Effects of EGFR-TKI on epidermal melanin unit integrity: Therapeutic implications for hypopigmented skin disorders.

Epidermal melanin unit integrity is crucial for skin homeostasis and pigmentation. Epidermal growth factor (EGF) receptor (EGFR) is a pivotal player in cell growth, wound healing, and maintaining skin homeostasis. However, its influence on skin pigmentation is relatively unexplored. This study investigates the impact and underlying mechanisms of EGFR inhibitors on skin pigmentation. We evaluated EGF and EGFR expression in various skin cells using quantitative real-time PCR, Western blot, and immunofluorescence. EGF and EGFR were predominantly expressed in epidermal keratinocytes, and treatment with the EGFR tyrosine kinase inhibitors (EGFR-TKIs) gefitinib and PD153035 significantly increased stem cell factor (SCF) and endothelin-1 (ET-1) expression in cultured keratinocytes. Enhanced melanocyte migration and proliferation were observed in co-culture, as evidenced by time-lapse live imaging and single-cell tracking assays. Furthermore, topical application of gefitinib to guinea pig dorsal skin induced increased pigmentation and demonstrated efficacy in mitigating rhododendrol-induced leukoderma. Suppression of EGF signaling indirectly enhanced skin pigmentation by upregulating SCF and ET-1 in epidermal keratinocytes. This novel mechanism highlights the pivotal role of EGF signaling in regulating skin pigmentation, and topical EGFR-TKI therapy at an appropriate dose may be a promising approach for depigmentation disorder management.

High α-diversity of skin microbiome and mycobiome in Japanese patients with vitiligo.

BACKGROUND: Vitiligo is an acquired pigmentary disorder characterized by depigmented patches on the skin that majorly impact patients' quality of life. Although its etiology involves genetic and environmental factors, the role of microorganisms as environmental factors in vitiligo pathology remains under-researched. OBJECTIVES: Our study explored the presence of characteristic bacterial and fungal flora in vitiligo-affected skin and investigated their potential roles in vitiligo pathogenesis. METHODS: We sequenced bacterial 16S rRNA and the fungal ITS1 region from skin swabs collected at frequently affected sites, namely the forehead and back, of patients with vitiligo. We analyzed bacterial and fungal flora in lesional and non-lesional areas of patients with vitiligo compared with corresponding sites in age- and sex-matched healthy subjects. RESULTS: Our findings revealed elevated α-diversity in both bacterial and fungal flora within vitiligo lesions compared with healthy controls. Notably, bacterial flora exhibited a distinctive composition in patients with vitiligo, and the proportional representation of Enterococcus was inversely correlated with the degree of vitiligo progression. Gammaproteobacteria, Staphylococcus spp., and Corynebacterium spp. were more abundant in vitiligo patients, with notable Staphylococcus spp. prevalence during the stable phase on the forehead. Conversely, the proportion of Malassezia sympodialis was lower and that of Malassezia globosa was higher in the progressive phase on the back of vitiligo patients. CONCLUSION: Our study identified some characteristic bacterial and fungal groups associated with vitiligo activity and prognosis, highlighting the potential roles of microorganisms in pathogenesis and offering insights into personalized disease-management approaches.

CXCL12 promotes CCR7 ligand-mediated breast cancer cell invasion and migration toward lymphatic vessels.

Chemokines are a family of cytokines that mediate leukocyte trafficking and are involved in tumor cell migration, growth, and progression. Although there is emerging evidence that multiple chemokines are expressed in tumor tissues and that each chemokine induces receptor-mediated signaling, their collaboration to regulate tumor invasion and lymph node metastasis has not been fully elucidated. In this study, we examined the effect of CXCL12 on the CCR7-dependent signaling in MDA-MB-231 human breast cancer cells to determine the role of CXCL12 and CCR7 ligand chemokines in breast cancer metastasis to lymph nodes. CXCL12 enhanced the CCR7-dependent in vitro chemotaxis and cell invasion into collagen gels at suboptimal concentrations of CCL21. CXCL12 promoted CCR7 homodimer formation, ligand binding, CCR7 accumulation into membrane ruffles, and cell response at lower concentrations of CCL19. Immunohistochemistry of MDA-MB-231-derived xenograft tumors revealed that CXCL12 is primarily located in the pericellular matrix surrounding tumor cells, whereas the CCR7 ligand, CCL21, mainly associates with LYVE-1+ intratumoral and peritumoral lymphatic vessels. In the three-dimensional tumor invasion model with lymph networks, CXCL12 stimulation facilitates breast cancer cell migration to CCL21-reconstituted lymphatic networks. These results indicate that CXCL12/CXCR4 signaling promotes breast cancer cell migration and invasion toward CCR7 ligand-expressing intratumoral lymphatic vessels and supports CCR7 signaling associated with lymph node metastasis.

