BackgroundLiver alterations, especially nonalcoholic fatty liver disease (NAFLD) are associated with Rheumatoid Arthritis (RA). This abnormality appears as asymptomatic and can progress to a severe liver damage rapidly. Chronic inflammation, treatment with methotrexate (MTX) or even autoimmunity are factors that might be involved, however the mechanisms related to this comorbidity in RA are not completely stablished yet.Objectives1) To evaluate the liver disease risk in RA patients through feasible indexes to be used in the daily clinical practice and its relationship with clinical features of the disease; 2) To analyze the impact of antibodies to citrullinated proteins antigens (ACPAs) on hepatic function; 3) To examine the influence of MTX treatment on classical parameters and new indexes of hepatic dysfunction in a cross-sectional and longitudinal cohort.Methods1) Cross-sectional study in 307 body mass index matched subjects: 55 healthy donors (HDs), 190 RA patients and 62 non-RA patients diagnosed with NAFLD through echography. Obese patients were excluded from the study. 2) Longitudinal study with 50 RA patients treated with MTX for 6 months. Clinical and laboratory parameters, subclinical liver disease biomarkers and 4 indexes to evaluate the presence of fibrosis and steatosis (APRI, “AST to platelet ratio index”; FIB-4, “fibrosis 4 score”; HSI, “hepatic steatosis index” and TyG, “triglycerides and glucosa index”) were measured. Association studies of hepatic dysfunction with clinical parameters were performed; A panel of 15 proteins directly involved in hepatic disease was analyzed in serum (C1QTNF1, IL7R, TIE1, CCL5, REG3A, CA3, LCN2, CCL14, NRP1, ICAM3, CD59, TIMP1, CNDP1, GNLY, IGFB6). 3) In vitro experiments were carried out in hepatocyte cell line (HEPG2) treated with ACPAs.ResultsUsing NALFD patients as positive controls for the four liver disease indexes, RA patients showed significantly higher levels of HSI and TyG biomarkers. In fact, a high proportion of these patients (42.7%) were estimated to suffer NALFD. The association studies in RA patients showed that liver disease biomarkers (HSI and TyG) were related to the insulin resistance state, inflammation, complement component 3(C3), disease activity, and the levels of ACPAs. Serum levels of CNDP1, CCL5, GNLY, TIMP-1, CD59 and CCL14 were significantly increased in RA patients and correlated with hepatic damage indexes. Treatment with ACPAs on hepatocytes promoted the secretion of C3 in parallel with a significant alteration of genes related to glucose and lipid metabolisms, inflammation, fibrosis and apoptosis. MTX treatment, from the point of cross-sectional approach, was not associated with an increase of hepatic enzymes, serum proteins nor liver disease indexes. Treatment with MTX for 6 months did not affect those levels either.Conclusion1) A high proportion of RA patients present an alteration in markers of NAFLD, associated with insulin resistance state, disease activity, inflammation, component C3 and ACPAs levels; 2) the autoantibodies could directly impact hepatocyte biology altering the expression of genes related to glucose and lipid metabolisms, inflammation, fibrosis and apoptosis, 3) Treatment with MTX did not promote any alteration in subclinical liver disease biomarkers after 6 months of treatment.Funded by Institute of Health Carlos III (PI20/00079).Disclosure of InterestsNone declared
Backgroundpatients with Spondyloarthritis (SpA) have an increased risk of cardiovascular disease. Taking into account the strong relationship between inflammation and CVD, there is an urgent need to identify different molecular drivers of CVD signs and their association with inflammation.Objectivesto investigate the alteration of CVD-related proteins in the plasma of SpA patients, their association with clinical features, and to evaluate their potential role as biomarkers for the identification of persistent inflammation.Methodsa cross-sectional study including 120 patients with SpA and 30 age-sex-matched healthy donors (HDs) was carried out. Clinical and laboratory parameters and CVD risk factors were recorded. To measure the presence of persistent inflammation, levels of c-reactive protein (CRP) were collected retrospectively for 5 years previous to the study, a patient was considered to have persistent inflammation with increased CRP in at least 50% of the measures during the previous 5 years. Levels of 92 proteins with a recognized