Comparative Analysis of Olive-Derived Phenolic Compounds' Pro-Melanogenesis Effects on B16F10 Cells and Epidermal Human Melanocytes.

Olive leaf contains plenty of phenolic compounds, among which oleuropein (OP) is the main component and belongs to the group of secoiridoids. Additionally, phenolic compounds such as oleocanthal (OL) and oleacein (OC), which share a structural similarity with OP and two aldehyde groups, are also present in olive leaves. These compounds have been studied for several health benefits, such as anti-cancer and antioxidant effects. However, their impact on the skin remains unknown. Therefore, this study aims to compare the effects of these three compounds on melanogenesis using B16F10 cells and human epidermal cells. Thousands of gene expressions were measured by global gene expression profiling with B16F10 cells. We found that glutaraldehyde compounds derived from olive leaves have a potential effect on the activation of the melanogenesis pathway and inducing differentiation in B16F10 cells. Accordingly, the pro-melanogenesis effect was investigated by means of melanin quantification, mRNA, and protein expression using human epidermal melanocytes (HEM). This study suggests that secoiridoid and its derivates have an impact on skin protection by promoting melanin production in both human and mouse cell lines.

lncRNA LINC00941 modulates MTA2/NuRD occupancy to suppress premature human epidermal differentiation.

Numerous long non-coding RNAs (lncRNAs) were shown to have a functional impact on cellular processes such as human epidermal homeostasis. However, the mechanism of action for many lncRNAs remains unclear to date. Here, we report that lncRNA LINC00941 regulates keratinocyte differentiation on an epigenetic level through association with the NuRD complex, one of the major chromatin remodelers in cells. We find that LINC00941 interacts with NuRD-associated MTA2 and CHD4 in human primary keratinocytes. LINC00941 perturbation changes MTA2/NuRD occupancy at bivalent chromatin domains in close proximity to transcriptional regulator genes, including the EGR3 gene coding for a transcription factor regulating epidermal differentiation. Notably, LINC00941 depletion resulted in reduced NuRD occupancy at the EGR3 gene locus, increased EGR3 expression in human primary keratinocytes, and increased abundance of EGR3-regulated epidermal differentiation genes in cells and human organotypic epidermal tissues. Our results therefore indicate a role of LINC00941/NuRD in repressing EGR3 expression in non-differentiated keratinocytes, consequentially preventing premature differentiation of human epidermal tissues.

Dendrite intercalation between epidermal cells tunes nociceptor sensitivity to mechanical stimuli in Drosophila larvae.

An animal's skin provides a first point of contact with the sensory environment, including noxious cues that elicit protective behavioral responses. Nociceptive somatosensory neurons densely innervate and intimately interact with epidermal cells to receive these cues, however the mechanisms by which epidermal interactions shape processing of noxious inputs is still poorly understood. Here, we identify a role for dendrite intercalation between epidermal cells in tuning sensitivity of Drosophila larvae to noxious mechanical stimuli. In wild-type larvae, dendrites of nociceptive class IV da neurons intercalate between epidermal cells at apodemes, which function as body wall muscle attachment sites, but not at other sites in the epidermis. From a genetic screen we identified miR-14 as a regulator of dendrite positioning in the epidermis: miR-14 is expressed broadly in the epidermis but not in apodemes, and miR-14 inactivation leads to excessive apical dendrite intercalation between epidermal cells. We found that miR-14 regulates expression and distribution of the epidermal Innexins ogre and Inx2 and that these epidermal gap junction proteins restrict epidermal dendrite intercalation. Finally, we found that altering the extent of epidermal dendrite intercalation had corresponding effects on nociception: increasing epidermal intercalation sensitized larvae to noxious mechanical inputs and increased mechanically evoked calcium responses in nociceptive neurons, whereas reducing epidermal dendrite intercalation had the opposite effects. Altogether, these studies identify epidermal dendrite intercalation as a mechanism for mechanical coupling of nociceptive neurons to the epidermis, with nociceptive sensitivity tuned by the extent of intercalation.

