determined using ICC (Fig. 5C,D). Increased expression in Arginase-1 and attenuation in the expression of NOS2 by CS-API treatment was noted in both activated wound mφ as well as naïve BMDM (Fig. 5C,D). Next, we determined whether CS-API was effective in influencing polarization in human macrophages. Treatment of human blood monocyte derived macrophages (hMDM) with CS-API resulted in macrophage polarization toward a pro-reparative phenotype as observed with mice wound mφ and BMDMs. Thus, the anti-inflammatory effect of CSO on macrophage polarization may be generalized across these species (Fig. S2A). Interestingly, CS-API treatment of hMDM, previously polarized towards pro-reparative phenotype, did not further induce the pro-reparative markers IL-10 or Arginase (Fig. S2B). These data recognize that the active component of CSO i.e., clostridial collagenases, directly influence mφ response and function across rodents and humans at the wound-site.  . CSO modifies cytokines release from murine wound macrophages contributing towards a proresolution milieu in acute wounds. Day 7 wound mφ were harvested from PVA sponges coated with CSO (300 mg), implanted subcutaneously in C57bl/6 mice. (A) Total RNA was isolated and mRNA expression of IL-10, TGF-β, TNF-α and IL-1β was measured using RTPCR. Data are expressed as mean ± SEM (n = 5); *p < 0.05 compared to day 7 wound mφ treated with equivalent amounts of white petrolatum (control). CSO, solid bars; control, blank bars. (B-E) The isolated wound mφ treated in vivo with CSO (300 mg) were cultured in presence of LPS (1 µg/ml). After 24 h of culture the media was collected and expression of cytokines were measured using ELISA: (B) IL-10, (C) total TGF-β, (D) IL-1β and (E) TNF-α. Data are expressed as % compared to white petrolatum (control) treated group, mean ± SEM (n = 5); *p < 0.05 compared to day 7 wound mφ treated with equal amounts of white petrolatum (control) treated with LPS. (F) Bone marrow derived macrophages (BMDM) from C57bl/6 mice were treated with recombinant mouse IL-10 for 1 h followed by activation with LPS (1 mg/ml, for 24 h). The release of TNF-α protein in culture media by BMDM was determined using ELISA. Data are mean ± SEM (n = 4); *p < 0.05 compared to control; † p < 0.05, compared to LPS treated group.
SCIeNTIFIC REpORtS | (2018) 8:1696 | DOI:10.1038/s41598-018-19879-w Molecular mechanisms of CSO mediated promotion of wound mφ heal polarization. Molecular regulation of mφ polarization is complex and involves a network of signaling molecules, transcription factors, epigenetic mechanisms, as well as posttranscriptional regulators. Canonical IRF/STAT signaling pathways are activated by IFNs and TLR signaling to skew mφ function towards M(LPS + IFNγ) phenotype via STAT1, while IL-4 and IL-13 treatments skew toward the M(IL-4) polarization/activity via STAT6 30 . CSO as well as its active constituent CS-API potently increased phosphorylation of STAT6 both in vivo in diabetic wound mφ and in vitro in an established mouse mφ cell line RAW 264.7 (Fig. 6A,B). To determine if the robust increase in STAT6 phosphorylation by CS-API Figure 2. CSO modifies cytokines release from murine wound macrophages contributing towards a proresolution milieu in chronic diabetic wounds. Day 7 wound mφ were harvested from PVA sponges coated with CSO (300 mg) implanted subcutaneously in db/db mice. (A) Total RNA was isolated and mRNA expression of IL-10, TGF-β, TNF-α and IL-1β was measured using RTPCR. Data are expressed as mean ± SEM (n = 5); *p < 0.05 compared to day 7 wound mφ treated with equivalent amounts of white petrolatum (control). CSO, solid bars; control, blank bars. (B-E) The isolated wound mφ treated in vivo with CSO (300 mg) were cultured in presence of LPS (1 µg/ml). After 24 h of culture the media was collected and expressions of cytokines were measured using ELISA: (B) IL-10, (C) total TGF-β, (D) IL-1β and (E) TNF-α. Data are expressed as % compared to white petrolatum (control) treated group, mean ± SEM (n = 5); *p < 0.05 compared to day 7 wound mφ treated with equal amounts of white petrolatum (control) treated with LPS.
