Brimonidine displays anti-inflammatory properties in the skin through the modulation of the vascular barrier function.

BACKGROUND: Rosacea is a chronic inflammatory skin disease. Characteristic vascular changes in rosacea skin include enlarged, dilated vessels of the upper dermis and blood flow increase. Brimonidine is approved for symptomatic relief of the erythema of rosacea. It acts by selectively binding to α2-adrenergic receptors present on smooth muscle in the peripheral vasculature, resulting in transient local vasoconstriction. OBJECTIVES: To provide further evidence of the anti-inflammatory potential of brimonidine across preclinical models of skin inflammation and its ability to decrease the neutrophil infiltration in human skin after ultraviolet light exposure. METHODS: The anti-inflammatory properties of brimonidine through modulation of the vascular barrier function were assessed using in vivo neurogenic vasodilation and acute inflammatory models and a well-described in vitro transmigration assay. A clinical study assessed the neutrophil infiltration in human skin after exposure to UV in 37 healthy Caucasian male subjects. RESULTS: In vitro, brimonidine affects the transmigration of human neutrophils through the endothelial barrier by modulating adhesion molecules. In vivo, in the mouse, topical treatment with brimonidine, used at a vasoconstrictive dose, confirmed its anti-inflammatory properties and prevented leucocyte recruitment (rolling and adhesion) mediated by endothelial cells. Topical pretreatment with brimonidine tartrate 0.33% gel once a day for 4 days significantly prevented neutrophil infiltration by 53.9% in human skin after exposure to UV light. CONCLUSION: Results from in vitro, in vivo and from a clinical study indicate that brimonidine impacts acute inflammation of the skin by interfering with neurogenic activation and/or recruitment of neutrophils.

Mid-infrared spectral microimaging of inflammatory skin lesions.

Skin is one of the most important organs of the human body because of its characteristics and functions. There are many alterations, either pathological or physiological, that can disturb its functioning. However, at present all methods used to investigate skin diseases, non-invasive or invasive, are based on clinical examinations by physicians. Thus, diagnosis, prognosis and therapeutic management rely on the expertise of the practitioner, the quality of the method and the accessibility of distinctive morphological characteristics of each lesion. To overcome the high sensitivity of these parameters, techniques based on more objective criteria must be explored. Vibrational spectroscopy has become as a key technique for tissue analysis in the biomedical research field. Based on a non-destructive light/matter interaction, this tool provides information about specific molecular structure and composition of the analyzed sample, thus relating to its precise physiopathological state and permitting to distinguish lesional from normal tissues. This label-free optical method can be performed directly on the paraffin-embedded tissue sections without chemical dewaxing. In this study, the potential of the infrared microspectroscopy, combined with data classification methods was demonstrated, to characterize at the tissular level different types of inflammatory skin lesions, and this independently from conventional histopathology.

A topical treatment containing heat-treated Lactobacillus johnsonii NCC 533 reduces Staphylococcus aureus adhesion and induces antimicrobial peptide expression in an in vitro reconstructed human epidermis model.

Staphylococcus aureus colonization is thought to contribute to the pathophysiology of atopic dermatitis (AD). AD patients exhibit reduced levels of cutaneous antimicrobial peptides (AMPs), which may explain their increased susceptibility to infections. Using an in vitro reconstructed human epidermis (RHE) model, we sought to determine whether topical application of a non-replicating probiotic, heat-treated Lactobacillus johnsonii NCC 533 (HT La1), could inhibit S. aureus adhesion to skin and boost cutaneous innate immunity. We found that application of HT La1 suspension to RHE samples reduced the binding of radiolabelled S. aureus by up to 74%. To investigate a potential effect of HT La1 on innate immunity, we analysed the expression of nine AMP genes, including those encoding beta defensins and S100 proteins, following topical application of HT La1 in suspension or in a daily moisturizer lotion. Analysed genes were induced by up to fourfold in a dose-dependent manner by HT La1 in suspension and by up to 2.4-fold by HT La1 in the moisturizer lotion. Finally, using ELISA and immunohistochemical detection, we evaluated the expression and secretion of the AMPs hBD-2 and psoriasin and determined that both proteins were induced by topical HT La1, particularly in the stratum corneum of the RHE. These findings demonstrate that a topically applied, non-replicating probiotic can modulate endogenous AMP expression and inhibit binding of S. aureus to an RHE model in vitro. Moreover, they suggest that a topical formulation containing HT La1 could benefit atopic skin by enhancing cutaneous innate immunity and reducing S. aureus colonization.