In vitro generation of epidermal keratinocytes from human CD34-positive hematopoietic stem cells.

The epidermis is largely composed of keratinocytes (KCs), and the proliferation and differentiation of KCs from the stratum basale to the stratum corneum is the cellular hierarchy present in the epidermis. In this study, we explore the differentiation abilities of human hematopoietic stem cells (HSCs) into KCs. Cultured HSCs positive for CD34, CD45, and CD133 with prominent telomerase activity were induced with keratinocyte differentiation medium (KDM), which is composed of bovine pituitary extract (BPE), epidermal growth factor (EGF), insulin, hydrocortisone, epinephrine, transferrin, calcium chloride (CaCl2), bone morphogenetic protein 4 (BMP4), and retinoic acid (RA). Differentiation was monitored through the expression of cytokeratin markers K5 (keratin 5), K14 (keratin 14), K10 (keratin 10), K1 (keratin 1), transglutaminase 1 (TGM1), involucrin (IVL), and filaggrin (FLG) on day 0 (D0), day 6 (D6), day 11 (D11), day 18 (D18), day 24 (D24), and day 30 (D30) using immunocytochemistry, fluorescence microscopy, flow cytometry, qPCR, and Western blotting. The results revealed the expression of K5 and K14 genes in D6 cells (early keratinocytes), K10 and K1 genes in D11-D18 cells (mature keratinocytes) with active telomerase enzyme, and FLG, IVL, and TGM1 in D18-D24 cells (terminal keratinocytes), and by D30, the KCs were completely enucleated similar to cornified matrix. This method of differentiation of HSCs to KCs explains the cellular order exists in the normal epidermis and opens the possibility of exploring the use of human HSCs in the epidermal differentiation.

C. elegans Afadin is required for epidermal morphogenesis and functionally interfaces with the cadherin-catenin complex and RhoGAP PAC-1/ARHGAP21.

During epithelial morphogenesis, the apical junctions connecting cells must remodel as cells change shape and make new connections with their neighbors. In the C. elegans embryo, new apical junctions form when epidermal cells migrate and seal with one another to encase the embryo in skin ('ventral enclosure'), and junctions remodel when epidermal cells change shape to squeeze the embryo into a worm shape ('elongation'). The junctional cadherin-catenin complex (CCC), which links epithelial cells to each other and to cortical actomyosin, is essential for C. elegans epidermal morphogenesis. RNAi genetic enhancement screens have identified several genes encoding proteins that interact with the CCC to promote epidermal morphogenesis, including the scaffolding protein Afadin (AFD-1), whose depletion alone results in only minor morphogenesis defects. Here, by creating a null mutation in afd-1, we show that afd-1 provides a significant contribution to ventral enclosure and elongation on its own. Unexpectedly, we find that afd-1 mutant phenotypes are strongly modified by diet, revealing a previously unappreciated parental nutritional input to morphogenesis. We identify functional interactions between AFD-1 and the CCC by demonstrating that E-cadherin is required for the polarized distribution of AFD-1 to cell contact sites in early embryos. Finally, we show that afd-1 promotes the enrichment of polarity regulator, and CCC-interacting protein, PAC-1/ARHGAP21 to cell contact sites, and we identify genetic interactions suggesting that afd-1 and pac-1 regulate epidermal morphogenesis at least in part through parallel mechanisms. Our findings reveal that C. elegans AFD-1 makes a significant contribution to epidermal morphogenesis and functionally interfaces with core and associated CCC proteins.

Upregulation of IDO gene expression reduces the immunogenicity of epidermal cells and strengthens the immune protection of epidermal cells during transplantation treatment of wounds.

