RESUMO
Hand eczema is a common inflammatory skin condition of the hands whose pathogenesis is largely unknown. More insight and knowledge of the disease on a more fundamental level might lead to a better understanding of the biological processes involved, which could provide possible new treatment strategies. We aimed to profile the transcriptome of lesional palmar epidermal skin of patients suffering from vesicular hand eczema using RNA-sequencing. RNA-sequencing was performed to identify differentially expressed genes in lesional vs. non-lesional palmar epidermal skin from a group of patients with vesicular hand eczema compared to healthy controls. Comprehensive real-time quantitative PCR analyses and immunohistochemistry were used for validation of candidate genes and protein profiles for vesicular hand eczema. Overall, a significant and high expression of genes/proteins involved in keratinocyte host defense and inflammation was found in lesional skin. Furthermore, we detected several molecules, both up or downregulated in lesional skin, which are involved in epidermal differentiation. Immune signalling genes were found to be upregulated in lesional skin, albeit with relatively low expression levels. Non-lesional patient skin showed no significant differences compared to healthy control skin. Lesional vesicular hand eczema skin shows a distinct expression profile compared to non-lesional skin and healthy control skin. Notably, the overall results indicate a large overlap between vesicular hand eczema and earlier reported atopic dermatitis lesional transcriptome profiles, which suggests that treatments for atopic dermatitis could also be effective in (vesicular) hand eczema.
Assuntos
Eczema/fisiopatologia , Dermatoses da Mão/fisiopatologia , Adulto , Idoso , Estudos de Casos e Controles , Eczema/genética , Feminino , Dermatoses da Mão/genética , Humanos , Masculino , Pessoa de Meia-Idade , Transcriptoma , Adulto JovemRESUMO
The epidermal compartment of the skin is regenerated constantly by proliferation of epidermal keratinocytes. Differentiation of a subset of these keratinocytes allows the epidermis to retain its barrier properties. Regulation of keratinocyte fate-whether to remain proliferative or terminally differentiate-is complex and not fully understood. The objective of our study was to assess if DNA methylation changes contribute to the regulation of keratinocyte fate. We employed genome-wide MethylationEPIC beadchip array measuring approximately 850 000 probes combined with RNA sequencing of in vitro cultured non-differentiated and terminally differentiated adult human primary keratinocytes. We did not observe a correlation between methylation status and transcriptome changes. Moreover, only two differentially methylated probes were detected, of which one was located in the TRIM29 gene. Although TRIM29 knock-down resulted in lower expression levels of terminal differentiation genes, these changes were minor. From these results, we conclude that-in our in vitro experimental setup-it is unlikely that changes in DNA methylation have an important regulatory role in terminal keratinocyte differentiation.
Assuntos
Diferenciação Celular/genética , Metilação de DNA/genética , Epigenoma/genética , Queratinócitos/metabolismo , Adulto , Proteínas de Ligação a DNA/genética , Humanos , Fatores de Transcrição/genéticaRESUMO
In biomedical research, cell culture contamination is one of the main culprits of experimental failure. Contamination sources and concomitant remedies are numerous and challenging to manage. We herein describe two cases of uncommon contamination of cell cultures that we encountered, and the successful determination and eradication strategies. The first case describes the infection with human adenovirus C that originated from pharyngeal tonsils used for isolation of primary tonsillar epithelial cells. It is known that viral contamination of in vitro cell cultures can occur symptomless and is therefore difficult to identify. The contamination was pervasive and persistent, as it was widely spread in flow cabinets and apparatus, and has caused a serious delay to our research projects and the inevitable loss of valuable (patient-derived) cell sources. Eradication was successful by formalin gas sterilization of the flow cabinet and elimination of all infected cell lines from our biobank after PCR-guided determination. Secondly, we encountered a spore-forming bacterium, namely Brevibacillus brevis, in our cell culture facility. This bacterium originated from contaminated tap water pipes and spread via regular aseptic culture techniques due to survival of the bacterial spores in 70% ethanol. B brevis overgrew the cultures within a few days after seeding of the primary cells. Chlorine solution effectively killed this spore-forming bacterium. Both cases of contamination were identified using DNA sequencing which enabled the deployment of targeted aseptic techniques for the elimination of the persistent contamination.
