RESUMO
The transmembrane protein claudin-1 is critical for formation of the epidermal barrier structure called tight junctions (TJ) and has been shown to be important in multiple disease states. These include neonatal ichthyosis and sclerosing cholangitis syndrome, atopic dermatitis and various viral infections. To develop a model to investigate the role of claudin-1 in different disease settings, we used CRISPR/Cas9 to generate human immortalized keratinocyte (KC) lines lacking claudin-1 (CLDN1 KO). We then determined whether loss of claudin-1 expression affects epidermal barrier formation/function and KC differentiation/stratification. The absence of claudin-1 resulted in significantly reduced barrier function in both monolayer and organotypic cultures. CLDN1 KO cells demonstrated decreases in gene transcripts encoding the barrier protein filaggrin and the differentiation marker cytokeratin-10. Marked morphological differences were also observed in CLDN1 KO organotypic cultures including diminished stratification and reduced formation of the stratum granulosum. We also detected increased proliferative KC in the basale layer of CLDN1 KO organotypic cultures. These results further support the role of claudin-1 in epidermal barrier and suggest an additional role of this protein in appropriate stratification of the epidermis.
Assuntos
Diferenciação Celular , Claudina-1 , Epiderme , Proteínas Filagrinas , Queratinócitos , Queratinócitos/metabolismo , Claudina-1/metabolismo , Claudina-1/genética , Humanos , Proteínas Filagrinas/metabolismo , Epiderme/metabolismo , Epiderme/patologia , Dermatopatias/genética , Dermatopatias/metabolismo , Junções Íntimas/metabolismo , Queratina-10/metabolismo , Queratina-10/genética , Técnicas de Inativação de Genes , Proliferação de Células , Sistemas CRISPR-CasRESUMO
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 ÁguaAssuntos
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
Three-dimensional human epidermal equivalents (HEEs) are a state-of-the-art organotypic culture model in preclinical investigative dermatology and regulatory toxicology. In this study, we investigated the utility of electrical impedance spectroscopy (EIS) for noninvasive measurement of HEE epidermal barrier function. Our setup comprised a custom-made lid fit with 12 electrode pairs aligned on the standard 24-transwell cell culture system. Serial EIS measurements for 7 consecutive days did not impact epidermal morphology, and readouts showed comparable trends with HEEs measured only once. We determined 2 frequency ranges in the resulting impedance spectra: a lower frequency range termed EISdiff correlated with keratinocyte terminal differentiation independent of epidermal thickness and a higher frequency range termed EISSC correlated with stratum corneum thickness. HEEs generated from CRISPR/Cas9-engineered keratinocytes that lack key differentiation genes FLG, TFAP2A, AHR, or CLDN1 confirmed that keratinocyte terminal differentiation is the major parameter defining EISdiff. Exposure to proinflammatory psoriasis- or atopic dermatitis-associated cytokine cocktails lowered the expression of keratinocyte differentiation markers and reduced EISdiff. This cytokine-associated decrease in EISdiff was normalized after stimulation with therapeutic molecules. In conclusion, EIS provides a noninvasive system to consecutively and quantitatively assess HEE barrier function and to sensitively and objectively measure barrier development, defects, and repair.
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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.
Assuntos
Diferenciação Celular , Epiderme , Proteínas Filagrinas , Queratinócitos , Receptores de Hidrocarboneto Arílico , Fator de Transcrição AP-2 , Receptores de Hidrocarboneto Arílico/metabolismo , Receptores de Hidrocarboneto Arílico/genética , Humanos , Fator de Transcrição AP-2/metabolismo , Fator de Transcrição AP-2/genética , Queratinócitos/metabolismo , Queratinócitos/citologia , Queratinócitos/fisiologia , Epiderme/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Transdução de Sinais , Células CultivadasAssuntos
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.
RESUMO
Late cornified envelope (LCE) proteins are small cationic epidermal proteins with antimicrobial properties, and the combined deletion of LCE3B and LCE3C genes is a risk factor for psoriasis that affects skin microbiome composition. In a yeast two-hybrid screen, we identified CYSRT1 as an interacting partner of members of all LCE groups except LCE6. These interactions were confirmed in a mammalian cell system by coimmunoprecipitation. CYSRT1 is a protein of unknown function that is specifically expressed in cutaneous and oral epithelia and spatially colocalizes with LCE proteins in the upper layers of the suprabasal epidermis. Constitutive CYSRT1 expression is present in fully differentiated epidermis and can be further induced in vivo by disruption of the skin barrier upon stratum corneum removal. Transcriptional regulation correlates to keratinocyte terminal differentiation but not to skin bacteria exposure. Similar to LCEs, CYSRT1 was found to have antibacterial activity against Pseudomonas aeruginosa. Comparative gene sequence analysis and protein amino acid alignment indicate that CYSRT1 is highly conserved among vertebrates and has putative antimicrobial activity. To summarize, we identified CYSRT1 in the outer skin layer, where it colocalizes with LCE proteins and contributes to the constitutive epidermal antimicrobial host defense repertoire.