BACKGROUND: Epidermal cell transplantation is a feasible treatment option for large wounds; however, sources of autologous epidermal cells are often limited. Allogeneic epidermal cells can be cultured conveniently; however, related immune rejection needs to be addressed. Herein, we hypothesized that the immunogenicity of epidermal cells with high indoleamine 2,3-dioxygenase (IDO) expression may be reduced by gene transfection. METHODS/RESULTS: To test this hypothesis, we obtained stable transfectants by transfecting epidermal stem cells with a lentiviral vector encoding the IDO gene and screening them for puromycin resistance (a marker for successful transfection). The phenotype tested using cell counting kit -8 and Transwell assays confirmed that IDO-transfected epidermal cells maintained their characteristics. Co-culture of IDO-transfected epidermal cells with allogeneic CD4+ T cells in vitro showed that the upregulation of IDO expression in epidermal cells inhibited the proliferation of CD4+ T cells (P < 0.001, P < 0.001, and P < 0.001, respectively) and promoted their apoptosis (P = 0.00028, P = 0.0006, and P = 0.00247, respectively) and transformation into functional regulatory T cells (Tregs) (P = 0.0051, P = 0.0132, and P = 0.0248, respectively) compared with Con, NC, and 1-MT groups. The increased proportion of Tregs may be related to the overexpression of IDO, which promoted the expression of transforming growth factor beta (TGF-ß) (P = 0.0001, P = 0.0013, and, P = 0.0009) and interleukin (IL) 10 (IL-10) (P = 0.0062, P = 0.0058, and P = 0.0119) while inhibited the expression of IL-2 (P = 0.0012, P = 0.0126, and P = 0.0066). We further verified these effects in vivo as transplanted IDO-transfected epidermal stem cells were effective in treating wounds in mice. On days 5 and 7, wounds treated with IDO cells healed faster than those in the other groups (day 5: P = 0.012 and P = 0.0136; day 7: P = 0.0242 and P = 0.0187, respectively), whereas this effect was significantly inhibited by 1-methyltryptophan (1-MT) (day 5: P = 0.0303; day 7: P = 0.0105). Immunofluorescence staining detected IDO and CD4+ Foxp3+ Tregs in the transplanted wounds, which may promote Foxp3+ Tregs in the wound tissue (day 5: P < 0.0001, P < 0.0001, and P < 0.0001; day 7: P < 0.0001, P < 0.0001, and P < 0.0001), respectively) and decrease CD4+ T cells (day 5: P < 0.0001, P < 0.0001, and P < 0.0001; day 7: P < 0.0001, P < 0.0001, and P < 0.0001). CONCLUSION: Our results suggest that the upregulation of IDO expression in epidermal stem cells can reduce their immunogenicity by promoting Tregs, thus inducing the immune protection of epidermal stem cells.

P75NTR regulates autophagy through the YAP-mTOR pathway to increase the proliferation of interfollicular epidermal cells and promote wound healing in diabetic mice.

Wound healing is delayed in diabetic patients. Increased autophagy and dysfunction of interfollicular epidermal (IFE) cells are closely associated with delayed healing of diabetic wounds. Autophagy plays an important role in all stages of wound healing, but its role in diabetic wound healing and the underlying molecular mechanisms are not clear. Here, we found that diabetic mice had delayed wound healing and increased autophagy in wounds compared with normal mice and that chloroquine, an inhibitor of autophagy, decreased the level of autophagy, improved the function of IFE cells, and accelerated wound healing in diabetic mice. Treatment of IFE cells with advanced glycosylation end products (AGEs) resulted in increased microtubule-associated protein chain (LC3) expression and decreased prostacyclin-62 (P62) expression, indicating increased autophagy in AGE-treated IFE cells. Moreover, P75NTR reduced autophagy in IFE cells in the presence of AGEs and significantly increased the proliferation of IFE cells. In addition, P75NTR participated in regulating autophagy in IFE cells and in wounds in diabetic mice through the YAP-mTOR signalling pathway, which increased the functional activity of the cells and the healing rate of wounds in diabetic mice. Thus, our study suggests that P75NTR protects IFE cells against AGEs by affecting autophagy and accelerating wound healing in diabetic mice, providing a basis for understanding the role of autophagy in diabetic wound healing.

