NRF2 Augments Epidermal Antioxidant Defenses and Promotes Atopy.

Atopic dermatitis is a chronic form of allergic contact dermatitis that is closely associated with a compromised epidermal barrier. Immunogenicity of a given electrophilic hapten after penetration of this barrier depends directly on biochemical reactions in the thiol-rich layer in the stratum granulosum. In response to electrophilic hapten, NF-erythroid 2-related factor 2 (NRF2) in keratinocytes efficiently induces the production of antioxidants. In this study, we show that the immunogenicity of a given hapten depends directly on the extent to which it induces antioxidant host defenses within the epidermal tissue. We found that allergic contact dermatitis did not develop in NRF2-deficient mice because of compromise of the epidermal innate immune responses that upregulate IL-1α. We also analyzed epidermal NRF2 in association with congenital disorders with features similar to atopic dermatitis in humans. Epidermal samples from patients with Netherton syndrome and peeling skin syndrome exhibited elevated levels of NRF2 and also elevated levels of its downstream target, small proline-rich protein 2. Taken together, these results suggest that the thiol-mediated biochemical responses in the stratum granulosum provide a critical link between defective epidermal barrier function and the development of atopy. Likewise, our results suggested that NRF2 may have a profound impact on the generation of cutaneous immunological memory.

Immune response to dermatomyositis-specific autoantigen, transcriptional intermediary factor 1γ can result in experimental myositis.

OBJECTIVES: To investigate whether autoimmunity to transcriptional intermediary factor 1 (TIF1)γ, a ubiquitous nuclear autoantigen for myositis-specific autoantibodies detected in patients with dermatomyositis (DM) is pathogenetic for inflammatory myopathy. METHODS: Wild-type, ß2-microglobulin-null, perforin-null, Igµ-null and interferon α/ß receptor (IFNAR)-null mice were immunised with recombinant human TIF1γ whole protein. A thymidine incorporation assay was performed using lymph node T cells from TIF1γ-immunised mice. Plasma was analysed using immunoprecipitation followed by western blot analysis and enzyme-linked immunosorbent assays. Femoral muscles were histologically and immunohistochemically evaluated. CD8+ or CD4+ T cells isolated from lymph node T cells or IgG purified from plasma were adoptively transferred to naïve mice. TIF1γ-immunised mice were treated with anti-CD8 depleting antibody and a Janus kinase inhibitor, tofacitinib. RESULTS: Immunisation with TIF1γ-induced experimental myositis presenting with necrosis/atrophy of muscle fibres accompanied by CD8+ T cell infiltration successfully in wild-type mice, in which TIF1γ-specific T cells and antihuman and murine TIF1γ IgG antibodies were detected. The incidence and severity of myositis were significantly lower in ß2-microglobulin-null, perforin-null, CD8-depleted or IFNAR-null mice, while Igµ-null mice developed myositis normally. Adoptive transfer of CD8+ T cells induced myositis in recipients, while transfer of CD4+ T cells or IgG did not. Treatment with tofacitinib inhibited TIF1γ-induced myositis. CONCLUSIONS: Here we show that TIF1γ is immunogenic enough to cause experimental myositis, in which CD8+ T cells and type I interferons, but not CD4+ T cells, B cells or antibodies, are required. This murine model would be a tool for understanding the pathologies of DM.

Nuclear Factor Erythroid 2-Related Factor 2 (Nrf2) Regulates Epidermal Keratinization under Psoriatic Skin Inflammation.

Psoriasis is an autoinflammatory/autoimmune skin disease and the epitome of an exaggerated primary inflammatory response in the surface barrier tissue. Despite the efficacy of dimethyl fumarate, an electrophilic drug for psoriasis management, there is a paucity of mechanistic evidence in vivo. In response to electrophiles, the Kelch-like erythroid cell-derived protein with cap-n-collar homology-associated protein 1/nuclear factor erythroid 2-related factor 2 (NRF2) system mediates a myriad of cytoprotective mechanisms, including the regulation of excessive inflammatory response and epidermal differentiation. Because the psoriasiform tissue reaction comprises neutrophil infiltration and parakeratotic scaling, it is hypothesized that Nrf2 not only regulates inflammatory responses but also maintains epidermal differentiation, a hallmark of epidermal homeostasis. By using the imiquimod-induced cutaneous inflammation model, an exaggerated inflammatory response and impaired epidermal differentiation in Nrf2-/- mice was detected. Dimethyl fumarate treatment in Nrf2+/+ mice attenuated a psoriasiform tissue reaction and rescued epidermal differentiation, which was not observed in Nrf2-/- mice. In accordance with the fact that psoriasis plaques form well-demarcated parakeratotic lesions in association with the psoriasiform tissue reaction, the lesional skin showed reduced expression levels of NRF2 and its downstream target genes compared with nonlesional skin. In conclusion, Nrf2 attenuates the psoriasiform tissue reaction and underscores the mechanistic legitimacy of the electrophile-based approach for the management of psoriasis.

