RESUMEN
OBJECTIVE: Class 3 semaphorins are reduced in the synovial tissue of RA patients and these proteins are involved in the pathogenesis of the disease. The aim of this study was to identify the transcription factors involved in the expression of class 3 semaphorins in the synovium of RA patients. METHODS: Protein and mRNA expression in synovial tissue from RA and individuals at risk (IAR) patients, human umbilical vein endothelial cells (HUVEC) and RA fibroblast-like synoviocytes (FLS) was determined by ELISA, immunoblotting and quantitative PCR. TCF-3, EBF-1 and HOXA5 expression was knocked down using siRNA. Cell viability, migration and invasion were determined using MTT, calcein, wound closure and invasion assays, respectively. RESULTS: mRNA expression of all class 3 semaphorins was significantly lower in the synovium of RA compared with IAR patients. In silico analysis suggested TCF-3, EBF-1 and HOXA5 as transcription factors involved in the expression of these semaphorins. TCF-3, EBF-1 and HOXA5 silencing significantly reduced the expression of several class 3 semaphorin members in FLS and HUVEC. Importantly, HOXA5 expression was significantly reduced in the synovium of RA compared with IAR patients and was negatively correlated with clinical disease parameters. Additionally, TNF-α down-regulated the HOXA5 expression in FLS and HUVEC. Finally, HOXA5 silencing enhanced the migratory and invasive capacities of FLS and the viability of HUVEC. CONCLUSION: HOXA5 expression is reduced during the progression of RA and could be a novel therapeutic strategy for modulating the hyperplasia of the synovium, through the regulation of class 3 semaphorins expression.
Asunto(s)
Artritis Reumatoide , Semaforinas , Sinoviocitos , Humanos , Semaforinas/genética , Células Cultivadas , Membrana Sinovial/metabolismo , Artritis Reumatoide/tratamiento farmacológico , Sinoviocitos/metabolismo , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Células Endoteliales de la Vena Umbilical Humana/patología , Factores de Transcripción/metabolismo , ARN Mensajero/metabolismo , Fibroblastos/metabolismo , Proliferación Celular , Proteínas de Homeodominio/metabolismo , Proteínas de Homeodominio/uso terapéuticoRESUMEN
OBJECTIVES: SSc is an autoimmune disease characterized by inflammation, vascular injury and excessive fibrosis in multiple organs. Secreted protein acidic and rich in cysteine (SPARC) is a matricellular glycoprotein that regulates processes involved in SSc pathology, such as inflammation and fibrosis. In vivo and in vitro studies have implicated SPARC in SSc, but it is unclear if the pro-fibrotic effects of SPARC on fibroblasts are a result of intracellular signalling or fibroblast interactions with extracellular SPARC hampering further development of SPARC as a potential therapeutic target. This study aimed to analyse the potential role of exogenous SPARC as a regulator of fibrosis in SSc. METHODS: Dermal fibroblasts from both healthy controls and SSc patients were stimulated with SPARC alone or in combination with TGF-ß1, in the absence or presence of a TGF receptor 1 inhibitor. mRNA and protein expression of extracellular matrix components and other fibrosis-related mediators were measured by quantitative PCR and western blot. RESULTS: Exogenous SPARC induced mRNA and protein expression of collagen I, collagen IV, fibronectin 1, TGF-ß and SPARC by dermal fibroblasts from SSc patients, but not from healthy controls. Importantly, exogenous SPARC induced the activation of the tyrosine kinase SMAD2 and pro-fibrotic gene expression induced by SPARC in SSc fibroblasts was abrogated by inhibition of TGF-ß signalling. CONCLUSION: These results indicate that exogenous SPARC is an important pro-fibrotic mediator contributing to the pathology driving SSc but in a TGF-ß dependent manner. Therefore, SPARC could be a promising therapeutic target for reducing fibrosis in SSc patients, even in late states of the disease.
