Rheumatoid Arthritis Synovial Tissue Research Articles

TNF-α/calreticulin dual signaling induced NLRP3 inflammasome activation associated with HuR nucleocytoplasmic shuttling in rheumatoid arthritis.

The present study was undertaken to validate whether TNF-α and calreticulin (CRT) serve as dual signaling to activate nucleotide-binding oligomerization domain-, leucine-rich repeat- and pyrin domain-containing 3 (NLRP3) inflammasome in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) and HUVECs. The effect of human antigen R (HuR) in NLRP3 inflammasome activation was also explored in RA FLS. Immunofluorescence was used to determine the expression of NLRP3 and adaptor protein apoptosis associated speck-like protein containing a CARD (ASC) in RA synovial tissue and HuR location in RA FLS. Western blot and quantitative real-time PCR were employed to measure the priming effect of NLRP3 inflammasome in cells and HuR expression in synovial tissue. The concentrations of IL-1β and IL-18 were detected by enzyme linked immunosorbent assay. Immunohistochemistry was used to visualize the expression of HuR in synovial tissue. HuR knockdown in RA FLS was achieved by siRNA-mediated gene silencing. Higher expression of NLRP3 and ASC in RA synovial tissue than those in osteoarthritis was detected. The staining of NLRP3, ASC and cleaved IL-1β were observed in FLS and vascular endothelial cells in RA synovium. Expression of NLRP3 and pro-IL-1β in RA FLS and HUVECs treated with TNF-α was increased. The pro-IL-18 expression was also enhanced in HUVECs, but not in RA FLS. TNF-α/CRT dual stimulation of cells gave rise to caspase-1 p20 expression and the secretion of IL-1β. The secreted IL-18 was also elevated in HUVECs but not in RA FLS. HuR expression was significantly elevated in RA synovial tissue. TNF-α initiated the nucleocytoplasmic shuttling of HuR in both FLS and HUVECs. The knockdown of HuR in FLS incubated with TNF-α led to reduced caspase-1 p20 protein expression and further resulted in decreased secretion of IL-1β in the presence of CRT. TNF-α/CRT dual signaling induced NLRP3 inflammasome activation, which could be suppressed by HuR knockdown presumably due to the block of HuR translocating from nucleus to cytoplasma.

MiRNA-506 inhibits rheumatoid arthritis fibroblast-like synoviocytes proliferation and induces apoptosis by targetting TLR4.

Fibroblast-like synoviocytes (FLSs) play a crucial role in rheumatoid arthritis (RA) pathogenesis. While miRNA (miR)-506 has been implicated in the progression of multiple diseases, its role in RA remains to be explored. The present study evaluated the function of miR-506 in the regulation of RA-FLSs. FLSs were prepared from RA and healthy synovial tissues. The expression of miR-506 was measured by quantitative real time PCR (qRT-PCR). The effects of miR-506 on RA-FLSs proliferation and apoptosis were detected by cell counting Kit-8 and flow cytometry assays, respectively. The determination of TNF-α, IL-6, and IL-1β concentrations in RA-FLSs supernatant were done by ELISA. The levels of miR-506 were detected to be significantly lower in the synovial tissues and FLSs of RA than in the synovial tissues and FLSs of healthy controls. The miR-506 up-regulation in RA-FLSs significantly inhibited the proliferation and promoted cell cycle arrest at the G0/G1 phase. The overexpression of miR-506 induced apoptosis, along with an increase in activities of caspase-3 and -8. A target gene Toll-like receptor 4 (TLR4) under the direct regulation of miR-506 was identified through the luciferase assay, qRT-PCR and western blot analysis. Forced overexpression of TLR4 in the rescue experiments showed that TLR4 effectively reversed the effect on proliferation and apoptosis in miR-506-overexpressing RA-FLSs. Thus, miR-506 may be a potential target for RA prevention and therapy of RA.

