Rheumatoid Arthritis Synovial Fibroblasts Research Articles

Impairment of chemical hypoxia-induced sphingosine kinase-1 expression and activation in rheumatoid arthritis synovial fibroblasts: A signature of exhaustion?

Sphingosine kinase 1 (SphK1) and 2 (SphK2) have been shown contribute to synovial inflammation in animal models of arthritis. However, low levels of intracellular sphingosine-1 phosphate (S1P) were reported in fibroblast-like synoviocytes (FLS) from patients in the end stage of rheumatoid arthritis (RA) compared to normal FLS. Moreover, the S1P receptor-mediated chemokine synthesis was altered in RAFLS in response to chemical hypoxia. Since the mechanisms responsible for low levels of intracellular S1P in RAFLS are not fully identified, we evaluated the contribution of SphKs to the S1P-induced synthesis of chemokines under conditions of chemical hypoxia. Our results show that a chemical hypoxia mimetic cobalt chloride (CoCl2) increased SphK1 expression and activation in normal FLS but not in RAFLS. Using selective inhibitors of SphKs and gene silencing approaches, we provide evidence that both SphK1 and SphK2 are involved in hypoxia-induced chemokine production in normal FLS. In contrast, only SphK2 mediates hypoxia-induced chemokine production in RAFLS. Moreover, CoCl2 increased S1P2 and S1P3 receptor mRNA levels in normal FLS but not in RAFLS. The data suggest that altered expression and/or activation of SphK1 combined with reduced induction of S1P receptor expression by CoCl2 impaired the CoCl2-mediated autocrine S1P receptor signaling loop and chemokine production in RAFLS.

Downregulated microRNA-135a ameliorates rheumatoid arthritis by inactivation of the phosphatidylinositol 3-kinase/AKT signaling pathway via phosphatidylinositol 3-kinase regulatory subunit 2.

Synovial fibroblasts (SFs) of rheumatoid arthritis (RA) are phenotypically aggressive, typically progressing into arthritic cartilage degradation. Throughout our study, we made explorations into the effects of microRNA-135a (miR-135a) on the SFs involved in RA by mediating the phosphatidylinositol 3-kinase (PI3K)/AKT signaling pathway via regulation of phosphatidylinositol 3-kinase regulatory subunit 2 (PIK3R2). The expression of PI3K was higher, the expression of PIK3R2 was lower, and AKT was phosphorylated in the RA synovial tissues, relative to the levels found in the normal synovial tissues. We predicted miR-135a to be a candidate miR targeting PIK3R2 using an online website, microRNA.org, which was verified with a dual-luciferase reporter gene assay. Subsequently, high miR-135a expression was observed in RA synovial tissues. To study the effect of the interaction between miR-135a and PIK3R2 in RA, the SFs isolated from RA samples were cultured and transfected with mimic, inhibitor, and small interfering RNA. The proliferation, invasion, and apoptosis of the SFs were detected after the transfection. The cells transfected with miR-135a inhibitor showed inhibited cell proliferation, migration, and invasion, while also displaying promoted cell apoptosis, G0/G1 cell ratio, and decreased S cell ratio, through upregulation of PIK3R2 and inactivation of the PI3K/AKT signaling pathway. These findings provided evidence that downregulation of miR-135a inhibits proliferation, migration, and invasion and promotes apoptosis of SFs in RA by upregulating the PIK3R2 coupled with inactivating the PI3K/AKT signaling pathway. The downregulation of miR-135a might be a potential target in the treatment of RA.

The Role of Extracellular Nucleic Acids in Rheumatoid Arthritis.

Chronic inflammation, synovial hyperplasia, and local hypoxia lead to synovial cell activation causing severe joint damage in chronic-inflammatory rheumatoid arthritis (RA). The proinflammatory and joint-destructive property of the increased release of extracellular nucleic acids has been demonstrated for extracellular mitochondrial DNA and oxidized DNA using an arthritis model. Microparticles derived from different cells are able to transport nucleic acids to distant cells and promote cellular activation in RA. In addition, extracellular RNA (eRNA) is present in the RA synovial lining layer, whereas eDNA could be detected in various areas of synovial tissue when compared to controls. The main source of eDNA is the formation of neutrophil extracellular traps (NETs) due to increased amounts of activated neutrophils in the synovial fluid in RA. A central cell type of joint destruction is the activated RA synovial fibroblast (RASF) characterized by increased production of proinflammatory factors, matrix-degrading enzymes, enhanced matrix adhesion, and cell migration. eRNA was shown to be released by RASF under hypoxia and RNase activity was increased in RA synovial fluid. In vitro, RNase-mediated reduction of eRNA decreased RASF adhesion to cartilage but not proliferation or adhesion to endothelial cells. In vivo, RNase1 treatment reduced RASF invasion into cartilage. Therefore, extracellular nucleic acids induced by (auto)immune responses in RA appear to promote inflammation and local joint destruction.

