GWAS for systemic sclerosis identifies six novel susceptibility loci including one in the Fcγ receptor region.

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.

A cysteine proteinase inhibitor ALLN alleviates bleomycin-induced skin and lung fibrosis.

BACKGROUND: Systemic sclerosis (SSc) is a connective tissue disease that is characterized by fibrosis in the skin and internal organs, such as the lungs. Activated differentiation of progenitor cells, which are mainly resident fibroblasts, into myofibroblasts is considered a key mechanism underlying the overproduction of extracellular matrix and the resultant tissue fibrosis in SSc. Calpains are members of the Ca2+-dependent cysteine protease family, whose enzymatic activities participate in signal transduction and tissue remodeling, potentially contributing to fibrosis in various organs. However, the roles of calpain in the pathogenesis of SSc remain unknown. This study aimed to examine the anti-fibrotic properties of N-acetyl-Leu-Leu-norleucinal (ALLN), one of the cysteine proteinase inhibitors that primarily inhibit calpain, in vitro and in vivo, to optimally translate into the therapeutic utility in human SSc. METHODS: Normal human dermal and lung fibroblasts pretreated with ALLN were stimulated with recombinant transforming growth factor beta 1 (TGF-ß1), followed by assessment of TGF-ß1/Smad signaling and fibrogenic molecules. RESULTS: ALLN treatment significantly inhibited TGF-ß1-induced phosphorylation and nuclear transport of Smad2/3 in skin and lung fibroblasts. TGF-ß1-dependent increases in α-smooth muscle actin (αSMA), collagen type I, fibronectin 1, and some mesenchymal transcription markers were attenuated by ALLN. Moreover, our findings suggest that ALLN inhibits TGF-ß1-induced mesenchymal transition in human lung epithelial cells. Consistent with these in vitro findings, administering ALLN (3 mg/kg/day) three times a week intraperitoneally remarkably suppressed the development of skin and lung fibrosis in a SSc mouse model induced by daily subcutaneous bleomycin injection. The number of skin- and lung-infiltrating CD3+ T cells decreased in ALLN-treated mice compared with that in control-treated mice. Phosphorylation of Smad3 and/or an increase in αSMA-positive myofibroblasts was significantly inhibited by ALLN treatment on the skin and lungs. However, no adverse effects were observed. CONCLUSIONS: Our results prove that calpains can be a novel therapeutic target for skin and lung fibrosis in SSc, considering its inhibitor ALLN.

The compound LG283 inhibits bleomycin-induced skin fibrosis via antagonizing TGF-ß signaling.

BACKGROUND: Systemic sclerosis (SSc) is a collagen disease that exhibits intractable fibrosis and vascular injury of the skin and internal organs. Transforming growth factor-ß (TGF-ß)/Smad signaling plays a central role in extracellular matrix (ECM) production by α-SMA-positive myofibroblasts. Myofibroblasts may be partially derived from various precursor cells in addition to resident fibroblasts. Recently, our high-throughput in vitro screening discovered a small compound, LG283, that may disrupt the differentiation of epithelial cells into myofibroblasts. This compound was originally generated as a curcumin derivative. METHODS: In this study, we investigated the effect of LG283 on inhibiting fibrosis and its mechanism. The action of LG283 on TGF-ß-dependent fibrogenic activity and epithelial-mesenchymal transition (EMT) was analyzed in vitro. The effects of LG283 were also examined in a bleomycin-induced skin fibrosis mouse model. RESULTS: LG283 suppressed TGF-ß-induced expression of ECM, α-SMA, and transcription factors Snail 1 and 2, and Smad3 phosphorylation in cultured human dermal fibroblasts. LG283 was also found to block EMT induction in cultured human epithelial cells. During these processes, Smad3 phosphorylation and/or expression of Snail 1 and 2 were inhibited by LG283 treatment. In the bleomycin-induced skin fibrosis model, oral administration of LG283 efficiently protected against the development of fibrosis and decrease of capillary vessels without significantly affecting cell infiltration or cytokine concentrations in the skin. No apparent adverse effects of LG283 were found. LG283 treatment remarkably inhibited the enhanced expression of α-SMA and phosphorylated Smad3, as well as those of Snail 1 and 2, in the bleomycin-injected skin. CONCLUSIONS: The LG283 compound exhibits antagonistic activity on fibrosis and vascular injury through inhibition of TGF-ß/Smad/Snail mesenchymal transition pathways and thus, may be a candidate therapeutic for the treatment of SSc. Although the involvement of EMT in the pathogenesis of SSc remains unclear, the screening of EMT regulatory compounds may be an attractive approach for SSc therapy.

