Yoichi Moroi

An environmental contaminant, benzo(a)pyrene, induces oxidative stress-mediated interleukin-8 production in human keratinocytes via the aryl hydrocarbon receptor signaling pathway

BACKGROUND Benzo(a)pyrene (BaP) is an environmental contaminant found in cigarette smoke. It is well known that cigarette smoking exacerbates interleukin-8 (IL-8)-related inflammatory skin diseases such as psoriasis, palmoplantar pustulosis and acne. Although BaP has been shown to exert its biological effects via the aryl hydrocarbon receptor (AhR) signaling pathway, the mechanism of its inflammatory effects on skin remains unanswered. OBJECTIVE To elucidate whether or not BaP cause AhR activation and subsequent oxidative stress leading to IL-8 production in normal human epidermal keratinocytes (NHEKs). METHODS NHEKs exposed to BaP were analyzed. Immunofluorescence, real-time PCR, Western blotting, ELISA, reactive oxygen species (ROS) detection using H2DCFDA and RNA interference using si (small interfering) RNA were employed. RESULTS Immunofluorescence analysis clearly demonstrated that BaP induced nuclear translocation of AhR from cytoplasm. The AhR activation subsequently induced CYP1A1 mRNA and protein expression in a dose-dependent manner. In addition, ROS and IL-8 production were coordinately augmented by BaP, whereas this was not the case in IL-1α, IL-6, TNF-α or GM-CSF production. Knockdown of AhR expression using siRNA transfection inhibited BaP-induced-ROS and IL-8 production, suggesting that these responses are strongly dependent on the AhR signaling pathway. Furthermore, the addition of N-acetyl cystein or catalase cancelled the IL-8 production by BaP, indicating that ROS production is essential for IL-8 production. RESULTS This data highlights AhR-ROS-dependent regulation of IL-8 in NHEKs by BaP, providing a plausible explanation, at least in part, for why cigarette smoking exacerbates IL-8-related skin diseases such as psoriasis, palmoplantar pustulosis and acne.

Identification of Ketoconazole as an AhR-Nrf2 Activator in Cultured Human Keratinocytes: The Basis of Its Anti-Inflammatory Effect

Ketoconazole (KCZ) has been shown to exhibit anti-inflammatory effects in addition to its inhibitory effects against fungi; however, the underlying molecular mechanism remains poorly understood. Aryl hydrocarbon receptor (AhR), a receptor that is activated by polycyclic aromatic hydrocarbons (PAHs) and halogenated aromatic hydrocarbons such as dioxin, is a sensor of the redox system against oxidative stress and regulates nuclear factor-erythroid 2-related factor-2 (Nrf2), a master switch of the redox machinery. To clarify whether KCZ modulates AhR-Nrf2 function leading to redox system activation, cultured human keratinocytes were treated with KCZ. Confocal microscopic analysis revealed that KCZ induced AhR nuclear translocation, resulting in the upregulation of CYP1A1 mRNA and protein expression. Furthermore, KCZ actively switched on Nrf2 nuclear translocation and quinone oxidoreductase 1 expression. Tumor necrosis factor-α- and benzo(a)pyrene (BaP)-induced reactive oxidative species (ROS) and IL-8 production were effectively inhibited by KCZ. Knockdown of either AhR or Nrf2 abolished the inhibitory capacity of KCZ on ROS and IL-8 production. In addition, KCZ-induced Nrf2 activation was canceled by AhR knockdown. Moreover, KCZ inhibited BaP-induced 8-hydroxydeoxyguanosine and IL-8 production. In conclusion, the engagement of AhR by KCZ exhibits the cytoprotective effect mediated by the Nrf2 redox system, which potently downregulates either cytokine-induced (AhR-independent) or PAH-induced (AhR-dependent) oxidative stress.

Induction of Efficient Antitumor Immunity Using Dendritic Cells Activated by Recombinant Sendai Virus and Its Modulation by Exogenous IFN-β Gene

Dendritic cell (DC)-based cancer immunotherapy has been paid much attention as a new and cancer cell-specific therapeutic in the last decade; however, little clinical outcome has been reported. Current limitations of DC-based cancer immunotherapy include sparse information about which DC phenotype should be administered. We here report a unique, representative, and powerful method to activate DCs, namely recombinant Sendai virus-modified DCs (SeV/DC), for cancer immunotherapy. In vitro treatment of SeV without any bioactive gene solely led DCs to a mature phenotype. Even though the expression of surface markers for DC activation ex vivo did not always reach the level attained by an optimized amount of LPS, superior antitumor effects to B16F1 melanoma, namely tumor elimination and survival, were obtained with use of SeV-GFP/DC as compared with those seen with LPS/DC in vivo, and the effect was enhanced by SeV/DC-expressing IFN-β (SeV-murine IFN-β (mIFN-β)/DC). In case of the treatment of an established tumor of B16F10 (7–9 mm in diameter), a highly malignant subline of B16 melanoma, SeV-modified DCs (both SeV-GFP/DC and SeV-mIFN-β/DC), but not immature DC and LPS/DC, dramatically improved the survival of animals. Furthermore, SeV-mIFN-β/DC but not other DCs could lead B16F10 tumor to the dormancy, associated with strongly enhanced CD8+ CTL responses. These results indicate that rSeV is a new and powerful tool as an immune booster for DC-based cancer immunotherapy that can be significantly modified by IFN-β, and SeV/DC, therefore, warrants further investigation as a promising alternative for cancer immunotherapy.

