Investigating the causes and consequences of miR-34a dysregulation in rheumatoid arthiritis

Rheumatoid arthritis (RA) is a chronic, inflammatory disorder, whereby synovial inflammation ultimately results in joint destruction. Although the joints are the main target tissues affected, RA is also associated with a number of co-morbidities - such as cardiovascular disease. A key cell type involved in the perpetuation of disease pathogenesis is the macrophage, but the mechanisms underlying inflammatory gene expression in these cells are not fully understood. One fascinating and rather novel area of research that could provide insights into the activation of macrophages in RA comprises the biology of microRNA. These small, non-coding RNA molecules are implicated in the post-transcriptional regulation of gene expression. Here, we show that the expression of one particular microRNA, miR-34a, is increased in synovial fluid CD14+ cells compared to matched peripheral blood cells from RA patients. We have demonstrated that miR-34a expression is increased in synovial tissues from RA patients compared to osteoarthritis comparators, and that a proportion of these miR-34a positive cells are CD68+ macrophages. Of particular interest, miR-34a was also up-regulated in peripheral blood CD14+ cells isolated from multiple drug-resistant RA patients compared to healthy controls. Using over and under-expression methodologies we were able to demonstrate that miR-34a over-expression reduces toll like receptor-induced cytokine production by macrophages, while miR-34a inhibition enhances cytokine production. The altered cytokine activities included TNFα and IL-6 that are both critically linked to disease pathogenesis, therefore we propose that miR-34a over-expression in RA macrophages represents a failed attempt to attenuate on-going inflammation. To further explore the mechanism of miR-34a action, a microarray was performed to investigate transcripts that were regulated in response to miR-34a over-expression in monocytes. This study uncovered several pathways, including interferon, metallothionein and chemokine pathways, wherein many members were down-regulated upon miR-34a over-expression. Future work will therefore aim to dissect the role of these pathways, and their relevance to miR-34a regulated macrophage and dendritic cell biology, and thus to the chronicity of synovitis

Regulation and function of SIRT1 in rheumatoid arthritis synovial fibroblasts

Rheumatoid arthritis (RA) is a chronic autoimmune disease characterized by inflammation and destruction of synovial joints. The function of sirtuin (SIRT)1 in RA is inconclusive. In human synovial cells, SIRT1 was shown to promote cytokine production and apoptosis resistance. However, deletion of SIRT1 aggravated inflammatory arthritis in mice and increased production of pro-inflammatory cytokines in murine macrophages. In the current study, we investigated the regulation, expression, and function of SIRT1 in RA, in particular its role in adhesion and proliferation of human RA synovial fibroblasts (RASF). We found that expression of SIRT1 was increased in vivo in synovial tissues of RA smokers and in vitro by stimulation of RASF with TNFα, but decreased upon treatment with cigarette smoke extract. Synovial tissues of RA smokers showed higher leukocytic infiltration that positively correlated with enhanced levels of SIRT1. Global transcriptome analysis revealed that SIRT1 modulates expression of genes involved in the regulation of inflammatory response and cell adhesion. In functional studies, silencing of SIRT1 reduced proliferation and leukocytic adhesion to RASF but showed inconsistent results in the regulation of adhesion to plastic. In conclusion, SIRT1 modulates the proliferative and potentially also adhesive properties of RASF and can therefore promote progression of RA. KEY MESSAGES: SIRT1 is upregulated by TNFα but decreased upon CSE treatment of RASF. Upregulation of SIRT1 in RA smokers correlates with increased leukocytic infiltration. SIRT1 modulates expression of genes regulating cell adhesion and inflammation. SIRT1 regulates proliferation of RASF

Quantification of human complement C2 protein using an automated turbidimetric immunoassay

Background: The measurement of complement components is clinically useful where a deficiency is suspected, or where excessive activation and consumption are present in disease. C2 deficiency carries an increased risk of developing systemic lupus erythematosus, recurrent infections and atherosclerosis. In this study, we have evaluated The Binding Site’s Human Complement C2 SPAPLUS® assay. Methods: Linearity was tested using 13 sample dilutions covering the standard measuring range. Within- and between-assay variabilities were calculated using five samples with different C2 concentrations. The correlation between C2 concentrations in EDTA-plasma and serum was assessed, as was the correlation between C2 measurements by the automated assay and radial immunodiffusion. C2 concentrations were compared with CH50 activity, and quantified in individuals with homozygous or heterozygous C2 deficiency, acquired angioedema and patients with chronic inflammatory conditions. Results: The assay was linear across the measuring range (3.8–42.3 mg/L). Intra- and interassay variability were 2.3%–3.8% and 0%–3.3%, respectively. Comparison between C2 measurements in EDTA-plasma and serum provided a strong correlation (p<0.0001, R2=0.82, slope 0.92), as did the correlation between the automated and radial immunodiffusion methods (p<0.0001, R2=0.89, slope 1.07). A positive correlation between C2 concentration and CH50 activity was demonstrated (p<0.0001, R2=0.48). Significant differences were observed between the median C2 concentrations obtained in healthy controls and the patient clinical samples, with homozygous C2-deficient patients giving below detectable results. Conclusions: This C2 SPAPLUS® assay allows the automated, rapid and precice quantification of complement C2 protein and could therefore be considered as a replacement for older, more time-consuming methods