A Lower Irradiation Dose of 308 nm Monochromatic Excimer Light Might Be Sufficient for Vitiligo Treatment: A Novel Insight Gained from In Vitro and In Vivo Analyses.

A 308 nm monochromatic excimer light (MEL) is widely used to treat patients with vitiligo. However, dose optimization still needs to be clarified. This study aimed to obtain objective evidence regarding various doses of MEL irradiation, induced cell level changes in vitro, and skin level alterations in vivo. Cultured human keratinocytes were irradiated with MEL using various doses. After irradiation at low doses, stem cell factor, endothelin-1, and glycoprotein nonmetastatic melanoma protein B, factors that activate and protect melanocytes, were found to be significantly elevated in keratinocytes. After irradiation using medium and high doses, inflammatory cytokines were induced. The amount of ATP released and the level of inflammasome activation, which are known to be related to interleukin-1ß activation, were also increased. The back skin of guinea pigs and mice were irradiated with MEL at varying doses. After irradiation, an increase of epidermal melanin and epidermal melanocytes was confirmed, using the minimal erythemal dose or less. In rhododendrol-induced leukoderma guinea pigs, a much lower dose of MEL irradiation was effective, when compared with the effective dose for control guinea pigs. Our results suggest that a lower irradiation dose of MEL might be sufficient and more suitable for repigmentation in vitiligo treatment.

GPNMB Extracellular Fragment Protects Melanocytes from Oxidative Stress by Inhibiting AKT Phosphorylation Independent of CD44.

Glycoprotein non-metastatic melanoma protein B (GPNMB) is a type I transmembrane glycoprotein that plays an important role in cancer metastasis and osteoblast differentiation. In the skin epidermis, GPNMB is mainly expressed in melanocytes and plays a critical role in melanosome formation. In our previous study, GPNMB was also found to be expressed in skin epidermal keratinocytes. In addition, decreased GPNMB expression was observed in the epidermis of lesional skin of patients with vitiligo. However, the exact role of keratinocyte-derived GPNMB and its effect on vitiligo is still unknown. In this study, we demonstrated that GPNMB expression was also decreased in rhododendrol-induced leukoderma, as seen in vitiligo. The extracellular soluble form of GPNMB (sGPNMB) was found to protect melanocytes from cytotoxicity and the impairment of melanogenesis induced by oxidative stress. Furthermore, the effect of rGPNMB was not altered by the knockdown of CD44, which is a well-known receptor of GPNMB, but accompanied by the suppressed phosphorylation of AKT but not ERK, p38, or JNK. In addition, we found that oxidative stress decreased both transcriptional GPNMB expression and sGPNMB protein expression in human keratinocytes. Our results suggest that GPNMB might provide novel insights into the mechanisms related to the pathogenesis of vitiligo and leukoderma.

Herb Sanqi-Derived Compound K Alleviates Oxidative Stress in Cultured Human Melanocytes and Improves Oxidative-Stress-Related Leukoderma in Guinea Pigs.

Sanqi, a traditional Chinese herb, is widely used for cardiovascular diseases, and its neuroprotective effects against oxidative stress were recently discovered. The purpose of this study was to investigate whether Sanqi-derived compound K (Sanqi-CK), an active metabolite of Sanqi, could protect melanocytes from oxidative stress. Cultured human primary skin epidermal melanocytes (HEMn-MPs) were treated with hydrogen peroxide (H2O2) in the presence or absence of Sanqi-CK. Sanqi-CK exhibited protective effects against H2O2-induced cell death by reducing oxidative stress. In addition, treatment with Sanqi-CK reversed the decreased glutathione reductase activity and decreased ratio of reduced glutathione (GSH)/oxidized glutathione (GSSG) seen in H2O2-treated melanocytes. Furthermore, topical application of Sanqi-CK alleviated leukoderma in guinea pigs, a disorder characterized by melanocyte cell death resulting from rhododendrol-induced oxidative stress. Taken together, these data suggest that Sanqi-CK protects melanocytes against oxidative stress, and its protective effects are associated with modulating the redox balance between GSH and GSSG and activating glutathione reductase. Thus, Sanqi-CK may be a good candidate for preventing melanocyte loss in oxidative-stress-associated pigmentary disorders.

Differential susceptibility between skin and vaginal mucosa in sensitization phase of allergic contact dermatitis in mice.

