Objective Arthralgia may precede the development of synovial inflammation in autoantibody-positive individuals at risk of developing rheumatoid arthritis (RA). A major pathway involved in pain is the prostaglandin (PG) E2 pathway. We investigated this pathway in the synovium of individuals with RA-specific autoantibodies and in early arthritis patients. Methods Nineteen autoantibody-positive individuals (IgM-rheumatoid factor and/or anti-cyclic citrullinated peptide antibodies) with arthralgia (n=15) and/or a positive family history of RA (n=8), who had been prospectively followed for at least 2 years, were included. In addition, we included early arthritis patients (disease-modifying antirheumatic drug naïve) who after 2 years follow up fulfilled classification criteria for RA (n=63), spondyloarthritis (SpA; n=14), or had unclassified arthritis (UA; n=27). In all subjects we assessed pain and performed synovial biopsy sampling by mini-arthroscopy at baseline. Tissue sections were examined by immunohistochemistry to detect and quantify PGE2 pathway enzymes expression levels (mPGES-1; COX-1 and -2; 15-PGDH). Results In both study groups synovial expression of PGE2 enzymes was not clearly related to pain sensation. Expression levels at baseline were not associated with the development of arthritis after follow up (6 out of 19 autoantibody-positive individuals). However, in early SpA patients the expression levels of mPGES-1 and COX-1 were significantly increased compared to RA and UA patients. Conclusion Pain in autoantibody-positive individuals without synovial inflammation who are at risk of developing RA and in early arthritis patients may be regulated by pathways other than the PGE2 pathway or originate at sites other than the synovium. In contrast, in SpA, the PGE2 pathway may be inherently linked to the pathophysiology/etiology of the disease.

Background Microsomal prostaglandin E synthase-1 (mPGES-1) is the terminal enzyme in the induced PGE2 production and well-recognised target for the development of novel anti-inflammatory drugs. Genetic deletion and inhibition of mPGES-1 appears to be protective in experimental models of arthritis. Targeting mPGES-1 alters eicosanoid profiles and may potentially affect the metabolism of other lipids. Fatty acids (FA) play essential roles as energy substrates, membrane structural components, second messengers and precursors of lipid mediators. Genetic deletion of mPGES-1 resulted in reduced levels of MUFA in the spleen suggesting a decreased activity/expression of stearoyl-CoA-desaturase (SCD). SCD has been implicated in modulation of inflammation. However whether mPGES-1 inhibition affects lipid metabolism in human cells is not known. Objective To investigate the effect of mPGES-1 deletion/inhibition on lipid metabolism in human cells. Methods A549 cells were induced with IL-1b and treated with mPGES-1 inhibitor (compound III) or COX-2 inhibitor (NS398). Eicosanoid profiles were analysed using LC-MS/MS and protein profiles were analysed using mass spectrometry based proteomics. FA composition of total lipids was determined using gas chromatography with flame ionization detector and sphingolipids were analysed by LC-MS/MS. Results Proteomic analysis of A549 cells identified the reduction of SCD levels in response to treatment with mPGES-1 or COX-2 inhibitors, as well as the suppression of the levels of a number of proteins involved in metabolism of fatty acids and sphingolipids. Conclusion Data reveals that down-regulation of COX-2/mPGES-1/ PGE2 axis affects the lipid metabolism in A549 cells. These effects have important implications regarding potential consequences of pharmacologic mPGES-1 inhibition.