Activated protein C targets immune cells and rheumatoid synovial fibroblasts to prevent inflammatory arthritis in mice.

To investigate whether activated protein C (APC), a physiological anticoagulant can inhibit the inflammatory/invasive properties of immune cells and rheumatoid arthritis synovial fibroblasts (RASFs) in vitro and prevent inflammatory arthritis in murine antigen-induced arthritis (AIA) and CIA models. RASFs isolated from synovial tissues of patients with RA, human peripheral blood mononuclear cells (PBMCs) and mouse thymus cells were treated with APC or TNF-α/IL-17 and the following assays were performed: RASF proliferation and invasion by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) and cell invasion assays, respectively; cytokines and signalling molecules using ELISA or western blot; Th1 and Th17 phenotypes in human PBMCs or mouse thymus cells by flow cytometry. The in vivo effect of APC was evaluated in AIA and CIA models. In vitro, APC inhibited IL-1β, IL-17 and TNF-α production, IL-17-stimulated cell proliferation and invasion and p21 and nuclear factor κB activation in RASFs. In mouse thymus cells and human PBMCs, APC suppressed Th1 and Th17 phenotypes. In vivo, APC inhibited pannus formation, cartilage destruction and arthritis incidence/severity in both CIA and AIA models. In CIA, serum levels of IL-1β, IL-6, IL-17, TNF-α and soluble endothelial protein C receptor were significantly reduced by APC treatment. Blocking endothelial protein C receptor, the specific receptor for APC, abolished the early or preventative effect of APC in AIA. APC prevents the onset and development of arthritis in CIA and AIA models via suppressing inflammation, Th1/Th17 phenotypes and RASF invasion, which is likely mediated via endothelial protein C receptor.

Receptor targeting drug delivery strategies and prospects in the treatment of rheumatoid arthritis.

Rheumatoid arthritis (RA), a chronic inflammatory disease, is characterized by cartilage damage, bone tissue destruction, morphological changes in synovial fluids, and synovial joint inflammation. The inflamed synovial tissue has potential for passive and active targeting because of enhanced permeability and retention effect and the existence of RA synovial macrophages and fibroblasts that selectively express surface receptors such as folate receptor β, CD44 and integrin αVβ. Although there are numerous interventions in RA treatment, they are not safe and effective. Therefore, it is important to develop new drug or drug delivery systems that specifically targets inflamed/swollen joints but attenuates other possible damages to healthy tissues. Recently some receptors such as toll-like receptors (TLRs), the nucleotide-binding oligomerization domain-like receptors, and Fc-γ receptor have been identified in synovial tissue and immune cells that are involved in induction or suppression of arthritis. Analysis of the TLR pathway has moreover suggested new insights into the pathogenesis of RA. In the present paper, we have reviewed drug delivery strategies based on receptor targeting with novel ligand-anchored carriers exploiting CD44, folate and integrin αVβ as well as TLRs expressed on synovial monocytes and macrophages and antigen presenting cells, for possible active targeting in RA. TLRs could not only open a new horizon for developing new drugs but also their antagonists or humanized monoclonal antibodies that block TLRS specially TLR4 and TLR9 signaling could be used as targeting agents to antigen presenting cells and dendritic cells. As a conclusion, common conventional receptors and multifunctional ligands that arte involved in targeting receptors or developing nanocarriers with appropriate ligands for TLRs can provide profoundly targeting drug delivery systems for the effective treatment of RA.

SPACIA1/SAAL1 Deletion Results in a Moderate Delay in Collagen-Induced Arthritis Activity, along with mRNA Decay of Cyclin-dependent Kinase 6 Gene.

