Halofuginone to treat fibrosis in chronic graft-versus-host disease and scleroderma.

Chronic graft-versus-host disease (cGvHD) and systemic sclerosis (scleroderma [SSc]) share clinical characteristics, including skin and internal organ fibrosis. Fibrosis, regardless of the cause, is characterized by extracellular matrix deposition, of which collagen type I is the major constituent. The progressive accumulation of connective tissue results in destruction of normal tissue architecture and internal organ failure. In both SSc and cGvHD, the severity of skin and internal organ fibrosis correlates with the clinical course of the disease. Thus, there is an unmet need for well-tolerated antifibrotic therapy. Halofuginone is an inhibitor of collagen type I synthesis in cells derived from various tissues and species and in animal models of fibrosis in which excess collagen is the hallmark of the disease. Halofuginone decreased collagen synthesis in the tight skin mouse (Tsk) and murine cGvHD, the 2 experimental systems that show many features resembling those of human GvHD. Inhibition of collagen synthesis by halofuginone is achieved by inhibiting transforming growth factor beta-dependent Smad3 phosphorylation. Dermal application of halofuginone caused a decrease in collagen content at the treated site of a cGvHD patient, and reduction in skin scores was observed in a pilot study with SSc patients. The results of the human studies provide basis for using halofuginone treatment for dermal fibrosis. As a first step toward future treatment of internal organ involvement, an oral administration study was performed in which halofuginone was well tolerated and plasma levels surpassed the predicted therapeutic exposure.     

Self reactive repertoire of tight skin mouse: immunochemical and molecular characterization of anti-topoisomerase I autoantibodies.

Tight skin (TSK) mice develop cutaneous hyperplasia accompanied by histopathological alterations of skin and collagen metabolism similar to those described in human scleroderma. Diffuse scleroderma, the most severe form of progressive systemic sclerosis, is associated with the production of autoantibodies specific for Scleroderma 70 antigen (topoisomerase I). Our studies show that there is an increase in the level of serum anti-topoisomerase I (topo I) autoantibodies in aged TSK mice. The monoclonal antibodies isolated from TSK mice bind to epitopes which interact with autoantibodies from scleroderma patients. A significant number of TSK monoclonal anti-topo I antibodies and serum immunoglobulin (Ig) from aged TSK mice bear a cross reactive idiotype (Id) recognized by a syngeneic monoclonal anti-Id antibody obtained from a 2 month-old TSK mouse. Analysis of V gene usage by monoclonal anti-topo I antibodies showed that the majority of these antibodies are encoded by VH genes derived from VHJ558 family pairing with VK genes from various families in a stochastic manner.     

High expression and autoinduction of monocyte chemoattractant protein-1 in scleroderma fibroblasts

Systemic sclerosis (SSc) is a connective tissue disease with unknown etiology characterized by excessive deposition of extracellular matrix in the skin as well as various internal organs. In the early stages of SSc, inflammatory infiltrates of mononuclear cells are found in the dermis, which is associated with increased collagen synthesis produced by activated fibroblasts. Monocyte chemoattractant protein-1 (MCP-1) is a predominant monocyte chemoattractant secreted by a variety of cell types, and recent in vivo and in vitro studies suggest the involvement of MCP-1 in tissue fibrosis. Here we demonstrate that cultured scleroderma fibroblasts, compared to fibroblasts from control skin, spontaneously express significantly elevated MCP-1 levels. Interestingly, exogenously administered MCP-1 stimulated autoinduction of MCP-1 mRNA. This effect was specific to scleroderma fibroblasts and abrogated by actinomycin D. These findings suggest that MCP-1 plays an important role in the induction of scleroderma by MCP-1 release from fibroblasts, which results in recruitment of monocytes to the skin. Moreover, increased responsiveness of scleroderma fibroblasts to MCP-1 could result in a continuation of the fibrotic response.     

Characterization of mutated protein encoded by partially duplicated fibrillin-1 gene in tight skin (TSK) mice.

Fibrillin-1 (Fbn-1) is a ubiquitous protein present in the extracellular matrix of various organs and it is a major component of microfibrils embedded in the core of elastic fibers. In humans, mutations or deletions of the Fbn-1 gene are associated with several genetic diseases. In addition, several microsatellite alleles near Fbn-1 gene were found associated with diffuse scleroderma. In TSK/+ mice, which develop a scleroderma-like syndrome, the Fbn-1 gene exhibits an inframe duplication of exons 17-40. In this study, we report that the synthesis and secretion of wild-type Fbn-1 in TSK/+ is higher than that of the mutated Fbn-1 protein excluding the possibility that TSK genetic defect is due to a loss of the wild allele. We also demonstrate for the first time that TGF-beta, which plays a crucial role in skin fibrosis, binds to both wild-type and mutated Fbn-1. The amount of bound TGF-beta was higher in mutated than wild-type Fbn-1 and appears related to the number of TGF-beta binding motifs.     

