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1.
Rheumatology (Oxford) ; 62(7): 2594-2600, 2023 07 05.
Artigo em Inglês | MEDLINE | ID: mdl-36342195

RESUMO

OBJECTIVE: Oligoarticular JIA disease progression and outcomes are variable. Our objective is to detect protein markers that would allow for earlier intervention to potentially halt disease progression. In this retrospective study of serial SF samples, elevated expression of CCL24, CXCL9 and CXCL10 was linked to the eventual need for advanced medications. METHODS: Serial SF samples were selected from patients with persistent and extended oligoarticular JIA. The samples were separated into two groups: those who did and did not receive advanced medications throughout their disease course. Protein antibody arrays and Luminex assays were performed to determine changes in protein expression. RESULTS: CCL24, CXCL9 and CXCL10 expression levels were significantly higher in patients who eventually required advanced treatment than in those who did not. The expression levels of CCL24 and CXCL9 were consistently elevated in paired samples of those who later received advanced medications. In the persistent oligoarticular JIA group, CXCL10 levels remained elevated over time in those who required advanced treatment. Conversely, CCL24 levels decreased in patients who did not require advanced treatment. In the extended samples, the levels of CCL24 and CXCL10 expression increased significantly over time in the patients who ultimately required advanced treatment. CONCLUSION: In patients with oligoarticular JIA, regardless of disease onset and progression, the consistent elevation of any or all three markers, the CCL24, CXCL9 and CXCL10 in SFs was associated with the future use of advanced therapy, which could be reflective of disease severity.


Assuntos
Artrite Juvenil , Líquido Sinovial , Humanos , Líquido Sinovial/metabolismo , Artrite Juvenil/diagnóstico , Estudos Retrospectivos , Progressão da Doença , Quimiocina CXCL9/metabolismo , Quimiocina CXCL10/metabolismo , Quimiocina CCL24/metabolismo
2.
Rheumatol Ther ; 11(1): 143-155, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38070102

RESUMO

INTRODUCTION: Fibroblast-like synoviocytes (FLS) play a critical role in inflammation that contributes to disease progression in juvenile idiopathic arthritis (JIA). In rheumatoid arthritis (RA), FLS express tumor necrosis factor alpha (TNFα). TNFα signaling has been shown to be upstream of transforming growth factor beta (TGFß) signaling. Overexpression of TNFα and TGFß, as well as related proteins, can cause increased inflammation in RA. In this study, we examine the effects of inhibiting TNFα signaling with adalimumab on FLS isolated from synovial fluid of patients with JIA. METHODS: Synovial fluid samples were selected from 41 patients in our repository. Of these samples, 23 were diagnosed with persistent oligoarticular JIA and 18 were diagnosed with extended oligoarticular JIA. All samples were taken prior to patients extending to a polyarticular course, or what we termed extended-to-be (ETB), and were on no medications or nonsteroidal anti-inflammatory drugs (NSAIDs) only at the time of arthrocentesis. For cell studies, FLS were isolated from synovial fluid and treated with adalimumab for 24 h. Protein antibody arrays were performed by RayBiotech, Inc. according to their protocols. RESULTS: In persistent FLS, TNFα (fold change [FC] = 1.2 p = 0.001), TGFß (FC = 1.5 p = 0.001), lymphotoxin alpha (LTα) (FC = 4.3 p = 0.015), soluble tumor necrosis factor receptor 1 (sTNFRI) (FC = 5.1 p = 0.008), and soluble tumor necrosis factor receptor 2 (sTNFRII) (FC = 3.8 p = 0.025) were significantly elevated in adalimumab treated cells compared to untreated cells. In ETB FLS, TNFα was significantly elevated (FC = 1.04 p = 0.023) while TGFß (FC = 1.03 p = 0.037) was significantly decreased in adalimumab-treated cells compared to untreated cells. CONCLUSIONS: This data suggests that, after only 24 h, adalimumab is effective at decreasing inflammation that occurs downstream of initial TNFα signaling in extended-to-be fibroblast-like synoviocytes.

