Lesional Skin Of Atopic Dermatitis Patients Research Articles

563 - Dupilumab improves skin lipid composition in atopic dermatitis irrespective of patient filaggrin (FLG) mutation status

Abstract Introduction/Background Type 2 inflammatory cytokines interleukin 4 (IL-4) and IL-13 play an important role in skin barrier disruption in atopic dermatitis (AD). Filaggrin and ceramides play a crucial role in skin barrier integrity. Loss of function mutations in the FLG gene are associated with the impaired skin barrier function and more severe AD.1,2 FLG mutations only affect a minority of AD patients; however, increased IL-4 and IL-13 cytokines are a common cause of reduced filaggrin expression in patients with AD, independent of FLG genotype. Objectives To explore whether dupilumab treatment improves skin barrier function in patients with or without FLG mutations. Methods In the BArrier function and LIpidomics STudy in Atopic Dermatitis (BALISTAD; NCT04447417), a 16-week study in patients with AD aged 12 to 65 years, adult patients with AD received dupilumab 300 mg every 2 weeks; adolescent patients with AD received dupilumab 200 mg every 2 weeks if their baseline weight was <60 kg and 300 mg if ≥60 kg. FLG mutations were evaluated in DNA from blood samples of consenting patients with AD and healthy volunteers. Transepidermal water loss (TEWL) was assessed longitudinally after 5 skin tape strippings (STS) from AD lesions (n = 26) and from the skin of healthy participants (n =26) (age: 12 to 63 years) over a 16-week course of dupilumab treatment. Quantitative N(C18)S-ceramide analysis of STS samples collected on Days 1, 15, 29, 57, and 85, and at Week 16 from AD lesions and from the skin of healthy participants was performed using liquid chromatography tandem mass spectrometry. Results At baseline, mean TEWL after 5 STS (TEWL5) was significantly higher in AD lesional skin than healthy skin (p<0.0001). The mean TEWL5 in AD lesions in subjects with FLG mutations (n = 6/19) was significantly higher at baseline than in AD subjects without mutations (P < 0.0001). Dupilumab treatment significantly reduced TEWL5 in AD lesional skin as early as Week 2 with a progressive decrease through Week 16 (P < 0.0001). Reduction in mean TEWL5 was similar from Week 2 to Week 16 in AD patients with and without FLG mutations. At Week 16, TEWL5 was comparable to healthy skin in the lesional skin of AD patients with and without FLG mutations (P > 0.05). AD skin lesions had increased levels of N(C18)S-ceramides at baseline (P < 0.0001); but no differences were noted in subjects with or without FLG mutations (P > 0.05). Dupilumab treatment significantly reduced levels of N(C18)S-ceramides in AD lesional skin as early as Week 2 with a progressive decrease through Week 16 (P < 0.0001). Dupilumab treatment decreased levels of N(C18)S-ceramides in STS samples similarly in subjects with and without FLG mutations from Week 2 to Week 16. Conclusions Dupilumab treatment normalizes TEWL5 and decreases levels of N(C18)S-ceramides in AD lesional skin of subjects with and without FLG mutations.

Preliminary studies on the role of mast cells in the phenotype and immune activation of type 2 inflammatory cytokine release of atopic dermatitis