Asunto(s)
Fibroblastos/metabolismo , Osteonectina/genética , Esclerodermia Sistémica/genética , Piel/patología , Factor de Crecimiento Transformador beta1/genética , Estudios de Casos y Controles , Células Cultivadas , Matriz Extracelular/genética , Proteínas de la Matriz Extracelular/genética , Fibrosis , Humanos , ARN Mensajero/genética , Transducción de Señal/genética , Piel/citología , Activación Transcripcional/genéticaRESUMEN
OBJECTIVE: To examine the role of Tie2 signalling in macrophage activation within the context of the inflammatory synovial microenvironment present in patients with RA and PsA. METHODS: Clinical responses and macrophage function were examined in wild-type and Tie2-overexpressing (Tie2-TG) mice in the K/BxN serum transfer model of arthritis. Macrophages derived from peripheral blood monocytes from healthy donors, RA and PsA patients, and RA and PsA synovial tissue explants were stimulated with TNF (10 ng/ml), angiopoietin (Ang)-1 or Ang-2 (200 ng/ml), or incubated with an anti-Ang2 neutralizing antibody. mRNA and protein expression of inflammatory mediators was analysed by quantitative PCR, ELISA and Luminex. RESULTS: Tie2-TG mice displayed more clinically severe arthritis than wild-type mice, accompanied by enhanced joint expression of IL6, IL12B, NOS2, CCL2 and CXCL10, and activation of bone marrow-derived macrophages in response to Ang-2 stimulation. Ang-1 and Ang-2 significantly enhanced TNF-induced expression of pro-inflammatory cytokines and chemokines in macrophages from healthy donors differentiated with RA and PsA SF and peripheral blood-derived macrophages from RA and PsA patients. Both Ang-1 and Ang-2 induced the production of IL-6, IL-12p40, IL-8 and CCL-3 in synovial tissue explants of RA and PsA patients, and Ang-2 neutralization suppressed the production of IL-6 and IL-8 in the synovial tissue of RA patients. CONCLUSION: Tie2 signalling enhances TNF-dependent activation of macrophages within the context of ongoing synovial inflammation in RA and PsA, and neutralization of Tie2 ligands might be a promising therapeutic target in the treatment of these diseases.
Asunto(s)
Artritis Experimental/metabolismo , Artritis Psoriásica/metabolismo , Artritis Reumatoide/metabolismo , Activación de Macrófagos/fisiología , Receptor TIE-2/metabolismo , Membrana Sinovial/metabolismo , Animales , Artritis Experimental/patología , Artritis Psoriásica/patología , Artritis Reumatoide/patología , Citocinas/metabolismo , Humanos , Inflamación/metabolismo , Inflamación/patología , Macrófagos/metabolismo , Ratones , Ratones Transgénicos , Transducción de Señal/fisiología , Líquido Sinovial/metabolismo , Membrana Sinovial/patologíaRESUMEN
Semaphorin (Sema)4A is a transmembrane glycoprotein that is elevated in several autoimmune diseases such as systemic sclerosis, rheumatoid arthritis and multiple sclerosis. Sema4A has a key role in the regulation of Thelper Th1 and Th2 differentiation and we recently demonstrated that CD4+ T cell activation induces the expression of Sema4A. However, the autocrine role of Sema4A on Th cell differentiation remains unknown. Naïve Th cells from healthy controls were cell sorted and differentiated into Th1, Th2 and Th17 in the presence or absence of a neutralizing antibody against the Sema4A receptor PlexinD1. Gene expression was determined by quantitative PCR and protein expression by ELISA and flow cytometry. We found that the expression of Sema4A is induced during Th1, Th2 and Th17 differentiation. PlexinD1 neutralization induced the differentiation of Th1 cells, while reduced the Th2 and Th17 skewing. These effects were associated with an upregulation of the transcription factor T-bet by Th1 cells, and to downregulation of GATA3 and RORγt in Th2 cells and Th17 cells, respectively. Finally, PlexinD1 neutralization regulates the systemic sclerosis patients serum-induced cytokine production by CD4+ T cells. Therefore, the autocrine Sema4A-PlexinD1 signaling acts as a negative regulator of Th1 skewing but is a key mediator on Th2 and Th17 differentiation, suggesting that dysregulation of this axis might be implicated in the pathogenesis of CD4+ T cell-mediated diseases.
