Holger Bang

Carbamylation of vimentin is inducible by smoking and represents an independent autoantigen in rheumatoid arthritis

Objectives Smoking has been connected to citrullination of antigens and formation of anti-citrullinated peptide antibodies (ACPAs) in rheumatoid arthritis (RA). Since smoking can modify proteins by carbamylation (formation of homocitrulline), this study was conducted to investigate these effects on vimentin in animal models and RA. Methods The efficiency of enzymatic carbamylation of vimentin was characterised. B-cell response was investigated after immunisation of rabbits with different vimentin isoforms. Effects of tobacco smoke exposure on carbamylation of vimentin and formation of autoantibodies were analysed in mice. The antibody responses against isoforms of vimentin were characterised with respect to disease duration and smoking status of patients with RA. Results Enzymatic carbamylation of vimentin was efficiently achieved. Subsequent citrullination of vimentin was not disturbed by homocitrullination. Sera from rabbits immunised with carbamylated vimentin (carbVim), in addition to carbVim also recognised human IgG-Fc showing rheumatoid factor-like reactivity. Smoke-exposed mice contained detectable amounts of carbVim and developed a broad immune response against carbamylated antigens. Although the prevalence of anti-carbamylated antibodies in smokers and non-smokers was similar, the titres of carbamylated antibodies were significantly increased in sera of smoking compared with non-smoking RA. CarbVim antibodies were observed independently of ACPAs in early phases of disease and double-positive patients for anti-mutated citrullinated vimentin (MCV) and anti-carbVim antibodies showed an extended epitope recognition pattern towards MCV. Conclusions Carbamylation of vimentin is inducible by cigarette smoke exposure. The polyclonal immune response against modified antigens in patients with RA is not exclusively citrulline-specific and carbamylation of antigens could be involved in the pathogenesis of disease. Trial registration number ISRCTN36745608; EudraCT Number: 2006-003146-41.

Antimodified protein antibody response pattern influences the risk for disease relapse in patients with rheumatoid arthritis tapering disease modifying antirheumatic drugs

Objective To perform a detailed analysis of the autoantibody response against post-translationally modified proteins in patients with rheumatoid arthritis (RA) in sustained remission and to explore whether its composition influences the risk for disease relapse when tapering disease modifying antirheumatic drug (DMARD) therapy. Methods Immune responses against 10 citrullinated, homocitrullinated/carbamylated and acetylated peptides, as well as unmodified vimentin (control) and cyclic citrullinated peptide 2 (CCP2) were tested in baseline serum samples from 94 patients of the RETRO study. Patients were classified according to the number of autoantibody reactivities (0–1/10, 2–5/10 and >5/10) or specificity groups (citrullination, carbamylation and acetylation; 0–3) and tested for their risk to develop relapses after DMARD tapering. Demographic and disease-specific parameters were included in multivariate logistic regression analysis for defining the role of autoantibodies in predicting relapse. Results Patients varied in their antimodified protein antibody response with the extremes from recognition of no (0/10) to all antigens (10/10). Antibodies against citrullinated vimentin (51%), acetylated ornithine (46%) and acetylated lysine (37%) were the most frequently observed subspecificities. Relapse risk significantly (p=0.011) increased from 18% (0–1/10 reactivities) to 34% (2–5/10) and 55% (>5/10). With respect to specificity groups (0–3), relapse risk significantly (p=0.021) increased from 18% (no reactivity) to 28%, 36% and finally to 52% with one, two or three antibody specificity groups, respectively. Conclusions The data suggest that the pattern of antimodified protein antibody response determines the risk of disease relapse in patients with RA tapering DMARD therapy. Trial registration number 2009-015740-42; Results.