Background Microsomal prostaglandin E synthase-1 (mPGES-1) is the terminal enzyme in the induced PGE2 production and well-recognized target for the development of novel anti-inflammatory drugs. Genetic deletion and inhibition of mPGES-1 appears to be protective in experimental models of arthritis. Targeting mPGES-1 alters eicosanoid profiles and may potentially affect the metabolism of other lipids. Fatty acids (FA) play essential roles as energy substrates, membrane structural components, second messengers and precursors of lipid mediators. Genetic deletion of mPGES-1 resulted in reduced levels of MUFA in the mouse spleen suggesting a decreased activity/expression of stearoyl-CoA-desaturase (SCD). SCD has been implicated in modulation of inflammation. However whether mPGES-1 inhibition affects lipid metabolism in human cells is not known. Objectives To investigate the effect of mPGES-1 inhibition on lipid metabolism in human cells. Methods A549 cells were induced with IL-1b and treated with mPGES-1 inhibitor (compound III) or COX-2 inhibitor (NS398). Eicosanoid profiles were analyzed using LC-MS/MS and protein profiles were analyzed using mass spectrometry based proteomics. FA composition of total lipids was determined using gas chromatography with flame ionization detector and sphingolipids were analysed by LC-MS/MS. Results Prostanoid profiles in supernatants from the IL-1b-induced cells treated with the COX-2 or mPGES-1 inhibitors were significantly different suggesting diverse effects of the inhibitors on down-stream pathways. Proteomic analysis of A549 cells identified the reduction of SCD levels in response to treatment with mPGES-1 or COX-2 inhibitors, as well as the suppression of the levels of a number of proteins involved in metabolism of fatty acids and sphingolipids. Conclusions The results suggest that down-regulation of COX-2/mPGES-1/PGE2 axis affects the lipid metabolism in A549 cells. These effects have important implications regarding potential consequences of pharmacologic mPGES-1 inhibition. Acknowledgements The Swedish Research Council, The Swedish Rheumatism Association, King Gustaf V 80 years Foundation, The Swedish Society of Medicine, Karolinska Institutet Foundation, The Swedish County Council and Marianne and Marcus Wallenberg foundation. Disclosure of Interest None declared DOI 10.1136/annrheumdis-2014-eular.5002

Background Synovial fibroblasts (SF) are activated in rheumatoid arthritis (RA) and show upregulation of enzymes of the PGE2 pathway. HMGB1 has been shown to promote inflammation together with ligands such as LPS and IL-1β. As HMGB1 is overexpressed extracellularly in synovitis, it might constitute a potential inducer of the mPGES-1/Cox/PGE2 axis in RASF. To verify this hypothesis, we investigated the cytokine and prostanoid-inducing capacity of HMGB1 alone or together with IL-1β in RASF cultures. We also studied receptor requirements for PGE2 induction. Objectives To characterize the involvement of the PGE2 pathway in the response of SF to HMGB1 alone or in combination with IL-1β. Methods SF obtained from RA patients were stimulated with recombinant HMGB1 or HMGB1 purified from thymus alone or together with low dose IL-1β (0.05 or 0.5 ng/ml). Pretreatment with IL-1RA was used to block IL-1RI-mediated signalling. IL-6, IL-8, RANTES and MCP-1 levels were measured by CBA and ELISA. Expression of enzymes of the PGE2 pathway (mPGES-1, Cox-1, Cox-2, 15-PGDH) were analysed by Western blot. Prostanoid levels were measured by EIA and LC-MS/MS. Results HMGB1 alone did not induce either cytokine production or PGE2 pathway up-regulation in SF. Low doses of IL-1β (0.05-0.5 ng/ml) resulted in limited effects on PGE2 production and cytokines. In contrast, stimulation of RASF with HMGB1 in combination with trace amounts of IL-1β (0.05 or 0.5 ng/ml) exerted synergistic effects on the induction of mPGES-1 and COX-2 expression as well as PGE2, IL-6, IL-8, MCP-1 and RANTES production when compared to IL-1b stimulation alone. PGE2 and cytokine production induced by HMGB1/IL-1β were completely abolished by pretreatment with IL-1RA, indicating that the synergistic effects were mediated through IL-1RI. Investigating the kinetics of the SF response, we showed that the induction of PGE2 precedes that of IL-6 and IL-8. In line with this result, NS-398, a Cox-2 inhibitor, reduced the IL-6 and IL-8 production after a 24h incubation, indicating that the IL-1β/HMGB1 combination upregulates the IL-6 and IL-8 production in part through prostanoid synthesis. Conclusions HMGB1 amplified the inflammatory response to suboptimal amounts of IL-1β resulting in the induction of the mPGES-1/Cox-2/PGE2 axis and the production of PGE2 and pro-inflammatory cytokines in SF. Therefore, upregulation of PGE2 synthesis constitutes an additional mechanism through which HMGB1 could perpetuate inflammatory and destructive activities in the arthritic joint. Disclosure of Interest None Declared

