The Kinetics of Humoral and Cellular Responses after the Booster Dose of COVID-19 Vaccine in Inflammatory Arthritis Patients.

Impaired immunogenicity of COVID-19 vaccinations in inflammatory arthritis (IA) patients results in diminished immunity. However, optimal booster vaccination regimens are still unknown. Therefore, this study aimed to assess the kinetics of humoral and cellular responses in IA patients after the COVID-19 booster. In 29 IA patients and 16 healthy controls (HC), humoral responses (level of IgG antibodies) and cellular responses (IFN-γ production) were assessed before (T0), after 4 weeks (T1), and after more than 6 months (T2) from the booster vaccination with BNT162b2. IA patients, but not HC, showed lower anti-S-IgG concentration and IGRA fold change at T2 compared to T1 (p = 0.026 and p = 0.031). Furthermore, in IA patients the level of cellular response at T2 returned to the pre-booster level (T0). All immunomodulatory drugs, except IL-6 and IL-17 inhibitors for the humoral and IL-17 inhibitors for the cellular response, impaired the immunogenicity of the booster dose at T2. Our study showed impaired kinetics of both humoral and cellular responses after the booster dose of the COVID-19 vaccine in IA patients, which, in the case of cellular response, did not allow the vaccination effect to be maintained for more than 6 months. Repetitive vaccination with subsequent booster doses seems to be necessary for IA patients.

Effectiveness of Soluble CTLA-4-Fc in the Inhibition of Bone Marrow T-Cell Activation in Context of Indoleamine 2.3-Dioxygenase (IDO) and CD4+Foxp3+ Treg Induction.

Background: Rheumatoid arthritis (RA) is a chronic autoimmune disease with systemic inflammation finally resulting in damaged joints. One of the RA development models suggests bone marrow (BM) as a place of inflammation development further leading to disease progression. We aimed to investigate the potential of CTLA-4-Fc molecule in inducing tolerogenic milieu in BM measured as indoleamine 2,3-dioxygenase (IDO) expression, CD4+Foxp3+ Treg induction, and T cell activation control. The expression of IDO-pathway genes was also examined in monocytes to estimate the tolerogenic potential in the periphery. Methods: Bone marrow mononuclear cells (BMMC) were stimulated by pro-inflammatory cytokines and CTLA-4-Fc. Next IDO expression, CD4+CD69+ and CD4+Foxp3+ percentage were estimated by PCR and FACS staining, respectively. Enzymatic activity of IDO was confirmed by HPLC in BM plasma and blood plasma. Genes expressed in IDO-pathway were analyzed by NGS in peripheral monocytes isolated from RA patients and healthy controls. Results: We found that CTLA-4-Fc and IFN-γ stimulation results in IDO production by BMMC. CTLA-4-Fc induced tryptophan catabolism can inhibit mitogen-induced CD4+ T cells activation without influencing CD8+ cells, but did not control CD25 nor Foxp3 expression in BM cells. Significantly higher expression of selected IDO-pathway genes was detected on peripheral monocytes isolated from RA as compared to healthy controls. Conclusion: This study sheds light on some immunosuppression aspects present or induced in BM. The potential of IDO-mediated pathways were confirmed in the periphery, what may represent the promising candidates for therapeutic strategies in RA.

Humoral and cellular immunogenicity of COVID-19 booster dose vaccination in inflammatory arthritis patients.

Introduction: Previous studies have shown a reduction in the effectiveness of primary COVID-19 vaccination in patients with rheumatic diseases. However, limited data is available regarding the effectiveness of the COVID-19 vaccine booster dose, especially on cellular response. The study aimed to assess the humoral and cellular immunogenicity of a booster dose in patients with inflammatory arthritis (IA). Patients and methods: 49 IA and 47 age and sex-matched healthy controls (HC) were included in a prospective cohort study. Both groups completed primary COVID-19 vaccination and after more than 180 days received a BNT162b2 booster shot. Humoral responses (level of IgG antibodies) and cellular responses (IFN-γ production) were assessed before and after 4 weeks from the booster dose of the vaccine. Results: After the booster dose, all participants showed an increased humoral response, although significantly reduced antibody levels were observed in IA patients compared to HC (p=0.004). The cellular response was significantly lower both before (p<0.001) and after (p<0.001) the booster dose in IA patients as compared to HC. Among the immunomodulatory drugs, only biological and targeted synthetic drugs lowered the humoral response after booster vaccination. However, the cellular response was decreased after all immunomodulatory drugs except IL-17 inhibitors and sulfasalazine. Conclusion: Our data indicate that patients with rheumatic diseases present lower humoral and cellular responses after the COVID-19 booster vaccine in comparison to HC. This may translate into a recommendation for subsequent booster doses of the COVID-19 vaccine for rheumatic patients.

