J. Jing

Short-course tocilizumab increases risk of hepatitis B virus reactivation in patients with rheumatoid arthritis: a prospective clinical observation

To investigate the impact of short‐course tocilizumab (TCZ) on hepatitis B virus (HBV) reactivation in rheumatoid arthritis (RA) patients.

Patients with Coexistence of Circulating Hepatitis B Surface Antigen and Its Antibody May Have a Strong Predisposition to Virus Reactivation During Immunosuppressive Therapy: A Hypothesis

Hepatitis B virus (HBV) reactivation is a well-recognized complication in patients who undergo immunosuppressive drug therapy. Although the recommendation of antiviral prophylaxis made by the American Gastroenterological Association in 2015 focuses on the risk stratification of different immunosuppressive drugs, risk factors for HBV reactivation are also worth identifying in clinical practice. Recent studies have shown that the uncommon serological pattern of coexistent circulating HBV surface antigen (HBsAg) and its antibody (anti-HBs) was associated with double mutations (A1762T/G1764A) in the basal core promoter (BCP) region of the HBV genome, which is critical for HBV replication. Here, we depicted rheumatoid arthritis (RA) patients with coexistent HBsAg and anti-HBs in our medical center, who developed HBV reactivation during immunosuppressive drug therapy. DNA sequencing analysis of the HBV genome revealed triple mutations (A1762T, G1764A, and T1753V) in the BCP region, which could further enhance the ability of HBV replication. Hence, a novel hypothesis is advanced for the first time that patients with coexistent HBsAg and anti-HBs may have a strong predisposition to HBV reactivation due to specific BCP mutations. This hypothesis would, if correct, justify the concurrent detection of HBsAg and anti-HBs in HBV screening in patients with rheumatic diseases and quickly recognize patients with high risk of HBV reactivation. Further controlled studies are needed to confirm this hypothesis.

FRI0023 Artesunate can inhibit migration and invasion of fibroblast-like synoviocytes via suppression of matrix metalloproteinase 9 in rheumatoid arthritis patients

Background Evidences show that antimalarial agents of artemisinin and its derivatives such as artesunate may inhibit proinflammatory cytokines secretion from human rheumatoid arthritis (RA) fibroblast-like synoviocytes (FLS) in vitro. It has also been demonstrated that artesunate may ameliorate the symptoms of arthritis and prevent joint damage in collagen induced arthritis rat, which suggests that artesunate may be used for RA treatment. Recent studies show that RA-FLS is critical for joint destruction in RA because it can migrate and attach to cartilage and bone, and then invade them by secreting proteases such as matrix metalloproteinases (MMP) 9 in RA. However, effects of artesunate on migration and invasion of RA-FLS are poorly understood. Objectives To investigated the effects of artesunate on migration and invasion of RA-FLS and its underlying mechanism. Methods Synovial tissues were obtained from active RA patients as well as osteoarthritis (OA) and noninflammatory orthopedic arthropathies (Orth.A) patients and immumohistochemical (IHC) staining were performed for MMP9 expression. FLS isolated from these patients were analyzed for MMP9 exprssion by western blot (WB) and incubated with artesunate at different concentrations (0μM, 20μM, 40μM and 60μM), methotrexate (MTX, 10nM) or hydroxychloroquine (HCQ, 20μM) for 24 hours. Effects of artesunate on migration and invasion capacity were detected by transwell and wound healing assays. MMP9 and PI3K/Akt signal transduction protein expression after artesunate treatment was measured by WB. Results (1) IHC staining showed that synovial MMP9 expressed in lining and sublining area with intense nuclear and endochylema staining in RA synovium and the percentage of MMP9+ cells was significantly higher in RA (n=32) than that in OA (n=6) or Orth.A (n=6, Figure 1A, B). (2) Migration and wound healing assays for 12 hours and invasion assay for 24 hours showed that RA-FLS possessed stronger capacity in migration and invasion than OA-FLS or Orth.A-FLS (Figure 1E, F). Artesunate inhabits the migration and invasion capacity of RA-FLS in a dose-dependent manner. MTX also has an inhibition effect on the migration and invasion of RA-FLS, but not HCQ (Figure 2A). (3) MMP9 expression in RA-FLS was significantly higher than that in OA-FLS or Orth.A-FLS (Figure 1C, D). 40μM or 60μM artesunate markedly inhibited the expression of MMP9 in RA-FLS by WB (Figure 2B). (4) WB analysis showed artesunate suppressed generation of phophso-Akt in a dose-dependent manner which indicated that Akt activity (phophso-Akt/Akt) in 40μM and 60μM artesunate treatment groups were significantly lower than that in untreated group (Figure 2C). Conclusions Artesunate could inhibit the migration and invasion capacity of RA-FLS and the expression of MMP9 through suppressing Akt activity. Acknowledgements This work was supported by National Natural Science Foundation of China (81671612 and 81471597), Research Project of Traditional Chinese Medicine Bureau of Guangdong Province (20161058) and Guangdong Natural Science Foundation (2014A030313074). Disclosure of Interest None declared

