The skin barrier defect underlying autosomal recessive congenital ichthyoses leads to excessive transepidermal water loss immediately after birth. Affected infants are often born as collodion babies reflecting a physical compensation for the defective permeability barrier. In the available knockout mouse models, however, extrauterine survival is limited to a few hours. These animals, therefore, do not allow evaluation of any postnatal therapy.

The corneocyte lipid envelope (CLE), a monolayer of u-hydroxyceramides whose function(s) remain(s) uncertain, is absent in patients with autosomal recessive congenital ichthyoses with mutations in enzymes that regulate epidermal lipid synthesis. Secreted lipids fail to transform into lamellar membranes in certain autosomal recessive congenital ichthyosis epidermis, suggesting the CLE provides a scaffold for the extracellular lamellae. However, because cornified envelopes are attenuated in these autosomal recessive congenital ichthyoses, the CLE may also provide a scaffold for subjacent cornified envelope formation, evidenced by restoration of cornified envelopes after CLE rescue. We provide multiple lines of evidence that the CLE originates as lamellar body-limiting membranes fuse with the plasma membrane: (i) ABCA12 patients and Abca12 e/e mice display normal CLEs; (ii) CLEs are normal in Netherton syndrome, despite destruction of secreted LB contents; (iii) CLEs are absent in VSP33B-negative patients; (iv) limiting membranes of lamellar bodies are defective in lipid-synthetic autosomal recessive congenital ichthyoses; and (v) lipoxygenases, lipase activity, and LIPN co-localize within putative lamellar bodies.

The -hydroxyceramide (-OH-Cer)-enriched, corneocyte lipid envelope (CLE) lies external to the cornified envelope (CE), but neither its functions nor origin are known. To address its functions, we assessed biopsies from recessive congenital ichthyosis (ARCI) patients and animals with compromised -OH-Cer generation. While both CLEs and CEs were attenuated, topical acylCer applications to ichthyin-deficient canines and transgenic rescue of fatp4 knockout mice restored both CLEs and CEs. The CLE also is required for lamellar membrane formation, because secreted lamellar body (LB)-derived lipids fail to transform into lamellar membranes in Alox12B-/-, despite unimpaired acylglucosylCer production/secretion. The CLE originates with insertion of LB limiting membranes into the plasma membrane during exocytosis, rather than from secreted LB contents, because: i) LB secretion yields normal CLEs in Harlequin ichthyosis and Abca12-/- mice; ii) CLEs are normal in Netherton syndrome, despite destruction of secreted LB contents; iii) limiting membranes of LBs were abnormal in ARCI patients/animal models; iv) 12R-Lox and Aloxe3, enzymes that generate -OH-Cer, immunolocalized to LB; v) CLEs are absent in ARC syndrome, where LB secretion is impaired. Thus, the CLE functions as a bidirectional scaffold, necessary for lamellar bilayer formation, originating with fusion of LB during exocytosis.

DEAR EDITOR, Autosomal recessive congenital ichthyoses (ARCIs) are a group of hereditary skin disorders. The disease phenotype is associated with an impaired epidermal barrier leading to increased transepidermal water loss, temperature instability and hypernatraemic dehydration in infancy. ARCIs are known to be caused by mutations in at least nine different genes, which encode proteins involved in the formation of the epidermal barrier. Two of these genes, ALOX12B and ALOXE3, code for the epidermal lipoxygenases 12R-LOX and eLOX-3, which are known to be indispensable for barrier function and lipid metabolism in the skin. Both eLOX-3and 12R-LOX-deficient mice die shortly after birth due to transepidermal water loss, and show dramatically

The skin is the primary barrier between the external environment and the internal milieu of the host protecting the body from physical and chemical insults and injury and preventing the loss of water. An active lipid metabolism and fatty acid-derived oxylipins are crucially involved in the structural integrity and functionality of the skin. Among them are lipoxygenases (LOX)-derived autacoids generated by an abundant and diverse cutaneous LOX metabolism. LOX products fulfill substantial functions in epithelial tissue homeostasis, inflammation as the general skin response to external damage, wound healing, and disease-related processes including numerous inflammatory skin conditions and the development of skin cancer. Recent results point to a critical role of a distinct LOX pathway in the development and maintenance of the epidermal barrier. This review focuses on the activities and mechanisms of actions of individual LOX-derived oxylipins, and the dysregulation of the corresponding LOX enzymes in diseased skin.