Propionibacterium acnes-induced IL-8 production may be mediated by NF-κB activation in human monocytes

Propionibacterium acnes (P. acnes) causes an inflammatory acne that is characterized by massive neutrophilic infiltration. IL-8 is thought to play an important role in the pathophysiology of P. acnes, although the mechanisms by which P. acnes up-regulates the release of IL-8, a neutrophilic chemokine, from target cells is not well understood. In this study, we investigated the mechanisms through which heat-killed P. acnes induces IL-8 production in THP-1 cells (a human monocytic cell line). We found that P. acnes is able to directly induce IL-8 production and IL-8 mRNA expression in human monocytic cells in a dose- and time-dependent manner through a mechanism requiring transcription factor NF-kappaB activation. Additionally, P. acnes-induced IL-8 secretion was inhibited by roxithromycin, a macrolide antibiotic, and its inhibitory effect seemed to be partially associated with the inhibition of P. acnes-induced NF-kappaB activation. This is the first study to show that NF-kappaB activation is involved in the IL-8 production of monocytic cells stimulated by P. acnes.

Clinicopathological features of cutaneous lesions of adult T-cell leukaemia/ lymphoma.

Background  Adult T‐cell leukaemia/lymphoma (ATLL) is a human malignancy associated with human T‐cell leukaemia virus type I (HTLV‐I). ATLL frequently involves the skin.

Clinicopathologic and prognostic significance of SATB1 in cutaneous malignant melanoma

BACKGROUND Special AT-rich sequence-binding protein-1 (SATB1), a new type of gene regulator, has been reported to be expressed in several human cancers and may have malignant potential. However, no data on SATB1 expression and its relationship to tumor progression in cutaneous malignant melanoma (CMM) has yet been reported. OBJECTIVE We examined the immunohistochemical expression of SATB1 in CMM to determine whether it could serve as a prognostic marker. METHODS A total of 97 samples of primary CMM and controls were immunostained for SATB1. The following clinicopathologic variables were evaluated: age, gender, subtype, SATB1 expression, Breslow thickness, Clark level, presence of ulceration, lymph node metastasis, distant metastasis, and survival. Statistical analyses were performed to assess for associations. Several parameters were analyzed for survival using the Kaplan-Meier method and Cox proportional-hazards model. RESULTS Forty cases (85.1%) of CMM showed positive staining for SATB1 by immunohistochemistry. The intensity of SATB1 staining was significantly higher in CMM than in nevus NV and normal skin (NS) (P < 0.01). High SATB1 expression was significantly correlated with Breslow thickness, Clark level, mortality, presence of ulceration, and lymph node metastasis (P < 0.01). Moreover, Kaplan-Meier analysis revealed that SATB1 overexpression was significantly associated with worse survival (P < 0.01). Further univariate analysis and multivariate regression analysis indicated that SATB1 expression was an independent prognostic marker for CMM (P = 0.03). CONCLUSIONS The overexpression of SATB1 correlated with metastatic potential of CMM and is a novel independent prognostic marker for predicting outcome.

Arylhydrocarbon receptor (AhR) activation in airway epithelial cells induces MUC5AC via reactive oxygen species (ROS) production.

The dioxins and dioxin-like compounds in cigarette smoke regulate various immunological responses via the arylhydrocarbon receptor (AhR). These environmental toxicants are known to cause bronchitis, asthma, chronic obstructive pulmonary disease (COPD), and lung cancer. Recent studies have demonstrated that AhR activation upregulates the expression of mucin 5AC, oligomeric mucus/gel-forming (MUC5AC) in the airway epithelial cell line. However, the mechanism for the production of mucin has not been clarified. In this study, we investigated the role and pathway of AhR in airway epithelial cells by using selective agonists and antagonists. After stimulation with or without benzopyrene (B[a]P), an AhR agonist, MUC5AC expression was measured by real-time RT-PCR. The mechanism of AhR-induced MUC5AC expression in airway epithelial cells was studied in terms of the production of cytokine and reactive oxygen species (ROS). Treatment with B[a]P increased ROS generation in NCI-H₂₉₂ cells. Furthermore, B[a]P-induced MUC5AC upregulation and mucin production were inhibited by AhR siRNA or the use of an antioxidative agent. These results suggest that the AhR-induced increase of mucin production is partially mediated by ROS generation. An antioxidant therapy approach may help to cure AhR-induced mucus hypersecretory diseases.