Asunto(s)
Comunicación Autocrina/inmunología , Péptidos y Proteínas de Señalización Intracelular/inmunología , Glicoproteínas de Membrana/inmunología , Semaforinas/inmunología , Células TH1/inmunología , Células Th17/inmunología , Células Th2/inmunología , Diferenciación Celular/inmunología , Citocinas/inmunología , Regulación de la Expresión Génica/inmunología , Humanos , Esclerodermia Sistémica/inmunología , Esclerodermia Sistémica/patología , Células TH1/patología , Células Th17/patología , Células Th2/patologíaRESUMEN
Angiopoietin-2 (Ang-2), a ligand of the tyrosine kinase receptor Tie2, is essential for vascular development and blood vessel stability and is also involved in monocyte activation. Here, we examined the role of Ang-2 on monocyte activation in patients with systemic sclerosis (SSc). Ang-2 levels were measured in serum and skin of healthy controls (HCs) and SSc patients by ELISA and array profiling, respectively. mRNA expression of ANG2 was analyzed in monocytes, dermal fibroblasts, and human pulmonary arterial endothelial cells (HPAECs) by quantitative PCR. Monocytes were stimulated with Ang-2, or with serum from SSc patients in the presence of a Tie2 inhibitor or an anti-Ang2 neutralizing antibody. Interleukin (IL)-6 and IL-8 production was analyzed by ELISA. Ang-2 levels were elevated in the serum and skin of SSc patients compared to HCs. Importantly, serum Ang-2 levels correlated with clinical disease parameters, such as skin involvement. Lipopolysaccharide (LPS) LPS, R848, and interferon alpha2a (IFN-α) stimulation up-regulated the mRNA expression of ANG2 in monocytes, dermal fibroblasts, and HPAECs. Finally, Ang-2 induced the production of IL-6 and IL-8 in monocytes of SSc patients, while the inhibition of Tie2 or the neutralization of Ang-2 reduced the production of both cytokines in HC monocytes stimulated with the serum of SSc patients. Therefore, Ang-2 induces inflammatory activation of SSc monocytes and neutralization of Ang-2 might be a promising therapeutic target in the treatment of SSc.
Asunto(s)
Angiopoyetina 2/metabolismo , Biomarcadores , Mediadores de Inflamación/metabolismo , Monocitos/metabolismo , Esclerodermia Sistémica/etiología , Esclerodermia Sistémica/metabolismo , Adulto , Anciano , Angiopoyetina 2/sangre , Estudios de Casos y Controles , Citocinas/metabolismo , Femenino , Fibroblastos/metabolismo , Humanos , Masculino , Persona de Mediana Edad , Esclerodermia Sistémica/patología , Piel/metabolismoRESUMEN
Objective: Class 3 semaphorins regulate diverse cellular processes relevant to the pathology of RA, including immune modulation, angiogenesis, apoptosis and invasive cell migration. Therefore, we analysed the potential role of class 3 semaphorins in the pathology of RA. Methods: Protein and mRNA expression in RA synovial tissue, SF and fibroblast-like synoviocytes (FLS) were determined by immunoblotting and quantitative PCR (qPCR). RA FLS migration and invasion were determined using wound closure and transwell invasion assays, respectively. PlexinA1, neuropilin-1 and neuropilin-2 expression was knocked down using small interfering RNA (siRNA). Activation of FLS intracellular signalling pathways was assessed by immunoblotting. Results: mRNA expression of semaphorins (Sema)3B, Sema3C, Sema3F and Sema3G was significantly lower in the synovial tissue of early arthritis patients at baseline who developed persistent disease compared with patients with self-limiting disease after 2 years follow-up. Sema3B and Sema3F expression was significantly lower in arthritis patients fulfilling classification criteria for RA compared with those who did not. FLS expression of Sema3A was induced after stimulation with TNF, IL-1ß or lipopolysaccharides (LPS), while Sema3B and Sema3F expression was downregulated. Exogenously applied Sema3A induced the migration and invasive capacity of FLS, while stimulation with Sema3B or Sema3F reduced spontaneous FLS migration, and platelet-derived growth factor induced cell invasion, effects associated with differential regulation of MMP expression and mediated by the PlexinA1 and neuropilin-1 and -2 receptors. Conclusion: Our data suggest that modulation of class 3 semaphorin signaling could be a novel therapeutic strategy for modulating the invasive behaviour of FLS in RA.
