Ryosuke Saigusa

Simultaneous downregulation of KLF5 and Fli1 is a key feature underlying systemic sclerosis

Systemic sclerosis (SSc) is manifested by fibrosis, vasculopathy and immune dysregulation. So far, a unifying hypothesis underpinning these pathological events remains unknown. Given that SSc is a multifactorial disease caused by both genetic and environmental factors, we focus on the two transcription factors, which modulate the fibrotic reaction and are epigenetically suppressed in SSc dermal fibroblasts, Friend leukemia integration 1 (Fli1) and Krüppel-like factor 5 (KLF5). In addition to Fli1 silencing-dependent collagen induction, simultaneous knockdown of Fli1 and KLF5 synergistically enhances expression of connective tissue growth factor. Notably, mice with double heterozygous deficiency of Klf5 and Fli1 mimicking the epigenetic phenotype of SSc skin spontaneously recapitulate all the three features of SSc, including fibrosis and vasculopathy of the skin and lung, B cell activation, and autoantibody production. These studies implicate the epigenetic downregulation of Fli1 and KLF5 as a central event triggering the pathogenic triad of SSc.

A possible contribution of endothelial CCN1 downregulation due to Fli1 deficiency to the development of digital ulcers in systemic sclerosis.

CCN1 is a pleiotropic molecule involved in angiogenesis and postnatal vasculogenesis, both of which are impaired in systemic sclerosis (SSc). To elucidate the potential role of CCN1 in the development of SSc, we investigated CCN1 expression in the lesional skin of SSc patients and SSc animal models and the clinical correlation of serum CCN1 levels. CCN1 expression was markedly decreased in dermal small blood vessels of SSc patients compared with those of healthy controls, while comparable between normal and SSc dermal fibroblasts. Transcription factor Fli1, whose deficiency due to epigenetic suppression is implicated in the pathogenesis of SSc, occupied the CCN1 promoter and gene silencing of Fli1 resulted in the reduction of CCN1 expression in human dermal microvascular endothelial cells. Consistently, CCN1 expression was suppressed uniformly and remarkably in dermal blood vessels of Fli1+/− mice and partially in those of endothelial cell‐specific Fli1 knockout mice. Furthermore, serum CCN1 levels were significantly decreased in SSc patients with previous and current history of digital ulcers as compared to those without. Collectively, these results suggest that endothelial CCN1 downregulation at least partially due to Fli1 deficiency may contribute to the development of digital ulcers in SSc patients. This study further supports the idea that epigenetic downregulation of Fli1 is a potential predisposing factor in the pathogenesis of SSc.

Multifaceted contribution of the TLR4-activated IRF5 transcription factor in systemic sclerosis

Significance The present study is, to our knowledge, the first demonstration of the molecular mechanisms underlying the association of IFN regulatory factor 5 (IRF5) expression with milder clinical manifestations of systemic sclerosis (SSc). It is speculated that endogenous ligands induce Toll-like receptor 4 signaling and promote IRF5 transcriptional regulation of its target gene promoters, which may be required for the development of SSc. Our present study supports this notion. Symptoms associated with SSc in humans were suppressed in mice deficient in the Irf5 gene. As such, this study offers previously unidentified insight into the complexity of SSc pathology, giving impetus to further clinical studies for the treatment of the disease. Systemic sclerosis (SSc) is a multisystem autoimmune disorder with clinical manifestations resulting from tissue fibrosis and extensive vasculopathy. A potential disease susceptibility gene for SSc is IFN regulatory factor 5 (IRF5), whose SNP is associated with milder clinical manifestations; however, the underlying mechanisms of this association remain elusive. In this study we examined IRF5-deficient (Irf5−/−) mice in the bleomycin-treated SSc murine model. We show that dermal and pulmonary fibrosis induced by bleomycin is attenuated in Irf5−/− mice. Interestingly, we find that multiple SSc-associated events, such as fibroblast activation, inflammatory cell infiltration, endothelial-to-mesenchymal transition, vascular destabilization, Th2/Th17 skewed immune polarization, and B-cell activation, are suppressed in these mice. We further provide evidence that IRF5, activated by Toll-like receptor 4 (TLR4), binds to the promoters of various key genes involved in SSc disease pathology. These observations are congruent with the high level of expression of IRF5, TLR4, and potential endogenous TLR4 ligands in SSc skin lesions. Our study sheds light on the TLR4-IRF5 pathway in the pathology of SSc with clinical implications of targeting the IRF5 pathways in the suppression of disease development.