A number of genodermatoses are characterized by distinct morphological markers which have been and are used for classification and diagnosis as well as for identifying causative gene mutations and pathogenetic pathways. Various types of animal models and organotypic cell cultures have been established to provide further insight into disease mechanisms and treatment. Selected examples are: (i) a spontaneous rat model for dominant epidermolysis bullosa revealing similar variability of anchoring fibril expression as in human patients; (ii) PNPLA1 as a new gene involved in autosomal recessive congenital ichthyosis pathology identified from a Golden Retriever breed spontaneously affected by lamellar ichthyosis; (iii) knockout mice for lipoxygenases expressing differential skin barrier defects and compensatory hyperkeratosis; (iv) a long-term skin-humanized mouse model for transglutaminase 1-deficient lamellar ichthyosis, obviously in some respects advantageous to organotypic cell cultures and successfully having been used for enzyme substitution therapy; (v) Persian cat with classical Ehlers-Danlos syndrome. Investigation of such monogenetic disease models can help to understand causal correlations between pathology and clinical manifestations and provide insights towards developing and evaluating novel causal therapies.

Background Autosomal recessive congenital ichthyoses (ARCIs) are keratinization disorders caused by impaired skin barrier function. Mutations in the genes encoding the lipoxygenases 12R-LOX and eLOX-3 are the second most common cause of ARCIs. In recent years, human skin equivalents recapitulating the ARCI phenotype have been established. Objectives To develop a murine organotypic tissue culture model for ARCI. Methods Epidermal keratinocytes were isolated from newborn 12R-LOX-deficient mice and cocultivated with mouse dermal fibroblasts embedded in a scaffold of native collagen type I. Results With this experimental set-up the keratinocytes formed a well-organized multilayered stratified epithelium resembling skin architecture in vivo. All epidermal layers were present and the keratinocytes within showed the characteristic morphological features. Markers for differentiation and maturation indicated regular epidermal morphogenesis. The major components of epidermal structures were expressed, and were obviously processed and assembled properly. In contrast to their wild-type counterparts, 12R-LOX-deficient skin equivalents showed abnormal vesicular structures in the upper epidermal layers correlating with altered lipid composition and increased transepidermal water loss, comparable with 12R-LOX-deficient mice. Conclusions The mouse skin equivalents faithfully recapitulate the 12R-LOX-deficient phenotype observed in vivo, classifying them as appropriate in vitro models to study molecular mechanisms involved in the development of ARCI and to evaluate novel therapeutic agents. In contrast to existing human three-dimensional skin models, the generation of these murine models is not constrained by a limited supply of material and does not depend on in vitro expansion and/or genetic manipulations that could result in inadvertent genotypic and phenotypic alterations.

Lipoxygenases (LOX) are key enzymes in the biosynthesis of a variety of highly active oxylipins which act as signaling molecules involved in the regulation of many biological processes. LOX are also able to oxidize complex lipids and modify membrane structures leading to structural changes that play a role in the maturation and terminal differentiation of various cell types. The mammalian skin represents a tissue with highly abundant and diverse LOX metabolism. Individual LOX isozymes are thought to play a role in the modulation of epithelial proliferation and/or differentiation as well as in inflammation, wound healing, inflammatory skin diseases and cancer. Emerging evidence indicates a structural function of a particular LOX pathway in the maintenance of skin permeability barrier. Loss-of-function mutations in the LOX genes ALOX12B and ALOXE3 have been found to represent the second most common cause of autosomal recessive congenital ichthyosis and targeted disruption of the corresponding LOX genes in mice resulted in neonatal death due to a severely impaired permeability barrier function. Recent data indicate that LOX action in barrier function can be traced back to the oxygenation of linoleate-containing ceramides which constitutes an important step in the formation of the corneocyte lipid envelope. This article is part of a Special Issue entitled The Important Role of Lipids in the Epidermis and their Role in the Formation and Maintenance of the Cutaneous Barrier. Guest Editors: Kenneth R. Feingold and Peter Elias.

