Fli1 Deficiency Research Articles

Transcription Factor Fli-1 Regulates T Cell Immunity and Tolerance in Gvh and GVL Responses

The E26 transformation-specific (ETS) transcription factor (TF) family member Friend Leukemia Virus Integration 1 (Fli-1) is linked to the oncogenesis of virally induced leukemia and Ewing's Sarcoma, as well as the development of autoimmune diseases including systemic lupus erythematosus and systemic sclerosis. A recent report presents compelling evidence that Fli-1 restrains CD8 effector T cell differentiation and protective immunity against cancer. How Fli-1 regulates primary T cell differentiation and function remain largely elusive. In this study, we investigated the role of Fli-1 in T-cell pathogenicity of graft-versus-host disease (GVHD) and validated Fli-1 as a therapeutic target. Using conditional knock-out models featured as homozygous (Fli-1KO) or heterozygous (Fli-1HET) deficiency of Fli-1 in T cells, we found that Fli-1 dynamically regulates different T-cell subsets in allogeneic responses and pathogenicity in the development of acute and chronic GVHD. Compared to Fli-1KO or Fli-1WT T cells, Fli-1HET T cells induced the mildest aGVHD and cGVHD supported by lowest Th1 and Th17 differentiation (Fig. 1 A-B). In addition, Fli-1HET T cells were able to maintain a strong graft-versus-leukemia (GVL) effect while inducing significantly attenuated GVHD. Fli-1-deficient CD8+ T cells expressed comparable levels of cytolytic molecules, reduced levels of exhaustion markers, and differentiated more into memory precursor effector cells compared to WT CD8 T cells after been transferred into allogeneic recipients. To further understand how Fli-1 regulates T-cell gene transcription and heterogeneity, we performed scRNA seq analysis of donor T cells, including Fli-1WT, Fli-1HET and Fli-1KO, isolated from recipient spleens. The scRNA seq analysis on the donor T cells indicated a major difference in the transcriptional regulation by Fli-1 in CD4 versus CD8 T cells and Fli-1 gene dose dependent modification in transcriptional pathways in Fli-1Het vs Fli-1KOT cells. Fli-1 deemed to play a distinct role in regulating gene transcription in CD4 versus CD8 T cells, in that more activation and less exhaustion gene pathways were enriched in CD8 Fli-1 deficient cells, while fewer genes in Th1/Th17 pathogenic pathways were enriched in CD4 Fli-1 deficient cells (Fig. 1 C-D). Overall, Fli-1 deficiency increased gene enrichment in OXPHOS pathway in both CD4 and CD8 T cells, and reduced gene enrichment in glycolysis pathway majorly in CD4 T cells (Fig. 1 E-F). Consistent with the lowest pathogenicity of Fli-1Het T cells for GVHD induction, Fli-1Het CD4 T cells had the lowest Th1/Th17 pathway gene enrichment and TCR pathway downstream gene expression. Camptothecin, topotecan or etoposide exhibited action as potent Fli-1 inhibitors at low-doses. For translational purpose, we tested these drugs and found that each of these drugs significantly attenuated GVHD severity while preserving the GVL effect. The efficacy of camptothecin or etoposide at low doses was dependent of Fli-1 expression on donor T cells. Mechanistically, blocking Fli-1 suppressed activated effector T cells while sparing Tregs and had no side effect on hematopoietic stem cells. Importantly, using the same treatment schedule as post-transplantation cyclophosphamide (PTCy), two doses of camptothecin or etoposide administered on day 3 and 4 were sufficient for the prevention of GVHD and leukemia relapse, in contrast to GVHD mortality in some of those treated with bendamustine and leukemia relapse in some of those treated with PTCy or cyclosporine (Fig. 1 G-I). In addition, the beneficial effect of camptothecin was extended in GVHD induced by human T cells in a xenograft model. In conclusion, we provide the first evidence that Fli-1 plays a crucial role in alloreactive CD4 T-cell activation and differentiation, and that targeting Fli-1 may be an attractive strategy for treating GVHD without compromising GVL effect. Figure 1View largeDownload PPTFigure 1View largeDownload PPT Close modal

