RESUMEN
Tissue repair is disturbed in fibrotic diseases like systemic sclerosis (SSc), where the deposition of large amounts of extracellular matrix components such as collagen interferes with organ function. LAIR-1 is an inhibitory collagen receptor highly expressed on tissue immune cells. We questioned whether in SSc, impaired LAIR-1-collagen interaction is contributing to the ongoing inflammation and fibrosis. We found that SSc patients do not have an intrinsic defect in LAIR-1 expression or function. Instead, fibroblasts from healthy controls and SSc patients stimulated by soluble factors that drive inflammation and fibrosis in SSc deposit disorganized collagen products in vitro, which are dysfunctional LAIR-1 ligands. This is dependent of matrix metalloproteinases and platelet-derived growth factor receptor signaling. In support of a non-redundant role of LAIR-1 in the control of fibrosis, we found that LAIR-1-deficient mice have increased skin fibrosis in response to repeated injury and in the bleomycin mouse model for SSc. Thus, LAIR-1 represents an essential control mechanism for tissue repair. In fibrotic disease, excessive collagen degradation may lead to a disturbed feedback loop. The presence of functional LAIR-1 in patients provides a therapeutic opportunity to reactivate this intrinsic negative feedback mechanism in fibrotic diseases.
Asunto(s)
Colágeno , Modelos Animales de Enfermedad , Fibroblastos , Fibrosis , Ratones Noqueados , Receptores Inmunológicos , Esclerodermia Sistémica , Animales , Humanos , Esclerodermia Sistémica/inmunología , Esclerodermia Sistémica/metabolismo , Esclerodermia Sistémica/patología , Ratones , Receptores Inmunológicos/metabolismo , Receptores Inmunológicos/genética , Colágeno/metabolismo , Fibroblastos/metabolismo , Bleomicina/efectos adversos , Piel/patología , Piel/metabolismo , Piel/inmunología , Transducción de Señal , Masculino , Femenino , Células CultivadasRESUMEN
BACKGROUND AND AIMS: Monocytes and dendritic cells (DC) are both key inflammatory cells, with recognized effects on cardiac repair. However, there are distinct subsets of monocytes with potential for beneficial or detrimental effects on heart failure (HF) pathogenesis. The connection between reverse cardiac remodelling, the potential anti-inflammatory effect of cardiac resynchronization therapy (CRT) and monocytes and DC homeostasis in HF is far from being understood. We hypothesized that monocytes and DC play an important role in cardiac reverse remodelling and CRT response. Therefore, we aimed to assess the potential role of baseline peripheral levels of blood monocytes and DC subsets and their phenotypic and functional activity for CRT response, in HF patients. As a secondary objective, we aimed to evaluate the impact of CRT on peripheral blood monocytes and DC subsets, by comparing baseline and post CRT circulating levels and phenotypic and functional activity. METHODS: Forty-one patients with advanced HF scheduled for CRT were included in this study. The quantification and phenotypic determination of classical (cMo), intermediate (iMo) and non-classical monocytes (ncMo), as well as of myeloid (mDC) and plasmacytoid DC (pDC) were performed by flow cytometry in a FACSCanto™II (BD) flow cytometer. The functional characterization of total monocytes and mDC was performed by flow cytometry in a FACSCalibur flow cytometer, after in vitro stimulation with lipopolysaccharide from Escherichia coli plus interferon (IFN)-γ, in the presence of Brefeldina A. Comparisons between the control and the patient group, and between responders and non-responders to CRT were performed. RESULTS: Compared to the control group, HF population presented a significantly lower frequency of pDC at baseline and a higher proportion of monocytes and mDC producing IL-6 and IL-1ß, both before and 6-months after CRT (T6). There was a remarkable decrease of cMo and an increase of iMo after CRT, only in responders. The responder group also presented higher ncMo values at T6 compared to the non-responder group. Both responders and non-responders presented a decrease in the expression of CD86 in all monocyte and DC populations after CRT. Moreover, in non-responders, the increased frequency of IL-6-producing DC persisted after CRT. CONCLUSION: Our study provides new knowledge about the possible contribution of pDC and monocytes subsets to cardiac reverse remodelling and response to CRT. Additionally, CRT is associated with a reduction on CD86 expression by monocytes and DC subsets and in their potential to produce pro-inflammatory cytokines, contributing, at least in part, for the well described anti-inflammatory effects of CRT in HF patients.