Objective: To investigate the role of mast cells in atopic dermatitis (AD) phenotype and the immune activation of type 2 inflammatory cytokine release. Methods: Nine AD skin samples were obtained from the Department of Dermatology, the Second Affiliated Hospital of Xi'an Jiaotong University, and nine healthy skin control samples were obtained from the surgical excision of excess normal skin by orthopedic surgery, the Second Affiliated Hospital of Xi'an Jiaotong University, which were subjected to toluidine blue staining and fluorescence staining to clarify the mast cell degranulation activation status of the AD skin lesions. We investigated whether MC903 could directly activate mast cells in vivo through the toe swelling and exudation assay in wild-type mice; we constructed the MC903-AD model using wild-type and KitW-sh/W-sh mast cell-deficient mice in order to investigate whether mast cells affected the phenotype, histopathology, and the level of type 2 inflammatory factors in AD mice; we extracted mouse peritoneal mast cells and the ability of MC903 to activate mast cells to release inflammatory mediators in vitro was explored by calcium imaging, tryptase and β-aminohexokinase release assays, and MCP-1 and CXCL-2 release assays. Results: The number of degranulated mast cells in an activated state was increased in skin lesions of AD patients compared to healthy controls, with (5.40±1.14) and (2.20±0.84), respectively (P<0.001). KitW-sh/W-sh mast cell-deficient AD mice had an attenuated phenotype with ADI scores of (5.50±1.05), compared to wild-type AD mice with (10.00±0.89) (P<0.001). The release of type 2 inflammatory factors in wild-type AD mice was higher than those in KitW-sh/W-sh mast cell-deficient AD mice, with IL-4 levels of (29.50±1.87) and (15.33±1.86) pg/mg (P<0.001), IL-13 levels were (6.32±0.25) and (3.93±0.22) pg/mg (P<0.001), IL-31 levels were (9.73±0.38) and (6.89±0.27) pg/mg (P<0.001), and TSLP levels were (206.00±4.43) and (99.00±4.86) pg/mg (P<0.001), respectively. MC903 could cause mast cell activation in wild-type mice, leading to increased swelling and exudation in the toes of mice, and MC903 could activate mast cells in vitro, leading to increased degranulation and release of inflammatory factors such as MCP-1 and CXCL-2. Conclusions: The number of activated mast cells was increased in skin lesions of AD patients than in healthy controls. KitW-sh/W-sh mast cell-deficient AD mice showed significantly reduced phenotype, histopathology, and type 2 inflammatory factor levels compared with wild-type AD mice. MC903 activates mast cells in vivo and in vitro. Mast cells play a key role in AD phenotype and immune activation.

Combining 16S Sequencing and qPCR Quantification Reveals Staphylococcus aureus Driven Bacterial Overgrowth in the Skin of Severe Atopic Dermatitis Patients.

Atopic dermatitis (AD) is an inflammatory skin disease with a microbiome dysbiosis towards a high relative abundance of Staphylococcus aureus. However, information is missing on the actual bacterial load on AD skin, which may affect the cell number driven release of pathogenic factors. Here, we combined the relative abundance results obtained by next-generation sequencing (NGS, 16S V1-V3) with bacterial quantification by targeted qPCR (total bacterial load = 16S, S. aureus = nuc gene). Skin swabs were sampled cross-sectionally (n = 135 AD patients; n = 20 healthy) and longitudinally (n = 6 AD patients; n = 6 healthy). NGS and qPCR yielded highly inter-correlated S. aureus relative abundances and S. aureus cell numbers. Additionally, intra-individual differences between body sides, skin status, and consecutive timepoints were also observed. Interestingly, a significantly higher total bacterial load, in addition to higher S. aureus relative abundance and cell numbers, was observed in AD patients in both lesional and non-lesional skin, as compared to healthy controls. Moreover, in the lesional skin of AD patients, higher S. aureus cell numbers significantly correlated with the higher total bacterial load. Furthermore, significantly more severe AD patients presented with higher S. aureus cell number and total bacterial load compared to patients with mild or moderate AD. Our results indicate that severe AD patients exhibit S. aureus driven increased bacterial skin colonization. Overall, bacterial quantification gives important insights in addition to microbiome composition by sequencing.

A CCR4 antagonist attenuates atopic dermatitis-like skin inflammation by inhibiting the recruitment and expansion of Th2 cells and Th17 cells.

CCR4 is a major trafficking receptor for T-helper (Th) 2 cells and Th17 cells and is considered as a potential therapeutic target for atopic dermatitis (AD). The CCR4 ligands CCL17 and CCL22 have been reported to be upregulated in the skin lesions of AD patients. Of note, thymic stromal lymphopoietin (TSLP), a master regulator of the Th2 immune response, promotes the expression of CCL17 and CCL22 in AD skin lesions. Here, we investigated the role of CCR4 in an AD mouse model induced by MC903, a TSLP inducer. Topical application of MC903 to ear skin increased the expression of not only TSLP but also CCL17, CCL22, the Th2 cytokine IL-4, and the Th17 cytokine IL-17A. Consistently, MC903 induced AD-like skin lesions as shown by increased epidermal thickness; increased infiltration of eosinophils, mast cells, type 2 innate lymphoid cells, Th2 cells, and Th17 cells; and elevated serum levels of total IgE. We also found increased expansion of Th2 cells and Th17 cells in the regional lymph nodes (LNs) of AD mice. Compound 22, a CCR4 inhibitor, ameliorated AD-like skin lesions with reduction of Th2 cells and Th17 cells in the skin lesions and regional LNs. We further confirmed that compound 22 diminished the expansion of Th2 cells and Th17 cells in the coculture of CD11c+ dendritic cells (DCs) and CD4+ T cells derived from the regional LNs of AD mice. Collectively, CCR4 antagonists may exhibit anti-allergic effects by inhibiting both the recruitment and expansion of Th2 cells and Th17 cells in AD.

