The Prolyl Endopeptidase PREP Is Involved in Filaggrinolysis and Cornification.

Filaggrin (FLG) is a well-known biomarker of atopic dermatitis and skin dryness. Its full proteolysis (or filaggrinolysis) produces the major constituents of the natural moisturizing factor. Some proteases/peptidases remain to be identified in this multistep process. Mining 16 omics analyses, we identified prolyl endopeptidase (PREP) as a candidate peptidase. Indirect immunofluorescence and confocal analysis demonstrated its localization in the granular and deep cornified layers, where it co-localized with FLG. Tandem mass spectroscopy and fluorescent quenching activity assays showed that PREP cleaved several synthetic peptides derived from the FLG sequence, at the carboxyl side of an internal proline. Deimination of these peptides increased PREP enzymatic efficiency. Specific inhibition of PREP in reconstructed human epidermis (RHEs) using benzyloxycarbonyl-Pro-Prolinal (ZPP) induced the accumulation of FLG monomers. Down-regulation of PREP expression in RHEs using RNA interference confirmed the impact of PREP on FLG metabolism, and highlighted a more general role of PREP in keratinocyte differentiation. Indeed, quantitative global proteomic, Western blotting and RT-qPCR analyses showed a strong reduction in the expression of bleomycin hydrolase, known to be involved in filaggrinolysis, and of several other actors of cornification like loricrin. Consequently, at the functional level, the trans-epidermal electric resistance was drastically reduced.

Human filaggrin monomer does not seem to be a proteasome target.

Absence of a functional proteasome in the suprabasal layers of the epidermis is responsible for keratosis linearis with ichthyosis congenital and sclerosing keratoderma syndrome. Patient epidermis shows hypergranulosis associated with abnormally shaped keratohyalin granules and abnormal distribution of filaggrin in the Stratum granulosum and Stratum corneum. This suggests that the proteasome is involved in the degradation of filaggrin. To test this hypothesis, the proteasome proteolytic activity was inhibited in 3D reconstructed human epidermis (RHE) with the specific clasto-lactacystin ß-lactone inhibitor. Confirming the efficacy of inhibition, ubiquitinated proteins accumulated in treated RHEs as compared to controls. Levels of urocanic acid (UCA) and pyrrolidone carboxylic acid (PCA), the end products of filaggrin degradation, were reduced. However, neither filaggrin accumulation nor appearance of filaggrin-derived peptides were observed. On the contrary, the amount of filaggrin was shown to decrease, and a similar tendency was observed for profilaggrin, its precursor. Accumulation of small cytoplasmic vesicles associated with a significant increase in autophagy markers indicated activation of the autophagy process upon proteasome inhibition. Taken together, these results suggest that the perturbation of UCA and PCA production after proteasome inhibition was probably due to down-regulation of filaggrin expression rather than to blocking of filaggrin proteolysis.

Deimination in epidermal barrier and hair formation.

Peptidylarginine deiminases (PADs) transform a protein arginine residue into the non-standard amino acid citrulline. This calcium-dependent post-translational modification of proteins is called citrullination or deimination. As described in this special issue, PADs play a role in various physiological processes, and PAD deregulations are involved in many human diseases. Three PADs are expressed in the epidermis, where their roles begin to be deciphered. PAD1 and PAD3 are involved in keratinocyte differentiation, particularly in the epidermal barrier function, keratins, filaggrin and filaggrin-related proteins being the most abundant deiminated epidermal proteins. Reduced amounts of deiminated proteins and PAD1 expression may be involved in the pathogenesis of psoriasis and atopic dermatitis, two very frequent and chronic skin inflammatory diseases. The trichohyalin/PAD3/transglutaminase three pathway is important for hair shaft formation. Mutations of the PADI3 gene, leading to a decreased activity or abnormal localization of the corresponding isotype, are the cause of a rare hair disorder called uncombable hair syndrome, and are associated with the central centrifugal cicatricial alopecia, a frequent alopecia mainly affecting women of African ancestry. This article is part of the Theo Murphy meeting issue 'The virtues and vices of protein citrullination'.

Down-regulation of peptidylarginine deiminase type 1 in reconstructed human epidermis disturbs nucleophagy in the granular layer and affects barrier function.