THU0025 Over-Expressed PGC-1β in CD14+ Monocytes of Rheumatoid Arthritis Patients and Its Down-Regulation Can Suppress Osteoclastogenesis and Bone Resorption through Inhibition of Cathepsin K and Trap

Background Peroxisome proliferator-activated receptor-gamma coactivator-1 (PGC-1) is a transcriptional coactivator that plays important roles in regulating energy metabolism and cytokine signaling pathways. Our previous study showed that down-regulating PGC-1β could inhibit rheumatoid arthritis fibroblast-like synoviocytes induced osteoclastogenesis by RAW264.7 (used as precursor of osteoclasts). Objectives To investigate the expression of PGC-1β in CD14+ monocytes from RA patients and its effect and underlying mechanism on osteoclastogenesis and bone resorption. Methods (1) Peripheral blood mononuclear cells (PBMCs) were collected from 3 patients with active RA and 3 healthy controls and CD14+ monocytes were selected by flow cytometry. PGC-1β expression was detected by immunofluorescence staining or flow cytometry. (2) PGC-1β in RAW264.7 was depleted by lentivirus short hairpin RNAs and then cultured with recombinant murine RANKL (50ng/ml) and M-CSF (25ng/ml). Tartrate-resistant acid phosphatase (TRAP) staining and western blot (WB) for detecting the expression of DC-STAMP, cathepsin K and TRAP were performed at day 7. Toluidine blue staining for examining resorption pits by reflected light microscopy and image analysis system was performed at day 14. (3) After PGC-1β knockdown in RAW264.7, cytoplasmic protein was extracted for WB determining the expression of JNK, p-JNK, p38, p-p38, p-ERK1/2, NFATc-1, NF-κB and IκB, while nuclear protein was extracted for WB determining the expression of PGC-1β, p- NF-κB, ERK1/2, c-jun and c-fos. Results (1) Immunofluorescence staining showed that the percentage of PGC-1β positive PBMCs in RA was significantly higher than that in healthy controls (94±3% vs. 57±7%, P=0.021, Figure 1A). Flow cytometric analyses showed that PGC-1β expression in CD14+ monocytes from RA patients was higher than that of healthy controls (Figure 1B) and the mean fluorescence intensity was significantly higher than that of healthy controls (274±23 vs. 167±15, P=0.034).(2) At day 7, the number of TRAP-positive multinucleated osteoclasts in sh-PGC-1β group was significantly lower than that in sh-GFP group (33.4 ± 3.4 vs. 192.5 ± 5.1 cells/per well, P=0.021, Figure 1C). At day 14, the pit area of bone resorption lacunae on the slices of sh-PGC-1β group was significantly reduced than that of sh-GFP group (13 ± 2 vs. 197 ± 15 μm2/per slice, P=0.012, Figure 1C). (3) WB assay showed that the protein expression of cathepsin K and TRAP,but not DC-STAMP, were obviously suppressed in sh-PGC-1β group (Figure 1D). Further analysis of signaling pathways showed the expression of NFATc1 and phosphorylate NF-κB p65 were also significantly decreased (Figure 1E). Conclusions Our results showed that over-expressed PGC-1β in CD14+ monocytes of RA patients and down-regulation of PGC-1β can suppress RANKL induced osteoclastogenesis and bone resorption through inhibition of cathepsin K and TRAP by NF-κB and NFATc1 pathway. Acknowledgement This work was supported by National Natural Science Foundation of China (81471597), Specialized Research Fund for the Doctoral Program of Higher Education (20130171110075) and Guangdong Natural Science Foundation (2014A030313074). Disclosure of Interest None declared

