RESUMO
Atopic dermatitis (AD) is a common T-helper 2 (Th2) lymphocyte-mediated chronic inflammatory skin disease characterized by disturbed epidermal differentiation (e.g., filaggrin (FLG) expression) and diminished skin barrier function. Therapeutics targeting the aryl hydrocarbon receptor (AHR), such as coal tar and tapinarof, are effective in AD, yet new receptor ligands with improved potency or bioavailability are in demand to expand the AHR-targeting therapeutic arsenal. We found that carboxamide derivatives from laquinimod, tasquinimod, and roquinimex can activate AHR signaling at low nanomolar concentrations. Tasquinimod derivative (IMA-06504) and its prodrug (IMA-07101) provided full agonist activity and were most effective to induce FLG and other epidermal differentiation proteins, and counteracted IL-4 mediated repression of terminal differentiation. Partial agonist activity by other derivatives was less efficacious. The previously reported beneficial safety profile of these novel small molecules, and the herein reported therapeutic potential of specific carboxamide derivatives, provides a solid rationale for further preclinical assertation.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Diferenciação Celular , Proteínas Filagrinas/genética , Queratinócitos/efeitos dos fármacos , Quinolonas/farmacologia , Receptores de Hidrocarboneto Arílico/metabolismo , Células Cultivadas , Regulação da Expressão Gênica , Células Hep G2 , Humanos , Interleucina-4 , Queratinócitos/metabolismo , Queratinócitos/fisiologia , Transdução de SinaisRESUMO
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
Skin in vitro models offer much promise for research, testing drugs, cosmetics, and medical devices, reducing animal testing and extensive clinical trials. There are several in vitro approaches to mimicking human skin behavior, ranging from simple cell monolayer to complex organotypic and bioengineered 3-dimensional models. Some have been approved for preclinical studies in cosmetics, pharmaceuticals, and chemicals. However, development of physiologically reliable in vitro human skin models remains in its infancy. This review reports on advances in in vitro complex skin models to study skin homeostasis, aging, and skin disease.
RESUMO
Therapeutic aryl hydrocarbon receptor (AHR) modulating agents gained attention in dermatology as non-steroidal anti-inflammatory drugs that improve skin barrier properties. By exploiting AHR's known ligand promiscuity, we generated novel AHR modulating agents by lead optimization of a selective AHR modulator (SAhRM; SGA360). Twenty-two newly synthesized compounds were screened yielding two novel derivatives, SGA360f and SGA388, in which agonist activity led to enhanced keratinocyte terminal differentiation. SGA388 showed the highest agonist activity with potent normalization of keratinocyte hyperproliferation, restored expression of skin barrier proteins and dampening of chemokine expression by keratinocytes upon Th2-mediated inflammation in vitro. The topical application of SGA360f and SGA388 reduced acute skin inflammation in vivo by reducing cyclooxygenase levels, resulting in less neutrophilic dermal infiltrates. The minimal induction of cytochrome P450 enzyme activity, lack of cellular toxicity and mutagenicity classifies SGA360f and SGA388 as novel potential therapeutic AHR ligands and illustrates the potential of medicinal chemistry to fine-tune AHR signaling for the development of targeted therapies in dermatology and beyond.
Assuntos
Receptores de Hidrocarboneto Arílico , Dermatopatias , Humanos , Receptores de Hidrocarboneto Arílico/metabolismo , Ligantes , Pele/metabolismo , Queratinócitos/metabolismo , Inflamação/metabolismo , Dermatopatias/tratamento farmacológicoRESUMO
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
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
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.
Assuntos
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
The three-dimensional culturing of human keratinocytes at the air-liquid interface yields a fully stratified epidermis including a functional stratum corneum and thus enables the study on epidermal structure and function in the context of biomedical, toxicological and pharmaceutical sciences. Here we provide a step-by-step detailed protocol for the isolation of human primary keratinocytes and the development of human epidermal equivalents generated from primary keratinocytes or immortalized keratinocytes (N/TERT-1; N/TERT-2G), including widely accepted procedures for the analysis of barrier function, tissue morphology, cell proliferation, and gene expression.