The hidden sentinel of the skin: An overview on the role of interleukin-13 in atopic dermatitis.

Recent evidence suggests that interleukin (IL)-13 is a crucial cytokine involved in the pathogenesis of atopic dermatitis (AD). It is a central driver of type-2 T-helper inflammation and is overexpressed in lesional skin of AD patients. Upon release in peripheral skin, IL-13 activates its receptors, recruits inflammatory cells, and modifies the skin microbiome. IL-13 also reduces the expression of epidermal barrier proteins and activates sensory nerve mediating the itch transmission signal. Novel therapeutics that target IL-13 seem to be efficacious and safe for the treatment of patients with moderate-to-severe AD. The aim of our manuscript is to review the role that IL-13 plays in AD immunopathogenesis.

Dermatophagoides farinae Extract Induces Interleukin 33-Mediated Atopic Skin Inflammation via Activation of RIP1

Receptor-interacting protein kinase (RIP) family 1 signaling has complex effects on inflammatory processes and cell death, but little is known concerning allergic skin diseases. We examined the role of RIP1 in Dermatophagoides farinae extract (DFE)-induced atopic dermatitis (AD)-like skin inflammation. RIP1 phosphorylation was increased in HKCs treated with DFE. Nectostatin-1, a selective and potent allosteric inhibitor of RIP1, inhibited AD-like skin inflammation and the expression of histamine, total IgE, DFE-specific IgE, IL-4, IL-5, and IL-13 in an AD-like mouse model. The expression of RIP1 was increased in ear skin tissue from a DFE-induced mouse model with AD-like skin lesions and in the lesional skin of AD patients with high house dust mite sensitization. The expression of IL-33 was down-regulated after RIP1 inhibition, and the levels of IL-33 were increased by over-expression of RIP1 in keratinocytes stimulated with DFE. Nectostatin-1 reduced IL-33 expression in vitro and in the DFE-induced mouse model. These results suggest that RIP1 can be one of the mediators that regulate IL-33-mediated atopic skin inflammation by house dust mites.

WFDC12-overexpressing contributes to the development of atopic dermatitis via accelerating ALOX12/15 metabolism and PAF accumulation

Atopic dermatitis (AD) is a chronic inflammatory skin disease characterized by eczema-like skin lesions, dry skin, severe itching, and recurrent recurrence. The whey acidic protein four-disulfide core domain gene WFDC12 is highly expressed in skin tissue and up-regulated in the skin lesions of AD patients, but its role and relevant mechanism in AD pathogenesis have not been studied yet. In this study, we found that the expression of WFDC12 was closely related to clinical symptoms of AD and the severity of AD-like lesions induced by DNFB in transgenic mice. WFDC12-overexpressing in the epidermis might promote the migration of skin-presenting cells to lymph nodes and increase Th cell infiltration. Meanwhile, the number and ratio of immune cells and mRNA levels of cytokines were significantly upregulated in transgenic mice. In addition, we found that ALOX12/15 gene expression was upregulated in the arachidonic acid metabolism pathway, and the corresponding metabolite accumulation was increased. The activity of epidermal serine hydrolase decreased and the accumulation of platelet-activating factor (PAF) increased in the epidermis of transgenic mice. Collectively, our data demonstrate that WFDC12 may contribute to the exacerbation of AD-like symptoms in DNFB-induced mouse model by enhancing arachidonic acid metabolism and PAF accumulation and that WFDC12 may be a potential therapeutic target for human atopic dermatitis.

Alterations of Epidermal Lipid Profiles and Skin Microbiome in Children With Atopic Dermatitis.