This study was performed to elucidate the molecular function of the synoviocyte proliferation-associated in collagen-induced arthritis (CIA) 1/serum amyloid A-like 1 (SPACIA1/SAAL1) in mice CIA, an animal model of rheumatoid arthritis (RA), and human RA-synovial fibroblasts (RASFs). SPACIA1/SAAL1-deficient mice were generated and used to create mouse models of CIA in mild or severe disease conditions. Cell cycle-related genes, whose expression levels were affected by SPACIA1/SAAL1 small interfering RNA (siRNA), were screened. Transcriptional and post-transcriptional effects of SPACIA1/SAAL1 siRNA on cyclin-dependent kinase (cdk) 6 gene expression were investigated in human RASFs. SPACIA1/SAAL1-deficient mice showed later onset and slower progression of CIA than wild-type mice in severe disease conditions, but not in mild conditions. Expression levels of cdk6, but not cdk4, which are D-type cyclin partners, were downregulated by SPACIA1/SAAL1 siRNA at the post-transcriptional level. The exacerbation of CIA depends on SPACIA1/SAAL1 expression, although CIA also progresses slowly in the absence of SPACIA1/SAAL1. The CDK6, expression of which is up-regulated by the SPACIA1/SAAL1 expression, might be a critical factor in the exacerbation of CIA.

ADAM15 in Apoptosis Resistance of Synovial Fibroblasts: Converting Fas/CD95 Death Signals Into the Activation of Prosurvival Pathways by Calmodulin Recruitment.

To investigate mechanisms underlying the capability of ADAM15 to transform FasL-mediated death-inducing signals into prosurvival activation of Src and focal adhesion kinase (FAK) in rheumatoid arthritis synovial fibroblasts (RASFs). Caspase 3/7 activity and apoptosis rate were determined in RASFs and ADAM15-transfected T/C28a4 cells upon Fas/CD95 triggering using enzyme assays and annexin V staining. Phosphorylated Src and FAK were analyzed by immunoblotting. Interactions of ADAM15 and CD95 with calmodulin (CaM), Src, or FAK were analyzed by pull-downs using CaM-Sepharose and coimmunoprecipitations with specific antibodies. Protein binding assays were performed using recombinant CaM and ADAM15. Immunofluorescence was performed to investigate subcellular colocalization of ADAM15, Fas/CD95, and CaM. The antiapoptotic effect of ADAM15 in FasL-stimulated cells was demonstrated either by increased apoptosis of cells transfected with an ADAM15 construct lacking the cytoplasmic domain compared to cells transfected with full-length ADAM15 or by reduced apoptosis resistance of RASFs upon RNA interference silencing of ADAM15. Fas ligation triggered a Ca2+ release-activated Ca2+ /calcium release-activated calcium channel protein 1 (CRAC/Orai1) channel-dependent CaM recruitment to Fas/CD95 and ADAM15 in the cell membrane. Simultaneously, Src associated with CaM was shown to become engaged in the ADAM15 complex also containing cytoplasmic-bound FAK. Accordingly, Fas ligation in RASFs led to ADAM15-dependent phosphorylation of Src and FAK, which was associated with increased survival. Pharmacologic interference with either the CaM inhibitor trifluoperazine or the CRAC/Orai inhibitor BTP-2 simultaneously applied with FasL synergistically enhanced Fas-mediated apoptosis in RASFs. ADAM15 provides a scaffold for formation of CaM-dependent prosurvival signaling complexes upon CRAC/Orai coactivation by FasL-induced death signals and a potential therapeutic target to break apoptosis resistance in RASFs.

MiR-3926 inhibits synovial fibroblasts proliferation and inflammatory cytokines secretion through targeting toll like receptor 5