role in CVD were analyzed in the plasma using proximity extension assay (PEA) technology (Olink Target 96 CVD III panel, Cobiomic Biosciences).ResultsSpA patients showed higher rates of CVD comorbidities compared to HDs. Plasma levels of TNF-R1, RARRES-2, CHI3L1, PGLYRP-1, CTSD, UPAR, IL2RA, TIMP-4, CTSB, GDF-15, MMP-9, and PDGF-A were significantly increased in SpA compared to HDs. Specifically, these proteins are also related to biological processes such as neutrophil degranulation, immune response, cell activation, atherosclerosis, apoptosis, and inflammatory response. Besides, a significant alteration of these CVD-related proteins in SpA was also associated with the presence of arterial hypertension, insulin resistance, obesity, hyperuricemia, and high levels of acute phase reactants. 36% of SpA patients displayed persistence of inflammation. Interestingly, SpA patients with persistent inflammation showed higher levels of ankylosing spondylitis disease activity (ASDAS) score, CRP, glucose, complement component 3, and lower levels of HDL-cholesterol and apolipoprotein A compared to SpA patients with non-persistent inflammation, suggesting the relationship of inflammation with metabolic alterations. In addition, 8 out of 12 CVD-related proteins altered in SpA patients were specifically changed in patients with persistent inflammation: MMP-9, RARRES-2, PGLYRP-1, UPAR, TNF-R1, PDGF-A, IL-2RA and GDF-15, highlighting levels of MMP-9 protein as a potential biomarker for persistent inflammation in SpA (AUC=0.796 p>0.0001).Conclusion1) SpA patients show an altered CVD proteome profile which is strongly associated with CVD risk factors and clinical inflammatory markers, 2) SpA patients with persistent inflammation display a deeper alteration in their plasma CVD protein pattern suggesting the link between subclinical CVD risk and the chronic inflammation, and 3) this study identifies novel potential biomarkers to distinguish SpA patients with persistent inflammation. Funded by ISCIII (PI20/00079, PMP21/00119, and RICOR-RD21/0002/0033) co-financed by ERDF.REFERENCES:NIL.Acknowledgements:NIL.Disclosure of InterestsNone Declared.
BackgroundSynovial membrane inflammation, driven by effector T-cell activation and altered inflammatory cytokine expression, is a key feature of this psoriatic arthritis. The proximity extension assay (PEA) technique could contribute to identifying novel protein biomarkers associated with PsA and to improving the biological understanding and the future management of PsA, leading to novel intervention targets.Objectives1) To characterize the inflammatory proteome of synovial fluid (SF) from patients with Psoriatic Arthritis (PsA) using a next generation proteomics technique, and 2) to evaluate its potential to stratify patients according to clinical features.MethodsInflammatory proteome profile of SF from thirteen PsA patients with active knee arthritis were analysed using PEA technology (Olink Target 96 Inflammation panel, Cobiomic Biosciences). Four patients with OA were included as control group.ResultsSeventy-nine inflammation-related proteins were detected in SF from PsA patients (SF-PsA). Unsupervised analyses of the molecular proteome profile in SF-PsA identified two specific phenotypes characterized by higher or lower levels of inflammation-related proteins. Clinically, SF-PsA with higher levels of inflammatory proteins also showed increased systemic inflammation and altered glucose and lipid metabolisms. Besides, SF from PsA patients showed 39 out of 79 proteins significantly altered compared to SF-OA specifically related to cell migration and inflammatory response. Among these, molecules such as TNFα, IL-17A, IL-6, IL-10, IL-8, ENRAGE, CCL20, TNFSF-14, OSM, IFNγ, MCP-3, CXCL-11, MCP4, CASP-8, CXCL-6, CD-6, ADA, CXCL-10, TNFβ and IL-7 showed the most significantly alteration.ConclusionThis is the first study that characterizes the inflammatory landscape of synovial fluid of PsA patients by analyzing a panel of 92 inflammatory proteins using PEA technology. Novel SF proteins have been described as potential pathogenic molecules involved in the pathogenesis of PsA. Despite the flare, inflammatory proteome could distinguish two different phenotypes related to systemic inflammation and lipid and glucose alterations.Acknowledgements:Funded by ISCIII (PI20/00079 and RD21/0002/0033) co-financed by ERDF.REFERENCES:NIL.Acknowledgements:NIL.Disclosure of InterestsNone Declared.
