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738 IL-27 facilitates skin wound healing through induction of epidermal proliferation and host defense
ABSTRACTS | Tissue Regeneration & Wound Healing 734
735
Skin regeneration properties of Bertholletia excelsa and Fevillea trilobata vegetable oils in an in vitro human fibroblast cell model FM Thomaz, PP Soldati and D Zimbardi Inovac¸a˜o, Natura Inovac¸a˜o e Tec. Prod. Ltda, Cajamar, Brazil Two Brazilian vegetable compounds, Bertholletia excelsa and Fevillea trilobata oils, were evaluated to understand their role in cellular mechanisms related to skin regeneration by a combination of in vitro cell culture and biochemical assays. The results showed that both oils were able to increase FGF2 levels (p<0.0001), a signaling marker induced after tissue damage and were also able to modulate the inflammatory process by a reduction of inflammatory cytokines IL-6 (p<0.05) and IL-12 (p<0.01) and an increase in antiinflammatory cytokine IL-10 (p<0.0001), important for the resolution of inflammatory phase. In addition, both treatments stimulated the fibroblast proliferation and the B. excelsa oil decreased the cell migration time (p< 0.01) and increased migration rate (p<0.0001). Finally, both oils were able to decrease elastase activity (p<0.05) and the B. excelsa oil also increased MMP-13 levels (p<0.001), contributing to tissue remodeling. Thus, both Brazilian natural oils have been demonstrated to act in key events of skin healing process with potential to being employed in anti-aging cosmetic formulations or for sensitized skin after aesthetical procedures.
Rescue potential of 25(OH)D on skin exposure to nitrogen mustard is a function of time and dose AM Binko, LM Das and KQ Lu Dermatology, Case Western Reserve University, Cleveland, OH Nitrogen Mustard (NM) is an alkylating agent known to cause severe blistering and systemic toxicity from cutaneous exposure. Our previous data shows that a single intraperitoneal injection of low dose 25-hydroxyvitamin D3 (25(OH)D) given 1hr after NM exposure rescues mice from pancytopenia and death. However timing of intervention with the appropriate dose of drug in cases of mass exposure either accidental or during warfare is critical to wound repair and survival. In this study we demonstrate that 5ng 25(OH)D administered up to 24hr post NM exposure is able to mediate 100% recovery in mice. Examination of gross wound images and lesion sizes measured up to 20 days demonstrated significant wound resolution with diminishing lesion size, prevention of severe weight loss and death. In contrast failure to intervene before 48 h proved too late at and beyond which there was 60% death between days 6 to 12. If left untreated for 72 to 96 h, all NM-exposed animals showed minimal wound healing, up to 25% loss in body weight that ultimately died or met euthanasia criteria despite intervention at these later time points. Since 48 h seemed to be the critical time at which 25(OH)D had no effect in exposed animals, we tested the effect of two consecutive doses of 5 ng of 25(OH)D at 48 and 72h post NM exposure. Treated animals showed marked improvement of lesion sizes with 100% recovery from mortality. However a 10-fold higher dose (50ng) of 25(OH)D administered at 48 h was not beneficial with only 66% mice surviving. 50 ng of 25(OH)D administered at 72 h resulted in all animals being dead by day 6 with exacerbated skin lesions. Further experiments need to be conducted to ascertain the time limit beyond which rescue from NM mediated toxic death is irreversible, irrespective of repeat doses of intervention. Taken together we propose that there is a finite time within which countermeasures have efficacy in the treatment of NM-induced skin injury.