Differential regulation of thymus- and activation-regulated chemokine induced by IL-4, IL-13, TNF-α and IFN-γ in human keratinocyte and fibroblast

Abstract The CC chemokine thymus- and activation-regulated chemokine (TARC/CCL17) acts on CC chemokine receptor 4 (CCR4), which is known to be selectively expressed in Th2 cells. In order to compare the regulatory profiles of TARC production by tumor necrosis factor-α (TNF-α), IFN-γ, interleukin-4 (IL-4) and IL-13 in keratinocytes and fibroblasts, HaCaT cells, a human keratinocyte cell line, and NG1RGB cells, a human skin fibroblast cell line, were used. The expression of TARC protein was measured using enzyme-linked immunosorbent assay (ELISA), and the mRNA level was detected by reverse transcriptase polymerase chain reaction (RT-PCR). The spontaneous expression of TARC protein and mRNA levels were augmented by TNF-α and IFN-γ and were inhibited by IL-4 and IL-13 in the keratinocytes. The fibroblasts expressed the TARC protein and mRNA only in the presence of IL-4+TNF-α or IL-13+TNF-α stimulation. IFN-γ further enhanced the IL-4+TNF-α or IL-13+TNF-α-induced TARC production in the fibroblasts. Thus, TNF-α and IFN-γ -induced TARC production was differentially regulated by IL-4 and IL-13 in human keratinocytes and fibroblasts.

Decrease in circulating Th17 cells correlates with increased levels of CCL17, IgE and eosinophils in atopic dermatitis

BACKGROUND Clinical significance of circulating CD4(+) T cell subsets, including T-helper (Th)1, Th2, Th17 and regulatory T (Treg) cells, in patients with atopic dermatitis (AD) remains unclear. No previous studies have simultaneously evaluated the four T cell subset profiles in AD. OBJECTIVE The aim of the present study was to explore whether the percentage of these four subsets of CD4(+) T cells correlate to the severity parameters of AD patients. METHODS Intracellular expression of interferon (IFN)-γ, interleukin (IL)-4, IL-17 and forkhead box P3 (Foxp3) in CD4(+) T cells was evaluated in peripheral blood mononuclear cells from normal controls and patient with AD as well as with chronic eczema using a flow cytometer. Serum CCL17 levels were measured as an objective severity parameter of AD together with percentage of eosinophils and serum IgE levels. RESULTS In AD patients, the number of Th1 (IFN-γ(+)) and Th17 (IL-17(+)) subsets was significantly decreased, but that of Th2 (IL-4(+)) and Treg (Foxp3(+)) subsets was similar to that of normal controls. The T cell subset profiles of patients with chronic eczema were not different with those of normal controls. The frequency of Th17cells, particularly that of the IFN-γ(nega)IL-17(+) subset, showed a significant negative correlation with CCL17, IgE and eosinophil levels in AD patients. This was, however, not the case in Th1, Th2 and Treg cells. CONCLUSION Decreased circulating Th17 cells might contribute to activity of AD.

Dosage and Adverse Effects of Topical Tacrolimus and Steroids in Daily Management of Atopic Dermatitis

Since 1999, combination therapy with tacrolimus and topical steroids has been widely used for the treatment of adolescent/adult‐type atopic dermatitis. In order to determine the clinical doses of topical tacrolimus and steroids for daily treatment of atopic dermatitis and to elucidate their beneficial and adverse effects, we analyzed the clinical data from 215 patients with atopic dermatitis who were more than 16 years old. Less than 70g of tacrolimus and less than 15 g of steroids were applied to 90% of the patients on the face and neck, and less than 75.8 g of tacrolimus and less than 322 g of steroids were applied to 90% of the patients on the trunk and extremities during the six‐month treatment period. Topical tacrolimus is much more frequently used on face and neck lesions (99.1%); in only 39.5% of cases was it used on the trunk and extremities. The majority of patients improved after six months of the combination topical therapy; however, atopic dermatitis was not controlled in 6% of the patients. The combination therapy did not seem to increase the risk of cutaneous infections; however, the incidence of herpes simplex infection on the face and neck was 2.8% at pre‐treatment and slightly increased to 4.7% during the therapy. The incidence of all steroid‐induced adverse effects was reduced both in frequency and intensity with a decrease in the dose of topical steroids through simultaneous tacrolimus application. Combination therapy with topical tacrolimus and steroids is useful for treating atopic dermatitis, but a small percentage of the patients still cannot be satisfactorily treated. For such patients, adjustments of the dose and rank of topical steroids and tacrolimus and other therapeutic adjuncts are necessary.