Epidermal barrier impairment predisposes for excessive growth of the allergy-associated yeast Malassezia on murine skin.

BACKGROUND: The skin barrier is vital for protection against environmental threats including insults caused by skin-resident microbes. Dysregulation of this barrier is a hallmark of atopic dermatitis (AD) and ichthyosis, with variable consequences for host immune control of colonizing commensals and opportunistic pathogens. While Malassezia is the most abundant commensal fungus of the skin, little is known about the host control of this fungus in inflammatory skin diseases. METHODS: In this experimental study, MC903-treated mice were colonized with Malassezia spp. to assess the host-fungal interactions in atopic dermatitis. Additional murine models of AD and ichthyosis, including tape stripping, K5-Nrf2 overexpression and flaky tail mice, were employed to confirm and expand the findings. Skin fungal counts were enumerated. High parameter flow cytometry was used to characterize the antifungal response in the AD-like skin. Structural and functional alterations in the skin barrier were determined by histology and transcriptomics of bulk skin. Finally, differential expression of metabolic genes in Malassezia in atopic and control skin was quantified. RESULTS: Malassezia grows excessively in AD-like skin. Fungal overgrowth could, however, not be explained by the altered immune status of the atopic skin. Instead, we found that by upregulating key metabolic genes in the altered cutaneous niche, Malassezia acquired enhanced fitness to efficiently colonise the impaired skin barrier. CONCLUSIONS: This study provides evidence that structural and metabolic changes in the dysfunctional epidermal barrier environment provide increased accessibility and an altered lipid profile, to which the lipid-dependent yeast adapts for enhanced nutrient assimilation. Our findings reveal fundamental insights into the implication of the mycobiota in the pathogenesis of common skin barrier disorders.

Atopic Dermatitis: The Fate of the Fat.

Atopic dermatitis (AD) is a chronic and relapsing inflammatory skin disease in which dry and itchy skin may develop into skin lesions. AD has a strong genetic component, as children from parents with AD have a two-fold increased chance of developing the disease. Genetic risk loci and epigenetic modifications reported in AD mainly locate to genes involved in the immune response and epidermal barrier function. However, AD pathogenesis cannot be fully explained by (epi)genetic factors since environmental triggers such as stress, pollution, microbiota, climate, and allergens also play a crucial role. Alterations of the epidermal barrier in AD, observed at all stages of the disease and which precede the development of overt skin inflammation, manifest as: dry skin; epidermal ultrastructural abnormalities, notably anomalies of the lamellar body cargo system; and abnormal epidermal lipid composition, including shorter fatty acid moieties in several lipid classes, such as ceramides and free fatty acids. Thus, a compelling question is whether AD is primarily a lipid disorder evolving into a chronic inflammatory disease due to genetic susceptibility loci in immunogenic genes. In this review, we focus on lipid abnormalities observed in the epidermis and blood of AD patients and evaluate their primary role in eliciting an inflammatory response.

Revisiting the Roles of Filaggrin in Atopic Dermatitis.

The discovery in 2006 that loss-of-function mutations in the filaggrin gene (FLG) cause ichthyosis vulgaris and can predispose to atopic dermatitis (AD) galvanized the dermatology research community and shed new light on a skin protein that was first identified in 1981. However, although outstanding work has uncovered several key functions of filaggrin in epidermal homeostasis, a comprehensive understanding of how filaggrin deficiency contributes to AD is still incomplete, including details of the upstream factors that lead to the reduced amounts of filaggrin, regardless of genotype. In this review, we re-evaluate data focusing on the roles of filaggrin in the epidermis, as well as in AD. Filaggrin is important for alignment of keratin intermediate filaments, control of keratinocyte shape, and maintenance of epidermal texture via production of water-retaining molecules. Moreover, filaggrin deficiency leads to cellular abnormalities in keratinocytes and induces subtle epidermal barrier impairment that is sufficient enough to facilitate the ingress of certain exogenous molecules into the epidermis. However, although FLG null mutations regulate skin moisture in non-lesional AD skin, filaggrin deficiency per se does not lead to the neutralization of skin surface pH or to excessive transepidermal water loss in atopic skin. Separating facts from chaff regarding the functions of filaggrin in the epidermis is necessary for the design efficacious therapies to treat dry and atopic skin.

