The aryl hydrocarbon receptor AhR links atopic dermatitis and air pollution via induction of the neurotrophic factor artemin.

Atopic dermatitis is increasing worldwide in correlation with air pollution. Various organic components of pollutants activate the transcription factor AhR (aryl hydrocarbon receptor). Through the use of AhR-CA mice, whose keratinocytes express constitutively active AhR and that develop atopic-dermatitis-like phenotypes, we identified Artn as a keratinocyte-specific AhR target gene whose product (the neurotrophic factor artemin) was responsible for epidermal hyper-innervation that led to hypersensitivity to pruritus. The activation of AhR via air pollutants induced expression of artemin, alloknesis, epidermal hyper-innervation and inflammation. AhR activation and ARTN expression were positively correlated in the epidermis of patients with atopic dermatitis. Thus, AhR in keratinocytes senses environmental stimuli and elicits an atopic-dermatitis pathology. We propose a mechanism of air-pollution-induced atopic dermatitis via activation of AhR.

Metabolic and pathologic profiles of human LSS deficiency recapitulated in mice.

Skin lesions, cataracts, and congenital anomalies have been frequently associated with inherited deficiencies in enzymes that synthesize cholesterol. Lanosterol synthase (LSS) converts (S)-2,3-epoxysqualene to lanosterol in the cholesterol biosynthesis pathway. Biallelic mutations in LSS have been reported in families with congenital cataracts and, very recently, have been reported in cases of hypotrichosis. However, it remains to be clarified whether these phenotypes are caused by LSS enzymatic deficiencies in each tissue, and disruption of LSS enzymatic activity in vivo has not yet been validated. We identified two patients with novel biallelic LSS mutations who exhibited congenital hypotrichosis and midline anomalies but did not have cataracts. We showed that the blockade of the LSS enzyme reaction occurred in the patients by measuring the (S)-2,3-epoxysqualene/lanosterol ratio in the forehead sebum, which would be a good biomarker for the diagnosis of LSS deficiency. Epidermis-specific Lss knockout mice showed neonatal lethality due to dehydration, indicating that LSS could be involved in skin barrier integrity. Tamoxifen-induced knockout of Lss in the epidermis caused hypotrichosis in adult mice. Lens-specific Lss knockout mice had cataracts. These results confirmed that LSS deficiency causes hypotrichosis and cataracts due to loss-of-function mutations in LSS in each tissue. These mouse models will lead to the elucidation of the pathophysiological mechanisms associated with disrupted LSS and to the development of therapeutic treatments for LSS deficiency.

The Antimicrobial Peptide Cathelicidin Exerts Immunomodulatory Effects via Scavenger Receptors.

An active form of cathelicidin antimicrobial peptide, LL-37, has immunomodulatory and stimulatory effects, though the specific pathways are not clear. The purpose of this study was to identify the cellular pathways by which LL-37 amplifies the inflammation induced by damage-associated molecular patterns (DAMPs). We performed DNA microarray, reverse transcription polymerase chain reaction, immunoblotting, and proximity ligation assays using cultured keratinocytes treated with LL-37 and/or the DAMP poly(I:C), a synthetic double-stranded RNA. In contrast to the combination of LL-37 and poly(I:C), LL-37 alone induced genes related to biological metabolic processes such as VEGFA and PTGS2 (COX-2). Inhibition of FPR2, a known receptor for cathelicidin, partially suppressed the induction of VEGFA and PTGS2. Importantly, VEGFA and PTGS2 induced by LL-37 alone were diminished by the knockdown of scavenger receptors including SCARB1 (SR-B1), OLR1 (SR-E1), and AGER (SR-J1). Moreover, LL-37 alone, as well as the combination of LL-37 and poly(I:C), showed proximity to the scavenger receptors, indicating that LL-37 acts via scavenger receptors and intermediates between them and poly(I:C). These results showed that the broad function of cathelicidin is generally dependent on scavenger receptors. Therefore, inhibitors of scavenger receptors or non-functional mock cathelicidin peptides may serve as new anti-inflammatory and immunosuppressive agents.