Asunto(s)
Artritis Reumatoide/genética , Regulación hacia Abajo , Fibroblastos/metabolismo , Regulación de la Expresión Génica , ARN Mensajero/genética , Semaforinas/genética , Sinoviocitos/metabolismo , Artritis Reumatoide/metabolismo , Artritis Reumatoide/patología , Movimiento Celular , Células Cultivadas , Femenino , Fibroblastos/patología , Humanos , Masculino , Semaforinas/biosíntesis , Membrana Sinovial/metabolismo , Membrana Sinovial/patología , Sinoviocitos/patologíaRESUMEN
Diagnosis of complex disease and response to treatment is often associated with multiple indicators, both clinical and laboratorial. With the use of biomarkers, various mechanisms have been unraveled which can lead to better and faster diagnosis, predicting and monitoring of response to treatment and new drug development. With the introduction of multiplex technology for immunoassays and the growing awareness of the role of immune-monitoring during new therapeutic interventions it is now possible to test large numbers of soluble mediators in small sample volumes. However, standardization of sample collection and laboratory assessments remains suboptimal. We developed a multiplex immunoassay for detection of 162 immune related proteins in human serum and plasma. The assay was split in panels depending on natural occurring concentrations with a maximum of 60 proteins. The aim of this study was to evaluate precision, accuracy, reproducibility and stability of proteins when repeated freeze-thaw cycles are performed of this in-house developed panel, as well as assessing the protein signature in plasma and serum using various anticoagulants. Intra-assay variance of each mediator was <10%. Inter-assay variance ranged between 1.6 and 37% with an average of 12.2%. Recoveries were similar for all mediators (mean 99.8 ± 2.6%) with a range between 89-107%. Next we measured all mediators in serum, EDTA plasma and sodium heparin plasma of 43 healthy control donors. Of these markers only 19 showed similar expression profiles in the 3 different matrixes. Only 5 mediators were effected by multiple freeze-thawing cycles. Principal component analysis revealed different coagulants cluster separately and that sodium heparin shows the most consistent profile.
Asunto(s)
Anticoagulantes/farmacología , Voluntarios Sanos , Inmunoproteínas/metabolismo , Adulto , Ácido Edético/farmacología , Femenino , Congelación , Heparina/farmacología , Humanos , Inmunoensayo , Límite de Detección , Masculino , Persona de Mediana Edad , Estabilidad Proteica , Estándares de Referencia , Reproducibilidad de los ResultadosRESUMEN
OBJECTIVES: Non-selective histone deacetylase (HDAC) inhibitors (HDACi) have demonstrated anti-inflammatory properties in both in vitro and in vivo models of rheumatoid arthritis (RA). Here, we investigated the potential contribution of specific class I and class IIb HDACs to inflammatory gene expression in RA fibroblast-like synoviocytes (FLS). METHODS: RA FLS were incubated with pan-HDACi (ITF2357, givinostat) or selective HDAC1/2i, HDAC3/6i, HDAC6i and HDAC8i. Alternatively, FLS were transfected with HDAC3, HDAC6 or interferon (IFN)-α/ß receptor alpha chain (IFNAR1) siRNA. mRNA expression of interleukin (IL)-1ß-inducible genes was measured by quantitative PCR (qPCR) array and signalling pathway activation by immunoblotting and DNA-binding assays. RESULTS: HDAC3/6i, but not HDAC1/2i and HDAC8i, significantly suppressed the majority of IL-1ß-inducible genes targeted by pan-HDACi in RA FLS. Silencing of HDAC3 expression reproduced the effects of HDAC3/6i on gene regulation, contrary to HDAC6-specific inhibition and HDAC6 silencing. Screening of the candidate signal transducers and activators of transcription (STAT)1 transcription factor revealed that HDAC3/6i abrogated STAT1 Tyr701 phosphorylation and DNA binding, but did not affect STAT1 acetylation. HDAC3 activity was required for type I IFN production and subsequent STAT1 activation in FLS. Suppression of type I IFN release by HDAC3/6i resulted in reduced expression of a subset of IFN-dependent genes, including the chemokines CXCL9 and CXCL11. CONCLUSIONS: Inhibition of HDAC3 in RA FLS largely recapitulates the effects of pan-HDACi in suppressing inflammatory gene expression, including type I IFN production in RA FLS. Our results identify HDAC3 as a potential therapeutic target in the treatment of RA and type I IFN-driven autoimmune diseases.
