Vitamin D3-Induced Promotor Dissociation of PU.1 and YY1 Results in FcεRI Reduction on Dendritic Cells in Atopic Dermatitis.

Atopic dermatitis (AD) is a severe inflammatory skin disease. Langerhans cells and inflammatory dendritic epidermal cells (IDEC) are located in the epidermis of AD patients and contribute to the inflammatory processes. Both express robustly the high-affinity receptor for IgE, FcεRI, and thereby sense allergens. A beneficial role of vitamin D3 in AD is discussed to be important especially in patients with allergic sensitization. We hypothesized that vitamin D3 impacts FcεRI expression and addressed this in human ex vivo skin, in vitro Langerhans cells, and IDEC models generated from primary human precursor cells. We show in this article that biologically active vitamin D3 [1,25(OH)2-D3] significantly downregulated FcεRI at the protein and mRNA levels of the receptor's α-chain, analyzed by flow cytometry and quantitative RT-PCR. We also describe the expression of a functional vitamin D receptor in IDEC. 1,25(OH)2-D3-mediated FcεRI reduction was direct and resulted in impaired activation of IDEC upon FcεRI engagement as monitored by CD83 expression. FcεRI regulation by 1,25(OH)2-D3 was independent of maturation and expression levels of microRNA-155 and PU.1 (as upstream regulatory axis of FcεRI) and transcription factors Elf-1 and YY1. However, 1,25(OH)2-D3 induced dissociation of PU.1 and YY1 from the FCER1A promotor, evaluated by chromatin immunoprecipitation. We show that vitamin D3 directly reduces FcεRI expression on dendritic cells by inhibiting transcription factor binding to its promotor and subsequently impairs IgE-mediated signaling. Thus, vitamin D3 as an individualized therapeutic supplement for those AD patients with allergic sensitization interferes with IgE-mediated inflammatory processes in AD patients.

The diagnosis and treatment of hereditary angioedema patients in Japan: A patient reported outcome survey.

BACKGROUND: The rate at which patients are accurately diagnosed with hereditary angioedema (HAE), as well as diagnosed patients access to modern treatments differs greatly among countries. Moreover, the severity and burden of HAE on patients have been reported mostly on the basis of physician-reported surveys. To gain insight into the real-world conditions of patients with HAE through a patient-reported survey in Japan and identify any unmet needs. METHODS: A questionnaire was distributed to 121 patients with HAE via a Japanese HAE patient organization during 2016-2017. Responses were collected from 70 patients (57.9%) and subjected to analysis. RESULTS: The average periods from the initial appearance of symptoms (e.g. edema) to a HAE diagnosis was 15.6 years (min-max, 0-53). Patients visited an average of 4.6 different departments until receiving a definitive diagnosis. The average age at the first visit was 25.6 years (3-73) and at diagnosis 32.8 years (0-73). Patients reported an average of 15.7 (0-100) attacks per year, but only 53.1% of attacks were treated. The days of hospitalization due to severe attacks was 14.3 (0-200) before diagnosis, but these declined to 4.3 (0-50) after diagnosis. In the treatment for attacks, 82% of the patients were treated with the plasma-derived C1 inhibitor concentrate, and 69% of the patients reported experiencing a therapeutic effect. CONCLUSIONS: There is a long gap between first attack and diagnosis of HAE, and the number of non-treated attacks is high in Japan. Steps are needed to improve the diagnostic and treatment environments to address these issues.

Staphylococcus aureus from atopic dermatitis skin accumulates in the lysosomes of keratinocytes with induction of IL-1α secretion via TLR9.

BACKGROUND: Staphylococcus aureus (S. aureus) is frequently detected in the skin of patients with atopic dermatitis (AD), and involved in the flare of AD. There are some evidence-specific strains of S. aureus affect the severity of AD. However, the mechanism of predominant colonization and the aggravation of dermatitis by certain strains of S. aureus in the patients with AD are still unknown. OBJECTIVE: To reveal the characteristics of S. aureus from patients with AD (S. aureus-AD), we analyzed the interaction of S. aureus-AD and keratinocytes in comparison with those of S. aureus laboratory strains (S. aureus-stand.). METHODS: We stimulated HaCaT cells, keratinocyte cell line, and human epidermal keratinocytes by heat-killed S. aureus strains, then evaluated immune response of keratinocytes by ELISA, immunofluorescence staining, and flow cytometry. RESULTS: Upon incubation with keratinocytes, three out of four strains of heat-killed S. aureus-AD were strongly agglutinated inside the cytoplasm. In the cells, they are located in lysosomes and promoted the secretion of interleukin-1α (IL-1α). These reactions were not observed by any of four strains of S. aureus-stand. and S. epidermidis and were abolished by the treatment of S. aureus with proteinase K. Moreover, the IL-1α secretion was diminished by the inhibition of Toll-like receptor 9 (TLR9). CONCLUSION: S. aureus-AD accumulates in lysosome of keratinocytes by means of bacterial cell wall proteins and induces IL-1α via TLR9.