Asunto(s)
Terapia de Resincronización Cardíaca , Insuficiencia Cardíaca , Humanos , Terapia de Resincronización Cardíaca/efectos adversos , Monocitos , Interleucina-6 , Insuficiencia Cardíaca/diagnóstico , Insuficiencia Cardíaca/terapia , Células Dendríticas , AntiinflamatoriosRESUMEN
BACKGROUND: T cells have been implicated in the development and progression of inflammatory processes in chronic heart failure (CHF). Cardiac resynchronization therapy (CRT) has beneficial effects on symptoms and cardiac remodeling in CHF. However, its impact on the inflammatory immune response remains controversial. We aimed to study the impact of CRT on T cells in heart failure (HF) patients. METHODS: Thirty-nine HF patients were evaluated before CRT (T0) and six months later (T6). Quantification of T cells, their subsets, and their functional characterization, after in vitro stimulation, were evaluated by flow cytometry. RESULTS: T regulatory (Treg) cells were decreased in CHF patients (healthy group (HG): 1.08 ± 0.50 versus (heart failure patients (HFP)-T0: 0.69 ± 0.40, P = 0.022) and remaining diminished after CRT (HFP-T6: 0.61 ± 0.29, P = 0.003). Responders (R) to CRT presented a higher frequency of T cytotoxic (Tc) cells producing IL-2 at T0 compared with non-responders (NR) (R: 36.52 ± 12.55 versus NR: 24.71 ± 11.66, P = 0.006). After CRT, HF patients presented a higher percentage of Tc cells expressing TNF-α and IFN-γ (HG: 44.50 ± 16.62 versus R: 61.47 ± 20.54, P = 0.014; and HG: 40.62 ± 15.36 versus R: 52.39 ± 18.66, P = 0.049, respectively). CONCLUSION: The dynamic of different functional T cell subpopulations is significantly altered in CHF, which results in an exacerbated pro-inflammatory response. Even after CRT, it seems that the inflammatory condition underlying CHF continues to evolve with the progression of the disease. This could be due, at least in part, to the inability to restore Treg cells levels. TRIAL REGISTRATION: Observational and prospective study with no trial registration.
Asunto(s)
Terapia de Resincronización Cardíaca , Insuficiencia Cardíaca , Humanos , Terapia de Resincronización Cardíaca/efectos adversos , Linfocitos T Reguladores , Estudios Prospectivos , Insuficiencia Cardíaca/diagnóstico , Insuficiencia Cardíaca/terapia , Corazón , Enfermedad Crónica , Resultado del TratamientoRESUMEN
OBJECTIVE: This study was undertaken to identify key disease pathways driving conventional dendritic cell (cDC) alterations in systemic sclerosis (SSc). METHODS: Transcriptomic profiling was performed on peripheral blood CD1c+ cDCs (cDC2s) isolated from 12 healthy donors and 48 patients with SSc, including all major disease subtypes. We performed differential expression analysis for the different SSc subtypes and healthy donors to uncover genes dysregulated in SSc. To identify biologically relevant pathways, we built a gene coexpression network using weighted gene correlation network analysis. We validated the role of key transcriptional regulators using chromatin immunoprecipitation (ChIP) sequencing and in vitro functional assays. RESULTS: We identified 17 modules of coexpressed genes in cDCs that correlated with SSc subtypes and key clinical traits, including autoantibodies, skin score, and occurrence of interstitial lung disease. A module of immunoregulatory genes was markedly down-regulated in patients with the diffuse SSc subtype characterized by severe fibrosis. Transcriptional regulatory network analysis performed on this module predicted nuclear receptor 4A (NR4A) subfamily genes (NR4A1, NR4A2, NR4A3) as the key transcriptional regulators of inflammation. Indeed, ChIP-sequencing analysis indicated that these NR4A members target numerous differentially expressed genes in SSc cDC2s. Inclusion of NR4A receptor agonists in culture-based experiments provided functional proof that dysregulation of NR4As affects cytokine production by cDC2s and modulates downstream T cell activation. CONCLUSION: NR4A1, NR4A2, and NR4A3 are important regulators of immunosuppressive and fibrosis-associated pathways in SSc cDCs. Thus, the NR4A family represents novel potential targets to restore cDC homeostasis in SSc.
