RESUMEN
The fibroblast-like synoviocytes (FLS) have a crucial role in the pathogenesis of Rheumatoid Arthritis (RA); however, its precise mechanisms remain partially unknown. The involvement of the fibroblast in activating adjuvant-induced arthritis (AA) has not been previously reported. The objective was to describe the participation of footpads' fibroblasts in the critical initial process that drives the AA onset. Wistar rats were injected with Complete Freund's Adjuvant (CFA) or saline solution in the hind paws' footpads and euthanized at 24 or 48 h for genetic and histological analyses. Microarrays revealed the differentially expressed genes between the groups. The CFA dysregulated RA-linked biological processes at both times. Genes of MAPK, Jak-STAT, HIF, PI3K-Akt, TLR, TNF, and NF-κB signaling pathways were altered 24 h before the arrival of immune cells (CD4, CD8, and CD68). Key markers TNF-α, IL-1ß, IL-6, NFκB, MEK-1, JAK3, Enolase, and VEGF were immunodetected in fibroblast in CFA-injected footpads at 24 h but not in the control group. Moreover, fibroblasts in the CFA inoculation site overexpressed cadherin-11, which is linked to the migration and invasion ability of RA-FLS. Our study shows that CFA induced a pathological phenotype in the fibroblast of the inoculation site at very early AA stages from 24 h, suggesting a prominent role in arthritis activation processes.
Asunto(s)
Artritis Experimental , Artritis Reumatoide , Sinoviocitos , Ratas , Animales , Sinoviocitos/metabolismo , Membrana Sinovial/patología , Adyuvante de Freund , Fosfatidilinositol 3-Quinasas/metabolismo , Ratas Wistar , Artritis Experimental/metabolismo , FN-kappa B/metabolismo , Fibroblastos/metabolismoRESUMEN
Lameness is a common condition in dairy cattle caused by infectious or noninfectious agents. Joint lesions are the second most common cause of lameness and can be diagnosed in association with the presentation of digit injuries. Fibroblast-like synoviocyte (FLS) are predominant cells of synovia and play a key role in the pathophysiology of joint diseases, thus increasing the expression of proinflammatory mediators. Tumor necrosis factor-alpha (TNF-α) is a potent proinflammatory cytokine involved in cyclooxygenase 2 (COX-2) and proinflammatory cytokine expression in FLS. Previously, TNF-α was demonstrated to increase hypoxia-inducible Factor 1 (HIF-1), a transcription factor that rewires cellular metabolism and increases the expression of interleukin (IL)-6 in bovine FLS (bFLS). Despite this, the proinflammatory effects of TNF-α in bFLS on metabolic reprogramming have been poorly studied. We hypothesized that TNF-α increases glycolysis and in this way controls the expression of IL-6, IL-8, and COX-2 in bFLS. Results first, gas chromatography/mass spectrometry (GC/MS)-based untargeted metabolomics revealed that bTNF-α altered the metabolism of bFLS, increasing glucose, isoleucine, leucine, methionine, valine, tyrosine, and lysine and decreasing malate, fumarate, α-ketoglutarate, stearate, palmitate, laurate, aspartate, and alanine. In addition, metabolic flux analysis using D-glucose-13C6 demonstrated an increase of pyruvate and a reduction in malate and citrate levels, suggesting a decreased flux toward the tricarboxylic acid cycle after bTNF-α stimulation. However, bTNF-α increased lactate dehydrogenase subunit A (LDHA), IL-6, IL-8, IL-1ß and COX-2 expression, which was dependent on glycolysis and the PI3K/Akt pathway. The use of FX11 and dichloroacetate (DCA), an inhibitor of LDHA and pyruvate dehydrogenase kinase (PDK) respectively, partially reduced the expression of IL-6. Our results suggest that bTNF-α induces metabolic reprogramming that favors glycolysis in bFLS and increases IL-6, IL-8, IL-1ß and COX-2/PGE2.