Background Microsomal prostaglandin E synthase-1 (mPGES-1) inhibition has been suggested as an alternative to cyclooxygenase (COX) inhibition in the treatment of pain and inflammation. This approach could potentially mitigate the gastro-intestinal and cardiovascular side effects associated to traditional non-steroidal anti-inflammatory drugs (NSAIDs) and Coxibs. Aim To characterise a selective inhibitor of mPGES-1 and study its impact on the prostanoid profile in various models of inflammation. Materials and Methods Potency and selectivity: Compound III was incubated with active enzymes of the prostanoid pathway (mPGES-1, COX-1/2, PGIS, H-PGDS). For potential cross-reactivity towards thromboxane synthase, compound III was tested in a thromboxane release assay in platelets. A549 cells: compound III was assayed in both short and long-term assays. Cells were stimulated with IL-1β/TNF-α or IL-1β alone and incubated in the presence of inhibitors. Whole blood assay/mouse peritoneal macrophages: blood and macrophages were induced with LPS and incubated with compound III for 24 h. Air pouch model: the inflammatory reaction was triggered by a carrageenan injection intra-pouch. Inhibitors were injected i.p. and exudates were collected after 6 h. Prostanoid analysis: Prostanoids were measured by mass spectrometry or enzyme immunoassay. Results Compound III has an IC50 of 0.4 µM and 1 µM in human and murine mPGES-1 respectively. It has no activity on other enzymes of the prostanoid pathway and did not inhibit thromboxane release from human platelets. In cellular assays, it reduced PGE2 production in A549 cells, mouse peritoneal macrophages and LPS-stimulated whole blood. In mouse peritoneal macrophages, compound III caused a shunt to the prostacyclin pathway. Lastly, we assayed compound III in the air pouch model to verify its impact on the prostanoid profile and compare it to the profile obtained in mPGES-1 k.o. mice. As opposed to mPGES-1 genetic deletion, which attenuated PGE2 induction and caused a shunt to the thromboxane pathway, mPGES-1 inhibition with compound III reduced PGE2 production and tended to decrease the levels of other prostanoids. Conclusions Compound III is an active and selective inhibitor of mPGES-1. Its impact on the prostanoid profile was assay dependent. However, in the air pouch, contrary to mPGES-1 gene deletion, compound III did not trigger shunting, a scenario more likely to represent the outcome of mPGES-1 inhibition at the inflammatory site.

PGE 2 is a potent lipid mediator of pain and oedema found elevated in RA . Microsomal prostaglandin E synthase‐1 ( mPGES ‐1) is a terminal enzyme of the PGE 2 pathway inducible by proinflammatory cytokines. mPGES ‐1 is markedly upregulated in RA synovial tissue despite antirheumatic treatments, suggesting that multiple inflammatory stimuli contribute to its induction. High‐mobility group box chromosomal protein 1 ( HMGB 1) is known to induce inflammation both by direct interaction with TLR 4 and by enhancement of other proinflammatory molecules signalling, through complex formation. The high expression of extracellular HMGB 1 within the inflamed synovium, implies its pro‐arthritogenic role in RA . We aimed to investigate the effects of IL ‐1β/ HMGB 1 complexes on mPGES ‐1 and other enzymes of the PGE 2 pathway in synovial fibroblasts ( SF s) from patients with arthritis. Furthermore, we studied the effect of COX ‐2 inhibition and IL‐1RI antagonism on prostanoid and cytokine production by SF s. Stimulation of SFs with HMGB1 in complex with suboptimal amounts of IL‐1β significantly increased mPGES ‐1 and COX ‐2 expressions as well as PGE 2 production, as compared to treatment with HMGB1 or IL‐1β alone. Furthermore, NS ‐398 reduced the production of IL‐6 and IL‐8, thus indicating that IL‐1β/HMGB1 complexes modulate cytokine production in part through prostanoid synthesis. Treatment with IL ‐1RA completely abolished the induced PGE 2 and cytokine production, suggesting an effect mediated through IL‐1RI. IL ‐1β/ HMGB 1 complexes promote the induction of mPGES ‐1, COX‐2 and PGE 2 in SF. The amplification of the PGE 2 biosynthesis pathway by HMGB 1 might constitute an important pathogenic mechanism perpetuating inflammatory and destructive activities in rheumatoid arthritis.