Assuntos
Anti-Infecciosos , Psoríase , Anti-Infecciosos/metabolismo , Proteínas Ricas em Prolina do Estrato Córneo/genética , Proteínas Ricas em Prolina do Estrato Córneo/metabolismo , Epiderme/metabolismo , Queratinócitos/metabolismo , Proteínas/metabolismo , Psoríase/genética , Psoríase/metabolismo , Pele/metabolismo , HumanosRESUMO
The aryl hydrocarbon receptor (AHR) is an evolutionary conserved environmental sensor identified as 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. Multi-omics analyses using human skin keratinocytes revealed that, upon ligand activation, AHR binds open chromatin to induce expression of transcription factors (TFs), e.g., Transcription Factor AP-2α (TFAP2A), as a swift response to environmental stimuli. The terminal differentiation program including upregulation of barrier genes, filaggrin 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 novel insights into the molecular mechanism behind AHR-mediated barrier function and potential novel targets for the treatment of skin barrier diseases.
RESUMO
BACKGROUND: Following descriptive studies on skin microbiota in health and disease, mechanistic studies on the interplay between skin and microbes are on the rise, for which experimental models are in great demand. Here, we present a novel methodology for microbial colonization of organotypic skin and analysis thereof. RESULTS: An inoculation device ensured a standardized application area on the stratum corneum and a homogenous distribution of bacteria, while preventing infection of the basolateral culture medium even during prolonged culture periods for up to 2 weeks at a specific culture temperature and humidity. Hereby, host-microbe interactions and antibiotic interventions could be studied, revealing diverse host responses to various skin-related bacteria and pathogens. CONCLUSIONS: Our methodology is easily transferable to a wide variety of organotypic skin or mucosal models and different microbes at every cell culture facility at low costs. We envision that this study will kick-start skin microbiome studies using human organotypic skin cultures, providing a powerful alternative to experimental animal models in pre-clinical research. Video Abstract.
Assuntos
Interações entre Hospedeiro e Microrganismos , Microbiota , Animais , Humanos , Pele/microbiologia , Epiderme , Modelos AnimaisRESUMO
Ever since the association between FLG loss-of-function variants and ichthyosis vulgaris and atopic dermatitis disease onset was identified, FLGs function has been under investigation. Intraindividual genomic predisposition, immunological confounders, and environmental interactions complicate the comparison between FLG genotypes and related causal effects. Using CRISPR/Cas9, we generated human FLG-knockout (ΔFLG) N/TERT-2G keratinocytes. FLG deficiency was shown by immunohistochemistry of human epidermal equivalent cultures. Next to (partial) loss of structural proteins (involucrin, hornerin, keratin 2, and transglutaminase 1), the stratum corneum was denser and lacked the typical basket weave appearance. In addition, electrical impedance spectroscopy and transepidermal water loss analyses highlighted a compromised epidermal barrier in ΔFLG human epidermal equivalents. Correction of FLG reinstated the presence of keratohyalin granules in the stratum granulosum, FLG protein expression, and expression of the proteins mentioned earlier. The beneficial effects on stratum corneum formation were reflected by the normalization of electrical impedance spectroscopy and transepidermal water loss. This study shows the causal phenotypical and functional consequences of FLG deficiency, indicating that FLG is not only central in epidermal barrier function but also vital for epidermal differentiation by orchestrating the expression of other important epidermal proteins. These observations pave the way to fundamental investigations into the exact role of FLG in skin biology and disease.
Assuntos
Sistemas CRISPR-Cas , Proteínas de Filamentos Intermediários , Humanos , Proteínas de Filamentos Intermediários/metabolismo , Proteínas Filagrinas , Queratinócitos/metabolismo , FenótipoRESUMO
Deletion of the late cornified envelope (LCE) genes LCE3B and LCE3C has recently been identified as a risk factor for psoriasis. Expression of 16 LCE genes of LCE groups 1, 2, 3, 5, and 6 was examined in vivo and in vitro. Quantitative PCR demonstrated that moderate to high LCE expression was largely confined to skin and a few oropharyngeal tissues. Genes of the LCE3 group demonstrated increased expression in lesional psoriatic epidermis and were induced after superficial injury of normal skin, whereas expression of members of other LCE groups was down-regulated under these conditions. Immunohistochemistry and immunoelectron microscopy demonstrated that LCE2 protein expression was restricted to the uppermost granular layer and the stratum corneum. Stimulation of in vitro reconstructed skin by several psoriasis-associated cytokines resulted in induction of LCE3 members. The data suggest that LCE proteins of groups 1, 2, 5, and 6 are involved in normal skin barrier function, whereas LCE3 genes encode proteins involved in barrier repair after injury or inflammation. These findings may provide clues to the mechanistic role of LCE3B/C deletion in psoriasis.
