Abstract Palmoplantar pustulosis (PPP) and dyshidrotic eczema (DE) are chronic vesiculopustular dermatoses with overlapping clinical presentations but distinct underlying biology. Although comparative transcriptomic and proteomic analyses between PPP and DE have been reported, they remain limited in number and scope, with no comprehensive understanding of their distinct molecular signatures. Moreover, their molecular mechanisms remain unclear, and currently available therapeutic options are limited. To clarify disease-specific epidermal programs underlying vesicle formation, we conducted Visium HD spatial transcriptomic analysis of FFPE lesional skin samples obtained from patients with PPP and DE, followed by immunohistochemical validation against normal palmoplantar skin controls. Spatial clustering identified a keratinocyte subpopulation adjacent to vesicles that exhibited distinct transcriptional programs in the two diseases. In PPP, vesicle-associated keratinocytes demonstrated marked downregulation of aquaporin-3 (AQP3) and E-cadherin, together with strong, spatially localized activation of JAK-STAT3 signaling. Conversely, DE exhibited diffuse AQP3 expression and more homogeneous activation of JAK-STAT3 signaling throughout the epidermis. These results indicate that, although PPP and DE share inflammatory pathways, they differ substantially in their spatial molecular architecture. Reduced AQP3 expression and localized STAT3 activation may contribute to vesicle formation in PPP, supporting our previous hypothesis that implicates intraepidermal sweat leakage as a pathogenic mechanism in PPP. Abstract Figure

Background Trehalose is a naturally occurring disaccharide found in invertebrates but cannot be synthesized by vertebrates. We previously reported that high-concentration trehalose induces a transient senescent-like state in fibroblasts, leading to cell cycle arrest and growth factor secretion via CDKN1A/p21, and this process promoted keratinocyte proliferation, enhancing capillary formation and wound closure in vivo . Objective This study aimed to investigate the effect of trehalose on human keratinocytes. Methods Previously published RNA-seq data of cytokine-untreated samples from our group of trehalose-treated human keratinocytes were re-analyzed, and an in vitro scratch assay was performed using cells treated with mitomycin C. Results The trehalose-treated group exhibited increased wound closure. A significantly increased secretion of vascular endothelial growth factor (VEGF) was observed in keratinocytes treated with high-concentration trehalose, which is one of the most crucial molecules inducing angiogenesis in the skin. Significant upregulation of mRNA level and protein secretion of VEGF was confirmed using qPCR and ELISA, respectively. Furthermore, treatment with axitinib, a VEGF receptor inhibitor, significantly suppressed trehalose-induced activation of keratinocyte migration. Additionally, the increase in trehalose-induced migration activity was significantly inhibited by the Jun N-terminal kinase (JNK) inhibitor SP600125 and the PI3K inhibitor LY294002. Conclusion Trehalose promotes wound healing via VEGF secretion from keratinocytes and the PI3K and JNK pathways. The findings of this study may lead to the development of novel therapeutic agents that can alter the wound healing process. IMPORTANT GUIDELINES CONTACT The Review Commons office can be contacted directly at: office{at}reviewcommons.org

Skin barrier dysfunction initiates or deteriorates various cutaneous problems, such as atopic dermatitis. At high concentrations, the nonreducing disaccharide trehalose (a-d-glucopyranosyl a-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.

Correction for 'Synthesis and photophysical properties of a new push-pull pyrene dye with green-to-far-red emission and its application to human cellular and skin tissue imaging' by Kazuki Inoue et al., J. Mater. Chem. B, 2022, 10, 1641-1649, https://doi.org/10.1039/D1TB02728J.

Conventional histopathological techniques, such as hematoxylin and eosin staining, are limited to 4-5 μm-thick tissue sections, restricting visualization to two-dimensional planes. Moreover, acquisition of three-dimensional horizontal images from the skin surface remains challenging, hindering precise assessment of tumor margins in skin lesions. This challenge is particularly pronounced in extramammary Paget's disease (EMPD), in which diffuse epidermal tumor cell spread complicates accurate evaluation of lesion extent. We hypothesized that combining horizontal sectioning with identification of individual tumor cells would enhance the determination of surgical margins. In this study, we developed a deep-imaging technique utilizing fluorescent solvatochromic dyes (LipiORDER® and HistoBright®) and two-photon microscopy to achieve high-resolution tumor margin visualization in EMPD. This technique enables identification of tumor cells in frozen and paraffin-embedded tissue blocks, as well as in live skin tissue under physiological conditions. Our novel approach holds substantial promise for improving the precision of surgical-margin assessment in EMPD and other cutaneous malignancies.