Circulating miRNA Correlates with Lipid Profile and Disease Activity in Psoriatic Arthritis, Rheumatoid Arthritis, and Ankylosing Spondylitis Patients.

This study aimed to investigate the associations of microRNA (miRs) signatures with cytokines, serum lipids, and disease activity in patients with psoriatic arthritis (PsA), ankylosing spondylitis (AS), and rheumatoid arthritis (RA). In total, 65 patients (PsA n = 25, AS n = 25, RA n = 15) and 25 healthy controls (HC) were enrolled into the study. The expression of miR-223-5p, miR-92b-3p, miR-485-3p, miR-10b-5p, let-7d-5p, miR-26a-2-3p, miR-146b-3p, and cytokines levels were measured in sera. DIANA-mirPath analysis was used to predict pathways targeted by the dysregulated miRs. Disease activity scores were calculated. Lipid profile, uric acid, glucose level, and C-reactive protein (CRP) concentrations were determined in the blood. Based on lipid profiles, the PsA group had hypertriglyceridaemia, and RA patients revealed mixed dyslipidaemia, while in AS, no specific changes were found. miR expression analysis revealed upregulation of miR-26a-2-3p and miR-10b-5p in PsA, miR-485-3p in AS, and let-7d-5p in RA. Several correlations between disease activity indexes, metabolites levels, and expression of miRs were observed in PsA, RA, and AS patients. Finally, in ROC analysis, miR-26a-2-3p/miR-485-3p, and let-7d-5p/miR-146b-3p tandems revealed high sensitivity and specificity in distinguishing between PsA, AS, and RA. Our study illustrates the superiority of miR expressions in distinguishing between RA, PsA, and AS. In PsA, a unique regulatory pathway exists through miR-26a-2-3p, miR-223-5p, miR-10b-5p, and miR-92b-3p that converges proatherogenic metabolism and disease activity.

Comprehensive microRNA and transcriptomic profiling of rheumatoid arthritis monocytes: role of microRNA-146b in pro-inflammatory progression.

OBJECTIVE: To explore global miRNA and transcriptomic profiling of monocytes from RA patients compared with healthy controls in order to predict which aberrantly expressed miRNA can negatively modulate inflammatory molecules. METHODS: Using next-generation sequencing, we have performed simultaneous global analysis of miRNA (miRNA-seq) and transcriptome (RNA-seq) of monocytes from RA patients and healthy controls. Global analysis of miRNA of SSc monocytes was also performed. Following differential analysis and negative correlation, miRNA-RNA pairs were selected. RESULTS: We found that 20 specific miRNA candidates are predicted to silence inflammatory mediators, out of 191 significantly changed miRNAs in RA monocytes. Based on the highest scoring in terms of negative correlation (r = -0.97, P = 1.75e-07, false discovery rate = 0.04) and the number of seeds in miRNA responsible for negative regulation, we selected miRNA-146b and its target gene anti-inflammatory retinoic acid receptor alpha (RARA). Similarly to next-generation sequencing, qPCR analysis also confirmed negative correlation between miRNA-146b and RARA expression (r = -0.45, P = 0.04). Additionally, miRNA-146b expression in RA monocytes significantly correlated with clinical parameters including DAS28 for RA with CRP (DAS28-CRP) and ESR (DAS28-ESR), whereas overexpression of miRNA-146b was able to functionally reduce RARA expression in the human monocytic cell line THP-1. Finally, circulating miRNA-146b expression in sera and SFs was significantly elevated in RA patients. CONCLUSIONS: Overall, in this study we have identified a new miRNA-146b candidate that is predicted to negatively regulate the anti-inflammatory RARA transcript, whereas circulating miRNA-146b level can be used as a biomarker predicting pro-inflammatory RA progression and disease activity.