amniotic fluid ectodysplasin A1 X-linked hypohidrotic ectodermal dysplasia Pathologies associated with genodermatoses and other genetic disorders can irremediably affect fetuses, making early-stage therapies desirable. Prenatal maternal drug administration, however, exposes mothers to potential drug toxicity and is limited by the variability in transplacental drug delivery. Alternative approaches to fetal treatment should entail low-risk drug delivery with reproducible pharmacokinetics. X-linked hypohidrotic ectodermal dysplasia (XLHED), the most common inherited disorder of ectoderm development, is caused by a lack of the signaling molecule ectodysplasin A1 (EDA1), which is essential for ectodermal placode formation and subsequent development of various skin appendages, glands, and teeth (Mikkola, 2009Mikkola M.L. Molecular aspects of hypohidrotic ectodermal dysplasia.Am J Med Genet A. 2009; 149: 2031-2036Crossref Scopus (155) Google Scholar). Patients with XLHED have less hair, fewer or no eccrine sweat, sebaceous and meibomian glands, and malformed or absent teeth. Insufficient thermoregulation can lead to perilous hyperthermic episodes during infancy (Blüschke et al., 2010Blüschke G. Nüsken K.D. Schneider H. Prevalence and prevention of severe complications of hypohidrotic ectodermal dysplasia in infancy.Early Hum Dev. 2010; 86: 397-399Abstract Full Text Full Text PDF PubMed Scopus (59) Google Scholar) and remains an important issue throughout life (Hammersen et al., 2011Hammersen J. Neukam V. Nüsken K.D. et al.Systematic evaluation of exertional hyperthermia in children with hypohidrotic ectodermal dysplasia: an observational study.Pediatr Res. 2011; 70: 297-301Crossref PubMed Scopus (18) Google Scholar). Many affected individuals suffer from recurrent airway and eye problems (Dietz et al., 2013Dietz J. Kaercher T. Schneider A.T. et al.Early respiratory and ocular involvement in X-linked hypohidrotic ectodermal dysplasia.Eur J Pediatr. 2013; 172: 1023-1031Crossref PubMed Scopus (39) Google Scholar). To date, only symptomatic treatment is available for these patients. Causative therapeutic approaches to such disorders are expected to be most effective if applied already in utero, with the additional benefit that immune tolerance of a replacement protein may be induced, facilitating postnatal reapplication (Schneider et al., 2002Schneider H. Mühle C. Douar A.M. et al.Sustained delivery of therapeutic concentrations of human clotting factor IX: a comparison of adenoviral and AAV vectors administered in utero.J Gene Med. 2002; 4: 46-53Crossref PubMed Scopus (55) Google Scholar; Waddington et al., 2003Waddington S.N. Buckley S.M.K. Nivsarkar M. et al.In utero gene transfer of human factor IX to fetal mice can induce postnatal tolerance of the exogenous clotting factor.Blood. 2003; 101: 1359-1366Crossref PubMed Scopus (91) Google Scholar). In the Tabby mouse, a well-characterized animal model of XLHED (Falconer, 1952Falconer D.S. A totally sex-linked gene in the house mouse.Nature. 1952; 169: 664-665Crossref PubMed Scopus (36) Google Scholar), prenatal exposure to EDA1 via serial intravenous administrations to the dam corrected developmental abnormalities to a far greater extent compared with postnatal administration (Gaide and Schneider, 2003Gaide O. Schneider P. Permanent correction of an inherited ectodermal dysplasia with recombinant EDA.Nat Med. 2003; 9: 614-618Crossref PubMed Scopus (167) Google Scholar). This approach may, however, be suboptimal for achieving reproducible therapeutic drug concentrations in human fetuses, and would expose the mother to high serum levels of an exogenous molecule. We hypothesized that EDI200, an EDA1 replacement protein consisting of the receptor-binding domain of EDA1 and the Fc part of IgG1, may enter the fetal circulation also after injection into the amniotic fluid (AF), because the fetus swallows AF regularly and the neonatal Fc receptor, which is present in rodent and human fetal intestine (Shah et al., 2003Shah U. Dickinson B.L. Blumberg R.S. et al.Distribution of the IgG Fc receptor, FcRn, in the human fetal intestine.Pediatr Res. 2003; 53: 295-301Crossref PubMed Scopus (106) Google Scholar), may facilitate intestinal absorption of Fc-containing proteins. AF could thus serve as a drug reservoir and provide for continuous drug uptake. Here, we report striking reversal of the XLHED phenotype of Tabby mice following a single intra-amniotic injection of EDI200. Long-term stability of this recombinant protein in AF, i.e. the retention of binding to its cognate receptor, was confirmed in vitro under various conditions (Supplementary Figure S1 online). Pharmacokinetics following intra-amniotic injection of 35 μg EDI200 per amniotic sac (=100 μg g-1 of estimated fetal body weight) was studied in wild-type mice at day 15 of gestation (E15). All maternal animals and 93% of the treated fetuses survived the procedure. EDI200 serum levels were measured at different time points