RESUMO
We aimed to compare a transient receptor potential vanilloid 2 (TRPV2) agonist with a TNF inhibitor, and to test the potential of their combination in collagen-induced arthritis (CIA) as a potential future strategy for rheumatoid arthritis (RA). Following the onset of CIA DBA1/j mice were started on treatment with either vehicle, etanercept (8 mg/kg three times a week), the TRPV2 agonist O1821 (20-30 mg/kg/day), or a combination of both. Mice were scored over a 61-day period. Synovial tissues were obtained for RNA sequencing. Mice on monotherapy with either O1821 or etanercept developed milder clinical disease. The O1821 protection was observed at an earlier time-point than in the etanercept group. The combination therapy group achieved a more robust and sustained reduction in disease severity than either monotherapy group. All treatment groups had reduced scores for synovial inflammation, synovial hyperplasia, and erosive changes, compared with controls, with the combination group achieving the most significant protection. RNA sequencing and pathway analyses of synovial tissues identified pathways and processes regulated by the TRPV2 agonist, such as chemotaxis and cytokine receptor signaling, including IL6R. The combination therapy affected additional pathways not seen in the monotherapy groups. In conclusion, the TRPV2 agonist achieved an overall similar reduction in arthritis severity and histology scores as etanercept, but the combination therapy achieved a more sustained disease control and more pronounced reduction in joint damage, suggesting a potential future option for improving disease control in RA. RNA sequencing analyses identified new pathways regulated by TRPV2, and also by the combination treatment.
Assuntos
Artrite Experimental , Artrite Reumatoide , Camundongos , Animais , Etanercepte/farmacologia , Etanercepte/uso terapêutico , Etanercepte/metabolismo , Inibidores do Fator de Necrose Tumoral , Artrite Reumatoide/patologia , Membrana Sinovial/metabolismo , Artrite Experimental/tratamento farmacológico , Artrite Experimental/metabolismo , Gravidade do Paciente , Canais de Cálcio/metabolismo , Canais de Cálcio/uso terapêutico , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/metabolismo , Canais de Cátion TRPV/uso terapêuticoRESUMO
Rhesus theta defensin-1 (RTD-1), a macrocyclic immunomodulatory host defense peptide from Old World monkeys, is therapeutic in pristane-induced arthritis (PIA) in rats, a model of rheumatoid arthritis (RA). RNA-sequence (RNA-Seq) analysis was used to interrogate the changes in gene expression in PIA rats, which identified 617 differentially expressed genes (DEGs) in PIA synovial tissue of diseased rats. Upstream regulator analysis showed upregulation of gene expression pathways regulated by TNF, IL1B, IL6, proinflammatory cytokines, and matrix metalloproteases (MMPs) involved in RA. In contrast, ligand-dependent nuclear receptors like the liver X-receptors NR1H2 and NR1H3 and peroxisome proliferator-activated receptor gamma (PPARG) were downregulated in arthritic synovia. Daily RTD-1 treatment of PIA rats for 1-5 days following disease presentation modulated 340 of the 617 disease genes, and synovial gene expression in PIA rats treated 5 days with RTD-1 closely resembled the gene signature of naive synovium. Systemic RTD-1 inhibited proinflammatory upstream regulators such as TNF, IL1, and IL6 and activated antiarthritic ligand-dependent nuclear receptor pathways, including PPARG, NR1H2, and NR1H3, that were suppressed in untreated PIA rats. RTD-1 also inhibited proinflammatory responses in IL-1ß-stimulated human RA fibroblast-like synoviocytes (FLS) in vitro and diminished expression of human orthologs of disease genes that are induced in rat PIA synovium. Thus, the antiarthritic mechanisms of systemic RTD-1 include homeostatic regulation of arthritogenic gene networks in a manner that correlates temporally with clinical resolution of rat PIA.
Assuntos
Artrite Reumatoide/tratamento farmacológico , Fibroblastos/metabolismo , Mediadores da Inflamação/antagonistas & inibidores , Peptídeos Cíclicos/farmacologia , Peptídeos Cíclicos/uso terapêutico , Membrana Sinovial/metabolismo , Transcriptoma/efeitos dos fármacos , alfa-Defensinas/farmacologia , alfa-Defensinas/uso terapêutico , Animais , Artrite Reumatoide/induzido quimicamente , Artrite Reumatoide/metabolismo , Linhagem Celular , Cercopithecidae , Citocinas/genética , Modelos Animais de Doenças , Feminino , Humanos , Imunossupressores/farmacologia , RNA-Seq , Ratos , Sinoviócitos/metabolismo , Terpenos/farmacologia , Regulação para CimaRESUMO
Constitutive proteasomes (c-20S) are ubiquitously expressed cellular proteases that degrade polyubiquitinated proteins and regulate cell functions. An isoform of proteasome, the immunoproteasome (i-20S), is highly expressed in human T cells, dendritic cells (DCs), and B cells, suggesting that it could be a potential target for inflammatory diseases, including those involving autoimmunity and alloimmunity. Here, we describe DPLG3, a rationally designed, noncovalent inhibitor of the immunoproteasome chymotryptic subunit ß5i that has thousands-fold selectivity over constitutive ß5c. DPLG3 suppressed cytokine release from blood mononuclear cells and the activation of DCs and T cells, diminished accumulation of effector T cells, promoted expression of exhaustion and coinhibitory markers on T cells, and synergized with CTLA4-Ig to promote long-term acceptance of cardiac allografts across a major histocompatibility barrier. These findings demonstrate the potential value of using brief posttransplant immunoproteasome inhibition to entrain a long-term response favorable to allograft survival as part of an immunomodulatory regimen that is neither broadly immunosuppressive nor toxic.