We aimed to investigate epidermal lipid profiles and their association with skin microbiome compositions in children with atopic dermatitis (AD). Specimens were obtained by skin tape stripping from 27 children with AD and 18 healthy subjects matched for age and sex. Proteins and lipids of stratum corneum samples from nonlesional and lesional skin of AD patients and normal subjects were quantified by liquid chromatography tandem mass spectrometry. Skin microbiome profiles were analyzed using bacterial 16S rRNA sequencing. Ceramides with nonhydroxy fatty acids (FAs) and C18 sphingosine as their sphingoid base (C18-NS-CERs) N-acylated with C16, C18 and C22 FAs, sphingomyelin (SM) N-acylated with C18 FAs, and lysophosphatidylcholine (LPC) with C16 FAs were increased in AD lesional skin compared to those in AD nonlesional skin and that of control subjects (all P < 0.01). SMs N-acylated with C16 FAs were increased in AD lesional skin compared to control subjects (P < 0.05). The ratio of NS-CERs with long-chain fatty acids (LCFAs) to short-chain fatty acids (SCFAs) (C24-32:C14-22), the ratio of LPC with LCFAs to SCFAs (C24-30:C16-22) as well as the ratio of total esterified omega-hydroxy ceramides to total NS-CERs were negatively correlated with transepidermal water loss (rho coefficients = -0.738, -0.528, and -0.489, respectively; all P < 0.001). The proportions of Firmicutes and Staphylococcus were positively correlated to SCFAs including NS ceramides (C14-22), SMs (C17-18), and LPCs (C16), while the proportions of Actinobacteria, Proteobacteria, Bacteroidetes, Corynebacterium, Enhydrobacteria, and Micrococcus were negatively correlated to these SCFAs. Our results suggest that pediatric AD skin shows aberrant lipid profiles, and these alterations are associated with skin microbial dysbiosis and cutaneous barrier dysfunction.

Bacterial membrane vesicles shape Staphylococcus aureus skin colonization and induction of innate immune responses.

Staphylococcus aureus colonization is abundant on the skin of atopic dermatitis (AD) patients where it contributes to skin inflammation. S. aureus produces virulence factors that distinguish it from commensal skin bacteria such as S. epidermidis and S. lugdunensis. However, it has remained unclear, which of these virulence factors have the strongest impact on AD. Membrane vesicles (MVs) are released by pathogenic bacteria and might play an essential role in the long-distance delivery of bacterial effectors such as virulence factors. We show that MVs are also released by skin commensals in a similar quantity and membrane lipid amount as those from pathogenic S. aureus. Interestingly, MVs from skin commensals can protect against S. aureus skin colonization by conditioning human skin for enhanced defence. In contrast, MVs released by S. aureus are able to induce CXCL8 and TNF-α in primary human keratinocytes, recruit neutrophils and induce neutrophil extracellular traps, which enhance S. aureus skin colonization. CXCL8 induction is TLR2- and NFkB-dependent and the induction level correlates with the membrane lipid and protein A content of the MVs. Interestingly, MVs of S. aureus strains from the lesional skin of AD patients show an enhanced membrane lipid and protein A content compared to the strains from the non-lesional sites and have an enhanced proinflammatory potential. Our data underline the complex interplay in host- and bacterial derived factors in S. aureus skin colonization and the important role of bacterial derived MVs and their membrane lipid and protein A content in skin inflammatory disorders.

122 Ex vivo infection of skin from atopic dermatitis patients reveals facilitated invasion of herpes simplex virus 1

During primary infection, herpes simplex virus 1 (HSV-1) enters human skin or mucosa and establishes lifelong latency in sensory neurons. The skin is normally well protected from invading pathogens as the epithelium forms effective barriers. How HSV-1 can overcome this barrier in vivo remains to be shown. One prominent example for successful HSV-1 skin invasion is eczema herpeticum, a disseminated HSV-1 infection and severe complication of atopic dermatitis (AD). In lesional skin of AD patients, the mechanical skin barrier consisting of the stratum corneum and tight junctions in the stratum granulosum is impaired.

205 The skin commensal Staphylococcus epidermidis induces inflammatory mediators in keratinocytes: Implications for atopic dermatitis