Abstract 12459: Downregulated Fli1 in Scleroderma Myeloid Cells May Contribute to Cardiac Fibrosis via a Galectin-3/mTOR Dependent Pathway

Introduction: Scleroderma (SSc) is an autoimmune fibrotic disease with high mortality and no effective therapies. Cardiac fibrosis in SSc is common and has a poor prognosis, but pathogenesis is unclear. Galectin-3 secretion by macrophages has been implicated in cardiac fibrosis, but its specific contribution to SSc cardiac disease has not been elucidated. We recently demonstrated that Fli1, a member of the Ets family of transcription factors, is decreased in SSc myeloid cells, and that Fli1 depleted macrophages have a profibrotic phenotype. We aim to evaluate the contribution of myeloid Fli1 to SSc cardiac fibrosis. Methods: Levels of galectin-3 were assessed by ELISA in serum, and qPCR and immunofluorescence in monocytes. Primary human cardiac fibroblasts were co-cultured with macrophages using inserts. Rapamycin (10nM) was used to treat the co-cultures. Western blot was used to assess protein levels of collagen and p70S6K. Collagen deposition in hearts from mice with conditional deletion of Fli1 in macrophages ( LysMCre/Fli1 fl/f l ) was assessed by picrosirius red. Results: SSc patients had higher expression of galectin-3 in monocytes and serum compared to healthy controls. Co-culture of human cardiac fibroblasts and Fli1 depleted macrophages resulted in induction of galectin-3 expression by macrophages, and activation of the mTOR/p70S6K pathway with enhanced collagen type I protein deposition by fibroblasts. These effects were blocked by the mTOR inhibitor Rapamycin, and by treatment of macrophages with siRNA against galectin-3. Deletion of Fli1 in macrophages via Cre-mediated recombination using LysMCre mice predisposed mice to develop cardiac fibrosis. Conclusions: These findings suggest that Fli1 deficiency in macrophages may contribute to SSc cardiac fibrosis, and that Rapamycin may be a potential therapeutic option for heart involvement in SSc.

Endothelial CCR6 expression due to FLI1 deficiency contributes to vasculopathy associated with systemic sclerosis

BackgroundWe 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.MethodsThe 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.ResultsCCL20 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.ConclusionsThe increased expression of endothelial CCR6 due to FLI1 deficiency may contribute to the development of SSc vasculopathy.

Fli1 deficiency suppresses RALDH1 activity of dermal dendritic cells and related induction of regulatory T cells: a possible role in scleroderma

BackgroundAldehyde 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.MethodsBleomycin (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.ResultsRALDH 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.ConclusionsFli1 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.

Serum vasohibin-1 levels: A potential marker of dermal and pulmonary fibrosis in systemic sclerosis.

Vasohibin-1 (VASH-1) is a potent anti-angiogenic factor mainly produced by endothelial cells. In addition, VASH-1 prevents TGF-β-dependent activation of renal fibroblasts. Since systemic sclerosis (SSc) is an autoimmune disease characterized by vasculopathy and fibrosis of multiple organs, VASH-1 may be involved in the development of this disease. In this study, we investigated the potential role of VASH-1 in SSc by evaluating the clinical correlation between serum VASH-1 levels and the expression of VASH-1 in SSc-involved skin. Serum VASH-1 levels were higher in SSc patients, especially those with diffuse cutaneous involvement, than in healthy controls and positively correlated with skin score. Furthermore, SSc patients with interstitial lung disease had significantly elevated levels of serum VASH-1 as compared to those without. Importantly, serum VASH-1 levels correlated inversely with both the percentage of predicted vital capacity and the percentage of predicted diffusion lung capacity for carbon monoxide and positively with serum KL-6 levels, but not serum surfactant protein D levels. In SSc-involved skin, VASH1 mRNA was remarkably upregulated compared with healthy control skin, but the major source of VASH-1 was not clear. Fli1 deficiency, a predisposing factor inducing SSc-like endothelial properties, did not affect VASH-1 expression in human dermal microvascular endothelial cells. Collectively, these results suggest that VASH-1 upregulation in the skin and sera is linked to dermal and pulmonary fibrotic changes in SSc, while the contribution of VASH-1 to SSc vasculopathy seems to be limited.