Mast cell chymase in synovial fluid of collagen-induced-arthritis rats regulates gelatinase release and promotes synovial fibroblasts proliferation via FAK/p21 signaling pathway

Mast cells (MCs) were accumulated in synovial tissue of rheumatoid arthritis (RA) patients. MC activation releases numerous mediators including MC-chymase. Synovial fibroblast shows a dramatic hyperplasia in RA articular cavity, but the effects of MC-chymase on synovial fibroblast are unknown. In this study, the collagen-induced-arthritis (CIA) rat model was employed to evaluate the dynamic changes of MC-chymase activity and other inflammatory factors during CIA development in-vivo. The fresh primary synovial fibroblasts from normal or CIA rats were extracted to compare the differences of these two cell-types, and to investigate the effects of MC-chymase on both-types of synovial fibroblast. The data showed that the dynamic changes in chymase activity in synovial fluids of CIA rats before and after immunizations were companied with the changes of tryptase, MMP-2/-9, TNF-α, IL-6, Co-II IgG, total protein, paw-thickness and body weight in-vivo. The baseline differences in cell grow rates, expression levels of p21, FAK, MMP-9 between normal and CIA-SFB have been seen. Compared to the normal synovial fibroblasts, CIA-SFB increased the percentage of the cells transferred to S or G2/M phases in cell cycle in-vivo; CIA-SFB become more proliferative with high-expression MMP-2/9 during CIA development. MC-chymase in-vitro promoted both types of synovial fibroblast proliferation and induced cell cycle changes, but CIA-synovial fibroblasts exhibited much stronger responses to MC-chymase stimulation by increased expression levels of p21, FAK, MMP-9 which associated with cell adhesion and migration. This study might give a new insight that the activated mast cell can be an important target cell for RA treatment and suggest that MC-chymase needs well attention for therapeutic aims.

Forkhead box o3a suppresses lipopolysaccharide-stimulated proliferation and inflammation in fibroblast-like synoviocytes through regulating tripartite motif-containing protein 3.

Fibroblast-like synoviocytes (FLS), synovial tissue-specific cells, are key effector cells during the pathogenesis of rheumatoid arthritis (RA). Our previous study has shown that tripartite motif-containing protein 3 (TRIM3) overexpression inhibits the proliferation and cytokine secretion of RA FLS. Experiments with gene knockout mice have suggested the important roles of forkhead box o3a (Foxo3a) in RA pathogenesis. The present study aimed to investigate the correlation between Foxo3a and TRIM3 during RA pathogenesis. The expression of Foxo3a and TRIM3 was reduced in RA synovial tissues in comparison to healthy controls, and Foxo3a messenger RNA (mRNA) expression in RA synovial tissues correlated positively with TRIM3 mRNA expression. We found that stimulation with lipopolysaccharide (LPS) caused the downregulation of Foxo3a and TRIM3 in FLS. Foxo3a or TRIM3 overexpression significantly attenuated the promoting effects of LPS on cell proliferation and the release of tumor necrosis factor-α, interleukin-6 (IL-6), and IL-1β. In addition, Foxo3a suppressed the inhibitory effects of LPS on the mRNA and protein levels of TRIM3, as well as the activity of TRIM3 promoter. Foxo3a or TRIM3 overexpression attenuated collagen-induced arthritis in rats. Furthermore, knockdown of TRIM3 significantly suppressed the effects of Foxo3a overexpression on LPS-activated FLS. In summary, our findings suggested that Foxo3a exerted inhibitory effects on LPS-induced proliferation and inflammation through increasing TRIM3 transcription. The decreased expression of Foxo3a may contribute to the RA pathogenesis.

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.

A Disintegrin and Metalloprotease 15 is Expressed on Rheumatoid Arthritis Synovial Tissue Endothelial Cells and may Mediate Angiogenesis.

A disintegrin and metalloprotease 15 (ADAM15) is involved in several malignancies. In this study, we investigated the role of ADAM15 in rheumatoid arthritis (RA) angiogenesis. Soluble ADAM15 (s-ADAM15) in serum from RA and normal (NL) subjects was measured using ELISA. To determine membrane-anchored ADAM15 (ADAM15) expression in RA synovial tissues, immunohistochemistry was performed. To examine the role of ADAM15 in angiogenesis, we performed in vitro Matrigel assays and monocyte adhesion assays using human umbilical vein endothelial cells (HUVECs) transfected with ADAM15 siRNA. Finally, to investigate whether angiogenic mediators were affected by ADAM15, cytokines in ADAM15 siRNA-transfected HUVEC-conditioned medium were measured. ADAM15 was significantly higher in RA serum than in NL serum. ADAM15 was also expressed on RAST endothelial cells. ADAM15 siRNA-treated HUVECs had decreased EC tube formation in response to RA synovial fluids compared with non-treated HUVECs. The adhesion index of ADAM15 siRNA-transfected HUVECs was significantly lower than the adhesion index of control siRNA-transfected HUVECs. ENA-78/CXCL5 and ICAM-1 were decreased in tumor necrosis factor (TNF)-α-stimulated ADAM15 siRNA-transfected HUVEC-conditioned medium compared with TNF-α-stimulated control siRNA-transfected HUVEC-conditioned medium. These data show that ADAM15 plays a role in RA angiogenesis, suggesting that ADAM15 might be a potential target in inflammatory diseases such as RA.