Clinical course of Japanese patients with early systemic sclerosis: A multicenter, prospective, observational study.

OBJECTIVES: To investigate the clinical course of Japanese patients with early diffuse cutaneous systemic sclerosis (dcSSc) and early SSc with interstitial lung disease (ILD). METHODS: We prospectively analyzed the clinical features of 207 Japanese patients with early dcSSc (n = 150) and limited cutaneous SSc (lcSSc) with ILD (n = 57) in 10 medical centers every year for 7 consecutive years. RESULTS: Mean modified Rodnan total skin thickness score (mRSS) was 18.3 and 67.4% of the cohort had ILD. Most patients started immunosuppressive therapy and vasodilators during 7 years (83.4% and 87.9%, respectively). Mean value of mRSS of total patients was significantly reduced from the initial registration after the first year. However, other parameters for physical function associated with skin sclerosis including fist closure, hand extension, and oral aperture were not so ameliorated during the study period. Health Assessment Questionnaire-disability index and serum KL-6 levels were constant throughout the course. Percent vital capacity and the presence of ILD, clinically suspected pulmonary arterial hypertension, and digital ulcers were gradually exacerbated during the period. CONCLUSION: In Japanese early dcSSc patients and SSc patients with ILD, mRSS was continuously reduced during 7 years of follow-up, but there was little improvement of physical disability and organ involvement.

Gene silencing of extracellular matrix protein 1 (ECM1) results in phenotypic alterations of dermal fibroblasts reminiscent of clinical features of lichen sclerosus.

BACKGROUND: Lichen sclerosus (LS) is an acquired inflammatory mucocutaneous disease affecting the anogenital area, characterized histologically by hyalinosis and thickened vessel walls in the dermis. The presence of serum autoantibodies against extracellular matrix protein 1 (ECM1) in LS patients may suggest its involvement in disease pathogenesis. OBJECTIVE: To examine if reduced ECM1 production by dermal fibroblasts contributes to the pathogenic features of LS. METHODS: Gene expression in ECM1 knockdown human dermal fibroblasts was analyzed by cDNA microarray. Functional enrichment for genes involved in cellular functions was conducted. Protein expression was analyzed by ELISA and confocal laser scanning microscopy using LS skin. RESULTS: Microarray analysis identified 3035 differentially expressed genes in ECM1 knockdown cells, wherein 1471 were upregulated genes related exclusively to cell adhesion, proliferation, apoptosis, intracellular signaling, and extracellular matrix organization. Further narrowing with criteria specific for localization and function of ECM1 identified 48 upregulated genes identified to have structural, fibrogenic, and carcinogenic properties. Of these, laminin-332 and collagen-IV displayed altered immunolabeling within the basement membrane zone (BMZ) and dermal vessels in LS skin, similar to that of collagen-VII, which exhibited unchanged transcription levels in ECM1-knockdown fibroblasts. Collagen-VII bound to recombinant ECM1 in a solid-phase immunoassay and colocalized with ECM1 in the skin BMZ. Further, ECM1-knockdown fibroblasts exhibited a marked delay in cell migration and gel contraction. CONCLUSION: In the absence of ECM1 expression in fibroblasts there is selective dysregulation and disassembly of structural and extracellular matrix molecules, which may result in microstructural abnormalities reminiscent of LS.

Potential Biomarkers in Systemic Sclerosis: A Literature Review and Update.

Systemic sclerosis (SSc) is a chronic autoimmune disease characterized by dysregulation of the immune system, vascular damage, and fibrosis of the skin and internal organs. Patients with SSc show a heterogeneous phenotype and a range of clinical courses. Therefore, biomarkers that are helpful for precise diagnosis, prediction of clinical course, and evaluation of the therapeutic responsiveness of disease are required in clinical practice. SSc-specific autoantibodies are currently used for diagnosis and prediction of clinical features, as other biomarkers have not yet been fully vetted. Krebs von den Lungen-6 (KL-6), surfactant protein-D (SP-D), and CCL18 have been considered as serum biomarkers of SSc-related interstitial lung disease. Moreover, levels of circulating brain natriuretic peptide (BNP) and N-terminal pro-brain natriuretic peptide (NT-proBNP) can provide diagnostic information and indicate the severity of pulmonary arterial hypertension. Assessment of several serum/plasma cytokines, chemokines, growth factors, adhesion molecules, and other molecules may also reflect the activity or progression of fibrosis and vascular involvement in affected organs. Recently, microRNAs have also been implicated as possible circulating indicators of SSc. In this review, we focus on several potential SSc biomarkers and discuss their clinical utility.

