E. Ossipova

IgG Antibodies to Cyclic Citrullinated Peptides Exhibit Profiles Specific in Terms of IgG Subclasses, Fc-Glycans and a Fab-Peptide Sequence

The Fc-glycan profile of IgG1 anti-citrullinated peptide antibodies (ACPA) in rheumatoid arthritis (RA) patients has recently been reported to be different from non-ACPA IgG1, a phenomenon which likely plays a role in RA pathogenesis. Herein we investigate the Fc-glycosylation pattern of all ACPA-IgG isotypes and simultaneously investigate in detail the IgG protein-chain sequence repertoire. IgG from serum or plasma (S/P, nu200a=u200a14) and synovial fluid (SF, nu200a=u200a4) from 18 ACPA-positive RA-patients was enriched using Protein G columns followed by ACPA-purification on cyclic citrullinated peptide-2 (CCP2)-coupled columns. Paired ACPA (anti-CCP2 eluted IgG) and IgG flow through (FT) fractions were analyzed by LC-MS/MS-proteomics. IgG peptides, isotypes and corresponding Fc-glycopeptides were quantified and interrogated using uni- and multivariate statistics. The Fc-glycans from the IgG4 peptide EEQFNSTYR was validated using protein A column purification. Relative to FT-IgG4, the ACPA-IgG4 Fc-glycan-profile contained lower amounts (pu200a=u200a0.002) of the agalacto and asialylated core-fucosylated biantennary form (FA2) and higher content (pu200a=u200a0.001) of sialylated glycans. Novel differences in the Fc-glycan-profile of ACPA-IgG1 compared to FT-IgG1 were observed in the distribution of bisected forms (nu200a=u200a5, pu200a=u200a0.0001, decrease) and mono-antennnary forms (nu200a=u200a3, pu200a=u200a0.02, increase). Our study also confirmed higher abundance of FA2 (pu200a=u200a0.002) and lower abundance of afucosylated forms (nu200a=u200a4, pu200a=u200a0.001) in ACPA-IgG1 relative to FT-IgG1 as well as lower content of IgG2 (pu200a=u200a0.0000001) and elevated content of IgG4 (pu200a=u200a0.004) in ACPA compared to FT. One λ-variable peptide sequence was significantly increased in ACPA (pu200a=u200a0.0001). In conclusion, the Fc-glycan profile of both ACPA-IgG1 and ACPA-IgG4 are distinct. Given that IgG1 and IgG4 have different Fc-receptor and complement binding affinities, this phenomenon likely affects ACPA effector- and immune-regulatory functions in an IgG isotype-specific manner. These findings further highlight the importance of antibody characterization in relation to functional in vivo and in vitro studies.

Light-activatable prodrugs based on hyaluronic acid biomaterials

Photosensitive in situ cross-linked hyaluronan (HA) hydrogels are prepared by modular chemoselective assembly from the biopolymer precursors and novel heterobifunctional linkers with middle photo-labile ortho-nitrobenzyl group and orthogonally reactive terminals. Starting from the thiol-modified HA and a linker with activated disulfide and hydrazide terminals, a photo-degradable HA hydrogel was prepared by the hydrazone cross-linking reaction. Moreover, a light-triggered drug-releasing hydrogel prodrug was constructed by an orthogonal conjugation of dopamine (DA) via a photo-labile linker to HA dually modified with thiol and hydrazide groups (hy-HA-SH) and a subsequent cross-linking with aldehyde-derivatized HA (HA-al). On-demand release of DA from the hydrogel was achieved upon exposure of the hydrogel to UV light whereas 11-fold less release of the drug was observed in the absence of light. The mechanical properties of the hydrogels, photodegradation kinetics, photorelease of the model drugs were studied by rheology, spectrophotometry, chromatography, and mass spectrometry. For the first time, integration of photolabile components into an actual polysaccharide of extracellular matrix was implemented for the light-controlled release of drug molecules.

Proteome analysis reveals an upregulation of heat-shock proteins in LPS-induced peritoneal macrophages from mPGES-1 knock-out mice

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.