Enhanced apoptosis by disruption of the STAT3-IκB-ζ signaling pathway in epithelial cells induces Sjögren's syndrome-like autoimmune disease.

Sjögren's syndrome (SS) is an autoimmune disease characterized by exocrinopathy that leads to dry eye and mouth. Although lymphocyte infiltration into exocrine glands and the generation of autoantibodies have been reported in SS, its pathogenic mechanism remains elusive. Here, we show that mice lacking the transcriptional regulator IκB-ζ developed SS-like inflammation characterized by lymphocyte-infiltrated dacryoadenitis and SS-associated autoantibodies. In particular, epithelial cells, but not hematopoietic cells, lacking IκB-ζ were essential for the development of inflammation. IκB-ζ-deficient epithelial cells in the lacrimal glands exhibited enhanced apoptosis even in the absence of lymphocytes. Administration of caspase inhibitors ameliorated the inflammation, indicating the critical role of caspase-mediated apoptosis. Furthermore, epithelial cell-specific STAT3-deficient mice developed SS-like inflammation with impaired IκB-ζ expression in the lacrimal glands. Thus, this study reveals a pathogenic mechanism of SS in which dysfunction of epithelial cells caused by disruption of STAT3-mediated IκB-ζ induction elicits the activation of self-reactive lymphocytes.

Functional Analysis of the Transcriptional Regulator IκB-ζ in Intestinal Homeostasis.

BACKGROUND: The Toll-like receptor signaling pathway contributes to the regulation of intestinal homeostasis through interactions with commensal bacteria. Although the transcriptional regulator IκB-ζ can be induced by Toll-like receptor signaling, its role in intestinal homeostasis is still unclear. AIMS: To investigate the role of IκB-ζ in gut homeostasis. METHODS: DSS-administration induced colitis in control and IκB-ζ-deficient mice. The level of immunoglobulins in feces was detected by ELISA. The immunological population in lamina propria (LP) was analyzed by FACS. RESULTS: IκB-ζ-deficient mice showed severe inflammatory diseases with DSS administration in the gut. The level of IgM in the feces after DSS administration was less in IκB-ζ-deficient mice compared to control mice. Upon administration of DSS, IκB-ζ-deficient mice showed exaggerated intestinal inflammation (more IFN-g-producing CD4+ T cells in LP), and antibiotic treatment canceled this inflammatory phenotype. CONCLUSION: IκB-ζ plays a crucial role in maintaining homeostasis in the gut.

Serum soluble CD163 and proinflammatory chemokines may be biomarkers of the onset of adverse events in dabrafenib plus trametinib combination therapy for advanced melanoma.

Various adverse events (AEs) have been reported to occur at a high rate in patients treated with dabrafenib plus trametinib (D + T) combination therapy. Among such AEs, the incidence of pyrexia was highest among the series of AEs in patients treated with D + T combination therapy. Although little is known about the mechanisms of pyrexia caused by D + T combination therapy, a recent report suggested that sCD163, as well as interferon-inducible chemokines (CXCL9, CXCL10, CXCL11), might correlate with pyrexia caused by encorafenib plus binimetinib combination therapy. In addition to these soluble factors, CXCL5 is a biomarker for predicting immune-related AEs in melanoma patients treated with nivolumab. From the above findings, we hypothesized that these soluble factors might also correlate with the onset of AEs in D + T combination therapy. The serum levels of sCD163 were increased in patients with pyrexia in parallel with their severity, whereas the serum levels of CXCL5 were increased in patients without pyrexia. Moreover, increased levels of CXCL9, CXCL10, and CXCL11 were prominent in patients with AEs over G2 levels. As these chemokines recruit Th1, Th17, and activated CD8+ T cells, increased serum levels of these chemokines might correlate with the positive feedback of inflammatory reactions related to AEs.

RB1 gene mutations are a distinct predictive factor in Merkel cell carcinoma.