Homeostatic Function of Dermokine in the Skin Barrier and Inflammation.

Dermokine is a chiefly skin-specific secreted glycoprotein localized in the upper epidermis, and its family consists of three splice variants in mice and five in humans. To investigate the pathophysiological impact of dermokine, we generated mice deficient for two (ßγ) or all dermokine isoforms (αßγ). Both variants, especially dermokine αßγ-deficient mice exhibited scale and wrinkle formation resembling ichthyosis accompanied by transepidermal water imbalance at the neonatal stage. Several dermokine αßγ-deficient mice died by postnatal day 21 when reared under low humidity. Moreover, the cornified envelope was vulnerable, and skin barrier lipid ceramides were reduced in the epidermis of dermokine αßγ-deficient mice. cDNA microarray and quantitative reverse transcriptase-PCR assays of the epidermis revealed the upregulation of small proline-rich protein and late cornified envelope family members, as well as antimicrobial peptides in the dermokine αßγ-deficient mice. These barrier gene signatures were similar to that seen in psoriasis, whereas recent studies demonstrated that congenital ichthyosis has gene profiles resembling psoriasis. In line with these findings, adult dermokine αßγ-deficient mice exhibited aggravated phenotypes in psoriasis-like dermatitis models but not in allergic dermatitis models. Dermokine may play a regulatory role in inflammatory dyskeratotic diseases, such as congenital ichthyosis and psoriasis, in the crosstalk between barrier dysfunction and inflammation.

Inhibition of the Progression of Skin Inflammation, Fibrosis, and Vascular Injury by Blockade of the CX3 CL1/CX3 CR1 Pathway in Experimental Mouse Models of Systemic Sclerosis.

OBJECTIVE: To assess the preclinical efficacy and mechanism of action of an anti-CX3 CL1 monoclonal antibody (mAb) in systemic sclerosis (SSc). METHODS: Cultured human dermal fibroblasts were used to evaluate the direct effect of anti-CX3 CL1 mAb on fibroblasts. In addition, bleomycin-induced and growth factor-induced models of SSc were used to investigate the effect of anti-CX3 CL1 mAb on leukocyte infiltration, collagen deposition, and vascular damage in the skin. RESULTS: Anti-CX3 CL1 mAb treatment significantly inhibited Smad3 phosphorylation (P < 0.05) and expression of type I collagen and fibronectin 1 (P < 0.01) in dermal fibroblasts stimulated with transforming growth factor ß1 (TGFß1). In the bleomycin model, daily subcutaneous bleomycin injection increased serum CX3 CL1 levels (P < 0.05) and augmented lesional CX3 CL1 expression. Simultaneous administration of anti-CX3 CL1 mAb or CX3 CR1 deficiency significantly suppressed the dermal thickness, collagen content, and capillary loss caused by bleomycin (P < 0.05). Injection of bleomycin induced expression of pSmad3 and TGFß1 in the skin, which was inhibited by anti-CX3 CL1 mAb. Further, the dermal infiltration of CX3 CR1+ cells, macrophages (inflammatory and alternatively activated [M2-like] subsets), and CD3+ cells significantly decreased following anti-CX3 CL1 mAb therapy (P < 0.05), as did the enhanced skin expression of fibrogenic molecules, such as thymic stromal lymphopoietin and secreted phosphoprotein 1 (P < 0.05). However, the treatment did not significantly reduce established skin fibrosis. In the second model, simultaneous anti-mCX3 CL1 mAb therapy significantly diminished the skin fibrosis induced by serial subcutaneous injection of TGFß and connective tissue growth factor (P < 0.01). CONCLUSION: Anti-CX3 CL1 mAb therapy may be a novel approach for treating early skin fibrosis in inflammation-driven fibrotic skin disorders such as SSc.

Dietary supplement product composed of natural ingredients as a suspected cause of erythema multiforme: A case report and identification for the confident false positivity of lymphocyte transformation test.

