RESUMEN
Ectopic lymphoid structures (ELSs) in the rheumatoid synovial joints sustain autoreactivity against locally expressed autoantigens. We recently identified recombinant monoclonal antibodies (RA-rmAbs) derived from single, locally differentiated rheumatoid arthritis (RA) synovial B cells, which specifically recognize fibroblast-like synoviocytes (FLSs). Here, we aimed to identify the specificity of FLS-derived autoantigens fueling local autoimmunity and the functional role of anti-FLS antibodies in promoting chronic inflammation. A subset of anti-FLS RA-rmAbs reacting with a 60 kDa band from FLS extracts demonstrated specificity for HSP60 and partial cross-reactivity to other stromal autoantigens (i.e., calreticulin/vimentin) but not to citrullinated fibrinogen. Anti-FLS RA-rmAbs, but not anti-neutrophil extracellular traps rmAbs, exhibited pathogenic properties in a mouse model of collagen-induced arthritis. In patients, anti-HSP60 antibodies were preferentially detected in RA versus osteoarthritis (OA) synovial fluid. Synovial HSPD1 and CALR gene expression analyzed using bulk RNA-Seq and GeoMx-DSP closely correlated with the lympho-myeloid RA pathotype, and HSP60 protein expression was predominantly observed around ELS. Moreover, we observed a significant reduction in synovial HSP60 gene expression followed B cell depletion with rituximab that was strongly associated with the treatment response. Overall, we report that synovial stromal-derived autoantigens are targeted by pathogenic autoantibodies and are associated with specific RA pathotypes, with potential value for patient stratification and as predictors of the response to B cell-depleting therapies.
Asunto(s)
Artritis Reumatoide , Autoantígenos , Chaperonina 60 , Centro Germinal , Artritis Reumatoide/inmunología , Artritis Reumatoide/patología , Animales , Humanos , Ratones , Autoantígenos/inmunología , Autoantígenos/genética , Centro Germinal/inmunología , Centro Germinal/patología , Chaperonina 60/inmunología , Chaperonina 60/genética , Autoanticuerpos/inmunología , Autoinmunidad , Masculino , Sinoviocitos/inmunología , Sinoviocitos/patología , Sinoviocitos/metabolismo , Artritis Experimental/inmunología , Artritis Experimental/patología , Femenino , Linfocitos B/inmunología , Linfocitos B/patología , Estructuras Linfoides Terciarias/inmunología , Estructuras Linfoides Terciarias/patologíaRESUMEN
Osteoarthritis (OA) is the most common form of arthritis, characterized by osteophyte formation, cartilage degradation, and structural and cellular alterations of the synovial membrane. Activated fibroblast-like synoviocytes (FLS) of the synovial membrane have been identified as key drivers, secreting humoral mediators that maintain inflammatory processes, proteases that cause cartilage and bone destruction, and factors that drive fibrotic processes. In normal tissue repair, fibrotic processes are terminated after the damage has been repaired. In fibrosis, tissue remodeling and wound healing are exaggerated and prolonged. Various stressors, including aging, joint instability, and inflammation, lead to structural damage of the joint and micro lesions within the synovial tissue. One result is the reduced production of synovial fluid (lubricants), which reduces the lubricity of the cartilage areas, leading to cartilage damage. In the synovial tissue, a wound-healing cascade is initiated by activating macrophages, Th2 cells, and FLS. The latter can be divided into two major populations. The destructive thymocyte differentiation antigen (THY)1â phenotype is restricted to the synovial lining layer. In contrast, the THY1+ phenotype of the sublining layer is classified as an invasive one with immune effector function driving synovitis. The exact mechanisms involved in the transition of fibroblasts into a myofibroblast-like phenotype that drives fibrosis remain unclear. The review provides an overview of the phenotypes and spatial distribution of FLS in the synovial membrane of OA, describes the mechanisms of fibroblast into myofibroblast activation, and the metabolic alterations of myofibroblast-like cells.
Asunto(s)
Fibroblastos , Fibrosis , Osteoartritis , Fenotipo , Sinoviocitos , Humanos , Osteoartritis/patología , Osteoartritis/inmunología , Osteoartritis/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patología , Fibroblastos/inmunología , Animales , Sinoviocitos/metabolismo , Sinoviocitos/patología , Sinoviocitos/inmunología , Membrana Sinovial/patología , Membrana Sinovial/inmunología , Membrana Sinovial/metabolismoRESUMEN
Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by synovial hyperplasia, pannus formation, and cartilage and bone destruction. Lysine-specific demethylase 1 (LSD1), an enzyme involved in transcriptional regulation, has an unclear role in synovial inflammation, fibroblast-like synoviocytes migration, and invasion during RA pathogenesis. In this study, we observed increased LSD1 expression in RA synovial tissues and in TNF-α-stimulated MH7A cells. SP2509, an LSD1 antagonist, directly reduced LSD1 expression and reversed the elevated levels of proteins associated with inflammation, apoptosis, proliferation, and autophagy induced by TNF-α. Furthermore, SP2509 inhibited the migratory capacity of MH7A cells, which was enhanced by TNF-α. In CIA models, SP2509 treatment ameliorated RA development, reducing the expression of pro-inflammatory cytokines and alleviating joint pathological symptoms. These findings underscore the significance of LSD1 in RA and propose the therapeutic potential of SP2509.
