Biomarkers For Rheumatoid Arthritis Research Articles

Quercetin mitigates rheumatoid arthritis by inhibiting adenosine deaminase in rats

Rheumatoid arthritis (RA) is a chronic inflammatory joint disease characterized by synovial proliferation and bone destruction. Adenosine deaminase (ADA) is a key inflammatory enzyme that increases joint stiffness and pain in RA. In this study, we evaluated the in-silico, and in vivo inhibitory effect of quercetin isolated from Egyptian Fenugreek on ADA enzyme activity. We also determined the combinatorial effect of quercetin on methotrexate mediated anti-inflammatory efficacy and toxicity. In-silico molecular docking was conducted and confirmed in an in vivo RA rat model. The results showed that the inhibition constant of quercetin on joint ADA by docking and in-vitro was 61.9 and 55.5 mM, respectively. Therefore, quercetin exhibits anti-inflammatory effect in a rat RA model as evidenced by reducing the specific activity of ADA in joint tissues, lower jaw volume, enhance body weight, downregulate ADA gene expression, reduce levels of RA cytokines interleukin-1β, interleukin-6, tumor necrosis factor-α, also, rheumatoid factor, C-reactive protein, and anti-cyclic citrullinated peptide RA biomarker levels. These findings demonstrate that the purified quercetin has a promising anti-inflammatory effect against RA disease through its inhibitory effects on the ADA enzyme. Furthermore, isolated quercetin improved the anti-inflammatory efficacy of methotrexate, reduced its toxic effects by increasing antioxidant enzymes and reducing oxidative stress.

Functional Analysis of Autoantibody Signatures in Rheumatoid Arthritis.

For the identification of antigenic protein biomarkers for rheumatoid arthritis (RA), we conducted IgG profiling on high density protein microarrays. Plasma IgG of 96 human samples (healthy controls, osteoarthritis, seropositive and seronegative RA, n = 24 each) and time-series plasma of a pristane-induced arthritis (PIA) rat model (n = 24 total) were probed on AIT’s 16k protein microarray. To investigate the analogy of underlying disease pathways, differential reactivity analysis was conducted. A total of n = 602 differentially reactive antigens (DIRAGs) at a significance cutoff of p < 0.05 were identified between seropositive and seronegative RA for the human samples. Correlation with the clinical disease activity index revealed an inverse correlation of antibodies against self-proteins found in pathways relevant for antigen presentation and immune regulation. The PIA model showed n = 1291 significant DIRAGs within acute disease. Significant DIRAGs for (I) seropositive, (II) seronegative and (III) PIA were subjected to the Reactome pathway browser which also revealed pathways relevant for antigen presentation and immune regulation; of these, seven overlapping pathways had high significance. We therefore conclude that the PIA model reflects the biological similarities of the disease pathogenesis. Our data show that protein array analysis can elucidate biological differences and pathways relevant in disease as well be a useful additional layer of omics information.

Immunomodulatory matrix-bound nanovesicles mitigate acute and chronic pristane-induced rheumatoid arthritis

Rheumatoid arthritis (RA) is an autoimmune disease characterized by chronic inflammation and destruction of synovial joints affecting ~7.5 million people worldwide. Disease pathology is driven by an imbalance in the ratio of pro-inflammatory vs. anti-inflammatory immune cells, especially macrophages. Modulation of macrophage phenotype, specifically an M1 to M2, pro- to anti-inflammatory transition, can be induced by biologic scaffold materials composed of extracellular matrix (ECM). The ECM-based immunomodulatory effect is thought to be mediated in part through recently identified matrix-bound nanovesicles (MBV) embedded within ECM. Isolated MBV was delivered via intravenous (i.v.) or peri-articular (p.a.) injection to rats with pristane-induced arthritis (PIA). The results of MBV administration were compared to intraperitoneal (i.p.) administration of methotrexate (MTX), the clinical standard of care. Relative to the diseased animals, i.p. MTX, i.v. MBV, and p.a. MBV reduced arthritis scores in both acute and chronic pristane-induced arthritis, decreased synovial inflammation, decreased adverse joint remodeling, and reduced the ratio of synovial and splenic M1 to M2 macrophages (p < 0.05). Both p.a. and i.v. MBV reduced the serum concentration of RA and PIA biomarkers CXCL10 and MCP-3 in the acute and chronic phases of disease (p < 0.05). Flow-cytometry revealed the presence of a systemic CD43hi/His48lo/CD206+, immunoregulatory monocyte population unique to p.a. and i.v. MBV treatment associated with disease resolution. The results show that the therapeutic efficacy of MBV is equal to that of MTX for the management of acute and chronic pristane-induced arthritis and, further, this effect is associated with modulation of local synovial macrophages and systemic myeloid populations.