after injection both in treated and untreated fetuses as well as in the dams. Intra-amniotic injection of EDI200 resulted in mean fetal serum levels of 9.0 and 1.2 μg ml-1 at 6 and 96 hours, respectively. After 6 hours, this corresponds to 180 ng of EDI200 per fetus or 0.5% of the injected protein, assuming a total serum volume of approximately 20 μl in an E15 mouse fetus. Interestingly, there was a low level of EDI200 transfer into the circulation of untreated siblings and that of the pregnant dam (Figure 1). The drug was partially and slowly redistributed from treated fetuses, in which EDI200 concentration diminished over time, to untreated siblings that witnessed a parallel increase in the serum levels (up to 0.57 μg ml-1). Maternal EDI200 serum levels remained <0.1 μg ml-1 at the time points investigated. Thus, intra-amniotic administration of EDI200 at E15 resulted in substantial fetal uptake with minimal maternal exposure. Download .pdf (.18 MB) Help with pdf files Supplementary Information This approach was then evaluated in pregnant Tabby mice with doses of 100, 10, and 1 μg g-1 of estimated fetal body weight. The surgical procedure was conducted under isofluran anesthesia plus perioperative analgesia with metamizole and was approved by the local government authorities. All treated Tabby mouse fetuses of the high and intermediate dose cohorts survived the E15 intra-amniotic injection and were born without complications. They were easily distinguishable from native Tabby mice already in the second week after birth. Later, normal eye opening, retro-auricular and guard hair as in wild-type mice, and a normally shaped tail tip (Figure 2a and b) were evident. Starch–iodine tests revealed regular sweat production at the paws (Figure 2c–e). Normal eccrine sweat glands (Figure 2f–h) were detected in footpads of these animals. In addition, size and shape of the molars resembled those of wild-type mice (Figure 2i–k). Thus, in contrast to a single maternal intravenous injection of 400 μg of EDI200 in pregnant Tabby mice at E15, which corrected the XLHED phenotype in the offspring only partially (unpublished own data), a single intra-amniotic dose of 3.5 μg or above resulted in complete phenotypic correction. No adverse effects were observed. All treated Tabby mice survived to adulthood and showed normal behavior and fertility. The lower dose of 1 μg g-1 body weight yielded only a partial restoration of normal ectoderm development, with less guard and/or tail hair and fewer sweat glands present (Supplementary Table S1 online). Interestingly, but less relevant to human singleton gestations, a dose-dependent correction was also observed for untreated littermates (Supplementary Table S1 online)—explained by partial leakage of the drug to neighboring fetuses. As expected from previous studies (Gaide and Schneider, 2003Gaide O. Schneider P. Permanent correction of an inherited ectodermal dysplasia with recombinant EDA.Nat Med. 2003; 9: 614-618Crossref PubMed Scopus (167) Google Scholar), the maternal Tabby phenotype was not visibly altered by EDI200 administration, regardless of the dose. All dams remained fertile and no adverse effects of the treatment could be detected during an observation period of 6–9 months. The EDA1 signaling pathway is well conserved among vertebrates (Pantalacci et al., 2008Pantalacci S. Chaumot A. Benoît G. et al.Conserved features and evolutionary shifts of the EDA signaling pathway involved in vertebrate skin appendage development.Mol Biol Evol. 2008; 25: 912-928Crossref PubMed Scopus (34) Google Scholar) and findings in animal models should therefore be transferable to human XLHED patients. Early corrective treatment would increase their life expectancy, would have a high impact on the quality of life, and substantially reduce medical expenses. Intra-amniotic drug delivery might be most beneficial if attempted during mid-gestation, when sweat gland development is not yet completed (Ersch and Stallmach, 1999Ersch J. Stallmach T. Assessing gestational age from histology of fetal skin: an autopsy study of 379 fetuses.Obstet Gynecol. 1999; 94: 753-757Crossref PubMed Scopus (0) Google Scholar). This approach is likely to have a good benefit/risk ratio, supported by the broad experience with amniocentesis, and may represent a novel paradigm for treatment of disorders in early human development. We thank Laure Willen (University of Lausanne) and Elisabeth Koppmann (University Hospital Erlangen) for excellent technical assistance. Most of the work was performed by Katharina Hermes in fulfillment of the requirements for obtaining the degree "Dr. med." from the Friedrich-Alexander-Universität Erlangen-Nürnberg. This study was supported by grants from the German Research Foundation (Schn 569/4 to HS), the Swiss National Science Foundation (31003A-138065 to PS), and Edimer Pharmaceuticals (to PS and HS). Supplementary material is linked to the online version of the paper at http://www.nature.com/jid

