Klára Prajzlerová

MicroRNAs (miRNAs) are small non-coding single-stranded RNAs of about 22 nucleotides in length that act as post-transcriptional regulators of gene expression. Depending on the complementarity between miRNA and target mRNA, cleavage, destabilization, or translational suppression of mRNA occurs within the RISC (RNA-induced silencing complex). As gene expression regulators, miRNAs are involved in a variety of biological functions. Dysregulation of miRNAs and their target genes contribute to the pathophysiology of many diseases, including autoimmune and inflammatory disorders. MiRNAs are also present extracellularly in their stable form in body fluids. Their incorporation into membrane vesicles or protein complexes with Ago2, HDL, or nucleophosmin 1 protects them against RNases. Cell-free miRNAs can be delivered to another cell in vitro and maintain their functional potential. Therefore, miRNAs can be considered mediators of intercellular communication. The remarkable stability of cell-free miRNAs and their accessibility in body fluid makes them potential diagnostic or prognostic biomarkers and potential therapeutic targets. Here we provide an overview of the potential role of circulating miRNAs as biomarkers of disease activity, therapeutic response, or diagnosis in rheumatic diseases. Many circulating miRNAs reflect their involvement in the pathogenesis, while for plenty, their pathogenetic mechanisms remain to be explored. Several miRNAs described as biomarkers were also shown to be of therapeutic potential, and some miRNAs are already tested in clinical trials.

Circulating miRNAs appear promising therapeutic and prognostic biomarkers. We aimed to investigate the predictive value of circulating miRNAs on the disease outcome following anti-TNF therapy in patients with ankylosing spondylitis (AS). Our study included 19 AS patients assessed at baseline (M0), after three (M3) and twelve months (M12) of therapy. Total RNA was isolated from plasma. A comprehensive analysis of 380 miRNAs using TaqMan Low Density Array (TLDA) was followed by a single assay validation of selected miRNAs. All AS patients had high baseline disease activity and an excellent response to anti-TNF therapy at M3 and M12. TLDA analysis revealed the dysregulation of 17 circulating miRNAs, including miR-145. Single assay validation confirmed that miR-145 is significantly downregulated at M3 compared to baseline. The decrease in the levels of miR-145 from M0 to M3 negatively correlated with the change in BASDAI from M0 to M3; and positively correlated with disease activity improvement from M3 to M12 as per BASDAI and ASDAS. The predictive value of the early change in miR-145 and levels of miR-145 at M3 were further validated by Receiver operating curves analysis. We show that the early change in circulating miR-145 may be a predictor for the future outcome of AS patients treated with TNF inhibitors. Patients with a more significant decrease in miR-145 levels may show further significant improvement of disease activity after 12 months. Monitoring the expression of miR-145 in plasma in AS patients may, therefore, influence our therapeutic decision-making.

Individuals carrying anti-citrullinated protein antibodies (ACPA) are considered at high risk of developing rheumatoid arthritis (RA). The altered expression of miRNAs contributes to the pathogenesis of RA. We aimed to identify differentially expressed miRNAs in the peripheral blood of ACPA-positive individuals with arthralgia at risk of RA compared to healthy controls (HC) and to determine their implications in the preclinical phase of RA. A comprehensive analysis of miRNAs revealed the dysregulation of miR-451 in peripheral blood mononuclear cells (PBMC) and plasma from RA-risk individuals. Higher miR-451 expression in PBMC from RA-risk individuals was further validated. Notably, miR-451 was previously shown to regulate CXCL16, a protein involved in RA pathogenesis. The expression of miR-451 in PBMC positively correlated with the CXCL16 mRNA, which could be secondary to the inflammation-induced expression of miR-451. Transfection of monocytes with pre-miR-451 in vitro resulted in the downregulation of CXCL16. Moreover, flow cytometry revealed a lower count of CXCL16-positive monocytes in RA-risk individuals. We propose that the constitutive or inflammation-induced upregulation of miR-451 in PBMC downregulates the expression of CXCL16, reduces the inflammatory milieu and thereby strives to delay the shift from the preclinical phase to the clinical manifestation of RA. This hypothesis warrants further investigation.

Individuals carrying antibodies against citrullinated proteins (ACPA) are at high risk of developing RA. EULAR provided a clinical definition of individuals with arthralgia suspicious for progression to RA (clinically suspect arthralgia, CSA). The alteration of monocyte subpopulations in patients with established RA has been previously described. We analysed peripheral blood monocyte subpopulations in individuals with arthralgia at risk of RA. We included 70 at-risk individuals, defined as having arthralgia without arthritis and being either ACPA⁺ or meeting the clinical CSA definition, 23 patients with early RA (ERA) and 19 healthy controls (HCs). Monocytes classified as classical (CD14⁺⁺CD16⁻), intermediate (CD14⁺⁺CD16^+/++) and nonclassical (CD14^−/+CD16⁺⁺) were analysed by flow cytometry. Of the 70 at-risk individuals, 46 were ACPA⁺ and 45 met the CSA definition. The at-risk individuals and, especially, ERA patients had a lower percentage of classical monocytes and a higher percentage of nonclassical monocytes than the HCs. ACPA positivity had no effect on the difference in the distribution of the monocyte subsets between at-risk individuals and ERA patients, but a difference was determined in those reaching the ERA phase. However, when compared with HCs, the shift of monocyte subsets was more significant in ACPA⁺ than in ACPA⁻ individuals with arthralgia. This trend was observed in individuals who did not meet the CSA definition. This finding was, however, determined by a selection bias, as these individuals were solely ACPA⁺. The shift from classical to nonclassical monocyte subpopulations was observed already in individuals at risk of developing RA.

