P-J Jakobsson

Effects of immunosuppressive treatment on microsomal prostaglandin E synthase 1 and cyclooxygenases expression in muscle tissue of patients with polymyositis or dermatomyositis

Objectives: To investigate the expression of microsomal prostaglandin E (PGE) synthase 1 (mPGES-1) and cyclooxygenase (COX) in muscle biopsies from patients with polymyositis or dermatomyositis before and after conventional immunosuppressive treatment. Methods: mPGES-1 and COX expression was evaluated by immunohistochemistry in muscle tissue from healthy individuals and from patients with polymyositis or dermatomyositis before and after conventional immunosuppressive treatment. The number of inflammatory cell infiltrates, T lymphocytes and macrophages was estimated before and after treatment. To localise the mPGES-1 expression double immunofluorescence was performed with antibodies against mPGES-1, CD3, CD68, CD163 and a fibroblast marker. A functional index was used to assess muscle function. Results: In patients with myositis, mPGES-1, COX-2 and COX-1 expression was significantly higher compared to healthy individuals and associated with inflammatory cells. Double immunofluorescence demonstrated a predominant expression of mPGES-1 in macrophages. Conventional immunosuppressive treatment resulted in improved but still lower muscle function than normal. A decreased number of CD68-positive macrophages and reduced COX-2 expression in muscle tissue was also seen. By contrast, following the same treatment no significant changes were observed in muscle tissue regarding number of infiltrates, T lymphocytes, CD163-positive macrophages or mPGES-1 protein levels. Conclusions: Increased expression of mPGES-1, COX-1 and COX-2 at protein level was observed in muscle tissue from patients with myositis compared to healthy individuals. Conventional immunosuppressive treatment led to a significant downregulation of COX-2 in myositis muscle tissue. However, the expression of mPGES-1 and COX-1 remained unchanged indicating a role of these enzymes in the chronicity of these diseases.

Dysregulations in circulating sphingolipids associate with disease activity indices in female patients with systemic lupus erythematosus: a cross-sectional study

Objective The objective of this study was to investigate the association of clinical and renal disease activity with circulating sphingolipids in patients with systemic lupus erythematosus. Methods We used liquid chromatography tandem mass spectrometry to measure the levels of 27 sphingolipids in plasma from 107 female systemic lupus erythematosus patients and 23 controls selected using a design of experiment approach. We investigated the associations between sphingolipids and two disease activity indices, the Systemic Lupus Activity Measurement and the Systemic Lupus Erythematosus Disease Activity Index. Damage was scored according to the Systemic Lupus International Collaborating Clinics damage index. Renal activity was evaluated with the British Island Lupus Activity Group index. The effects of immunosuppressive treatment on sphingolipid levels were evaluated before and after treatment in 22 female systemic lupus erythematosus patients with active disease. Results Circulating sphingolipids from the ceramide and hexosylceramide families were increased, and sphingoid bases were decreased, in systemic lupus erythematosus patients compared to controls. The ratio of C16:0-ceramide to sphingosine-1-phosphate was the best discriminator between patients and controls, with an area under the receiver-operating curve of 0.77. The C16:0-ceramide to sphingosine-1-phosphate ratio was associated with ongoing disease activity according to the Systemic Lupus Activity Measurement and the Systemic Lupus Erythematosus Disease Activity Index, but not with accumulated damage according to the Systemic Lupus International Collaborating Clinics Damage Index. Levels of C16:0- and C24:1-hexosylceramides were able to discriminate patients with current versus inactive/no renal involvement. All dysregulated sphingolipids were normalized after immunosuppressive treatment. Conclusion We provide evidence that sphingolipids are dysregulated in systemic lupus erythematosus and associated with disease activity. This study demonstrates the utility of simultaneously targeting multiple components of a pathway to establish disease associations.

Neutralisation of ACPA – A Way to Go?

