scholarly journals Splicing machinery is impaired in rheumatoid arthritis, associated with disease activity and modulated by anti-TNF therapy

2021 ◽  
pp. annrheumdis-2021-220308
Author(s):  
Alejandro Ibáñez-Costa ◽  
Carlos Perez-Sanchez ◽  
Alejandra María Patiño-Trives ◽  
Maria Luque-Tevar ◽  
Pilar Font ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Synovial Fluid ◽  
Disease Activity ◽  
Synovial Fibroblasts ◽  
Data Set ◽  
Altered Expression ◽  
Healthy Donors ◽  
Inflammatory Profile ◽  
Splicing Machinery

ObjectivesTo characterise splicing machinery (SM) alterations in leucocytes of patients with rheumatoid arthritis (RA), and to assess its influence on their clinical profile and therapeutic response.MethodsLeucocyte subtypes from 129 patients with RA and 29 healthy donors (HD) were purified, and 45 selected SM elements (SME) were evaluated by quantitative PCR-array based on microfluidic technology (Fluidigm). Modulation by anti-tumour necrosis factor (TNF) therapy and underlying regulatory mechanisms were assessed.ResultsAn altered expression of several SME was found in RA leucocytes. Eight elements (SNRNP70, SNRNP200, U2AF2, RNU4ATAC, RBM3, RBM17, KHDRBS1 and SRSF10) were equally altered in all leucocytes subtypes. Logistic regressions revealed that this signature might: discriminate RA and HD, and anti-citrullinated protein antibodies (ACPAs) positivity; classify high-disease activity (disease activity score-28 (DAS28) >5.1); recognise radiological involvement; and identify patients showing atheroma plaques. Furthermore, this signature was altered in RA synovial fluid and ankle joints of K/BxN-arthritic mice. An available RNA-seq data set enabled to validate data and identified distinctive splicing events and splicing variants among patients with RA expressing high and low SME levels. 3 and 6 months anti-TNF therapy reversed their expression in parallel to the reduction of the inflammatory profile. In vitro, ACPAs modulated SME, at least partially, by Fc Receptor (FcR)-dependent mechanisms. Key inflammatory cytokines further altered SME. Lastly, induced SNRNP70-overexpression and KHDRBS1-overexpression reversed inflammation in lymphocytes, NETosis in neutrophils and adhesion in RA monocytes and influenced activity of RA synovial fibroblasts.ConclusionsOverall, we have characterised for the first time a signature comprising eight dysregulated SME in RA leucocytes from both peripheral blood and synovial fluid, linked to disease pathophysiology, modulated by ACPAs and reversed by anti-TNF therapy.

scholarly journals POS0388 INTERLEUKIN 40 (IL-40) IS UP-REGULATED IN RHEUMATOID ARTHRITIS (RA) AND ASSOCIATED WITH DISEASE ACTIVITY, LEVELS OF AUTOANTIBODIES AND CHEMOKINES

2021 ◽  
Vol 80 (Suppl 1) ◽  
pp. 424.1-424
Author(s):  
A. Navrátilová ◽  
L. Andres Cerezo ◽  
H. Hulejova ◽  
V. Becvar ◽  
D. Tegzová ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
B Cells ◽  
Synovial Fluid ◽  
Disease Activity ◽  
B Cell ◽  
Synovial Tissue ◽  
Lupus Erythematosus ◽  
Synovial Fibroblasts ◽  
Clinical Activity

