Human Keratinocyte Monolayers Research Articles

Plasma activated water as a pre-treatment strategy in the context of biofilm-infected chronic wounds

Healing and treatment of chronic wounds are often complicated due to biofilm formation by pathogens. Here, the efficacy of plasma activated water (PAW) as a pre-treatment strategy has been investigated prior to the application of topical antiseptics polyhexamethylene biguanide, povidone iodine, and MediHoney, which are routinely used to treat chronic wounds. The efficacy of this treatment strategy was determined against biofilms of Escherichia coli formed on a plastic substratum and on a human keratinocyte monolayer substratum used as an in vitro biofilm-skin epithelial cell model. PAW pre-treatment greatly increased the killing efficacy of all the three antiseptics to eradicate the E. coli biofilms formed on the plastic and keratinocyte substrates. However, the efficacy of the combined PAW-antiseptic treatment and single treatments using PAW or antiseptic alone was lower for biofilms formed in the in vitro biofilm-skin epithelial cell model compared to the plastic substratum. Scavenging assays demonstrated that reactive species present within the PAW were largely responsible for its anti-biofilm activity. PAW treatment resulted in significant intracellular reactive oxygen and nitrogen species accumulation within the E. coli biofilms, while also rapidly acting on the microbial membrane leading to outer membrane permeabilisation and depolarisation. Together, these factors contribute to significant cell death, potentiating the antibacterial effect of the assessed antiseptics.

Sepia Melanin-Loaded Primary Human Gingival Keratinocytes: An In Vitro Model for Studies on Pigmented Gingiva

The objective of this study is the development of an in vitro cell culture model of pigmented gingival keratinocytes to provide a unique tool to assess oral care products such as toothpaste and evaluate whether pigmented gingival cells might be less susceptible than unpigmented cells to cytotoxicity by any toothpaste. Sepia melanin at various concentrations was added to primary human gingival keratinocyte (HGK) monolayers to identify the concentration at which melanin is sufficiently phagocytosed in the absence of cytotoxicity; this concentration was subsequently used to generate pigmented HGK model. Extracts from three commercial adult toothpastes (Crest 3D White, Sensodyne, and Colgate Optic) at different dilutions were evaluated in pigmented and unpigmented HGKs for cytotoxicity over a 24 h duration by MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] assay. Results demonstrated that HGKs showed a concentration-dependent uptake of sepia melanin with a significant linear correlation of pigment uptake. Moreover, the melanin was distributed perinuclearly in the cells, that was similar to the distribution of physiological gingiva in vivo. Further experiments were conducted with 25 µg/mL sepia melanin as higher concentrations induced cytotoxicity. Evaluation of three commercial toothpastes on unpigmented and pigmented HGKs showed no differential effects at any dilution. In summary, a model of pigmented HGKs with the ability to create a controlled level of pigmentation was demonstrated. Examination of extracts from three commercial toothpastes revealed similar cytotoxicity to both pigmented and unpigmented HGKs. In conclusion, this study shows that the artificially pigmented HGK model is an easy and low-cost tool that mimics the in vivo gingival pigmentation. Moreover, the evaluated toothpastes showed similar cytotoxicity to pigmented and unpigmented HGKs, suggesting that the presence of melanin did not impart any protective effects. Further studies to employ this pigmented HGK model to evaluate a large number of oral care products and include repeated exposures and longer duration are warranted.

Characterization of a live Cutibacterium acnes subspecies defendens strain XYCM42 and clinical assessment as a topical regimen for general skin health and cosmesis.