Background and Objectives In a previous study, we have identified endogeneously citrullinated sites in fibrinogen from RA synovial tissue (Hermansson, et al, 2010 in Proteomics-Clin Appl). Within the alpha chain, Arg573 and Arg591 were found citrullinated with an occupancy rate in the range of 1–2% and in the β-chain, Arg72 and Arg74 were also found citrullinated. We now demonstrate that these citrullinated residues are autoantigenic as well as demonstrate that peptides containing these epitopes can be used as probes for development of ACPA neutralising compounds. Materials and Methods The autoantigenic potential was investigated using the Phadia’s ImmunoCAP ISAC® system. Citrullinated and unmodified fibrinogen peptides were immobilised onto a glass slide in an arrayed fashion and serum from 404 CCP positive and 532 CCP negative RA patients and 461 healthy controls from the EIRA cohort were tested. We also assayed the identified citrulline fibrinogen peptides for their ability to prevent purified ACPA (Ossipova, et al, 2012 submitted) to bind to CCP (CCPlus® ELISA, Euro-Diagnostica AB). Peptides were individually or in combinations incubated with different ACPA pools and the blocking efficiency was expressed as percent of inhibition and IC50. Corresponding arginine peptides were used as controls. Results We found that 31% (87% are CCP positive) of patients were positive to Cit573 peptide. For the Cit591 peptide, the corresponding numbers were 10% (65%), for the Cit74 peptide 28% (68%) and for the Cit72 peptide 20% (68%). Interestingly, citrullinated 573 and Cit591 peptides revealed a maximum of 77% and 48% ACPA inhibition, respectively. When equally mixed, these peptides displayed an additive higher degree of ACPA neutralisation (84%). In contrast, Cit74 and Cit72 peptides reached a more modest maximum inhibition of 26% and 30%, respectively. This experiment was repeated using a different set of ACPA pool and then the efficiencies were lower for Cit573 (47%) but similar for Cit591 (51%). Logically, the efficiency of specific citrullinated compounds will depend on the individual ACPA specificities. Conclusions Here we demonstrate extensive autoantibody reactivity against in vivo citrullinated fibrinogen epitopes found in RA synovial membranes. These peptides can now be used as additional biomarkers for studies of ACPA sub-specificity profiles as recently reported (Brink, et al, 2012 A&R, in press). We also demonstrate that these citrullinated peptides can be used as neutralising agents blocking a significant portion of ACPA binding to CCP. These results open novel possibilities for the design of personalised ACPA blockers preventing for instance the osteoclastogenesis and bone loss induced by ACPA (Harre, et al, 2012 JCI).

Inflammation increases MMP levels via PGE 2 in human vascular wall and plasma of obese women

Background and objectives:Matrix metalloproteinases (MMPs) are involved in several inflammatory processes including obesity-related vascular diseases and graft failure of coronary artery (CA) bypass grafts [internal mammary artery (IMA), saphenous vein (SV)]. In these inflammatory conditions, the release of prostaglandin E2 (PGE2) is increased via the activity of inducible microsomal PGE synthase-1 (mPGES-1). Our aim was to investigate whether MMPs and their endogenous inhibitor (TIMPs) may be regulated by PGE2 under inflammatory conditions in human vasculature and perivascular adipose tissue (PVAT), as well as in plasma of obese patients.Methods:MMP-1,-2 and TIMP-1,-2 densities were measured in human plasma (n = 68) as well as in supernatants of human vascular wall (IMA n = 16, SV n = 14, CA n = 13) and their PVAT. The effects of inflammation and mPGES-1 inhibitor (Compound III, 10 µM) on MMPs regulation were evaluated. The correlations between PGE2 and several parameters were calculated in plasma from patients with or without obesity.Results:The vascular wall and PVAT from SV exhibited the greatest MMP-1,-2 release. An increase of MMP-1,-2 and/or a decrease of TIMP-1 quantities have been detected under inflammation only in vascular wall not in PVAT. These changes under inflammation were completely reversed by inhibition of mPGES-1. In obesity, C-reactive protein (CRP), biomarker of inflammation, and PGE2 levels were increased. PGE2 contents were positively correlated with some anthropometric parameters and plasmatic CRP in both genders, while the correlation with the plasmatic MMP-1 density was significant only in women.Conclusions:The greater MMP activity observed in SV may contribute to the increased prevalence of graft failure. Under inflammation, the greater mPGES-1 and PGE2 levels lead to enhanced MMP activity in human vascular walls. The positive association between PGE2 and MMP-1 or CRP has been observed in plasma of women. We suggest that mPGES-1 inhibitors could prevent graft failure and obesity-related vascular remodeling mostly in women.

