Unraveling heterogeneity within ACPA-negative rheumatoid arthritis: the subgroup of patients with a strong clinical and serological response to initiation of DMARD treatment favor disease resolution

Background Rheumatoid arthritis (RA) is a heterogeneous disease, as evidenced by the differences in long-term outcomes. This applies especially to anti-citrullinated protein antibodies (ACPA)-negative RA, where a proportion achieves sustained DMARD-free remission (SDFR; sustained absence of synovitis after DMARD cessation). Differentiation of RA patients who will achieve SDFR can guide personalized treatment/tapering strategies. Although this subgroup remains scarcely discerned, previous research demonstrated that these RA patients are characterized by an early clinical response (DAS remission after 4 months) after DMARD start. We studied whether, in addition to this clinical response, a specific biomarker response can further distinguish the subgroup of RA patients most likely to achieve SDFR. Methods In 266 RA patients, levels of 12 biomarkers (SAA/CRP/MMP-1/MMP-3/resistin/leptin/IL-6/TNF-R1/YKL-40/EGF/VEGF/VCAM-1), in the first 2 years after diagnosis, were studied in relation to SDFR, stratified for ACPA status. Subsequently, biomarkers associated with SDFR development were combined with early DAS remission to study its additional value in defining subgroups. Since most biomarker levels are not routinely measured in clinical practice, we explored how this subgroup can be clinically recognized. Results ACPA-negative RA patients achieving SDFR were characterized by high baseline levels and stronger decline in MMP-1/MMP-3/SAA/CRP after DMARD-start, respectively 1.30×/1.44×/2.12×/2.24× stronger. This effect was absent in ACPA-positive RA. In ACPA-negative RA, a strong biomarker decline is associated with early DAS remission. The combination of both declines (clinical, biomarker) was present in a subgroup of ACPA-negative RA patients achieving SDFR. This subgroup can be clinically recognized by the combination of high baseline CRP levels (≥ 3 times ULN), and early DAS remission (DAS4 months < 1.6). This latter was replicated in independent ACPA-negative RA patients. Conclusions ACPA-negative RA patients with early DAS remission and a strong biomarker response (or baseline CRP levels ≥ 3× ULN) are most likely to achieve SDFR later on. This could guide personalized decisions on DMARD tapering/cessation in ACPA-negative RA.

Association between inflammatory cytokines and immune–checkpoint molecule in rheumatoid arthritis

Background Anti-citrullinated peptide antibodies (ACPA) and inflammatory cytokines play important roles in the development of rheumatoid arthritis (RA). T cell immunoglobulin and mucin–domain containing–3 (TIM–3) is an immune-checkpoint molecule involved in inhibitory signaling. Galectin–9 (Gal–9) mediated ligation of TIM–3 induces the amelioration of autoimmune diseases. TIM–3 is expressed in synovial osteoclasts and involved in the rheumatoid bone destruction. The aim of this study was to investigate the relationships between inflammatory cytokines and immune–checkpoint molecules in RA patients. Methods Serum levels of interleukin–6 (IL–6), tumor necrosis factor–α (TNF–α), soluble TIM–3 (sTIM–3) and Gal–9 were determined by ELISA. Patients were stratified into two groups based on ACPA titers: low-medium ACPA (ACPA <200 U/mL) and high ACPA (ACPA ≥200 U/mL). Serum levels of cytokines or immune-checkpoint molecules were evaluated between RA patients with low-medium ACPA titers and high ACPA titers. Results Elevated serum levels of inflammatory cytokines were correlated with DAS28–ESR in RA patients. Although serum levels of sTIM–3 were elevated in RA patients, significant correlations between sTIM–3 and cytokines (IL–6 or TNF–α) were observed exclusively in RA patients with low-medium ACPA titers (<200 U/mL). Serum levels of IL–6 and TNF–α levels were significantly correlated with elevated Gal–9 levels regardless of ACPA status. A significant correlation between IL–6 and Gal–9 was observed in RA patients without advanced joint damage. Conversely, a significant correlation between TNF–α and Gal–9 was observed in RA patients with advanced joint damage. Conclusions Our data indicated that there are positive correlations between circulating inflammatory cytokines and checkpoint molecules in RA patients and these interactions can be modulated by ACPA status or joint damage stage.