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Miembro 2 del Grupo A de la Subfamilia 4 de Receptores Nucleares , Esclerodermia Sistémica , Humanos , Miembro 2 del Grupo A de la Subfamilia 4 de Receptores Nucleares/genética , Miembro 2 del Grupo A de la Subfamilia 4 de Receptores Nucleares/metabolismo , Regulación de la Expresión Génica , Expresión Génica , Esclerodermia Sistémica/genética , Fibrosis , Glicoproteínas/metabolismo , Antígenos CD1/genéticaRESUMEN
Fibrosis is a major cause of mortality worldwide, characterized by myofibroblast activation and excessive extracellular matrix deposition. Systemic sclerosis is a prototypic fibrotic disease in which CXCL4 is increased and strongly correlates with skin and lung fibrosis. Here we aim to elucidate the role of CXCL4 in fibrosis development. CXCL4 levels are increased in multiple inflammatory and fibrotic mouse models, and, using CXCL4-deficient mice, we demonstrate the essential role of CXCL4 in promoting fibrotic events in the skin, lungs, and heart. Overexpressing human CXCL4 in mice aggravates, whereas blocking CXCL4 reduces, bleomycin-induced fibrosis. Single-cell ligand-receptor analysis predicts CXCL4 to affect endothelial cells and fibroblasts. In vitro, we confirm that CXCL4 directly induces myofibroblast differentiation and collagen synthesis in different precursor cells, including endothelial cells, by stimulating endothelial-to-mesenchymal transition. Our findings identify a pivotal role of CXCL4 in fibrosis, further substantiating the potential role of neutralizing CXCL4 as a therapeutic strategy.
Asunto(s)
Matriz Extracelular/patología , Miofibroblastos/metabolismo , Factor Plaquetario 4/metabolismo , Fibrosis Pulmonar/patología , Esclerodermia Sistémica/patología , Animales , Bleomicina/toxicidad , Línea Celular , Colágeno/biosíntesis , Modelos Animales de Enfermedad , Células Endoteliales/citología , Células Endoteliales/metabolismo , Transición Epitelial-Mesenquimal/fisiología , Células Endoteliales de la Vena Umbilical Humana , Humanos , Pulmón/patología , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Miofibroblastos/citología , Pericitos/metabolismo , Factor Plaquetario 4/genética , Células del Estroma/citología , Células del Estroma/metabolismoRESUMEN
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
OBJECTIVE: SSc is a complex disease characterized by vascular abnormalities and inflammation culminating in hypoxia and excessive fibrosis. Previously, we identified chemokine (C-X-C motif) ligand 4 (CXCL4) as a novel predictive biomarker in SSc. Although CXCL4 is well-studied, the mechanisms driving its production are unclear. The aim of this study was to elucidate the mechanisms leading to CXCL4 production. METHODS: Plasmacytoid dendritic cells (pDCs) from 97 healthy controls and 70 SSc patients were cultured in the presence of hypoxia or atmospheric oxygen level and/or stimulated with several toll-like receptor (TLR) agonists. Further, pro-inflammatory cytokine production, CXCL4, hypoxia-inducible factor (HIF) -1α and HIF-2α gene and protein expression were assessed using ELISA, Luminex, qPCR, FACS and western blot assays. RESULTS: CXCL4 release was potentiated only when pDCs were simultaneously exposed to hypoxia and TLR9 agonist (P < 0.0001). Here, we demonstrated that CXCL4 production is dependent on the overproduction of mitochondrial reactive oxygen species (mtROS) (P = 0.0079) leading to stabilization of HIF-2α (P = 0.029). In addition, we show that hypoxia is fundamental for CXCL4 production by umbilical cord CD34 derived pDCs. CONCLUSION: TLR-mediated activation of immune cells in the presence of hypoxia underpins the pathogenic production of CXCL4 in SSc. Blocking either mtROS or HIF-2α pathways may therapeutically attenuate the contribution of CXCL4 to SSc and other inflammatory diseases driven by CXCL4.