Merkel cell carcinoma (MCC) is a rare cutaneous neuroendocrine carcinoma that tends to show local recurrence and metastasis. Typically, MCC is polyomavirus (MCPyV)-associated and cytokeratin 20 (CK20) positive. However, little is known about this tumor and its origins. Here, we aimed to determine the developmental origins of MCC and to identify prognostic clinicopathologic factors. Initial examinations revealed that CK20 and MCPyV expression (CK20+, MCPyV+ (60%); CK20+, MCPyV- (10%); CK20-, and MCPyV- (30%)) did not affect overall survival. With RB1 gene sequencing of FFPE specimens, which covered an entire exon, all RB1 mutation-positive cases showed positive regional lymph node and/or distant metastases (8/8 cases, 100%), whereas the frequency of the metastasis was statistically significantly lower in RB1 mutation-negative cases, (10/16 cases, 62%, P = 0.033). The results were also confirmed with immunohistochemistry, and either RB1 alterations, entire exon sequencing, or immunohistochemistry was associated with the metastasis (P = 0.007). RB1 alterations may be used to access the aggressive clinical course of MCC.

The IL-1 promoter-driven luciferase reporter cell line THP-G1b can efficiently predict skin-sensitising chemicals.

IL-1 functions as an essential pro-inflammatory mediator for the sensitisation of allergic contact dermatitis (ACD). However, studies conducted to date have typically used a limited number of haptens and examined their effects only on murine ACD or murine dendritic cells (DCs). It therefore remains unclear whether IL-1α and/or IL-1ß is produced in ACD induced by haptens other than those commonly used in mouse ACD models, and whether they are essential for sensitisation leading to ACD in humans. In addition, it is unclear whether human DCs also produce IL-1α or IL-1ß after stimulation by haptens in general. Here, we first demonstrated that 10 haptens (3 extreme, 1 strong, 3 moderate and 3 weak) increased both IL-1α mRNA and IL-1ß mRNA expression by the human monocyte cell line THP-1, a commonly used surrogate of DCs in in vitro skin sensitisation tests. Next, we constructed an in vitro skin sensitisation test using a stable IL-1ß reporter cell line, THP-G1b, and evaluated whether 88 haptens and 34 non-haptens increase IL-1ß reporter activity. We found that 94% of 77 haptens evaluated after considering their applicability domain and solubility in the chosen media stimulated reporter activity. These studies demonstrated that most haptens, irrespective of their potency, increased IL-1ß mRNA expression by THP-1 cells, confirming that human DCs also produce IL-1ß after stimulation by most haptens. The luciferase assay using THP-G1b cells is thus another skin sensitisation test based on the adverse outcome pathway with reasonable performance.

The modified IL-8 Luc assay, an in vitro skin sensitisation test, can significantly improve the false-negative judgment of lipophilic sensitizers with logKow values > 3.5.

False-negative judgment due to poor chemical solubility is a problem with in vitro skin sensitisation tests. Water-insoluble chemicals are typically dissolved in DMSO in most sensitisation tests but precipitate when diluted with medium beyond their solubility in water. Such tests lack procedures to rule out false-negative judgments due to poor solubility. The IL-8 Luc assay (OECD442E) is unique in that if chemicals do not dissolve at 20 mg/mL in medium and have no effect on IL-8 luciferase activity (IL8LA), they are classified as indeterminate. The purpose of the present study was to reduce the number of indeterminate chemicals and improve assay performance. The IL-8 Luc assay can simultaneously examine glyceraldehyde 3-phosphate dehydrogenase luciferase activity (GAPLA) and IL8LA, and thus we examined the correlation between the reduction of GAPLA (defined as Inh-GAPLA) and the reduction of propidium iodide (PI)-excluding cells for three sensitizers and three non-sensitizers. We observed a significant correlation between luciferase activity driven by the GAPDH promoter of THP-G8 cells and the number of viable cells. Furthermore, chemicals providing an Inh-GAPLA value below 0.8 always reduced the ratio of PI-excluding cells to less than 0.6. Using the modified criteria, indeterminate chemicals are judged as negative if they provide Inh-GAPLA values below 0.8. This modification reduced the number of indeterminate chemicals and increased specificity, highlighting the unique advantage of the IL-8 Luc assay.