Background:Interleukin 40 (IL-40) is newly identified B cell - associated cytokine implicated in humoral immune responses and in B cell development. As B cells play a pivotal role in autoimmunity, we aimed to investigate the function of IL-40 in rheumatoid arthritis (RA).Objectives:The aim of our study was to determine the function of IL-40 in RA.Methods:IL-40 expression in the synovial tissue was determined by immunohistochemistry and immunofluorescence (n=4-5). IL-40 was analysed in the serum/synovial fluid of patients with RA (n=69), systemic lupus erythematosus (SLE; n=69), osteoarthritis (OA; n=44), and in healthy controls (HC; n=25). Given the association of IL-40 with B cells, we analysed the effect of rituximab therapy on the serum IL-40 in 19 patients with RA after 16 and 24 weeks of the therapy. The clinical activity of patients with RA was assessed according to the 28 joint count Disease Activity Score (DAS28). Levels of C-reactive protein (CRP) and autoantibodies were measured by routine laboratory techniques. In vitro experiments were performed in RA synovial fibroblasts (n=9). Levels of cytokines and inflammatory mediators were determined in serum, synovial fluid and supernatants using ELISA or multiplex immunoassay.Results:IL-40 was overexpressed in RA synovial tissue compared to OA, particularly by synovial fibroblasts and immune cells such as B and T lymphocytes, macrophages and neutrophils. The levels of IL-40 were significantly higher in the synovial fluid of RA patients compared to OA (33.2 (6.6-68.9) vs. 0.7 (0.1-2.4) ng/ml; p<0.0001). In addition, IL-40 was increased in the serum of RA patients compared to SLE, OA or HC (4.8 (1.7-24.9) vs. 1.4 (1.0-1.9), 1.6 (0.6-3.1) or 1.5 (0.7-2.7) ng/ml; p<0.0001 for all) and decreased after 16 (p<0.01) and 24 weeks (p<0.001) in a subgroup of rituximab treated patients with RA. IL-40 levels in RA patients correlated with autoantibodies rheumatoid factor (IgM) and anti-citrullinated protein antibody (ACPA) in the serum (p<0.0001 and p<0.01) as well as in the synovial fluid (p<0.0001 and p<0.001). IL-40 in RA synovial fluid was also significantly associated with DAS28 (p<0.05), synovial fluid leukocyte count (p<0.01), number of swollen joints (p<0.05) and neutrophil attractants IL-8 (p<0.01) and MIP-1α (p<0.01). RA synovial fibroblasts exposed to recombinant IL-40 increased secretion of IL-8 (p<0.01), MCP-1 (p<0.05) and MMP-13 (p<0.01) compared to unstimulated cells in in vitro conditions.Conclusion:Our results show for the first time that IL-40 is elevated in RA and decreases following B-cell depletion therapy. The association of IL-40 with autoantibodies and chemokines may imply its potential involvement in RA development. Moreover, IL-40 up-regulates the secretion of chemokines and MMP-13 by synovial fibroblasts, indicating its role in the regulation of inflammation and tissue destruction in RA.Acknowledgements:Supported by MHCR 023728 a SVV 260 523Disclosure of Interests:None declared

scholarly journals Evaluation of the degree of clinical rheumatoid arthritis activity based on the concentrations of cytokines TNF-alpha, IL-12, IL-15, and IL-18 in serum and synovial fluid

2006 ◽  
Vol 63 (1) ◽  
pp. 21-26 ◽  
Author(s):  
Ljiljana Petrovic-Rackov
Keyword(s):  
Rheumatoid Arthritis ◽  
Synovial Fluid ◽  
Disease Activity ◽  
Tnf Alpha ◽  
Control Group ◽  
High Degree ◽  
Rheumatoid Arthritis Activity ◽  
Active Disease

Bacground/Aim. Experimental in vitro and in vivo investigations in a mouse model have proved that TNF-alpha, IL-12, IL- 15 and IL-18 participate in the pathogenesis of erosive inflammatory arthritis. The aim of this research was to determine the clinical significance of cytokines in the evaluation of the activity of rheumatoid arthritis (RA). Methods. Inside a 4-year period we followed-up 64 patients with RA as newly occurred or in the phase of worsening. We observed the clinical manifestation of the disease upon wluch we divided the patients in to 3 groups: the patients with low active RA, patients with moderate active RA, and the patients with wild active RA. The control group (n = 25 patients) included the patients with osteoarthrosis (OA), and arthritis of the knee. In the samples of serum of all of the patients the concentrating of cytokines TNF-alpha, IL-12, IL-15, and IL-18 were determined using the immunoenzymatic methods in mice for human interleukines. By comparing the concentrations in 30 patients with the high, 14 patients with moderate, and 20 patients with the mild activity of RA it was determined that the patients with the high degree of the disease activity, had significantly high (p < 0.01; p < 0.05) concentrations of the examined cytokines in blood and synovial fluid as compared to the patients with the moderate and mild active disease. There was a relationship (p < 0.01) between the concentrations of cytokines in blood and synovial fluid with the quantity of the Disease Activity Score in 28 joints. Conclusions. Cytokines concentrations could be good indicators of the degree of the general activity of RA. This research could contribute to the interpretation of insufficiently well known views of the pathogenesis role and significance of citokines in an active disease.