Asunto(s)
Factor Plaquetario 4/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Esclerodermia Sistémica , Receptor Toll-Like 9 , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Células Dendríticas/metabolismo , Humanos , Hipoxia/metabolismo , Subunidad alfa del Factor 1 Inducible por HipoxiaRESUMEN
Compelling evidence shows the involvement of plasmacytoid dendritic cells (pDCs) in systemic sclerosis (SSc) pathogenesis. This study investigated whether microRNAs (miRNAs) are involved in the dysregulation of pDCs in SSc patients already at early stages. RNA from circulating pDCs was isolated from two independent cohorts of SSc patients with different disease phenotypes, and individuals with Raynaud's phenomenon, for microRNA profiling and RNA-sequencing analysis. Proteomic analysis was exploited to identify novel direct miRNA targets at the protein level. Twelve and fifteen miRNAs were differentially expressed in at least one group of patients compared to healthy controls in discovery cohort I and II, respectively. Of note, miR-126 and miR-139-5p were upregulated in both preclinical and definite SSc patients and correlated with the expression of type I interferon (IFN)-responsive genes. Toll-like receptor 9 (TLR9) stimulation of healthy pDCs upregulated the expression of both miRNAs, similarly to what was observed in patients. The proteomic analysis identified USP24 as a novel target of miR-139-5p. The expression level of USP24 was inversely correlated with miR-139-5p expression in SSc patients and induced by TLR9 stimulation in healthy pDCs. These findings demonstrated that the miRNA profile is altered in pDCs of SSc patients already at early stages of the disease and indicate their potential contribution to pDC activation observed in patients.
RESUMEN
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
Inhibitory receptors are crucial immune regulators and are essential to prevent exacerbated responses, thus contributing to immune homeostasis. Leukocyte associated immunoglobulin like receptor 1 (LAIR-1) is an immune inhibitory receptor which has collagen and collagen domain containing proteins as ligands. LAIR-1 is broadly expressed on immune cells and has a large availability of ligands in both circulation and tissues, implicating a need for tight regulation of this interaction. In the current study, we sought to examine the regulation and function of LAIR-1 on monocyte, dendritic cell (DC) and macrophage subtypes, using different in vitro models. We found that LAIR-1 is highly expressed on intermediate monocytes as well as on plasmacytoid DCs. LAIR-1 is also expressed on skin immune cells, mainly on tissue CD14+ cells, macrophages and CD1c+ DCs. In vitro, monocyte and type-2 conventional DC stimulation leads to LAIR-1 upregulation, which may reflect the importance of LAIR-1 as negative regulator under inflammatory conditions. Indeed, we demonstrate that LAIR-1 ligation on monocytes inhibits toll like receptor (TLR)4 and Interferon (IFN)-α- induced signals. Furthermore, LAIR-1 is downregulated on GM-CSF and IFN-γ monocyte-derived macrophages and monocyte-derived DCs. In addition, LAIR-1 triggering during monocyte derived-DC differentiation results in significant phenotypic changes, as well as a different response to TLR4 and IFN-α stimulation. This indicates a role for LAIR-1 in skewing DC function, which impacts the cytokine expression profile of these cells. In conclusion, we demonstrate that LAIR-1 is consistently upregulated on monocytes and DC during the inflammatory phase of the immune response and tends to restore its expression during the resolution phase. Under inflammatory conditions, LAIR-1 has an inhibitory function, pointing toward to a potential intervention opportunity targeting LAIR-1 in inflammatory conditions.