Assuntos
Técnicas de Cultura de Células , Células Epidérmicas/citologia , Queratinócitos/citologia , Pele/citologia , Animais , Biomarcadores , Diferenciação Celular , Linhagem Celular Transformada , Separação Celular/métodos , Células Cultivadas , Imunofluorescência , Expressão Gênica , Humanos , Imuno-Histoquímica , Queratinócitos/metabolismo , CamundongosRESUMO
Skin colonization by Staphylococcus aureus and its relative abundance is associated with atopic dermatitis (AD) disease severity and treatment response. Low levels of antimicrobial peptides in AD skin may be related to the microbial dysbiosis. Therapeutic targeting of the skin microbiome and antimicrobial peptide expression can, therefore, restore skin homeostasis and combat AD. In this study, we analyzed the cutaneous microbiome composition in 7 patients with AD and 10 healthy volunteers upon topical coal tar or vehicle treatment. We implemented and validated a Staphylococcus-specific single-locus sequence typing approach combined with classic 16S ribosomal RNA marker gene sequencing to study the bacterial composition. During coal tar treatment, Staphylococcus abundance decreased, and Propionibacterium abundance increased, suggesting a shift of the microbiota composition toward that of healthy controls. We, furthermore, identified a hitherto unknown therapeutic mode of action of coal tar, namely the induction of keratinocyte-derived antimicrobial peptides via activation of the aryl hydrocarbon receptor. Restoring antimicrobial peptide levels in AD skin via aryl hydrocarbon receptor-dependent transcription regulation can be beneficial by creating a (anti)microbial milieu that is less prone to infection and inflammation. This underscores the importance of coal tar in the therapeutic aryl hydrocarbon receptor armamentarium and highlights the aryl hydrocarbon receptor as a target for drug development.
Assuntos
Anti-Infecciosos/farmacologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/agonistas , Alcatrão/farmacologia , Dermatite Atópica/tratamento farmacológico , Disbiose/tratamento farmacológico , Microbiota/efeitos dos fármacos , Receptores de Hidrocarboneto Arílico/agonistas , Pele/microbiologia , Administração Cutânea , Adulto , Anti-Infecciosos/uso terapêutico , Peptídeos Catiônicos Antimicrobianos/imunologia , Peptídeos Catiônicos Antimicrobianos/metabolismo , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Biópsia , Linhagem Celular , Alcatrão/uso terapêutico , Dermatite Atópica/imunologia , Dermatite Atópica/microbiologia , Dermatite Atópica/patologia , Disbiose/imunologia , Disbiose/microbiologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/imunologia , Técnicas de Silenciamento de Genes , Voluntários Saudáveis , Humanos , Queratinócitos , Masculino , Microbiota/imunologia , Pessoa de Meia-Idade , Cultura Primária de Células , Propionibacterium/imunologia , Propionibacterium/isolamento & purificação , Receptores de Hidrocarboneto Arílico/genética , Receptores de Hidrocarboneto Arílico/metabolismo , Pele/efeitos dos fármacos , Pele/imunologia , Pele/patologia , Creme para a Pele/farmacologia , Creme para a Pele/uso terapêutico , Staphylococcus aureus/imunologia , Staphylococcus aureus/isolamento & purificação , Adulto JovemRESUMO
Despite recent advances in understanding microbial diversity in skin homeostasis, the relevance of microbial dysbiosis in inflammatory disease is poorly understood. Here we perform a comparative analysis of skin microbial communities coupled to global patterns of cutaneous gene expression in patients with atopic dermatitis or psoriasis. The skin microbiota is analysed by 16S amplicon or whole genome sequencing and the skin transcriptome by microarrays, followed by integration of the data layers. We find that atopic dermatitis and psoriasis can be classified by distinct microbes, which differ from healthy volunteers microbiome composition. Atopic dermatitis is dominated by a single microbe (Staphylococcus aureus), and associated with a disease relevant host transcriptomic signature enriched for skin barrier function, tryptophan metabolism and immune activation. In contrast, psoriasis is characterized by co-occurring communities of microbes with weak associations with disease related gene expression. Our work provides a basis for biomarker discovery and targeted therapies in skin dysbiosis.
Assuntos
Dermatite Atópica/genética , Interações entre Hospedeiro e Microrganismos/genética , Microbiota/genética , Psoríase/genética , Pele/metabolismo , Pele/microbiologia , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Estudos de Casos e Controles , Dermatite Atópica/microbiologia , Disbiose/genética , Feminino , Expressão Gênica , Perfilação da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Psoríase/microbiologia , RNA Ribossômico 16S , Adulto JovemRESUMO
The strong societal urge to reduce the use of experimental animals, and the biological differences between rodent and human skin, have led to the development of alternative models for healthy and diseased human skin. However, the limited availability of primary keratinocytes to generate such models hampers large-scale implementation of skin models in biomedical, toxicological, and pharmaceutical research. Immortalized cell lines may overcome these issues, however, few immortalized human keratinocyte cell lines are available and most do not form a fully stratified epithelium. In this study we compared two immortalized keratinocyte cell lines (N/TERT1, N/TERT2G) to human primary keratinocytes based on epidermal differentiation, response to inflammatory mediators, and the development of normal and inflammatory human epidermal equivalents (HEEs). Stratum corneum permeability, epidermal morphology, and expression of epidermal differentiation and host defence genes and proteins in N/TERT-HEE cultures was similar to that of primary human keratinocytes. We successfully generated N/TERT-HEEs with psoriasis or atopic dermatitis features and validated these models for drug-screening purposes. We conclude that the N/TERT keratinocyte cell lines are useful substitutes for primary human keratinocytes thereby providing a biologically relevant, unlimited cell source for in vitro studies on epidermal biology, inflammatory skin disease pathogenesis and therapeutics.