Rheumatoid arthritis synovial fibroblasts (RASFs) play a key role in the pathogenesis of rheumatoid arthritis (RA). This study was aimed to investigate the effects of miR-3926 on the biological activities of RASFs. The results showed that miR-3926 was significantly down-regulated in RASFs and RA synovial tissue. Overexpression of miR-3926 significantly inhibited RASFs proliferation and decreased the secretion of inflammatory cytokines including TNF-α, IL-1β and IL-6 in RASFs. TLR5 was identified to be a direct target of miR-3926. TLR5 showed an opposite expression trends with miR-3926 in RASFs and RA synovial tissue. Overexpression of miR-3926 led to a reduction of endogenous TLR5 in RASFs, whereas down-regulation of miR-3926 increased TLR5 expression. Knocking down of TLR5 significantly inhibited RASFs proliferation and inflammatory cytokines secretion. Rescue experiments with a miR-3926-resistant variant of TLR5 showed that overexpression of TLR5 restored RASFs proliferation and inflammatory cytokines secretion in miR-3926-overexpressing RASFs. In conclusion, miR-3926 is downregulated in RA synovial tissues and its overexpression caused the inhibitory effects on RASF proliferation and inflammatory cytokines secretion by targeting TLR5. The miR-3926/TLR5 pathway may represent a novel target for prevention and treatment of RA.

Interactions between rheumatoid arthritis synovial fibroblast migration and endothelial cells.

Leukocytes travel within the circulation and enter connective tissues by interactions with endothelium of postcapillary venules mediated by cell adhesion molecules, summarized as the leukocyte adhesion cascade. In the severe combined immunodeficient (SCID) mouse model, rheumatoid arthritis (RA) synovial fibroblasts (SF) migrated to distant cartilage through the vasculature. Therefore, RASF adhesion toward endothelial cells (EC) and E- and P-selectins were analyzed. Cell-to-cell binding assays between SF and EC were performed. Interactions of SF with tumor necrosis factor α (TNFα)-activated EC or selectins were analyzed in flow adhesion assays. Immunohistochemistry for E-selectin ligand CD15s was performed. CD15s induction in RASF by human serum or media was evaluated. Wild-type and E-/-/ P-/- Selectin-SCID mice were used for inverse-wrap surgery. After laser-mediated microdissection, real-time PCR for E-/P-selectin/vascular cell adhesion molecule 1 was performed. Adhesion between SF/EC under static conditions was highest in Roswell Park Memorial Institute-cultured RASF to TNFαα-activated human umbilical vein endothelial cells (2.25-fold) and RASF adhesion was higher toward venous than arterial EC (Dulbecco's modified eagle medium P=0.0419, RPMI P=0.0119). In flow chamber assays, RASF adhesion to E-selectin was higher than to P-selectin (e.g. 0.9dyncm-2 P=0.0001). Osteoarthritis synovial fibroblasts showed lower rolling/adhesion properties (e.g. 0.5dyncm-2 , P=0.0010). RASF adhesion to TNFαα-activated EC was increased (e.g. 0.9dyncm-2 , P=0.0061). CD15s induction in RASF was strongest in RA serum. Vimentin/CD15s double-positive cells were detectable. In E-/P-selectin-deficient mice, contralateral invasion was reduced (P=0.023). E- and P-selectin, and vascular cell adhesion molecule 1 expression in EC of implants was confirmed. Our data indicate that the milieu within vessels induces CD15s which enables RASF to interact with E-selectin/EC under flow. Therefore, RASF may migrate to distant sites and leave the vasculature similarly to leukocytes.

Myostatin induces tumor necrosis factor-α expression in rheumatoid arthritis synovial fibroblasts through the PI3K-Akt signaling pathway.

In rheumatoid arthritis (RA), a chronic inflammatory disease, loss of muscle mass is an important contributor to the loss of muscle strength in RA patients. Myostatin, a myokine involved in the process of muscle hypertrophy and myogenesis, enhances osteoclast differentiation and inflammation. Here, we investigated the mechanisms of myostatin in RA synovial inflammation. We found a positive correlation between myostatin and tumor necrosis factor-α (TNF-α), a well-known proinflammatory cytokine, in RA synovial tissue. Our in vitro results also showed that myostatin dose-dependently induced TNF-α expression through the phosphatidylinositol 3-kinase (PI3K)-Akt-AP-1 signaling pathway. Myostatin treatment of human MH7A cells stimulated AP-1-induced luciferase activity and activation of the c-Jun binding site on the TNF-α promoter. Our results indicated that myostatin increases TNF-α expression via the PI3K-Akt-AP-1 signaling pathway in human RA synovial fibroblasts. Myostatin appears to be a promising target in RA therapy.

JAK/STAT Blockade Alters Synovial Bioenergetics, Mitochondrial Function, and Proinflammatory Mediators in Rheumatoid Arthritis.

