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Introduction: Basal cell carcinoma (BCC) is a common skin cancer that rarely metastasizes but can deeply infiltrate local tissues. The small number of unresectable BCC cases makes it difficult to conduct clinical trials, resulting in delays in the development of effective drugs for BCC. Cancer gene panel testing has led to an increasing number of new treatment proposals for patients with solid tumors for whom no standard treatment is available. Case Presentation: We described a case of unresectable BRCA1 L63*-mutated BCC invading the parietal bone, which was successfully treated with cisplatin and fluorouracil. Conclusion: Our present case suggests that comprehensive mutation analysis by gene panel testing is important in advanced BCC because genes other than those involved in the hedgehog signaling pathway can be driver genes.

Abstract Psoriasis is a chronic inflammatory disease that sometimes necessitates therapeutic intervention with biologics. Autoantibody production during treatment with tumor necrosis factor (TNF) inhibitors is a recognized phenomenon, however, the production of autoantibodies associated with antiphospholipid syndrome (APS) has not been comprehensively evaluated in patients with psoriasis. This study was conducted to assess the prevalence of APS-associated autoantibodies in patients with psoriasis treated with different biologics and to investigate the potential associations between autoantibody production and clinical or serological parameters. Patients with psoriasis undergoing biologics treatments were enrolled in this study, and were categorized based on the type of biologics administered, TNF, interleukin (IL)-17, or IL-23 inhibitors. Clinical and serological data were collected and analyzed in conjunction with data on APS autoantibodies. TNF inhibitors were associated with a higher frequency of APS autoantibodies compared to IL-17 and IL-23 inhibitors. Notably, the presence of APS autoantibodies correlated with concurrent arthritis and higher disease severity at treatment initiation in patients treated with TNF inhibitors. Elevated Psoriasis Area and Severity Index scores and anti-nuclear antibody titers higher than × 320 were predictors of APS autoantibody production. Despite the higher autoantibody rates, clinical symptoms of APS were absent in these patients. This study provides the first comprehensive evidence of an increased frequency of APS autoantibodies associated with TNF inhibitor treatment in patients with psoriasis. The observed association between APS autoantibody positivity and TNF inhibitor treatment or clinical parameters suggests a potential immunomodulatory interplay between autoimmunity and inflammation in the pathogenesis of psoriasis.

Introduction: Capicua transcriptional repressor (CIC)-DUX4 rearranged sarcoma is a subtype of CIC-rearranged sarcomas composed of undifferentiated Wilms’ tumor 1 (WT1)+, CD99+ round cells with recurrent CIC gene rearrangement. The diagnosis of CIC-rearranged sarcoma remains challenging, and the prognosis of CIC-rearranged sarcomas is poor. Case Presentation: In this report, we described a case of CIC-DUX4 rearranged sarcoma presenting in the skin, expressing WT1 and CD99 in a dot-like pattern. In addition, the assessment of genomic alterations using genome panel testing was useful to confirm the accurate diagnosis. Conclusion: Our present case suggests that widespread use of genomic panel testing in the future may lead to early treatment and improve the prognosis of CIC-rearranged sarcomas.

Aging is a high-risk factor for obstructive and fibrotic lung diseases. Fibrotic lung disease leading to decreased lung function is characterized by interstitial remodeling and tissue scarring (sclerosis), with destruction of alveoli and excess deposition of type I collagen, an extracellular matrix component secreted by fibroblasts. Therefore, regulating transforming growth factor-β (TGF-β) as a profibrotic signal is essential to suppress pulmonary fibrosis. In pulmonary fibrosis, TGF-β signaling is mediated by Smad and YAP/TAZ, and TAZ linked to the pathology of pulmonary function is observed in lung fibroblasts from patients with idiopathic pulmonary fibrosis. Although fibrosis is thought to be irreversible, it is an interventional condition. Decorin (DCN) blocks TGF-β signaling in pulmonary fibrosis, although there are no cellular pharmacological methods to stimulate DCN secretion. We previously showed that chicken eggshell membrane (ESM, a well-known wound-healing material) promotes dcn gene expression in fibroblasts. In this study, we investigated whether ESM stimulates DCN secretion as an endogenous mediator and ameliorates pulmonary fibrosis. Decorin secretion was significantly enhanced in the WI-38 lung fibroblast culture supernatants supplemented with ESM. This effect was increased with major component lysozyme and maximally promoted in experiments with lysozyme and ovotransferrin (the two main proteins in soluble ESM) at a 16:1 concentration ratio, the ratio in the ESM extract. Decorin secretion by ESM modulates TGF-β signaling in lung fibroblasts by reducing TAZ and pSmad2 nuclear localization. Decorin siRNA experiments confirmed that nuclear localization of TAZ is DCN-dependent. In a mouse model of bleomycin-induced pulmonary fibrosis, all fibrotic markers of ESM treatment group such as hydroxyproline (a collagen deposition marker), and both evaluation of fibrosis density by automated thresholding of picrosirius red-stained lung tissue scan images and Ashcroft fibrosis scores, and also the nuclear localization of TAZ were reduced after 2 weeks compared with control group. Furthermore, long-term (22 week) ESM consumption by healthy individuals significantly improved vital capacity and the forced expiratory volume in 1 s to forced vital capacity ratio (FEV1/FVC). This study reveals that ESM, a well-established wound-healing material, may be a potential preventive medicine for pulmonary fibrosis.