Assuntos
Adenovírus Humanos , Brevibacillus , Cultura Primária de Células , Tonsila Faríngea/citologia , Tonsila Faríngea/virologia , Adenovírus Humanos/isolamento & purificação , Brevibacillus/isolamento & purificação , DNA Bacteriano/análise , DNA Viral/análise , Descontaminação/métodos , Células Epiteliais , Contaminação de Equipamentos , Humanos , Engenharia Sanitária , Análise de Sequência de DNA , Microbiologia da ÁguaRESUMO
PURPOSE: We aimed to assess the biological and clinical significance of the human cysteine protease inhibitor cystatin M/E, encoded by the CTS6 gene, in diseases of human hair and skin. METHODS: Exome and Sanger sequencing was performed to reveal the genetic cause in two related patients with hypotrichosis. Immunohistochemical, biophysical, and biochemical measurements were performed on patient skin and 3D-reconstructed skin from patient-derived keratinocytes. RESULTS: We identified a homozygous variant c.361C>T (p.Gln121*), resulting in a premature stop codon in exon 2 of CST6 associated with hypotrichosis, eczema, blepharitis, photophobia and impaired sweating. Enzyme assays using recombinant mutant cystatin M/E protein, generated by site-directed mutagenesis, revealed that this p.Gln121* variant was unable to inhibit any of its three target proteases (legumain and cathepsins L and V). Three-dimensional protein structure prediction confirmed the disturbance of the protease/inhibitor binding sites of legumain and cathepsins L and V in the p.Gln121* variant. CONCLUSION: The herein characterized autosomal recessive hypotrichosis syndrome indicates an important role of human cystatin M/E in epidermal homeostasis and hair follicle morphogenesis.
Assuntos
Alopecia/congênito , Cistatina M/deficiência , Cistatina M/genética , Inibidores de Cisteína Proteinase/metabolismo , Dermatopatias/genética , Alopecia/genética , Criança , Consanguinidade , Feminino , Humanos , Mutação com Perda de Função , Masculino , Sequenciamento do ExomaRESUMO
The skin barrier is an important shield regulating the outside-in as well as inside-out penetration of water, nutrients, ions and environmental stimuli. We can distinguish four different barrier compartments: the physical, chemical, immunological and microbial skin barrier. Well-functioning of those is needed to protect our body from the environment. To better understand the function and the contribution of barrier dysfunction in skin diseases, 3D skin or epidermal models are a valuable tool for in vitro studies. In this review, we summarize the development and application of different skin models in skin barrier research. During the last years, enormous effort was made on optimizing these models to better mimic the in vivo composition of the skin, by fine-tuning cell culture media, culture conditions and including additional cells and tissue components. Thereby, in vitro barrier formation and function has been improved significantly. Moreover, in this review we point towards changes and chances for in vitro 3D skin models to be used for skin barrier research in the nearby future.
