Proteins subjected to post-translational modifications, such as citrullination, carbamylation, acetylation or malondialdehyde (MDA)-modification are targeted by autoantibodies in seropositive rheumatoid arthritis (RA). Epidemiological and experimental studies have both suggested the pathogenicity of such humoral autoimmunity, however, molecular mechanisms triggered by anti-modified protein antibodies have remained to be identified. Here we describe in detail the pathways induced by anti-MDA modified protein antibodies that were obtained from synovial B cells of RA patients and that possessed robust osteoclast stimulatory potential and induced bone erosion in vivo. Anti-MDA antibodies boosted glycolysis in developing osteoclasts via an FcγRI, HIF-1α and MYC-dependent mechanism and subsequently increased oxidative phosphorylation. Osteoclast development required robust phosphoglyceride and triacylglyceride biosynthesis, which was also enhanced by anti-MDA by modulating citrate production and expression of the glycerol-3-phosphate dehydrogenase 1 (GPD1) and glycerol-3-phosphate acyltransferase 2 (GPAT2) genes. In summary, we described novel metabolic pathways instrumental for osteoclast differentiation, which were targeted by anti-MDA antibodies, accelerating bone erosion, a central component of RA pathogenesis.

OBJECTIVES:Our knowledge about the effect of tocilizumab (TCZ) on the synovium in rheumatoid arthritis (RA) is limited. The aim of this study was to investigate the effect of TCZ on citrullination and on inflammation in the synovial tissue and in the peripheral blood.METHODS:15 patients with RA underwent synovial biopsy before and 8 weeks after TCZ initiation. Clinical evaluation was performed at baseline and at 8 weeks. Using immunohistochemistry, we evaluated the expression of CD68, CD3, CD20, osteoprotegerin (OPG) and receptor activator for nuclear factor-κB ligand (RANKL) before and after treatment with TCZ. We also analysed the expression of protein arginine deiminase (PAD)-2 and PAD-4 enzymes in the synovial tissue and protein citrullination patterns with the help of anticitrullinated protein antibody (ACPA) clones 1325:04C03 and 1325:01B09. Serum levels of interleukin-6 (IL-6), IL-8, RANKL, OPG and C-terminal crosslinked telopeptide type II collagen were measured by ELISA. Paired-wise Wilcoxon signed-rank test was used to compare median values before and after treatment.RESULTS:Disease activity in patients was reduced from baseline to 8 weeks. Although PAD-2 and PAD-4 expressions remained unchanged after TCZ treatment, the binding of one ACPA clone decreased in the synovial tissue. TCZ did not affect the number of CD68+ macrophages or CD20+ B cells but induced significant decrease in the number of CD3+ T cells. RANKL and OPG expression remained unchanged in the synovial tissue. A significant increase in the levels of IL-6 and RANKL was observed in the serum. This increase was statistically significant in patients who responded to TCZ (achieving Clinical Disease Activity Index low disease activity or remission) but not in non-responders.CONCLUSIONS:TCZ reduced synovial T-cell counts but not macrophages. A significant increase of serum IL-6 was observed in responders.