AB1064 Exploring cerebrospinal fluid proteome in fibromyalgia

Background Fibromyalgia (FM) is a heterogeneous disease of unknown etiology characterised by chronic widespread pain that affects up to 4% of population. Overlapping and heterogeneous symptoms of various chronic pain conditions complicate their diagnosis, emphasising the need for more specific biomarkers to improve the diagnosis and understand the disease mechanisms. Cerebrospinal fluid (CSF) flows in the ventricles within the brain and diffuses over the brain and spinal cord. Due to direct contact of CSF with CNS, content of CSF reflects biochemical changes in CNS making it an excellent source for biomarker discovery. Objectives In current study we aim to explore CSF proteome of FM patients utilising quantitative proteomics method based on stable isotope labelling of CSF peptides combined with multivariate data analysis (MVDA) in order to monitor the dynamics of the proteome while comparing to the CSF proteomes in patients with rheumatoid arthritis (RA) and other neurological diseases (OND) and define the potential biomarker candidates in FM. We also investigate, which protein products have been found in human CSF with respect to known “pain” genes,1 human CSF proteome explored if these proteins represent any clear subgrouping of “pain proteins”. Methods CSF samples from patients with FM, RA and control OND group were collected by lumbar puncture and equal aliquots were subsequently reduced, alkylated and digested by trypsin. Obtained peptides were labelled by stable isotopes and mixed prior sample fractionation. The degree of sample complexity was reduced by off-line peptide separation using HPLC instrumentation. Obtained 80 peptide fractions were combined into 10 fractions across the gradient area. Fractions were analysed by LC-MS/MS, proteins in acquired data were identified and quantified, and data was analysed using MVDA. Results Out of the 1422 proteins identified, 855 proteins were included in the quantitative data analysis. Comparing FM, RA and OND groups to each other using univariate testing we found 53 statistical significant proteins (q-value <0.05). Six out of these have been reported as “pain proteins” (Complement C4-A, Prostaglandin-H2 d-isomerase, Apolipoprotein D, Granulins, Pro-cathepsin H, and BMP and activin membrane-bound inhibitor homolog). Conclusions We have employed quantitative proteomics methods combined with extensive bioinformatics data analysis to investigate differences in proteome profiles in CSFs obtained from patients with FM, and identified six differentially expressed pain proteins of various functions in CSF of FM patients. The involvement of these proteins in the disease pathogenesis as well use of the identified proteins as potential biomarkers should be investigated. Reference [1] Ultsch A, Kringel D, Kalso E, Mogil JS, Lötsch J. A data science approach to candidate gene selection of pain regarded as a process of learning and neural plasticity. Pain. 2016. Acknowledgements The research leading to these results has received funding from the European Union Seventh Framework Programme (FP7/2007 – 2013) under grant agreement no 6u200902u2009919 (GLORIA), Swedish Medical Research Council, Åke Wiberg Foundation and Magnus Bergvalls Foundation and Uppsala Berzelii Technology Centre for Neurodiagnostics, with financing from the Swedish Governmental Agency for Innovation Systems. Disclosure of Interest None declared

A9.6 Effect of mPGES-1 targeting on lipid metabolism in human cells

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. Methodsxa0 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.

FRI0101 Lung changes are present in ACPA positive RA already at disease onset

Background We have previously demonstrated that smoking induces citrullination in the lungs of healthy smokers and we know that ACPA develops in RA patients many years before disease onset. Objectives In this study we aimed to investigate the hypothesis that lung changes in association with smoking and ACPA might be a primary rather than a secondary extra articular manifestation in RA. Methods 103 RA patients with symptom duration less than 1 year at the time of diagnosis and naive to DMARD treatment were included in the RA cohort. A second non-RA cohort matched for age, smoking status and gender (n=43) was investigated with an identical protocol. X-ray, (high resolution computer tomography (HRCT) and dynamic spirometry were performed in both cohorts. HRCT changes were defined as presence of infiltrates, fibrosis, emphysema and bronchiectasis. Bronchoscopy was performed in a subgroup of RA patients (n=21) and BAL samples as well as mucosal large bronchial biopsies were retrieved. ELISA was used to detect local production of IgA and IgG ACPA in the BAL fluid. Mass spectrometry was used for identification of citrullinated epitopes in 6 of the lung biopsies and additional 8 synovial RA biopsies. immunohistocehmsitry was performed for detection peptydilamino deiminase (PAD) enzymes in the lungs biopseis. Contingency tables and chi-square test as well as a generalized linear model were used for analysis of the clinical data. Man-Whitney test was used to analyze differences in immunohistochemistry double blind semi-quantitative scores between independent groups. Results 44% of the RA patients had identifiable HRCT changes as compared with only 23% in the healthy non-RA controls. ACPA presence but not smoking status was associated with HRCT changes. A majority of serum ACPA positive RA patients subjected to lung bronchoscopy had detectable levels of ACPA in the BAL fluids. Mass spectometry identified 5 proteins in the synovium (in total 8 sites) and 4 in the lungs (in total 6 sites) containing citrullinated residues. Two vimentin derived citrullinated peptides were present in a majority of both synovial and lung biopsies with slightly higher citrullinated/unmodified peptides ratios in the smokers as compared to non-smokers (median ratio of 0.03 in smokers and 0.02 in non smokers for one of the peptides and a median ratio of 4.5 in the smokers and 0.04 in the non smokers for the second vimentin peptide). Immunohistochemistry demonstrated a significant increase in expression of both PAD2 and PAD4 in the lungs of current smokers independent of the ACPA status. Results on citrullinated protein expression are pending. Conclusions We demonstrate that HRCT abnormalities, shared citrullinated epitopes with the joints as well as local production of ACPA are present in the in RA lungs already at disease onset. We propose that site-specific extra articular changes (such as in the lungs) are the intitiating event of the specific immune response in ACPA positive RA. Disclosure of Interest None Declared

