Aberrant serine protease activities in atopic dermatitis.

Atopic dermatitis (AD) is a chronic inflammatory skin disease; the three major factors responsible for AD, i.e., epidermal barrier dysfunction, allergic inflammation, and itching, interact with each other to form a pathological condition. Excessive protease activities are characteristic abnormalities that affect the epidermal barrier in patients with AD. In normal skin, epidermal serine protease activities are controlled by kallikrein-related peptidases (KLKs) and their inhibitors, including lympho-epithelial Kazal-type-related inhibitor (LEKTI). In AD lesions, KLKs are excessively expressed, which results in the enhancement of epidermal serine protease activities and facilitates the invasion by allergens and microorganisms. In addition, some KLKs can activate protease-activated receptor 2 (PAR2) in epidermal keratinocytes and peripheral nerves, resulting in the induction of inflammation and itching. Furthermore, in AD patients with single nucleotide polymorphism (SNP) such as E420K and D386N of SPINK5 which encodes LEKTI, LEKTI function is attenuated, resulting in the activation of KLKs and easy invasion by allergens and microorganisms. Further analysis is needed to elucidate the detailed mechanism underlying the control of serine protease activities, which may lead to the development of new therapeutic and prophylactic agents for AD.

Sphingomyelin Deacylase, the Enzyme Involved in the Pathogenesis of Atopic Dermatitis, Is Identical to the ß-Subunit of Acid Ceramidase.

A ceramide deficiency in the stratum corneum (SC) is an essential etiologic factor for the dry and barrier-disrupted skin of patients with atopic dermatitis (AD). Previously, we reported that sphingomyelin (SM) deacylase, which hydrolyzes SM and glucosylceramide at the acyl site to yield their lysoforms sphingosylphosphorylcholine (SPC) and glucosylsphingosine, respectively, instead of ceramide and/or acylceramide, is over-expressed in AD skin and results in a ceramide deficiency. Although the enzymatic properties of SM deacylase have been clarified, the enzyme itself remains unidentified. In this study, we purified and characterized SM deacylase from rat skin. The activities of SM deacylase and acid ceramidase (aCDase) were measured using SM and ceramide as substrates by tandem mass spectrometry by monitoring the production of SPC and sphingosine, respectively. Levels of SM deacylase activity from various rat organs were higher in the order of skin > lung > heart. By successive chromatography using Phenyl-5PW, Rotofor, SP-Sepharose, Superdex 200 and Shodex RP18-415, SM deacylase was purified to homogeneity with a single band of an apparent molecular mass of 43 kDa with an enrichment of > 14,000-fold. Analysis by MALDI-TOF MS/MS using a protein spot with SM deacylase activity separated by 2D-SDS-PAGE allowed its amino acid sequence to be determined and identified as the ß-subunit of aCDase, which consists of α- and ß-subunits linked by amino bonds and a single S-S bond. Western blotting of samples treated with 2-mercaptoethanol revealed that, whereas recombinant human aCDase was recognized by antibodies to the α-subunit at ~56 kDa and ~13 kDa and the ß-subunit at ~43 kDa, the purified SM deacylase was detectable only by the antibody to the ß-subunit at ~43 kDa. Breaking the S-S bond of recombinant human aCDase with dithiothreitol elicited the activity of SM deacylase with ~40 kDa upon gel chromatography. These results provide new insights into the essential role of SM deacylase expressed as an aCDase-degrading ß-subunit that evokes the ceramide deficiency in AD skin.

Social defeat stress exacerbates atopic dermatitis through downregulation of DNA methyltransferase 1 and upregulation of C-C motif chemokine receptor 7 in skin dendritic cells.