JAK1 signaling in dendritic cells promotes peripheral tolerance in autoimmunity through PD-L1-mediated regulatory Tcell induction.

Dendritic cells (DCs) induce peripheral Tcell tolerance, but cell-intrinsic signaling cascades governing their stable tolerogenesis remain poorly defined. Janus Kinase 1 (JAK1) transduces cytokine-receptor signaling, and JAK inhibitors (Jakinibs), including JAK1-specific filgotinib, break inflammatory cycles in autoimmunity. Here, we report in heterogeneous DC populations of multiple secondary lymphoid organs that JAK1 promotes peripheral Tcell tolerance during experimental autoimmune encephalomyelitis (EAE). Mice harboring DC-specific JAK1 deletion exhibit elevated peripheral CD4+ Tcell expansion, less regulatory Tcells (Tregs), and worse EAE outcomes, whereas adoptive DC transfer ameliorates EAE pathogenesis by inducing peripheral Tregs, programmed cell death ligand 1 (PD-L1) dependently. This tolerogenic program is substantially reduced upon the transfer of JAK1-deficient DCs. DC-intrinsic IFN-γ-JAK1-STAT1 signaling induces PD-L1, which is required for DCs to convert CD4+ Tcells into Tregs invitro and attenuated upon JAK1 deficiency and filgotinib treatment. Thus, DC-intrinsic JAK1 promotes peripheral tolerance, suggesting potential unwarranted DC-mediated effects of Jakinibs in autoimmune diseases.

Analysis of rheumatoid factor and acute phase proteins using microarrays in patients with rheumatoid arthritis

One of the biomarkers of biggest clinical importance in rheumatoid arthritis (RA) is rheumatoid factor (IgM RF). The rheumatoid factor has insufficient sensitivity and specificity, therefore, to increase the diagnostic information of the test, acute phase proteins were used as concomitant biomarkers. Using biological microchips, we measured IgM RF, C-reactive protein (CRP) and Serum amyloid protein A (SAA) in patients with RA (n = 60), ankylosing spondylitis (AS) (n=55), systemic lupus erythematosus (SLE) (n=20) and healthy donors (HD) (n=9). It was shown that the medians of IgM RF concentrations are significantly higher (p<0.01) in patients with RA compared to patients suffering from other diseases and healthy donors. CRP and SAA were also significantly increased (p<0.05) in patients with RA and AS compared with SLE and HD. It has been shown that the complex determination of three biomarkers in differentiating RA patients with the comparison group had a higher diagnostic sensitivity than the isolated determination of IgM RF, while the addition of SAA makes the greatest contribution to improving the diagnostic characteristics of the biomarker panel: the use of a logistic regression model based on IgM RF and SAA allowed to increase the diagnostic sensitivity of the analysis from 58.3% to 65%. Thus, the developed microarray-based method can be used to detect and elucidate the diagnostic characteristics of RA biomarkers; however, further use requires validation of the obtained results on an expanded sampling.

Will 14-3-3η Be a New Diagnostic and Prognostic Biomarker in Rheumatoid Arthritis? A Prospective Study of Its Utility in Early Diagnosis and Response to Treatment.

Results Serum14-3-3η levels were significantly higher in all RA patients than in controls (P < 0.001), its sensitivity was 86.7% and 88.3% in early and established RA patients with a significant difference with RF and ACCP at early disease, and the specificity was 96.7%. There was a significant reduction of 14-3-3η levels 6 months after treatment in the first group (p=0.004), and there was a significant positive correlation between serum 14-3-3η levels and parameters of disease activity and severity. Conclusion 14-3-3η could be a novel, potent, and efficacious diagnostic, and prognostic marker for RA with high sensitivity, that may become a new therapeutic target for RA.