FRI0367 New autoimmune targets in idiopathic inflammatory myopathies - an antigen bead array approach

Background The Idiopathic Inflammatory Myopathies (IIM) is a group of rare systemic inflammatory diseases characterized by severe organ involvement and premature mortality. Several myositis specific auto-antibodies (MSAs) have been recognized and associated with specific clinical manifestations and prognosis, still many patients are autoantibody negative. Identification of new autoimmune targets will be helpful in improving diagnosis, better stratifying into subgroups, prediction of prognosis, tailoring treatment and to understand underlying biological pathways. Objectives To identify new autoimmune targets in IIM by antigen bead array (1). Methods A bead array with 354 antigens was used to explore the autoimmune reactivity in 881 plasma samples from patients with IIM (N=225), Systemic Lupus Erythematosus (SLE) (N=350) and population controls (N=306). The antigens were selected from initial screenings of 160 SLE-samples on a total of 5760 antigens on planar arrays, and a first verification bead array with 355 antigens. The IIM samples represented three groups of patients with distinct diagnoses: Dermatomyositis (DM, N=83), Polymyositis (PM, N=111) and Inclusion Body Myositis (IBM, N=31), who were regularly followed at the Rheumatology Unit of the Karolinska University Hospital from January 2003 until March 2014. Based on 2 possible levels of cutoff, each sample was classified as reactive to each single antigen (Ag) at low or high cut off or non-reactive. Results In general, depending on the cutoff stringency, 86–88% of the 354 selected antigens showed reactivity in at least one sample with no difference between IIM, SLE and controls. Comparing PM, DM, IBM according to the number of samples which showed reactivity towards each single Ag, reactivity at high cut off towards NADH dehydrogenase 1 α subcomplex 11 (NDUFA11), poly(A) RNA polymerase D4 (PAPD4), CD163, l(3)mbt-like 1 (L3MBTL1) and calcium release-activated calcium modulator 2 (ORAI2) was discovered with higher frequencies in the IBM samples compared to PM and DM. In the group of IIM patients testing negative for all the known MSAs increased reactivity at high cut off was observed towards E3 ubiquitin protein ligase 2 (SIAH2), leiomodin 2 (LMO2) and RAD23 homolog A (RAD23A). In the group of IIM patients with history of malignancy and no evidence for anti-p155/140 antibodies the antigens early B-cell factor 2 (EBF2), POU class 6 homebox 1 (POUF61) and growth differentiation factor 7 (GDF7) revealed high serum reactivity. In IIM patients with interstitial lung disease increased reactivity at high cut off was found towards zinc finger protein 688 (ZNF688) and prostaglandin D2 receptor (PTGDR). A high frequency of known target reactivities (MSAs) was also confirmed. Conclusions Reactivity towards autoantigens corresponding to human proteins was present in plasma samples from IIM, controls, and SLE. Potentially new autoimmune targets have been discovered in IIM subgroups, although further validation in independent cohorts is needed. References Ayoglu B1 et al. Anoctamin 2 identified as an autoimmune target in multiple sclerosis. Proc Natl Acad Sci U S A. 2016 Feb 23;113(8):2188–93. Disclosure of Interest None declared

A8.07 Characterising effects of epigenetic regulation in assays using peripheral blood mononuclear cells from patients with inflammatory diseases