Altered Properties of Endothelial Cells and Mesenchymal Stem Cells Underlying the Development of Scleroderma-like Vasculopathy in KLF5+/- ;Fli-1+/- Mice.

In prevous studies, we established a new animal model, KLF5+/- ;Fli-1+/- mice, in which fundamental pathologic features of systemic sclerosis (SSc) are broadly recapitulated. SSc vasculopathy is believed to occur as a result of impaired vascular remodeling, but its detailed mechanism of action remains unknown. To address this, the present study investigated the properties of dermal microvascular endothelial cells (DMECs), bone marrow-derived endothelial progenitor cells (BM-EPCs), and bone marrow-derived mesenchymal stem cells (BM-MSCs), a precursor of pericytes, in KLF5+/- ;Fli-1+/- mice. Neovascularization and angiogenesis were assessed in KLF5+/- ;Fli-1+/- mice by in vivo Matrigel plug assay and in vitro tube formation assay, respectively. The properties of mouse BM-EPCs and BM-MSCs were assessed with in vitro studies. Dermal vasculature was visualized in vivo by injecting the mice with fluorescein isothiocyanate-conjugated dextran. Neovascularization was diminished in skin-embedded Matrigel plugs from KLF5+/- ;Fli-1+/- mice. DMECs from KLF5+/- ;Fli-1+/- mice showed defective tubulogenic activity, decreased expression of VE-cadherin and CD31, and an imbalance in the expression of Notch1/Dll4, suggesting that angiogenesis and anastomosis are disturbed. KLF5+/- ;Fli-1+/- mouse BM-MSCs exhibited enhanced proliferation and migration and increased collagen production following stimulation with transforming growth factor β1, indicating that these cells differentiate preferentially into myofibroblasts rather than pericytes. KLF5+/- ;Fli-1+/- mouse BM-EPCs displayed a transition toward mesenchymal cells, suggesting that vasculogenesis is impaired. Wound healing was delayed in KLF5+/- ;Fli-1+/- mice (mean ± SD healing time 15.67 ± 0.82 days versus 13.50 ± 0.84 days; P = 0.0017), and the vascular network was poorly developed in wound scar tissue. The characteristics observed in the KLF5+/- ;Fli-1+/- mouse model - specifically, impaired neovascularization and vascular maturation - are similar to those observed in human SSc, and could be at least partially attributable to the induction of SSc-like properties in DMECs, BM-EPCs, and BM-MSCs. These findings indicate the critical contribution of Klf5 and Fli1 deficiency in vascular cells and related cell precursors to the development of SSc vasculopathy.

Fli1 deficiency induces endothelial adipsin expression, contributing to the onset of pulmonary arterial hypertension in systemic sclerosis.

Adipsin, or complement factor D, is a serine proteinase catalysing complement factor C3 breakdown, leading to the production of opsonin (C3b), membrane attack complex (C5b-C9) and anaphylatoxins (C3a and C5a). Since adipsin is potentially associated with pulmonary arterial hypertension in SSc, we investigated adipsin expression in dermal small vessels of SSc-involved skin, the mechanism regulating adipsin expression in endothelial cells, and the correlation of serum adipsin levels with SSc clinical symptoms. Adipsin expression was assessed by immunohistochemistry with skin sections of SSc and healthy subjects. mRNA levels of target genes and transcription factor binding to the ADIPSIN promoter were evaluated by quantitative reverse transcription PCR and chromatin immunoprecipitation, respectively. Serum adipsin levels were determined by enzyme-linked immunosorbent assay. Adipsin expression was remarkably increased in dermal small vessels of SSc-involved skin as compared with those of healthy control skin. Consistent with the notion that Fli1 deficiency induces SSc-like phenotypes in various types of cells, FLI1 siRNA enhanced adipsin expression at protein and mRNA levels and Fli1 bound to the ADIPSIN promoter in human dermal microvascular endothelial cells. Serum adipsin levels were significantly lower in diffuse cutaneous SSc patients than in limited cutaneous SSc patients and healthy controls, and were associated positively with elevated right ventricular systolic pressure and inversely with interstitial lung disease by multivariate regression analysis. Adipsin is up-regulated at least partially by Fli1 deficiency in endothelial cells, potentially contributing to the development of pulmonary vascular involvement in SSc.