A Microfluidic Platform for Detection and Quantification of Two Biomarkers for Rheumatoid Arthritis

A Microfluidic Platform for Detection and Quantification of Two Biomarkers for Rheumatoid Arthritis

The Role of Serum Chitinase-3-Like 1 Protein (YKL-40) Level and its Correlation with Proinflammatory Cytokine in Patients with Rheumatoid Arthritis

Chitinase-3-like 1 protein (YKL-40) is a glycoprotein primarily produced in the arthritic joint and plays a crucial role in inflammatory processes. The aim of the study is to establish the role of YKL-40 as a biomarker for rheumatoid arthritis (RA) compared to proinflammatory biomarkers and disease activity. The study included 58 patients and 18 control. Diseases activity score (DAS-28) and clinical disease activity index (CDAI) were measured. Serum level of YKL-40, tumor necrosis factor-α (TNF-α), interleukin-1B (IL-1β), erythrocyte sedimentation (ESR), rheumatoid factor (RF), C-reactive protein (CRP), and anti-citrullinated protein antibody (ACPA) were assessed. The results showed that the median serum YKL-40 level which was 5.42 ng/ml, the TNF-a level which was 123.6 ng/ml, and the IL-1B level which was 204.365 pg/dl were significantly higher in patients compared to control 3.28 (1.58–4.99) ng/ml, 56.47 (9.38–77.01) ng/ml and 67.887 (15.493–122.689)pg/dl respectively. There was no correlation between serum YKL-40 and disease activity, while there were significant associations observed with TNF-α (r=0.5,p =0.0001) and IL-1β (r=0.41, p=0.001). YKL-40 indicating good levels of RA prediction at 2.47 ng/ml, sensitivity 87.9%, specificity 58.6%, accuracy 78.1%, PPV 80.9%, and NPV 70.8%. In conclusion, a higher level of serum YKL-40 is found in RA patients. The findings from our study suggest a strong association between YKL-40 and the proinflammatory biomarkers. As a result, these biomarkers together might play a role in RA pathogenesis, and YKL-40 may be used as a potential diagnostic biomarker for RA.

Predictive Value of Beclin1 in the Pathogenesis of Rheumatoid Arthritis in the Indian Population

Background: Rheumatoid arthritis (RA) is a systemic autoimmune disease leading to the destruction of articular cartilage and deformity of joints if not detected early. There is an unmet need to find out a highly sensitive and specific biomarker for RA. This was the first study designed in Indian setting to assess whether it can be used as a biomarker in the diagnosis of RA in the Indian population. Aim: To correlate serum Beclin1 in the pathogenesis of rheumatoid arthritis in the Indian population. Patients and Methods: Observational analytical study was conducted for 18 months at AIIMS Rishikesh, Department of Biochemistry in collaboration with the Department of Rheumatology. Beclin1 serum expression levels were estimated by the enzyme-linked immunosorbent assay and beclin1 mRNA expression was assessed by a real-time polymerase chain reaction from peripheral blood mononuclear cells (PBMCs). Beclin1 expression was compared by Mann–Whitney U-test using SPSS 22 version. Cut-off values of Beclin1 for screening of cases were analysed by receiver operating characteristic test. Results: Age- and sex-matched 38 RA patients (5 males and 33 females) and 39 controls (8 males and 31 females) were recruited in the study. Patients with the American College of Rheumatology/European League Against Rheumatism score ≥6 were recruited in this study. Serum level of beclin1 was significantly (P ≤ 0.001) lower in cases (6.30 [2.82]) compared to healthy controls (11.43 [4.62]) which were corroborated with mRNA expression. The optimal cut-off value for detecting RA cases was 7.25 with 89.7% sensitivity and 79.8% specificity. Conclusion: Beclin1 may be involved in the pathogenesis of RA and may be considered a diagnostic marker for RA cases.

Immunosensing the rheumatoid arthritis biomarker through bifunctional aldehyde-amine linkers on an iron oxide nanoparticle seeded voltammetry sensor

