BackgroundOur group have recently shown that (Sema)phorin3B is reduced in RA patients and plays a protective role in an arthritis mouse model, through the reduction of inflammatory mediators and the synovial fibroblast invasiveness and was associated with a reduction of CD68 positive synovial macrophages (Igea, 2022). On the other hand, MerTK is a receptor protein kinase involved in the resolution of inflammation and plays a protective role in arthritis mice models (Waterborg, 2018). A recent study has shown that a population of synovial tissue macrophages characterized by the expression of MerTK are associated with remission maintenance and with an anti-inflammatory phenotype of RA FLS (Alivernini, 2020).ObjectivesThe aim of this study is to determine the role of Sema3B in the phenotypic characteristics of RA macrophages and the implication of MerTK.MethodsPeripheral blood monocytes from RA patients (n=10) were differentiated into macrophages by culturing in the presence of IFN-γ (10 ng/mL) for 6 days. Afterwards, macrophages were stimulated for 24 h with LPS (10 ng/mL), Sema3B (200 ng/mL) or the combination of both mediators. The expression of pro- and anti-inflammatory mediators was determined by quantitative PCR (qPCR) and ELISA. The expression of MerTK and macrophage surface markers was measured by flow cytometry.ResultsSema3B did not modulate the macrophage expression of pro-inflammatory mediatorsIL1B, IL6, IL12B, IL23, TNF, CCL2, CXCL10andCD86, but significantly reduced the LPS-induced expression of these mediators (Figure 1A), as well as the protein secretion of IL6, IL12p70 and TNF. In addition, Sema3B alone, but not in combination with LPS, significantly induced the gene and protein expression of MerTK and the secretion of Resolvin D1, a MerTK-mediated lipid involved in resolution of inflammation (Figure 1B). Moreover, Sema3B reduced the expression of the M1 marker CD64, while induced the expression of the M2 marker CD163.Figure 1.Sema3B enhances anti-inflammatory activity in macrophages of RA patients (n=6). (A) Significative reduction of the genetic expression of pro-inflammatory mediators. (B) Significative increase ofMERTKand ResolvinD1. Macrophages differentiated from monocytes of RA patients during 6 days (IFN-γ 10 ng/mL) and stimulated with Sema3B (200 ng/ml), LPS (10 ng/mL) and their combination for 24 hours. Symbols represent individual patients; bars show the mean ± SEM. *P < 0.05 and **P < 0.01.ConclusionOur data demonstrate that Sema3B modulates the macrophage phenotype of RA macrophages, inducing a skewing towards an anti-inflammatory/pro-resolving phenotype, likely in a MerTK-dependent manner. Therefore, here we identified a new mechanism involved in the protective role of Sema3B in RA pathogenesis.References[1] Alivernini, S.et al. Nat. Med.26, 1295–1306 (2020).[2] Igea, A.et al. Arthritis Rheumatol.74(6), 972–983(2022).[3] Waterborg CEJ, et al.Front. Immunol. 9:742 (2018).AcknowledgementsAll patients involved in this study.Disclosure of InterestsNone Declared.
BackgroundSeveral studies have shown that different semaphorin family members are involved in the pathogenesis of rheumatoid arthritis (RA). On one hand, our group has demonstrated that (Sema)phorin 3B and Sema3F are reduced in RA patients and play a protective role. On the other hand, Sema4A and Sema4D are increased in RA patients and are associated with inflammatory processes (Garcia, 2019; Igea, 2022).ObjectivesThe aim of this study is to determine the role of Sema4B in the pathogenesis of RA.MethodsGene expression of Sema4B was obtained from the gene expression array available in Gene Expression Omnibus-NCBI (GSE77298). Fibroblasts-like synoviocytes from RA patients (RA-FLS) (n=8) were stimulated 4 and 24 h with Sema4B (200ng/mL), TNF (10 ng/mL) or the combination of both. Peripheral blood monocytes from RA patients (n=12) were differentiated into M1 macrophages by culturing in the presence of IFN-γ (10 ng/mL) for 6 days. Afterwards, macrophages were stimulated 24 h with Sema4B (200ng/mL), LPS (10 ng/mL) or the combination of both. The expression of inflammatory mediators was determined by quantitative PCR (qPCR) and ELISA. Viability and migration of FLS were determined using calcein assays and wound closure assays, respectively.ResultsSema4B expression was significantly higher in the synovial tissue and FLS of RA patients compared to healthy controls (HC) and osteoarthritis patients (OA), respectively. A significantly higher expression ofSEMA4Bin the synovium and FLS of RA patients compared to, respectively, was found. Interestingly, TNF stimulation induced the expression ofSEMA4Bby RA-FLS. Functional studies showed that Sema4B did not affect the viability of FLS but increased their migratory capacity. Moreover, Sema4B alone did not induce the expression of inflammatory mediators (data non shown), but significantly enhanced the TNF-induced expression ofIL6, IL8, TNF, CCL2andMMP3(Figure 1A) and the secretion of TNF. Finally, Sema4B alone did not modulate the expression of inflammatory mediators in macrophages, but significantly enhanced the LPS-mediated expression ofTNF,CCL2, andMMP1(Figure 