Tight-skin Mouse Research Articles

Abstract 14651: Noncoding RNA Drug, TY1, Attenuates Cardiac Fibrosis in Two Mouse Models of Systemic Sclerosis

Introduction: Systemic sclerosis (SSc) is a connective tissue disease characterized by inflammation and fibrosis of the skin and vital organs. The disease has no specific treatment options and a high mortality (~30% at 3 years). We have previously demonstrated anti-fibrotic and anti-inflammatory properties of EV-YF1, a small Y RNA abundant in extracellular vesicles secreted by cardiosphere-derived cells (CDCs). Here we describe the therapeutic bioactivity of a shorter, engineered derivative, TY1. Methods: 5-7-week-old female C57BL/6J wild type mice (n=100 total) were injected with phosphate-buffered saline (PBS; WT) or 100 μL bleomycin (1 mg/ml) subcutaneously every other day for 3 weeks. Animals then received either vehicle (formulation only, in PBS), TY1, or scrambled TY1 (as an RNA control) via oral gavage. Endpoints include exercise capacity, lung weight, systolic and diastolic function by echocardiography, and histologic quantification of fibrosis in heart and skin. To confirm the antifibrotic findings in an inflammation-independent model, 4-6-week-old Tight-skin (tsk-1) mice (n=18 total) received oral TY1, scramble or vehicle for 4 weeks. Results: Diastolic dysfunction was evident in bleomycin mice, but was normalized by TY1 ingestion (E/E’ ratios in WT: 18.91, vehicle: 32.29, scramble: 36.19, TY1: 20.65 p <0.0001). Likewise, lung congestion and exercise intolerance were ameliorated by TY1 ingestion (p<0.0001). In both models (bleomycin-induced and tsk-1), skin and cardiac fibrosis were greater in vehicle or scramble groups than in WT, but were normalized by TY1 after 4 weeks (p <0.001). Conclusions: These results demonstrate the therapeutic bioactivity of a bioinspired new chemical entity in two complementary mouse models of SSc. Thus, TY1 merits investigation as a potential novel therapeutic for SSc.

Fibrillin-1 mutant mouse captures defining features of human primary open glaucoma including anomalous aqueous humor TGF beta-2

Primary open angle glaucoma (POAG) features an optic neuropathy, elevated aqueous humor (AH) TGFβ2, and major risk factors of central corneal thickness (CCT), increasing age and intraocular pressure (IOP). We examined Tight skin (Tsk) mice to see if mutation of fibrillin-1, a repository for latent TGFβ, is associated with characteristics of human POAG. We measured: CCT by ocular coherence tomography (OCT); IOP; retinal ganglion cell (RGC) and optic nerve axon counts by microscopic techniques; visual electrophysiologic scotopic threshold responses (STR) and pattern electroretinogram (PERG); and AH TGFβ2 levels and activity by ELISA and MINK epithelial cell-based assays respectively. Tsk mice had open anterior chamber angles and compared with age-matched wild type (WT) mice: 23% thinner CCT (p < 0.003); IOP that was higher (p < 0.0001), more asymmetric (p = 0.047), rose with age (p = 0.04) and had a POAG-like frequency distribution. Tsk mice also had RGCs that were fewer (p < 0.04), declined with age (p = 0.0003) and showed increased apoptosis and glial activity; fewer optic nerve axons (p = 0.02); abnormal axons and glia; reduced STR (p < 0.002) and PERG (p < 0.007) visual responses; and higher AH TGFβ2 levels (p = 0.0002) and activity (p = 1E−11) especially with age. Tsk mice showed defining features of POAG, implicating aberrant fibrillin-1 homeostasis as a pathogenic contributor to emergence of a POAG phenotype.