Monitoring UV-induced signalling pathways in an ex vivo skin organ culture model using phospho-antibody array.

We investigated UV-induced signalling in an ex vivo skin organ culture model using phospho-antibody array. Phosphorylation modulations were analysed in time-course experiments following exposure to solar-simulated UV and validated by Western blot analyses. We found that UV induced P-p38 and its substrates, P-ERK1/2 and P-AKT, which were previously shown to be upregulated by UV in cultured keratinocytes and in vivo human skin. This indicates that phospho-antibody array applied to ex vivo skin organ culture is a relevant experimental system to investigate signalling events following perturbations. As the identified proteins are components of pathways implicated in skin tumorigenesis, UV-exposed skin organ culture model could be used to investigate the effect on these pathways of NMSC cancer drug candidates. In addition, we found that phospho-HCK is induced upon UV exposure, producing a new candidate for future studies investigating its role in the skin response to UV and UV-induced carcinogenesis.

Topical Treatment of Rosacea with Ivermectin Inhibits Gene Expression of Cathelicidin Innate Immune Mediators, LL-37 and KLK5, in Reconstructed and Ex Vivo Skin Models.

INTRODUCTION: Numerous intrinsic and extrinsic factors have been associated with the pathophysiology of rosacea, including dysregulation of innate immunity. A high level of cathelicidin antimicrobial peptides (e.g., LL-37) has been shown in the facial skin of patients with rosacea. Excessive production of both LL-37 and KLK5, the serine protease responsible for its cleavage, has been suggested to play a role in the pathophysiology of rosacea. Ivermectin 10 mg/g cream, indicated for the treatment of inflammatory lesions of rosacea, is reported to have dual anti-parasitic and anti-inflammatory properties. However, the exact mechanism of action of ivermectin cream in the treatment of rosacea is unknown. METHODS: This study aimed to evaluate the effect of ivermectin on the expression of KLK5 and the subsequent effect on the maturation process of cathelicidins. Experimental studies were performed either on normal human epidermal keratinocytes (NHEK), reconstructed human epidermis (RHE) or on human skin ex vivo stimulated with calcitriol (1α,25-dihydroxyvitamin D3), which is known to induce KLK5 and LL-37 expression. RESULTS: The results show that ivermectin is able to inhibit KLK5 and CAMP gene expression and protein secretion in NHEK cells stimulated with calcitriol. Those results were confirmed in 3D models of the skin (RHE and skin ex vivo). The anti-inflammatory effects of ivermectin were associated with an inhibition of IL-8, IL-6 and MCP-1 (CCL2) secretion from NHEK cells. CONCLUSIONS: These results suggest that ivermectin can prevent the inflammatory effects of rosacea triggered by abnormal LL-37 processing, through the inhibition of KLK5 gene expression in the epidermis. FUNDING: Nestlé Skin Health R&D.

IL-17/Th17 pathway is activated in acne lesions.

The mechanisms of inflammation in acne are currently subject of intense investigation. This study focused on the activation of adaptive and innate immunity in clinically early visible inflamed acne lesions and was performed in two independent patient populations. Biopsies were collected from lesional and non-lesional skin of acne patients. Using Affymetrix Genechips, we observed significant elevation of the signature cytokines of the Th17 lineage in acne lesions compared to non-lesional skin. The increased expression of IL-17 was confirmed at the RNA and also protein level with real-time PCR (RT-PCR) and Luminex technology. Cytokines involved in Th17 lineage differentiation (IL-1ß, IL-6, TGF-ß, IL23p19) were remarkably induced at the RNA level. In addition, proinflammatory cytokines and chemokines (TNF-α, IL-8, CSF2 and CCL20), Th1 markers (IL12p40, CXCR3, T-bet, IFN-γ), T regulatory cell markers (Foxp3, IL-10, TGF-ß) and IL-17 related antimicrobial peptides (S100A7, S100A9, lipocalin, hBD2, hBD3, hCAP18) were induced. Importantly, immunohistochemistry revealed significantly increased numbers of IL-17A positive T cells and CD83 dendritic cells in the acne lesions. In summary our results demonstrate the presence of IL-17A positive T cells and the activation of Th17-related cytokines in acne lesions, indicating that the Th17 pathway is activated and may play a pivotal role in the disease process, possibly offering new targets of therapy.

Characterization of ABC transporters in human skin.

