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.

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.

Bcl-3 induced by IL-22 via STAT3 activation acts as a potentiator of psoriasis-related gene expression in epidermal keratinocytes.

IL-22 induces STAT3 phosphorylation and mediates psoriasis-related gene expression. However, the signaling mechanism leading from pSTAT3 to the expression of these genes remains unclear. We focused on Bcl-3, which is induced by STAT3 activation and mediates gene expression. In cultured human epidermal keratinocytes, IL-22 increased Bcl-3, which was translocated to the nucleus with p50 via STAT3 activation. The increases in CXCL8, S100As and human ß-defensin 2 mRNA expression caused by IL-22 were abolished by siRNA against Bcl-3. Although CCL20 expression was also augmented by IL-22, the knockdown of Bcl-3 increased its level. Moreover, the combination of IL-22 and IL-17A enhanced Bcl-3 production, IL-22-induced gene expression, and the expression of other psoriasis-related genes, including those encoding IL-17C, IL-19, and IL-36γ. The expression of these genes (except for CCL20) was also suppressed by the knockdown of Bcl-3. Bcl-3 overexpression induced CXCL8 and HBD2 expression but not S100As expression. We also compared Bcl-3 expression between psoriatic skin lesions and normal skin. Immunostaining revealed strong signals for Bcl-3 and p50 in the nucleus of epidermal keratinocytes from psoriatic skin. The IL-22-STAT3-Bcl-3 pathway may be important in the pathogenesis of psoriasis.

Heparinoid suppresses Der p-induced IL-1ß production by inhibiting ERK and p38 MAPK pathways in keratinocytes.

Epidermal keratinocytes initiate skin inflammation by activating immune cells. The skin barrier is disrupted in atopic dermatitis (AD) and epidermal keratinocytes can be exposed to environmental stimuli, such as house dust mite (HDM) allergens. We showed previously that HDM allergens activate the NLRP3 inflammasome of keratinocytes, thereby releasing pro-inflammatory cytokines. Heparinoid is an effective moisturizer for atopic dry skin. However, a recent report showed that heparinoid treatment can improve inflammation of lichen planus. Therefore, we hypothesized that it acts on epidermal keratinocytes not only as a moisturizer, but also as a suppressant of the triggers of skin inflammation. We found that HDM allergen-induced interleukin (IL)-1ß release from keratinocytes was inhibited significantly by heparinoid pretreatment without affecting cell viability. However, heparinoid did not affect caspase-1 release, suggesting that heparinoid did not affect HDM allergen-induced inflammasome activation. Heparinoid treatment not only decreased intracellular levels of pro-IL-1ß, but also suppressed IL-1ß messenger RNA (mRNA) expression in keratinocytes. Among the intracellular signalling pathways, the activation of extracellular signal-regulated kinase and p38 pathways, which are required for IL-1ß expression in keratinocytes, was inhibited by heparinoid treatment. The inhibitory effect of heparinoid on IL-1ß mRNA expression was also confirmed with living skin equivalents. Our results demonstrated that heparinoid suppresses the initiation of keratinocyte-mediated skin inflammation.

The microbiome of the "sterile" pustules in palmoplantar pustulosis.

The skin microbiome influences skin pathophysiology. Palmoplantar pustulosis (PPP) is a chronic skin disease characterized by infectious-like pustules on the palms and soles. These pustules are thought to be sterile because bacterial cultures obtained from the pustules are negative. However, culture methods are limited in their ability to identify all bacteria on the skin. We hypothesized that the "sterile" pustules of PPP do not lack bacteria, but rather contain a microbiome. To test this hypothesis, we identified bacteria in "sterile" pustules using non-culture methods. We conducted Sanger and 16S rRNA sequencing using primers specific to the V1-V2 region in PPP-pustulovesicles (PVs) (n = 43) and pompholyx vesicle fluids (n = 15). Sanger sequencing identified some Staphylococcus, Propionibacterium, Streptococcus and Pyrinomonas species in PPP-PVs but failed to identify any bacteria in most of the pompholyx vesicles. 16S rRNA sequencing of PPP-PVs indicated the presence of a microbiome that included various phyla, including Firmicutes, Proteobacteria, Actinobacteria and Bacteroidetes. At the genus level, smokers had higher levels of Staphylococcus in PPP-PVs compared with non-smokers. These results indicate that a microbiome exists in "sterile" pustules of PPP and that PPP smokers had higher levels of Staphylococcus in pustules. It is therefore necessary to reconsider the pathogenesis of PPP from the perspective of the microbiome.

A Successful Switch From Ustekinumab to an Extended Dosing Interval of Guselkumab Without Induction in a Patient With Psoriasis Vulgaris.