Loricrin: Past, Present, and Future.

The terminal differentiation of the epidermis is a complex physiological process. During the past few decades, medical genetics has shown that defects in the stratum corneum (SC) permeability barrier cause a myriad of pathological conditions, ranging from common dry skin to lethal ichthyoses. Contrarily, molecular phylogenetics has revealed that amniotes have acquired a specialized form of cytoprotection cornification that provides mechanical resilience to the SC. This superior biochemical property, along with desiccation tolerance, is attributable to the proper formation of the macromolecular protein-lipid complex termed cornified cell envelopes (CE). Cornification largely depends on the peculiar biochemical and biophysical properties of loricrin, which is a major CE component. Despite its quantitative significance, loricrin knockout (LKO) mice have revealed it to be dispensable for the SC permeability barrier. Nevertheless, LKO mice have brought us valuable lessons. It is also becoming evident that absent loricrin affects skin homeostasis more profoundly in many more aspects than previously expected. Through an extensive review of aggregate evidence, we discuss herein the functional significance of the thiol-rich protein loricrin from a biochemical, genetic, pathological, metabolic, or immunological aspect with some theoretical and speculative perspectives.

Correlation between blood cell count and outcome of melanoma patients treated with anti-PD-1 antibodies.

BACKGROUND: Anti-programmed cell death protein 1 monoclonal antibodies (αPD-1mAbs) have been shown to be effective for advanced malignant melanoma. Treatment with αPD-1mAbs can also cause immune-related adverse events (irAEs). However, clinical predictive factors for treatment responses or irAE risk remain unclear. OBJECTIVE: To identify useful blood biomarkers for response and occurrence of irAEs with αPD-1mAbs treatment. METHODS: We retrospectively collected data from patients with melanoma treated with αPD-1mAbs at the University of Tsukuba Hospital. Clinical data including age, sex, clinical type, metastatic site, treatment course, blood laboratory tests, irAEs and treatment outcome were collected. RESULTS: Multivariate logistic regression analysis showed that increased baseline neutrophil-lymphocyte ratio (NLR) was significantly associated with poor response (odds ratio [OR]: 2.638, P = 0.0227, cutoff value = 2.8). Similarly, multivariate Cox regression analysis revealed that NLR at baseline were significantly associated with shorter progression survival (hazard ratio: 1.343, P = 0.0095). As for irAEs, logistic regression analysis revealed that baseline absolute eosinophil count was positively associated with occurrence of endocrine irAEs (OR: 1.601, P = 0.045, cutoff value = 240/µL). Additionally, a higher relative eosinophil count at 1 month was significantly correlated with occurrence of endocrine irAEs (OR: 1.229, P = 0.0296, cutoff value = 3.2%). CONCLUSION: Our results suggested that NLR > 2.8 could be a useful baseline biomarker for indicating poor response to αPD-1mAbs treatment and that absolute eosinophil count >240/µL at baseline and relative eosinophil count at 1 month >3.2% could be useful biomarkers to predict endocrine irAEs in patients receiving αPD-1mAbs.

Unusual Bone Lesions with Osteonecrosis Mimicking Bone Metastasis of Squamous Cell Carcinoma in Recessive Dystrophic Epidermolysis Bullosa.

Some bone lesions are reported to mimic bone metastasis on imaging tests. Herein, we report a case of a 55-year-old Japanese man who presented with a skin tumor on the left lower extremity. He also had a history of recurrent generalized cutaneous blister and erosion formation since childhood. His skin lesions were diagnosed as cutaneous squamous cell carcinoma complicated by recessive dystrophic epidermolysis bullosa. Magnetic resonance imaging of the left lower extremity detected multiple focal bone lesions mimicking bone metastases in the left femur and tibia. However, bone biopsy revealed that the bone lesions were osteonecrosis without tumor cells. We suggest that cancer-induced osteonecrosis should be included in the differential diagnosis of bone lesions suspected of being metastases on magnetic resonance imaging.