To examine the effects of tofacitinib on metabolic activity, mitochondrial function, and proinflammatory mechanisms in rheumatoid arthritis (RA). Ex vivo RA synovial explants and primary RA synovial fibroblasts (RASFs) were cultured with 1 μM tofacitinib. RASF bioenergetics were assessed using an XF24 analyzer, and key metabolic genes were assessed by reverse transcription-polymerase chain reaction (RT-PCR) analysis. Mitochondrial function was assessed using specific cell fluorescent probes and by mitochondrial gene arrays. Mitochondrial mutagenesis was quantified using a mitochondrial random mutation capture assay, and lipid peroxidation was quantified by enzyme-linked immunosorbent assay (ELISA). The effect of tofacitinib on spontaneous release of proinflammatory mediators from RA whole tissue synovial explants was quantified by ELISAs/MSD multiplex assays, and metabolic markers were quantified by RT-PCR. Finally, RASF invasion, matrix degradation, and synovial outgrowths were assessed by transwell invasion/Matrigel outgrowth assays and ELISA. Tofacitinib significantly decreased mitochondrial membrane potential, mitochondrial mass, and reactive oxygen species production by RASFs and differentially regulated key mitochondrial genes. Tofacitinib significantly increased oxidative phosphorylation, ATP production, and the maximal respiratory capacity and the respiratory reserve in RASFs, an effect paralleled by a decrease in glycolysis and the genes for the key glycolytic enzymes hexokinase 2 (HK2), glycogen synthase kinase 3α (GSK-3α), lactate dehydrogenase A, and hypoxia-inducible factor 1α. Tofacitinib inhibited the effect of oncostatin M (OSM) on interleukin-6 (IL-6) and monocyte chemotactic protein 1 and reversed the effects of OSM on RASF cellular metabolism. Using RA whole tissue synovial explants, we found that tofacitinib inhibited the key metabolic genes for glucose transporter 1, 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3, 3'-phosphoinositide-dependent protein kinase 1, HK2, and GSK-3α, the proinflammatory mediators IL-6, IL-8, IL-1β, intercellular adhesion molecule 1, vascular endothelial growth factor, and TIE-2, and RASF outgrowth from synovial explants, RASF invasion, and matrix metalloproteinase 1 activity. This study demonstrates that JAK/STAT signaling mediates the complex interplay between inflammation and cellular metabolism in RA pathogenesis.

E2F2 directly regulates the STAT1 and PI3K/AKT/NF-\u03baB pathways to exacerbate the inflammatory phenotype in rheumatoid arthritis synovial fibroblasts and mouse embryonic fibroblasts

BackgroundExpression of E2F transcription factor 2 (E2F2), a transcription factor related to the cell cycle, is abnormally high in rheumatoid arthritis synovial fibroblasts (RASFs). Deregulated expression of E2F2 leads to abnormal production of proinflammatory cytokines, such as interleukin (IL)-1α, IL-1β, and tumor necrosis factor (TNF)-α in RASFs. However, the underlying mechanism by which E2F2 regulates expression of IL-1α, IL-1β, and TNF-α has not been fully elucidated. This study aimed to elucidate this mechanism and confirm the pathological roles of E2F2 in rheumatoid arthritis (RA).MethodsE2f2 knockout (KO) and wild-type (WT) mice were injected with collagen to induce RA. Cytokine production was assessed by quantitative real-time polymerase chain reaction (qRT-PCR) and enzyme-linked immunosorbent assay (ELISA). Western blot and qRT-PCR were performed to evaluate the effect of E2F2 on signaling pathway activity. Chromatin immunoprecipitation (ChIP)-PCR and luciferase assays were used to detect the transcriptional activity of target genes of E2F2. Nuclear translocation of STAT1 and p65 were assayed by Western blot, co-immunoprecipitation (co-IP), and immunofluorescence experiments.ResultsThe occurrence and severity of collagen-induced arthritis were decreased in E2f2-KO mice compared with WT mice. The expression of IL-1α, IL-1β, and TNF-α was also suppressed in mouse embryonic fibroblasts (MEFs) from E2f2-KO mice and RASFs with E2F2 knocked down. Mechanistically, we found that E2F2 can upregulate the expression of STAT1 and MyD88 through direct binding to their promoters, facilitate the formation of STAT1/MyD88 complexes, and consequently activate AKT. However, silencing STAT1/MyD88 or inactivating AKT significantly attenuated the induction of IL-1α, IL-1β, and TNF-α caused by the introduction of E2F2.ConclusionsThis study confirms the pathological role of E2F2 in RA and found that the E2F2-STAT1/MyD88-Akt axis is closely related with the inflammatory phenotype in RASFs.