3.
Pediatr Rheumatol Online J ; 22(1): 6, 2024 Jan 02.
Artigo em Inglês | MEDLINE | ID: mdl-38166938

RESUMO

BACKGROUND: Juvenile Idiopathic Arthritis (JIA) induces growth disturbances in affected joints. Fibroblast-like synoviocytes (FLS) play a crucial role in JIA pathogenesis. FLS overexpress bone morphogenetic protein 4 (BMP4) and have a chondrocyte-like phenotype. FLS contribute directly to joint growth disturbances through endochondral bone formation. We investigated the ability of methotrexate to inhibit BMP4 expression and alter the hypertrophic chondrocyte-like phenotype of JIA FLS. METHODS: We selected primary cells from three subjects with persistent oligoarticular JIA, three subjects who eventually extended to a polyarticular disease course, which we termed extended-to-be (ETB), and three subjects who had polyarticular arthritis at time of diagnosis. We treated cells with methotrexate and two BMP4 inhibitors: noggin and chordin. We measured protein concentration from three chondrocyte cell markers: collagen II, aggrecan, and collagen X as well as BMP4. RESULTS: ColX, marker of chondrocyte hypertrophy, was significantly increased in polyarticular FLS when compared to both persistent FLS and ETB FLS, making polyarticular FLS the most like hypertrophic chondrocytes. Methotrexate caused significant decreases in BMP4 and ColX expression in persistent, ETB, and polyarticular FLS when compared to respective untreated cells. Ligand-binding BMP4 antagonists, noggin and chordin, caused significant decreases in ColX expression in FLS from all three disease courses and significant increases in collagen II protein, an early chondrocyte marker, when compared to respective untreated cells. CONCLUSIONS: Methotrexate, the first-line therapy in the treatment of JIA, mimics BMP4 antagonists by effectively lowering BMP4 and ColX expression in FLS. Inhibiting FLS from undergoing hypertrophy could prevent these cells from contributing to joint growth disturbances via endochondral bone formation.


Assuntos
Artrite Juvenil , Proteína Morfogenética Óssea 4 , Metotrexato , Humanos , Artrite Juvenil/metabolismo , Proteína Morfogenética Óssea 4/antagonistas & inibidores , Proteína Morfogenética Óssea 4/metabolismo , Condrócitos/metabolismo , Colágeno/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patologia , Hipertrofia/metabolismo , Metotrexato/farmacologia , Fenótipo
4.
Arthritis Res Ther ; 24(1): 225, 2022 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-36167601

RESUMO

BACKGROUND: Fibroblast-like synoviocytes (FLS) play a crucial role in JIA pathogenesis; however, the mechanisms by which they contribute to disease progression are not well described. Previous studies demonstrated that rheumatoid arthritis FLS are heterogeneous, and subpopulations with transformed, aggressive phenotypes cause invasive and destructive disease activity. We employ single-cell RNA-sequencing (scRNA-seq) to investigate JIA FLS heterogeneity and gene expression that distinguishes JIA subtypes. METHODS: JIA FLS cell lines from three persistent oligoarticular, three pre-extension oligoarticular, and three polyarticular subtypes were cultured. scRNA-seq was performed by Genewiz according to 10 × Genomics Chromium protocols. SeuratR package was used for QC, analysis, and exploration of data. RESULTS: FLS are heterogeneous and have characteristics of fibroblasts, chondrocytes, and smooth muscle cells. The chondrocyte-like subpopulation is the predominant cell type and percentages of this subpopulation increase with disease severity. Despite overlapping subpopulations, the chondrocyte-like cells have unique genetic fingerprints that distinguish between JIA subtypes. LRRC15, GREM1, and GREM2 are overexpressed in chondrocyte-like cells from persistent oligoarticular JIA FLS compared to pre-extension oligoarticular JIA FLS. S100A4, TIMP3, and NBL1 are overexpressed in pre-extension oligoarticular JIA FLS compared to polyarticular JIA FLS. CRLF1, MFAP5, and TNXB are overexpressed in persistent oligoarticular JIA FLS compared to polyarticular JIA FLS. CONCLUSIONS: We found biologically relevant differences in gene expression between JIA subtypes that support a critical role for FLS in pathogenesis. We also demonstrate that gene expression within the chondrocyte-like subpopulation can be used to distinguish between these subtypes.