Antibodies to Citrullinated Proteins (ACPA) Associate with Markers of Osteoclast Activation and Bone Destruction in the Bone Marrow of Patients with Rheumatoid Arthritis.

Normalizing bone metabolism is a challenge in rheumatoid arthritis (RA). Studies in mice suggest that anti-citrullinated protein antibodies (ACPAs) can trigger osteoclast activation and bone resorption in the bone marrow. However, data on the presence and role of ACPAs in human bone marrow are scarce. We investigated whether ACPAs can contribute to osteoclast activation and bone erosion in RA bone marrow. Anti-cyclic citrullinated peptide antibodies (anti-CCP Abs), osteoclast activation indicators-the tartrate-resistant acid phosphatase 5b (TRAP5b) and cathepsin K, and bone degradation marker-C-terminal telopeptide of type I collagen (CTX-I) were measured in the bone marrow and peripheral blood of RA patients using ELISAs. We found that ACPAs present in RA bone marrow was associated with increased amounts of TRAP5b, cathepsin K and CTX-I in this location. Levels of IL-8, the key mediator of anti-citrullinated protein antibody (ACPA)-induced bone resorption, were also elevated in bone marrow containing anti-CCP Abs and positively correlated with TRAP5b and cathepsin K concentrations. Higher levels of TRAP5b, cathepsin K, CTX-I and IL-8 in bone marrow compared to peripheral blood indicate local generation of these molecules. Our results complement data from animal studies and highlight the relevance of ACPAs and bone marrow in bone resorption in RA.

Biologic Drugs for Rheumatoid Arthritis in the Context of Biosimilars, Genetics, Epigenetics and COVID-19 Treatment.

Rheumatoid arthritis (RA) affects around 1.2% of the adult population. RA is one of the main reasons for work disability and premature retirement, thus substantially increasing social and economic burden. Biological disease-modifying antirheumatic drugs (bDMARDs) were shown to be an effective therapy especially in those rheumatoid arthritis (RA) patients, who did not adequately respond to conventional synthetic DMARD therapy. However, despite the proven efficacy, the high cost of the therapy resulted in limitation of the widespread use and unequal access to the care. The introduction of biosimilars, which are much cheaper relative to original drugs, may facilitate the achievement of the therapy by a much broader spectrum of patients. In this review we present the properties of original biologic agents based on cytokine-targeted (blockers of TNF, IL-6, IL-1, GM-CSF) and cell-targeted therapies (aimed to inhibit T cells and B cells properties) as well as biosimilars used in rheumatology. We also analyze the latest update of bDMARDs' possible influence on DNA methylation, miRNA expression and histone modification in RA patients, what might be the important factors toward precise and personalized RA treatment. In addition, during the COVID-19 outbreak, we discuss the usage of biologicals in context of effective and safe COVID-19 treatment. Therefore, early diagnosing along with therapeutic intervention based on personalized drugs targeting disease-specific genes is still needed to relieve symptoms and to improve the quality of life of RA patients.

Small Molecule Inhibitors in the Treatment of Rheumatoid Arthritis and Beyond: Latest Updates and Potential Strategy for Fighting COVID-19.

The development of biological disease-modifying antirheumatic drugs (bDMARDs) and target synthetic DMARDs (tsDMARDs), also known as small molecule inhibitors, represent a breakthrough in rheumatoid arthritis (RA) treatment. The tsDMARDs are a large family of small molecules targeting mostly the several types of kinases, which are essential in downstream signaling of pro-inflammatory molecules. This review highlights current challenges associated with the treatment of RA using small molecule inhibitors targeting intracellular JAKs/MAPKs/NF-κB/SYK-BTK signaling pathways. Indeed, we have provided the latest update on development of small molecule inhibitors, their clinical efficacy and safety as a strategy for RA treatment. On the other hand, we have highlighted the risk and adverse effects of tsDMARDs administration including, among others, infections and thromboembolism. Therefore, performance of blood tests or viral infection screening should be recommended before the tsDMARDs administration. Interestingly, recent events of SARS-CoV-2 outbreak have demonstrated the potential use of small molecule inhibitors not only in RA treatment, but also in fighting COVID-19 via blocking the viral entry, preventing of hyperimmune activation and reducing cytokine storm. Thus, small molecule inhibitors, targeting wide range of pro-inflammatory singling pathways, may find wider implications not only for the management of RA but also in the controlling of COVID-19.