Staphylococcus aureus in atopic dermatitis: Strain-specific cell wall proteins and skin immunity.

Atopic dermatitis (AD) is a common chronic skin disease. The presence of the bacterium Staphylococcus aureus (S. aureus) is frequently detected on skin affected with AD. In this review, we focused on the characteristics of S. aureus strains isolated from AD skin, particularly the proteins on the cell surface that modulates the interactions between Langerhans cell, keratinocyte, and S. aureus. The skin microbiome plays an important role in maintaining homeostasis of the skin, and colonization of S. aureus in AD is considered to be deeply involved in the clinical manifestation and pathogenesis of skin flares. Colonizing S. aureus strains in AD harbor different surface proteins at the strain level, which are indicated as clonal complexes. Moreover, the cell wall proteins of S. aureus affect skin adhesion and induce altered immune responses. S. aureus from AD skin (AD strain) exhibits internalization into keratinocytes and induces imbalanced Th1/Th2 adaptive immune responses via Langerhans cells. AD strain-derived cell wall proteins and secreted virulence factors are expected to represent therapeutic targets. In addition, the microbiome on the AD skin surface is associated with skin immunity; thus, microbiome-based immunotherapy, whose mechanism of action completely differs from that of typical steroid ointments, are expected to be developed in the future.

Activation of Human Peripheral Basophils in Response to High IgE Antibody Concentrations without Antigens.

Basophils and mast cells have high affinity IgE receptors (FcεRI) on their plasma membrane and play important roles in FcεRI-associated allergic diseases, such as pollen allergy, food allergy, chronic spontaneous urticarial (CSU), and atopic dermatitis (AD). To date, several reports have revealed that high IgE antibody concentrations activate mast cells-which reside in tissue-in the absence of any antigens (allergens). However, IgE antibody-induced activation of basophils-which circulate in blood-has not been reported. Here, we investigated whether IgE antibodies may regulate functions of human peripheral basophils without antigens in vitro. We successfully removed IgE antibodies bound to FcεRI on the surface of human peripheral basophils by treating with 0.1% lactic acid. We also demonstrated that high IgE antibody concentrations (>1 µM) induced histamine release, polarization, and CD203c upregulation of IgE antibody-stripped basophils. Thus, high IgE antibody concentrations directly activate basophils, which express IgE-free FcεRI on the cell surface. This mechanism may contribute to the pathogenesis of patients with AD and CSU who have higher serum IgE concentrations compared to healthy donors.

A Staphylococcus epidermidis strain inhibits the uptake of Staphylococcus aureus derived from atopic dermatitis skin into the keratinocytes.

BACKGROUND: Various bacterial species form a microbiome in the skin. In the past, dead Staphylococcus aureus derived from atopic dermatitis (AD) are taken up by keratinocytes; however, whether live S. aureus can be taken up by keratinocytes is unknown. OBJECTIVE: This study aimed to examine whether live AD strains of S. aureus internalize into the keratinocytes and how the internalization changes under conditions in which other bacterial species including S. epidermidis are present. METHODS: HaCaT cells were cultured with live S. aureus and S. epidermidis (live or heat-treated) or their culture supernatants. After coculture, the change in the amount of S. aureus in the cytoplasm of HaCaT cells was analyzed using, a high-throughput imaging system, Opera Phenix™. RESULTS: Live S. aureus were taken up in the cytoplasm of HaCaT cells. Coculturing live S. aureus with live S. epidermidis or the culture supernatants decreased the abundance of S. aureus in the cytoplasm. The heat-treated culture supernatants of live S. epidermidis or culture supernatants of other S. strains did not decrease the abundance of S. aureus in the cytoplasm. CONCLUSION: Live S. aureus was internalized into the cytoplasm of HaCaT cells as does heat-treated S. aureus. In addition, the heat-sensitive substances secreted by coculture with S. epidermidis and keratinocytes inhibited the uptake of S. aureus by keratinocytes.

Burden of illness seen in hereditary angioedema in Japanese patients: Results from a patient reported outcome survey.