1,25(OH)2D3 and dexamethasone additively suppress synovial fibroblast activation by CCR6+ T helper memory cells and enhance the effect of tumor necrosis factor alpha blockade

BackgroundDespite recent improvements in the treatment of rheumatoid arthritis (RA), an insufficient treatment response and the development of treatment resistance in many patients illustrates the need for new therapeutic strategies. Chronic synovial inflammation could be suppressed by targeting RA synovial fibroblast (RASF) activation by, for example, interleukin (IL)-17A-producing CCR6+ T helper memory (memTh) cells. Here, we modulated this interaction by combining the active vitamin D metabolite 1,25(OH)2D3 with dexamethasone (DEX) and explored the potential therapeutic applications.MethodsCCR6+ memTh cells from peripheral blood mononuclear cells (PBMCs) of healthy donors or treatment-naive early RA patients were cultured alone or with RASF from established RA patients for 3 days and treated with or without 1,25(OH)2D3, DEX, or etanercept. Treatment effects were assessed using enzyme-linked immunosorbent assay (ELISA) and flow cytometry.Results1,25(OH)2D3, and to lesser extent DEX, reduced production of the pro-inflammatory cytokines IL-17A, IL-22, and interferon (IFN)γ in CCR6+ memTh cells. Tumor necrosis factor (TNF)α was only inhibited by the combination of 1,25(OH)2D3 and DEX. In contrast, DEX was the strongest inhibitor of IL-6, IL-8, and tissue-destructive enzymes in RASF. As a result, 1,25(OH)2D3 and DEX additively inhibited inflammatory mediators in CCR6+ memTh-RASF cocultures. Interestingly, low doses of mainly DEX, but also 1,25(OH)2D3, combined with etanercept better suppressed synovial inflammation in this coculture model compared with etanercept alone.ConclusionThis study suggests that 1,25(OH)2D3 and DEX additively inhibit synovial inflammation through targeting predominantly CCR6+ memTh cells and RASF, respectively. Furthermore, low doses of DEX and 1,25(OH)2D3 enhance the effect of TNFα blockade in inhibiting RASF activation, thus providing a basis to improve RA treatment.

Small RNAs detected in exosomes derived from the MH7A synovial fibroblast cell line with TNF-α stimulation.

Rheumatoid arthritis (RA) is an autoimmune disease that causes the chronic inflammation of the joints. Intercellular communication containing synovial fibroblasts seems to play a major role in RA pathogenesis. In this study, to better understand intercellular communication related to RA pathogenesis, we identified exosomal microRNAs (miRNAs) derived from synovial fibroblasts. Exosomes were collected from an RA synovial fibroblast (RASF) cell line, namely, MH7A, with or without stimulation by tumor necrosis factor alpha (TNF-α). We used small RNA sequencing to analyze the profile of small RNAs, including miRNAs, in MH7A exosomes and cells. By using differential expression analysis, we identified four miRNAs (miR-155-5p, miR-146a-5p, miR-323a-5p, and miR-1307-3p) that are upregulated in exosomes with TNF-α stimulation. The identification of miR-155-5p and miR-146a-5p which have been reported in RA patients demonstrated the validity of our experimental model. Other two miRNAs were newly identified. miR-323a-5p was predicted to target the protein encoding gene CD6, which attenuates T-cell activation signals, and miR-1307-3p was predicted to target the protein encoding gene N-myc downstream-regulated gene 2 (NDRG2), which inhibits osteoclast-related gene expression. The results suggested that these miRNAs might be involved in RA pathogenesis. We hope our results will help us understand the role of RASF exosomes in RA pathogenesis.

4-(Hydroxymethyl)catechol Extracted From Fungi in Marine Sponges Attenuates Rheumatoid Arthritis by Inhibiting PI3K/Akt/NF-κB Signaling.

