Small non-coding RNAs have emerged as possible biomarkers for various diseases including autoimmune diseases. A number of studies have demonstrated that the expression of specific microRNAs (miRNAs) is dysregulated in rheumatoid arthritis (RA). So far, all studies on miRNAs in RA patients have been performed using either microarray or reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analyses. Compared to RT-qPCR and microarray analyses, next-generation sequencing (NGS) allows the genome-wide analysis of small RNAs and the differentiation between miRNAs that differ by a single nucleotide. The application of NGS to the analysis of small RNAs circulating in sera of RA patients has not been reported. This study provides a global overview of the circulating small RNAs in the sera of RA patients and healthy subjects and identifies differences between these groups using NGS. Several classes of small RNAs, including hY RNA-derived fragments, tRNA-derived fragments and miRNAs, were determined. Differentially expressed individual small RNAs were verified by RT-qPCR. The levels of two miRNAs, miR-223-3p and miR-16-5p, were significantly lower in the sera from early RA patients than in those from established RA patients and healthy controls. In contrast, the serum level of miR-16-5p was higher in patients with established RA than in healthy control samples. These miRNAs may not only serve as biomarkers, but may also shed more light on the pathophysiology of RA.

Introduction: The pathophysiology of systemic sclerosis (SSc) is closely linked to overactive TGFβ signaling. TGFβ is produced and circulates in latent form, making its activation crucial for signaling. This activation can be mediated via integrins. We investigated the balance between active and latent TGFβ in serum of SSc patients and investigated if this correlates with integrin expression on monocytes.Methods: A TGFβ/SMAD3-or BMP/SMAD1/5-luciferase reporter construct was expressed in primary human skin fibroblasts. Both acidified and non-acidified sera of ten SSc patients and ten healthy controls were tested on these cells to determine total and active TGFβ and BMP levels respectively. A pan-specific TGFβ1/2/3 neutralizing antibody was used to confirm TGFβ signaling. Monocytes of 20 SSc patients were isolated using CD14+ positive selection, and integrin gene expression was measured using qPCR. Integrin expression was modulated using rhTGFβ1 or a small molecule inhibitor of TGFBR1: SB-505124.Results: SSc sera induced 50% less SMAD3-reporter activity than control sera. Serum acidification increased reporter activity, but a difference between healthy control and SSc serum was no longer observed, indicating that total TGFβ levels were not different. Addition of a pan-specific TGFβ1/2/3 neutralizing antibody fully inhibited SMAD3reporter activity of both acidified and not-acidified control and SSc sera. Both HC and SSc sera induced similar SMAD1/5-reporter activity, and acidification increased this, but not differently between groups. Interestingly, expression of two integrin alpha subunits ITGA5 and ITGAV was significantly reduced in monocytes obtained from SSc patients. Furthermore, ITGB3, ITGB5, and ITGB8 expression was also reduced in SSc monocytes. Stimulation of monocytes with TGFβ1 induced ITGA5 and ITGAV but lowered ITGB8 expression, whereas the use of the TGFβ receptor inhibitor SB-505124 had the opposite effect.Conclusion: Total TGFβ serum levels are not different between SSc patients and controls, but TGFβ activity is. This coincides with a reduced expression of TGFβ-activating integrins in monocytes of SSc patients. Because TGFβ regulates expression of these integrins in monocytes, a negative feedback mechanism possibly underlies these observations.

Background Rheumatoid arthritis (RA) is characterised by the presence of autoantibodies and infiltration of B and T-cells in synovial joints. Accordingly, targeting B-cells by rituximab treatment has been shown to be beneficial for a subset of RA patients. Despite a rapid depletion of B-cells in peripheral blood, B-cells may persist in synovial tissue and bone marrow. Since B-cell differentiation occurs in secondary lymphoid tissue we investigated the effect of rituximab treatment on B and T-cell subsets in lymphoid biopsies of RA patients. Methods Fourteen patients with active RA (baseline Disease Activity Score 28 joint assessment (DAS28) was 5.3 (4.7–6.0 [median, IQR]) were treated with intravenous infusions of 1000 mg of rituximab on day 1 and day 15. Premedication with methylprednisolone was omitted to study the specific effects of rituximab. Ultrasound-guided inguinal lymph node biopsies were obtained before start of rituximab treatment and four weeks after the first infusion and immediately processed for flow cytometry analyses. For comparison 5 lymph node biopsies from healthy controls were analysed. Results Lymph node B-cell subsets in RA patients were not significantly increased compared to healthy controls, while frequencies of early activated T-cells, CD3+CD69+ and CD3+CD25+CD69+ were significantly increased (p = 0.01 and p = 0.02 respectively). The frequencies of switched memory B-cells (CD27+IgD-) correlated significantly with the frequency of early activated T-cells (CD3+CD69+ p = 0.02, r = 0.65; CD3+CD25+CD69+ p = 0.002, r = 0.80). EULAR response after 24 weeks was as follows: 29% good, 50% moderate and 21% non-responders. Four weeks after treatment there was a complete depletion of B-cells in peripheral blood (cut off < 0.0001 × 109/Litre) in 9 of the 13 patients measured. In lymph nodes, 4 weeks after rituximab treatment, total CD19+ B-cells were significantly but incompletely depleted (p = 0.0004). CD19 frequency before treatment was 21.3% (13.8–32.4 [median, IQR]) and CD19 frequency after treatment was 9.7% (1.3–14.9 [median, IQR]). Especially naive (CD27-IgD+; p = 0.0006) and unswitched memory B-cells (CD27+IgD+; p = 0.0012) significantly decreased, while switched memory B-cells (CD27+IgD-) and double negative B-cells (CD27-IgD-) persisted. Also, activated T-cells (CD3+CD25+CD69+ T; p = 0.03) significantly decreased. No correlation was found between lymphoid B or T-cell subset depletion and response to treatment. Conclusion This study provides for the first time detailed insight in the effects of rituximab therapy on lymph node immune cells in RA patients. Of interest, rituximab not only affects the number of lymphoid B-cells but also reduced the number of activated lymphoid T-cells suggesting that lymphoid B-cells contribute to lymphoid T-cell activation in RA.