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Células Dendríticas/metabolismo , Inflamación/metabolismo , Monocitos/metabolismo , Receptores Inmunológicos/metabolismo , Células Cultivadas , Citocinas/metabolismo , Células Dendríticas/inmunología , Regulación de la Expresión Génica , Humanos , Inflamación/genética , Inflamación/inmunología , Mediadores de Inflamación/metabolismo , Macrófagos/inmunología , Macrófagos/metabolismo , Monocitos/inmunología , Receptores Inmunológicos/genética , Transducción de SeñalRESUMEN
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
OBJECTIVE: To analyze how monocyte and macrophage exposure to CXCL4 induces inflammatory and fibrotic processes observed in Systemic sclerosis (SSc) patients. METHODS: In six independent experiments, monocytes of healthy controls (HC) and SSc patients were stimulated with CXCL4, TLR-ligands, IFNÉ or TGFß and the secretion of cytokines in the supernatant was assessed by multiplex immunoassays. PDGF-BB production by monocyte-derived macrophages was quantified using immunoassays. The number of monocytes and PDGF-BB in circulation was quantified in HC and SSc patients with the Sysmex XT-1800i haematology counter and immunoassays. Intracellular PDGF-BB was quantified in monocytes by Western blot. PDGF-receptor inhibition was achieved using siRNA-mediated knockdown or treatment with Crenolanib. The production of inflammatory mediators and extracellular matrix (ECM) components by dermal fibroblasts was analyzed by qPCR, ELISA and ECM deposition assays. RESULTS: SSc and HC monocytes released PDGF-BB upon stimulation with CXCL4. Conversely, TLR ligands, IFNÉ or TGFß did not induce PDGF-bb release. PDGF-BB plasma levels were significantly (P = 0.009) higher in diffuse SSc patients (n = 19), compared with HC (n = 21). In healthy dermal fibroblasts, PDGF-BB enhanced TNFÉ-induced expression of inflammatory cytokines and increased ECM production. Comparable results were observed in fibroblasts cultured in supernatant taken from macrophages stimulated with CXCL4. This effect was almost completely abrogated by inhibition of the PDGF-receptor using Crenolanib. CONCLUSION: Our findings demonstrate that CXCL4 can drive fibroblast activation indirectly via PDGF-BB production by myeloid cells. Hence, targeting PDGF-BB or CXCL4-induced PDGF-BB release could be clinically beneficial for patients with SSc.
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Becaplermina/metabolismo , Fibroblastos/inmunología , Inflamación/inmunología , Macrófagos/inmunología , Monocitos/inmunología , Factor Plaquetario 4/metabolismo , Esclerodermia Sistémica/inmunología , Adulto , Anciano , Bencimidazoles/farmacología , Células Cultivadas , Citocinas/metabolismo , Femenino , Humanos , Masculino , Persona de Mediana Edad , Piperidinas/farmacología , Receptores del Factor de Crecimiento Derivado de Plaquetas/antagonistas & inhibidoresRESUMEN
Systemic sclerosis (SSc), systemic lupus erythematosus (SLE) and primary Sjögrens syndrome (pSS) are clinically distinct systemic autoimmune diseases (SADs) that share molecular pathways. We quantified the frequency of circulating immune-cells in 169 patients with these SADs and 44 healty controls (HC) using mass-cytometry and assessed the diagnostic value of these results. Alterations in the frequency of immune-cell subsets were present in all SADs compared to HC. Most alterations, including a decrease of CD56hi NK-cells in SSc and IgM+ Bcells in pSS, were disease specific; only a reduced frequency of plasmacytoid dendritic cells was common between all SADs Strikingly, hierarchical clustering of SSc patients identified 4 clusters associated with different clinical phenotypes, and 9 of the 12 cell subset-alterations in SSc were also present during the preclinical-phase of the disease. Additionally, we found a strong association between the use of prednisone and alterations in B-cell subsets. Although differences in immune-cell frequencies between these SADs are apparent, the discriminative value thereof is too low for diagnostic purposes. Within each disease, mass cytometry analyses revealed distinct patterns between endophenotypes. Given the lack of tools enabling early diagnosis of SSc, our results justify further research into the value of cellular phenotyping as a diagnostic aid.
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Citometría de Flujo/métodos , Lupus Eritematoso Sistémico/inmunología , Esclerodermia Sistémica/inmunología , Síndrome de Sjögren/inmunología , Adulto , Anciano , Femenino , Humanos , Lupus Eritematoso Sistémico/diagnóstico , Masculino , Persona de Mediana Edad , Fenotipo , Esclerodermia Sistémica/diagnóstico , Síndrome de Sjögren/diagnósticoRESUMEN
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.
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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
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.
