Junctions and Inflammation in the Skin

Abstract

The skin forms a life-sustaining barrier between the organism and physical environment. The physical barrier of skin is mainly localized in the stratum corneum (SC); however, nucleated epidermis also contributes to the barrier through tight, gap, and adherens junctions (AJs), as well as through desmosomes and cytoskeletal elements. Many inflammatory diseases, such as atopic dermatitis (AD) and psoriasis, are associated with barrier dysfunction. It is becoming increasingly clear that the skin barrier function is not only affected by inflammatory signals but that defects in structural components of the barrier may be the initiating event for inflammatory diseases. This view is supported by findings that mutations in filaggrin, a key structural epidermal barrier protein, cause the inflammatory skin disease AD, and that a loss of AJ components, namely epidermal p120 catenin or α-catenin results in skin inflammation.     

Mice deficient in involucrin, envoplakin, and periplakin have a defective epidermal barrier

The cornified envelope is assembled from transglutaminase cross-linked proteins and lipids in the outermost epidermal layers and is essential for skin barrier function. Involucrin, envoplakin, and periplakin form the protein scaffold on which the envelope assembles. To examine their combined function, we generated mice deficient in all three genes. The triple knockouts have delayed embryonic barrier formation and postnatal hyperkeratosis (abnormal accumulation of cornified cells) resulting from impaired desquamation. Cornified envelopes form but are ultrastructurally abnormal, with reduced lipid content and decreased mechanical integrity. Expression of proteases is reduced and the protease inhibitor, serpina1b, is highly upregulated, resulting in defective filaggrin processing and delayed degradation of desmoglein 1 and corneodesmosin. There is infiltration of CD4⁺ T cells and a reduction in resident γδ⁺ T cells, reminiscent of atopic dermatitis. Thus, combined loss of the cornified envelope proteins not only impairs the epidermal barrier, but also changes the composition of T cell subpopulations in the skin.     

Loss-of-Function Mutations in CAST Cause Peeling Skin,Leukonychia, Acral Punctate Keratoses,Cheilitis, and Knuckle Pads

Calpastatin is an endogenous specific inhibitor of calpain, a calcium-dependent cysteine protease. Here we show that loss-of-function mutations in calpastatin (CAST) are the genetic causes of an autosomal-recessive condition characterized by generalized peeling skin, leukonychia, acral punctate keratoses, cheilitis, and knuckle pads, which we propose to be given the acronym PLACK syndrome. In affected individuals with PLACK syndrome from three families of different ethnicities, we identified homozygous mutations (c.607dup, c.424A>T, and c.1750delG) in CAST, all of which were predicted to encode truncated proteins (p.Ile203Asnfs^∗8, p.Lys142^∗, and p.Val584Trpfs^∗37). Immunohistochemistry shows that staining of calpastatin is reduced in skin from affected individuals. Transmission electron microscopy revealed widening of intercellular spaces with chromatin condensation and margination in the upper stratum spinosum in lesional skin, suggesting impaired intercellular adhesion as well as keratinocyte apoptosis. A significant increase of apoptotic keratinocytes was also observed in TUNEL assays. In vitro studies utilizing siRNA-mediated CAST knockdown revealed a role for calpastatin in keratinocyte adhesion. In summary, we describe PLACK syndrome, as a clinical entity of defective epidermal adhesion, caused by loss-of-function mutations in CAST.     