Interleukin-15 as a Biomarker Candidate of Rheumatoid Arthritis Development.

There is a need for definite diagnosis of rheumatoid arthritis (RA) at its earliest stages of development in order to introduce early and effective treatment. Here we assessed whether serum interleukin-15 (IL-15) can serve as a new biomarker of RA development in patients with undifferentiated arthritis (UA). Interleukin-15, IgM-rheumatoid factor (RF) and anti-cyclic citrullinated peptide antibodies (anti-CCP Abs) were measured in UA patients at inclusion. Six months later, the diagnosis was re-evaluated, and statistical analysis was performed. We found that at the UA stage, IL-15 was more prevalent in patients who progressed to RA than RF or anti-CCP Abs (83.3% vs. 61.1% and 66.7%, respectively). Interleukin-15 showed higher sensitivity (77.8%) than both autoantibodies and higher specificity (80.9%) than anti-CCP Abs in identification of UA patients who developed RA. The diagnostic utility of IL-15 was comparable to that of RF (AUC: 0.814 vs. 0.750, p > 0.05), but higher than that of anti-CCP Abs (AUC: 0.814 vs. 0.684, p = 0.04). The combined use of IL-15, RF and anti-CCP Abs yielded higher diagnostic accuracy for RA than autoantibodies determination only. Our results indicate that IL-15 can be used as a biomarker of RA development in patients with UA.

CD4+FOXP3+ T Cells in Rheumatoid Arthritis Bone Marrow Are Partially Impaired.

There is evolving evidence that dysregulation of immune homeostasis in the bone marrow (BM) adjacent to the inflamed joints is involved in the pathogenesis of. In this study, we are addressing the phenotype and function of regulatory T cells (Tregs) residing in the BM of patients with rheumatoid arthritis (RA) and osteoarthritis (OA). BM and peripheral blood samples were obtained from RA and OA patients undergoing hip replacement surgery. The number and phenotype of Tregs were analyzed by flow cytometry and immunohistochemistry. The function of Tregs was investigated ex vivo, addressing their suppressive activity on effector T cells. [3H]-Thymidine incorporation assay and specific enzyme-linked immunosorbent assay were used for quantification of cell proliferation and pro-inflammatory (TNF, IFN-γ) cytokine release, respectively. Significantly lower numbers of CD4+FOXP3+ T cells were found in the BM of patients with RA compared to control patients with OA. High expression of CD127 (IL-7 receptor) and relatively low expression of CXCR4 (receptor for stromal cell-derived factor CXCL12) are characteristics of the CD4+FOXP3+ cells residing in the BM of RA patients. The BM-resident Tregs of RA patients demonstrated a limited suppressive activity on the investigated immune response. Our results indicate that the reduced number and impaired functional properties of CD4+FOXP3+ T cells present in the BM of RA patients may favor the inflammatory process, which is observed in RA BM.

Global miRNA and mRNA expression profiles identify miRNA-26a-2-3p-dependent repression of IFN signature in systemic sclerosis human monocytes.

Dysregulation in type I IFN and IFN-stimulated genes (ISGs) induced by monocytes is one of the key features of systemic sclerosis (SSc) pathogenesis. Abnormalities in microRNA (miRNA) expression are related to excessive IFN production, however the role of miRNA remains largely elusive in SSc monocytes. This study explores global miRNA-mRNA profiling of SSc monocytes and functional attenuation of IFN and ISGs by specific miRNAs. Global sequencing of mRNA (mRNA-seq) and miRNA (miRNA-seq) samples were performed simultaneously on healthy controls and SSc monocytes. Following computational analysis, selected miRNAs-mRNA candidates were validated, correlated with clinical parameters, and tested by functional assays. Transcriptomics data and qPCR analysis confirmed IFN signature in SSc but not in rheumatoid arthritis monocytes. Based on miRNA-seq analysis, five miRNAs were selected for further validation. Only the expression patterns of miRNA-26a-2-3p and miRNA-485-3p were confirmed and negatively correlated with clinical parameters. Exogenous delivery of miRNA-26a-2-3p to TLR-stimulated monocytic THP-1 cells specifically inhibited ISGs but not inflammasome activity in functional assays. In conclusion, our miRNA-mRNA co-sequencing and functional analysis identify miRNA-26a-2-3p as a new candidate, which is predicated to negatively regulate ISGs. This implies that reduced expression of miRNA-26a-2-3 may be involved in pathogenic IFN signature in SSc monocytes.