Optimization of the IL-2 Luc assay for immunosuppressive drugs: a novel in vitro immunotoxicity test with high sensitivity and predictivity.

We have reported that the IL-2 Luc assay can detect the effects of chemicals on IL-2 promoter activity by using a dual reporter cell line, 2H4 cells that measure IL-2 promoter-driven luciferase activity (IL2LA) and GAPDH promoter-driven luciferase activity (GAPLA). Since the IL-2 Luc assay cannot detect immunosuppressive drugs that are antimitotic towards rapidly proliferating cells, we attempted to establish a new assay to detect these chemicals by taking advantage of the dual reporter cell properties of 2H4 cells. We first determined the optimal incubation time with drugs and the seeding cell density, and confirmed that the change in GAPLA and IL2LA levels reflects the change in cell count and IL-2 production of 2H4 cells after drug treatment. We designed the IL-2 luciferase lymphotoxicity test (IL-2 Luc LTT) to detect the antimitotic effects of chemicals by modifying the protocol and criteria of the IL-2 Luc assay. To determine the performance of the IL-2 Luc LTT and that of the combination of the IL-2 Luc LTT and the IL-2 Luc assay, we examined 46 drugs: 19 immunosuppressive drugs with different mechanisms of action, 12 anti-cancer drugs, and 15 non-immunosuppressive drugs. The performances of the IL-2 Luc LTT, the IL-2 Luc assay and their combination were 43.3%, 61.3%, and 93.3%, respectively, for sensitivity, 84.6%, 53.3%, and 50.0%, respectively, for specificity, and 55.8%, 58.7%, and 79.5%, respectively, for accuracy. These results demonstrated that the combination of these two assays is promising for the detection of immunosuppressive drugs with different mechanisms of action.

Serum CCL22 levels decreased in parallel with disease activity in CCR4-positive mycosis fungoides treated with mogamulizumab.

Mogamulizumab is a humanized anti-C-C chemokine receptor type (CCR)4 antibody that shows cytotoxicity against CCR4+ lymphoma cells via antibody-dependent cell-mediated cytotoxicity in advanced cutaneous T cell lymphoma (CTCL) patients. The production levels of ligands for CCR4, that is, Chemokine (C-C motif) ligand (CCL)17 and CCL22, are important for the assessment of the disease activity in CTCL patients. We evaluated the serum levels of CCL17, CCL19, CCL22, C-X-C motif chemokine ligand (CXCL)10, and CXCL13, which are ligands for CCR4, CCR7, CCR4, C-X-C Motif Chemokine Receptor (CXCR)3, and CXCR5, respectively, at baseline and 4 weeks after the administration of mogamulizumab in five patients with mycosis fungoides. The serum levels of CCL22 were significantly decreased in patients who responded to mogamulizumab, but no differences were identified in the serum levels of CCL17, CCL19, CXCL10, or CXCL13. Immunofluorescence staining revealed that the majority of CCL22-producing cells were cluster of differentiation (CD)163+ tumor-associated macrophages, and they were surrounded by CCR4+ CTCL cells. Our present data suggested that the serum CCL22 level may be a predictive marker of the efficacy of mogamulizumab for the treatment of CCR4+ CTCL.

Successful treatment of mogamulizumab-resistant mycosis fungoides with mogamulizumab plus etoposide combined therapy: Investigation of the immunomodulatory effects of etoposide on the tumor microenvironment.

Mogamulizumab shows cytotoxicity against CCR4+ lymphoma cells by antibody-dependent cell-mediated cytotoxicity (ADCC) in advanced cutaneous T-cell lymphoma (CTCL) patients. Although mogamulizumab is used as one of the anchor drugs for the treatment of advanced CTCL, its efficacy is unsatisfactory, especially in mycosis fungoides (MF). Therefore, additional drugs to enhance the antitumor effects of mogamulizumab are needed to further optimize its use for the treatment of MF. In this report, two cases of mogamulizumab-resistant MF successfully treated with additional administration of etoposide are presented. Moreover, the possible mechanisms of mogamulizumab-etoposide combined therapy for the treatment of MF were investigated based on the modulation of chemokine profiles in vivo using an EL-4 mouse T-cell lymphoma model. Intraperitoneal administration of etoposide significantly increased the mRNA expressions of CCL17, CXCL5, and CXCL10, suggesting that CCR4+ CTCL cells gather around the tumor-associated macrophagess. Furthermore, the immunomodulatory effects of etoposide on the mRNA expressions of these chemokines were validated using monocyte-derived M2 macrophages in vitro. Since mogamulizumab shows cytotoxicity against CCR4+ lymphoma cells by ADCC that depends on the contact between the lymphoma cells and the effector cells, these chemokines could enhance the therapeutic effect of mogamulizumab.