Abstract Here we report the largest Asian genome-wide association study (GWAS) for systemic sclerosis performed to date, based on data from Japanese subjects and comprising of 1428 cases and 112,599 controls. The lead SNP is in the FCGR/FCRL region, which shows a penetrating association in the Asian population, while a complete linkage disequilibrium SNP, rs10917688, is found in a cis-regulatory element for IRF8. IRF8 is also a significant locus in European GWAS for systemic sclerosis, but rs10917688 only shows an association in the presence of the risk allele of IRF8 in the Japanese population. Further analysis shows that rs10917688 is marked with H3K4me1 in primary B cells. A meta-analysis with a European GWAS detects 30 additional significant loci. Polygenic risk scores constructed with the effect sizes of the meta-analysis suggest the potential portability of genetic associations beyond populations. Prioritizing the top 5% of SNPs of IRF8 binding sites in B cells improves the fitting of the polygenic risk scores, underscoring the roles of B cells and IRF8 in the development of systemic sclerosis. The results also suggest that systemic sclerosis shares a common genetic architecture across populations.

Introduction: Chemoradiotherapy with taxanes is well-recognized as a first-line therapy for cutaneous angiosarcoma (CAS), but second-line therapy for CAS is still controversial. Case Presentation: In this report, we described a 75-year-old Japanese case of recurrent, tumor mutation burden-high CAS on the scalp treated with pembrolizumab. Our present case survived for 1 year despite of taxane refractory CAS with mediastinal lymph node metastasis, though the administration of anti-PD-1 Abs alone could not fully suppress the tumor progression of CAS. Conclusion: Since various factors such as pro-angiogenic molecules are correlated with the tumor progression in CAS, the administration of anti-PD-1 Abs alone could not fully suppress the tumor progression of CAS. Further novel anticancer drugs are needed in the future for the treatment of CAS.

Abstract The papular variant of elastolytic giant cell granuloma (EGCG) is a rare variant of EGCG, and it is occasionally associated with metabolic comorbidities, including diabetes mellitus (DM). Although the mechanisms of developing EGCG are still unknown, one of the possible mechanisms for ECGC developing in a DM patient might be explained by hyperglycaemia‐induced inflammation. In this report, we described a case of papular interstitial EGCG mainly composed of matrix metalloproteinase (MMP)‐9 bearing cells, concomitant with lichen amyloidosus in a type II DM patient.

Abstract Objectives Graft‐versus‐host disease (GVHD) is an important complication of bone marrow transplantation. Recent reports suggest the significance of T‐cell subsets (Th1, Th17, and cytotoxic CD8+ T cells) as well as CD163+ macrophages in the development of cutaneous GVHD. CD163+ macrophages produce various chemokines to establish the immunological microenvironment following stimulation by stromal factors in lesional skin. Thus, the purpose of this study is to determine the main source of IFN‐inducible chemokines in the lesional skin of GVHD. Methods We employed immunohistochemical (IHC) staining for CD163 as well as interferon (IFN)‐inducible chemokines (CXCL9, CXCL10, CXCL11) to determine if the main source of IFN‐inducible chemokines in the lesional skin of GVHD was CD163+ macrophages. Moreover, we investigated the possible cytokine profiles of lesional skin in GVHD by evaluating phospho‐signal transducer and activator of transcription (pSTAT) expression in epidermal keratinocytes. Results Immunohistochemical staining of serial sections for CD163 revealed that CXCL9‐expressing cells, CXCL10‐expressing cells, and CXCL11‐expressing cells were detected in adjacent to CD163+ TAMs in the dermis. In contrast, there were no CCL17‐expressing cells or CCL22‐expressing cells in the dermis. The nuclei of epidermal keratinocytes in GVHD expressed pSTAT1, pSTAT3, and pSTAT5B. Conclusions The chemokine expression patterns on CD163+ macrophages matched the expected phosphorylation pattern of epidermal STATs. Our present study suggested that CD163 + macrophages may be a therapeutic target in GVHD.

Olaparib is recently approved as an anti-tumor agent for several cancers, including castration-resistant prostate cancer, which inhibits poly (adenosine diphosphate-ribose) polymerase, a DNA repair factor. Since olaparib is a newly approved drug, there are few reports of skin disorders that may be triggered by olaparib administration. In this report, we present a case with an olaparib-induced drug eruption presenting multiple purpuras on the patient’s fingers and fingertips. The present case suggests that olaparib might induce purpura as nonallergic drug eruption.

