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- Circiumaru, A, et al.
(author)
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SPECIFIC ACPA REACTIVITIES AND INFLAMMATORY BIOMARKERS ALONG WITH ULTRASOUND TENOSYNOVITIS ARE ASSOCIATED WITH ARTHRITIS ONSET IN A POPULATION AT RISK FOR RHEUMATOID ARTHRITIS
- 2020
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In: ANNALS OF THE RHEUMATIC DISEASES. - : BMJ. - 0003-4967 .- 1468-2060. ; 79, s. 1247-1247
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Conference paper (other academic/artistic)abstract
- Anti-citrullinated protein antibodies (ACPA) are characteristic markers for rheumatoid arthritis (RA), developing years before disease onset. Early clinical and biological biomarkers could provide useful information on the onset of RA in predisposed individuals.Objectives:The aim of the study was to investigate whether ACPA along with inflammatory markers and musculoskeletal ultrasound changes could predict arthritis development in individuals at risk for RA.Methods:ACPA-positive individuals with musculoskeletal complaints were referred from primary care to a rheumatology clinic, recruited in the Risk-RA research program and followed-up for up to 3 years, between April 2014 and October 2019. All individuals lacked arthritis both at clinical examination by a trained rheumatologist and ultrasound assessment of hands and feet and any other symptomatic joints (according to EULAR-OMERACT definition). Blood samples were collected at inclusion and were analyzed for 15 ACPA fine specificities (by custom made peptide array), 92 inflammation-associated protein biomarkers (by multiplex immunoassay with Olink extension technology) and HLA-SE (DR low resolution kit). Statistical analysis used univariate and multivariate models with backwards selection and cox regression.Results:268 individuals with a median age of 48 (36-58) were recruited, out of which 212 (79%) were females. 75 (28%) developed arthritis within 11 months of follow-up while the median follow-up for those not developing arthritis was 21 months (14-28). Increased ACPA levels, shorter symptom duration and RF positivity were the main differences between individuals developing arthritis and those who did not. In univariate models, the presence of HLA-SE, specific ACPA reactivities, certain inflammatory markers and ultrasound-detected tenosynovitis were associated with arthritis development. In multivariate analysis the presence of anti-cit-fillagrin (HR 2.1 (95% CI 1.2-3.7, p 0.01), IL6 levels (HR 1.4 (95% CI 1.2-1.7, p 0.0001) and tenosynovitis (HR 2.9 (95% CI 1.7-5.0, p 0.0001) remained significant predictors for arthritis onset.Conclusion:Certain ACPA reactivities together with inflammatory markers and ultrasound-detected tenosynovitis predict arthritis development in predisposed individuals for developing RA.Disclosure of Interests:None declared
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2. |
- Jenning, M, et al.
(author)
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Bacterial citrullinated epitopes generated by Porphyromonas gingivalis infection-a missing link for ACPA production
- 2020
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In: Annals of the rheumatic diseases. - : BMJ. - 1468-2060 .- 0003-4967. ; 79:9, s. 1194-1202
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Journal article (peer-reviewed)abstract
- Porphyromonas gingivalis (P.g.) is discussed to be involved in triggering self-reactive immune responses. The aim of this study was to investigate the autocitrullinated prokaryotic peptidylarginine deiminase (PPAD) from P.g. CH2007 (RACH2007-PPAD) from a rheumatoid arthritis (RA) patient and a synthetic citrullinated PPAD peptide (CPP) containing the main autocitrullination site as potential targets for antibody reactivity in RA and to analyse the possibility of citrullinating native human proteins by PPAD in the context of RA.MethodsRecombinant RACH2007-PPAD was cloned and expressed in Escherichia coli. Purified RACH2007-PPAD and its enzymatic activity was analysed using two-dimensional electrophoresis, mass spectrometry, immunoblot and ELISA. Autoantibody response to different modified proteins and peptides was recorded and bioinformatically evaluated.ResultsRACH2007-PPAD was capable to citrullinate major RA autoantigens, such as fibrinogen, vimentin, hnRNP-A2/B1, histone H1 and multiple peptides, which identify a common RG/RGG consensus motif. 33% of RA patients (n=30) revealed increased reactivity for α-cit-RACH2007-PPAD before RA onset. 77% of RA patients (n=99) presented α-cit-specific signals to CPP amino acids 57–71 which were positively correlated to α-CCP2 antibody levels. Interestingly, 48% of the α-CPP-positives were rheumatoidfactor IgM/anti-citrullinated peptide/protein antibodies (ACPA)-negative. Anti-CPP and α-RACH2007-PPAD antibody levels increase with age. Protein macroarrays that were citrullinated by RACH2007-PPAD and screened with RA patient sera (n=6) and controls (n=4) uncovered 16 RACH2007-PPAD citrullinated RA autoantigens and 9 autoantigens associated with lung diseases. We showed that the α-CPP response could be an important determinant in parenchymal changes in the lung at the time of RA diagnosis (n=106; p=0.018).ConclusionsRACH2007-PPAD induced internal citrullination of major RA autoantigens. Anti-RACH2007-PPAD correlates with ACPA levels and interstitial lung disease autoantigen reactivity, supporting an infection-based concept for induction of ACPAs via enzymatic mimicry.