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.

Bright polymethine dyes, typified by carbocyanines, are employed in various fluorescence techniques such as the 3D visualization of living cell morphology and the tracking of extracellular vesicles in the blood vessels of a zebrafish. However, they often exhibit low photostability, particularly for dyes with red-shifted absorption/fluorescence wavelengths due to extended polymethine length, and limited photofunctionality. This limitation restricts their utility in specific applications requiring high-power excitation and/or a wash-free approaches. This study introduces novel merocyanine dyes, MCPY3 and MCPY5, comprising a newly developed pyrene-fused dioxaborine and polymethine chain. Despite their minimal polymethine lengths, their absorption/fluorescence wavelengths reside in the red to near infra-red regions due to the substantial pi-conjugation system of pyrene. Moreover, they exhibit a considerably superior photostability to carbocyanine dyes and fluorogenic behavior between low (ON) and high (OFF) polar solvents, while maintaining brightness comparable to carbocyanine. Leveraging these advantages, the hydrophilic analogs of MCPY3, MCPY3S, were applied to two-photon microscopy imaging of the skin tissues on the finger of a living mouse. The dye clearly visualized the individual cell morphology in the epidermis and the elastin within the dermis, highlighting the potential of the new dye as a valuable tool for fundamental dermatological and histological studies. We have developed novel merocyanines, MCPY3 and MCPY5, based on pyrene-fused dioxaborine and polymethine chain. These dyes exhibit red to near-infrared emission depending on polymethine length, considerably superior photostability to carbocyanines, and fluorogenic behavior, along with high brightness. The hydrophilic and red-emissive derivative, MCPY3S, was successfully applied to fluorescence imaging of skin tissues on the finger of a living mouse.

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 β-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.

In extramammary Paget's disease (EMPD), Paget cells are sometimes detected outside the clinical border (subclinical extension) with a skipping pattern. Therefore, “mapping biopsy” is performed to evaluate the clinical border before complete resection. To increase the proportion of Paget cells in biopsy specimens, it is necessary to increase the number of biopsies. However, this is associated with increased pain and discomfort to the patient. The tumor border decision is also a critical factor for prognosis. If we could detect the EMPD tumor cells from the skin surface directly without cutting the patient skin, we could check numerous points where the mapping biopsy is necessary for detecting the tumor border, and it may bring a good prognosis with a skin surgery. Recently, we have established a new fluorescent three-dimensional deep-imaging technique using two-photon excitation fluorescence microscopy for skin lesions.1, 2 The new technique is very good at describing the skin tissue structure in the epidermis and dermis, for example, acrosyringium, individual keratinocytes, and nerve fibre.1 Hence, to resolve the abovementioned issue, we evaluated whether a new fluorescent three-dimensional deep-imaging technique using two-photon excitation fluorescence microscopy for skin lesions is applicable for mapping biopsy in EMPD. EMPD samples resected during surgery were used in this study. Briefly, skin tissue samples were collected via 2-mm disposal biopsy punches from six areas around the main EMPD lesion. After fixation with 4% paraformaldehyde in PBS for 30 min, the samples were processed for propidium-iodide staining, and optically cleared using the transparency-enhancing ilLUmination of Cleared organs to IDentify target molecules method (LUCID),3 and newly established push-pull pyrene probe bearing a ketone acceptor group (PK), which is a solvatochromic dye.4 The samples were evaluated by two-photon excitation fluorescent microscopy (TPM), as described previously.1 All procedures were performed with prior approval from the Ethics Committees of Ehime University Graduate School of Medicine. This study was conducted in accordance with the principles of the Declaration of Helsinki. As shown in Figure 1A, typical histopathological findings of EMPD tumor cells include clear cytoplasm and a “shotgun” pattern on haematoxylin and eosin staining (HE) in the epidermis. In the same sagittal slice view, TPM confirmed similar LUCID/PK staining of the EMPD tumor cells (Figure 1B). The tumor cells had a dark, clear cytoplasm and nucleus, and there were tumor nests in the epidermis (Figure 1B, Video S1). On horizontal slices, EMPD tumor cells could be detected in the epidermis (Figure 1C) and dermis (Figure 1D, Video S2), suggesting that this tumor was already an invasive carcinoma as opposed to an in situ lesion. The major advantage of this new method is the ability of deep imaging to show the whole area from the surface to the deep lesion. This new technique has several advantages over the routine pathological procedure with HE staining. First, although we could obtain cell and tissue information with slices only 5 μm thick per slide via HE staining, our method can determine histological information over a thickness of 100–500 μm, which allowed us to obtain a three-dimensional image of each tissue slice. In addition, the tissue slices do not require processing; the thin tissue can be directly observed after LUCID/PK staining. Second, preparing the HE pathological specimen usually takes at least 1 week because a number of procedures are required, including making a paraffin block, cutting slices, staining with HE, dehydration, and mounting. However, the new technique only requires that the sample be placed in the LUCID/PK staining cocktail for several hours (overnight if needed), with no requirement for histopathological preparation. Two-photon microscopy is a very useful application of multiphoton microscopy in dermatological studies.5 Conventional skin tissue slices have autoimmunofluorescence and the basic structure of the epidermis, which can reveal the dermis and skin appendages. However, the resolution of this procedure is inferior to conventional HE staining. Therefore, a new technique using TPM with immunohistochemistry was developed that can reveal the detailed structure of skin appendages.6 Two-photon microscopy with immunohistochemical analysis has already been applied to EMPD,7 and the results revealed the EMPD tumor border histologically. Compared to their method, our new technique requires only a LUCID/PK cocktail and TPM; there is no requirement for immunohistochemical staining or sample processing, such as making paraffin blocks or slices. Our procedure also has s higher resolution and is very easy to perform in any laboratory where TPM is available. Furthermore, confocal laser microscopy can detect the signal in tissue (<100 μm thick). Further experiments are underway to establish the non-invasive “mapping biopsy” technique. MM, RK, and YN involved in conceptualization; MM, RK, and TT involved in data curation; MM and RK involved in methodology and formal analysis, writing—original draft preparation, writing—review and editing, and funding acquisition; MM, RK, and TT involved in validation, investigation, and visualization; RK, TI involved in technical supervision and advisory. The English in this document has been checked by at least two professional editors, both native speakers of English. For a certificate, please see: http://www.textcheck.com/certificate/RycH7j. The authors have no conflict of interest to declare. This study was supported by JSPS KAKENHI Grant number 20 K08691, Grant-in-Aid for Scientific Research on Innovative Areas_Platforms for Advanced Technologies and Research Resources “Advanced Bioimaging Support,” and AMED under Grant Number JP22ym0126810. Research data are not shared. Video S1. EMPD lesion in the epidermis with three-dimensional observation. Video S2. EMPD lesion scanned from the surface to the dermis. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

