A fluorescence imaging technique suggests that sweat leakage in the epidermis contributes to the pathomechanism of palmoplantar pustulosis.

Sweat is an essential protection system for the body, but its failure can result in pathologic conditions, including several skin diseases, such as palmoplantar pustulosis (PPP). As reduced intraepidermal E-cadherin expression in skin lesions was confirmed in PPP skin lesions, a role for interleukin (IL)-1-rich sweat in PPP has been proposed, and IL-1 has been implicated in the altered E-cadherin expression observed in both cultured keratinocytes and mice epidermis. For further investigation, live imaging of sweat perspiration on a mouse toe-pad under two-photon excitation microscopy was performed using a novel fluorescent dye cocktail (which we named JSAC). Finally, intraepidermal vesicle formation which is the main cause of PPP pathogenesis was successfully induced using our "LASER-snipe" technique with JSAC. "LASER-snipe" is a type of laser ablation technique that uses two-photon absorption of fluorescent material to destroy a few acrosyringium cells at a pinpoint location in three-dimensional space of living tissue to cause eccrine sweat leakage. These observatory techniques and this mouse model may be useful not only in live imaging for physiological phenomena in vivo such as PPP pathomechanism investigation, but also for the field of functional physiological morphology.

Nuclear IL-33 Plays an Important Role in EGFR-Mediated Keratinocyte Migration by Regulating the Activation of Signal Transducer and Activator of Transcription 3 and NF-κB.

Nuclear IL-33 levels are high at the epidermal edges of skin wounds and facilitate wound healing. However, IL-33-mediated regulation of keratinocyte (KC) biology during wound healing remains poorly understood. During skin-wound healing, KC migration and re-epithelialization are mediated predominantly by EGFR signaling activation and depend on the function of signal transducer and activator of transcription 3 (STAT3). We found that migrating KCs at the leading edges of mouse skin wounds exhibited concomitant induction and nuclear colocalization of IL-33 and phosphorylated STAT3. In cultured human KCs, activation of EGFR signaling caused rapid elevation of nuclear IL-33, which directly interacts with phosphorylated STAT3, promoting STAT3 activation. In vitro KC migration and wound-healing assays revealed that high nuclear IL-33 levels were required for KC migration and wound closure. KC mobility associated with a lack of suprabasal epidermal keratins and extracellular matrix degradation mediated by matrix metalloproteinases (MMPs) control cell migration at the intracellular and extracellular levels, respectively. In EGFR-activated KCs, nuclear IL-33 mediated keratin 1 and 10 downregulation and MMP9 upregulation by promoting STAT3 activation and limited MMP1, MMP3, and MMP10 induction by suppressing NF-κB transactivation. Thus, epidermal nuclear IL-33 is involved in KC migration and wound closure by regulating the STAT3 and NF-κB pathways.

Highly concentrated trehalose induces prohealing senescence-like state in fibroblasts via CDKN1A/p21.

Trehalose is the nonreducing disaccharide of glucose, evolutionarily conserved in invertebrates. The living skin equivalent (LSE) is an organotypic coculture containing keratinocytes cultivated on fibroblast-populated dermal substitutes. We demonstrated that human primary fibroblasts treated with highly concentrated trehalose promote significantly extensive spread of the epidermal layer of LSE without any deleterious effects. The RNA-seq analysis of trehalose-treated 2D and 3D fibroblasts at early time points revealed the involvement of the CDKN1A pathway, the knockdown of which significantly suppressed the upregulation of DPT, ANGPT2, VEGFA, EREG, and FGF2. The trehalose-treated fibroblasts were positive for senescence-associated ß-galactosidase. Finally, transplantation of the dermal substitute with trehalose-treated fibroblasts accelerated wound closure and increased capillary formation significantly in the experimental mouse wounds in vivo, which was canceled by the CDKN1A knockdown. These data indicate that high-concentration trehalose can induce the senescence-like state in fibroblasts via CDKN1A/p21, which may be therapeutically useful for optimal wound repair.

Ceramide Analysis in Combination With Genetic Testing May Provide a Precise Diagnosis for Self-Healing Collodion Babies.