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b2 integrin-dependent activation of NOX2 oxidase in wound macrophages is required for physiological wound healing A Kuegler1, S Schatz1, S Vander Beeken1, D Jiang1, A Ru¨ck2, B De Geest3, P Hawkins4, K Scharffetter-Kochanek1 and S Anca1 1 Dermatology and Allergic Diseases, University of Ulm, Ulm, Germany, 2 Core Facility, University of Ulm, Ulm, Germany, 3 Department of Pharmaceutics, Ghent University, Ghent, Belgium and 4 Inositide Laboratory, The Babraham Institute, Cambridge, United Kingdom We here studied whether reactive oxygen species (ROS) released upon activation of the phagocyte NADPH oxidase NOX2 in wound macrophages (Mf) are required for proper wound healing and, if so, whether the leukocyte adhesion molecules b2 integrins control ROS release. b2 integrin-deficient (CD18-/-) mice revealed a severely impaired wound healing with impaired granulation tissue formation and angiogenesis due to insufficient transforming growth factor-b1 (TGF-b1) release from CD18-/- Mf. Targeting the NOX2 activator rotenone to wound Mf fully restored reduced ROS levels measured by in vivo imaging with redox sensitive dye, reconstituted impaired TGF-b1 activation and granulation tissue formation as assessed by ELISA and Western blot analyses of wound tissue lysates, and eventually rescued wound healing of CD18-/- mice. This effect was completely abolished by the NOX2 inhibitor ebselen, indicating that NOX2 activation with ROS release are impaired and causal for delayed wound healing in CD18-/- conditions. NADPH-based fluorescence lifetime imaging demonstrated that NOX2 assembly and activation were severely impaired at CD18-/macrophages membrane upon adhesion to apoptotic cells. Moreover, mice lacking the p40phox NADPH subunit (p40phox-/-) with impaired oxidase function presented significantly reduced ROS levels and defective wound healing, very much resembling wound closure of CD18-/- mice. Impaired TGF-b1 activation by p40phox-/- wound Mf was causal for reduced angiogenesis, myofibroblasts differentiation and wound contraction of p40phox-/- mice. Injection of p40phox-/- Mf around wound margins could not compensate b2 integrin deficiency and restore the impaired wound healing of CD18-/- mice, supporting a central role of the b2 integrin-NADPH oxidase pathway in macrophages for tissue repair and inflammation.
Deferoxamine in wound healing CN Tchanque-Fossuo1,2, SE Dahle1,3, SR Buchman4 and RR Isseroff1 1 Dermatology, University of California Davis, Sacramento, CA, 2 Dermatology, VANCHS, Mather, CA, 3 Surgery, Podiatry Section, VANCHS, Mather, CA and 4 Plastic Surgery, University of Michigan, Ann Arbor, MI Chronic wounds (CW) are a societal burden and medical challenge. Even innovative tissue regenerative therapies heal half of treated patients. There is a need for new approaches to improve healing. Chronic hypoxia and iron play a significant role in the pathogenicity of CW, including pressure ulcer (PU), venous leg ulcers (VLU) and diabetic foot ulcer (DFU). In CW, there is diminished oxygen tension due to local vasculature compromise. We speculate that tissue ischemia with vessel occlusion in PU lead to increase iron deposition as seen in VLU from erythrocytes leakage and in DFU due to ferritin synthesis due to insulin resistance. Iron release initiates a pathophysiologic process,including reactive oxygen species generation, lipids peroxidation, and unrestrained pro-inflammatory M1 macrophage phenotype, which sustain the chronic inflammatory state of CW. We postulate that topical use of Deferoxamine (DFO), which simultaneously acts as an iron-chelator and effective HIF-1a inducer and stabilizer, is a potential therapy for wound healing. Topical application of DFO improved wound healing in treated db/db mice with increased granulation tissue, neovascularization, and significant elevation in local VGEF expression. DFO demonstrated a significant reversal of radiation-induced hypovascularity in our in vivo murine model of pathologic fracture repair in radiated bone compared to the radiated untreated. Transdermal delivery of DFO for DFU prevention in an animal wound healing model was reported. DFO can prevent iron-release in PU, VLU, DFU; thus DFO indirectly inhibits the deleterious cascade of iron-induced free radicals that overload the wound’s antioxidant capacities and perpetuate the inflammatory cycle. DFO-induced HIF-1a stabilization can circumvent the chronic hypoxic milieu of wounds, stimulate new vessels, and potentiate cell motility and recruitment of endothelial precursors. Altogether, DFO has the potential to provide a safe, novel therapeutic tool to optimize healing in CW.
