Matthias Schmuth

Comprehensive analysis of the gene encoding filaggrin uncovers prevalent and rare mutations in ichthyosis vulgaris and atopic eczema

We recently reported two common filaggrin (FLG) null mutations that cause ichthyosis vulgaris and predispose to eczema and secondary allergic diseases. We show here that these common European mutations are ancestral variants carried on conserved haplotypes. To facilitate comprehensive analysis of other populations, we report a strategy for full sequencing of this large, highly repetitive gene, and we describe 15 variants, including seven that are prevalent. All the variants are either nonsense or frameshift mutations that, in representative cases, resulted in loss of filaggrin production in the epidermis. In an Irish case-control study, the five most common European mutations showed a strong association with moderate-to-severe childhood eczema (χ2 test: P = 2.12 × 10−51; Fishers exact test: heterozygote odds ratio (OR) = 7.44 (95% confidence interval (c.i.) = 4.9–11.3), and homozygote OR = 151 (95% c.i. = 20–1,136)). We found three additional rare null mutations in this case series, suggesting that the genetic architecture of filaggrin-related atopic dermatitis consists of both prevalent and rare risk alleles.

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.

Abnormal skin barrier in the etiopathogenesis of atopic dermatitis.

Purpose of reviewMany recent studies have revealed the key roles played by Th1/Th2 cell dysregulation, IgE production, mast cell hyperactivity, and dendritic cell signaling in the pathogenesis of atopic dermatitis. Accordingly, current therapy has been largely directed towards ameliorating Th2-mediated inflammation and/or pruritus. We will review here emerging evidence that the inflammation in atopic dermatitis results from inherited and acquired insults to the barrier and the therapeutic implications of this new paradigm. Recent findingsRecent molecular genetic studies have shown a strong association between mutations in FILAGGRIN and atopic dermatitis, particularly in Northern Europeans. But additional acquired stressors to the barrier are required to initiate inflammation. Sustained hapten access through a defective barrier stimulates a Th1 → Th2 shift in immunophenotype, which in turn further aggravates the barrier. Secondary Staphylococcus aureus colonization not only amplifies inflammation but also further stresses the barrier in atopic dermatitis. SummaryThese results suggest a new ‘outside-to-inside, back to outside’ paradigm for the pathogenesis of atopic dermatitis. This new concept is providing impetus for the development of new categories of ‘barrier repair’ therapy.

Filaggrin Genotype in Ichthyosis Vulgaris Predicts Abnormalities in Epidermal Structure and Function

Although it is widely accepted that filaggrin (FLG) deficiency contributes to an abnormal barrier function in ichthyosis vulgaris and atopic dermatitis, the pathomechanism of how FLG deficiency provokes a barrier abnormality in humans is unknown. We report here that the presence of FLG mutations in Caucasians predicts dose-dependent alterations in epidermal permeability barrier function. Although FLG is an intracellular protein, the barrier abnormality occurred solely via a paracellular route in affected stratum corneum. Abnormal barrier function correlated with alterations in keratin filament organization (perinuclear retraction), impaired loading of lamellar body contents, followed by nonuniform extracellular distribution of secreted organelle contents, and abnormalities in lamellar bilayer architecture. In addition, we observed reductions in corneodesmosome density and tight junction protein expression. Thus, FLG deficiency provokes alterations in keratinocyte architecture that influence epidermal functions localizing to the extracellular matrix. These results clarify how FLG mutations impair epidermal permeability barrier function.

