Impact of fucosyltransferase 1-mediated epidermal blood group antigen H on anti-inflammatory response in atopic dermatitis.

The presence of the blood group H2 antigen on the membrane of red blood cells determines blood type O in individuals and this H2 antigen serves as a precursor to the A and B antigens expressed in blood types A and B, respectively. However, the specific involvement of ABH antigens in skin diseases is unknown. Therefore, we aim to investigate the expression of ABH antigens in skin tissue of patients with atopic dermatitis (AD) and MC903-induced AD-like mice. We demonstrated that the expression of ABH antigen is primarily located in the granular and horny layers of the skin in healthy control individuals. However, in patients with AD, the expression of the ABH antigen was absent or diminished in these layers, while the H2 antigen expression increased in the spinous layers of the affected skin lesions. Then, we investigated the biological function of blood group H antigen mediated by fucosyltransferase 1 (Fut1) in the skin, utilizing an AD mouse model induced by MC903 in wild-type (WT) and Fut1-knockout mice. After the application of MC903, Fut1-deficient mice, with no H2 antigen expression on their skin, exhibited more severe clinical signs, increased ear swelling, and elevated serum IgE levels compared with those of WT mice. Additionally, the MC903-induced thickening of both the epidermis and dermis was more pronounced in Fut1-deficient mice than that in WT mice. Furthermore, Fut1-deficient mice showed a significantly higher production of interleukin-4 (IL-4) and IL-6 in skin lesions compared with that of their WT counterparts. The expression of chemokines, particularly Ccl2 and Ccl8, was notably higher in Fut1-deficient mice compared with those of WT mice. The infiltration of CD4+ T cells, eosinophils, and mast cells into the lesional skin was significantly elevated in Fut1-deficient mice compared with that in WT mice. These findings demonstrate the protective role of H2 antigen expression against AD-like inflammation and highlight its potential therapeutic impact on AD through the regulation of blood group antigens.

Mapping the immune cell landscape of severe atopic dermatitis by single-cell RNA-seq.

BACKGROUND: Efforts to profile atopic dermatitis (AD) tissues have intensified, yet comprehensive analysis of systemic immune landscapes in severe AD remains crucial. METHODS: Employing single-cell RNA sequencing, we analyzed over 300,000 peripheral blood mononuclear cells from 12 severe AD patients (Eczema area and severity index (EASI) > 21) and six healthy controls. RESULTS: Results revealed significant immune cell shifts in AD patients, including increased Th2 cell abundance, reduced NK cell clusters with compromised cytotoxicity, and correlated Type 2 innate lymphoid cell proportions with disease severity. Moreover, unique monocyte clusters reflecting activated innate immunity emerged in very severe AD (EASI > 30). While overall dendritic cells (DCs) counts decreased, a distinct Th2-priming subset termed "Th2_DC" correlated strongly with disease severity, validated across skin tissue data, and flow cytometry with additional independent severe AD samples. Beyond the recognized role of Th2 adaptive immunity, our findings highlight significant innate immune cell alterations in severe AD, implicating their roles in disease pathogenesis and therapeutic potentials. CONCLUSION: Apart from the widely recognized role of Th2 adaptive immunity in AD pathogenesis, alterations in innate immune cells and impaired cytotoxic cells have also been observed in severe AD. The impact of these alterations on disease pathogenesis and the effectiveness of potential therapeutic targets requires further investigation.

Uncovering the impact of UV radiation on mitochondria in dermal cells: a STED nanoscopy study.

Mitochondria are essential organelles in cellular energy metabolism and other cellular functions. Mitochondrial dysfunction is closely linked to cellular damage and can potentially contribute to the aging process. The purpose of this study was to investigate the subcellular structure of mitochondria and their activities in various cellular environments using super-resolution stimulated emission depletion (STED) nanoscopy. We examined the morphological dispersion of mitochondria below the diffraction limit in sub-cultured human primary skin fibroblasts and mouse skin tissues. Confocal microscopy provides only the overall morphology of the mitochondrial membrane and an indiscerptible location of nucleoids within the diffraction limit. Conversely, super-resolution STED nanoscopy allowed us to resolve the nanoscale distribution of translocase clusters on the mitochondrial outer membrane and accurately quantify the number of nucleoids per cell in each sample. Comparable results were obtained by analyzing the translocase distribution in the mouse tissues. Furthermore, we precisely and quantitatively analyzed biomolecular distribution in nucleoids, such as the mitochondrial transcription factor A (TFAM), using STED nanoscopy. Our findings highlight the efficacy of super-resolution fluorescence imaging in quantifying aging-related changes on the mitochondrial sub-structure in cells and tissues.