Metabolites of the epidermal lipoxygenase-3 (eLOX-3) are involved in various metabolic pathways. Most unexpectedly, intra-amniotic delivery of eLOX-3 to mice at gestational day 14.5, both via an adenoviral vector and as recombinant protein, resulted in fetal growth restriction and intrauterine death. Periodic acid-Schiff staining and RT-PCR analysis of placentae from fetuses exposed to eLOX-3 indicated a lack of glycogen trophoblasts in the junctional zone. Placenta-specific gene expression was altered. Thus, the observed prenatal toxicity of eLOX-3 could be due to a strong effect on placental development.

Loss-of-function mutations in the lipoxygenase (LOX) genes ALOX12B and ALOXE3 are the second most common cause of autosomal recessive congenital ichthyosis. The encoded proteins, 12R-LOX and epidermal LOX-3 (eLOX-3), act in sequence to convert fatty acid substrates via R-hydroperoxides to specific epoxyalcohol derivatives and have been proposed to operate in the same metabolic pathway during epidermal barrier formation. Here, we show that eLOX-3 deficiency in mice results in early postnatal death, associated with similar but somewhat less severe barrier defects and morphological changes than reported earlier for the 12R-LOX-knockout mice. Skin lipid analysis demonstrated that the severity of barrier failure is related to the loss of covalently bound ceramides in both 12R-LOX- and eLOX-3-null mice, confirming a proposed functional linkage of the LOX pathway to ceramide processing and formation of the corneocyte lipid envelope. Furthermore, analysis of free oxygenated fatty acid metabolites revealed strongly reduced levels of hepoxilin metabolites in eLOX-3-deficient epidermis, indicating an additional function of eLOX-3 in mammalian skin as a hepoxilin synthase linked to the 12S-LOX pathway. Journal of Investigative Dermatology (2013) 133, 172-180; doi:10.1038/jid.2012.250; published online 26 July 2012