Halofuginone inhibits NF-kappaB and p38 MAPK in activated T cells

Halofuginone, a low molecular weight plant alkaloid, inhibits collagen alpha1 (I) gene expression in several animal models and in patients with fibrotic disease, including scleroderma and graft-versus-host disease. In addition, halofuginone has been shown to inhibit angiogenesis and tumor progression. It was demonstrated recently that halofuginone inhibits transforming growth factor-beta (TGF-beta), an important immunomodulator. The present study was undertaken to explore the effects of halofuginone on activated T cells. Peripheral blood T cells were activated by anti-CD3 monoclonal antibodies in the absence and presence of halofuginone and assessed for nuclear factor (NF)-kappaB activity, production of tumor necrosis factor alpha (TNF-alpha) and interferon-gamma (IFN-gamma), T cell apoptosis, chemotaxis, and phosphorylation of p38 mitogen-activated protein kinase (MAPK). A delayed-type hypersensitivity (DTH) model was applied to investigate the effect of halofuginone on T cells in vivo. Preincubation of activated peripheral blood T cells with 10-40 ng/ml halofuginone resulted in a significant dose-dependent decrease in NF-kappaB activity (80% inhibition following incubation with 40 ng halofuginone, P = 0.002). In addition, 40 ng/ml halofuginone inhibited secretion of TNF-alpha, IFN-gamma, interleukin (IL)-4, IL-13, and TGF-beta (P < 0.005). Similarly, halofuginone inhibited the phosphorylation of p38 MAPK and apoptosis in activated T cells (P = 0.0001 and 0.005, respectively). In contrast, T cell chemotaxis was not affected. Halofuginone inhibited DTH response in mice, indicating suppression of T cell-mediated inflammation in vivo. Halofuginone inhibits activated peripheral blood T cell functions and proinflammatory cytokine production through inhibition of NF-kappaB activation and p38 MAPK phosphorylation. It also inhibited DTH response in vivo, making it an attractive immunomodulator and anti-inflammatory agent.     

UVA1 irradiation inhibits fibroblast proliferation and alleviates pathological changes of scleroderma in a mouse model

The purpose of the present study was to compare the effects of different doses of ultraviolet radiation A1 (UVA1) on human fibroblast proliferation and collagen level in a mouse model of scleroderma,so as to identify appropriate irradiation doses for clinical treatment of scleroderma.Monolayer from human fibroblasts was cultured in vitro,and a mouse model of scleroderma was established by subcutaneous injection of 100 μL of 400 μg/mL bleomycin into the back of BALB/c mice for 4 weeks.The mouse models and human fibroblasts were divided into UVA1exposed (100,60 and 20 J/cm 2) and UVA-unexposed groups.At 0,24 and 48 h after exposure,cell proliferation and levels of hydroxyproline and collagen were detected.UVA1 irradiation was performed 3 times weekly for 10 weeks,and the pathological changes of skin tissues,skin thickness and collagen level were observed after phototherapy.Cell proliferation and the levels of hydroxyproline and collagen were inhibited after phototherapy,and there was a significant difference between the UVA1-exposed cells and UVA1-unexposed cells (P < 0.001).In addition,UVA1 phototherapy improved dermal sclerosis and softened the skin,and there were significant differences between the high-dose UVA1 group and the model group,and the negative group (P < 0.05).It is concluded that UVA1 radiation can reduce cell proliferation,and decrease hydroxyproline and collagen levels in a dose-dependent manner in vitro.High-dose UVA1 phototherapy has marked therapeutic effect on scleroderma in the mouse model.Decreased collagen level may be related to the reduced number and activity of cells,as well as inhibition of collagen synthesis.     

Pharmacological blockade of A2A receptors prevents dermal fibrosis in a model of elevated tissue adenosine

Adenosine is a potent modulator of inflammation and tissue repair. We have recently reported that activation of adenosine A(2A) receptors promotes collagen synthesis by human dermal fibroblasts and that blockade or deletion of this receptor in mice protects against bleomycin-induced dermal fibrosis, a murine model of scleroderma. Adenosine deaminase (ADA) is the principal catabolic enzyme for adenosine in vivo, and its deficiency leads to the spontaneous development of pulmonary fibrosis in mice. The aim of this study was to characterize further the contributions of endogenous adenosine and adenosine A(2A) receptors to skin fibrosis. Taking advantage of genetically modified ADA-deficient mice, we herein report a direct fibrogenic effect of adenosine on the skin, in which increased collagen deposition is accompanied by increased levels of key mediators of fibrosis, including transforming growth factor beta1, connective tissue growth factor, and interleukin-13. Pharmacological treatment of ADA-deficient mice with the A(2A) receptor antagonist ZM-241385 prevented the development of dermal fibrosis in this model of elevated tissue adenosine, by reducing dermal collagen content and expression of profibrotic cytokines and growth factors. These data confirm a fibrogenic role for adenosine in the skin and reveal A(2A) receptor antagonists as novel therapeutic agents for the modulation of dermal fibrotic disorders.     