The chronic inflammatory skin disease atopic dermatitis (AD) is characterized by a dysbiosis of the microbiota. This is reflected by a decreased microbial diversity and the abundance of specific potential pathogenic strains such as Staphylococcus (S.) aureus. Staphylococcus (S.) epidermidis is an abundant member of the cutaneous microbiota and is considered generally as an apathogenic commensal that exhibits beneficial effects in AD, e.g. by controlling the growth of S. aureus. However, S. epidermidis is also frequently present in skin lesions of AD patients and a correlation between abundance of S. epidermidis and severity of AD has been reported. We therefore hypothesized that S. epidermidis may exhibit proinflammatory activities. To evaluate this hypothesis we stimulated cultured human keratinocytes and 3D skin models with different S. epidermidis isolates derived either from healthy skin or from lesional skin of AD patients. Real-time PCR and ELISA analyses revealed a significant induced expression of the AD-relevant cytokine thymic stromal lymphopoietin (TSLP) in keratinocytes and 3D skin after stimulation with S. epidermidis strains. S. epidermidis strains derived from lesional AD skin induced a higher TSLP expression than strains derived from healthy skin. Similar results were also obtained with the pro-inflammatory cytokines IL-17c and TNF-alpha. Both, S. epidermidis strains derived from healthy skin and strains derived from AD skin activated the inflammation-associated transcription factor NF-kappaB. Moreover, expression of the barrier-associated protein filaggrin was downregulated by S. epidermidis. Together, the observed S. epidermidis-mediated induction of proinflammatory mediators and downregulation of filaggrin in keratinocytes and 3D skin models may explain why AD patients carrying a high number of S. epidermidis are associated with a more severe inflamed disease state.

A novel function of NLRP3 independent of inflammasome as a key transcription factor of IL-33 in epithelial cells of atopic dermatitis

Atopic dermatitis (AD) is a common chronic pruritic inflammatory skin disorder characterized by recurrent eczematous lesions. Interleukin (IL)−33, a cytokine of the IL-1 family, was found to play an important role in the pathogenesis of AD. As a key component of the inflammasome, NLRP3 has been mostly described in myeloid cells that to mediate inflammasome activation conducted proinflammatory cytokine production of the IL-1 family. However, the role of NLRP3 inflammasome in the pathogenesis of AD, as well as IL-33 processing are highly controversial. Whether NLRP3 can mediate IL-33 expression and secretion independently of the inflammasome in the epithelium of AD has remained unclear. In this article, we found the mRNA expression of Il33 and Nlrp3 were notably increased in the lesional skin of AD patients compared to healthy controls. We then found a significant positive correlation between the expression of Nlrp3 and Il33 in the epithelium of MC903-mediated AD mice model, but no changes were observed for Il36α, Il36γ, Il1β, or Il18 mRNA expression, as well as IL-1β or IL-18 production. Overexpression of NLRP3 in human immortalized epithelial cells increased IL-33 expression, whereas siRNA targeting NLRP3 abolished IL-33 expression. In addition, inhibition of NLRP3 inflammasome activation or caspase-1 activity with MCC950 or VX-765 showed no effect on the expression and secretion of IL-33 in AD mice. Unlike myeloid cells, NLRP3 predominantly located in the nucleus of epithelial cells, which could directly bind to Il33 specific-promoters and transactivate it through an interaction with transcription factor IRF4. Furthermore, NLRP3 deficient mice exhibited a significant alleviated epidermis inflammation and decreased mRNA expression and secretion of IL-33 in MC903-mediated AD mice without interfering with TSLP and IL-1β production. Our results demonstrate a novel ability of NLRP3 to function as a crucial transcription factor of IL-33 in epithelium independently of inflammasome that to mediate the pathological process of AD.

The Role of New IL-1 Family Members (IL-36 and IL-38) in Atopic Dermatitis, Allergic Asthma, and Allergic Rhinitis.

Since the discovery of its very first member in 1974, the IL-1 family has expanded into a group of 11 potent molecules which are essential in both innate and acquired immunity. Pro-inflammatory cytokines IL-36α, IL-36β, and IL-36γ and their receptor antagonists IL-36Ra and IL-38, which belong to the IL-36 subfamily, are some of the most recently identified members. Recent studies show that these members possess pro-inflammatory and anti-inflammatory activities and may take part in the pathogenesis of allergy. In this review, the involvement and importance of these newly described IL-1 family members in the most common allergic diseases, i.e., atopic dermatitis (AD), allergic asthma, and allergic rhinitis, will be discussed. Dysregulation of IL-36 and IL-38 was observed in the skin and respiratory tract of AD, allergic rhinitis, and allergic asthma individuals. Although the upregulation in IL-36α and IL-36γ observed in the lesional skin of AD patients was unexpectedly small, IL-36 may play an important role in AD pathogenesis especially upon Staphylococcus aureus colonization. While IL-36γ regulates eosinophils to induce an inflammatory response in allergic rhinitis, IL-36α was found to regulate Th17 immunity. IL-36 receptor antagonists, IL-36Ra and IL-38, however, both show promising anti-inflammatory activities against allergic asthma. Of note, IL-38 in allergic asthmatic children is significantly lower than their healthy counterparts, while the anti-inflammatory effects of IL-38 in allergic asthma exacerbation upon viral-like infection were demonstrated in in vitro, HDM-induced, and humanized mice models. Dysregulated expression of IL-36 and IL-38 observed in allergic patients and mice models revealed that they may have essential roles in the pathogenesis in AD, allergic rhinitis, and allergic asthma, especially during the host defense against pathogens at inflammatory sites. Their receptor antagonists, IL-36Ra and IL-38, could also be promising biologics in the control of allergy. Since allergic diseases are phenotypically complex, contradictory data obtained in different studies may be explained if further stratification of disease endotypes is explored. Genetically modified mice model and investigation in anti-IL-36 treatment may be useful to characterize the therapeutic potential of these cytokines in the regulation of allergy in the future.