DNA methylation is an epigenetic modification that regulates gene transcription. DNA methyltransferase 1 (DNMT1) plays an important role in DNA methylation. However, the involvement of DNMT1 and DNA methylation in the pathogenesis of atopic dermatitis (AD) remains unclear. In this study, microarray analysis revealed that peripheral blood mononuclear cells of AD patients with low DNMT1 expression (DNMT1-low) highly expressed dendritic cell (DC) activation-related genes. Also, DNMT1-low AD patients exhibited a higher itch score compared to AD patients with high DNMT1 expression (DNMT1-high). By using an AD-like mouse model induced by the application of Dermatophagoides farinae body ointment, we found that Dnmt1 expression was decreased, while the expression of C-C chemokine receptor type 7 (Ccr7) was upregulated in mouse skin DCs. Furthermore, mice exposed to social defeat stress exhibited Dnmt1 downregulation and Ccr7 upregulation in skin DCs. Additionally, dermatitis and itch-related scratching behavior were exacerbated in AD mice exposed to stress. The relationship between low DNMT1 and itch induction was found in both human AD patients and AD mice. In mouse bone marrow-derived DCs, Ccr7 expression was inhibited by 5-aza-2-deoxycytidine, a methylation inhibitor. Furthermore, in mouse skin DCs, methylation of CpG sites in Ccr7 was modified by either AD induction or social defeat stress. Collectively, these findings suggest that social defeat stress exacerbates AD pathology through Dnmt1 downregulation and Ccr7 upregulation in mouse skin DCs. The data also suggest a role of DNMT1 downregulation in the exacerbation of AD pathology.

Pseudoephedrine alleviates atopic dermatitis-like inflammatory responses in vivo and in vitro.

AIMS: Atopic dermatitis is a chronic inflammatory disease characterized by eczematous lesions and has become a serious health problem worldwide. Pseudoephedrine (PSE) is a nasal decongestant to treat the common cold. PSE has been reported that is beneficial to allergic diseases. However, whether PSE has the potential in atopic dermatitis remains to be elucidated. MAIN METHODS: Male BALB/c mice were challenged with 2,4-dinitrochlorobenzene (DNCB) to induce atopic dermatitis-like lesion and orally administrated with PSE for two weeks. The skin hydration and the scratching behavior were detected. The skin lesions and histopathological changes were evaluated and inflammatory factors levels were detected. Human Keratinocytes (HaCaT cells) were stimulated by TNF-α/IFN-γ after PSE-pretreatment. The transcriptions of inflammatory factors were detected. KEY FINDINGS: PSE decreased skin lesion area and skin thickness in atopic dermatitis mice. PSE improved skin hydration and scratching. Histologically, PSE reduced mast cell and CD4+ cell infiltration. PSE suppressed serum TNF-α and IgE levels, reducing cytokines (IL-1ß, IL-4, IL-6, IL-13, IL-33, TSLP, and IL-23) and neutrophil migration factors (CCL2 and MMP-9) in skin tissues. In addition, PSE inhibited TNF-α/IFN-γ-induced release of inflammatory factors (TNF-α, IL-1ß, and IL-23) in HaCaT cells. Furthermore, PSE suppressed the activation of MAPKs and NF-κB signaling pathways in vivo and in vitro. SIGNIFICANCE: These results demonstrate that PSE could inhibit inflammatory responses in atopic dermatitis models. PSE may serve as a viable alternatives drug for the treatment of atopic dermatitis.

Baricitinib: therapeutic potential for moderate to severe atopic dermatitis.

INTRODUCTION: Atopic dermatitis (AD) is a chronic inflammatory skin disease mediated by multiple signals including janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway. Current therapeutic armamentarium consists of a limited number of drugs which may result in the insufficient management of AD. Preclinical evidence regarding inhibition of JAK/STAT led to the development of a promising class of therapeutics, namely, JAK inhibitors. Baricitinib, a novel JAK1/JAK2 inhibitor is currently under investigation in AD clinical trials. AREAS COVERED: This review offers an overview of Baricitinib and examines clinical efficacy and safety data in patients with moderate-to-severe AD. EXPERT OPINION: Baricitinib showed promising preliminary data in terms of efficacy in phase II and III trials, with a very rapid onset of response and great improvements of itch and sleep disturbances. These aforementioned aspects combined with the advantage of an oral formulation have reduced drug production costs compared to biologic agents and could lead to consideration of baricitinib as a first line systemic treatment. Also, in some countries, it could be a therapeutic option in the case of contraindication or failure of conventional systemic drugs prior to biologic therapies. Data related to long-term safety and efficacy will be important to refine the place-in-therapy of this drug.