Background: Anti-citrullinated protein antibodies (ACPAs) play an important role in rheumatoid arthritis (RA) pathogenesis. We hypothesized that the effect of these antibodies is mediated by their binding to synovial fibroblasts and inducing an increased mobility of fibroblasts 1 . Objectives: In our study, we analyzed and compared fibroblast modulation by ACPA pools obtained from different patients or by a set of monoclonal ACPAs with different fine specificity that were obtained from different tissue sites. Methods: Synovial fibroblasts were isolated from RA patients synovial tissue biopsies. Individual polyclonal ACPA and control IgGs were purified from sera of four ACPA-positive RA patients by affinity purification on protein G and CCP-2 columns. Monoclonal antibodies were derived from memory B cell isolated from blood2, synovial fluid or bronchoalveolar lavage of RA patients. Whole antibodies and F(ab’)2 fragments were tested in fibroblast migration using IncuCyte live-cell analysis. Blocking experiments were performed with soluble citrullinated proteins in SF migration. Cross-reactivity of the antibodies to citrullinated and acetylated epitopes was tested using PAD inhibitors (Cl-amidine and GSK199), histone acetyltransferases (anacardic acid) and deacetylases (trichostatin A). Binding patterns of monoclonal ACPAs, both whole and F(ab’)2 fragments were analyzed in synovial biopsies obtained from both healthy donors and RA patients. Results: Three out of four tested individual ACPA were able to promote fibroblast migration. Five out of nine tested monoclonal ACPAs stimulated fibroblast migration. One of these antibodies, clone 1325:01B09 is characterized by cross-reactivity to citrullinated, homocitrullinated and acetylated targets. The effect of 1325:01B09 on fibroblast migration was completely abolished by Cl-amidine or by pre-incubating the antibody with citrullinated fibrinogen or histone but not citrullinated enolase or vimentin. Despite the cross-reactivity to acetylated epitopes, neither anacardic acid nor trichostatin A could modulate the 1325:01B09 effect on fibroblast migration. F(ab’)2 fragments of this antibody stimulated fibroblast migration and labelled podoplanin-positive fibroblasts in inflamed RA synovium similarly to the intact antibody, indicating an Fc-independent effect. Conclusion: The effect on fibroblast mobility was likely to be mediated by binding to citrullinated epitopes but not through Fc receptors. Detection of fibroblast modulating ACPAs in majority of RA patients indicated that fibroblasts might be key cellular targets in disease pathogenesis, although individual variability might exist in the composition of ACPA cellular targets. References: [1]Sun M, Rethi B, Krishnamurthy A, et al. Anticitrullinated protein antibodies facilitate migration of synovial tissue-derived fibroblasts. Ann Rheum Dis 2019;78(12):1621-31. doi: 10.1136/annrheumdis-2018-214967 [published Online First: 2019/09/05] [2]Amara K, Lena Israelsson, Ragnhild Stålesen, et al. A Refined Protocol for Identifying Citrulline-specific Monoclonal Antibodies from Single Human B Cells from Rheumatoid Arthritis Patient Material. Bio-protocol 2019;9(16) Disclosure of Interests: Meng Sun: None declared, Bence Réthi: None declared, Akilan Krishnamurthy: None declared, Vijay Joshua: None declared, Alexandra Circiumaru: None declared, Marianne Engström: None declared, Caroline Grönwall: None declared, Vivianne Malmström Grant/research support from: VM has had research grants from Janssen Pharmaceutica, Khaled Amara: None declared, Lars Klareskog: None declared, Heidi Wähämaa: None declared, Anca Catrina: None declared

Background:Malondialdehyde (MDA) is a highly reactive compound produced by lipid-peroxidation in situations associated with oxidative stress. MDA can irreversibly modify proteins residues such as lysine, arginine and histidine. In addition, MDA adducts can further react with acetaldehyde to generate malondialdehyde-acetaldehyde (MAA) modifications. Such modifications can give rise to immunogenic neo-epitopes that are recognized by autoantibodies. In fact, anti-MDA/MAA IgG antibodies are significantly increased in the serum of patients with autoimmune diseases, such as rheumatoid arthritis (RA) (1) and systemic lupus erythematosus (2). Recently, we have shown that anti-MDA/MAA IgG antibodies are able to promote osteoclast (OC) differentiationin vitro(1).Objectives:To investigate the molecular mechanisms triggered by anti-MDA/MAA autoantibodies during osteoclastogenesis.Methods:OCs were generated from monocyte-derived macrophages in the presence of the