Self-healing collodion baby (SHCB), also called "self-improving collodion baby", is a rare mild variant of autosomal recessive congenital ichthyosis and is defined as a collodion baby who shows the nearly complete resolution of scaling within the first 3 months to 1 year of life. However, during the neonatal period, it is not easy to distinguish SHCB from other inflammatory forms of autosomal recessive congenital ichthyosis, such as congenital ichthyosiform erythroderma. Here, we report a case study of two Japanese SHCB patients with compound heterozygous mutations, c.235G>T (p.(Glu79∗))/ c.1189C>T (p.(Arg397Cys)) and c.1295A>G (p.(Tyr432Cys))/ c.1138delG (p.(Asp380Thrfs∗3)), in CYP4F22, which encodes cytochrome P450, family 4, subfamily F, polypeptide 22 (CYP4F22). Immunohistochemically, inflammation with the strong expression of IL-17C, IL-36γ, and TNF-α was seen in the skin at birth. CYP4F22 is an ultra-long-chain FA ω-hydroxylase responsible for ω-O-acylceramide (acylceramide) production. Among the epidermal ceramides, acylceramide is a key lipid in maintaining the epidermal permeability barrier function. We found that the levels of ceramides with ω-hydroxy FAs including acylceramides and the levels of protein-bound ceramides were much lower in stratum corneum samples obtained by tape stripping from SHCB patients than in those from their unaffected parents and individuals without SHCB. Additionally, our cell-based enzyme assay revealed that two mutants, p.(Glu79∗) and p.(Arg397Cys), had no enzyme activity. Our findings suggest that genetic testing coupled with noninvasive ceramide analyses using tape-stripped stratum corneum samples might be useful for the early and precise diagnosis of congenital ichthyoses, including SHCB.

EGFR ligands synergistically increase IL-17A-induced expression of psoriasis signature genes in human keratinocytes via IκBζ and Bcl3.

Various epidermal growth factor receptor (EGFR) ligands are highly expressed in the epidermis of psoriasis lesions, and abnormal EGFR activation appears to be involved in the pathogenesis of psoriasis. However, how EGFR signaling contributes to the development of psoriasis is unclear. Interleukin (IL)-17A, a critical effector of the IL-23/IL-17A pathway, increases the expression of psoriasis signature genes in keratinocytes and plays an essential role in the pathogenesis of psoriasis by inducing IκBζ, a critical transcriptional regulator in psoriasis. In this study, we stimulated primary human keratinocytes with IL-17A and various EGFR ligands to investigate whether EGFR ligands regulate the expression of psoriasis signature genes. In cultured normal human keratinocytes and a living skin equivalent, EGFR ligands did not induce psoriasis-related gene expression, but significantly enhanced the IL-17A-mediated induction of various psoriasis signature genes, including antimicrobial peptides, cytokines, and chemokines. This was dependent on an EGFR activation-mediated synergistic increase in IL-17A-induced IκBζ expression and was partially mediated by the EGFR-dependent upregulation of Bcl3. Therefore, EGFR ligands can act as synergistic agents of IL-17A signaling by stimulating the epidermal production of psoriasis signature genes in psoriasis lesions. This study reveals a potential mechanism by which EGFR signaling contributes to the pathogenesis of psoriasis.

TSLP Impairs Epidermal Barrier Integrity by Stimulating the Formation of Nuclear IL-33/Phosphorylated STAT3 Complex in Human Keratinocytes.

Atopic dermatitis (AD) is an inflammatory skin disease characterized by skin barrier dysfunction. Although T helper type 2 cytokines downregulate the expression of epidermal barrier proteins, the signaling mechanism underlying these effects remains unclear. IL-33, a chromatin-associated cytokine, is highly expressed in the nuclei of epidermal keratinocytes in AD skin; however, it is unclear whether this expression promotes the development of AD. TSLP, an epithelial cells-derived pro‒T helper type 2 cytokine, is elevated in the epidermis of patients with AD. TSLP affects the pathogenesis of AD by activating T helper type 2 responses and impairing epidermal barrier integrity. In this study, we stimulated postconfluent human keratinocytes and living skin equivalent with TSLP to investigate the role of nuclear IL-33 in TSLP-induced epidermal barrier defects. We observed that TSLP reduced the levels of FLG, hBD2, S100A7, and claudin-1, which required nuclear IL-33 expression. Similar to the T helper type 2 cytokines IL-4, IL-13, and IL-31, TSLP was shown to upregulate IL-33 expression and triggered the formation of nuclear IL-33/phosphorylated signal transducer and activator of transcription 3 complex, which bound to the FLG promoter, thereby inhibiting transcription. Moreover, nuclear IL-33 acted as a cofactor of signal transducer and activator of transcription 3 in the TSLP-induced transcriptional repression of hBD2, S100A7, and claudin-1. Therefore, epidermal nuclear IL-33 may be a key regulator of TSLP-mediated epidermal barrier dysfunction.