Estrogens Counteract the Profibrotic Effects of TGF-β and their Inhibition Exacerbates Experimental Dermal Fibrosis

Systemic sclerosis primarily affects women. This sex bias raises the question on the role female hormones could play in the development of fibrosis, which is largely unknown. Our aim was to evaluate the effects of estrogens in the development of experimental dermal fibrosis, in the mouse models of bleomycin-induced dermal fibrosis and tight skin (Tsk-1) mice, and on the activation of dermal fibroblasts by transforming growth factor-β (TGF-β). Estrogen inhibition, obtained through gene inactivation for the estrogen receptor-αknockout or treatment with tamoxifen, exacerbated skin fibrosis in the bleomycin model and in the Tsk-1 mice. In the dermal fibroblasts, treatment with 17-β-estradiol significantly decreased the stimulatory effects of TGF-β on collagen synthesis and myofibroblast differentiation, decreased the activation of canonical TGF-β signaling, and markedly reduced the expression of the TGF-β target genes. Tamoxifen reversed the inhibitory effects of estrogens by restoring Smad2/3 phosphorylation and TGF-β-induced collagen synthesis. Our results demonstrate a beneficial effect of estrogens in dermal fibrosis. Estrogens reduce the TGF-β-dependent activation of dermal fibroblasts, and estrogen inhibition leads to a more severe experimental dermal fibrosis. These findings are consistent with the prominent development of systemic sclerosis in postmenopausal women and the greater severity of the disease in men.

Interleukin-4 and interleukin-13 as possible therapeutic targets in systemic sclerosis

Systemic sclerosis (SSc) is an autoimmune disease characterized by fibrosis of skin and internal organs. Its pathogenesis, which is still poorly understood, features three main pathogenic moments: an initial diffuse vasculopathy followed by low-grade inflammation and a subsequent tissue fibrosis. Numerous evidences support the role of a Th2-oriented immune response during both the inflammatory and the fibrotic phase of SSc.Levels of IL-4, IL-13 and CXCL4 are higher in the serum of SSc patients compared to healthy controls. Fibrotic tissue in SSc displays a Th2 polarized CD4+ cell infiltration, influencing fibroblast phenotype and inducing collagen and extra cellular matrix protein synthesis. In tight skin mice the administration of neutralizing anti-IL-4 antibodies prevents the development of dermal fibrosis. Back-crossing these mice onto a genetic background that cannot respond to IL-4 prevents skin sclerosis. In SSc, CD8+ T lymphocytes secrete IL-13 and mediate dermal fibrosis and have skin-homing receptors. Incubation with healthy dermal fibroblasts results in elevation of extracellular matrix, which can be reduced with anti-IL13 antibodies. Specifically, IL-4 and IL-13 take part in the inflammatory phase, contribute to the transition from the inflammatory to the fibrotic phase and maintain a profibrotic state in affected organs, mediating the interaction between T cells and fibroblasts. Blocking the cross-talk between these cell types by acting on the soluble cytokines, on their receptors on cell surfaces or on intracellular signaling pathways could constitute a new therapeutic approach.

Fibrillin-1 mutant mice (tight skin) show increased spontaneous and trauma-induced osteoarthritis development

Purpose: Osteoarthritis development is a major chronic disorder affected >8million British people. Despite the high prevalence of OA, there are currently no therapies to slow disease progression. Fibrillin-1 is an extracellular matrix protein found in elastic fibres. One of its main roles is to control growth factor bioavailability, which play vital functions in joint homeostasis. Fibrillin-1 mutations, as those found in Tight Skin mice (TSK), can increase TGFß signalling, and leads to tight skin, myocardial hypertrophy, marfan-like skeletal phenotype and lung emphysema. The aim of this study is to assess OA development in TSK mice in spontaneous and in trauma-induced models. Methods: Immunohistochemistry for Fibrillin-1 was performed in CBA and Str/ort mice (spontaneous OA), and in trauma-induced OA. For the ageing model, knees from 35wk-old TSK and littermate control male mice were collected, microCT scanned and used for histology. The non-invasive loading model was used and the right knee of male TSK and WT mice were loaded i) a single time at 7 or 9N loads and joints analysed 1 weeks later; or ii) repetitively for 2 weeks to induce OA progression, and joints assessed 6 weeks after the last loading episode. Histology was performed on these joints after decalcification (10% formic acid), serial sections cut across the whole joint at 6μm, and AC degradation severity assessed (OARSI grading system). Results: Fibrillin-1 was found in the pericellular matrix of chondrocytes in both the growth plate (resting and hypertrophic cells) and in the uncalcified articular cartilage in healthy CBA mice. During the development of OA in Str/ort mice and in response to mechanical trauma, Fibrillin-1 immunolabelling was decreased. TSK mice showed spontaneous increased joint space mineralised tissue volume (including meniscus and ligament calcification) and increased AC degradation compared to WT at 35wks of age. In contrast, no significant differences were seen in tibial epiphyseal bone (medial and lateral; BV/TV, Trabecular Thickness, Trabecular Separation, Trabecular number). A single loading episode at both 9 and 7N produced AC lesions in the lateral femur in both WT and TSK mice, however severity of lesions created were more severe in TSK mice (preliminary data). Repetitive loading episodes lead to slight increase in mineralised joint space tissue between TSK and litter mate WT controls (preliminary data), which from our previous studies has shown to be correlated with OA severity. Conclusions: This study shows that Fibrillin-1 is decreased from the pericellular matrix during OA development in spontaneous and trauma-induced OA, suggesting increased Fibrillin-1 degradation. In addition, mutations in Fibrillin-1 in the TSK mouse lead to increased spontaneous and trauma-induced OA development. These data suggest that normal Fibrillin-1 expression and function may play an important role in joint homeostasis and that abnormal expression or mutations promote OA development initiation and progression.