For in vivo two-photon fluorescence microscopy (2PM) imaging, the development of techniques that can improve the observable depth and temporal resolution is an important challenge to address biological and biomedical concerns such as vascular dynamics in the deep brain (typically the hippocampal region) of living animals. Improvements have been achieved through two approaches: an optical approach using a highly tissue-penetrating excitation laser oscillating in the second near-infrared wavelength region (NIR-II, 1100-1350 nm) and a chemical approach employing fluorescent probes with high two-photon brightness (characterized by the product of the two-photon absorption cross section, σ2, and the fluorescence quantum yield, Φ). To integrate these two approaches, we developed a fluorescent dye exhibiting a sufficiently high σ2Φ value of 68 Goeppert-Mayer units at 1100 nm. When a nanoemulsion encapsulating >1000 dye molecules per particle and a 1100 nm laser were employed for 2PM imaging, capillary blood vessels in almost the entire hippocampal CA1 region of the mouse brain (approximately 1.1-1.5 mm below the surface) were clearly visualized at a frame rate of 30 frames s-1 (averaged over eight frames, practically 3.75 frames s-1). This observable depth and frame rate are much higher than those in previous reports on 2PM imaging. Furthermore, this nanoemulsion allowed for the visualization of blood vessels at a depth of 1.8 mm, corresponding to the hippocampal dentate gyrus. These results highlight the advantage of combining bright probes with NIR-II lasers. Our probe is a promising tool for studying the vascular dynamics of living animals and related diseases.