Assuntos
Sobrevivência de Enxerto , Transplante de Coração/métodos , Imunossupressores/farmacologia , Complexo de Endopeptidases do Proteassoma/metabolismo , Inibidores de Proteassoma/farmacologia , Animais , Linhagem Celular Tumoral , Proliferação de Células , Citocinas/imunologia , Células Dendríticas/citologia , Células Dendríticas/imunologia , Células Hep G2 , Humanos , Memória Imunológica , Leucócitos Mononucleares/citologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Linfócitos T/imunologiaRESUMO
Fibroblast-like synoviocytes (FLSs) are a key cell type involved in rheumatoid arthritis (RA) progression. We previously identified the KCa1.1 potassium channel (Maxi-K, BK, Slo 1, KCNMA1) as a regulator of FLSs and found that KCa1.1 inhibition reduces disease severity in RA animal models. However, systemic KCa1.1 block causes multiple side effects. In this study, we aimed to determine whether the KCa1.1 ß1-3-specific venom peptide blocker iberiotoxin (IbTX) reduces disease severity in animal models of RA without inducing major side effects. We used immunohistochemistry to identify IbTX-sensitive KCa1.1 subunits in joints of rats with a model of RA. Patch-clamp and functional assays were used to determine whether IbTX can regulate FLSs through targeting KCa1.1. We then tested the efficacy of IbTX in ameliorating disease in two rat models of RA. Finally, we determined whether IbTX causes side effects including incontinence or tremors in rats, compared with those treated with the small-molecule KCa1.1 blocker paxilline. IbTX-sensitive subunits of KCa1.1 were expressed by FLSs in joints of rats with experimental arthritis. IbTX inhibited KCa1.1 channels expressed by FLSs from patients with RA and by FLSs from rat models of RA and reduced FLS invasiveness. IbTX significantly reduced disease severity in two rat models of RA. Unlike paxilline, IbTX did not induce tremors or incontinence in rats. Overall, IbTX inhibited KCa1.1 channels on FLSs and treated rat models of RA without inducing side effects associated with nonspecific KCa1.1 blockade and could become the basis for the development of a new treatment of RA.
Assuntos
Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/metabolismo , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Terapia de Alvo Molecular , Peptídeos/farmacologia , Bloqueadores dos Canais de Potássio/farmacologia , Venenos de Escorpião/química , Animais , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/antagonistas & inibidores , Peptídeos/uso terapêutico , Bloqueadores dos Canais de Potássio/uso terapêutico , Ratos , Sinoviócitos/efeitos dos fármacos , Sinoviócitos/metabolismoRESUMO
OBJECTIVES: While new treatments for rheumatoid arthritis (RA) have markedly improved disease control by targeting immune/inflammatory pathways, current treatments rarely induce remission, underscoring the need for therapies that target other aspects of the disease. Little is known about the regulation of disease severity and joint damage, which are major predictors of disease outcome, and might be better or complementary targets for therapy. In this study, we aimed to discover and characterise a new arthritis severity gene. METHODS: An unbiased and phenotype-driven strategy including studies of unique congenic rat strains was used to identify new arthritis severity and joint damage genes. Fibroblast-like synoviocytes (FLS) from rats and patients with RA expressing or not Huntingtin-interacting protein 1 (HIP1) were studied for invasiveness, morphology and cell signalling. HIP1 knockout mice were used in in vivo confirmatory studies. Paired t-test was used. RESULTS: DNA sequencing and subcongenic strains studied in pristane-induced arthritis identified a new amino acid changing functional variant in HIP1. HIP1 was required for the increased invasiveness of FLS from arthritic rats and from patients with RA. Knocking down HIP1 expression reduced receptor tyrosine kinase-mediated responses in RA FLS, including RAC1 activation, affecting actin cytoskeleton and cell morphology and interfering with the formation of lamellipodia, consistent with reduced invasiveness. HIP1 knockout mice were protected in KRN serum-induced arthritis and developed milder disease. CONCLUSION: HIP1 is a new arthritis severity gene and a potential novel prognostic biomarker and target for therapy in RA.