Asunto(s)
Artritis Reumatoide , Sinoviocitos , Bovinos , Animales , Sinoviocitos/metabolismo , Factor de Necrosis Tumoral alfa/metabolismo , Interleucina-6/metabolismo , Membrana Sinovial/patología , Dinoprostona/metabolismo , Interleucina-8/metabolismo , Malatos/metabolismo , Artritis Reumatoide/patología , Ciclooxigenasa 2/metabolismo , Cojera Animal , Fosfatidilinositol 3-Quinasas/metabolismo , Citocinas/metabolismo , Células Cultivadas , Fibroblastos/metabolismoRESUMEN
SUMMARY: Meniscus tear is an important injury affecting the quality of life. This work is aimed to investigate the activity of CD68 and ADAMTS-5 in cells in synovial fluid in male and female patients with meniscal tear. In this study ,18 male and 22 female patients with meniscal tears were included. Local pain sensation during patients' physical examination, swelling, performing daily activities and difficulty in running-walking complaints were determined. 5 cc synovial fluids were aspirated from the lateral suprapatellar pouch part of the knees with meniscal pain. After routine histological follow-up of the samples, they were embedded in paraffin and sectioned with microtome and 5 micrometer thickness. CD68 and ADAMTS-5 primary antibodies were used for immunohistochemical analysis. Sections were taken and evaluated with a stylish microscope. The distribution of blood cells after meniscus tear was higher in female patients than in male patients. CD68 distribution in female patients appeared higher than in male patients. CD68 expression was high in macrophage cell cytoplasm. ADAMTS-5 expression was higher in female patients in degenerative cells and apoptotic cells. ADAMTS-5 is an important metallo-protein involved in the development of apoptotic signal and extracellular matrix synthesis in patients with ADAMTS-5 meniscus tear, and it may be an important criterion for the treatment developed after injury. CD68 and ADAMTS-5 activity was thought to be one of the important signal pathways that can be identified in the treatment of meniscus tear.
RESUMEN: La rotura del menisco es una lesión importante que afecta la calidad de vida. El objetivo fue investigar la actividad de CD68 y ADAMTS-5 en células del líquido sinovial en pacientes masculinos y femeninos con desgarro meniscal. Se incluyeron 18 pacientes masculinos y 22 femeninos con desgarros meniscales. Se determinó la sensación de dolor local durante el examen físico de los pacientes, la hinchazón, la realización de actividades diarias y la dificultad al correr y caminar. Se aspiraron 5 cc de líquido sinoviale de la parte de la bolsa suprapatelar lateral de las rodillas de los pacientes con dolor meniscal. Después del seguimiento histológico de rutina, las muestras se incluyeron en parafina y se seccionaron con un micrótomo de grosor de 5 micrómetros. Para el análisis inmunohistoquímico se usaron los anticuerpos primarios CD68 y ADAMTS-5. La distribución de las células sanguíneas después del desgarro del menisco fue mayor en pacientes femeninos que en pacientes masculinos. La distribución de CD68 en pacientes femeninos fue más alta que en pacientes masculinos. Además la expresión de CD68 fue alta en el citoplasma de los macrófagos. La expresión de ADAMTS-5 fue mayor en pacientes femeninos en las células degenerativas y células apoptóticas. ADAMTS-5 es una metaloproteína importante en el desarrollo de la señal apoptótica y la síntesis de matriz extracelular en pacientes con rotura de menisco ADAMTS-5, y puede ser un criterio importante para el tratamiento después de la lesión. La actividad de CD68 y ADAMTS-5 era una de las vías de señal importantes que se pueden identificar en el tratamiento de la rotura del menisco.
Asunto(s)
Humanos , Masculino , Femenino , Lesiones de Menisco Tibial/metabolismo , Lesiones de Menisco Tibial/patología , Articulación de la Rodilla/metabolismo , Articulación de la Rodilla/patología , Líquido Sinovial/química , Inmunohistoquímica , Antígenos CD/análisis , Sinoviocitos/metabolismo , Proteína ADAMTS5/análisis , Articulación de la Rodilla/citologíaRESUMEN
Mice are widely used to assess the pathogenesis of diseases. An experimental model of gout consists of the injection of uric acid crystals into joints of mice, which reproduce inflammation and functional changes of the human disease. Uric acid crystals activate synoviocytes culminating in the release of IL-1ß and neutrophil recruitment, key inflammatory elements in gouty arthritis. Since MIF plays an important role in orchestrating gout inflammation, we detail valuable procedures to investigate uric acid crystal-induced joint inflammation in mice and give options for further understanding the functions of MIF in gouty arthritis in vivo and in vitro.