THU0234 INFLUENCE OF THE SPLICING MACHINERY DYSREGULATION ON THE PATHOGENESIS OF LUPUS AND THE KIDNEY INVOLVEMENT

2020 ◽  
Vol 79 (Suppl 1) ◽  
pp. 344.1-344
Author(s):  
C. Lopez-Pedrera ◽  
M. Luque-Tévar ◽  
A. Ibañez-Costa ◽  
A. M. Patiño-Trives ◽  
M. A. Aguirre ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Lupus Nephritis ◽  
Renal Involvement ◽  
Research Support ◽  
Altered Expression ◽  
Kidney Involvement ◽  
Inflammatory Profile ◽  
Potential Biomarkers ◽  
Splicing Machinery

Background:Objectives:The aim of this study was to evaluate whether alterations in the splicing machinery of immune cells from Systemic Lupus Erythematosus (SLE) patients could influence the development and activity of the disease and the kidney involvement.Methods:The study was conducted in 41 SLE patients and 34 healthy donors (HD). Monocytes, lymphocytes and neutrophils were purified by immunomagnetic selection. Then, selected elements of the splicing machinery and a set of genes related to inflammation, renal and cardiovascular disease (including interleukins, adipocytokines, chemokines, and oxidative stress markers, among others) were evaluated using a microfluidic qPCR array (Fluidigm). Besides, the inflammatory profile in plasma, including the analysis of 27 proteins, was evaluated by using the Bioplex assay. In parallel, an extensive clinical/serological evaluation was performed; comprising disease activity and cardiovascular and renal involvement along with autoantibodies, complement factors, and acute phase reactants. Correlation and association studies and logistic models among those clinical and analytical parameters were developed. Mechanistic in vitro studies were carried out by incubation of HD-leukocytes with anti-dsDNA-IgG purified from SLE patients and changes promoted in both, splicing machinery and leukocyte inflammatory profile, were assessed.Results:A significantly altered expression of spliceosome components was found in all the leukocyte subsets: 27, 12 and 11 components were differentially expressed in monocytes, lymphocytes and neutrophils, respectively, in SLE patient’s vs HD. In parallel, a number of genes codifying for proteins involved in inflammation, fatty acid metabolism, oxidative stress and migration were found altered and correlations with spliceosome components were further identified. Besides, those statistical analyses demonstrated multiple links among altered spliceosome components and the clinical profile of these patients, such as the activity of the disease (SLEDAI), the occurrence of obstetric complications and the presence of arterial hypertension. Logistic regression and ROC curve analyses identified a signature composed in each leukocyte subset by 3 altered spliceosome components that could differentiate between SLE and HDs. Remarkably, the levels of a high number of those altered components were associated to the presence of lupus nephritis (LN). Moreover, ROC curve analyses allowed to identify several cell-specific spliceosome components as potential biomarkers of renal disease. In patients with LN we could also identify a distinctive inflammatory profile in plasma in relation to patients without renal involvement, which further correlated with the altered expression of a number of spliceosome components in each leukocyte subset. Lastly, the in vitro treatment of HD leukocytes with anti-dsDNA promoted the alteration of several spliceosome components found also altered in vivo in SLE lymphocytes, monocytes and neutrophils.Conclusion:1)The splicing machinery is greatly altered in leukocytes from SLE patients, regulated, at least partially, by anti-dsDNA antibodies and closely related to the activity of the disease, including obstetrical complications, hypertension and lupus nephritis. 2)Specific components of the spliceosome in SLE leukocytes subsets might be used as potential biomarkers to typify the disease, particularly kidney involvement.Acknowledgments :Funded by ISCIII (PI18/00837 and RIER RD16/0012/0015) co-funded with FEDER.Disclosure of Interests: :Chary Lopez-Pedrera Grant/research support from: ROCHE and Pfizer., María Luque-Tévar: None declared, Alejandro Ibañez-Costa: None declared, Alejandra M. Patiño-Trives: None declared, Maria A Aguirre: None declared, Pérez Sánchez Laura: None declared, Maria del Carmen Abalos-Aguilera: None declared, Iván Arias de la Rosa: None declared, Pedro Seguí Azpilcueta: None declared, Mario Espinosa: None declared, Nuria Barbarroja Puerto Grant/research support from: ROCHE and Pfizer., Speakers bureau: ROCHE and Celgene., Eduardo Collantes-Estévez Grant/research support from: ROCHE and Pfizer., Speakers bureau: ROCHE, Lilly, Bristol and Celgene., Justo Pastor Castaño Fuentes: None declared, Raúl Miguel Luque Huertas: None declared, Carlos Perez-Sanchez: None declared