Assuntos
Artrite Experimental/patologia , Artrite Reumatoide/patologia , Proteínas de Ligação a DNA/fisiologia , Fibroblastos/fisiologia , Membrana Sinovial/patologia , Animais , Artrite Experimental/genética , Artrite Experimental/metabolismo , Artrite Reumatoide/genética , Artrite Reumatoide/metabolismo , Biomarcadores/metabolismo , Movimento Celular/fisiologia , Células Cultivadas , Proteínas de Ligação a DNA/genética , Humanos , Camundongos Knockout , Polimorfismo de Nucleotídeo Único , Prognóstico , RNA Interferente Pequeno/genética , Ratos , Receptores do Fator de Crescimento Derivado de Plaquetas/fisiologia , Transdução de Sinais , Sinoviócitos/metabolismo , Sinoviócitos/patologia , Sinoviócitos/fisiologia , Proteínas rac1 de Ligação ao GTP/fisiologiaRESUMO
Large-conductance calcium-activated potassium channel (KCa1.1; BK, Slo1, MaxiK, KCNMA1) is the predominant potassium channel expressed at the plasma membrane of rheumatoid arthritis fibroblast-like synoviocytes (RA-FLSs) isolated from the synovium of patients with RA. It is a critical regulator of RA-FLS migration and invasion and therefore represents an attractive target for the therapy of RA. However, the molecular mechanisms by which KCa1.1 regulates RA-FLS invasiveness have remained largely unknown. Here, we demonstrate that KCa1.1 regulates RA-FLS adhesion through controlling the plasma membrane expression and activation of ß1 integrins, but not α4, α5, or α6 integrins. Blocking KCa1.1 disturbs calcium homeostasis, leading to the sustained phosphorylation of Akt and the recruitment of talin to ß1 integrins. Interestingly, the pore-forming α subunit of KCa1.1 coimmunoprecipitates with ß1 integrins, suggesting that this physical association underlies the functional interaction between these molecules. Together, these data outline a new signaling mechanism by which KCa1.1 regulates ß1-integrin function and therefore invasiveness of RA-FLSs.-Tanner, M. R., Pennington, M. W., Laragione, T., Gulko, P. S., Beeton, C. KCa1.1 channels regulate ß1-integrin function and cell adhesion in rheumatoid arthritis fibroblast-like synoviocytes.
Assuntos
Artrite Reumatoide/metabolismo , Adesão Celular/fisiologia , Integrina beta1/metabolismo , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/metabolismo , Sinoviócitos/fisiologia , Cálcio/metabolismo , Regulação da Expressão Gênica/fisiologia , Humanos , Integrina beta1/genética , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/genética , Proteínas Proto-Oncogênicas c-akt/genética , Proteínas Proto-Oncogênicas c-akt/metabolismoRESUMO
Magnesium has been suggested to have anti-inflammatory properties in short-term, mostly in vitro studies. To examine the effect of dietary magnesium modifications in arthritis severity and joint damage DA rats were placed on one of three diet regimens before the induction of autoimmune pristane-induced arthritis (PIA): a 4 wk low-magnesium diet, normal diet, and a magnesium-supplemented diet. The diets were switched to a normal diet 14 days after the induction of PIA (typical time of disease onset). Arthritis severity was scored for 38 days, and joints were examined by histology and quantitative PCR for proinflammatory genes. Rats on the low-magnesium diet were significantly and reproducibly protected and had 70% lower median arthritis severity score, with preservation of normal joint histology without erosive changes. Rats on the normal or magnesium-supplemented diets were not protected and developed equally severe and erosive disease. While the dietary modifications were at disease onset (day 14 postinduction), the protective effect of the short-term low-magnesium diet persisted, suggesting a lasting effect on a critical pathogenic pathway. Rats on the low-magnesium diet had significant reduction in synovial tissue expression of IL-6, RORA, and RORC, which are genes required for the development of Th17 T cells. This study revealed a novel role for dietary magnesium in the regulation of autoimmune arthritis and opens new possibilities for the treatment of autoimmune diseases such as rheumatoid arthritis and psoriatic arthritis with short courses of dietary or drug-induced modulations of magnesium levels.
Assuntos
Artrite Reumatoide/tratamento farmacológico , Artrite Reumatoide/imunologia , Magnésio/uso terapêutico , Animais , Interleucina-6/genética , Interleucina-6/metabolismo , Masculino , Membro 1 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Membro 1 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/genética , Membro 3 do Grupo F da Subfamília 1 de Receptores Nucleares/metabolismo , Ratos , Membrana Sinovial/metabolismo , Células Th17/metabolismoRESUMO
Little is known about the regulation of arthritis severity and joint damage in rheumatoid arthritis (RA). Fibroblast-like synoviocytes (FLS) have a central role in joint damage and express increased levels of the cation channel Trpv2. We aimed at determining the role of Trpv2 in arthritis. Treatment with Trpv2-specific agonists decreased the in vitro invasiveness of FLS from RA patients and arthritic rats and mice. Trpv2 stimulation suppressed IL-1ß-induced expression of MMP-2 and MMP-3. Trpv2 agonists, including the new and more potent LER13, significantly reduced disease severity in KRN serum- and collagen-induced arthritis, and reduced histologic joint damage, synovial inflammation, and synovial blood vessel numbers suggesting anti-angiogenic activity. In this first in vivo use of Trpv2 agonists we discovered a new central role for Trpv2 in arthritis. These new compounds have the potential to become new therapies for RA and other diseases associated with inflammation, invasion, and angiogenesis.
