Melanie Hupe

FILAGGRIN DEFICIENCY CONFERS A PARACELLULAR BARRIER ABNORMALITY THAT REDUCES INFLAMMATORY THRESHOLDS TO IRRITANTS AND HAPTENS

BACKGROUND Mutations in the human filaggrin gene (FLG) are associated with atopic dermatitis (AD) and are presumed to provoke a barrier abnormality. Yet additional acquired stressors might be necessary because the same mutations can result in a noninflammatory disorder, ichthyosis vulgaris. OBJECTIVE We examined here whether FLG deficiency alone suffices to produce a barrier abnormality, the basis for the putative abnormality, and its proinflammatory consequences. METHODS By using the flaky-tail mouse, which lacks processed murine filaggrin because of a frameshift mutation in the gene encoding profilaggrin that mimics some mutations in human AD, we assessed whether FLG deficiency provokes a barrier abnormality, further localized the defect, identified its subcellular basis, and assessed thresholds to irritant- and hapten-induced dermatitis. RESULTS Flaky-tail mice exhibit low-grade inflammation with increased bidirectional, paracellular permeability of water-soluble xenobiotes caused by impaired lamellar body secretion and altered stratum corneum extracellular membranes. This barrier abnormality correlates with reduced inflammatory thresholds to both topical irritants and haptens. Moreover, when exposed repeatedly to topical haptens at doses that produce no inflammation in wild-type mice, flaky-tail mice experience a severe AD-like dermatosis with a further deterioration in barrier function and features of a T(H)2 immunophenotype (increased CRTH levels plus inflammation, increased serum IgE levels, and reduced antimicrobial peptide [mBD3] expression). CONCLUSIONS FLG deficiency alone provokes a paracellular barrier abnormality in mice that reduces inflammatory thresholds to topical irritants/haptens, likely accounting for enhanced antigen penetration in FLG-associated AD.

Psychological stress downregulates epidermal antimicrobial peptide expression and increases severity of cutaneous infections in mice

The skin is the first line of defense against microbial infection, and psychological stress (PS) has been shown to have adverse effects on cutaneous barrier function. Here we show that PS increased the severity of group A Streptococcus pyogenes (GAS) cutaneous skin infection in mice; this was accompanied by increased production of endogenous glucocorticoids (GCs), which inhibited epidermal lipid synthesis and decreased lamellar body (LB) secretion. LBs encapsulate antimicrobial peptides (AMPs), and PS or systemic or topical GC administration downregulated epidermal expression of murine AMPs cathelin-related AMP and beta-defensin 3. Pharmacological blockade of the stress hormone corticotrophin-releasing factor or of peripheral GC action, as well as topical administration of physiologic lipids, normalized epidermal AMP levels and delivery to LBs and decreased the severity of GAS infection during PS. Our results show that PS decreases the levels of 2 key AMPs in the epidermis and their delivery into LBs and that this is attributable to increased endogenous GC production. These data suggest that GC blockade and/or topical lipid administration could normalize cutaneous antimicrobial defense during PS or GC increase. We believe this to be the first mechanistic link between PS and increased susceptibility to infection by microbial pathogens.

Urea Uptake Enhances Barrier Function and Antimicrobial Defense in Humans by Regulating Epidermal Gene Expression

Urea is an endogenous metabolite, known to enhance stratum corneum hydration. Yet, topical urea anecdotally also improves permeability barrier function, and it appears to exhibit antimicrobial activity. Hence, we hypothesized that urea is not merely a passive metabolite, but a small-molecule regulator of epidermal structure and function. In 21 human volunteers, topical urea improved barrier function in parallel with enhanced antimicrobial peptide (LL-37 and β-defensin-2) expression. Urea both stimulates expression of, and is transported into keratinocytes by two urea transporters, UT-A1 and UT-A2, and by aquaporin 3, 7 and 9. Inhibitors of these urea transporters block the downstream biological effects of urea, which include increased mRNA and protein levels for: (i) transglutaminase-1, involucrin, loricrin and filaggrin; (ii) epidermal lipid synthetic enzymes, and (iii) cathelicidin/LL-37 and β-defensin-2. Finally, we explored the potential clinical utility of urea, showing that topical urea applications normalized both barrier function and antimicrobial peptide expression in a murine model of atopic dermatitis (AD). Together, these results show that urea is a small-molecule regulator of epidermal permeability barrier function and antimicrobial peptide expression after transporter uptake, followed by gene regulatory activity in normal epidermis, with potential therapeutic applications in diseased skin.

The neuroendocrine peptide catestatin is a cutaneous antimicrobial and induced in the skin after injury.