Deimination is a post-translational modification catalyzed by a family of enzymes named peptidylarginine deiminases (PADs). PADs transform arginine residues of protein substrates into citrulline. Deimination has been associated with numerous physiological and pathological processes. In human skin, three PADs are expressed (PAD1-3). While PAD3 is important for hair shape formation, the role of PAD1 is less clear. To decipher the main role(s) of PAD1 in epidermal differentiation, its expression was down-regulated using lentivirus-mediated shRNA interference in primary keratinocytes and in three-dimensional reconstructed human epidermis (RHE). Compared to normal RHEs, down-regulation of PAD1 caused a drastic reduction in deiminated proteins. Whereas proliferation of keratinocytes was not affected, their differentiation was disturbed at molecular, cellular and functional levels. The number of corneocyte layers was significantly reduced, expression of filaggrin and cornified cell envelope components, such as loricrin and transglutaminases, was down-regulated, epidermal permeability increased and trans-epidermal-electric resistance diminished drastically. Keratohyalin granule density decreased and nucleophagy in the granular layer was disturbed. These results demonstrate that PAD1 is the main regulator of protein deimination in RHE. Its deficiency alters epidermal homeostasis, affecting the differentiation of keratinocytes, especially the cornification process, a special kind of programmed cell death.

Targeting TGF-ß1/miR-21 Pathway in Keratinocytes Reveals Protective Effects of Silymarin on Imiquimod-Induced Psoriasis Mouse Model.

Epidermal cells integrate multiple signals that activate the signaling pathways involved in skin homeostasis. TGF-ß1 signaling pathway upregulates microRNA (miR)-21-5p in keratinocytes and is often deregulated in skin diseases. To identify the bioactive compounds that enable to modulate the TGF-ß1/miR-21-5p signaling pathway, we screened a library of medicinal plant extracts using our miR-ON RILES luciferase reporter system placed under the control of the miR-21-5p in keratinocytes treated with TGF-ß1. We identified silymarin, a mixture of flavonolignans extracted from Silybum marianum (L.) Gaertn., as the most potent regulator of miR-21-5p expression. Using Argonaute 2 immunoprecipitation and RT-qPCR, we showed that silymarin regulates the expression of miR-21-5p through a noncanonical TGF-ß1 signaling pathway, whereas RNA-sequencing analysis revealed three unexpected transcriptomic signatures associated with keratinocyte differentiation, cell cycle, and lipid metabolism. Mechanistically, we demonstrated that SM blocks cell cycle progression, inhibits keratinocyte differentiation through repression of Notch3 expression, stimulates lipid synthesis via activation of PPARγ signaling and inhibits inflammatory responses by suppressing the transcriptional activity of NF-κB. We finally showed that topical application of silymarin alleviates the development of imiquimod-induced psoriasiform lesions in mice by abrogating the altered expression levels of markers involved in inflammation, proliferation, differentiation, and lipid metabolism.

An Inflamed and Infected Reconstructed Human Epidermis to Study Atopic Dermatitis and Skin Care Ingredients.

Atopic dermatitis (AD), the most common inflammatory skin disorder, is a multifactorial disease characterized by a genetic predisposition, epidermal barrier disruption, a strong T helper (Th) type 2 immune reaction to environmental antigens and an altered cutaneous microbiome. Microbial dysbiosis characterized by the prevalence of Staphylococcus aureus (S. aureus) has been shown to exacerbate AD. In recent years, in vitro models of AD have been developed, but none of them reproduce all of the pathophysiological features. To better mimic AD, we developed reconstructed human epidermis (RHE) exposed to a Th2 pro-inflammatory cytokine cocktail and S. aureus. This model well reproduced some of the vicious loops involved in AD, with alterations at the physical, microbial and immune levels. Our results strongly suggest that S. aureus acquired a higher virulence potential when the epidermis was challenged with inflammatory cytokines, thus later contributing to the chronic inflammatory status. Furthermore, a topical application of a Castanea sativa extract was shown to prevent the apparition of the AD-like phenotype. It increased filaggrin, claudin-1 and loricrin expressions and controlled S. aureus by impairing its biofilm formation, enzymatic activities and inflammatory potential.

Corneal epithelium in keratoconus underexpresses active NRF2 and a subset of oxidative stress-related genes.