Epithelial Fli1 deficiency drives systemic autoimmunity and fibrosis: Possible roles in scleroderma

Systemic sclerosis (SSc), or scleroderma, is a multisystem autoimmune disorder characterized by vasculopathy and fibrosis in the skin and internal organs, most frequently in the esophagus and lungs. Hitherto, studies on SSc pathogenesis centered on immune cells, vascular cells, and fibroblasts. Although dysregulated keratinocytes in SSc have been recently reported, the contribution of epithelial cells to pathogenesis remains unexplored. In this study, we demonstrated the induction of SSc-like molecular phenotype in keratinocytes by gene silencing of transcription factor Friend leukemia virus integration 1 (Fli1), the deficiency of which is implicated in SSc pathogenesis. Keratin 14–expressing epithelial cell–specific Fli1 knockout mice spontaneously developed dermal and esophageal fibrosis with epithelial activation. Furthermore, they developed remarkable autoimmunity with interstitial lung disease derived from thymic defects with down-regulation of autoimmune regulator (Aire). Importantly, Fli1 directly regulated Aire expression in epithelial cells. Collectively, epithelial Fli1 deficiency might be involved in the systemic autoimmunity and selective organ fibrosis in SSc. This study uncovers unidentified roles of dysregulated epithelial cells in SSc pathogenesis.

Association of anti-RNA polymerase III antibody and malignancy in Japanese patients with systemic sclerosis

Patients with systemic sclerosis (SSc) have an increased risk of malignancy compared with the general population. Recently, SSc patients with anti‐RNA polymerase III antibody have been reported to have an increased risk of malignancy as compared with those with other disease‐specific autoantibodies in US, European and Australian populations. Therefore, we studied the relationship between disease‐specific autoantibodies and malignancy in 261 Japanese SSc patients. The prevalence of malignancy was significantly higher in patients with anti‐RNA polymerase III antibody (7/22, 31.8%) than in those with anti‐topoisomerase I antibody (2/82, 2.4%) and in those with anticentromere antibody (8/137, 5.8%). Importantly, among seven patients with anti‐RNA polymerase III antibody and malignancy, three patients (42.9%) developed malignancy from 6 months before to 12 months after SSc onset. Thus, malignancy complication in Japanese SSc patients with anti‐RNA polymerase III antibody is as high as that in other races, suggesting that SSc patients with anti‐RNA polymerase III antibody share the same pathological process among different ethnic groups.

Increased expression of chemerin in endothelial cells due to Fli1 deficiency may contribute to the development of digital ulcers in systemic sclerosis

OBJECTIVES Chemerin is a member of adipocytokines with a chemoattractant effect on plasmacytoid dendritic cells and macrophages and pro-angiogenic properties. We investigated the potential role of chemerin in the development of SSc. METHODS Chemerin expression was evaluated by immunostaining and/or real-time quantitative RT-PCR in human and murine skin. The mechanisms regulating chemerin expression in dermal fibroblasts and endothelial cells were examined using the gene silencing technique and chromatin immunoprecipitation. Serum chemerin levels were determined by ELISA in 64 SSc patients and 19 healthy subjects. RESULTS In SSc lesional skin, chemerin was up-regulated in small blood vessels, while it was down-regulated in fibroblasts surrounded with thickened collagen bundles. The decreased expression of chemerin was significantly reversed by TGF-β1 antisense oligonucleotide in cultured SSc dermal fibroblasts and chemerin expression was markedly decreased in dermal fibroblasts of bleomycin-treated mice. Gene silencing of transcription factor Fli1, which binds to the chemerin promoter, induced chemerin expression in human dermal microvascular endothelial cells and Fli1(+/-) mice exhibited elevated chemerin expression in dermal blood vessels. Serum chemerin levels inversely correlated with estimated glomerular filtration rate in SSc patients with renal dysfunction. In SSc patients with normal renal function, patients with digital ulcers had higher serum chemerin levels than those without. CONCLUSION Chemerin is down-regulated in SSc dermal fibroblasts by autocrine TGF-β, while it is up-regulated in SSc dermal blood vessels through endothelial Fli1 deficiency. Increased chemerin expression in dermal blood vessels may be associated with the development of digital ulcers in SSc.