B-cell Deficiency does not Abrogate Development of Cutaneous Hyperplasia in Mice Inheriting the Defective Fibrillin-1 Gene

Tight-skin (TSK) mouse, the experimental model for scleroderma, develops cutaneous hyperplasia, cardiac hypertrophy, pulmonary emphysema and autoimmunity against scleroderma target autoantigens. The cutaneous hyperplasia is associated with the accumulation of microfibrils and elastic fibers in the middle and deep dermis. Fibrillin-1 (Fbn-1) is a major component of the 10–12 nm microfibrils found in the extracellular matrix. In this study we report the identification of a genetic marker in the Fbn-1 gene that can distinguish the mutant phenotype. TSK mice exhibit an unique polymorphism in the Fbn-1 gene. RNA analysis, PCR analysis and sequence determination of the mutant gene showed that the Fbn-1 gene polymorphism is due to intragenic duplication of a segment of the gene coding for 3.0 Kb of mRNA sequence (10 Kb of the genome). Histological analysis of skin samples from F1 progeny obtained by crossing TSK mice with J_H−/−, RAG2−/− or vit/vit showed a significant correlation between the inheritance of the defective Fbn-1 gene and the development of cutaneous hyperplasia. Further, our results also show that in mice deficient in mature B cells inheriting the defective Fbn-1 gene, development of cutaneous hyperplasia is not abrogated. Thus, production of auto-antibodies or the presence of mature B lymphocytes do not play an integral role in the pathogenesis of cutaneous hyperplasia.     

Stimulatory autoantibodies to the PDGF receptor: a link to fibrosis in scleroderma and a pathway for novel therapeutic targets

Systemic sclerosis (scleroderma) is a complex disease characterized by excessive deposition of collagen and abnormalities of blood vessels. In addition, activation of the immune system is a central feature of scleroderma as shown by mononuclear cell infiltration of the skin, autoantibody production and release of inflammatory cytokines. The pathogenesis of the disease is poorly understood and the molecular events underlying the main clinical features are not known. The detection of agonistic autoantibodies targeting PDGF receptor in serum of patients with scleroderma may indicate a novel link between phenotypic features of the disease and a specific signalling pathway. Agonistic PDGF receptor antibodies induce in vitro the scleroderma phenotype in normal human fibroblasts and, thus, link autoimmunity to fibrosis. These findings pave the way to novel therapeutic strategies.     

B Lymphocyte signaling established by the CD19/CD22 loop regulates autoimmunity in the tight-skin mouse

Systemic sclerosis (SSc) is characterized by fibrosis and autoimmmunity. Peripheral blood B cells from SSc patients specifically overexpress CD19, a critical cell-surface signal transduction molecule in B cells. CD19 deficiency in B cells also attenuates skin fibrosis in the tight-skin (TSK/+) mouse, a genetic model for SSc. Herein we analyzed two transgenic mouse lines that overexpress CD19. Remarkably, 20% increase of CD19 expression in mice spontaneously induced SSc-specific anti-DNA topoisomerase I (topo I) antibody (Ab) production, which was further augmented by 200% overexpression. In TSK/+ mice overexpressing CD19, skin thickness did not increase, although anti-topo I Ab levels were significantly augmented, indicating that abnormal CD19 signaling influences autoimmunity in TSK/+ mice and also that anti-topo I Ab does not have a pathogenic role. The molecular mechanisms for abnormal CD19 signaling were further assessed. B-cell antigen receptor crosslinking induced exaggerated calcium responses and augmented activation of extracellular signal-regulated kinase in TSK/+ B cells. CD22 function was specifically impaired in TSK/+ B cells. Consistently, CD19, a major target of CD22-negative regulation, was hyperphosphorylated in TSK/+ B cells. These findings indicate that reduced inhibitory signal provided by CD22 results in abnormal activation of signaling pathways including CD19 in TSK/+ mice and also suggest that this disrupted B cell signaling contribute to specific autoantibody production.     