Background: S100A4 is a member of calcium binding S100 protein family well known for its role in cancer progression and metastasis. Nevertheless, S100A4 also serves as a negative regulator of bone formation. Dickkopf-1 (DKK-1), marker of bone remodelling, is also implicated in the process of syndesmophyte formation in ankylosing spondylitis. The aim of our study was to evaluate plasma levels of S100A4 in patients with axial spondyloarthritis and to determine the potential association of S100A4 with disease severity, clinical manifestations and with bone changes in a cross-sectional study. Methods: Fifty-eight patients with axial spondyloarthritis and 40 healthy controls were studied. Biological samples were analysed for S100A4 and Dickkopf-1. Disease activity was assessed according to the Bath Ankylosing Spondylitis Disease Activity Index. C-reactive protein (CRP) was used as a marker of inflammation. Radiographic damage was assessed using the modified Stoke Ankylosing Spondylitis Spinal Score (mSASSS). Results: The plasma levels of S100A4 were significantly higher in patients with axial spondyloarthritis compared to heathy controls (p < 0.0001). The levels of S100A4 were higher in early stages of the disease and lower in patients with the presence of syndesmophytes (p = 0.009). Furthermore, we found weak but significant inverse correlation of plasma S100A4 with the mSASSS (r = − 0.363, p = 0.030). Levels of S100A4 were negatively associated with disease duration (r = − 0.404, p = 0.002) and positively with Dickkopf-1 binding capacity (r = 0.312, p = 0.023). Conclusions: This is the first study showing elevated circulating levels of S100A4 in patients with axial spondyloarthritis, particularly in early stages of the disease prior to spinal involvement, and its significantly lower levels in patients with syndesmophytes. The role of S100A4 in the pathogenesis of axial spondyloarthritis can be suggested.

Dysregulation of miRNAs and their target genes contributes to the pathophysiology of autoimmune diseases. Circulating miRNAs may serve as diagnostic/prognostic biomarkers. We aimed to investigate the association between circulating miRNAs, disease activity and spinal involvement in patients with axial spondyloarthritis (AxSpA). Total RNA was isolated from the plasma of patients with non-radiographic (nr)AxSpA, patients with ankylosing spondylitis (AS) and healthy controls (HC) via phenol-chloroform extraction. A total of 760 miRNAs were analysed with TaqMan^® Low Density Arrays, and the expression of 21 miRNAs was assessed using single assays. Comprehensive analysis demonstrated the differential expression of miRNAs among patients with progressive spinal disease. Of the 21 miRNAs selected according to their expression patterns, the levels of miR-625-3p were significantly different between nr-AxSpA patients and HCs. We found no correlation between miRNA levels and Bath Ankylosing Spondylitis Disease Activity Index (BASDAI) in nr-AxSpA patients. Selected miRNAs, such as miR-29a-3p, miR-146a-5p or miR-222-3p with an established role in extracellular matrix formation and inflammation were associated with spinal changes and/or disease activity assessed by BASDAI in AS patients, including miR-625-3p reflecting disease activity in AS with spinal involvement. Our data indicate that circulating miRNAs play a role in the pathogenesis of AxSpA and are also suggestive of their potential as biomarkers of disease progression. We hypothesize that differential systemic levels of miRNA expression reflect miRNA dysregulation at sites of spinal inflammation or bone formation where these molecules contribute to the development of pathophysiological features typical of AxSpA.

