PADI2 Polymorphisms Are Significantly Associated With Rheumatoid Arthritis, Autoantibodies Serologic Status and Joint Damage in Women from Southern Mexico

The enzymes of the family peptidylarginine deiminases (PADs) have an important role in the pathogenesis of rheumatoid arthritis (RA) due to their association with the anti-citrullinated protein antibodies (ACPA) production. To evaluate the association between single-nucleotide polymorphisms (SNPs) in the PADI2 gene and RA susceptibility, related clinical parameters, and the serologic status of autoantibodies in a women population with RA from southern Mexico, a case-control study was conducted (case n=229; control n=333). Sociodemographic characteristics were evaluated, along with clinical parameters, inflammation markers, the levels of ACPAs as anti-cyclic citrullinated peptides (anti-CCPs), anti-modified citrullinated vimentin (anti-MCV), and rheumatoid factor (RF). Genomic DNA was extracted from peripheral blood, and three SNPs of the PADI2 gene (rs1005753, rs2057094, and rs2235926) were performed by qPCR using TaqMan probes. The data analysis reveals that the carriers of the T allele for rs2057094 and rs2235926 presented an earlier onset of the disease (β= -3.26; p = 0.03 and β = -4.13; p = 0.015, respectively) while the carriers of the T allele for rs1005753 presented higher levels of anti-CCPs (β= 68.3; p = 0.015). Additionally, the T allele of rs2235926 was associated with a positive RF (OR = 2.90; p = 0.04), anti-MCV (OR = 2.92; p = 0.05), and with the serologic status anti-CCP+/anti-MCV+ (OR = 3.02; p = 0.03), and anti-CCP+/anti-MCV+/RF+ (OR = 3.79; p = 0.004). The haplotypes GTT (OR =1.52; p = 0.027) and TTT (OR = 1.32; p = 0.025) were associated with the presence of RA. In addition, in this study the haplotype TTT is linked to the presence of radiographic joint damage defined by a Sharp-van der Heijde score (SHS) ≥2 (OR = 1.97; p = 0.0021) and SHS ≥3 (OR = 1.94; p = 0.011). The haplotype TTT of SNPs rs1005753, rs2057094, and rs2235926 of the PADI2 gene confers genetic susceptibility to RA and radiographic joint damage in women from southern Mexico. The evidence reveals that SNPs of the PADI2 gene favors the presence of a positive serologic status in multiple autoantibodies and the clinical manifestations of RA at an early onset age.

POS0465 DIFFERENT CLINICAL RELEVANCE OF ANTI-CITRULLINATED PROTEINS ANTIBODIES IN RA PATIENTS

Background:Anti-citrullinated proteins antibodies (ACPA) are a broad group of antibodies, including antibodies to citrullinated fibrinogen, antibodies to cyclic citrullinated peptide (anti-CCP), antibodies to modified citrullinated vimentin (anti-MCV), antibodies to citrullinated α-enolase.Objectives:To find a potential relationship between ACPA and disease activity, bone destruction, and ACPAs responses to various therapeutic regimens.Methods:The study included 232 patients (pts) with rheumatoid arthritis (RA); 90 pts (74 women, Me;IQR age 53.0 (38.0–58.5) years had early RA, with mean disease duration 5.0 (4.0–9.0) months, DAS 28 5.3 (4.4–6.1)); 142 pts had advanced stage of the disease (123 women, median age 51.0 (43.0–60.0) years, disease duration 56.0 (24.0–96.0) months, DAS 28 6.2 (5.5–6.8)). Pts with early RA received methotrexate (MTX) subcutaneously at average 17.5 mg dose once weekly. Pts with advanced RA received the following anti-B-cell therapy: 34 pts - rituximab (RTX); 20 pts - RTX biosimilar; 43 pts - tocilizumab (TCZ) in combination with conventional DMARDs. Serum anti-CCP and anti-MCV concentrations were measured using ELISA.Results:77 (85.6%) pts with early RA were high positive for anti-CCP, and 29 (70.7%) pts - high positive for anti-MCV. A positive correlation was found between anti-MCV and DAS 28 (r=0.4, p=0.04). As for advanced RA, 78 (80.4%) pts were high positive for anti-CCP, and 70 (79.5%) - for anti-MCV. There was a positive correlation between anti-MCV concentration and SDAI (r=0.4, p=0.02), as well as CDAI (r=0.4, p=0.02). No significant correlations were found between the anti-CCP levels and activity indices, anti-CCP and acute-phase parameters in both early and advanced RA groups. Higher total Sharp scores (96.5 (65.0-122.0)) were found in pts high positive for anti-MCV (n=79), compared to low-positive/negative (57.0 (31.0–88.0), respectively, (n=27, p<0.05). anti-MCV levels dropped significantly in pts on RTX and TCZ therapy at weeks 12 and 24 after initiation of treatment, while high anti-CCP concentration persisted throughout the treatment (Table 1)Conclusion:anti-MCV levels correlated with inflammatory activity and development of bone destruction, and were decreasing in pts on treatment. Anti-CCP was less responsive, showed minor changes during treatment, therefore its’ thorough monitoring was not feasible.Disclosure of Interests:None declared