Rheumatoid arthritis (RA) is a progressive autoimmune disease specific to synovial joints; it causes joint damage and other systemic abnormalities, thereby leading to physical disability and early mortality. Marine sponge-derived fungi, Pestalotiopsis sp., secrete immunosuppressive compounds in the culture broth. In the present study, we isolated 4-(hydroxymethyl)catechol (4-HMC) from these fungal species, and evaluated its anti-RA effects using a murine collagen-induced arthritis model and tumor necrosis factor-α-stimulated human RA synovial fibroblasts. Oral 4-HMC administration decreased the clinical arthritis score, paw thickness, histologic and radiologic changes, and serum IgG1 and IgG2a levels. It prevented the proliferation of helper T (Th) 1/Th17 CD4+ lymphocytes isolated from inguinal lymph nodes, thereby reducing inflammatory cytokine production in CIA mice. It decreased the expression of inflammatory mediators, including cytokines and matrix metalloproteinases (MMPs), both in vitro and in vivo. We observed that 4-HMC suppresses Th immune responses and MMP expression to inhibit inflammatory cytokine production in human RA synovial fibroblasts by modulating the PI3K/Akt/NF-κB pathway. These results verify the anti-RA potential of 4-HMC.

Apremilast Ameliorates Experimental Arthritis via Suppression of Th1 and Th17 Cells and Enhancement of CD4+Foxp3+ Regulatory T Cells Differentiation.

Apremilast is a novel phosphodiesterase 4 (PDE4) inhibitor suppressing immune and inflammatory responses. We assessed the anti-inflammatory effects of Apremilast in type II collagen (CII)-induced arthritis (CIA) mouse model. To determine whether Apremilast can ameliorate arthritis onset in this model, Apremilast was given orally at day 14 after CII immunization. Bone erosion was measured by histological and micro-computed tomographic analysis. Anti-mouse CII antibody levels were measured by enzyme-linked immunosorbent assay, and Th17, Th1 cells, and CD4+Foxp3+ regulatory T (Treg) cells were assessed by flow cytometry in the lymph nodes. Human cartilage and rheumatoid arthritis (RA) synovial fibroblasts (RASFs) implantation in the severe combined immunodeficiency mouse model of RA were used to study the role of Apremilast in the suppression of RASF-mediated cartilage destruction in vivo. Compared with untreated and vehicle control groups, we found that Apremilast therapy delayed arthritis onset and reduced arthritis scores in the CIA model. Total serum IgG, IgG1, IgG2a, and IgG2b were all decreased in the Apremilast treatment groups. Moreover, Apremilast markedly prevented the development of bone erosions in CIA mice by CT analysis. Furthermore, in the Apremilast treated group, the frequency of Th17 cells and Th1 cells was significantly decreased while Treg cells’ frequency was significantly increased. The high dose of Apremilast (25 mg/kg) was superior to low dose (5 mg/kg) in treating CIA. Apremilast treatment reduced the migratory ability of RASFs and their destructive effect on cartilage. Compared with the model group, Apremilast treatment significantly reduced the RASFs invasion cartilage scores in both primary implant and contralateral implant models. Our data suggest that Apremilast is effective in treating autoimmune arthritis and preventing the bone erosion in the CIA model, implicating its therapeutic potential in patients with RA.

Tetraspanin CD82 affects migration, attachment and invasion of rheumatoid arthritis synovial fibroblasts

Tetraspanins function as membrane adaptors altering cell-cell fusion, antigen presentation, receptor-mediated signal transduction and cell motility via interaction with membrane proteins including other tetraspanins and adhesion molecules such as integrins....

MiR-124a inhibits proliferation and invasion of rheumatoid arthritis synovial fibroblasts.

To investigate the impact of miR-124Aa on the proliferation, invasion and cytokine excretion of rheumatoid arthritis synovial fibroblasts (RASFs) in patients with rheumatoid arthritis (RA). RASFs were separated for in-vitro culture, and transfected using lipidosome that connected with chemically synthesized miR-124a mimic or miR-124a inhibitor. Then, MTT, transwell chamber, and flow-cytometry were used to detect the impact on the proliferation, invasion, and apoptosis of RASFs; RT-PCR and Western-blotting were employed to measure the effect of miR-124a on the expressions of matrixmetalloproteinase3/13 (MMP3/13) and interleukin1β (IL-1β) of RASFs. miR-124a significantly suppresses the proliferation of RASFs, while inhibits the invasion of RASFs. The flow cytometry indicated that miR-124a showed no significant effect on the apoptosis of RASFs. Finally, miR-124a downregulates the expressions of MMP3/13 and IL-1β. MiR-124a is of great significance for the onset of RA by inhibiting the proliferation and invasion of RASFs possibly through downregulating the expression of MMP3/13 and IL-1β.