Asunto(s)
Artritis Reumatoide , Histona Demetilasas , Artritis Reumatoide/metabolismo , Artritis Reumatoide/inmunología , Histona Demetilasas/metabolismo , Humanos , Animales , Ratones , Sinoviocitos/metabolismo , Sinoviocitos/patología , Sinoviocitos/inmunología , Progresión de la Enfermedad , Factor de Necrosis Tumoral alfa/metabolismo , Movimiento Celular , Línea Celular , Masculino , Membrana Sinovial/patología , Membrana Sinovial/inmunología , Membrana Sinovial/metabolismo , Apoptosis , Artritis Experimental/inmunología , Citocinas/metabolismo , Autofagia , Proliferación Celular/efectos de los fármacosRESUMEN
Objectives: IL26 levels are elevated in the blood and synovial fluid of patients with inflammatory arthritis. IL26 can be produced by Th17 cells and locally within joints by tissue-resident cells. IL26 induces osteoblast mineralization in vitro. As osteoproliferation and Th17 cells are important factors in the pathogenesis of axial spondyloarthritis (axSpA), we aimed to clarify the cellular sources of IL26 in spondyloarthritis. Methods: Serum, peripheral blood mononuclear cells (n = 15-35) and synovial tissue (n = 3-9) of adult patients with axSpA, psoriatic arthritis (PsA) and rheumatoid arthritis (RA) and healthy controls (HCs, n = 5) were evaluated by ELISA, flow cytometry including PrimeFlow assay, immunohistochemistry and immunofluorescence and quantitative PCR. Results: Synovial tissue of axSpA patients shows significantly more IL26-positive cells than that of HCs (p < 0.01), but numbers are also elevated in PsA and RA patients. Immunofluorescence shows co-localization of IL26 with CD68, but not with CD3, SMA, CD163, cadherin-11, or CD90. IL26 is elevated in the serum of RA and PsA (but not axSpA) patients compared with HCs (p < 0.001 and p < 0.01). However, peripheral blood CD4+ T cells from axSpA and PsA patients show higher positivity for IL26 in the PrimeFlow assay compared with HCs. CD4+ memory T cells from axSpA patients produce more IL26 under Th17-favoring conditions (IL-1ß and IL-23) than cells from PsA and RA patients or HCs. Conclusion: IL26 production is increased in the synovial tissue of SpA and can be localized to CD68+ macrophage-like synoviocytes, whereas circulating IL26+ Th17 cells are only modestly enriched. Considering the osteoproliferative properties of IL26, this offers new therapeutic options independent of Th17 pathways.
Asunto(s)
Antígenos CD , Artritis Psoriásica , Interleucinas , Sinoviocitos , Humanos , Artritis Psoriásica/inmunología , Artritis Psoriásica/metabolismo , Sinoviocitos/metabolismo , Sinoviocitos/inmunología , Sinoviocitos/patología , Masculino , Adulto , Femenino , Antígenos CD/metabolismo , Interleucinas/metabolismo , Interleucinas/sangre , Persona de Mediana Edad , Antígenos de Diferenciación Mielomonocítica/metabolismo , Espondiloartritis Axial/inmunología , Células Th17/inmunología , Células Th17/metabolismo , Membrana Sinovial/inmunología , Membrana Sinovial/metabolismo , Membrana Sinovial/patología , Articulaciones/patología , Articulaciones/inmunología , Articulaciones/metabolismo , Artritis Reumatoide/inmunología , Artritis Reumatoide/metabolismo , Artritis Reumatoide/sangre , Artritis Reumatoide/patologíaAsunto(s)
Artritis Reumatoide , Fibroblastos , Inmunomodulación , Sinoviocitos , Artritis Reumatoide/inmunología , Artritis Reumatoide/patología , Humanos , Fibroblastos/inmunología , Sinoviocitos/inmunología , Sinoviocitos/metabolismo , Sinoviocitos/patología , Animales , Membrana Sinovial/inmunología , Membrana Sinovial/patologíaRESUMEN
Osteoarthritis (OA) is now widely acknowledged as a low-grade inflammatory condition, in which the intrinsic immune system plays a significant role in its pathogenesis. While the involvement of macrophages and T cells in the development of OA has been extensively reviewed, recent research has provided mounting evidence supporting the crucial contribution of NK cells in both the initiation and advancement of OA. Accumulated evidence has emerged in recent years indicating that NK cells play a critical role in OA development and progression. This review will outline the ongoing understanding of the utility of NK cells in the etiology of OA, focusing on how NK cells interact with chondrocytes, synoviocytes, osteoclasts, and other immune cells to influence the course of OA disease.