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IL-27 facilitates skin wound healing through induction of epidermal proliferation and host defense B Yang, R Sanchez Lagunes, T Phillips, J Suwanpradid and A MacLeod Dermatology, Duke University, Durham, NC Skin wound repair requires a coordinated program of epithelial cell proliferation and differentiation as well as microbial resistance. Recent studies from our lab and others demonstrate that IL-27 is produced by activated antigen-presenting cells in the skin upon skin damage and is shown to exert both pro-inflammatory and anti-inflammatory effects. The potential functional role of IL-27 in wound repair is unknown. We hypothesize that IL-27 plays a major role in wound healing by regulating wound re-epithelialization and cutaneous antimicrobial function. To test this hypothesis, we characterized the functional role of IL-27 and its receptor IL-27Ra in wounded skin. We established a mouse model of cutaneous wound healing and demonstrate that IL-27 is rapidly and transiently elevated in the skin after wounding. Both macrophages and dendritic cells produced IL-27 at the wound edge. Genetic ablation of Il27Ra attenuated wound healing (p<0.05). Furthermore, in an in vitro scratch assay, recombinant IL-27 (100ng/ml) significantly accelerated wound closure compared to vehicle-treated keratinocytes at 24 and 48 hrs. (P<0.0001) and this effect appeared to be dependent on JAK/STAT3 signaling. IL-27 stimulation led to strong induction of Ki67 expression, whereas IL-27 stimulation of human keratinocytes resulted in suppression of loricirin and filaggrin expression (p<0.01). To test whether IL-27 influences antimicrobial host defense function, we examined whether IL-27 could stimulate an antibacterial and antiviral response. IL-27 did not affect expression of anti-bacterial human beta defensin 2 (HBD2), but potently increased the antiviral oligoadenylate synthase 2 (OAS2). Together, our data suggest a previously unrecognized role for IL-27 in regulating epithelial cell proliferation and antiviral host defense in the wound healing response. Our results may have major implications in our understanding of how keratinocyte proliferation and protective antiviral immunity is regulated in wound healing and in other inflammatory and hyper-proliferative skin diseases.
S130 Journal of Investigative Dermatology (2016), Volume 136
Hyaluronan controls adipogenesis following skin injury T Dokoshi, L Zhang, RL Gallo, C Adase, T Takahashi, JA Sanford, MR Williams, E Sato and T Nakatsuji Department of Dermatology, University of California San Diego, San Diego, CA Skin immune defense and repair is a coordinated response to a variety of danger signals and involves the complex interactions of several different cell types. Dermal adipogenesis is a previously unknown but critical event in this process that participates in antimicrobial defense and inflammation. We have previously shown that high-molecular-weight hyaluronan (HMW-HA) undergoes breakdown into small fragments after injury where it can then activate endothelial cells, macrophages and DCs via TLR4. In this study we investigated if HA may also influence adipogenesis and thus regulate the wound repair process. Maturation of 3T3L1 cells into adipocytes was accompanied by increased HMW-HA seen by Gel Electrophoresis and expression of HAS2 mRNA measured by qPCR (p ¼0.006). Conversely, digestion of HMW-HA by addition of hyaluronidase inhibited differentiation into mature adipocytes as seen by decreased mRNA for zfp521(p¼0.02), zfp423(p¼0.01) and Adiponectin (p ¼0.004), as well as decreased accumulation of lipid droplets and enhanced cell proliferation. The inhibition of early adipogenesis was not due to HA fragments since the addition of HA fragments of 6.4kDa, 50kDa, 150kDa and 1000kD had no effect. In vivo, tissue specific expression of hyaluronidase 1 (HYAL1) in mice prior to injury resulted in less mature dermal fat tissue as seen by immunohistochemistry and lower expression of mRNA for C/EBPa (p¼0.002) but not Adiponeqtin(p¼0.13) or PPARg(p¼0.27). In normal mice, HA immunostaining was lost 3 days following injury coincident with an increase in expression of HAS2 mRNA (p¼0.0008) and mRNA for the hyaluronidase KIAA1199 (p¼0.01). The loss of HMW-HA in vivo occurred after the expected increase in adipogenesis immediately after injury. Taken together, these results suggest that HA plays a regulatory role during wound repair by controlling dermal adipogenesis. HMW-HA facilitates fat maturation early in the wound repair process, and hyaluronidase action stops this event later in the wound healing process.
