RESUMEN
Interleukin-23 (IL-23) plays a pivotal role in rheumatoid arthritis (RA). IL-23 and microRNA-223 (miR-223) are both up-regulated and mediate osteoclastogenesis in mice with collagen-induced arthritis (CIA). The aim of this study was to examine the association between IL-23 and miR-223 in contributing to osteoclastogenesis and arthritis. Levels of IL-23p19 in joints of mice with CIA were determined. Lentiviral vectors expressing short hairpin RNA (shRNA) targeting IL-23p19 and lisofylline (LSF) were injected intraperitoneally into arthritic mice. Bone marrow-derived macrophages (BMMs) were treated with signal transducers and activators of transcription 4 (STAT4) specific shRNA and miR-223 sponge carried by lentiviral vectors in response to IL-23 stimulation. Treatment responses were determined by evaluating arthritis scores and histopathology in vivo, and detecting osteoclast differentiation and miR-223 levels in vitro. The binding of STAT4 to the promoter region of primary miR-223 (pri-miR-223) was determined in the Raw264.7 cell line. IL-23p19 expression was increased in the synovium of mice with CIA. Silencing IL-23p19 and inhibiting STAT4 activity ameliorates arthritis by reducing miR-223 expression. BMMs from mice in which STAT4 and miR-223 were silenced showed decreased osteoclast differentiation in response to IL-23 stimulation. IL-23 treatment increased the expression of miR-223 and enhanced the binding of STAT4 to the promoter of pri-miR-223. This study is the first to demonstrate that IL-23 promotes osteoclastogenesis by transcriptional regulation of miR-223 in murine macrophages and mice with CIA. Furthermore, our data indicate that LSF, a selective inhibitor of STAT4, should be an ideal therapeutic agent for treating RA through down-regulating miR-223-associated osteoclastogenesis.
Asunto(s)
Artritis Experimental , Artritis Reumatoide , Subunidad p19 de la Interleucina-23/metabolismo , MicroARNs , Animales , Artritis Experimental/tratamiento farmacológico , Artritis Reumatoide/metabolismo , Interleucina-23/genética , Interleucina-23/metabolismo , Ratones , MicroARNs/metabolismo , Osteoclastos/metabolismo , Osteogénesis , ARN Interferente Pequeño/metabolismoRESUMEN
BACKGROUND: Systemic maternal inflammation and neonatal hyperoxia arrest alveolarization in neonates. The aims were to test whether human mesenchymal stem cells (MSCs) reduce lung inflammation and improve lung development in perinatal inflammation- and hyperoxia-induced experimental bronchopulmonary dysplasia. METHODS: Pregnant Sprague-Dawley rats were intraperitoneally injected with lipopolysaccharide (LPS, 0.5 mg/kg/day) on Gestational Days 20 and 21. Human MSCs (3×10(5) and 1×10(6) cells) in 0.03 ml normal saline (NS) were administered intratracheally on Postnatal Day 5. Pups were reared in room air (RA) or an oxygen-enriched atmosphere (O2) from Postnatal Days 1 to 14, and six study groups were obtained: LPS+RA+NS, LPS+RA+MSC (3×10(5) cells), LPS+RA+MSC (1×10(6) cells), LPS+O2+NS, LPS+O2+MSC (3×10(5) cells), and LPS+O2+MSC (1×10(6) cells). The lungs were excised for cytokine, vascular endothelial growth factor (VEGF) and connective tissue growth factor (CTGF) expression, and histological analyses on Postnatal Day 14. RESULTS: Body weight was significantly lower in rats reared in hyperoxia than in those reared in RA. The LPS+O2+NS group exhibited a significantly higher mean linear intercept (MLI) and collagen density and a significantly lower vascular density than the LPS+RA+NS group did. Administering MSC to hyperoxia-exposed rats improved MLI and vascular density and reduced tumor necrosis factor-α and interleukin-6 levels and collagen density to normoxic levels. This improvement in lung development and fibrosis was accompanied by an increase and decrease in lung VEGF and CTGF expression, respectively. CONCLUSION: Human MSCs attenuated perinatal inflammation- and hyperoxia-induced defective alveolarization and angiogenesis and reduced lung fibrosis, likely through increased VEGF and decreased CTGF expression.