scholarly journals POS0419 ABERRANT SPLICEOSOME AND ALTERED EXPRESSION OF IFN-RESPONSE RELATED GENES ARE HALLMARKS OF MONOCYTES FROM LUPUS PATIENTS WITH RENAL DISEASE

2021 ◽  
Vol 80 (Suppl 1) ◽  
pp. 438.2-439
Author(s):  
A. M. Patiño-Trives ◽  
C. Perez-Sanchez ◽  
A. Ibañez-Costa ◽  
M. Luque-Tévar ◽  
M. D. C. Abalos-Aguilera ◽  
...  
Keyword(s):  
Renal Disease ◽  
Renal Involvement ◽  
Aberrant Expression ◽  
Altered Expression ◽  
Inflammatory Status ◽  
Inflammatory Profile ◽  
Active Release ◽  
Splicing Machinery

Background:To date, novel mechanisms such as the involvement of splicing machinery components in lupus nephropathy and its interplay with the transcriptome in innate immune cells have not been evaluated.Objectives:1- To identify altered transcriptomic signatures associated with the immune response of monocytes from SLE patients and its association with clinical features. 2- To evaluate the role of the spliceosome linked to the transcriptomic profile of SLE monocytes. 3- To analyze mechanistically the impact of anti-dsDNA antibodies (Ab) and the modulation of the spliceosome in the SLE monocytes activity.Methods:Sixty SLE patients and forty healthy donors (HD) were included in the study. Infiltration rate of myeloid cells and its association with clinical features were analyzed in kidney biopsies by Immunohistochemistry. In parallel, circulating monocytes were purified from peripheral blood by immune-magnetic selection. The expression of a set of 770 genes related to autoimmune/inflammatory diseases was evaluated using NanoString Technologies. The levels of the main 45 components of the splicing machinery were further analyzed in these samples using a microfluidic qPCR array (Fluidigm). An extensive clinical/serological evaluation was also performed, comprising disease activity, renal involvement parameters, autoAb profile, and the systemic inflammatory status (27-plex Assay). Finally, in vitro studies involving anti-dsDNA-IgG Ab treatment and over/down-expression of splicing machinery components were carried out to analyze their effects in the monocyte activity.Results:Infiltration of CD68 expressing cells was confirmed in kidney biopsies and associated with parameters of kidney failure (C3/C4, chronic index), highlighting the key role of the myeloid compartment in lupus nephropathy. Gene expression profiling recognized 156 genes differentially expressed in SLE monocytes compared with HDs, including 87 genes up-regulated and 69 down-regulated. Functional analysis showed that most dysregulated genes were associated with the IFN response (i.e. IFIT1, IFI44, IFI44L, RSAD2). In parallel, the altered expression of 27 spliceosome components was demonstrated in SLE monocytes compared with HD, including 3 up-regulated and 24 down-regulated. Correlation studies demonstrated that the aberrant expression of splicing machinery components was linked to the altered interferon signature and the plasma inflammatory profile. This aberrant profile at molecular level was associated with the disease activity status, anti-dsDNA positivity and C3/C4 levels. Interestingly, SLE patients with renal disease displayed a simultaneous alteration of both, the IFN and the spliceosome signatures in monocytes, along with an enlarged pro-inflammatory profile in plasma. Logistic regression models