ABSTRACT
BACKGROUND: For one half-century, cultures of human epidermal keratinocytes have opened new paths of research in skin biology and dermatology. Either performed with serum and feeder layer, in serum-free conditions, or in autocrine conditions, cells cultured as monolayers became research materials for basic science and dermatology, as well as a source for grafting, particularly to treat severely burned patients. More recently, tissue reconstruction at air-liquid interface has opened new perspectives for in vitro toxicology, studies of epidermal barrier, and modeling skin diseases. SUMMARY: This review presents a brief retrospective of the emergence of keratinocyte-based culture techniques. It also presents opportunities and eventual problems that researchers might encounter when exploring the skin using such procedures. KEY MESSAGES: While methodologies in tissue culture evolve, the multiplicity of procedures concomitantly increases, requiring to make some selective but difficult choice. Keeping tracks of technological evolution in epidermal cell culture should help choosing the adequate methodology for a specific investigation or innovating with new, more dedicated ones.
Subject(s)
Epidermis , Keratinocytes , Humans , Retrospective Studies , Cells, Cultured , Keratinocytes/metabolism , Epidermis/metabolism , Epidermal CellsABSTRACT
Fungal infections of the skin, known as dermatophytoses, are initiated at the epidermal barrier and lead to dysfunctions of the stratum corneum and cornified skin appendages. Dermatophytosis affects a significant part of the human population and, despite the availability of effective treatments, its prevalence is still increasing. Numerous dermatophyte species are able to induce lesions in both animals and humans, with different clinical pictures and host inflammatory responses. The understanding of the infectious process and of tissue responses has been impeded by discrepancies between observations in vivo or in research models. Indeed, cells cultured as monolayers do not undergo the keratinization process required to study the adherence and invasion of dermatophytes. Animal models lack relevance to study human dermatophytosis because of species-specific differences in the development of lesions and inflammatory responses. This review focuses on the recent development of cultured human skin equivalents, which partly overcomes those limitations and allows improved understanding of the pathogenesis of dermatophytosis in human being, especially the impacts of infection on epidermal barrier integrity.
Subject(s)
Arthrodermataceae/pathogenicity , Dermatomycoses/metabolism , Epidermis/physiology , Skin/microbiology , Animals , Epidermis/microbiology , Fungi , Genetic Predisposition to Disease , Humans , In Vitro Techniques , Keratins/chemistry , Risk Factors , Skin Physiological Phenomena , Tinea , TrichophytonABSTRACT
Dermatophytosis is a superficial fungal infection of keratinized structures that exhibits an increasing prevalence in humans and is thus requesting novel prophylactic strategies and therapies. However, precise mechanisms used by dermatophytes to adhere at the surface of the human epidermis and invade its stratum corneum are still incompletely identified, as well as the responses provided by the underlying living keratinocytes during the infection. We hereby report development of an in vitro model of human dermatophytosis through infection of reconstructed human epidermis (RHE) by arthroconidia of the anthropophilic Trichophyton rubrum species or of the zoophilic Microsporum canis and Arthroderma benhamiae species. By modulating density of arthroconidia in the inoculum and duration of exposure to such pathogens, fungal infection limited to the stratum corneum was obtained, mimicking severe but typical in vivo situation. Fungal elements in infected RHE were monitored over time by histochemical analysis using periodic-acid Schiff-staining or quantified by qPCR-detection of fungal genes inside RHE lysates. This model brings improvements to available ones, dedicated to better understand how dermatophytes and epidermis interact, as well as to evaluate preventive and therapeutic agents. Indeed, miconazole topically added to RHE was demonstrated to inhibit fungal infection in this model.