Fatty acyl availability modulates cardiolipin composition and alters mitochondrial function in HeLa cells.

The molecular assembly of cells depends not only on the balance between anabolism and catabolism but to a large degree on the building blocks available in the environment. For cultured mammalian cells, this is largely determined by the composition of the applied growth medium. Here, we study the impact of lipids in the medium on mitochondrial membrane architecture and function by combining LC-MS/MS lipidomics and functional tests with lipid supplementation experiments in an otherwise serum-free and lipid-free cell culture model. We demonstrate that the composition of mitochondrial cardiolipins strongly depends on the lipid environment in cultured cells and favors the incorporation of essential linoleic acid over other fatty acids. Simultaneously, the mitochondrial respiratory complex I activity was altered, whereas the matrix-localized enzyme citrate synthase was unaffected. This raises the question on a link between membrane composition and respiratory control. In summary, we found a strong dependency of central mitochondrial features on the type of lipids contained in the growth medium. This underlines the importance of considering these factors when using and establishing cell culture models in biomedical research. In summary, we found a strong dependency of central mitochondrial features on the type of lipids contained in the growth medium.

Keratinocyte-derived IL-1ß induces PPARG downregulation and PPARD upregulation in human reconstructed epidermis following barrier impairment.

Peroxisome proliferator-activated receptors (PPARs) are a family of nuclear hormone receptors. In skin, PPARs modulate inflammation, lipid synthesis, keratinocyte differentiation and proliferation and thus are important for skin barrier homeostasis. Accordingly, PPAR expression is altered in various skin conditions that entail epidermal barrier impairment, that is atopic dermatitis (AD) and psoriasis. Using human epidermal equivalents (HEEs), we established models of acute epidermal barrier impairment devoid of immune cells. We assessed PPAR and cytokine expression after barrier perturbation and examined effects of keratinocyte-derived cytokines on PPAR expression. We show that acetone or SDS treatment causes graded impairment of epidermal barrier function. Furthermore, we demonstrate that besides IL-1ß and TNFα, IL-33 and TSLP are highly relevant markers for acute epidermal barrier impairment. Both SDS- and acetone-mediated epidermal barrier impairment reduce PPARG expression levels, whereas only SDS enhances PPARD expression. In line with findings in IL-1ß and TNFα-treated HEEs, abrogation of IL-1 signalling restores PPARG expression and limits the increase of PPARD expression in SDS-induced epidermal barrier impairment. Thus, following epidermal barrier perturbation, keratinocyte-derived IL-1ß and partly TNFα modulate PPARG and PPARD expression. These results emphasize a role for PPARγ and PPARß/δ in acute epidermal barrier impairment with possible implications for diseases such as AD and psoriasis.

PPARdelta in Affected Atopic Dermatitis and Psoriasis: A Possible Role in Metabolic Reprograming.

Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors expressed in the skin. Three PPAR isotypes, α (NRC1C1), ß or δ (NRC1C2) and γ (NRC1C3), have been identified. After activation through ligand binding, PPARs heterodimerize with the 9-cis-retinoic acid receptor (RXR), another nuclear hormone receptor, to bind to specific PPAR-responsive elements in regulatory regions of target genes mainly involved in organogenesis, cell proliferation, cell differentiation, inflammation and metabolism of lipids or carbohydrates. Endogenous PPAR ligands are fatty acids and fatty acid metabolites. In past years, much emphasis has been given to PPARα and γ in skin diseases. PPARß/δ is the least studied PPAR family member in the skin despite its key role in several important pathways regulating inflammation, keratinocyte proliferation and differentiation, metabolism and the oxidative stress response. This review focuses on the role of PPARß/δ in keratinocytes and its involvement in psoriasis and atopic dermatitis. Moreover, the relevance of targeting PPARß/δ to alleviate skin inflammation is discussed.

Filaggrin null mutations are associated with altered circulating Tregs in atopic dermatitis.