Herein, we discuss a new pyrene-based push-pull dye (PC) and our investigation of its photophysical properties and applicability to biological studies. The newly synthesized dye exhibits highly polarity-sensitive fluorescence over a significantly wide range (i.e., the green to far-red region), accompanied by high fluorescence quantum yields (ΦFL > 0.70 in most organic solvents) and superior photostability to that of the commonly used Nile Red (NR) dye, which also fluoresces in the green to red region. When human prostate cancer cells stained with PC were imaged using a confocal laser scanning fluorescence microscope, PC was found to selectively stain the lipid droplets. Under the cell conditions where the formation of droplets was inhibited, PC could be distributed to both the remaining droplets and the intercellular membranes, which could be distinguished based on the fluorescence solvatochromic function of PC. Furthermore, PC efficiently stained normal human skin tissue blocks treated with a transparency-enhancing agent and enabled clear visualization of individual cells in each tissue architecture by means of two-photon fluorescence microscopy (2PM). Interestingly, PC provides bright 2PM images under tissue-penetrative 960 nm excitation, realizing much clearer and deeper tissue imaging than conventional pyrene dyes and NR. These results suggest that PC could replace several commonly used dyes in various biological applications, particularly the rapid and accurate diagnosis of tissue diseases, typified by biopsy.

Palmoplantar pustulosis (PPP) vesicles arise from eccrine sweat in the acrosyringium and subsequently develop into pustulovesicles and pustules [ [1] Murakami M. Ohtake T. Horibe Y. et al. Acrosyringium is the main site of the vesicle/pustule formation in palmoplantar pustulosis. J. Invest. Dermatol. 2010; 130: 2010-2016 Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar , [2] Murakami M. Terui T. Palmoplantar pustulosis: current understanding of disease definition and pathomechanism. J. Dermatol. Sci. 2020; 98: 13-19 Abstract Full Text Full Text PDF PubMed Scopus (14) Google Scholar ]. However, it is very difficult to determine whether a PPP vesicle lies within or around an acrosyringium because of the technical limitations of routine histopathological examination. Recently, we developed a high-quality fluorescence imaging system using transparency-enhancing technology [ [3] Mizutani H. Ono S. Ushiku T. et al. Transparency-enhancing technology allows three-dimensional assessment of gastrointestinal mucosa: a porcine model. Pathol. Int. 2018; 68: 102-108 Crossref PubMed Scopus (11) Google Scholar ] and an improved fluorescent solvatochromic probe that changes emission spectra and fluorescent intensity depending on the hydrophobicity of lipid surrounding the probe [ [4] Valanciunaite J. Kempf E. Seki H. et al. Polarity mapping of cells and embryos by improved fluorescent solvatochromic pyrene probe. Anal. Chem. 2020; 92: 6512-6520 Crossref PubMed Scopus (15) Google Scholar ]. In other words, fluorescent solvatochromic probe that varies its fluorescence color (wavelength) and intensity depending on its location between plasma and intracellular membranes. This new technique makes it possible to obtain high-quality, three-dimensional (3D) epidermal images, particularly of acrosyringia [ [5] Murakami M. Kawakami R. Niko Y. et al. High-quality fluorescence imaging of the human acrosyringium using a transparency: enhancing technique and an improved, fluorescent solvatochromic pyrene probe. Acta Histochem. Cytochem. 2020; 53: 131-138 Crossref PubMed Scopus (1) Google Scholar ]. We obtained 3D images of PPP vesicles to directly explore the associations between PPP vesicles/pustules and the acrosyringium. We evaluated six biopsy specimens obtained from patients at Ehime University Hospital diagnosed with PPP based on their clinical features, histopathologies, and disease courses (supplement table) [ [6] Teymouri M. Halabchi F. Mirshahi M. et al. Comparison of plantar pressure distribution between three different shoes and three common movements in futsal. PLos One. 2017; 12e0187359 Crossref PubMed Scopus (6) Google Scholar ]. All procedures were approved by the Ethics Committee of Ehime University, and the study adhered to all relevant principles of the Declaration of Helsinki. The sample preparation procedure prior to fluorescence microscopy has been described previously [ [5] Murakami M. Kawakami R. Niko Y. et al. High-quality fluorescence imaging of the human acrosyringium using a transparency: enhancing technique and an improved, fluorescent solvatochromic pyrene probe. Acta Histochem. Cytochem. 2020; 53: 131-138 Crossref PubMed Scopus (1) Google Scholar ]. Briefly, punch biopsy samples 2 mm in diameter were fixed in 4 % (v/v) paraformaldehyde in phosphate buffer (pH 7.4), stained with propidium iodide in phosphate-buffered saline, immersed in 2 μM pyrene probe solution [ [4] Valanciunaite J. Kempf E. Seki H. et al. Polarity mapping of cells and embryos by improved fluorescent solvatochromic pyrene probe. Anal. Chem. 2020; 92: 6512-6520 Crossref PubMed Scopus (15) Google Scholar ], and subjected to optical clearance using a transparency-enhancing technology (OCTET) [ [3] Mizutani H. Ono S. Ushiku T. et al. Transparency-enhancing technology allows three-dimensional assessment of gastrointestinal mucosa: a porcine model. Pathol. Int. 2018; 68: 102-108 Crossref PubMed Scopus (11) Google Scholar ] for 48 h at room temperature. Images were acquired by two-photon excitation microscopy (TPM: Nikon A1RMP + and Spectra physics MaiTai DeepSee eHP) as described previously [ [5] Murakami M. Kawakami R. Niko Y. et al. High-quality fluorescence imaging of the human acrosyringium using a transparency: enhancing technique and an improved, fluorescent solvatochromic pyrene probe. Acta Histochem. Cytochem. 2020; 53: 131-138 Crossref PubMed Scopus (1) Google Scholar ]. In briefly, Excitation laser was set at 960 nm and emission was divided by using dichroic mirrors at 495 nm and 560 nm. After TPM, all samples were processed for routine histopathology, embedded in paraffin blocks, cut into 3-μm-thick slices, stained with hematoxylin and eosin, and viewed under a microscope.