Assuntos
Artrite Juvenil , Sinoviócitos , Artrite Juvenil/patologia , Cromo/metabolismo , Fibroblastos/metabolismo , Humanos , Proteínas de Membrana/metabolismo , RNA/metabolismo , Análise de Célula Única , Sinoviócitos/metabolismo
5.
Pediatr Rheumatol Online J ; 19(1): 72, 2021 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-33980237

RESUMO

BACKGROUND: We examined influences of conditioned media from chondrocytes (Ch) on juvenile idiopathic arthritis synovial fibroblasts (JFLS) and potential for JFLS to undergo endochondral bone formation (EBF). METHODS: Primary cells from three control fibroblast-like synoviocytes (CFLS) and three JFLS were cultured in Ch-conditioned media and compared with untreated fibroblast-like synoviocytes (FLS). RNA was analyzed by ClariomS microarray. FLS cells cultured in conditioned media were exposed to either TGFBR1 inhibitor LY3200882 or exogenous BMP4 and compared with FLS cultured in conditioned media from Ch (JFLS-Ch). Media supernatants were analyzed by ELISA. RESULTS: In culture, JFLS downregulate BMP2 and its receptor BMPR1a while upregulating BMP antagonists (NOG and CHRD) and express genes (MMP9, PCNA, MMP12) and proteins (COL2, COLX, COMP) associated with chondrocytes. Important TGFß superfamily member gene expression (TGFBI, MMP9, COL1A1, SOX6, and MMP2) is downregulated when JFLS are cultured in Ch-conditioned media. COL2, COLX and COMP protein expression decreases in JFLS-Ch. BMP antagonist protein (NOG, CHRD, GREM, and FST) secretion is significantly increased in JFLS-Ch. Protein phosphorylation increases in JFLS-Ch exposed to exogenous BMP4, and chondrocyte-like phenotype is restored in BMP4 presence, evidenced by increased secretion of COL2 and COLX. Inhibition of TGFBR1 in JFLS-Ch results in overexpression of COL2. CONCLUSIONS: JFLS are chondrocyte-like, and Ch-conditioned media can abrogate this phenotype. The addition of exogenous BMP4 causes JFLS-Ch to restore this chondrocyte-like phenotype, suggesting that JFLS create a microenvironment favorable for endochondral bone formation, thereby contributing to joint growth disturbances in juvenile idiopathic arthritis.


Assuntos
Proteína Morfogenética Óssea 4 , Transtornos do Crescimento , Osteogênese , Receptor do Fator de Crescimento Transformador beta Tipo I , Sinoviócitos/metabolismo , Proteínas da Superfamília de TGF-beta/metabolismo , Artrite Juvenil/complicações , Artrite Juvenil/metabolismo , Proteína Morfogenética Óssea 4/antagonistas & inibidores , Proteína Morfogenética Óssea 4/metabolismo , Células Cultivadas , Microambiente Celular/efeitos dos fármacos , Microambiente Celular/fisiologia , Condrócitos/fisiologia , Meios de Cultivo Condicionados/farmacologia , Regulação da Expressão Gênica , Transtornos do Crescimento/etiologia , Transtornos do Crescimento/metabolismo , Humanos , Osteogênese/efeitos dos fármacos , Osteogênese/fisiologia , Receptor do Fator de Crescimento Transformador beta Tipo I/antagonistas & inibidores , Receptor do Fator de Crescimento Transformador beta Tipo I/metabolismo , Transdução de Sinais/efeitos dos fármacos
6.
Pediatr Rheumatol Online J ; 18(1): 89, 2020 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-33198759

RESUMO

BACKGROUND: To examine critical interactions between juvenile idiopathic arthritis synovial fibroblasts (JFLS) and chondrocytes (Ch), and their role in bony overgrowth seen in patients with juvenile idiopathic arthritis (JIA). METHODS: Control (CFLS) and JFLS were cultured in synoviocyte media containing recombinant BMP4. Ch were cultured in either CFLS or JFLS conditioned-media without stimulation. Media supernatants were analyzed by ELISA. RNA from conditioned media experiment was analyzed by ClariomS microarray. RESULTS: As expected, genes expressed in untreated JFLS and CFLS cultured in synoviocyte media were similar to each other and this expression differed from untreated Ch cultured in chondrocyte media. JFLS favor BMP ligand gene expression while downregulating TGFß receptors' expression. Noggin and chordin, antagonists with high affinity for BMP4, are JFLS- but not Ch-preferred regulators of BMP signaling. Compared to Ch, JFLS overexpress collagen X (COLX), a marker of chondrocyte hypertrophy. Exogenous BMP4 causes JFLS to significantly decrease expression of noggin and collagen II (COL2), a marker of chondrocyte proliferation, and causes overexpression of COLX and alkaline-phosphatase (ALP). Chondrocytes cultured in JFLS-conditioned media (Ch-JFLS) express BMP genes and favor chordin protein expression over other antagonists. Ch-JFLS have significantly increased expression of COL2 and significantly decreased expression of COLX. CONCLUSIONS: These data suggest JFLS, in the presence of BMP4, undergo hypertrophy and that JFLS-conditioned media influence chondrocytes to become highly proliferative. To the authors' knowledge, no prior study has shown that JFLS and chondrocytes play a direct role in the bony overgrowth in joints of patients with JIA and that BMPs or regulation of these growth factors influence the interaction between two prominent synovial cell types.