POS0368 CITRULLINATION INDUCES EPIGENETIC MEMORY OF THE INNATE IMMUNE SYSTEM

Background:During trained immunity, monocytes and macrophages undergo a functional and transcriptional reprogramming toward activation, which is induced by a priming stimulus and results in enhanced responsiveness to subsequent triggers. Monocytes from patients with rheumatoid arthritis (RA) display features consistent with a trained immunity phenotype. Citrullinated proteins as citrullinated vimentin (c-vimentin), which function as damage-associated patterns in RA, may be implicated in the process of trained immunity.Objectives:We aimed to investigate if c-vimentin induces trained immunity in vitro in healthy individuals.Methods:Monocytes were isolated from the peripheral blood (EDTA blood, n=22; buffy coats, n=6) from healthy donors by Ficoll-paque centrifugation and negative selection using CD3/CD19/CD56 magnetic beads. The cells were stimulated with c-vimentin (0.1 μg/ml) for 24h and re-stimulated 5 days later with the lipopolysaccharide of E.coli (LPS) (10 ng/ml). Protein as well as lactate release were estimated in cell culture supernatants at day 6 by ELISA. RT-PCR and/or Western Blotting were applied to measure mRNA and/or protein expression. The Ligand-receptor glycocapture technology LRC-TRiCEPS was used to identify candidate cell surface targets of c-vimentin. The methylation of histone H3 at lysine 4 (H3K4) was examined by chromatin immunoprecipitation.Results:Priming with citrullinated vimentin induced training in human monocytes, as suggested by the significantly increased levels of secreted interleukin-6 (IL-6), upon restimulation with LPS (1.29-fold increase, n=22, p<0.001). Likewise, the release of chemokines CXCL1 and CCL20/Macrophage Inflammatory Protein 3a was significantly increased (1.81-fold and 2.32-fold increase, respectively, n=14, both p<0.001). LRC-TRiCEPS enabled the identification of STING cell surface receptor for the ligand c-vimentin. Indeed, c-vimentin induced activation of TBK1, which is implicated in the STING signaling pathway, by phosphorylation, while STING inhibition with the covalent small molecule H151 (2μM) abolished this effect. Besides, H151 inhibited trained immunity by decreasing IL-6 release and expression (1.61-fold and 1.93-fold decrease, respectively, n=5). Trained monocytes also displayed high lactate production (primed vs. unprimed cells, n=9, p=0.004), reflecting a shift in metabolism with an increase in glycolysis. By inhibiting the metabolic pathway of glycolysis by 2-deoxyglucose (11mM), the induction of trained immunity could be counteracted (5.32-fold decrease in IL-6 release, n=7, p=0.016). Finally, c-vimentin induced H3K4 methylation with increased levels of this mark in the promoter of the IL-6 gene. By modulating the function of epigenetic enzymes with methylthioadenosine (1mM), which specifically inhibits histone methyltransferase, trained immunity was reversed (8.43-fold decrease in IL-6 release, n=6, p=0.031).Conclusion:Citrullinated vimentin induces epigenetic modifications and metabolic changes in monocytes, probably through a STING and TBK1-dependent activation, resulting in enhanced cytokine and chemokine production upon restimulation. Inhibition of the STING signaling pathway may be a novel therapeutic target for myeloid activation in RA.Disclosure of Interests:None declared