that integrated the concomitant alteration of some splicing machinery components and IFNs genes identified lupus nephritis patients with high accuracy. Mechanistic studies showed that in vitro treatment of monocytes from HDs with anti-dsDNA promoted a concomitant deregulation of the IFN signature and the expression of several spliceosome components (i.e. PTB, RBM17, RNU6ATAC). Finally, the over/down-expression of selected spliceosome components (PTB and RBM17) in monocytes from SLE patients reduced the active release of inflammatory cytokines and their adhesion capacity.Conclusion:1) Monocytes from SLE patients with renal involvement exhibit a remarkable alteration of genes associated with the IFN response, further linked with the aberrant expression of several splicing machinery components. 2) Anti-dsDNA promoted the dysregulation in monocytes of both the IFN and spliceosome signatures, along with an active release of pro-inflammatory mediators. 3) The modulation of key splicing components in monocytes from SLE patients reduce their pro-inflammatory status and migration capacity. Ongoing studies may provide novel biomarkers and therapeutic tools to treat lupus nephropathy.Acknowledgements:Funded by ISCIII, PI18/00837 and RIER RD16/0012/0015 co-funded with FEDERDisclosure of Interests:None declared

scholarly journals Optimized “In Vitro” Culture Conditions for Human Rheumatoid Arthritis Synovial Fibroblasts

2014 ◽  
Vol 2014 ◽  
pp. 1-9 ◽  
Author(s):  
Claudia Casnici ◽  
Donatella Lattuada ◽  
Noemi Tonna ◽  
Katia Crotta ◽  
Claudio Storini ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Synovial Fluid ◽  
Proinflammatory Cytokines ◽  
Synovial Fibroblasts ◽  
In Vitro Studies ◽  
Tnf Alpha ◽  
Purinergic Signalling ◽  
Human Rheumatoid Arthritis ◽  
Cells Cultured

The composition of synovial fluid in rheumatoid arthritis (RA) is complex and strongly influences the microenvironment of joints and it is an inseparable element of the disease. Currently, “in vitro” studies are performed on RA cells cultured in the presence of either recombinant proinflammatory cytokines-conditioned medium or medium alone. In this study, we evaluated the use of synovial fluid, derived from RA patients, as optimal culture condition to perform “in vitro” studies on RA synovial fibroblasts. We observed that synovial fluid is more effective in inducing cell proliferation with respect to TNF-alpha or culture medium alone. Spontaneous apoptosis in fibroblasts was also decreased in response to synovial fluid. The expression of proinflammatory cytokines in the presence of synovial fluid was significantly elevated with respect to cells cultured with TNF-alpha or medium, and the overall morphology of cells was also modified. In addition, modulation of intracellular calcium dynamics elicited in response to synovial fluid or TNF-alpha exposure is different and suggests a role for the purinergic signalling in the modulation of the effects. These results emphasize the importance of using RA synovial fluid in “in vitro” studies involving RA cells, in order to reproduce faithfully the physiopathological environmental characteristic of RA joints.

scholarly journals Impaired microRNA processing in neutrophils from rheumatoid arthritis patients confers their pathogenic profile. Modulation by biological therapies