Abatacept blocks anti-citrullinated protein antibody and rheumatoid factor mediated cytokine production in human macrophages in IDO-dependent manner

BackgroundThe anti-inflammatory effect of abatacept is most pronounced in patients with high-titer autoantibodies (including anticitrullinated protein antibodies [ACPA] and rheumatoid factor [RF]). Considering that autoantibodies trigger inflammatory cytokine production by monocytes and that abatacept binds to monocytes, influencing their functional state, we hypothesized that abatacept may effectively inhibit the production of several different cytokines by ACPA- or RF-challenged monocytes.MethodsPeripheral blood CD68+ monocytes stimulated with macrophage colony-stimulating factor for 24 h were exposed to random immunoglobulin G alone (negative control), purified ACPA, purified RF, or lipopolysaccharide (positive control) in cell culture plates coated with citrullinated vimentin (to allow ACPA immune complex formation). Stimulations were done in the presence or absence of abatacept or tumor necrosis factor (TNF) antibody (adalimumab) with or without indoleamine 2,3-dioxygenase (IDO) inhibitor 1-methyl-d-tryptophan. Supernatants were analyzed for key proinflammatory cytokines TNF-α, interleukin (IL)-1β, IL-6, IL-8, and chemokine (C-C motif) ligand 2 (CCL2) after 24 h.ResultsExposure to ACPA or RF significantly induced the production of TNF-α (20-fold and 27-fold, respectively), IL-1β (each 4-fold), IL-6 (12-fold and 11-fold, respectively), IL-8 (43-fold and 30-fold, respectively), and CCL2 (each 4-fold) in human monocytes. Abatacept inhibited this autoantibody-mediated upregulation of cytokines, reducing TNF-α by > 75%, IL-1β by > 65%, IL-6 and IL-8 by > 80%, and CCL2 by > 60%. In contrast, a TNF inhibitor did not influence autoantibody-induced proinflammatory cytokine production. IDO inhibition reversed the effect of abatacept and again permitted the induction of cytokine production by ACPA and RF.ConclusionsThese data show that abatacept interferes with autoantibody-mediated cytokine production by monocytes through induction of IDO. This inhibitory effect on the production of several effector cytokines in RA may explain the fast anti-inflammatory effect of abatacept as well as its preferential efficacy in patients with high-titer ACPA and RF.

SAT0201 Abatacept but not tnf inhibitors block autoantibody-mediated cytokine production by monocytes

Background The anti-inflammatory effect of abatacept (CTLA4-Ig) is most pronounced in patients with high-titer autoantibodies (including anti-citrullinated protein antibodies, ACPA, and rheumatoid factor, RF) even exceeding the effect of TNF inhibitors (TNFi)1. Considering that autoantibodies trigger inflammatory cytokine production by monocytes2 and that abatacept bind to monocytes influencing their functional state3 we hypothesized that abatacept, in contrast to TNFi, may effectively inhibit the production of several different cytokines by ACPA-or RF-challenged monocytes. Objectives (i) To test whether abatacept inhibits the production of TNFa, IL-1b, IL-6 and IL-8 by monocytes exposed to ACPA or RF, (ii) to compare these effects of abatacept with those of TNFi and (iii) to investigate whether the effect of abatacept on cytokine production is based on IDO induction in monocytes. Methods CD68+ monocytes were isolated from peripheral blood and stimulated with MCSF for 24 hours before exposing them to random IgG alone (negative control), 10mg/mL purified anti-citrullinated vimentin antibodies (ACPA), 10mg/mL RF or LPS (positive control) in cell culture plates coated with citrullinated vimentin (to allow ACPA immune complex formation). ACPA and RF stimulation was done in the presence or absence of abatacept or TNF-antibody (adalimumab) with or without IDO inhibitor 1-MT. Supernatants were analyzed for four key pro-inflammatory cytokines TNFa, IL-1b, IL-6 and IL-8 by cytokine array (R&D Proteome Profiler) after 24h. Results Exposure to ACPA or RF dramatically induced the production of TNFa (20 fold and 27-fold, respectively) IL-1b (each 4-fold), IL-6 (12-fold and 11-fold, respectively) IL-8 (43-fold and 30-fold, respectively) in human monocytes. Abatacept significantly inhibited this up-regulation of inflammatory cytokine production with TNFa reduced by 79%, IL-1b by 74%, IL-6 by 88% and IL-8 by 83%. In contrast, TNFi did not influence autoantibody-induced production of IL-1b, IL-6 and IL-8. Inhibition of IDO by 1-MT reversed the effect of abatacept and unlocked cytokine production in the presence of ACPA and RF. Conclusions These data show that abatacept interferes with autoantibody mediated cytokine production by induction of IDO. The fact that several different effector cytokines are inhibited simultaneously may explain the strong anti-inflammatory effect of abatacept in RA patients with high-titer ACPA and RF. References Sokolove J, et al. Impact of baseline anti-cyclic citrullinated peptide-2 antibody concentration on efficacy outcomes following treatment with subcutaneous abatacept or adalimumab: 2-year results from the AMPLE trial. Ann Rheum Dis. 2016;75:709–14. Clavel C, et al Among human macrophages polarised to different phenotypes, the M-CSF-oriented cells present the highest pro-inflammatory response to the rheumatoid arthritis-specific immune complexes containing ACPA. Ann Rheum Dis. 2016;75:2184–2191. Bozec A, et al. T cell costimulation molecules CD80/86 inhibit osteoclast differentiation by inducing the IDO/tryptophan pathway. Sci Transl Med. 2014;6:235ra60. Acknowledgements This project was supported by an unrestricted research grant from BMS and the IMI project BTCure. Disclosure of Interest None declared