When formulating topical products to treat skin diseases and addressing general skin health and cosmesis, most of the focus has traditionally been placed on how any given ingredient may impact the structure, function, and health of human skin elements. However, recent research is beginning to highlight the importance of the skin microbiome in relation to certain skin conditions and general cosmesis. Cutibacterium acnes is one of the most prolific skin-specific bacterial species. Research has shown that the species is divided into subspecies, some of which are thought to be beneficial to the skin. This paper aims to determine the efficacy of strainXYCM42, a C. acnes subspecies defendens derived strain designed to improve the health and appearance of the skin. In vitro studies were performed on human keratinocyte and fibroblast monolayers, human peripheral blood mononuclear cells (PBMC), and skin explants to elucidate the effects of live XYCM42 cells and their ferment on human skin cells and tissues. Subsequently, clinical studies were performed using XYCM42-based topical regimens designed to deliver and support the engraftment of live XYCM42 cells onto subjects' skin. Two studies were performed, a 3-week pilot study (n=10) and a 8-week pivotal study (n=121). In the latter, 32 subjects were enrolled for an in-clinic portion for efficacy evaluation, with clinic visits occurring at Baseline, Week 1, Week 4, and Week 8. In vitro data suggest that XYCM42 and its ferment filtrate have potential to provide benefits to the skin via antioxidant, anti-inflammatory, and select antimicrobial activities. Clinical observation demonstrated that a XYCM42-containing regimen supports a healthy skin environment, promotes increased skin hydration, decreases erythema, calms the skin, and regulates sebum production. These studies provide further evidence that specific strains of C. acnes, such as XYCM42, have a more beneficial function regarding skin health and appearance than was previously thought. Appropriate use of formulations derived from symbiotic strains within the skin microbiome can support the development of novel, beneficial topicals.

Regulation of gingival keratinocyte monocyte chemoattractant protein-1-induced protein (MCPIP)-1 and mucosa-associated lymphoid tissue lymphoma translocation protein (MALT)-1 expressions by periodontal bacteria, lipopolysaccharide, and interleukin-1β.

The aim of this study was to evaluate oral bacteria- and interleukin (IL)-1β-induced protein and mRNA expression profiles of monocyte chemoattractant protein-1-induced protein (MCPIP)-1 and mucosa-associated lymphoid tissue lymphoma translocation protein (MALT)-1 in human gingival keratinocyte monolayers and organotypic oral mucosal models. Human gingival keratinocyte (HMK) monolayers were incubated with Porphyromonas gingivalis, Fusobacterium nucleatum, P. gingivalis lipopolysaccharide (LPS) and IL-1β. The protein levels of MCPIP-1 and MALT-1 were examined by immunoblots and mRNA levels by qPCR. MCPIP-1 and MALT-1 protein expression levels were also analyzed immunohistochemically using an organotypic oral mucosal model. One-way analysis of variance followed by Tukey correction was used in statistical analyses. In keratinocyte monolayers, MCPIP-1 protein expression was suppressed by F. nucleatum and MALT-1 protein expression was suppressed by F. nucleatum, P. gingivalis LPS and IL-1β. P. gingivalis seemed to degrade MCPIP-1 and MALT-1 at all tested time points and degradation was inhibited when P. gingivalis was heat-killed. MCPIP-1 mRNA levels were increased by P. gingivalis, F. nucleatum, and IL-1β, however, no changes were observed in MALT-1 mRNA levels. Gingival keratinocyte MCPIP-1 and MALT-1 mRNA and protein expression responses are regulated by infection and inflammatory mediators. These findings suggest that periodontitis-associated bacteria-induced modifications in MCPIP-1 and MALT-1 responses can be a part of periodontal disease pathogenesis.

Mechanical coupling of supracellular stress amplification and tissue fluidization during exit from quiescence

Cellular quiescence is a state of reversible cell cycle arrest that is associated with tissue dormancy. Timely regulated entry into and exit from quiescence is important for processes such as tissue homeostasis, tissue repair, stem cell maintenance, developmental processes, and immunity. However, little is known about processes that control the mechanical adaption to cell behavior changes during the transition from quiescence to proliferation. Here, we show that quiescent human keratinocyte monolayers sustain an actinomyosin-based system that facilitates global cell sheet displacements upon serum-stimulated exit from quiescence. Mechanistically, exposure of quiescent cells to serum-borne mitogens leads to rapid amplification of preexisting contractile sites, leading to a burst in monolayer tension that subsequently drives large-scale displacements of otherwise motility-restricted monolayers. The stress level after quiescence exit correlates with the level of quiescence depth at the time of activation, and a critical stress magnitude must be reached to overcome the cell sheet displacement barrier. The study shows that static quiescent cell monolayers are mechanically poised for motility, and it identifies global stress amplification as a mechanism for overcoming motility restrictions in confined confluent cell monolayers.