Subject(s)
Antifungal Agents/pharmacology , Cell Culture Techniques , Keratinocytes/microbiology , Microbial Sensitivity Tests/methods , Mitosporic Fungi/drug effects , Models, Biological , Tinea/microbiology , Arthrodermataceae/drug effects , Arthrodermataceae/genetics , Cells, Cultured , DNA, Fungal/genetics , Epidermal Cells , Epidermis/microbiology , Humans , Keratinocytes/cytology , Miconazole/pharmacology , Microsporum/drug effects , Microsporum/genetics , Mitosporic Fungi/genetics , Real-Time Polymerase Chain Reaction , Spores, Fungal/drug effects , Spores, Fungal/isolation & purification , Spores, Fungal/pathogenicity , Tinea/drug therapy , Trichophyton/drug effects , Trichophyton/geneticsABSTRACT
BACKGROUND: Plasmodium vivax malaria is a major public health problem in French Guiana. Some cases of resistance to chloroquine, the first-line treatment used against P. vivax malaria, have been described in the Brazilian Amazon region. The aim of this study is to investigate a possible dispersion of chloroquine-resistant P. vivax isolates in French Guiana. The genotype, polymorphism and copy number variation, of the P. vivax multidrug resistance gene-1 (pvmdr1) have been previously associated with modification of the susceptibility to chloroquine. METHODS: The pvmdr1 gene polymorphism was evaluated by sequencing and copy number variation was assessed by real-time PCR, in P. vivax isolates obtained from 591 symptomatic patients from 1997 to 2013. RESULTS: The results reveal that 1.0% [95% CI 0.4-2.2] of French Guiana isolates carry the mutations Y976F and F1076L, and that the proportion of isolates with multiple copies of pvmdr1 has significantly decreased over time, from 71.3% (OR = 6.2 [95% CI 62.9-78.7], p < 0.0001) in 1997-2004 to 12.8% (OR = 0.03 [95% CI 9.4-16.9], p < 0.0001) in 2009-2013. A statistically significant relationship was found between Guf-A (harboring the single mutation T958M) and Sal-1 (wild type) alleles and pvmdr1 copy number. CONCLUSIONS: Few P. vivax isolates harboring chloroquine-resistant mutations in the pvmdr1 gene are circulating in French Guiana. However, the decrease in the prevalence of isolates carrying multiple copies of pvmdr1 might indicate that the P. vivax population in French Guiana is evolving towards a decreased susceptibility to chloroquine.
Subject(s)
Gene Dosage , Malaria, Vivax/parasitology , Multidrug Resistance-Associated Proteins/genetics , Plasmodium vivax/genetics , Plasmodium vivax/isolation & purification , Polymorphism, Genetic , Protozoan Proteins/genetics , Adolescent , Adult , Aged , Alleles , Antimalarials/pharmacology , Child , Child, Preschool , Chloroquine/pharmacology , Drug Resistance , Female , French Guiana , Genotype , Humans , Infant , Male , Middle Aged , Mutation, Missense , Real-Time Polymerase Chain Reaction , Sequence Analysis, DNA , Young AdultABSTRACT
Increasing resistance of dermatophytes against antifungals creates global public health problems, rendering essential a better understanding of virulence mechanisms and factors determining host-specificity of dermatophytes. Since dermatophytes switch from a saprophytic to a parasitic lifestyle by reprogramming gene expression, reliable experimental models are needed to investigate the pathogenesis of dermatophytosis. Here, a relevant mouse model of Trichophyton benhamiae dermatophytosis was assessed, together with a model based on reconstructed human epidermis (RHE), allowing their respective validation regarding fungal gene expressed during infection. The use of a standardized inoculum induced a natural-like superficial infection in mice. The severity and persistence of lesions enabled the assessment of infection markers, including mouse-specific pro-inflammatory molecules and fungal genes previously reported as potential virulence factors. Upregulated expression of fungal genes, including those encoding subtilisins, in infected RHE revealed that dermatophytes deploy similar processes as those observed during in vivo infection. The RHE model was then used to compare infections by anthropophilic Trichophyton rubrum and zoophilic T. benhamiae. Therefore, these two models represent complementary analytical tools to study the pathogenesis of acute dermatophytoses. In addition, we have identified certain fungal markers of infection and highlighted the existence of different mechanisms deployed by zoophilic versus anthropophilic dermatophytes.