Beta-Hemolysin Promotes Skin Colonization by Staphylococcus aureus

Colonization by Staphylococcus aureus is a characteristic feature of several inflammatory skin diseases and is often followed by epidermal damage and invasive infection. In this study, we investigated the mechanism of skin colonization by a virulent community-acquired methicillin-resistant S. aureus (CA-MRSA) strain, MW2, using a murine ear colonization model. MW2 does not produce a hemolytic toxin, beta-hemolysin (Hlb), due to integration of a prophage, ϕSa3mw, inside the toxin gene (hlb). However, we found that strain MW2 bacteria that had successfully colonized murine ears included derivatives that produced Hlb. Genome sequencing of the Hlb-producing colonies revealed that precise excision of prophage ϕSa3mw occurred, leading to reconstruction of the intact hlb gene in their chromosomes. To address the question of whether Hlb is involved in skin colonization, we constructed MW2-derivative strains with and without the Hlb gene and then subjected them to colonization tests. The colonization efficiency of the Hlb-producing mutant on murine ears was more than 50-fold greater than that of the mutant without hlb. Furthermore, we also showed that Hlb toxin had elevated cytotoxicity for human primary keratinocytes. Our results indicate that S. aureus Hlb plays an important role in skin colonization by damaging keratinocytes, in addition to its well-known hemolytic activity for erythrocytes.     

Highlighting Kathleen Green and Mario Delmar,Guest Editors of Special Issue (part 2): Junctional Targets of Skin and Heart Disease

Abstract

Cell Communication and Adhesion has been fortunate to enlist two pioneers of epidermal and cardiac cell junctions, Kathleen Green and Mario Delmar, as Guest Editors of a two part series on junctional targets of skin and heart disease. Part 2 of this series begins with an overview from Dipal Patel and Kathy Green comparing epidermal desmosomes to cardiac area composita junctions, and surveying the pathogenic mechanisms resulting from mutations in their components in heart disease. This is followed by a review from David Kelsell on the role of desmosomal mutation in inherited syndromes involving skin fragility. Agnieszka Kobeliak discusses how structural deficits in the epidermal barrier intersect with the NFkB signaling pathway to induce inflammatory diseases such as psoriasis and atopic dermatitis. Farah Sheikh reviews the specialized junctional components in cardiomyocytes of the cardiac conduction system and Robert Gourdie discusses how molecular complexes between sodium channels and gap junction proteins within the perijunctional microdomains within the intercalated disc facilitate conduction. Glenn Radice evaluates the role of N-cadherin in heart. Andre Kleber and Chris Chen explore new approaches to study junctional mechanotransduction in vitro with a focus on the effects of connexin ablation and the role of cadherins, respectively. To complement this series of reviews, we have interviewed Werner Franke, whose systematic documentation the tissue-specific complexity of desmosome composition and pioneering discovery of the cardiac area composita junction greatly facilitated elucidation of the role of desmosomal components in the pathophysiology of human heart disease.     

Identification of Lympho-Epithelial Kazal-Type Inhibitor 2 in Human Skin as a Kallikrein-Related Peptidase 5-Specific Protease Inhibitor

Kallikreins-related peptidases (KLKs) are serine proteases and have been implicated in the desquamation process of the skin. Their activity is tightly controlled by epidermal protease inhibitors like the lympho-epithelial Kazal-type inhibitor (LEKTI). Defects of the LEKTI-encoding gene serine protease inhibitor Kazal type (Spink)5 lead to the absence of LEKTI and result in the genodermatose Netherton syndrome, which mimics the common skin disease atopic dermatitis. Since many KLKs are expressed in human skin with KLK5 being considered as one of the most important KLKs in skin desquamation, we proposed that more inhibitors are present in human skin. Herein, we purified from human stratum corneum by HPLC techniques a new KLK5-inhibiting peptide encoded by a member of the Spink family, designated as Spink9 located on chromosome 5p33.1. This peptide is highly homologous to LEKTI and was termed LEKTI-2. Recombinant LEKTI-2 inhibited KLK5 but not KLK7, 14 or other serine proteases tested including trypsin, plasmin and thrombin. Spink9 mRNA expression was detected in human skin samples and in cultured keratinocytes. LEKTI-2 immune-expression was focally localized at the stratum granulosum and stratum corneum at palmar and plantar sites in close localization to KLK5. At sites of plantar hyperkeratosis, LEKTI-2 expression was increased. We suggest that LEKTI-2 contributes to the regulation of the desquamation process in human skin by specifically inhibiting KLK5.     