Advanced melanoma patients with liver metastases show a limited response to immunotherapy by the induction of regulatory T cells and depletion of effector cells, which leads to a poor prognosis. Tumor-associated macrophages (TAMs) induce apoptosis of activated antigen-specific CD8+ T cells in melanomas, leading to induction of tolerance to immune checkpoint inhibitors. In addition, TAMs produce various chemokines, and several serum pro-inflammatory chemokines measured at baseline are useful for the prediction of the efficacy of immunomodulatory drugs. In this study, serum levels of CCL22, CXCL5, and CXCL10 were evaluated by ELISA at baseline in 10 melanoma patients, 5 with liver metastases and 5 with lung metastases, treated with anti-PD1 Abs. Serum levels of CCL22, but not CXCL5 and CXCL10, were increased in patients with liver metastases compared to those with lung metastases or historical controls. The present data suggest that elevated serum CCL22 levels might be a biomarker for liver metastases in melanoma patients.

A20 gene variants are linked with systemic sclerosis (SS), but the mechanisms are unclear. Here, the authors show that A20 expression is reduced in SS skin and lungs, that its ablation in mice induces SS, and that show that fibrosis can be ameliorated by induction of A20.

Abstract Bexarotene is often administered to phototherapy‐resistant early cutaneous T‐cell lymphoma (CTCL) patients as one of the first‐line therapies in real‐world practice. Since bexarotene reduces the expression of CCR4 in CTCL cells and CCL22 to decrease serum CCL22 levels, bexarotene inhibits the migration of CTCL cells, as well as other CCR4+ cells, such as cytotoxic T cells and regulatory T cells, in the lesional skin of CTCL. In this report, the efficacy of bexarotene in 28 cases of CTCL, as well as its correlations with immunohistochemical profiles of tumour‐infiltrating leucocytes (TILs), was retrospectively investigated. The overall response rate at 1 and 4 months for the total cohort was 70.8% (95% CI, 50.6%–86.3%) and 47.8% (95% CI, 29.2%–67.0%), respectively. The disease control rate for the total cohort at 4 months was 65.2% (95% CI, 44.8%–81.3%). The mean event‐free survival for all patients was 4.1 months (0.3–68.5 months). In addition, the immunoreactive cells were calculated using digital microscopy, suggesting that the ratio of CD25+ cells among TILs was significantly increased in patients who responded to bexarotene (p = 0.0209), whereas there were no significant differences in the ratios of CD8+ cells, granulysin+ cells, and Foxp3+ cells among TILs between responder and non‐responder patients. Collectively, the ratio of CD25 expression among TILs might be a predictive biomarker for the efficacy of bexarotene.

Primary cutaneous anaplastic large cell lymphoma (PCALCL) is a rare variant of cutaneous T cell lymphoma (CTCL) characterized by CD30-expressing large atypical cells with kidney-shaped nuclei called hallmark cells. Since PCALCL is a rare variant of CTCL, the treatment of PCALCL is still controversial. In this report, a case of PCALCL successfully treated with denileukin diftitox as second-line therapy is described. Interestingly, the administration of denileukin diftitox decreased CD8+ T cells, CD25+ cells, granulysin-bearing lymphocytes, and CD163+ macrophages. The present case suggests that denileukin diftitox might induce an anti-lymphoma effect as well as modulate the tumor microenvironment.

Encorafenib plus binimetinib combination therapy is one of the first-line therapies for advanced melanoma, and it is known to cause a different profile of adverse events (AEs) than dabrafenib plus trametinib combination therapy. Of such AEs, tubulointerstitial nephritis caused by BRAF plus MEK inhibitors combination therapy is limited. In this report, a case of tubulointerstitial nephritis that developed in a rheumatoid arthritis patient with advanced melanoma treated with encorafenib plus dabrafenib combination therapy is presented.

Abstract Linear childhood discoid lupus erythematosus (DLE) is a rare variant of DLE with a linear distribution that preferentially occurs on the face and scalp of children. We report a rare case of linear childhood DLE on the Blaschko's line of his left upper arm of a 7‐year‐old Japanese boy with 4‐year history of the disease. He was positive for anti‐single‐stranded DNA antibodies, but still showed no systemic symptoms during the 3‐year follow‐up period. We reviewed previous reports on linear childhood DLE and found that patients with nonfacial/nonscalp skin lesions are significantly more likely to be positive for autoantibodies.