ABSTRACT
The Reconstructed Human Epidermis (RHE) model derived from epidermal keratinocytes offers an ethical and scientific alternative to animal experimentation, particularly in cutaneous toxicology and dermatological research, where the elimination of animal cruelty is of paramount importance. Thus, we compared commercially available chemically defined animal origin-free (cdAOF) supplements, designed for regenerative medicine, to the widely utilized supplement (human keratinocyte growth supplement), which contains growth factors and bovine pituitary extract. Herein we present the extended characterization of RHE derived from newborn, adult, and immortalized N/telomerase reverse transcriptase keratinocytes under cdAOF conditions. Culture of RHE in the cdAOF media produced histological features that were similar to that produced using human keratinocyte growth supplement, with the exception that the basal keratinocytes were less cylindrical. Additionally, immunolocalization of involucrin in the basal layer and increased mRNA expression of several inflammatory-proliferative markers were observed under cdAOF conditions. In RHEs cultured in cdAOF media, expression and immunolocalization of other expected markers of keratinization were similar, while monitoring of barrier function (transepithelial electrical resistance) revealed results that were statistically equal to, or lower than those observed in RHE cultured in human keratinocyte growth supplement. Our study indicates that reconstruction of RHE was accomplished under cdAOF culture conditions and that further refinement could promote an expanded use beyond regenerative medicine, for in vitro toxicology applications.
ABSTRACT
Dermatophytoses are superficial infections of human and animal keratinized tissues caused by filamentous fungi named dermatophytes. Because of a high and increasing incidence, as well as the emergence of antifungal resistance, a better understanding of mechanisms involved in adhesion and invasion by dermatophytes is required for the further development of new therapeutic strategies. In the last years, several in vitro and in vivo models have emerged to study dermatophytosis pathogenesis. However, the procedures used for the growth of fungi are quite different, leading to a highly variable composition of inoculum for these models (microconidia, arthroconidia, hyphae), thus rendering difficult the global interpretation of observations. We hereby optimized growth conditions, including medium, temperature, atmosphere, and duration of culture, to improve the sporulation and viability and to favour the production of arthroconidia of several dermatophyte species, including Trichophyton rubrum and Trichophyton benhamiae. The resulting suspensions were then used as inoculum to infect reconstructed human epidermis in order to validate their ability to adhere to and to invade host tissues. By this way, this paper provides recommendations for dermatophytes culture and paves the way towards a standardized procedure for the production of infective spores usable in in vitro and in vivo experimental models.
ABSTRACT
TSG-6 is a soluble protein secreted in the extracellular matrix by various cell types in response to inflammatory stimuli. TSG-6 interacts with extracellular matrix molecules, particularly hyaluronan (HA), and promotes cutaneous wound closure in mice. Between epidermal cells, the discrete extracellular matrix contains HA and a tiny amount of TSG-6. However, challenges imposed to keratinocytes in reconstructed human epidermis revealed strong induction of TSG-6 expression, after exposure to T helper type 2 cytokines to recapitulate the atopic dermatitis phenotype or after fungal infection that causes secretion of cytokines and antimicrobial peptides. After both types of challenge, enhanced release of TSG-6 happens simultaneously with increased HA production. TSG-6 deficiency in N/TERT keratinocytes was created by inactivating TNFAIP6 using CRISPR/Cas9. Some TSG-6 -/- keratinocytes analyzed through scratch assays tend to migrate more slowly but produce reconstructed human epidermis that exhibits normal morphology and differentiation. Few significant alterations were noticed by transcriptomic analysis. Nevertheless, reduced HA content in TSG-6 -/- reconstructed human epidermis was observed, along with enhanced HA release into the culture medium, and this phenotype was even more pronounced after the challenging conditions. Reintroduction of cells producing TSG-6 in reconstructed human epidermis reduced HA leakage. Our results show a role for TSG-6 in sequestering HA between epidermal cells in response to inflammation.