AB0043 Affinity purification and characterization of human acpas

Background Autoimmunity in rheumatoid arthritis (RA) is characterized by autoantibodies to citrullinated proteins/peptides (ACPA)1. These antibodies, present in 60-70% of patients, antedate clinical onset and associate with an erosive disease course, suggesting a direct pathogenic involvement in disease initiation and progression2. Objectives With this study, we aimed to develop an efficient method for the purification of human ACPA, and to characterize their frequency and fine-specificity pattern in synovial fluid (SF) and plasma of RA patients. Methods SF and plasma samples were collected with informed consent and ethical approval from patients (fulfilling the ACR criteria for RA) with high anti-CCP antibody levels. SF samples (n=36) were first treated with hyaluronidase to decrease viscosity, then proteins were precipitated with ammonium sulfate, dissolved and further dialysed against phosphate buffered saline (PBS), before the IgG fractions were purified on ProteinG columns (GE Healthcare, Uppsala, Sweden). Plasma samples (n=10) were diluted in PBS before applied to the ProteinG column. ACPAs were further purified using CCP2 affinity columns, kindly provided by Euro-Diagnostica. Recovery and purity of total IgG and anti-CCP IgG were analysed using SDS-PAGE, Nanodrop (Thermo Scientific, Wilmington, DE, USA) and the CCP2-ELISA kit. Fine-specificity of the purified ACPAs were investigated using in-house ELISAs, with peptides from citrullinated α-enolase (CEP-1), -vimentin (Cit-vim), -fibrinogen (Cit-fib) and -collagen type II (Cit-C1). Results Anti-CCP IgG could efficiently be purified from SF and plasma, using ProteinG-, followed by CCP2-, columns. No CCP IgG response could be detected in the flow-through fractions. Higher concentrations of total IgG were found in plasma (13,6 mg/ml) compared to SF (4,2 mg/ml), while a higher percentage of CCP-specific IgG was detected in SF (3%), compared to plasma (2%). The purified anti-CCP IgG fractions cross-reacted with CEP-1, Cit-vim, Cit-fib and Cit-C1, while no reactivity to these citrullinated antigens were detected in the IgG flow-through fractions. Anti-CCP IgG dilution curves (starting at 10 μg/ml of purified antibodies) demonstrated differences in affinity between patients, which may correspond to the different ACPA-fine specificity patterns seen in patients. Conclusions The described methodology efficiently purifies ACPAs with multiple specificities, which will allow for their use in in vivo and in vitro studies, to further elucidate their arthritogenic and pathogenic capacity. In addition, the ACPAs will be tools for future immunoprecipitation-, immunoblotting- and immunohistochemistry experiments. References Schellekens, G. A. et al., The diagnostic properties of rheumatoid arthritis antibodies recognizing a cyclic citrullinated peptide. Arthritis Rheum 43(1), 155 (2000). van der Helm-van Mil AH, Verpoort KN, Breedveld FC, Toes RE, Huizinga TW, Antibodies to citrullinated proteins and differences in clinical progression of rheumatoid arthritis. Arthritis Res Ther 7(5), R949 (2005). Disclosure of Interest None Declared

ACPA response against fibrinogen epitopes citrullinated in vivo in the synovial membrane in RA patients detected with an autoantibody microarray