Cooperative actin filament nucleation by the Arp2/3 complex and formins maintains the homeostatic cortical array in Arabidopsis epidermal cells.

Precise control over how and where actin filaments are created leads to the construction of unique cytoskeletal arrays within a common cytoplasm. Actin filament nucleators are key players in this activity and include the conserved actin-related protein 2/3 (Arp2/3) complex as well as a large family of formins. In some eukaryotic cells, these nucleators compete for a common pool of actin monomers and loss of one favors the activity of the other. To test whether this mechanism is conserved, we combined the ability to image single filament dynamics in the homeostatic cortical actin array of living Arabidopsis (Arabidopsis thaliana) epidermal cells with genetic and/or small molecule inhibitor approaches to stably or acutely disrupt nucleator activity. We found that Arp2/3 mutants or acute CK-666 treatment markedly reduced the frequency of side-branched nucleation events as well as overall actin filament abundance. We also confirmed that plant formins contribute to side-branched filament nucleation in vivo. Surprisingly, simultaneous inhibition of both classes of nucleator increased overall actin filament abundance and enhanced the frequency of de novo nucleation events by an unknown mechanism. Collectively, our findings suggest that multiple actin nucleation mechanisms cooperate to generate and maintain the homeostatic cortical array of plant epidermal cells.

Human filaggrin monomer does not seem to be a proteasome target.

Absence of a functional proteasome in the suprabasal layers of the epidermis is responsible for keratosis linearis with ichthyosis congenital and sclerosing keratoderma syndrome. Patient epidermis shows hypergranulosis associated with abnormally shaped keratohyalin granules and abnormal distribution of filaggrin in the Stratum granulosum and Stratum corneum. This suggests that the proteasome is involved in the degradation of filaggrin. To test this hypothesis, the proteasome proteolytic activity was inhibited in 3D reconstructed human epidermis (RHE) with the specific clasto-lactacystin ß-lactone inhibitor. Confirming the efficacy of inhibition, ubiquitinated proteins accumulated in treated RHEs as compared to controls. Levels of urocanic acid (UCA) and pyrrolidone carboxylic acid (PCA), the end products of filaggrin degradation, were reduced. However, neither filaggrin accumulation nor appearance of filaggrin-derived peptides were observed. On the contrary, the amount of filaggrin was shown to decrease, and a similar tendency was observed for profilaggrin, its precursor. Accumulation of small cytoplasmic vesicles associated with a significant increase in autophagy markers indicated activation of the autophagy process upon proteasome inhibition. Taken together, these results suggest that the perturbation of UCA and PCA production after proteasome inhibition was probably due to down-regulation of filaggrin expression rather than to blocking of filaggrin proteolysis.

From epidermal cells to functional pores: Understanding stomatal development.

Stomata, small hydromechanical valves in the leaf epidermis, are fundamental in regulating gas exchange and water loss between plants and the environment. Stomatal development involves a series of coordinated events ranging from the initial cell division that determines the meristemoid mother cells to forming specialized structures such as guard cells. These events are orchestrated by the transcription factors SPEECHLESS, FAMA, and MUTE through signaling networks. The role of plant hormones (e.g., abscisic acid, jasmonic acid, and brassinosteroids) in regulating stomatal development has been elucidated through these signaling cascades. In addition, environmental factors, such as light availability and CO2 concentration, also regulate the density and distribution of stomata in leaves, ultimately affecting overall water use efficiency. In this review, we highlight the mechanisms underlying stomatal development, connecting key signaling processes that activate or inhibit cell differentiation responsible for forming guard cells in the leaf epidermis. The factors responsible for integrating transcription factors, hormonal responses, and the influence of climatic factors on the signaling network that leads to stomatal development in plants are further discussed. Understanding the intricate connections between these factors, including the metabolic regulation of plant development, may enable us to maximize plant productivity under specific environmental conditions in changing climate scenarios.