ABSTRACT
Despite the threatening incidence of dermatophytosis, information is still lacking about the consequences of infection on epidermal barrier functions and about the keratinocyte responses that alert immune components. To identify the mechanisms involved, arthroconidia of the anthropophilic dermatophyte Trichophyton rubrum were prepared to infect reconstructed human epidermis (RHE) in vitro. Integrity of the barrier was monitored during infection by measurements of transepithelial electrical resistance and dye-permeation through the RHE. Expression and release of pro-inflammatory cytokines and antimicrobial peptides by keratinocytes inserted into the RHE were assessed, respectively, by quantitative reverse transcriptase-PCR (to analyze mRNA content in tissue extracts) and by ELISA (to detect proteins in culture media). Results reveal that infection by T. rubrum is responsible for disruption of the epidermal barrier, including loss of functional tight junctions. It additionally causes simultaneous expression and release of cytokines and antimicrobial peptides by keratinocytes. Potential involvement of the p38 mitogen-activated protein kinase signaling pathway was evaluated during infection by targeted inhibition of its activity. Intriguingly, among several p38 mitogen-activated protein kinase inhibitors, PD169316 alone was able to inhibit growth of T. rubrum on Sabouraud agar and to suppress the process of infection on RHE. This suggests that PD169316 acts on a specific target in dermatophytes themselves.
Subject(s)
Arthrodermataceae/drug effects , Arthrodermataceae/isolation & purification , Imidazoles/pharmacology , Tinea/drug therapy , Culture Media , Cytokines/metabolism , Epidermal Cells/cytology , Epidermal Cells/drug effects , Humans , In Vitro Techniques , Keratinocytes/cytology , Keratinocytes/drug effects , Microscopy, Electron/methods , RNA, Messenger/metabolism , Real-Time Polymerase Chain Reaction/methods , Reference Values , Sensitivity and Specificity , Tinea/diagnosisABSTRACT
BACKGROUND: Care for malaria patients in endemic areas has been improved through the increasing use of Rapid Diagnostic Tests (RDTs). Most RDTs target the histidine-rich protein-2 antigen (PfHRP2) to detect P. falciparum, as it is abundant and shows great heat stability. However, their use in South America has been widely questioned following a recent publication that pinpoints the high prevalence of Peruvian field isolates lacking the gene encoding this protein. In the remote rural health centers of French Guiana, RDTs are the main diagnosis tools. Therefore, a study of PfHRP2 RDT performances and pfhrp2 genotyping was conducted to determine whether a replacement of the current pLDH-based kit could be considered. METHODS: The performance study compared the SD Malaria Ag test P.f/Pan® kit with the current gold standard diagnosis by microscopy. The prevalence of pfhrp2 and pfhrp3 deletions were evaluated from 221 P. falciparum isolates collected between 2009 and 2011 in French Guiana. RESULTS: Between January 2010 and August 2011, 960 suspected cases of malaria were analyzed using microscopy and RDTs. The sensitivity of the SD Malaria Ag test P.f/Pan® for detection of P. falciparum was 96.8% (95% CI: 90.9-99.3), and 86.0% (95% CI: 78.9-91.5) for the detection of P. vivax. No isolates (95% CI: 0-4.5) lacking either exon of the pfhrp2 gene were identified among the 221 P. falciparum isolates analyzed, but 7.4% (95% CI: 2.8-15.4) lacked the exon 2 part of the pfhrp3 gene. CONCLUSIONS: Field isolates lacking either exon of the pfhrp2 gene are absent in this western part of South America. Despite its sensibility to detect P. vivax, the SD Malaria Ag test P.f/Pan® kit is a satisfying alternative to microscopy in remote health centers, where it is difficult to provide highly skilled microscopists and to maintain the necessary equipment.