Immunoprofiling of early, untreated rheumatoid arthritis using mass cytometry reveals an activated basophil subset inversely linked to ACPA status

Background Autoantibody production is a hallmark of rheumatoid arthritis (RA). Anti-citrullinated protein antibodies (ACPA) are highly disease-specific, and their presence is associated with more severe disease and poor prognosis compared to ACPA-negative patients. However, the immune cell composition associated with antibody-positive/negative disease is incompletely defined. Mass cytometry (MC) is a high-dimensional technique offering new possibilities in the determination of the immune cell composition in rheumatic diseases. Here, we set up a broad phenotyping panel to study the immune cell profile of early untreated RA to investigate if specific immune cell subsets are associated with ACPA+ versus ACPA− RA. Methods Freshly obtained PBMCs of early, untreated RA patients (8 ACPA+ and 7 ACPA−) were analysed using a 36-marker MC panel, including markers related to various immune lineages. Data were processed using Cytosplore for dimensional reduction (HSNE) and clustering. Groups were compared using Cytofast. A second validation cohort of cryopreserved PBMCs obtained from early RA patients (27 ACPA+ and 20 ACPA−) was used to confirm MC data by flow cytometry (FC). FC data were processed and analysed using both an unsupervised analysis pipeline and through manual gating. Results MC indicated no differences when comparing major immune lineages (i.e. monocytes, T and B cells), but highlighted two innate subsets: CD62L+ basophils (p = 0.33) and a subset of CD16− NK cells (p = 0.063). Although the NK cell subset did not replicate by FC, FC replication confirmed the difference in CD62L+ basophil frequency when comparing ACPA+ to ACPA− patients (mean 0.32% vs. 0.13%; p = 0.01). Conclusions Although no differences in major lineages were found between early ACPA+ and ACPA− RA, this study identified the reduced presence of activated basophils in ACPA-negative disease as compared to ACPA-positive disease and thereby provides the first evidence for a connection between activated basophils and ACPA status.

The Role of Autoimmunity on the Relation Between Erosions and Bone Mineral Density in Rheumatoid Arthritis: A Clinical Research

IntroductionBoth erosions and osteoporosis are present in rheumatoid arthritis and are related to RANK-L pathway activation. The aim of the study was to evaluate the relationship between erosion and bone mineral density (BMD) in RA and whether it can be driven by autoimmunity.Patients and methodsPatients followed in the Department of Rheumatology between January 2008 and May 2019 satisfied the 1987 ACR or 2010 ACR-EULAR criteria. Erosions were evaluated by the modified Sharp/van der Heidje erosion score (SHSe) on radiographs and bone mineral density (BMD) in g/cm2 and by the T-score at the hip on DXA. The presence and titers of ACPA as well as rheumatoid factor (RF) and anti-nuclear antibodies (ANAs) were recorded at intervals of less than 2 years for both DXA and radiography.ResultsA total of 149 patients met the inclusion criteria. A total of 61.1% were ACPA positive, 79.9% were erosive and 10.7% had a hip T-score ≤-2.5. ACPA status but not titers was associated with a higher erosion score (63.0 (53.2) for ACPA + vs. 45.5 (44.1) for ACPA – (p= 0.04)). ACPA titers were associated with lower BMD at the hip (value -0.216; p=0.01) but not with T-score. A higher erosion score was associated with a lower BMD (R2: 0,049 and value: -0.222; p=0.009) and T-score (R2: 0,158 and value -0.397; p<0.0001) at the hip. In linear regression, erosion and systemic bone loss were still associated with but not driven by ACPA status or titer. RF and ANA did not demonstrate any role in this association.ConclusionWe showed that the relationship between erosion and bone mineral density associated with RA does not seem to be driven by ACPA or other autoimmunity parameters. However, the presence of ACPA or erosion should lead to osteoporosis assessment.

