RESUMEN
Nonobese diabetic (NOD) mice spontaneously develop lacrimal and salivary gland autoimmunity similar to human Sjögren syndrome. In both humans and NOD mice, the early immune response that drives T-cell infiltration into lacrimal and salivary glands is poorly understood. In NOD mice, lacrimal gland autoimmunity spontaneously occurs only in males with testosterone playing a role in promoting lacrimal gland inflammation, while female lacrimal glands are protected by regulatory T cells (Tregs). The mechanisms of this male-specific lacrimal gland autoimmunity are not known. Here, we studied the effects of Treg depletion in hormone-manipulated NOD mice and lacrimal gland gene expression to determine early signals required for lacrimal gland inflammation. While Treg-depletion was not sufficient to drive dacryoadenitis in castrated male NOD mice, chemokines (Cxcl9, Ccl19) and other potentially disease-relevant genes (Epsti1, Ubd) were upregulated in male lacrimal glands. Expression of Cxcl9 and Ccl19, in particular, remained significantly upregulated in the lacrimal glands of lymphocyte-deficient NOD-severe combined immunodeficiency (SCID) mice and their expression was modulated by type I interferon signaling. Notably, Ifnar1-deficient NOD mice did not develop dacryoadenitis. Together these data identify disease-relevant genes upregulated in the context of male-specific dacryoadenitis and demonstrate a requisite role for type I interferon signaling in lacrimal gland autoimmunity in NOD mice.
Asunto(s)
Dacriocistitis/metabolismo , Interferón Tipo I/metabolismo , Síndrome de Sjögren/metabolismo , Animales , Células Cultivadas , Quimiocina CCL19/metabolismo , Quimiocina CXCL9/metabolismo , Femenino , Aparato Lagrimal/metabolismo , Aparato Lagrimal/patología , Masculino , Ratones , Ratones Endogámicos NOD , Ratones SCID , Transducción de Señal , Linfocitos T Reguladores/metabolismoRESUMEN
Follistatin-like protein 1 (FSTL-1) possesses several newly identified roles in mammalian biology, including interleukin (IL)-17-driven inflammation, though the mechanism underlying FSTL-1 influence on IL-17-mediated cytokine production is unknown. Using parallel in vitro bone marrow stromal cell models of FSTL-1 suppression, we employed unbiased microarray analysis to identify FSTL-1-regulated genes and pathways that could influence IL-17-dependent production of IL-6 and granulocyte colony-stimulating factor. We discovered that FSTL-1 modulates Il17rc gene expression. Specifically, FSTL-1 was necessary for Il17rc gene transcription, IL-17RC surface protein expression and IL-17-dependent cytokine production. This work identifies a mechanism by which FSTL-1 influences IL-17-driven inflammatory signaling in vitro and reveals a novel function for FSTL-1, as a modulator of gene expression. Thus enhanced understanding of the interplay between FSTL-1 and IL-17-mediated inflammation may provide insight into potential therapeutic targets of IL-17-mediated diseases and warrants ongoing study of in vivo models and clinical scenarios of FSTL-1-influenced diseases.