Background Lesions of atopic dermatitis have fewer Th17 cells than those of psoriasis, resulting in frequent skin infections. Expression of CCL20, a chemokine that is important for recruiting Th17 cells, is suppressed in the lesions of atopic dermatitis. We previously reported that IL-4 induces the expression of cytokine-inducible SH2-containing protein 1 (CIS1), a member of the CIS/SOCS family, in epidermal keratinocytes. Objective To investigate whether CIS1 influences CCL20 production in epidermal keratinocytes. Methods Expression of CIS1 was examined in atopic dermatitis skin and in cultured keratinocytes. The effects of overexpression of CIS1 on CCL20 production by IL-17A, and on signaling pathways inhibited by CIS1, were assessed in vitro. Results Expression of CIS1 was enhanced in the basal layer of the lesional epidermis of skin with atopic dermatitis. When CIS1 was expressed in keratinocytes using adenoviral vectors, IL-17A-induced CCL20 expression, but not HBD2 or S100A7 expression, was significantly suppressed. TNF-α/IL-1-induced CCL20 production was not altered by CIS1. Overexpression of CIS1 attenuated IL-17A-induced ERK phosphorylation. ERK phosphorylation was mediated by the Act1 and Src family kinase pathways. CIS1 overexpression suppressed Src phosphorylation. Among the Src family kinases, the Yes kinase may have an important role because knockdown of Yes in epidermal keratinocytes resulted in suppression of ERK phosphorylation and CCL20 mRNA expression by IL-17A. Conclusion CIS1 induced by Th2 cytokines has the ability to change the response of epidermal keratinocytes to IL-17A by suppression of Src family kinases.

Trehalose is the nonreducing disaccharide of glucose, evolutionarily conserved in invertebrates, but does not exist in vertebrates. 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 data and Ingenuity pathway analysis of the differentially expressed genes of trehalose-treated 2D and 3D fibroblasts at early time points revealed the involvement of the CDKN1A pathway, which is necessary for the marked upregulation of growth factors including DPT. By contrast, the mRNA-seq data of LSEs 2-weeks after air exposure indicated that gene expression profiles are similar for untreated and trehalose-treated cells in both keratinocytes and fibroblasts. The trehalose-treated fibroblasts were positive for senescence-associated β-galactosidase with the significantly downregulated expressions of LMNB1. Finally, we demonstrated that transplantation of the dermal substitute with trehalose-treated fibroblasts accelerated wound closure and increased capillary formation significantly in the experimental mouse wounds in vivo . These data indicate that high-concentration trehalose can induce the beneficial senescence-associated secretory phenotype in fibroblasts via CDKN1A/p21, which may be therapeutically useful for optimal wound repair.

Two-photon, excitation fluorescent microscopy featuring autofluorescence or immunofluorescence, combined with optical clearance using a transparency-enhancing technique, allows deep imaging of three-dimensional (3D) skin structures. However, it remains difficult to obtain high-quality images of individual cells or 3D structures. We combined a new dye with a transparency-enhancing technology and performed high-quality structural analysis of human epidermal structures, especially the acrosyringium. Human fingertip skin samples were collected, formalin-fixed, embedded in both frozen and paraffin blocks, sliced, stained with propidium iodide, optically cleared using a transparency-enhancing technique, and stained with a new fluorescent, solvatochromic pyrene probe. Microscopy revealed fine skin features and detailed epidermal structures including the stratum corneum (horny layer), keratinocytes, eccrine sweat glands, and peripheral nerves. Three-dimensional reconstruction of an entire acrosyringium was possible in one sample. This new fluorescence microscopy technique yields high-quality epidermal images and will aid in histopathological analyses of skin disorders.