1B), as well as the TNF protein secretion.Figure 1.Sema4B enhances pro-inflammatory activity in FLS (n=6) and macrophages (n=10) of RA patients. (A) Significative increment of the genetic expression of pro-inflammatory mediators in RA-FLS stimulated with Sema4B (200 ng/ml), TNF (10 ng/mL) and their combination for 24 hours. (B) Significative increase of the genetic expression of pro-inflammatory mediators in M1 macrophages. Macrophages differentiated from monocytes of RA patients during 6 days (IFN-γ 10 ng/mL) and stimulated with Sema4B (200 ng/ml), LPS (10 ng/mL) and their combination for 24 hours. Symbols represent individual patients; bars show the mean ± SEM. *P < 0.05.ConclusionOur data demonstrate that, in an inflammatory context, Sema4B induces FLS migration and the production of inflammatory mediators by FLS and macrophages. These results suggest that Sema4B is involved in inflammatory processes observed in the RA synovium and might be a potential therapeutic target in the treatment of RA.References[1]Garcia S. Role of Semaphorins in Immunopathologies and Rheumatic Diseases.Int J Mol Sci. 2019;20(2):374. Published 2019 Jan 16. doi:10.3390/ijms20020374[2]Igea A, Carvalheiro T, Malvar-Fernández B, et al. Central Role of Semaphorin 3B in a Serum-Induced Arthritis Model and Reduced Levels in Patients With Rheumatoid Arthritis.Arthritis Rheumatol. 2022;74(6):972-983. doi:10.1002/art.42065AcknowledgementsAll patients involved in this study.Disclosure of InterestsNone Declared.
BackgroundEndothelial cell (EC) dysfunction is a hallmark of Systemic Lupus Erythematosus (SLE) and has been generally accepted to be one of the important factors contributing to the higher risk of thrombosis and atherosclerotic events observed in SLE patients. Although the presence of traditional factors (smoking, diabetes, increased age, obesity) and the presence of autoantibodies are associated with atherosclerosis and thrombotic events, they do not completely explain the higher risk of these events in SLE, suggesting the existence of other mechanism/factors.Tie2 is a tyrosine kinase receptor essential for vascular development and blood vessel remodeling through interaction with its ligands angiopoietin-1 (Ang-1) and Ang-2. In homeostatic conditions, both Ang-1 and Ang-2 activate Tie2 signaling and induce vascular stabilization in a Tie1-dependent manner. However, inflammatory processes induce Tie1 cleavage, leading to the inhibition of Ang-1-induced Tie2 activation, and to the increase of Ang-2 now acting as a Tie2 antagonist, culminating in vascular dysfunction and EC activation [1,2]. Importantly, this process has been implicated in both atherosclerosis and thrombosis.ObjectivesAs type I Interferons (IFN-α and IFN-β) are key cytokines in the pathogenesis of SLE, the aim of this study is to determine whether these cytokines induce Tie2 signaling-mediated endothelial cell dysfunction.MethodsSerum levels of Ang-1, Ang-2 and sTie1 in SLE patients (n=48) and healthy control (HC, n=29) were measured by ELISA. Human Umbilical Vein EC (HUVEC) were stimulated with SLE serum (20%), IFN-α and IFN-β (1000 IU) for 5, 15, 30 min and 1, 2, 4, 6, 8, 12, 24, 48 and 72 hours and mRNA and protein expression of Ang-1, Ang-2, Tie1 and Tie2 were determined by quantitative PCR (qPCR) and ELISA, respectively. The phosphorylation of Tie2 was determined byWestern Blotand HUVEC viability by calcein assay. The angiogenic capacity was measured by tube formation assay. Silencing assays were performed with siRNAs addressed to IFNAR1 and Tie1 receptors.ResultsType I IFNs, mainly IFN-β, significantly reducedTIE1andTIE2levels. IFN-β stimulation significantly increased the secretion of the Tie1 ectodomain (sTie1). Both IFNs significantly reduced protein secretion of Ang-1 after 24h of stimulation. Also, IFN-β induced Ang-2 secretion at early time points (<4h). Furthermore, IFN-α and IFN-β stimulation reduced Tie2 activation (Figure 1). Both type I IFNs significantly reduced the viability of HUVEC. SLE serum increased Ang-2 and sTie1 secretion levels in HUVEC at early time points (<1h). We found reduced levels of Ang-1 and elevated Ang-2 and sTie1 in SLE patients compared to HC. Also, IFN-β induced tube formation at short times (4h) and decreased at 24h. Remarkably, this effect was reversed by silencing the receptors Tie1 and IFNAR1.Figure 1.Ang-1, Ang-2, Tie1 and Tie2 mRNA expression and protein secretion in HUVEC stimulated with IFN-α or IFN-β (1000 IU/ml) for the indicated time points. Means and SEM are shown. * p<0.05, ** p<0.01 and **** p<0.0001. Also, representative immunoblot of Tie2 phosphorilation in HUVEC stimulated with IFN-α or IFN-β (1000 IU/ml) for 24 h is shown.ConclusionOur results demonstrate that type I IFNs play a relevant role in the stability of endothelial cells by inhibiting Tie2 signaling, suggesting that these processes may be implicated in the cardiovascular events observed in SLE patients.References[1]Kim, M.et al.Journal of Clinical Investigation126,3511–3525 (2016).[2]Saharinen, P., Eklund, L. & Alitalo, K.Nature Reviews Drug Discovery16,635–661 (2017).Acknowledgements:NIL.Disclosure of InterestsNone Declared.