cytokines RANK-L and M-CSF. The development of OCs was monitored by light microscopy following tartrate-resistant acid phosphatase (TRAP) staining and erosion area on synthetic calcium phosphate-coated plates. Three different recombinant human monoclonal anti-MDA/MAA antibodies, cloned from single synovial B cells of RA patients, control antibodies and Fab fragments of the antibodies were added to OC cultures. Glycolysis was inhibited by 2-deoxyglucose, and Fc-gamma receptor I or II by anti-CD64 or anti-CD16 neutralizing antibodies. IL-8 levels were measured by enzyme linked immunosorbent assay. Cellular metabolism was monitored using Seahorse XF Analyzer (extracellular acidification rate and oxygen consumption) and a colorimetric L-Lactate assay.Results:Lactic acid production correlated with the osteoclastogenetic effect of some but not all anti-MDA/MAA antibodies on OCs (R=0.4758, p=0.0252) suggesting an antibody-mediated regulation of glycolysis. Further, extracellular acidification (ECAR) and oxygen consumption (OCR) rate of the developing OCs were increased by the osteoclastogenic anti-MDA/MAA clones (maximum increase of 54% for the ECAR and 78% for the OCR by clone 146+:01G07, and maximum increase of 28% for the ECAR and 39% for the OCR by clone 1103:01H05), but not by the non-osteoclastogenetic anti-MDA/MAA clones or control antibodies. The glycolysis inhibitor 2-deoxyglucose completely abolished the osteoclastogenetic effect of the anti-MDA/MAA clones at drug concentrations that did not influenced baseline OC development. Fab2 fragments of the osteocalstogenetic anti-MDA/MAA clones had no effect on OC development and metabolism. In accordance with this, Fc-gamma receptor I neutralizing antibodies completely abolished the osteocalstogenetic effect of the anti-MDA/MAA clones. The osteoclastogenetic effect of the anti-MDA/MAA antibodies was independent of IL-8 production. In contrast to anti MDA/MAA antibodies, ACPA-mediated osteoclastogenesis was independent of glycolysis and Fc-gamma receptors but dependent on IL-8.Conclusion:Our results describe a novel glycolysis-dependent mechanism by which anti-MDA/MAA antibodies promote osteoclast development that is different from the one previously described for ACPA.References:[1] C. Grönwall et al. Journal of Autoimmunity 84 (2017) 29-45.[2] C. Wang et al. Arthritis and Rheumatism 62 (2010) 2064-2072Disclosure of Interests:Koji Sakuraba: None declared, Akilan Krishnamurthy: None declared, Alexandra Circiumaru: None declared, Meng Sun: None declared, Vijay Joshua: None declared, Marianne Engström: None declared, Xiaowei Zheng: None declared, Cheng Xu: None declared, Khaled Amara: None declared, Vivianne Malmström Grant/research support from: VM has had research grants from Janssen Pharmaceutica, Sergiu-Bogdan Catrina: None declared, Caroline Grönwall: None declared, Bence Réthi: None declared, Anca Catrina: None declared

OBJECTIVES:Rheumatoid arthritis (RA)-specific anti-citrullinated protein/peptide antibodies (ACPAs) might contribute to bone loss and arthralgia before the onset of joint inflammation. We aimed to dissect additional mechanisms by which ACPAs might contribute to development of joint pathology.METHODS:Fibroblast-like synoviocytes (FLS) were isolated from the synovial membrane of patients with RA. The FLS cultures were stimulated with polyclonal ACPAs (anti-CCP-2 antibodies) purified from the peripheral blood of patients with RA or with monoclonal ACPAs derived from single synovial fluid B cells. We analysed how ACPAs modulate FLS by measuring cell adhesion and mobility as well as cytokine production. Expression of protein arginine deiminase (PAD) enzymes and protein citrullination were analysed by immunofluorescence, and signal transduction was studied using immunoblotting.RESULTS:Challenge of FLS by starvation-induced stress or by exposure to the chemokine interleukin-8 was essential to sensitise the cells to ACPAs. These challenges led to an increased PAD expression and protein citrullination and an ACPA-mediated induction of FLS migration through a mechanism involving phosphoinositide 3-kinase activation. Inhibition of the PAD enzymes or competition with soluble citrullinated proteins or peptides completely abolished the ACPA-induced FLS migration. Different monoclonal ACPAs triggered distinct cellular effects in either fibroblasts or osteoclasts, suggesting unique roles for individual ACPA clones in disease pathogenesis.CONCLUSION:We propose that transient synovial insults in the presence of a certain pre-existing ACPA repertoire might result in an ACPA-mediated increase of FLS migration.