Fucosylation deficiency enhances imiquimod-induced psoriasis-like skin inflammation by promoting CXCL1 expression.

Psoriasis is a multifaceted chronic inflammatory skin disease; however, its underlying molecular mechanisms remain unclear. In this study, we explored the role of fucosylation in psoriasis using an imiquimod-induced psoriasis-like mouse model. ABH antigen and fucosyltransferase 1 (Fut1) expression was reduced in the granular layer of lesional skin of patients with psoriasis. In particular, the blood group H antigen type 2 (H2 antigen)-a precursor of blood group A and B antigens-and FUT1 were highly expressed throughout the spinous layer in both patients with psoriasis and the skin of imiquimod-treated mice. Upon the application of imiquimod, Fut1-deficient mice, which lacked the H2 antigen, exhibited higher clinical scores based on erythema, induration, and scaling than those of wild-type mice. Imiquimod-treated Fut1-deficient mice displayed increased skin thickness, trans-epidermal water loss, and Gr-1+ cell infiltration compared with wild-type mice. Notably, the levels of CXCL1 protein and mRNA were significantly higher in Fut1-deficient mice than those in wild-type mice; however, there were no significant differences in other psoriasis-related markers, such as IL-1ß, IL-6, IL-17A, and IL-23. Fut1-deficient primary keratinocytes treated with IL-17A also showed a significant increase in both mRNA and protein levels of CXCL1 compared with IL-17A-treated wild-type primary keratinocytes. Further mechanistic studies revealed that this increased Cxcl1 mRNA in Fut1-deficient keratinocytes was caused by enhanced Cxcl1 mRNA stabilization. In summary, our findings indicated that fucosylation, which is essential for ABH antigen synthesis in humans, plays a protective role in psoriasis-like skin inflammation and is a potential therapeutic target for psoriasis.

Functionally similar genes exhibit comparable/similar time-course expression kinetics in the UV-induced photoaged mouse model.

Skin photoaging induced by ultraviolet (UV) irradiation contributes to the formation of thick and coarse wrinkles. Humans are exposed to UV light throughout their lives. Therefore, it is crucial to determine the time-sequential effects of UV on the skin. In this study, we irradiated the mouse back skin with UV light for eight weeks and observed the changes in gene expressions via microarray analysis every week. There were more downregulated genes (514) than upregulated genes (123). The downregulated genes had more functional diversity than the upregulated genes. Additionally, the number of downregulated genes did not increase in a time-dependent manner. Instead, time-dependent kinetic patterns were observed. Interestingly, each kinetic cluster harbored functionally enriched gene sets. Since collagen changes in the dermis are considered to be a major cause of photoaging, we hypothesized that other gene sets contributing to photoaging would exhibit kinetics similar to those of the collagen-regulatory genes identified in this study. Accordingly, co-expression network analysis was conducted using 11 well-known collagen-regulatory seed genes to predict genes with similar kinetics. We ranked all downregulated genes from 1 to 504 based on their expression levels, and the top 50 genes were suggested to be involved in the photoaging process. Additionally, to validate and support our identified top 50 gene lists, we demonstrated that the genes (FN1, CCDC80, PRELP, and TGFBR3) we discovered are downregulated by UV irradiation in cultured human fibroblasts, leading to decreased collagen levels, which is indicative of photoaging processes. Overall, this study demonstrated the time-sequential genetic changes in chronically UV-irradiated skin and proposed 50 genes that are involved in the mechanisms of photoaging.

Single-cell RNA sequencing identifies distinct transcriptomic signatures between PMA/ionomycin- and αCD3/αCD28-activated primary human T cells.