is the underlying cause for the induction in mφ heal mφ polarization, RAW 264.7 mφ were treated with a specific pharmacological inhibitor of STAT6, AS1517499. AS1517499, or 4-(benzylamino)-2-pyrimidine-5-carboxamide, is a potent and selective inhibitor of STAT6 31 . Pretreatment of mφ with STAT6 inhibitor abrogated the effect of  implanted subcutaneously in db/db mice on day 7 post-implantations. Total RNA was isolated and mRNA expression of Arginase-1, CD206, YM1 and VEGF was measured using RTPCR. CSO, solid bars; control, blank bars. (B) Total protein isolated from day 7 wound mφ of db/db mice, treated with CSO in vivo, was subjected to capillary electrophoresis immunoassay to measure expression of Arginase 1 protein. (C) Arginase activity of in vivo CSO treated (300 mg) day 7 wound mφ of db/db mice were measured by Arginase activity assay kit (Colorimetric). (D) mRNA expression of CD74 was measured in day 7 wound mφ of db/db mice treated with CSO (300 mg). (E) PMA-induced superoxide anion production in day 3 wound mφ of db/db mice treated in vivo with CSO (300 mg) was measured. For all figure parts, data are expressed as mean ± SEM (n = 3-6); *p < 0.05 compared to wound mφ treated with equal amounts of white petrolatum (control). (F) Immunocytochemistry (ICC) images of Arginase-1 (red) and NOS2 (red) protein expression in d7 wound mφ from db/db animals. Day 7 wound mφ were harvested from PVA sponges coated with CSO (300 mg) implanted subcutaneously. Counter staining was performed using DAPI (nuclear, blue). Scale bar, 20 μm.
SCIeNTIFIC REpORtS | (2018) 8:1696 | DOI:10.1038/s41598-018-19879-w CS-API on mφ polarization, eliminating or reducing the shift towards mφ heal phenotype for different markers ( Fig. 6C-F), pointing towards a key role of STAT6 in CS-API and CSO mediated mφ heal polarization of mφ at the wound-site. In addition to a key role of STAT6 pathway, a moderate attenuation in LPS inducible NF-κB activation was noted in CS-API treated cultured macrophages (Fig. 6G). In our efforts to further understand the underlying  . CS-API activates STAT6 while inhibiting NF-κB pathways in macrophage polarization. (A) Wound mφ were harvested from PVA sponges coated with CSO (300 mg) implanted subcutaneously in db/db mice on day 7 post-implantations. Phosphorylation of STAT6 was measured using a cell-based ELISA kit. Data are expressed as mean ± SEM (n = 5); *p < 0.05 compared to white petrolatum (control) treated group. (B) Cultured mouse mφ were treated with CS-API (250 ng/ml) for 24 hours. Phosphorylation of STAT6 was measured using a cell-based ELISA kit. Data are expressed as mean ± SEM (n = 5); *p < 0.05 compared to control. (C-F) Cultured mouse mφ were treated with STAT6 inhibitor AS 1517499 (5 nM) for 1 hour followed by CS-API (250 ng/mL) treatment for 24 hours. mRNA expression of (C) IL-10 (D) Arginase-1 (E) NOS2 and (F) IL-12 was measured. Data are expressed as mean ± SEM (n = 3-4); *p < 0.05 compared to control, † p < 0.05, compared to CS-API treated group. (G) Cultured mouse mφ were treated with CS-API (250 ng/ml) for 24 hours. DNA binding activity of LPS (1 µg/ml, 3 h) inducible NF-κB was measured using an ELISA-based (Trans-AM) method. Data are expressed as mean ± SEM (n = 5); *p < 0.05 compared to control.

Discussion
Mφ are critical mediators of innate immune response and key facilitators of mounting/resolving the wound inflammatory response 16,42 . In response to altering wound microenvironment, considerable plasticity in these (A) Cultured mouse mφ were treated with CS-API (250 ng/ml) for 24 hours. PGE 2 level in the media was measured using ELISA. Data are expressed as mean ± SEM (n = 3); *p < 0.05 compared to control. (B) Cultured mouse mφ were treated with EP4 inhibitor MF498 (50 nM) for 1 hour followed by CS-API (250 ng/ml) for 24 hours. Phosphorylation of STAT6 was measured using cell-based ELISA. Data are expressed as mean ± SEM (n = 3); *p < 0.05 compared to control, † p < 0.05, compared to CS-API treated group. (C-F) Cultured mouse mφ were treated with EP4 inhibitor MF498 (50 nM) for 1 hour followed by CS-API (250 ng/ml) for 24 hours. mRNA expression of (C) IL-10 (D) Arginase-1 (E) NOS2 and (F) IL-12 was measured. Data are expressed as mean ± SEM (n = 3); *p < 0.05 compared to control, † p < 0.05, compared to CS-API treated group.