Successful treatment of unresectable recurrent cutaneous squamous cell carcinoma of the scalp with meningeal invasion with nivolumab monotherapy.

Although the number of cutaneous squamous cell carcinoma (cSCC) cases is increasing, the effectiveness of systemic therapy for the treatment of advanced cSCC is limited. Since cSCC possesses a high tumor mutation burden (TMB) compared to other cancer species, and since high TMB correlated with increased neoantigens and the efficacy of anti-PD1 antibodies (Abs) in various cancers, cSCC could be a target for anti-PD1 Abs monotherapy. In this report, we describe a case of unresectable recurrent cSCC of the scalp with meningeal invasion, but highly expressed programmed death-ligand 1 (PD-L1), treated with nivolumab monotherapy.

Malassezia-derived aryl hydrocarbon receptor ligands enhance the CCL20/Th17/soluble CD163 pathogenic axis in extra-mammary Paget's disease.

Malassezia yeast play a role in the pathogenesis of chronic dermatitis, especially in apocrine areas, by polarizing the local immunologic background to a Th2/Th17 state through aryl hydrocarbon receptor (AhR)-dependent pathways. Extra-mammary Paget's disease (EMPD) is an adenocarcinoma of apocrine origin, and except for cases associated with Malassezia yeast and their metabolites, the lesions typically develop in areas not exposed to environmental material. The purpose of this study was to investigate (a) the immunomodulatory effects of Malassezia metabolites on normal human keratinocytes (NHKCs), focusing on interleukin (IL)-17 and related cytokines/chemokines (IL-23, IL-36γ, CCL20), (b) the expression of these factors in lesion-affected skin in EMPD and (c) the activation of tumor-associated macrophages (TAMs) by these factors. Malassezia metabolites augmented the expression of cytochrome P450, family 1, subfamily A, polypeptide 1 (CYP1A1), CCL20 and IL-36γ mRNA in NHKCs in vitro. In lesion-affected skin of patients with EMPD, epidermal keratinocytes expressed CYP1A1 and CCL20. In addition, Paget cells expressed CCL20 and IL-23. IL-17-producing cells were distributed adjacent to Paget cells. Compared to healthy donors, patients with EMPD exhibited significantly increased serum levels of soluble (s)CD163, CXCL5, CXCL10 and CCL20. In addition, serum levels of sCD163 decreased significantly following tumor resection. Our study demonstrates a possible mechanism for the development of EMPD involving AhR-mediated signalling by epidermal keratinocytes and RANKL-induced recruitment of Th17 cells and TAMs.

A novel technique to diagnose non-melanoma skin cancer by thermal conductivity measurements: Correlations with cancer stromal factors.

The skin surface temperature reflects the physiological state of the human body. Quantitative methods of identification of skin cancers based on accurate measurement of effective thermal conductivity (ETC) are among the promising diagnostic tools for differentiating non-invasive and invasive melanomas before surgical treatment. To validate these findings, in this report, the diagnostic methods for invasive and non-invasive extramammary Paget's disease (EMPD) and squamous cell carcinoma (SCC) were further tested by measuring the absolute value of skin surface temperature and the ETC of the skin. In addition, to investigate the stromal factors that might affect ETC, immunohistochemical staining for LL37, periostin (POSTN), MMP12, and MMP28 was performed. The invasive SCC and EMPD group showed a relatively higher skin surface temperature compared to the in situ SCC group. The non-invasive EMPD and SCC group showed significantly lower values of ETC at lesions, whereas the invasive EMPD group showed significantly higher ETC values at lesions compared to healthy skin. Immunohistochemical staining showed that the percentage of LL37-producing cells was significantly increased in invasive EMPD and SCC compared to that in non-invasive EMPD and SCC. Moreover, Spearman's rank correlation test showed a significant inverse correlation between the percentage of MMP12-positive cells and increased levels of ETC-expressing areas in EMPD and SCC (r = -.5997). The present study suggested that differences in ETC could be a novel high-accuracy diagnostic technique for non-melanoma skin cancer, especially for detecting dermal invasion of SCC and EMPD.