Systemically administered wound-homing peptide accelerates wound healing by modulating syndecan-4 function.

CAR (CARSKNKDC) is a wound-homing peptide that recognises angiogenic neovessels. Here we discover that systemically administered CAR peptide has inherent ability to promote wound healing: wounds close and re-epithelialise faster in CAR-treated male mice. CAR promotes keratinocyte migration in vitro. The heparan sulfate proteoglycan syndecan-4 regulates cell migration and is crucial for wound healing. We report that syndecan-4 expression is restricted to epidermis and blood vessels in mice skin wounds. Syndecan-4 regulates binding and internalisation of CAR peptide and CAR-mediated cytoskeletal remodelling. CAR induces syndecan-4-dependent activation of the small GTPase ARF6, via the guanine nucleotide exchange factor cytohesin-2, and promotes syndecan-4-, ARF6- and Cytohesin-2-mediated keratinocyte migration. Finally, we show that genetic ablation of syndecan-4 in male mice eliminates CAR-induced wound re-epithelialisation following systemic administration. We propose that CAR peptide activates syndecan-4 functions to selectively promote re-epithelialisation. Thus, CAR peptide provides a therapeutic approach to enhance wound healing in mice; systemic, yet target organ- and cell-specific.

The R2R3-MYB transcription factor EVER controls the emission of petunia floral volatiles by regulating epicuticular wax biosynthesis in the petal epidermis.

The epidermal cells of petunia (Petunia × hybrida) flowers are the main site of volatile emission. However, the mechanisms underlying the release of volatiles into the environment are still being explored. Here, using cell-layer-specific transcriptomic analysis, reverse genetics by virus-induced gene silencing and clustered regularly interspaced short palindromic repeat (CRISPR), and metabolomics, we identified EPIDERMIS VOLATILE EMISSION REGULATOR (EVER)-a petal adaxial epidermis-specific MYB activator that affects the emission of volatiles. To generate ever knockout lines, we developed a viral-based CRISPR/Cas9 system for efficient gene editing in plants. These knockout lines, together with transient-suppression assays, revealed EVER's involvement in the repression of low-vapor-pressure volatiles. Internal pools and annotated scent-related genes involved in volatile production and emission were not affected by EVER. RNA-Seq analyses of petals of ever knockout lines and EVER-overexpressing flowers revealed enrichment in wax-related biosynthesis genes. Liquid chromatography/gas chromatography-MS analyses of petal epicuticular waxes revealed substantial reductions in wax loads in ever petals, particularly of monomers of fatty acids and wax esters. These results implicate EVER in the emission of volatiles by fine-tuning the composition of petal epicuticular waxes. We reveal a petunia MYB regulator that interlinks epicuticular wax composition and volatile emission, thus unraveling a regulatory layer in the scent-emission machinery in petunia flowers.

Requisite instruments for the establishment of three-dimensional epidermal human skin equivalents-A methods review.

Human skin equivalents (HSEs) are three-dimensional skin organ culture models raised in vitro. This review gives an overview of common techniques for setting up HSEs. The HSE consists of an artificial dermis and epidermis. 3T3-J2 murine fibroblasts, purchased human fibroblasts or freshly isolated and cultured fibroblasts, together with other components, for example, collagen type I, are used to build the scaffold. Freshly isolated and cultured keratinocytes are seeded on top. It is possible to add other cell types, for example, melanocytes, to the HSE-depending on the research question. After several days and further steps, the 3D skin can be harvested. Additionally, we show possible markers and techniques for evaluation of artificial skin. Furthermore, we provide a comparison of HSEs to human skin organ culture, a model which employs human donor skin. We outline advantages and limitations of both models and discuss future perspectives in using HSEs.