Growing and sustainable consumption of health-care products raises a controversial issue underlying the reliability of an in vitro diagnostic approach for adverse skin reaction. This report aimed to: (i) discuss the causative nature of a commercial dietary supplement composed of natural ingredients, particularly an Euglena-containing product, suspicious for erythema multiforme in our exemplified case; and (ii) to address the assay suitability of the lymphocyte transformation test (LTT) for identifying allergic reaction to any ingredient(s) of the product. A Japanese elderly man developed erythema multiforme after intake of a commercially available natural dietary product, whose LTT was positive. His clinical course and positive LTT suggested a provisional diagnosis of natural dietary product-induced eruption. We conducted an inquiry survey for the standard LTT with any commercial products containing Euglena in three major Japanese laboratory services and identified 22 subjects, almost all of whom (21/22, 95.6%) showed a positive LTT for any Euglena-containing products as a suspected causative. Seven normal healthy volunteers who had no intake history of Euglena-containing products showed an equivalent LTT positivity rate with the same product taken by our case; culprit components of the product included Euglena, Angelica keiskei, Barley grass and Chlorella. A cell-free culture system and enzyme-linked immunoassay suggest that the high LTT positivity relies on the non-specific lymphoproliferative activity, and not contamination of uncharacterized microorganisms and endotoxins. Because of the constitutive false positivity of LTT, this assay is unreliable for in vitro supportive diagnosis of adverse skin events caused by dietary products containing particular natural ingredients, as well as herbal materials.

A Case of Erythema Multiforme Major Developed after Sequential Use of Two Immune Checkpoint Inhibitors, Nivolumab and Ipilimumab, for Advanced Melanoma: Possible Implication of Synergistic and/or Complementary Immunomodulatory Effects.

Immune checkpoint inhibitors, such as ipilimumab and nivolumab, reverse the imbalance of antitumor self-tolerance and enhance T-cell responses. Currently, ipilimumab and nivolumab have a reported therapeutic impact on unresectable or metastatic melanomas; however, they also induce immune-related adverse events (irAEs). Ipilimumab-induced cutaneous irAEs are mostly low grade and manageable, although all-grade rash may occur in approximately 45% of all patients. We here report the case of a young woman with erythema multiforme major, which developed after sequential use of these 2 immune checkpoint inhibitors for advanced melanoma of the scalp. Initially, she received 12 cycles of nivolumab monotherapy followed by ipilimumab. A week later, multiple erythematous papulo-erythemas appeared on almost her entire body, with high-grade fever, mucosal involvements, and dyspnea. Immunohistochemistry using the lesioned skin revealed lymphocytic infiltration predominantly positive for CD8, contrasting with those for CD4 and Foxp3. Ipilimumab was stopped but she continued to receive empirical antibiotics; additionally, she was treated with intravenous steroid pulse therapy and immunoglobulin, followed by oral prednisolone. Her symptoms subsided rapidly, allowing a restart of nivolumab monotherapy alone. In our case, the long-standing preceding nivolumab monotherapy may synergistically and/or complementary have contributed to - in combination with the later administration of ipilimumab - recover antigen-responsive T-cell immunity, which is similar to the concept of immune reconstitution inflammatory syndrome, resulting in the establishment of an underlying immunopathology of erythema multiforme and life-threatening airway obstruction.

Sublamina densa-type linear IgA bullous dermatosis with IgA autoantibodies specific for type VII collagen: a case report and clinicopathological review of 32 cases.

Linear IgA bullous dermatosis (LABD) is a rare autoimmune bullous disorder characterized by linear deposits of IgA at the basement membrane zone(BMZ) and/or by circulating IgA anti-BMZ antibodies. Comparing with other immuno-bullous diseases, LABD represents a heterogeneous disease entitywith diversity of pathogenic IgA autoantibodies to different hemidesmosomal antigens and an association with malignancies and occasional drug use. We herein present an 82-year-old Japanese man with LABD, whose indirect immunofluorescence using 1M NaCl-split skin showed positive staining for IgA at the dermal side alone. Fluorescence overlay antigen mapping using laser scanning confocal microscopy (FOAM-LSCM) was employed to examine the in vivo bound patient's IgA, which was specific for type VII collagen (COL7), a prominent antigen of the sublamina densa. One year later, he developed malignant lymphoma, suggesting the diagnosis of paraneoplastic LABD. We reviewed 32 cases of sublamina-densa type LABD with anti-COL7 IgA antibodies thus far reported in the literature to compare the clinicopathological characteristics of this rare disease variant and emphasize that COL7 is the main autoantigen in sublamina densa disease.