Immunologists have activated T cells in vitro using various stimulation methods, including phorbol myristate acetate (PMA)/ionomycin and αCD3/αCD28 agonistic antibodies. PMA stimulates protein kinase C, activating nuclear factor-κB, and ionomycin increases intracellular calcium levels, resulting in activation of nuclear factor of activated T cell. In contrast, αCD3/αCD28 agonistic antibodies activate T cells through ZAP-70, which phosphorylates linker for activation of T cell and SH2-domain-containing leukocyte protein of 76 kD. However, despite the use of these two different in vitro T cell activation methods for decades, the differential effects of chemical-based and antibody-based activation of primary human T cells have not yet been comprehensively described. Using single-cell RNA sequencing (scRNA-seq) technologies to analyze gene expression unbiasedly at the single-cell level, we compared the transcriptomic profiles of the non-physiological and physiological activation methods on human peripheral blood mononuclear cell-derived T cells from four independent donors. Remarkable transcriptomic differences in the expression of cytokines and their respective receptors were identified. We also identified activated CD4 T cell subsets (CD55+) enriched specifically by PMA/ionomycin activation. We believe this activated human T cell transcriptome atlas derived from two different activation methods will enhance our understanding, highlight the optimal use of these two in vitro T cell activation assays, and be applied as a reference standard when analyzing activated specific disease-originated T cells through scRNA-seq.

Disruption of the endopeptidase ADAM10-Notch signaling axis leads to skin dysbiosis and innate lymphoid cell-mediated hair follicle destruction.

Hair follicles (HFs) function as hubs for stem cells, immune cells, and commensal microbes, which must be tightly regulated during homeostasis and transient inflammation. Here we found that transmembrane endopeptidase ADAM10 expression in upper HFs was crucial for regulating the skin microbiota and protecting HFs and their stem cell niche from inflammatory destruction. Ablation of the ADAM10-Notch signaling axis impaired the innate epithelial barrier and enabled Corynebacterium species to predominate the microbiome. Dysbiosis triggered group 2 innate lymphoid cell-mediated inflammation in an interleukin-7 (IL-7) receptor-, S1P receptor 1-, and CCR6-dependent manner, leading to pyroptotic cell death of HFs and irreversible alopecia. Double-stranded RNA-induced ablation models indicated that the ADAM10-Notch signaling axis bolsters epithelial innate immunity by promoting ß-defensin-6 expression downstream of type I interferon responses. Thus, ADAM10-Notch signaling axis-mediated regulation of host-microbial symbiosis crucially protects HFs from inflammatory destruction, which has implications for strategies to sustain tissue integrity during chronic inflammation.

A new treatment protocol of microfocused ultrasound for lower eyelid fat bulging.

BACKGROUND: Microfocused ultrasound (MFU) causes tissue tightening by producing thermal injury zones and is used to treat various age-related changes including lower eyelid fat bulging. OBJECTIVE: To investigate the efficacy of a new treatment protocol of MFU for lower eyelid fat bulging. METHODS AND MATERIALS: We reviewed the medical records of all patients who began MFU for lower eyelid fat bulging from March 2017 to September 2018. MFU was performed in two steps to tighten the lower eyelid dermis and orbital septum. Data on age, sex, bulging severity, and the number of treatment sessions were obtained. Associations of these variables with treatment response were determined through an ordinal logistic regression analysis. RESULTS: Among 191 enrolled patients, 119 (62.3%) and 47 (24.6%) achieved fair and good responses, respectively. In the multivariable analysis, multiple treatment sessions (odds ratio (OR) 6.618; 95% confidence interval (CI) 3.242-13.513; p < .001), moderate bulging (OR 4.328; 95% CI 1.755-10.671; p = .001), and severe bulging (OR 7.570; 95% CI 2.537-22.585; p < .001) were associated with greater treatment response. There were no serious adverse events. CONCLUSIONS: The new treatment protocol of MFU is an effective and safe strategy for lower eyelid fat bulging.

Microfluidic skin chip with vasculature for recapitulating the immune response of the skin tissue.

There is a considerable need for cell-based in vitro skin models for studying dermatological diseases and testing cosmetic products, but current in vitro skin models lack physiological relevance compared to human skin tissue. For example, many dermatological disorders involve complex immune responses, but current skin models are not capable of recapitulating the phenomena. Previously, we reported development of a microfluidic skin chip with a vessel structure and vascular endothelial cells. In this study, we cocultured dermal fibroblasts and keratinocytes with vascular endothelial cells, human umbilical vascular endothelial cells. We verified the formation of a vascular endothelium in the presence of the dermis and epidermis layers by examining the expression of tissue-specific markers. As the vascular endothelium plays a critical role in the migration of leukocytes to inflammation sites, we incorporated leukocytes in the circulating media and attempted to mimic the migration of neutrophils in response to external stimuli. Increased secretion of cytokines and migration of neutrophils was observed when the skin chip was exposed to ultraviolet irradiation, showing that the microfluidic skin chip may be useful for studying the immune response of the human tissue.

Targeted therapy guided by single-cell transcriptomic analysis in drug-induced hypersensitivity syndrome: a case report.