Strigolactones promote the localization of the ABA exporter ABCG25 at the plasma membrane in root epidermal cells of Arabidopsis thaliana.

The phytohormones strigolactones crosstalk with abscisic acid (ABA) in acclimation to osmotic stress, as ascertained in leaves. However, our knowledge about underground tissues is limited, and lacking in Arabidopsis: whether strigolactones affect ABA transport across plasma membranes has never been addressed. We evaluated the effect of strigolactones on the localization of ATP BINDING CASSETTE G25 (ABCG25), an ABA exporter in Arabidopsis thaliana. Wild-type, strigolactone-insensitive, and strigolactone-depleted seedlings expressing a green fluorescent protein:ABCG25 construct were treated with ABA or strigolactones, and green fluorescent protein was quantified by confocal microscopy in different subcellular compartments of epidermal root cells. We show that strigolactones promote the localization of an ABA transporter at the plasma membrane by enhancing its endosomal recycling. Genotypes altered in strigolactone synthesis or perception are not impaired in ABCG25 recycling promotion by ABA, which acts downstream or independent of strigolactones in this respect. Additionally, we confirm that osmotic stress decreases strigolactone synthesis in A. thaliana root cells, and that this decrease may support local ABA retention under low water availability by allowing ABCG25 internalization. Thus, we propose a new mechanism for ABA homeostasis regulation in the context of osmotic stress acclimation: the fine-tuning by strigolactones of ABCG25 localization in root cells.

HD-Zip IV transcription factors: Drivers of epidermal cell fate integrate metabolic signals.

The leaf epidermis comprises the outermost layer of cells that protect plants against environmental stresses such as drought, ultraviolet radiation, and pathogen attack. Research over the past decades highlights the role of class IV homeodomain leucine-zipper (HD-Zip IV) transcription factors (TFs) in driving differentiation of various epidermal cell types, such as trichomes, guard cells, and pavement cells. Evolutionary origins of this family in the charophycean green algae and HD-Zip-specific gene expression in the maternal genome provide clues to unlocking their secrets which include ties to cell cycle regulation. A distinguishing feature of these TFs is the presence of a lipid binding pocket that integrates metabolic information with gene expression. Identities of metabolic partners are beginning to emerge, uncovering feedback loops to maintain epidermal cell specification. Discoveries of associated molecular mechanisms are revealing fascinating links to phospholipid and sphingolipid metabolism and mechanical signaling.

Vitamin D receptor cross-talk with p63 signaling promotes epidermal cell fate.

The vitamin D receptor with its ligand 1,25 dihydroxy vitamin D3 (1,25D3) regulates epidermal stem cell fate, such that VDR removal from Krt14 expressing keratinocytes delays re-epithelialization of epidermis after wound injury in mice. In this study we deleted Vdr from Lrig1 expressing stem cells in the isthmus of the hair follicle then used lineage tracing to evaluate the impact on re-epithelialization following injury. We showed that Vdr deletion from these cells prevents their migration to and regeneration of the interfollicular epidermis without impairing their ability to repopulate the sebaceous gland. To pursue the molecular basis for these effects of VDR, we performed genome wide transcriptional analysis of keratinocytes from Vdr cKO and control littermate mice. Ingenuity Pathway analysis (IPA) pointed us to the TP53 family including p63 as a partner with VDR, a transcriptional factor that is essential for proliferation and differentiation of epidermal keratinocytes. Epigenetic studies on epidermal keratinocytes derived from interfollicular epidermis showed that VDR is colocalized with p63 within the specific regulatory region of MED1 containing super-enhancers of epidermal fate driven transcription factor genes such as Fos and Jun. Gene ontology analysis further implicated that Vdr and p63 associated genomic regions regulate genes involving stem cell fate and epidermal differentiation. To demonstrate the functional interaction between VDR and p63, we evaluated the response to 1,25(OH)2D3 of keratinocytes lacking p63 and noted a reduction in epidermal cell fate determining transcription factors such as Fos, Jun. We conclude that VDR is required for the epidermal stem cell fate orientation towards interfollicular epidermis. We propose that this role of VDR involves cross-talk with the epidermal master regulator p63 through super-enhancer mediated epigenetic dynamics.