Background Anti-citrullinated protein antibodies (ACPA) play an important role in rheumatoid arthritis (RA) pathogenesis. They recognize a wide number of citrullinated targets and show limited cross-reactivity to acetylated ones(1). Objectives We aimed to investigate the effect of monoclonal ACPAs with different binding patterns on RA synovial tissue derived fibroblasts (SF). Methods Synovial fibroblasts were isolated from synovial tissue of RA patients by enzymatic digestion. ACPA and control monoclonal antibodies (mAbs) were derived from single B cells isolated from RA patients. Two monoclonal ACPA (1325:01B09 and 1325:05C06) showed cross-reactivity to acetylated-targets while four of them (1325:04C03, 1325:07E07, 14CFCT2D09, 14CFCT2H12) did not(1). mAbs were tested in synovial fibroblast migration (Incucyte live time image analysis) and osteoclast formation (TRAP) assays. The role of mAbs cross-reactivity was tested in fibroblast migration assays using inhibitors of peptidylarginine deiminases (Cl-amidine), histone acetyltransferase (anacardic acid) and histone deacetylase (trichostatin A). Binding patterns of monoclonal ACPAs were tested in synovial biopsies obtained from both healthy donors and RA patients. Results One acetylation cross-reactive ACPA (1325:01B09) and two with no cross reactivity (14CFCT2D09 14CFCT2H12) significantly enhanced fibroblast migration (mean±SD fold increase of 1.9±0.5, 1.7±0.4 and 1.8±0.6, respectively) compared to control mAb 1362:01E02 (p<0.05), while having no effect on osteoclast formation. In contrast, one acetylation non-cross-reactive ACPA (1325:04C03) promoted osteoclastogenesis (mean±SD fold increase of 1.6±0.05) compared to control mAb 1362:01E02 (p<0.05), while having no effect on fibroblast migration. Cl-amidine completely abolished the effect of 1325:01B09, while neither anacardic acid nor trichostatin A had any effect. Moreover, the fibroblast-promoting ACPA (1325:01B09) but not the osteoclastogenic ACPA1325:04C03 co-localize with CD55-positive SF in the inflamed rheumatoid synovium. No detectable signals were found in healthy synovium. Conclusion Monoclonal ACPAs have distinct cellular effects on synovial fibroblasts and osteoclasts that are not related to cross reactivity towards acetylated targets References [1] K. A. Lloyd, G. Wigerblad, P. Sahlstrom, M. G. Garimella, K. Chemin, J. Steen, P. J. Titcombe, B. Marklein, D. Zhou, R. Stalesen, E. Ossipova, C. Lundqvist, O. Ekwall, J. Ronnelid, D. L. Mueller, M. C. I. Karlsson, M. J. Kaplan, K. Skriner, L. Klareskog, F. Wermeling, V. Malmstrom, C. Gronwall, Differential ACPA Binding to Nuclear Antigens Reveals a PAD-Independent Pathway and a Distinct Subset of Acetylation Cross-Reactive Autoantibodies in Rheumatoid Arthritis. Front Immunol9, 3033 (2018). Disclosure of Interests Meng Sun Grant/research support from: Yes, but not for presented project., Akilan Krishnamurthy: None declared, Alexandra Circiumaru: None declared, Marianne Engström: None declared, Johanna Steen: None declared, Philip Titcombe: None declared, Vivianne Malmström: None declared, Heidi Wähämaa: None declared, Lars Klareskog Grant/research support from: Yes, but not for the presented study., Bence Réthi: None declared, Anca Catrina Grant/research support from: Yes, but not for the presented study.

Career situation of first and presenting author Young investigator. Introduction CCL17 and CCL22 are chemokines that bind to the receptor CCR4, which is expressed on various immune cells as well as on neurons. CCL17 has been shown to mediate the pro-inflammatory and algesic actions of GM-CSF in murine arthritis models1 and CCL22 could activate nociceptive neurons in cell culture2 or induce hyperthermia by acting on the hypothalamus.3 Our previous studies have indicated that chemokine production by osteoclasts (OCs) might contribute to bone damage and arthralgia in the presence of anti-citrullinated protein antibodies.4 5 Objectives We analyzed the expression and potential roles of CCL17 and CCL22 during rheumatoid arthritis (RA). Methods We compared CCL17 and CCL22 levels in the sera of individuals at risk of developing RA, in patients at early stages of RA and in healthy controls. We also studied the production of these molecules in OC cultures. We analyzed whether CCL22 can induce arthralgia or affect OC development. Results Serum levels of CCL22 were elevated in individuals at risk of developing RA and in early untreated RA, when compared to healthy controls. On the contrary, CCL17 levels showed no significant difference between the studied cohorts. In the group of RA patients, higher CCL22 concentrations were associated with smoking. In OC cultures CCL22 production was triggered by M-CSF or GM-CSF and CCL22 levels correlated with both of these cytokines in the synovial fluid of RA patients. CCL22 induced arthralgia when injected into the ankle joints of mice and it stimulated OC differentiation in cell culture. Conclusions The early increase of CCL22 might contribute to the development of RA, potentially by inducing pain and osteoclastogenesis. Inflammatory cytokines, like GM-CSF and M-CSF, and also by environmental triggers such as smoking can contribute to the increase of CCL22. References Achuthan A, et al. J Clin Invest 2016. Oh SB, et al. J Neurosci 2001. Osborn O, et al. Cytokine 2011. Krishnamurthy A, et al. Ann Rheum Dis 2016. Wigerblad G, et al. Ann Rheum Dis 2016. Disclosure of Interest B. Réthi: None declared, A. Krishnamurthy: None declared, A. Circiumaru: None declared, K. Sakurabas: None declared, V. Joshua: None declared, M. Sun: None declared, C. Szu-Ying: None declared, M. Engström: None declared, H. Wähämaa: None declared, A. Hensvold: None declared, J. Smith Employee of: GSK, A. Catrina: None declared.