Eruptive xanthomas in a patient with soft-drink diabetic ketosis and apolipoprotein E4/2.

Soft-drink diabetic ketosis, characterized by acute onset ketosis induced by excessive ingestion of sugar-containing drinks, is often seen in obese, young patients, even with undiagnosed type 2 diabetes. We herein report a 15-year-old obese patient with the apolipoprotein E4/2 phenotype, in whom eruptive xanthomas lead to a diagnosis of soft-drink diabetic ketosis. He developed multiple asymptomatic yellowish papules on the auricles, back, buttocks and the extensor surfaces of the elbows and knees. He initially visited a dermatology clinic and his blood triglyceride and HbA1c levels were found to be 6,490 mg/dL and 16.5%, respectively. He was referred to our hospital for treatment of hyperglycemia and hypertyriglyceridemia. On admission, he had ketonuria and increased blood levels of 3-hydroxybutylate and acetoacetate. He habitually drank 1-3 litters of sweet beverages daily to quench his thirst. Therefore, "soft-drink diabetic ketosis" was diagnosed. Severe hypertriglyceridemia was considered to have been a consequence of impaired insulin action and his apolipoprotein E4/2 phenotype. We treated the diabetic ketosis and hypertriglyceridemia with intensive insulin therapy and a fat-restricted diet. At discharge, he no longer required insulin therapy and his blood glucose levels were controlled with metformin and voglibose. Along with amelioration of the hyperglycemia, triglyceride levels decreased to 247 mg/dL without administration of anti-hyperlipidemia agents. The eruptive xanthoma lesions gradually diminished in size and number and eventually disappeared by 12 months. This case provides an instructive example of eruptive xanthomas serving as a sign of severe dysregulation, not only of lipid, but also glucose, metabolism.

Minocycline decreases Th2 chemokines from M2 macrophages: Possible mechanisms for the suppression of bullous pemphigoid by traditional bullous disease drugs.

Minocycline/tetracycline is clinically used for the treatment of bullous pemphigoid (BP), and its clinical benefits are superior to those of prednisolone when considering adverse events. Although the clinical benefits of minocycline/tetracycline are well known, its immunosuppressive mechanisms are still unclear. In this study, we investigated the immunomodulatory effects of traditional anti-BP drugs (minocycline, nicotinic acid amide, dexamethasone and cyclosporine) on CD163+ M2 macrophages in vitro, with special focus on the production of CCL18 and CCL22, both of which are produced by CD163+ M2 macrophages in the lesional skin of BP and are increased in the serum of BP patients. Minocycline decreased the production of CCL22, CCL24 and CCL26 as well as CCL2 from M2 macrophages. CCL18 from M2 macrophages was decreased by dexamethasone and cyclosporine, but not decreased by minocycline. These data suggest that the clinical benefit of minocycline is partially explained by its suppressive effects against the production of specific Th2 chemokines from M2 macrophages, which should contribute to the recruitment of Th2 cells and eosinophils in the lesional skin of BP patients.

Cytotoxic antimelanoma drugs suppress the activation of M2 macrophages.