An immunosensor was developed on an interdigitated electrode (IDE) by voltammetry sensing for the early identification of the autoimmune disease ‘rheumatoid arthritis (RA)’ by detecting the biomarker anti-cyclic citrullinated peptide antibody (anti-CCP). Higher immobilization of cyclic citrullinated peptide (CCP) as a probe was achieved by using green synthesized iron oxide nanoparticles (IONPs). Field-emission scanning electron microscopy and field-emission transmission electron microscopy observations revealed that the polydispersed material displayed multifaceted features. X-ray photoelectron spectroscopy analysis confirms the occurrence of Fe, O, and C groups on the synthesized IONPs. IONPs were immobilized with a probe on IDE through bifunctional aldehyde-amine linkers. Due to the elevated occupancy of CCP and the highly efficient electric transfer from IONPs, higher changes in current are observed upon binding of anti-CCP with CCP. In the linear range from 8 to 250 pg/mL, the sensitivity and detection limit of anti-CCP were 8 and 15 pg/mL, respectively, with a regression coefficient of y = 1E−06x−3E−07; R2 = 0.9637. Control experiments with nonimmune antibody and anti-carcinoembryonic antigen indicate the specific detection of anti-CCP. Furthermore, spiking of anti-CCP in human serum does not interfere, representing the specific detection of anti-CCP. This CCP-immobilized IDE through IONP helps to quantify anti-CCP levels in the biological fluid for diagnosing RA.

Identification and Validation of ATF3 Serving as a Potential Biomarker and Correlating With Pharmacotherapy Response and Immune Infiltration Characteristics in Rheumatoid Arthritis.

Background: Although disease-modifying antirheumatic drugs (DMARDs) have significantly improved the prognosis of patients with rheumatoid arthritis (RA), approximately 40% of RA patients have limited response. Therefore, it was essential to explore new biomarkers to improve the therapeutic effects on RA. This study aimed to develop a new biomarker and validate it by an in vitro study. Methods: The RNA-seq and the clinicopathologic data of RA patients were downloaded from Gene Expression Omnibus (GEO) databases. Differentially expressed genes were screened in the GPL96 and GPL570 databases. Then, weighted gene co-expression network analysis (WGCNA) was used to explore the most correlated gene modules to normal and RA synovium in the GPL96 and GPL570 databases. After that, the differentially expressed genes were intersected with the correlated gene modules to find the potential biomarkers. The CIBERSORT tool was applied to investigate the relationship between activated transcription factor 3 (ATF3) expression and the immune cell infiltration, and Gene Set Enrichment Analysis (GSEA) was used to investigate the related signaling pathways of differentially expressed genes in the high and low ATF3 groups. Furthermore, the relationships between ATF3 expression and clinical parameters were also explored in the GEO database. Finally, the role of ATF3 was verified by in vitro cell experiments. Results: We intersected the differentially expressed genes and the most correlated gene modules in the GPL570 and GPL96 databases and identified that ATF3 is a significant potential biomarker and correlates with some clinical–pharmacological variables. Immune infiltration analysis showed that activated mast cells had a significant infiltration in the high ATF3 group in the two databases. GSEA showed that metabolism-associated pathways belonged to the high ATF3 groups and that inflammation and immunoregulation pathways were enriched in the low ATF3 group. Finally, we validated that ATF3 could promote the proliferation, migration, and invasion of RA fibroblast-like synoviocyte (FLS) and MH7A. Flow cytometry showed that ATF3 expression could decrease the proportion of apoptotic cells and increase the proportion of S and G2/M phase cells. Conclusion: We successfully identified and validated that ATF3 could serve as a novel biomarker in RA, which correlated with pharmacotherapy response and immune cell infiltration.

Epigenome association study for DNA methylation biomarkers in buccal and monocyte cells for female rheumatoid arthritis

Genetics (i.e., mutations) has been assumed to be the major factor in rheumatoid arthritis (RA) etiology, but accounts for a minority of the variance in disease risk for RA. In contrast to genetics, the environment can have dramatic impacts on epigenetics that associate with disease etiology. The current study used buccal cells and purified blood monocytes from two different clinical cohorts involving Caucasian or African American female populations with or without arthritis. The differential DNA methylation regions (DMRs) between the control and RA populations were identified with an epigenome-wide association study. The DMRs (i.e., epimutations) identified in the buccal cells and monocytes were found to be distinct. The DMR associated genes were identified and many have previously been shown to be associated with arthritis. Observations demonstrate DNA methylation epimutation RA biomarkers are cell type specific and similar findings were observed with the two racial background populations. Rheumatoid arthritis susceptibility epigenetic diagnosis appears feasible and may improve the clinical management of RA and allowpreventative medicine considerations.

Anti-SARS-CoV-2 mRNA vaccines as inducers of humoral response against apolipoprotein A-1?

