Myrtus communis and Celastrol enriched plant cell culture extracts control together the pivotal role of Cutibacterium acnes and inflammatory pathways in acne.

INTRODUCTION: Acne is a multifactorial inflammatory disease of the pilosebaceous unit in which Cutibacterium acnes is one of the main triggers. A strong predominance of C. acnes phylotype IA1 is present in acne skin with higher biofilm organization and virulence, promoting local immuno-inflammation, especially the Th17 pathway. OBJECTIVES: We evaluated the single and combined pharmacological properties of the plant extracts, Myrtus communis (Myrtacine®) and Celastrol enriched plant cell culture (CEE) extracts on the C. acnes/Th17 pathway. METHODS: The effect of Myrtacine® on the virulence of C. acnes phylotype IA1 was quantified according to the expression of several related genes. The activity of Myrtacine® and CEE on the inflammatory cascade was assessed using monocytes-derived dendritic cells (Mo-DC) stimulated with membranes or biofilms of the C. acnes phylotype IA1. Finally, the effect of CEE on the Th17 pathway was studied using C. acnes stimulated sebocyte 2D cultures and 3D skin tissue models containing preactivated Th17 cells. RESULTS: Myrtacine® had an anti-virulence effect, evident as a significant and strong inhibition of the expression of several virulence factor genes by 60%-95% compared to untreated controls. Myrtacine® and CEE significantly inhibited proinflammatory cytokine (IL-6, IL-8, IL-12p40 and TNF-α) production by Mo-DC in response to C. acnes phylotype IA1. Interestingly, these two ingredients resulted in synergistic inhibition of most cytokines when used in combination. Finally, we demonstrated an inhibitory effect of CEE, in solution or formulated at 0.3%, specifically on IL-17 release by Th17 lymphocytes in a C. acnes-stimulated sebocyte 2D cultures and by Th17-lymphocytes integrated in a 3D skin models. CONCLUSIONS: 2D and 3D models were developed to represent relevant and specific pathways involved in acne. Myrtacine® and CEE were shown to alter one or more of these pathways, indicating their potential beneficial effects on this disease.

Comprehensive characterization of the structure and properties of human stratum corneum relating to barrier function and skin hydration: modulation by a moisturizer formulation.

The stratum corneum (SC) is key in the maintenance of the biomechanical barrier and hydration of skin. Our previous investigations showed beneficial effects of a combination of emollients on water capture and retention and protein and lipid organization, all of which are linked to dryness and dry skin damage. Here, we show how a formulation containing an emollient combination ("Trio") and its basal formulation (placebo) impacted the descriptors of SC hydration in SC layers. Only the Trio formulation-not its placebo formulation-modified SC biomechanical drying stress behaviour and imparted a high capacity to protect it from dehydration. This was in accordance with findings at the molecular level using Raman analyses and at the structural level using cryo-scanning electron microscopy (SEM). After topical application, only the Trio formulation profoundly increased lateral packing of lipids and their compactness. Cryo-SEM showed that, unlike the placebo formulation, the Trio formulation prevented the water loss when applied before the dehydration process. In conclusion, these studies demonstrate that stresses in the SC due to dehydration can be alleviated using a formulation containing emollients that interact with the SC lipid components.

Effect of thermal spring water on human dendritic cell inflammatory response.

BACKGROUND: Hydrotherapy appears as a valuable therapeutic tool in the management of patients suffering from chronic skin inflammatory diseases. Nevertheless, the underlying immune mechanisms of these beneficial effects remain poorly understood. To better understand the biological effects of thermal spring water on the immune system, we investigated the effects of Avène thermal spring water (ASW) on dendritic cells as key cells participating in the control of the immune response. METHODS: Dendritic cells (DCs) were generated from human monocytes and matured with LPS in ASW-based culture medium or in dexamethasone supplemented culture medium as an anti-inflammatory treatment. The phenotypes and abilities of these DCs to produce cytokines and induce allogeneic T cell response was next assessed. RESULTS: We showed that ASW modulated the differentiation of monocytes into DCs and impacted the DC maturation upon LPS priming. We observed a reduction of the CD83, CD86, CD1a and HLA-DR molecule expression and a decrease of IL-12 and IL-23 production whereas IL-10 production was increased. LPS-primed DCs generated in presence of ASW exhibited a reduced capacity to induce naive CD4+ T cell proliferation and IFN-γ and IL-17 production. CONCLUSION: Our study showed that ASW is endowed with an immunomodulatory potential. ASW limits the DC stimulatory capacity of Th1 and Th17 cell responses by impairing their maturation, IL-12 and IL-23 production and accessory cell function.