DNA Methylation as a Future Therapeutic and Diagnostic Target in Rheumatoid Arthritis.

Rheumatoid arthritis (RA) is a long-term autoimmune disease of unknown etiology that leads to progressive joint destruction and ultimately to disability. RA affects as much as 1% of the population worldwide. To date, RA is not a curable disease, and the mechanisms responsible for RA development have not yet been well understood. The development of more effective treatments and improvements in the early diagnosis of RA is direly needed to increase patients' functional capacity and their quality of life. As opposed to genetic mutation, epigenetic changes, such as DNA methylation, are reversible, making them good therapeutic candidates, modulating the immune response or aggressive synovial fibroblasts (FLS-fibroblast-like synoviocytes) activity when it is necessary. It has been suggested that DNA methylation might contribute to RA development, however, with insufficient and conflicting results. Besides, recent studies have shown that circulating cell-free methylated DNA (ccfDNA) in blood offers a very convenient, non-invasive, and repeatable "liquid biopsy", thus providing a reliable template for assessing molecular markers of various diseases, including RA. Thus, epigenetic therapies controlling autoimmunity and systemic inflammation may find wider implications for the diagnosis and management of RA. In this review, we highlight current challenges associated with the treatment of RA and other autoimmune diseases and discuss how targeting DNA methylation may improve diagnostic, prognostic, and therapeutic approaches.

Survival of lymphocytes is not restricted by IDO-expressing fibroblast from rheumatoid arthritis patients.

Objective: Rheumatoid arthritis (RA) is characterized by expansion of fibroblast-like synoviocytes (FLS) in inflamed joints and activation of lymphocytes. Tryptophan (trp) is an essential amino acid indispensable for the biosynthesis of proteins and critical for survival of lymphocytes. Indoleamine 2,3-dioxygenase (IDO) that initiates the degradation of trp and tryptophanyl-tRNA synthetase (TTS) essential for tryptophan synthesis, regulate trp bioavailability. Here, we tested the hypothesis that triggered by cytokines, enhanced IDO activity modulate regulatory function of otherwise non-tolerogenic FLS isolated from RA patients. Materials and methods: IDO and TTS mRNA expression were evaluated by RT-PCR. IDO enzymatic activity was confirmed using HPLC. Resting or PHA-activated PBMC from healthy volunteers and RA patients were co-cultured with IDO expressing untreated (FLSC) or IFNγ-treated (FLSIFNγ) RA FLS. Lymphocyte survival and proliferation were evaluated by flow cytometry analysis and tritiated thymidine incorporation, respectively. Results: RA FLSIFNγ produce functionally active IDO and constitutively express TTS. RA FLSC and FLSIFNγ increased survival of resting lymphocytes in both studied groups, and decreased proliferation of healthy, but not RA, PBMC. Only FLSIFNγ diminished survival of activated CD3+CD4-, but not CD3+CD4+, healthy T cells and similar tendency was observed in rheumatoid cells. Importantly, IDO inhibitor, 1-methyl-DL-tryptophan (1-MT), failed to reverse this effect. PBMC, irrespective of their state (resting versus activated) or origin (healthy or RA), expressed high level of TTS mRNA. Conclusions: We suggest that RA FLS express functionally active IDO but control survival and expansion of healthy cells in IDO-independent mechanism and exert weaker, if any, suppressive effect on rheumatoid cells.

Different Secretory Activity of Articular and Subcutaneous Adipose Tissues from Rheumatoid Arthritis and Osteoarthritis Patients.