Role of Galectin-9 in Atopic Dermatitis - Is It Mediated Through E Selectin? A Clinical and Immunohistochemical Study.

BackgroundAtopic dermatitis (AD) is a recognized T helper (Th)2, allergic, skin disease. Galectin-9 (gal-9) is a member of galectin family. It alters T-cell balance resulting in Th2 polarization. These Th2 cells yield various cytokines that may influence E selectin expression. Therefore, we hypothesized that gal-9 may have an active role in AD and this role could be mediated through E selectin.ObjectiveTo assess this hypothesis, immunohistochemical expression of gal-9 and E selectin was investigated in skin lesions, from atopic dermatitis patients, and compared.MethodsTwenty-two atopic dermatitis patients and ten controls were included in this case-control study. SCORAD score was used to evaluate atopic dermatitis severity. Biopsies from skin lesions of AD patients and matched sites of controls were taken and stained immunohistochemically by gal-9 and E selectin polyclonal antibodies.ResultsCompared to controls, atopic dermatitis patients exhibited a significant increased gal-9 H score, percent of expression, cellular localization (P˂0.001) and intensity (P=0.04) as well as dermal cellular infiltrate (P˂0.001). Also, there were significant elevations in E selectin H score (P=0.002), percent of expression (P=0.001) and cellular localization (P<0.001) as well as dermal inflammatory infiltrates in AD cases than controls. In AD, 20 cases showed co expression of both gal-9 and E selectin in the epidermis with insignificant correlation between their H scores.Study LimitationsThis study only included a small number of studied subjects.ConclusionGalectin-9 and E selectin participates independently in atopic dermatitis pathogenesis, that may help in development of new therapeutic agents in atopic dermatitis management program.

Transglutaminase 3 Promotes Skin Inflammation in Atopic Dermatitis by Activating Monocyte-Derived Dendritic Cells via DC-SIGN

Atopic dermatitis (AD) is often concomitant with increased levels of IgE against not only foreign allergens but also autoallergens. AD patients with autoallergy are likely to be more severe and difficult to treat, and self-reactive IgE might be a contributing factor in the pathogenesis of AD. However, how autoallergens are recognized by the immune system and what immune responses are induced subsequently remain largely unknown. We found that the serum level of IgE against transglutaminase 3 (TGase3) was significantly higher in AD patients than in healthy individuals and was positively correlated with disease severity. The expression of TGase3 in the lesional skin of AD patients was markedly increased compared with that of the controls, and Th2 cytokines and/or allergen promoted the expression of TGase3 in keratinocytes. TGase3 bond monocytes-derived dendritic cells (MoDCs) via dendritic cell-specific ICAM-3-grabbing non-integrin (DC-SIGN), which resulted in the production of IL-6 and activation of the NF-κB signaling pathway in MoDCs; and TGase3-treated MoDCs facilitated Th1 polarization. Moreover, skin inflammation in the mouse model of MC903-induced AD was attenuated when TGase3 was inhibited. In conclusion, TGase3 was revealed as an autoallergen in AD andactively involved in skin inflammation; TGase3-targeting might be a therapeutic strategy for the treatment of AD.

Mast Cells Stimulated with Peptidoglycan from Staphylococcus aureus Augment the Development of Th1 Cells.

