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Despite recent advances in understanding skin scarring, mechanisms triggering hypertrophic scar formation are still poorly understood. In the present study, we investigate mature human hypertrophic scars and developing scars in mice at single cell resolution. Compared to normal skin, we find significant differences in gene expression in most cell types present in scar tissue. Fibroblasts show the most prominent alterations in gene expression, displaying a distinct fibrotic signature. By comparing genes upregulated in murine fibroblasts during scar development with genes highly expressed in mature human hypertrophic scars, we identify a group of serine proteases, tentatively involved in scar formation. Two of them, dipeptidyl-peptidase 4 (DPP4) and urokinase (PLAU), are further analyzed in functional assays, revealing a role in TGFβ1-mediated myofibroblast differentiation and over-production of components of the extracellular matrix in vitro. Topical treatment with inhibitors of DPP4 and PLAU during scar formation in vivo shows anti-fibrotic activity and improvement of scar quality, most prominently after application of the PLAU inhibitor BC-11. In this study, we delineate the genetic landscape of hypertrophic scars and present insights into mechanisms involved in hypertrophic scar formation. Our data suggest the use of serine protease inhibitors for the treatment of skin fibrosis.

Burn injuries elicit a unique and dynamic stress response which can lead to burn injury progression. Though neutrophils represent crucial players in the burn-induced immunological events, the dynamic secretion pattern and systemic levels of neutrophil-derived factors have not been investigated in detail so far. Serum levels of neutrophil elastase (NE), myeloperoxidase (MPO), citrullinated histone H3 (CitH3), and complement factor C3a were quantified in burn victims over 4 weeks post injury. Furthermore, the potential association with mortality, degree of burn injury, and inhalation trauma was evaluated. In addition, leukocyte, platelet, neutrophil, and lymphocyte counts were assessed. Lastly, we analyzed the association of neutrophil-derived factors with clinical severity scoring systems. Serum levels of NE, MPO, CitH3, and C3a were remarkably elevated in burn victims compared to healthy controls. Leukocyte and neutrophil counts were significantly increased on admission day and day 1, while relative lymphocytes were decreased in the first 7 days post burn trauma. Though neutrophil-derived factors did not predict mortality, patients suffering from 3rd degree burn injuries displayed increased CitH3 and NE levels. Accordingly, CitH3 and NE were elevated in cases with higher abbreviated burn severity indices (ABSI). Taken together, our data suggest a role for neutrophil activation and NETosis in burn injuries and burn injury progression. Targeting exacerbated neutrophil activation might represent a new therapeutic option for severe cases of burn injury.

Recently, a specific Schwann cell type with profibrotic and tissue regenerative properties that contributes to keloid formation has been identified. In the present study, we reanalyzed published single-cell RNA sequencing (scRNA-seq) studies of keloids, healthy skin, and normal scars to reliably determine the specific gene expression profile of keloid-specific Schwann cell types in more detail. We were able to confirm the presence of the repair-like, profibrotic Schwann cell type in the datasets of all three studies and identified a specific gene-set for these Schwann cells. In contrast to keloids, in normal scars, the number of Schwann cells was not increased, nor was their gene expression profile distinctly different from that of Schwann cells of normal skin. In addition, our bioinformatics analysis provided evidence for a role of transcription factors of the AP1, STAT, and KLF families, and members of the IER genes in the dedifferentiation process of keloidal Schwann cells. Together, our analysis strengthens the role of the profibrotic Schwann cell type in the formation of keloids. Knowledge of the exact gene expression profile of these Schwann cells will facilitate their identification in other organs and diseases.