Background and objectives Systemic inflammatory diseases, such as systemic lupus erythematosus (SLE) and myositis, have largely unknown aetiology and represent a disease area with majorunmet medical needs. Treatment often give a clinical effect, but not in all patients; and symptoms often remain. In collaboration with the Structural Genomics Consortium (SGC), we investigate cellular effects of chemical probes, which are drug-like molecules that can enter cells and which selectively inhibit potential new drug targets at therapeutically relevant doses. The effects we investigate are of two types, 1) either effects on expression of molecules which have been shown to be of pathological relevance in systemic inflammatory diseases, or 2) novel effects using unbiased analysis of the proteome. We have investigated cellular effects of 39 different probes which bind and inhibit epigenetic enzymes and regulators, such as bromodomains and histone methyltransferases. Material and methods Peripheral blood mononuclear cells (PBMC) from healthy donors were incubated in presence of titrated doses (typically 0,01 – 10uM) of 39 different chemical probes for 1, 3 and 6 days. Cell viability was determined using the Fluorometricmicroculture cytotoxicity assay which is based on measurements of FDA hydrolysis, or by flow cytometry using the live/dead marker viability IR® combined with markers for T cells, B cells and monocytes. Results The viability of PBMC was affected by some of the epigenetic inhibitors and if so, viability decreased after 1–3 days of incubation. The two methods gave comparable results. In cases where the probes caused an increased cell death, it was typically seen in T cells, monocytes as well as in B cells. Planned studies We aim to investigate disease relevant effects of these epigenetic chemical probes using PBMC from patients with SLE or myositis, in particular type I interferon responsiveness and B cell maturation in vitro. In addition, proteomic analysis of cells cultured in presence of these probes will be investigated in an effort to describe novel effects of inhibiting specific epigenetic regulators.

A6.15 Characterisation of systemic lupus erythematosus subgroups with features of antiphospholipid or sjögrens´s syndrome utilising affinity proteomics

Background and objectives Systemic Lupus Erythematosus (SLE) is an autoimmune disease that covers a wide range of phenotypes, from subtle symptoms to life-threatening conditions. The heterogeneous presentation of SLE is a major obstacle in clinical trials and the lack of biomarkers also hampers accurate diagnosis and choice of treatment. In this study we explored biochemical pathways in two suggested subgroups of SLE utilising affinity proteomics in order to characterise subgroups and identify biomarkers for personalised medicine. Materials and methods We have utilised Karolinska SLE cohort consisting of 320 well-characterised SLE patients and 320 individually matched population based controls in a cross-sectional study. SLE subgroups were defined only based on patients´ autoantibody profile: an Antiphospholipid Syndrome-like (APS-like, n = 55) and a Sjögrens Syndrome-like (SS-like, n = 58) subgroup. Based on literature and clinical knowledge we have made a targeted selection of 281 proteins targeted by in total 367 antibodies from the Human Protein Atlas. Antibodies were covalently coupled to colour-coded magnetic beads. EDTA-plasma samples were labelled with biotin and screened in a 384 bead array format. The read-out was made by adding a streptavidin fluorophore and using the Luminex-technology. Results We identified differences in protein profiles comparing APS-like and SS-like SLE subgroups: The top most significantly different proteins were Integrin beta 1 (p = 9.8e-8, Log2 fold change=1.5), renin (p = 2.7e-5, Log2 fold change=0.5), glutamic-oxaloacetic transaminase 1 (p = 2.9e-5, Log2 fold change=0.5) and Apolipoprotein M (p = 3.4e-5, Log2 fold change=0.3) that all were increased in SS-like SLE, while Apolipoprotein H (β2-GPI, p = 6.6e-5, Log2 fold change=-0.3) were increased in the APS-like subgroup. Conclusions The two suggested SLE subgroups were defined exclusively on autoantibody profile as the APS-like and the SS-like subgroups. Our results demonstrate that the two subgroups differ in their protein profiles and that these observations indicate underlying pathogenic differences between SS-like and APS-like SLE. A common aetiology of the presence of β2-GPI in both APS and in APS-like SLE is suggested. Therefore, we believe that stratification of SLE patients should be further explored towards personalised medicine.