735
Skin regeneration properties of Bertholletia excelsa and Fevillea trilobata vegetable oils in an in vitro human fibroblast cell model FM Thomaz, PP Soldati and D Zimbardi Inovac¸a˜o, Natura Inovac¸a˜o e Tec. Prod. Ltda, Cajamar, Brazil Two Brazilian vegetable compounds, Bertholletia excelsa and Fevillea trilobata oils, were evaluated to understand their role in cellular mechanisms related to skin regeneration by a combination of in vitro cell culture and biochemical assays. The results showed that both oils were able to increase FGF2 levels (p<0.0001), a signaling marker induced after tissue damage and were also able to modulate the inflammatory process by a reduction of inflammatory cytokines IL-6 (p<0.05) and IL-12 (p<0.01) and an increase in antiinflammatory cytokine IL-10 (p<0.0001), important for the resolution of inflammatory phase. In addition, both treatments stimulated the fibroblast proliferation and the B. excelsa oil decreased the cell migration time (p< 0.01) and increased migration rate (p<0.0001). Finally, both oils were able to decrease elastase activity (p<0.05) and the B. excelsa oil also increased MMP-13 levels (p<0.001), contributing to tissue remodeling. Thus, both Brazilian natural oils have been demonstrated to act in key events of skin healing process with potential to being employed in anti-aging cosmetic formulations or for sensitized skin after aesthetical procedures.
Rescue potential of 25(OH)D on skin exposure to nitrogen mustard is a function of time and dose AM Binko, LM Das and KQ Lu Dermatology, Case Western Reserve University, Cleveland, OH Nitrogen Mustard (NM) is an alkylating agent known to cause severe blistering and systemic toxicity from cutaneous exposure. Our previous data shows that a single intraperitoneal injection of low dose 25-hydroxyvitamin D3 (25(OH)D) given 1hr after NM exposure rescues mice from pancytopenia and death. However timing of intervention with the appropriate dose of drug in cases of mass exposure either accidental or during warfare is critical to wound repair and survival. In this study we demonstrate that 5ng 25(OH)D administered up to 24hr post NM exposure is able to mediate 100% recovery in mice. Examination of gross wound images and lesion sizes measured up to 20 days demonstrated significant wound resolution with diminishing lesion size, prevention of severe weight loss and death. In contrast failure to intervene before 48 h proved too late at and beyond which there was 60% death between days 6 to 12. If left untreated for 72 to 96 h, all NM-exposed animals showed minimal wound healing, up to 25% loss in body weight that ultimately died or met euthanasia criteria despite intervention at these later time points. Since 48 h seemed to be the critical time at which 25(OH)D had no effect in exposed animals, we tested the effect of two consecutive doses of 5 ng of 25(OH)D at 48 and 72h post NM exposure. Treated animals showed marked improvement of lesion sizes with 100% recovery from mortality. However a 10-fold higher dose (50ng) of 25(OH)D administered at 48 h was not beneficial with only 66% mice surviving. 50 ng of 25(OH)D administered at 72 h resulted in all animals being dead by day 6 with exacerbated skin lesions. Further experiments need to be conducted to ascertain the time limit beyond which rescue from NM mediated toxic death is irreversible, irrespective of repeat doses of intervention. Taken together we propose that there is a finite time within which countermeasures have efficacy in the treatment of NM-induced skin injury.