INTRODUCTION: Mechanisms underlying skin sensitization in allergic contact dermatitis have been actively studied using the murine contact hypersensitivity (CHS) model. However, much less is known about sensitization at the vaginal mucosa (VM). METHODS: We developed a CHS model with VM sensitization and epicutaneous elicitation at the ear. We then examined the proliferation activity of lymphocytes, the frequencies of T cells and the differentiation of hapten-specific T cells in draining lymph nodes (dLNs) after sensitization. RESULTS: Hapten-specific CHS responses to 2,4-dinitrofluorobenzene (DNFB), 2,4,6-trinitrochrolobenzene, and oxazolone assessed by ear swelling suggested that the VM would be an inductive site of CHS to haptens. In the comparisons of CHS responses to each of the three haptens examined, the lower responses in VM-sensitized mice were observed than skin-sensitized mice (e.g., DNFB-induced responses, -56%; p < .001, at 48 h after challenge). Consistent with the CHS responses, the DNFB-induced proliferation of cells in dLNs examined by 5-bromo-2'-deoxyuridine assay was lower (-62%; p < .001) in VM-sensitized mice than skin-sensitized mice. On the other hand, between skin and VM sensitization, no significant differences were observed in the frequencies of interferon-γ-producing CD4+ and CD8+ effector, and regulatory T cells in dLNs after sensitization. We also observed no significant differences with respect to differentiation of hapten-specific T cells based on the examination of cytokine production from dLN cells stimulated in vitro with 2,4-dinitrobenzene sulfonate. CONCLUSION: These findings suggested that the lower T cell proliferation after VM sensitization is important for the lower CHS responses with VM sensitization than skin sensitization.

An acid-hydrolyzed wheat protein activates the inflammatory and NF-κB pathways leading to long TSLP transcription in human keratinocytes.

Hydrolyzed wheat proteins (HWPs) contained in cosmetics have occasionally caused immediate-type hypersensitivity following repeated skin exposure. Although the Cosmetic Ingredient Review Expert Panel concluded that < 3,500 Da HWP is safe for use in cosmetics, it remains biologically unknown how allergenic HWPs evoke immediate-type allergy percutaneously. Keratinocyte-derived thymic stromal lymphopoietin (TSLP) induces type 2 immune responses, which play an essential role in the pathogenesis of immediate-type allergy. Previously, we demonstrated that protein allergens in cultured human keratinocytes strongly induced long-form TSLP (loTSLP) transcription. However loTSLP-regulating signaling by HWP is poorly understood. In this study, we performed global gene expression analysis by microarray to investigate how the allergenic HWP acts on epidermal keratinocytes and the induction of loTSLP. Compared to human serum albumin (HSA), allergenic HWP induced a distinct gene expression pattern and preferentially activated various inflammatory pathways (High Mobility Group Box 1, Interleukin [IL]-6, IL-8, and acute phase response signaling). We identified 85 genes as potential nuclear factor-kappa B (NF-κB) target genes in GP19S-treated cells, compared with 29 such genes in HSA-treated cells. In addition, HWP specifically altered IL-17 signaling pathways in which transcription factors, NF-κB and activator protein-1, were activated. NF-κB signaling may be an important factor for HWP-induced inflammatory loTSLP transcription via inhibition assay. In conclusion, allergenic HWP caused an easily sensitizable milieu of activated inflammatory pathways and induced NF-κB-dependent loTSLP transcription in keratinocytes.

Local Epidermal Endocrine Estrogen Protects Human Melanocytes against Oxidative Stress, a Novel Insight into Vitiligo Pathology.

As the outermost barrier of the body, skin is a major target of oxidative stress. In the brain, estrogen has been reported synthesized locally and protects neurons from oxidative stress. Here, we explored whether estrogen is also locally synthesized in the skin to protect from oxidative stress and whether aberrant local estrogen synthesis is involved in skin disorders. Enzymes and estrogen receptor expression in skin cells were examined first by quantitative real-time PCR and Western blot analyses. Interestingly, the estrogen synthesis enzyme was mainly localized in epidermal keratinocytes and estrogen receptors were mainly expressed in melanocytes among 13 kinds of cultured human skin cells. The most abundant estrogen synthesis enzyme expressed in the epidermis was 17ß-hydroxysteroid dehydrogenase 1 (HSD17ß1) localized in keratinocytes, and the most dominant estrogen receptor expressed in the epidermis was G protein-coupled estrogen receptor 1 (GPER1) in melanocytes. To investigate whether keratinocyte-derived estradiol could protect melanocytes from oxidative stress, cultured human primary epidermal melanocytes (HEMn-MPs) were treated with H2O2 in the presence or absence of 17ß estradiol or co-cultured with HSD17ß1 siRNA-transfected keratinocytes. Keratinocyte-derived estradiol exhibited protective effects against H2O2-induced cell death. Further, reduced expression of HSD17ß1 in the epidermis of skin from vitiligo patients was observed compared to the skin from healthy donors or in the normal portions of the skin in vitiligo patients. Our results suggest a possible new target for interventions that may be used in combination with current therapies for patients with vitiligo.