A potential contribution of antimicrobial peptide LL-37 to tissue fibrosis and vasculopathy in systemic sclerosis.

LL‐37 is an antimicrobial peptide with pleiotropic effects on the immune system, angiogenesis and tissue remodelling. These are cardinal pathological events in systemic sclerosis (SSc).

Fli1 deficiency contributes to the downregulation of endothelial protein C receptor in systemic sclerosis: a possible role in prothrombotic conditions.

Endothelial protein C receptor (EPCR), expressed predominantly on endothelial cells, plays a critical role in the regulation of the coagulation system and also mediates various cytoprotective effects by binding and activating protein C. So far, the role of EPCR has not been studied in systemic sclerosis (SSc).

Glycyrrhizin Ameliorates Fibrosis, Vasculopathy, and Inflammation in Animal Models of Systemic Sclerosis

Systemic sclerosis (SSc) is a multisystem inflammatory and vascular disease resulting in extensive tissue fibrosis. Glycyrrhizin, clinically used for chronic hepatic diseases and itching dermatitis, modulates the pathological processes of inflammation, vasculopathy, and fibrosis in human diseases and their animal models. Therefore, we investigated a potential impact of glycyrrhizin on the key pathological manifestations of SSc, including inflammation, vasculopathy, and tissue fibrosis, with bleomycin-treated mice mimicking the fibrotic and inflammatory components of SSc and endothelial cell-specific Fli1-knockout mice recapitulating SSc vasculopathy. Glycyrrhizin significantly ameliorated dermal fibrosis in bleomycin-treated mice, which was partly attributable to blockade of transforming growth factor-β signaling in dermal fibroblasts through the down-regulation of thrombospondin 1, a latent transforming growth factor-β receptor, and transcription factors Smad3 and Ets1. Furthermore, bleomycin-dependent induction of T helper type 2-skewed immune polarization, M2 macrophage infiltration, and endothelial-to-mesenchymal transition were greatly suppressed in mice administered glycyrrhizin. Glycyrrhizin also improved vascular permeability of endothelial cell-specific Fli1-knockout mice by increasing the expression of molecules regulating vascular integrity. These results indicate that glycyrrhizin ameliorates bleomycin-induced dermal fibrosis through the inhibition of fibroblast activation, T helper type 2-skewed immune polarization, M2 macrophage infiltration, and endothelial-to-mesenchymal transition and improves endothelial Fli1 deficiency-dependent vascular disintegrity, implying its potential as a disease-modifying drug for SSc.

A potential contribution of altered cathepsin L expression to the development of dermal fibrosis and vasculopathy in systemic sclerosis.

Cathepsin L (CTSL) is a lysosomal proteolytic enzyme involved in inflammation and vascular and extracellular matrix remodelling, which are the three cardinal pathological events associated with systemic sclerosis (SSc). To elucidate the potential role of CTSL in the development of SSc, we here investigated CTSL expression in the lesional skin of patients with SSc and SSc animal models and the clinical correlation of serum CTSL levels. CTSL expression was elevated in dermal small vessels of SSc patients compared with those of healthy controls. Consistently, CTSL mRNA levels were increased in SSc lesional skin samples, but not in cultivated SSc dermal fibroblasts, compared with corresponding control samples from healthy individuals. Serum CTSL levels were significantly higher in SSc patients than in healthy controls and inversely correlated with skin score. Furthermore, the elevation of serum CTSL levels was linked to SSc vasculopathy. Supporting these results, Ctsl mRNA levels were decreased in the skin of bleomycin‐treated mice, an SSc animal model recapitulating its fibrotic aspect, and CTSL expression was enhanced in dermal small vessels of endothelial cell‐specific Fli1 knockout mice, reminiscent of SSc vasculopathy. Importantly, gene silencing of FLI1 induced CTSL mRNA expression and Fli1 occupied the CTSL promoter in human dermal microvascular endothelial cells. Collectively, these results suggest that endothelial CTSL up‐regulation partially due to Fli1 deficiency may contribute to the development of vasculopathy, while the decrease in dermal CTSL expression is likely associated with dermal fibrosis in SSc.