High humidity aggravates the severity of arthritis in collagen-induced arthritis mice by upregulating xylitol and L-pyroglutamic acid

Background Humidity was an unfavorable factor for patients with rheumatoid arthritis (RA). RA disease activity was severe in high humidity conditions. However, there is no evidence to demonstrate the effects of humidity on arthritis in the animal experiments and explore its relevant mechanism. Methods Using the DBA/1 mice, this study addressed the effects of a high humidity (80 ± 5%) on arthritis in collagen-induced arthritis (CIA) mice. Then, this study used the gas chromatography-mass spectrometer (GC-MS) to explore alterations in serum metabolome caused by the high humidity. Furthermore, xylitol and L-pyroglutamic acid, which were both significantly upregulated by the high humidity, were selected to further study their effects on arthritis in the CIA mice. Results The high humidity (80 ± 5%) could aggravate arthritis variables including increasing arthritis score and swelling, serum autoantibodies (anti-COII and anti-CCP), and proinflammatory cytokines (IL-6, IL-17A, and G-CSF). In addition, the high humidity could cause significant alterations in serum metabolome in the CIA mice. Xylitol and L-pyroglutamic acid were the representative serum metabolites that were significantly upregulated by the high humidity. Further experiments demonstrated that the supplementation of 0.4 mg/mL xylitol in drinking water after inducing the CIA model and 2.0 mg/mL in drinking water before inducing the CIA model could both aggravate arthritis in the CIA mice. Conclusions These data demonstrated that high humidity was not beneficial for arthritis development and its mechanism might be associated with xylitol and L-pyroglutamic acid.

Epigenetic Regulation (Including Micro-RNAs, DNA Methylation and Histone Modifications) of Rheumatoid Arthritis: A Systematic Review

The inflammatory reaction in rheumatoid arthritis (RA) is controlled by major epigenetic modifications that modulate the phenotype of synovial and immune cells. The aim of this work was to perform a systematic review focusing on miR expression, DNA methylation and histone modifications in RA. We demonstrated that, in human samples, the expressions of miR-155, miR-146a and miR-150 were significantly decreased while the expression of miR-410-3p was significantly increased in the RA group. Moreover, miR-146a significantly decreased pro-autoimmune IL-17 cytokine expression in RA. In a murine model, miR-34a inhibition can ameliorate the arthritis score. However, this evidence remain critically insufficient to support current therapeutic applications in RA patients.

Acetylated Diacylglycerol 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol in Autoimmune Arthritis and Interstitial Lung Disease in SKG Mice

Acetylated diacylglycerol 1-palmitoyl-2-linoleoyl-3-acetyl-rac-glycerol (PLAG) is a lipid molecule from the antlers of sika deer that might reduce inflammation by effectively controlling neutrophil infiltration, endothelial permeability and inflammatory chemokine production. Therefore, we evaluated the modulatory effect of PLAG on arthritis and interstitial lung disease (ILD) in an autoimmune arthritis model. We injected curdlan into SKG mice and PLAG was orally administered every day from 3 weeks to 20 weeks after the curdlan injection. The arthritis score was measured every week after the curdlan injection. At 20 weeks post-injection, the lung specimens were evaluated with H&E, Masson’s trichrome and multiplexed immunofluorescent staining. Serum cytokines were also analyzed using a Luminex multiple cytokine assay. PLAG administration decreased the arthritis score until 8 weeks after the curdlan injection. However, the effect was not sustained thereafter. A lung histology revealed severe inflammation and fibrosis in the curdlan-induced SKG mice, which was attenuated in the PLAG-treated mice. Furthermore, immunofluorescent staining of the lung tissue showed a GM-CSF+ neutrophil accumulation and a decreased citrullinated histone 3 expression after PLAG treatment. PLAG also downregulated the levels of IL-6 and TNF-α and upregulated the level of sIL-7Rα, an anti-fibrotic molecule. Our results indicate that PLAG might have a preventative effect on ILD development through the resolution of NETosis in the lung.