A potential contribution of trappin-2 to the development of vasculopathy in systemic sclerosis.

Trappin-2/pre-elafin is an endogenous inhibitor of human neutrophil elastase involved in inflammation, innate immunity and vascular remodelling, which consist of the complex pathological process of systemic sclerosis (SSc). To clarify the potential role of trappin-2 in SSc. Serum trappin-2 levels were determined by enzyme-linked immunosorbent assay in 51 SSc and 18 healthy subjects. Trappin-2 expression was evaluated in SSc lesional skin and cultured endothelial cells treated with FLI1 siRNA by immunohistochemistry, reverse transcription-real-time PCR and/or immunoblotting. Friend leukaemia virus integration 1 (Fli1) binding to the PI3 promoter was assessed by chromatin immunoprecipitation. Since serum trappin-2 levels inversely correlated with estimated glomerular filtration rate in SSc patients with renal dysfunction, SSc patients with normal renal function were analysed. Although serum trappin-2 levels were comparable between diffuse cutaneous SSc, limited cutaneous SSc and control subjects, the prevalence of digital ulcers or elevated right ventricular systolic pressure (RVSP) was significantly higher in SSc patients with elevated serum trappin-2 levels than in those with normal levels. Furthermore, serum trappin-2 levels were significantly increased in SSc patients with digital ulcers or elevated RVSP compared to those without. Moreover, serum trappin-2 levels positively correlated with RVSP values in SSc patients. Importantly, trappin-2 expression was enhanced in small vessels of SSc lesional skin. In cultured endothelial cells, trappin-2 expression was elevated by gene silencing of FLI1 at mRNA and protein levels and Fli1 occupied the PI3 promoter. Endothelial trappin-2 up-regulation partially due to Fli1 deficiency can be associated with the development of SSc vasculopathy.

Increased expression of aquaporin-1 in dermal fibroblasts and dermal microvascular endothelial cells possibly contributes to skin fibrosis and edema in patients with systemic sclerosis

BackgroundAquaporin-1 (AQP1), a water channel protein controlling the water contents of cells and tissues, exerts pleiotropic effects on various biological activities, including inflammation, angiogenesis, and extracellular matrix remodeling, by regulating cell behaviors and tissue water balance. ObjectiveTo investigate AQP1 roles in systemic sclerosis (SSc) which is characterized by autoimmune inflammation, vasculopathy, and tissue fibrosis. MethodsAQP1 expression was evaluated by immunohistochemistry and quantitative reverse transcription PCR in skin samples from human and animal models and by immunoblotting in cultured cells. Fli1 binding to the AQP1 promoter was evaluated by chromatin immunoprecipitation. Cell migration was assessed by scratch assay. ResultsDermal fibroblasts and endothelial cells highly expressed AQP1 in SSc lesional skin, and AQP1 expression in dermal fibroblasts and endothelial cells positively correlated with the degrees of tissue fibrosis and edema, respectively. Consistently, SSc dermal fibroblasts up-regulated AQP1 compared with normal dermal fibroblasts in vitro. Furthermore, TGF-β stimulation induced AQP1 expression in normal dermal fibroblasts, while TGF-β1 antisense oligonucleotide suppressed AQP1 expression in SSc dermal fibroblasts. In endothelial cells, Fli1 deficiency resulted in AQP1 up-regulation in vivo and in vitro and Fli1 bound to the AQP1 promoter. Importantly, SSc dermal fibroblasts and FLI1 siRNA-treated endothelial cells had a pro-migratory property, which was remarkably diminished by gene silencing of AQP1. ConclusionAQP1 is up-regulated in SSc dermal fibroblasts and SSc endothelial cells at least partially due to autocrine TGF-β stimulation and Fli1 deficiency, respectively, possibly contributing to inflammation, vasculopathy, and tissue fibrosis by regulating tissue edema and cell migration.

CXCL13 produced by macrophages due to Fli1 deficiency may contribute to the development of tissue fibrosis, vasculopathy and immune activation in systemic sclerosis.