Assuntos
Proteínas Ricas em Prolina do Estrato Córneo/biossíntese , Regulação da Expressão Gênica , Psoríase/diagnóstico , Psoríase/genética , Estudos de Casos e Controles , Proteínas Ricas em Prolina do Estrato Córneo/metabolismo , Deleção de Genes , Frequência do Gene , Humanos , Imuno-Histoquímica/métodos , Inflamação , Microscopia de Fluorescência/métodos , Microscopia Imunoeletrônica/métodos , Psoríase/patologia , Risco , Pele/metabolismoRESUMO
Absent in melanoma 2 (AIM2) is a double-stranded DNA receptor, and its activation initiates an interleukin-1 beta processing inflammasome. AIM2 is implicated in host defense against several pathogens, but could hypothetically also contribute to autoinflammatory or autoimmune diseases, such as is the case for NLRP3. Using thoroughly characterised antibodies, we analysed AIM2 expression in human tissues and primary cells. A strong epidermal upregulation of AIM2 protein expression was observed in several acute and chronic inflammatory skin disorders, such as psoriasis, atopic dermatitis, venous ulcera, contact dermatitis, and experimental wounds. We also found AIM2 induction by interferon-gamma in submerged and three-dimensional in vitro models of human epidermis. Our data highlight the dynamics of epidermal AIM2 expression, showing Langerhans cell and melanocyte-restricted expression in normal epidermis but a pronounced induction in subpopulations of epidermal keratinocytes under inflammatory conditions.
Assuntos
Dermatite de Contato/metabolismo , Epiderme/metabolismo , Proteínas Nucleares/metabolismo , Psoríase/metabolismo , Adulto , Animais , Especificidade de Anticorpos , Doença Crônica , Proteínas de Ligação a DNA , Dermatite de Contato/imunologia , Dermatite de Contato/patologia , Epiderme/imunologia , Epiderme/patologia , Humanos , Proteínas Nucleares/imunologia , Psoríase/imunologia , Psoríase/patologia , CoelhosRESUMO
Psoriasis and atopic dermatitis are chronic inflammatory skin diseases characterized by keratinocyte (KC) hyperproliferation and epidermal acanthosis (hyperplasia). The milieu of disease-associated cytokines and soluble factors is considered a mitogenic factor; however, pinpointing the exact mitogens in this complex microenvironment is challenging. We employed organotypic human epidermal equivalents, faithfully mimicking native epidermal proliferation and stratification, to evaluate the proliferative effects of a broad panel of (literature-based) potential mitogens. The KC GF molecule, the T-helper 2 cytokines IL-4 and IL-13, and the psoriasis-associated cytokine IL-17A caused acanthosis by hyperplasia through a doubling in the number of proliferating KCs. In contrast, IFN-γ lowered proliferation, whereas IL-6, IL-20, IL-22, and oncostatin M induced acanthosis not by hyperproliferation but by hypertrophy. The T-helper 2âcytokineâmediated hyperproliferation was Jak/signal transducer and activator of transcription 3 dependent, whereas IL-17A and KC GF induced MAPK/extracellular signalâregulated kinase kinase/extracellular signalâregulated kinaseâdependent proliferation. This discovery that key regulators in atopic dermatitis and psoriasis are direct KC mitogens not only adds evidence to their crucial role in the pathophysiological processes but also highlights an additional therapeutic pillar for the mode of action of targeting biologicals (e.g., dupilumab) or small-molecule drugs (e.g., tofacitinib) by the normalization of KC turnover within the epidermal compartment.
RESUMO
Cystatin M/E (CST6) is a nonredundant, epithelium-specific protease inhibitor with a presumed role in epidermal differentiation and tumor suppression. We have previously reported that cystatin M/E deficiency in Cst6(-/-) mice causes neonatal lethality because of excessive transepidermal water loss. Biochemical evidence suggests that cystatin M/E controls the activity of legumain, cathepsin L, cathepsin V, and transglutaminase-3. Using a genetic approach we sought to define the role of cystatin M/E in epithelial biology by identification of its target proteases and their downstream functions. Ablation of cathepsin L in a Cst6(-/-) background (Cst6(-/-)Ctsl(-/-) double-knockout mice) restored viability and resulted in normalization of stratum corneum morphology. Ablation of legumain or transglutaminase-3 in Cst6(-/-) mice, however, did not rescue the lethal phenotype. Intriguingly, both Cst6(-/-)Ctsl(-/-) and Cst6(-/-)Ctsl(+/-) mice were viable, but the absence of cystatin M/E caused scarring alopecia in adult animals. In the cornea of Cst6(-/-)Ctsl(+/-) mice, we observed keratitis, hyperplasia, and transition to a cornified epithelium. Evidence is provided that activation of cathepsin D and transglutaminase-1 are downstream events, dependent of cathepsin L activity. We conclude that a tightly regulated balance between cathepsin L and cystatin M/E is essential for tissue integrity in epidermis, hair follicles, and corneal epithelium.