A9.01 Characterisation of extracellular histidyl-trnasynthethase in myositis

Background and objectives Histidyl-transfer RNA synthetase (HisRS) is a major autoantigen in myositis with lung involvement. Simultaneous presence of anti-HisRS and anti-Ro52 antibodies has been demonstrated in patients with myositis. We investigated the presence of HisRS in extracellular compartments such as plasma, sera and bronchoalveolar lavage (BAL). In addition, the occurrence of anti-HisRS antibody isotypes as well as other auto-reactivities was evaluated in BAL and sera from patients with myositis. Materials and methods HisRS was measured in sera, plasma and BAL from myositis (anti-HisRS antibody positive, anti-Jo1+ and negative, anti-Jo1-), sarcoidosis, rheumatoid arthritis (RA) patients and healthy controls (HC) by dot-blot, western-blot, immunoprecipitation and mass spectrometry. The presence in BAL and sera of anti-Jo1 isotypes and autoantibodies to other reactivities was analysed by ELISA and addressable laser bead immunoassay. Results HisRS was detected in sera, plasma and BAL fluid of patients with myositis, sarcoidosis and RA and in HC. HisRS systemic levels were elevated in anti-Jo1+ myositis in comparison to anti-Jo1- myositis, sarcoidosis, RA or HC sera. In HC BAL HisRS was detected in significant levels. Experiments demonstrated the presence of a factor in BAL with high binding capacity for HisRS and HisRS complexed with anti-HisRS-N-terminal antibody. Immune complexes (IC) containing HisRS and C1q were not the binding factor. However, several anti-HisRS isotypes (anti-Jo1 IgG, IgM and IgA) as well as anti-Ro52IgG were identified in both BAL and sera. Furthermore, a positive correlation between the presence of anti-Jo1 IgG and anti-Ro52IgG in BAL was identified. Conclusions HisRS was detected both in blood and BAL fluid. The identification of extracellular HisRS, anti-HisRSisotypes and anti-Ro52 in myositis BAL may provide additional clues for the development of autoimmunity in the lungs.