736
737
b2 integrin-dependent activation of NOX2 oxidase in wound macrophages is required for physiological wound healing A Kuegler1, S Schatz1, S Vander Beeken1, D Jiang1, A Ru¨ck2, B De Geest3, P Hawkins4, K Scharffetter-Kochanek1 and S Anca1 1 Dermatology and Allergic Diseases, University of Ulm, Ulm, Germany, 2 Core Facility, University of Ulm, Ulm, Germany, 3 Department of Pharmaceutics, Ghent University, Ghent, Belgium and 4 Inositide Laboratory, The Babraham Institute, Cambridge, United Kingdom We here studied whether reactive oxygen species (ROS) released upon activation of the phagocyte NADPH oxidase NOX2 in wound macrophages (Mf) are required for proper wound healing and, if so, whether the leukocyte adhesion molecules b2 integrins control ROS release. b2 integrin-deficient (CD18-/-) mice revealed a severely impaired wound healing with impaired granulation tissue formation and angiogenesis due to insufficient transforming growth factor-b1 (TGF-b1) release from CD18-/- Mf. Targeting the NOX2 activator rotenone to wound Mf fully restored reduced ROS levels measured by in vivo imaging with redox sensitive dye, reconstituted impaired TGF-b1 activation and granulation tissue formation as assessed by ELISA and Western blot analyses of wound tissue lysates, and eventually rescued wound healing of CD18-/- mice. This effect was completely abolished by the NOX2 inhibitor ebselen, indicating that NOX2 activation with ROS release are impaired and causal for delayed wound healing in CD18-/- conditions. NADPH-based fluorescence lifetime imaging demonstrated that NOX2 assembly and activation were severely impaired at CD18-/macrophages membrane upon adhesion to apoptotic cells. Moreover, mice lacking the p40phox NADPH subunit (p40phox-/-) with impaired oxidase function presented significantly reduced ROS levels and defective wound healing, very much resembling wound closure of CD18-/- mice. Impaired TGF-b1 activation by p40phox-/- wound Mf was causal for reduced angiogenesis, myofibroblasts differentiation and wound contraction of p40phox-/- mice. Injection of p40phox-/- Mf around wound margins could not compensate b2 integrin deficiency and restore the impaired wound healing of CD18-/- mice, supporting a central role of the b2 integrin-NADPH oxidase pathway in macrophages for tissue repair and inflammation.
Deferoxamine in wound healing CN Tchanque-Fossuo1,2, SE Dahle1,3, SR Buchman4 and RR Isseroff1 1 Dermatology, University of California Davis, Sacramento, CA, 2 Dermatology, VANCHS, Mather, CA, 3 Surgery, Podiatry Section, VANCHS, Mather, CA and 4 Plastic Surgery, University of Michigan, Ann Arbor, MI Chronic wounds (CW) are a societal burden and medical challenge. Even innovative tissue regenerative therapies heal half of treated patients. There is a need for new approaches to improve healing. Chronic hypoxia and iron play a significant role in the pathogenicity of CW, including pressure ulcer (PU), venous leg ulcers (VLU) and diabetic foot ulcer (DFU). In CW, there is diminished oxygen tension due to local vasculature compromise. We speculate that tissue ischemia with vessel occlusion in PU lead to increase iron deposition as seen in VLU from erythrocytes leakage and in DFU due to ferritin synthesis due to insulin resistance. Iron release initiates a pathophysiologic process,including reactive oxygen species generation, lipids peroxidation, and unrestrained pro-inflammatory M1 macrophage phenotype, which sustain the chronic inflammatory state of CW. We postulate that topical use of Deferoxamine (DFO), which simultaneously acts as an iron-chelator and effective HIF-1a inducer and stabilizer, is a potential therapy for wound healing. Topical application of DFO improved wound healing in treated db/db mice with increased granulation tissue, neovascularization, and significant elevation in local VGEF expression. DFO demonstrated a significant reversal of radiation-induced hypovascularity in our in vivo murine model of pathologic fracture repair in radiated bone compared to the radiated untreated. Transdermal delivery of DFO for DFU prevention in an animal wound healing model was reported. DFO can prevent iron-release in PU, VLU, DFU; thus DFO indirectly inhibits the deleterious cascade of iron-induced free radicals that overload the wound’s antioxidant capacities and perpetuate the inflammatory cycle. DFO-induced HIF-1a stabilization can circumvent the chronic hypoxic milieu of wounds, stimulate new vessels, and potentiate cell motility and recruitment of endothelial precursors. Altogether, DFO has the potential to provide a safe, novel therapeutic tool to optimize healing in CW.