Association Between Inflammatory Cytokines and Immune-Checkpoint Molecule in Rheumatoid Arthritis

BackgroundRheumatoid arthritis (RA) is a heterogeneous inflammatory disease. Both anti-citrullinated peptide antibodies (ACPA) and inflammatory cytokines play an important role in the development of RA. The aim of this study was to investigate the association between inflammatory cytokines and co-inhibitory checkpoint molecules in patients with RA.MethodsOne hundred and thirty-two Japanese patients with established RA were enrolled. Serum levels of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF- α) were determined by ELISA. Patients were stratified into two groups based on ACPA titers: low-medium ACPA (ACPA <200 U/mL) and high ACPA (ACPA ≥200 U/mL). Serum levels of cytokines or co-inhibitory checkpoint molecules were compared according to the status of ACPA titers and joint progression stage.ResultsBaseline serum levels of TNF-α and IL-6 were significantly elevated in RA patients; however, the two cytokines showed no correlation with each other. Serum levels of IL-6 or TNF-α showed a significant correlation with DAS28-CRP, independent of ACPA status. Although serum levels of soluble T-cell immunoglobulin and mucin-domain containing-3 (sTIM-3) were elevated in RA patients, significant correlation of sTIM-3 with IL-6 or TNF-α was only observed in RA patients with low-medium levels of ACPA titers (<200 U/mL). Serum levels of IL-6 and TNF-α were significantly correlated with elevated levels of galectin-9 (Gal-9) independent of ACPA status. Whereas, a significant correlation between IL-6 and Gal-9 was observed only in RA patients without advanced joint damage (Stage I). Conversely, significant correlation between TNF-α and Gal-9 was observed only in RA patients with advanced joint damage (Stage II–IV).ConclusionsRA patients may be differentiated based on the interplay between serum cytokines and co-inhibitory molecules. RA patients with minimal joint damage (Stage I) appear to show positive correlation between Gal-9 and IL-6. Conversely, RA patients with advanced joint damage (Stage II–IV) appear to show positive correlation between Gal-9 and TNF-α. Serum levels of cytokines and immune-checkpoint molecules may be useful markers for predicting the immune phenotype and further personalized treatment of RA.

POS0387 ACPA STATUS CORRELATES WITH DIFFERENTIAL IMMUNE PROFILE OF RHEUMATOID ARTHRITIS PATIENTS

Background:Rheumatoid arthritis (RA) is a progressive erosive autoimmune disease that affects 1% of the world population. Anti-citrullinated protein autoantibodies (ACPA) are routinely used for the diagnosis of RA, however 20-30% of patients are ACPA negative. ACPA status is a delineator of RA disease endotypes with similar clinical manifestation but potentially different pathophysiology. Elucidating the underlying mechanisms of disease pathogenesis could inform a treat to target approach for both ACPA-positive and ACPA-negative RA patients.Objectives:To identify peripheral blood and synovial tissue immune population differences that associate with RA disease endotype.To identify unique RA patient synovial tissue gene signatures and enriched pathways that correlate with ACPA status.Methods:Detailed high dimensionality flow cytometric analysis with supervised and unsupervised algorithm analysis of ACPApos and ACPAneg RA patient peripheral blood and synovial tissue single cell suspensions. Ex vivo peripheral blood and synovial tissue T cell stimulation and cytokine production characterisation. RNAseq analysis with specific pathway enrichment analysis of APCApos and ACPAneg RA patient synovial tissue biopsies.Results:Detailed profiling based on high dimensionality flow cytometric analysis of key peripheral blood and synovial tissue immune populations including B cells, T follicular helper (Tfh) cells, T peripheral helper cells (Tph) and CD4 T cell proinflammatory cytokine responses with supervised and unsupervised algorithm analysis revealed unique RA patient peripheral blood B cell and Tfh cell profiles. ACPApos RA patients were characterised by significantly (*P=0.03) increased frequency of Tfh (CXCR5+CD4+) cells and distinct clustering influenced by increased switched (IgD-CD27+) and DN (IgD-CD27-) memory B cells compared to APCAneg RA patients. Surprisingly synovial tissue B cell subpopulation distribution was similar between ACPAneg and ACPApos RA patients, with significant accumulation of switched and double negative memory B cells, highlighting a key role for specific B cell subsets in both disease endotypes. Interestingly, synovial tissue CD4 T cell proinflammatory cytokine (TNF-α, IFN-γ, IL-2, GM-CSF, IL-17A, IL-22, IL-4) production was markedly different between ACPAneg and APCApos RA patients with hierarchical clustering and PCA analysis revealing endotype specific cytokine profiles with ACPAneg RA patient synovial T cells showing increased TNF-α (P=0.01) expression. RNAseq analysis of RA patient synovial tissue revealed significant disease endotype specific gene signatures with specific enrichment for B cell receptor signalling and T cell specific pathways in ACPApos compared to ACPAneg RA patients. Additionally, significantly different chemokine receptor expression based on RA patient ACPA status was observed with increased CXCR3 (P<0.001), CCR7 (P=0.002), and CCR2 (P=0.004) but decreased CXCR7 (P=0.007) expression in APCApos compared to ACPAneg RA patient synovial biopsies.Conclusion:ACPA status associates with unique synovial tissue immune cell and gene profile signatures highlighting differences in the underlying immunological mechanisms involved, therefore reinforcing the need for a treat to target approach for both endotypes of RA.Figure 1.RNAseq analysis of synovial tissue biopsies revealed specific T cell related pathway enrichment in ACPA positive compared to ACPA negative RA patients (n=50, analysis performed with the DESq2 and pathfindeR pipelines in R).Disclosure of Interests:Achilleas Floudas: None declared, Mary Canavan: None declared, Trudy McGarry Employee of: Novartis, Vinod Krishna Employee of: Janssen, Sunil Nagpal Employee of: Janssen, GSK, Douglas Veale Speakers bureau: Abbvie, Janssen, Novartis, MSD, Pfizer, UCB, Consultant of: Abbvie, Janssen, Novartis, MSD, Pfizer, UCB, Grant/research support from: Janssen, Abbvie, Pfizer, UCB, Ursula Fearon Speakers bureau: Abbvie, Grant/research support from: Janssen, Abbvie, Pfizer, UCB