Calreticulin induced endothelial ICAM-1 up-regulation associated with tristetraprolin expression alteration through PI3K/Akt/eNOS/p38 MAPK signaling pathway in rheumatoid arthritis

The present study was undertaken to determine whether extracellular calreticulin (CRT) participates in the regulation of ICAM-1in rheumatoid arthritis (RA) and further explore the potential mechanism. Our results showed that ICAM-1 and VCAM-1 levels were positively correlated with CRT levels in RA serum and synovial fluid, respectively. In RA synovial tissue, increased co-expressions of CRT and ICAM-1 in vascular endothelium and perivascular areas and elevated co-location of CRT and VCAM-1 localized predominantly to lining layer were observed compared to those in OA. In in vitro HUVECs model, enhanced ICAM-1expression and increased phosphorylation levels of Akt and eNOS were detected in the presence of CRT. Increased phosphorylated eNOS was significantly inhibited by a PI3K inhibitor LY294002 and elevated ICAM-1expression was partially blocked by the inhibitors of both PI3K and eNOS (L-NAME). It has been certified that the RNA-binding protein TTP targets AU-rich elements in the ICAM-1 3′-UTR and suppresses ICAM-1 expression. Knocking down TTP in HUVECs led to an increased induction of ICAM-1 by CRT. We have currently known that activation of p38 downstream kinase MK-2 leads to phosphorylation and inactivation of human TTP. The block of p38 MAPK/MK-2 signaling led to decreased protein expression and mRNA stability of TTP and ICAM-1. Furthermore, L-NAME and/or LY294002 pre-treated HUVECs manifested decreased p38 and MK-2 phosphorylation, which was accompanied by reduced TTP and ICAM-1 protein expression as well as decreased mRNA stability. Our results suggested that CRT could promote ICAM-1 expression in endothelial cells through PI3K/Akt/eNOS/p38 MAPK signaling mediated TTP accumulation, probably in an inactive form, which may provide a possible proinflammatory mechanism of CRT in RA.

Silencing Long Non-Coding RNA NEAT1 Attenuates Rheumatoid Arthritis Through Downregulation of microRNA-129 and microRNA-204 via the AMPK Signaling Pathway

Background: The participation of long noncoding RNAs (lncRNAs) and microRNAs (miRs) in the progression of rheumatoid arthritis (RA) continues to be a topical subject of investigation. The aim of the current study was to discuss the action of lncRNA nuclear paraspeckle assembly transcript 1 (NEAT1) in fibroblast-like synoviocyte (FLS) proliferation and synovitis in RA. Methods: A rat model of RA was established. The expression of NEAT1 in the synovial tissues of RA patients and FLSs from the RA rat model was detected by RT-qPCR. Then, dual luciferase reporter assay was done to investigate the relationship among miR-129/204 and MAPK. The binding relationship between miR-204 and lncRNA NEAT1 was evaluated by RIP assay, and miR-129 promotor methylation was determined by MSP. The expression of lncRNA NEAT1, miR-129 or miR-204 were altered in FLSs, and the extent of ERK1/2 phosphorylation was detected. Besides, FLS synovitis and proliferation were detected by ELISA and EdU assay, respectively. LncRNA NEAT1 was silenced and miR-129/miR-204 was overexpressed in RA rats to explore their roles in vivo. Results: LncRNA NEAT1 was upregulated and miR-129 and miR-204 was poorly expressed in RA synovial tissues and FLSs. MAPK1 was a target gene of both miR-129 and miR-204. LncRNA NEAT1 bound to miR-204 and promoted miR-129 promoter methylation. Silencing lncRNA NEAT1 or overexpressing miR-129/miR-204 enhanced miR-129/miR204 expression, but reduced extent of ERK1/2 phosphorylation, proliferation of FLSs and synovitis in RA. Conclusion: Collectively, silencing lncRNA NEAT1 upregulated miR-129 and miR-204 to inhibit MAPK signaling pathway so as to reduce FLS synovitis in RA. Funding Statement: This study was supported by Youth Innovation in Medical Research in Sichuan Province; Health Department of Sichuan Province and Education Department of Sichuan Province (No. 18ZB0640). Declaration of Interests: The authors declare that they have no conflict of interest. Ethics Approval Statement: All patients in this study signed informed consent. The experiment was officially recognized by the Ethics Committee of The Affiliated Hospital of Southwest Medical University, strictly in line with the Declaration of Helsinki. All animal measures were under the direction of experimental animals announced by the Ministry of Science and Technology of the People's Republic of China. This study minimized the pain of experimental animals as much as possible.