MO223MEMBRANOUS NEPHROPATHY ASSOCIATED TO ANKYLOSING SPONDYLITIS: CASE REPORT

Background and Aims Ankylosing Spondylitis (AS) is a chronic inflammatory rheumatic disease diagnosed by the presence of the HLA-B27 antigen with joint and extra-articular manifestations. Its pathogenesis was initially based on auto-inflammatory phenomena, mainly involving innate immunity. However, many studies carried out in the recent years focus on its adaptive immunity aspect, especially autoimmune. These mechanisms seem to interact with each other, resulting in a complex pathophysiology. The general autoimmune characteristics of AS were investigated by Yuan and al, who reviewed recent reports of autoantibodies levels in AS patients. Their analysis shows that none of the autoantibodies considered in the study meet the criteria to be considered as a biomarker for the disease (including antibodies Anti- :CD74; beta2-Microglobulin; mutated Citrullinated Vimentin (MCV); Heat Shock Protein 65 (HSP65); 14-3-3 eta autoantibodies (14-3-3η); 1A-dependent autoantibody magnesium anti-protein phosphatase (PPM1A); sclerostin (SOST); and Anti-microbial antibodies). Renal involvement in AS is dominated by amyloidosis and IgA nephropathy. In rare cases, this disease has been associated with Membranous Nephropathy (MN). The pathogenetic link between the two disorders remains obscure. However, the recent connection of AS to autoantibodies potentially indicates the involvement of immune complexes formed from these autoantibodies in the development of MN. Method We report the case of a 36-years-old woman with a 7 years history of sacroiliitis, who also developed a pedal edema in May 2020. AS was diagnosed in 2014 by the presence of positive HLA-B27, and clinical and radiological manifestations of bilateral sacroiliitis without extra renal manifestations treated by indometacin during periods of pain . A pure nephrotic syndrome was revealed with a 24h proteinuria of 12g / 24h.The patient’s Albumin levels were at 16g/l, without HTA or hematuria, and with a correct renal function (Creatinine at 60mmol/l). A renal biopsy showed a MN with type I polytypical. Light microscopy showed a thickening of capillary loops, while IF staining revealed granular deposits of IgG along the capillary wall. Investigations of further secondary MNs were negative, and the patient was aPLA2R-negative. Results A treatment by corticosteroids was initiated prior biopsy to her referral , which resulted in pain relief and urinary protein reduction (prot 24h 1.5g/24h). Given her good response to this initial treatment, corticotherapy was maintained. As the levels of inflammation and discomfort were low, the patient did not wish to be treated by biotherapy. Conclusion This case suggests a secondary MN in association with AS.The discovery of new autoantibodies associated with AS opens up promising perspectives, and could potentially lead to the characterization of biomarkers for screening and monitoring this disease. However, more studies are needed in order to improve our understanding of the role played by possible immune complex diseases, (in particular MN), in relation to this Ankylosing spondilitis.