Psoriasis vulgaris, also known as plaque-type psoriasis, is the most common form of psoriasis. It is characterized by erythematous plaques covered with scales. Among the available treatments, the fully human monoclonal antibodies ustekinumab (UST) and guselkumab (GUS) have low immunogenicity. Additionally, GUS has not been found to have a significant risk of inducing the development of clinically relevant neutralizing antibodies. Therefore, we sometimes consider switching to GUS when UST is insufficiently effective. However, switching to another biological agent usually requires an induction phase, potentially incurring additional costs. We herein present the first case of a successful transition from UST 90 mg to an extended dosing interval of GUS without an induction phase. This approach may be a viable and cost-saving option, especially for patients with relatively low disease activity.

Paradoxical Eczema Associated With Interleukin-17A Inhibitor Use in a Patient With Generalized Pustular Psoriasis Accompanied by Asthma.

Although rare, paradoxical eczema (PE) is an adverse event associated with the use of biological agents to treat psoriasis, particularly in patients with atopic predispositions. We report the first case of severe PE induced by secukinumab in a patient with generalized pustular psoriasis (GPP) and asthma. A woman in her 50s with a history of interstitial nephritis attributable to Sjogren's syndrome experienced a flare-up of GPP after discontinuing mycophenolate mofetil and was hospitalized. Treatment with secukinumab accompanied by an increased prednisolone level afforded rapid improvement, but she subsequently developed widespread, itchy, serous papules and erythema. A biopsy confirmed that the erythema was an eczematous reaction, thus PE. Her condition improved after switching from secukinumab to deucravacitinib with a temporary increase in the prednisolone level; no recurrence of GPP or PE was observed for 11 months. Elevated serum levels of interleukin (IL)-17A, IL-22, and the Th2 chemokine TARC recorded at the onset time of PE suggested that these mediators contributed to the observed pathology. Our case highlights the need for careful consideration when prescribing IL-17 inhibitors to patients with GPP, particularly those with atopic predispositions, given the potential activation of the Th2 axis and thus severe eczematous reactions. Further research is required to understand the essential nature of PE in patients with GPP and the roles of IL-17A and IL-22 in this context.

Effects of hot bath therapy on cholinergic urticaria with hypohidrosis or anhidrosis.

Cholinergic urticaria with hypohidrosis or anhidrosis (CUHA) can impair quality of life due to itching, tingling, and reduced sweating. Current treatment options for CUHA include antihistamines, pulsed steroids, and sweat-promoting therapies such as exercise or hot baths. However, the efficacy of these therapies, particularly hot bath therapy, has yet to be established. We evaluated the efficacy of hot bath therapy in patients with CUHA. We enrolled eight patients who underwent hot bath therapy between January 2010 and August 2022. Patients had a half-body bath in a bathtub filled with hot water (40-43°C) for 30-60 minutes daily for 3-7 days. After treatment, pain improved in three (42.9%) patients, urticaria improved in four (50%) patients, and anhidrosis improved in five (62.5%) patients without any severe adverse events. Because hot bath therapy is easily performed, it should be considered a treatment option for patients with CUHA.

Trehalose Prevents IL-4/IL-13-Induced Skin Barrier Impairment by Suppressing IL-33 Expression and Increasing NRF2 Activation in Human Keratinocytes In Vitro.

Skin barrier dysfunction initiates or deteriorates various cutaneous problems, such as atopic dermatitis. At high concentrations, the nonreducing disaccharide trehalose (α-d-glucopyranosyl α-d-glucopyranoside) induces a transient senescence-like state in fibroblasts and promotes wound repair. In this study, we investigated the effect of trehalose on normal human keratinocytes and demonstrated its specific role in the skin barrier. RNA-sequencing analysis revealed that trehalose regulates the expression of many skin barrier-associated genes. T helper 2 cytokines IL-4/IL-13 were observed to downregulate several differentiation markers (FLG, loricrin, keratin 1, and keratin 10) and epidermal antimicrobial proteins in monolayer-cultured keratinocytes and living skin equivalents and impaired skin barrier function in living skin equivalents, all of which were significantly upregulated or restored by trehalose. Trehalose inhibited IL-33 expression and reduced nuclear IL-33 levels by activating MAPK/extracellular signal-regulated kinase kinase 5-extracellular signal-regulated kinase 5 and suppressing extracellular signal-regulated kinase kinase 1/2-extracellular signal-regulated kinase pathway. It also increased NRF2 activation to trigger antioxidant enzyme production through JNK, thus neutralizing IL-4/IL-13-mediated oxidative stress. Trehalose prevented IL-4/IL-13-mediated signal transducer and activator of transcription 3/signal transducer and activator of transcription 6 activation and restored IL-4/IL-13-suppressed skin barrier molecules through IL-33 downregulation and NRF2 activation. This study demonstrated that trehalose may play a role in skin barrier repair in atopic dermatitis.

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.

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.

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.