Asunto(s)
Artritis Reumatoide/metabolismo , Fibroblastos/metabolismo , Histona Desacetilasas/fisiología , Mediadores de Inflamación/metabolismo , Sinoviocitos/metabolismo , Acetilación , Adulto , Anciano , Artritis Reumatoide/genética , Artritis Reumatoide/inmunología , Células Cultivadas , Regulación hacia Abajo/fisiología , Femenino , Regulación de la Expresión Génica/inmunología , Regulación de la Expresión Génica/fisiología , Histona Desacetilasas/genética , Humanos , Interferón beta/biosíntesis , Interleucina-1beta/biosíntesis , Interleucina-1beta/genética , Masculino , Persona de Mediana Edad , Fosforilación , Factor de Transcripción STAT1/metabolismo , Membrana Sinovial/metabolismo , Sinoviocitos/inmunologíaRESUMEN
OBJECTIVES: Epigenetic modifications play an important role in the regulation of gene transcription and cellular function. Here, we examined if pro-inflammatory factors present in the inflamed joint of patients with rheumatoid arthritis (RA) could regulate histone deacetylase (HDAC) expression and function in fibroblast-like synoviocytes (FLS). METHODS: Protein acetylation in synovial tissue was assessed by immunohistochemistry. The mRNA levels of HDAC family members and inflammatory mediators in the synovial tissue and the changes in HDAC expression in RA FLS were measured by quantitative (q) PCR. FLS were either transfected with HDAC5 siRNA or transduced with adenoviral vector encoding wild-type HDAC5 and the effects of HDAC5 manipulation were examined by qPCR arrays, ELISA and ELISA-based assays. RESULTS: Synovial class I HDAC expression was associated with local expression of tumour necrosis factor (TNF) and matrix metalloproteinase-1, while class IIa HDAC5 expression was inversely associated with parameters of disease activity (erythrocyte sedimentation rate, C-reactive protein, Disease Activity Score in 28 Joints). Interleukin (IL)-1ß or TNF stimulation selectively suppressed HDAC5 expression in RA FLS, which was sufficient and required for optimal IFNB, CXCL9, CXCL10 and CXCL11 induction by IL-1ß, associated with increased nuclear accumulation of the transcription factor, interferon regulatory factor 1(IRF1). CONCLUSIONS: Inflammatory cytokines suppress RA FLS HDAC5 expression, promoting nuclear localisation of IRF1 and transcription of a subset of type I interferon response genes. Our results identify HDAC5 as a novel inflammatory mediator in RA, and suggest that strategies rescuing HDAC5 expression in vivo, or the development of HDAC inhibitors not affecting HDAC5 activity, may have therapeutic applications in RA treatment.