High global antioxidant protection and stimulation of the collagen synthesis of new anti-aging product containing an optimized active mix.

To assess the in vitro efficacy on antioxidant potential, protection against global oxidative stress, and effect on collagen neosynthesis of minimalist formula (Peptide-C ampoules product) containing 10% natural vitamin C, rice and lupin bio-peptides, hyaluronic acid, and Vichy volcanic mineralizing water (active mix). In-tube quantitative tests ("in-tube screening") assessed global antioxidant properties, anti-lipid peroxidation, anti-protein glycosylation, and metalloproteinase inhibition (anti-collagenase, anti-elastase, and anti-hyaluronidase activity) properties of the formula. Protection against oxidative stress was evaluated on human keratinocyte monolayer cultures, and collagen neosynthesis was quantified on fibroblast monolayer cultures treated with supernatants from product-treated reconstructed human epidermis. Product (5% concentration) showed high antioxidant ability (blocking 99.0% oxidation), protection against oxidative stress damage (51.8% lipid peroxidation and 37.8% protein glycosylation decreases), and inhibition of hyaluronidase (21.9%), elastase (47.1%), and collagenase (61.8%). The protective effect was validated on human keratinocyte monolayer cultures in the presence of active mix (0.025%). Oxidative stress (ROS) was reduced by 99.0%, while global oxidative stress (RMS) induced by pollution, UVA radiation, and a combination of both factors was reduced by 48.94%, 8.7%, and 96.28%, respectively. The product increased collagen neosynthesis (11.21%) by cellular dialogue in fibroblasts incubated with product/mix-treated-RHE supernatants. The combination of ingredients in the product showed high global antioxidant capacity, as well as a protective effect against oxidative stress induced by UVA, pollution, or both combined factors and an ability to stimulate collagen neosynthesis in in vitro studies, which support the clinical efficacy of this product.

The eIF2 kinase GCN2 directs keratinocyte collective cell migration during wound healing via coordination of reactive oxygen species and amino acids

Healing of cutaneous wounds requires the collective migration of epithelial keratinocytes to seal the wound bed from the environment. However, the signaling events that coordinate this collective migration are unclear. In this report, we address the role of phosphorylation of eukaryotic initiation factor 2 (eIF2) and attendant gene expression during wound healing. Wounding of human keratinocyte monolayers in vitro led to the rapid activation of the eIF2 kinase GCN2. We determined that deletion or pharmacological inhibition of GCN2 significantly delayed collective cell migration and wound closure. Global transcriptomic, biochemical, and cellular analyses indicated that GCN2 is necessary for maintenance of intracellular free amino acids, particularly cysteine, as well as coordination of RAC1-GTP-driven reactive oxygen species (ROS) generation, lamellipodia formation, and focal adhesion dynamics following keratinocyte wounding. In vivo experiments using mice deficient for GCN2 validated the role of the eIF2 kinase during wound healing in intact skin. These results indicate that GCN2 is critical for appropriate induction of collective cell migration and plays a critical role in coordinating the re-epithelialization of cutaneous wounds.

2-Hydroxychalcone as a Potent Compound and Photosensitizer Against Dermatophyte Biofilms.