Rheumatoid arthritis (RA) and osteoarthritis (OA) are characterized by joint and systemic high- or low-grade inflammation, respectively. Adipose tissue (AT) may contribute to the pathogenesis of these diseases. To address this issue, we investigated whether basal and pro-inflammatory cytokine (IL-1ß)-triggered release of adipocytokines (TNF, IL-6, IL-10, IL-1Ra, TGFß, CCL2/MCP-1, CCL5/RANTES, MMP-3) from subcutaneous (ScAT) and intraarticular (AAT) adipose tissues of RA and OA patients mirror differences between these diseases in an intensity of systemic and local inflammation. We found that in both diseases basal adipocytokine release was usually higher from AAT than ScAT, reflecting stronger local than systemic inflammation. However, ScAT secreted considerable amounts of pro- and anti-inflammatory factors as well. Spontaneous secretion of some adipocytokines (MMP-3 and/or TNF, CCL2/MCP-1, IL-1Ra) was higher in osteoarthritis than rheumatoid ATs and probably caused by weaker anti-inflammatory treatment of OA patients. By contrast, reactivity of ATs to IL-1ß was significantly lower in OA than RA and IL-1ß antagonist (IL-1Ra) could be responsible for this because we found its overproduction in OA ATs. Interestingly, higher reactivity of ScAT than AAT to IL-1ß was a characteristic for OA while reactivity of rheumatoid ScAT and AAT to this stimulus was equal. We conclude that differences between OA and RA in reactivity of AAT and ScAT to pro-inflammatory stimulus mimicking in vivo condition reflect dissimilarity in an intensity of disease-specific inflammation and thus support contribution of ATs to these pathological processes. Moreover, we propose that more efficient anti-inflammatory mechanism(s) are preserved in ATs of OA than RA patients.

Cartilage and bone damage in rheumatoid arthritis.

Rheumatoid arthritis (RA), which is a chronic inflammatory disease with a multifactorial aetiology, leads to partial or permanent disability in the majority of patients. It is characterised by persistent synovitis and formation of pannus, i.e. invasive synovial tissue, which ultimately leads to destruction of the cartilage, subchondral bone, and soft tissues of the affected joint. Moreover, inflammatory infiltrates in the subchondral bone, which can lead to inflammatory cysts and later erosions, play an important role in the pathogenesis of RA. These inflammatory infiltrates can be seen in magnetic resonance imaging (MRI) as bone marrow oedema (BME). BME is observed in 68-75% of patients in early stages of RA and is considered a precursor of rapid disease progression. The clinical significance of synovitis and bone marrow oedema as precursors of erosions is well established in daily practice, and synovitis, BME, cysts, hyaline cartilage defects and bone erosions can be detected by ultrasonography (US) and MRI. A less explored subject is the inflammatory and destructive potential of intra- and extra-articular fat tissue, which can also be evaluated in US and MRI. Finally, according to certain hypotheses, hyaline cartilage damage may trigger synovitis and lead to irreversible joint damage, and MRI may be used for preclinical detection of cartilage biochemical abnormalities. This review discusses the pathomechanisms that lead to articular cartilage and bone damage in RA, including erosion precursors such as synovitis and osteitis and panniculitis, as well as the role of imaging techniques employed to detect early cartilage damage and bone erosions.

Changes in MiRNA-5196 Expression as a Potential Biomarker of Anti-TNF-α Therapy in Rheumatoid Arthritis and Ankylosing Spondylitis Patients.

