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Objective: Optineurin (OPTN) is an autophagy adaptor/receptor that acts as an intrinsic negative regulator of osteoclast differentiation. RANKL expressed by rheumatoid arthritis synovial fibroblasts (RASFs) is primarily responsible for the development of bone erosions in patients with RA. The aim of the present study was to explore the role of OPTN in the pathogenesis of joint destruction in RA. Methods: RASFs were left untreated or incubated with tumor necrosis factor (TNF) or interferon‐γ (IFNγ), and expression of OPTN by RASFs was analyzed by reverse transcription–quantitative polymerase chain reaction (RT‐qPCR) and Western blotting. Expression of RANKL and osteoprotegerin (OPG) was evaluated in cultures of OPTN‐reduced RASFs with or without TNF or IFNγ treatment. OPTN‐reduced RASFs were cocultured with monocytes and stained for tartrate‐resistant acid phosphatase (TRAP). IκBα, NF‐κB1, and RelA protein levels were measured to evaluate NF‐κB signaling. Expression of messenger RNA (mRNA) for matrix metalloproteinase 3 (MMP3), interleukin‐6 (IL6), GATA binding protein 3 (GATA3), carbohydrate sulfotransferase 15 (CHST15), hyaluronan synthase 1 (HAS1), and GATA1 was analyzed by RT‐qPCR. Results: In RASFs incubated with TNF or IFNγ, OPTN expression was up‐regulated and RANKL expression was increased, and these effects were further pronounced in OPTN‐reduced RASFs (all P < 0.05 versus controls). OPG mRNA levels remained unchanged. Monocytes cocultured with OPTN‐reduced RASFs differentiated to a greater extent into TRAP+ multinucleated cells compared to monocytes cocultured with control RASFs (P < 0.05). IκBα degradation and nuclear NF‐κB1 expression following TNF treatment were both prolonged in OPTN‐reduced RASFs (each P < 0.05 versus controls). MMP3 mRNA levels were up‐regulated, while GATA3, CHST15, and HAS1mRNA levels were down‐regulated in OPTN‐reduced RASFs (each P < 0.05 versus controls). Conclusion: OPTN plays a protective role in RA when it is up‐regulated in RASFs in the presence of proinflammatory cytokines. Absence of OPTN might worsen RA by generating a joint‐destructive state, as indicated by evidence of increased RANKL expression on RASFs and subsequent osteoclast differentiation. [ABSTRACT FROM AUTHOR]

The treatment of rheumatoid arthritis (RA) is now entering a new era, the era of Janus kinase (JAK) inhibitors. JAK inhibitors target multiple cytokines including IL-6 and exhibit a beneficial treatment effect in patients with RA and inadequate response to conventional synthetic or biologic disease-modifying anti-rheumatic drugs. Since the treatment effect of JAK inhibitors is promising even for patients refractory to anti-IL-6 therapy, it needs to be considered how multiple cytokines play roles in the pathogenesis of RA. It is also worth noting that an increased risk of herpes zoster is specifically related to the use of JAK inhibitors. Among cytokines targeted by JAK inhibitors, the current review focuses on IFN-γ, particularly on its role in synovial biology, autoimmunity, bone metabolism, pain, and varicella zoster virus infection. Recent studies provided new insights into IFN-γ in the pathogenesis of RA, which may account for the efficacy of JAK inhibitors. [ABSTRACT FROM AUTHOR]