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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
OBJECTIVES: Systemic sclerosis (SSc) is an autoimmune disease with unknown pathogenesis manifested by inflammation, vasculopathy and fibrosis in skin and internal organs. Type I interferon signature found in SSc propelled us to study plasmacytoid dendritic cells (pDCs) in this disease. We aimed to identify candidate pathways underlying pDC aberrancies in SSc and to validate its function on pDC biology. METHODS: In total, 1193 patients with SSc were compared with 1387 healthy donors and 8 patients with localised scleroderma. PCR-based transcription factor profiling and methylation status analyses, single nucleotide polymorphism genotyping by sequencing and flow cytometry analysis were performed in pDCs isolated from the circulation of healthy controls or patients with SSc. pDCs were also cultured under hypoxia, inhibitors of methylation and hypoxia-inducible factors and runt-related transcription factor 3 (RUNX3) levels were determined. To study Runx3 function, Itgax-Cre:Runx3f/f mice were used in in vitro functional assay and bleomycin-induced SSc skin inflammation and fibrosis model. RESULTS: Here, we show downregulation of transcription factor RUNX3 in SSc pDCs. A higher methylation status of the RUNX3 gene, which is associated with polymorphism rs6672420, correlates with lower RUNX3 expression and SSc susceptibility. Hypoxia is another factor that decreases RUNX3 level in pDC. Mouse pDCs deficient of Runx3 show enhanced maturation markers on CpG stimulation. In vivo, deletion of Runx3 in dendritic cell leads to spontaneous induction of skin fibrosis in untreated mice and increased severity of bleomycin-induced skin fibrosis. CONCLUSIONS: We show at least two pathways potentially causing low RUNX3 level in SSc pDCs, and we demonstrate the detrimental effect of loss of Runx3 in SSc model further underscoring the role of pDCs in this disease.
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Subunidad alfa 3 del Factor de Unión al Sitio Principal/genética , Células Dendríticas/metabolismo , Regulación de la Expresión Génica , ARN/genética , Esclerodermia Sistémica/genética , Piel/patología , Animales , Subunidad alfa 3 del Factor de Unión al Sitio Principal/biosíntesis , Células Dendríticas/patología , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Fibroblastos/metabolismo , Fibroblastos/patología , Fibrosis/genética , Fibrosis/metabolismo , Fibrosis/patología , Humanos , Ratones , Esclerodermia Sistémica/metabolismo , Esclerodermia Sistémica/patología , Piel/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.
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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
BACKGROUND AND OBJECTIVE: Systemic sclerosis (SSc) is a severe autoimmune disease, in which the pathogenesis is dependent on both genetic and epigenetic factors. Altered gene expression in SSc monocytes, particularly of interferon (IFN)-responsive genes, suggests their involvement in SSc development. We investigated the correlation between epigenetic histone marks and gene expression in SSc monocytes. METHODS: Chromatin immunoprecipitation followed by sequencing (ChIPseq) for histone marks H3K4me3 and H3K27ac was performed on monocytes of nine healthy controls and 14 patients with SSc. RNA sequencing was performed in parallel to identify aberrantly expressed genes and their correlation with the levels of H3K4me3 and H3K27ac located nearby their transcription start sites. ChIP-qPCR assays were used to verify the role of bromodomain proteins, H3K27ac and STATs on IFN-responsive gene expression. RESULTS: 1046 and 534 genomic loci showed aberrant H3K4me3 and H3K27ac marks, respectively, in SSc monocytes. The expression of 381 genes was directly and significantly proportional to the levels of such chromatin marks present near their transcription start site. Genes correlated to altered histone marks were enriched for immune, IFN and antiviral pathways and presented with recurrent binding sites for IRF and STAT transcription factors at their promoters. IFNα induced the binding of STAT1 and STAT2 at the promoter of two of these genes, while blocking acetylation readers using the bromodomain BET family inhibitor JQ1 suppressed their expression. CONCLUSION: SSc monocytes have altered chromatin marks correlating with their IFN signature. Enzymes modulating these reversible marks may provide interesting therapeutic targets to restore monocyte homeostasis to treat or even prevent SSc.