Th22 Cells Promote Osteoclast Differentiation via Production of IL-22 in Rheumatoid Arthritis.

T helper (Th) cells can differentiate into functionally distinct subsets and play a pivotal role in inflammatory and autoimmune diseases such as rheumatoid arthritis (RA). Th22 cells have been identified as a new subset secreting interleukin (IL)-22. Although elevated levels of IL-22 in the synovial fluids of RA patients were reported, its pathological roles remain unclear. Here, we demonstrated that IL-22 was characteristically produced from CD3+CD4+CC-chemokine receptor (CCR)4+CCR6+CCR10+ cells and their ability of the production of IL-22 markedly exceeded that of other Th subsets and the subset, thereby, designated Th22 cells. Th22 cells were efficiently induced by the stimulation with tumor necrosis factor-α, IL-6, and IL-1β. Th22 cells were markedly infiltrated in synovial tissue in patients with active RA, but not in patients with osteoarthritis (OA). CCL17, CCL20, and CCL28, which are chemokine ligands of CCR4, CCR6, and CCR10, respectively, were abundantly expressed in RA synovial tissue compared to OA. By in vitro Trans-well migration assay, Th22 cells efficiently migrated toward CCL28. Co-culture of Th22 cells, which were sorted from peripheral blood, with monocytes in the presence of macrophage colony-stimulating factor and receptor activator of nuclear factor (NF)-κB ligand induced osteoclasts formation more efficiently than that of either Th1 cells or Th17 cells. Furthermore, IL-22 markedly augmented osteoclast differentiation by promoting nuclear factor of activated T cells c1 expression in CD14+ monocytes. Contrarily, the addition of IFN-γ to the culture significantly decreased osteoclasts number, whereas IL-17 had marginal effects. IL-22 neutralizing antibody inhibited osteoclast formation in the co-culture of Th22 cells with CD14+ monocytes. Collectively, the results indicated that Th22 cells, which co-express chemokine receptors CCR4, CCR6, and CCR10, possess strong potency of tissue migration and accumulate into inflamed synovial tissues where the ligands such as CCL28 are highly expressed. Thus, Th22 cells have the capacity to promote osteoclast differentiation through production of IL-22 and thus play a pivotal role in bone destruction in patients with RA.

IL4-10 fusion protein: a novel immunoregulatory drug combining activities of interleukin 4 and interleukin 10.