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)‐1β 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‐1β, but also suppressed IL‐1β 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‐1β expression in keratinocytes, was inhibited by heparinoid treatment. The inhibitory effect of heparinoid on IL‐1β mRNA expression was also confirmed with living skin equivalents. Our results demonstrated that heparinoid suppresses the initiation of keratinocyte‐mediated skin inflammation.

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.

BACKGROUND:High mobility group box 1 (HMGB1) is a nuclear protein that stabilizes DNA and facilitates gene transcription. Additionally, cell stress or death induces the release of HMGB1 outside the cell membrane, where HMGB1 functions as an alarmin, causing an inflammatory response in combination with other cytokines, damage-associated molecular patterns (DAMPs), and pathogen-associated molecular patterns (PAMPs).OBJECTIVE:To evaluate the effect of reduced-HMGB1 (previously termed chemoattractive-HMGB1) on polyinosine-polycytidylic acid [poly(I:C)]-induced inflammation in normal human keratinocytes (NHKs).METHODS:We focused on downstream components of the poly(I:C)-Toll-like receptor 3 (TLR3), retinoic acid-inducible gene-I (RIG-I), and melanoma differentiation-associated protein 5 (MDA5) pathways, including IκBα, nuclear factor (NF)-κB p65, mitogen-activated protein kinase (MAPK), and interferon regulatory factor 3 (IRF3), and assessed whether these pathways are involved in the suppression of poly(I:C)-induced inflammation in NHKs by HMGB1. An immunoprecipitation was performed to know whether HMGB1 could bind to poly(I:C), and immunofluorescence staining and flow cytometric analysis were performed to check whether reduced-HMGB interferes with cellular uptake of poly(I:C) translocation (possibly by endocytosis).RESULTS:Application of exogenous HMGB1 before, but not after, exerted a suppressive effect on poly(I:C)-induced inflammation in NHKs. In addition, reduced-HMGB1, but not disulfide-HMGB1, exerted a suppressive effect on poly(I:C)-induced inflammation in NHKs, suggesting the importance of the redox status of exogenous HMGB1. Pre-treatment with reduced-HMGB1 inhibited the phosphorylation of IκBα, NF-κB p65, and IRF3 induced by poly(I:C) stimulation in NHKs; however, phosphorylation of p38, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK) was unaffected. Disulfide-HMGB1 formed a complex with poly(I:C), as did reduced- and oxidized-HMGB1, albeit to a lesser extent. Immunofluorescence staining and flow cytometric analysis indicated that reduced-HMGB interferes with cellular uptake of poly(I:C) translocation (possibly by endocytosis).CONCLUSION:These findings suggest that pre-treatment with reduced-HMGB1 ameliorates poly(I:C)-mediated inflammation in NHKs.

We previously reported that the early vesicle of the palmoplantar pustulosis (PPP) vesicle originated from eccrine sweat in the acrosyringium and that the PPP vesicle contains the antimicrobial peptide human cathelicidin-18/LL-37. The concentration of LL-37 was sufficient to induce the subsequent inflammation in lesions and human keratinocytes, and the PPP vesicles contained additional small fragments of human cathelicidin-18, of approximately 7 kDa, which have not been identified. The aim of the present study was to clarify the additional processed forms found in PPP vesicles and their physiological effects on normal keratinocytes and sweat gland cells. Lesional PPP vesicles were collected from PPP patients, and endogenous human cathelicidin-18/LL-37 was depleted using a LL-37 antibody affinity column. A designed recombinant human cathelicidin-18 peptide was prepared and incubated with the depleted PPP vesicle fluid to confirm the additional processed form. In-gel digestion analysis and protein sequencing confirmed the additional form as TLN58. TLN-58 up-regulated IL-17C, IL-8, IL-23, IL-1 alpha, and IL-1 beta mRNA and protein expression in normal human keratinocytes and also showed antibacterial activity against Staphylococcus aureus, Staphylococcus epidermidis, and group A Streptococcus species, similar to LL-37. This additional form could be involved in the continued inflammation in PPP lesions.