Flare-up of generalized pustular psoriasis combined with systemic capillary leak syndrome after coronavirus disease 2019 mRNA vaccination.

Generalized pustular psoriasis (GPP) is characterized by acute flare-ups induced by various factors, but few reports have described GPP onset or flare-up induced by vaccination. To our knowledge, only three such cases following coronavirus disease 2019 (COVID-19) vaccination have been reported. We herein report a case of GPP flare-up after COVID-19 mRNA vaccination. A 65-year-old man with GPP controlled by infliximab presented with widespread pustular erythema, fever, and malaise following his second COVID-19 mRNA vaccination. A skin eruption was apparent at the injection site. He also exhibited systemic capillary leak syndrome (SCLS), which responded rapidly to secukinumab and systemic corticosteroids. Two biopsies, one of which was of the injection site, revealed not only findings typical of GPP, but also a dermal mixed-cell infiltration with eosinophils, and microthrombi in the small dermal vessels. The latter findings have been observed in cutaneous lesions induced by both COVID-19 infection and vaccination. This is the first case of a GPP flare-up accompanied by SCLS induced by a COVID-19 mRNA vaccine. Also, this is the first flare-up induced by the second vaccine dose, and the first such report including detailed histological data, including for the injection site.

Locus-specific induction of gene expression from heterochromatin loci during cellular senescence.

Senescence is a fate-determined state, accompanied by reorganization of heterochromatin. Although lineage-appropriate genes can be temporarily repressed through facultative heterochromatin, stable silencing of lineage-inappropriate genes often involves the constitutive heterochromatic mark, histone H3 lysine 9 trimethylation (H3K9me3). The fate of these heterochromatic genes during senescence is unclear. In the present study, we show that a small number of lineage-inappropriate genes, exemplified by the LCE2 skin genes, are derepressed during senescence from H3K9me3 regions in fibroblasts. DNA FISH experiments reveal that these gene loci, which are condensed at the nuclear periphery in proliferative cells, are decompacted during senescence. Decompaction of the locus is not sufficient for LCE2 expression, which requires p53 and C/EBPß signaling. NLRP3, which is predominantly expressed in macrophages from an open topologically associated domain (TAD), is also derepressed in senescent fibroblasts due to the local disruption of the H3K9me3-rich TAD that contains it. NLRP3 has been implicated in the amplification of inflammatory cytokine signaling in senescence and aging, highlighting the functional relevance of gene induction from 'permissive' H3K9me3 regions in senescent cells.

Nuclear IL-33 Plays an Important Role in IL-31‒Mediated Downregulation of FLG, Keratin 1, and Keratin 10 by Regulating Signal Transducer and Activator of Transcription 3 Activation in Human Keratinocytes.

IL-33, a chromatin-associated multifunctional cytokine, is implicated in the pathogenesis of atopic dermatitis (AD), an inflammatory skin disorder characterized by skin barrier dysfunction. IL-33 accumulates in the nuclei of epidermal keratinocytes (KCs) in AD lesions. However, it is unclear whether nuclear IL-33 directly contributes to the pathogenesis of AD. IL-31, a pruritogenic cytokine primarily produced by T helper type 2 cells, is elevated in AD lesions and promotes AD development by suppressing KC differentiation and inducing itching. In this study, we investigated the involvement of nuclear IL-33 in IL-31‒mediated suppression of KC differentiation. In monolayer cultures and living skin equivalent, IL-31 increased the expression of full-length IL-33 and the phosphorylation of signal transducer and activator of transcription 3 (STAT3) in the nuclei of human KCs, which in turn downregulated the expression of differentiation markers. We found that IL-31 and IL-4/IL-13 use very similar mechanisms to inhibit KC differentiation: nuclear IL-33 combines with phosphorylated STAT3 and functions as a STAT3 transcription cofactor, promoting phosphorylated STAT3 binding to the FLG promoter to inhibit its transcription; moreover, the nuclear IL-33/phosphorylated STAT3 complex drives the downregulation of keratin 1 and keratin 10 by reducing the availability of the transcription factor RunX1. Therefore, nuclear IL-33 plays an important role in IL-31‒mediated differentiation suppression by regulating STAT3 activation in human KCs.