Assuntos
Artrite/induzido quimicamente , Canais de Cálcio/metabolismo , Fibroblastos/fisiologia , Membrana Sinovial/citologia , Canais de Cátion TRPV/metabolismo , Animais , Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Canais de Cálcio/genética , Canabidiol/análogos & derivados , Canabidiol/farmacologia , Canabinoides/farmacologia , Colágeno/toxicidade , Humanos , Camundongos , Interferência de RNA , RNA Interferente Pequeno , Ratos , Membrana Sinovial/efeitos dos fármacos , Canais de Cátion TRPV/agonistas , Canais de Cátion TRPV/genética , Terpenos/toxicidade , Técnicas de Cultura de TecidosRESUMO
Receptor tyrosine kinases (RTKs) have an important role in arthritis severity and in models of rheumatoid arthritis (RA), but their regulation is not fully understood. The dual specificity phosphatase 6 (DUSP6) has been implicated in the regulation of RTK signaling, but never in the context of arthritis and autoimmunity. We used the KRN serum-induced arthritis (KSIA) model of RA and showed that DUSP6-/- mice were protected and had a 50% lower maximum arthritis score (p = 0.006) and reduced joint damage than C57BL/6 DUSP6+/+ controls. Serum levels of interleukin (IL) 10 were significantly increased (>2-fold), and IL6 decreased in DUSP6-/- mice. DUSP6-/- mice had increased numbers of IL10+ cells including Tr1 regulatory cells (p < 0.01). Introduction of the IL10-/- into DUSP6-/- (double knockout [KO]) reversed the DUSP6-/- protection. In conclusion, this study reports a pro-arthritic role for DUSP6. This discovery has the potential to generate a previously unknown target for therapies for RA and inflammatory diseases.
RESUMO
Background: Huntingtin-interacting protein-1 (HIP1) is a new arthritis severity gene implicated in the regulation of the invasive properties of rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS). These invasive properties of FLS strongly correlate with radiographic and histology damage in patients with RA and rodent models of arthritis. While HIP1 has several intracellular functions, little is known about its binding proteins, and identifying them has the potential to expand our understanding of its role in cell invasion and other disease-contributing phenotypes, and potentially identify new targets for therapy. Methods: FLS cell lines from arthritic DA (highly invasive) and from arthritis-protected congenic rats R6 (minimally invasive), which differ in an amino-acid changing HIP1 SNP, were cultured and lysed, and proteins were immunoprecipitated with an anti-HIP1 antibody. Immunoprecipitates were analyzed by mass spectrometry. Differentially detected (bound) proteins were selected for functional experiments using siRNA knockdown in human RA FLS to examine their effect in cell invasiveness, adhesion, cell migration and proliferation, and immunofluorescence microscopy. Results: Proteins detected included a few known HIP1-binding proteins and several new ones. Forty-five proteins differed in levels detected in the DA versus R6 congenic mass spectrometry analyses. Thirty-two of these proteins were knocked down and studied in vitro, with 10 inducing significant changes in RA FLS phenotypes. Specifically, knockdown of five HIP1-binding protein genes (CHMP4BL1, COPE, KIF1C, YWHAG, and YWHAH) significantly decreased FLS invasiveness. Knockdown of KIF1C also reduced RA FLS migration. The binding of four selected proteins to human HIP1 was confirmed. KIF1C colocalized with lamellipodia, and its knockdown prevented RA FLS from developing an elongated morphology with thick linearized actin fibers or forming polarized lamellipodia, all required for cell mobility and invasion. Unlike HIP1, KIF1C knockdown did not affect Rac1 signaling. Conclusion: We have identified new HIP1-binding proteins and demonstrate that 10 of them regulate key FLS phenotypes. These HIP1-binding proteins have the potential to become new therapeutic targets and help better understand the RA FLS pathogenic behavior. KIF1C knockdown recapitulated the morphologic changes previously seen in the absence of HIP1, but did not affect the same cell signaling pathway, suggesting involvement in the regulation of different processes.