Multifaceted Analyses of Epidermal Serine Protease Activity in Patients with Atopic Dermatitis.

The serine proteases kallikrein-related peptidase (KLK) 5 and KLK7 cleave cell adhesion molecules in the epidermis. Aberrant epidermal serine protease activity is thought to play an important role in the pathogenesis of atopic dermatitis (AD). We collected the stratum corneum (SC) from healthy individuals (n = 46) and AD patients (n = 63) by tape stripping and then measuring the trypsin- and chymotrypsin-like serine protease activity. We also analyzed the p.D386N and p.E420K of SPINK5 variants and loss-of-function mutations of FLG in the AD patients. The serine protease activity in the SC was increased not only in AD lesions but also in non-lesions of AD patients. We found, generally, that there was a positive correlation between the serine protease activity in the SC and the total serum immunoglobulin E (IgE) levels, serum thymus and activation-regulated chemokine (TARC) levels, and peripheral blood eosinophil counts. Moreover, the p.D386N or p.E420K in SPINK5 and FLG mutations were not significantly associated with the SC's serine protease activity. Epidermal serine protease activity was increased even in non-lesions of AD patients. Such activity was found to correlate with a number of biomarkers of AD. Further investigations of serine proteases might provide new treatments and prophylaxis for AD.

Korean red ginseng water extract alleviates atopic dermatitis-like inflammatory responses by negative regulation of mitogen-activated protein kinase signaling pathway in vivo.

Atopic dermatitis (AD) is a chronic inflammatory skin disease. Korean red ginseng is a Korean traditional medicine. In this study, we estimated the effects of Korean red ginseng water extract (RGE) in the 1-chloro-2,4-dinitrobenzene (DNCB)-induced BALB/c mouse model which develops AD-like lesions. After RGE administration (100, 200, and 400 mg/kg) to DNCB-induced mice there were improvements in the dermatitis score and skin pH, a decrease in trans-epidermal water loss, and improved skin hydration. RGE also significantly inhibited eosinophil infiltration, increased filaggrin protein levels, and decreased serum IgE levels, epidermal thickness, mast cell infiltration, and ceramidase release. Compared with that in DNCB-induced mice, RGE effectively decreased the mRNA expression levels of interleukin-6 (IL-6), thymic stromal lymphopoietin (TSLP), and tumor necrosis factor-α (TNF-α), as well as the protein level of thymus and activation-regulated chemokine (TARC). These inhibitory RGE effects are mediated by inhibiting the phosphorylation of mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 MAPK. Furthermore, we confirmed that RGE suppresses interferon-γ (IFN-γ) and TNF-α-induced expression of macrophage-derived chemokine (MDC) and TARC genes in human keratinocyte (HaCaT) cells. Taken together, these results demonstrate that RGE may exert anti-atopic related to responses by suppression the expression of inflammatory mediators, cytokines, and chemokines via downregulation of MAPK signaling pathways, suggesting that RGE may be an effective therapeutic approach for prevention of AD-like disease.

The Role of Kallikrein-Related Peptidases in Atopic Dermatitis.

Excessive protease activity is a characteristic abnormality that affects the epidermal barrier in patients with atopic dermatitis (AD). Kallikrein-related peptidases (KLKs) are excessively expressed in AD lesions, and it is suggested that the abnormal action of KLKs is involved in the skin barrier dysfunction in AD. In other words, overexpressed KLKs disrupt the normal barrier function, and due to that breakdown, external substances that can become antigens of AD easily invade the epidermis, resulting in dermatitis, coupled with the induction of Th2 cytokines. Further investigations are required to elucidate the role of KLKs in AD; this knowledge could contribute to the design of new therapeutic and prophylactic drugs for AD.