Asunto(s)
Proteínas Relacionadas con la Folistatina/genética , Interleucina-17/metabolismo , Células Madre Mesenquimatosas/fisiología , ARN Mensajero/genética , Receptores de Interleucina/metabolismo , Animales , Células Cultivadas , Técnicas de Cultivo de Embriones , Proteínas Relacionadas con la Folistatina/metabolismo , Regulación de la Expresión Génica , Técnicas de Inactivación de Genes , Inflamación/genética , Ratones , Ratones Endogámicos C57BL , Análisis de Secuencia por Matrices de Oligonucleótidos , Estabilidad del ARN , ARN Interferente Pequeño/genética , Receptores de Interleucina/genética , Transducción de SeñalRESUMEN
Follistatin-like protein 1 (FSTL-1) is overexpressed in a number of inflammatory conditions characterized by elevated IL-1ß. Here, we found that FSTL-1 serum concentration was increased threefold in patients with bacterial sepsis and fourfold following administration of LPS to mice. To test the contribution of FSTL-1 to IL-1ß secretion, WT and FSTL-1-deficient mice were injected with LPS. While LPS induced IL-1ß in the sera of WT mice, it was low or undetectable in FSTL-1-deficient mice. Monocytes/macrophages, a key source of IL-1ß, do not normally express FSTL-1. However, FSTL-1 was found in tissue macrophages after injection of LPS into mouse footpads, demonstrating that macrophages are capable of taking up FSTL-1 at sites of inflammation. In vitro, intracellular FSTL-1 localized to the mitochondria. FSTL-1 activated the mitochondrial electron transport chain, increased the production of ATP (a key activator of the nod-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome) and IL-1ß secretion. FSTL-1 also enhanced transcription of the NLRP3 and procaspase 1 genes, two components of the NLRP3 inflammasome. Adenovirus-mediated overexpression of FSTL-1 in mouse paws led to activation of the inflammasome complex and local secretion of IL-1ß and IL-1ß-related proinflammatory cytokines. These results suggest that FSTL-1 may act on the NLRP3 inflammasome to promote IL-1ß secretion from monocytes/macrophages.
Asunto(s)
Proteínas Portadoras/inmunología , Proteínas Relacionadas con la Folistatina/inmunología , Inflamasomas/inmunología , Interleucina-1beta/inmunología , Macrófagos/inmunología , Monocitos/inmunología , Animales , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Caspasa 1/genética , Caspasa 1/inmunología , Caspasa 1/metabolismo , Femenino , Proteínas Relacionadas con la Folistatina/genética , Proteínas Relacionadas con la Folistatina/metabolismo , Humanos , Inflamasomas/genética , Inflamasomas/metabolismo , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Macrófagos/citología , Macrófagos/metabolismo , Masculino , Ratones , Ratones Noqueados , Persona de Mediana Edad , Monocitos/citología , Monocitos/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR , Células U937RESUMEN
OBJECTIVES: Chondrocytes, the only cells in the articular cartilage, play a pivotal role in osteoarthritis (OA) because they are responsible for maintenance of the extracellular matrix (ECM). Follistatin-like protein 1 (FSTL1) is a secreted protein found in mesenchymal stem cells (MSCs) and cartilage but whose function is unclear. FSTL1 has been shown to modify cell growth and survival. In this work, we sought to determine whether FSTL1 could regulate chondrogenesis and chondrogenic differentiation of MSCs. METHODS: To study the role of FSTL1 in chondrogenesis, we used FSTL1 knockout (KO) mice generated in our laboratory. Proliferative capacity of MSCs, obtained from skulls of E18.5 embryos, was analysed by flow cytometry. Chondrogenic differentiation of MSCs was carried out in a pellet culture system. Gene expression differences were assessed by microarray analysis and real-time PCR. Phosphorylation of Smad3, p38 MAPK and Akt was analysed by western blotting. RESULTS: The homozygous FSTL1 KO embryos showed extensive skeletal defects and decreased cellularity in the vertebral cartilage. Cell proliferation of FSTL1-deficient MSCs was reduced. Gene expression analysis in FSTL1 KO MSCs revealed dysregulation of multiple genes important for chondrogenesis. Production of ECM proteoglycans and collagen II expression were decreased in FSTL1-deficient MSCs differentiated into chondrocytes. Transforming growth factor ß signalling in FSTL1 KO cells was significantly suppressed. CONCLUSIONS: FSTL1 is a potent regulator of chondrocyte proliferation, differentiation and expression of ECM molecules. Our findings may lead to the development of novel strategies for cartilage repair and provide new disease-modifying treatments for OA.