The skin of patients with atopic dermatitis (AD) is superficially colonized by Staphylococcus aureus. We have previously found that percutaneous permeation of peptidoglycan (PEG) from S. aureus increases the number of mast cells in the dermis, as seen in skin lesions of AD patients. The purpose of the present study was to clarify the influence of PEG on T helper type 1 (Th1)/ T helper type 2 (Th2) cell development mediated by mast cells. Mast cells were induced by long-term culture of murine spleen cells in medium supplemented with tumor necrosis factor (TNF)- a. Ovalbumin (OVA) peptide-pulsed mast cells were incubated with naïve Th cells in the presence or absence of PEG. Five days later, Th cells in the culture were stimulated with phorbol 12-myristate 13-acetate (PMA) and ionomycin, and Th1/Th2 cytokine production was investigated by enzyme-linked immunosorbent assay. It was confirmed that the mast cells we obtained had surface expression of I-Ad, worked as antigen-presenting cells, and induced Th1 cell and Th2 cell development. The stimulation of mast cells with PEG enhanced the development of Th1 cells but not that of Th2 cells. The increase of Th1 cell development stimulated by PEG was associated with an increase in the expression of Notch ligand Delta 1 in the mast cells. Furthermore, treatment of mast cells with the macrolide antibiotic josamycin suppressed Th1 cell development and this was correlated with a reduction of both Delta 1 expression and interleukin (IL)-12 production in mast cells. Colonization of S. aureus on the lesioned skin of AD patients contributes to not only an increase in the number of mast cells but also Th1 cell development mediated by mast cells in the dermis and subsequent induction of chronic inflammation, which is characterized by up-regulation of the Th1 cytokine, interferon (IFN)- g. Therefore, application of josamycin to the lesional skin of AD patients may provide relief from chronic inflammation mediated by mast cells.

401 The inhibitory effect of a histamine 4 receptor antagonist on chemokine production by monocyte-derived Langerhans cells in patients with atopic dermatitis

Histamine4 receptor (H4R) is a member of the histamine receptors which exists on Th17 cells, NK cells, keratinocytes and dendritic cells. H4R was recently demonstrated to be functionally expressed on Langerhans cells (LCs), while H4 signaling was found to participate in the itch response. We performed in vitro experiments to investigate the production of CCL17 and CCL22 production by MoLCs. We next examined the effects of an H4R antagonist (JNJ7777120) on the production of Th2 chemokines by the MoLCs of atopic dermatitis (AD) patients. We examined the inhibitory effect of the H4R antagonist on the production of CCL17 and CCL22 by the MoLCs of AD patients. The application of the H4R antagonist significantly inhibited the production of both the CCL17 and CCL22 chemokines by MoLCs of AD patients. We examined the inhibitory effect of the H4R antagonist on PGN-stimulated CCL17 and CCL22 production by the MoLCs of AD patients. We detected inhibitory effect of H4R antagonist on CCL17 and CCL22 production by PGN-treated MoLCs. Significant inhibitory effects were observed at 33nM concentration for the chemokine production. We demonstrated the following new findings. First, an H4R antagonist (JNJ7777120) inhibited CCL17 and CCL22 production by the MoLCs of AD patients. Next the PGN-induced chemokine production in AD patients was suppressed by the H4R antagonists. Finally, the H4R antagonist inhibited the PGN-induced phosphorylated p38 MAP kinase activity in the MoLCs of AD patients. We also confirmed the enhancement of CCL17 and CCL22 production by the MoLCs of AD patients in comparison to HCs. Our data demonstrate, for the first time, that an H4R antagonist significantly inhibited the CCL17 and CCL22 production by the MoLCs of AD patients. Thus, our data strongly suggest that H4R signals can modulate Th2 cells recruitment in the lesional skin of AD patients.

The Expression and Regulation of Interleukin-33 in Human Epidermal Keratinocytes: A New Mediator of Atopic Dermatitis and Its Possible Signaling Pathway.