POS0392 PRESENCE OF FOUR SERUM AUTOANTIBODIES ASSOCIATES WITH THE ACPA STATUS IN EARLY RHEUMATOID ARTHRITIS

Background:The presence of anti-citrullinated protein antibodies (ACPAs) is a hallmark of rheumatoid arthritis (RA) that precede the development of the disease by years and is used for its clinical diagnosis. However, there are RA subjects that test negative for ACPA and thus the early diagnosis on these patients may be delayed. Furthermore, the presence or absence of ACPA in RA supports the hypothesis that on these two subsets of patients underlie different pathogenesis and clinical outcomes.Objectives:In this work, we searched for serum autoantibodies useful to assist the early diagnosis of ACPA-seronegative RA and its management.Methods:We profiled the serum autoantibody repertoire of 80 ACPA-seronegative and 80 ACPA-seropositive RA subjects from the Swedish population-based Epidemiological Investigation of RA (EIRA) cohort. A suspension bead array platform built on protein fragments within Human Protein Atlas and selected from an initial untargeted screening using arrays containing 2660 total antigens was employed to identify IgG and IgA serum autoantibodies. A validation phase on antigen suspension bead arrays was carried out on another set of samples from EIRA containing 386 ACPA-seropositive, 358 ACPA-seronegative and 372 randomly selected control subjects of the same age and sex. A sample-specific threshold based on 20 times the median absolute deviation plus the median of all signals was selected to determine the reactivity of samples. The Wilcoxon rank sum test and Fisher’s test were applied for the comparison of autoantibody levels and reactivity frequencies between the groups.Results:Our data revealed four antigens associated with the ACPA status (Table 1). Testis-specific Y-encoded-like protein 4 (TSPYL4) showed significantly higher IgG reactivity frequency in ACPA-seronegative subjects compared to ACPA-seropositive (8% vs. 3%; P<0.05). Significant differences at IgG autoantibody levels (P<0.05) were also observed between ACPA-seronegative subjects and controls for this specific antigen. Significantly higher IgG autoantibody levels (P<0.05) towards another antigen, dual specificity mitogen-activated protein kinase kinase 6 (MAP2K6), were also observed in ACPA-seronegative subjects compared to ACPA-seropositive and controls. In contrast, we found significantly higher IgG autoantibody levels (P<0.05) in ACPA-seropositive individuals compared to ACPA-seronegative and controls towards two antigens, anosmin-1 (ANOS-1) and muscle related coiled-coil protein (MURC). ANOS-1 shows also significantly higher IgG reactivity frequency in ACPA-seropositive individuals compared to ACPA-seronegative and controls (22%, 9% and 6% respectively; P<0.05). Interestingly, three out of the four antigens discovered to be associated with the ACPA status in early RA are highly expressed in lungs and heart, two of the main extraarticular sites affected in RA. No significant differences were observed at IgA levels for any of the antigens analyzed.Table 1.Scheme of the different phases of the study, the features within each phase and the results. The reactivity to four antigens allows to distinguish ACPA-seronegative (ACPA-), ACPA seropositive (ACPA+) and controls.PhasesUntargeteddiscoveryTargeteddiscoveryTargetedvalidationNumber of samples80 ACPA-80 ACPA-358 ACPA-372 Controls80 ACPA+80 ACPA+386 ACPA+Antigen arrayplatformPlanararraysSuspensionbead array 1Suspensionbead array 2Number of antigens26606227Number of candidatebiomarkers6227 4 (TSPYL4,MAP2K6,ANOS1,MURC)Conclusion:Upon further validation in other early RA sample cohorts, our data suggest the measurement of these four autoantibodies may be useful for the early diagnosis of ACPA-seronegative RA and give insight into the pathogenesis of the different RA subsets.Characters from table content including title and footnotes:Disclosure of Interests:None declared