BackgroundThe use of high throughput techniques such as transcriptomic sequencing has recently made considerable progress in the identification of molecular profiles involved in the pathogenesis of chronic autoinflammatory diseases. To date, only a few studies have been carried out in axial spondyloarthritis (axSpA), which would allow the identification of new therapeutic targets and disease biomarkers.Objectives1) To identify clusters of highly correlated genes enriched in biological functions and specific molecular pathways involved in the pathogenesis of axSpA. 2) To study the association between the molecular signatures identified and the clinical-analytical profile of the disease.MethodsCross-sectional study including 75 axSpA patients from the CASTRO cohort who underwent an exhaustive clinical evaluation including disease activity and functional limitation, structural damage, and spinal mobility. Additionally, analytical parameters were measured and the carotid intima-media thickness was evaluated by carotid eco-doppler. Mononuclear cells were purified from peripheral blood, and RNA was isolated. RNA from 25 axSpA patients was sequenced using the Illumina platform. For the identification of patient subgroups and the generation of co-expressed gene modules, the “hierarchical clustering” and WGCNA (“Weight gene correlation network analysis”) methodologies were used, respectively. Functional analysis of the genes conforming each module was carried out to identify enriched pathways and functions using the EnrichR platform. Hub genes were measured through high throughput PCR (Fluidigm Biomark HD) in a validation cohort of 50 axSpA patients. Association and correlation studies between the molecular and clinical profiles were performed.In vitrostudies in peripheral blood mononuclear cells (PBMCs) from patients belonging to different molecular clusters were performed.ResultsUnsupervised analysis of the transcriptome revealed the presence of two “clusters” of axSpA patients, clearly differentiated by their molecular and clinical profiles. Specifically, the molecular analysis distinguished patients with longer disease duration, greater disease activity, radiographic damage, and cardiovascular risk. WGCNA identified 11 highly co-expressed modules. Among them, six were differentially expressed between the two clusters, being responsible for the molecular and clinical distinction of those groups. The functional analysis of these 6 gene modules revealed the enrichment of these genes in pathways related to inflammation, oxidative metabolism, proliferation of B and T lymphocytes, immune response, and the increase of cell survival. Finally, key genes were identified within each module (“hub genes”), whose expression was associated with a more active phenotype of the disease such as ALOX5, GAB2, PSMD13, CASP8, NOTCH e ITGA4. Hub genes were validated in an additional cohort of 50 axSpA patients and correlated with mSASSS. Treatment of PBMCs from patients belonging to the two different clusters with autologous serum induced a different expression of genes involved in cell activation and inflammation.Conclusion1) The whole transcriptomic analysis by RNAseq in peripheral mononuclear cells from axSpA patients distinguished, in an unsupervised manner, subgroups of patients with distinctive clinical profiles. 2)The analysis of gene modules identified new pathways and molecular functions potentially involved in the pathophysiology of the disease. Funded by ISCIII (PMP21/00119) co-financed by ERDF, Andalusian Foundation of Rheumatology (FAR).REFERENCES:NIL.Acknowledgements:NIL.Disclosure of InterestsNone Declared.
BackgroundIn recent years, the increased prevalence of liver abnormalities in Psoriatic Arthritis (PsA) has acquired relevance. The liver dysfunction in PsA might be induced by inflammatory components in parallel with the presence of metabolic complications or due to the potential hepatotoxicity of the treatments administered.Objectives1) to analyze the impact of PsA serum on hepatocytes and 2) to investigate the effect of methotrexate, anti-PDE-4, and tofacitinib on hepatocytes treated with PsA serum.Methodsserum from 12 healthy donors (HDs) and 20 age and sex-matched PsA patients were collected to performin vitroexperiments with the hepatocyte cell line (HEPG2). Cells were treated with HDs serum or PsA serum alone or combined with methotrexate (50µM), roflumilast (1nM), or tofacitinib (250 nM) for 24 hours. The expression of genes related to inflammation, immune response, apoptosis, and liver dysfunction was analyzed by RT-PCR. Cell stress protein array was performed. The levels of 92 inflammatory proteins were determined on the treated hepatocytes using PEA technology (Olink Target 96 Inflammation panel, Cobiomic Biosciences). STRING platform (version 11.5) was used to determine the functional protein association networks.ResultsPsA patients showed a mean disease activity in PsA (DAPSA) of 27.10 ±12.52 (moderate-high disease activity) and significantly elevated levels of c-reactive protein and erythrocyte sedimentation rate with respect to HDs. In hepatocytes, the treatment with PsA serum induced the mRNA expression of ADAM-17, CHUK, CTSD, FAF-1, GDF-15, IFGR-1, IL-6, IL-8, MMP-1, STAT-3, TGF-β and TFPI