Interleukin (IL)-33 is a novel member of the IL-1 superfamily of cytokines that has recently become a focal point for research into the pathogenesis of atopic dermatitis (AD). However, the expression and regulation of IL-33 in human epidermal keratinocytes have not well been delineated. The aim of this study was to evaluate IL-33 and its receptor ST2L expression in skin lesions of AD patients and explored the signal transduction mechanisms leading to the IL-33 expression and of the IL-33's pharmacological action in keratinocytes from AD patients (ADKs) and those from healthy controls (NHEKs). We performed immunocytochemistry, reverse transcription-polymerase chain reaction, Western blotting, and enzyme-linked immunosorbent assay to investigate ADKs compared with NHEKs. We found that IL-33/ST2L were positively expressed in the skin lesions of AD patients and a high expression of IL-33 was induced in keratinocytes by IL-4 plus interferon [IFN]-γ or IFN-γ alone at the mRNA and protein levels. Meanwhile, IFN-γ induced IL-33 expression through extracellular signal-regulated kinase (ERK), p38 mitogen-activated protein kinase (MAPK), epidermal growth factor receptor (EGFR), and Janus kinase/signal transducer and activator of transcription. The expression of ST2L was increased in a time- and dose-dependent manner in both types of cells incubated with IL-33, and was especially increased in ADKs compared with NHEKs. We examined the cytokine IL-6 and the chemokines CXCL-8/IL-8, CCL-20, CCL-17, CCL-5, and CCL-2 in keratinocytes, which showed increased expression in a time- and dose-dependent manner in ADKs when induced by IL-33. Furthermore, 4 signaling pathways (ERK, p38 MAPK, c-Jun N-terminal kinase, and nuclear factor-κB) were involved in the IL-33-mediated induction in both cells. In conclusion, IL-33 is closely interlinked in AD skins and keratinocytes. IL-33 plays an important role in the pathogenesis of immune inflammatory responses in AD, which might be a possible therapeutic target in the treatment of AD.

The crucial role of IL-22 and its receptor in thymus and activation regulated chemokine production and T-cell migration by house dust mite extract.

House dust mite (HDM) is known as one of the factors that causes atopic dermatitis (AD). Interleukin (IL)-22 and thymus and activation regulated chemokine (TARC) are related to skin inflammatory disease and highly expressed in AD lesions. However, the effects of HDM on IL-22 production in T cells and on TARC production and IL-22Rα receptor expression in keratinocytes are unknown. To identify the role of HDM in keratinocytes and T cells, we investigated IL-22Rα expression and TARC production in the human keratinocyte cell line HaCaT and IL-22 production in T cells treated with HDM extract as well as their roles in HDM-induced skin inflammation. HDM extract not only increased IL-22Rα expression and TARC production in HaCaT but also enhanced IL-22, tumor necrosis factor (TNF)-α and interferon (IFN)-γ production in T cells. The HDM extract-induced IL-22 from T cells significantly increased the production of IL-1α, IL-6 and TARC in HaCaT cells. In addition, we found that TARC produced in HDM extract-treated HaCaT induced T-cell recruitment. These results suggest that there is a direct involvement of HDM extract-induced IL-22 in TARC production and T-cell migration. Taken together, TARC production in HaCaT through the interaction between IL-22 and IL-22Rα facilitates T-cell migration. These data show one of the reasons for inflammation in the skin lesions of AD patients.

Profile of skin barrier proteins (filaggrin, claudins 1 and 4) and Th1/Th2/Th17 cytokines in adults with atopic dermatitis.

Atopic dermatitis (AD) in adults and profile of skin barrier proteins and inflammatory cytokines. Evaluation of the expression of skin barrier proteins such as filaggrin, claudins 1 and 4 and of circulating inflammatory cytokines (Th1/Th2/Th17) in adults with AD. Thirty-three adult patients with AD diagnosed according to the Hanifin & Rajkacriteria, and 25 healthy controls were enrolled in the study. AD severity was measured by Eczema Area and Severity Index (EASI). Laboratory assays included immunohistochemistry analysis of skin barrier proteins, such as filaggrin, claudins 1 and 4 and interleukin-17 (IL-17) from skin samples and determination of circulating cytokine levels (IL-2, 4, 5, 6, 10, 17A, TNF and IFN-γ) by flow cytometry (Cytometric Bead Array). We observed a reduced expression of filaggrin and claudin 1 in lesional skin of AD patients, when compared to controls. There was an inverse correlation of filaggrin expression and disease severity. In addition, IL-17 expression was enhanced in AD patients. Similarly, higher levels of inflammatory cytokines (IL-2, 5, 6, 10, 17A and IFN-γ) were found in AD patients. Our data reinforce the role of an altered skin barrier in the pathogenesis of AD. Our results show not only reduced expression of filaggrin and claudin 1 in lesional atopic skin but also inverse correlation of filaggrin expression and disease severity. Moreover, elevation of in situ IL-17 and of circulating interleukin levels in AD emphasize the systemic, inflammatory profile of this defective skin barrier dermatosis.