Haematologica ◽  
2020 ◽  
Vol 105 (9) ◽  
pp. 2250-2261 ◽  
Author(s):  
Ivan Arias De la Rosa ◽  
Carlos Perez-Sanchez ◽  
Patricia Ruiz-Limon ◽  
Alejandra Patiño-Trives ◽  
Carmen Torres-Granados ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Synovial Fluid ◽  
Interleukin 6 ◽  
Mirna Biogenesis ◽  
Biological Therapies ◽  
Protein Antigens ◽  
Mrna Targets ◽  
Healthy Donors ◽  
Inflammatory Profile ◽  
Citrullinated Protein

The aim of this study was to investigate the microRNA (miRNA) expression pattern in neutrophils from rheumatoid arthritis (RA) patients and its contribution to their pathogenic profile and to analyze the effect of specific autoantibodies or inflammatory components in the regulation of miRNA in RA neutrophils and its modulation by biological therapies. Neutrophils were isolated from paired peripheral blood (PB) and synovial fluid samples of 40 patients with RA and from PB of 40 healthy donors. A miRNA array was performed using nCounter technology. Neutrophils from healthy donors were treated in vitrowith antibodies to citrullinated protein antigens isolated from RA patients and tumor necrosis factor-a (TNF-a) or interleukin-6. A number of cytokines and chemokines were analyzed. In vitro treatments of RA-neutrophils with tocilizumab or infliximab were carried out. Transfections with pre-miRNA and DICER downregulation experiments were further performed. RA-neutrophils showed a global downregulation of miRNA and genes involved in their biogenesis, alongside with an upregulation of various potential mRNA targets related to migration and inflammation. Decreased levels of miRNA and DICER correlated with autoimmunity, inflammation and disease activity. Citrullinated protein antigens and TNF-a decreased the expression of numerous miRNA and their biogenesis-related genes, increasing their potential mRNA targets. Infliximab reversed those effects. Transfections with pre-miRNA-223, -126 and -148a specifically modulated genes regulating inflammation, survival and migration whereas DICER depletion influenced the inflammatory profile of neutrophils. Taken together RA-neutrophils exhibited a global low abundance of miRNA induced by autoantibodies and inflammatory markers, which potentially contributed to their pathogenic activation. miRNA biogenesis was significantly impaired in RAneutrophils and further associated with a greater downregulation of miRNA mainly related to migration and inflammation in synovial fluid neutrophils. Finally, anti-TNF-a and anti-interleukin-6 receptor treatments can modulate miRNA levels in the neutrophils, minimizing their inflammatory profile.

scholarly journals The correlation of syndecan-4 with disease activity and serological characteristics of rheumatoid arthritis

2021 ◽  
Author(s):  
Juan Zhao ◽  
Xia Ye ◽  
Zhuoli Zhang
Keyword(s):  
Rheumatoid Arthritis ◽  
Synovial Fluid ◽  
Disease Activity ◽  
Rheumatoid Factor ◽  
Synovial Tissue ◽  
Synovial Fibroblasts ◽  
Healthy Controls ◽  
Immunosorbant Assay ◽  
Enzyme Linked Immunosorbant Assay

Abstract [Objectives]To investigate the expression of syndecan-4 in serum, synovial fluid (SF) and synovium in rheumatoid arthritis (RA) by comparing with osteoarthritis (OA) patients, and to analyze the correlation of syndecan-4 with disease activity of RA .[Methods]Syndecan-4 in sera of 60 RA patients, 20 OA patients, 20 healthy controls, and in paired SF of 23 RA patients were tested by enzyme linked immunosorbant assay (ELISA). The expressions of syndecan-4 in synovium of 5 RA patients and 5 OA patients were detected by immunohistochemistry. The expressions of syndecan-4 of cultured synovial fibroblasts from RA and OA patients were detected by immunofluorescence. The correlation between serum syndecan-4 concentration and disease activity were analyzed in 60 RA patients.[Results]The serum syndedcan-4 concentration was significantly higher in RA patients than in OA patients and healthy controls, and was higher in rheumatoid factor (RF)-positive RA patients than in RF-negative ones. Syndecan-4 concentration in SF of RA patients was comparable with OA patients. Syndecan-4 expression in synovial tissue was similar between RA and OA patients. The syndecan-4 concentration was significantly lower in synovial fluid than in serum, either in RA or in OA patients. The serum and SF syndecan-4 concentrations were both positively correlated with RA disease activity scores.[Conclusion]The serum syndecan-4 concentration is higher in RA patients than in OA patients, and significantly higher in RF-positive RA patients than in RF-negative ones. The serum and SF syndecan-4 concentrations were both positively correlated with disease activity of RA patients.