Hereditary angioedema (HAE) is a potentially life-threatening rare disease, which is mainly caused by the deficiency or dysfunction of C1-esterase inhibitor, and characterized by spontaneous, recurrent episodes of edema in various parts of the body including internal organs and the laryngeal area. Delayed diagnosis and treatment increase the burdens and risks of this condition. The current study aimed to understand the burden of illness for HAE patients in Japan before and after diagnosis through a patient reported outcome survey. A survey instrument was distributed to 121 adult patients with HAE by a patient organization via HAE treating physicians between July and November in 2016. Seventy patients (57.9%) returned the questionnaire. Patients reported high levels of medical resource utilization, including emergency procedures and services. Episodes of receiving laparotomy were somewhat less after diagnosis with HAE than before, but no apparent difference in episodes of tracheotomy between before and after the diagnosis. The economic burden, including direct and indirect medical costs, was highest before diagnosis, but still perceived as substantial after diagnosis. Patients reported disruption of work and school life, with 40% reporting that they miss 10 or more days from work or education per year. Sixty percent of patients reported that HAE affected their daily activities. We concluded that HAE is associated with considerable physical, social, economic and psycho-social burdens even after diagnosis, and that higher attack frequency is associated with a heavy disease burden for patients in Japan.

Uptake of Staphylococcus aureus by keratinocytes is reduced by interferon-fibronectin pathway and filaggrin expression.

Staphylococcus aureus (S. aureus) is frequently detected in the skin of patients with atopic dermatitis (AD). AD skin-derived strains of S. aureus (AD strain) are selectively internalized into keratinocytes (HaCaT cells) compared to standard strains. However, the mechanism of AD strain internalization by keratinocytes and effect of the skin environment on internalization remain unclear. HaCaT cells were exposed to heat-killed AD or standard strains of fluorescently labeled S. aureus, with or without interferon (IFN)-γ, interleukin (IL)-4, and IL-13 cytokines, for 24 h. Filaggrin and fibronectin expression in HaCaT cells was knocked down using small interfering RNA. The amount of internalized S. aureus was evaluated using a cell imaging system. The effects of INF-γ, IL-4, and S. aureus exposure on mRNA expression in HaCaT cells were analyzed using single-cell RNA sequencing. AD strains adhered to HaCaT cells in approximately 15 min and were increasingly internalized for up to 3 h (2361 ± 467 spots/100 cells, mean ± SD), whereas the standard strain was not (991 ± 71 spots/100 cells). In the presence of IFN-γ, both the number of internalized strains and fibronectin expression significantly decreased compared to in the control, whereas Th2 cytokines had no significant effects. The number of internalized AD strains was significantly higher in filaggrin knockdown and lower in fibronectin knockdown HaCaT cells compared to in the control. RNA sequencing revealed that IFN-γ decreased both fibronectin and filaggrin expression. Keratinocyte internalization of the AD strain may be predominantly mediated by the INF-γ-fibronectin pathway and partially regulated by filaggrin expression.

A survey on subtypes and clinical characteristics of 1061 patients with urticaria in the primary care institutes in Japan.

The prevalence of urticaria has been reported mostly in Europe and North America. However, precise information regarding its subtypes and clinical characteristics in primary care practice, especially in Asian countries, are scant. Patients with urticaria and/or angioedema who visited nine primary clinics of accredited dermatologists and allergologists in Japan were recruited from October to November 2020. The information of age, sex, disease duration, urticaria control test (UCT), and concomitant urticaria subtypes were collected. A total of 1061 patients participated. The number of patients was high in the 20 to 50 age groups with a peak in the 40s. The most frequent urticaria subtype was chronic spontaneous urticaria (CSU) followed by dermographism, acute spontaneous urticaria (ASU), angioedema, and cholinergic urticaria (CholU) (66.8%, 22.7%, 18.9%, 14.1% and 5.7% in all patients with urticaria). CSU development increased with age from the 20s to 50s, especially in females. Dermographism had a peak in the 40s. ASU had bimodal peaks in childhood and in the 30s. CholU was common in males in the 10-20s. Most angioedema patients were female with an increase in their 30s. Angioedema was solely present in 14 of 1061 participants (1.3%), while 136 (12.8%) had angioedema concomitant with urticaria. UCT showed poorly controlled urticaria with lower scores in patients with concomitant CSU and other subtypes than in those with CSU alone. Urticaria tends to develop in young to middle-aged females. The most common urticaria subtype is CSU, while the number of patients with CholU is high and that of angioedema is low in Japan.