BackgroundBelimumab (BLM) is a recombinant human IgG-1λ monoclonal antibody that inhibits B-cell activating factor. It is approved for the treatment of systemic lupus erythematosus (SLE). It is effective in reducing disease activity, flares, damage prevention and also as a steroid-sparing agent. A treat to target (T2T) approach in the care of SLE patients is important in terms of improving short and long-term outcomes.ObjectivesTo evaluate belimumab (BLM) effectiveness in SLE patients from a Spanish multicenter registry.MethodsA longitudinal retrospective multicenter cohort including SLE patients treated with belimumab from 18 Spanish rheumatology departments. Demographic, clinical and serological data were collected at baseline, 6, 12 and in the last visit available. Changes in SLEDAI-2K; LLDAS and DORIS 2021 states and global response according to physician criteria were compared between visits, as well as changes in damage and glucocorticoids used. T-test was used for numerical variables and the Fisher’s test for categorical variables.Results324 patients were included: 295 (91%) females with a mean (±SD) age of 42.4 (±12.9) years. Mean follow-up was 3,8 (±2.7) years and mean time with BLM was 2.7 (±2.4) years. At baseline, mean SLEDAI-2K was 10.4 (±5.25), 68.2% had elevated anti-double-stranded DNA (anti-dsDNA) antibodies and 69.8% had complement consumption. BLM was initiated concomitant to other DMARD in 67.9% (n=220) of patients.Mean reduction in SLEDAI-2K score was 5.0 (± 5.1), 6.1 (±5.5) and 7.13 (±5.3) points at 6, 12 months and in the last visit, respectively (p<0.05 for all comparisons). Rates of achievement of LLDAS, DORIS and clinical response according to physician criteria, significantly increased from baseline to 6, 12 months, and to the last visit (Table 1). Anti-dsDNA antibodies and inflammatory markers (ESR, CRP), significantly decreased from baseline to 6, 12 months and in the last visit. Complements increased over the follow up but without statistical significance.A total of 107 (45,9%) patients discontinued GC. At 6 months, 58.9% (n=155) of patients reduced the dose of GC with respect to baseline and 72.8% (n=131) of patients did it at the last visit.Mean (±SD) prednisone dose was significantly reduce over the visits: 12.3 (±12.16); 7.42 (±5.36); 5.8 (±4.42) and 4.7 (±3.7) mg/day at baseline, 6 and 12 months and in the last visit, respectively. Median (IQR) SDI score at the end of the observation period did not change from baseline visit: 0 (0-1) and 0 (0-1), respectively (p=0.97). Neither were changes observed in the percentage of patients with damage between the beginning and the end of the observation period: at baseline 47.5% (n=152) patients presented damage and, in the last visit, 45.6% (n=99).Table 1.Clinical response and changes in GC dose.Disease activity Mean (±SD) or number (%); n=324Baseline6 months12 monthsLast visitSLEDAI reduction; mean (±SD)10 (± 5.25)5.0 (± 5.1) *6.1 (± 5.5) *7.13 (± 5.3) *LLDAS6 (1.9%)137(45.8%) *145 (62%) *177 (73.1%) *DORIS6 (1.9%)72 (24%) *85 (36.3%) *127 (52.5%) *Response according to physician212 (65.4%) *185 (57.1%) *165 (50.9%) *Number of swollen joints; mean (±SD)3.3 (±3.6)1.2 (±2.8) *0.69 (±1.95) *0.55 (±1.82) *Prednisone dose (mg/day); mean (±SD)12.3 (± 12.16) *7.4 (± 5,36) *5.8 (± 4,42) *4.75 (±3.74) *SLEDAI:Systemic Lupus Erythematosus Disease Activity Index. cSLEDAI: clinical SLEDAI; LLDAS:Lupus Low Disease Activity State. DORIS:Definition of remission in SLE. *p<0.05Figure 1.Rates of therapeutic targets attained by patients in treatment with Belimumab.6 months12 monthsLast visitDiscontinued4574130Ongoing279250194ConclusionReal-world data of SLE patients confirm belimumab efficacy in real world, reducing clinical and serological activity in the short and medium-term. Add-on therapy with BLM leads to high rates of LLDAS and DORIS at 6 months, that continue increasing over time. BLM has an important GC sparing effect and prevents organ damage accrual. All these data shows that BLM is useful to achieve the therapeutic goals of a T2T strategy.REFERENCES:NIL.Acknowledgements:NIL.Disclosure of InterestsNone Declared.