Asunto(s)
Condrocitos , Células Asesinas Naturales , Osteoartritis , Humanos , Osteoartritis/inmunología , Células Asesinas Naturales/inmunología , Animales , Condrocitos/inmunología , Osteoclastos/inmunología , Sinoviocitos/inmunología , Sinoviocitos/metabolismo , Comunicación Celular/inmunología , Macrófagos/inmunologíaRESUMEN
Rheumatoid arthritis (RA) is a chronic immune-mediated joint inflammatory disorder associated with aberrant activation of fibroblast-like synoviocytes (FLS). Recently, FLS gained importance due to its crucial role in RA pathogenesis, and thus, targeting FLS is suggested as an attractive treatment strategy for RA. FLS-targeted approaches may be combined with disease-modifying antirheumatic drugs (DMARDs) and natural phytochemicals to improve efficacy in RA control and negate immunosuppression. In this study, we assessed the therapeutic effectiveness of DD NP HG in primary RA-FLS cells isolated from the synovial tissue of FCA-induced RA rats. We observed that DD NP HG had good biosafety for healthy FLS cells and, at higher concentrations, a mild inhibitory effect on RA-FLS. The combination therapy (DD NP HG) of MTX NP and PEITC NE in RA-FLS showed a higher rate of apoptosis with significantly reduced LPS-induced expression of pro-inflammatory cytokines (TNF-α, IL-17A, and IL-6) in arthritic FLS. Further, the gene expression studies showed that DD NP HG significantly down-regulated the mRNA expression of IL-1ß, RANKL, NFATc1, DKK1, Bcl-xl, Mcl-1, Atg12, and ULK1, and up-regulated the mRNA expression of OPG, PUMA, NOXA and SQSTM1 in LPS-stimulated RA-FLS cells. Collectively, our results demonstrated that DD NP HG significantly inhibited the RA-FLS proliferation via inducing apoptosis, down-regulating pro-inflammatory cytokines, and further enhancing the expression of genes associated with bone destruction in RA pathogenesis. A nanotechnology approach is a promising strategy for the co-delivery of dual drugs to regulate the RA-FLS function and achieve synergistic treatment of RA.
Asunto(s)
Apoptosis , Artritis Reumatoide , Autofagia , Fibroblastos , Nanopartículas , Sinoviocitos , Sinoviocitos/efectos de los fármacos , Sinoviocitos/metabolismo , Sinoviocitos/inmunología , Artritis Reumatoide/tratamiento farmacológico , Artritis Reumatoide/metabolismo , Animales , Apoptosis/efectos de los fármacos , Ratas , Autofagia/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/efectos de los fármacos , Sinergismo Farmacológico , Citocinas/metabolismo , Células Cultivadas , Humanos , Metotrexato/farmacología , Antirreumáticos/farmacología , Antirreumáticos/uso terapéutico , Inflamación/tratamiento farmacológico , Masculino , Modelos Animales de EnfermedadRESUMEN
G-protein coupled receptor (GPCR) kinases (GRKs) and hypoxia-inducible factor-1α (HIF-1α) play key roles in rheumatoid arthritis (RA). Several studies have demonstrated that HIF-1α expression is positively regulated by GRK2, suggesting its posttranscriptional effects on HIF-1α. In this study, we review the role of HIF-1α and GRK2 in RA pathophysiology, focusing on their proinflammatory roles in immune cells and fibroblast-like synoviocytes (FLS).We then introduce several drugs that inhibit GRK2 and HIF-1α, and briefly outline their molecular mechanisms. We conclude by presenting gaps in knowledge and our prospects for the pharmacological potential of targeting these proteins and the relevant downstream signaling pathways.Future research is warranted and paramount for untangling these novel and promising roles for GRK2 and HIF-1α in RA.
Asunto(s)
Artritis Reumatoide , Quinasa 2 del Receptor Acoplado a Proteína-G , Subunidad alfa del Factor 1 Inducible por Hipoxia , Sinoviocitos , Humanos , Artritis Reumatoide/tratamiento farmacológico , Artritis Reumatoide/genética , Artritis Reumatoide/inmunología , Hipoxia/genética , Hipoxia/inmunología , Subunidad alfa del Factor 1 Inducible por Hipoxia/antagonistas & inhibidores , Subunidad alfa del Factor 1 Inducible por Hipoxia/genética , Subunidad alfa del Factor 1 Inducible por Hipoxia/inmunología , Inflamación/tratamiento farmacológico , Inflamación/genética , Inflamación/inmunología , Sinoviocitos/inmunología , Quinasa 2 del Receptor Acoplado a Proteína-G/antagonistas & inhibidores , Quinasa 2 del Receptor Acoplado a Proteína-G/genética , Quinasa 2 del Receptor Acoplado a Proteína-G/inmunologíaRESUMEN
BACKGROUND: Phospholipase C-like 1 (PLCL1), a protein that lacks catalytic activity, has similar structures to the PLC family. The aim of this research was to find the function and underlying mechanisms of PLCL1 in fibroblast-like synoviocyte (FLS) of rheumatoid arthritis (RA). METHODS: In this study, we first analyzed the expression of PLCL1 in the synovial tissue of RA patients and K/BxN mice by immunohistochemical staining. Then silencing or overexpressing PLCL1 in FLS before stimulating by TNF-α. The levels of IL-6, IL-1ß and CXCL8 in FLS and supernatants were detected by Western Blot (WB), Real-Time Quantitative PCR and Enzyme Linked Immunosorbent Assay. We used INF39 to specifically inhibit the activation of NLRP3 inflammasomes, and detected the expression of NLRP3, Cleaved Caspase-1, IL-6 and IL-1ß in FLS by WB. RESULT: When PLCL1 was silenced, the level of IL-6, IL-1ß and CXCL8 were down-regulated. When PLCL1 was overexpressed, the level of IL-6, IL-1ß and CXCL8 were unregulated. The previous results demonstrated that the mechanism of PLCL1 regulating inflammation in FLS was related to NLRP3 inflammasomes. INF39 could counteract the release of inflammatory cytokines caused by overexpression of PLCL1. CONCLUSION: Result showed that the function of PLCL1 in RA FLS might be related to the NLRP3 inflammasomes. We finally confirmed our hypothesis with the NLRP3 inhibitor INF39. Our results suggested that PLCL1 might promote the inflammatory response of RA FLS by regulating the NLRP3 inflammasomes.