scholarly journals IL-40: A New B Cell-Associated Cytokine Up-Regulated in Rheumatoid Arthritis Decreases Following the Rituximab Therapy and Correlates With Disease Activity, Autoantibodies, and NETosis

2021 ◽  
Vol 12 ◽  
Author(s):  
Adela Navrátilová ◽  
Lucie Andrés Cerezo ◽  
Hana Hulejová ◽  
Viktor Bečvář ◽  
Michal Tomčík ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Synovial Fluid ◽  
Disease Activity ◽  
B Cell ◽  
Synovial Tissue ◽  
Lupus Erythematosus ◽  
Synovial Fibroblasts ◽  
Tissue Destruction ◽  
Humoral Immune ◽  
B Cell Homeostasis

BackgroundInterleukin 40 (IL-40) is a newly identified B cell-associated cytokine implicated in humoral immune responses and B cell homeostasis. As B cells play a pivotal role in autoimmunity, we investigated the function of IL-40 in rheumatoid arthritis (RA).MethodsIL-40 expression was determined in the synovial tissue from RA and osteoarthritis (OA) patients. IL-40 was analysed in the serum/synovial fluid of patients with RA (n=50), systemic lupus erythematosus (SLE, n=69), OA (n=44), and healthy controls (HC, n=50). We assessed the changes of IL-40 levels in RA patients following the B cell depletion by rituximab (n=29) or after the TNF inhibition by adalimumab (n=25). We examined the relationship between IL-40, disease activity, autoantibodies, cytokines, and NETosis markers. Effect of IL-40 on synovial fibroblasts was determined.ResultsIL-40 was overexpressed in RA synovial tissue, particularly by synovial lining and infiltrating immune cells. The levels of IL-40 were up-regulated in the synovial fluid of RA versus OA patients (p&lt;0.0001). Similarly, IL-40 was increased in the serum of RA patients compared to HC, OA, or SLE (p&lt;0.0001 for all) and decreased after 16 and 24 weeks (p&lt;0.01 and p&lt;0.01) following rituximab treatment. No significant effect of adalimumab on IL-40 was observed. IL-40 levels in RA patients correlated with rheumatoid factor-IgM and anti-cyclic citrullinated peptides (anti-CCP) in the serum (p&lt;0.0001 and p&lt;0.01), as well as in the synovial fluid (p&lt;0.0001 and p&lt;0.001). Synovial fluid IL-40 was also associated with disease activity score DAS28 (p&lt;0.05), synovial fluid leukocyte count (p&lt;0.01), neutrophil attractants IL-8 (p&lt;0.01), MIP-1α (p&lt;0.01), and markers of neutrophil extracellular traps externalization (NETosis) such as proteinase 3 (p&lt;0.0001) and neutrophil elastase (p&lt;0.0001). Synovial fibroblasts exposed to IL-40 increased the secretion of IL-8 (p&lt;0.01), MCP-1 (p&lt;0.05), and MMP-13 (p&lt;0.01) compared to the unstimulated cells.ConclusionsWe show the up-regulation of IL-40 in RA and its decrease following B cell depleting therapy. The association of IL-40 with autoantibodies, chemokines, and markers of NETosis may imply its potential involvement in RA development. Moreover, IL-40 up-regulates the secretion of chemokines and MMP-13 in synovial fibroblasts, indicating its role in the regulation of inflammation and tissue destruction in RA.

scholarly journals AB0043 EFFECT OF ANTI-INFLAMMATORY MEDICATION ON INTERACTION OF SYNOVIAL FIBROBLASTS WITH MACROPHAGES IN RHEUMATOID ARTHRITIS