Abstract Background We have recently demonstrated that serum CCL20 levels positively correlate with mean pulmonary arterial pressure in patients with systemic sclerosis (SSc). Considering a proangiogenic effect of CCL20 on endothelial cells via CCR6, the CCL20/CCR6 axis may contribute to the development of SSc vasculopathy. Therefore, we explored this hypothesis using clinical samples, cultured cells, and murine SSc models. Methods The expression levels of CCL20 and CCR6 in the skin, mRNA levels of target genes, and the binding of transcription factor FLI1 to the target gene promoter were evaluated by immunostaining, quantitative reverse transcription PCR, and chromatin immunoprecipitation, respectively. Vascular permeability was evaluated by Evans blue dye injection in bleomycin-treated mice. Angiogenic activity of endothelial cells was assessed by in vitro angiogenesis assay. Results CCL20 expression was significantly elevated in dermal fibroblasts of patients with early diffuse cutaneous SSc, while CCR6 was significantly up-regulated in dermal small vessels of SSc patients irrespective of disease subtypes and disease duration. In human dermal microvascular endothelial cells, FLI1 siRNA induced the expression of CCR6, but not CCL20, and FLI1 bound to the CCR6 promoter. Importantly, vascular permeability, a representative SSc-like vascular feature of bleomycin-treated mice, was attenuated by Ccr6 siRNA treatment, and CCR6 siRNA suppressed the angiogenic activity of human dermal microvascular endothelial cells assayed by in vitro tube formation. Conclusions The increased expression of endothelial CCR6 due to FLI1 deficiency may contribute to the development of SSc vasculopathy.

Systemic sclerosis (SSc) disease involves multisystem fibrosis and autoimmunity with limited treatment options. Here the authors demonstrate that IL-31 and IL-31RA are overexpressed in dermal fibroblasts from SSc patients and show that fibrosis and cytokine release can be reduced upon blocking of IL-31/IL-31RA.

Abstract Antimelanoma differentiation‐associated gene 5 antibody (anti‐MDA5 Ab)‐positive dermatomyositis frequently develops life‐threatening rapidly progressive interstitial lung disease (RP‐ILD). The longitudinal dynamics of antibody titers reflects treatment responses, and negative conversion of anti‐MDA5 Ab leads to the prevention of RP‐ILD relapse. Case 1 finally achieved negative conversion of anti‐MDA5 Ab after 18‐month immunosuppressive therapy despite early diagnosis from skin manifestations. Case 2 showed re‐elevation of anti‐MDA5 Ab after tacrolimus discontinuation; immediate restart of tacrolimus recovered it without relapse. These cases suggest that anti‐MDA5 Ab monitoring is essential to determine therapeutic strategy in dermatomyositis patients with RP‐ILD during both initial and maintenance phases.

Abstract Background Aldehyde dehydrogenase 1 family member A1 (RALDH1)-producing dermal dendritic cells (DCs), a conventional DC subset regulating skin fibrosis, are decreased in the involved skin of patients with systemic sclerosis (SSc). In this study, we investigated the contribution of Fli1 deficiency, a potential predisposing factor of SSc, to the phenotypical alteration of RALDH1-producing dermal DCs by using SSc model mice and SSc skin samples. Methods Bleomycin (BLM)-induced skin fibrosis was generated with Fli1 +/− and wild-type mice. The proportions of DC and CD4+ T cell subsets were determined by flow cytometry in the dermis of BLM-treated mice. Fli1 expression in dermal DCs was evaluated by immunofluorescence with skin samples of SSc and healthy control subjects. Results RALDH activity of dermal DCs was significantly decreased in BLM-treated Fli1 +/− mice compared with BLM-treated wild-type mice, whereas the proportion of CD103−CD11b− dermal DCs, a major DC subset producing RALDH1 in response to BLM injection, was comparable between groups. Relevant to this finding, the proportion of regulatory T cells (Tregs) in the dermis was decreased in BLM-treated Fli1 +/− mice relative to BLM-treated wild-type mice, while the proportions of Th1, Th2 and Th17 cells were unaltered. In the involved skin of SSc patients, Fli1 was downregulated in CD11c+ cells, including dermal DCs. Conclusions Fli1 deficiency inhibits RALDH1 activity of CD103−CD11b− dermal DCs and related induction of Tregs in BLM-treated mice. Considering Fli1 reduction in SSc dermal DCs, Fli1deficiency may impair the dermal DC-Treg system, contributing to the development of skin fibrosis in SSc.

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.

Despite antigen affinity of B cells varying from cell to cell, functional analyses of antigen-reactive B cells on individual B cells are missing due to technical difficulties. Especially in the field of autoimmune diseases, promising pathogenic B cells have not been adequately studied to date because of its rarity. In this study, functions of autoantigen-reactive B cells in autoimmune disease were analyzed at the single-cell level. Since topoisomerase I is a distinct autoantigen, we targeted systemic sclerosis as autoimmune disease. Decreased and increased affinities for topoisomerase I of topoisomerase I-reactive B cells led to anti-inflammatory and pro-inflammatory cytokine production associated with the inhibition and development of fibrosis, which is the major symptom of systemic sclerosis. Furthermore, inhibition of pro-inflammatory cytokine production and increased affinity of topoisomerase I-reactive B cells suppressed fibrosis. These results indicate that autoantigen-reactive B cells contribute to the disease manifestations in autoimmune disease through their antigen affinity.