compared to HDs serum, genes directly associated with biological processes which might impact liver function, such as inflammatory response, immune system, apoptotic process and even directly associated with non-alcoholic fatty liver disease. In addition, PsA serum promoted the expression of 22 proteins mainly involved in cell stress in hepatocytes compared to HDs serum treatment. These proteins are also associated with lipids and atherosclerosis, apoptosis, regulation of inflammatory response, hepatic diseases, and regulation of the extracellular matrix. Surprisingly, 33 out of 92 inflammatory-related proteins were significantly increased in hepatocytes treated with PsA serum compared to HDs serum (36% of proteins). On the other hand, the treatment with anti-PDE-4 promoted the reduction of 11 out of 33 proteins altered by the treatment with PsA serum, proteins mainly involved in leukocyte migration and the immune system. The treatment with anti-JAK reduced levels of 20 out of 33 proteins altered by the treatment with PsA serum. However, the treatment with methotrexate significantly increased the expression of 5 out of 33 proteins altered by the treatment with PsA serum.Conclusion1) PsA serum characterized by the presence of inflammatory components profoundly impacts the hepatocyte function promoting the alteration in proteins and genes involved in the inflammatory response, immune system, and the apoptotic and fibrotic process; 2) the treatment with anti-PDE-4 or anti-JAK is able to partially reverse the deleterious inflammatory effect of PsA serum produced in the hepatocytes.Funded by ISCIII (PI20/00079 and RICOR-RD21/0002/0033), the Andalusian government (1381035-F) co-financed by ERDF.REFERENCES:NIL.Acknowledgements:NIL.Disclosure of InterestsNone Declared.
BackgroundLiver abnormalities, particularly non-alcoholic fatty liver disease (NAFLD) are strongly associated with psoriatic arthritis (PsA). In the last decades, the possible hepatotoxic effects of methotrexate in PsA have been studied, however, up to date, the results are controversial.Objectives1) to evaluate the liver damage risk in PsA patients and its relationship with clinical features of the disease; 2) to determine the influence of cumulative doses of methotrexate byin vivoandin vitroapproaches.Methodsa cross-sectional study was performed on 326 subjects: 155 PsA patients, 87 NAFLD non-PsA patients, and 84 healthy donors (HDs). Clinical and laboratory parameters, liver disease biomarkers, and several indexes to evaluate the risk of suffering hepatic steatosis or fibrosis were evaluated. Cumulative doses of methotrexate were calculated retrospectively. Mechanistic studies were carried out on hepatocytes (HEPG2 cell line) treated with cumulative doses of methotrexate for up to 72 hours and 150µM. The changes in the levels of 92 inflammatory proteins on the hepatocytes were analyzed using PEA technology (Olink Target 96 Inflammation panel, Cobiomic Biosciences).Resultsusing a NAFLD non-PsA BMI, age and sex-matched cohort, a cut-off value of hepatic steatosis index (HSI) (35.68; AUC=0.865p<0.0001) was calculated to distinguish NAFLD patients from HDs. Taking this value into account, PsA patients showed a significantly high risk of suffering NAFLDvsHDs, 65% respect to 22%. In addition, PsA patients with a high risk for steatosis displayed a significant prevalence of insulin resistance, obesity, type 2 diabetes mellitus, and arterial hypertension compared to PsA patients without that risk. Interestingly, these PsA patients also showed significantly elevated levels of triglycerides, complement component 3, DAPSA, c-reactive protein, and erythrocyte sedimentation rate. Next, PsA patients were divided into two groups depending on mean cumulative doses of methotrexate (<1.5 gr for no hepatotoxic risk group and >1.5 gr for the hepatotoxic risk group). Patients with more than 1.5gr of methotrexate cumulative dose showed significantly lower levels of acute phase reactants and no significant differences in metabolic profile or liver disease biomarkers. However, fibrosis-4 score (FIB-4) was significantly elevated in the group of hepatotoxic risk and positively correlated with cumulative doses. Although, this association was influenced by the age variable and not due to the levels of transaminases or platelets. On the other hand,in vitrotreatment of hepatocytes with methotrexate for 24 h induced the expression of 19 inflammatory proteins out of 92. These changes were not significantly altered after 72 hours of treatment with methotrexate added each 24h.Conclusion1) PsA patients with a high risk of liver disease show elevated rates of cardiometabolic comorbidities, inflammatory clinical markers and disease activity suggesting the link between metabolic alterations and the clinical features of PsA with the development of NAFLD, 2) PsA patients with high cumulative doses of methotrexate had no alteration of hepatic enzymes and indexes and 3) High cumulative doses of methotrexatein vitrodid not show a significant impact on hepatocyte cells compared to low doses.Funded by ISCIII (PI20/00079 and RICOR-RD21/0002/0033), the Andalusian government (1381035-F) co-financed by ERDF.REFERENCES:NIL.Acknowledgements:NIL.Disclosure of InterestsNone Declared.