EGFR-mediated epidermal stem cell motility drives skin regeneration through COL17A1 proteolysis.

Skin regenerative capacity declines with age, but the underlying mechanisms are largely unknown. Here we demonstrate a functional link between epidermal growth factor receptor (EGFR) signaling and type XVII collagen (COL17A1) proteolysis on age-associated alteration of keratinocyte stem cell dynamics in skin regeneration. Live-imaging and computer simulation experiments predicted that human keratinocyte stem cell motility is coupled with self-renewal and epidermal regeneration. Receptor tyrosine kinase array identified the age-associated decline of EGFR signaling in mouse skin wound healing. Culture experiments proved that EGFR activation drives human keratinocyte stem cell motility with increase of COL17A1 by inhibiting its proteolysis through the secretion of tissue inhibitor of metalloproteinases 1 (TIMP1). Intriguingly, COL17A1 directly regulated keratinocyte stem cell motility and collective cell migration by coordinating actin and keratin filament networks. We conclude that EGFR-COL17A1 axis-mediated keratinocyte stem cell motility drives epidermal regeneration, which provides a novel therapeutic approach for age-associated impaired skin regeneration.

Case of pemphigoid vegetans with autoantibodies against the BP180 C-terminal domain and BP230 antigen.

Pemphigoid vegetans is a rare variant of bullous pemphigoid characterized by vegetative and purulent lesions of the groin, axillae, thighs, hands, eyelids, and perioral regions. The clinical features and histological findings of pemphigus vegetans and immunohistochemical characteristics of bullous pemphigoid are shown. An 86-year-old woman presented with vegetative lesions in the vulva and groin, and blisters on the head, neck, axillae, and thighs. Although clinically suspected as pemphigus vegetans, skin biopsy showed subepidermal clefts with eosinophil infiltration and eosinophilic pustules in the epidermis. Direct immunofluorescence analysis showed linear deposition of immunoglobulin (Ig)G along the basement membrane zone. Indirect immunofluorescence using 1 mol/L NaCl salt-split skin showed linear deposition of IgG on the epidermal side. Chemiluminescent enzyme immunoassay and enzyme-linked immunosorbent assay were positive for BP180 and BP230. Immunoblotting of recombinant protein of the BP180 C-terminal domain showed positive reactivity. Pemphigoid vegetans was diagnosed and treated successfully with oral corticosteroids. This is the first case of pemphigoid vegetans reported to date with detection of autoantibodies against both BP180 C-terminal domain and BP230.

Nuclear IL-33 Plays an Important Role in the Suppression of FLG, LOR, Keratin 1, and Keratin 10 by IL-4 and IL-13 in Human Keratinocytes.

IL-33 is a chromatin-associated multifunctional cytokine implicated in the pathogenesis of atopic dermatitis (AD), an inflammatory skin disorder characterized by skin barrier dysfunction. The previous reports show that IL-33 is highly detected in the nucleus of epidermal keratinocytes in AD lesions compared with that in unaffected or normal skin. However, it is unclear whether intracellular IL-33 directly contributes to the pathogenesis of AD. T helper type 2 cytokines IL-4 and IL-13 that are elevated in AD lesions suppress keratinocyte differentiation to impair skin barrier function. We investigated whether intracellular IL-33 is involved in IL-4 and IL-13 function. In monolayer culture and living skin equivalent analyses, IL-4 and IL-13 increased the expression of full-length IL-33 in the nucleus of keratinocytes by activating the MAPK/extracellular signal‒regulated kinase kinase/extracellular signal‒regulated kinase signaling pathway, which is necessary for the inhibition of differentiation markers FLG, LOR, keratin 1, and keratin 10. The nuclear IL-33 functions as a transcription cofactor of signal transducer and activator of transcription 3, increasing the binding of phosphorylated signal transducer and activator of transcription 3 to FLG promoter, thereby inhibiting its transcription, and it inhibits the expression of transcription factor RUNX1 by signal transducer and activator of transcription 3 and signal transducer and activator of transcription 6, thereby downregulating LOR, keratin 1, and keratin 10. Thus, the elevated nuclear IL-33 in the epidermis of AD lesions may be involved in the pathogenesis of AD by inhibiting keratinocyte differentiation and skin barrier function.