Atopic dermatitis (AD) is a chronic inflammatory skin disease with a complex pathogenesis. Although regulatory T cells (Tregs) have previously been studied in AD, their role remains controversial, likely owing to patient heterogeneity. Thus, we recruited adult AD patients and age-matched healthy controls, and assessed their filaggrin (FLG) genotype, serum IgE level, and eczema area and severity index (EASI). We found increased proportions of all circulating Treg subpopulations in AD patients. Moreover, we show positive correlations between circulating Tregs and serum IgE FLG null mutations limited the expansion of both memory and effector Tregs and enhanced that of recently thymus-emigrated Tregs. Furthermore, proportions of circulating Th2- or Th17-Tregs but not Th1-Tregs were increased in AD patients, and accentuated by FLG null mutations, thereby mimicking the immune deviation observed in Th cell populations. Moreover, ICOS+ Tregs showed reduced production of interleukin-10, suggesting impaired immunosuppression in AD. The level of demethylation of FOXP3i1, which reflects the stability of FOXP3 expression, was similar in the blood and skin of AD patients and healthy controls. Overall, these results show that Tregs may participate into AD pathogenesis and that FLG null mutations exert further modifications on specific subpopulations of circulating Tregs.

In Vivo Applicability of Neosartorya fischeri Antifungal Protein 2 (NFAP2) in Treatment of Vulvovaginal Candidiasis.

As a consequence of emerging numbers of vulvovaginitis cases caused by azole-resistant and biofilm-forming Candida species, fast and efficient treatment of this infection has become challenging. The problem is further exacerbated by the severe side effects of azoles as long-term-use medications in the recurrent form. There is therefore an increasing demand for novel and safely applicable effective antifungal therapeutic strategies. The small, cysteine-rich, and cationic antifungal proteins from filamentous ascomycetes are potential candidates, as they inhibit the growth of several Candida spp. in vitro; however, no information is available about their in vivo antifungal potency against yeasts. In the present study, we investigated the possible therapeutic application of one of their representatives in the treatment of vulvovaginal candidiasis, Neosartorya fischeri antifungal protein 2 (NFAP2). NFAP2 inhibited the growth of a fluconazole (FLC)-resistant Candida albicans strain isolated from a vulvovaginal infection, and it was effective against both planktonic cells and biofilm in vitro We observed that the fungal cell-killing activity of NFAP2 is connected to its pore-forming ability in the cell membrane. NFAP2 did not exert cytotoxic effects on primary human keratinocytes and dermal fibroblasts at the MIC in vitro. In vivo murine vulvovaginitis model experiments showed that NFAP2 significantly decreases the number of FLC-resistant C. albicans cells, and combined application with FLC enhances the efficacy. These results suggest that NFAP2 provides a feasible base for the development of a fundamental new, safely applicable mono- or polytherapeutic topical agent for the treatment of superficial candidiasis.

Xenobiotic Receptors and Their Mates in Atopic Dermatitis.

Atopic dermatitis (AD) is the most common inflammatory skin disease worldwide. It is a chronic, relapsing and pruritic skin disorder which results from epidermal barrier abnormalities and immune dysregulation, both modulated by environmental factors. AD is strongly associated with asthma and allergic rhinitis in the so-called 'atopic march.' Xenobiotic receptors and their mates are ligand-activated transcription factors expressed in the skin where they control cellular detoxification pathways. Moreover, they regulate the expression of genes in pathways involved in AD in epithelial cells and immune cells. Activation or overexpression of xenobiotic receptors in the skin can be deleterious or beneficial, depending on context, ligand and activation duration. Moreover, their impact on skin might be amplified by crosstalk among xenobiotic receptors and their mates. Because they are activated by a broad range of endogenous molecules, drugs and pollutants owing to their promiscuous ligand affinity, they have recently crystalized the attention of researchers, including in dermatology and especially in the AD field. This review examines the putative roles of these receptors in AD by critically evaluating the conditions under which the proteins and their ligands have been studied. This information should provide new insights into AD pathogenesis and ways to develop new therapeutic interventions.

