Jeroen van Smeden

Increase in short-chain ceramides correlates with an altered lipid organization and decreased barrier function in atopic eczema patients

A hallmark of atopic eczema (AE) is skin barrier dysfunction. Lipids in the stratum corneum (SC), primarily ceramides, fatty acids, and cholesterol, are crucial for the barrier function, but their role in relation to AE is indistinct. Filaggrin is an epithelial barrier protein with a central role in the pathogenesis of AE. Nevertheless, the precise causes of AE-associated barrier dysfunction are largely unknown. In this study, a comprehensive analysis of ceramide composition and lipid organization in nonlesional SC of AE patients and control subjects was performed by means of mass spectrometry, infrared spectroscopy, and X-ray diffraction. In addition, the skin barrier and clinical state of the disease were examined. The level of ceramides with an extreme short chain length is drastically increased in SC of AE patients, which leads to an aberrant lipid organization and a decreased skin barrier function. Changes in SC lipid properties correlate with disease severity but are independent of filaggrin mutations. We demonstrate for the first time that changes in ceramide chain length and lipid organization are directly correlated with the skin barrier defects in nonlesional skin of AE patients. We envisage that these insights will provide a new therapeutic entry in therapy and prevention of AE.

LC/MS analysis of stratum corneum lipids: ceramide profiling and discovery

Ceramides (CERs) in the upper layer of the skin, the stratum corneum (SC), play a key role in the skin barrier function. In human SC, the literature currently reports 11 CER subclasses that have been identified. In this paper, a novel quick and robust LC/MS method is presented that allows the separation and analysis of all known human SC CER subclasses using only limited sample preparation. Besides all 11 known and identified subclasses, a 3D multi-mass chromatogram shows the presence of other lipid subclasses. Using LC/MS/MS with an ion trap (IT) system, a Fourier transform-ion cyclotron resonance system, and a triple quadrupole system, we were able to identify one of these lipid subclasses as a new CER subclass: the ester-linked ω-hydroxy fatty acid with a dihydrosphingosine base (CER [EOdS]). Besides the identification of a new CER subclass, this paper also describes the applicability and robustness of the developed LC/MS method by analyzing three (biological) SC samples: SC from human dermatomed skin, human SC obtained by tape stripping, and SC from full-thickness skin explants. All three biological samples showed all known CER subclasses and slight differences were observed in CER profile.

TNF-α and Th2 Cytokines Induce Atopic Dermatitis–Like Features on Epidermal Differentiation Proteins and Stratum Corneum Lipids in Human Skin Equivalents

Atopic dermatitis (AD) is a chronic inflammatory skin disease in which the skin barrier function is disrupted. In this inflammatory AD environment, cytokines are upregulated, but the cytokine effect on the AD skin barrier is not fully understood. We aimed to investigate the influence of Th2 (IL-4, IL-13, IL-31) and pro-inflammatory (tumor necrosis factor alpha (TNF-α)) cytokines on epidermal morphogenesis, proliferation, differentiation, and stratum corneum lipid properties. For this purpose, we used the Leiden epidermal model (LEM) in which the medium was supplemented with these cytokines. Our results show that IL-4, IL-13, IL-31, and TNF-α induce spongiosis, augment TSLP secretion by keratinocytes, and alter early and terminal differentiation-protein expression in LEMs. TNF-α alone or in combination with Th2 cytokines decreases the level of long chain free fatty acids (FFAs) and ester linked ω-hydroxy (EO) ceramides, consequently affecting the lipid organization. IL-31 increases long chain FFAs in LEMs but decreases relative abundance of EO ceramides. These findings clearly show that supplementation with TNF-α and Th2 cytokines influence epidermal morphogenesis and barrier function. As a result, these LEMs show similar characteristics as found in AD skin and can be used as an excellent tool for screening formulations and drugs for the treatment of AD.

