Efficacy of combined peroxisome proliferator-activated receptor-α ligand and glucocorticoid therapy in a murine model of atopic dermatitis.

Although topical glucocorticoids (GCs) show potent anti-inflammatory activity in inflamed skin, they can also exert numerous harmful effects on epidermal structure and function. In contrast, topical applications of ligands of peroxisome proliferator-activated receptor-α (PPARα) not only reduce inflammation but also improve cutaneous barrier homeostasis. Therefore, we examined whether sequential topical GCs followed by topical Wy14643 (a ligand of PPARα) might be more effective than either alone for atopic dermatitis (AD) in a hapten (oxazolone (Ox))-induced murine model with multiple features of AD (Ox-AD). Despite expected anti-inflammatory benefits, topical GC alone induced (i) epidermal thinning; (ii) reduced expression of involucrin, loricrin, and filaggrin; and (iii) allowed outside-to-inside penetration of an epicutaneous tracer. Although Wy14643 alone yielded significant therapeutic benefits in mice with mild or moderate Ox-AD, it was less effective in severe Ox-AD. Yet, topical application of Wy14643 after GC was not only significantly effective comparable with GC alone, but it also prevented GC-induced structural and functional abnormalities in permeability barrier homeostasis. Moreover, rebound flares were largely absent after sequential treatment with GC and Wy14643. Together, these results show that GC and PPARα ligand therapy together is not only effective but also prevents development of GC-induced side effects, including rebound flares, in murine AD

Fatty acid transport protein 4 is required for incorporation of saturated ultralong-chain fatty acids into epidermal ceramides and monoacylglycerols

Fatty acid transport protein 4 (FATP4) is an acyl-CoA synthetase that is required for normal permeability barrier in mammalian skin. FATP4 (SLC27A4) mutations cause ichthyosis prematurity syndrome, a nonlethal disorder. In contrast, Fatp4-/- mice die neonatally from a defective barrier. Here we used electron microscopy and lipidomics to characterize defects in Fatp4-/- mice. Mutants showed lamellar body, corneocyte lipid envelope, and cornified envelope abnormalities. Lipidomics identified two lipids previously speculated to be present in mouse epidermis, sphingosine β-hydroxyceramide and monoacylglycerol; mutants displayed decreased proportions of these and the two ceramide classes that carry ultralong-chain, amide-linked fatty acids (FAs) thought to be critical for barrier function, unbound ω-O-acylceramide and bound ω-hydroxyceramide, the latter constituting the major component of the corneocyte lipid envelope. Other abnormalities included elevated amounts of sphingosine α-hydroxyceramide, phytosphingosine non-hydroxyceramide, and 1-O-acylceramide. Acyl chain length alterations in ceramides also suggested roles for FATP4 in esterifying saturated non-hydroxy and β-hydroxy FAs with at least 25 carbons and saturated or unsaturated ω-hydroxy FAs with at least 30 carbons to CoA. Our lipidomic analysis is the most thorough such study of the Fatp4-/- mouse skin barrier to date, providing information about how FATP4 can contribute to barrier function by regulating fatty acyl moieties in various barrier lipids

Ichthyosis in Sjögren–Larsson syndrome reflects defective barrier function due to abnormal lamellar body structure and secretion

Sjögren–Larsson syndrome is a genetic disease characterized by ichthyosis, mental retardation, spasticity and mutations in the ALDH3A2 gene coding for fatty aldehyde dehydrogenase, an enzyme necessary for oxidation of fatty aldehydes and fatty alcohols. We investigated the cutaneous abnormalities in 9 patients with Sjögren–Larsson syndrome to better understand how the enzymatic deficiency results in epidermal dysfunction. Histochemical staining for aldehyde oxidizing activity was profoundly reduced in the epidermis. Colloidal lanthanum perfusion studies showed abnormal movement of tracer into the extracellular spaces of the stratum corneum consistent with a leaky water barrier. The barrier defect could be attributed to the presence of abnormal lamellar bodies, many with disrupted limiting membranes or lacking lamellar contents. Entombed lamellar bodies were present in the cytoplasm of corneocytes suggesting blockade of lamellar body secretion. At the stratum granulosum–stratum corneum interface, non-lamellar material displaced or replaced secreted lamellar membranes, and in the stratum corneum, the number of lamellar bilayers declined and lamellar membrane organization was disrupted by foci of lamellar/non-lamellar phase separation. These studies demonstrate the presence of a permeability barrier abnormality in Sjögren–Larsson syndrome, which localizes to the stratum corneum interstices and can be attributed to abnormalities in lamellar body formation and secretion