Atopic dermatitis induces the expansion of thymus-derived regulatory T cells exhibiting a Th2-like phenotype in mice.

Atopic dermatitis (AD) is a widespread inflammatory skin disease with an early onset, characterized by pruritus, eczematous lesions and skin dryness. This chronic relapsing disease is believed to be primarily a result of a defective epidermal barrier function associated with genetic susceptibility, immune hyper-responsiveness of the skin and environmental factors. Although the important role of abnormal immune reactivity in the pathogenesis of AD is widely accepted, the role of regulatory T cells (Tregs) remains elusive. We found that the Treg population is expanded in a mouse model of AD, i.e. mice topically treated with vitamin D3 (VitD). Moreover, mice with AD-like symptoms exhibit increased inducible T-cell costimulator (ICOS)-, cytotoxic T-lymphocyte antigen-4 (CTLA-4)- and Glycoprotein-A repetitions predominant receptor (GARP)-expressing Tregs in skin-draining lymph nodes. Importantly, the differentiation of Tregs into thymus-derived Tregs is favoured in our mouse model of AD. Emigrated skin-derived dendritic cells are required for Treg induction and Langerhans cells are responsible for the biased expansion of thymus-derived Tregs . Intriguingly, thymus-derived Tregs isolated from mice with AD-like symptoms exhibit a Th2 cytokine profile. Thus, AD might favour the expansion of pathogenic Tregs able to produce Th2 cytokines and to promote the disease instead of alleviating symptoms.

Role of PPAR, LXR, and PXR in epidermal homeostasis and inflammation.

Epidermal lipid synthesis and metabolism are regulated by nuclear hormone receptors (NHR) and in turn epidermal lipid metabolites can serve as ligands to NHR. NHR form a large superfamily of receptors modulating gene transcription through DNA binding. A subgroup of these receptors is ligand-activated and heterodimerizes with the retinoid X receptor including peroxisome proliferator-activated receptor (PPAR), liver X receptor (LXR) and pregnane X receptor (PXR). Several isotypes of these receptors exist, all of which are expressed in skin. In keratinocytes, ligand activation of PPARs and LXRs stimulates differentiation, induces lipid accumulation, and accelerates epidermal barrier regeneration. In the cutaneous immune system, ligand activation of all three receptors, PPAR, LXR, and PXR, has inhibitory properties, partially mediated by downregulation of the NF-kappaB pathway. PXR also has antifibrotic effects in the skin correlating with TGF-beta inhibition. In summary, ligands of PPAR, LXR and PXR exert beneficial therapeutic effects in skin disease and represent promising targets for future therapeutic approaches in dermatology. 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.

Skin response to a carcinogen involves the xenobiotic receptor pregnane X receptor.

Skin is in daily contact with potentially harmful molecules from the environment such as cigarette smoke, automobile emissions, industrial soot and groundwater. Pregnane X receptor (PXR) is a transcription factor expressed in liver and intestine that is activated by xenobiotic chemicals including drugs and environmental pollutants. Topical application of the tumor initiator 7,12-dimethylbenz(a)anthracene (DMBA) enhances Pxr, Cyp1a1, Cyp1b1 and Cyp3a11, but not Ahr expression in the skin. Surprisingly, DMBA-induced Pxr upregulation is largely impaired in Langerin(+) cell-depleted skin, suggesting that DMBA mainly triggers Pxr in Langerin(+) cells. Furthermore, PXR deficiency protects from DNA damage in epidermal cells but to a lesser extent than aryl hydrocarbon receptor (AHR) deficiency. Interestingly, skin exposure to low doses of DMBA induces migration of PXR-deficient but not of wild-type and AHR-deficient Langerhans cells (LCs). PXR-humanized mice show a marked increase in DNA damage to epidermal cells after topical application of DMBA, demonstrating relevance of these findings in human tissue. This is the first report suggesting that carcinogens might trigger PXR in epidermal cells, particularly in LCs, thus leading to DNA damage. Further studies are required to better delineate the role of PXR in cutaneous carcinogenesis.

Epidermal barrier in hereditary ichthyoses, atopic dermatitis, and psoriasis.

