Elena Ossipova

Induction of osteoclastogenesis and bone loss by human autoantibodies against citrullinated vimentin

Autoimmunity is complicated by bone loss. In human rheumatoid arthritis (RA), the most severe inflammatory joint disease, autoantibodies against citrullinated proteins are among the strongest risk factors for bone destruction. We therefore hypothesized that these autoantibodies directly influence bone metabolism. Here, we found a strong and specific association between autoantibodies against citrullinated proteins and serum markers for osteoclast-mediated bone resorption in RA patients. Moreover, human osteoclasts expressed enzymes eliciting protein citrullination, and specific N-terminal citrullination of vimentin was induced during osteoclast differentiation. Affinity-purified human autoantibodies against mutated citrullinated vimentin (MCV) not only bound to osteoclast surfaces, but also led to robust induction of osteoclastogenesis and bone-resorptive activity. Adoptive transfer of purified human MCV autoantibodies into mice induced osteopenia and increased osteoclastogenesis. This effect was based on the inducible release of TNF-α from osteoclast precursors and the subsequent increase of osteoclast precursor cell numbers with enhanced expression of activation and growth factor receptors. Our data thus suggest that autoantibody formation in response to citrullinated vimentin directly induces bone loss, providing a link between the adaptive immune system and bone.

Shared immunological targets in the lungs and joints of patients with rheumatoid arthritis: identification and validation

Objectives Immunological events in the lungs might trigger production of anti-citrullinated protein antibodies during early rheumatoid arthritis (RA). We investigated the presence of shared immunological citrullinated targets in joints and lungs of patients with RA. Patients and methods Proteins extracted from bronchial (n=6) and synovial (n=7) biopsy specimens from patients with RA were investigated by mass spectrometry-based proteomics. One candidate peptide was synthesised and used to investigate by ELISA the presence of antibodies in patients with RA (n=393), healthy controls (n=152) and disease controls (n=236). HLA-DRB1 shared epitope (SE) alleles were detected in patients with RA. Results Ten citrullinated peptides belonging to seven proteins were identified, with two peptides shared between the synovial and bronchial biopsy samples. Further analysis, using accurate mass and retention time, enabled detection of eight citrullinated peptides in synovial and seven in bronchial biopsy specimens, with five peptides shared between the synovial and bronchial biopsy specimens. Two citrullinated vimentin (cit-vim) peptides were detected in the majority of synovial and lung tissues. Antibodies to a synthesised cit-vim peptide candidate (covering both cit-vim peptides identified in vivo) were present in 1.8% of healthy controls, 15% of patients with RA, and 3.4% of disease controls. Antibodies to cit-vim peptide were associated with the presence of the SE alleles in RA. Conclusions Identical citrullinated peptides are present in bronchial and synovial tissues, which may be used as immunological targets for antibodies of patients with RA. The data provide further support for a link between lungs and joints in RA and identify potential targets for immunity that may mediate this link.

MS analysis of rheumatoid arthritic synovial tissue identifies specific citrullination sites on fibrinogen

Purpose: Citrullination is a post‐translational modification of arginine residues to citrulline catalyzed by peptidyl arginine deiminases. Induced expression of citrullinated proteins are frequently detected in various inflammatory states including arthritis; however, direct detection of citrullination in arthritic samples has not been successfully performed in the past.

Endurance Exercise Improves Molecular Pathways of Aerobic Metabolism in Patients With Myositis

Endurance exercise demonstrates beneficial effects in polymyositis/dermatomyositis (PM/DM); however, the molecular effects of exercise on skeletal muscle are incompletely understood. We undertook this controlled pilot study to investigate the effects of a 12‐week endurance exercise training program on the molecular profile of skeletal muscle in patients with established PM/DM compared to a nonexercised control group of patients with established PM/DM.

Targeting of anti-citrullinated protein/peptide antibodies in rheumatoid arthritis using peptides mimicking endogenously citrullinated fibrinogen antigens

IntroductionWe have previously identified endogenously citrullinated peptides derived from fibrinogen in rheumatoid arthritis (RA) synovial tissues. In this study, we have investigated the auto-antigenicity of four of those citrullinated peptides, and explored their feasibility to target anti-citrullinated protein/peptide antibodies (ACPA).MethodsThe autoantigenic potential of the fibrinogen peptides was investigated by screening 927 serum samples from the Epidemiological Investigation of RA (EIRA) cohort on a peptide microarray based on the ImmunoCAP ISAC® system. In order to assay for ACPA blocking, two independent pools of purified ACPA were incubated with the respective targeting peptide prior to binding to cyclic citrullinated peptide (CCP)2 using the CCPlus® ELISA kit.ResultsTwo peptides derived from the fibrinogen α chain, Arg573Cit (563-583) and Arg591Cit (580-600), referred to as Cit573 and Cit591, and two peptides from the fibrinogen β chain, Arg72Cit (62-81) and Arg74Cit (62-81) (Cit72 and Cit74), displayed 65 %, 15 %, 35 %, and 53 % of immune reactivity among CCP2-positive RA sera, respectively. In CCP2-negative RA sera, a positive reactivity was detected in 5 % (Cit573), 6 % (Cit591), 8 % (Cit72), and 4 % (Cit74). In the competition assay, Cit573 and Cit591 peptides reduced ACPA binding to CCP2 by a maximum of 84 % and 63 % respectively. An additive effect was observed when these peptides were combined. In contrast, Cit74 and Cit72 were less effective. Cyclization of the peptide structure containing Cit573 significantly increased the blocking efficiency.ConclusionsHere we demonstrate extensive autoantibody reactivity against in vivo citrullinated fibrinogen epitopes, and further show the potential use of these peptides for antagonizing ACPA.