Asunto(s)
Artritis Reumatoide/metabolismo , Citocinas/genética , Fibroblastos/metabolismo , Histona Desacetilasas/metabolismo , Membrana Sinovial/citología , Adulto , Anciano , Artritis Reumatoide/genética , Sedimentación Sanguínea , Proteína C-Reactiva/análisis , Epigénesis Genética , Femenino , Humanos , Factor 1 Regulador del Interferón/genética , Interleucina-1beta/metabolismo , Masculino , Metaloproteinasa 1 de la Matriz/metabolismo , Persona de Mediana Edad , ARN Mensajero/metabolismo , Índice de Severidad de la Enfermedad , Transcripción Genética , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
Endothelial cell (EC) dysfunction is a hallmark of Systemic Lupus Erythematosus (SLE) and Tie2 is a receptor essential for vascular stability. Inflammatory processes promote inhibition of Tie2 homeostatic activation, driving vascular dysfunction. In this work we determined whether type I Interferons (IFN) induce Tie2 signalling-mediated endothelial dysfunction in patients with SLE. Serum levels of Angiopoietin (Ang)-1, Ang-2 and soluble (s)Tie1 in patients with SLE and healthy controls were measured by ELISA. Monocytes from patients with SLE and Human Umbilical Vein EC (HUVEC) were stimulated with IFN-α, IFN-ß (1000 I.U.) or SLE serum (20%). mRNA and protein expression, phosphorylation and translocation were determined by quantitative PCR, ELISA, Western Blot, flow cytometry and confocal microscopy. Viability and angiogenic capacity were determined by calcein and tube formation assays. We found that sTie1 and Ang-2 serum levels were increased and Ang-1 decreased in patients with SLE and were associated with clinical characteristics. Type I IFN significantly decreased Ang-1 and increased Ang-2 in monocytes from patients with SLE. Type I IFN increased sTie1 and Ang-2 secretion and reduced Tie2 activation in HUVEC. Functionally, type I IFN significantly reduced EC viability and impaired angiogenesis in a Tie2 signalling-dependent manner. Finally, SLE serum increased Ang-2 and sTie1 secretion and significantly decreased tube formation. Importantly, Tie1 and IFNAR1 knockdown reversed these effects in tube formation. Overall, type I IFN play an important role in the stability of EC by inhibiting Tie2 signalling, suggesting that these processes may be implicated in the cardiovascular events observed in patients with SLE.
Asunto(s)
Interferón Tipo I , Lupus Eritematoso Sistémico , Receptor TIE-2 , Humanos , Células Endoteliales de la Vena Umbilical Humana/metabolismo , Interferón Tipo I/farmacología , Receptor TIE-2/metabolismo , Transducción de SeñalRESUMEN
Previous works from our group show that Semaphorin3B (Sema3B) is reduced in RA and plays a protective role in a mouse arthritis model. In turn, MerTK plays a protective function in murine arthritis models, is expressed by synovial tissue macrophages and is linked to remission in patients with RA. In this study, we examined the role of Sema3B in the phenotypic characteristics of RA macrophages and the implication of MerTK. Peripheral blood monocytes from RA patients were differentiated into IFN-γ (RA MØIFN-γ) or M-CSF (RA MØM-CSF) macrophages and stimulated with LPS, Sema3B or their combination. Alternatively, RA fibroblast like synoviocytes (FLS) were stimulated with RA MØIFN-γ and RA MØM-CSF supernatants. Gene expression was determined by qPCR and protein expression and activation by flow cytometry, ELISA and western blot. Sema3B down-regulated the expression of pro-inflammatory mediators, in both RA MØIFN-γ and RA MØM-CSF. We observed a similar reduction in RA FLS stimulated with the supernatant of Sema3B-treated RA MØIFN-γ and RA MØM-CSF. Sema3B also modulated cell surface markers in macrophages towards an anti-inflammatory phenotype. Besides, MerTK expression and activation was up-regulated by Sema3B, just as GAS6 expression, Resolvin D1 secretion and the phagocytic activity of macrophages. Importantly, the inhibition of MerTK and neuropilins 1 and 2 abrogated the anti-inflammatory effect of Sema3B. Our data demonstrate that Sema3B modulates the macrophage characteristics in RA, inducing a skewing towards an anti-inflammatory/pro-resolving phenotype in a MerTK-dependant manner. Therefore, here we identify a new mechanism supporting the protective role of Sema3B in RA pathogenesis.