Assuntos
Artrite Reumatoide , Proteínas de Ligação a DNA , Fibroblastos , Cinesinas , Fenótipo , Sinoviócitos , Animais , Humanos , Ratos , Artrite Reumatoide/metabolismo , Artrite Reumatoide/patologia , Artrite Reumatoide/genética , Movimento Celular , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Fibroblastos/metabolismo , Cinesinas/genética , Cinesinas/metabolismo , Sinoviócitos/metabolismo , Sinoviócitos/patologiaRESUMO
Rheumatoid arthritis (RA) is a common autoimmune and inflammatory disease characterized by inflammation and hyperplasia of the synovial tissues. RA pathogenesis involves multiple cell types, genes, transcription factors (TFs) and networks. Yet, little is known about the TFs, and key drivers and networks regulating cell function and disease at the synovial tissue level, which is the site of disease. In the present study, we used available RNA-seq databases generated from synovial tissues and developed a novel approach to elucidate cell type-specific regulatory networks on synovial tissue genes in RA. We leverage established computational methodologies to infer sample-specific gene regulatory networks and applied statistical methods to compare network properties across phenotypic groups (RA versus osteoarthritis). We developed computational approaches to rank TFs based on their contribution to the observed phenotypic differences between RA and controls across different cell types. We identified 18 (fibroblast-like synoviocyte), 16 (T cells), 19 (B cells) and 11 (monocyte) key regulators in RA synovial tissues. Interestingly, fibroblast-like synoviocyte (FLS) and B cells were driven by multiple independent co-regulatory TF clusters that included MITF, HLX, BACH1 (FLS) and KLF13, FOSB, FOSL1 (B cells). However, monocytes were collectively governed by a single cluster of TF drivers, responsible for the main phenotypic differences between RA and controls, which included RFX5, IRF9, CREB5. Among several cell subset and pathway changes, we also detected reduced presence of Natural killer T (NKT) cells and eosinophils in RA synovial tissues. Overall, our novel approach identified new and previously unsuspected Key driver genes (KDG), TF and networks and should help better understanding individual cell regulation and co-regulatory networks in RA pathogenesis, as well as potentially generate new targets for treatment.
Assuntos
Artrite Reumatoide , Redes Reguladoras de Genes , Membrana Sinovial , Humanos , Artrite Reumatoide/genética , Artrite Reumatoide/imunologia , Membrana Sinovial/metabolismo , Membrana Sinovial/imunologia , Membrana Sinovial/patologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Perfilação da Expressão Gênica , Biologia Computacional/métodos , Sinoviócitos/metabolismo , Osteoartrite/genética , Osteoartrite/metabolismo , Regulação da Expressão Gênica , Linfócitos B/imunologia , Linfócitos B/metabolismo , TranscriptomaRESUMO
Rheumatoid arthritis (RA) is a common autoimmune and inflammatory disease characterized by inflammation and hyperplasia of the synovial tissues. RA pathogenesis involves multiple cell types, genes, transcription factors (TFs) and networks. Yet, little is known about the TFs, and key drivers and networks regulating cell function and disease at the synovial tissue level, which is the site of disease. In the present study, we used available RNA-seq databases generated from synovial tissues and developed a novel approach to elucidate cell type-specific regulatory networks on synovial tissue genes in RA. We leverage established computational methodologies to infer sample-specific gene regulatory networks and applied statistical methods to compare network properties across phenotypic groups (RA versus osteoarthritis). We developed computational approaches to rank TFs based on their contribution to the observed phenotypic differences between RA and controls across different cell types. We identified 18,16,19,11 key regulators of fibroblast-like synoviocyte (FLS), T cells, B cells, and monocyte signatures and networks, respectively, in RA synovial tissues. Interestingly, FLS and B cells were driven by multiple independent co-regulatory TF clusters that included MITF, HLX, BACH1 (FLS) and KLF13, FOSB, FOSL1 (synovial B cells). However, monocytes were collectively governed by a single cluster of TF drivers, responsible for the main phenotypic differences between RA and controls, which included RFX5, IRF9, CREB5. Among several cell subset and pathway changes, we also detected reduced presence of NKT cell and eosinophils in RA synovial tissues. Overall, our novel approach identified new and previously unsuspected KDG, TF and networks and should help better understanding individual cell regulation and co-regulatory networks in RA pathogenesis, as well as potentially generate new targets for treatment.
RESUMO
RNA-sequencing and differential gene expression studies have significantly advanced our understanding of pathogenic pathways underlying Rheumatoid Arthritis (RA). Yet, little is known about cell-specific regulatory networks and their contributions to disease. In this study, we focused on fibroblast-like synoviocytes (FLS), a cell type central to disease pathogenesis and joint damage in RA. We used a strategy that computed sample-specific gene regulatory networks (GRNs) to compare network properties between RA and osteoarthritis FLS. We identified 28 transcription factors (TFs) as key regulators central to the signatures of RA FLS. Six of these TFs are new and have not been previously implicated in RA, and included BACH1, HLX, and TGIF1. Several of these TFs were found to be co-regulated, and BACH1 emerged as the most significant TF and regulator. The main BACH1 targets included those implicated in fatty acid metabolism and ferroptosis. The discovery of BACH1 was validated in experiments with RA FLS. Knockdown of BACH1 in RA FLS significantly affected the gene expression signatures, reduced cell adhesion and mobility, interfered with the formation of thick actin fibers, and prevented the polarized formation of lamellipodia, all required for the RA destructive behavior of FLS. This is the first time that BACH1 is shown to have a central role in the regulation of FLS phenotypes, and gene expression signatures, as well as in ferroptosis and fatty acid metabolism. These new discoveries have the potential to become new targets for treatments aimed at selectively targeting the RA FLS.