Blocking Jak/STAT signalling using tofacitinib inhibits angiogenesis in experimental arthritis

Objective During rheumatoid arthritis (RA), the angiogenic processes, occurring with pannus-formation, may be a therapeutic target. JAK/STAT-pathway may play a role and the aim of this work was to investigate the inhibiting role of a JAK-inhibitor, tofacitinib, on the angiogenic mechanisms occurring during RA. Methods After ethical approval, JAK-1, JAK-3, STAT-1, STAT-3 and VEGF expression was evaluated on RA-synovial-tissues. In vitro, endothelial cells (ECs), stimulated with 20 ng/ml of VEGF and/or 1 μM of tofacitinib, were assessed for tube formation, migration and proliferation, by Matrigel, Boyden chamber assay and ki67 gene-expression. In vivo, 32 mice received collagen (collagen-induced arthritis (CIA)) and 32 mice PBS (control). At day 19, CIA and controls mice were divided: 16 mice receiving vehicle and 16 mice receiving tofacitinib. At day 35, the arthritis score, the thickness of paw joints and the serum levels of VEGF and Ang-2 were evaluated. Results The expression of JAK-1, JAK-3, STAT-1, STAT-3 and VEGF in synovial tissue of RA-patients were significantly higher than healthy controls. In vitro, tofacitinib inhibited the ECs ability to form vessels, to proliferate and to migrate. In vivo, administration of tofacitinib prevented the increase of the arthritis score, the paw thickness, the synovial vessels and VEGF and Ang-2 serum-accumulation, when compared to CIA without tofacitinib. Conclusions We explored the anti-angiogenic role of tofacitinib, reporting its ability to inhibit in vitro the angiogenic mechanisms of ECs and in vivo the formation of new synovial vessels, occurring in CIA model. These findings suggest that the therapeutic effect of tofacitinib during RA may be also related to its anti-angiogenic activity.

AB0062 TIME COURSE OF INTESTINAL PERMEABILITY AND BACTERIAL TRANSLOCATION IN THE MODEL OF ADJUVANT-INDUCED ARTHRITIS

Background:Intestinal inflammation, dysbiosis, intestinal permeability (IP) and bacterial translocation (BT) have been identified in patients with spondyloarthritis but the time at which they appear and their contribution to the pathogenesis of the disease is still a matter of debate.Objectives:To investigate the time-course of intestinal inflammation, IP and BT in a rat model of reactive arthritis, a subgroup of SpA, the adjuvant-induced arthritis model.Methods:Adjuvant-induced arthritis (AIA) was induced in 6-week-old male Lewis rats by an injection at the base of the tail of Mycobacterium butyricum with incomplete Freund’s adjuvant (Day (D) 0). Control rats received saline using the same procedure. Body weights and a clinical arthritis score were daily assessed. A group of AIA and control rats (n=15 per group) were euthanized at three different times of arthritis: D4 for the pre-arthritic phase (AIA-preclinical), D11 for the onset of arthritis (AIA-onset) and D28 for the acute phase (AIA-acute). In each group (AIA and control, n=15 per group)), IP was assessed by measuring plasma levels of zonulin (ELISA) and ileal mRNA expression of zonulin and occludin (RT-qPCR), BT was studied by measuring bacterial endotoxins (or LPS, by LCMS2 method), soluble CD-14 (sCD14, ELISA) and ileal mRNA expression of TLR-4, and intestinal inflammation was assessed by measuring ileal mRNA expression of IL-8, IL-33, IL-17, IL-23p19 and TNF-α (RT-qPCR). Joint damage was assessed by the determination of a clinical and radiographic score of hind paws.Results:Body weights of AIA rats decreased from D4 to D28 as compared to controls, in parallel to the development of a severe clinical and radiographic arthritic disease from D11 and D28. Compared to control rats, AIA induced an increase in plasma zonulin levels at D4, D11 but not at D28. Ileal mRNA zonulin overexpression occurred at D11 while occludin was unchanged. As early as Day 4 (preclinical phase), mRNA of IL-8, IL-33 and IL-17 were overexpressed in ileum from AIA. At Day 11 (onset), overexpression of IL-8 persisted and mRNA of TNF-α and IL-23p19 increased in AIA. Neither LPS levels nor ileal mRNA expression of TLR-4 were changed by arthritis whatever the phase of arthritis. By contrast, blood levels of sCD-14 was significantly increased in the AIA group at all stages of arthritis. No correlation was found between clinical and radiographic arthritis scores and zonulin or LPS levels. Conversely, a negative correlation was observed between intestinal IL-8 mRNA expression and arthritis score (r=-0.3, p=0.02).Conclusion:In an animal model of SpA, intestinal inflammation and increased intestinal permeability occur prior to joint inflammation, suggesting a role of these disorders in the pathogenesis of this disease.Acknowledgements:I would like to thank the Société Française de Rhumatologie for its support in this work.Disclosure of Interests:None declared

Troxerutin-mediated C9 inhibition is a disease-modifying treatment for inflammatory arthritis.