CXCL13, a chemokine for B cells, follicular T cells, T helper 17 cells, and regulatory T cells, is reported to contribute to the development of systemic sclerosis (SSc), reflecting aberrant activation of immune system. To better understand the role of CXCL13 in SSc, we investigated the influence of Fli1 deficiency, a potential predisposing factor of this disease, on CXCL13 expression and assessed the clinical correlation of serum CXCL13 levels by multivariate regression analysis. Haploinsufficient loss of Fli1 remarkably induced CXCL13 expression in murine peritoneal macrophages, while gene silencing of FLI1 did not affect the expression of CXCL13 in human dermal fibroblasts and human dermal microvascular endothelial cells. Serum CXCL13 levels were elevated in SSc patients compared with healthy controls and correlated positively with skin score and negatively with pulmonary function test results. SSc patients with elevated serum CXCL13 levels had longer disease duration, diffuse cutaneous involvement, interstitial lung disease (ILD), heart involvement, pulmonary arterial hypertension, Raynaud's phenomenon, pitting scars, digital ulcers, telangiectasia, and high serum IgG levels more frequently than the other patients. In particular, serum CXCL13 levels were associated with ILD and digital ulcers by multivariate regression analysis. Taken together, these results indicate that CXCL13 expression is upregulated by Fli1 deficiency in macrophages, potentially contributing to the development of tissue fibrosis, vasculopathy and immune activation in SSc, especially ILD and digital ulcers.

Fli1-haploinsufficient dermal fibroblasts promote skin-localized transdifferentiation of Th2-like regulatory T cells

BackgroundFriend 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.MethodsCytokine 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.ResultsTh2- 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.ConclusionsFli1 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.

A possible implication of reduced levels of LIF, LIFR, and gp130 in vasculopathy related to systemic sclerosis.

Leukemia inhibitory factor (LIF) is a member of IL-6 family, which serves as a potent chemoattractant for neutrophils as well as a potent angiostatic factor. LIF has been implicated in various autoimmune inflammatory diseases, but its role still remains elusive in systemic sclerosis (SSc). Therefore, we investigated the potential role of LIF in the development of SSc by evaluating the clinical correlation of serum LIF levels, the expression of LIF and its receptors in skin samples, and in vitro experiments with human dermal microvascular endothelial cells. Serum LIF levels were significantly decreased in patients with SSc, especially in those with disease duration of<1year compared with healthy controls. As for clinical correlation, SSc patients with digital ulcers exhibited serum LIF levels significantly lower than those without. In immunohistochemistry, the expression of LIF and its receptors, LIF receptor and gp130, was remarkably decreased in dermal blood vessels of SSc lesional skin relative to those of healthy control skin. Furthermore, gene silencing of transcription factor Fli1, whose deficiency is involved in the development of SSc vasculopathy, suppressed the expression of LIF, LIF receptor, and gp130 and Fli1 bound to the promoters of those genes in human dermal microvascular endothelial cells. Collectively, these results suggest that decreased serum LIF levels may be associated with vasculopathy in SSc and that Fli1 deficiency may contribute to the inhibition of LIF-dependent biological effects on SSc endothelial cells by suppressing the expression of LIF, LIF receptor, and gp130.

381 Fli1 deficiency promotes CXCL13 expression from macrophages, contributing to the development of systemic sclerosis

381 Fli1 deficiency promotes CXCL13 expression from macrophages, contributing to the development of systemic sclerosis

305 Fli1 deficiency alters the phenotype of dermal dendritic cells, promoting the development of dermal fibrosis in bleomycin-treated mice

305 Fli1 deficiency alters the phenotype of dermal dendritic cells, promoting the development of dermal fibrosis in bleomycin-treated mice

The impact of transcription factor Fli1 deficiency on the regulation of angiogenesis.