Topical Peroxisome Proliferator Activated Receptor Activators Accelerate Postnatal Stratum Corneum Acidification

Previous studies have shown that pH declines from between 6 and 7 at birth to adult levels (pH 5.0–5.5) over 5–6 days in neonatal rat stratum corneum (SC). As a result, at birth, neonatal epidermis displays decreased permeability barrier homeostasis and SC integrity, improving days 5–6. We determined here whether peroxisome proliferator-activated receptor (PPAR) activators accelerate postnatal SC acidification. Topical treatment with two different PPARα activators, clofibrate and WY14643, accelerated the postnatal decline in SC surface pH, whereas treatment with PPARγ activators did not and a PPARβ/δ activator had only a modest effect. Treatment with clofibrate significantly accelerated normalization of barrier function. The morphological basis for the improvement in barrier function in PPARα-treated animals includes accelerated secretion of lamellar bodies and enhanced, postsecretory processing of secreted lamellar body contents into mature lamellar membranes. Activity of β-glucocerebrosidase increased after PPARα-activator treatment. PPARα activator also improved SC integrity, which correlated with an increase in corneodesmosome density and increased desmoglein-1 content, with a decline in serine protease activity. Topical treatment of newborn animals with a PPARα activator increased secretory phospholipase A2 activity, which likely accounts for accelerated SC acidification. Thus, PPARα activators accelerate neonatal SC acidification, in parallel with improved permeability homeostasis and SC integrity/cohesion. Hence, PPARα activators might be useful to prevent or treat certain common neonatal dermatoses

Lack of the Vitamin D Receptor is Associated with Reduced Epidermal Differentiation and Hair Follicle Growth

The active vitamin D metabolite, 1,25-dihydroxyvitamin D, acting through the vitamin D receptor, regulates the expression of genes in a variety of vitamin D-responsive tissues, including the epidermis. To investigate the role of the vitamin D receptor in mediating epidermal differentiation, we examined the histomorphology and expression of differentiation markers in the epidermis of vitamin D receptor knockout mice generated by gene targeting. The homozygous knockout mouse displayed a phenotype that closely resembles vitamin D-dependent rickets type II in humans, including the development of rickets and alopecia. Hair loss developed by 3mo after birth and gradually led to nearly total hair loss by 8mo. Histologic analysis of the skin of homozygous knockout mice revealed dilation of the hair follicles with the formation of dermal cysts starting at the age of 3wk. These cysts increased in size and number with age. Epidermal differentiation markers, including involucrin, profilaggrin, and loricrin, detected by immunostaining and in situ hybridization, showed decreased expression levels in homozygous knockout mice from birth until 3wk, preceding the morphologic changes observed in the hair follicles. Keratin 10 levels, however, were not reduced. At the ultrastructural level, homozygous knockout mice showed increased numbers of small dense granules in the granular layer with few or no surrounding keratin bundles and a loss of keratohyalin granules. Thus, both the interfollicular epidermis and the hair follicle appear to require the vitamin D receptor for normal differentiation. The temporal abnormalities between the two processes reflect the apparent lack of requirement for the vitamin D receptor during the anagen phase of the first (developmental) hair cycle, but with earlier effects on the terminal differentiation of the interfollicular epidermis

Pathogenesis of the cutaneous phenotype in inherited disorders of cholesterol metabolism: Therapeutic implications for topical treatment of these disorders