BACKGROUND:A relationship between rheumatoid arthritis (RA) and periodontitis has been suggested from findings that individuals with RA are prone to have advanced periodontitis and vice versa. In search of possible common pathogenetic features of these two diseases, we investigated the presence of citrullinated proteins and expression of endogenous peptidylarginine deiminases (PAD2 and PAD4), in periodontal tissue of individuals with periodontitis and healthy controls, in relation to the periodontal pathogens Porphyromonas gingivalis (P. gingivalis) and Aggregatibacter actinomycetemcomitans (A. actinomycetemcomitans), producing leukotoxin as virulence factor. These two oral bacteria have been suggested to be linked to anti-citrullinated protein antibodies in patients with RA.METHODS:Gingival tissue biopsies were obtained from 15 patients with periodontitis and 15 individuals without periodontal disease. Presence of CD3-positive lymphocytes, citrullinated proteins, PAD2, PAD4, P. gingivalis as well as A. actinomycetemcomitans and Mannheimia haemolytica produced leukotoxins were analysed by immunohistochemistry, followed by triple-blind semi-quantitative analysis. Mann-Whitney and Fisher's exact tests were used to analyse differences between groups. PADI2 and PADI4 mRNA levels were assessed by RT-qPCR and analysed using Wilcoxon signed rank test.RESULTS:Increased staining of citrullinated proteins was observed in gingival connective tissue from subjects with periodontitis (80%, 12/15) compared to healthy gingival tissue (27%, 4/15), whereas no differences were observed in gingival epithelium. There was also an increased staining of the citrullinating enzymes PAD2 and PAD4 in gingival connective tissue of patients with periodontitis whereas similar levels of PAD2 and PAD4 were observed in the gingival epithelium of the two groups. Similarly, the mRNA levels of PADI2 and PADI4 were also increased in the gingival tissue of patients with periodontitis compared to healthy controls. Furthermore, presence of P. gingivalis and leukotoxins was comparable in both epithelium and connective tissue, from the different investigated individuals with and without periodontitis, and there were no correlations between the presence of periodontal pathogens and the expression of citrullinated proteins or PAD enzymes.CONCLUSION:Chronic gingival inflammation is associated with increased local citrullination and PAD2 and PAD4 expression in periodontitis. The increased citrullination and PAD2 and PAD4 expression in periodontitis were, however, independent of the presence of periodontal pathogen P. gingivalis and A. actinomycetemcomitans leukotoxin.