The endoplasmic reticulum-associated protein reticulon 1A (RTN1A) is primarily expressed in neuronal tissues but was recently identified also specifically in cells of the dendritic cell (DC) lineage, including epidermal Langerhans cells (LCs) and dermal DCs in human skin. In this study, we found that in mice major histocompatibility complex class II (MHCII)+CD207+ LCs but not dermal DCs express RTN1A. Further, RTN1A expression was identified in CD45+MHCII+CD207+ cells of the lymph node and spleen but not in the thymus. Of note, RTN1A was expressed in CD207low LCs in adult skin as well as emigrated LCs and DCs in lymph nodes and marginally in CD207hi cells. Ontogeny studies revealed that RTN1A expression occurred before the appearance of the LC markers MHCII and CD207 in LC precursors, while dermal DC and T cell precursors remained negative during skin development. Analogous to the expression of RTN1A in neural tissue, we identified expression of RTN1A in skin nerves. Immunostaining revealed co-localization of RTN1A with nerve neurofilaments only in fetal but not in newborn or adult dermis. Our findings suggest that RTN1A might be involved in the LC differentiation process given its early expression in LC precursors and stable expression onward. Further analysis of the RTN1A expression pattern will enable the elucidation of the functional roles of RTN1A in both the immune and the nervous system of the skin.

Objectives Burn injuries elicit a unique and dynamic stress response which can lead to burn injury progression. Though neutrophils represent crucial players in the burn-induced immunological events, the dynamic secretion pattern and systemic levels of neutrophil-derived factors have not been investigated in detail so far. Methods Serum levels of neutrophil elastase (NE), myeloperoxidase (MPO), citrullinated histone H3 (CitH3), and complement factor C3a were quantified in burn victims over 4 weeks post injury. Furthermore, the potential association with mortality, degree of burn injury, and inhalation trauma was evaluated. In addition, leukocyte, platelet, neutrophil, and lymphocyte counts were assessed. Lastly, we analyzed the association of neutrophil-derived factors with clinical severity scoring systems. Results Serum levels of NE, MPO, CitH3, and C3a were remarkably elevated in burn victims compared to healthy controls. Leukocyte and neutrophil counts were significantly increased on admission day and day 1, while relative lymphocytes were decreased in the first 7 days post burn trauma. Though neutrophil-derived factors did not predict mortality, patients suffering from 3rd degree burn injuries displayed increased CitH3 and NE levels. Accordingly, CitH3 and NE were elevated in cases with higher abbreviated burn severity indices (ABSI). Conclusions Taken together, our data suggest a role for neutrophil activation and NETosis in burn injuries and burn injury progression. Targeting exacerbated neutrophil activation might represent a new therapeutic option for severe cases of burn injury.

Neutrophil extracellular trap (NET)-formation represents an important defence mechanism for the rapid clearance of infections. However, exaggerated NET formation has been shown to negatively affect tissue-regeneration after injury. As our previous studies revealed the strong tissue-protective and regenerative properties of the secretome of stressed peripheral blood mononuclear cells (PBMCsec), we here investigated the influence of PBMCsec on the formation of NETs. The effect of PBMCsec on NET formation was assessed ex vivo in ionomycin stimulated neutrophils derived from healthy donors using flow cytometry, image stream analysis, and quantification of released extracellular DNA. The effect of PBMCsec on molecular mechanisms involved in NET formation, including Ca-flux, protein kinase C activity, reactive oxygen species production, and protein arginine deiminase 4 activity, were analysed. Our results showed that PBMCsec significantly inhibited NET formation. Investigation of the different biological substance classes found in PBMCsec revealed only a partial reduction in NET formation, suggesting a synergistic effect. Mechanistically, PBMCsec treatment did not interfere with calcium signalling and PKC-activation, but exerted anti-oxidant activity, as evidenced by reduced levels of reactive oxygen species and upregulation of heme oxygenase 1 and hypoxia inducible-factor 1 in PBMCsec-treated neutrophils. In addition, PBMCsec strongly inhibited the activation of protein arginine deiminase 4 (PAD4), ultimately leading to the inhibition of NET formation. As therapeutics antagonizing excessive NET formation are not currently available, our study provides a promising novel treatment option for a variety of conditions resulting from exaggerated NET formation.

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