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales , Artritis Reumatoide , Proteína con Dominio Pirina 3 de la Familia NLR , Fosfoinositido Fosfolipasa C , Sinoviocitos , Proteínas Adaptadoras Transductoras de Señales/inmunología , Animales , Artritis Reumatoide/inmunología , Artritis Reumatoide/patología , Fibroblastos/metabolismo , Humanos , Inflamasomas/metabolismo , Inflamación , Interleucina-6/inmunología , Ratones , Proteína con Dominio Pirina 3 de la Familia NLR/inmunología , Fosfoinositido Fosfolipasa C/inmunología , Sinoviocitos/inmunología , Sinoviocitos/patologíaRESUMEN
Dehydroevodiamine (DHE) is an effective natural active substance extracted from Euodiae Fructus, which is a widely used herbal drug in traditional Chinese medicine. The focus of this study was to test the possibility of using DHE in the treatment of rheumatoid arthritis (RA) diseases. A rat model of adjuvant-induced arthritis (AIA) was generated using Complete Freund's Adjuvant (CFA). Body weight changes, arthritis scores, ankle pathology, tumor necrosis factor-alpha (TNF-α), interleukin-1ß(IL-1ß), interleukin-6 (IL-6), and interleukin-17 (IL-17) secretion, as well as matrix metalloproteinase (MMP) expression in joint tissue, were measured as indicators of viability of DHE medicated AIA rats. Human fibroblast-like synoviocytes (MH7A cells) were connected to check these impacts. The results confirmed that DHE administration had an excellent therapeutic impact on the AIA rat model, substantially relieving joint swelling, inhibiting synovial pannus hyperplasia, and decreasing joint scores. In addition, the serum enzyme-linked immunosorbent assay (ELISA) showed that DHE treatment reduced the expression of pro-inflammatory factors in AIA rats. The immunohistochemical results showed that DHE treatment could reduce the synthesis of MMPs such as matrix metalloproteinase-1(MMP-1) and matrix metalloproteinase-3 (MMP-3) in the ankle tissue of AIA rats. In vitro, DHE inhibited cell proliferation, mRNA transcription, protein synthesis of proinflammatory factors such as IL-1ßand IL-6, and matrix metalloproteinases such as MMP-1 and MMP-3. Furthermore, DHE inhibited the phosphorylation levels of p38, JNK, and ERK proteins in TNF-α-treated MH7A cells.This work assessed the effect of DHE in AIA rats and revealed its mechanism in vitro.
Asunto(s)
Alcaloides/administración & dosificación , Antiinflamatorios/administración & dosificación , Artritis Experimental/tratamiento farmacológico , Adyuvante de Freund/efectos adversos , Sinoviocitos/citología , Alcaloides/farmacología , Animales , Antiinflamatorios/farmacología , Artritis Experimental/inducido químicamente , Artritis Experimental/genética , Artritis Experimental/inmunología , Peso Corporal/efectos de los fármacos , Línea Celular , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Citocinas/genética , Citocinas/metabolismo , Modelos Animales de Enfermedad , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Metaloproteinasas de la Matriz/metabolismo , Ratas , Sinoviocitos/efectos de los fármacos , Sinoviocitos/inmunologíaRESUMEN
Lysophosphatidylserine (lysoPS) is known to regulate immune cell functions. Phospholipase A1 member A (PLA1A) can generate this bioactive lipid through hydrolysis of sn-1 fatty acids on phosphatidylserine (PS). PLA1A has been associated with cancer metastasis, asthma, as well as acute coronary syndrome. However, the functions of PLA1A in the development of systemic autoimmune rheumatic diseases remain elusive. To investigate the possible implication of PLA1A during rheumatic diseases, we monitored PLA1A in synovial fluids from patients with rheumatoid arthritis and plasma of early-diagnosed arthritis (EA) patients and clinically stable systemic lupus erythematosus (SLE) patients. We used human primary fibroblast-like synoviocytes (FLSs) to evaluate the PLA1A-induced biological responses. Our results highlighted that the plasma concentrations of PLA1A in EA and SLE patients were elevated compared to healthy donors. High concentrations of PLA1A were also detected in synovial fluids from rheumatoid arthritis patients compared to those from osteoarthritis (OA) and gout patients. The origin of PLA1A in FLSs and the arthritic joints remained unknown, as healthy human primary FLSs does not express the PLA1A transcript. Besides, the addition of recombinant PLA1A stimulated cultured human primary FLSs to secrete IL-8. Preincubation with heparin, autotaxin (ATX) inhibitor HA130 or lysophosphatidic acid (LPA) receptor antagonist Ki16425 reduced PLA1A-induced-secretion of IL-8. Our data suggested that FLS-associated PLA1A cleaves membrane-exposed PS into lysoPS, which is subsequently converted to LPA by ATX. Since primary FLSs do not express any lysoPS receptors, the data suggested PLA1A-mediated pro-inflammatory responses through the ATX-LPA receptor signaling axis.