SCIeNTIFIC REpORtS | (2018) 8:1696 | DOI:10.1038/s41598-018-19879-w cells allows them to acquire and exhibit diverse functional/activation/polarization states 20,43,44 . The factors or cues in the wound microenvironment that drive such diversity are vaguely understood at present. This study provides maiden evidence demonstrating that a commonly used enzymatic wound debridement agent CSO that contains clostridial collagenases as the active ingredient is a powerful inducer of mφ heal functional state in acute and chronic diabetic wounds. CSO treatment to wounds also promoted a pro-resolving environment that may be beneficial for chronic wounds known to have persistent inflammation as one of the major underlying factors driving the chronicity of these wounds.
The last decade has witnessed a considerable development in understanding of intrinsic mechanisms that regulate the inflammatory response. Such understanding has led to the design of therapies that promote mechanisms favoring resolution of inflammation. Remarkably, a widely used enzymatic debridement agent, CSO, promoted a pro-resolving wound environment by augmenting production of IL-10 by wound-site mφ in both acute and diabetic chronic wounds. IL-10 is one of the most prominent pro-resolution cytokines that is released by cells of monocytic lineage 45 . The pro-resolution property of IL-10 is attributed to its potent inhibitory effect on the production of a large spectrum of pro-inflammatory cytokines such as IL-1β, IL-6, and TNF-α 46,47 . Forced overproduction of IL-10 in wounds promoted regenerative adult wound healing by decreasing the inflammatory response 48 . Presence of multifold high levels of pro-inflammatory cytokines is a hallmark of chronic wounds 10,49 including diabetic ulcers 50,51 . We and others have reported that mφ from diabetic wounds produce significantly attenuated levels of IL-10 as compared to mφ from non-diabetic wounds. CSO significantly improved the production of IL-10 in diabetic wound mφ underscoring the potential to promote resolution of inflammation in chronic diabetic wounds.
Hallmarks of mφ cell biology include plasticity and dynamicity such that these cells can exist within an inflammatory environment in diverse activation and function states 16,52 . Such diverse activation forms are part of a continuum, the two terminals of this continuum are defined by the classically activated or inflammatory mφ, and reparative or alternatively activated mφ 17,[52][53][54] . The classically activated mφ produces factors that favor a pro-inflammatory milieu (mφ inf ). These factors include IL-12, TNF-α, IL-6, IL-1β, and nitric oxide (NO) in response to microbial pathogens or LPS 55 . The alternatively activated mφ are known to favor a pro-resolution and reparative (mφ heal ) milieu by producing IL-10, TGF-β, Arginase-1 (Arg1), CD206 (mannose receptor) and Clec7a (dectin-1) 55 . The ratio of nitric oxide synthase iNOS to Arg 1 is commonly used to determine the functional status of mφ 22,56,57 . The M(LPS + IFNγ) & M(IL-4) paradigm represents two contrasting pathways for utilization of an amino acid. Arginine, via increased iNOS, in M(LPS + IFNγ) mφ is metabolized to: arginine →NO + citrulline. In contrast, for M (IL-4) mφ, arginine → ornithine + urea 58 . NO, a product of iNOS, is a reactive radical, the toxicity of which is greatly enhanced upon reaction with superoxide to form peroxynitrite (ONOO − ) 59 . Ornithine, generated as a result of arginase metabolism, serves as a substrate for multiple enzymes including: (i) ornithine decarboxylase (ODC), a rate-limiting enzyme in the synthesis of polyamines, and (ii) ornithine aminotransferase (OAT), an enzyme that catalyzes the conversion to proline 27 . Although the role of Arginase in fibrosis has been questioned in regulation of fibrosis 60 , polyamines are essential for cell growth and development and have been shown to exert inhibitory effect against pro-inflammatory cytokines 27 . Proline is essential for the synthesis and maturation of collagen. A critical role of Arginase-expressing and ornithine-producing mφ heal has been reported in facilitating wound healing 58 . A potent increase in Arginase-expressing mφ heal in wounds treated with CSO indicates an alternative action of this proven debridement agent in facilitating wound healing. It is now recognized that multiple subtypes of the anti-inflammatory mφ heal exist at the repair-site 54 . In addition to mφ heal phenotype that may promote collagen synthesis, collagenolytic subtypes may exist 61,62 . The current study did not