Assuntos
Artrite Juvenil , Proteína Morfogenética Óssea 4 , Proteínas de Transporte , Condrócitos , Hiperostose/metabolismo , Sinoviócitos , Artrite Juvenil/metabolismo , Artrite Juvenil/patologia , Proteína Morfogenética Óssea 4/genética , Proteína Morfogenética Óssea 4/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Comunicação Celular , Diferenciação Celular/genética , Células Cultivadas , Condrócitos/metabolismo , Condrócitos/patologia , Colágeno/metabolismo , Regulação da Expressão Gênica , Humanos , Sinoviócitos/metabolismo , Sinoviócitos/patologia
7.
Pediatr Rheumatol Online J ; 16(1): 3, 2018 Jan 08.
Artigo em Inglês | MEDLINE | ID: mdl-29310668

RESUMO

BACKGROUND: Our intent was to identify differences between the transcriptome of fibroblast-like synoviocytes (FLS) in oligoarticular juvenile idiopathic arthritis (JIA) before extension when compared to persistent subtype of JIA, when the two are clinically indistinguishable. Additionally, we sought to determine if differences between the transcriptomes of FLS from extended-to-be and polyarticular course JIA could be detected. Our hypothesis was that intrinsic differences in the transcriptome of the FLS from extended-to-be JIA would distinguish them from persistent oligoarticular JIA, before the course is clinically apparent. METHODS: Global gene expression was defined in cultured FLS from 6 controls, 12 JIA with persistent course, 7 JIA prior to extension (extended-to-be), 4 JIA with extended course and 6 polyarticular onset, using Affymetrix Human GeneChips 133plus2.0. RESULTS: Bioconductor Linear Models for Microarray Analysis revealed 22 probesets with differential expression between persistent and extended-to-be FLS at 15% FDR, however only 2 probesets distinguished extended-to-be from extended and none distinguished extended-to-be and polyarticular at 15% FDR. Differences in extended and polyarticular gene expression profiles were not detected. Confirmation of select genes was done on the RNA level by RT-qPCR and on the protein level in synovial fluid by ELISA. CONCLUSIONS: The transcriptome of FLS from extended-to-be juvenile idiopathic arthritis is distinct from persistent course before a clinical distinction can be made. Additionally, the transcriptome of extended-to-be and polyarticular course, including those who have already extended, are indistinguishable. These gene expression data suggest that FLS already reflect a polyarticular behavior early in disease course, suggesting that extended-to-be may be "latent polyarticular" at onset. These differences can be used to develop early biomarkers of disease course, allowing for better-informed treatment decisions.


Assuntos
Artrite Juvenil/metabolismo , Biomarcadores/metabolismo , Sinoviócitos/metabolismo , Adolescente , Artrite Juvenil/diagnóstico , Artrite Juvenil/genética , Técnicas de Cultura de Células , Criança , Pré-Escolar , Progressão da Doença , Ensaio de Imunoadsorção Enzimática , Feminino , Humanos , Masculino , Análise em Microsséries , Reação em Cadeia da Polimerase em Tempo Real , Líquido Sinovial/citologia , Transcriptoma/genética
8.
Proteomics Clin Appl ; 11(5-6)2017 05.
Artigo em Inglês | MEDLINE | ID: mdl-28012239