SummaryThe objective of this study was to test the capacity of a newly developed fusion protein of interleukin 4 (IL‐4) and IL‐10 [IL4‐10 fusion protein (FP)] to shift multiple pro‐inflammatory pathways towards immune regulation, and to inhibit pro‐inflammatory activity in arthritis models. The effects of IL4‐10 FP in comparison with IL‐4, IL‐10 and IL‐4 plus IL‐10 on pro‐ and anti‐inflammatory mediators, T cells and immunoglobulin (Ig) receptors in favour of immunoregulatory activity were studied. In addition, the capacity of IL4‐10 FP to inhibit pro‐inflammatory activity in ex‐vivo and in‐vivo arthritis models was investigated. IL4‐10 FP robustly inhibited pro‐inflammatory cytokine [IL‐1β, tumour necrosis factor (TNF)‐α, IL‐6 and IL‐8] production in whole blood cultures, mediated by both the IL‐10 and the IL‐4 moiety. IL4‐10 fusion protein induced IL‐1 receptor antagonist (IL‐1RA) production and preserved soluble TNF receptor (sTNFR) levels, strongly increasing IL‐1RA/IL‐1β and sTNFR/TNF‐α ratios. In addition, IL4‐10 FP strongly inhibited T helper (Th) type 1 and 17 cytokine secretion, while maintaining FoxP3 expression and up‐regulating Th2 activity. In addition, while largely leaving expression of activating Fc gamma receptor (FcγR)I, III and Fc epsilon receptor (FcεR) unaffected, it significantly shifted the FcγRIIa/FcγRIIb ratio in favour of the inhibitory FcγRIIb. Moreover, IL4–10 FP robustly inhibited secretion of pro‐inflammatory cytokines by rheumatoid arthritis synovial tissue and suppressed experimental arthritis in mice, without inducing B cell hyperactivity. IL4‐10 fusion protein is a novel drug, signalling cells to induce immunoregulatory activity that overcomes limitations of IL‐4 and IL‐10 stand‐alone therapy, and therefore has therapeutic potential for inflammatory diseases such as rheumatoid arthritis.

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.

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.

MiR-92a inhibits fibroblast-like synoviocyte proliferation and migration in rheumatoid arthritis by targeting AKT2

Growing data have indicated that the miR-17-92 cluster is implicated in inflammatory response and rheumatoid arthritis (RA). This study was aimed to investigate the effects of miR-92a on the proliferation and migration of rheumatoid arthritis fibroblast-like synoviocytes (RA-FLSs). Our results showed that miR-92a was significantly down-regulated in RA synovial tissue and RA-FLSs, whereas the protein level of AKT2 is increased. Restoration of miR-92a suppressed the proliferation and migration of RA-FLSs. Down-regulation of miR-92a promotes proliferation and migration of normal human FLSs. Dual luciferase reporter gene assay showed that miR-92a could specifically bind with the 30UTR of AKT2 and significantly repressed the luciferase activity. Down-regulation or up-regulation of miR-92a significantly increased or decreased the protein and phosphorylation levels of AKT2. siRNA-mediated down-regulation of AKT2 significantly prevented cell proliferation and migration of RA-FLSs, which were similar to the effects induced by overexpression of miR-92a. Moreover, AKT2 overexpression rescued miR-92a-mediated suppressive effect on proliferation and migration of RA-FLS. Thus, miR-92a could inhibit the proliferation and migration of RA-FLSs through regulation of AKT2 expression.

Identification of differentially expressed genes in synovial tissue of rheumatoid arthritis and osteoarthritis in patients.

Rheumatoid arthritis (RA) and osteoarthritis (OA) are the common joints disorder in the world. Although they have showed the analogous clinical manifestation and overlapping cellular and molecular foundation, the pathogenesis of RA and OA were different. The pathophysiologic mechanisms of arthritis in RA and OA have not been investigated thoroughly. Thus, the aim of study is to identify the potential crucial genes and pathways associated with RA and OA and further analyze the molecular mechanisms implicated in genesis. First, we compared gene expression profiles in synovial tissue between RA and OA from the National Center of Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) database. Gene Expression Series (GSE) 1919, GSE55235, and GSE36700 were downloaded from the GEO database, including 20 patients of OA and 21 patients of RA. Differentially expressed genes (DEGs) including "CXCL13," "CD247," "CCL5," "GZMB," "IGKC," "IL7R," "UBD///GABBR1," "ADAMDEC1," "BTC," "AIM2," "SHANK2," "CCL18," "LAMP3," "CR1," and "IL32." Second, Gene Ontologyanalyses revealed that DEGs were significantly enriched in integral component of extracellular space, extracellular region, and plasma membrane in the molecular function group. Signaling pathway analyses indicated that DEGs had common pathways in chemokine signaling pathway, cytokine-cytokine receptor interaction, and cytosolic DNA-sensing pathway. Third, DEGs showed the complex DEGs protein-protein interactionnetwork with the Coexpression of 83.22%, Shared protein domains of 8.40%, Colocalization of 4.76%, Predicted of 2.87%, and Genetic interactions of 0.75%. In conclusion, the novel DEGs and pathways between RA and OA identified in this study may provide new insight into the underlying molecular mechanisms of RA.