Keratoconus (KC) is a multifactorial progressive ectatic disorder characterized by local thinning of the cornea, leading to decreased visual acuity due to irregular astigmatism and opacities. Despite the evolution of advanced imaging methods, the exact etiology of KC remains unknown. Our aim was to investigate the involvement of corneal epithelium in the pathophysiology of the disease. Corneal epithelial samples were collected from 23 controls and from 2 cohorts of patients with KC: 22 undergoing corneal crosslinking (early KC) and 6 patients before penetrating keratoplasty (advanced KC). The expression of genes involved in the epidermal terminal differentiation program and of the oxidative stress pathway was assessed by real time PCR analysis. Presence of some of the differentially expressed transcripts was confirmed at protein level using immunofluorescence on controls and advanced KC additional corneal samples. We found statistically significant under-expression in early KC samples of some genes known to be involved in the mechanical resistance of the epidermis (KRT16, KRT14, SPRR1A, SPRR2A, SPRR3, TGM1 and TGM5) and in oxidative stress pathways (NRF2, HMOX1 and HMOX2), as compared to controls. In advanced KC samples, expression of SPRR2A and HMOX1 was reduced. Decreased expression of keratin (KRT)16 and KRT14 proteins was observed. Moreover, differential localization was noted for involucrin, another protein involved in the epidermis mechanical properties. Finally, we observed an immunofluorescence staining for the active form of NRF2 in control epithelia that was reduced in KC epithelia. These results suggest a defect in the mechanical resistance and the oxidative stress defense possibly mediated via the NRF2 pathway in the corneal keratoconic epithelium.

Cold Atmospheric Plasma Jet Treatment Improves Human Keratinocyte Migration and Wound Closure Capacity without Causing Cellular Oxidative Stress.

Cold Atmospheric Plasma (CAP) is an emerging technology with great potential for biomedical applications such as sterilizing equipment and antitumor strategies. CAP has also been shown to improve skin wound healing in vivo, but the biological mechanisms involved are not well known. Our study assessed a possible effect of a direct helium jet CAP treatment on keratinocytes, in both the immortalized N/TERT-1 human cell line and primary keratinocytes obtained from human skin samples. The cells were covered with 200 µL of phosphate buffered saline and exposed to the helium plasma jet for 10−120 s. In our experimental conditions, micromolar concentrations of hydrogen peroxide, nitrite and nitrate were produced. We showed that long-time CAP treatments (≥60 s) were cytotoxic, reduced keratinocyte migration, upregulated the expression of heat shock protein 27 (HSP27) and induced oxidative cell stress. In contrast, short-term CAP treatments (<60 s) were not cytotoxic, did not affect keratinocyte proliferation and differentiation, and did not induce any changes in mitochondria, but they did accelerate wound closure in vitro by improving keratinocyte migration. In conclusion, these results suggest that helium-based CAP treatments improve wound healing by stimulating keratinocyte migration. The study confirms that CAP could be a novel therapeutic method to treat recalcitrant wounds.

Assessment of the Genetic Spectrum of Uncombable Hair Syndrome in a Cohort of 107 Individuals.

Importance: Uncombable hair syndrome (UHS) is a rare hair shaft anomaly that manifests during infancy and is characterized by dry, frizzy, and wiry hair that cannot be combed flat. Only about 100 known cases have been reported so far. Objective: To elucidate the genetic spectrum of UHS. Design, Setting, and Participants: This cohort study includes 107 unrelated index patients with a suspected diagnosis of UHS and family members who were recruited worldwide from January 2013 to December 2021. Participants of all ages, races, and ethnicities were recruited at referral centers or were enrolled on their own initiative following personal contact with the authors. Genetic analyses were conducted in Germany from January 2014 to December 2021. Main Outcomes and Measures: Clinical photographs, Sanger or whole-exome sequencing and array-based genotyping of DNA extracted from blood or saliva samples, and 3-dimensional protein modeling. Descriptive statistics, such as frequency counts, were used to describe the distribution of identified pathogenic variants and genotypes. Results: The genetic characteristics of patients with UHS were established in 80 of 107 (74.8%) index patients (82 [76.6%] female) who carried biallelic pathogenic variants in PADI3, TGM3, or TCHH (ie, genes that encode functionally related hair shaft proteins). Molecular genetic findings from 11 of these 80 individuals were previously published. In 76 (71.0%) individuals, the UHS phenotype were associated with pathogenic variants in PADI3. The 2 most commonly observed PADI3 variants account for 73 (48.0%) and 57 (37.5%) of the 152 variant PADI3 alleles in total, respectively. Two individuals carried pathogenic variants in TGM3, and 2 others carried pathogenic variants in TCHH. Haplotype analyses suggested a founder effect for the 4 most commonly observed pathogenic variants in the PADI3 gene. Conclusions and Relevance: This cohort study extends and gives an overview of the genetic variant spectrum of UHS based on molecular genetic analyses of the largest worldwide collective of affected individuals, to our knowledge. Formerly, a diagnosis of UHS could only be made by physical examination of the patient and confirmed by microscopical examination of the hair shaft. The discovery of pathogenic variants in PADI3, TCHH, and TGM3 may open a new avenue for clinicians and affected individuals by introducing molecular diagnostics for UHS.