BackgroundCOVID‐19 and some anti‐SARS‐CoV‐2 vaccines trigger a humoral autoimmune response against a broad range of endogenous components, which may affect recipients’ prognosis in predisposed individuals. Autoantibodies directed against apolipoprotein A‐1 (AAA1 IgG) the major protein fraction of High Density Lipoprotein have been shown to be raised in COVID‐19 and in rheumatoid arthritis (RA) patients and other populations where they have been associated with poorer outcomes. We wanted to assess the impact of anti‐SARS‐CoV‐2 mRNA‐based vaccination on AAA1 autoimmune biomarkers in RA patients.Methods20 healthy controls and 77 RA mRNA‐based vaccinated patients were collected at baseline, 3 weeks after the first vaccination, 2 and 8 weeks after the second vaccination. AAA1 and SARS‐CoV‐2 serologies were measured by immunoassays. Systemic and local symptoms occurring during the vaccination protocol were recorded.ResultsmRNA‐based vaccination induced a significant increase in median AAA1 IgG levels in both healthy controls and RA patients overtime. However, in both populations, these medians trend did not translate into significant increase in AAA1 IgG seropositivity rates despite evolving from 5 to 10% in healthy controls, and from 9 to 12.9% in RA patients. No associations were retrieved between AAA1 IgG and symptoms of any kind during the vaccination protocol.ConclusionsmRNA‐based vaccination seems to induce a light AAA1 IgG response in immunocompetent individuals within 2 months after the last injection. Although we did not observe any warning signs, the formal demonstration of the harmlessness of such biological warrants further studies.

Measurement of synovium and serum dual specificity phosphatase 22 level: Their inter‐correlation and potency as biomarkers in rheumatoid arthritis

BackgroundDual specificity phosphatase 22 (DUSP22), also named as Jun N‐terminal kinase pathway associated phosphatase recently, is reported to be closely engaged in immune and inflammation regulation. This study aimed to investigate the interaction between synovium DUSP22 and serum DUSP22 levels and to explore their correlation with rheumatoid arthritis (RA) risk, inflammation, and disease activity.MethodsSynovium and serum samples from 42 RA patients with knee involvement underwent arthroscopy, and 20 knee trauma patients were collected. Besides, serum samples from 40 healthy controls were also obtained. Synovium DUSP22 expression was detected by reverse transcription quantitative polymerase chain reaction, while serum DUSP22 level was detected by enzyme‐linked immunosorbent assay.ResultsSynovium DUSP22 level was greatly decreased in RA patients compared to trauma controls (p < 0.001), and it was negatively correlated with tender joint count (TJC) (r = −0.318, p = 0.040), C‐reactive protein (CRP) (r = −0.330, p = 0.033), and Lysholm score (r = −0.423, p = 0.005) in RA patients. Serum DUSP22 level was lowest in RA patients, followed by trauma controls, then highest in healthy controls (p < 0.001). Serum DUSP22 level was negatively associated with TJC (r = −0.438, p = 0.004), swollen joint count (SJC) (r = −0.372, p = 0.015), CRP (r = −0.391, p = 0.011), and disease activity score in 28 joints (DAS28ESR) score (r = −0.406, p = 0.008), and it increased after treatment (p = 0.001) in RA patients. In addition, serum DUSP22 level positively related to synovium DUSP22 level in RA patients (r = 0.394, p = 0.010).ConclusionSynovium and serum DUSP22 are intercorrelated and insufficiently expressed in RA patients; meanwhile, their deficiency correlates with increased systemic inflammation, disease activity, and joint dysfunction.

AGE/Non-AGE Glycation: An Important Event in Rheumatoid Arthritis Pathophysiology.

Rheumatoid arthritis (RA) is a chronic inflammatory, autoimmune disease that gradually affects the synovial membrane and joints. Many intrinsic and/or extrinsic factors are crucial in making RA pathology challenging throughout the disease. Substantial enzymatic or non-enzymatic modification of proteins driving inflammation has gained a lot of interest in recent years. Endogenously modified glycated protein influences disease development linked with AGEs/non-AGEs and is reported as a disease marker. In this review, we summarized current knowledge of the differential abundance of glycated proteins by compiling and analyzing a variety of AGE and non-AGE ligands that bind with RAGE to activate multi-faceted inflammatory and oxidative stress pathways that are pathobiologically associated with RA-fibroblast-like synoviocytes (RA-FLS). It is critical to comprehend the connection between oxidative stress and inflammation generation, mediated by glycated protein, which may bind to the receptor RAGE, activate downstream pathways, and impart immunogenicity in RA. It is worth noting that AGEs and non-AGEs ligands play a variety of functions, and their functionality is likely to be more reliant on pathogenic states and severity that may serve as biomarkers for RA. Screening and monitoring of these differentially glycated proteins, as well as their stability in circulation, in combination with established pre-clinical characteristics, may aid or predict the onset of RA.

