Epidermal Reactions to Injury with Implications for Innate Immunity

Abstract The epidermis is one of our primary interfaces towards the external milieu. Following injury, the physical barrier function of the skin is destroyed and the epidermis is left vulnerable to microbial invasion. Inducible innate immune response mechanisms exist to keep the wound site free from infection, thus allowing the wound to heal and the epidermis to re-establish its barrier function. The main focus of the present thesis has been to investigate the regulatory mechanisms of some of these responses. In paper I we present an injury-induced mechanism for increasing the production of antimicrobial peptides (AMPs) in the skin mediated by the epidermal growth factor receptor (EGFR) transactivation process. In paper II we further highlight the importance of this mechanism by demonstrating that it is responsible for the bulk of the expression of the AMPs known to be induced in epidermis during the proliferative phase of wound healing. In addition we show that the EGFR-mediated increase in interleukin-8 (IL-8) production represents the primary source of chemotactic activity towards neutrophils generated in injured human epidermis. Thus a novel molecular link between cutaneous injury and neutrophil accumulation is provided. In paper III we disclose a possible role of the prion protein as an AMP in host defense, based on its antimicrobial properties and EGFR-dependent induction in response to injury. In paper IV we furthermore report an increased expression of several protease inhibitors during wound healing and a change in gene expression in the epidermal tissue representing a shift in the apoptotic balance. The shift indicates a reduced sensitivity to the extrinsic pathway of apoptosis and concomitantly, an apparently increased sensitivity to the intrinsic pathway of apoptosis. Taken together we hypothesize that this represents an epidermal response to cope with the external detrimental effects of inflammation while safeguarding itself against the increased risk of malignant transformation accompanying the increased proliferation. Finally we identify a highly significant overrepresentation of transcription factor binding sites for forkhead box O1 (FOXO1), FOXO4 and STAT5A, in the most differentially expressed genes in injured skin. This indicates, for the first time, that these transcription factors might play a major role in the wound healing process

Injury Is a Major Inducer of Epidermal Innate Immune Responses during Wound Healing

We examined the importance of injury for the epidermal innate immune response in human skin wounds. We found that injury, independent of infiltrating inflammatory cells, generated prominent chemotactic activity toward neutrophils in injured skin because of IL-8 production. Furthermore, injury was a major inducer of the expression of antimicrobial (poly)peptides (AMPs) in skin wounds. In human skin, these injury-induced innate immune responses were mediated by activation of the epidermal growth factor receptor (EGFR). Consequently, inhibition of the EGFR blocked both the chemotactic activity generated in injured skin and the expression of the majority of the AMPs. The importance of injury was confirmed in mouse experiments in vivo, in which injury independent of infection was a potent inducer of AMPs in skin wounds. To our knowledge, these data thereby provide a previously unreported molecular link between injury and neutrophil accumulation and identify the molecular background for the vast expression of IL-8 and AMPs in wounded epidermis. Conceptually, these data show that the growth factor response elicited by injury is important for the recruitment of neutrophils in skin wounds

The use of a rigid disc to protect exposed structures in wounds treated with negative pressure wound therapy: Effects on wound bed pressure and microvascular blood flow.

There are increasing reports of deaths and serious complications associated with the use of negative pressure wound therapy (NPWT). Bleeding may occur in patients when NPWT is applied to a wound with exposed blood vessels or vascular grafts, possibly due to mechanical deformation and hypoperfusion of the vessel walls. Recent evidence suggests that using a rigid barrier disc to protect underlying tissue can prevent this mechanical deformation. The aim of this study was to examine the effect of rigid discs on the tissue exposed to negative pressure with regard to tissue pressure and microvascular blood flow. Peripheral wounds were created on the backs of eight pigs. The pressure and microvascular blood flow in the wound bed were measured when NPWT was applied. The wound was filled with foam, and rigid discs of different designs were inserted between the wound bed and the foam. The discs were created with or without channels (to accommodate exposed sensitive structures such as blood vessels and nerves), perforations, or a porous dressing that covered the underside of the discs (to facilitate pressure transduction and fluid evacuation). When comparing the results for pressure transduction to the wound bed, no significant differences were found using different discs covered with dressing, whereas pressure transduction was lower with bare discs. Microvascular blood flow in the wound bed decreased by 49 ± 7% when NPWT was applied to control wounds. The reduction in blood flow was less in the presence of a protective disc (e.g., -6 ± 5% for a dressing-covered, perforated disc, p = 0.006). In conclusion, NPWT causes hypoperfusion of superficial tissue in the wound bed. The insertion of a rigid barrier counteracts this effect. The placement of a rigid disc over exposed blood vessels or nerves may protect these structures from rupture and damage

A Rigid Disc for Protection of Exposed Blood Vessels During Negative Pressure Wound Therapy

Background. There are increasing reports of serious complications and deaths associated with negative pressure wound therapy (NPWT). Bleeding may occur when NPWT is applied to a wound with exposed blood vessels. Inserting a rigid disc in the wound may protect these structures. The authors examined the effects of rigid discs on wound bed tissue pressure and blood flow through a large blood vessel in the wound bed during NPWT. Methods. Wounds were created over the femoral artery in the groin of 8 pigs. Rigid discs were inserted. Wound bed pressures and arterial blood flow were measured during NPWT. Results. Pressure transduction to the wound bed was similar for control wounds and wounds with discs. Blood flow through the femoral artery decreased in control wounds. When a disc was inserted, the blood flow was restored. Conclusions. NPWT causes hypoperfusion in the wound bed tissue, presumably as a result of mechanical deformation. The insertion of a rigid barrier alleviates this effect and restores blood flow

The influence on wound contraction and fluid evacuation of a rigid disc inserted to protect exposed organs during negative pressure wound therapy.