subcategorize the mφ heal at the wound-site. STAT6 (signal transducers and activators of transcription 6) a member of STAT family of transcription factors 63 , directly regulates transcription of many genes associated with M(IL-4) mφ phenotype 17,64 . This study noted that CS-API potently activated STAT6 and inhibited NF-κB as a central mechanism to promote M(IL-4) or mφ heal while attenuating mφ inf polarization. Abrogating the activation of STAT6 by pharmacological inhibitors resulted in the loss of function of CS-API in facilitating mφ heal polarization recognizing STAT6 as one of the primary pathways for CSO action on influencing mφ polarization. STAT6 activation occurs via IL-4R mediated canonical 65,66 as well as non-canonical pathways [32][33][34][35][36][37][38][39][40] . Our laboratory has earlier reported that PGE 2 in mφ acts via an EP4-cAMP pathway 41 . PGE 2 is known to drive mφ towards mφ heal polarization via a cAMP/CREB pathway [67][68][69] . Inhibition of PGE 2 receptor EP4 using specific pharmacological inhibitor (MF498), resulted in significant reduction in CS-API mediated STAT6 activation and a significant induction of mφ heal phenotype. These new data implicate a novel alternative PGE 2 -EP4 pathway for STAT6 activation and a significant induction of mφ heal phenotype following CS-API treatment.
The NF-κB family of transcription factors regulate the expression of numerous genes implicated in immunity and inflammation 70 .We have previously reported a novel miR-21-NFκB-PTEN mediated pathway in the regulation of wound macrophage polarization 46 .
The principle active ingredients in CSO are collagenases derived from a specific propriety strain of Clostridium histolyticum 3,8 . First reported in 1953, clostridial collagenases are encoded by col G and col H genes for class I and class II type of collagenases, respectively 71 . Unlike mammalian matrix metalloproteinases (MMPs) that cleave native collagen into specific three-quarter and one-quarter fragments, clostridial collagenases breakdown repeating Gly-X-Y collagen sequences at distinct Y-Gly bond containing sites 71 . A recent study has identified novel collagen fragments, and collagen-associated peptides derived from thrombospondin-1, multimerin-1, fibronectin, and tenascin-C, generated from CSO-digested human dermal capillary endothelial and fibroblastic extracellular matrix (ECM) 8 . It is plausible that such degradation products of CSO are implicated in the mechanism of action reported in this study. Additionally, a direct effect of CS-API on mφ receptors/pathways leading to induction of mφ heal polarization may not be ruled out. Taken together, this work sheds new light on what is currently utilized as a standard wound debridement agent in clinics. Findings of this work reposition CSO as a potential agent that may be effective in resolving wound inflammation, including wounds of diabetics, via STAT6 and NF-κB pathways.

Materials and Methods
Isolation of murine wound mφ and bone marrow-derived macrophages (BMDM). All animal studies have been approved by, and all methods were performed in accordance with the guidelines and regulations set by The Ohio State University's Institutional Animal Care and Use Committee (IACUC). Male C57BL/6 mice (8-12 weeks old) were obtained from Harlan Laboratories (Envigo). Mice homozygous for spontaneous mutation of the leptin receptor (Leprdb) (BKS.Cg-m +/+ Leprdb/J, or db/db; stock no 000642) were procured from Jackson Laboratories. For wound mφ isolation, circular (8 mm) sterile PVA sponges loaded with (300 mg/sponge) CSO (Smith & Nephew, Inc., Fort Worth, TX) or white petrolatum vehicle (control) (Fougera Pharmaceuticals Inc., NY) were implanted subcutaneously on the backs of 8 to 12 week-old mice 15 . The incisional wounds were sutured and closed after implantation of the PVA sponges. Sponge-infiltrated wound mφ were isolated after 3 or 7 days post-implantation, as previously described 15,25,41 . In brief, following induction of anesthesia, subcutaneously implanted PVA sponges were harvested and wound cell suspension was generated by repeated compression of the sponges. The cell suspension was then subjected to magnetic cell sorting using mouse anti-CD11b tagged microbeads (Miltenyi Biotec, Auburn, CA). This procedure yields a purified (>95%) population of wound mφ as determined by F4/80 staining 15,25,41 . Murine bone marrow-derived macrophages (BMDM) were isolated as previously described 41 . ELISA. Levels of cytokines secreted by wound mφ were measured using commercially available ELISA kits 41,46 .