Therapeutic options for treating advanced melanoma have progressed rapidly in recent decades. Until 6 years ago, the regimen for treating advanced melanoma consisted mainly of cytotoxic agents such as dacarbazine and type I interferons. Since 2014, anti-programmed cell death 1 (PD1) antibodies have been recognized as anchor drugs for treating advanced melanoma, with or without additional combination drugs such as ipilimumab, but the efficacies of these immunotherapies are not fully satisfactory. In this review, we describe the development of the currently available anti-PD1 Abs-based immunotherapies for advanced melanoma, focusing on their efficacy and immune-related adverse events (AEs), as well as clinical trials still ongoing for the future treatment of advanced melanoma.

Systemic inflammatory response markers, including neutrophil-to-lymphocyte ratio, platelet-to-lymphocyte ratio and monocyte-to-lymphocyte ratio, are useful prognostic factors for various malignant tumours. The aim of this study was to investigate the clinical relevance of these markers in primary cutaneous angiosarcoma. Twenty-six patients were retrospectively divided into 2 groups according to pretreatment peri­pheral blood cell counts or systemic inflammatory response marker levels; overall survival and progression-free survival were compared. Univariate analysis found that high neutrophil count (> 3.1×109/l), high neutrophil-to-lymphocyte ratio (> 2.4), high platelet-to-lymphocyte ratio (> 175) and low lymphocyte count (≤ 1.3×109/l) were related to shorter overall survival, while high neutrophil and low lymphocyte groups had shorter progression-free survival. In multivariate analysis, high neutrophil count and high neutrophil-to- lymphocyte ratio (hazard ratio 7.44 and 5.04, 95% confidence interval 1.48–37.2 and 1.26–20.1, respectiv­ely) were identified as independent prognostic factors for poor overall survival. These results indicate that systemic inflammatory response markers serve as prognostic predictors in primary cutaneous angiosarcoma, as well as in other types of soft-tissue sarcoma.

Abstract Background Previous studies have shown the relationship between higher skin thickness score and the existence of organ involvements in systemic sclerosis (SSc). Here, we firstly investigated the correlation between skin thickness score and quantitative measurements of each organ involvement in Japanese patients with SSc. Methods All Japanese SSc patients hospitalized to our clinic for initial evaluation of SSc were selected. Skin thickness was evaluated by modified Rodnan total skin thickness score (mRSS). Relationship between mRSS and prevalence or incidence of organ involvements was examined by logistic analyses. Correlation between mRSS and quantitative measurements of organ involvements was examined by correlation analyses and regression analyses. Results We recruited 198 patients into our study. The mean disease duration was 7.3 years with the mean follow-up duration of 3.2 years. Multivariate logistic regression analyses revealed that higher mRSS is related to higher prevalence of interstitial lung disease (P

In many malignancies, dysregulation of the Notch pathways, composed of 4 Notch receptors (Notch1–4) and 5 Notch ligands (Jagged1–2, Delta-like ligand-1, 3–4), is associated with their development. In mycosis fungoides, interaction between Notch1 and Jagged1 is known to activate the Notch pathways and promote the proliferation of tumour cells. However, the involvement of other Notch ligands has not been reported. This study investigated the roles of Delta-like ligand 4 in mycosis fungoides. Delta-like ligand 4 mRNA levels in lesional skin of patients with mycosis fungoides were significantly elevated compared with those of normal controls, and correlated with disease-specific mortality. Immunohistochemical staining demonstrated prominent expression of Delta-like ligand 4 on vascular endothelial cells and tumour cells in mycosis fungoides lesional skin. In addition, Delta-like ligand 4 augmented the proliferation of cutaneous T-cell lym­phoma cell lines. These results suggest that enhanced Delta-like ligand 4 expression may contribute directly to the progression of mycosis fungoides through proliferating tumour cells.

Abstract Background Friend leukemia virus integration 1 (Fli1) deficiency, a predisposing factor of systemic sclerosis (SSc), induces SSc-like phenotypes in various cell types. A recent study demonstrated the transdifferentiation of T helper type 2 cell (Th2)-like regulatory T cells (Tregs) in SSc lesional skin through interleukin (IL)-33 produced by fibroblasts. Therefore, we investigated the role of Fli1 deficiency in dermal fibroblast-mediated transdifferentiation of Tregs. Methods Cytokine expression was assessed in Tregs by flow cytometry and in skin samples and cultivated cells by immunostaining, immunoblotting, and/or qRT-PCR. Fli1 binding to the target gene promoters was examined by chromatin immunoprecipitation. Murine dermal fibroblasts and Tregs were cocultured with or without blocking antibodies against target cytokines. Results Th2- and Th17-like cell proportions in skin-homing Tregs were increased in bleomycin-treated Fli1 +/− mice compared with bleomycin-treated wild-type mice, whereas Th1-, Th2-, and Th17-like cell proportions in splenic Tregs were comparable. Fli1 +/− fibroblasts overproduced IL-33 and IL-6, in particular IL-33, and Fli1 occupied the IL33 and IL6 promoters in dermal fibroblasts. Importantly, the IL-4-producing cell proportion was significantly higher in wild-type Tregs cocultured with Fli1 +/− fibroblasts than in those cocultured with wild-type fibroblasts, which were canceled by neutralizing anti-IL-33 antibody. Under the same coculture condition, an increased tendency of IL-17A-producing cell proportion, which was possibly mediated by IL-6, was evident. Conclusions Fli1 haploinsufficiency increases the proportions of Th2- and Th17-like Tregs in bleomycin-induced profibrotic skin conditions, in which IL-33-producing dermal fibroblasts contribute to Th2-like Treg transdifferentiation, suggesting a critical role of Fli1 deficiency in the interaction of dermal fibroblasts with immune cells in pathological skin fibrosis.