Asunto(s)
Susceptibilidad a Enfermedades , Gota/etiología , Gota/metabolismo , Factores Inhibidores de la Migración de Macrófagos/genética , Animales , Células Cultivadas , Modelos Animales de Enfermedad , Fibroblastos/metabolismo , Fibroblastos/patología , Gota/patología , Factores Inhibidores de la Migración de Macrófagos/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Macrófagos/patología , Ratones , Membrana Sinovial/metabolismo , Membrana Sinovial/patología , Sinoviocitos/metabolismoRESUMEN
Osteoarthritis (OA) is one of the main locomotor disorders in horses. Although nonsteroidal anti-inflammatory drugs are the first-line treatment for OA, opioids could also be used. In previous studies, opioids showed promising anti-inflammatory and analgesic effects. In this study, we aimed to investigate the effects of two opioids (morphine and methadone) against inflammation in lipopolysaccharide (LPS)-stimulated synoviocytes by analyzing microsomal prostaglandin E synthase-1 (mPGES-1) and prostaglandin-endoperoxide synthase 2 (PTGS2) expression. Synoviocytes were obtained from the joints at the distal limbs of dead animals. The cytotoxic effects of LPS, morphine, and methadone were investigated by using a cell viability assay with crystal violet dye. Synoviocytes were treated with LPS, LPS plus morphine, or LPS plus methadone for 3, 6, and 12â¯h, and mPGES-1 and PTGS2 expression was measured using real-time polymerase chain reaction. LPS, and morphine did not affect the viability of synoviocytes, even at high concentrations. LPS treatment increased mPGES-1 and PTGS2 expression, whereas morphine inhibited the increase in mPGES-1 and PTGS2 expression in LPS-stimulated synoviocytes. Methadone did not inhibit mPGES-1 or PTGS2 expression. These results suggest that morphine may exhibit anti-inflammatory effect; therefore, it might be beneficial for the treatment of OA.
Asunto(s)
Ciclooxigenasa 2/química , Inflamación/tratamiento farmacológico , Lipopolisacáridos/toxicidad , Metadona/farmacología , Microsomas/enzimología , Morfina/farmacología , Prostaglandina-E Sintasas/antagonistas & inhibidores , Sinoviocitos/efectos de los fármacos , Analgésicos Opioides/farmacología , Animales , Células Cultivadas , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Caballos , Inflamación/inducido químicamente , Inflamación/enzimología , Masculino , Sinoviocitos/inmunología , Sinoviocitos/metabolismo , Sinoviocitos/patologíaRESUMEN
Robust correlation between the severity of rheumatoid arthritis (RA) and cigarette smoking has been clinically demonstrated. Nevertheless, cigarette compounds responsible for this toxic effect and their mechanisms have not been described. Considering that hydroquinone (HQ) is an abundant, pro-oxidative compound of the matter particle phase of cigarette smoke, we investigated whether HQ exposure during the initial phase of collagen-induced arthritis (CIA) could aggravate the disease. For this purpose, male Wistar rats were exposed to aerosolized HQ (25â¯ppm), saline or 5% ethanol solution (HQ vehicle) for 1â¯h per day during 14 days. CIA was induced through s.c. injection of bovine collagen Type II (0.4â¯mg/100⯵L) at days seven and 14 of exposure. Clinical signs of disease and the cell profile and chemical mediators in the synovial fluid and membrane were analysed at day 35 after the beginning of exposure. HQ exposure aggravated CIA-related paw edema and increased the cell infiltrate and interleukin-6 (IL-6) levels in the synovial fluid, promoted intense tissue collagen deposition and enhanced synoviocyte proliferation and higher frequency of aryl hydrocarbon receptor (AhR+) and interleukin (IL-17+) neutrophils in the synovial membrane. in vitro data also highlighted that neutrophils expressed increased levels of AhR, IL-17 and reactive oxygen species (ROS) generation. However, only AhR expression and ROS generation were blocked by in vitro treatment with AhR antagonist. Therefore, we conclude that in vivo HQ exposure at the early phase of AR onset worsens RA, leading to high frequency of AhR/IL-17+ neutrophils into the joint.