Background mPGES-1 is considered an attractive alternative target for anti-inflammatory treatment with improved selectivity and safety compared to NSAIDs. Genetic deletion or pharmacological inhibition of mPGES-1 activity or expression down-regulate inflammation and pain in experimental models of arthritis. However, a detailed understanding of the molecular mechanisms and pathways affected by deletion/inhibition of mPGES-1 is essential before mPGES-1 inhibitors can safely be applied in the clinics. Objectives To investigate the effect of mPGES-1 deletion on the levels of bioactive lipid mediators such as the eicosanoids (down-stream cascade) and fatty acids (up-stream cascade). Methods Peritoneal macrophages (PM) from wild type (WT) and knock-out (mPGES-1-/-, KO) mice were induced with LPS for 16 h. Cells were harvested for gene expression analysis and supernatants were collected for eicosanoid analysis. Gene expression profiling in WT and KO macrophages was performed using the microarray analysis (Applied Biosystems). Eicosanoid profiling of approximately 30 compounds was performed using LC-MS/MS. Fatty acid composition of total lipids in spleen and brain homogenates of WT and KO mice were determined using GC/FID. Results Microarray analysis revealed that genetic deletion of mPGES-1 affected expression of genes related to lipid metabolism and mainly associated with eicosanoid, fatty acid and phospho- lipid metabolism, (e.g., Pla1A, Ptgis, Fabp3, Cept1, Comt etc). Compared to WT, mPGES-1 deficient PM displayed a markedly attenuated increase in PGE2 production upon LPS stimulation, and exhibited significantly increased levels of PGD2 metabolites such as 15-deoxy-∆12,14 PGJ2 and 15-deoxy-∆12,14 PGD2 (p < 0.05). There were significant differences in the fatty acid composition in spleen (e.g., palmitoleic acid decreased in KO, p < 0.05) and brain (e.g., myristic acid decreased in KO, p < 0.05) of KO and WT mice, suggesting that feed-back mechanisms are alternated by the deletion of m-PGES-1. Conclusions Data reveals that mPGES-1 deficient PMs displayed shunting towards PGD2 pathway, i.e., towards anti-inflammatory metabolites, upon LPS stimulation compared to WT PMs. Moreover, mPGES-1 depletion alters the fatty acid composition of tissue lipids. These effects of inducible PGE2 on lipid metabolism have important implications for future mPGES-1 inhibitors and deserve further investigation.

mPGES-1 is considered an alternative target for anti-inflammatory treatment with improved selectivity and safety compared to NSAIDs. mPGES-1 depletion not only suppresses inflammation via absence of inducible PGE2 but might also cause an activation of anti-inflammatory pathways. We studied effects of mPGES-1 deletion on the eicosanoid and fatty acid (FA) profiles in mice. In LPS-induced peritoneal macrophages from mPGES-1 knock-out (mPGES-1-/-, KO) mice PGE2 production was markedly attenuated, whereas levels of PGD2 metabolites (15-deoxy-Δ(12,14) PGJ2 and 15-deoxy-Δ(12,14) PGD2) were increased compared to wild type mice. The levels of oxidized fatty acid 13-HODE were also significantly up-regulated in KO macrophages. Significant differences in the total lipid FA composition (decrease in monounsaturated FA and increase in eicosadienoic acid) were detected in spleen of KO and WT mice. These effects of mPGES-1 deletion on eicosanoid and fatty acid metabolism have important implications for future mPGES-1 inhibitors and deserve further investigation.

The aim of this study was to assess inflammation and the presence and relative levels of cytokines, which may be involved in regulating early human Achilles tendon healing.

Microsomal prostaglandin E synthase-1 (mPGES-1) inhibition has been suggested as an alternative to cyclooxygenase (COX) inhibition in the treatment of pain and inflammation. We characterized a selective inhibitor of mPGES-1 activity (compound III) and studied its impact on the prostanoid profile in various models of inflammation. Compound III is a benzoimidazole, which has a submicromolar IC50 in both human and rat recombinant mPGES-1. In cellular assays, it reduced PGE2 production in A549 cells, mouse macrophages and blood, causing a shunt to the prostacyclin pathway in the former two systems. Lastly, we assayed compound III in the air pouch model to verify its impact on the prostanoid profile and compare it to the profile obtained in mPGES-1 k.o. mice. As opposed to mPGES-1 genetic deletion, which attenuated PGE2 production and caused a shunt to the thromboxane pathway, mPGES-1 inhibition with compound III reduced PGE2 production and tended to decrease the levels of other prostanoids.