RESUMEN
OBJECTIVE: The heterogeneous nature of mesenchymal stem cells (MSCs) and the absence of known MSC-specific biomarkers make it challenging to define MSC phenotypes and characteristics. In this study, we compared the phenotypic and functional features of human placenta-derived MSCs with those of human dermal fibroblasts in vitro in order to identify a biomarker that can be used to increase the purity of MSCs in a primary culture of placenta-derived cells. MATERIALS AND METHODS: Liquid chromatography-tandem mass spectrometry analysis was used to analyze and compare the proteome of human placenta-derived MSCs with that of fibroblasts. Quantitative real-time polymerase chain reaction, immunofluorescence, and flow cytometry were used to determine expression levels of EphA2 in placenta-derived MSCs. EphA2-positive cells were enriched by magnetic-activated cell sorting or with a cell sorter. An shRNA-mediated EphA2 knockdown was used to assess the role of EphA2 in MSC response to Tumor necrosis factor (TNF)-α stimulation. RESULTS: Analysis of proteomics data from MSCs and fibroblasts resulted in the identification of the EphA2 surface protein biomarker, which could reliably distinguish MSCs from fibroblasts. EphA2 was significantly upregulated in placenta-derived MSCs when compared to fibroblasts. EphA2 played an important role in MSC migration in response to inflammatory stimuli, such as TNF-α. EphA2-enriched MSCs were also more responsive to inflammatory stimuli in vitro when compared to unsorted MSCs, indicating a role for EphA2 in the immunomodulatory functionality of MSCs. CONCLUSION: EphA2 can be used to distinguish and isolate MSCs from a primary culture of placenta-derived cells. EphA2-sorted MSCs exhibited superior responsiveness to TNF-α signaling in an inflammatory environment compared with unsorted MSCs or MSC-like cells.
Asunto(s)
Células Madre Mesenquimatosas/metabolismo , Placenta/citología , Receptor EphA2/metabolismo , Cordón Umbilical/citología , Adulto , Biomarcadores/metabolismo , Movimiento Celular , Células Cultivadas , Cromatografía Liquida , Femenino , Fibroblastos/metabolismo , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Humanos , Persona de Mediana Edad , Embarazo , Reacción en Cadena en Tiempo Real de la Polimerasa , Espectrometría de Masas en Tándem , Regulación hacia Arriba , Adulto JovenRESUMEN
OBJECTIVE: The transcription factor Snail is involved in various biologic functions. We hypothesized that this molecule regulates tumor necrosis factor α (TNFα)-mediated synovial fibroblast activation in the rheumatoid joint. The aim of this study was to examine the role of Snail in the expression of cadherin-11 (Cad-11) and myofibroblast markers, interleukin-6 (IL-6) production, and the invasive ability of cells. METHODS: Synovium samples were obtained from patients with rheumatoid arthritis (RA) and from rats with collagen-induced arthritis (CIA). Synovial fibroblasts were treated with TNFα or a Wnt signaling inducer, and the joints of rats with CIA were injected with a TNFα antagonist. Modulation of Snail expression in the synovial fibroblasts and joints was performed by lentiviral vector-mediated transfer of complementary DNA or short hairpin RNA. RESULTS: The expression of Snail and Cad-11 was higher in synovium and synovial fibroblasts from patients with RA compared with patients with osteoarthritis and was increased in rats with CIA. TNFα stimulation or activation of Wnt signaling up-regulated the expression of Snail, Cad-11, and α-smooth muscle actin (α-SMA) in synovial fibroblasts, and anti-TNFα therapy down-regulated the expression of Snail, Cad-11, and α-SMA in the joints of rats with CIA. Although synovial fibroblast transfectants in which Snail was overexpressed showed increased expression of Cad-11 and α-SMA and enhanced TNFα-mediated invasive capacity and IL-6 production, synovial fibroblast transfectants from rats with CIA in which Snail was silenced showed decreased expression and had the opposite effect on these functions. Normal joints in which Snail was overexpressed had hyperplastic synovium, with increased expression of Cad-11, α-SMA, and IL-6. Silencing Snail expression ameliorated arthritis, with reduced Cad-11 expression and reduced levels of extracellular matrix deposition in the joints of rats with CIA, whereas overexpression of Snail exacerbated arthritis, with increased Cad-11 expression and increased levels of extracellular matrix deposition. CONCLUSION: Our results demonstrate that Snail regulates TNFα-mediated activation of synovial fibroblasts in the rheumatoid joint. These findings may contribute to the pharmacologic development of therapeutics targeting synovial fibroblasts in patients with RA.