We aimed to investigate the involvement of macroautophagy/autophagy in autoimmunity in rheumatoid arthritis (RA) through citrullination of VIM (vimentin) and its interaction with MHC class II in synovial fibroblasts (SFs). The cell surface expression of MHC class II and B7 costimulatory molecules on SFs was analyzed by flow cytometry after treatment with IFNG/IFN-γ (interferon gamma). Intracellular citrullinated autoantigens in SFs were analyzed by immunoblotting using serum from anti-citrullinated peptide antibodies (ACPA)-positive patient as a primary antibody. SFs were incubated in serum-free medium or treated with proteasome inhibitor MG132 to induce autophagy. An autophagy inhibitor 3-methyladenin (3-MA) was used. Intracellular citrullinated VIM (cVIM) was evaluated by immunoblotting and immunocytochemistry. The interaction between MHC class II and cVIM was evaluated with co-immunoprecipitation and proximity ligation assay (PLA). We demonstrated that MHC class II, CD274/B7-H1 and PDCD1LG2/B7-DC were expressed on SFs following treatment with IFNG whereas CD276/B7-H3 was detected on SFs regardless of the presence of IFNG. ACPA-positive sera recognized a 54 kDa protein in SFs. By immunoprecipitation, the 54 kDa protein recognized by RA sera was revealed to be cVIM. Following induction of autophagy, intracellular cVIM was increased in SFs but the effect was canceled by 3-MA. The interaction between MHC class II and cVIM was demonstrated by co-immunoprecipitation. Furthermore, PLA revealed the significant increase of MHC class II-cVIM interaction following induction of autophagy. Our findings suggest that SFs may contribute to the autoimmunity in RA through citrullination of VIM and its interaction with MHC class II promoted by autophagy. Abbreviations: 3-MA: 3-methyladenine; ACPA: anti-citrullinated peptide antibodies; anti-CCP: anti-cyclic citrullinated peptide antibody; cVIM: citrullinated VIM; BECN1: beclin1; DAPI: 4ʹ,6-diamidino-2-phenylindole; FBS: fetal bovine serum; HLA: human leukocyte antigen; IFNG/IFN-γ: interferon gamma; IL6: interleukin 6; IP: immunoprecipitation; MAP1LC3/LC3: microtubule associated protein 1 light chain 3; MFI: mean fluorescence index; MHC: major histocompatibility complex; OA: osteoarthritis; PADI: peptidyl arginine deiminase; PepA: pepstatin A; PBS: phosphate-buffered saline; PtdIns3K: phosphatidylinositol 3-kinase; RA: rheumatoid arthritis; SFs: synovial fibroblasts; siRNA: small interfering RNA; VIM: vimentin [ABSTRACT FROM AUTHOR]

Ultrasonography is useful for assessment of synovitis in the hand of rheumatoid arthritis (RA) patients. The aim of this study was to investigate the predictive value of the quantitative power Doppler (PD) signal assessment in the subchondral bone region of the metacarpophalangeal (MCP) joint in patients with RA showing radiographic progression of the hand by comparing with those of previously reported scoring systems. Twenty-two patients (20 women) with RA who underwent power Doppler ultrasonography (PDUS) of the bilateral one to five MCP joints at baseline were included in the study. Radiography of both hands was performed at baseline and at 1 year. PDUS of the synovial space was evaluated according to semi-quantitative scoring (0–3) and quantitative measurement (0–100%). The PD signal in the subchondral bone region was qualitatively (0, 1) and quantitatively (mm2) assessed. The performance of PDUS assessment was compared using the area under the curve (AUC) of the receiver operating characteristic (ROC) curve and the risk ratio (RR). As a predictor for radiographic progression, the quantitative PD signal assessment in the subchondral bone region (AUC = 0.842, p < 0.01) was equivalent to quantitative vascularity (AUC = 0.817, p < 0.05) and semi-quantitative scoring (AUC = 0.754, p < 0.05). As for the RR of the PD signal in the subchondral bone region for radiographic progression, the quantitative PD signal assessment was 5.40 (p < 0.01), whereas the qualitative PD signal assessment was 1.60 (p = 0.204). Quantitative PD signal assessment in the subchondral bone region can predict radiographic progression in the hand of RA patients. [ABSTRACT FROM AUTHOR]

Brain activity is reported to be associated with individual pain susceptibility and inflammatory status, possibly contributing to disease activity assessment in inflammatory arthritis (IA) including rheumatoid arthritis (RA) and spondyloarthritis (SpA). However, what alteration of brain function associated with disease activity and therapeutic effectiveness in IA remains unclear. We aimed to identify the alterations of brain functional connectivity (FC) shared in both RA and SpA, and evaluate its relationship to anti-rheumatic treatment response using functional magnetic resonance imaging (MRI). Structural and resting-state functional MRI data were acquired from patients with IA, patients with osteoarthritis (OA) and heathy controls (HCs). Two datasets were adopted to derive (51 IA, 56 OA, and 17 HCs) and validate (31 IA) the observations. 33 IA patients in the derivation dataset and all the patients in validation dataset required biological treatment and were clinically evaluated before and after therapy. Via whole-brain pair-wise FC analyses, we analyzed IA-specific FC measures relevant to therapeutic response to biologics. The value of FC between left insular cortex (IC) and anterior cingulate cortex (ACC) was significantly low in IA patients compared with OA patients and HCs. We demonstrated that the FC between left anterior long insular gyrus as a subdivision of IC and ACC was significantly associated with therapeutic response to biologics regarding the improvement of patients' global assessment (PGA) in both derivation and validation datasets. Disease-specific resting-state FC provides a means to assess the therapeutic improvement of PGA and would be a clinical decision-making tool with predictability for treatment response in both RA and SpA. [Display omitted] [ABSTRACT FROM AUTHOR]