RESUMO
Cia4 is a locus on rat chromosome 7 that regulates disease severity and joint damage in models of rheumatoid arthritis, including pristane-induced arthritis (PIA). To identify molecular processes regulated by Cia4, synovial tissues from MHC-identical DA (severe erosive) and DA.F344(Cia4) congenics (mild nonerosive) rats were collected at preclinical and recent onset stages following the induction of PIA and analyzed for gene expression levels. Il6 levels were significantly higher in DA compared with congenics on day 10 (135-fold) after PIA induction (preclinical stage) and remained increased on days 14 (47.7-fold) and 18 (29.41-fold). Il6 increased before Il1b suggesting that Il6 could be driving Il1b expression and early synovial inflammation; 187 genes had significantly different expression levels and included inflammatory mediators increased in DA such Slpi (10.94-fold), Ccl7 (5.17-fold), and Litaf (2.09-fold). Syk or NF-κB activating and interacting genes, including Cd74 Ccl21, were increased in DA; 59 genes implicated in cancer-related phenotypes were increased in DA. Genes involved in cell metabolism, transport across membranes, and tissue protection such as Dgat1, Dhcr7, and Slc1a1 were increased in DA.F344(Cia4) congenics; 21 genes differentially expressed or expressed in only one of the strains were located within the Cia4 interval and could be the gene accounting for the arthritis effect. In conclusion, the Cia4 interval contains at least one new arthritis gene that regulates early Il6, Il1b expression, and other inflammatory mediators. This gene regulates the expression of cancer genes that could mediate the development of synovial hyperplasia and invasion, and cartilage and bone destruction.
Assuntos
Artrite/genética , Artrite/patologia , Loci Gênicos/genética , Interleucina-1beta/genética , Interleucina-6/genética , NF-kappa B/metabolismo , Animais , Animais Congênicos , Bovinos , Cromossomos de Mamíferos/genética , Feminino , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Redes Reguladoras de Genes , Mediadores da Inflamação/metabolismo , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Articulações/metabolismo , Articulações/patologia , Masculino , NF-kappa B/genética , Análise de Sequência com Séries de Oligonucleotídeos , Ratos , Ratos Endogâmicos F344 , Reprodutibilidade dos Testes , Membrana Sinovial/metabolismo , Membrana Sinovial/patologia , TerpenosRESUMO
Little is known about the genes regulating disease severity and joint damage in rheumatoid arthritis (RA). In the present study we analyzed the gene expression characteristics of synovial tissues from four different strains congenic for non-MHC loci that develop mild and nonerosive arthritis compared with severe and erosive DA rats. DA.F344(Cia3d), DA.F344(Cia5a), DA.ACI(Cia10), and DA.ACI(Cia25) rats developed mild arthritis compared with DA. We found 685 genes with significantly different expression between congenics and DA, independent of the specific congenic interval, suggesting that these genes represent a new nongenetic core group of mediators of arthritis severity. This core group includes genes not previously implicated or with unclear role in arthritis severity, such as Tnn, Clec4m, and Spond1 among others, increased in DA. The core genes also included Scd1, Selenbp1, and Slc7a10, increased in congenics. Genes implicated in nuclear receptor activity, xenobiotic and lipid metabolism were also increased in the congenics, correlating with protection. Several disease mediators were among the core genes reduced in congenics, including IL-6, IL-17, and Ccl2. Analyses of upstream regulators (genes, pathways, or chemicals) suggested reduced activation of Stat3 and TLR-related genes and chemicals in congenics. Additionally, cigarette smoking was among the upstream regulators activated in DA, while p53 was an upstream regulator activated in congenics. We observed congenic-specific differential expression and detection in each individual strain. In conclusion, this new nongenetically regulated core genes of disease severity or protection in arthritis should provide new insight into critical pathways and potential new environmental risk factor for arthritis.
Assuntos
Artrite Reumatoide/metabolismo , Cápsula Articular/metabolismo , Animais , Animais Congênicos , Artrite Reumatoide/genética , Artrite Reumatoide/fisiopatologia , Expressão Gênica , Masculino , Ratos , Especificidade da EspécieRESUMO
Fibroblast-like synoviocytes (FLS) play important roles in the pathogenesis of rheumatoid arthritis (RA). Potassium channels have regulatory roles in many cell functions. We have identified the calcium- and voltage-gated KCa1.1 channel (BK, Maxi-K, Slo1, KCNMA1) as the major potassium channel expressed at the plasma membrane of FLS isolated from patients with RA (RA-FLS). We further show that blocking this channel perturbs the calcium homeostasis of the cells and inhibits the proliferation, production of VEGF, IL-8, and pro-MMP-2, and migration and invasion of RA-FLS. Our findings indicate a regulatory role of KCa1.1 channels in RA-FLS function and suggest this channel as a potential target for the treatment of RA.