Topical Enzyme-Replacement Therapy Restores Transglutaminase 1 Activity and Corrects Architecture of Transglutaminase-1-Deficient Skin Grafts

Transglutaminase-1 (TG1)-deficient autosomal-recessive congenital ichthyosis (ARCI) is a rare and severe genetic skin disease caused by mutations in TGM1. It is characterized by collodion babies at birth, dramatically increased transepidermal water loss (TEWL), and lifelong pronounced scaling. The disease has a tremendous burden, including the problem of stigmatization. Currently, no therapy targeting the molecular cause is available, and the therapeutic situation is deplorable. In this study, we developed the basis for a causative therapy aiming at the delivery of the enzyme to the inner site of the keratinocytes’ plasma membrane. We prepared sterically stabilized liposomes with encapsulated recombinant human TG1 (rhTG1) and equipped with a highly cationic lipopeptide vector to mediate cellular uptake. The liposomes overcame the problems of insufficient cutaneous delivery and membrane penetration and provided excellent availability and activity of rhTG1 in primary keratinocytes. To demonstrate the general feasibility of this therapeutic approach in a humanized context, we used a skin-humanized mouse model. Treatment with rhTG1 liposomes resulted in considerable improvement of the ichthyosis phenotype and in normalization of the regenerated ARCI skin: in situ monitoring showed a restoration of TG1 activity, and cholesterol clefts vanished ultrastructurally. Measurement of TEWL revealed a restoration of epidermal barrier function. We regard this aspect as a major advance over available nonspecific approaches making use of, for example, retinoid creams. We conclude that this topical approach is a promising strategy for restoring epidermal integrity and barrier function and provides a causal cure for individuals with TG1 deficiency.     

Atopic Dermatitis-Like Disease and Associated Lethal Myeloproliferative Disorder Arise from Loss of Notch Signaling in the Murine Skin

Background

The Notch pathway is essential for proper epidermal differentiation during embryonic skin development. Moreover, skin specific loss of Notch signaling in the embryo results in skin barrier defects accompanied by a B-lymphoproliferative disease. However, much less is known about the consequences of loss of Notch signaling after birth.

Methodology and Principal Findings

To study the function of Notch signaling in the skin of adult mice, we made use of a series of conditional gene targeted mice that allow inactivation of several components of the Notch signaling pathway specifically in the skin. We demonstrate that skin-specific inactivation of Notch1 and Notch2 simultaneously, or RBP-J, induces the development of a severe form of atopic dermatitis (AD), characterized by acanthosis, spongiosis and hyperkeratosis, as well as a massive dermal infiltration of eosinophils and mast cells. Likewise, patients suffering from AD, but not psoriasis or lichen planus, have a marked reduction of Notch receptor expression in the skin. Loss of Notch in keratinocytes induces the production of thymic stromal lymphopoietin (TSLP), a cytokine deeply implicated in the pathogenesis of AD. The AD-like associated inflammation is accompanied by a myeloproliferative disorder (MPD) characterized by an increase in immature myeloid populations in the bone marrow and spleen. Transplantation studies revealed that the MPD is cell non-autonomous and caused by dramatic microenvironmental alterations. Genetic studies demontrated that G-CSF mediates the MPD as well as changes in the bone marrow microenvironment leading to osteopenia.

Significance

Our data demonstrate a critical role for Notch in repressing TSLP production in keratinocytes, thereby maintaining integrity of the skin and the hematopoietic system.     

Immunolocalization of the Tumor-Sensitive Calmodulin-Like Protein CALML3 in Normal Human Skin and Hyperproliferative Skin Disorders

Background and Objective

Calmodulin-like protein CALML3 is an epithelial-specific protein regulated during keratinocyte differentiation in vitro. CALML3 expression is downregulated in breast cancers and transformed cell lines making it an attractive marker for tumor formation. The objective of this study was to survey CALML3 localization in normal epidermis and in hyperproliferative skin diseases including actinic keratosis, squamous and basal cell carcinoma as well as verruca and psoriasis and to compare CALML3 immunoreactivity with the proliferation marker Ki-67.

Methods

Paraffin-embedded tissue sections from normal human skin and hyperproliferative skin disorders were examined by immunohistochemistry and analyzed for localization and expression of CALML3 and Ki-67.