BackgroundTreat to Target strategies are necessary in Systemic Lupus Erythematosus (SLE). They are difficult to establish due to the heterogeneity of the disease. The current definitions of Lupus Low Disease Activity State (LLDAS) according to the Asia Pacific Lupus Collaboration (APLC) and remission according to Definition of Remission in SLE (DORIS) 2021 are difficult to achieve and maintain over time.ObjectivesTo evaluate the concordance between the LLDAS and the clinical status according to the rheumatologist opinion and reasons of disagreement. To explore modifications in LLDAS definition that best fit with the expert´s opinion.MethodsProspective multicenter study of SLE patients (ACR 1997 Classification Criteria or clinical diagnosis by the physician) from seven Spanish Rheumatology Departments. Statistical analysis: descriptive cross-sectional (at the time of recruitment) analysis of the demographic and clinical characteristics, treatments; remission and LLDAS and the subjective evaluations of SLE activity by the rheumatologist. Analysis of the level of agreement between expert opinion and the definition of LLDAS and its modification were evaluated using Cohen’s kappa.ResultsDEMOGRAPHIC, DISEASE CHARACTERISTICS AND TREATMENTS. Five hundred and eight were included (92% women; mean age (±SD): 50.4 years (±13.7)). Mean SLEDAI-2K (±SD) was 2.84 (±3.31). A total of 406 (79.9%) patients presented SLEDAI-2K≤4. A total of 317 (74.1%) patients were on antimalarial treatment. Two hundred and twenty-two (43.7%) patients were on some type of immunosuppressive or biological therapy. More than half of patients were not taking glucocorticoids (n=310, 61%). A total of 38 patients (7.5%) were taking doses of prednisone higher than 7.5mg/day.REMISSION/LLDAS 267 (54.4%) patients were in remission and 304 (62.7%) patients were in LLDAS. According to the expert opinion of the rheumatologist, remission was the most frequent state considered (n=206, 41.6%); followed by low activity (n=153, 30.9%); serologically active (n=71, 14.3%); moderate activity (n=55, 11.1%) and high activity (n=10, 2%).AGREEMENT Overall agreement between expert opinion and the definition of LLDAS was 71.4 % with a Cohen’s kappa of 0.3. The majority of the cases (96.1%) that fulfilled the definition of LLDAS, were classified by the expert as remission, serologically active or low activity. Only 12 (3,9%) patients were classified by the expert as moderate or high activity. Of the patients that did not fulfill the definition of LLDAS, 126 out of 179 (70.4%) patients were classified by the expert as remission/low disease activity (Figure 1). The main reasons for discordance in the group that did not fulfill the definition of LLDAS were the presence of new clinical features compared to previous visit and a SLEDAI 2-K >4, in 74 (58.7%) and 59 (46.8%) patients, respectively. The LLDAS adjustment that meant a significant increase in the agreement was the exclusion of the comparative features with the previous visit, with an increase in the agreement to 82.6% (95% CI: 81.61-83.96%). The modification of prednisone to 5mg/daily dose, did not represent a significant change in agreement from the original definition.Figure 1.Comparison of LLDAS and expert opinionConclusionAt a given point in time, almost two thirds of SLE patients were in remission or in LLDAS. There is a good correlation between LLDAS and the physician’s opinion, particularly for those patients who fulfill LLDAS definition. However, the agreement is not so good for patients who don’t, these being excessively classified by the physician as remission or low activity. The main LLDAS items causing this disagreement were a SLEDAI-2K >4 and the appearance of different clinical manifestations from the previous evaluation. On the contrary, physician assessment by the PGA adequately fits the LLDAS definition. The modification of the LLDAS definition excluding the comparison with previous assessment increases the agreement with the expert opinion to 82.6%.Disclosure of InterestsNone declared
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