Pathogenic roles of B lymphocytes in systemic sclerosis

Systemic sclerosis (SSc) is a collagen disease characterized by autoimmunity and excessive extracellular matrix deposition in the skin and visceral organs. Although the pathogenic relationship between systemic autoimmunity and the clinical manifestations of SSc remains unknown, SSc patients show a variety of abnormal immune activation including the production of disease-specific autoantibodies and cytokine production. Many recent studies have demonstrated that immune cells, including T cells, B cells, and macrophages, have a variety of immunological abnormalities in SSc. So far, several groups and our group reported that B cells play a critical role in systemic autoimmunity and disease expression through various functions, such as cytokine production, lymphoid organogenesis, and induction of other immune cell activation in addition to autoantibody production. Recent studies show that B cells from SSc patients demonstrate an up-regulated CD19 expression, a crucial regulator of B cell activation, which induces chronic hyper-reactivity of memory B cells and SSc-specific autoantibody production and also causes fibrosis of several organs. Furthermore, in SSc-model mice, such as tight-skin mice, bleomycin-induced SSc model mice, and DNA topoisomerase I and complete Freund’s adjuvant-induced SSc model mice, have abnormal B cell activation which associates with skin and lung fibrosis. Indeed, B cell depletion therapy using anti-CD20 Ab, Rituximab, is considered to one potential beneficial treatment for patients with SSc. However, there is no direct evidence which can explain how B cells, especially autoantigen-reactive B cells, progress or regulate disease manifestations of SSc. Collectively, B cell abnormalities in SSc is most likely participating in fibrosis and tissue damage of SSc. If the relationship between SSc-specific tissue damage and B cell abnormalities is revealed, these findings lead to novel effective therapy for SSc.

Vasodilation of Isolated Vessels and the Isolation of the Extracellular Matrix of Tight-skin Mice

The interferon regulatory factor 5 (IRF5) is crucial for cells to determine if they respond in a pro-inflammatory or anti-inflammatory fashion. IRF5's ability to switch cells from one pathway to another is highly attractive as a therapeutic target. We designed a decoy peptide IRF5D with a molecular modeling software for designing small molecules and peptides. IRF5D inhibited IRF5, reduced alterations in extracellular matrix, and improved endothelial vasodilation in the tight-skin mouse (Tsk/+). The Kd of IRF5D for recombinant IRF5 is 3.72 ± 0.74 x 10-6 M as determined by binding experiments using biolayer interferometry experiments. Endothelial cells (EC) proliferation and apoptosis were unchanged using increasing concentrations of IRF5D (0 to 100 µg/mL, 24 h). Tsk/+ mice were treated with IRF5D (1 mg/kg/d subcutaneously, 21 d). IRF5 and ICAM expressions were decreased after IRF5D treatment. Endothelial function was improved as assessed by vasodilation of facialis arteries from Tsk/+ mice treated with IRF5D compared to Tsk/+ mice without IRF5D treatment. As a transcription factor, IRF5 traffics from the cytosol to the nucleus. Translocation was assessed by immunohistochemistry on cardiac myocytes cultured on the different cardiac extracellular matrices. IRF5D treatment of the Tsk/+ mouse resulted in a reduced number of IRF5 positive nuclei in comparison to the animals without IRF5D treatment (50 µg/mL, 24 h). These findings demonstrate the important role that IRF5 plays in inflammation and fibrosis in Tsk/+ mice.