The insufficiency of Friend leukaemia virus integration 1 (Fli1), a member of the Ets family transcription factors, is implicated in the pathogenesis of vasculopathy associated with systemic sclerosis (SSc). Fli1 deficiency accelerates early steps of angiogenesis, including detachment of pre-existing pericytes and extracellular matrix degradation by endothelial proteinases, but the impact of Fli1 deficiency on the other steps of angiogenesis has not been investigated. Therefore, we evaluated the effect of Fli1 deficiency on migration, proliferation, cell survival and tube formation of human dermal microvascular endothelial cells (HDMECs). HDMECs transfected with FLI1 siRNA exhibited a greater migratory property in scratch assay and transwell migration assay and a higher proliferation rate in BrdU assay than HDMECs transfected with non-silencing scrambled RNA. In flow cytometry-based apoptosis assay, FLI1 siRNA-transduced HDMECs revealed the decreased number of annexin and propidium iodide-double-positive apoptotic cells compared with control cells, reflecting the promotion of cell survival. On the other hand, tubulogenic activity on Matrigel was remarkably suppressed in Fli1-deficient HDMECs relative to control cells. These results indicate that Fli1 deficiency promotes migration, proliferation and cell survival, while abating tube formation of endothelial cells, suggesting that Fli1 deficiency is potentially attributable to the development of both proliferative obliterative vasculopathy (occlusion of arterioles and small arteries) and destructive vasculopathy (loss of small vessels) characteristic of SSc vasculopathy.

Systemic Sclerosis Dermal Fibroblasts Suppress Th1 Cytokine Production via Galectin-9 Overproduction due to Fli1 Deficiency

Dermal fibroblasts promote skin-localized transdifferentiation of regulatory T cells to T helper (Th) type 2-like cells in systemic sclerosis (SSc). However, the entire effect of SSc dermal fibroblasts on immune cells still remains unknown. Because galectin-9 induces Th2 cytokine-predominant immune imbalance by negatively regulating Th1/Th17 cells in inflammatory diseases, we investigated the contribution of galectin-9 to Th immune balance in SSc lesional skin. We used human clinical samples and Fli1+/- mice because Fli1 deficiency induces SSc-like phenotypes in various cell types. Galectin-9 was overexpressed in SSc dermal fibroblasts invivo and invitro. Serum galectin-9 levels were significantly elevated in SSc patients and positively correlated with skin score. Galectin-9 was up-regulated by autocrine endothelin stimulation and Fli1 deficiency, and Fli1 occupied the LGALS9 promoter in dermal fibroblasts. Co-culture of splenic CD4+ T cells with Fli1+/- dermal fibroblasts significantly increased IL-4-producing cell proportion, and this effect was cancelled in parallel with the increased interferon-γ production when Fli1+/- dermal fibroblasts were transfected with Lgals9 small interfering RNA. Furthermore, Lgals9 small interfering RNA suppressed dermal collagen deposition by increasing interferon-γ production of skin-infiltrating CD4+ T cells in bleomycin-treated mice. These results suggest that SSc dermal fibroblasts suppress interferon-γ expression of skin-infiltrating CD4+ T cells through galectin-9 overproduction, promoting skin fibrosis development.

Fli1 Deficiency Induces CXCL6 Expression in Dermal Fibroblasts and Endothelial Cells, Contributing to the Development of Fibrosis and Vasculopathy in Systemic Sclerosis.

CXCL6, a chemokine with proangiogenic property, is reported to be involved in vasculopathy associated with systemic sclerosis (SSc). We investigated the contribution of CXCL6 to SSc development by focusing on the association of friend leukemia virus integration 1 (Fli1) deficiency, a potential predisposing factor of SSc, with CXCL6 expression and clinical correlation of serum CXCL6 levels. mRNA levels of target genes and the binding of Fli1 to the CXCL6 promoter were evaluated by quantitative reverse transcription-PCR and chromatin immunoprecipitation, respectively. Serum CXCL6 levels were determined by ELISA. FLI1 siRNA significantly enhanced CXCL6 mRNA expression in human dermal fibroblasts and human dermal microvascular endothelial cells, while Fli1 haploinsufficiency significantly suppressed CXCL6 mRNA expression in murine peritoneal macrophages stimulated with lipopolysaccharide. Supporting a critical role of Fli1 deficiency to induce SSc-like phenotypes, CXCL6 mRNA expression was higher in SSc dermal fibroblasts than in normal dermal fibroblasts. Importantly, Fli1 bound to the CXCL6 promoter in dermal fibroblasts, endothelial cells, and THP-1 cells. In patients with SSc, serum CXCL6 levels correlated positively with the severity of dermal and pulmonary fibrosis and were elevated in association with cardiac and pulmonary vascular involvement and cutaneous vascular symptoms, including Raynaud phenomenon, digital ulcers (DU)/pitting scars, and telangiectasia. Especially, serum CXCL6 levels were associated with DU/pitting scars and heart involvement by multiple regression analysis. CXCL6 expression is upregulated by Fli1 deficiency in fibroblasts and endothelial cells, potentially contributing to the development of fibrosis and vasculopathy in the skin, lung, and heart of SSc.