Long form of thymic stromal lymphopoietin of keratinocytes is induced by protein allergens.

A growing body of evidence suggests that epicutaneous sensitization of protein allergens induces immediate-type hypersensitivity (IHS) following induction of Type 2 immune responses in animals and humans. Thymic stromal lymphopoietin (TSLP) derived from keratinocytes is a cytokine that can activate dendritic cells and has been implicated in development of inflammatory Type 2 helper T-cells. However, there is no direct evidence that allergens directly regulate TSLP expression in keratinocytes. This study aimed to evaluate the response of TSLP to protein allergens in cultured human keratinocytes and to identify appropriate endpoints for IHS. The transcription of long-form TSLP (loTSLP) was strongly induced by ovalbumin, wheat gluten (WG), acid-hydrolyzed WG (acid-HWG), and extracts from feces of Dermatophagoides pteronyssinus and D. farina, and trypsin, but not by rare allergens, human serum albumin (HSA), or extracts of mite bodies. In acid-HWG, loTSLP mRNA was significantly augmented by acid hydrolysis of WG for 0.5 h compared to WG. However, prolonged acid hydrolysis attenuated this induction similarly to that reported in previous animal studies. These results suggested that intense loTSLP transcriptional induction was a characteristic of a high-allergenic protein. Additionally, TSLP production was induced by exposure to ovalbumin, WG, and acid-HWG in combination with a trio of cytokines, i.e. interleukin (IL)-4, IL-13, and tumor necrosis factor (TNF)-α. However, no TSLP protein was detected following exposure to HSA, even in the presence of these cytokines. With acid-HWG, TSLP protein release was consistent with loTSLP transcription. Thus, intense loTSLP transcriptional induction and TSLP protein expression are each effective indicators that can be used for in vitro screening of IHS.

Anaphylactic augmentation by epicutaneous sensitization to acid-hydrolyzed wheat protein in a guinea pig model.

Recent reports suggest that hydrolyzed wheat protein (HWP) variants such as Glupearl® 19S (GP19S) induce immediate-type hypersensitivity via epicutaneous (EC) sensitization. The identification of strong allergens is a key step in product assessment before commercial launch. However, few reports have described the estimation of actual and potential anaphylactic sensitizing capacity. In this study we assessed the strength of both the actual and potential anaphylactic sensitizing capacity by investigating the immediate-type hypersensitivity inducing potential of HWP compared with gluten. We assessed these strengths via the EC route using an EC or intradermal (ID) sensitization method. We quantified the strength of immediate-type hypersensitivity by evaluating the titer of serum antibodies isolated from sensitized subjects using passive cutaneous anaphylaxis (PCA) reactions. We also evaluated the cross-reactivity between GP19S and gluten. GP19S and gluten applied by both the sensitization methods induced obvious IgG1-mediated PCA reactions. GP19S had stronger sensitizing potential than gluten, according to the serum titers and dye spot diameters. The difference in antibody titers between GP19S and gluten was 16-fold for the EC method versus 2-fold for the ID method. GP19S cross-reacted with gluten. Acid hydrolysis of gluten increased anaphylactic sensitizing capacity in the EC method. To our knowledge, our study is the first to quantitatively confirm that HWP and gluten can induce immediate-type hypersensitivity through an intact skin. These findings suggest that acid-HWP imposes a higher risk of EC sensitization than gluten because of the ease with which the former confers a sensitizing effect through the intact skin.

Depigmentation of the skin induced by 4-(4-hydroxyphenyl)-2-butanol is spontaneously re-pigmented in brown and black guinea pigs.

Chemically induced depigmentation of the skin, which occurs following exposure (application or inhalation) to a depigmenting agent, is a disease with clinical findings similar to vitiligo. Recently, skin depigmentation possibly resulting from exposure to 4-(4-hydroxyphenyl)-2-butanol (HPB) was reported in humans. However, the role of HPB as the causative material of this skin depigmentation was not clear. To evaluate whether HPB has the potential for skin depigmentation, we characterized its effects on the skin of pigmented guinea pigs. Following exposure to 30% HPB 3 times/day for about 20 days, we found that obvious skin depigmentation was induced in brown and black guinea pigs. In the depigmented skin, there was a marked reduction in melanin pigment, and decreased numbers of DOPA and S-100 positive epidermal melanocytes were observed histologically. In addition, the depigmentation gradually recovered spontaneously and the number of melanocytes in the skin also increased after terminating the application of HPB. Complete re-pigmentation needed 31 to 70 days to return to the original baseline level. These data indicate that skin depigmentation is induced by the toxicity of HPB to epidermal melanocytes, and that the induced skin depigmentation can recover by terminating the application of HPB.