Epithelia establish a microbial barrier against infection through the production of antimicrobial peptides (AMPs). In this study, we investigated whether catestatin (Cst), a peptide derived from the neuroendocrine protein chromogranin A (CHGA), is a functional AMP and is present in the epidermis. We show that Cst is antimicrobial against relevant skin microbes, including gram-positive and gram-negative bacteria, yeast, and fungi. The antimicrobial mechanism of Cst was found to be similar to other AMPs, as it was dependent on bacterial charge and growth conditions, and induced membrane disruption. The potential relevance of Cst against skin pathogens was supported by the observation that CHGA was expressed in keratinocytes. In human skin, CHGA was found to be proteolytically processed into the antimicrobial fragment Cst, thus enabling its AMP function. Furthermore, Cst expression in murine skin increased in response to injury and infection, providing potential for increased protection against infection. These data demonstrate that a neuroendocrine peptide has antimicrobial function against a wide assortment of skin pathogens and is upregulated upon injury, thus demonstrating a direct link between the neuroendocrine and cutaneous immune systems. JID JOURNAL CLUB ARTICLE: For questions, answers, and open discussion about this article please go to http://network.nature.com/group/jidclub.

3D In Vitro Model of a Functional Epidermal Permeability Barrier from Human Embryonic Stem Cells and Induced Pluripotent Stem Cells

Summary Cornification and epidermal barrier defects are associated with a number of clinically diverse skin disorders. However, a suitable in vitro model for studying normal barrier function and barrier defects is still lacking. Here, we demonstrate the generation of human epidermal equivalents (HEEs) from human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). HEEs are structurally similar to native epidermis, with a functional permeability barrier. We exposed a pure population of hESC/iPSC-derived keratinocytes, whose transcriptome corresponds to the gene signature of normal primary human keratinocytes (NHKs), to a sequential high-to-low humidity environment in an air/liquid interface culture. The resulting HEEs had all of the cellular strata of the human epidermis, with skin barrier properties similar to those of normal skin. Such HEEs generated from disease-specific iPSCs will be an invaluable tool not only for dissecting molecular mechanisms that lead to epidermal barrier defects but also for drug development and screening.

Is the Filaggrin–Histidine–Urocanic Acid Pathway Essential for Stratum Corneum Acidification?

TO THE EDITOR Acidification of the surface of the stratum corneum (SC), the acid mantle, was initially thought to be important in the defense against infection. The growth of pathogenic microorganisms, such as Staphylococcus aureus and Streptococcus pyogenes, is inhibited by an acidic skin pH whereas the growth of resident (normal) skin flora is stimulated (Puhvel et al., 1975; Korting et al., 1990, 1992). However, recent studies have shown that acidification of the SC has additional functions, including regulating several key SC functions. A major function of the skin is to form a permeability barrier between the dry external environment and the moist interior of the body (Elias, 2007). This permeability barrier resides in the extracellular lipid membranes of the SC, and studies have shown that an acidified SC is required for the formation of a functionally competent permeability barrier (Mauro et al., 1998; Fluhr et al., 2001; Hachem et al., 2003). Specifically, in the SC b-glucocerebrosidase and acid sphingomyelinase metabolize glucosylceramides and sphingomyelin, respectively, to ceramides, which is the major family of lipids in the extracellular membranes that mediate permeability barrier function (Feingold, 2007). Both the enzymes require an acidic milieu for optimal enzymatic activity; hence, when the pH of the SC increases, the metabolism of glucosylceramides and sphingomyelin to ceramides is impaired, resulting in abnormal permeability barrier homeostasis (Holleran et al., 1992, 1993; Feingold, 2007). In addition, an acidic SC pH inhibits the activity of serine proteases thereby maintaining the cohesiveness and integrity of the SC (Hachem et al., 2005). With an increase in SC pH, the activities of these serine proteases are stimulated resulting in the degradation of corneodesmosomes and a decrease in SC integrity and cohesion (Fluhr et al., 2004b; Hachem et al., 2005). Thus, an acidic SC is important in regulating the metabolism and function of the SC, and alterations in SC pH could have numerous adverse effects. A variety of different pathways are postulated to contribute to the acid mantle Abbreviation: SC, stratum corneum

Endoplasmic reticulum Ca2+ depletion activates XBP1 and controls terminal differentiation in keratinocytes and epidermis.

Background  Endoplasmic reticulum (ER) Ca2+ depletion, previously shown to signal pathological stress responses, has more recently been found also to trigger homeostatic physiological processes such as differentiation. In keratinocytes and epidermis, terminal differentiation and barrier repair require physiological apoptosis and differentiation, as evidenced by protein synthesis, caspase 14 expression, lipid secretion and stratum corneum (SC) formation.