[National prevalence survey of hereditary angioedema in Japan].

BACKGROUND: The prevalence of hereditary angioedema (HAE) is estimated to be approximately 1 case per 50000 persons in English literatures. However, neither disease prevalence nor epidemiologic features of HAE has been surveyed in Japan. METHODS: A nation-wide prevalence survey of HAE in Japan was conducted in 2009. We mailed questionnaires to hospitals with 200 or more beds (1389 hospitals and 5240 departments including Dermatology, Otolaryngology, Emergency Medical Care, Internal Medicine, Pulmonary Medicine, Allergy & Rheumatology), to ask numbers, disease types, symptoms and treatments of angioedema of patients visited to their hospitals. RESULTS: In total, 1128 replies were obtained and 411 patients of angioedema including 52 HAE type1 or type2 patients were reported. In the HAE type1 or type2 patients, 54% patients have experienced cutaneous swelling on face, 42% patients have experienced throat discomfort and 37% had experienced abdominal symptoms. In acute attacks of HAE, 29% patients had been treated with C1-inhibitor concentrates. CONCLUSION: The prevalence of HAE in Japan may be lower than the estimated prevalence mainly in Europe and North America. Many patients with HAE may not be appropriately treated especially for their acute attacks. Further studies by genomic analysis should be performed to reveal the penetrance of the C1 inhibitor gene deficiency and occurrence of HAE type3 in Japan.

Complicated cutaneous leishmaniasis caused by an imported case of Leishmania tropica in Japan: a case report.

BACKGROUND: Leishmaniasis is not endemic in Japan, and imported cases are rare. However, there are increasing concerns regarding imported cases of cutaneous leishmaniasis from endemic countries to Japan. This report describes a case of imported cutaneous leishmaniasis that was diagnosed and treated in Japan. CASE PRESENTATION: A 53-year-old Pakistani man presented with skin lesions on both malleoli of his right ankle and the dorsum of the left foot. The skin lesions manifested as erythematous nodules surrounding an ulcer in the center of the lesion. The lesions of the malleoli of his right ankle each measured 3 × 3 cm, and the lesion on the top of his left foot measured 5 × 4 cm. He had been living and working in Japan but had a history of a visit to Pakistan for about 2 months in 2018. The skin lesions were biopsied. Giemsa and hematoxylin and eosin staining of biopsy samples showed amastigotes of Leishmania in macrophages, and the presence of Leishmania was confirmed by skin tissue culture. Polymerase chain reaction using biopsy specimens identified Leishmania parasites, and DNA sequence analysis revealed that the species was Leishmania tropica. The patient was treated with intravenous liposomal amphotericin B for 6 days. The erythema disappeared, and the erythematous nodules resolved within 3 weeks. CONCLUSION: This is the first report of imported cutaneous leishmaniasis caused by L. tropica from Pakistan, and it is interesting that all three testing modalities showed positive results in this case.

Comparison of the time course of atopic dermatitis from birth to 19 years old among generations of patients in Japan.

Atopic dermatitis (AD) mostly develops in early childhood and tends to resolve with age. However, its time course in severity before and after adolescence varies widely among patients. To investigate the course of disease severity from birth to 19 years old of adult patients with AD, we conducted a nationwide Web-based survey of 3090 Japanese adult subjects diagnosed with AD, using a questionnaire to choose a pattern that most resembled their own out of 10 courses of AD severity. Patients in the 20s and 30s age groups tended to choose the option "gradually improved" or "improved by the age of 12", but patients in the 40s or older age group tended to choose the option "aggravation between the age of 12 and 19". Those who chose "AD development at age 20 or older" increased as the generation was older. This survey revealed that the time course of AD severity from birth to 19 years old varies depending on the generation targeted in this study. It is presumed that the acquired factors affecting the natural history of AD have changed over the past 50 years in Japan.

Survey of actual conditions of erythema marginatum as a prodromal symptom in Japanese patients with hereditary angioedema.