Citrullinated vimentin mediates development and progression of lung fibrosis

The mechanisms by which environmental exposures contribute to the pathogenesis of lung fibrosis are unclear. Here, we demonstrate an increase in cadmium (Cd) and carbon black (CB), common components of cigarette smoke (CS) and environmental particulate matter (PM), in lung tissue from subjects with idiopathic pulmonary fibrosis (IPF). Cd concentrations were directly proportional to citrullinated vimentin (Cit-Vim) amounts in lung tissue of subjects with IPF. Cit-Vim amounts were higher in subjects with IPF, especially smokers, which correlated with lung function and were associated with disease manifestations. Cd/CB induced the secretion of Cit-Vim in an Akt1- and peptidylarginine deiminase 2 (PAD2)–dependent manner. Cit-Vim mediated fibroblast invasion in a 3D ex vivo model of human pulmospheres that resulted in higher expression of CD26, collagen, and α-SMA. Cit-Vim activated NF-κB in a TLR4-dependent fashion and induced the production of active TGF-β1, CTGF, and IL-8 along with higher surface expression of TLR4 in lung fibroblasts. To corroborate ex vivo findings, mice treated with Cit-Vim, but not Vim, independently developed a similar pattern of fibrotic tissue remodeling, which was TLR4 dependent. Moreover, wild-type mice, but not PAD2−/− and TLR4 mutant (MUT) mice, exposed to Cd/CB generated high amounts of Cit-Vim, in both plasma and bronchoalveolar lavage fluid, and developed lung fibrosis in a stereotypic manner. Together, these studies support a role for Cit-Vim as a damage-associated molecular pattern molecule (DAMP) that is generated by lung macrophages in response to environmental Cd/CB exposure. Furthermore, PAD2 might represent a promising target to attenuate Cd/CB-induced fibrosis.

Clinical Significance of Anti-Modified Citrullinated Vimentin Antibodies in Palindromic Rheumatism

Objective This study evaluated anti-modified citrullinated vimentin (anti-MCV) performance in determining the clinical picture and outcomes of palindromic rheumatism (PR). Methods In a retrospective study, patients with PR with at least 1 year of follow-up diagnosed according to clinical criteria were enrolled. Anti-MCV antibodies were measured, and levels &gt;20 IU/mL were considered positive. Disease prognosis was assessed according to patients acquiring remission and preventing PR from developing into rheumatoid arthritis (RA) or other diseases. Results Seventy-six patients with PR with a mean follow-up of 30.57 months (median = 21 months; minimum = 12 months; maximum = 48 months) were included in the study. Anti-MCV antibodies were positive in 69.7% of patients. Metacarpophalangeal (MCP) joint involvement and positive anti-cyclic citrullinated peptides were significantly higher in patients who were anti-MCV-positive, whereas ankle joint involvement was significantly lower. No significant correlation was observed between the anti-MCV titer and the severity of attacks. Remission in patients who were anti-MCV-positive and negative was 75.5% and 78.3%, respectively, with no significant difference. Evolution to RA was observed in only 3.8% of patients who were anti-MCV-positive. No patients who were anti-MCV-negative developed RA. Conclusion Except for MCP and ankle joint involvement, anti-MCV was not helpful in determining the clinical picture and outcome of PR.

New laboratory criterion for the differential diagnosis of sarcoidosis and pulmonary tuberculosis

In some cases there is a problem of differential diagnosis of sarcoidosis (SD) and tuberculosis (TB) because of the similarities in clinical, X-ray and laboratory features. The aim of this study was to search for new differential diagnostic criteria for sarcoidosis and tuberculosis by calculating the index, based on the level of autoantibodies to modified citrullinated vimentin (anti-MCV) and the level of B-cell subpopulations. These parameters were measured in patients with sarcoidosis (n = 93), tuberculosis (n = 28) and healthy donors (n = 40) using the ELISA and cytometry. The absence of a statistically significant difference when comparing the level of anti-MCV, the number of B-cells in SD and TB suggests that these changes may be characteristic of granulomatous diseases. The use of the formula Ds=([B-naïve%]\[B-memory%])*([B-CD38%]+[B-CD5%])/[anti-MCV] might allow to differentiate SD with an increase in the calculated index of more than 5 units with a sensitivity of 80.00% and specificity of 93.10% (AUC = 0.926).