AB0189 Anti-Modified Protein Antibody Response Pattern Influences The Risk for Disease Relapse in Rheumatoid Arthritis Patients Tapering Disease Modifying Anti-Rheumatic Drugs

Background Autoimmunity is still present in rheumatoid arthritis patients in sustained disease remission. In the absence of inflammation the pattern of autoimmunity against post-translationally modified proteins could potentially impact the course of disease of rheumatoid arthritis patients, espepcially their risk to experience relapse of disease when disease modifying anti-rheumatic drugs (DMARDs) are tapered or stopped Objectives To perform a detailed analysis of the autoantibody response against post-translationally modified proteins in rheumatoid arthritis (RA) patients in sustained remission and to test whether its composition influences the risk for disease relapse when tapering DMARD therapy. Methods Immune responses against 10 citrullinated, homocitrullinated/carbamylated and acetylated peptides, as well as unmodified vimentin (control) and cyclic citrullinated peptide 2 (CCP2) were tested in baseline serum samples from 94 patients of the RETRO study. Patients were classified according to the number of autoantibody reactivities (0–1/10, 2–5/10 and >5/10) or specificity groups (citrullination, carbamylation and acetylation; 0 to 3) and tested for their risk to develop relapses after DMARD tapering. Demographic and disease-specific parameters were included in multivariate logistic regression analysis for defining the role of autoantibodies in predicting relapse. Results Patient varied in their anti-modified protein antibody response with the extremes from recognition of no (0/10) to all antigens (10/10). Antibodies against citrullinated vimentin (51%), acetylated ornithine (46%) and acetylated lysine (37%) were the most frequently observed sub-specificities. Relapse risk significantly (p=0.011) increased from 18% (0–1/10 reactivities) to 34% (2–5/10) and 55% (>5/10). With respect to specificity groups (0 to 3), relapse risk significantly (p=0.021) increased from 18% (no reactivity) to 28%, 36% and finally to 52% with one, two or three antibody specificity groups, respectively. Conclusions The data suggest that the pattern of anti-modified protein antibody response determines the risk of disease relapse in RA patients tapering DMARD therapy. Disclosure of Interest C. Figueiredo: None declared, H. Bang Employee of: Organtec Diagnostica, J. Cobra: None declared, M. Englbrecht: None declared, A. Hueber: None declared, J. Haschka: None declared, B. Manger: None declared, A. Kleyer: None declared, M. Reiser: None declared, S. Finzel: None declared, H.-P. Tony: None declared, S. Kleinert: None declared, J. Wendler: None declared, F. Schuch: None declared, M. Ronneberger: None declared, M. Feuchtenberger: None declared, M. Fleck: None declared, K. Manger: None declared, W. Ochs: None declared, M. Schmitt-Haendle: None declared, H.-M. Lorenz: None declared, H. Nuesslein: None declared, R. Alten: None declared, J. Henes: None declared, K. Krueger: None declared, J. Rech: None declared, G. Schett: None declared