Asunto(s)
Artritis/patología , Fibroblastos/patología , Gota/patología , Lupus Eritematoso Sistémico/patología , Fosfolipasas A1/metabolismo , Hidrolasas Diéster Fosfóricas/metabolismo , Receptores del Ácido Lisofosfatídico/metabolismo , Sinoviocitos/patología , Artritis/genética , Artritis/inmunología , Artritis/metabolismo , Estudios de Casos y Controles , Femenino , Fibroblastos/inmunología , Fibroblastos/metabolismo , Gota/genética , Gota/inmunología , Gota/metabolismo , Humanos , Lupus Eritematoso Sistémico/genética , Lupus Eritematoso Sistémico/inmunología , Lupus Eritematoso Sistémico/metabolismo , Masculino , Fosfolipasas A1/genética , Hidrolasas Diéster Fosfóricas/genética , Receptores del Ácido Lisofosfatídico/genética , Líquido Sinovial/inmunología , Líquido Sinovial/metabolismo , Sinoviocitos/inmunología , Sinoviocitos/metabolismoRESUMEN
The production and oxidation mechanism of reactive oxygen species (ROS) are out of balance in rheumatoid arthritis (RA). However, the correlation between ROS and T cell subsets in RA remains unclear. Peripheral blood mononuclear cells (PBMCs) from patients with RA (n = 40) and healthy controls (n = 10) were isolated from whole blood samples. Synovial tissues (n = 3) and synovial fluid (n = 10) were obtained from patients with RA. The repartition of T cell subsets and expression of ROS and cytokines were examined according to RA severity. Fibroblast-like synoviocytes (FLSs) from patients with RA were stimulated with PBMCs and the expression of inflammation-related molecules were measured by RT-PCR and cytokine array. Regulatory T cells from patients with moderate (5.1 > DAS28 ≥ 3.2) RA showed the highest expression of mitochondrial ROS among the groups based on disease severity. Although ROS levels steadily increased with RA severity, there was a slight decline in severe RA (DAS28 ≥ 5.1) compared with moderate RA. The expression of inflammatory cytokines in RA FLSs were significantly inhibited when FLSs were co-cultured with PBMCs treated with ROS inhibitor. These findings provide a novel approach to suppress inflammatory response of FLSs through ROS regulation in PBMCs.
Asunto(s)
Artritis Reumatoide/sangre , Artritis Reumatoide/inmunología , Fibroblastos/inmunología , Estrés Oxidativo , Especies Reactivas de Oxígeno/metabolismo , Sinoviocitos/inmunología , Linfocitos T Colaboradores-Inductores/inmunología , Linfocitos T Reguladores/inmunología , Adulto , Anciano , Anciano de 80 o más Años , Artritis Reumatoide/patología , Estudios de Casos y Controles , Células Cultivadas , Técnicas de Cocultivo , Citocinas/metabolismo , Femenino , Humanos , Inflamación/metabolismo , Masculino , Persona de Mediana Edad , Mitocondrias/metabolismo , Índice de Severidad de la Enfermedad , Líquido Sinovial/metabolismoRESUMEN
ES-62 is the major secreted protein of the parasitic filarial nematode, Acanthocheilonema viteae. The molecule exists as a large tetramer (MW, ~240kD), which possesses immunomodulatory properties by virtue of multiple phosphorylcholine (PC) moieties attached to N-type glycans. By suppressing inflammatory immune responses, ES-62 can prevent disease development in certain mouse models of allergic and autoimmune conditions, including joint pathology in collagen-induced arthritis (CIA), a model of rheumatoid arthritis (RA). Such protection is associated with functional suppression of "pathogenic" hyper-responsive synovial fibroblasts (SFs), which exhibit an aggressive inflammatory and bone-damaging phenotype induced by their epigenetic rewiring in response to the inflammatory microenvironment of the arthritic joint. Critically, exposure to ES-62 in vivo induces a stably-imprinted CIA-SF phenotype that exhibits functional responses more typical of healthy, Naïve-SFs. Consistent with this, ES-62 "rewiring" of SFs away from the hyper-responsive phenotype is associated with suppression of ERK activation, STAT3 activation and miR-155 upregulation, signals widely associated with SF pathogenesis. Surprisingly however, DNA methylome analysis of Naïve-, CIA- and ES-62-CIA-SF cohorts reveals that rather than simply preventing pathogenic rewiring of SFs, ES-62 induces further changes in DNA methylation under the inflammatory conditions pertaining in the inflamed joint, including targeting genes associated with ciliogenesis, to programme a novel "resolving" CIA-SF phenotype. In addition to introducing a previously unsuspected aspect of ES-62's mechanism of action, such unique behaviour signposts the potential for developing DNA methylation signatures predictive of pathogenesis and its resolution and hence, candidate mechanisms by which novel therapeutic interventions could prevent SFs from perpetuating joint inflammation and destruction in RA. Pertinent to these translational aspects of ES-62-behavior, small molecule analogues (SMAs) based on ES-62's active PC-moieties mimic the rewiring of SFs as well as the protection against joint disease in CIA afforded by the parasitic worm product.