Remodeling of elastic fiber components in scleroderma skin

The skin of patients with scleroderma is characterized by an excess accumulation of collagen in the extracellular matrix of the fibrotic reticular dermis. Elastic fibers are also disrupted in this disease, however, in contrast to collagen, relatively few studies have provided information concerning the changes that occur to elastic fiber components in scleroderma. In the present study, the extracellular matrix in scleroderma skin was examined with a specific focus on the integrity of elastic fibers. Electron microscopic observations confirmed an excess of 10 nm microfibrils present in small bundles independent of amorphous elastin in the fibrotic reticular dermis. In the same area, a population of stellate-shaped fibroblasts was identified in close association with the dermal elastic fibers. In contrast to the uniform black appearance of the elastic fibers seen in the papillary dermis and in areas of the reticular dermis not infiltrated by these cells, the elastic fibers apposed to the cells were mottled in density and often almost electron-lucent. These observations suggest that the elastic fibers in the reticular dermis were being actively degraded. Results from this study provide evidence for disintegration of elastic fibers in the skin of scleroderma patients and suggest the possibility that degradation products from the elastic matrix in the diseased tissues may act as a feedback signal for increased matrix production.     

In vivo investigations on anti-fibrotic potential of proteasome inhibition in lung and skin fibrosis

In systemic sclerosis (SSc), a disease characterized by fibrosis of the skin and internal organs, the occurrence of interstitial lung disease is responsible for high morbidity and mortality. We previously demonstrated that proteasome inhibitors (PI) show anti-fibrotic properties in vitro by reducing collagen production and favoring collagen degradation in a c-jun N-terminal kinase (JNK)-dependent manner in human fibroblasts. Therefore, we tested whether PI could control fibrosis development in bleomycin-induced lung injury, which is preceded by massive inflammation. We extended the study to test PI in TSK-1/+ mice, where skin fibrosis develops in the absence of overt inflammation. C57Bl/6 mice received bleomycin intratracheally and were treated or not with PI. Lung inflammation and fibrosis were assessed by histology and quantification of hydroxyproline content, type I collagen mRNA, and TGF-beta at Days 7, 15, and 21, respectively. Histology was used to detect skin fibrosis in TSK-1/+mice. The chymotryptic activity of 20S proteasome was assessed in mice blood. JNK and Smad2 phosphorylation were evaluated by Western blot on lung protein extracts. PI reduced collagen mRNA levels in murine lung fibroblasts, without affecting their viability in vitro. In addition, PI inhibited the chymotryptic activity of proteasome and enhanced JNK and TGF-beta signaling in vivo. PI failed to prevent bleomycin-induced lung inflammation and fibrosis and to attenuate skin fibrosis in TSK-1/+mice. In conclusion, our results provide direct evidence that, despite promising in vitro results, proteasome blockade may not be a strategy easily applicable to control fibrosis development in diseases such as lung fibrosis and scleroderma.     

Cultured fibroblasts in avian scleroderma, an autoimmune fibrotic disease, display an activated phenotype.

University of California, Davis, line 200 and 206 chickens spontaneously develop an autoimmune syndrome that has many features analogous to human scleroderma, including dermal fibrosis, antinuclear antibodies and antibodies to type II collagen. These birds also have thymic subcapsular epithelial defects and an abnormality in T cell calcium influx and proliferation in response to both T cell receptor-dependent and -independent activators. To determine whether fibroblast activation is a contributing factor to development of skin fibrosis in line 200/206 chickens, as it is in human scleroderma, we studied the collagen, non-collagenous protein and glycosaminoglycan (GAG) production of 34 separate fibroblast lines derived from the normal and fibrotic skin of line 200 and 206 chickens and from the skin of control chicken lines 058 and 254. The mean +/- SEM 24-h incorporation of 3H-proline or 3H-glucosamine into extracellular collagen, non-collagenous protein or GAG by first passage fibroblast lines derived from the fibrotic skin of diseased birds was 1,526 +/- 136, 859 +/- 82 and 25.7 +/- 1.3 dpm/10(3) cells, respectively, while fibroblast lines derived from the skin of control birds produced only 341 +/- 36, 343 +/- 42 and 15.2 +/- 1.4 dpm/10(3) cells. Similar differences in results were recorded for cell-associated production, and when collagen and non-collagenous protein production were assessed using non-radioactive electrophoretic methods. The activated phenotype of the fibroblast lines derived from the fibrotic skin of diseased birds persisted through 10 cell doublings in tissue culture. However, the ratio of type I:III collagen and the profile of GAG types produced were similar in all fibroblast lines studied. These results suggest that fibroblast activation is responsible for the skin fibrosis observed in this avian model of scleroderma.