Assuntos
Alternativas ao Uso de Animais , Modelos Biológicos , Pele/metabolismo , Humanos , Técnicas In Vitro , Microbiota , Permeabilidade , Pele/microbiologia , Junções ÍntimasRESUMO
Deficiency of the cysteine protease inhibitor cystatin M/E (Cst6) in mice leads to disturbed epidermal cornification, impaired barrier function, and neonatal lethality. We report the rescue of the lethal skin phenotype of ichq (Cst6-deficient; Cst6-/-) mice by transgenic, epidermis-specific, reexpression of Cst6 under control of the human involucrin (INV) promoter. Rescued Tg(INV-Cst6)Cst6ichq/ichq mice survive the neonatal phase, but display severe eye pathology and alopecia after 4 mo. We observed keratitis and squamous metaplasia of the corneal epithelium, comparable to Cst6-/-Ctsl+/- mice, as we have reported in other studies. We found the INV promoter to be active in the hair follicle infundibulum; however, we did not observe Cst6 protein expression in the lower regions of the hair follicle in Tg(INV-Cst6)Cst6ichq/ichq mice. This result suggests that unrestricted activity of proteases is involved in disturbance of hair follicle biology, eventually leading to baldness. Using quenched activity-based probes, we identified mouse cathepsin B (CtsB), which is expressed in the lower regions of the hair follicle, as an additional target of mouse Cst6. These data suggest that Cst6 is necessary to control CtsB activity in hair follicle morphogenesis and highlight Cst6-controlled proteolytic pathways as targets for preventing hair loss.-Oortveld, M. A. W., van Vlijmen-Willems, I. M. J. J., Kersten, F. F. J., Cheng, T., Verdoes, M., van Erp, P. E. J., Verbeek, S., Reinheckel, T., Hendriks, W. J. A. J., Schalkwijk, J., Zeeuwen, P. L. J. M. Cathepsin B as a potential cystatin M/E target in the mouse hair follicle.
Assuntos
Catepsina B/metabolismo , Diferenciação Celular/fisiologia , Cistatina M/metabolismo , Epiderme/metabolismo , Folículo Piloso/metabolismo , Alopecia/metabolismo , Animais , Catepsina L/metabolismo , Células Cultivadas , Cistatina M/deficiência , Humanos , Camundongos , Pele/metabolismoRESUMO
BACKGROUND: Defensins are antimicrobial peptides that exert immunomodulatory and chemotactic functions. Based on these properties and their high expression levels in the skin, they are likely to affect skin inflammation, infection, and wound healing. This may lead to therapeutic applications in (burn) wound healing. OBJECTIVE: We aimed to investigate the effects of human ß-defensins (hBDs) on keratinocytes and fibroblasts, 2 major skin cell types involved in skin regeneration. METHODS: Monolayer keratinocyte and fibroblast cultures were exposed to recombinant hBDs, and we overexpressed hBD2 and hBD3 in keratinocytes of reconstructed epidermal equivalents by lentiviral transduction. The effects were measured by immunohistochemistry, quantitative real-time PCR, and migration assays. Kinome analyses were performed on cultured keratinocytes to investigate the signal transduction events elicited by hBD stimulation. RESULTS: We found that hBD3 induced the expression of cytokines and chemokines in keratinocytes, which was not observed in fibroblasts. hBD2, however, stimulated cell migration only in fibroblasts, which was not found for hBD3. Both defensins are likely to exert receptor-mediated effects in keratinocytes, as witnessed by changes in protein kinase activation following stimulation by hBD2 and hBD3. Kinome analysis suggested that protein kinase C activation was a common event for both defensins. We observed, however, considerable differences in keratinocyte responses between stimulation by exogenous recombinant defensins and endogenous defensins expressed following lentiviral transduction. CONCLUSION: Defensins exert modest biological effects on skin cells that are potentially beneficial in wound healing, but many questions regarding the biological mechanisms of action and relevance for the in vivo situation are still remaining.