Assuntos
Membrana Celular/metabolismo , Regulação da Expressão Gênica , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/biossíntese , Febre Reumática/metabolismo , Líquido Sinovial/metabolismo , Membrana Sinovial/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Cálcio/metabolismo , Membrana Celular/patologia , Proliferação de Células , Precursores Enzimáticos/biossíntese , Feminino , Gelatinases/biossíntese , Células HEK293 , Homeostase , Humanos , Interleucina-8/biossíntese , Subunidades alfa do Canal de Potássio Ativado por Cálcio de Condutância Alta/antagonistas & inibidores , Masculino , Pessoa de Meia-Idade , Febre Reumática/patologia , Membrana Sinovial/patologia , Fator A de Crescimento do Endotélio Vascular/biossínteseRESUMO
Chemokines facilitate the recruitment of inflammatory cells into tissues, contributing to target organ injury in a wide range of inflammatory and autoimmune diseases. Targeting either single chemokines or chemokine receptors alters the progression of disease in animal models of rheumatoid arthritis and lupus with varying degrees of efficacy but clinical trials in humans have been less successful. Given the redundancy of chemokine-chemokine receptor interactions, targeting of more than one chemokine may be required to inhibit active inflammatory disease. To test the effects of multiple-chemokine blockade in inflammation, we generated an adenovirus expressing bovine herpesvirus 1 glycoprotein G (BHV1gG), a viral chemokine antagonist that binds to a wide spectrum of murine and human chemokines, fused to the Fc portion of murine IgG2a. Administration of the adenovirus significantly inhibited thioglycollate-induced migration of polymorphonuclear leukocytes into the peritoneal cavity of BALB/c mice and reduced both clinical severity and articular damage in K/BxN serum transfer-induced arthritis. However, treatment with BHV1gG-Ig fusion protein did not prevent monocyte infiltration into the peritoneum in the thioglycollate model and did not prevent renal monocyte infiltration or nephritis in lupus-prone NZB/W mice. These observations suggest that the simultaneous inhibition of multiple chemokines by BHV1gG has the potential to interfere with acute inflammatory responses mediated by polymorphonuclear leukocytes, but is less effective in chronic inflammatory disease mediated by macrophages.
Assuntos
Movimento Celular/imunologia , Inflamação/imunologia , Monócitos/imunologia , Neutrófilos/imunologia , Proteínas Virais/imunologia , Animais , Artrite Experimental/imunologia , Artrite Experimental/prevenção & controle , Cálcio/imunologia , Cálcio/metabolismo , Bovinos , Movimento Celular/efeitos dos fármacos , Quimiocinas/metabolismo , Herpesvirus Bovino 1/genética , Soros Imunes/imunologia , Fragmentos Fc das Imunoglobulinas/genética , Fragmentos Fc das Imunoglobulinas/imunologia , Inflamação/metabolismo , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos , Camundongos SCID , Monócitos/efeitos dos fármacos , Neutrófilos/efeitos dos fármacos , Ligação Proteica , Proteínas Recombinantes de Fusão/imunologia , Proteínas Recombinantes de Fusão/farmacologia , Tioglicolatos/imunologia , Tioglicolatos/farmacologia , Proteínas Virais/metabolismo , Proteínas Virais/farmacologiaRESUMO
OBJECTIVE: Cia3 is a locus on rat chromosome 4 that regulates severity and joint damage in collagen- and pristane-induced arthritis (CIA and PIA). This study was undertaken to refine the Cia3 gene-containing interval toward gene identification and obtain insights into its mode of action. METHODS: Five DA.F344(Cia3) subcongenic rat strains were generated and studied using the PIA and CIA models. Levels of antibodies against type II collagen (both allo- and autoantibodies) were measured. Joints and synovial tissue were collected 32 days after the induction of PIA (chronic stage) for histologic and quantitative polymerase chain reaction analysis of interleukin-1ß (IL-1ß) and matrix metalloproteinase (MMP) levels. RESULTS: Three subcongenic strains sharing the centromeric Cia3d interval were protected and 2 subcongenic strains sharing the telomeric Cia3g interval, which did not overlap with Cia3d, were also protected, developing significantly less severe CIA and PIA. Normal joint architecture was preserved in DA.F344(Cia3) and DA.F344(Cia3d) congenic rats with PIA, while DA rats had pronounced synovial hyperplasia, angiogenesis, inflammatory infiltration, and bone or cartilage erosions. The DA.F344(Cia3d) and DA.F344(Cia3g) strains had significantly lower synovial levels of IL-1ß (5-fold and nearly 2-fold, respectively [the latter not reaching statistical significance]), MMP-1 (expressed predominantly in DA rats), MMP-3 (79-fold and 8-fold, respectively), and MMP-14 (21-fold and 1.4-fold, respectively) and reduced levels of pathogenic autoantibodies against type II collagen, compared with DA rats. CONCLUSION: We have identified 2 new arthritis severity and articular damage loci within Cia3. These loci regulate pathogenic processes in 2 different models of rheumatoid arthritis, and the identification of these genes has the potential to generate new targets for therapies aimed at reducing disease severity and articular damage, and may additionally have prognostic value.