AB0216 Overexpression of Synovial Lining Peroxisome Proliferator-Activated Receptor-Gamma Coactivator-1β Involved in Joint Destruction through Upregulating MMP-3 in Rheumatoid Arthritis

Background Our previous study showed that suppression of peroxisome proliferator-activated receptor-gamma coactivator-1β (PGC-1β) alleviated the secretion of matrix metalloproteinase (MMP)-3 from fibroblast-like synoviocytes and inhibited osteoclastogenesis in vitro. However, little was known about PGC-1β with joint destruction in RA patients. Objectives To investigate the expression and correlation of synovial PGC-1β with joint destruction in RA patients. Methods Eighty-one patients with active RA were recruited and followed up for one year. Serum MMP3 was detected by ELISA. Synovial tissue was obtained by closed-needle biopsy for H&E and immunohistochemistry staining for PGC-1β, MMP-3, CD3, CD20, CD38, CD68 and CD15. X-ray assessment of hand/wrist was repeated at baseline and one year and radiographic progression was defined as TSS≥0.5 unit. Results (1) PGC-1β expression in RA was observed with intense nuclear staining in lining cells and sublining inflammatory cells (including macrophages, lymphocytes and plasma cells) which were significantly higher than osteoarthritis (OA) or orthopedic arthropathies (Orth.A, Fig. 1A). (2) The percentage of lining PGC-1β+ cells significantly correlated with serum and synovial MMP-3 (r=0.394 and 0.376, both P<0.01), as well as CRP, ESR, CD15+ neutrophils and CD68+ macrophages in sublining area of RA synovium (r=0.266–0.356, all P<0.05), and the percentage of sublining PGC-1β+ cells significantly correlated with CRP, ESR, total synovitis score, CD3+ T cells, CD20+ B cells and CD38+ plasma cells, CD68+ macrophages in sublining area of RA synovium (r=0.225-0.428, all P<0.05). (3) Forty-three (53%) RA patients had erosive disease (2013 EULAR definition) at baseline and their percentage of lining and sublining PGC-1β+ cells was significantly higher than non-erosive patients (both P<0.05, Fig. 1B). Both percentages of lining and sublining PGC-1β+ cells were significantly correlated with mTSS, joint space narrowing and erosion subscore (r=0.228-0.261, all P<0.05). (4) Thirty-nine patients had finished one year follow-up and 23% of them had radiographic progression. The percentage of lining PGC-1β+ cells was significantly higher in progressive patients than non-progressive patients [83% (79%–91%) vs 75% (65%–82%), P=0.002]. ROC curve analysis showed that the tradeoff value of lining PGC-1β+ cells for predicting 1-year radiographic progression was 78% with sensitivity 89% and specificity 70% (AUC=0.833, 95% CI: 0.689–0.978, P=0.003). (5) Serum and synovial MMP-3 were significantly higher in patients with high synovial lining PGC-1β than that in patients with low synovial lining PGC-1β (both P<0.05, Fig. 1C–E). Multivariate logistic regression analysis showed that high lining PGC-1β+ cells was a significant predictor of 1-year radiographic progression after adjustment for synovial and serum MMP-3 (OR: 15.002, 95% CI: 1.43–156.698, P=0.024, Fig. 1C). Conclusions Overexpression of synovial lining PGC-1β might play important roles in aggravating joint destruction through upregulating MMP-3 in RA. Acknowledgement This work was supported by National Natural Science Foundation of China (81471597), Specialized Research Fund for the Doctoral Program of Higher Education (20130171110075) and Guangdong Natural Science Foundation (2014A030313074). Disclosure of Interest None declared