Anticancer agent ABT-737 possesses anti-atopic dermatitis activity via blockade of caspase-1 in atopic dermatitis in vitro and in vivo models.

OBJECTIVE: Previous studies reported that depletion of Bcl-2 has a protective effect against allergic diseases. Furthermore, recently our study showed that anticancer drug has antiallergic inflammatory effect. An anticancer agent ABT-737 is an inhibitor of Bcl-2 and has an anti-inflammatory effect. However, the antiallergic inflammatory activity of ABT-737 is still unknown. Here, we aimed to explore the anti-atopic dermatitis (AD) activity and the mechanism of ABT-737 in AD models. MATERIALS AND METHODS: HaCaT cells were used for in vitro experiments. To evaluate the effect of ABT-737 in vivo model, BalB/c mice were orally administered ABT-737 for 6 weeks in 2,4-dinitrofluorobenzene (DNFB)-induced AD-like murine model. Major assays were enzyme-linked immunosorbent assay, reverse transcription-PCR, caspase-1 assay, histamine assay, and H&E staining. RESULTS: ABT-737 significantly decreased thymic stromal lymphopoietin (TSLP) secretion and caspase-1 activity in activated HaCaT cells. In DNFB-induced AD mice, oral administration of ABT-737 alleviated clinical severity and scratching behavior. ABT-737 decreased levels of AD-related biomarkers including IgE, histamine, TSLP, and inflammatory cytokines. In addition, ABT significantly reduced caspase-1 activity in skin lesions of AD mice. DISCUSSION AND CONCLUSIONS: ABT-737 elicited an anti-AD activity via suppression of caspase-1 activation in AD in vitro and in vivo models. Therefore, this study provides important information regarding the use of anticancer drugs for controlling allergic inflammatory diseases.

Inflammatory cytokine-mediated induction of serine racemase in atopic dermatitis.

Serine racemase (SR) is an enzyme that catalyses the synthesis of d-serine, an endogenous coagonist for N-methyl-D-aspartate (NMDA)-type glutamate receptor in the central nervous system. Our previous study demonstrated that SR was expressed in the epidermis of wild-type (WT) mice but not in SR knockout (KO) mice. In addition, SR immune-reactivity was only found in the granular and cornified layers of the epidermis in WT mice. These findings suggested that SR is involved in the differentiation of epidermal keratinocytes and the formation of the skin barrier. However, its role in skin barrier dysfunction such as atopic dermatitis (AD) remains elusive. AD is a chronic inflammatory disease of skin, and the clinical presentation of AD has been reported to be occasionally associated with psychological factors. Therefore, this study examined the content of d-serine in stratum corneum in AD patients and healthy controls using a tape-stripping method. Skin samples were collected from the cheek and upper arm skin of AD patient's lesion and healthy individuals. The d-serine content was significantly increased in the involved skin of AD in comparison with healthy individuals. An immunohistochemical analysis also revealed an increased SR expression in the epidermis of AD patients. Furthermore, the SR expression in cultured human keratinocytes was significantly increased by the stimulation with tumour necrosis factor -α or macrophage migration inhibitory factor. Taken together, these findings suggest that d-serine expressed particularly strongly in AD lesional skin and that the SR expression in the keratinocytes is linked to inflammatory cytokines.

[What's new in dermatological treatment?] / Quoi de neuf en thérapeutique dermatologique ?

On a therapeutic point of view, 2017 in dermatology could be summarized in one disease, one pathway and in one number : atopic dermatitis, JAK inhibitors and 23. 2017 will be the year of the first registration of a biologic treatment in atopic dermatitis, dupilumab, with numerous other drugs currently in development. JAK inhibitors show promising results in several difficult-to-treat conditions, such as alopecia areata, vitiligo or atopic dermatitis, but still warrant confirmation in upcoming controlled trials. Monoclonal antibodies targeting IL-23 have confirmed in phase III studies their great efficacy in controlling psoriasis and will be soon available in practice, illustrating well the optimal link between bench side and bed in this emblematic inflammatory dermatological condition.

