An Inflamed and Infected Reconstructed Human Epidermis to Study Atopic Dermatitis and Skin Care Ingredients.

Atopic dermatitis (AD), the most common inflammatory skin disorder, is a multifactorial disease characterized by a genetic predisposition, epidermal barrier disruption, a strong T helper (Th) type 2 immune reaction to environmental antigens and an altered cutaneous microbiome. Microbial dysbiosis characterized by the prevalence of Staphylococcus aureus (S. aureus) has been shown to exacerbate AD. In recent years, in vitro models of AD have been developed, but none of them reproduce all of the pathophysiological features. To better mimic AD, we developed reconstructed human epidermis (RHE) exposed to a Th2 pro-inflammatory cytokine cocktail and S. aureus. This model well reproduced some of the vicious loops involved in AD, with alterations at the physical, microbial and immune levels. Our results strongly suggest that S. aureus acquired a higher virulence potential when the epidermis was challenged with inflammatory cytokines, thus later contributing to the chronic inflammatory status. Furthermore, a topical application of a Castanea sativa extract was shown to prevent the apparition of the AD-like phenotype. It increased filaggrin, claudin-1 and loricrin expressions and controlled S. aureus by impairing its biofilm formation, enzymatic activities and inflammatory potential.

Development of new 3D human ex vivo models to study sebaceous gland lipid metabolism and modulations.

OBJECTIVES: Sebaceous glands maintain skin homeostasis by producing sebum. Low production can induce hair loss and fragile skin. Overproduction provokes seborrhoea and may lead to acne and inflammatory events. To better study sebaceous gland maintenance, sebocyte maturation, lipid production and ageing or inflammatory processes, we developed innovative 3D ex vivo models for human sebaceous glands. MATERIALS AND METHODS: Culture conditions and analytical methods optimized on sebocyte monolayers were validated on extracted sebaceous glands and allowed the development of two 3D models: (a) "air-liquid" interface and (b) human fibronectin-coated "sandwich" method. Lipid production was assessed with microscopy, fluorometry or flow cytometry analysis after Nile Red staining. Specific lipids (particularly squalene and peroxidized squalene) were measured by Gas or liquid Chromatography and Mass spectrometry. RESULTS: This study allowed us to select appropriate conditions and design Seb4Gln culture medium inducing sebocyte proliferation and neutral lipid production. The "air-liquid" model was appropriate to induce sebocyte isolation. The "sandwich" model enabled sebaceous gland maintenance up to 42 days. A treatment with Insulin Growth Factor-1 allowed validation of the model as we succeeded in mimicking dynamic lipid overproduction. CONCLUSION: Functional sebocyte maturation and physiological maintenance were preserved up to 6 weeks in our models. Associated with functional assays, they provide a powerful platform to mimic physiological skin lipid metabolism and to screen for active ingredients modulating sebum production.

Understanding Solar Skin Elastosis-Cause and Treatment.

Photoageing, also called actinic ageing, is the main cause of prematurely aged skin. Our expertise in elastic fibers has led us to discover a process triggered in response to ultraviolet (UV) light and which upsets the balance of elastin fibers: there is too much elastin and insufficient lysyl oxidase (LOXL1) enzyme to form functional elastic fibers. This imbalance then leads to an accumulation of nonfunctional elastin, which forms aggregates. In addition to this imbalance, UV rays also induce elafin synthesis by fibroblasts. Known to be a marker of elastotic aggregates, elafin crystallizes the elastin fibers and stimulates the formation of aggregates that cannot be naturally eliminated by the skin. We developed a Hamamelis virginiana leaf extract that was able to restore both the balance between elastin and LOXL1 and to decrease the elafin synthesis to fight and correct the damage. This specific Hamamelis virginiana extract increased LOXL1 expression by twofold and decreased elafin synthesis. As a consequence, elastic fibers became functional and aggregates of unfunctional fibers decreased. The specific Hamamelis extract activity was confirmed in vivo with decreasing wrinkles and improving skin firmness.