Background Tocilizumab (TCZ) is an anti-IL6R monoclonal antibody approved for the treatment of Rheumatoid Arthritis (RA). There is limited data on synovial tissue histology changes. Objectives The aim of this study was to evaluate the effect of TCZ on synovial cell populations and on citrullination. Methods 15 patients with RA, according to ACR 1987 criteria, independent of disease duration, were included. Synovial biopsies were obtained before and after 8 weeks of treatment. We evaluated by immunohistochemistry (IHC) expression of citrullinated proteins (CP) and protein arginine deiminase (PAD) enzymes in synovial tissue before and after treatment (1325:C03, 1325:B09, PAD2, PAD4). Negative controls were used for each antibody. Expression of CD68, CD3, CD20 and CD55 was also evaluated. Evaluation of all IHC variables was performed by two blinded independent observers using a semiquantitative score on a 0–3 scale (0, no staining; 1, low amounts of staining; 2, moderate amounts of staining; 3, high amounts of staining). Paired-wised Wilcoxon Signed Ranks Test was used to compare the median values. Results The median (IQR) age, disease duration, N. prior biologic DMARDs and DAS28 at baseline was 66,58–79 4 (1–13), 1 (0–2), 6 (5–7), respectively. 93% were female, 53% were RF +and 60% ACPA+, 53% had concomitant glucocorticoids and only 27% had concomitant conventional synthetic DMARDs. Significant reductions in DAS28, swollen and tender joint count (SJC and TJC, respectively), and acute phase reactants (ESR and CRP) were observed between baseline and 8 weeks (table 1). By IHC, TCZ induced significant decrease in the number of CD3, C03 and CD55, but not in the number of the other CP, PAD2, PAD4 and CD68 (table 1). Abstract Sat0054 – Table 1 Baseline 8 weeks p-value(Wilcoxon Signed Ranks Test) DAS28 BL 5.9 (4.7–6.8) 2.98 (2.0–3.8) 0.028 SJC BL 9 (3–14) 1 (0–4.25) 0.009 TJC BL 104–15 1 (0–2.25) 0.017 ESR BL 3415–69 6 (5–16) 0.001 CRP BL 115–27 1 (1–2) 0.005 CD68 2 (1–3) 2 (0.75–3) 0.41 CD3 2 (1–3) 1 (0–2) 0.046 CD55 2 (2–3) 1.5 (1–2) 0.05 C03 (cit) 1 (0–1) 0 (0–1) 0.025 B09 (cit) 2 (1–2) 2 (1–2) 0.20 PAD2 3 (2–3) 2 (1.5–3) 0.16 PAD4 2 (2–3) 2 (2–3) 0.37 Conclusions Treatment with tocilizumab reduced the number of synovial tissue T cells. This was observed in the responders but not in non-responders. Significant reduction in citrullination as assessed by expression of 1325:C03 but not 1325:B09. No significant reduction in macrophages was observed. Acknowledgements This study was funded by Roche. Disclosure of Interest None declared

Background We have shown previously in various experimental models that anti-citrullinated protein antibodies (ACPAs) can contribute to bone erosion and arthralgia through IL-8 dependent mechanisms. Objectives In cell cultures osteoclasts (OCs) played a prominent role in the ACPA-induced IL-8 secretion and therefore we decided to characterize in detail the autocrine regulation of OC differentiation by IL-8 in the presence or absence of ACPAs. Methods Peripheral blood CD14-positive monocytes were used to generate OCs in the presence of M-CSF and RANKL. Expression levels of IL-8 and CXCR1/2 were monitored during OCs maturation using intracellular flow cytometry, cytometric bead array, immunohistochemistry and RT-PCR. Inhibition of IL-8 and its receptors were performed in OC cultures using IL-8 neutralizing antibodies and small molecule CXCR1 and CXCR2 antagonists (Reparixin, SB-332235), in the presence or absence of polyclonal ACPAs. OC numbers were counted using light microscope after TRAP staining. Results Macrophages and developing OCs secreted high levels of IL-8 in response to the cytokine M-CSF. IL-8 expression kinetic was characterized by transient peaks when new cytokine was added, followed by very low level of steady-state production. In the presence of ACPAs IL-8 production increased 3–4 fold during late phases of OC differentiation. With the help of IL-8 neutralizing antibodies or inhibitors acting on the IL-8 binding chemokine receptors CXCR1 and CXCR2 we showed that the M-CSF induced IL-8 plays a crucial role in OC differentiation in the presence of exogenous RANK-L. In the presence of ACPAs OC differentiation was further enhanced in response to the ACPA-mediated increase in IL-8 production. The CXCR1 and CXCR2 inhibitors Reparixin and SB-332235 significantly inhibited the RANKL-mediated osteoclastogenesis at 100μM and 10μM respectively, both in the presence or absence of ACPAs. By using the inhibitors at low concentrations we could counteract the ACPA-mediated increase in OC development without interfering with steady state OC differentiation. Conclusions Our findings further support an important role for IL-8 in bone biology and in the ACPA-mediated OC differentiation both under physiological conditions and in the presence of ACPAs. Together with previous data showing in vivo, our data indicated that small molecule CXCR1 and CXCR2 antagonists could provide novel therapeutic tools for targeting OCs in ACPA+ individuals. Disclosure of Interest None declared