Vasodilation of Isolated Vessels and the Isolation of the Extracellular Matrix of Tight-skin Mice.

The interferon regulatory factor 5 (IRF5) is crucial for cells to determine if they respond in a pro-inflammatory or anti-inflammatory fashion. IRF5's ability to switch cells from one pathway to another is highly attractive as a therapeutic target. We designed a decoy peptide IRF5D with a molecular modeling software for designing small molecules and peptides. IRF5D inhibited IRF5, reduced alterations in extracellular matrix, and improved endothelial vasodilation in the tight-skin mouse (Tsk/+). The Kd of IRF5D for recombinant IRF5 is 3.72 ± 0.74 x 10-6 M as determined by binding experiments using biolayer interferometry experiments. Endothelial cells (EC) proliferation and apoptosis were unchanged using increasing concentrations of IRF5D (0 to 100 µg/mL, 24 h). Tsk/+ mice were treated with IRF5D (1 mg/kg/d subcutaneously, 21 d). IRF5 and ICAM expressions were decreased after IRF5D treatment. Endothelial function was improved as assessed by vasodilation of facialis arteries from Tsk/+ mice treated with IRF5D compared to Tsk/+ mice without IRF5D treatment. As a transcription factor, IRF5 traffics from the cytosol to the nucleus. Translocation was assessed by immunohistochemistry on cardiac myocytes cultured on the different cardiac extracellular matrices. IRF5D treatment of the Tsk/+ mouse resulted in a reduced number of IRF5 positive nuclei in comparison to the animals without IRF5D treatment (50 µg/mL, 24 h). These findings demonstrate the important role that IRF5 plays in inflammation and fibrosis in Tsk/+ mice.

Development of spinal deformities in the tight-skin mouse

Tight-skin (TSK) mice are commonly used as an animal model to study the pathogenesis of Marfan syndrome (MFS), but little is known of their skeletal phenotype and in particular of the development of the spinal deformities, common in MFS. Here we examined growth of the axial skeletons of TSK and wild-type(B6) mice during their period of rapid growth. The whole bodies of mice, 4–12 weeks of age, were scanned after sacrifice, by micro-computed tomography (microCT). We reconstructed three-dimensional models of the spine and ribs, and measured vertebral body heights and rib lengths using the Mac-based image-processing software “OsiriX”. Although the TSK mice were smaller than the B6 mice at 4 weeks, they experienced an early growth spurt and by 8 weeks the height, but not the width, of the vertebral body was significantly greater in the TSK mice than the B6 mice. Measurement of the angles of scoliotic and kyphotic curves post-mortem in the mice was problematic, hence we measured changes that develop in skeletal elements in these disorders. As a marker of kyphosis, we measured anterior wedging of the vertebral bodies; as a marker for scoliosis we measured asymmetries in rib length. We found, unlike in the B6 mice where the pattern was diffuse, wedging in TSK mice was directly related to spinal level and peaked steeply at the thoracolumbar junction. There was also significant asymmetry in length of the ribs in the TSK mice, but not in the B6 mice. The TSK mice thus appear to exhibit spinal deformities seen in MFS and could be a useful model for gaining understanding of the mechanisms of development of scoliosis and kyphosis in this disorder.

Mesenchymal stem cell transplantation in tight-skin mice identifies miR-151-5p as a therapeutic target for systemic sclerosis.

Systemic sclerosis (SSc), an autoimmune disease, may cause significant osteopenia due to activation of the IL4Rα/mTOR pathway. Mesenchymal stem cell transplantation (MSCT) can ameliorate immune disorders in SSc via inducing immune tolerance. However, it is unknown whether MSCT rescues osteopenia phenotype in SSc. Here we show that MSCT can effectively ameliorate osteopenia in SSc mice by rescuing impaired lineage differentiation of the recipient bone marrow MSCs. Mechanistically, we show that donor MSCs transfer miR-151-5p to the recipient bone marrow MSCs in SSc mice to inhibit IL4Rα expression, thus downregulating mTOR pathway activation to enhance osteogenic differentiation and reduce adipogenic differentiation. Moreover, systemic delivery of miR-151-5p is capable of rescuing osteopenia, impaired bone marrow MSCs, tight skin, and immune disorders in SSc mice, suggesting that miR-151-5p may be a specific target for SSc treatment. Our finding identifies a previously unrecognized role of MSCT in transferring miRNAs to recipient stem cells to ameliorate osteopenia via rescuing a non-coding RNA pathway.