AB0150 ACPA-STATUS AND RHEUMATOID ARTHRITIS: MUCH MORE TO BE REVEALED!

Background:Anti-citrullinated protein antibodies (ACPA) are commonly associated with Rheumatoid arthritis (RA). RA is, therefore, classified as immunopositive or immunonegative, with disparate mechanisms in predisposition.Objectives:Our study aimed to determine the baseline characteristics and differences of ACPA-positive and ACPA-negative RA.Methods:We conducted a cross-sectional study including 224 patients with RA. All patients fulfilled the 2010 American College of Rheumatology/European League Against Rheumatism RA classification criteria. The patients were divided according to their ACPA status into two groups: ACPA-positive group (G1) and ACPA-negative group (G2). We compared clinical, radiological, and laboratory findings between the two groups, as well as extra-articular manifestations, comorbidities including fractures and osteoporosis. The Fracture Risk Assessment Tool (FRAX) was used to estimate the 10-year probability of major osteoporotic fracture (MOF) and also hip fracture (FH).Results:Of the 224 patients, 31.6% were negative for ACPA (n=71). Female predominance was found in both groups with a sex ratio of 0.25 (p=0.203). ACPA-negative subjects were younger (57±11 versus 59±12 years) (p= 0.305).The initial presentation of RA was different between the two groups without reaching statistical significance. In the ACPA-negative group, alteration of general condition was more frequent (16.9% in G2 versus 13.7% in G1) (p=0.533), with a tendency to oligo-articular onset (18.5% in G2 versus 6.7% in G1) (p=0.737). ACPA-positivity was more associated with an acute start of symptoms (10.4% in G1 versus 8.4% in G2) (p=0.639)There was no significant difference in the mean DAS28-VS (5.2±1.1 in G2 versus 5.5±1.3 in G1) and DAS28-CRP levels (5±1 in G2 versus 5.3±1.2 in G1) (p=0.069 and p=0.098 respectively).ACPA-positive RA was, however, significantly associated with more structural joint damage: erosions (55.9±53 in G1 versus 78±36 in G2, p=0.01), joint space narrowing (50.4±45.5 in G1 versus 33.1±36.6 in G2, p=0.003), Sharp/van der Heijde radiographic score (126.6 ±103.2 in G1 versus 88.8±81.5 in G2, p=0.004). ACPA-positive RA patients had more atlantoaxial dislocation: 20.2% in G1 versus 7% in G2 (p=0.012). There was no significant difference in hip involvement (9.8% in G1 versus 14% G2) (p=0.344).There were no significant differences in extra-articular manifestations between the two groups: Rheumatoid nodules (10.4% in G1 versus 18.3% in G2) (p=0.891), Sjögren’s syndrome (16.3% in G1 versus 16.9% in G2)(p=0.715), amyloidosis (0.6% in G1) (p=1), pulmonary fibrosis (5.8% in G1 versus 4.2% in G2) (p=0.757), neurological signs (4.5% in in G1 versus 5.6% in G2) (p=0.733), anaemia (5.8% in G1 versus 1.4% in G2) (p=0.175).When analyzing comorbidities, no significant differences were found: diabetes (10.4% in G1 versus 18.3% in G2) (p=0.103), cardiovascular diseases (19.6% in G1) (p=1), neurological diseases (0.06% in G1 versus 1.4% in G2) (p=0.534), dysthyroidism (2.6% in G1 versus 5.6% in G2) (p=0.267), dyslipidemia (3.2% in G1 versus 4.2% in G2) (p=0.711), cancer (1.3% in G1) (p=1). There were neither significant differences in the prevalence of fracture (21.5% in G1 versus 18.3% in G2) (p=0.574), and osteoporosis (23.6% in G1 versus 29.5% in G2) (p=0.347), between the two groups. However, ACPA-positive patients presented with a significantly higher FRAX score of MOF (2±2.8 in G1 versus 1.2±1 in G2) (p=0.006), and FRAX score of FH (0,9±1.8 in G1 versus 0.3±0.5 in G2) (p=0.003).Conclusion:ACPA status appears to influence both the clinical presentation and radiological progression of RA patients. ACPA-positive patients present with an acute start of symptoms, with more structural damages and atlantoaxial dislocation. Comorbidities, including osteoporosis, does not seem affected by ACPA status. However, regardless of osteoporosis, ACPA-positivity is associated with a higher probability of major osteoporotic and hip fractures. Further research is needed to clarify this relationship.Disclosure of Interests:None declared