Objective To investigate the role of autophagy in the regulation of cell death in rheumatoid arthritis synovial fibroblasts (RASFs). Methods RASFs and osteoarthritis synovial fibroblasts (OASFs) were treated with thapsigargin (TG), an inducer of endoplasmic reticulum (ER) stress, and MG132, a proteasome inhibitor. Then, 3-methyladenine was used as an autophagy inhibitor and bafilomycin A1 as a lysosome inhibitor. Polyubiquitinated proteins, p62, and autophagy induction were evaluated by immunoblotting, immunofluorescence microscopy, and immunohistochemistry, respectively. OASFs were transfected with small interfering RNA targeting autophagy-linked FYVE protein (ALFY). Cell death was evaluated by flow cytometry and a caspase 3 activity assay. Results In RASFs, the induction of autophagy by TG and MG132 was increased compared to that in OASFs. Whereas autophagy promoted a caspase 3-independent induction of cell death under ER stress, autophagy had a protective role in apoptosis induced by proteasome inhibition. Treatment of RASFs with 3-methyladenine blocked TG-induced cell death. ER stress induced a strong accumulation of p62-positive polyubiquitinated protein aggregates, accompanied by the formation of large vacuoles in RASFs but not OASFs. Furthermore, TG-induced p62 protein expression was increased, whereas TG-induced ALFY expression was reduced, in RASFs compared to OASFs. ALFY knockdown promoted the accumulation of p62, the formation of polyubiquitinated protein aggregates, and cell death. Conclusion Our data provide the first evidence of a dual role of autophagy in the regulation of death pathways in RASFs. A reduced expression of ALFY and the formation of p62-positive polyubiquitinated protein aggregates promote cell death in RASFs under severe ER stress. [ABSTRACT FROM AUTHOR]

Power Doppler ultrasonography (PDUS) and MRI are independently useful to predict structural damage in patients with rheumatoid arthritis (RA). We hypothesize that there is a complementary relationship between these modalities. The aim of this study is, therefore, to investigate the usefulness of the predictive value of composite assessment of PDUS and contrast-enhanced MRI in radiographic outcomes in patients with RA. 20 patients (17 females and 3 males) with RA on disease-modifying antirheumatic drugs underwent PDUS and MRI of both hands at baseline. Radiography of the bilateral hands was performed at baseline and at 1 year. Articular synovitis on PDUS was evaluated according to quantitative measurement. Synovitis, bone marrow edema and bone erosion were scored according to the RA MRI scoring method. The changes of joint space narrowing and bone erosion on radiograph were assessed by the Sharp/van der Heijde method. We applied t-statistics to combine the assessment of quantitative PDUS with semiquantitative MRI. Structural damage progression for radiography was not correlated with any evaluations for MRI, while it showed significant correlation with synovitis on PDUS (rs = 0.597, p = 0.005). The composite assessment of both modalities (synovitis for PDUS and bone marrow edema for MRI) was correlated with structural damage progression on radiograph (rs = 0.792, p < 0.0001). Composite assessment of PDUS and MRI may have a stronger predictive value in radiographic progression than PDUS or MRI alone in RA. Composite assessment of PDUS and MRI may be an effective predictor of structural damage in RA. [ABSTRACT FROM AUTHOR]