Thematic Review Series: Skin Lipids. Peroxisome proliferator-activated receptors and liver X receptors in epidermal biology

The epidermis is a very active site of lipid metabolism, and all peroxisome proliferator-activated receptor (PPAR) and liver X receptor (LXR) isoforms are expressed in the epidermis. Activation of PPARα, -β/δ, or -γ or LXRs stimulates keratinocyte differentiation. Additionally, activation of these receptors also improves permeability barrier homeostasis by a number of mechanisms, including stimulating epidermal lipid synthesis, increasing lamellar body formation and secretion, and increasing the activity of enzymes required for the extracellular processing of lipids in the stratum corneum, leading to the formation of lamellar membranes that mediate permeability barrier function. The stimulation of keratinocyte differentiation and permeability barrier formation also occurs during fetal development, resulting in accelerated epidermal development. PPAR and LXR activation regulates keratinocyte proliferation and apoptosis, and studies have shown that these receptors play a role in cutaneous carcinogenesis. Lastly, PPAR and LXR activation is anti-inflammatory, reducing inflammation in animal models of allergic and irritant contact dermatitis. Because of their broad profile of beneficial effects on skin homeostasis, PPAR and LXR have great potential to serve as drug targets for common skin diseases such as psoriasis, atopic dermatitis, and skin cancer.

Production of IL-12 by Human Monocyte-Derived Dendritic Cells Is Optimal When the Stimulus Is Given at the Onset of Maturation, and Is Further Enhanced by IL-4

Dendritic cells produce IL-12 both in response to microbial stimuli and to T cells, and can thus skew T cell reactivity toward a Th1 pattern. We investigated the capacity of dendritic cells to elaborate IL-12 with special regard to their state of maturation, different maturation stimuli, and its regulation by Th1/Th2-influencing cytokines. Monocyte-derived dendritic cells were generated with GM-CSF and IL-4 for 7 days, followed by another 3 days ± monocyte-conditioned media, yielding mature (CD83+/dendritic cell-lysosome-associated membrane glycoprotein+) and immature (CD83−/dendritic cell-lysosome-associated membrane glycoprotein−) dendritic cells. These dendritic cells were stimulated for another 48 h, and IL-12 p70 was measured by ELISA. We found the following: 1) Immature dendritic cells stimulated with CD154/CD40 ligand or bacteria (both of which concurrently also induced maturation) secreted always more IL-12 than already mature dendritic cells. Mature CD154-stimulated dendritic cells still made significant levels (up to 4 ng/ml). 2) Terminally mature skin-derived dendritic cells did not make any IL-12 in response to these stimuli. 3) Appropriate maturation stimuli are required for IL-12 production: CD40 ligation and bacteria are sufficient; monocyte-conditioned media are not. 4) Unexpectedly, IL-4 markedly increased the amount of IL-12 produced by both immature and mature dendritic cells, when present during stimulation. 5) IL-10 inhibited the production of IL-12. Our results, employing a cell culture system that is now being widely used in immunotherapy, extend prior data that IL-12 is produced most abundantly by dendritic cells that are beginning to respond to maturation stimuli. Surprisingly, IL-12 is only elicited by select maturation stimuli, but can be markedly enhanced by the addition of the Th2 cytokine, IL-4.

Topical corticosteroid therapy for acute radiation dermatitis: a prospective, randomized, double-blind study

Summary Background Radiation dermatitis is a common side‐effect of radiation therapy, but there is no current consensus about its appropriate therapy.