Drug-induced hypersensitivity syndrome/drug reaction with eosinophilia and systemic symptoms (DiHS/DRESS) is a potentially fatal multiorgan inflammatory disease associated with herpesvirus reactivation and subsequent onset of autoimmune diseases1-4. Pathophysiology remains elusive and therapeutic options are limited. Cases refractory to corticosteroid therapy pose a clinical challenge1,5 and approximately 30% of patients with DiHS/DRESS develop complications, including infections and inflammatory and autoimmune diseases1,2,5. Progress in single-cell RNA sequencing (scRNA-seq) provides an opportunity to dissect human disease pathophysiology at unprecedented resolutions6, particularly in diseases lacking animal models, such as DiHS/DRESS. We performed scRNA-seq on skin and blood from a patient with refractory DiHS/DRESS, identifying the JAK-STAT signaling pathway as a potential target. We further showed that central memory CD4+ T cells were enriched with DNA from human herpesvirus 6b. Intervention via tofacitinib enabled disease control and tapering of other immunosuppressive agents. Tofacitinib, as well as antiviral agents, suppressed culprit-induced T cell proliferation in vitro, further supporting the roles of the JAK-STAT pathway and herpesviruses in mediating the adverse drug reaction. Thus, scRNA-seq analyses guided successful therapeutic intervention in the patient with refractory DiHS/DRESS. scRNA-seq may improve our understanding of complicated human disease pathophysiology and provide an alternative approach in personalized medicine.

IL-17A-Producing Innate Lymphoid Cells Promote Skin Inflammation by Inducing IL-33-Driven Type 2 Immune Responses.

Atopic dermatitis (AD) is a chronic, pruritic, inflammatory skin disease characterized by type 2 cytokines secreted by T helper type 2 cells and group 2 innate lymphoid cells. Despite a high degree of heterogeneity, AD is still explained by type 2 immunity, and the role of IL-17A, which is increased in acute, pediatric, or Asian patients with AD, remains poorly understood. Here, we aimed to investigate the role of IL-17A-producing group 3 innate lymphoid cells (ILC3s), which are unexplored immune cells, in the pathogenesis of AD. We found that the numbers of ILC3s in the skin of AD-induced mice were increased, and that neutralizing IL-17A delayed development of AD. Moreover, adoptive transfer of ILC3s accelerated the symptoms of AD. Mechanically, ILC3s induced IL-33 production by nonimmune skin cells, keratinocytes, and fibroblasts, which promoted type 2 immune responses. Because AD has a complex pathophysiology and a broad spectrum of clinical phenotypes, the presence of ILC3s in the skin and their interaction with nonimmune skin cells could explain the pathogenesis of cutaneous AD.

Comparison of the Gut Microbiota of Centenarians in Longevity Villages of South Korea with Those of Other Age Groups.

Several studies have attempted to identify factors associated with longevity and maintenance of health in centenarians. In this study, we analyzed and compared the gut microbiota of centenarians in longevity villages with the elderly and adults in the same region and urbanized towns. Fecal samples were collected from centenarians, elderly, and young adults in longevity villages, and the gut microbiota sequences of elderly and young adults in urbanized towns of Korea were obtained from public databases. The relative abundance of Firmicutes was found to be considerably higher in subjects from longevity villages than those from urbanized towns, whereas Bacteroidetes was lower. Age-related rearrangement of gut microbiota was observed in centenarians, such as reduced proportions of Faecalibacterium and Prevotella, and increased proportion of Escherichia, along with higher abundances of Akkermansia, Clostridium, Collinsella, and uncultured Christensenellaceae. Gut microbiota of centenarians in rehabilitation hospital were also different to those residing at home. These differences could be due to differences in diet patterns and living environments. In addition, phosphatidylinositol signaling system, glycosphingolipid biosynthesis, and various types of N-glycan biosynthesis were predicted to be higher in the gut microbiota of centenarians (corrected p < 0.05). These three metabolic pathways of gut microbiota can be associated with the immune status and healthy gut environment of centenarians. Although further studies are necessary to validate the function of microbiota between groups, this study provides valuable information on centenarians' gut microbiota.

Inhibition of N-glycosylation by tunicamycin attenuates cell-cell adhesion via impaired desmosome formation in normal human epidermal keratinocytes.