Multidrug Resistance Decreases with Mutations of Melanosomal Regulatory Genes

Whereas resistance to chemotherapy has long impeded effective treatment of metastatic melanoma, the mechanistic basis of this resistance remains unknown. One possible mechanism of drug resistance is alteration of intracellular drug distribution either by drug efflux or sequestration into intracellular organelles. Melanomas, as well as primary melanocytes from which they arise, have intracellular organelles, called melanosomes, wherein the synthesis and storage of the pigment melanin takes place. In this study, comparisons of congenic cells with and without functional molecules regulating melanosome formation show that sensitivity to the chemotherapeutic agent cis-diaminedichloroplatinum II (cis-platin) significantly increases with the mutation of genes regulating melanosome formation, concomitant disruption of melanosome morphology, and loss of mature melanosomes. Absence of the melanosomal structural protein gp100/Pmel17 causes increased cis-platin sensitivity. Independent mutations in three separate genes that regulate melanosome biogenesis (Dtnbp1, Pldn, Vps33a) also result in increased cis-platin sensitivity. In addition, a mutation of the gene encoding the integral melanosomal protein tyrosinase, resulting in aberrant melanosome formation, also causes increased cis-platin sensitivity. Furthermore, sensitivity to agents in other chemotherapeutic classes (e.g., vinblastine and etoposide) also increased with the mutation of Pldn. In contrast, a mutation in another melanosomal regulatory gene, Hps1, minimally affects melanosome biogenesis, preserves the formation of mature melanosomes, and has no effect on cis-platin or vinblastine response. Together, these data provide the first direct evidence that melanosomal regulatory genes influence drug sensitivity and that the presence of mature melanosomes likely contributes to melanoma resistance to therapy.

Cellular Changes that Accompany Shedding of Human Corneocytes

Corneocyte desquamation has been ascribed to either: 1) proteolytic degradation of corneodesmosomes (CD); 2) disorganization of extracellular lamellar bilayers; and/or 3) ‘swell-shrinkage-slough’ (SSS) from hydration/dehydration. To address the cellular basis for normal exfoliation, we compared changes in lamellar bilayer architecture and CD structure in DSquame® strips from the 1st vs. 5th stripping (‘outer’ vs. ‘mid’-stratum corneum [SC], respectively) from 9 normal adult forearms. Strippings were either processed for standard EM or for ruthenium (Ru-V)- or osmium-tetroxide (Os-V) vapor fixation, followed by immediate epoxy embedment, an artifact-free protocol that to our knowledge is previously unreported. CDs are largely intact in the mid-SC, but replaced by electron-dense (hydrophilic) clefts (lacunae) that expand laterally, splitting lamellar arrays in the outer SC. Some undegraded DSG1/DSC1 redistribute uniformly into corneocyte envelopes (CEs) in the outer SC (shown by proteomics, Z-stack confocal imaging and immunoEM). CEs then thicken, likely facilitating exfoliation by increasing corneocyte rigidity. In vapor-fixed images, hydration only altered the volume of the extracellular compartment, expanding lacunae further separating membrane arrays. During dehydration, air replaced water, maintaining the expanded extracellular compartment. Hydration also provoked degradation of membranes by activating contiguous acidic ceramidase activity. Together, these studies identify several parallel mechanisms that orchestrate exfoliation from the surface of normal human skin.

Topical hesperidin improves epidermal permeability barrier function and epidermal differentiation in normal murine skin

Abstract:  Orange peel extract appears to exhibit beneficial effects on skin whitening, inflammation, UVB protection, as well as keratinocyte proliferation. In the present study, we determine whether topical hesperidin influences epidermal permeability barrier function and its underlying mechanisms. Hairless mice were treated topically with 2% hesperidin or 70% ethanol alone twice daily for 6 days. At the end of treatment, basal transepidermal water loss (TEWL) was measured 2 and 4 h post barrier disruption. Epidermal proliferation and differentiation were evaluated by immunohistochemical staining and Western blot analysis. Additionally, lamellar body density and secretion were assessed by electron microscopy. Although there were no significant differences in basal barrier function, in comparison with control animals, topical hesperidin significantly accelerated barrier recovery at both 2 and 4 h after acute barrier abrogation. Enhanced barrier function in hesperidin‐treated skin correlated with stimulation of both epidermal proliferation and differentiation, as well as enhanced lamellar body secretion. These results indicate that topical hesperidin enhances epidermal permeability barrier homeostasis at least in part due to stimulation of epidermal proliferation, differentiation, as well as lamellar body secretion.