Dermatophytes, fungi that cause dermatophytosis, can invade keratinized tissues in humans and animals. The biofilm-forming ability of these fungi was described recently, and it may be correlated with the long treatment period and common recurrences of this mycosis. In this study, we evaluated the anti-dermatophytic and anti-biofilm activity of 2-hydroxychalcone (2-chalcone) in the dark and photodynamic therapy (PDT)-mediated and to determine its mechanism of action. Trichophyton rubrum and Trichophyton mentagrophytes strains were used in the study. The antifungal susceptibility test of planktonic cells, early-stage biofilms, and mature biofilms were performed using colorimetric methods. Topographies were visualized by scanning electron microscopy (SEM). Human skin keratinocyte (HaCat) monolayers were also used in the cytotoxicity assays. The mechanisms of action of 2-chalcone in the dark and under photoexcitation were investigated using confocal microscopy and the quantification of ergosterol, reactive oxygen species (ROS), and death induction by apoptosis/necrosis. All strains, in the planktonic form, were inhibited after treatment with 2-chalcone (minimum inhibitory concentration (MIC) = 7.8-15.6 mg/L), terbinafine (TRB) (MIC = 0.008–0.03 mg/L), and fluconazole (FLZ) (1–512 mg/L). Early-stage biofilm and mature biofilms were inhibited by 2-chalcone at concentrations of 15.6 mg/L and 31.2 mg/L in all tested strains. However, mature biofilms were resistant to all the antifungal drugs tested. When planktonic cells and biofilms (early-stage and mature) were treated with 2-chalcone-mediated PDT, the inhibitory concentrations were reduced by four times (2–7.8 mg/L). SEM images of biofilms treated with 2-chalcone showed cell wall collapse, resulting from a probable extravasation of cytoplasmic content. The toxicity of 2-chalcone in HaCat cells showed higher IC50 values in the dark than under photoexcitation. Further, 2-chalcone targets ergosterol in the cell and promotes the generation of ROS, resulting in cell death by apoptosis and necrosis. Overall, 2-chalcone-mediated PDT is a promising and safe drug candidate against dermatophytes, particularly in anti-biofilm treatment.

Curcuma amarissima Extract Activates Growth and Survival Signal Transduction Networks to Stimulate Proliferation of Human Keratinocyte.

Simple SummaryLike many plants in the family of Zingiberaceae, Curcuma amarissima has been traditionally used to induce healing and tissue regeneration. However, there is no scientific evidence to explain how Curcuma amarissima works to accelerate wound healing. Our data clearly proved that Curcuma amarissima extract could potentially accelerate the closure of scratch wounds of human keratinocytes by stimulating cell proliferation. The potential mechanisms underlying these effects were defined to be associated with the activated signal transduction pathways relevant to cell proliferation and survival. This strongly suggests the ability of Curcuma amarissima to enhance the process of keratinocyte reepithelization during wound healing. Our current study provides convincing evidence that supports the possibility to develop an effective wound-healing promoting agent from this plant.Many medicinal plants have been used to treat wounds. Here, we revealed the potential wound healing effects of Curcuma amarissima (CA). Our cell viability assay showed that CA extract increased the viability of HaCaT cells that were cultured in the absence of serum. This increase in cell viability was proved to be associated with the pharmacological activities of CA extract in inducing cell proliferation. To further define possible molecular mechanisms of action, we performed Western blot analysis and immunofluorescence study, and our data demonstrated that CA extract rapidly induced ERK1/2 and Akt activation. Consistently, CA extract accelerated cell migration, resulting in rapid healing of wounded human keratinocyte monolayer. Specifically, the CA-induced increase of cell monolayer wound healing was blocked by the MEK inhibitor (U0126) or the PI3K inhibitor (LY294002). Moreover, CA extract induced the expression of Mcl-1, which is an anti-apoptotic protein, supporting that CA extract enhances human keratinocyte survival. Taken together, our study provided convincing evidence that Curcuma amarissima can promote proliferation and survival of human keratinocyte through stimulating the MAPK and PI3K/Akt signaling cascades. These promising data emphasize the possibility to develop this plant as a wound healing agent for the potential application in regenerative medicine.