Asunto(s)
Antiinflamatorios/farmacología , Artritis Experimental/prevención & control , Epigénesis Genética , Fibroblastos/metabolismo , Proteínas del Helminto/farmacología , Inflamación/prevención & control , Sinoviocitos/metabolismo , Acanthocheilonema/metabolismo , Animales , Artritis Experimental/etiología , Artritis Experimental/metabolismo , Artritis Experimental/patología , Células Cultivadas , Metilación de ADN , Fibroblastos/efectos de los fármacos , Fibroblastos/inmunología , Inflamación/etiología , Inflamación/metabolismo , Inflamación/patología , Masculino , Ratones , Ratones Endogámicos DBA , Sinoviocitos/efectos de los fármacos , Sinoviocitos/inmunologíaRESUMEN
Fibroblast-like synoviocytes (FLS) play an important pathological role in persistent inflammatory joint diseases such as rheumatoid arthritis (RA). These cells have primarily been characterized in the RA synovial membrane. Here we aim to phenotypically and functionally characterize cultured synovial fluid-derived FLS (sfRA-FLS). Paired peripheral blood mononuclear cells (PBMC) and sfRA-FLS from patients with RA were obtained and monocultures of sfRA-FLS and autologous co-cultures of sfRA-FLS and PBMC were established. The in situ activated sfRA-FLS were CD34-, CD45-, Podoplanin+, Thymocyte differentiation antigen-1+. SfRA-FLS expressed uniform levels of NFкB-related pathway proteins and secreted several pro-inflammatory cytokines dominated by IL-6 and MCP-1. In a co-culture model with autologous PBMC, the ICAM-1 and HLA-DR expression on sfRA-FLS and secretion of IL-1ß, IL-6, and MCP-1 increased. In vivo, human sfRA-FLS were cartilage invasive both at ipsilateral and contralateral implantation site. We conclude that, sfRA-FLS closely resemble the pathological sublining layer FLS subset in terms of surface protein expression, cytokine production and leukocyte cross-talk potential. Further, sfRA-FLS are comparable to tissue-derived FLS in their capabilities to invade cartilage at implantation sites but also spread tissue destruction to a distant site. Collectively, sfRA-FLS can serve as a an easy-to-obtain source of pathological sublining FLS in RA.
Asunto(s)
Artritis Reumatoide/metabolismo , Artritis Reumatoide/patología , Biomarcadores , Sinoviocitos/metabolismo , Artritis Reumatoide/etiología , Línea Celular , Células Cultivadas , Citocinas/metabolismo , Progresión de la Enfermedad , Susceptibilidad a Enfermedades , Fibroblastos/metabolismo , Fibroblastos/patología , Humanos , Inmunofenotipificación , Mediadores de Inflamación/metabolismo , Leucocitos Mononucleares/inmunología , Leucocitos Mononucleares/metabolismo , Fenotipo , Líquido Sinovial/citología , Líquido Sinovial/metabolismo , Sinoviocitos/inmunología , Sinoviocitos/patologíaRESUMEN
Recent evidence has suggested that synovial inflammation and macrophage polarization were involved in the pathogenesis of osteoarthritis (OA). Additionally, high-molecular-weight hyaluronic acid (HMW-HA) was often used clinically to treat OA. GRP78, an endoplasmic reticulum (ER) stress chaperone, was suggested to contribute to the hyperplasia of synovial cells in OA. However, it was still unclear whether HMW-HA affected macrophage polarization through GRP78. Therefore, we aimed to identify the effect of HMW-HA in primary synovial cells and macrophage polarization and to investigate the role of GRP78 signaling. We used IL-1ß to treat primary synoviocytes to mimic OA, and then treated them with HMW-HA. We also collected conditioned medium (CM) to culture THP-1 macrophages and examine the changes in the phenotype. IL-1ß increased the expression of GRP78, NF-κB (p65 phosphorylation), IL-6, and PGE2 in primary synoviocytes, accompanied by an increased macrophage M1/M2 polarization. GRP78 knockdown significantly reversed the expression of IL-1ß-induced GRP78-related downstream molecules and macrophage polarization. HMW-HA with GRP78 knockdown had additive effects in an IL-1ß culture. Finally, the synovial fluid from OA patients revealed significantly decreased IL-6 and PGE2 levels after the HMW-HA treatment. Our study elucidated a new form of signal transduction for HMW-HA-mediated protection against synovial inflammation and macrophage polarization and highlighted the involvement of the GRP78-NF-κB signaling pathway.