Proteinase activated receptor-2 counterbalances the vascular effects of endothelin-1 in fibrotic tight-skin mice.

The majority of the severe vascular complications in fibrosis are a consequence of a deregulated activity of mediators controlling vasomotor tone. One of the most important of these mediators is endothelin-1 (ET-1). Here, we have investigated the role of proteinase-activated receptor 2 (PAR2) in the vascular dysfunction in a model of fibrosis, using tight-skin (Tsk) mice. Aortas were collected from Tsk, transgenic over-expressing PAR2 (TgPAR2), PAR2 deficient (PAR2-/- ) or the corresponding WT mice. Histological and immunohistochemistry analysis for α-smooth muscle actin, PAR2 and ET-1 receptors were performed on aorta sections. Vascular responses to phenylephrine, ET-1 and PAR2 activating peptide (PAR2-AP) were assessed on aortic rings. In aortas from Tsk mice, responses to phenylephrine were reduced, contractions to ET-1 were increased and vasorelaxation to PAR2-AP was enhanced. These alterations matched changes observed in whole vessel architecture such as vascular fibre re-organization, increased collagen deposition and enhanced α-smooth muscle actin expression. Expression of both ETA receptors and PAR2 was enhanced in Tsk mice. Antagonism of PAR2 potentiated vascular effects of ET-1, whereas antagonism of ETA receptors increased vasorelaxation induced by PAR2-AP. In TgPAR2 mice, responses to ET-1 and ET-1 plasma levels were reduced. Conversely, PAR2-/- mice showed enhanced ET-1 induced contraction in aortic rings and higher circulating ET-1 levels. Our data show that PAR2 counterbalanced enhanced contractions to ET-1 in aortas from Tsk mice. PAR2 could represent a possible target for novel drugs in the treatment of vascular complications in fibrosis. This article is part of a themed section on Targeting Inflammation to Reduce Cardiovascular Disease Risk. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v174.22/issuetoc and http://onlinelibrary.wiley.com/doi/10.1111/bcp.v82.4/issuetoc.

An IRF5 Decoy Peptide Reduces Myocardial Inflammation and Fibrosis and Improves Endothelial Cell Function in Tight-Skin Mice

Interferon regulatory factor 5 (IRF5) has been called a “master switch” for its ability to determine whether cells mount proinflammatory or anti-inflammatory responses. Accordingly, IRF5 should be an attractive target for therapeutic drug development. Here we report on the development of a novel decoy peptide inhibitor of IRF5 that decreases myocardial inflammation and improves vascular endothelial cell (EC) function in tight-skin (Tsk/+) mice. Biolayer interferometry studies showed the Kd of IRF5D for recombinant IRF5 to be 3.72 ± 0.74x10-6M. Increasing concentrations of IRF5D (0–100 μg/mL, 24h) had no significant effect on EC proliferation or apoptosis. Treatment of Tsk/+ mice with IRF5D (1mg/kg/d subcutaneously, 21d) reduced IRF5 and ICAM-1 expression and monocyte/macrophage and neutrophil counts in Tsk/+ hearts compared to expression in hearts from PBS-treated Tsk/+ mice (p<0.05). EC-dependent vasodilatation of facialis arteries isolated from PBS-treated Tsk/+ mice was reduced (~15%). IRF5D treatments (1mg/kg/d, 21d) improved vasodilatation in arteries isolated from Tsk/+ mice nearly 3-fold (~45%, p<0.05), representing nearly 83% of the vasodilatation in arteries isolated from C57Bl/6J mice (~55%). IRF5D (50μg/mL, 24h) reduced nuclear translocation of IRF5 in myocytes cultured on both Tsk/+ cardiac matrix and C57Bl/6J cardiac matrix (p<0.05). These data suggest that IRF5 plays a causal role in inflammation, fibrosis and impaired vascular EC function in Tsk/+ mice and that treatment with IRF5D effectively counters IRF5-dependent mechanisms of inflammation and fibrosis in the myocardium in these mice.

Treatment with abatacept prevents experimental dermal fibrosis and induces regression of established inflammation-driven fibrosis

ObjectiveActivated T cells are the main component of the inflammatory skin infiltrates that characterise systemic sclerosis (SSc). Our aim was to investigate the efficacy of abatacept, which tempers T-cell activation,...