Effects of dextran sulfate, 4-t-butylcyclohexanol, pongamia oil and hesperidin methyl chalcone on inflammatory and vascular responses implicated in rosacea.

BACKGROUND: Rosacea is a chronic facial skin disorder characterized by inflammation and vascular abnormalities. The pathophysiology of rosacea involves increased activation of the capsaicin receptor, TRPV1, the vascular endothelial growth factor (VEGF) pathway, and cathelicidin LL-37, MMP-9, and KLKs. We evaluated the activity of four compounds (dextran sulfate, 4-t-butylcyclohexanol [BCH; TRP-regulin®], pongamia oil, and hesperidin methyl chalcone [HMC]) on inflammatory and vascular responses implicated in rosacea. MATERIALS AND METHODS: The anti-inflammatory activity of dextran sulfate was evaluated on PGE2 production after PMA stimulation of NCTC-2544 keratinocytes, and on normal human epidermal keratinocytes (NHEKs) after proinflammatory stimulation to mimic a rosacea environment. The anti-angiogenic activity of dextran sulfate was measured by analyzing pseudotube formation in co-cultured human microvascular endothelial cells/normal human dermal fibroblasts. HMC modulation of vascular responses and IL-8 cytokine production after SP stimulation was evaluated in human skin explants. We also assessed the effect of BCH on TRPV1 activation, and the effect of combined BCH and pongamia oil on the inflammatory response of NHEKs. RESULTS: Dextran sulfate strongly and significantly inhibited PMA-induced PGE2 production, inhibited KLK5 and MMP-9 mRNA expression, and IL-8, IL-1α and VEGF production, and displayed a highly significant inhibitory effect on VEGF-induced pseudotube formation. In SP-stimulated human skin explants, HMC significantly decreased the proportion of dilated vessels, total vessel area, and IL-8 production. BCH significantly and dose-dependently inhibited TRPV1 activation, and BCH and pongamia oil inhibited CXCL1 and CXCL6 mRNA expression and IL-8 production in NHEKs. Combined BCH/pongamia oil inhibited IL-8 production synergistically. CONCLUSION: These in vitro results showed that dextran sulfate, BCH, pongamia oil and HMC, possess complementary soothing and anti-redness properties, supporting their combination in Avène redness-relief cosmetic products for sensitive skin prone to redness, and for topical adjunctive rosacea treatment.

Measurement of the biomechanical function and structure of ex vivo drying skin using raman spectral analysis and its modulation with emollient mixtures.

An important aspect of the biomechanical behaviour of the stratum corneum (SC) is the drying stresses that develop with water loss. These stresses act as a driving force for damage in the form of chapping and cracking. Betasitosterol is a plant sterol with a structure similar to cholesterol, a key component in the intercellular lipids of the outermost layer of human skin, the SC. Cholesterol plays an important role in stabilizing the SC lipid structure, and altered levels of cholesterol have been linked with SC barrier abnormalities. Betasitosterol is currently applied topically to skin for treatment of wounds and burns. However, it is unknown what effect betasitosterol has on the biomechanical barrier function of skin. Here, by analysing the drying stress profile of SC generated during a kinetics of dehydration, we show that betasitosterol, in combination with two emollient molecules, isocetyl stearoyl stearate (ISS) and glyceryl tri-2-ethylhexanoate (GTEH), causes a significant modulation of the drying stress behaviour of the SC by reducing both the maximal peak stress height and average plateau of the drying stress profile. Raman spectra analyses demonstrate that the combination of betasitosterol with the two emollients, ISS and GTEH, allows a high water retention capacity within the SC, while the lipid conformational order by increasing the amount of trans conformers. Our study highlights the advantage of combining a biomechanical approach together with Raman spectroscopy in engineering a suitable combination of molecules for alleviating dryness and dry skin damage.