Several skin disorders are associated with impaired skin barrier function. Primary dysfunction is caused by monogenic defects in key components of the epidermis (for example ichthyoses). Secondary barrier impairment occurs in inflammatory dermatoses marked by disturbed epidermal homeostasis (eczema, psoriasis, etc.). In these disorders, inflammation impedes the synthesis or maintenance of skin barrier components. Recent evidence suggests a combination of primary and secondary barrier dysfunction in atopic dermatitis and, to a lesser extent, also in psoriasis. In the future, subtypes of atopic dermatitis may likely be defined, in which one or the other is prevalent.

Connexins in epidermal health and diseases: insights into their mutations, implications, and therapeutic solutions.

The epidermis, the outermost layer of the skin, serves as a protective barrier against external factors. Epidermal differentiation, a tightly regulated process essential for epidermal homeostasis, epidermal barrier formation and skin integrity maintenance, is orchestrated by several players, including signaling molecules, calcium gradient and junctional complexes such as gap junctions (GJs). GJ proteins, known as connexins facilitate cell-to-cell communication between adjacent keratinocytes. Connexins can function as either hemichannels or GJs, depending on their interaction with other connexons from neighboring keratinocytes. These channels enable the transport of metabolites, cAMP, microRNAs, and ions, including Ca2+, across cell membranes. At least ten distinct connexins are expressed within the epidermis and mutations in at least five of them has been linked to various skin disorders. Connexin mutations may cause aberrant channel activity by altering their synthesis, their gating properties, their intracellular trafficking, and the assembly of hemichannels and GJ channels. In addition to mutations, connexin expression is dysregulated in other skin conditions including psoriasis, chronic wound and skin cancers, indicating the crucial role of connexins in skin homeostasis. Current treatment options for conditions with mutant or altered connexins are limited and primarily focus on symptom management. Several therapeutics, including non-peptide chemicals, antibodies, mimetic peptides and allele-specific small interfering RNAs are promising in treating connexin-related skin disorders. Since connexins play crucial roles in maintaining epidermal homeostasis as shown with linkage to a range of skin disorders and cancer, further investigations are warranted to decipher the molecular and cellular alterations within cells due to mutations or altered expression, leading to abnormal proliferation and differentiation. This would also help characterize the roles of each isoform in skin homeostasis, in addition to the development of innovative therapeutic interventions. This review highlights the critical functions of connexins in the epidermis and the association between connexins and skin disorders, and discusses potential therapeutic options.

Skin Barrier- and Immune Response-Related Biomarkers of Solar UVR Exposure Comparing Indoor and Outdoor Workers.

Outdoor workers have increased risk of developing keratinocyte cancer due to accumulated skin damage resulting from chronic and excessive exposure to UVR. This study aims to identify potential noninvasive biomarkers to assess chronic UVR exposure. We analyzed stratum corneum biomarkers collected from 2 skin locations and 2 occupational groups with contrasting solar UVR exposure: the forehead and retroauricular skin among outdoor workers and indoor workers. Using a linear mixed model adjusting for age and skin phototype, we compared biomarkers between both skin sites in indoor and outdoor workers. We measured markers of the immune response and skin barrier, including cytokines, GFs, 15-hydroxyeicosatetraenoic acid, cis- and trans-urocanic acid, and corneocyte topography, indicated by circular nano objects. Differences between the 2 skin sites were found for cis-urocanic acid, total urocanic acid, IL-1α, IL-1RA, IL-1RA/IL-1α, IL-18, 15-hydroxyeicosatetraenoic acid, CCL4, and circular nano objects. The levels of cis-urocanic acid and CCL4 also differed between indoor and outdoor workers. These findings underscore changes in both immune response and skin barrier induced by UVR. They indicate the potential utility of stratum corneum biomarkers in detecting both chronic UVR exposure in occupational setting and aiding in the development of preventive measures.

Excessive Production of Hydrogen Peroxide in Mitochondria Contributes to Atopic Dermatitis.