The nuclear receptor peroxisome proliferator-activated receptor gamma (PPAR gamma) is essential for adipogenesis. Although several fatty acids and their derivatives are known to bind and activate PPAR gamma, the nature of the endogenous ligand(s) promoting the early stages of adipocyte differentiation has remained enigmatic. Previously, we showed that lipoxygenase (LOX) activity is involved in activation of PPAR gamma during the early stages of adipocyte differentiation. Of the seven known murine LOXs, only the unconventional LOX epidermis-type lipoxygenase 3 (eLOX3) is expressed in 3T3-L1 preadipocytes. Here, we show that forced expression of eLOX3 or addition of eLOX3 products stimulated adipogenesis under conditions that normally require an exogenous PPAR gamma ligand for differentiation. Hepoxilins, a group of oxidized arachidonic acid derivatives produced by eLOX3, bound to and activated PPAR gamma. Production of hepoxilins was increased transiently during the initial stages of adipogenesis. Furthermore, small interfering RNA-mediated or retroviral short hairpin RNA-mediated knockdown of eLOX3 expression abolished differentiation of 3T3-L1 preadipocytes. Finally, we demonstrate that xanthine oxidoreductase (XOR) and eLOX3 synergistically enhanced PPAR gamma-mediated transactivation. Collectively, our results indicate that hepoxilins produced by the concerted action of XOR and eLOX3 may function as PPAR gamma activators capable of promoting the early PPAR gamma-dependent steps in the conversion of preadipocytes into adipocytes.

In recent years several new genes for autosomal recessive congenital ichthyosis (ARCI) have been identified. However, little is known about the molecular epidemiology and pathophysiology of this genetically and clinically heterogeneous group of severe disorders of keratinization. ARCI is characterized by intense scaling of the whole integument often associated with erythema. We and others have shown that mutations in ALOX12B and ALOXE3, coding for the lipoxygenases 12R-LOX and eLOX-3 predominantly synthesized in the epidermis, can underlie this rare condition. Here we have surveyed a large group of 250 patients with ARCI for mutations in these two genes. We have identified 11 different previously unreported mutations in ALOX12B and ALOXE3 in 21 ARCI patients from 19 unrelated families and demonstrated that mutations in the two genes are the second most common cause for ARCI in this cohort of patients. Examination of the molecular data revealed allelic heterogeneity for ALOX12B and two mutational hotspots in ALOXE3. Functional analysis of all missense mutations and a splice site mutation demonstrated that complete loss of function of the enzymes underlies the phenotype. Our findings further establish the pivotal role of the 12-lipoxygenase pathway during epidermal differentiation.

12R-lipoxygenase (12R-LOX) represents a key enzyme of a recently identified eicosanoid pathway in the skin that plays an essential role in the establishment and/or maintenance of the epidermal barrier function. Genetic studies show that loss-of-function mutations in ALOX12B, encoding 12R-LOX, and in ALOXE3, encoding another closely related LOX involved in this pathway, are the second most common cause for autosomal recessive congenital ichthyosis (ARCI). To investigate the pathomechanism of ARCI and the function of 12R-LOX, we recently generated a 12R-LOX knockout model. 12R-LOX-deficient mice die rapidly after birth from severe barrier dysfunction without exhibiting an obvious cutaneous phenotype. Thus, we analyzed the adult phenotype of 12R-LOX(-/-) skin transplanted onto nude mice. 12R-LOX(-/-) skin develops an ichthyosiform appearance with thickening of the epidermis, hyperproliferation, hypergranulosis, focal parakeratosis, and severe hyperkeratosis. The adult mutant mouse skin phenotype closely reproduces the ichthyosis phenotype seen in patients with ALOX12B mutations. Western blot analysis revealed restoration of profilaggrin processing that used to be disturbed in neonatal mutant skin and overexpression of filaggrin, involucrin, and repetin. The results indicate that 12R-LOX knockout mice may represent a useful animal model for a detailed analysis of mechanisms involved in ARCI forms that are associated with impaired LOX metabolism.