Assuntos
Fibroblastos/efeitos dos fármacos , Queratinócitos/efeitos dos fármacos , beta-Defensinas/genética , beta-Defensinas/farmacologia , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Células Cultivadas , Citocinas , Ativação Enzimática/efeitos dos fármacos , Fibroblastos/metabolismo , Humanos , Queratinócitos/metabolismo , Proteína Quinase C/metabolismo , Proteínas Recombinantes/farmacologia , Transdução de Sinais/efeitos dos fármacos , beta-Defensinas/metabolismoRESUMO
The diversity and dynamics of the skin microbiome in health and disease have been studied recently, but adequate model systems to study skin microbiotas in vitro are largely lacking. We developed an in vitro system that mimics human stratum corneum, using human callus as substrate and nutrient source for bacterial growth. The growth of several commensal and pathogenic bacterial strains was measured for up to one week by counting colony-forming units or by quantitative PCR with strain-specific primers. Human skin pathogens were found to survive amidst a minimal microbiome consisting of 2 major skin commensals: Staphylococcus epidermidis and Propionibacterium acnes. In addition, complete microbiomes, taken from the backs of healthy volunteers, were inoculated and maintained using this system. This model may enable the modulation of skin microbiomes in vitro and allow testing of pathogens, biological agents and antibiotics in a medium-throughput format.
Assuntos
Calo Ósseo/microbiologia , Propionibacterium acnes/crescimento & desenvolvimento , Pele/microbiologia , Staphylococcus epidermidis/crescimento & desenvolvimento , Humanos , Técnicas In Vitro , Testes de Sensibilidade Microbiana , Microbiota , Reação em Cadeia da Polimerase , Propionibacterium acnes/efeitos dos fármacos , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/crescimento & desenvolvimento , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/crescimento & desenvolvimento , Staphylococcus epidermidis/efeitos dos fármacos , Células-Tronco , Streptococcus pyogenes/efeitos dos fármacos , Streptococcus pyogenes/crescimento & desenvolvimento , Tetraciclina/farmacologiaRESUMO
BACKGROUND: Previous research revealed heterogeneity in the perfusion intensity within clinically homogenous-appearing plaques, without differences in erythema. In addition, an increased perfusion was found within the perilesional skin. This raises the question whether the heterogeneity in perfusion found both inside and outside a lesion influences the expression levels of genes and proteins involved in the pathogenesis of psoriasis. OBJECTIVES: To correlate the perfusion intensity to mRNA and protein expression of genes associated with the pathogenesis of psoriasis and to visualize the dynamics of the perfusion intensity over time using laser Doppler perfusion imaging. METHODS: Fourteen patients with plaque psoriasis were included. The superficial microcirculation and clinical local scores (single usability metric, SUM, scores) were analysed in one representative lesion every 2 weeks. After 8 weeks 4 biopsies were taken, one from a highly perfused area (hotspot) and one from a low perfusion area (coldspot) of the lesional skin, one biopsy from the highly perfused perilesional skin and one from the distant uninvolved skin. RESULTS: Statistically significant differences in mRNA and protein expression, including IL-17 and TBX21/T-Bet, were found between hotspots and coldspots, and between the highly perfused perilesional and the uninvolved skin. Hotspots tend to remain on the same location during 8 weeks of follow-up. CONCLUSIONS: Within homogenous-appearing psoriatic plaques, there are remarkable differences in mRNA and protein levels, which are correlated with the perfusion intensity and can be detected by using laser Doppler perfusion imaging. In addition, differences in mRNA and protein expression between the highly perfused perilesional skin and the uninvolved skin were found, indicating that several biological changes occur well before clinical changes become manifest.