Detection and characterization of bacterial nucleic acids in culture-negative synovial tissue and fluid samples from rheumatoid arthritis or osteoarthritis patients

Human intestinal microbes can mediate development of arthritis – Studies indicate that certain bacterial nucleic acids may exist in synovial fluid (SF) and could be involved in arthritis, although the underlying mechanism remains unclear. To characterize potential SF bacterial nucleic acids, we used 16S rRNA gene amplicon sequencing to assess bacterial nucleic acid communities in 15 synovial tissue (ST) and 110 SF samples from 125 patients with rheumatoid arthritis (RA) and 16 ST and 42 SF samples from 58 patients with osteoarthritis (OA). Our results showed an abundant diversity of bacterial nucleic acids in these clinical samples, including presence of Porphyromonas and Bacteroides in all 183 samples. Agrobacterium, Comamonas, Kocuria, Meiothermus, and Rhodoplanes were more abundant in synovial tissues of rheumatoid arthritis (STRA). Atopobium, Phascolarctobacterium, Rhodotorula mucilaginosa, Bacteroides uniformis, Rothia, Megasphaera, Turicibacter, Leptotrichia, Haemophilus parainfluenzae, Bacteroides fragilis, Porphyromonas, and Streptococcus were more abundant in synovial tissues of osteoarthritis (STOA). Veillonella dispar, Haemophilus parainfluenzae, Prevotella copri and Treponema amylovorum were more abundant in synovial fluid of rheumatoid arthritis (SFRA), while Bacteroides caccae was more abundant in the synovial fluid of osteoarthritis (SFOA). Overall, this study confirms existence of bacterial nucleic acids in SF and ST samples of RA and OA lesions and reveals potential correlations with degree of disease.

PIM-1 kinase is a novel regulator of proinflammatory cytokine-mediated responses in rheumatoid arthritis fibroblast-like synoviocytes.

This study investigated the expression of proviral-integration site for Moloney murine leukaemia virus (PIM) -1 kinase in RA synovium and RA fibroblast-like synoviocytes (FLSs) along with its impact on RA-FLS aggressiveness. The expression of PIM kinases was assessed in synovial tissues by immunohistochemistry and double IF. After PIM-1 inhibition using either small-interfering RNA or the chemical inhibitor AZD1208, we performed proliferation and migration assays and measured the levels of MMPs and IL-6 released from RA-FLSs under stimulation with proinflammatory cytokines (TNF-α, S100A4 and IL-6/soluble IL-6 receptor). Additionally, PIM-1-associated downstream signalling pathways were analysed by immunoblotting. Three isoforms of PIM kinases were immunodetected in the synovial tissues from patients with RA or OA. Specifically, PIM-1 and PIM-3 were upregulated in RA synovium and PIM-1 was expressed in T cells, macrophages and FLSs. Additionally, upon stimulation of RA-FLSs with TNF-α, S100A4 and IL-6/sIL-6R, PIM-1 and PIM-3, but not PIM-2, were significantly inducible. Moreover, PIM-1 knockdown or AZD1208 treatment significantly suppressed basal or cytokine-induced proliferation and migration of RA-FLS and the secretion of MMPs from stimulated RA-FLSs. PIM-1 knockdown significantly affected the phosphorylation levels of extracellular signal-regulated kinase and cAMP responsive element binding protein in RA-FLSs. PIM-1 was upregulated in RA synovial tissues and RA-FLSs and its inhibition significantly reduced the proliferation, migration and MMP production of RA-FLSs in vitro. These findings suggest PIM-1 as a novel regulator of the aggressive and invasive behaviour of RA-FLSs and indicate its potential as a target for RA treatment.

Synovial GATA1 mediates rheumatoid arthritis progression via transcriptional activation of NOS2 signaling.

Transcriptional regulation of inducible nitric oxide synthase (iNOS) is critically involved in the pathogenesis and progression of rheumatoid arthritis (RA); however, the specific transcription factors that control this process remain largely unidentified. In the present study, it was discovered that expression of the key erythroid factor, globin transcription factor 1 (GATA1), is significantly greater in human RA synovial tissues than in osteoarthritis (OA) tissues. IL 6 was found to induce synovial GATA1 expression in a signal transducer and activator of transcription 3-dependent manner. Functionally, knockdown of GATA1 expression using specific small interfering RNA treatment was found to compromise immunoreaction-elicited expression of proinflammatory cytokines and thus impair invasiveness of the human fibroblast-like synovial cell line MH7A, whereas introduction of exogenous GATA1 was found to promote production of proinflammatory cytokines, leading to greater aggressiveness of MH7A cells. Mechanistically, GATA1 acts as the transcriptional coactivator of NOS2 (the gene encoding iNOS) transcription. Collectively, these data suggest that synovial GATA1 is an essential contributor to development and exacerbation of RA, presumably by inducing NOS2 transcription.