Background Anti-citrullinated proteins antibodies (ACPAs) injected in mice induce IL-8 dependent bone loss and arthralgia, but no synovial changes. We hypothesized that additional stimulus, sensitizing the synovial compartment to ACPA effects, is needed for the transition from bone to synovial pathology. Methods Synovial fibroblasts (SFs) were isolated from synovial tissue of RA patients by enzymatic digestion. Polyclonal ACPA and other non-ACPA IgGs were separated from peripheral blood of RA patients by affinity purification on a cyclic citrullinated peptide (CCP)-2 column. SF migration capacity was tested by scratch-assays in starved and non-starved cultures treated with ACPAs, with or without presence of IL-8. The results were evaluated by NIH ImageJ software. SF adhesion was analyzed by xCELLigence System Real-Time Cell Analyzer (ACEA bioscience). Peptidylarginine deiminases (PAD) expression and protein citrullination were evaluated by immunohistochemistry. The role of signaling pathways in the ACPA-mediated SF modulation was analyzed by using specific signal inhibitors and by monitoring protein phosphorylations using western blot. Results Serum starvation of SF increased citrullinated proteins and PAD expression. Starved but not non-starved SF showed an increased mobility index following polyclonal ACPA stimulation to a mean±SD fold increase of 2.6±0.5. This effect was abolished by PAD inhibition as well as ACPA blocking with citrullinated but not native fibrinogen. Exogenous pro-inflamamatory cytokines (IL-8 and TNF) synergistically increased SF mobility when added together with ACPA. Phosphorylation and inhibition studies of intracellular signalling pathways in starved SF indicated an important role for PI3K-mediated signals in the ACPA-induced increase of SF mobility. Conclusions We demonstrated that additional stimuli (such as stress-induced citrullination and cytokine priming) were needed for SF to react upon ACPA stimulation. This is an indirect proof supporting the idea that a synovial insult that will normally resolve unobserved, might be essential for the transition towards chronic synovial changes in the presence of ACPA. Disclosure of Interest None declared

Introduction Rheumatoid arthritis (RA) is an autoimmune disease with unknown etiopathogenesis where systemic autoimmunity precedes clinical disease onset. Adaptive immunity is initiated in lymphoid tissue where lymph node stromal cells (LNSC) play a crucial role in shaping the immune response and maintaining peripheral tolerance. Objectives We developed an experimental model for studying the functional capacities of human LNSC during the earliest phases of RA and compared their cellular and molecular characteristics to LNSC from healthy volunteers. Methods ACPA +RA patients (n=24), ACPA +RA risk individuals (n=23) and seronegative healthy controls (n=14;HC) underwent ultrasound-guided inguinal lymph node biopsy. Human LNSCs were isolated and expanded in vitro for cellular (flow cytometry), molecular (methylome, transcriptome and microRNA) and functional analyses. Results Key LN chemokines CCL19, CCL21 and CXCL13 were induced in LNSCs upon stimulation with TNFα and lymphotoxin α1β2, but to a lesser extent in LNSCs from RA patients. RNA sequencing was performed on LNSC of HC (n=5), ACPA +RA risk individuals (n=6) and ACPA +RA patients (n=4). Of interest, LNSC from ACPA +RA risk individuals and ACPA +RA patients revealed a common significantly differential expressed gene signature compared with HC LNSC. Pathway analysis of this common signature showed, among others, significant enrichment of pathways affecting actin cytoskeleton, focal adhesion and cell junction. Accordingly, in a gel contraction assay LNSC from ACPA +RA risk individuals and RA patients showed impaired collagen contraction compared to healthy LNSC. In RA LNSC a significant enrichment was observed for genes involved in TGFb signalling while in RA-risk LNSC cell cycle genes were differentially expressed compared with HC. DNA methylation analyses revealed common differentially methylated CpG sites (DMS) in LNSC from ACPA +RA patients (n=5) and ACPA +RA risk individuals (n=3) compared with HC (n=4). These DMS were significantly hypomethylated and associated with antigen processing and presentation (HLA-DRB1). Conclusions This data point towards alterations in the cytoskeleton and antigen-processing and presentation in LNSC from ACPA+RA risk individuals and RA patients. Further studies are required to investigate the influence of this LNSC abnormality on immune responses. Disclosure of interest C. Ospelt: None declared, E. Karouzakis: None declared, J. Hähnlein: None declared, H. Semmelink: None declared, R. Gay: None declared, P.-P. Tak Employee of: Currently: Senior Vice President R and D Pipeline, Global Development Leader and Chief Immunology Officer, GSK, D. Gerlag Employee of: Currently: Head Clinical Unit Cambridge at GSK, S. Gay: None declared, L. Van Baarsen: None declared