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Epigénesis Genética , Código de Histonas/genética , Monocitos/inmunología , Esclerodermia Sistémica/genética , Adulto , Anciano , Azepinas/farmacología , Estudios de Casos y Controles , Ensamble y Desensamble de Cromatina/genética , Ensamble y Desensamble de Cromatina/inmunología , Femenino , Perfilación de la Expresión Génica/métodos , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/genética , Regulación de la Expresión Génica/inmunología , Histonas/genética , Humanos , Interferón-alfa/inmunología , Masculino , Persona de Mediana Edad , Terapia Molecular Dirigida/métodos , Factor de Transcripción STAT1/metabolismo , Factor de Transcripción STAT2/metabolismo , Esclerodermia Sistémica/inmunología , Triazoles/farmacologíaRESUMEN
PURPOSE: IL-17-producing T cells have been implicated in the inflammatory milieu of chronic heart failure (CHF), which implies a dismal prognosis in affected patients. The aim of this study was to evaluate the impact of cardiac resynchronization therapy (CRT) on the frequency and functional activity of Th17 and Tc17 cells, as well as, on IL-17 mRNA expression in patients with CHF. METHODS: Twenty-eight patients with CHF, analyzed before CRT (T0) and 6 months later (T6), and 15 healthy controls (HC) were enrolled in this study. Circulating Th17 and Tc17 cells were evaluated by flow cytometry. The quantification of IL-17A mRNA expression was performed by real-time PCR. RESULTS: Circulating Tc17 cells tended to be higher in CHF patients submitted to CRT than in HC (0.92% (0.24-3.32) versus 0.60% (0.09-3.68), although not reaching statistical significance. The frequency of Tc17 cells in CHF patients significantly decreases after CRT reaching levels similar to those of HC (0.92% (0.24-3.32) at T0 versus 0.56% (0.21-4.20) at T6, P < 0.05), mainly due to responders to CRT. Additionally, the expression of IL-17 mRNA was detected in a few number of responder patients at T0 (27%) and only detected in one responder at T6 (7%). Conversely, in non-responders, the proportion of patients exhibiting IL-17 mRNA expression increases from baseline (17%) to T6 (42%). No significant differences were observed in Th17 cells between HC, CHF patients in T0 and patients in T6. CONCLUSION: The inflammatory response mediated by circulating IL-17 producing cells seems to be suppressed by CRT, particularly in responders.
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Terapia de Resincronización Cardíaca , Insuficiencia Cardíaca/sangre , Insuficiencia Cardíaca/terapia , Interleucina-17/sangre , Anciano , Biomarcadores/sangre , Estudios de Casos y Controles , Femenino , Humanos , Mediadores de Inflamación/sangre , Masculino , Persona de Mediana Edad , Estudios Prospectivos , Células Th17RESUMEN
OBJECTIVE: MicroRNAs (miRNAs) are regulatory molecules, which have been addressed as potential biomarkers and therapeutic targets in rheumatic diseases. Here, we investigated the miRNA signature in the serum of systemic sclerosis (SSc) patients and we further assessed their expression in early stages of the disease. METHODS: The levels of 758 miRNAs were evaluated in the serum of 26 SSc patients as compared to 9 healthy controls by using an Openarray platform. Three miRNAs were examined in an additional cohort of 107 SSc patients and 24 healthy donors by single qPCR. MiR-483-5p expression was further analysed in the serum of patients with localized scleroderma (LoS) (nâ¯=â¯22), systemic lupus erythematosus (SLE) (nâ¯=â¯33) and primary Sjögren's syndrome (pSS) (nâ¯=â¯23). The function of miR-483-5p was examined by transfecting miR-483-5p into primary human dermal fibroblasts and pulmonary endothelial cells. RESULTS: 30 miRNAs were significantly increased in patients with SSc. Of these, miR-483-5p showed reproducibly higher levels in an independent SSc cohort and was also elevated in patients with preclinical-SSc symptoms (early SSc). Notably, miR-483-5p was not differentially expressed in patients with SLE or pSS, whereas it was up-regulated in LoS, indicating that this miRNA could be involved in the development of skin fibrosis. Consistently, miR-483-5p overexpression in fibroblasts and endothelial cells modulated the expression of fibrosis-related genes. CONCLUSIONS: Our findings showed that miR-483-5p is up-regulated in the serum of SSc patients, from the early stages of the disease onwards, and indicated its potential function as a fine regulator of fibrosis in SSc.