Troxerutin (TXR) is a phytochemical reported to possess anti-inflammatory and hepatoprotective effects. In this study, we aimed to exploit anti-arthritic properties of TXR using an adjuvant induced arthritic (AIA) rat model. AIA induced rats showed highest arthritis score at disease onset and by oral administration of TXR (50, 100, 200 mg/kg body weight), reduced to basal level in a dose dependent manner. Isobaric tag for relative and absolute quantitative (iTRAQ) proteomics tool was employed to identify deregulated joint homogenate proteins in AIA and TXR treated rats to decipher probable mechanism of the TXR action in arthritis. iTRAQ analysis identified a set of 434 joint homogenate proteins with 65 deregulated proteins (log2 case/control ≥ 1.5) in AIA. Expressions of a set of important proteins (AAT, T-kininogen, vimentin, desmin, and nucleophosmin) that could classify AIA from healthy were validated using Western blot analysis. Western blot data corroborated proteomics findings. In silico protein-protein interaction study of joint homogenate proteome revealed that complement component 9 (C9), the major building blocks of the membrane attack complex (MAC) responsible for sterile inflammation, gets perturbed in AIA. Our dosimetry study suggests that a TXR dose of 200 mg/kg body weight for 15 days is sufficient to bring the arthritis score to basal levels in AIA rats. We have shown the importance of TXR as an anti-arthritis agent in AIA model and after additional investigation its arthritis ameliorating properties could be exploited for clinical usability.

Anti-Arthritic and Anti-Inflammatory Potential of Spondias mangifera Extract Fractions: An In Silico, In Vitro and In Vivo Approach

The fruits of Spondias mangifera (S. mangifera) have traditionally been used for the management of rheumatism in the northeast region of India. The present study explores the probable anti-arthritis and anti-inflammatory potential of S. mangifera fruit extract’s ethanolic fraction (EtoH-F). To support this study, we first approached the parameters in silico by means of the active constituents of the plant (beta amyrin, beta sitosterol, oleonolic acid and co-crystallised ligands, i.e., SPD-304) via molecular docking on COX-1, COX-2 and TNF-α. Thereafter, the absorption, distribution, metabolism, excretion and toxicity properties were also determined, and finally experimental activity was performed in vitro and in vivo. The in vitro activities of the plant extract fractions were evaluated by means of parameters like 1,1-Diphenyl-2- picrylhydrazyl (DPPH), free radical-reducing potential, albumin denaturation, and protease inhibitory activity. The in vivo activity was evaluated using parameters like COX, TNF-α and IL-6 inhibition assay and arthritis score in Freund Adjuvant (CFA) models at a dose of 400 mg/kg b.w. per day of different fractions (hexane, chloroform, alcoholic). The molecular docking assay was performed on COX-1, COX-2 and TNF-α. The results of in vitro studies showed concentration-dependent reduction in albumin denaturation, protease inhibitors and scavenging activity at 500 µg/mL. Administration of the S. mangifera alcoholic fraction at the abovementioned dose resulted in a significant reduction (p < 0.01) in arthritis score, paw diameters, TNF-α, IL-6 as compared to diseased animals. The docking results showed that residues show a critical binding affinity with TNF-α and act as the TNF-α antagonist. The alcoholic fraction of S. mangifera extract possesses beneficial effects on rheumatoid arthritis as well as anti-inflammatory potential, and can further can be used as a possible agent for novel target-based therapies for the management of arthritis.

LINC02085 Regulates Cell Growth and Inflammatory Response in Rheumatoid Arthritis by Regulating PI3K/ AKT Signaling Pathway