ADAM-17 is expressed on rheumatoid arthritis fibroblast-like synoviocytes and regulates proinflammatory mediator expression and monocyte adhesion

BackgroundTo examine the expression of ADAM-17 in rheumatoid arthritis (RA) biological fluids and the role it plays in monocyte adhesion to RA fibroblast-like synoviocytes (FLSs).MethodsADAM-17 expression was measured by enzyme-linked immunosorbent assays (ELISAs) in serum from normal (NL) subjects, osteoarthritis (OA) patients, and RA patients. We also analyzed the correlation between ADAM-17 and disease activity score 28 (DAS28) in RA. To determine expression of ADAM-17 in RA synovial tissues (STs) and RA FLS, we performed immunofluorescence analyses. To determine the role of ADAM-17 in RA, we transfected RA FLSs with small interfering RNA (siRNA) against ADAM-17. THP-1 adhesion to ADAM-17 siRNA-transfected RA FLSs was measured. Finally, adhesion molecules on ADAM-17 siRNA-transfected RA FLSs were measured using cell surface ELISAs.ResultsADAM-17 in RA serum was significantly higher than that in NL and OA serum and correlated with DAS28. ADAM-17 in RA synovial fluids was higher than that in OA synovial fluids. ADAM-17 was expressed on RA cells lining STs and RA FLSs. THP-1 adhesion to ADAM-17 siRNA-transfected RA FLSs was decreased compared with that to control siRNA-transfected RA FLSs. ICAM-1 on TNF-α-stimulated ADAM-17 siRNA-transfected RA FLSs was significantly decreased compared with that on control siRNA-transfected RA FLSs.ConclusionsThese data indicate that ADAM-17 is expressed on RA STs and plays a role in RA inflammation by regulating monocyte adhesion to RA FLSs. ADAM-17 might be an important inflammatory mediator in inflammatory diseases such as RA.

Acetazolamide protects rat articular chondrocytes from IL-1β-induced apoptosis by inhibiting the activation of NF-κB signal pathway.

Because the excessive apoptosis of articular chondrocytes contributes to extracellular matrix (ECM) loss and cartilage damage in rheumatoid arthritis (RA), inhibiting chondrocyte apoptosis might be a promising strategy for RA. Aquaporin1 (AQP1) is overexpressed in RA cartilage and synovial tissues, and play a vital pathogenic role in RA development. Particularly, we previously reported that acetazolamide (AZ) as an AQP1 inhibitor suppressed secondary inflammation and promoted ECM production in cartilage of adjuvant-induced arthritis rats. Here, we investigated the antiapoptotic effect of AZ on interleukin-1β (IL-1β)-induced apoptosis, a classic in vitro model of chondrocyte apoptosis. AZ treatment could inhibit IL-1β-induced apoptosis, evidenced by increasing cell viability, relieving apoptotic nuclear morphology, decreasing apoptosis rates, and restoring mitochondrial membrane potential. Additionally, AZ reversed IL-1β-induced decrease of Bcl-2 protein and reduced IL-1β-induced increases of Bax and caspase 3 protein, accompanied by inhibiting IκBα degradation and phosphorylation in cytoplasm, reducing NF-κB p65 protein level in nucleus and preventing NF-κB p65 translocation from cytoplasm to nucleus. In conclusion, our findings indicated that AZ could effectively attenuate IL-1β-induced chondrocyte apoptosis mediated by regulating the protein levels of apoptosis-related genes and inhibiting the activation of NF-κB signal pathway, suggesting that AZ might be of potential clinical interest in RA treatment.

Hexokinase 2 as a novel selective metabolic target for rheumatoid arthritis

ObjectivesRecent studies indicate that glucose metabolism is altered in rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS). Hexokinases (HKs) catalyse the first step in glucose metabolism, and HK2 constitutes the principal HK...