Machine learning to identify immune-related biomarkers of rheumatoid arthritis based on WGCNA network.

This study was designed to identify the potential diagnostic biomarkers of rheumatoid arthritis (RA) and to explore the potential pathological relevance of immune cell infiltration in this disease. Three previously published datasets containing gene expression data from 35 RA patients and 29 controls (GSE55235, GSE55457, and GSE12021) were downloaded from the GEO database, after which a weighted correlation network analysis (WGCNA) approach was utilized to clarify differentially abundant genes. Candidate biomarkers of RA were then identified via the use of a LASSO regression model and support vector machine recursive feature elimination (SVM-RFE) analyses. Data were validated based upon the area under the receiver operating characteristic curve (AUC) values, with hub genes being identified as those with an AUC > 85% and a P value < 0.05. Lastly, the CIBERSORT algorithm was used to assess immune cell infiltration of RA tissues, and correlations between immune cell infiltration and disease-related diagnostic biomarkers were assessed. The green-yellow module containing 87 genes was found to be highly correlated with RA positivity. FADD, CXCL2, and CXCL8 were identified as potential RA diagnostic biomarkers (AUC > 0.85), and these results were validated using the GSE77298 dataset. Immune cell infiltration analyses revealed the expression of hub genes to be correlated with mast cells, monocytes, activated NK cells, CD8 T cells, resting dendritic cells, and plasma cells. These data indicate that FADD, CXCL2, and CXCL8 are valuable diagnostic biomarkers of RA, offering new insight that can guide future studies of RA incidence and progression.

Bruton's Tyrosine Kinase (BTK) Inhibitors and Autoimmune Diseases: Making Sense of BTK Inhibitor Specificity Profiles and Recent Clinical Trial Successes and Failures.

Clinical development of BTK kinase inhibitors for treating autoimmune diseases has lagged behind development of these drugs for treating cancers, due in part from concerns over the lack of selectivity and associated toxicity profiles of first generation drug candidates when used in the long term treatment of immune mediated diseases. Second generation BTK inhibitors have made great strides in limiting off-target activities for distantly related kinases, though they have had variable success at limiting cross-reactivity within the more closely related TEC family of kinases. We investigated the BTK specificity and toxicity profiles, drug properties, disease associated signaling pathways, clinical indications, and trial successes and failures for the 13 BTK inhibitor drug candidates tested in phase 2 or higher clinical trials representing 7 autoimmune and 2 inflammatory immune-mediated diseases. We focused on rheumatoid arthritis (RA), multiple sclerosis (MS), and systemic lupus erythematosus (SLE) where the majority of BTK nonclinical and clinical studies have been reported, with additional information for pemphigus vulgaris (PV), Sjogren’s disease (SJ), chronic spontaneous urticaria (CSU), graft versus host disease (GVHD), and asthma included where available. While improved BTK selectivity versus kinases outside the TEC family improved clinical toxicity profiles, less profile distinction was evident within the TEC family. Analysis of genetic associations of RA, MS, and SLE biomarkers with TEC family members revealed that BTK and TEC family members may not be drivers of disease. They are, however, mediators of signaling pathways associated with the pathophysiology of autoimmune diseases. BTK in particular may be associated with B cell and myeloid differentiation as well as autoantibody development implicated in immune mediated diseases. Successes in the clinic for treating RA, MS, PV, ITP, and GVHD, but not for SLE and SJ support the concept that BTK plays an important role in mediating pathogenic processes amenable to therapeutic intervention, depending on the disease. Based on the data collected in this study, we propose that current compound characteristics of BTK inhibitor drug candidates for the treatment of autoimmune diseases have achieved the selectivity, safety, and coverage requirements necessary to deliver therapeutic benefit.

Analysis of gut microbiota and metabolites in patients with rheumatoid arthritis and identification of potential biomarkers.