BACKGROUND: Hereditary angioedema (HAE) is a rare but life-threatening condition. HAE types I and II (HAE-1/2) result from C1-inhibitor (C1-INH) deficiency. However, recent genetic analysis has established a new type of HAE with normal C1-INH (HAEnC1-INH). The mutations of factor XII, plasminogen, angiopoietin 1, and kininogen 1 genes may be the cause of HAEnC1-INH. Nevertheless, other causative molecules (HAE-unknown) may be involved. The Japanese therapeutic environment for HAE has been improving owing to the self-subcutaneous injection of icatibant, which was approved for the treatment of acute attack and enables early therapy. Erythema marginatum (EM) is a visible prodromal symptom which occasionally occurs prior to an angioedema attack; hence, recognizing the risk of an acute attack is important for early treatment. However, the detailed characteristics of EM remain unclear. In this study, we first investigated the clinical manifestations of EM in Japanese patients with HAE. METHODS: A 20-point survey was developed and distributed to 40 physicians to gather clinical data on EM from patients with HAE. RESULTS: Data on 68 patients with HAE (58 patients with HAE-1/2 and 10 patients with HAE-unknown) were collected. Of the patients with HAE-1/2, 53.4% experienced EM, whereas 43.1% did not. The forearm was the most frequent area of EM (64.5%), followed by the abdomen (29.0%) and upper arm and precordium (19.3%). Of the HAE-1/2 patients with EM, 41.9% always had angioedema following EM, while 29.0% always had colocalization of EM with angioedema. Moreover, 3.2% showed a correlation between the awareness of EM and severity of an angioedema attack. In 60.9% of HAE-1/2 patients with EM, the interval between the awareness of EM and appearance of angioedema was <3 h. Of the patients with HAE-unknown, 30.0% also experienced EM. CONCLUSION: We confirmed that more than one-half of Japanese patients with HAE-1/2 and one-third of those with HAE-unknown develop EM as the prodromal symptom of an angioedema attack. Physicians should communicate the significance of EM to patients with HAE to prepare them for possible imminent attacks.

The practice of active patient involvement in rare disease research using ICT: experiences and lessons from the RUDY JAPAN project.

BACKGROUND: The role of patients in medical research is changing, as more emphasis is being placed on patient involvement, and patient reported outcomes are increasingly contributing to clinical decision-making. Information and communication technology provides new opportunities for patients to actively become involved in research. These trends are particularly noticeable in Europe and the US, but less obvious in Japan. The aim of this study was to investigate the practice of active involvement of patients in medical research in Japan by utilizing a digital platform, and to analyze the outcomes to clarify what specific approaches could be put into practice. METHODS: We developed the RUDY JAPAN system, an ongoing rare disease medical research platform, in collaboration with the Rare and Undiagnosed Diseases Study (RUDY) project in the UK. After 2 years of preparation, RUDY JAPAN was launched in December 2017. Skeletal muscle channelopathies were initially selected as target diseases, and hereditary angioedema was subsequently added. Several approaches for active patient involvement were designed through patient-researcher collaboration, namely the Steering Committee, questionnaire development, dynamic consent, and other communication strategies. We analyzed our practices and experiences focusing on how each approach affected and contributed to the research project. RESULTS: RUDY JAPAN has successfully involved patients in this research project in various ways. While not a part of the initial decision-making phase to launch the project, patients have increasingly been involved since then. A high level of patient involvement was achieved through the Steering Committee, a governance body that has made a major contribution to RUDY JAPAN, and the process of the questionnaire development. The creation of the Patient Network Forum, website and newsletter cultivated dialogue between patients and researchers. The registry itself allowed patient participation through data input and control of data usage through dynamic consent. CONCLUSIONS: We believe the initial outcomes demonstrate the feasibility and utility of active patient involvement in Japan. The collaboration realized through RUDY JAPAN was enabled by digital technologies. It allowed busy patients and researchers to find the space to meet and work together for the Steering Committee, questionnaire development and various communication activities. While the practice of active patient involvement in Japan is still in its early stages, this research confirms its viability if the right conditions are in place. (331 words).

Peritoneal injection of fucoidan suppresses the increase of plasma IgE induced by OVA-sensitization.

We previously reported that fucoidan, a dietary fiber purified from seaweed, inhibited IgE production by B cells in vitro. In this study, we examined the effect of fucoidan on IgE production in vivo. The OVA-induced increase of plasma IgE was significantly suppressed when fucoidan was intraperitoneally, but not orally, administered prior to the first immunization with OVA. The production of IL-4 and IFN-gamma in response to OVA in spleen cells isolated from OVA-sensitized mice treated with fucoidan in vivo was lower than that from mice treated without fucoidan. Moreover, the flow cytometric analysis and ELISpot assay revealed that the administration of fucoidan suppressed a number of IgE-expressing and IgE-secreting B cells, respectively. These results indicate that fucoidan inhibits the increase of plasma IgE through the suppression of IgE-producing B cell population, and the effect of fucoidan in vivo is crucially dependent on the route and timing of its administration.