RESUMO

PURPOSE: The goal is to investigate the specific contribution of fibroblast-like synoviocytes (FLS) to the inflammatory milieu of the synovium in juvenile idiopathic arthritis (JIA) through detection of secreted proteins. EXPERIMENTAL DESIGN: Expression of 89 cytokines and chemokines is determined on unprocessed synovial fluid from controls and JIA patients using antibody arrays. Supernatants from pure cell cultures of FLS grown from synovial fluids or tissues from JIA and controls are also examined for protein expression. Ingenuity Pathway Analysis (IPA) is revealed top pathways and upstream regulators of significant proteins. RESULTS: Protein studies is revealed that JIA FLS release pro-inflammatory cytokines and chemokines, including IL-4, IL-6, IL-17, CXCL1, and CXCL6, and lose expression of important regulator signals, such as IL-10 and TIMP2. Of the 84 proteins differentially expressed between controls and JIA in the synovial fluid, 1/3 (29 proteins) are differentially expressed in the cell culture supernatants of JIA and control FLS. ELISA of cell culture supernatants and synovial fluid confirmed seven key proteins. CONCLUSION AND CLINICAL RELEVANCE: JIA FLS are central to perpetuation of inflammation in JIA, including trafficking of inflammatory cells and effects on the extracellular matrix. These cells express key disease-specific chemokines that, with further refinement, may allow us to tailor therapy appropriately.


Assuntos
Artrite Juvenil/metabolismo , Artrite Juvenil/patologia , Quimiocinas/metabolismo , Fibroblastos/patologia , Transdução de Sinais , Sinoviócitos/metabolismo , Sinoviócitos/patologia , Adolescente , Quimiocinas/biossíntese , Criança , Pré-Escolar , Feminino , Humanos , Inflamação/metabolismo , Masculino , NF-kappa B/metabolismo , Líquido Sinovial/metabolismo
9.
Arthritis Rheumatol ; 66(5): 1352-62, 2014 May.
Artigo em Inglês | MEDLINE | ID: mdl-24782191

RESUMO

OBJECTIVE: This study was designed to investigate the pathogenic contributions of fibroblast-like synoviocytes (FLS) to juvenile idiopathic arthritis (JIA) by identifying pathways with dysregulated gene expression in FLS from patients with oligoarticular JIA. METHODS: FLS were derived from synovial fluid obtained by arthrocentesis from patients with JIA undergoing intraarticular steroid injections and from orthopedic control patients. Gene expression profiles of the JIA and control FLS were obtained using the Affymetrix platform, with application of Ingenuity Pathway Analysis and Gene Set Enrichment Analysis software to define gene sets in dysregulated pathways and networks of potential pathologic relevance in this disease. Biologically relevant differentially expressed genes were confirmed by RNA and protein analysis. RESULTS: Exploration of global gene expression profiles of the JIA FLS revealed important dysregulated pathways, including the transforming growth factor ß (TGFß) signaling, as well as endochondral bone formation, cartilage formation, and ß-catenin networks. Importantly, bone morphogenetic protein 4 (BMP-4) was significantly overexpressed in the JIA FLS. FLS from patients with oligoarticular JIA exhibit a chondrocyte phenotype, as evidenced by expression of type II collagen and aggrecan. CONCLUSION: Dysregulation of the pathways involved in the pathogenesis of oligoarticular JIA were revealed through gene expression profiling. JIA FLS displayed dysregulated TGFß signaling and exhibited a hypertrophic chondrocyte phenotype. These characteristics, along with contributions from the ß-catenin network may have implications for endochondral bone formation and local growth disturbances in oligoarticular JIA. Overexpression of BMP-4 in FLS from patients with oligoarticular JIA in particular may play an important role in disease pathogenesis, with a direct effect on functional outcome and with implications for future treatment.


Assuntos
Artrite Juvenil/metabolismo , Proteína Morfogenética Óssea 4/metabolismo , Condrócitos/patologia , Hiperostose/metabolismo , Fenótipo , Transdução de Sinais/fisiologia , Membrana Sinovial/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Adolescente , Agrecanas/genética , Agrecanas/metabolismo , Artrite Juvenil/patologia , Artrite Juvenil/fisiopatologia , Proteína Morfogenética Óssea 4/genética , Estudos de Casos e Controles , Diferenciação Celular/fisiologia , Criança , Pré-Escolar , Condrócitos/metabolismo , Colágeno Tipo II/genética , Colágeno Tipo II/metabolismo , Feminino , Perfilação da Expressão Gênica , Humanos , Hiperostose/patologia , Hiperostose/fisiopatologia , Masculino , Osteogênese/fisiologia , Líquido Sinovial/metabolismo , Membrana Sinovial/patologia , Fator de Crescimento Transformador beta/genética , beta Catenina/genética , beta Catenina/metabolismo
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