Poly-ADP ribose polymerase-14 limits severity of allergic skin disease.

Poly-ADP ribose polymerase-14 (PARP14 or ARTD8) was initially identified as a transcriptional co-activator for signal transducer and activator of transcription 6 (Stat6), where the presence of interleukin-4 (IL-4) and activated Stat6 induces the enzymatic activity of PARP14 that promotes T helper type 2 differentiation and allergic airway disease. To further our understanding of PARP14 in allergic disease, we studied the function of PARP14 in allergic inflammation of skin using mice that express constitutively active Stat6 in T cells (Stat6VT) and develop spontaneous inflammation of the skin. We mated Stat6VT mice to Parp14-/- mice and observed that approximately 75% of the Stat6VT × Parp14-/- mice develop severe atopic dermatitis (AD)-like lesions, compared with about 50% of Stat6VT mice, and have increased morbidity compared with Stat6VT mice. Despite this, gene expression in the skin and the cellular infiltrates was only modestly altered by the absence of PARP14. In contrast, we saw significant changes in systemic T-cell cytokine production. Moreover, adoptive transfer experiments demonstrated that decreases in IL-4 production reflected a cell intrinsic role for PARP14 in Th2 cytokine control. Hence, our data suggest that although PARP14 has similar effects on T-cell cytokine production in several allergic disease models, the outcome of those effects is distinct, depending on the target organ of disease.

Increased filaggrin-metabolizing enzyme activity in atopic skin: a pilot study using a canine model of atopic dermatitis.

BACKGROUND: Filaggrin (FLG) and its metabolites are essential for skin barrier function and hydration of the stratum corneum. Alteration of the FLG metabolism could be the basis for an abnormal skin barrier in allergic dogs. OBJECTIVES: To investigate the expression and distribution of calpain-1, caspase-14, furin and matriptase, four enzymes involved in FLG metabolism, in the skin of atopic and healthy beagles. METHODS: Skin biopsies were collected from four healthy and four atopic beagles before and after allergen exposure. The dogs were challenged for three consecutive days to mimic an acute exposure, or once weekly to mimic a chronic exposure to allergens. Skin biopsies were taken on days 0 (nonlesional), 3 and 10 in the "acute" model and on days 0 (nonlesional), 14 and 28 in the "chronic" model. Four healthy dogs were used as controls. Indirect immunofluorescence was used to analyse the distribution and the expression of FLG enzymes in a semi-quantitative manner. Five consecutive pictures/section were taken and the intensity analysed tracing the epidermis and using ImageJ on the traced areas. The enzymes' expression was compared between healthy and atopic nonlesional skin (Day 0) and over time in each group. RESULTS: All enzymes were expressed in all layers of the epidermis. A significantly higher expression of calpain-1 (P = 0.028), caspase 14 (P = 0.028) and matriptase (P = 0.028) was evident in atopic compared to control dogs on Day 0. No differences over time were seen for any enzyme analysed. CONCLUSIONS AND CLINICAL IMPORTANCE: This preliminary study suggests an abnormal catabolism of FLG in canine atopic skin.

JAK inhibitors in dermatology: The promise of a new drug class.

New molecularly targeted therapeutics are changing dermatologic therapy. Janus kinase-signal transducer and activator of transcription (JAK-STAT) is an intracellular signaling pathway upon which many different proinflammatory signaling pathways converge. Numerous inflammatory dermatoses are driven by soluble inflammatory mediators, which rely on JAK-STAT signaling, and inhibition of this pathway using JAK inhibitors might be a useful therapeutic strategy for these diseases. Growing evidence suggests that JAK inhibitors are efficacious in atopic dermatitis, alopecia areata, psoriasis, and vitiligo. Additional evidence suggests that JAK inhibition might be broadly useful in dermatology, with early reports of efficacy in several other conditions. JAK inhibitors can be administered orally or used topically and represent a promising new class of medications. The use of JAK inhibitors in dermatology is reviewed here.

Ceramide synthase 4 is highly expressed in involved skin of patients with atopic dermatitis.