In this study, we analysed the expression level of sera circulating miRNA-5196 in rheumatoid arthritis (RA) and ankylosing spondylitis (AS) patients before and after tumor necrosis factor (TNF)-α therapy as biomarkers predicting positive treatment outcome. We enrolled 10 RA patients, 13 AS patients, and 12 healthy individuals in the study. The expression of miRNA-5196 was measured by real-time polymerase chain reaction before and after anti-TNF-α therapy. Disease activity of RA patients was assessed using disease activity score 28 (DAS28), whereas ankylosing spondylitis DAS (ASDAS) was used in AS patients. MiRNA-5196 expression was significantly higher in patients with RA and AS before TNF-α therapy than in those following anti-TNF-α therapy and healthy controls. Changes in miRNA-5196 expression positively correlated with delta DAS28 or delta ASDAS, respectively, following TNF-α therapy. In contrast, changes in C-reactive protein (CRP) levels in RA and AS patients did not positively correlate with DAS28 or ASDAS changes. Receiver-operating characteristic analysis showed better diagnostic accuracy of miRNA-5196 expression both in RA (area under curve (AUC) = 0.87, p = 0.055) and AS patients (AUC = 0.90, p = 0.050) compared to CRP levels in RA (AUC = 0.75, p = 0.201) and AS patients (AUC = 0.85, p = 0.086) upon biologic therapy treatment. Finding novel biomarkers, including miRNA-5196 which allow to predict and monitor anti-TNF-α response, would be of clinical value especially during the early phase of RA or AS development.

Monocyte-related biomarkers of rheumatoid arthritis development in undifferentiated arthritis patients - a pilot study.

OBJECTIVES: Enhanced/disturbed activities of monocytes are crucial for perpetuation and for development of rheumatoid arthritis (RA). Therefore, knowledge about monocyte activities and regulation of molecular pathways operating within monocytes early in the course of RA development may help to predict the progression to the full-blown disease. We aimed to investigate the profile of miRNAs expression in circulating monocytes and monocyte-related cytokines in sera of individuals at undifferentiated arthritis (UA) stage, wich could serve as new biomarkers for RA development. MATERIAL AND METHODS: Magnetically sorted monocytes from peripheral blood of 20 UA patients served for total RNA isolation. RNA samples were used for microRNA profiling. Concentrations of CCL3/MIP-1α, M-CSF, CCL2/MCP-1, IL-6, TNF-α and IL-15 in sera of UA patients were measured using ELISA assays. Verification of diagnosis after 4 years of follow-up led to the identification of patients who developed RA (UA→RA patients) and patients who remained still in UA phase (UA → UA patients). Comparisons between patients groups were performed using two-tailed Mann-Whitney U test. RESULTS: We identified 50 miRNAs in monocytes with the largest variation of expression across all patients samples. From these selected miRNAs, expression of miR-642b-5p, miR-483-3p, miR-371b-5p were significantly up-regulated and miR-25-3p and miR-378d were significantly down-regulated in UA → RA vs. UA → UA patients. This specific pattern of miRNAs expression in circulating monocytes paralleled elevated IL-15 and M-CSF concentrations in sera of UA patients who progressed to RA. CONCLUSIONS: Results of our pilot study indicate that altered activity of monocytes can be detected at early stages of RA. We found new miRNA candidates differentially expressed in peripheral blood monocytes and elevated concentrations of IL-15 and M-CSF involved in monocyte activity and differentiation in patients with UA who subsequently developed RA, in comparison to UA patients who did not progress to RA after 4 years follow-up.

Monocyte alterations in rheumatoid arthritis are dominated by preterm release from bone marrow and prominent triggering in the joint.

OBJECTIVE: Rheumatoid arthritis (RA) accompanies infiltration and activation of monocytes in inflamed joints. We investigated dominant alterations of RA monocytes in bone marrow (BM), blood and inflamed joints. METHODS: CD14+ cells from BM and peripheral blood (PB) of patients with RA and osteoarthritis (OA) were profiled with GeneChip microarrays. Detailed functional analysis was performed with reference transcriptomes of BM precursors, monocyte blood subsets, monocyte activation and mobilisation. Cytometric profiling determined monocyte subsets of CD14++CD16-, CD14++CD16+ and CD14+CD16+ cells in BM, PB and synovial fluid (SF) and ELISAs quantified the release of activation markers into SF and serum. RESULTS: Investigation of genes differentially expressed between RA and OA monocytes with reference transcriptomes revealed gene patterns of early myeloid precursors in RA-BM and late myeloid precursors along with reduced terminal differentiation to CD14+CD16+monocytes in RA-PB. Patterns associated with tumor necrosis factor/lipopolysaccharide (TNF/LPS) stimulation were weak and more pronounced in RA-PB than RA-BM. Cytometric phenotyping of cells in BM, blood and SF disclosed differences related to monocyte subsets and confirmed the reduced frequency of terminally differentiated CD14+CD16+monocytes in RA-PB. Monocyte activation in SF was characterised by the predominance of CD14++CD16++CD163+HLA-DR+ cells and elevated concentrations of sCD14, sCD163 and S100P. CONCLUSION: Patterns of less mature and less differentiated RA-BM and RA-PB monocytes suggest increased turnover with accelerated monocytopoiesis, BM egress and migration into inflamed joints. Predominant activation in the joint indicates the action of local and primary stimuli, which may also promote adaptive immune triggering through monocytes, potentially leading to new diagnostic and therapeutic strategies.