Asunto(s)
Diferenciación Celular/fisiología , Proliferación Celular/fisiología , Condrocitos/citología , Condrogénesis/fisiología , Proteínas Relacionadas con la Folistatina/fisiología , Células Madre Mesenquimatosas/citología , Animales , Células Cultivadas , Condrocitos/efectos de los fármacos , Condrocitos/fisiología , Colágeno Tipo II/metabolismo , Matriz Extracelular/metabolismo , Proteínas Relacionadas con la Folistatina/deficiencia , Proteínas Relacionadas con la Folistatina/genética , Células Madre Mesenquimatosas/efectos de los fármacos , Células Madre Mesenquimatosas/fisiología , Ratones , Ratones Noqueados , Modelos Animales , Proteoglicanos/metabolismo , Transducción de Señal/efectos de los fármacos , Factor de Crecimiento Transformador beta/farmacologíaRESUMEN
OBJECTIVE: FSTL1 is a secreted glycoprotein that exacerbates murine arthritis and is overexpressed in human arthritis. The aim of this study was to determine the mechanism by which FSTL1 promotes arthritis. METHODS: Collagen-induced arthritis was induced in mice hypomorphic for FSTL1, generated with a gene trap-targeted mutant embryonic stem cell line. Arthritis was assessed by measuring paw swelling and using a qualitative arthritis index. Bone marrow-derived mesenchymal stromal cells from hypomorphic mice, as well as mouse stromal ST2 cells transduced with short hairpin RNA to suppress FSTL1 expression, were stimulated with interleukin-1ß (IL-1ß), tumor necrosis factor α, and IL-17. The monocyte cell line U937, which does not express FSTL1, was transfected with a plasmid encoding FSTL1 and stimulated with phorbol myristate acetate and lipopolysaccharide. Cell supernatants were assayed for IL-6, IL-8, monocyte chemotactic protein 1 (MCP-1), and FSTL1 by enzyme-linked immunosorbent assay. RESULTS: FSTL1 hypomorphic mice had reduced levels of FSTL1 compared to littermate controls. Following induction of arthritis, a significant correlation was observed between serum FSTL1 levels and both paw swelling and the arthritis index. Similar correlations were observed between the amount of FSTL1 produced by mesenchymal stromal cells, stromal ST2 cells, and monocytes and the secretion of IL-6, IL-8, and MCP-1. CONCLUSION: These findings demonstrate that FSTL1 up-regulates proinflammatory mediators important in the pathology of arthritis, and that serum levels of FSTL1 correlate with severity of arthritis. The latter supports the possibility that FSTL1 might be a target for treatment of certain forms of arthritis.
Asunto(s)
Artritis Experimental/metabolismo , Quimiocinas/metabolismo , Citocinas/metabolismo , Proteínas Relacionadas con la Folistatina/metabolismo , Animales , Artritis Experimental/patología , Línea Celular , Articulaciones/metabolismo , Articulaciones/patología , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/patología , Ratones , Monocitos/metabolismo , Monocitos/patología , Índice de Severidad de la EnfermedadRESUMEN
OBJECTIVE: To examine both the source of follistatin-like protein 1 (FSTL-1) and the factors that induce its expression in arthritis, and to determine whether juvenile rheumatoid arthritis (JRA) is characterized by overexpression of FSTL-1. METHODS: FSTL-1 expression patterns were analyzed by immunohistochemical staining of joint tissue derived from mice with collagen-induced arthritis. Induction of FSTL-1 secretion was assessed in osteoblasts, adipocytes, and human fibroblast-like synoviocytes in response to transforming growth factor beta (TGFbeta), interleukin-1beta (IL-1beta), tumor necrosis factor alpha (TNFalpha), and IL-6. In addition, sera and synovial fluid from children with oligoarticular, polyarticular, or systemic-onset JRA were assayed for FSTL-1 using a custom enzyme-linked immunosorbent assay. FSTL-1 concentrations in these patients were assessed for correlations with the erythrocyte sedimentation rate (ESR) and platelet count. RESULTS: Immunohistochemical staining of murine joint sections demonstrated expression of FSTL-1 in all cell types of the mesenchymal lineage, including osteocytes, chondrocytes, adipocytes, and fibroblasts. FSTL-1 could be induced in osteoblasts, adipocytes, and human fibroblast-like synoviocytes by TGFbeta, IL-1beta, TNFalpha, and IL-6. The IL-1beta response was significantly greater than the TNFalpha response (P < 0.05). In human serum and synovial fluid, only those samples from children with the systemic-onset JRA subtype had elevated concentrations of FSTL-1. The synovial fluid concentrations of FSTL-1 were 2-3-fold higher than the serum concentrations. The elevation in serum FSTL-1 concentrations seen in children with systemic-onset JRA correlated closely with elevations in the ESR and platelet count. CONCLUSION: These findings demonstrate that the arthritic joint matrix is a major source of FSTL-1 and that IL-1beta is a central mediator of FSTL-1 secretion. Furthermore, FSTL-1 may represent a useful biomarker of disease activity in systemic-onset JRA.