INTRODUCTION:Rheumatoid arthritis (RA) is a chronic inflammatory disease in which prostaglandin E2 (PGE2) displays an important pathogenic role. The enzymes involved in its synthesis are highly expressed in the inflamed synovium, while little is known about 15- prostaglandin dehydrogenase (15-PGDH) that metabolizes PGE2. Here we aimed to evaluate the localization of 15-PGDH in the synovial tissue of healthy individuals or patients with inflammatory arthritis and determine the influence of common RA therapy on its expression.METHODS:Synovial tissue specimens from healthy individuals, psoriatic arthritis, ostheoarthritis and RA patients were immunohistochemically stained to describe the expression pattern of 15-PGDH. In addition, the degree of enzyme staining was evaluated by computer analysis on stained synovial biopsies from two groups of RA patients, before and after RA specific treatment with either intra-articular glucocorticoids or oral methotrexate therapy. Prostaglandins derived from the cyclooxygenase (COX) pathway were determined by liquid-chromatography mass spectrometry in supernatants from interleukin (IL) 1β-activated fibroblast-like synoviocytes (FLS) treated with methotrexate.RESULTS:15-PGDH was present in healthy and inflamed synovial tissue, mainly in lining macrophages, fibroblasts and vessels. Intra-articular glucocorticoids showed a trend towards reduced 15-PGDH expression in RA synovium (p = 0.08) while methotrexate treatment left the PGE2 pathway unaltered both in biopsies ex vivo and in cultured FLS.CONCLUSIONS:Early methotrexate therapy has little influence on the expression of 15-PGDH and on any of the PGE2 synthesizing enzymes or COX-derived metabolites. Thus therapeutic strategies involving blocking induced PGE2 synthesis may find a rationale in additionally reducing local inflammatory mediators.

Background Microsomal prostaglandin E synthase-1 (mPGES-1) is the terminal enzyme in the induced PGE2 production at the sites of inflammation and well-recognised target for the development of novel anti-inflammatory drugs that can reduce symptoms of inflammation in rheumatic diseases. Genetic deletion of mPGES-1 in arthritic mice reduces inflammation, humoral immune response and protects them from pain and joint destruction. However, molecular mechanisms and signalling pathways involved in anti-inflammatory effects of selective mPGES-1 inhibition/deletion at sites of inflammation have not been explored. Objective To study effects of mPGES-1 deletion on total proteins expression and eicosanoid profile upon lipopolysaccharide (LPS)-induced macrophage activation. Methods Peritoneal macrophages (PM) from wild type (WT) and knock-out (mPGES-1−/−, knockout (KO)) mice were induced with LPS for 16 h. Supernatants were harvested for eicosanoid analysis and proteins were isolated from the cells. Proteomics approach using high resolution mass spectrometry coupled to nanoflow-liquid chromatography has been employed to determine the identity and the relative abundance of expressed proteins. Eicosanoid profiling was performed using liquid chromatography-mass spectrometry. Results Compared to wild-type, mPGES-1 deficient PM displayed a markedly attenuated increase in PGE2 production upon LPS stimulation, and exhibited increased levels of prostaglandin D2 metabolite and prostaglandin F2 α. Comparative proteomic analysis of LPS-induced macrophages from WT and mPGES-1 KO mice identified 248 proteins with different molecular and cellular functions including cell death, cellular movement, cellular growth and proliferation, cellular function and maintenance and protein synthesis. In PM from KO mice, 109 proteins were upregulated, 101 proteins were downregulated and 38 proteins were not changed compared with PM from WT mice. Interestingly, among the top 10 proteins upregulated in macrophages from KO mice the authors identified two heat shock proteins with immune-modulatory properties, Hsp70-5 and Hsp70-8. Hsp70-5 (the immunoglobulin binding protein, BiP) is known to be upregulated by cyclopentenone prostaglandins and suppress experimental arthritis in mice. Hsp70-8 (heat chock cognate 70, Hsc70) has been shown to modulate dendritic cells function. The expression of Hsp70-5 and Hsp70-8 in LPS-induced macrophages from WT and KO mice was confirmed by Western blot. Conclusion The data reveal that inhibition of mPGES-1 activity in pro-inflammatory conditions resulted in the upregulation of heat-shock proteins with anti-inflammatory and immuno-modulatory properties.