The course of systemic sclerosis-associated interstitial lung disease (SSc-ILD) varies among individuals. Red blood cell distribution width (RDW) has been reported to be a predictor of idiopathic pulmonary fibrosis. However, there are no studies on the relationship between RDW and SSc-ILD. We conducted a retrospective study of 28 patients who were diagnosed with SSc-ILD on their first visit to our hospital and were followed-up for 5 years. The correlation between the changes in RDW, KL-6, and SP-D (ΔRDW, ΔKL-6, ΔSP-D) and the changes in percent-predicted forced lung volume and % carbon monoxide diffusion (Δ%FVC, Δ%DLco) was investigated. ΔRDW at 1 year after diagnosis was significantly inversely correlated with Δ%FVC at 5 years after diagnosis (r = −0.51, p < 0.001) and Δ%DLco at 5 years after diagnosis (r = −0.47, p < 0.001), whereas ΔKL-6 and ΔSP-D at 1 year were not correlated with Δ%FVC or Δ%DLco at 5 years. In the group of SSc-ILD patients with RDW increase in the first year after diagnosis, %FVC and %DLco were significantly lower than baseline at 3-, 4-, and 5-year assessments. In the group of patients without RDW increase in the first year, %FVC and %DLco did not decrease during the follow-up period. In conclusion, the changes in RDW in the first year after diagnosis may be useful surrogate markers to predict the long-term course of SSc-ILD.

Abstract Skin fibrosis in systemic sclerosis (SSc) is accompanied by attrition of dermal white adipose tissue (dWAT) and reduced levels of circulating adiponectin. Since adiponectin has potent regulatory effects on fibroblasts, we sought to assess adiponectin signaling in SSc skin biopsies, and evaluate fibrosis in mice with adiponectin gain- and loss-of-function mutations. Furthermore, we investigated the effects and mechanism of action of agonist peptides targeting adiponectin receptors in vitro and in vivo. We found that adiponectin pathway activity was significantly reduced in a subset of SSc skin biopsies. Mice lacking adiponectin mounted an exaggerated dermal fibrotic response, while transgenic mice with constitutively elevated adiponectin showed selective dWAT expansion and protection from skin and peritoneal fibrosis. Adiponectin receptor agonists abrogated ex vivo fibrotic responses in explanted normal and SSc fibroblasts and in 3D human skin equivalents, in part by attenuating focal adhesion complex assembly, and prevented and reversed experimentally-induced organ fibrosis in mice. These results implicate aberrant adiponectin pathway activity in skin fibrosis, identifying a novel function for this pleiotropic adipokine in regulation of tissue remodeling. Restoring adiponectin signaling in SSc patients therefore might represent an innovative pharmacological strategy for intractable organ fibrosis.

Abstract Skin fibrosis is a devastating clinical condition commonly seen in skin-restricted and systemic disorders. The goal of skin fibrosis treatment is the restoration of abnormally activated dermal fibroblasts producing the excessive amount of extracellular matrix, which is generally a final consequence of the complex disease process including the activation of vascular and immune systems. Among various skin fibrotic conditions, the molecular mechanisms underlying dermal fibroblast activation have been mostly well studied in systemic sclerosis (SSc). SSc is a multisystem autoimmune and vascular disease resulting in extensive fibrosis of the skin and various internal organs. Since SSc pathogenesis is believed to include all the critical components regulating tissue fibrosis, the studies on anti-fibrotic drugs against SSc provide us much useful information regarding the strategy for the treatment of various skin fibrotic conditions. In the recent decade, as is the case with other autoimmune and inflammatory diseases, the molecular targeting therapy with monoclonal antibody has been clinically well examined in SSc. Promising clinical outcomes are so far reported in tocilizumab (an anti-IL-6 receptor antibody), rituximab (an anti-CD20 antibody), and fresolimumab (an anti-TGF-β antibody). The analysis of gene expression profiles in skin lesions of SSc patients treated with tocilizumab or fresolimumab revealed a critical role of monocyte-macrophage lineage cells in the development of skin fibrosis and the involvement of IL-6 and TGF-β in the activation of those cells. Considering that B cells modulate the differentiation and activation of macrophages, favorable clinical outcomes of rituximab treatment imply the central role of B cell/monocyte-macrophage lineage cell axis in the pathogenesis of SSc. This scenario may be applicable at least partly to other skin fibrotic conditions. In this review article, the currently available data on these drugs are summarized and the future directions are discussed.