Asunto(s)
Artritis Experimental/metabolismo , Artritis Juvenil/metabolismo , Proteínas Relacionadas con la Folistatina/metabolismo , Articulaciones/metabolismo , Osteoblastos/metabolismo , Líquido Sinovial/metabolismo , Adolescente , Animales , Biomarcadores/metabolismo , Células Cultivadas , Niño , Condrocitos/efectos de los fármacos , Condrocitos/metabolismo , Citocinas/metabolismo , Citocinas/farmacología , Femenino , Humanos , Inmunohistoquímica , Articulaciones/efectos de los fármacos , Masculino , Ratones , Ratones Endogámicos DBA , Osteoblastos/efectos de los fármacos , Líquido Sinovial/efectos de los fármacos , Membrana Sinovial/efectos de los fármacos , Membrana Sinovial/metabolismo , Adulto JovenRESUMEN
OBJECTIVE: We sought to determine whether follistatin-like protein 1 (FSTL1), a protein produced by articular chondrocytes, promotes healthy articular cartilage and prevents chondrocytes from undergoing terminal differentiation to hypertrophic cells. METHODS: In vitro experiments were performed with immortalized human articular chondrocytes. The cells were transduced with a lentivirus encoding human FSTL1 small hairpin RNA or with an adenovirus encoding FSTL1. A quantitative polymerase chain reaction was used for gene expression analysis. Protein expression was assessed by Western blotting. Co-immunoprecipitation was used to identify interacting partners of FSTL1. FSTL1 expression in human articular cartilage was analyzed using confocal microscopy. RESULTS: Downregulation of FSTL1 expression in transforming growth factor ß (TGFß)-stimulated chondrocyte pellet cultures led to chondrocyte terminal differentiation characterized by poor production of cartilage extracellular matrix and altered expression of genes and proteins involved in cartilage homeostasis, including MMP13, COL10A1, RUNX2, COL2A1, ACAN, Sox9, and phospho-Smad3. We also showed that FSTL1 interacts with TGFß receptor proteins, Alk1 and endoglin, suggesting a potential mechanism for its effects on chondrocytes. Transduction of chondrocytes with an FSTL1 transgene increased COL2A1 expression, whereas it did not affect MMP13 expression. FSTL1 protein expression was decreased in human osteoarthritic cartilage in situ. CONCLUSION: Our data suggest that FSTL1 plays an important role in maintaining healthy articular cartilage and the FSTL1 pathway may represent a therapeutic target for degenerative diseases of cartilage.
RESUMEN
Follistatin-like protein 1 (FSTL1) is a secreted glycoprotein produced mainly by cells of mesenchymal origin. FSTL1 has been shown to play an important role during embryogenesis; FSTL1-deficient mice die at birth from multiple developmental abnormalities. In the last decade, FSTL1 has been identified as a novel inflammatory protein, enhancing synthesis of proinflammatory cytokines and chemokines by immune cells in vitro and in vivo. FSTL1 mediates proinflammatory events in animal models of inflammatory diseases, particularly in collagen-induced arthritis in mice. FSTL1 is elevated in various inflammatory conditions and decreased during the course of treatment. FSTL1 may therefore be a valuable biomarker for such diseases. Moreover, a variety of experiments suggest that targeting of FSTL1 may be useful in the treatment of diseases in which inflammation plays a central role.