Rheumatoid arthritis bone marrow environment supports Th17 response.

BACKGROUND: Rheumatoid arthritis (RA) is a systemic, autoimmune disease leading to joint destruction and ultimately disability. Bone marrow (BM) is an important compartment in RA, where pathological processes from "outside the joint" can occur. IL-17 is a cytokine that exerts proinflammatory effects and participates in the process of bone destruction. It is believed that IL-17 is involved in pathogenesis of RA. However, little is known about the biology of this cytokine in BM. In the present study we investigated Th17-related cytokines in RA BM. METHODS: BM samples were obtained from RA and osteoarthritis (OA) patients during total hip replacement surgery. Levels of IL-17AF, IL-17AA, IL-17FF, IL-1ß, IL-6, IL-23, TGF-ß and CCL20 in BM plasma were determined by specific enzyme-linked immunosorbent assay tests. Percentage of IL-17-producing cells in BM was evaluated by flow cytometry. The effect of IL-15 stimulation on IL-17 production by BM mononuclear cells was examined in vitro. RESULTS: Increased levels of IL-17AF were observed in BM plasma of RA patients in comparison to OA patients. Increased concentrations of IL-1ß, IL-6 and CCL20 were observed in RA compared to OA BM plasma. Concordant with these findings, significantly increased percentages of CD3+CD4+IL-17+ and CD3+CD4+IL-17+IFN-γ+ cells were present in RA BM in comparison to OA BM samples. Finally, abundant in RA BM, IL-15 increased IL-17 production by cultured BM mononuclear cells. CONCLUSIONS: In the course of RA, the BM microenvironment can promote the development of Th17 cell responses and overproduction of IL-17AF that may lead to increased inflammation and tissue destruction in RA BM.

The role of microRNA-5196 in the pathogenesis of systemic sclerosis.

BACKGROUND: Systemic sclerosis (SSc) is a chronic autoimmune disease characterised by tissue fibrosis and immune abnormalities. Recent evidence suggests that activated circulating monocytes from patients with SSc play an important role in early stages of SSc pathogenesis due to enhanced expression of tissue inhibitor of metalloproteinases 1 (TIMP-1), IL-8 and reactive oxygen species (ROS) induction. However, the exact factors that contribute to chronic inflammation and subsequently fibrosis progression are still unknown. MATERIALS AND METHODS: The expression pattern of IL-8, TIMP-1, AP-1 transcription factor-Fra2 and ROS induction in peripheral blood monocytes following DZNep (histone methyltransferase inhibitor) and TLR8 agonist stimulation was investigated. Exogenous microRNA-5196, which is predicted to bind 3'UTR of Fra2 gene, was delivered to reverse profibrotic phenotype in monocytes. Expression of circulating microRNA-5196 was correlated with SSc parameters. RESULTS: DZNep + TLR8 agonist stimulation enhanced profibrotic TIMP-1, IL-8 and ROS generation in HC and SSc monocytes. As opposed by the decrease of miRNA-5196 and antioxidant SOD1 expression in SSc monocytes. Exogenous delivery of microRNA-5196 reduced Fra2 and TIMP-1 expression suggesting that it may be used as a potential modulator of fibrogenesis in SSc. Circulating microRNA-5196 was significantly increased in SSc and positively correlated with CRP level but not with Rodnan skin score or ESR. CONCLUSIONS: These results suggest that microRNA-5196 can be used as a potential biomarker characterising SSc. Overall, this study may open new possibilities for the development of microRNA-5196-based diagnostics and therapy in early phases of SSc.