Biphasic influence of Staphylococcus aureus on human epidermal tight junctions.

Bacterial infections (e.g., with Staphylococcus aureus) are serious problems in skin with a compromised barrier, such as in patients with atopic dermatitis. Previously, it was shown that tight junction (TJ) proteins are influenced by staphylococcal infection, and TJ function is impaired after infection of the keratinocyte cell line HaCaT. However, functional studies in cells or models more similar to human skin are missing. Therefore, we investigated bacterial colonialization and infection with live S. aureus in primary human keratinocytes and reconstructed human epidermis (RHE). We show that short-term inoculation results in increased TJ barrier function-which could not be seen in HaCaT cells-hinting at an early protective effect. This is accompanied by occludin phosphorylation and sustained localization of occludin and claudin-4 at cell membranes. Long-term incubation resulted in decreased presence of claudin-1 and claudin-4 at cell membranes and decreased TJ barrier function. The agr regulon of S. aureus plays a role in the increasing but not in the decreasing effect. Proinflammatory cytokines, which are produced as a result of S. aureus inoculation, influence both phases. In summary, we show here that S. aureus can have short-term promoting effects on the TJ barrier, while in the long term it results in disturbance of TJs.

IL-1ß induces thymic stromal lymphopoietin and an atopic dermatitis-like phenotype in reconstructed healthy human epidermis.

Atopic dermatitis (AD) is a common skin inflammatory disease characterized by the production of thymic stromal lymphopoietin (TSLP) and marked TH 2 polarization. Recent studies suggest that IL-1ß contributes to the development of AD skin inflammation. Here, we have investigated the impact of IL-1ß signalling on the epidermal homeostasis of both healthy subjects and AD patients [with functional filaggrin (FLG) alleles], with particular attention to TSLP production and keratinocyte differentiation. In healthy reconstructed human epidermis (RHE), IL-1ß promoted (i) robust secretion of TSLP in an NF-κB-dependent manner and (ii) a significant decrease in the expression of filaggrin and other proteins of the epidermal differentiation complex. These effects were prevented by treatment of RHE with the anti-IL-1ß mAb canakinumab and by the IL-1 receptor antagonist anakinra. Interestingly, RHE generated from AD donors behaved like that of healthy individuals and showed comparable responses to IL-1ß signals. Collectively, our results suggest that IL-1ß may be an early key mediator for the acquisition of an AD phenotype through induction of TSLP and alteration of the epidermal homeostasis. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Acefylline activates filaggrin deimination by peptidylarginine deiminases in the upper epidermis.

BACKGROUND: Peptidylarginine deiminases (PADs) catalyze deimination (or citrullination), a calcium-dependent post-translational modification involved in several physiological processes and human diseases, such as rheumatoid arthritis and cancer. Deimination of filaggrin (FLG) by PAD1 and PAD3 during the last steps of keratinocyte differentiation is a crucial event for the epidermis function and homeostasis. This allows the complete degradation of FLG, leading to the production of free amino acids and their derivatives that are essential for epidermal photoprotection and moisturizing of the stratum corneum. OBJECTIVE: To increase the flux of this catabolic pathway, we searched for activators of PADs. METHODS: A large chemical library was screened first in silico and then by using an automated assay based on an indirect colorimetric measurement of recombinant human PAD activity. Potential activators were then confirmed using a recombinant human FLG as a substrate, and secondly after topical application at the surface of three-dimensional reconstructed human epidermis. RESULTS: The data obtained after the library screening pointed to xanthine derivatives as potential PAD activators. Among seven xanthine derivatives tested at 50-300µM, caffeine, theobromine and acefylline proved to be the most potent enhancers of in vitro deimination of FLG by PAD1 and PAD3. After topical application of a gel formulation containing 3% acefylline at the surface of reconstructed epidermis, immunoblotting analysis showed an increase in the total amount of deiminated proteins, and confocal microscopy showed an enhanced deimination in the stratum corneum. This demonstrated the activation of PADs in living cells. CONCLUSION: As a PAD activator, acefylline will be useful to study the role of deimination and could be proposed to increase or correct the hydration of the cornified layers of the epidermis.