Thymoquinone-Loaded Polymeric Films and Hydrogels for Bacterial Disinfection and Wound Healing

The purpose of this study was to synthesize and characterize novel biocompatible topical polymeric film and hydrogel systems that have the potential to deliver the antibacterial agent thymoquinone (TQ) directly to the skin target site to manage the local wound infection and thereby wound healing. The polyvinyl pyrrolidone (PVP) matrix-type films containing TQ were prepared by the solvent casting method. In vitro skin permeation studies on human cadaver skin produced a mean flux of 2.3 µg TQ/cm2/h. Human keratinocyte monolayers subjected to a scratch wound (an in vitro wound healing assay) showed 85% wound closure at day 6 in the TQ group (100 ng/mL TQ) as compared to 50% in the vehicle control group (p = 0.0001). In a zone-of-inhibition (ZOI) assay, TQ-containing films and hydrogels completely wiped out Staphylococcus aureus in 10 cm diameter Tryptic Soy Agar plates while 500 µg/mL gentamicin containing filters gave 10 mm of ZOI. In an ex vivo model, TQ-containing films eradicated bacterial colonization on human cadaver skin. Furthermore, in a full-thickness wound infection model in mice, TQ-containing films showed significant activity in controlling Staphylococcus aureus infection, thereby disinfecting the skin wound. In summary, TQ-containing PVP films and hydrogels developed in this study have the potential to treat and manage wound infections.

Molecular Mechanisms at the Basis of Pharmaceutical Grade Triticum vulgare Extract Efficacy in Prompting Keratinocytes Healing.

Background: It has been shown that many plant- or microbial-derived oligos and polysaccharides may prompt tissue repair. Among the different extracts that have been studied, the aqueous one of Triticum vulgare (TVE) that was obtained from a whole germinated plant has been proven to have different biological properties that are useful in the process of wound healing. Nevertheless, with the long tradition of its use in pharmaceutical cream and ointments, especially in Italy, a new protocol was recently proposed (and patented) to improve the extraction process. Methods: In a simplified in vitro model, human keratinocyte monolayers were scratched and used to run time lapse experiments by using time lapse video microscopy (TLVM) to quantify reparation rate while considering a dose–response effect. Contemporarily, the molecular mechanisms that are involved in tissue repair were studied. In fact, key biomarkers that are involved in remodeling, such as MMP-2 and MMP-9, and in matrix structure assembly, such as collagen I, elastin, integrin αV and aquaporin 3, were evaluated with gene expression analyses (RT-PCR) and protein quantification in western blotting. Results: All TVE doses tested on the HaCat-supported cell proliferation. TVE also prompted cell migration in respect to the control, correctly modulating the timing of metalloproteases expression toward a consistent and well-assessed matrix remodeling. Furthermore, TVE treatments upregulated and positively modulated the expression of the analyzed biomarkers, thus resulting in a better remodeling of dermal tissue during healing. Conclusions: The in vitro results on the beneficial effects of TVE on tissue elasticity and regeneration may support a better understanding of the action mechanism of TVE as active principles in pharmaceutical preparation in wound treatment.

Isolation of Epidermal Keratinocytes from Human Skin: The Scratch-Wound Assay for Assessment of Epidermal Keratinocyte Migration.

The migration of epidermal keratinocytes is the basis for skin reepithelialization during wound healing. The in vitro scratch-wound assay using monolayers of primary human epidermal keratinocytes is a straightforward and effective method to assess their migratory capacity. The mechanical scratch of a confluent monolayer directly disrupts the adhesion of the keratinocytes to one another and to the underlying matrix, resembling the physical trauma of a wound in an in vitro assay. The keratinocytes will undergo an epithelial-to-mesenchymal transition, which will confer an ability to migrate toward each other to cover the gap by restructuring cell-cell and cell-extracellular matrix connections. However, a good scratch-wound method and protocol to ensure scratch reproducibility is essential, particularly when using primary cell cultures where donor variability may also impact on results.

