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Cancer-associated fibroblasts (CAFs) interact reciprocally with tumor cells through various signaling pathways in many cancer types, including cutaneous squamous cell carcinoma. Among normal fibroblast subtypes, papillary fibroblasts (PFs) and reticular fibroblasts (RFs) respond distinctly to tumor cell signaling, eventuating the differentiation of RFs rather than PFs into CAFs. The regulation of subtype differentiation in fibroblasts remains poorly explored. In this study, we assessed the differences between PFs, RFs, and CAFs and examined the effects of small-molecule inhibitors targeting the TGFβ, phosphoinositide 3-kinase/protein kinase B/mTOR, and NOTCH pathways on the tumor-promoting property of CAFs and CAF reprogramming in 2-dimensional and 3-dimensional cultures. Blocking TGFβ and phosphoinositide 3-kinase strongly deactivated and concurrently induced a PF phenotype in RFs and CAFs. Three-dimensional coculturing of a cutaneous squamous cell carcinoma cell line MET2 with RFs or CAFs led to enhanced tumor invasion, RF–CAF transition, and cytokine production, which were further repressed by blocking TGFβ and phosphoinositide 3-kinase/mTOR pathways but not NOTCH pathway. In conclusion, the study identified biomarkers for PFs, RFs, and CAFs and displayed different effects of blocking key signaling pathways in CAFs and tumor cell–CAF interplay. These findings prompted a CAF-to-PF therapeutic strategy and provided perspectives of using included inhibitors in CAF-based cancer therapy.

Ubiquitin-specific protease 15 (USP15) plays a significant role in regulating various biological processes in several autoimmune diseases and cancers. However, its role in psoriatic keratinocytes (KCs) has not been extensively studied. In this study, we described that USP15 promotes proliferation and inflammation in KCs by stabilizing squamous cell carcinoma antigen 2. We discovered that the expression of USP15 and squamous cell carcinoma antigen 2 was elevated in lesions from patients with clinical psoriasis and an imiquimod-induced psoriatic dermatitis mouse model. USP15 was able to bind, deubiquitinate, and stabilize squamous cell carcinoma antigen 2. Knocking down USP15 resulted in reduced KC inflammation and impaired KC viability and clonogenicity. Topically applying USP15 small interfering RNA significantly ameliorated imiquimod-induced psoriatic dermatitis and reduced the infiltration of T cells and neutrophils. In addition, we determined that IL-22 was a key cytokine that upregulated the expression of USP15. These findings provide insights regarding the mechanisms involved in the proliferation and inflammation of KCs mediated by IL-22, suggesting a potential IL-22–USP15–squamous cell carcinoma antigen 2 axis in the pathogenesis of psoriatic KCs.

Objective:Full-thickness skin models comprise a three-dimensional dermal equivalent based on an animal-derived collagen matrix that harbors fibroblasts and an epidermal equivalent formed by keratinocytes. The functionality of both equivalents is influenced by many factors, including extracellular matrix composition and resident cell type. Animal-derived collagens differ in amino acid composition and physicochemical properties from human collagens. This composition could alter the functionality of the dermal equivalent and epidermal morphogenesis with the barrier formation in full-thickness models (FTMs). By replacement of animal-derived collagen for human collagen, we generated and characterized the animal material-free human collagen full-thickness models (hC-FTMs) that better mimic native dermal tissue.Materials and Methods:An isolation procedure to obtain soluble collagen from human abdominal dermis was developed. Both FTMs and hC-FTMs were generated with primary human fibroblasts and keratinocytes. Immunohistochemical analyses with biomarkers for the dermal matrix composition, basement membrane (BM) formation, epidermal proliferation, differentiation, and activation were performed. The stratum corneum (SC) lipid composition was studied with liquid chromatography–mass spectrometry. Lipid lamellar organization was determined by small-angle X-ray diffraction.Results:The FTMs and hC-FTMs exhibit many similarities, including the dermal matrix structure, BM formation, epidermal basal layer proliferation, and execution of differentiation programs. The SC contains a similar number of corneocyte layers and the same level of lipids. The ceramide chain length distribution and ceramide subclass profile showed only minor differences. Subsequently, this led to an unaltered lamellar organization.Conclusion:The animal material-free hC-FTM is generated successfully using collagens isolated from human abdominal dermis. Utilization of human collagens revealed that (epi-)dermal morphogenesis and lipid barrier formation resembled that of original FTMs. The hC-FTMs contain a dermal equivalent that mimics the native stromal tissue to a higher extent. Therefore these in vitroskin models can be used as promising tool for research purposes that contribute to animal-free experimentation.