Assuntos
Microcirculação , Psoríase/metabolismo , Psoríase/fisiopatologia , Pele/irrigação sanguínea , Pele/metabolismo , Adulto , Idoso , Complexo CD3/genética , Complexo CD3/metabolismo , Elafina/genética , Elafina/metabolismo , Feminino , Expressão Gênica , Humanos , Interleucina-17/genética , Interleucina-17/metabolismo , Interleucina-8/genética , Interleucina-8/metabolismo , Queratina-16/genética , Queratina-16/metabolismo , Masculino , Pessoa de Meia-Idade , Molécula-1 de Adesão Celular Endotelial a Plaquetas/genética , Molécula-1 de Adesão Celular Endotelial a Plaquetas/metabolismo , RNA Mensageiro/metabolismo , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , beta-Defensinas/genética , beta-Defensinas/metabolismoAssuntos
Candidíase Mucocutânea Crônica/imunologia , Interferon gama/imunologia , Queratinócitos/metabolismo , Queratinócitos/patologia , Fator de Transcrição STAT1/genética , Candidíase Mucocutânea Crônica/genética , Candidíase Mucocutânea Crônica/metabolismo , Células Cultivadas , Proteínas Ricas em Prolina do Estrato Córneo/metabolismo , Mutação com Ganho de Função , Humanos , Transdução de Sinais/imunologia , Pele/imunologiaRESUMO
Deletion of two members of the late cornified envelope (LCE) family, LCE3B and LCE3C (LCE3C_LCE3B-del), has been identified as risk factor for psoriasis with a possible role in skin barrier function. Moreover, genetic interaction between LCE3C_LCE3B-del and HLA-C*06, located in the psoriasis susceptibility regions 4 and 1 (PSORS4 and 1), has been reported in several populations. Because of high linkage disequilibrium between the PSORS1 genes HLA-C*06 and corneodesmosin (CDSN), both genes are potentially involved in psoriasis. As corneodesmosin and LCE proteins are both constituents of the stratum corneum, we investigated potential direct protein-protein interactions between six LCE proteins and two corneodesmosin sequence variants. Partial colocalization of LCE2 and CDSN was observed in normal and psoriasis skin using immunofluorescence microscopy. Co-expression of eCFP-LCE and mRFP-CDSN proteins in COS-1 cells and human adult keratinocytes, and GST pull-down results did not provide evidence for direct interactions between LCE proteins and CDSN variants.
Assuntos
Proteínas Ricas em Prolina do Estrato Córneo/metabolismo , Glicoproteínas/metabolismo , Animais , Células COS , Chlorocebus aethiops , Proteínas Ricas em Prolina do Estrato Córneo/química , Proteínas Ricas em Prolina do Estrato Córneo/genética , Variação Genética , Glicoproteínas/química , Glicoproteínas/genética , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Queratinócitos/metabolismo , Desequilíbrio de Ligação , Mapeamento de Interação de Proteínas , Psoríase/genética , Psoríase/metabolismo , Fatores de Risco , Pele/metabolismoRESUMO
This study aims to investigate associations between observed clinical parameters and known genetic risk factors of psoriasis in a well-defined prospective cohort of paediatric patients with plaque psoriasis (n = 151). Significant associations were found for paediatric-onset psoriasis with ERAP1 (p = 0.002), IL23R (p = 0.01), LCE3C_LCE3B-del (p = 0.00049) and HLA-C*06 (p = 3.15 × 10(-30)). Psoriasis severity was associated with the single nucleotide polymorphisms tagging IFIH1 and ERAP1 (p < 0.05). An onset before 10 years of age was associated with IL12B (p = 0.02). Nail psoriasis was more often seen in HLA-C*06-negative patients (p = 0.008). Remarkably, family history is clearly not associated with HLA-C*06 in this specific group. The large proportion of patients with a positive family history in HLA-C*06 negative patients (and the lack of correlation between the two) indicates that other genes, either alone or interaction between two or more genes, may have significant effects on heritability.
Assuntos
Antígenos HLA-C/genética , Psoríase/genética , Adolescente , Idade de Início , Estudos de Casos e Controles , Criança , Pré-Escolar , Feminino , Estudos de Associação Genética , Predisposição Genética para Doença , Hereditariedade , Humanos , Masculino , Países Baixos/epidemiologia , Linhagem , Fenótipo , Estudos Prospectivos , Psoríase/diagnóstico , Psoríase/epidemiologia , Psoríase/imunologia , Sistema de Registros , Fatores de Risco , Índice de Gravidade de DoençaRESUMO
The aryl hydrocarbon receptor (AHR) is an evolutionary conserved environmental sensor identified as an indispensable regulator of epithelial homeostasis and barrier organ function. Molecular signaling cascade and target genes upon AHR activation and their contribution to cell and tissue function are however not fully understood. Multiomics analyses using human skin keratinocytes revealed that upon ligand activation, AHR binds open chromatin to induce expression of transcription factors, for example, TFAP2A, as a swift response to environmental stimuli. The terminal differentiation program, including upregulation of barrier genes, FLG and keratins, was mediated by TFAP2A as a secondary response to AHR activation. The role of AHR-TFAP2A axis in controlling keratinocyte terminal differentiation for proper barrier formation was further confirmed using CRISPR/Cas9 in human epidermal equivalents. Overall, the study provides additional insights into the molecular mechanism behind AHR-mediated barrier function and identifies potential targets for the treatment of skin barrier diseases.