Keratinocyte Monolayers on Hyaluronic Acid Membranes as "Upside-Down" Grafts Reconstitute Full-Thickness Wounds.

BackgroundSkin replacement by means of cultured epithelial keratinocytes is a well-accepted method. However, several clinical drawbacks of sheet autografts (CEA – cultured epithelial autografts) have stimulated various efforts to optimize cell culture and cell delivery. Recent developments include use of cell monolayers instead of a fully differentiated epithelium, as well as use of various biomaterials to grow and transport the cultured cells.To optimize the transfer of human keratinocytes directly to the recipient wound bed, we used an “upside-down” technique, delivering cultured cells directly to the wound with the carrier material on top.Material/MethodsSubconfluent second-passage human keratinocyte monolayers on esterified hyaluronic acid membranes (KHAMC – Keratinocyte-Hyaluronic-Acid-Membrane-Composites) were transplanted either as upside-down grafts or as upside-up grafts onto standardized full-thickness wounds in athymic nude mice versus controls with the cell-free membrane alone.ResultsIn the upside-down group, 14 days after grafting, a multi-layered, differentiating epidermis was found, whereas the wounds in the upside-up group and in the control group were not completely closed up to day 21. Persistence of human keratinocytes was shown in the upside-down group only, from day 7 until day 35 after grafting.ConclusionsThis study confirms that upside-down grafting of subconfluent monolayers of serum-free cultured human keratinocytes on esterified hyaluronic acid membranes is a suitable means to transfer actively proliferative keratinocytes, and reduces wound contraction. Compared to standard grafting protocols of cultured epithelium, such as CEA sheet grafts, it is easier to apply, does not need enzymatic detachment of cells from the culture dish, and limits the number of production steps required.

629 The ATP-dependent chromatin remodeler BRG1 controls epidermal keratinocytes migration during human cutaneous wound healing

SWI/SNF ATP-dependent chromatin remodelling complexes alter nucleosome structure, positioning and chromatin compaction state resulting in target gene activation or repression. The SWI/SNF complexes contain either BRG1 or BRM as the core ATPase together with other common and variable subunits. BRG1 is required for epidermal terminal differentiation in both mice and human; and for hair follicle stem cell activation during mouse hair regeneration and cutaneous wound healing. However, the role of SWI/SNF complexes in human skin wound healing remains unknown. Here, we demonstrate that genes encoding SWI/SNF complex subunits are expressed in human epidermis, and BRG1 is substantially upregulated in hyper-proliferating and migrating keratinocytes in healing cutaneous wounds. siRNA mediated BRG1 gene suppression leads to the substantial retardation of the wound closure in the full thickness human excision wounded skin tissue culture model and in scratched human primary keratinocyte monolayers. Furthermore, we showed that the retardation in the keratinocyte migration with no changes in cellular proliferation or apoptosis. Micro-array based transcriptome analysis revealed that BRG1 is required for full induction of genes encoding several signaling molecules, transcription factors and structural proteins involved in changes in cellular adhesion and cytoskeletal organization, driving keratinocyte migration during skin wound healing, including the stress cytokeratins 16 and 17. In summary, our data uncovered an essential role of the BRG1 chromatin remodeler in induction of the transcription programme driving keratinocyte migration during skin wound closure and re-epithelisation.

Production of progenitor cells from primary human epithelial cell monolayer cultures.