Effects of Hydroxydecine(®) (10-hydroxy-2-decenoic acid) on skin barrier structure and function in vitro and clinical efficacy in the treatment of UV-induced xerosis.

10-Hydroxy-2-decenoic acid, a natural fatty acid only found in royal jelly, may be of value in correcting skin barrier dysfunction. We evaluated the activity of Hydroxydecine(®), its synthetic counterpart, in vitro on the regulation of epidermal differentiation markers, ex vivo on the inflammatory response and restoration of skin barrier function, and in vivo on UV-induced xerosis in healthy human volunteers. In cultured normal human keratinocytes, Hydroxydecine(®) induced involucrin, transglutaminase-1 and filaggrin protein production. In topically Hydroxydecine(®)-treated skin equivalents, immunohistochemical analysis revealed an increase in involucrin, transglutaminase-1 and filaggrin staining. In a model of thymic stromal lymphopoietin (TSLP)-induced inflamed epidermis, a Hydroxydecine(®)-containing emulsion inhibited TSLP release. In a model of inflammation and barrier impairment involving human skin explants maintained alive, Hydroxydecine(®) balm restored stratum corneum cohesion and significantly increased filaggrin expression, as shown by immunohistochemistry. It also decreased pro-inflammatory cytokine secretion (IL-4, IL-5 and IL-13). In healthy volunteers with UV-induced xerosis, the hydration index increased by +28.8% (p<0.01) and +60.4% (p<0.001) after 7 and 21 days of treatment with Hydroxydecine(®) cream, respectively. Hydroxydecine(®) thus proved its efficacy in activating keratinocyte differentiation processes in vitro, restoring skin barrier function and reducing inflammation ex vivo, and hydrating dry skin in vivo.

Binding of alpha2ML1 to the low density lipoprotein receptor-related protein 1 (LRP1) reveals a new role for LRP1 in the human epidermis.

BACKGROUND: The multifunctional receptor LRP1 has been shown to bind and internalize a large number of protein ligands with biological importance such as the pan-protease inhibitor alpha2-macroglobulin (alpha2M). We recently identified Alpha2ML1, a new member of the alpha2M gene family, expressed in epidermis. alpha2ML1 might contribute to the regulation of desquamation through its inhibitory activity towards proteases of the chymotrypsin family, notably KLK7. The expression of LRP1 in epidermis as well as its ability to internalize alpha2ML1 was investigated. METHODS AND PRINCIPAL FINDINGS: In human epidermis, LRP1 is mainly expressed within the granular layer of the epidermis, which gathers the most differentiated keratinocytes, as shown by immunohistochemistry and immunofluorescence using two different antibodies. By using various experimental approaches, we show that the receptor binding domain of alpha2ML1 (RBDl) is specifically internalized into the macrophage-like cell line RAW and colocalizes with LRP1 upon internalization. Coimmunoprecipitation assays demonstrate that RBDl binds LRP1 at the cell surface. Addition of RAP, a universal inhibitor of ligand binding to LRP1, prevents RBDl binding at the cell surface as well as internalization into RAW cells. Silencing Lrp1 expression with specific siRNA strongly reduces RBDl internalization. CONCLUSIONS AND SIGNIFICANCE: Keratinocytes of the upper differentiated layers of epidermis express LRP1 as well as alpha2ML1. Our study also reveals that alpha2ML1 is a new ligand for LRP1. Our findings are consistent with endocytosis by LRP1 of complexes formed between alpha2ML1 and proteases. LRP1 may thus control desquamation by regulating the biodisponibility of extracellular proteases.

Large-scale identification of human genes implicated in epidermal barrier function.