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3. |
- Sakuraba, K, et al.
(author)
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METABOLIC CHANGES INDUCED BY ANTI-MALONDIALDEHYDE/MALINDIALDEHYDE-ACETALDEHYDE ANTIBODIES PROMOTE OSTEOCLAST DEVELOPMENT
- 2020
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In: ANNALS OF THE RHEUMATIC DISEASES. - : BMJ. - 0003-4967 .- 1468-2060. ; 79, s. 934-935
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Conference paper (other academic/artistic)abstract
- 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
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4. |
- Sakuraba, K, et al.
(author)
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METABOLIC CHANGES INDUCED BY ANTI-MALONDIALDEHYDE/MALINDIALDEHYDE- ACETALDEHYDE ANTIBODIES PROMOTE OSTEOCLAST DEVELOPMENT
- 2021
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In: ANNALS OF THE RHEUMATIC DISEASES. - : BMJ. - 0003-4967 .- 1468-2060. ; 80, s. 429-429
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Conference paper (other academic/artistic)abstract
- Malondialdehyde (MDA) is a highly reactive compound generated during lipid-peroxidation in conditions associated with oxidative stress. MDA can irreversibly modify proteins (e.g. lysine, arginine and histidine residues). In addition, acetaldehyde can further react with MDA adducts to form malondialdehyde-acetaldehyde (MAA) modification. Such protein modifications can lead 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). Interestingly, anti-MDA/MAA antibodies have been shown to promote osteoclast (OC) differentiation in vitro suggesting a potential role for these autoantibodies in bone damage associated with RA (1).Objectives:Little is known about the molecular mechanisms activated by autoantibodies in RA. Here, we elucidate the pathways specifically triggered by anti-MDA/MAA autoantibodies in developing osteoclasts.Methods:Recombinant human monoclonal anti-MDA/MAA antibodies, which were previously cloned from single synovial B cells of RA patients, were added to different OC assays. OCs were generated from monocyte-derived macrophages in the presence of the cytokines RANK-L and M-CSF. OC development was monitored by light microscopy following tartrate-resistant acid phosphatase staining and by erosion assays using calcium phosphate-coated plates. Bone morphometrics were studied in anti-MDA/MAA-injected mice using X-ray microscopy. Cellular metabolism was analyzed by mass spectrometry, Seahorse XF Analyzer and a colorimetric L-Lactate assay.Results:Anti-MDA/MAA antibodies induced a robust OC differentiation in vitro and bone loss in vivo. The anti-MDA/MAA antibodies acted on developing OCs by increasing glycolysis via an Fcγ receptor I-mediated pathway and the upregulation of the transcription factors HIF-1α, Myc and CHREBP. Such regulation of cellular metabolism was exclusively observed in the presence of the osteoclastogenic anti-MDA/MAA clones, whereas other RA-associated autoantibodies (anti-MDA/MAA or anti-citrullinated protein antibodies) had no effect on metabolism. The anti-MDA/MAA treatment induced a shift in the tricarboxylic acid (TCA) cycle activity in developing OCs, leading to the accumulation of citrate and aconitate.Conclusion:We described a novel type of autoantibody-induced pathway in RA, which might contribute to increased OC activation and a consequent bone loss. Anti-MDA/MAA antibodies promoted osteoclast development by increasing glycolysis and by modulating the TCA cycle through a signaling pathway that included Fcγ receptor I and a network of transcription factors acting on glycolysis. A TCA cycle bias towards citrate production suggests that the anti-MDA/MAA antibodies might stimulate OCs via increasing lipid biosynthesis in the cells.References:[1]Grönwall C. et al. J. Autoimmunity 84 (2017): 29-45.Acknowledgements:This Project has received funding from FOREUM, Foundation for Research in Rheumatology, from the European Research Council (ERC) grant agreement CoG 2017 - 7722209_PREVENT RA, the EU/EFPIA Innovative Medicine Initiative grant agreement 777357_RTCure, the Konung Gustaf V:s och Drottning Victorias Frimurarestiftelse and Knut and Alice Wallenberg Foundation.Disclosure of Interests:Koji Sakuraba: None declared, Akilan Krishnamurthy: None declared, Alexandra Circiumaru: None declared, Vijay Joshua: None declared, Heidi Wähämaa: None declared, Marianne Engström: None declared, Meng Sun: None declared, Xiaowei Zheng: None declared, Cheng Xu: None declared, Khaled Amara: None declared, Vivianne Malmström Grant/research support from: collaboration with Pfizer, unrelated to the abstract, Sergiu-Bogdan Catrina: None declared, Caroline Grönwall: None declared, Bence Réthi: None declared, Anca Catrina Grant/research support from: collaboration with BMS and Pfizer, unrelated to the present abstract
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5. |
- Sun, M, et al.
(author)
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DIVERSITY OF ANTI-CITRULLINATED PROTEIN ANTIBODY COMPOSITIONS INFLUENCE SYNOVIAL FIBROBLAST REACTIVITY
- 2020
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In: ANNALS OF THE RHEUMATIC DISEASES. - : BMJ. - 0003-4967 .- 1468-2060. ; 79, s. 569-570
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Conference paper (other academic/artistic)abstract
- 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 fibroblasts1.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
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