Asunto(s)
Artritis Experimental/inducido químicamente , Colágeno Tipo II , Hidroquinonas/toxicidad , Membrana Sinovial/efectos de los fármacos , Sinoviocitos/efectos de los fármacos , Aerosoles , Animales , Artritis Experimental/metabolismo , Artritis Experimental/patología , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Hidroquinonas/administración & dosificación , Mediadores de Inflamación/metabolismo , Exposición por Inhalación , Interleucina-17/metabolismo , Masculino , Infiltración Neutrófila/efectos de los fármacos , Neutrófilos/efectos de los fármacos , Neutrófilos/metabolismo , Neutrófilos/patología , Ratas Wistar , Especies Reactivas de Oxígeno/metabolismo , Receptores de Hidrocarburo de Aril/metabolismo , Líquido Sinovial/metabolismo , Membrana Sinovial/metabolismo , Membrana Sinovial/patología , Sinoviocitos/metabolismo , Sinoviocitos/patología , Factores de TiempoRESUMEN
Rheumatoid arthritis (RA) is an autoimmune disease that leads to joint destruction. The fibroblast-like synoviocytes (FLS) has a central role on the disease pathophysiology. The present study aimed to examine the role of gastrin-releasing peptide (GRP) and its receptor (GRPR) on invasive behavior of mice fibroblast-like synoviocytes (FLS), as well as to evaluate GRP-induced signaling on PI3K/AKT pathway. The expression of GRPR in FLS was investigated by immunocytochemistry, western blot (WB) and qRT-PCR. The proliferation and invasion were assessed by SRB and matrigel-transwell assay after treatment with GRP and/or RC-3095 (GRPR antagonist), and/or Ly294002 (inhibitor of PI3K/AKT pathway). Finally, AKT phosphorylation was assessed by WB. GRPR protein was detected in FLS and the exposure to GRP increased FLS invasion by nearly two-fold, compared with untreated cells (p<0.05), while RC-3095 reversed that effect (p<0.001). GRP also increased phosphorylated AKT expression in FLS. When Ly294002 was added with GRP, it prevented the GRP-induced increased cell invasiveness (p<0.001). These data suggest that GRPR expression in FLS and that exogenous GRP are able to activate FLS invasion. This effect occurs at least in part through the AKT activation. Therefore, understanding of the GRP/GRPR pathway could be relevant in the development of FLS-targeted therapy for RA.
Asunto(s)
Artritis Reumatoide/tratamiento farmacológico , Péptido Liberador de Gastrina/administración & dosificación , Receptores de Bombesina/genética , Sinoviocitos/metabolismo , Animales , Artritis Reumatoide/genética , Artritis Reumatoide/patología , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Cromonas/administración & dosificación , Fibroblastos/efectos de los fármacos , Péptido Liberador de Gastrina/genética , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Ratones , Morfolinas/administración & dosificación , Fosfatidilinositol 3-Quinasas/genética , Fosforilación/genética , Proteínas Proto-Oncogénicas c-akt/genética , Transducción de Señal/efectos de los fármacos , Sinoviocitos/efectos de los fármacos , Sinoviocitos/patologíaRESUMEN
BACKGROUND: Gout is the most common inflammatory arthropathy of metabolic origin and it is characterized by intense inflammation, the underlying mechanisms of which are unknown. The aim of this study was to evaluate the oxidative stress in human fibroblast-like synoviocytes (FLS) exposed to monosodium urate (MSU) crystals, which trigger an inflammatory process. METHODS: Human FLS isolated from synovial tissue explants were stimulated with MSU crystals (75 µg/mL) for 24 h. Cellular viability was evaluated by crystal violet staining, apoptosis was assessed using Annexin V, and the cellular content of reactive oxygen species (ROS) and nitrogen species (RNS) (O2 (-), H2O2, NO) was assessed with image-based cytometry and fluorometric methods. In order to determine protein oxidation levels, protein carbonyls were detected through oxyblot analysis, and cell ultrastructural changes were assessed by transmission electron microscopy. RESULTS: The viability of FLS exposed to MSU crystals decreased by 30 % (P < 0.05), while apoptosis increased by 42 % (P = 0.01). FLS stimulated with MSU crystals exhibited a 2.1-fold increase in H2O2 content and a 1.5-fold increase in O2 (-) and NO levels. Oxyblots revealed that the spots obtained from FLS protein lysates exposed to MSU crystals exhibited protein carbonyl immunoreactivity, which reflects the presence of oxidatively modified proteins. Concomitantly, MSU crystals triggered the induction of changes in the morphostructure of FLS, such as the thickening and discontinuity of the endoplasmic reticulum, and the formation of vacuoles and misfolded glycoproteins. CONCLUSIONS: Our results prove that MSU crystals induce the release of ROS and RNS in FLS, subsequently oxidizing proteins and altering the cellular oxidative state of the endoplasmic reticulum, which results in FLS apoptosis.