Fibroblast-like synoviocytes (FLS) play critical roles in rheumatoid arthritis (RA). Itaconate (ITA), an endogenous metabolite derived from the tricarboxylic acid (TCA) cycle, has attracted attention because of its anti-inflammatory, antiviral, and antimicrobial effects. This study evaluated the effect of ITA on FLS and its potential to treat RA. ITA significantly decreased FLS proliferation and migration in vitro, as well as mitochondrial oxidative phosphorylation and glycolysis measured by an extracellular flux analyzer. ITA accumulates metabolites including succinate and citrate in the TCA cycle. In rats with type II collagen-induced arthritis (CIA), intra-articular injection of ITA reduced arthritis and bone erosion. Irg1 -deficient mice lacking the ability to produce ITA had more severe arthritis than control mice in the collagen antibody-induced arthritis. ITA ameliorated CIA by inhibiting FLS proliferation and migration. Thus, ITA may be a novel therapeutic agent for RA. • ITA inhibited the proliferation and migration of RA-FLS. • ITA inhibited glycolysis and mitochondrial OXPHOS in RA-FLS. • Irg1 -deficient mice had more severe arthritis than control mice in the CAIA. • Intra-articular injection of ITA ameliorated CIA in rats. [ABSTRACT FROM AUTHOR]

We investigated the effect of miR-9 on fibroblast-like synoviocytes (FLS) from RA patients and animal arthritis model. The binding of miR-9 to NF-κB1 3'UTR was analyzed by luciferase reporter assay and immunoprecipitation. ChIP assay and luciferase promoter assay were performed to identify the binding of NF-κB1 to RANKL promoter and its activity. FLS were treated with miR-9/anti-miR-9 to evaluate cell proliferation and the expression of RANKL. Therapeutic effect of intra-articular miR-9 was evaluated in type-II collagen-induced arthritis in rats. miR-9 bound to the 3'-UTR of NF-κB1 and downregulated NF-κB1. NF-κB1 bound to RANKL promoter and increased the promoter activity of RANKL. RANKL was downregulated by miR-9. Proliferation of FLS was increased by miR-9 inhibitor. miR-9 dampened experimental arthritis by lowering inflammatory state, reducing RANKL and osteoclasts formation. Our findings revealed miR-9-NF-κB1-RANKL pathway in RA-FLS, further, miR-9 ameliorated inflammatory arthritis in vivo which propose therapeutic implications of miR- 9 in RA. Known: miR-9 has lower expression in arthritis cartilage tissue. Normally, miR-9 binds to NF-κB1 or protegenin and promotes the survival and proliferation rate of chondrocyte as well as inhibits apoptosis. Loss of miR-9 will against this progress and hence reduce the cell numbers of chondrocytes [ 14 , 33 ]. However, miR-9 could also bind to MCPIP and upregulate the IL-6 expression which is the main cytokines in the progression of arthritis [ 35 ]. Bone fracture in vivo study has lower miR-9 expression which can promote the osteoclast precursor migration and increase the survival of osteoclast [ 34 ]. TNF-α induces IL-6 expression and the combination of IL-6/sIL-6R directly increases the expression of RANKL in RA-FLS [ 28 ]. Novel: In our study, we discover the role of NF-κB1 as one of the transcription factors to RANKL promoter, i.e. TNF-α and IFN-γ synergistically activates NF-κB signaling and hence upregulates RANKL expression in mRNA levels and protein levels (purple arrows). MiR-9 binds to NF-κB1 3'UTR and suppresses translational effect or cleaves NF-κB1 mRNA therefore suppresses the downstream of NF-κB1 signaling (red arrows). NF-κB1/p105 is processed by proteasome and produces NF-κB1/p50 which then enters the nucleus and acts as a transcription factor. RANKL expression and inflammation of RA are then indirectly downregulated by miR-9. Inhibiting miR-9 increases the proliferation of RA-FLS, which contributes to the pathophysiology of RA (green arrow) IL-6: interleukin-6; MCPIP: monocyte chemoattractant protein-induced protein 1; NF-κB1: nuclear factor kappa B; RA-FLS: rheumatoid arthritis fibroblast-like synoviocytes; RANKL: receptor activator nuclear factor kappa b ligand. Unlabelled Image • miR-9 down-regulates NF-κB1 in fibroblast-like synoviocytes (FLS) from RA patients. • NF-κB1 was identified as a transcription factor for RANK ligand in RA-FLS • miR-9 ameliorated collagen-induced arthritis animal model. [ABSTRACT FROM AUTHOR]