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.

Myeloid Fli1 deficiency induces tissue fibrosis, vasculopathy, and immune abnormalities recapitulating systemic sclerosis

Systemic sclerosis (SSc) is a multisystem chronic disease characterized by immune abnormalities, vasculopathy, and tissue fibrosis. In SSc lesional skin, transcription factor Friend leukemia virus integration 1 (Fli1) is constitutively down-regulated in various cell types, especially by an epigenetic mechanism in dermal fibroblasts, and Fli1 deficiency in dermal fibroblasts and endothelial cells induces an SSc-like phenotype in each cell type. However, it has still remained to be elucidated whether Fli1 deficiency in immune cells has an impact on the pathogenesis of SSc. As a part of studies to address this issue, we focused our interests on myeloid cells in this study. We generated myeloid cell-specific Fli1 deficient (Fli1 Mye-KO) mice (LysM-Cre+/−Fli1flox/flox) and examined the phenotypic changes in these mice. Fli1 Mye-KO mice showed increased dermal thickness and collagen production through activation of dermal fibroblasts. As for vasculopathy, Fli1 Mye-KO mice showed stenosis, dilatation, and regression of dermal blood vessels as visualized by FITC-conjugated dextran injection. Increased permeability of dermal blood vessels was also confirmed by leakage of Evans blue dye into interstitial spaces in Fli1 Mye-KO mice. Histopathological analysis of the skin of Fli1 Mye-KO mice showed decreased pericyte coverage in dermal blood vessels, leading to endothelial hyperplasia, vascular dilatation, and decreased stability of blood vessels. Regarding immune dysfunction, Fli1 Mye-KO mice showed production of anti-nuclear antibodies. Thus, Fli1 deficiency in myeloid cells may contribute to the induction of three cardinal pathological features of SSc, including dermal fibrosis, vascular injury, and immune abnormalities.

The impact of Fli1 deficiency in B cells on the development of systemic sclerosis

Fli1 is a potential predisposing factor of systemic sclerosis (SSc), whose deficiency is involved in its fibrotic and vascular processes. Given that B cells from Klf5+/−; Fli1+/− mice, spontaneously developing SSc-like tissue fibrosis and vasculopathy, represent SSc-like features such as higher CD 19 expression and autoantibody production, Fli1 deficiency may also contribute to its immunological aspect, such as B cell activation. Here, we confirmed decreased Fli1 expression in SSc-derived B cells and further explored the role of Fli1-deficient B cells in SSc utilizing B cell-specific Fli1 knockout (BcKO) mice. Fli1-deficient B cells revealed lower expression of CD22, an inhibitory receptor, and higher expression of BAFF receptor than control B cells. Consistently, Fli1-deficient B cells were constitutively activated with higher IL-6 secretion and autoantibody production. Furthermore, the proportion of regulatory B cells was decreased and Th17 skewed immune polarization was noted in Fli1 BcKO mice. As for the fibrotic and vascular aspects, of note, tissue fibrosis and SSc-like vascular changes were evident in the skin and lung of Fli1 BcKO mice. Relevant to tissue fibrosis, TGF-beta and IL-6 expression was elevated in the fibrotic tissue and cultivated Fli1-deficient B cells. More importantly, the direct interaction with Fli1-deficient B cells activated pro-angiogenic gene program and altered the expression of cell adhesion molecules promoting Th2/Th17 cell infiltration in endothelial cells. Collectively, Fli1-deficient B cells induces endothelial cell activation and Th17 skewed immune polarization, eventually leading to SSc-like tissue fibrosis and vasculopathy, indicating a pivotal contribution of Fli1 deficiency in B cells to the pathogenesis of SSc.