Together with regulatory T cells (Tregs), tumor-associated macrophages (TAMs) play roles in maintaining the tumor microenvironment. Although cytotoxic antimelanoma drugs such as dacarbazine (DTIC), nimustine hydrochloride (ACNU) and vincristine (VCR) have been used for the treatment of malignant melanoma as adjuvant therapy in Japan, the detailed mechanisms of their immunomodulatory effects are not fully understood. As the majority of TAMs are alternatively activated M2 macrophages that favour tumor development, the aim of this study was to elucidate the immunomodulatory effects of these reagents on human monocyte-derived M2 macrophages. First, mRNA expressions and protein production of immune checkpoint molecules, PD-L1 and chemokines by CD163+ CD206+ M2 macrophages derived from peripheral blood mononuclear cells were investigated to determine the immunomodulatory effects of DTIC, ACNU, and VCR. DTIC and VCR significantly decreased PD-L1 mRNA expression, which was confirmed by flow cytometry. Moreover, the mRNA expression and production of CCL22 were significantly decreased by DTIC, which suggested that DTIC might suppress the recruitment of Tregs in the tumor site. Furthermore, the decreased expression of PD-L1 and production of CCL22 were validated in vivo, using the B16F10 mouse melanoma model, leading to abrogation of the suppressive function of T-cell proliferation. The present report suggests one of the possible antimelanoma mechanisms of DAV combination chemotherapy for melanoma patients.

Profiling the immunotoxicity of chemicals based on in vitro evaluation by a combination of the Multi-ImmunoTox assay and the IL-8 Luc assay.

We established a luciferase reporter assay system, the Multi-ImmunoTox Assay (MITA), which can evaluate the effects of chemicals on the promoter activities of four cytokines: IL-2, IFN-γ, IL-1ß, and IL-8. We previously reported that MITA correctly reflected the change in mRNA of human whole-blood cells treated with dexamethasone, cyclosporine, FK506, or several other immunosuppressive drugs. In this study, we combined MITA with the IL-8 Luc assay to detect skin sensitization chemicals (OECD 442E) (modified MITA: mMITA) and established a data set of 60 chemicals examined by mMITA. Using the mMITA results, chemicals can be classified based on the lowest observed effect level (LOEL) of chemicals in suppressing or augmenting the promoter activities of the four cytokines. Moreover, we demonstrated that K-means clustering and hierarchical clustering of the 60 chemicals based on the LOEL for their effects on IL-2 and IL-8 promoter activities and the judgment by the IL-8 Luc assay resulted in the same 6-cluster solution: cluster 1 with preferential suppression of IL-8, cluster 2 with suppression of IL-2 and a positive IL-8 Luc assay result, cluster 3 with suppression of both IL-2 and IL-8, cluster 4 with no effects on IL-2 or IL-8 and a negative IL-8 Luc assay result, cluster 5 with suppression of both IL-2 and IL-8 and a negative IL-8 Luc assay result, and cluster 6 with preferential suppression of IL-8. These data suggest that mMITA is a promising novel high-throughput approach for detecting unrecognized immunological effects of chemicals and for profiling their immunotoxic effects.

Artificial Pigmented Human Skin Created by Muse Cells.

The skin composes physiological and chemical barrier and renews skin component cells throughout the human life. Melanocytes locate in the basal layer of the epidermis and produce melanin to protect the skin from ultraviolet. Melanin plays key roles in determining human skin and hair color. Melanocyte dysfunction observed in albinism and vitiligo not only causes cosmetic problems but also increases risk of skin cancer. As rejuvenate therapy, embryonic stem (ES) cells and induced pluripotent stem (iPS) cells have been reported to generate melanocytes. Other than ES and iPS cells, human skin tissues maintain pluripotent stem cells, named multilineage-differentiating stress-enduring (Muse) cells. We employ Muse cells isolated from human fibroblasts and adipose tissue to differentiate into melanocytes (Muse-MC). Muse-MC express melanocyte-related molecules, such as tyrosinase and DCT, and show tyrosinase activity. We also succeeded to differentiate Muse cells into fibroblasts and keratinocytes and created three-dimensional (3D) reconstituted skin with Muse cell-derived melanocytes, fibroblasts, and keratinocytes. The 3D reconstituted skin of Muse cell-derived cells coordinately showed epidermis layers and Muse-MC localized in the basal layer of the epidermis. Thus Muse cells in the human skin can be a source of rejuvenation medicine for the skin reconstruction.