The importance of free fatty acid chain length for the skin barrier function in atopic eczema patients

An important feature of atopic eczema (AE) is a decreased skin barrier function. The stratum corneum (SC) lipids – comprised of ceramides (CERs), free fatty acids (FFAs) and cholesterol – fulfil a predominant role in the skin barrier function. In this clinical study, the carbon chain length distribution of SC lipids (FFAs and CERs) and their importance for the lipid organization and skin barrier function were examined in AE patients and compared with control subjects. A reduction in FFA chain length and an increase in unsaturated FFAs are observed in non‐lesional and lesional SC of AE patients. The reduction in FFA chain length associates with a reduced CER chain length, suggesting a common synthetic pathway. The lipid chain length reduction correlates with a less dense lipid organization and a decreased skin barrier function. All changes are more pronounced in lesional SC compared with non‐lesional skin. No association was observed between lipid properties and filaggrin mutations, an important predisposing factor for developing AE. The results of this study demonstrate an altered SC lipid composition and signify the importance of these changes (specifically regarding the CER and FFA chain lengths) for the impaired skin barrier function in AE. This provides insights into epidermal lipid metabolism as well as new opportunities for skin barrier repair.

Intercellular Skin Barrier Lipid Composition and Organization in Netherton Syndrome Patients

Netherton syndrome (NTS) is a rare genetic skin disease caused by mutations in the serine protease inhibitor Kazal-type 5 gene, which encodes the lympho-epithelial Kazal-type-related inhibitor. NTS patients have profoundly impaired skin barrier function. As stratum corneum (SC) lipids have a crucial role in the skin barrier function, we investigated the SC lipid composition and organization in NTS patients. We studied the SC lipid composition by means of mass spectrometry, and the lipid organization was examined by infrared spectroscopy and X-ray diffraction. Decreased free fatty acid (FFA) chain length and increased levels of monounsaturated FFAs were observed in the SC of NTS patients compared with controls. Furthermore, the level of short-chain ceramides (CERs) was enhanced in NTS patients and a strong reduction in long-chain CER levels was seen in several patients. The changes in lipid composition modified the lipid organization leading to an increased disordering of the lipids compared with the controls. In addition, in a subgroup of patients the organization of the lipid layers changed dramatically. The altered FFA and CER profiles in NTS patients corresponded to changes in the expression of enzymes involved in SC lipid processing. The observed changes in lipid composition, lipid organization, and enzyme expression are likely to contribute to the barrier dysfunction in NTS.

Increased Presence of Monounsaturated Fatty Acids in the Stratum Corneum of Human Skin Equivalents

Previous results showed that our in-house human skin equivalents (HSEs) differ in their stratum corneum (SC) lipid organization compared with human SC. To elucidate the cause of the altered SC lipid organization in the HSEs, a recently developed liquid chromatography/mass spectrometry method was used to study the free fatty acid (FFA) and ceramide composition in detail. In addition, the SC lipid composition of the HSEs and human skin was examined quantitatively with high-performance thin-layer chromatography. Our results reveal that all our HSEs have an increased presence of monounsaturated FFAs compared with human SC. Moreover, the HSEs display the presence of ceramide species with a monounsaturated acyl chain, which are not detected in human SC. All HSEs also exhibit an altered expression of stearoyl-CoA desaturase, the enzyme that converts saturated FFAs to monounsaturated FFAs. Furthermore, the HSEs show the presence of 12 ceramide subclasses, similar to native human SC. However, the HSEs have increased levels of ceramides EOS and EOH and ceramide species with short total carbon chains and a reduced FFA level compared with human SC. The presence of unsaturated lipid chains in HSE offers new opportunities to mimic the lipid properties of human SC more closely.

Knock‐down of filaggrin does not affect lipid organization and composition in stratum corneum of reconstructed human skin equivalents

Human skin mainly functions as an effective barrier against unwanted environmental influences. The barrier function strongly relies on the outermost layer of the skin, the stratum corneum (SC), which is composed of corneocytes embedded in an extracellular lipid matrix. The importance of a proper barrier function is shown in various skin disorders such as atopic dermatitis (AD), a complex human skin disorder strongly associated with filaggrin (FLG) null mutations, but their role in barrier function is yet unclear. To study the role of FLG in SC barrier properties in terms of SC lipid organization and lipid composition, we generated an N/TERT‐based 3D‐skin equivalent (NSE) after knock‐down of FLG with shRNA. In these NSEs, we examined epidermal morphogenesis by evaluating the expression of differentiation markers keratin 10, FLG, loricrin and the proliferation marker ki67. Furthermore, the SC was extensively analysed for lipid organization, lipid composition and SC permeability. Our results demonstrate that FLG knock‐down (FLG‐KD) did not affect epidermal morphogenesis, SC lipid organization, lipid composition and SC permeability for a lipophilic compound in NSEs. Therefore, our findings indicate that FLG‐KD alone does not necessarily affect the functionality of a proper barrier function.