SAT0189 Characterization of Extracellular Histidyl-TRNA Synthetase in Myositis

Background Histidyl-transfer RNA synthetase (HisRS) is a major autoantigen in myositis with lung involvement1–4. Simultaneous presence of anti-HisRS and anti-Ro52 antibodies has been demonstrated in patients with myositis5–7. Objectives We investigated the presence of HisRS in the extracellular compartments plasma, sera and bronchoalveolar lavage fluid (BAL). In addition, the occurrence of anti-HisRS antibody isotypes as well as other auto-reactivities was evaluated in BAL and sera from patients with myositis. Methods HisRS was measured in sera, plasma and BAL from myositis (anti-HisRS antibody positive, anti-Jo1+ and negative, anti-Jo1-), sarcoidosis, rheumatoid arthritis (RA) patients and healthy controls (HC) by dot-blot, western-blot, immunoprecipitation and mass spectrometry. The presence in BAL and sera of anti-Jo1 isotypes and other ANA-associated autoantibodies was analysed by ELISA and addressable laser bead immunoassay. Results HisRS was detected in sera, plasma and BAL of patients with myositis, sarcoidosis and RA and in HC. HisRS systemic levels were elevated in anti-Jo1+ myositis (14 out of 20 sera) compared to anti-Jo1- myositis (10/18), sarcoidosis (0/8) and RA (3/15) patients, and HC (5/23). In BAL, significant levels of HisRS were detected in 6/8 HC and 5/8 sarcoidosis, compared to 4/8 myositis (2 anti-Jo1+ and 2 anti-Jo1-). Our results demonstrated the presence of a factor in BAL with high binding capacity for HisRS and HisRS complexed with anti-HisRS-N-terminal antibody. C1q-binding immune complexes (IC) containing HisRS were not the binding factor. However, anti-Jo1 isotypes as well as anti-Ro52 IgG were identified in both BAL (5/8 patients were positive for anti-Jo1 IgG, 3/8 for anti-Jo1 IgA, 3/8 for anti-Jo1 IgM and 4/8 for anti-Ro52 IgG) and sera (5/8 for anti-Jo1 IgG, 3/8 for anti-Jo1 IgA, 4/8 for anti-Jo1 IgM and 3/8 for anti-Ro52 IgG) from patients with myositis. Furthermore, a positive correlation between the presence of anti-Jo1 IgG and anti-Ro52 IgG in BAL was identified (r2=0,881; p=0,007). Conclusions HisRS was detected both in blood and BAL fluid. The identification of extracellular HisRS, anti-Jo1 isotypes and anti-Ro52 in myositis BAL may provide additional clues for the development of autoimmunity in the lungs. References Bernstein RM., et al. Br Med J (Clin Res Ed). 1984 Jul 21;289(6438):151–2; Marguerie C., et al. Q J Med. 1990 Oct;77(282):1019–38; Hervier B., et al. Eur Respir J. 2013 Nov;42(5):1271–82; Hamaguchi Y., et al. PLoS One. 2013;8(4):e60442; La Corte R., et al. Autoimmunity. 2006 May;39(3):249–53; Brouwer R., et al. Ann Rheum Dis. 2001 Feb;60(2):116–23; Rutjes SA., et al. Clin Exp Immunol. 1997 Jul;109(1):32–40. Disclosure of Interest None declared

A2.22 Influence of TNF on the proteome of rheumatoid arthritis synovial fibroblasts

Background and objectives Rheumatoid arthritis (RA) is a chronic autoimmune systemic disorder that affects primarily the joints and leads to their destruction. Rheumatoid arthritis synovial fibroblasts (RASFs) localised in the RA synovium play a crucial role in the destructions of joints by production of pro-inflammatory cytokines, matrix-degrading enzymes or via production of matrix metalloproteinases (MMPs). Activated leukocytes or adipocytes secrete Tumour Necrosis Factor (TNF), which in turn stimulates RASFs resulting in production of MMPs, maintenance of inflammation, and induction of osteoclastogenesis. For better understanding on how TNF contributes to RASFs stimulation and to reveal pathways activated by TNF in RASFs, we investigate the effect of TNF-alpha on the proteome of human RASFs obtained from synovial tissues of patients with RA. Materials and Methods Expanded in cell culture, RASFs (passages 5 and 7) obtained from two CCP- donors, were stimulated with TNF (10 ng/ml, 24 h). Cells were harvested and lysed in buffer containing SDS. Quantitative proteome analysis utilising iTRAQ (Isobaric Tagging for Relative and Absolute Quantification) - labelling was employed to the TNF treated and untreated cells in order to detect gene expression at the protein level. Pathway analysis was performed by using Ingenuity Systems Pathway Analysis (IPA) program. Results Out of 1028 profiled proteins, 14 proteins exhibited elevated expression and 28 proteins showed reduced expression upon stimulation with TNF-alpha. The following canonical pathways (top 5) were activated: EIF2 Signalling, Regulation of eIF4 and p70S6K Signalling, mTOR Signalling, Remodelling of Epithelial Adherens Junctions and Protein Ubiquitination Pathway. IPA analysis showed that TNF-alpha increased cell-to-cell signalling and interaction and tissue development by increasing attachment of cells, promotion of cell death of connective tissue cells, induction of reactive oxygen species, increased apoptosis and decreased degradation of oxygen peroxide. Conclusions Quantitative proteomic analysis of RASFs stimulated with TNF-alpha revealed upregulation of several genes involved in inflammatory processes in RA and bone destruction as well as activation of several pathways, which play an important role in protein folding and osteoclastgenesis.