High disease activity is associated with higher blood pressure in recent onset rheumatoid arthritis patients, post-hoc analyses of IMPROVED study

Funding Acknowledgements Type of funding sources: None. Background Rheumatoid arthritis (RA) is a chronic autoimmune disease with a high prevalence of cardiovascular morbidity and mortality. Purpose: purpose of our project was to investigate the association between disease activity and systolic and diastolic blood pressure (SBP, DBP) in patients with recent-onset rheumatoid arthritis (RA 2010 criteria) or undifferentiated arthritis (UA) who were treated to target disease activity score (DAS)&lt;1.6 in the IMPROVED study. Methods: The associations between disease activity and SBP/DBP were tested for 610 patients (364 RA, 246 UA), cross-sectionally and over time. GEE analyses were performed with both SBP and DBP as outcome measures and disease activity categories (DAS&lt;1.6;&gt;1.6 but ≤2.4; &gt;2.4), CRP level, treatment arms or the number of visits on a certain drug as potential predictors in separate analyses. Separate analyses tested potential contributions of gender, anti-cyclic citrullinated peptide antibodies (ACPA) status, and fulfilling the 2010 ACR/EULAR (American college of rheumatology/ European league against rheumatism) classification criteria. In addition association of BP with various levels of disease activity was tested with T-test. Results: At the baseline mean (SD) SBP was 133 (20) and DBP mean (SD) was 80 (10). SBP &gt; 140mm Hg was observed in 40% of patients and DBP &gt; 90 mm Hg in 21% of patients. SBP and DBP statistically significantly decreased during 5 years follow up (mainly during year 1), but the difference in mm Hg was small. Estimates from GEE analysis showed that patients with high DAS &gt;2.4 (HDAS) had a statistically significantly higher SBP (average 3 mm Hg higher, 95% CI 1.7; 4.2, p &lt; 0.01), than the patients in with DAS ≤2.4. ANOVA analyses showed a statistically significant association between SBP and DAS. In addition, post hoc analyses showed that patients with HDAS had a statistically significantly higher SBP (mean (SD) 132 (19) than the patients with DAS &lt; 1.6 (remission) (mean (SD) 129 (20), p &lt; 0.01), and patients in LDAS but DAS≥1.6 had a statistically significantly higher SBP (mean (SD) 131 (19) than the patients in remission (mean (SD) 129 (20), p = 0.02) (Figure 1), whereas no association was found between DAS category and DBP. Gender, ACPA status or fulfilling the 2010 classification criteria did not influence the relation between DAS and blood pressure. Conclusions: In patients with RA or UA, a higher DAS is associated with higher blood pressure, but the clinical impact is unclear. Abstract Figure 1