BACKGROUND: Most identified drug transporters belong to the ATP-binding cassette (ABC) and solute carrier (SLC) families. Recent research indicates that these transporters play an important role in the absorption, distribution and excretion of drugs, and are involved in clinically relevant drug-drug interactions for systemic drugs. However, very little is known about the role of drug transporters in human skin, especially in the disposition of topically applied drugs, and their involvement in drug-drug interactions. The aim of this work was to characterize the ABC transporters in human skin. METHODS: Expressions of ABCB1 multidrug resistance protein 1 (MDR1) also known as P-gp, ABCC1 and ABCC2 multidrug resistance-associated protein 1 and 2 (MRP1 and MRP2), and ABCG2 brest cancer resistance protein (BCRP) in human skin tissues were analyzed by quantitative real-time polymerase chain reaction (RT-PCR). The modulations of ABCB1 and ABCC1 expressions were analyzed after ex vivo treatment of human skin with rifampicin and dexamethasone. The localization of the major transporter MRP1 in human skin was analyzed by immunohistochemistry. Finally, functional analysis of MRP1 in human skin was performed using different specific substrates and inhibitors. RESULTS: The expressions of ABCB1, ABCC1, ABCC2, and ABCG2 were all detected in human skin, of which the expression of ABCC1 was considered the most important. The comparison of human skin with human hepatocytes and kidneys shows that the expression of ABCC1 increased 15-fold in skin than in hepatocytes. Immunohistochemistry revealed marked expressions of MRP1 within the hair follicle, sweat gland and muscle, as well as moderate expression in the basal epidermis. Functional analysis demonstrated that the skin absorptions of rhodamine 123, [3H]-vinblastine, and [3H]-LTC4 were markedly decreased in the presence of MRP1 inhibitors (verapamil and MK571), thus supporting the role of MRP1 in the uptake of compounds from the epidermal compartment as well as their secretion into the bloodstream and sweat ducts. CONCLUSIONS: The present findings are the first to demonstrate the involvement of MRP1 in drug uptake in human skin.

In vitro assessment of eye irritancy using the Reconstructed Human Corneal Epithelial SkinEthic HCE model: application to 435 substances from consumer products industry.

The 7th amendment of the EU Cosmetics Directive led to the ban of eye irritation testing for cosmetic ingredients in animals, effective from March 11th 2009. Over the last 20years, many efforts have been made to find reliable and relevant alternative methods. The SkinEthic HCE model was used to evaluate the in vitro eye irritancy potential of substances from a cosmetic industry portfolio. An optimized protocol based on a specific 1-h treatment and a 16-h post-treatment incubation period was first assessed on a set of 102 substances. The prediction model (PM) based on a 50% viability cut-off, allowed to draw up two classes (Irritants and Non-Irritants), with good associated sensitivity (86.2%) and specificity (83.5%). To check the robustness of the method, the evaluated set was expanded up to 435 substances. Final performances maintained a high level and were characterized by an overall accuracy value > 82% when using EU or GHS classification rules. Results showed that the SkinEthic HCE test method is a promising in vitro tool for the prediction of eye irritancy. Optimization datasets were shared with the COLIPA Eye Irritation Project Team and ECVAM experts, and reviewed as part of an ongoing progression to enter an ECVAM prospective validation study for eye irritation.

A characteristic subset of psoriasis-associated genes is induced by oncostatin-M in reconstituted epidermis.

The pathological manifestations of psoriasis are orchestrated by many secreted proteins, but only a handful, tumor necrosis factor-alpha, IFN-gamma and IL-1, have been studied in great detail. Oncostatin-M (OsM) has also been found in psoriatic skin and we hypothesized that it makes a unique and characteristic contribution to the psoriatic processes. To define in-depth the molecular effects of OsM in epidermis, we used high-density DNA microarrays for transcriptional profiling of OsM-treated human skin equivalents. We identified 374 unambiguously OsM-regulated genes, out of 22,000 probed. OsM suppressed the expression of the "classical" epidermal differentiation markers, but strongly and specifically induced the S100A proteins. Cytoskeletal and complement proteins, proteases, and their inhibitors were also induced by OsM. Interestingly, a large set of genes was induced by OsM at early time points but suppressed later; these genes are known regulatory targets of IFN and thus provide a nexus between the OsM and IFN pathways. OsM induces IL-4 and suppresses the T-helper 1-type and IL-1-responsive signals, potentially attenuating the psoriatic pathology. The data suggest that OsM plays a unique role in psoriasis, different from all other, more thoroughly studied cytokines.