Lipid to protein ratio plays an important role in the skin barrier function in patients with atopic eczema

The barrier function of the skin is primarily provided by the stratum corneum (SC), the outermost layer of the skin. Skin barrier impairment is thought to be a primary factor in the pathogenesis of atopic eczema (AE). Filaggrin is an epidermal barrier protein and common mutations in the filaggrin gene strongly predispose for AE. However, the role of filaggrin mutations in the decreased skin barrier in AE is not fully understood. It was recently shown that changes in SC lipid composition and organization play a role in the reduced skin barrier in AE.

Quantitative analysis of ceramides using a novel lipidomics approach with three dimensional response modelling.

In the outermost layer of the skin, the stratum corneum (SC), ceramides form a diverse and essential pool of lipids. Due to their diversity and the limited availability of synthetic standards it is challenging to quantitatively analyse all SC ceramides independently. We aim to perform a detailed analysis of ceramides on SC harvested from in vivo and ex vivo skin, therefore, a LC/MS method was developed in which all steps from sample acquisition until data analysis were examined and optimized. Improving extraction efficiency of ceramides resulted in an increase in efficiency from 71.5% to 99.3%. It was shown that sample harvesting by tape-stripping in vivo was accurate and precise. A full scan MS method was developed, compatible with all sample types, enabling simultaneously qualitative and quantitative data analysis. A novel three dimensional response model was constructed to quantify all detected ceramides from full scan data using a limited amount of synthetic ceramides. The application is demonstrated on various SC sample types. When ex vivo SC was regenerated during human skin culture, increases are observed in the amount of the ceramide sphingosine subclasses, in mono unsaturated ceramides (which have an cis-double bond in the acyl chain), and ceramides with a short C34 carbon chain (ceramides with a total carbon chain of 34 carbon atoms), compared with native human skin. These changes in ceramide levels are also often encountered in diseased skin.

Altered expression of epidermal lipid bio-synthesis enzymes in atopic dermatitis skin is accompanied by changes in stratum corneum lipid composition

BACKGROUND The barrier dysfunction in atopic dermatitis (AD) skin correlates with stratum corneum (SC) lipid abnormalities including reduction of global lipid content, shorter ceramide (CER) as well as free fatty acid (FFA) chain length and altered CER subclass levels. However, the underlying cause of these changes in lipid composition has not been fully investigated. AIM We investigated whether the expression of CER and FFA biosynthesis enzymes are altered in AD skin compared with control skin and determine whether changes in enzyme expression can be related with changes in lipid composition. METHODS In AD patients and controls the expression of enzymes involved in the biosynthesis of FFAs and CERs was analyzed in relation to the SC lipid composition. These enzymes include stearoyl CoA desaturase (SCD), elongase 1 (ELOVL1) and ELOVL6 involved in FFA synthesis and β-glucocerebrosidase (GBA), acid-sphingomyelinase (aSmase), ceramide synthase 3 (CerS3) involved in CER synthesis. In TH2 treated human skin equivalents (AD HSEs) mimicking lesional AD skin, the mRNA expression of these enzymes was investigated. RESULTS The results reveal an altered expression of SCD and ELOVL1 in AD lesional skin. This was accompanied by functional changes displayed by increased unsaturated FFAs (SCD) and reduced FFA C22-C28 (ELOVL1) in AD lesional skin. The expression of GBA, aSmase and CerS3 were also altered in lesional skin. The CER composition in AD lesional skin showed corresponding changes such as increased CER AS and NS (aSmase) and decreased esterified ω-hydroxy CERs (CerS3). In support of the results from AD skin, the AD HSEs showed reduced mRNA ELOVL1, GBA and a Smase levels. CONCLUSION This study shows that alterations in the expression of key enzymes involved in SC lipid synthesis contribute to changes in the lipid composition in AD skin and inflammation may influence expression of these enzymes.