Asunto(s)
Artritis Reumatoide , Glicoproteínas de Membrana , Semaforinas , Tirosina Quinasa c-Mer , Humanos , Artritis Reumatoide/metabolismo , Células Cultivadas , Factor Estimulante de Colonias de Macrófagos/metabolismo , Macrófagos/metabolismo , Fenotipo , Tirosina Quinasa c-Mer/metabolismo , Glicoproteínas de Membrana/genética , Semaforinas/genéticaRESUMEN
OBJECTIVE: Semaphorin 3B (Sema3B) decreases the migratory and invasive capacities of fibroblast-like synoviocytes (FLS) in rheumatoid arthritis (RA) and suppresses expression of matrix metalloproteinases. We undertook this study to examine the role of Sema3B in a mouse model of arthritis and its expression in RA patients. METHODS: Clinical responses, histologic features, and FLS function were examined in wild-type (WT) and Sema3B-/- mice in a K/BxN serum transfer model of arthritis. Protein and messenger RNA expression of Sema3B in mouse joints and murine FLS, as well as in serum and synovial tissue from patients with arthralgia and patients with RA, was determined using enzyme-linked immunosorbent assay, immunoblotting, quantitative polymerase chain reaction, and RNA sequencing. FLS migration was determined using a wound closure assay. RESULTS: The clinical severity of serum-induced arthritis was significantly higher in Sema3B-/- mice compared to WT mice. This was associated with increased expression of inflammatory mediators and increased migratory capacity of murine FLS. Administration of recombinant mouse Sema3B reduced the clinical severity of serum-induced arthritis and the expression of inflammatory mediators. Sema3B expression was significantly lower in the synovial tissue and serum of patients with established RA compared to patients with arthralgia. Serum Sema3B levels were elevated in patients with arthralgia that later progressed to RA, but not in those who did not develop RA; however, these levels drastically decreased 1 and 2 years after RA development. CONCLUSION: Sema3B expression plays a protective role in a mouse model of arthritis. In RA patients, expression levels of Sema3B in the serum depend on the disease stage, suggesting different regulatory roles in disease onset and progression.
Asunto(s)
Artritis Reumatoide , Glicoproteínas de Membrana , Semaforinas , Sinoviocitos , Animales , Artralgia/genética , Artralgia/metabolismo , Artralgia/patología , Artritis Reumatoide/genética , Artritis Reumatoide/metabolismo , Artritis Reumatoide/patología , Células Cultivadas , Fibroblastos/metabolismo , Fibroblastos/patología , Humanos , Mediadores de Inflamación/metabolismo , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Ratones , Semaforinas/genética , Semaforinas/metabolismo , Membrana Sinovial/metabolismo , Sinoviocitos/metabolismo , Sinoviocitos/patologíaRESUMEN
Immune system CD4 T-cells with high cell-surface CD26 expression show anti-tumoral properties. When engineered with a chimeric antigen receptor (CAR), they incite strong responses against solid cancers. This subset was originally associated to human CD4 T helper cells bearing the CD45R0 effector/memory phenotype and later to Th17 cells. CD26 is also found in soluble form (sCD26) in several biological fluids, and its serum levels correlate with specific T cell subsets. However, the relationship between glycoprotein sCD26 and its dipeptidyl peptidase 4 (DPP4) enzymatic activity, and cell-surface CD26 expression is not well understood. We have studied ex vivo cell-surface CD26 and in vitro surface and intracellular CD26 expression and secretome's sCD26 in cultured CD4 T cells under different polarization conditions. We show that most human CD26negative CD4 T cells in circulating lymphocytes are central memory (TCM) cells while CD26high expression is present in effector Th1, Th2, Th17, and TEM (effector memory) cells. However, there are significant percentages of Th1, Th2, Th17, and Th22 CD26 negative cells. This information may help to refine the research on CAR-Ts. The cell surface CD45R0 and CD26 levels in the different T helper subsets after in vitro polarization resemble those found ex vivo. In the secretomes of these cultures there was a significant amount of sCD26. However, in all polarizations, including Th1, the levels of sCD26 were lower (although not significantly) compared to the Th0 condition (activation without polarization). These differences could have an impact on the various physiological functions proposed for sCD26/DPP4.