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.

Dupilumab-Associated Adverse Events During Treatment of Allergic Diseases.

Among the new biological therapies for atopic diseases, dupilumab is a fully human monoclonal antibody directed against IL-4Rα, the common chain of interleukin-4 and interleukin-13 receptors. Dupilumab showed clinical improvements in patients with atopic dermatitis, asthma, and chronic rhinosinusitis and is currently under development for other indications. While dupilumab is considered to be well tolerated, a number of recent publications have reported various adverse events. This review aims to summarize the current knowledge about these adverse events, which may help clinicians to improve the follow-up of patients on dupilumab. Injection-site reactions are the most common reported adverse event. However, dupilumab has also been shown to cause ophthalmic complications (e.g., dry eyes, conjunctivitis, blepharitis, keratitis, and ocular pruritus), head and neck dermatitis, onset of psoriatic lesions, progression of cutaneous T-cell lymphoma exacerbation, alopecia areata, hypereosinophilia, and arthritis. Most are managed during dupilumab treatment continuation, but some (e.g., severe conjunctivitis) may result in a discontinuation of treatment. Their molecular origin is unclear and requires further investigations. Among other hypothesis, it has been suggested that T helper (Th)2-mediated pathway inhibition may worsen Th1/Th17-dependent immune responses. An ophthalmological examination for the presence of potential predictive indicators of ophthalmic adverse events is recommended before initiation of dupilumab therapy.

IL-22 Downregulates Peptidylarginine Deiminase-1 in Human Keratinocytes: Adding Another Piece to the IL-22 Puzzle in Epidermal Barrier Formation.

Increased presence of IL-22+ cells in the skin is a characteristic finding in skin barrier defects, such as psoriasis and atopic dermatitis. However, mechanistic insight into effects of IL-22 on epidermal functioning is yet to be elucidated. One crucial step during epidermal differentiation is deimination or citrullination. Here, we show reduced levels of peptidylarginine deiminase 1, an enzyme that converts peptidylarginine into citrulline in lesional psoriatic skin. IL-22 signaling through the IL-22 receptor complex was found to suppress expression of peptidylarginine deiminase 1 in epidermal keratinocytes. Subsequently, total peptidylarginine deiminase activity and extent of protein deimination in keratinocytes treated with IL-22 were reduced together with a significant decrease in deimination of keratin 1 and FLG, both important for epidermal differentiation. Vitamin D and acitretin partly restored the peptidylarginine deiminase 1 defect caused by IL-22. Collectively, we show that IL-22 downregulates deimination, thus identifying a potential target for treatment of skin barrier defects.

Cold helium plasma jet does not stimulate collagen remodeling in a 3D human dermal substitute.

Cold Atmospheric Plasma (CAP) is an emerging physical approach displaying encouraging antitumor and wound healing effects both in vitro and in vivo. In this study, we assessed the potential of direct CAP to remodel skin collagens using an original tissue-engineered human dermal substitute model rich in endogenous extracellular matrix (ECM) covered with 600 µl of culture medium and treated with CAP for 30 and 120 s. Our results indicated that Reactive Oxygen and Nitrogen Species (RONS) such as H2O2, NO3- and NO2- were produced in the medium during treatment. It appeared that in the CAP-treated dermal substitutes 1) cell viability was not altered, 2) pro-collagen I secretion was not modified over 48 h of culture after treatment, 3) global activity of matrix metalloproteinases MMPs was not modulated over 48 h after treatment, and 4) no change in hydroxyproline content was observed over 5 days after treatment. In order to confirm the efficiency of our device, we showed that the plasma-activated culture medium induced cell apoptosis and growth delay using a 3D human tumor spheroid model. In conclusion, no effect of direct CAP treatment was monitored on dermal ECM production and degradation, indicating that CAP does not stimulate collagen remodeling at the tissue scale.

The upper epidermis of atopic dogs is altered at the functional and structural levels.