Atopic dermatitis (AD) is a complex disease characterized by chronic recurring eczema and pruritus. In addition, patients with AD display increased cutaneous and systemic levels of oxidative damage markers, whose source remains elusive. In this study, we investigated oxidative and mitochondrial stress in AD epidermis. The levels of superoxide dismutase 2 and hydrogen peroxide are augmented in the mitochondria of flaky tail (ft/ft) mouse keratinocytes, which is associated with the inhibition of the glutathione system and catalase. Furthermore, reduced levels of glutathione peroxidase 4 are associated with accumulation of malondialdehyde, 4-hydroxy-2-nonenal, and oxidized phosphatidylcholines in ft/ft epidermis. Cytochrome c is markedly increased in ft/ft epidermis, hence showing mitochondrial stress. Topical application of MitoQ, which is a mitochondrial-targeting antioxidant, to ft/ft mouse skin reduced damage to macromolecules and inflammation and restored epidermal homeostasis. Absence of alteration in the expression of superoxide dismutase 2, catalase, and glutathione peroxidase 4 and limited lipid peroxidation as well as oxidized phosphatidylcholines in the epidermis of Flg-/- mice suggest that FLG deficiency marginally contributes to oxidative stress in ft/ft epidermis. Increased superoxide dismutase 2, lipid peroxidation, and cytochrome c in the epidermis of patients with AD, associated with reduced antioxidant response in primary AD keratinocytes, corroborate mitochondrial dysfunction and lack of cellular adjustment to oxidative stress in AD epidermis.

Lack of IL-2 in PPAR-α-deficient mice triggers allergic contact dermatitis by affecting regulatory T cells.

The aim of the present work was to decipher the cellular basis of the immunoregulatory role of peroxisome proliferator-activated receptor (PPAR)-α in cutaneous hypersensitivity reactions. After challenge with a contact allergen, we observed augmented hypersensitivity reactions with increased numbers of activated T lymphocytes in the skin of PPAR-α-/- mice. Furthermore, following antigen challenge, the percentages of Tregs in the blood, draining lymph nodes and skin were decreased in these mice. PPAR-α deficiency impaired the production of IL-2 in lymph nodes, whereas TGF-ß levels remained unchanged. Injection of IL-2 into PPAR-α-/- mice restored the Treg population in the skin-draining lymph nodes of allergen-challenged mice. In vivo induction of Tregs from WT CD4+ CD25- T cells was impaired when adoptively transferred into PPAR-α-/- mice as compared with transfer into WT mice, and reversed by injection of IL-2 into PPAR-α-/- mice. Furthermore, the suppressive capacity of PPAR-α-/- Tregs was impaired when compared to WT Tregs in vitro and in co-adoptive transfer experiments. Finally, injection of IL-2 to PPAR-α-/- mice decreased skin inflammation to a level similar to WT mice. In conclusion, the pro-inflammatory skin phenotype of PPAR-α-/- mice is due to lack of IL-2-mediated Treg induction in these mice.

Modulation of T lymphocyte function by the pregnane X receptor.

The pregnane X receptor (PXR) is a ligand-activated transcription factor regulating genes central to drug and hormone metabolism in the liver. Previous reports indicated that PXR is expressed in PBMC, but the role of PXR in immune cells remains unknown. In this paper, we report increased PXR expression in mouse and human T lymphocytes upon immune activation. Furthermore, pharmacologic activation of PXR inhibits T lymphocyte proliferation and anergizes T lymphocytes by decreasing the expression of CD25 and IFN-gamma and decreasing phosphorylated NF-kappaB and MEK1/2. Although these effects are preceded by an increase of suppressor of cytokine signaling 1, a master switch for IFN-gamma expression, in a PXR-dependent manner, T-bet expression remains unchanged. Conversely, PXR-deficient mice exhibit an exaggerated T lymphocyte proliferation and increased CD25 expression. Furthermore, PXR-deficient lymphocytes produce more IFN-gamma and less of the anti-inflammatory cytokine IL-10. In summary, these results reveal a novel immune-regulatory role of PXR in T lymphocytes and identify suppressor of cytokine signaling 1 as an early signal in PXR-mediated T lymphocyte suppression.