Primary keratinocytes derived from human epidermis are widely used in tissue engineering and regenerative medicine. An important aspect in clinical applications is the preservation of human skin keratinocyte stem cells. However, it is difficult to expand the number of human skin keratinocyte stem cells, which are undifferentiated and highly proliferative in culture by using standard cell culture methods. It is even more difficult to identify them, since universal specific markers for human skin keratinocyte stem cells have not been identified. In this paper, we show a method to produce a large number of primary progenitor human skin keratinocytes by using our novel culture techniques. Primary human skin keratinocyte monolayers are cultured using twice the volume of medium without serum and lacking essential fatty acids. Once the cells reach 70-80% confluence, they begin to float up into the overlying medium and are called "epithelial pop-up keratinocytes (ePUKs)" allowing the cells to be passaged without the use of trypsin. We analyzed the properties of ePUKs by cell size, cell viability, immunocytofluorescence biomarker staining, and cell cycle phase distribution by fluorescence-activated cell sorting (FACS). Our results showed that these ePUKs appear to be progenitor epithelial cells, which are small in size, undifferentiated, and have a high proliferative capacity. We believe that ePUKs are suitable for use in medical applications requiring a large number of primary human progenitor skin keratinocytes.

Interleukin-17A plays a fundamental role in the expression of psoriasis signature genes in keratinocytes through the induction of IκB-ζ

In psoriasis lesions, a diverse mixture of cytokines is upregulated which influence each other generating a complex inflammatory situation. Although this is the case, the inhibition of Interleukin-17A (IL-17A) alone showed unprecedented clinical results in psoriasis patients, indicating that IL-17A is a critical inducer of psoriasis pathogenesis. To identify IL-17A-driven keratinocyte-intrinsic signaling pathways and clarify the interplay between IL-17A with other cytokines, we treated monolayers of normal human epidermal keratinocytes in vitro with a mixture of 6 cytokines (IL-17A, TNF-α, IL-17C, IL-22, IL-36γ and IFN-γ) involved in psoriasis, to mimic the inflammatory milieu in psoriasis lesions. Microarray and gene set enrichment analysis revealed that this cytokine mixture induced similar gene expression changes with the previous transcriptome studies using psoriasis lesions. Importantly, we identified a set of IL-17A-regulated genes in keratinocytes, which recapitulate typical psoriasis genes exemplified by DEFB4A, S100A7, CCL20, CXCL1, IL23 A, IL19 and CSF3, based on differences in the expression profiles of cells stimulated with 6 cytokines versus cells stimulated with only 5 cytokines lacking IL-17A. Furthermore a specific IL-17A-induced gene, NFKBIZ, which encodes IκB-ζ, a transcriptional regulator for NF-κB, was demonstrated to have a significant role for IL-17A-induced gene expression. Thus, we present novel in vitro data from normal human keratinocytes that would help elucidating the IL-17A-driven keratinocyte activation in psoriasis.

Nanoparticle-mediated interplay of chitosan and melatonin for improved wound epithelialisation

Herein, we propose an innovative approach to improving wound healing. Our strategy is to deliver melatonin locally at the wound site by means of lecithin/chitosan nanoparticles. We used four types of chitosan that differed in terms of molecular weight and/or deacetylation degree. Melatonin encapsulation efficiency, nanoparticle size, zeta potential, biocompatibility and in vitro drug release were studied as a function of the type of chitosan used in preparation. The nanoparticles were evaluated in terms of their potential to promote wound epithelialisation via an in vitro scratch assay using a human keratinocyte (HaCaT) monolayer. The model wounds were treated with nanoparticle suspensions at a chitosan concentration of 5μgml−1, which was based on preceding cell biocompatibility studies. Nanoparticles prepared with different types of chitosan showed similar effect on the keratinocyte proliferation/migration. Nanoparticle-mediated interplay of chitosan and melatonin was shown to be crucial for improved wound epithelialisation.

IL-17A plays a central role in the expression of psoriasis signature genes through the induction of IκB-ζ in keratinocytes