Assuntos
Artrite Experimental/imunologia , Colágeno/toxicidade , Imunossupressores/toxicidade , Terpenos/toxicidade , Animais , Animais Congênicos , Artrite Experimental/induzido quimicamente , Artrite Experimental/patologia , Autoanticorpos/imunologia , Biomarcadores/metabolismo , Colágeno/imunologia , Colágeno Tipo II/imunologia , Progressão da Doença , Expressão Gênica , Marcadores Genéticos , Interleucina-1beta/genética , Interleucina-1beta/metabolismo , Articulações/efeitos dos fármacos , Articulações/patologia , Articulações/fisiopatologia , Metaloproteases/genética , Metaloproteases/metabolismo , Locos de Características Quantitativas/genética , Ratos , Ratos Endogâmicos F344 , Índice de Gravidade de Doença , Especificidade da Espécie , Membrana Sinovial/efeitos dos fármacos , Membrana Sinovial/patologia , Membrana Sinovial/fisiopatologia , Terpenos/imunologiaRESUMO
BACKGROUND: Rheumatoid arthritis (RA) is a common autoimmune disease with emerging environmental and microbiome risk factors. The western diet is typically deficient in magnesium (Mg), and there is some evidence suggesting that Mg may have anti-inflammatory properties. But the actual role of Mg supplementation in arthritis or in T cell subsets has not been explored. METHODS: We investigated the role of a high Mg diet in two different mouse models of RA induced with the KRN serum, and collagen-induced arthritis. We also characterized the phenotypes of splenocytes, gene expression, and an extensive intestinal microbiome analyses including fecal material transplantation (FMT). FINDINGS: The high Mg diet group was significantly protected with reduced arthritis severity and joint damage, and reduced expression of IL-1ß, IL-6, and TNFα. The high Mg group also had increased numbers of Foxp3+ Treg cells and IL-10-producing T cells. The high Mg protective effect disappeared in IL-10 knockout mice. FMT from the high Mg diet mice recreated the phenotypes seen in the diet-treated mice, with reduced arthritis severity, increased Foxp3+ Treg, and increased IL-10-producing T cells. Intestinal microbiome analyses using 16S rDNA sequencing revealed diet-specific changes, including reduced levels of RA-associated Prevotella in the high Mg group, while increasing levels of Bacteroides and other bacteria associated with increased production of short-chain fatty acids. Metagenomic analyses implicated additional pathways including L-tryptophan biosynthesis and arginine deiminase. INTERPRETATION: We describe a new role for Mg in suppressing arthritis, in expanding Foxp3+ T reg cells and in the production of IL-10, and show that these effects are mediated by the intestinal microbiome. Our discoveries suggest a novel strategy for modifying the intestinal microbiome to treat RA and other autoimmune and inflammatory diseases. FUNDING: None.
Assuntos
Artrite Reumatoide , Microbioma Gastrointestinal , Camundongos , Animais , Linfócitos T Reguladores , Magnésio/metabolismo , Magnésio/farmacologia , Interleucina-10/genética , Interleucina-10/metabolismo , Citocinas/metabolismo , Artrite Reumatoide/metabolismo , Camundongos Knockout , Células Th17 , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismoRESUMO
Background: Glucose metabolism, specifically, hexokinase 2 (HK2), has a critical role in rheumatoid arthritis (RA) fibroblast-like synoviocyte (FLS) phenotype. HK2 localizes not only in the cytosol but also in the mitochondria, where it protects mitochondria against stress. We hypothesize that mitochondria-bound HK2 is a key regulator of RA FLS phenotype. Methods: HK2 localization was evaluated by confocal microscopy after FLS stimulation. RA FLSs were infected with Green fluorescent protein (GFP), full-length (FL)-HK2, or HK2 lacking its mitochondrial binding motif (HK2ΔN) expressing adenovirus (Ad). RA FLS was also incubated with methyl jasmonate (MJ; 2.5 mM), tofacitinib (1 µM), or methotrexate (1 µM). RA FLS was tested for migration and invasion and gene expression. Gene associations with HK2 expression were identified by examining single-cell RNA sequencing (scRNA-seq) data from murine models of arthritis. Mice were injected with K/BxN serum and given MJ. Ad-FLHK2 or Ad-HK2ΔN was injected into the knee of wild-type mice. Results: Cobalt chloride (CoCl2) and platelet-derived growth factor (PDGF) stimulation induced HK2 mitochondrial translocation. Overexpression of the HK2 mutant and MJ incubation reversed the invasive and migrative phenotype induced by FL-HK2 after PDGF stimulation, and MJ also decreased the expression of C-X-C Motif Chemokine Ligand 1 (CXCL1) and Collagen Type I Alpha 1 Chain (COL1A1). Of interest, tofacitinib but not methotrexate had an effect on HK2 dissociation from the mitochondria. In murine models, MJ treatment significantly decreased arthritis severity, whereas HK2FL was able to induce synovial hypertrophy as opposed to HK2ΔN. Conclusion: Our results suggest that mitochondrial HK2 regulates the aggressive phenotype of RA FLS. New therapeutic approaches to dissociate HK2 from mitochondria offer a safer approach than global glycolysis inhibition.