The incidence of cancers in atopic dermatitis (AD) is not increased, although the Th2-dominant environment is known to downregulate tumor immunity. To gain mechanistic insights regarding tumor immunity in AD, we utilized CCL17 transgenic (TG) mice overexpressing CCL17, which is a key chemokine in AD. Tumor formation and lung metastasis were accelerated in CCL17 TG mice when melanoma cells were injected subcutaneously or intravenously. Flow cytometric analysis showed increases in regulatory T cells (Tregs) in lymph nodes in CCL17 TG mice with high mRNA levels of IL-10 and Foxp3 in tumors, suggesting that Tregs attenuated tumor immunity. The frequency of myeloid-derived suppressor cells (MDSCs), however, was significantly decreased in tumors of CCL17 TG mice, suggesting that decreased MDSCs might promote tumor immunity. Expression of CXCL17, a chemoattractant of MDSCs, was decreased in tumors of CCL17 TG mice. Depletion of Tregs by the anti-CD25 antibody markedly reduced tumor volumes in CCL17 TG mice, suggesting that tumor immunity was accelerated by the decrease in MDSCs in the absence of Tregs. Thus, CCL17 attenuates tumor immunity by increasing Tregs and Th2 cells, while it decreases MDSCs through reductions in CXCL17, which may work as a “safety-net” to reduce the risk of malignant tumors in the Th2-dominant environment.

Emerging evidence has demonstrated that Toll-like receptors (TLRs) are associated with autoimmune diseases. In this study, we investigated the role of TLR2 in psoriasis using imiquimod-induced psoriasis-like dermatitis. Although TLR2 signaling is known to play a critical role in the induction of proinflammatory cytokines by immune cells, such as dendritic cells (DCs), macrophages, and monocytes, TLR2 deficiency unexpectedly exacerbated psoriasiform skin inflammation. Importantly, messenger RNA (mRNA) levels of Foxp-3 and IL-10 in the lesional skin were significantly decreased in TLR2 KO mice compared with wild-type mice. Furthermore, flow cytometric analysis of the lymph nodes revealed that the frequency of regulatory T cells (Tregs) among CD4-positive cells was decreased. Notably, stimulation with Pam3CSK4 (TLR2/1 ligand) or Pam2CSK4 (TLR2/6 ligand) increased IL-10 production from Tregs and DCs and the proliferation of Tregs. Finally, adoptive transfer of Tregs from wild-type mice reduced imiquimod-induced skin inflammation in TLR2 KO mice. Taken together, our results suggest that TLR2 signaling directly enhances Treg proliferation and IL-10 production by Tregs and DCs, suppressing imiquimod-induced psoriasis-like skin inflammation. Enhancement of TLR2 signaling may be a new therapeutic strategy for psoriasis.

OBJECTIVE: To assess the utility of circulating adhesion molecule levels as a prognostic indicator of disease progression in systemic sclerosis (SSc) patients with early onset disease. METHODS: Ninety-two Japanese patients with early onset SSc presenting with diffuse skin sclerosis and/or interstitial lung disease were registered in a multicentre, observational study. Concentrations of intercellular adhesion molecule (ICAM) -1, E-selectin, L-selectin, and P-selectin in serum samples from all patients were measured by enzyme-linked immunosorbent asssay (ELISA). In 39 patients, adhesion molecule levels were measured each year for four years. The ability of baseline adhesion molecule levels to predict subsequent progression and severity in clinical and laboratory features were evaluated statistically. RESULTS: At their first visit, serum levels of ICAM-1, E-selection, P-selectin were significantly elevated and serum L-selectin levels were significantly reduced in patients with SSc compared with healthy controls. Overall, serum ICAM-1 levels at each time point were significantly inversely associated with the %vital capacity (VC) of the same time and subsequent years by univariate analysis. The initial serum ICAM-1 levels were significantly inversely associated with the %VC at the fourth year by multiple regression analysis. The initial serum P-selectin levels were significantly associated with the health assessment questionnaire disability index (HAQ-DI) at the fourth year by multiple regression analysis. Initial adhesion molecule levels were not significantly associated with other clinical features including skin thickness score. Baseline adhesion molecule levels were not significantly associated with subsequent rate of change of clinical parameters. CONCLUSION: In patients with SSc, serum levels of ICAM-1 and P-selectin may serve as prognostic indicators of respiratory dysfunction and physical disability, respectively. Further longitudinal studies of larger populations are needed to confirm these findings.