SAT0048 Overexpression of CCL18 Promotes Migration and Activation of Fibroblast-like Synoviocytes in Rheumatoid Arthritis

Background CC chemokine ligand 18 (CCL18) has been reported overexpressed in numerous inflammatory disorders since 1997. However, its functional receptor, phosphatidylinositol transfer membrane-associated phosphatidylinositol transfer protein 3 (PITPNM3), has not been discovered until 2011 by our colleagues. Multiple functions of CCL18 besides its chemotactic activity were then identified, including promoting cytokine production, migration/invasion of cancer cells, type I collagen production by human fibroblasts of lung or skin. The effect on the pathogenesis of rheumatoid arthritis (RA) remains unclear. Objectives To explore the expression of CCL18 in RA patients and its effect on RA fibroblast-like synoviocytes (FLS). Methods CCL18 in serum and synovial fluid was measured by ELISA. Synovial tissue was obtained by closed-needle biopsy for H&E and immunohistochemistry staining, and FLS culture in vitro. Expression of PITPNM3 on cell membrane was detected by western blot. Cell proliferation was identified by cell counting kit (CCK-8) and migration ability was analyzed by transwell assay. Cytokines (IL-1β, IL-6, IL-17A, TNF-α, GM-CSF), chemokines (CCL2, CCL3, CCL5, CCL8) and MMP3 in culture supernatant were measured by Cytometric Bead Array (CBA) or ELISA. Results (1) Serum CCL18 of 51 active RA patients was 112 ng/mL (IQR, 85–132), which was significantly higher than healthy controls (n=11, 64 ng/mL (IQR, 36–70), p<0.001, Figure 1A). Eighteen RA patients had synovial fluid and CCL18 in synovial fluid was 1229 ng/mL (IQR, 934–1540) which was significantly higher than corresponding serum level (p<0.001) and synovial fluid CCL18 of osteoarthritis patients (n=7, 33 ng/mL (IQR, 25–229), p=0.001). (2) Immunohistochemistry staining showed CCL18-positive cells in lining and sublining area of RA synovium (n=48), which were significantly greater than osteoarthritis synovium (both p=0.001, Figure 1B∼D). Double-immunocytofluorescent staining showed vimentin-positive RA-FLS in vitro expressed PITPNM3 (Figure 2A) and western blot of membrane protein showed the expression of PITPNM3 protein on RA-FLS. (3) RA-FLS was incubated with different concentrations of CCL18 (0, 100, 250, 500 ng/mL) and CCK-8 proliferation assay showed no significant impact throughout 9 days (Figure 2B). Transwell assay showed that the migration ability of RA-FLS was significantly enhanced after stimulation of CCL18 (500 ng/mL) for 48 hours (Figure 2C). IL-6, CCL2 and MMP3 in culture supernatant were significantly increased after stimulation of CCL18 (500 ng/mL) for 48 hours (Figure 2D∼F). Conclusions Our preliminary results show overexpression of CCL18 in synovial tissue and fluid of RA patients can stimulate migration and activation of RA-FLS probably through the interaction between CCL18 and PITPNM3. Acknowledgement This work was supported by National Natural Science Foundation of China (81471597), Specialized Research Fund for the Doctoral Program of Higher Education (20130171110075) and Guangdong Natural Science Foundation (2014A030313074). Disclosure of Interest None declared