Barrier function and ultrastructure characteristics of epidermis in patients with primary cutaneous amyloidosis.

Previous studies on primary cutaneous amyloidosis (PCA) have mainly focused on exploring genetic mutation and components of amyloid in patients with PCA. However, studies on skin barrier function in PCA patients are scarce. Here, we detected the skin barrier function in PCA patients and healthy people by using noninvasive techniques and characterized ultrastructural features of PCA lesions compared with healthy people using transmission electron microscopy (TEM). The expression of proteins related to skin barrier function was examined by immunohistochemistry staining. A total of 191 patients with clinically diagnosed PCA and 168 healthy individuals were enrolled in the study. Our analysis revealed that all investigated lesion areas displayed higher transepidermal water loss and pH values, and lower Sebum levels and stratum corneum hydration levels in PCA patients compared with the same site area in healthy individuals. The TEM results showed that the intercellular spaces between the basal cells were enlarged and the number of hemidesmosomes decreased in PCA lesions. Immunohistochemical staining showed that the expression of integrin α6 and E-cadherin in PCA patients was less than that in healthy controls, while no differences in the expression of loricrin and filaggrin were observed. Our study revealed that individuals with PCA displayed skin barrier dysfunction, which may be related to alterations in epidermal ultrastructure and a decrease in the skin barrier-related protein E-cadherin. However, the molecular mechanisms underlying skin barrier dysfunction in PCA remain to be elucidated.

Ovol1/2 loss-induced epidermal defects elicit skin immune activation and alter global metabolism.

Skin epidermis constitutes the outer permeability barrier that protects the body from dehydration, heat loss, and myriad external assaults. Mechanisms that maintain barrier integrity in constantly challenged adult skin and how epidermal dysregulation shapes the local immune microenvironment and whole-body metabolism remain poorly understood. Here, we demonstrate that inducible and simultaneous ablation of transcription factor-encoding Ovol1 and Ovol2 in adult epidermis results in barrier dysregulation through impacting epithelial-mesenchymal plasticity and inflammatory gene expression. We find that aberrant skin immune activation then ensues, featuring Langerhans cell mobilization and T cell responses, and leading to elevated levels of secreted inflammatory factors in circulation. Finally, we identify failure to gain body weight and accumulate body fat as long-term consequences of epidermal-specific Ovol1/2 loss and show that these global metabolic changes along with the skin barrier/immune defects are partially rescued by immunosuppressant dexamethasone. Collectively, our study reveals key regulators of adult barrier maintenance and suggests a causal connection between epidermal dysregulation and whole-body metabolism that is in part mediated through aberrant immune activation.

Human epidermis organotypic cultures, a reproducible system recapitulating the epidermis in vitro.

The translatability of research is highly dependent on models that recapitulate human tissues and organs. Here, we describe a procedure for the generation of human epidermis organotypic cultures (HEOCs) from primary keratinocytes isolated from foreskin and adult skin as well as from an immortalized keratinocyte cell line (KerTr). We tested several media conditions to develop a defined HEOC growing and expansion media. We characterized the HEOCs and show that in optimal culture conditions they express the proliferation marker Ki67, the basement membrane protein collagen 17 (col17) and the epidermal differentiation markers keratin 15 (K15), keratin 14 (K14), keratin 5 (K5), keratin 10 (K10), keratin 1 (K1), transglutaminase 1 (TGM1), transglutaminase 3 (TGM3) and filaggrin (FLG). Thus, they recapitulate the human epidermis and are stratified from the basal layer to the stratum corneum. These HEOC can be generated reproducibly on a large scale, making it an invaluable model for screening therapeutic compounds and also for the study of pathologies affecting the epidermis.