In psoriasis lesions, a diverse mixture of cytokines is up-regulated that influence each other generating a complex inflammatory situation. Although this is the case, the inhibition of IL-17A alone showed unprecedented clinical results in patients, indicating that IL-17A is a critical inducer of psoriasis pathogenesis. To elucidate IL-17A-driven keratinocyte-intrinsic signaling pathways, we treated monolayers of normal human epidermal keratinocytes in vitro with a mixture of six cytokines (IL-17A, TNF-α, IL-17C, IL-22, IL-36γ and IFN-γ) involved in psoriasis to mimic the inflammatory milieu in psoriasis lesions. Microarray and gene set enrichment analysis revealed that this cytokine mixture induced similar gene expression changes with the previous transcriptome studies using psoriasis lesions. Importantly, we identified a set of IL-17A-regulated genes in keratinocytes, which recapitulate typical psoriasis genes exemplified by DEFB4A, S100A7, IL19 and CSF3, based on the differences in the expression profiles of cells stimulated with six cytokines versus cells stimulated with only five cytokines lacking IL-17A. Furthermore, a specific IL-17A-induced gene, NFKBIZ, which encodes IκB-ζ, a transcriptional regulator for NF-κB, was demonstrated to have a significant role for IL-17A-induced gene expression. Thus, we present novel in vitro data from normal human keratinocytes that would help elucidating the IL-17A-driven keratinocyte activation in psoriasis.

Lactobacillus rhamnosus GG Lysate Increases Re-Epithelialization of Keratinocyte Scratch Assays by Promoting Migration.

A limited number of studies have investigated the potential of probiotics to promote wound healing in the digestive tract. The aim of the current investigation was to determine whether probiotic bacteria or their extracts could be beneficial in cutaneous wound healing. A keratinocyte monolayer scratch assay was used to assess re-epithelialization; which comprises keratinocyte proliferation and migration. Primary human keratinocyte monolayers were scratched then exposed to lysates of Lactobacillus (L) rhamnosus GG, L. reuteri, L. plantarum or L. fermentum. Re-epithelialization of treated monolayers was compared to that of untreated controls. Lysates of L. rhamnosus GG and L. reuteri significantly increased the rate of re-epithelialization, with L. rhamnosus GG being the most efficacious. L. reuteri increased keratinocyte proliferation while L. rhamnosus GG lysate significantly increased proliferation and migration. Microarray analysis of L. rhamnosus GG treated scratches showed increased expression of multiple genes including the chemokine CXCL2 and its receptor CXCR2. These are involved in normal wound healing where they stimulate keratinocyte proliferation and/or migration. Increased protein expression of both CXCL2 and CXCR2 were confirmed by ELISA and immunoblotting. These data demonstrate that L. rhamnosus GG lysate accelerates re-epithelialization of keratinocyte scratch assays, potentially via chemokine receptor pairs that induce keratinocyte migration.

Hyaluronan Metabolism in Human Keratinocytes and Atopic Dermatitis Skin Is Driven by a Balance of Hyaluronan Synthases 1 and 3

Hyaluronan (HA) is a glycosaminoglycan synthesized directly into the extracellular matrix by three hyaluronan synthases (HAS1, HAS2, and HAS3). HA is abundantly synthesized by keratinocytes but its epidermal functions remain unclear. We used culture models to grow human keratinocytes as autocrine monolayers or as reconstructed human epidermis (RHE) to assess HA synthesis and HAS expression levels during the course of keratinocyte differentiation. In both the models, epidermal differentiation downregulates HAS3 mRNA expression while increasing HAS1 without significant changes in hyaluronidase expression. HA production correlates with HAS1 mRNA expression level during normal differentiation. To investigate the regulation of HAS gene expression during inflammatory conditions linked to perturbed differentiation, lesional and non-lesional skin biopsies of atopic dermatitis (AD) patients were analyzed. HAS3 mRNA expression level increases in AD lesions compared with healthy and non-lesional skin. Simultaneously, HAS1 expression decreases. Heparin-binding EGF-like growth factor (HB-EGF) is upregulated in AD epidermis. An AD-like HAS expression pattern is observed in RHE incubated with HB-EGF. These results indicate that HAS1 is the main enzyme responsible for HA production by normal keratinocytes and thus, must be considered as an actor of normal keratinocyte differentiation. In contrast, HAS3 can be induced by HB-EGF and seems mainly involved in AD epidermis.