Results

CALML3 was strongly expressed in differentiating layers of normal skin, staining the periphery in suprabasal cells and exhibiting nuclear localization in the stratum granulosum. CALML3 nuclear localization was inversely correlated to Ki-67 staining in each disease, indicating that CALML3 nuclear presence is related to terminal cell differentiation and postmitotic state.

Conclusions

Increased CALML3 expression in suprabasal layers is characteristic for differentiating keratinocytes in normal epidermis, and nuclear expression of CALML3 inversely correlates with expression of the proliferation marker Ki-67. This suggests that CALML3 is a useful marker for normal and benign hyperplastic epidermal development, whereas the loss of nuclear CALML3 indicates progression to a proliferative and potentially malignant phenotype.     

Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function and physiology of their in vivo tissue counterparts. This is exemplified by organotypic skin cultures which faithfully recapitulates the epidermal differentiation and stratification program. Primary human epidermal keratinocytes are genetically manipulable through retroviruses where genes can be easily overexpressed or knocked down. These genetically modified keratinocytes can then be used to regenerate human epidermis in organotypic skin cultures providing a powerful model to study genetic pathways impacting epidermal growth, differentiation, and disease progression. The protocols presented here describe methods to prepare devitalized human dermis as well as to genetically manipulate primary human keratinocytes in order to generate organotypic skin cultures. Regenerated human skin can be used in downstream applications such as gene expression profiling, immunostaining, and chromatin immunoprecipitations followed by high throughput sequencing. Thus, generation of these genetically modified organotypic skin cultures will allow the determination of genes that are critical for maintaining skin homeostasis.     

CYLD regulates keratinocyte differentiation and skin cancer progression in humans

CYLD is a gene mutated in familial cylindromatosis and related diseases, leading to the development of skin appendages tumors. Although the deubiquitinase CYLD is a skin tumor suppressor, its role in skin physiology is unknown. Using skin organotypic cultures as experimental model to mimic human skin, we have found that CYLD acts as a regulator of epidermal differentiation in humans through the JNK signaling pathway. We have determined the requirement of CYLD for the maintenance of epidermal polarity, keratinocyte differentiation and apoptosis. We show that CYLD overexpression increases keratinocyte differentiation while CYLD loss of function impairs epidermal differentiation. In addition, we describe the important role of CYLD in the control of human non-melanoma skin cancer progression. Our results show the reversion of the malignancy of human squamous cell carcinomas that express increased levels of CYLD, while its functional inhibition enhances the aggressiveness of these tumors which progress toward spindle cell carcinomas. We have found that the mechanisms through which CYLD regulates skin cancer progression include the control of tumor differentiation, angiogenesis and cell survival. These findings of the role of CYLD in human skin cancer prognosis make our results relevant from a therapeutic point of view, and open new avenues for exploring novel cancer therapies.     

Persistent Release of IL-1s from Skin Is Associated with Systemic Cardio-Vascular Disease,Emaciation and Systemic Amyloidosis: The Potential of Anti-IL-1 Therapy for Systemic Inflammatory Diseases

The skin is an immune organ that contains innate and acquired immune systems and thus is able to respond to exogenous stimuli producing large amount of proinflammatory cytokines including IL-1 and IL-1 family members. The role of the epidermal IL-1 is not limited to initiation of local inflammatory responses, but also to induction of systemic inflammation. However, association of persistent release of IL-1 family members from severe skin inflammatory diseases such as psoriasis, epidermolysis bullosa, atopic dermatitis, blistering diseases and desmoglein-1 deficiency syndrome with diseases in systemic organs have not been so far assessed. Here, we showed the occurrence of severe systemic cardiovascular diseases and metabolic abnormalities including aberrant vascular wall remodeling with aortic stenosis, cardiomegaly, impaired limb and tail circulation, fatty tissue loss and systemic amyloid deposition in multiple organs with liver and kidney dysfunction in mouse models with severe dermatitis caused by persistent release of IL-1s from the skin. These morbid conditions were ameliorated by simultaneous administration of anti-IL-1α and IL-1β antibodies. These findings may explain the morbid association of arteriosclerosis, heart involvement, amyloidosis and cachexia in severe systemic skin diseases and systemic autoinflammatory diseases, and support the value of anti-IL-1 therapy for systemic inflammatory diseases.     