738
739
IL-27 facilitates skin wound healing through induction of epidermal proliferation and host defense B Yang, R Sanchez Lagunes, T Phillips, J Suwanpradid and A MacLeod Dermatology, Duke University, Durham, NC Skin wound repair requires a coordinated program of epithelial cell proliferation and differentiation as well as microbial resistance. Recent studies from our lab and others demonstrate that IL-27 is produced by activated antigen-presenting cells in the skin upon skin damage and is shown to exert both pro-inflammatory and anti-inflammatory effects. The potential functional role of IL-27 in wound repair is unknown. We hypothesize that IL-27 plays a major role in wound healing by regulating wound re-epithelialization and cutaneous antimicrobial function. To test this hypothesis, we characterized the functional role of IL-27 and its receptor IL-27Ra in wounded skin. We established a mouse model of cutaneous wound healing and demonstrate that IL-27 is rapidly and transiently elevated in the skin after wounding. Both macrophages and dendritic cells produced IL-27 at the wound edge. Genetic ablation of Il27Ra attenuated wound healing (p<0.05). Furthermore, in an in vitro scratch assay, recombinant IL-27 (100ng/ml) significantly accelerated wound closure compared to vehicle-treated keratinocytes at 24 and 48 hrs. (P<0.0001) and this effect appeared to be dependent on JAK/STAT3 signaling. IL-27 stimulation led to strong induction of Ki67 expression, whereas IL-27 stimulation of human keratinocytes resulted in suppression of loricirin and filaggrin expression (p<0.01). To test whether IL-27 influences antimicrobial host defense function, we examined whether IL-27 could stimulate an antibacterial and antiviral response. IL-27 did not affect expression of anti-bacterial human beta defensin 2 (HBD2), but potently increased the antiviral oligoadenylate synthase 2 (OAS2). Together, our data suggest a previously unrecognized role for IL-27 in regulating epithelial cell proliferation and antiviral host defense in the wound healing response. Our results may have major implications in our understanding of how keratinocyte proliferation and protective antiviral immunity is regulated in wound healing and in other inflammatory and hyper-proliferative skin diseases.
S130 Journal of Investigative Dermatology (2016), Volume 136
Hyaluronan controls adipogenesis following skin injury T Dokoshi, L Zhang, RL Gallo, C Adase, T Takahashi, JA Sanford, MR Williams, E Sato and T Nakatsuji Department of Dermatology, University of California San Diego, San Diego, CA Skin immune defense and repair is a coordinated response to a variety of danger signals and involves the complex interactions of several different cell types. Dermal adipogenesis is a previously unknown but critical event in this process that participates in antimicrobial defense and inflammation. We have previously shown that high-molecular-weight hyaluronan (HMW-HA) undergoes breakdown into small fragments after injury where it can then activate endothelial cells, macrophages and DCs via TLR4. In this study we investigated if HA may also influence adipogenesis and thus regulate the wound repair process. Maturation of 3T3L1 cells into adipocytes was accompanied by increased HMW-HA seen by Gel Electrophoresis and expression of HAS2 mRNA measured by qPCR (p ¼0.006). Conversely, digestion of HMW-HA by addition of hyaluronidase inhibited differentiation into mature adipocytes as seen by decreased mRNA for zfp521(p¼0.02), zfp423(p¼0.01) and Adiponectin (p ¼0.004), as well as decreased accumulation of lipid droplets and enhanced cell proliferation. The inhibition of early adipogenesis was not due to HA fragments since the addition of HA fragments of 6.4kDa, 50kDa, 150kDa and 1000kD had no effect. In vivo, tissue specific expression of hyaluronidase 1 (HYAL1) in mice prior to injury resulted in less mature dermal fat tissue as seen by immunohistochemistry and lower expression of mRNA for C/EBPa (p¼0.002) but not Adiponeqtin(p¼0.13) or PPARg(p¼0.27). In normal mice, HA immunostaining was lost 3 days following injury coincident with an increase in expression of HAS2 mRNA (p¼0.0008) and mRNA for the hyaluronidase KIAA1199 (p¼0.01). The loss of HMW-HA in vivo occurred after the expected increase in adipogenesis immediately after injury. Taken together, these results suggest that HA plays a regulatory role during wound repair by controlling dermal adipogenesis. HMW-HA facilitates fat maturation early in the wound repair process, and hyaluronidase action stops this event later in the wound healing process.