Methylation of LOXL1 Promoter by DNMT3A in Aged Human Skin Fibroblasts.

Lysyl oxidase-like 1 (LOXL1) is an amino-oxidase involved in maturation of elastic fibers. Its downregulation has been associated with elastic fibers repair loss in aging aorta, lung, ligament, and skin. Several evidences of LOXL1 epigenetic silencing by promoter methylation were reported in cancer and cutis laxa syndrome. We hypothesized that this mechanism could be implicated in skin aging process, as far as elastic fibers are also concerned. Anti-DNMT3A chromatin immunoprecipitation was conducted with nuclear extracts from skin fibroblasts isolated from young and elderly individuals, and showed a higher level of DNMT3A protein binding to the LOXL1 promoter in older cells concomitantly to the decrease of LOXL1 mRNA expression and the increase of LOXL1 promoter methylation. Using luciferase reporter assay driven by LOXL1 promoter in HEK293 cells, we demonstrated that LOXL1 transcriptional activity was dramatically reduced when a recombinant DNMT3A was concomitantly overexpressed. LOXL1 promoter transcriptional activity was restored in the presence of a broad-spectrum inhibitor of DNMT activity, 5-aza-2'-deoxycytidine. Finally, to assess whether the interplay between DNMT3A and LOXL1 promoter could be targeted to increase LOXL1 mRNA expression level, an Origanum majorana extract was selected among 43 plant extracts as a new inhibitor of human DNMT3A activity to restore LOXL1 secretion without cytotoxicity in aged skin fibroblasts.

In vitro glycation of an endothelialized and innervated tissue-engineered skin to screen anti-AGE molecules.

Glycation is one of the major processes responsible for skin aging through induction of the detrimental formation of advanced glycation end-products (AGEs). We developed an innovative tissue-engineered skin combining both a capillary-like and a nerve networks and designed a protocol to induce continuous AGEs formation by a treatment with glyoxal. We determined the optimal concentration of glyoxal to induce AGEs formation identified by carboxymethyl-lysin expression while keeping their toxic effects low. We showed that our tissue-engineered skin cultured for 44 days and treated with 200 µm glyoxal for 31 days displayed high carboxymethyl-lysine expression, which induced a progressively increased alteration of its capillary and nerve networks between 28 and 44 days. Moreover, it produced an epidermal differentiation defect evidenced by the lack of loricrin and filaggrin expression in the epidermis. These effects were almost completely prevented by addition of aminoguanidine 1.5 mm, an anti-glycation compound, and only slightly decreased by alagebrium 500 µm, an AGE-breaker molecule. This tissue-engineered skin model is the first one to combine a capillary and nerve network and to enable a continuous glycation over a long-term culture period. It is a unique tool to investigate the effects of glycation on skin and to screen new molecules that could prevent AGEs formation.

Exosomes bearing HLA-DR1 molecules need dendritic cells to efficiently stimulate specific T cells.

Exosomes are small vesicles (60-100 nm) secreted by various cell types upon the fusion of endosomal compartments with the plasma membrane. Exosomes from antigen-presenting cells (APC), such as B lymphocytes and dendritic cells (DC), bear MHC class II molecules. In addition, the injection of DC-derived exosomes was reported to elicit potent T cell responses in vivo. Here, we analyzed the activation of specific T cells by MHC class II-bearing exosomes in vitro. The rat mast cell line, RBL-2H3, was engineered to express human class II molecules uniformly loaded with an antigenic peptide [HLA-DR1-hemagglutinin (HA)]. These cells secreted exosomes bearing DR1 class II molecules upon stimulation by a calcium ionophore or IgE receptor cross-linking. Exosomes bearing DR1-HA(306-318) complexes activated HA/DR1-specific T cells only weakly, whereas the cross-linking of such exosomes to latex beads increased stimulation of specific T cells. By contrast, the incubation of free exosomes with DC resulted in the highly efficient stimulation of specific T cells. Thus, exosomes bearing MHC class II complexes must be taken up by professional APC for efficient T cell activation.