Proteomic Analysis of Loricrin Knockout Mouse Epidermis.

The crosslinked envelope of the mammalian epidermal corneocyte serves as a scaffold for assembly of the lipid barrier of the epidermis. Thus, deficient envelope crosslinking by keratinocyte transglutaminase (TGM1) is a major cause of the human autosomal recessive congenital ichthyoses characterized by barrier defects. Expectations that loss of some envelope protein components would also confer an ichthyosis phenotype have been difficult to demonstrate. To help rationalize this observation, the protein profile of epidermis from loricrin knockout mice has been compared to that of wild type. Despite the mild phenotype of the knockout, some 40 proteins were incorporated into envelope material to significantly different extents compared to those of wild type. Nearly half were also incorporated to similarly altered extents into the disulfide bonded keratin network of the corneocyte. The results suggest that loss of loricrin alters their incorporation into envelopes as a consequence of protein-protein interactions during cell maturation. Mass spectrometric protein profiling revealed that keratin 1, keratin 10, and loricrin are prominent envelope components and that dozens of other proteins are also components. This finding helps rationalize the potential formation of functional envelopes, despite loss of a single component, due to the availability of many alternative transglutaminase substrates.

Recent advances in dermatomyositis-specific autoantibodies.

PURPOSE OF REVIEW: In dermatomyositis, disease-specific autoantibodies now cover more than 70% of patients. These autoantibodies closely correlate with distinct clinical manifestations. In the past few years, extensive evidence has been accumulated on clinical significance of dermatomyositis-specific autoantibodies including autoantibodies against melanoma differentiation antigen 5 (MDA5), transcriptional intermediary factor 1 (TIF1), nuclear matrix protein 2 (NXP2), and small ubiquitin-like modifier activating enzyme (SAE). RECENT FINDINGS: Anti-MDA5 antibodies are found with high specificity in clinically amyopathic dermatomyositis presenting rapidly progressive interstitial lung disease (ILD) especially in Asian population. Similar tendency has been reported in the US/Europe, although the frequency of positivity and the type of ILD may differ. Anti-TIF1 antibodies are present in juvenile and adult dermatomyositis patients with close correlation with malignancy in adult population. Anti-NXP2 antibodies share similar phenotype with anti-TIF1 antibodies, except that anti-NXP2 antibodies are associated with calcinosis and severe muscle disease. Although numbers are still small, patients with anti-SAE antibodies tend to present skin disease first and then progress to muscle weakness with systematic symptoms including dysphagia. Moreover, distinct cutaneous manifestations and muscle histopathology findings for each autoantibody have been reported. SUMMARY: 'Autoantibody-based classification' of dermatomyositis subsets is now a useful strategy for comprehending the heterogeneous spectrum of dermatomyositis.

NF1 gene silencing induces upregulation of vascular endothelial growth factor expression in both Schwann and non-Schwann cells.

Neurofibromatosis type I (NF1) is associated with typical hypervascular tumors, including neurofibroma, glioma, malignant peripheral nerve sheath tumors (MPNST) and glomus tumors. Previously, we and other groups reported that neurofibromas showed high-level expression of vascular endothelial growth factor (VEGF), a potent angiogenic factor involved in neovascularization. However, the molecular mechanism underlying the upregulation of VEGF in neurofibromas remains unclear. In this study, we examined the effects of Nf1 gene silencing on VEGF expression in Schwann cell and non-Schwann cell line and the upstream mTOR-HIF-1α - VEGF pathway in Schwann cell line. The results indicated that Nf1 gene silencing by lentiviral-mediated RNA interference resulted in elevated expression of VEGF, HIF-1α and phosphorylated mTOR at the protein level. The results obtained from Nf1 gene silencing in murine Schwann cell line analogously suggest that NF1 gene haploinsufficiency in human tumor Schwann cells may directly elicit upregulation of VEGF expression without the tumor microenvironment by activation of the mTOR-HIF-1α - VEGF pathway. We also showed that interleukin-6 is upregulated in Nf1 gene knock-down Schwann cells at the protein level.

Pituitary tumor-transforming gene 1 as a proliferation marker lacking prognostic value in cutaneous squamous cell carcinoma.