Mutations in COX7B Cause Microphthalmia with Linear Skin Lesions,an Unconventional Mitochondrial Disease

Microphthalmia with linear skin lesions (MLS) is an X-linked dominant male-lethal disorder associated with mutations in holocytochrome c-type synthase (HCCS), which encodes a crucial player of the mitochondrial respiratory chain (MRC). Unlike other mitochondrial diseases, MLS is characterized by a well-recognizable neurodevelopmental phenotype. Interestingly, not all clinically diagnosed MLS cases have mutations in HCCS, thus suggesting genetic heterogeneity for this disorder. Among the possible candidates, we analyzed the X-linked COX7B and found deleterious de novo mutations in two simplex cases and a nonsense mutation, which segregates with the disease, in a familial case. COX7B encodes a poorly characterized structural subunit of cytochrome c oxidase (COX), the MRC complex IV. We demonstrated that COX7B is indispensable for COX assembly, COX activity, and mitochondrial respiration. Downregulation of the COX7B ortholog (cox7B) in medaka (Oryzias latipes) resulted in microcephaly and microphthalmia that recapitulated the MLS phenotype and demonstrated an essential function of complex IV activity in vertebrate CNS development. Our results indicate an evolutionary conserved role of the MRC complexes III and IV for the proper development of the CNS in vertebrates and uncover a group of mitochondrial diseases hallmarked by a developmental phenotype.     

Darier disease : A disease model of impaired calcium homeostasis in the skin

The importance of extracellular calcium in epidermal differentiation and intra-epidermal cohesion has been recognized for many years. Darier disease (DD) was the first genetic skin disease caused by abnormal epidermal calcium homeostasis to be identified. DD is characterized by loss of cell-to-cell adhesion and abnormal keratinization. DD is caused by genetic defects in ATP2A2 encoding the sarco/endoplasmic reticulum Ca²⁺-ATPase isoform 2 (SERCA2). SERCA2 is a calcium pump of the endoplasmic reticulum (ER) transporting Ca²⁺ from the cytosol to the lumen of ER. ATP2A2 mutations lead to loss of Ca²⁺ transport by SERCA2 resulting in decreased ER Ca²⁺ concentration in Darier keratinocytes. Here, we review the role of SERCA2 pumps and calcium in normal epidermis, and we discuss the consequences of ATP2A2 mutations on Ca²⁺ signaling in DD. This article is part of a Special Issue entitled: 11th European Symposium on Calcium.     

Scabies Mites Alter the Skin Microbiome and Promote Growth of Opportunistic Pathogens in a Porcine Model

Background

The resident skin microbiota plays an important role in restricting pathogenic bacteria, thereby protecting the host. Scabies mites (Sarcoptes scabiei) are thought to promote bacterial infections by breaching the skin barrier and excreting molecules that inhibit host innate immune responses. Epidemiological studies in humans confirm increased incidence of impetigo, generally caused by Staphylococcus aureus and Streptococcus pyogenes, secondary to the epidermal infestation with the parasitic mite. It is therefore possible that mite infestation could alter the healthy skin microbiota making way for the opportunistic pathogens. A longitudinal study to test this hypothesis in humans is near impossible due to ethical reasons. In a porcine model we generated scabies infestations closely resembling the disease manifestation in humans and investigated the scabies associated changes in the skin microbiota over the course of a mite infestation.