RESUMO
The emergence of resistance against current antibiotics calls for the development of new compounds to treat infectious diseases. Synthetic pantothenamides are pantothenate analogs that possess broad-spectrum antibacterial activity in vitro in minimal media. Pantothenamides were shown to be substrates of the bacterial coenzyme A (CoA) biosynthetic pathway, causing cellular CoA depletion and interference with fatty acid synthesis. In spite of their potential use and selectivity for bacterial metabolic routes, these compounds have never made it to the clinic. In the present study, we show that pantothenamides are not active as antibiotics in the presence of serum, and we found that they were hydrolyzed by ubiquitous pantetheinases of the vanin family. To address this further, we synthesized a series of pantetheinase inhibitors based on a pantothenate scaffold that inhibited serum pantetheinase activity in the nanomolar range. Mass spectrometric analysis showed that addition of these pantetheinase inhibitors prevented hydrolysis of pantothenamides by serum. We found that combinations of these novel pantetheinase inhibitors and prototypic pantothenamides like N5-Pan and N7-Pan exerted antimicrobial activity in vitro, particularly against Gram-positive bacteria (Staphylococcus aureus, Staphylococcus epidermidis, Streptococcus pneumoniae, and Streptococcus pyogenes) even in the presence of serum. These results indicate that pantothenamides, when protected against degradation by host pantetheinases, are potentially useful antimicrobial agents.
Assuntos
Antibacterianos/farmacologia , Bactérias Gram-Positivas/efeitos dos fármacos , Ácido Pantotênico/farmacologia , Antibacterianos/química , Testes de Sensibilidade Microbiana , Ácido Pantotênico/análogos & derivados , Ácido Pantotênico/química , Staphylococcus aureus/efeitos dos fármacos , Streptococcus pneumoniae/efeitos dos fármacos , Streptococcus pyogenes/efeitos dos fármacosAssuntos
Células Epidérmicas/metabolismo , Epiderme/metabolismo , Proteínas de Filamentos Intermediários/deficiência , Queratinócitos/metabolismo , Alelos , Animais , Diferenciação Celular , Proliferação de Células , Proteínas Filagrinas , Técnicas de Inativação de Genes , Humanos , Camundongos , PermeabilidadeRESUMO
In atopic dermatitis (AD), chronic skin inflammation is associated with skin barrier defects and skin microbiome dysbiosis including a lower abundance of Gram-positive anaerobic cocci (GPACs). We here report that, through secreted soluble factors, GPAC rapidly and directly induced epidermal host-defense molecules in cultured human keratinocytes and indirectly via immune-cell activation and cytokines derived thereof. Host-derived antimicrobial peptides known to limit the growth of Staphylococcus aureus-a skin pathogen involved in AD pathology-were strongly upregulated by GPAC-induced signaling through aryl hydrocarbon receptor (AHR)-independent mechanisms, with a concomitant AHR-dependent induction of epidermal differentiation genes and control of pro-inflammatory gene expression in organotypic human epidermis. By these modes of operandi, GPAC may act as an "alarm signal" and protect the skin from pathogenic colonization and infection in the event of skin barrier disruption. Fostering growth or survival of GPAC may be starting point for microbiome-targeted therapeutics in AD.