For the assay, wound mφ isolated from CSO or vehicle treated groups were seeded in 6-well or 12-well plates followed by activation with LPS (1 µg/ml) for 24 h. After 24 h, the media was collected and the cytokine levels in culture media were measured using commercially available ELISA kits (R & D Systems, Minneapolis, MN) as per manufacturer's instructions.

Isolation of RNA, reverse transcription and quantitative RT-PCR (qRT-PCR). mirVana RNA iso-
lation kit (Ambion, Austin, TX), was used according to the manufacturer's instructions to extract total RNA as described 72 . Gene expression was measured by real-time qPCR assay using DNA intercalation dye SYBR Green-I (Applied Biosystems, CA) as described previously 15,25,41,46,73,74 . Arginase activity assay. Arginase activity was determined with Arginase activity assay kit per manufacturer's recommendation (Abcam, Cambridge, MA). In this assay, Arginase reacts with arginine through an intermediary product reacts with the OxiRed ™ Probe to generate a colored product that can be read at OD 570 nm. For the assay, cell lysates or standards are mixed with a reaction cocktail provided with the kit followed by incubation at 37 °C for 30 mins. The colored product was read at 570 nm with a Plate Reader. The activity was normalized using equal amount of proteins in the cell lysate. The activity was expressed in μmol/mg of protein.
Superoxide anion production. Superoxide anion production was measured using a standard luminol based LumiMax Superoxide anion detection kit (Agilent technologies, Santa Clara, CA) 72 . The principle of the assay is that superoxide anion oxidizes luminol in a reaction that produces photons of light that are readily measured with a standard luminometer.
Capillary Electrophoresis Immunoassay. Capillary electrophoresis immunoassay was performed using the Simon (Protein Simple, Santa Clara, CA) according to the manufacturer's protocol. The sample loading, electrophoresis and immunodetection was performed using a fully automated capillary electrophoresis system as described 75 . Capillary electrophoresis immunoassay was carried out at room temperature, and using default instrument settings (stacking/sample load time, 12 sec/8 sec; separation time, 40 min; blocking time, 15 min; primary antibody, 120 min; secondary antibody, 60 min). Arginase-1 (BD Biosciences, CA; dilution 1:30), and β-actin (Sigma Aldrich, MO; dilution 1:100) primary antibody were used for immuno-detection. The digital images were analyzed using Compass software (Protein Simple).
Identification of wound cell populations by flow cytometry. Cytometry markers used to identify leukocyte subsets included: Alexa Fluor ® 488-F4/80, PerCP-Ly6G, APC-Ly6C, PE-CCR2 (Biolegend), PE-CD4 and FITC-CD25 (Miltenyi). Cells were surface-stained for 30 minutes in staining buffer (1 × DPBS/1% BSA) as described previously 43 . Cells were then gated using forward and side scatter characteristics for leukocytes with at least 2,000 gated events recorded using BDTM LSR II flow cytometry (BD Biosciences) and analyzed using DNA binding of NF-κB. Nuclear protein extracts from mφ were prepared using the nuclear protein extraction kit (Active Motif, Carlsbad, CA). Binding of NF-κB family of proteins to their consensus sites was determined using an ELISA-based Trans-AM NF-κB kit (Active Motif, Carlsbad, CA) following manufacturer's instructions as previously described 46 .
SCIeNTIFIC REpORtS | (2018) 8:1696 | DOI:10.1038/s41598-018-19879-w Statistics. Data are reported as mean ± SEM as indicated in the respective figure legends. Student's t test (two-tailed) was used to determine significant differences. Comparisons among multiple groups were tested using ANOVA, and p < 0.05 was considered statistically significant.
Disclosures. The authors declare that CSO, CS-API and partial research funding was provided by Smith & Nephew, Inc. ER and LS are current employees of Smith & Nephew, Inc (R&D, Fort, Worth).