Non-melanoma skin cancer is the most frequently occurring type of cancer worldwide and is caused by epidermal carcinogenesis and malignant progression that involve dysregulated expression of proto-oncogenes and tumor suppressor genes. The proto-oncogene pituitary tumor-transforming gene 1 (PTTG1) is a ubiquitously expressed transcription factor that can promote enhanced proliferation of cultured epidermal keratinocytes. To investigate the potential roles of PTTG1 in epidermal carcinogenesis and malignant progression, the expression of PTTG1 was analysed by immunohistochemistry along with Ki67, keratin 10 (K10) and p53 in tissue samples of cutaneous squamous cell carcinomas (SCC), actinic keratoses (AK) and Bowen's disease (BD). Expression levels of PTTG1 were compared among these disease groups to test for correlations with proliferation, differentiation capacity or the existence of mutated tumor suppressor genes in each disease group. In each disease group, the expression levels of PTTG1 correlated positively with those of Ki67, although the differentiation status, measured by K10 expression, did not show any correlation. In contrast, the existence of mutated p53 proteins showed a positive correlation only in the SCC group. Moreover, the expression levels of PTTG1 in SCC did not correlate with known prognostic factors such as TNM staging or tumor thickness. These results suggest that PTTG1 may represent a proliferation marker associated with mutated p53 proteins but is not an informative predictor of poor clinical outcomes in SCC.

GATA-3 regulates differentiation-specific loricrin gene expression in keratinocytes.

Loricrin is a major component of the epidermal cornified cell envelope and is expressed only in terminally differentiated keratinocytes. This cell differentiation-specific expression pattern suggests specific regulatory mechanisms for activation of loricrin gene transcription in differentiated keratinocytes. Here, we identified a positive regulatory element in the proximal promoter region of the loricrin gene involved in activation of its expression in differentiated keratinocytes. A database search indicated that this sequence contained a GATA-3 binding motif. Constructs with point mutations in the GATA-3 binding motif showed decreased reporter activity, indicating that GATA-3 positively regulates loricrin gene transcription. Western blotting analysis indicated that GATA-3 is more abundant in differentiated than in undifferentiated keratinocytes. Cotransfection experiments indicated that GATA-3 activates transcription of the loricrin gene in a cooperative manner with c-Fos and Sp1. These findings indicate that GATA-3 contributes to keratinocyte differentiation-specific activation of loricrin gene transcription via interaction with c-Fos and Sp1.

Malignant blue nevus arising in a giant congenital cellular blue nevus in an infant.

Giant congenital blue nevus (GCBN) is rare and usually occurs on the scalp. Malignant blue nevus (MBN) is also rare and has a poor prognosis. We report a case of MBN arising in a GCBN on the back. There have been three previous reports of MBN associated with GCBN on the trunk; our case had the earliest onset of MBN arising in a GCBN.

The Role of KEAP1-NRF2 System in Atopic Dermatitis and Psoriasis.

The Kelch-like erythroid cell-derived protein with cap'n'collar homology-associated protein 1 (KEAP1)-nuclear factor erythroid-2-related factor 2 (NRF2) system, a thiol-based sensor-effector apparatus, exerts antioxidative and anti-inflammatory effects and maintains skin homeostasis. Thus, NRF2 activation appears to be a promising treatment option for various skin diseases. However, NRF2-mediated defense responses may deteriorate skin inflammation in a context-dependent manner. Atopic dermatitis (AD) and psoriasis are two common chronic inflammatory skin diseases caused by a defective skin barrier, dysregulated immune responses, genetic predispositions, and environmental factors. This review focuses on the role of the KEAP1-NRF2 system in the pathophysiology of AD and psoriasis and the therapeutic approaches that utilize this system.

NRF2 in the Epidermal Pigmentary System.

Melanogenesis is a major part of the environmental responses and tissue development of the integumentary system. The balance between reduction and oxidation (redox) governs pigmentary responses, for which coordination among epidermal resident cells is indispensable. Here, we review the current understanding of melanocyte biology with a particular focus on the "master regulator" of oxidative stress responses (i.e., the Kelch-like erythroid cell-derived protein with cap'n'collar homology-associated protein 1-nuclear factor erythroid-2-related factor 2 system) and the autoimmune pigment disorder vitiligo. Our investigation revealed that the former is essential in pigmentogenesis, whereas the latter results from unbalanced redox homeostasis and/or defective intercellular communication in the interfollicular epidermis (IFE). Finally, we propose a model in which keratinocytes provide a "niche" for differentiated melanocytes and may "imprint" IFE pigmentation.