FRI0088 Cyclic citrullinated peptides from the sequence of modified vimentin (MCV) are targeted by different antibodies subclasses in patients with rheumatoid arthritis

Background The determination of antibodies (abs) against citrullinated antigens (ACPA) nowadays belongs to the diagnostic tests included in the classification criteria for rheumatoid arthritis (RA). However, the standard cyclic citrullinated peptide antigen (CCP) is artificial and not expressed in the targeted tissues. Recently, mutated and citrullinated vimentin (MCV) was isolated and characterized as another modified autoantigen in RA. It is of interest, whether cyclic peptides from the sequence of MCV are also recognized as antigenic epitopes. Objectives To analyse reactivity against cyclic citrullinated peptides derived from the sequence of MCV in a cohort of patients with early (ERA) and established (RA). Methods Immunglobulin subclass (IgA, IgG and IgM) ab against MCV-cyclic epitopes (MCE, Orgentec) were investigated in a cohort of well characterized Russian patients (n=206) with RA using ELISA. Sensitivity of MCE-reactivity was compared to commercially available ELISAs for anti-CCP IgG (Medipan), anti-RF (IgA, IgG and IgG), and anti-MCV IgG ab (Orgentec). Patients were classified according to the duration of disease as ERA (<12 months; n=34) or established RA (>12 months; n=172). Results Abs isotypes IgG, IgA and IgM against MCE were observed in 64.7%, 23% and 17.6% of patients with ERA, as well as in 68.6%, 27.3% and 13.7% of patients with RA, respectively. Anti-MCV ab IgG were found positive in 61.8% of patients with ERA, and in 55.8% of patients with RA. Thus, the frequency of IgG-ab against MCE was even higher compared to the complete MCV antigen. In comparison, anti-CCP IgG ab and RF IgG, IgA and IgM were detectable in 73.5%, 70.6, 64.7%, and 35.3% of patients with ERA, and in 80.2%, 71.5%, 68.6%, and 41.9% of patients with RA, respectively. Single positive results for CCP IgG ab were observed in 8.8% of ERA and 10.5% of RA patients. By summarizing all MCE subclass abs, single positive results were observed in 11.8% of ERA and 2.3% of RA patients. The highest IgG ACPA titers were measured for the anti-CCP (mean ERA 2274.9 U/ml and RA 1890.1 U/ml) and the anti-MCE IgG ab (mean ERA 516.0 U/ml and RA 591.6 U/ml) followed by anti-MCV ab (mean ERA 338.0 U/ml and RA 443.7 U/ml). Conclusions The antigenic epitopes of MCV are maintained by using cyclic citrullinated peptides. Antibody reactivity against MCE includes all subclasses, with a predominance of IgG-ab. For diagnostic purposes, the standard CCP ELISA provided the highest sensitivity in the analysed cohort. Disclosure of Interest O. Derganowa: None Declared, L. Martinez-Gamboa: None Declared, K. Egerer: None Declared, H. Bang Employee of: Orgentec, D. Roggenbuck Employee of: Medipan, I. Esaulenko: None Declared, G. Burmester: None Declared, T. Chernykh: None Declared, E. Feist: None Declared