BackgroundPatients with rheumatoid arthritis (RA) have a high risk of suffering cardiovascular disease. The cardiovascular and cardiometabolic proteome analysis in RA patients may provide disease biomarkers and insights into the biological pathways that contribute to cardiovascular risk.Objectives1) To evaluate the changes in the cardiometabolic and cardiovascular serum proteome in two cohorts of active RA patients: newly diagnosed (naïve-treated) and well-established disease and its relationship with the clinical characteristics and 2) to analyze the modulation of the levels of these proteins by methotrexate and tofacitinib.Methods1) Cross-sectional study in two cohorts of RA: patients at diagnosis (n=25) and patients with established disease (evolution time>25 years, n=25), age and sex-matched with healthy donors (n=25). 2) Longitudinal study was performed on 25 RA patients treated with methotrexate and 25 RA patients treated with Tofacitinib combined with DMARDs for 6 months. Clinical and analytical variables were recorded. Proteomic profiling of 184 proteins (cardiometabolic and cardiovascular II panels) was performed in the serum using Olink Proteomics technology.ResultsThere were no statistical differences between the RA at diagnosis and RA-established disease groups in age and DAS28. The evolution time average in the RA-established disease group was 37.48±11.79 years. One hundred and seven proteins were significantly increased in the serum of RA patients at diagnosis, among these 47 proteins were elevated in RA patients with established disease. The PCA analysis showed that the alteration of these proteins could discriminate between RA patients and healthy donors. The molecules that more significantly increased in the RA at diagnosis group were SAA4, ST6GAL1, CCL18, TNC, IGLC2, IL6, SORT1, TNFRSF10A, AGRP, and CD4. In addition, the top ten molecules significantly elevated in RA patients with the established disease were SAA4, CCL18, ST6GAL1, CRTAC1, ANGPTL3, TNFRSF10A, LEP, Gal-9, SORT1, and TRAIL-R2. The increase of these proteins was associated with the inflammation shown by the correlation with C reactive protein (CRP) levels and DAS28. The elevated levels of SAA4 (serum amyloid A protein-4) discriminated between RA patients and healthy donors with high sensitivity and specificity (AUC=0.95).Treatment with methotrexate in RA patients at diagnosis reduced the levels of 64 proteins (50% of the altered proteins) while tofacitinib modulated the expression of 30 proteins (50% of the altered proteins) in RA patients with established disease after 6 months. These reductions were associated with a decrease in CRP and DAS28 levels.Conclusion1)The cardiovascular and cardiometabolic serum proteome is altered in RA patients at diagnosis and after a long time of the disease evolution and chronic treatments, 2) SAA4 may represent a biomarker for the diagnosis of RA with high sensitivity and specificity, 3) Methotrexate and tofacitinib specifically modulate the alteration on the serum proteome after six months of treatment alongside the reduction of clinical inflammatory and disease activity markers.Funded by Pfizer-IRS-Aspire 2019.REFERENCES:NIL.Acknowledgements:NIL.Disclosure of InterestsNuria Barbarroja Puerto Grant/research support from: Pfizer, Carlos Perez-Sanchez: None declared, Iván Arias de la Rosa: None declared, Laura Cuesta López: None declared, Miriam Ruiz-Ponce: None declared, Antonio Gonzalez: None declared, Eva Perez-Pampín: None declared, Chamaida Plasencia: None declared, Ana Martínez-Feito: None declared, Rafaela Ortega Castro: None declared, Jerusalem Calvo Gutierrez: None declared, Eduardo Collantes Estevez: None declared, Chary Lopez-Pedrera: None declared, Alejandro Escudero Contreras Grant/research support from: Pfizer.
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