N-Glycosylation affects protein functions such as location, stability, and susceptibility to proteases. Desmosomes in keratinocytes are essential to maintain epidermal tissue integrity to protect against environmental insults. However, it is not yet known whether N-glycosylation affects desmosomal functions in primary keratinocytes. Tunicamycin is an inhibitor of N-glycosylation that has been a useful tool in glycobiology. Therefore, we investigated the effect of inhibiting N-glycosylation by tunicamycin treatment on desmosomes in primary keratinocytes. In our experiments, cell-cell adhesive strength was reduced in tunicamycin-treated primary keratinocytes. TEM showed that desmosome formation was impaired by tunicamycin. Desmogleins (Dsgs) 1 and 3, which constitute the core structure of desmosomes, were well transported to the cell-cell borders, but the amount decreased and showed an aberrant distribution at the cell borders in tunicamycin-treated keratinocytes. The stability of both desmoglein proteins was also reduced, and they were degraded through both proteasomal and lysosomal pathways, although inhibiting degradation did not restore the cell-cell adhesion. Finally, tunicamycin induced desmosomal instability, enhancing their disassembly. In conclusion, these results indicate that N-glycosylation is critical to the desmosome complex to maintain cell-cell adhesive strength in primary keratinocytes.

Urban particulate matter in air pollution penetrates into the barrier-disrupted skin and produces ROS-dependent cutaneous inflammatory response in vivo.

BACKGROUND: Particulate matter (PM) is an integral part of air pollution, which is a mixture of particles suspended in the air. Recently, it has been reported that PM is associated with increased risks of skin diseases, especially atopic dermatitis in children. However, it is unclear if PM directly goes into the skin and what mechanisms are involved in response to PM. OBJECTIVE: To see whether PM could penetrate into the barrier-disrupted skin, produce reactive oxygen species (ROS), and elicit an inflammatory response. METHODS: We collected PMs during a winter in Seoul and used cultured keratinocytes for in vitro study and tape-stripped BALB/c mice for in vivo study. RESULTS: Keratinocyte cytotoxicity increased in a dose-dependent manner by PM treatment. IL-8 and MMP-1 mRNA expression and protein levels were significantly increased compared to control by qPCR and ELISA, respectively. Cellular ROS production was increased by PM treatment, and antioxidant N-acetyl cysteine pretreatment prevented induction of inflammatory cytokines IL-8 and MMP-1. In PM-treated keratinocytes, electron-dense subcellular particles were observed by transmission electron microscopy. PM was observed inside hair follicles in both intact and barrier-disrupted skin in vivo. Additionally, intercellular penetration of PM was seen in the barrier-disrupted skin. Repeated PM application induced epidermal thickening and dermal inflammation with neutrophil infiltration. Finally, N-acetyl cysteine could ameliorate skin inflammation induced by PM application. CONCLUSION: PM penetrates into the barrier-disrupted skin, causing inflammation, demonstrating detrimental effects in the skin.

Imiquimod-applied Interleukin-10 deficient mice better reflects severe and persistent psoriasis with systemic inflammatory state.

Previous studies have shown that imiquimod-induced psoriasis-like skin inflammation in mice resembles phenotypic changes and cytokine profiles of human psoriasis. However, a psoriasis animal model reflecting the chronic inflammatory course and comorbidities has not yet been established. We aimed to evaluate the imiquimod-applied interleukin (IL)-10 deficient mouse model in comparison with previous models. IL-10 deficient and wild-type (WT) mice received either imiquimod or vehicle cream for 12 days and were sacrificed on day 15. For earlier time point data, either imiquimod or vehicle cream was applied for 2 days, and the mice were sacrificed on day 3. Imiquimod-applied IL-10 deficient mice showed more persistent psoriasis-like inflammation and higher severity index than did WT between day 8 and 15. Histopathologically, they demonstrated significantly thicker epidermis and larger number of CD45+, myeloperoxidase+ and IL-17+ cell counts on day 15. Quantitative reverse transcription-polymerase chain reaction with skin tissue revealed significantly higher imiquimod-induced IL-23p19 expression in imiquimod-applied IL-10 deficient mice on day 15. IL-10 deficient mice also showed significantly higher serum levels of imiquimod-induced IL-17A and tumor necrosis factor-α by enzyme-linked immunosorbent assay on day 15. Furthermore, IL-10 deficient mice showed more prominent increase of spleen weight and decrease of body weight in response to imiquimod application on day 3 and 15. In conclusion, IL-10 deficient mice model with imiquimod application may better reflect severe and persistent psoriasis with systemic inflammatory state.