Identification of shared citrullinated immunological targets in the lungs and joints of patients with rheumatoid arthritis

Background The authors have previously demonstrated that smoking induces citrullination in the lungs of healthy smokers and they know that anticitrullinated protein antibodies (ACPA) develop in rheumatoid arthritis (RA) patients many years before disease onset. It was hypothesised that shared citrullinated targets are present in the lungs and joints of RA affected individuals and sought to investigate this by full-proteome analysis of synovial and lung biopsies of RA patients. Material and methods Proteins were extracted from synovial (n=7, five females and two males, median age 58, 66.7% ACPA positive) and lung (n=6, four females and two males, median age 63, 66.7% ACPA positive) biopsies of RA patients. Synovial biopsies were obtained at the time of open surgery from patients with long-standing RA (mean disease duration 24 years). Large bronchi biopsies were obtained by bronchoscopy from patients with newly diagnosed RA (three smokers and three non-smokers) with symptom duration less than 1 year. The proteins were reduced, alkylated and digested with Lys-C, separated by reverse-phase nanoflow-chromatography and analysed by LTQ-Velos-Orbitrap using multiple fragmentation methods. The data were searched against the human International Protein Index database using the Mascot search engine and all citrullinated peptides were manually verified. The degree of modification was quantified manually. The final results were expressed as ratios of citrullinated versus non-modified peptides. Results Over 3300 peptides and 500 proteins were identified in the different samples. The overall protein profiles varied between patients. Five of the identified proteins in the synovium (in total eight sites) and four in the lungs (in total four sites) contained citrullinated residues. Two vimentin derived citrullinated peptides were present in a majority of synovial and lung biopsies with slightly higher citrullinated/unmodified peptides ratios in smokers 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). While non-modified and citrullinated fibrinogen α-chain derived peptides were present in various amounts in the synovium, only the unmodified sites could be detected in the lungs of a subset of the patients (three out of six). Conclusions The authors demonstrate the presence of shared in vivo citrullinated proteins in the joints and lungs of RA individuals, providing further support for the important pathogenic link between joints and lungs in development of RA.

Mass spectrometry-based proteomics identifies UPF1 as a critical gene expression regulator in MonoMac 6 cells.

5-Lipoxygenase (5-LO) catalyzes the two initial steps in the biosynthesis of leukotrienes, a group of inflammatory lipid mediators derived from arachidonic acid. Recently, we have demonstrated that 5-LO mRNA expression is regulated by alternative splicing and nonsense-mediated mRNA decay (NMD). In addition to this, 5-LO protein expression was reduced on translational level in UPF1 knockdown cells, suggesting that UPF1 has a positive influence on 5-LO translation. Therefore, a mass spectrometry-based proteomics study was performed to identify compartment-specific protein expression changes upon UPF1 knockdown in differentiated and undifferentiated MM6 cells. The proteomics analysis revealed that the knockdown of UPF1 results in numerous protein changes in the microsomal fraction (~21%) but not in the cytosolic fraction (<1%). The results suggest that UPF1 is a critical gene expression regulator in a compartment-specific way. During differentiation by TGFβ and calcitriol, the majority of UPF1 regulated proteins were adjusted to normal level. This indicates that the translational regulation by UPF1 can potentially be cell differentiation-dependent.

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).

02.48 Inhibition of in vitro b cell maturation and igg secretion by new chemical probes in assays using blood cells from patients with sle and iim

Background Systemic inflammatory diseases, such as systemic lupus erythematosus (SLE) and idiopathic inflammatory myositis (IIM), have largely unknown aetiology and represent a disease area with major unmet 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 have investigated in vitro B-cell effects of about 50 different chemical probes which bind and inhibit epigenetic enzymes and regulators, such as bromodomains and histone methyltransferases, as well as kinases and other intracellular protein targets. These probes are small molecules that can enter cells and selectively inhibit potential new drug-targets at therapeutically relevant doses. The read out are expression of molecules which have been shown to be of pathological relevance in systemic inflammatory diseases. Materials and methods Peripheral blood mononuclear cells (PBMC) from patients with SLE or IIM and healthy controls were incubated in presence of different chemical probes at 1 or 0,1 uM for 6 days in culture medium containing B-cell stimulating factors; IL4, IL10, IL21, sCD40L and CpG2006. Cell viability was determined by flow cytometry using the live/dead marker viability IR® and B-cell maturation was investigated using markers for memory B cells (CD27), plasmablasts (CD38) and surface IgG. Secretion of IgG was quantified by ELISA. Results The percentage of memory B cells, plasmablasts and IgG-expressing B cells, as well as IgG secretion, was induced by the B-cell stimulating factors. These parameters were suppressed by a set of the epigenetic probes, as well as by some of the probes targeting other intracellular proteins. A few of the probes decreased cell viability. Both B cells from SLE and IIM were affected. Conclusions We have found a set of chemical probes, with documented target selectivity and potent inhibitory effects of these targets, which affect in vitro B-cell maturation and secretion of IgG. Further analysis is required to better understand the pathways that are affected.

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.