Backgroundand objectives Citrullination is a post-translational modification of proteins catalysed by specific peptidylarginine deiminases, which modify arginine residues to citrulline. Citrullinated proteins are frequently detected in various inflammatory states including rheumatoid arthritis, and a lot of attention has been drawn to the presence of auto-antibodies targeting proteins containing citrulline. Using high resolution mass spectrometry (MS) the authors have first identified citrullinated peptides corresponding to amino acids 559–575 of fibrinogen α-chain and corresponding to amino acids 52–77 of the fibrinogen β-chain in synovial tissues from patient with rheumatoid arthritis (RA) (Hermansson et al Proteomics: Clinical Applications 2010). In this work the authors have also identified citrullinated peptide corresponding to amino acids 580–599 of fibrinogen α-chain but decided do not include it to publication due to possible deamidation of asparagine or glutamine, which leads to the same mass shift as deamidation of arginine. In this follow-up study The authors test sera from RA patients for anticitrullinated protein antibodies (ACPA) response against MS identified citrullinated fibrinogen peptides. Material and methods In this work a microarray assay based on Phadia´s ImmunoCAP ISAC system, where reactivity to more than 100 antigens can be analysed simultaneously using small volumes of sera, was employed. Four synthetised fibrinogen peptides (containing citrulline at the positions 573 (Fib 573), 591 (Fib 591) within the α-chain and at the positions 72 (Fib 72) and 74 (Fib 74) within the β-chain, respectively) were immobilised onto a chemically modified glass slide in an arrayed fashion. After binding of antigens to the glass surface diluted serum from 936 RA patients (404 cyclic citrullinated peptide (CCP) positive and 532 CCP negative) and 461 healthy controls from the Epidemiological Investigations in Rheumatoid Arthritis (EIRA) case control cohort were applied into reactions sites on the glass slides, followed by fluorescent-labeled anti-human IgG antibody and scanning in a laser scanner. Cutoff values were determined as the 98th percentile of the healthy control responses. Results The authors found that 31% (among them 87% are CCP positive) of patients were positive to Fib573 peptide. For the Fib2591 peptide, the corresponding figures were 10% (65%), for the Fib74 peptide 28% (68%) and for the Fib72 peptide 20% (68%). Conclusions Here the authors show extensive autoantibody reactivity against fibrinogen epitopes that are citrullinated in vivo in RA synovial membranes. A substantial part of the ACPA peptide reactivities were found among anti-CCP2 negative RA patients. The results suggest the use of these citrullinated fibrinogen peptides as biomarkers in RA.

Humoral immune response against fibrinogen epitopes citrullinated in vivo in rheumatoid arthritis synovial tissue detected by autoantibody multiplexing

Background The anticitrullinated protein/peptide antibody (ACPA) response in rheumatoid arthritis (RA) might be driven by key autoantigens citrullinated in the target organ. We have recently by using high definition mass spectrometry defined fibrinogen sequences which are citrullinated in RA synovial tissue.1 Objective To use a newly developed multiplexing assay to investigate the humoral immune response against such in vivo citrullinated fibrinogen epitopes. Methods The Phadia ImmunoCAP ISAC chip was used to bind four synthetic fibrinogen peptides containing citrulline as shown in vivo corresponding to the α chain positions 573 and 591, the β chain positions 72 and 74, as well as their arginine-containing control peptides. 848 RA patients and 150 controls from the Epidemiological Investigations in Rheumatoid Arthritis cohort were investigated. Peptide-specific ELISA tests were developed for validation of the microarray in a smaller group of RA sera (n=123) and controls (n=20). Results Using the 98th percentile among the controls, 13.3% (113/848) of the RA patients had antibodies against the citrullinated 573 peptide. There was a substantial reactivity also to the arginine control peptide both in RA patients and controls, not generally observed for other arginine-containing control peptides. After subtraction of the arginine response, the net immune response discriminated better between patients and controls, and using the 98th percentile for the net citrulline response among HC, 35.5% (301/848) of the RA patients were autoantibody positive. The area under the receiver operator characteristics (ROC) curve was 0.610 for uncorrected values, but after subtraction of the readout for arginine controls the AUC increased to 0.800. The microarray showed good correlation to autoantibody levels measured with ELISA (r=0.83). The four peptides showed considerably divergent responses in ROC curve analyses both before and after correction for arginine control peptides, and the remaining three citrullinated peptides are currently validated. Conclusion This study proves a substantial autoantibody reactivity against fibrinogen epitopes citrullinated in vivo in RA synovial tissue. Autoantibody multiplexing can be used for the screening humoral reactivities against large numbers of potential autoantigenic epitopes, for example, those found citrullinated in vivo. This technique also allows easy correction for autoantibody reactivity against the arginine-containing peptide backbone normally not performed with ACPA ELISA tests. Concerning the fibrinogen 573 peptide, such correction increased the diagnostic performance of autoantibody measurement considerably.