Oxidation-associated malondialdehyde (MDA) modification of proteins can generate immunogenic neo-epitopes that are recognized by autoantibodies. In health, IgM antibodies to MDA-adducts are part of the natural antibody pool, while elevated levels of IgG anti-MDA antibodies are associated with inflammatory and autoimmune conditions. Yet, in human autoimmune disease IgG anti-MDA responses have not been well characterized and their potential contribution to disease pathogenesis is not known. Here, we investigate MDA-modifications and anti-MDA-modified protein autoreactivity in rheumatoid arthritis (RA). While RA is primarily associated with autoreactivity to citrullinated antigens, we also observed increases in serum IgG anti-MDA in RA patients compared to controls. IgG anti-MDA levels significantly correlated with disease activity by DAS28-ESR and serum TNF-alpha, IL-6, and CRP. Mass spectrometry analysis of RA synovial tissue identified MDA-modified proteins and revealed shared peptides between MDA-modified and citrullinated actin and vimentin. Furthermore, anti-MDA autoreactivity among synovial B cells was discovered when investigating recombinant monoclonal antibodies (mAbs) cloned from single B cells, and 3.5% of memory B cells and 2.3% of plasma cells were found to be anti-MDA positive. Several clones were highly specific for MDA-modification with no cross-reactivity to other antigen modifications such as citrullination, carbamylation or 4-HNE-carbonylation. The mAbs recognized MDA-adducts in a variety of proteins including albumin, histone 2B, fibrinogen and vimentin. Interestingly, the most reactive clone, originated from an IgG1-bearing memory B cell, was encoded by near germline variable genes, and showed similarity to previously reported natural IgM. Other anti-MDA clones display somatic hypermutations and lower reactivity. Importantly, these anti-MDA antibodies had significant in vitro functional properties and induced enhanced osteoclastogenesis, while the natural antibody related high-reactivity clone did not. We postulate that these may represent distinctly different facets of anti-MDA autoreactive responses.

Background and objectives Circulating levels of IL-8 is related to the bone loss associated with breast cancer metastasis. Recently, we have shown that anti-citrullinated protein antibodies (ACPA) can induce osteoclastogenesis. We aimed to investigate the role of IL-8 and its receptors in mediating osteoclastogenesis in presence or absence of ACPAs Methods IL-8 levels was measured by ELISA in serum of risk RA patients and synovial fluid samples of RA patients. CD14 positive monocytes were used to generate osteoclasts with M-CSF and RANKL. Inhibition of IL-8 and its receptor were tested with IL-8 neutralising antibody or small molecule CXCR1/2 anta-agonist (Reparixin and SCH-527123) on osteoclasts (OC) in the presence or absence of ACPA. The number of the OC’s were counted in light microscope after TRAP staining. Cell counting kit 8 (CCK8) assay was performed to detect cytotoxicity. CXCR1 and CXCR2 expression was analysed using flow cytometry and immunohistochemistry during different days of osteoclasts maturation. Results Increased IL-8 levels were observed in the serum of ACPA positive at risk RA individuals and in synovial fluid of established ACPA positive RA patients. Exogenous IL-8 dose dependently increased OC numbers in presence of RANKL. IL-8 neutralising antibody inhibited the OC numbers even in the presence of ACPA. Reparixin and SCH-527123 significantly inhibited RANKL mediated osteoclastogenesis at 100 µM concentration. No cytotoxicity of the drugs were detected with CCK8 assay. With flow cytometry, we observed CXCR1 and CXCR2 expression in the OC precursors at day 0 and in presence of RANKL the expression was completely lost at day 1 and it reappeared at day 4. Immunohistochemistry staining confirmed the CXCR1/2 expression on OC precursors. Conclusions Our data provides insights in to the importance of IL-8 autocrine loop in RANKL and ACPA mediated OC development. IL-8 receptor blockade might be a novel way of targeting osteoclasts in RA.