B lymphocytes in systemic sclerosis: Abnormalities and therapeutic targets.

Systemic sclerosis (SSc) is a connective tissue disease characterized by excessive extracellular matrix deposition in the skin and visceral organs with an autoimmune background. Although the pathogenic relationship between systemic autoimmunity and the clinical manifestations remains unknown, SSc patients have immunological abnormalities including the production of disease-specific autoantibodies. Recent studies have demonstrated that B cells play a crucial role in systemic autoimmunity and disease expression via various functions in addition to autoantibody production. Recent studies show that B cells from SSc patients demonstrate an upregulated CD19 signaling pathway, which is a crucial regulator of B-cell activation, that induces SSc-specific autoantibody production in SSc. In addition, B cells from SSc patients exhibit an overexpression of CD19. Consistently, in CD19 transgenic mice, CD19 overexpression induces SSc-specific autoantibody production. SSc patients have also intrinsic B-cell abnormalities characterized by chronic hyperreactivity of memory B cells, possibly due to CD19 overexpression. Similarly, B cells from a tight-skin mouse, a genetic model of SSc, show augmented CD19 signaling and chronic hyperreactivity. Furthermore, in bleomycin-induced SSc model mice, endogenous ligands for Toll-like receptors, induced by bleomycin treatment, stimulate B cells to produce various fibrogenic cytokines and autoantibodies. Remarkably, CD19 loss results in inhibition of B-cell hyperreactivity and elimination of autoantibody production, which is associated with improvement of fibrosis. Taken together, altered B-cell function may result in tissue fibrosis, as well as autoimmunity, in SSc. Although further studies and greater understanding are needed, B cells are potential therapeutic target in SSc.

Abnormal B lymphocyte activation and function in systemic sclerosis.

Systemic sclerosis (SSc) is characterized by tissue fibrosis and autoimmunity. Although the pathogenic relationship between autoimmunity and clinical manifestations of SSc remains unknown, SSc patients display abnormal immune responses including the production of disease-specific autoantibodies. Previous studies have demonstrated that B cells play a critical role in systemic autoimmunity and disease expression through various functions such as induction of the activation of other immune cells in addition to autoantibody production. CD19 is a crucial regulator of B cell activation. Recent studies demonstrated that B cells from SSc patients showed an up-regulated CD19 signaling pathway that induced SSc-specific autoantibody production in SSc mouse models. CD19 transgenic mice lost tolerance for autoantigen and generated autoantibodies spontaneously. B cells from SSc patients exhibited an overexpression of CD19 that induced SSc-specific autoantibody production in transgenic mice. Moreover, SSc patients displayed intrinsic B cell abnormalities characterized by chronic hyper-reactivity of memory B cells, which was possibly due to CD19 overexpression. Similarly, B cells from a tight-skin mouse, a genetic model of SSc, showed augmented CD19 signaling. In bleomycin-induced SSc mouse models, endogenous ligands for toll-like receptor 4 induced by bleomycin stimulated B cells to produce various fibrogenic cytokines and autoantibodies. Remarkably, the loss of CD19 resulted in the inhibition of B cell hyper-reactivity and autoantibody production, which are associated with improvements in fibrosis and a parallel decrease in fibrogenic cytokine production by B cells. Taken together, the findings suggest that altered B cell function may result in tissue fibrosis as well as autoimmunity in SSc.

Amelioration of tissue fibrosis by toll-like receptor 4 knockout in murine models of systemic sclerosis.