Molecular geneticists tend to conceptualize disease pathogenesis from the mutated gene outward, an approach that does not take into account the impact of barrier requirements in determining disease phenotype. An ‘outside-to-inside’ perspective has provided quite different explanations for the ichthyoses, including several of the disorders of distal cholesterol metabolism. Elucidation of responsible pathogenic mechanisms also is pointing to appropriate, pathogenesis (pathway)-based therapeutic strategies. In the case of the lipid metabolic disorders, it takes full advantage of new molecular, genetic and cellular pathogenesis information to correct or bypass the metabolic abnormality. This approach fully exploits the unique accessibility of the skin to a topical approach. Moreover, since it will utilize topical lipids and lipid-soluble, and often generic, lipid-soluble drugs, these treatments should be readily transported across the stratum corneum. If successful, this approach could initiate an entirely new departure for the therapy of the ichthyoses. Finally, because these agents are relatively safe and inexpensive, this form of treatment has the potential to be widely-deployed, even in the developing world

Serine Protease Signaling of Epidermal Permeability Barrier Homeostasis

Evidence is growing that protease-activated receptor-2 (PAR-2) plays a key role in epithelial inflammation. We hypothesized here that PAR-2 plays a central role in epidermal permeability barrier homeostasis by mediating signaling from serine proteases (SP) in the stratum corneum (SC). Since the SC contains tryptic- and chymotryptic-like activity, we assessed the influence of SP activation/inhibition on barrier function. Acute barrier disruption increases SP activity and blockade by topical SP inhibitors (SPI) accelerates barrier recovery after acute abrogation. This improvement in barrier function is due to accelerated lamellar body (LB) secretion. Since tryptic SP signal certain downstream responses through PAR-2, we assessed its potential role in mediating the negative effects of SP on permeability barrier. Firstly, PAR-2 is expressed in the outer nucleated layers of the epidermis and most specifically under basal condition to the lipid raft (LR) domains. Secondly, tape stripping-induced barrier abrogation provokes PAR-2 activation, as shown by receptor internalization (i.e. receptor movement from LR to cytolpasmic domains). Thirdly, topical applications of PAR-2 agonist peptide, SLIGRL, delay permeability barrier recovery and inhibit LB secretion, while, conversely, PAR-2 knockout mice display accelerated barrier recovery kinetics and enhanced LB secretion, paralleled by increased LR formation and caveolin-1 expression. These results demonstrate first, the importance of SP/SPI balance for normal permeability barrier homeostasis, and second, they identify PAR-2 as a novel signaling mechanism of permeability barrier, that is, of response linked to LB secretion

Exosomes from Human Adipose Tissue-Derived Mesenchymal Stem Cells Promote Epidermal Barrier Repair by Inducing de Novo Synthesis of Ceramides in Atopic Dermatitis.

Atopic dermatitis (AD) is a multifactorial, heterogeneous disease associated with epidermal barrier disruption and intense systemic inflammation. Previously, we showed that exosomes derived from human adipose tissue-derived mesenchymal stem cells (ASC-exosomes) attenuate AD-like symptoms by reducing multiple inflammatory cytokine levels. Here, we investigated ASC-exosomes' effects on skin barrier restoration by analyzing protein and lipid contents. We found that subcutaneous injection of ASC-exosomes in an oxazolone-induced dermatitis model remarkably reduced trans-epidermal water loss, while enhancing stratum corneum (SC) hydration and markedly decreasing the levels of inflammatory cytokines such as IL-4, IL-5, IL-13, TNF-α, IFN-γ, IL-17, and TSLP, all in a dose-dependent manner. Interestingly, ASC-exosomes induced the production of ceramides and dihydroceramides. Electron microscopic analysis revealed enhanced epidermal lamellar bodies and formation of lamellar layer at the interface of the SC and stratum granulosum with ASC-exosomes treatment. Deep RNA sequencing analysis of skin lesions demonstrated that ASC-exosomes restores the expression of genes involved in skin barrier, lipid metabolism, cell cycle, and inflammatory response in the diseased area. Collectively, our results suggest that ASC-exosomes effectively restore epidermal barrier functions in AD by facilitating the de novo synthesis of ceramides, resulting in a promising cell-free therapeutic option for treating AD