Rheumatoid arthritis (RA) is an autoimmune disease described by joint destruction, synovitis and pannus formation. The gut microbiota acts as an environmental factor that plays an important role in RA, but little research regarding the etiopathogenic mechanisms of the microbiome in RA has been carried out. We used an integrated approach of 16S rRNA gene sequencing and ultrahigh-performance liquid chromatography-mass spectrometry-based metabolomics to analyze the structure and diversity of the intestinal flora and metabolites of the gut microbiota in RA patients compared with healthy subjects. In this study, α-diversity analysis of the gut microbiota showed that there was no significant difference between the healthy control (HC) and RA groups. However, β-diversity analysis showed that there was a significant difference between the two groups. Further analysis of alteration of the gut microbiota revealed that at the phylum level, the relative abundance of p_Bacteroidetes was significantly decreased in the RA group, while that of Verrucomicrobia and Proteobacteria was significantly increased in the RA group. At the genus level, Bacteroides, Faecalibacterium and some probiotics were decreased in the RA group, while 97 genera, including Lactobacillus, Streptococcus and Akkermansia, were increased in the RA group. Seventy-four differentially abundant metabolites were identified between the HC and RA groups, and we identified two potential biomarkers (9,12-octadecadiynoic acid and 10Z-nonadecenoic acid) in RA.

Identification of a Novel Serological Marker in Seronegative Rheumatoid Arthritis Using the Peptide Library Approach.

Rheumatoid arthritis (RA) is a systemic autoimmune disease characterized by chronic inflammation mainly affecting the joints leading to cartilage and bone destruction. The definition of seropositive or seronegative RA is based on the presence or absence of rheumatoid factor (RF) and anti-citrullinated peptide antibodies (ACPAs). Other autoantibodies have been identified in the last decade such as antibodies directed against carbamylated antigens, peptidyl-arginine deiminase type 4 and v-Raf murine sarcoma viral oncogene homologue B. In order to identify relevant autoantigens, we screened a random peptide library (RPL) with pooled IgGs obtained from 50 patients with seronegative RA. Patients’ sera were then used in an ELISA test to identify the most frequently recognized peptide among those obtained by screening the RPL. Sera from age- and sex-matched healthy subjects were used as controls. We identified a specific peptide (RA-peptide) recognized by RA patients’ sera, but not by healthy subjects or by patients with other immune-mediated diseases. The majority of sera from seronegative and seropositive RA patients (73.8% and 63.6% respectively) contained IgG antibodies directed against the RA-peptide. Interestingly, this peptide shares homology with some self-antigens, such as Protein-tyrosine kinase 2 beta, B cell scaffold protein, Liprin-alfa1 and Cytotoxic T lymphocyte protein 4. Affinity purified anti-RA-peptide antibodies were able to cross react with these autoantigens. In conclusion, we identified a peptide that is recognized by seropositive and, most importantly, by seronegative RA patients’ sera, but not by healthy subjects, conferring to this epitope a high degree of specificity. This peptide shares also homology with other autoantigens which can be recognized by autoantibodies present in seronegative RA sera. These newly identified autoantibodies, although present also in a percentage of seropositive RA patients, may be considered as novel serum biomarkers for seronegative RA, which lacks the presence of RF and/or ACPAs.

TNF induces glycolytic shift in fibroblast like synoviocytes via GLUT1 and HIF1A

TNF is a central cytokine in the pathogenesis of rheumatoid arthritis (RA). Elevated level of TNF causes local inflammation that affects immune cells and fibroblast-like synoviocytes (FLS). Nowadays, only 20–30% of patients experience remission after the standard of care therapy—antibodies against TNF. Interestingly, responders show reduced levels of GLUT1 and GAPDH, highlighting a potential link to cellular metabolism. The aim of the study was to investigate whether TNF directly affects the metabolic phenotype of FLS. Real-time respirometry displayed TNF-induced upregulation of glycolysis along with a modest increase of oxidative phosphorylation in FLS from healthy donors. In addition, TNF stimulation enhanced HIF1A and GLUT1 expression. The upregulation of HIF1A and GLUT1 reflects their enriched level in FLS from RA patients (RA-FLS). The inhibition of TAK1, HIF1a and hexokinase deciphered the importance of TNF/TAK1/HIF1A/glycolysis signaling axis. To prove that inhibition of glycolysis reduced the pathogenic phenotype, we showed that 2-deoxyglucose, a hexokinase inhibitor, partially decreased secretion of RA biomarkers. In summary, we identified a direct role of TNF on glycolytic reprogramming of FLS and confirmed the potency of immunometabolism for RA. Further studies are needed to evaluate the therapeutic impact especially regarding non-responder data.