Background: The present study explored the possible functions and the underlying mechanism of long Non-coding RNA LINC02085 in rheumatoid arthritis (RA). Methods: Primary fibroblast-like synoviocytes (FLS) were separated from synovial tissues and was established cell lines, then cultured for subsequent cell experiments by transfecting different vectors. Rat with AA were injected with sh-LINC02085. The progression of AA was explored by measuring arthritis score and histologic analysis. ELISA analysis was employed to detect the levels of inflammatory cytokines. CCK8 assay, migration and invasion assays were used to evaluate the proliferation, migration and invasion abilities of cells, respectively. Besides, the levels of the the PI3K/AKT pathway-related proteins were measured by WB and IF. Results: The expression level of LINC02085 was significant high in patients with RA, and positively associated with clinical indexes. We found that LINC02085 was upregulated in RA -FLS and TNF-αstimulated. And overexpression of LINC02085 could promote proliferation, migration and invasion induced by TNF-α, through upregulating the levels of TNF-αand TNFAIP2 and promoting the activation of PI3K/AKT pathway. Whereas downexpression of LINC02085 received the opposite results. Knockdown of LINC02085 significantly ameliorated the progression of AA reflected by decreased arthritis score and cartilage destruction. Conclusion: The present study revealed that LINC02085 could regulate cell growth and inflammatory response of RA-FLS by activating the PI3K/ AKT signaling pathway, subsequently playing important roles in promoting the occurrence and development of RA.

Therapeutic Effects of Bruton’s Tyrosine Kinase Inhibitor HM71224 as Monotherapy and in Combination with Methotrexate on Collagen-Induced Arthritis in Rats

Background: The purpose of this study was to determine the potential effects of HM71224, a Bruton’s tyrosine kinase (BTK) inhibitor, as a monotherapy and in combination with methotrexate (MTX) in rats with collagen-induced arthritis (CIA) and to evaluate the effects of drug-drug interactions in combination therapy.Methods: The therapeutic effect of HM71224 and the combination effects with MTX were evaluated in CIA rats. Arthritis was induced through immunization by type II collagen in Lewis rats. The therapeutic effects were evaluated by arthritis score, paw volume, body weight changes, and histopathological examination. The effective dose levels (ED) were determined from arthritis score. Bone erosion, synovial inflammation, and cartilage degradation were assessed by staining with hematoxylin and eosin (H&E) and safranin-O in ankle joints. The drug-drug interaction between HM71224 and MTX was investigated by the comparison of plasma levels and monitoring of liver enzymes, creatinine and blood cell counts in CIA rats. Results: HM71224 dose dependently ameliorated the clinical signs, paw volume and body weight loss on the development of arthritis in CIA rats, and ED50 and ED90 values were 1.0 and 2.5 mg/kg, respectively. The combination of HM71224 with MTX decreased the arthritis score, bone erosion, synovitis and cartilage degradation compared with HM71224 and MTX alone; however, no drug-drug interactions in the plasma and no abnormalities in the liver enzymes, creatinine or blood cell counts were observed. Conclusion: BTK inhibition by HM71224 prevented the development of arthritis and the combination therapy with MTX produced additive therapeutic effects with no drug-drug interactions in CIA rats. Therefore, we suggest that HM71224 may be useful as a monotherapy and in combination with MTX for the treatment of patients with rheumatoid arthritis.

Troxerutin acts on complement mediated inflammation to ameliorate arthritic symptoms in rats

Troxerutin (TXR), is a phytochemical reported to possess anti-inflammatory and hepatoprotective effects. In this study, we aimed to exploit anti-arthritic properties of TXR using an adjuvant induced arthritic (AIA) rat model. AIA induced rats showed highest arthritis score at disease onset and by oral administration of TXR (50, 100, 200 mg/kg body weight), reduced to basal level in a dose dependent manner. Isobaric tag for relative and absolute quantitative (iTRAQ) proteomics tool was employed to identify deregulated joint homogenate proteins in AIA and TXR treated rats to decipher probable mechanism of the TXR action in arthritis. iTRAQ analysis identified a set of 434 joint homogenate proteins with 65 deregulated proteins (log2 case/control ≥1.5) in AIA. Expressions of a set of important proteins (AAT, T-kininogen, vimentin, desmin, and nucleophosmin) that could classify AIA from healthy were validated using Western blot analysis. Western blot data corroborated proteomics findings. In silico protein-protein interaction study of joint homogenate proteome revealed that complement component 9, the major building blocks of the membrane attack complex (MAC) responsible for sterile inflammation, gets perturbed in AIA. Our dosimetry study suggests that a TXR dose of 200 mg/kg body weight for 15 days is sufficient to bring the arthritis score to basal levels in AIA rats. We have shown the importance of TXR as an anti-arthritis agent in AIA model and after additional investigation its arthritis ameliorating properties could be exploited for clinical usability.