Microsomal prostaglandin E synthase (mPGES)-1 inhibition has been proposed as an alternative to cyclooxygenase (COX) inhibition in the treatment of pain and inflammation. This novel approach could potentially mitigate the gastro-intestinal and cardiovascular side effects seen after long-term treatment with traditional non-steroidal anti-inflammatory drugs (NSAIDs) and Coxibs respectively. Several human mPGES-1 inhibitors have been developed in the recent years. However, they were all shown to be considerably less active on rodent mPGES-1, precluding the study of mPGES-1 inhibition in rodent models of inflammation and pain. The aim of this study was to characterize the new mPGES-1 inhibitor compound II, a pyrazolone that has similar potency on rat and human recombinant mPGES-1, in experimental models of inflammation. In cell culture, compound II inhibited PGE2 production in synovial fibroblasts from patients with rheumatoid arthritis (RASF) and in rat peritoneal macrophages. In vivo, compound II was first characterized in the rat air pouch model of inflammation where treatment inhibited intra-pouch PGE2 production. Compound II was also investigated in a rat adjuvant-induced arthritis model where it attenuated both the acute and delayed inflammatory responses. In conclusion, compound II represents a valuable pharmacological tool for the study of mPGES-1 inhibition in rat models.

Dysregulation of TNF is an important pathophysiological phenotype for many diseases. Recently, certain genetically regulated loci have been identified to regulate several inflammatory diseases. We hypothesized that a region on rat chromosome 4 known to regulate experimental autoimmune encephalomyelitis, experimental arthritis and experimental autoimmune neuritis harbors a gene regulating central inflammatory molecules, such as TNF. We therefore mapped TNF production using linkage analysis in the 12th generation of an advanced intercross line between DA and PVG.AV1 rats, which differ in susceptibility to several inflammatory conditions. A single TNF-regulating quantitative trait locus with a logarithm of odds score of 6.2 was identified and its biological effect was confirmed in a congenic rat strain. The profound TNF regulation mapped in congenic strains to the macrophage population. Several TLR signaling cascades led to the same reduced proinflammatory phenotype in congenic macrophages, indicating control of a convergence point for innate inflammatory activity. The decreased TNF potential and reduced proinflammatory macrophage phenotype in congenic rats was also associated with reduced clinical severity in experimental autoimmune encephalomyelitis, pristane-induced arthritis and sepsis experimental models. Determination of genes and mechanisms involved in this genetically determined TNF regulation will be valuable in understanding disease pathogenesis and aid treatment development.

The inducible cyclooxygenase isoform (COX-2) is associated with inflammation, tumorigenesis, as well as with physiological events. Despite efforts deployed in order to understand the biology of this multi-faceted enzyme, much remains to be understood. Nucleobindin (Nuc), a ubiquitous Ca2+-binding protein, possesses a putative COX-binding domain. In this study, we investigated its expression and subcellular localization in human neutrophils, its affinity for COX-2 as well as its possible impact on PGE2 biosynthesis. Complementary subcellular localization approaches including nitrogen cavitation coupled to Percoll fractionation, immunofluorescence, confocal and electron microscopy collectively placed Nuc, COX-2, and all of the main enzymes involved in prostanoid synthesis, in the Golgi apparatus and endoplasmic reticulum of human neutrophils. Immunoprecipitation experiments indicated a high affinity between Nuc and COX-2. Addition of human recombinant (hr) Nuc to purified hrCOX-2 dose-dependently caused an increase in PGE2 biosynthesis in response to arachidonic acid. Co-incubation of Nuc with COX-2-expressing neutrophil lysates also increased their capacity to produce PGE2. Moreover, neutrophil transfection with hrNuc specifically enhanced PGE2 biosynthesis. Together, these results identify a COX-2-associated protein which may have an impact in prostanoid biosynthesis.