Bleomycin-induced fibrosis and the tight skin (TSK/+) mouse are well-established experimental murine models of human systemic sclerosis (SSc). Growing evidence has demonstrated the pivotal role of Toll-like receptors (TLRs) in several autoimmune inflammatory diseases, including SSc. This study was undertaken to determine the role of TLR-4 in the fibrotic processes in these murine models. We generated a murine model of bleomycin-induced SSc using TLR-4(-/-) mice and TLR-4(-/-) ;TSK/+ mice. The mechanisms by which TLR-4 contributes to pathologic tissue fibrosis were investigated in these 2 models by histologic examination, hydroxyproline assay, enzyme-linked immunosorbent assay, real-time polymerase chain reaction, and flow cytometry. Dermal and lung fibrosis was attenuated in bleomycin-treated TLR-4(-/-) mice compared with their wild-type counterparts. Inflammatory cell infiltration, expression of various inflammatory cytokines, and pathologic angiogenesis induced by bleomycin treatment were suppressed with TLR-4 deletion. Furthermore, the increased expression of interleukin-6 (IL-6) in fibroblasts, endothelial cells, and immune cells in response to bleomycin in vivo and to lipopolysaccharide in vitro was notably abrogated in the absence of TLR-4. Moreover, TLR-4 deletion was associated with alleviated B cell activation and skew toward a Th2/Th17 response against bleomycin treatment. Importantly, in TSK/+ mice, another SSc murine model, TLR-4 abrogation attenuated hypodermal fibrosis. These results indicate the pivotal contribution of TLR-4 to the pathologic tissue fibrosis of SSc murine models. Our results indicate the critical role of TLR-4 signaling in the development of tissue fibrosis, suggesting that biomolecular TLR-4 targeting might be a potential therapeutic approach to SSc.

OP0139 Targeting IL-6 by Both Passive or Active Immunization Strategies Prevents Inflammation-Driven Skin Fibrosis

BackgroundInterleukin 6 (IL-6) is a pleiotropic cytokine involved in inflammatory and autoimmune processes. Preliminary data have suggested that IL-6 might contribute to systemic sclerosis (SSc).ObjectivesOur aim was to compare the...

Inhibiting interferon regulatory factor 5 attenuates endothelial mesenchymal transition (1152.5)

Introduction: Systemic Sclerosis (SSc) is an autoimmune connective tissue disease characterized by oxidative stress, fibrosis and inflammation of skin and internal organs. Tight skin (Tsk/+) mice are an established model of SSc that displays inflammatory and fibrotic features that are consistent with SSc. When endothelial cells (ECs) are cultured on extracellular matrix (ECM) isolated from the skin of Tsk/+ mice they undergo endothelial mesenchymal transition (EndoMT). 4F, an apoA‐I mimetic that inhibits atherosclerosis by many mechanisms, not only decreases myocardial fibrosis in Tsk/+ mice but also decreases interferon regulator factor 5 (IRF5), a transcription factor considered a molecular switch for autoimmunity. The goal of the present study was to determine if directly targeting IRF5 reduces inflammation and oxidative stress in ECs cultured on cardiac ECM of Tsk/+ mice. Methods: KEP20 was designed and developed to specifically inhibit IRF5.The oxidative state of cardiac ECM was determined using the advanced oxidized protein product (AOPP) assay and western blot analysis for 3‐Nitrotyrosine and advanced glycation end products (AGE). ECs were cultured on Tsk/+ cardiac ECM and effects on expression of TGF β, Twist, collagen‐I in ECs ± KEP20 were determined by western blot analysis. Results: Tsk/+ ECM increase the levels of AOPP (13%), 3‐Nitrotyrosine (21%), and AGE (86%) in EC cultures as compared to EC maintained on cardiac ECM C57BL/6J. KEP20 treatments (50µg/mL) of EC cultured on Tsk/+ ECM reduces expression of TGFβ (221%), Twist (35 ± 14.51%), Collagen‐I (137%) compared ECs on Tsk/+ ECM. KEP20 also reduces Twist by 73%, TGFβ by 34% and collagen‐I by 56% compared to the levels in ECs on Tsk/+ ECM. Conclusion: These data indicate inhibiting IRF5 prevents Tsk/+ cardiac matrix from inducing EndoMT as well as increasing EC inflammation and fibrosis and demonstrate that IRF5 is a novel therapeutic target for SSc.

THU0057 Inhibition of Heat Shock Protein 90 (Hsp90) Prevents Fibrosis by Targeting Canonical TGF-B Signaling

BackgroundHeat shock protein 90 (Hsp90) has a crucial role in folding and conformational stabilization of TGF-β receptors and also of Src kinases, which are intracellular mediators of the pro-fibrotic effects...

OP0228 Protective Effect of LPA1 and 3 Receptor Antagonism in Experimental Skin Fibrosis is Linked to LPA Activity in Dermal Fibroblasts of SSC Patients

BackgroundLysophosphatidic acid (LPA) is a phospholipid generated by phospholipase D-mediated cleavage of phosphatidylcholine, mainly in adipocytes, fibroblasts or activated platelets. Knock down studies of the LPA1 receptor in lung and...