BACKGROUND: During epidermal differentiation, keratinocytes progressing through the suprabasal layers undergo complex and tightly regulated biochemical modifications leading to cornification and desquamation. The last living cells, the granular keratinocytes (GKs), produce almost all of the proteins and lipids required for the protective barrier function before their programmed cell death gives rise to corneocytes. We present here the first analysis of the transcriptome of human GKs, purified from healthy epidermis by an original approach. RESULTS: Using the ORESTES method, 22,585 expressed sequence tags (ESTs) were produced that matched 3,387 genes. Despite normalization provided by this method (mean 4.6 ORESTES per gene), some highly transcribed genes, including that encoding dermokine, were overrepresented. About 330 expressed genes displayed less than 100 ESTs in UniGene clusters and are most likely to be specific for GKs and potentially involved in barrier function. This hypothesis was tested by comparing the relative expression of 73 genes in the basal and granular layers of epidermis by quantitative RT-PCR. Among these, 33 were identified as new, highly specific markers of GKs, including those encoding a protease, protease inhibitors and proteins involved in lipid metabolism and transport. We identified filaggrin 2 (also called ifapsoriasin), a poorly characterized member of the epidermal differentiation complex, as well as three new lipase genes clustered with paralogous genes on chromosome 10q23.31. A new gene of unknown function, C1orf81, is specifically disrupted in the human genome by a frameshift mutation. CONCLUSION: These data increase the present knowledge of genes responsible for the formation of the skin barrier and suggest new candidates for genodermatoses of unknown origin.

A novel protease inhibitor of the alpha2-macroglobulin family expressed in the human epidermis.

In the course of a large scale analysis of late-expressed genes in the human epidermis, we identified a new member of the alpha(2)-macroglobulin (alpha2M) protease inhibitor family, A2ML1 (for alpha(2)-macroglobulin-like 1). Like A2M and PZP, A2ML1 is located on chromosome 12p13.31. A2ML1 encodes a protein of 1454 amino acids, which fits the characteristics of alpha2Ms: 1) strong conservation in amino acid sequence including most of cysteine positions with alpha2M; 2) a putative central bait domain; 3) a typical thiol ester sequence. Northern blot and reverse transcriptase-PCR studies revealed a single 5-kb A2ML1 mRNA, mainly in the epidermis granular keratinocytes. A2ML1 is also transcribed in placenta, thymus, and testis. By Western blot analysis, alpha2ML1 is detected as a monomeric, approximately 180-kDa protein in human epidermis. In vitro keratinocyte differentiation is associated with increased expression levels. By immunohistochemistry, alpha2ML1 was detected within keratinosomes in the granular layer of the epidermis, and as a secreted product in the extracellular space between the uppermost granular layer and the cornified layer. Recombinant alpha2ML1 displayed inhibitory activity toward chymotrypsin, papain, thermolysin, subtilisin A, and to a lesser extent, elastase but not trypsin. Incubation with chymotrypsin and the chymotrypsin-like kallikrein 7 protease indicated that alpha2ML1 binds covalently to these proteases, a feature shared with other members of the family. Therefore, alpha2ML1 is the first alpha2M family member detected in the epidermis. It may play an important role during desquamation by inhibiting extracellular proteases.

Characterization and expression analysis of the Spink5 gene, the mouse ortholog of the defective gene in Netherton syndrome.

The human SPINK5 gene, encoding the putative 15-domain serine protease inhibitor LEKTI, was identified as the defective gene in the severe autosomal recessive ichthyosiform skin disorder known as Netherton syndrome and as a candidate susceptibility gene for atopic disease. Here we report mapping of the murine Spink5 gene to chromosome 18 and its characterization. We show that, unlike in humans, transcription of the mouse Spink5 gene generates two mRNAs that differ in the 3' untranslated region. The encoded protein, which is detected in differentiated primary cultured keratinocytes and mouse skin as an approximately 130-kDa glycosylated precursor, displays approximately 60% identity with its human counterpart but lacks the human LEKTI domain 6. As in the human, mouse Lekti represents a marker of epithelial differentiation, strongly expressed in the granular layer of the epidermis, in suprabasal layers of stratified epithelia, and in thymic Hassall's bodies. Our data indicate that mouse Spink5/Lekti, like its human counterpart, is involved in the control of epithelial tissue homeostasis, but also highlight specific features of the murine gene and protein.