A cytokine protein-protein interaction network for identifying key molecules in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a chronic inflammatory disease of the synovial joints. Though the current RA therapeutics such as disease-modifying antirheumatic drugs (DMARDs), nonsteroidal anti-inflammatory drugs (NSAIDs) and biologics can halt the progression of the disease, none of these would either dramatically reduce or cure RA. So, the identification of potential therapeutic targets and new therapies for RA are active areas of research. Several studies have discovered the involvement of cytokines in the pathogenesis of this disease. These cytokines induce signal transduction pathways in RA synovial fibroblasts (RASF). These pathways share many signal transducers and their interacting proteins, resulting in the formation of a signaling network. In order to understand the involvement of this network in RA pathogenesis, it is essential to identify the key transducers and their interacting proteins that are part of this network. In this study, based on a detailed literature survey, we have identified a list of 12 cytokines that induce signal transduction pathways in RASF. For these cytokines, we have built a signaling network using the protein-protein interaction (PPI) data that was obtained from public repositories such as HPRD, BioGRID, MINT, IntAct and STRING. By combining the network centrality measures with the gene expression data from the RA related microarrays that are available in the open source Gene Expression Omnibus (GEO) database, we have identified 24 key proteins of this signaling network. Two of these 24 are already drug targets for RA, and of the remaining, 12 have direct PPI links to some of the current drug targets of RA. Therefore, these key proteins seem to be crucial in the pathogenesis of RA and hence might be treated as potential drug targets.

Oxidative stress impairs energy metabolism in primary cells and synovial tissue of patients with rheumatoid arthritis

BackgroundIn this study, we examined the effect of oxidative stress on cellular energy metabolism and pro-angiogenic/pro-inflammatory mechanisms of primary rheumatoid arthritis synovial fibroblast cells (RASFC) and human umbilical vein endothelial cells (HUVEC).MethodsPrimary RASFC and HUVEC were cultured with the oxidative stress inducer 4-hydroxy-2-nonenal (4-HNE), and extracellular acidification rate, oxygen consumption rate, mitochondrial function and pro-angiogenic/pro-inflammatory mechanisms were assessed using the Seahorse analyser, complex I–V activity assays, random mutation mitochondrial capture assays, enzyme-linked immunosorbent assays and functional assays, including angiogenic tube formation, migration and invasion. Expression of angiogenic growth factors in synovial tissue (ST) was assessed by IHC in patients with rheumatoid arthritis (RA) undergoing arthroscopy before and after administration of tumour necrosis factor inhibitors (TNFi).ResultsIn RASFC and HUVEC, 4-HNE-induced oxidative stress reprogrammed energy metabolism by inhibiting mitochondrial basal, maximal and adenosine triphosphate-linked respiration and reserve capacity, coupled with the reduced enzymatic activity of oxidative phosphorylation complexes III and IV. In contrast, 4-HNE elevated basal glycolysis, glycolytic capacity and glycolytic reserve, paralleled by an increase in mitochondrial DNA mutations and reactive oxygen species. 4-HNE activated pro-angiogenic responses of RASFC, which subsequently altered HUVEC invasion and migration, angiogenic tube formation and the release of pro-angiogenic mediators. In vivo markers of angiogenesis (vascular endothelial growth factor, angiopoietin 2 [Ang2], tyrosine kinase receptor [Tie2]) were significantly associated with oxidative damage and oxygen metabolism in the inflamed synovium. Significant reduction in ST vascularity and Ang2/Tie2 expression was demonstrated in patients with RA before and after administration of TNFi.ConclusionsOxidative stress promotes metabolism in favour of glycolysis, an effect that may contribute to acceleration of inflammatory mechanisms and subsequent dysfunctional angiogenesis in RA.