The use of a rigid disc as a barrier between the wound bed and the wound filler during negative pressure wound therapy (NPWT) has been suggested to prevent damage to exposed organs. However, it is important to determine that the effects of NPWT, such as wound contraction and fluid removal, are maintained during treatment despite the use of a barrier. This study was performed to examine the effect of NPWT on wound contraction and fluid evacuation in the presence of a rigid disc. Peripheral wounds were created on the backs of eight pigs. The wounds were filled with foam, and rigid discs of different designs were inserted between the wound bed and the foam. Wound contraction and fluid evacuation were measured after application of continuous NPWT at -80 mmHg. Wound contraction was similar in the presence and the absence of a rigid disc (84 ± 4% and 83 ± 3%, respectively, compared with baseline). Furthermore, the rigid disc did not affect wound fluid removal compared with ordinary NPWT (e.g. after 120 seconds, 71 ± 4 ml was removed in the presence and 73 ± 3 ml was removed in the absence of a disc). This study shows that a rigid barrier may be placed under the wound filler to protect exposed structures during NPWT without affecting wound contraction and fluid removal, which are two crucial features of NPWT

Transcription factor binding site analysis identifies FOXO transcription factors as regulators of the cutaneous wound healing process.

The search for significantly overrepresented and co-occurring transcription factor binding sites in the promoter regions of the most differentially expressed genes in microarray data sets could be a powerful approach for finding key regulators of complex biological processes. To test this concept, two previously published independent data sets on wounded human epidermis were re-analyzed. The presence of co-occurring transcription factor binding sites for FOXO1, FOXO3 and FOXO4 in the majority of the promoter regions of the most significantly differentially expressed genes between non-wounded and wounded epidermis implied an important role for FOXO transcription factors during wound healing. Expression levels of FOXO transcription factors during wound healing in vivo in both human and mouse skin were analyzed and a decrease for all FOXOs in human wounded skin was observed, with FOXO3 having the highest expression level in non wounded skin. Impaired re-epithelialization was found in cultures of primary human keratinocytes expressing a constitutively active variant of FOXO3. Conversely knockdown of FOXO3 in keratinocytes had the opposite effect and in an in vivo mouse model with FOXO3 knockout mice we detected significantly accelerated wound healing. This article illustrates that the proposed approach is a viable method for identifying important regulators of complex biological processes using in vivo samples. FOXO3 has not previously been implicated as an important regulator of wound healing and its exact function in this process calls for further investigation

Injury-induced innate immune response in human skin mediated by transactivation of the epidermal growth factor receptor

We found that sterile wounding of human skin induced epidermal expression of the antimicrobial (poly)peptides human β-defensin–3, neutrophil gelatinase–associated lipocalin, and secretory leukocyte protease inhibitor through activation of the epidermal growth factor receptor. After skin wounding, the receptor was activated by heparin-binding epidermal growth factor that was released by a metalloprotease-dependent mechanism. Activation of the epidermal growth factor receptor generated antimicrobial concentrations of human β-defensin–3 and increased the activity of organotypic epidermal cultures against Staphylococcus aureus. These data demonstrate that sterile wounding initiates an innate immune response that increases resistance to overt infection and microbial colonization

Wound healing rate in FOXO3 and FOXO3 LysM-cre knockout mice compared to control.

<p>A) Quantitative RT-PCR using mRNA from non-wounded and <i>in vivo</i> wounded mouse skin was performed. All data was normalized to GAPDH expression (Glyceraldehyde 3-phosphate dehydrogenase) that was used as housekeeping gene. A one-way analysis of variance was performed. Significant differences were found day 1 after wounding for FOXO3 and FOXO4 when performing a Dunnett's Multiple Comparison Test against non-wounded control. (*<i>P</i><0.05, **<i>P</i><0.01). Error bar denotes mean ±SD (n = 3). B) Graph displays wound size in mm² over time. Values from each mouse represent an average of 4 wounds induced by 6 mm punches through folded dorsal skin. A two-way ANOVA was performed to detect differences over time between FOXO3 knock out mice and C57bl/6 control mice using Bonferroni post-testing to detect differences at each time point. An overall difference was detected over time <i>P</i><0.023 and post-testing generated significant results for day 1 to day 4. (*<i>P</i><0.05, **<i>P</i><0.01, ***<i>P</i><0.001) (n = 4) C) Bacterial loads in the wound beds day 10 after wounding. D) Wound healing rate in FOXO3 LysM-cre mice and control mice over time. E) Group A streptococcal (GAS) survival in neutrophil killing assays using neutrophils isolated from either FOXO3 LysM-cre mice or C57bl/6 control mice. Percent survival is expressed relative to GAS survival with no neutrophils present.</p