2021 ◽  
Vol 80 (Suppl 1) ◽  
pp. 1054.1-1054
Author(s):  
M. Schmeller ◽  
M. Diller ◽  
R. Hasseli ◽  
A. Knothe ◽  
S. Rehart ◽  
...  
Keyword(s):  
Rheumatoid Arthritis ◽  
Mononuclear Cells ◽  
Joint Inflammation ◽  
Synovial Fibroblasts ◽  
Therapeutic Effects ◽  
Synergistic Activity ◽  
M1 Macrophages ◽  
Healthy Donors ◽  
Proinflammatory Activity ◽  
Anti Inflammatory

Background:One of the key mechanisms in the pathogenesis of rheumatoid arthritis (RA) is the interaction of macrophages and synovial fibroblasts during joint inflammation. Increased synergistic proinflammatory activity of both cell types leads to the release of high levels of proinflammatory cytokines, especially of interleukin-6 (IL-6), and of matrix degrading enzymes. If this mechanism is uncontrolled, progressive destruction of articular cartilage and bone will take place.In active disease, immediate anti-inflammatory treatment with glucocorticoids is usually replaced by disease-modifying anti-rheumatic drugs (DMARDS), especially by methotrexate (MTX) and biologics such as TNF-α- or IL-6-inhibitors. This led to great improvements in prognosis and outcome for RA patients. However, about 40% of patients experience no remission or suffer from side effects of medication. To optimize established substances and to develop new treatment strategies, it is necessary to understand the mechanisms underlying the limited therapeutic effects.Objectives:Evaluation of the effect of prednisolone, MTX, adalimumab, tocilizumab on IL-6 secretion by RA synovial fibroblasts (RASF) and macrophages.Methods:RA synovium was used for RASF isolation. Peripheral blood mononuclear cells (PBMCs) were isolated from blood of healthy donors and RA patients by using Ficoll© medium followed by density gradient centrifugation. Mononuclear cells were seeded on six well plates (6x10^6/well) and incubated for one week. Then they were stimulated with Interferon-у (20 ng/ml) and LPS (50 ng/ml) for 48h to initiate differentiation into proinflammatory M1 macrophages. The M1 macrophages were co-cultured with RASF (100.000/well) and different treatments added (prednisolone: 10, 25, 50, 75, 100 nM, 1 µM; adalimumab: 100, 500 µg/ml; tocilizumab: 1, 5 µg/ml; MTX: 0,5, 1, 5, 10, 100 nM, 1µM). After 24h culture supernatants were collected and IL-6- and TNFα-ELISAs were performed.Results:IL-6 concentrations of untreated controls were comparable, regardless whether M1 macrophages from healthy donors or RA-patients were used for co-culture. Prednisolone reduced co-culture-induced IL-6 up to 56% (p<0.001) in co-culture of RASF and M1 macrophages of healthy donors and up to 60% (p<0.001) in co-culture of RASF and RA M1 macrophages. Adalimumab reduced IL-6 up to 28% (p<0.05) in M1 of healthy donors and up to 45% (p<0.01) in RA M1 macrophage co-cultures. A minor reduction by 10-20% of IL-6 was observed with tocilizumab and no significant effect could be achieved after treatment with MTX.Conclusion:Prednisolone and adalimumab clearly decrease but do not eliminate proinflammatory synergistic activity of RASF and M1 macrophages. These results confirm the clinical observation, that there is a large number of RA-patients that independent of anti-inflammatory treatment still suffer from low-level joint inflammation.The synergistic proinflammatory activity of M1 macrophages and RASF seems to be a complex and multifactorial mechanism that is difficult to eliminate by a single treatment substance. Since it is one of the key mechanisms in RA pathogenesis, there is a critical need to investigate how therapy effects could be optimized. This study confirmed RASFs as one of the leading effector cells of increased synergistic proinflammatory activity, thus underlining their promising role as a treatment target in rheumatoid arthritis.Disclosure of Interests:None declared

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