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Catepsina L/metabolismo , Córnea/metabolismo , Cistatina M/metabolismo , Epiderme/metabolismo , Cabelo/metabolismo , Homeostase , Animais , Sequência de Bases , Western Blotting , Catepsina L/genética , Cistatina M/genética , Cisteína Endopeptidases/genética , Primers do DNA , Imunofluorescência , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Reação em Cadeia da PolimeraseAssuntos
Antibacterianos/administração & dosagem , Técnicas de Cultura de Células/métodos , Queratinócitos/citologia , Queratinócitos/efeitos dos fármacos , Pele/citologia , Pele/efeitos dos fármacos , Antibacterianos/toxicidade , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Meios de Cultura/efeitos adversos , Meios de Cultura/química , Células Epidérmicas , Epiderme/efeitos dos fármacos , Epiderme/crescimento & desenvolvimento , Humanos , Imageamento Tridimensional , Modelos Biológicos , Pele/crescimento & desenvolvimentoRESUMO
Terminally differentiating epidermal keratinocytes express a large number of structural and antimicrobial proteins that are involved in the physical barrier function of the stratum corneum and provide innate cutaneous host defense. Late cornified envelope (LCE) genes, located in the epidermal differentiation complex on chromosome 1, encode a family of 18 proteins of unknown function, whose expression is largely restricted to epidermis. Deletion of two members, LCE3B and LCE3C (LCE3B/C-del), is a widely-replicated psoriasis risk factor that interacts with the major psoriasis-psoriasis risk gene HLA-C*06. Here we performed quantitative trait locus analysis, utilizing RNA-seq data from human skin and found that LCE3B/C-del was associated with a markedly increased expression of LCE3A, a gene directly adjacent to LCE3B/C-del. We confirmed these findings in a 3-dimensional skin model using primary keratinocytes from LCE3B/C-del genotyped donors. Functional analysis revealed that LCE3 proteins, and LCE3A in particular, have defensin-like antimicrobial activity against a variety of bacterial taxa at low micromolar concentrations. No genotype-dependent effect was observed for the inside-out or outside-in physical skin barrier function. Our findings identify an unknown biological function for LCE3 proteins and suggest a role in epidermal host defense and LCE3B/C-del-mediated psoriasis risk.
Assuntos
Proteínas Ricas em Prolina do Estrato Córneo/genética , Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Psoríase/genética , Psoríase/imunologia , Antibacterianos/imunologia , Biópsia por Agulha , Células Cultivadas/citologia , Células Cultivadas/metabolismo , Feminino , Predisposição Genética para Doença , Genótipo , Humanos , Imuno-Histoquímica , Queratinócitos , Desequilíbrio de Ligação , Masculino , Polimorfismo de Nucleotídeo Único , Psoríase/patologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Papel (figurativo)RESUMO
The use of tissue-engineered human skin equivalents (HSE) for fundamental research and industrial application requires the expansion of keratinocytes from a limited number of skin biopsies donated by adult healthy volunteers or patients. A pharmacological inhibitor of Rho-associated protein kinases, Y-27632, was recently reported to immortalize neonatal human foreskin keratinocytes. Here, we investigated the potential use of Y-27632 to expand human adult keratinocytes and evaluated its effects on HSE development and in vitro gene delivery assays. Y-27632 was found to significantly increase the life span of human adult keratinocytes (up to five to eight passages). The epidermal morphology of HSEs generated from high-passage, Y-27632-treated keratinocytes resembled the native epidermis and was improved by supplementing Y-27632 during the submerged phase of HSE development. In addition, Y-27632-treated keratinocytes responded normally to inflammatory stimuli, and could be used to generate HSEs with a psoriatic phenotype, upon stimulation with relevant cytokines. Furthermore, Y-27632 significantly enhanced both lentiviral transduction efficiency of primary adult keratinocytes and epidermal morphology of HSEs generated thereof. Our study indicates that Y-27632 is a potentially powerful tool that is used for a variety of applications of adult human keratinocytes.