ABSTRACTWe previously found the LL-37-derived peptide P60.4Ac to be effective against methicillin-resistant Staphylococcus aureus(MRSA) on human epidermal models (EMs). The goal of this study was to identify the preferred carrier for this peptide for topical application on skin and mucosal surfaces. We prepared P60.4Ac in three formulations, i.e., a water-in-oil cream with lanolin (Softisan 649), an oil-in-water cream with polyethylene glycol hexadecyl ether (Cetomacrogol), and a hydroxypropyl methylcellulose (hypromellose) 4000 gel. We tested the antimicrobial efficacy of the peptide in these formulations against mupirocin-resistant and -sensitive MRSA strains on EMs and bronchial epithelial models (BEMs). The cytotoxic effects of formulated P60.4Ac on these models were determined using histology and WST-1 and lactate dehydrogenase assays. Moreover, we assessed the stability of the peptide in these formulations with storage for up to 3 months. Killing of MRSA by P60.4Ac in the two creams was less effective than that by P60.4Ac in the hypromellose gel. In agreement with those findings, P60.4Ac in the hypromellose gel was highly effective in eradicating the two MRSA strains from EMs. We found that even 0.1% (wt/wt) P60.4Ac in the hypromellose gel killed >99% of the viable planktonic bacteria and >85% of the biofilm-associated bacteria on EMs. Hypromellose gels containing 0.1% and 0.5% (wt/wt) P60.4Ac effectively reduced the numbers of viable MRSA cells from BEMs by >90%. No cytotoxic effects of P60.4Ac in the hypromellose gel with up to 2% (wt/wt) P60.4Ac on keratinocytes in EMs and in the hypromellose gel with up to 0.5% (wt/wt) P60.4Ac on epithelial cells in BEMs were observed. High-performance liquid chromatography analysis showed that P60.4Ac was stable in the Softisan cream and the hypromellose gel but not in the Cetomacrogol cream. We conclude that P60.4Ac formulated in hypromellose gel is both stable and highly effective in eradicating MRSA from colonized EMs and BEMs.

ABSTRACTBurn wound infections are often difficult to treat due to the presence of multidrug-resistant bacterial strains and biofilms. Currently, mupirocin is used to eradicate methicillin-resistant Staphylococcus aureus(MRSA) from colonized persons; however, mupirocin resistance is also emerging. Since we consider antimicrobial peptides to be promising candidates for the development of novel anti-infective agents, we studied the antibacterial activities of a set of synthetic peptides against different strains of S. aureus, including mupirocin-resistant MRSA strains. The peptides were derived from P60.4Ac, a peptide based on the human cathelicidin LL-37. The results showed that peptide 10 (P10) was the only peptide more efficient than P60.4Ac, which is better than LL-37, in killing MRSA strain LUH14616. All three peptides displayed good antibiofilm activities. However, both P10 and P60.4Ac were more efficient than LL-37 in eliminating biofilm-associated bacteria. No toxic effects of these three peptides on human epidermal models were detected, as observed morphologically and by staining for mitochondrial activity. In addition, P60.4Ac and P10, but not LL-37, eradicated MRSA LUH14616 and the mupirocin-resistant MRSA strain LUH15051 from thermally wounded human skin equivalents (HSE). Interestingly, P60.4Ac and P10, but not mupirocin, eradicated LUH15051 from the HSEs. None of the peptides affected the excretion of interleukin 8 (IL-8) by thermally wounded HSEs upon MRSA exposure. In conclusion, the synthetic peptides P60.4Ac and P10 appear to be attractive candidates for the development of novel local therapies to treat patients with burn wounds infected with multidrug-resistant bacteria.

