The healthy human epidermis provides physical protection and is impenetrable for pathogenic microbes. Nevertheless, commensal and pathogen bacteria such as Staphylococcus aureus are able to colonize the skin surface, which may subsequently lead to infection. To identify and characterize regulatory elements facilitating adaptation of S. aureus to the human skin environment we used ex vivo tissue explants and quantified S. aureus gene transcription during co-culture. This analysis provided evidence for a significant downregulation of the global virulence regulator agr upon initial contact with skin, regardless of the growth phase of S. aureus prior to co-culture. In contrast, the alternative sigma factor B (sigB) and the antimicrobial peptide-sensing system (graRS) were expressed during early colonization. Consistently, sigB target genes such as the clumping factor A (clfA) and fibrinogen and fibronectin binding protein A (fnbA) were strongly upregulated upon skin contact. At later timepoints of the adhesion process, wall teichoic acid (WTA) synthesis was induced. Besides the expression of adhesive molecules, transcription of molecules involved in immune evasion were increased during late colonization (staphylococcal complement inhibitor and staphylokinase). Similar to nasal colonization, enzymes involved in cell wall metabolism (sceD and atlA) were highly transcribed. Finally, we detected a strong expression of proteases from all three catalytic classes during the entire colonization process. Taken together, we here present an ex vivo skin colonization model that allows the detailed characterization of the bacterial adaptation to the skin environment.

Microneedling therapy is a widely used technique in dermatology. However, little is known about the underlying molecular effects of this therapy on extracellular matrix remodeling, wound healing, and inflammation. The aim of this study was to examine morphological and molecular changes caused by microneedling treatment in a standardized in vitro full-thickness 3D model of human skin.

Secreted frizzled related protein 1 (SFRP1) functions as an important inhibitor of the Wnt pathway and is a known tumor suppressor gene, which is epigenetically silenced in a variety of tumors e.g. in breast cancer. However, it is still unclear how SFRP1 exactly affects the Wnt pathway. Our aim was to decipher SFRP1 involvement in biochemical signaling in dependency of different breast cancer subtypes and to identify novel SFRP1-regulated genes. We generated SFRP1 over-expressing in vitro breast cancer models, reflecting the two major subtypes by using basal-like BT20 and luminal-like HER2-positive SKBR3 cells. DNA microarray expression profiling of these models revealed that SFRP1 expression potentially modulates Bone morphogenetic protein- and Smoothened signaling (p<0.01), in addition to the known impact on Wnt signaling. Importantly, further statistical analysis revealed that in dependency of the cancer subtype model SFRP1 may affect the canonical and non-canonical Wnt pathway (p<0.01), respectively. While SFRP1 re-expression generally mediated distinct patterns of transcriptionally induced or repressed genes in BT20 and SKBR3 cells, brain derived neurotrophic factor (BDNF) was identified as a SFRP1 induced gene in both cell lines. Although BDNF has been postulated as a putative oncogene, the co-regulation with SFRP1 indicates a potential suppressive function in breast cancer. Indeed, a positive correlation between SFRP1 and BDNF protein expression could be shown (p<0.001) in primary breast cancer samples. Moreover, TCGA dataset based analysis clearly underscores that BDNF mRNA is down-regulated in primary breast cancer samples predicting a poor prognosis of these patients. In line, we functionally provide evidence that stable BDNF re-expression in basal-like BT20 breast cancer cells blocks tumor cell proliferation. Hence, our results suggest that BDNF might rather mediate suppressive than promoting function in human breast cancer whose mode of action should be addressed in future studies.

Non-melanoma skin cancer (NMSC) is the most common cancer in Caucasians worldwide. We investigated the pathophysiological role of MIF and its homolog D-DT in UVB- and chemically induced NMSC using Mif-/-, D-dt-/- and Mif-/-/D-dt-/- mice on a hairless SKH1 background. Knockout of both cytokines showed similar attenuating effects on inflammation after acute UVB irradiation and tumor formation during chronic UVB irradiation, without additive protective effects noted in double knockout mice, indicating that both cytokines activate a similar signaling threshold. In contrast, genetic deletion of Mif and D-dt had no major effects on chemically induced skin tumors. To get insight into the contributing mechanisms, we used an in vitro 3D skin model with incorporated macrophages. Application of recombinant MIF and D-DT led to an accumulation of macrophages within the epidermal part that could be reversed by selective inhibitors of MIF and D-DT pathways. In summary, our data indicate that MIF and D-DT contribute to the development and progression of UVB- but not chemically induced NMSC, a role at least partially accounted by effects of both cytokines on epidermal macrophage accumulation. These data highlight that MIF and D-DT are both potential therapeutic targets for the prevention of photocarcinogenesis but not chemical carcinogenesis.

Ablative fractional laser treatment leads to a loss of matrix metalloproteinase-3 (MMP-3) expression; therefore, in the present in vitro study, we addressed the role of MMP-3 and its regulation by calcium pantothenate in wound healing processes at the molecular level. Utilizing confocal laser microscopy, we investigated MMP-3 protein expression in fractional ablative CO2 laser-irradiated skin models. In addition, we established full-thickness 3D skin models using fibroblasts and keratinocytes with a MMP-3 knockdown that were irradiated with a fractional ablative Er:YAG laser to set superficial injuries with standardized dimensions and minimal thermal damage to the surrounding tissue. We revealed an upregulation of MMP-3 protein expression in laser-irradiated skin models receiving aftercare treatment with calcium pantothenate. Skin models with MMP-3 knockdown exhibited a slower wound closure after laser treatment compared to controls. Gene expression profiling detected an MMP-3 knockdown-dependent upregulation of cytokines and chemokines (e.g. IL-36B, CXCL17, IL-37, CXCL5), antimicrobial peptides (e.g., S100A7, S100A12), epidermal crosslinking enzymes (TGM5), and differentiation markers (e.g., LOR, KRT1, FLG2). We also detected a downregulation of cathepsin V and MMP-10, both of which play a prominent role in wound healing processes. After fractional ablative laser injury, an aftercare treatment with calcium pantothenate accelerated wound closure in MMP-3 expressing models faster than in MMP-3 knockdown models. Our data substantiate a major role of MMP-3 in wound healing processes after ablative laser treatments. For the first time, we could show that calcium pantothenate exerts its wound healing-promoting effects at least partly via MMP-3.

Psoriasis is a chronic skin disease affecting 2-3% of the global population. The proinflammatory IL-17A is a key cytokine in psoriasis. Accumulating evidence has revealed that IL-36γ plays also a pathogenic role. To understand more precisely the role of the IL-17A-IL-36γ cytokine network in skin pathology, we used an ear injection model. We injected IL-17A or IL-36γ alone and in combination into the ear pinnae of mice. This resulted in a significant increase in ear thickness measured over time. Histological evaluation of IL-17A + IL-36γ-treated skin showed a strong acanthosis, hyperparakeratosis and infiltration of neutrophils. The same histological features were found in mice after injection of IL-36γ alone, but to a lesser extent. IL-17A alone was not able to induce psoriasis-like changes. Genes encoding proteins of the S100 family, antimicrobial peptides and chemo-attractants for neutrophils were upregulated in the IL-17A + IL-36γ group. A much weaker expression was seen after the injection of each cytokine alone. These results strengthen the hypothesis that IL-17A and IL-36γ drive psoriatic inflammation via a synergistic interaction. Our established intradermal ear injection model can be utilized in the future to monitor effects of various inhibitors of this cytokine network.