Methodology/Principal Findings

In a 21 week trial, skin scrapings were collected from pigs infected with S. scabies var. suis and scabies-free control animals. A total of 96 skin scrapings were collected before, during infection and after acaricide treatment, and analyzed by bacterial 16S rDNA tag-encoded FLX-titanium amplicon pyrosequencing. We found significant changes in the epidermal microbiota, in particular a dramatic increase in Staphylococcus correlating with the onset of mite infestation in animals challenged with scabies mites. This increase persisted beyond treatment from mite infection and healing of skin. Furthermore, the staphylococci population shifted from the commensal S. hominis on the healthy skin prior to scabies mite challenge to S. chromogenes, which is increasingly recognized as being pathogenic, coinciding with scabies infection in pigs. In contrast, all animals in the scabies-free cohort remained relatively free of Staphylococcus throughout the trial.

Conclusions/Significance

This is the first experimental in vivo evidence supporting previous assumptions that establishment of pathogens follow scabies infection. Our findings provide an explanation for a biologically important aspect of the disease pathogenesis. The methods developed from this pig trial will serve as a guide to analyze human clinical samples. Studies building on this will offer implications for development of novel intervention strategies against the mites and the secondary infections.     

Occupational Skin Diseases in the San Francisco Bay Area

From answers by one-third of the practicing dermatologists in the San Francisco Bay Area to a questionnaire on occupational skin diseases, contact dermatitis due to irritants and sensitizers was found to rank first. Poison oak, which is the leading reported cause on “Doctor''s First Report of Work Injury” received by the California Department of Industrial Relations, was sixth on the list of the survey, trailing solvents, cleansing agents, petroleum products and epoxy resins. A history of atopic dermatitis was often noted in current cases of occupational diseases of the skin.Avoidance of exposure or limiting the contact with pathogenic substances—through engineering changes, observation of working conditions by physicians, education of workers—appeared to be the best preventive measures.     

Genome-wide Comparative Analysis of Atopic Dermatitis and Psoriasis Gives Insight into Opposing Genetic Mechanisms

Atopic dermatitis and psoriasis are the two most common immune-mediated inflammatory disorders affecting the skin. Genome-wide studies demonstrate a high degree of genetic overlap, but these diseases have mutually exclusive clinical phenotypes and opposing immune mechanisms. Despite their prevalence, atopic dermatitis and psoriasis very rarely co-occur within one individual. By utilizing genome-wide association study and ImmunoChip data from >19,000 individuals and methodologies developed from meta-analysis, we have identified opposing risk alleles at shared loci as well as independent disease-specific loci within the epidermal differentiation complex (chromosome 1q21.3), the Th2 locus control region (chromosome 5q31.1), and the major histocompatibility complex (chromosome 6p21–22). We further identified previously unreported pleiotropic alleles with opposing effects on atopic dermatitis and psoriasis risk in PRKRA and ANXA6/TNIP1. In contrast, there was no evidence for shared loci with effects operating in the same direction on both diseases. Our results show that atopic dermatitis and psoriasis have distinct genetic mechanisms with opposing effects in shared pathways influencing epidermal differentiation and immune response. The statistical analysis methods developed in the conduct of this study have produced additional insight from previously published data sets. The approach is likely to be applicable to the investigation of the genetic basis of other complex traits with overlapping and distinct clinical features.     

Discrimination Between Paraffin-Embedded and Frozen Skin Sections Using Synchrotron Infrared Microspectroscopy

The difference between paraffin-embedded and frozen skin sections is always questionable. Ten patients of early stage mycosis fungoides, ten patients with psoriasis and ten normal controls were included in this study. Aim of this study is to differentiate between paraffin-embedded and frozen skin sections in inflammatory and malignant dermatoses using synchrotron infrared microspectroscopy (SIRM). It was found that epidermal beta sheets in paraffin-embedded sections were higher in a highly significant manner than frozen sections (P < 0.001). Also, epidermal nucleic acids in paraffin-embedded sections were lower in a highly significant manner than frozen sections (P < 0.001). However, when various skin diseases were compared with the control. It was found that the difference between paraffin-embedded and frozen skin sections were almost similar. In conclusion SIRM is a unique promising diagnostic technique and it seems that frozen processing preserve skin tissue more, this was represented by less apoptosis (beta sheets) and more nucleic acids than paraffin processing. However, there are still many advantages of both approaches over the other depending on the goal of the study.