Human skin equivalents (HSEs) are three-dimensional culture models that are used as a model for native human skin. In this study the barrier properties of two novel HSEs, the fibroblast-derived matrix model (FDM) and the Leiden epidermal model (LEM), were compared with the full-thickness collagen model (FTM) and human skin. Since the main skin barrier is located in the lipid regions of the upper layer of the skin, the stratum corneum (SC), we investigated the epidermal morphology, expression of differentiation markers, SC permeability, lipid composition, and lipid organization of all HSEs and native human skin. Our results demonstrate that the barrier function of the FDM and LEM improved compared with that of the FTM, but all HSEs are more permeable than human skin. Further, the FDM and LEM have a relatively lower free fatty acid content than the FTM and human skin. Several similarities between the FDM, LEM and FTM were observed: (1) the morphology and the expression of the investigated differentiation markers were similar to those observed in native human skin, except for the observed expression of keratin 16 and premature expression of involucrin that were detected in all HSEs, (2) the lipids in the SC of all HSEs were arranged in lipid lamellae, similar to human skin, but show an increase in the number of lipid lamellae in the intercellular regions and (3) the SC lipids of all HSEs show a less densely packed lateral lipid organization compared with human SC. These findings indicate that the HSEs mimic many aspects of native human skin, but differ in their barrier properties.

Recessive epidermolysis bullosa simplex (REBS) is characterized by generalized cutaneous blistering in response to mechanical trauma. This results from fragility of the basal keratinocytes that lack keratin tonofilaments because of homozygote null mutation in the keratin 14 gene. REBS patients display in addition focal dyskeratotic skin lesions with histology of epidermolytic hyperkeratosis (EHK) and tonofilament clumping in the suprabasal layers of the epidermis. In this study we examined whether it is possible to mimic in vitrothe bullous and dyskeratotic cellular phenotype. For this purpose, fibroblasts from nondyskeratotic (K14−/−) and dyskeratotic (K14−/−) skin of a REBS patient and fibroblasts from a healthy donor (K14+/+) were isolated and incorporated into collagen matrices. Subsequently, fresh biopsies originating from the nondyskeratotic and dyskeratotic skin of the patient and from a healthy donor were placed onto the collagen matrices and cultured at the air-liquid interface. Epidermal morphogenesis was evaluated on the basis of tissue morphology and the expression of a series of keratins. The results of the present study indicate that basal cell vacuolization in REBS can be mimicked in vitrobut not the EHK. Fibroblasts seem to play an important regulatory role in establishing the REBS phenotype. These findings suggest that wild-type fibroblasts may enhance the stability of K14−/− keratinocytes in vitro.

During aging, skin accumulates senescent cells. The transient presence of senescent cells, followed by their clearance by the immune system, is important in tissue repair and homeostasis. The persistence of senescent cells that evade clearance contributes to the age-related deterioration of the skin. The senescence-associated secretory phenotype of these cells contains immunomodulatory molecules that facilitate clearance but also promote chronic damage. Here, we investigated the epilipidome—the oxidative modifications of phospholipids—of senescent dermal fibroblasts, because these molecules are among the bioactive lipids that were recently identified as senescence-associated secretory phenotype factors. Using replicative- and stress- induced senescence protocols, we identified lysophosphatidylcholines as universally elevated in senescent fibroblasts, whereas other oxidized lipids displayed a pattern that was characteristic for the used senescence protocol. When we tested the lysophosphatidylcholines for senescence-associated secretory phenotype activity, we found that they elicit chemokine release in nonsenescent fibroblasts but also interfere with toll-like receptor 2 and 6/CD36 signaling and phagocytic capacity in macrophages. Using matrix-assisted laser desorption/ionization Fourier transform ion cyclotron resonance mass spectrometry imaging, we localized two lysophosphatidylcholine species in aged skin. This suggests that lysophospholipids may facilitate immune evasion and low-grade chronic inflammation in skin aging.