BACKGROUND: Ceramide is a crucial lipid in the stratum corneum (SC) which maintains the barrier function and hydration of the skin. In atopic dermatitis (AD) patients who have defective skin barrier function, ceramide levels are altered. We previously reported that although the amount of total ceramide was lower in involved skin compared with uninvolved skin of AD patients and with healthy control skin, the amounts of smaller ceramide species of Cer[NS] (<40 total carbons, which are total carbons of both sphingoid base and amide-linked fatty acid), especially Cer[NS] with 34 total carbons (C34-Cer[NS]), were higher. However, the enzyme(s) that produces the higher levels of smaller ceramide species in involved skin of AD patients was unclear. OBJECTIVE: To identify the enzyme(s) that produces higher levels of smaller ceramide species of Cer[NS] in the involved skin of AD patients. METHODS: Eight female Caucasian subjects who were diagnosed with AD on their arms (age range: 21-45 years) were enroled in this study. We compared ceramide levels in the SC and the expression levels of enzymes involved in ceramide metabolism using real-time PCR and immunohistochemistry between involved and uninvolved skin of AD patients. RESULTS: Level of mRNA encoding ceramide synthase 4 (CERS4), which is one of the enzymes that synthesize ceramide from a sphingoid base and an amide-linked fatty acid, was significantly higher in involved skin than in uninvolved skin (P < 0.01). Additionally, the protein expression level of CERS4 in the epidermis was also higher in involved skin compared with uninvolved skin. The expression level of CERS4 correlated with the amount of C34-Cer[NS] (P < 0.01) and the skin hydration value (P < 0.05). CONCLUSIONS: The elevated expression level of CERS4 contributes to the increase of C34-Cer[NS] and the impaired SC barrier function in involved skin of AD patients.

Hyperactivation of JAK1 tyrosine kinase induces stepwise, progressive pruritic dermatitis.

Skin homeostasis is maintained by the continuous proliferation and differentiation of epidermal cells. The skin forms a strong but flexible barrier against microorganisms as well as physical and chemical insults; however, the physiological mechanisms that maintain this barrier are not fully understood. Here, we have described a mutant mouse that spontaneously develops pruritic dermatitis as the result of an initial defect in skin homeostasis that is followed by induction of a Th2-biased immune response. These mice harbor a mutation that results in a single aa substitution in the JAK1 tyrosine kinase that results in hyperactivation, thereby leading to skin serine protease overexpression and disruption of skin barrier function. Accordingly, treatment with an ointment to maintain normal skin barrier function protected mutant mice from dermatitis onset. Pharmacological inhibition of JAK1 also delayed disease onset. Together, these findings indicate that JAK1-mediated signaling cascades in skin regulate the expression of proteases associated with the maintenance of skin barrier function and demonstrate that perturbation of these pathways can lead to the development of spontaneous pruritic dermatitis.

A Promoter Polymorphism of the Vitamin D Metabolism Gene Cyp24a1 is Associated with Severe Atopic Dermatitis in Adults.

Atopic dermatitis (AD) is a chronic inflammatory skin disease in which genetic and environmental factors result in impaired epidermal barrier functioning and an altered immune response. Vitamin D influences these 2 pathomechanisms, and beneficial results have been suggested in AD. The aim of this study was to investigate the potential roles of the 2 essential vitamin D metabolizing enzymes. The frequencies of 6 common polymorphisms in the genes encoding the vitamin D synthesizing enzyme Cyp27b1 or the inactivating enzyme Cyp24a1 were assessed in 281 patients with AD and 278 healthy donors in a case-control setting. The Cyp24a1 rs2248359-major C allele was significantly over-represented in patients with AD compared with controls, which was more pronounced in patients with severe AD. In addition, haplotypes of the Cyp24a1 and Cyp27b1 genes were associated with AD. These data support that vitamin D mediates beneficial functions in AD and suggest that future studies on the impact of vitamin D on AD should consider the individual genotypes of the vitamin D metabolizing enzymes.