Fli1, a member of the Ets transcription factor family, is a key repressor of the human α2(I) collagen (COL1A2) gene. Although our previous studies have delineated that TGF-β induces displacement of Fli1 from the COL1A2 promoter through sequential post-translational modifications, the detailed mechanism by which Fli1 functions as a potent transcriptional repressor of the COL1A2 gene has not been fully investigated. To address this issue, we carried out a series of experiments especially focusing on protein-protein interaction and epigenetic transcriptional regulation. The combination of tandem affinity purification and mass spectrometry identified HDAC1 as a Fli1 interacting protein. Under quiescent conditions, HDAC1 induced deacetylation of Fli1 resulting in an increase of Fli1 DNA binding ability and p300 enhanced this process by promoting the formation of a Fli1-HDAC1-p300 complex. TGF-β-induced phosphorylation of Fli1 at threonine 312 led to disassembly of this protein complex. In quiescent dermal fibroblasts Fli1, HDAC1, and p300 occupied the -404 to -237 region, including the Fli1 binding site, of the COL1A2 promoter. TGF-β induced Fli1 and HDAC1 dissociation from the COL1A2 promoter, while promoting Ets1 and p300 recruitment. Furthermore, acetylation levels of histone H3 around the Fli1 binding site in the COL1A2 promoter inversely correlated with the DNA occupancy of Fli1 and HDAC1, while positively correlating with that of Ets1 and p300. In the functional studies, HDAC1 overexpression magnified the inhibitory effect of Fli1 on the COL1A2 promoter. Moreover, pharmacological blockade of HDAC1 by entinostat enhanced collagen production in dermal fibroblasts. Collectively, these results indicate that under quiescent conditions Fli1 recruits HDAC1/p300 to the COL1A2 promoter and suppresses the expression of the COL1A2 gene by chromatin remodeling through histone deacetylation. TGF-β-dependent phosphorylation of Fli1 at threonine 312 is a critical step regulating the remodeling of the Fli1 transcription repressor complex, leading to transcriptional activation of the COL1A2 gene.

BACKGROUND: Bortezomib is a proteasome inhibitor that has shown impressive efficacy in the treatment of multiple myeloma. In mice, the addition of dextran sulfate sodium (DSS) to drinking water leads to acute colitis that can serve as an experimental animal model for human ulcerative colitis. METHODOLOGY/PRINCIPAL FINDINGS: Bortezomib treatment was shown to potently inhibit murine DSS-induced colitis. The attenuation of DSS-induced colitis was associated with decreased inflammatory cell infiltration in the colon. Specifically, bortezomib-treated mice showed significantly decreased numbers of CD4(+) and CD8(+) T cells in the colon and mesenteric lymph nodes. Bortezomib treatment significantly diminished interferon (IFN)-γ expression in the colon and mesenteric lymph nodes. Furthermore, cytoplasmic IFN-γ production by CD4(+) and CD8(+) T cells in mesenteric lymph nodes was substantially decreased by bortezomib treatment. Notably, bortezomib enhanced T cell apoptosis by inhibiting nuclear factor-κB activation during DSS-induced colitis. CONCLUSIONS/SIGNIFICANCE: Bortezomib treatment is likely to induce T cell death, thereby suppressing DSS-induced colitis by reducing IFN-γ production.

Cathepsin B (CTSB) is a proteolytic enzyme potentially modulating angiogenic processes and extracellular matrix remodeling. While matrix metalloproteinases are shown to be implicated in tissue fibrosis and vasculopathy associated with systemic sclerosis (SSc), the role of cathepsins in this disease has not been well studied. The aim of this study is to evaluate the roles of CTSB in SSc. Serum pro-CTSB levels were determined by enzyme-linked immunosorbent assay in 55 SSc patients and 19 normal controls. Since the deficiency of transcription factor Fli1 in endothelial cells is potentially associated with the development of SSc vasculopathy, cutaneous CTSB expression was evaluated by immunostaining in Fli1(+/-) and wild type mice as well as in SSc and control subjects. The effects of Fli1 gene silencing and transforming growth factor-β (TGF-β) on CTSB expression were determined by real-time PCR in human dermal microvascular endothelial cells (HDMECs) and dermal fibroblasts, respectively. Serum pro-CTSB levels were significantly higher in limited cutaneous SSc (lcSSc) and late-stage diffuse cutaneous SSc (dcSSc) patients than in healthy controls. In dcSSc, patients with increased serum pro-CTSB levels showed a significantly higher frequency of digital ulcers than those with normal levels. CTSB expression in dermal blood vessels was increased in Fli1(+/-) mice compared with wild type mice and in SSc patients compared with healthy controls. Consistently, Fli1 gene silencing increased CTSB expression in HDMECs. In cultured dermal fibroblasts from early dcSSc, CTSB expression was decreased compared with normal fibroblasts and significantly reversed by TGF-β1 antisense oligonucleotide. In conclusion, up-regulation of endothelial CTSB due to Fli1 deficiency may contribute to the development of SSc vasculopathy, especially digital ulcers, while reduced expression of CTSB in lesional dermal fibroblasts is likely to be associated with skin sclerosis in early dcSSc.