Asunto(s)
Chaperón BiP del Retículo Endoplásmico/metabolismo , Ácido Hialurónico/farmacología , Inflamación/prevención & control , Interleucina-1beta/efectos adversos , Macrófagos/inmunología , FN-kappa B/metabolismo , Osteoartritis/prevención & control , Anciano , Anciano de 80 o más Años , Citocinas/metabolismo , Chaperón BiP del Retículo Endoplásmico/genética , Humanos , Inflamación/inducido químicamente , Inflamación/inmunología , Inflamación/patología , Activación de Macrófagos , Persona de Mediana Edad , Peso Molecular , FN-kappa B/genética , Osteoartritis/inducido químicamente , Osteoartritis/inmunología , Osteoartritis/patología , Transducción de Señal , Sinoviocitos/efectos de los fármacos , Sinoviocitos/inmunología , Sinoviocitos/metabolismo , Sinoviocitos/patologíaRESUMEN
Objective: Antibodies against carbamylated proteins (anti-CarP) are associated with poor prognosis and the development of bone erosions in rheumatoid arthritis (RA). RA neutrophils externalize modified autoantigens through the formation of neutrophil extracellular traps (NETs). Increased levels of the cathelicidin LL37 have been documented in the synovium of RA patients, but the cellular source remains unclear. We sought to determine if post-translational modifications of LL37, specifically carbamylation, occur during NET formation, enhance this protein's autoantigenicity, and contribute to drive bone erosion in the synovial joint. Methods: ELISA and Western blot analyses were used to identify carbamylated LL37 (carLL37) in biological samples. Anti-carLL37 antibodies were measured in the serum of HLA-DRB1*04:01 transgenic mice and in human RA synovial fluid. Results: Elevated levels of carLL37 were found in plasma and synovial fluid from RA patients, compared to healthy controls. RA NETs release carLL37 and fibroblast-like synoviocytes (FLS) internalized NET-bound carLL37 and loaded it into their MHCII compartment. HLA-DRB1*04:01 transgenic mice immunized with FLS containing NETs developed autoantibodies against carLL37. Anti-carLL37 antibodies were present in RA sera and synovial fluid and they correlated with radiologic bone erosion scores of the hands and feet in RA patients. CarLL37-IgG immune complexes enhanced the ability of monocytes to differentiate into osteoclasts and potentiated osteoclast-mediated extracellular matrix resorption. Conclusions: NETs are a source of carLL37 leading to induction of anti-carbamylated autoantibody responses. Furthermore, carLL37-IgG immune complexes may be implicated in the bone damage characteristic of RA. These results support that dysregulated NET formation has pathogenic roles in RA.
Asunto(s)
Artritis Reumatoide/etiología , Artritis Reumatoide/patología , Autoanticuerpos/inmunología , Autoantígenos/inmunología , Resorción Ósea/inmunología , Resorción Ósea/patología , Catelicidinas/inmunología , Animales , Artritis Reumatoide/metabolismo , Resorción Ósea/metabolismo , Trampas Extracelulares/inmunología , Humanos , Ratones , Osteoclastos/inmunología , Osteoclastos/metabolismo , Líquido Sinovial/inmunología , Sinoviocitos/inmunología , Sinoviocitos/metabolismo , Sinoviocitos/patologíaRESUMEN
OBJECTIVE: Nitidine chloride (NC), a natural small molecular compound from traditional Chinese herbal medicine zanthoxylum nitidum, has been shown to exhibit anti-tumor effect. However, its role in autoimmune diseases such as rheumatoid arthritis (RA) is unknown. Here, we investigate the effect of NC in controlling fibroblast-like synoviocytes (FLS)-mediated synovial inflammation and joint destruction in RA and further explore its underlying mechanism(s). METHODS: FLSs were separated from synovial tissues obtained from patients with RA. Protein expression was analyzed by Western blot or immunohistochemistry. Gene expression was measured using quantitative RT-PCR. ELISA was used to measure the levels of cytokines and MMPs. Cell proliferation was detected using EdU incorporation. Migration and invasion were evaluated by Boyden chamber assay. RNA sequencing analysis was used to identify the target of NC. Collagen-induced arthritis (CIA) model was used to evaluate the in vivo effect of NC. RESULTS: NC treatment reduced the proliferation, migration, invasion, and lamellipodia formation but not apoptosis of RA FLSs. We also demonstrated the inhibitory effect of NC on TNF-α-induced expression and secretion of IL-6, IL-8, CCL-2, MMP-1 and MMP-13. Furthermore, we identified KCNH1, a gene that encodes ether-à-go-go-1 channel, as a novel targeting gene of NC in RA FLSs. KCNH1 expression was increased in FLSs and synovial tissues from patients with RA compared to healthy controls. KCNH1 knockdown or NC treatment decreased the TNF-α-induced phosphorylation of AKT. Interestingly, NC treatment ameliorated the severity of arthritis and reduced synovial KCNH1 expression in mice with CIA. CONCLUSIONS: Our data demonstrate that NC treatment inhibits aggressive and inflammatory actions of RA FLSs by targeting KCNH1 and sequential inhibition of AKT phosphorylation. Our findings suggest that NC might control FLS-mediated rheumatoid synovial inflammation and joint destruction, and be a novel therapeutic agent for RA.
Asunto(s)
Artritis Experimental/tratamiento farmacológico , Artritis Reumatoide/tratamiento farmacológico , Benzofenantridinas/farmacología , Canales de Potasio Éter-A-Go-Go/antagonistas & inhibidores , Membrana Sinovial/efectos de los fármacos , Animales , Artritis Experimental/inmunología , Artritis Experimental/patología , Artritis Reumatoide/inmunología , Artritis Reumatoide/patología , Benzofenantridinas/uso terapéutico , Células Cultivadas , Canales de Potasio Éter-A-Go-Go/genética , Canales de Potasio Éter-A-Go-Go/metabolismo , Femenino , Fibroblastos/inmunología , Fibroblastos/metabolismo , Técnicas de Silenciamiento del Gen , Voluntarios Sanos , Humanos , Masculino , Ratones , Persona de Mediana Edad , Cultivo Primario de Células , Membrana Sinovial/inmunología , Membrana Sinovial/patología , Sinoviocitos/efectos de los fármacos , Sinoviocitos/inmunologíaRESUMEN
TNF is a central cytokine in the pathogenesis of rheumatoid arthritis (RA). Elevated level of TNF causes local inflammation that affects immune cells and fibroblast-like synoviocytes (FLS). Nowadays, only 20-30% of patients experience remission after the standard of care therapy-antibodies against TNF. Interestingly, responders show reduced levels of GLUT1 and GAPDH, highlighting a potential link to cellular metabolism. The aim of the study was to investigate whether TNF directly affects the metabolic phenotype of FLS. Real-time respirometry displayed TNF-induced upregulation of glycolysis along with a modest increase of oxidative phosphorylation in FLS from healthy donors. In addition, TNF stimulation enhanced HIF1A and GLUT1 expression. The upregulation of HIF1A and GLUT1 reflects their enriched level in FLS from RA patients (RA-FLS). The inhibition of TAK1, HIF1a and hexokinase deciphered the importance of TNF/TAK1/HIF1A/glycolysis signaling axis. To prove that inhibition of glycolysis reduced the pathogenic phenotype, we showed that 2-deoxyglucose, a hexokinase inhibitor, partially decreased secretion of RA biomarkers. In summary, we identified a direct role of TNF on glycolytic reprogramming of FLS and confirmed the potency of immunometabolism for RA. Further studies are needed to evaluate the therapeutic impact especially regarding non-responder data.