Calgizzarin (S100A11) is a member of the S100 protein family that acts in different tumors by regulating a number of biologic functions. Recent data suggest its association with low-grade inflammation in osteoarthritis (OA). The aim of our study is to compare S100A11 expression in the synovial tissues, synovial fluid and serum of patients with rheumatoid arthritis (RA) and osteoarthritis (OA) and to characterize the potential association between S100A11 and disease activity. S100A11 protein expression was detected in synovial tissue from patients with RA (n = 6) and patients with OA (n = 6) by immunohistochemistry and immunofluorescence. Serum and synovial fluid S100A11 levels were measured by ELISA in patients with RA (n = 40) and patients with OA (n = 34). Disease activity scores in 28 joints based on C-reactive protein (DAS28-CRP) were used to assess disease activity. Cytokine content in peripheral blood mononuclear cells (PBMCs), synovial fibroblasts (SFs) and synovial fluid was analysed by ELISA, western blotting or cytometric bead array. S100A11 expression was significantly up-regulated in the synovial lining and sublining layers (p < 0.01) and vessels (p < 0.05) of patients with RA compared to patients with OA, and was associated with fibroblasts and T cells. S100A11 was significantly increased in synovial fluid (p < 0.0001) but not in serum (p = 0.158) from patients with RA compared to patients with OA when adjusted for age and sex. Synovial fluid S100A11 correlated with DAS28 (r = 0.350, p = 0.027), serum CRP (r = 0.463, p = 0.003), synovial fluid leukocyte count (r = 0.677, p < 0.001), anti-cyclic citrullinated peptide antibodies (anti-CCP) (r = 0.424, p = 0.006) and IL-6 (r = 0.578, p = 0.002) and IL-8 (r = 0.740, p < 0.001) in synovial fluid from patients with RA. PBMCs and SFs isolated from patients with RA synthesized and spontaneously secreted higher levels of S100A11 in comparison with PBMCs and SFs from patients with OA (p = 0.011 and 0.03, respectively). S100A11 stimulated the production of the pro-inflammatory cytokine IL-6 by PBMCs (p < 0.05) and SFs (p < 0.01). Our data provide the first evidence of S100A11 up-regulation and its association with inflammation and disease activity in patients with RA.

MicroRNAs (miRNAs) are small RNAs that regulate gene expression by targeting mRNA. It was proved that some miRNAs are significantly deregulated in rheumatoid arthritis (RA). MicroRNA-125b negatively regulates expression of TNF-α, which plays a crucial role in RA pathogenesis. The aim of this study was to determine the treatment outcome of patients with early RA based on the expression of circulating and cellular miR-125b. Total RNA was isolated from the plasma and peripheral blood mononuclear cells (PBMCs) of 58 patients with early RA before and three months after treatment initiation and of 54 age- and sex-matched healthy controls (HC). The expression of miR-125b was measured by TaqMan quantitative PCR. The treatment responders were defined as patients achieving remission or low disease activity (28-joint count disease activity score (DAS28) <3.2). Receiver operating characteristic (ROC) curve and stepwise backward multivariable logistic regression analyses of miR-125b expression were used to predict the disease outcome at three and six months after initiation of treatment. The expression of miR-125b in the PBMCs and plasma of treatment-naïve early RA patients was significantly lower than that of HC and increased significantly after three months of treatment, particularly in responders. However, only the cellular expression of miR-125b was inversely correlated with disease activity. MiR-125b expression in PBMCs was higher in responders than in non-responders after three months (p = 0.042). Using ROC analysis, the cellular expression of miR-125b, but not the disease activity at baseline, predicted the treatment response after three months of therapy (area under the curve 0.652 (95 % CI 0.510 to 0.793); p = 0.048). The expression of miR-125b in PBMCs of treatment-naïve patients may present a novel biomarker for monitoring the treatment outcome during the early phase of RA.

Background We have previously shown differential expression of some circulating miRNAs in patients with early RA (ERA) and their potential as markers of disease activity and treatment response. Objectives We aimed to investigate differentially expressed circulating miRNAs in clinically suspect arthralgia patients with positive antibodies to citrullinated peptide antigens (ACPA) who are at high risk of developing RA and compared them to ERA and established RA. Methods The study included 20 ACPA+ arthralgia patients, 25 treatment naïve ERA patients who met the 2010 ACR/EULAR criteria (disease duration Results Patients with arthralgia showed no clinical and ultrasound evidence of arthritis (grey scale and power Doppler for single joint ≤1) Of these, all were ACPA+, CRP 7.4±19.2 mg/l. Treatment naïve ERA patients had active disease (DAS28 5.7±1.3, CRP 20.7±23.2 mg/l), 60% were ACPA+. Patients with established RA all had high disease activity (DAS28 6.6±0.9, CRP 28.1±31.3 mg/l), 73% were ACPA+, had failed DMARDs therapy and were due to commence anti-TNF treatment. Out of the 380 miRNAs analysed by TLDA, 128 miRNAs were detected in HC, 129 in arthralgia, 44 in ERA and 25 in RA patients. TLDA analysis revealed 1.7x higher levels of miR-126 in ACPA+ arthralgia patients compared to HC, and 27x and 63x lower levels in ERA and RA compared to HC, respectively. These data were suggestive of significant dysregulation of miR-126 in patients with active polyarthritis compared to ACPA+ arthralgia. However, further validation showed no difference in miR-126 levels among HC, arthralgia and ERA patients but the levels were 2.6x lower in RA patients compared to HC (p=0.0037). The levels of miR-126 significantly negatively correlated with DAS28 (r=-0.460; p=0.047), SDAI (r=-0.535; p=0.018), SJC (r=-0.504; p=0.028), and TJC (r=-0.517; p=0.023) in treatment naïve ERA patients, but not in those with established disease. There was no correlation with CRP in any patient group. Conclusions Higher miR-126 levels reflect lower disease activity in treatment naïve ERA patients. We hypothesize that significant downregulation of miR-126 levels in RA patients may be modified by previous exposure to multiple treatments. Acknowledgement MHCR 023728 project Disclosure of Interest None declared