Co-Regulation and Interdependence of the Mammalian Epidermal Permeability and Antimicrobial Barriers

Human epidermis elaborates two small cationic, highly hydrophobic antimicrobial peptides (AMP), β-defensin 2 (hBD2), and the carboxypeptide cleavage product of human cathelicidin (hCAP18), LL-37, which are co-packaged along with lipids within epidermal lamellar bodies (LBs) before their secretion. Because of their colocalization, we hypothesized that AMP and barrier lipid production could be coregulated by altered permeability barrier requirements. mRNA and immunostainable protein levels for mBD3 and cathelin-related antimicrobial peptide (CRAMP) (murine homologues of hBD2 and LL-37, respectively) increase 1–8hours after acute permeability barrier disruption and normalize by 24hours, kinetics that mirror the lipid metabolic response to permeability barrier disruption. Artificial permeability barrier restoration, which inhibits the lipid-synthetic response leading to barrier recovery, blocks the increase in AMP mRNA/protein expression, further evidence that AMP expression is linked to permeability barrier function. Conversely, LB-derived AMPs are also important for permeability barrier homeostasis. Despite an apparent increase in mBD3 protein, CRAMP−/− mice delayed permeability barrier recovery, attributable to defective LB contents and abnormalities in the structure of the lamellar membranes that regulate permeability barrier function. These studies demonstrate that (1) the permeability and antimicrobial barriers are coordinately regulated by permeability barrier requirements and (2) CRAMP is required for permeability barrier homeostasis

Fetal Epidermal Differentiation and Barrier Development In Vivo is Accelerated by Nuclear Hormone Receptor Activators1

Nuclear receptors which interact with the retinoid X receptor are involved in the regulation of epidermal differentiation and development. We have recently shown that activators of the peroxisome proliferator-activated receptor and of the farnesoid X-activated receptor accelerate epidermal barrier maturation in fetal rat skin in vitro. In this study we asked whether cutaneous development in utero was affected by peroxisome proliferator-activated receptor or farnesoid X-activated receptor activators, or by an activator of another retinoid X receptor partner, liver X receptor. Activators of the peroxisome proliferator-activated receptor (clofibrate or linoleic acid), farnesoid X-activated receptor (farnesol or juvenile hormone III), or liver X receptor (22R-hydroxycholesterol), were injected into the amniotic fluid of fetal rats on gestational day 17. Fetal epidermal barrier function and morphology was assessed on day 19. Whereas vehicle-treated fetal rats displayed no measurable barrier (transepidermal water loss > 10 mg per cm2 per h), a measurable barrier was induced by the intra-amniotic administration of all activators tested (transepidermal water loss range 4.0–8.5 mg per cm2 per h). By light microscopy, control pups lacked a well-defined stratum corneum, whereas a distinct stratum corneum and a thickened stratum granulosum were present in treated pups. By electron microscopy, the extracellular spaces of the stratum corneum in control pups revealed a paucity of mature lamellar unit structures, whereas these structures filled the stratum corneum interstices in treated pups. Additionally, protein and mRNA levels of loricrin and filaggrin, two structural proteins of stratum corneum, were increased in treated epidermis, as were the activities of two lipid catabolic enzymes critical to stratum corneum function, β-glucocerebrosidase and steroid sulfatase. Finally, peroxisome proliferator-activated receptor-α and -δ and liver X receptor-α and -β mRNAs were detected in fetal epidermis by reverse transcriptase–polymerase chain reaction and northern analyses. The presence of these receptors and the ability of their activators to stimulate epidermal barrier and stratum corneum development suggest a physiologic role for peroxisome proliferator-activated receptor and liver X receptor and their endogenous ligands in the regulation of cutaneous development