BACKGROUND: The pathogenesis of human atopic dermatitis (AD) is complex. Like humans, dogs develop spontaneous AD so this species could be a useful model of study. However, AD has been less characterised in dogs than in humans. OBJECTIVES: To compare the epidermis of normal and spontaneously atopic dogs at the functional and structural levels. ANIMALS: Six healthy and five atopic laboratory Beagle dogs. METHODS AND MATERIALS: Dogs were clinically characterised by general examination, Canine Atopic Dermatitis Extent and Severity Index, 4th iteration (CADESI-04) evaluation and trans-epidermal water loss (TWEL) measurement. Skin biopsies were taken from healthy skin from normal dogs and on nonlesional and lesional skin from atopic dogs. Samples were analysed using transmission electron microscopy (TEM). Cornified envelopes were extracted and examined for their visual aspects (smooth versus ruffled). RESULTS: CADESI-04 and TWEL were significantly higher in atopic dogs. Healthy and nonlesional skin could be distinguished from lesional skin by histopathological evaluation. TEM examination revealed abnormal morphology of the stratum corneum (SC) in atopic skin. The SC compactum corneocyte layer was larger. Thicker and wrinkled corneocytes were more prominent (P = 0.005) in the lesional skin. Similar changes were observed in the nonlesional skin, but less pronounced. The proportion of immature ruffled envelopes was increased in atopic samples (P < 0.05), both from lesional and nonlesional areas. CONCLUSIONS: The morphology of the SC was altered in the lesional and apparently nonlesional skin of spontaneously atopic dogs.

Atopic dermatitis: Role of the skin barrier, environment, microbiome, and therapeutic agents.

Atopic dermatitis (AD) is a chronic, inflammatory skin disorder characterized by eczematous and pruritic skin lesions. In recent decades, the prevalence of AD has increased worldwide, most notably in developing countries. The enormous progress in our understanding of the complex composition and functions of the epidermal barrier allows for a deeper appreciation of the active role that the skin barrier plays in the initiation and maintenance of skin inflammation. The epidermis forms a physical, chemical, immunological, neuro-sensory, and microbial barrier between the internal and external environment. Not only lesional, but also non-lesional areas of AD skin display many morphological, biochemical and functional differences compared with healthy skin. Supporting this notion, genetic defects affecting structural proteins of the skin barrier, including filaggrin, contribute to an increased risk of AD. There is evidence to suggest that natural environmental allergens and man-made pollutants are associated with an increased likelihood of developing AD. A compromised epidermal barrier predisposes the skin to increased permeability of these compounds. Numerous topical and systemic therapies for AD are currently available or in development; while anti-inflammatory therapy is central to the treatment of AD, some existing and novel therapies also appear to exert beneficial effects on skin barrier function. Further research on the skin barrier, particularly addressing epidermal differentiation and inflammation, lipid metabolism, and the role of bacterial communities for skin barrier function, will likely expand our understanding of the complex etiology of AD and lead to identification of novel targets and the development of new therapies.

Deimination, Intermediate Filaments and Associated Proteins.

Deimination (or citrullination) is a post-translational modification catalyzed by a calcium-dependent enzyme family of five peptidylarginine deiminases (PADs). Deimination is involved in physiological processes (cell differentiation, embryogenesis, innate and adaptive immunity, etc.) and in autoimmune diseases (rheumatoid arthritis, multiple sclerosis and lupus), cancers and neurodegenerative diseases. Intermediate filaments (IF) and associated proteins (IFAP) are major substrates of PADs. Here, we focus on the effects of deimination on the polymerization and solubility properties of IF proteins and on the proteolysis and cross-linking of IFAP, to finally expose some features of interest and some limitations of citrullinomes.

Update on canine filaggrin: a review.

Human filaggrin (FLG) plays a key role in epidermal barrier function, and loss-of-function mutations of its gene are primarily responsible for the development of human atopic dermatitis (AD). FLG expression is also reduced in the epidermis of atopic patients, due to the transcriptional effect of Th2 type cytokines. Canine atopic dermatitis (CAD) is a prevalent skin disease that shares many clinical and pathogenic features with its human homologue. The aim of this review is discuss current knowledge on canine filaggrin (Flg) in both healthy and atopic dogs, as compared to the human protein. Although the molecular structures of the two proteins, as deduced from the sequences of their gene, are different, their sites of expression and their proteolytic processing in the normal epidermis are similar. Concerning the expression of Flg in CAD, conflicting results have been published at the mRNA level and little accurate information is available at the protein level. It derives from a large precursor, named profilaggrin (proFLG), formed by several FLG units and stored in keratohyalin granules of the stratum granulosum. Canine and human proFLG sequences display little amino acid similarity (33% as shown using the Basic Local Alignment Search Tool (BLAST)) except at the level of the S100 homologous part of the N-terminus (75%). Genetic studies in the dog are at an early stage and are limited by the variety of breeds and the small number of cases included. Many questions remain unanswered about the involvement of Flg in CAD pathogenesis.