Asunto(s)
Artritis Reumatoide/inmunología , Transportador de Glucosa de Tipo 1/inmunología , Subunidad alfa del Factor 1 Inducible por Hipoxia/inmunología , Sinoviocitos/inmunología , Factor de Necrosis Tumoral alfa/inmunología , Células Cultivadas , Humanos , Sinoviocitos/citologíaRESUMEN
Fibroblast-like synoviocytes (FLS) play an important role in maintaining joint homeostasis and orchestrating local inflammatory processes. When activated during injury or inflammation, FLS undergo transiently increased bioenergetic and biosynthetic demand. We aimed to identify metabolic changes which occur early in inflammatory disease pathogenesis which might support sustained cellular activation in persistent inflammation. We took primary human FLS from synovial biopsies of patients with very early rheumatoid arthritis (veRA) or resolving synovitis, and compared them with uninflamed control samples from the synovium of people without arthritis. Metabotypes were compared using NMR spectroscopy-based metabolomics and correlated with serum C-reactive protein levels. We measured glycolysis and oxidative phosphorylation by Seahorse analysis and assessed mitochondrial morphology by immunofluorescence. We demonstrate differences in FLS metabolism measurable after ex vivo culture, suggesting that disease-associated metabolic changes are long-lasting. We term this phenomenon 'metabolic memory'. We identify changes in cell metabolism after acute TNFα stimulation across disease groups. When compared to FLS from patients with early rheumatoid arthritis, FLS from patients with resolving synovitis have significantly elevated mitochondrial respiratory capacity in the resting state, and less fragmented mitochondrial morphology after TNFα treatment. Our findings indicate the potential to restore cell metabotypes by modulating mitochondrial function at sites of inflammation, with implications for treatment of RA and related inflammatory conditions in which fibroblasts play a role.
Asunto(s)
Artritis Reumatoide/inmunología , Fibroblastos/inmunología , Inflamación/inmunología , Sinoviocitos/inmunología , Factor de Necrosis Tumoral alfa/metabolismo , Adulto , Anciano , Artritis Reumatoide/metabolismo , Artritis Reumatoide/patología , Células Cultivadas , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Humanos , Inflamación/metabolismo , Inflamación/patología , Masculino , Persona de Mediana Edad , Fosforilación Oxidativa , Análisis de Regresión , Sinoviocitos/metabolismo , Sinoviocitos/patología , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
OBJECTIVE: To investigate the role of PF-06650833, a highly potent and selective small-molecule inhibitor of interleukin-1-associated kinase 4 (IRAK4), in autoimmune pathophysiology in vitro, in vivo, and in the clinical setting. METHODS: Rheumatoid arthritis (RA) inflammatory pathophysiology was modeled in vitro through 1) stimulation of primary human macrophages with anti-citrullinated protein antibody immune complexes (ICs), 2) RA fibroblast-like synoviocyte (FLS) cultures stimulated with Toll-like receptor (TLR) ligands, as well as 3) additional human primary cell cocultures exposed to inflammatory stimuli. Systemic lupus erythematosus (SLE) pathophysiology was simulated in human neutrophils, dendritic cells, B cells, and peripheral blood mononuclear cells stimulated with TLR ligands and SLE patient ICs. PF-06650833 was evaluated in vivo in the rat collagen-induced arthritis (CIA) model and the mouse pristane-induced and MRL/lpr models of lupus. Finally, RNA sequencing data generated with whole blood samples from a phase I multiple-ascending-dose clinical trial of PF-06650833 were used to test in vivo human pharmacology. RESULTS: In vitro, PF-06650833 inhibited human primary cell inflammatory responses to physiologically relevant stimuli generated with RA and SLE patient plasma. In vivo, PF-06650833 reduced circulating autoantibody levels in the pristane-induced and MRL/lpr murine models of lupus and protected against CIA in rats. In a phase I clinical trial (NCT02485769), PF-06650833 demonstrated in vivo pharmacologic action pertinent to SLE by reducing whole blood interferon gene signature expression in healthy volunteers. CONCLUSION: These data demonstrate that inhibition of IRAK4 kinase activity can reduce levels of inflammation markers in humans and provide confidence in the rationale for clinical development of IRAK4 inhibitors for rheumatologic indications.