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Artritis Experimental/fisiopatología , Artritis Reumatoide/fisiopatología , Fibroblastos/fisiología , Articulaciones/fisiopatología , Membrana Sinovial/fisiopatología , Factores de Transcripción/fisiología , Factor de Necrosis Tumoral alfa/fisiología , Actinas/metabolismo , Animales , Artritis Experimental/metabolismo , Artritis Experimental/patología , Artritis Reumatoide/metabolismo , Artritis Reumatoide/patología , Biomarcadores/metabolismo , Cadherinas/metabolismo , Modelos Animales de Enfermedad , Fibroblastos/efectos de los fármacos , Fibroblastos/patología , Humanos , Interleucina-6/metabolismo , Articulaciones/metabolismo , Articulaciones/patología , Masculino , Osteoartritis/metabolismo , Osteoartritis/patología , Osteoartritis/fisiopatología , Ratas , Ratas Sprague-Dawley , Factores de Transcripción de la Familia Snail , Membrana Sinovial/efectos de los fármacos , Membrana Sinovial/patología , Factor de Necrosis Tumoral alfa/farmacología , Vía de Señalización Wnt/fisiologíaRESUMEN
OBJECTIVE: MicroRNA (miRNA) plays a role in autoimmune diseases. MiRNA-223 (miR-223) is up-regulated in patients with rheumatoid arthritis (RA) and is involved in osteoclastogenesis, which contributes to erosive disease. The aim of this study was to test the feasibility of using lentiviral vectors expressing the miR-223 target sequence (miR-223T) to suppress miR-223 activity as a therapeutic strategy in a mouse model of collagen-induced arthritis (CIA). METHODS: Levels of miR-223 in the synovial tissue of patients with RA or osteoarthritis (OA), as well as in the ankle joints of mice with CIA, were determined by quantitative reverse transcriptase-polymerase chain reaction (RT-PCR). Lentiviral vectors expressing miR-223T (LVmiR-223T) or luciferase short hairpin RNA (LVshLuc) as a control vector were injected intraperitoneally into mice with CIA. Treatment responses and disease-related bone mineral density were monitored. Levels of nuclear factor 1A (NF-1A), a direct target of miR-223, and macrophage colony-stimulating factor receptor (M-CSFR), which is critical for osteoclastogenesis, were measured by immunohistochemistry and quantitative RT-PCR. Osteoclasts were assessed by tartrate-resistant acid phosphatase staining. RESULTS: MiR-223 expression was significantly higher in the synovium of RA patients and in the ankle joints of mice with CIA as compared to OA patients and normal mice. LVmiR-223T treatment reduced the arthritis score, histologic score, miR-223 expression, osteoclastogenesis, and bone erosion in mice with CIA. Down-regulation of miR-223 with concomitant increases in NF-1A levels and decreases in M-CSFR levels was detected in the synovium of LVmiR-223T-treated mice. CONCLUSION: This study is the first to demonstrate that lentivirus-mediated silencing of miR-223 can reduce disease severity of experimental arthritis. Furthermore, our results indicate that inhibition of miR-223 activity should be further explored as a therapeutic strategy in RA.
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Artritis Experimental/genética , MicroARNs/genética , Membrana Sinovial/metabolismo , Animales , Articulación del Tobillo/metabolismo , Articulación del Tobillo/patología , Artritis Experimental/metabolismo , Artritis Experimental/patología , Artritis Reumatoide/genética , Artritis Reumatoide/metabolismo , Artritis Reumatoide/patología , Densidad Ósea/genética , Modelos Animales de Enfermedad , Silenciador del Gen , Humanos , Lentivirus , Ratones , MicroARNs/metabolismo , Osteoartritis/genética , Osteoartritis/metabolismo , Osteoartritis/patología , Osteoclastos/metabolismo , Osteoclastos/patología , Membrana Sinovial/patologíaRESUMEN
Cellular behaviour is controlled by numerous processes, including intracellular signalling pathways that are triggered by the binding of ligands with cell surface receptors. Multivalent ligands have multiple copies of a recognition element that binds to receptors and influences downstream signals. Nanoparticle-ligand complexes may form multivalent structures to crosslink receptors with high avidity and specificity. After conjugation onto gold nanoparticles, galectin-1 (Au-Gal1) bound with higher affinity to Jurkat cells to promote CD45 clustering and inhibition of its phosphatase activity, resulting in enhancement of apoptosis via caspase-dependent pathways. Au-Gal1 injected intra-articularly into rats with collagen-induced arthritis (CIA) promoted apoptosis of CD4+ T cells and reduced pro-inflammatory cytokine levels in the ankle joints as well as ameliorated clinical symptoms of arthritis. These observed therapeutic effects indicate that the multivalent structure of nanoparticle-ligands can regulate the distribution of cell surface receptors and subsequent intracellular signalling, and this may provide new insights into nanoparticle applications.
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Apoptosis/efectos de los fármacos , Artritis Experimental/tratamiento farmacológico , Galectina 1/administración & dosificación , Oro/administración & dosificación , Nanopartículas del Metal/administración & dosificación , Receptores de Superficie Celular/efectos de los fármacos , Animales , Articulación del Tobillo/diagnóstico por imagen , Articulación del Tobillo/patología , Artritis Experimental/inducido químicamente , Artritis Experimental/diagnóstico por imagen , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/metabolismo , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Citocinas/efectos de los fármacos , Citocinas/metabolismo , Galectina 1/química , Oro/química , Humanos , Inyecciones Intraarticulares , Células Jurkat/efectos de los fármacos , Células Jurkat/metabolismo , Masculino , Nanopartículas del Metal/química , Radiografía , Ratas , Ratas Sprague-Dawley , Receptores de Superficie Celular/metabolismoRESUMEN
Cellular mechanisms involving the enhancement of interferon (IFN) signaling by ribavirin remain poorly understood. Here, we identified a novel role of ribavirin in the communication between p53 and the mammalian target of rapamycin (mTOR) signaling. Ribavirin activates p53 by stimulating mTOR and promoting the interaction between mTOR and p53. Activated p53 stimulates the transcription of IFN regulatory factor 9 and subsequently enhances IFN signaling. Furthermore, ribavirin-induced activation of mTOR and p53 enhances IFN-dependent signaling for the IFN-alpha/ribavirin combined treatment. We conclude that ribavirin enhances activities of mTOR and p53, which may account for its antiviral and antitumor effects.