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Dipeptidil Peptidasa 4/genética , Subgrupos de Linfocitos T/inmunología , Células TH1/inmunología , Células Th17/inmunología , Dipeptidil Peptidasa 4/inmunología , Regulación de la Expresión Génica/genética , Regulación de la Expresión Génica/inmunología , Humanos , Antígenos Comunes de Leucocito/genética , Antígenos Comunes de Leucocito/inmunología , Células T de Memoria/inmunología , Neoplasias/genética , Neoplasias/inmunología , Neoplasias/patología , Células TH1/metabolismo , Células Th17/metabolismo , Células Th2/inmunología , Células Th2/metabolismoRESUMEN
OBJECTIVE: To analyze the potential role of semaphorin 4A (Sema4A) in inflammatory and fibrotic processes involved in the pathology of systemic sclerosis (SSc). METHODS: Sema4A levels in the plasma of healthy controls (n = 11) and SSc patients (n = 20) were determined by enzyme-linked immunosorbent assay (ELISA). The expression of Sema4A and its receptors in monocytes and CD4+ T cells from healthy controls and SSc patients (n = 6-7 per group) was determined by ELISA and flow cytometry. Th17 cytokine production by CD4+ T cells (n = 5-7) was analyzed by ELISA and flow cytometry. The production of inflammatory mediators and extracellular matrix (ECM) components by dermal fibroblast cells (n = 6) was analyzed by quantitative polymerase chain reaction, ELISA, Western blotting, confocal microscopy, and ECM deposition assay. RESULTS: Plasma levels of Sema4A, and Sema4A expression by circulating monocytes and CD4+ T cells, were significantly higher in SSc patients than in healthy controls (P < 0.05). Inflammatory mediators significantly up-regulated the secretion of Sema4A by monocytes and CD4+ T cells from SSc patients (P < 0.05 versus unstimulated SSc cells). Functional assays showed that Sema4A significantly enhanced the expression of Th17 cytokines induced by CD3/CD28 in total CD4+ T cells as well in different CD4+ T cell subsets (P < 0.05 versus unstimulated SSc cells). Finally, Sema4A induced a profibrotic phenotype in dermal fibroblasts from both healthy controls and SSc patients, which was abrogated by blocking or silencing the expression of Sema4A receptors. CONCLUSION: Our findings indicate that Sema4A plays direct and dual roles in promoting inflammation and fibrosis, 2 main features of SSc, suggesting that Sema4A might be a novel therapeutic target in SSc.
Asunto(s)
Linfocitos T CD4-Positivos/inmunología , Citocinas/inmunología , Fibroblastos/metabolismo , Fibrosis/metabolismo , Inflamación/metabolismo , Monocitos/inmunología , Esclerodermia Sistémica/metabolismo , Semaforinas/metabolismo , Adulto , Western Blotting , Estudios de Casos y Controles , Ensayo de Inmunoadsorción Enzimática , Matriz Extracelular/metabolismo , Matriz Extracelular/patología , Femenino , Fibroblastos/inmunología , Fibroblastos/patología , Fibrosis/patología , Humanos , Inflamación/inmunología , Masculino , Microscopía Confocal , Persona de Mediana Edad , Reacción en Cadena de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Esclerodermia Sistémica/inmunología , Esclerodermia Sistémica/patología , Piel/citología , Células Th17/inmunologíaRESUMEN
Prolactin (PRL) is a neuroendocrine hormone that can promote inflammation. We examined the synovial tissue and fluid levels of PRL in patients with inflammatory arthritis, PRL expression in differentiated MÏs from patients with arthritis and from healthy donors, and the effects of different stimuli on PRL production by MÏs. PRL levels were measured in paired synovial fluid (SF) and peripheral blood of patients with rheumatoid arthritis (RA, n = 19), psoriatic arthritis (PsA, n = 11), and gout (n = 11). Synovial-tissue PRL mRNA expression was measured by quantitative PCR in patients with RA (n = 25), PsA (n = 11), and gout (n = 12) and in MÏs differentiated in SF of patients with RA, PsA, other subtypes of spondyloarthritis (SpA), and gout. Synovial-tissue PRL mRNA expression correlated significantly with clinical disease parameters in patients with RA and PsA, including erythrocyte sedimentation rate (ESR, r = 0.424; P = 0.049) and disease activity score evaluated in 28 joints (DAS28, r = 0.729; P = 0.017). Synovial-tissue PRL expression was similar in RA, PsA, and gout. PRL mRNA expression was detected in monocyte-derived MÏs from patients with RA and was significantly higher (P ≤ 0.01) in MÏs differentiated in pooled SF from patients with RA and PsA compared with SpA or gout. PRL production by MÏ differentiation in the SF from patients with RA was not further regulated by stimulation with CD40L, IgG, LPS, or TNF. PRL is produced locally in the synovium of patients with inflammatory arthritis. The production of PRL by MÏs was increased by unknown components of RA and PsA SF, where it could contribute to disease progression.