Basis for the permeability barrier abnormality in lamellar ichthyosis

Abstract: The basis for the permeability barrier abnormality in lamellar ichthyosis (LI) is not known. LI is caused by mutations in the gene that encodes the enzyme, transglutaminase 1 (TGI), which is responsible for assembly of the cornified envelope (CE). TG1 also has been suggested recently to catalyze the covalent attachment of omega‐hydroxyceramides (omega‐OHCer) to the CE, forming the corneocyte‐lipid envelope (CLE). We first assessed the barrier function and the permeability pathway of the water‐soluble tracer, colloidal lanthanum, across the stratum corneum (SC) in patients with LI with absent (n = 4) or low (n = 2) TG1 activity/protein. Increased movement of tracer through the SC correlated with increased transcutaneous water loss, and tracer remained restricted to the SC interstices. Enhanced extracellular permeability, in turn, was explicable by truncation and fragmentation of extracellular lamellar membrane arrays. The resultant clefts in the SC interstices represent the likely pathway for increased water permeability. Moreover, tracer movement remained restricted to the interstices, despite the demonstration of increased corneocyte fragility associated with widespread variations in CE structure. Regardless of variability in CE structure, however, CLE structure and bound omega‐OHCer content were normal. The normal CLE in LI may explain both the restriction of tracer to the SC interstices, as well as the presence of foreshortened membrane arrays with near‐normal interlamellar dimensions. Finally, the demonstration of a normal CLE in LI also raises questions about the putative role of TG1 in forming the CLE. These results demonstrate: (1) the extracellular nature of increased permeability in LI; (2) discontinuities in extracellular membrane structures that account for the enhanced permeability in LI; (3) that these membrane abnormalities are both associated with and explained by abnormalities in the subjacent CE scaffold; and (4) an intact CLE is present in LI, despite abnormalities in the CE, which may restrict water movement to the SC interstices in LI.

Expression of the C-C chemokine MIP-3α/CCL20 in human epidermis with impaired permeability barrier function

Abstract: External assault to the skin is followed by an epidermal response including synthesis of DNA, lipids, cytokines and migration of antigen presenting cells. MIP‐3α (CCL20, LARC, Exodus‐1, Scya20) is a recently described C‐C chemokine, predominantly expressed in extralymphoid tissue, which is known to direct migration of dendritic cell precursors and memory lymphocytes to sites of antigen invasion. We assessed the expression of MIP‐3α in human skin using semi‐quantitative polymerase chain reaction. In vivo, MIP‐3α mRNA was constitutively expressed at low levels in untreated human epidermis. After acute disruption of the epidermal permeabiltiy barrier MIP‐3α mRNA was upregulated in the epidermal fraction, whereas dermal MIP‐3α mRNA levels remained unchanged. In vitro, MIP‐3α was increased in cultured keratinocytes treated with IL‐1α and TNF‐α and was present in immature and mature dendritic cells, THP‐1 monocytic cells and activated T cells. Finally, skin biopsies from patients with psoriasis, contact dermatitis and mycosis fungoides showed abundant expression. In biopsies from atopic dermatitis and graft vs. host disease a weak signal was present, whereas no expression was found in scleroderma and toxic epidermal necrolysis. We conclude that regulation of MIP‐3α mRNA is part of the epidermal response to external assault. Its upregulation may represent a danger signal for increased immunosurveillance in barrier disrupted skin and inflammatory skin conditions with impaired barrier function to counteract potential antigen invasion.

Filaggrin mutations p.R501X and c.2282del4 in ichthyosis vulgaris

Ichthyosis vulgaris (IV) is the most common hereditary disorder of cornification in humans, characterized by generalized fine scaling of the skin, palmar hyperlinearity with or without keratosis pilaris and atopy. Recently, the molecular basis of IV was ascribed to loss-of-function mutations in the gene encoding filaggrin (FLG), namely p.R501X and c.2282del4. Homozygotes and compound heterozygotes were severely affected whereas heterozygotes showed mild disease or were asymptomatic, suggesting semidominant inheritance with incomplete penetrance in heterozygotes. We report the presence of FLG mutations in 15 out of 21 IV patients with a marked generalized scaling phenotype, including eight affected members of a four-generation family. In this group of patients not only homozygous and compound heterozygous, but also heterozygous patients for p.R501X and c.2282del4 display a pronounced phenotype, whereas in none of six individuals these two mutations were detectable despite decreased filaggrin expression on immunohistochemistry in two patients, indicating that other mutations in FLG and/or in other genes remain to be identified. In contrast, two additional p.R501X heterozygotes from the extended family are asymptomatic. In a control population from west-Austria a combined p.R501X and c.2282del4 carrier frequency of 6/110 (5.45%) was observed. We confirm that these FLG variants are common, but our results point to the existence of additional modifiers.