Quantification of human C1 esterase inhibitor protein using an automated turbidimetric immunoassay

BACKGROUND: Impaired levels or function of C1 inhibitor (C1-INH) results in angioedema due to increased bradykinin. It is important to distinguish between angioedema related to C1-INH deficiency and that caused by other mechanisms, as treatment options are different. In hereditary (HAE) and acquired (AAE) angioedema, C1-INH concentration is measured to aid patient diagnosis. Here, we describe an automated turbidimetric assay to measure C1-INH concentration on the Optilite® analyzer. METHODS: Linearity, precision, and interference were established over a range of C1-INH concentrations. The 95th percentile reference interval was generated from 120 healthy adult donors. To compare the Optilite C1-INH assay with a predicate assay used in a clinical laboratory, samples sent for C1-INH investigation were used. The predicate results were provided to allow comparison. RESULTS: The Optilite C1-INH assay was linear across the measuring range at the standard sample dilution. Intra and interassay variability was <6%. The 95th percentile adult reference interval for the assay was 0.21-0.38 g/L. There was a strong correlation between the Optilite concentrations and those generated with the predicate assay (R2 = 0.94, P < 0.0001, slope y = 0.83x). All patients with Type I HAE (n = 24) and AAE (n = 3) tested had concentrations below the measuring range in both assays, while all patients with unspecified angioedema (UAE), not diagnosed with HAE or AAE had values within the reference range. CONCLUSION: The Optilite assay allows the automated and precise quantification of C1-INH concentrations in patient samples. It could therefore be used as a tool to aid the investigation of patients with angioedema

MicroRNA-34a dependent regulation of AXL controls the activation of dendritic cells in inflammatory arthritis

Current treatments for rheumatoid arthritis (RA) do not reverse underlying aberrant immune function. A genetic predisposition to RA, such as HLA-DR4 positivity, indicates that dendritic cells (DC) are of crucial importance to pathogenesis by activating auto-reactive lymphocytes. Here we show that microRNA-34a provides homoeostatic control of CD1c+ DC activation via regulation of tyrosine kinase receptor AXL, an important inhibitory DC auto-regulator. This pathway is aberrant in CD1c+ DCs from patients with RA, with upregulation of miR-34a and lower levels of AXL compared to DC from healthy donors. Production of pro-inflammatory cytokines is reduced by ex vivo gene-silencing of miR-34a. miR-34a-deficient mice are resistant to collagen-induced arthritis and interaction of DCs and T cells from these mice are reduced and do not support the development of Th17 cells in vivo. Our findings therefore show that miR-34a is an epigenetic regulator of DC function that may contribute to RA

Mir-34a in Rheumatoid Arthritis: characterisation of elevated synovial expression and association with treatment resistance

Background/Purpose: Cells of the monocyte/macrophage lineage are critical to RA pathogenesis: unravelling mechanisms underlying macrophage inflammatory gene expression should elucidate novel disease-associated pathways and thereby biomarkers and therapeutic targets. MicroRNA (miR) comprise small, non-coding RNA species that are key regulators of mammalian gene expression. We sought to define the expression and regulation of novel miR species that regulate RA macrophage biology.&lt;p&gt;&lt;/p&gt; Methods: Blood (PB; healthy controls and RA patients with defined treatment characteristics) or synovial fluid (SF; RA) CD14+ cells were purified using histopaque centrifugation and autoMACS bead separation. CD14+ cells were matured with M-CSF or GM-CSF for up to 7 days, or stimulated with various TLR ligands. For T cell -macrophage interaction, CD4+ T cells were cultured with TNF, IL-2, IL-6, prior to 24hr co-culture with M-CSF-matured CD14+ cells. miR expression and copy number was variously quantified in cells or tissues via TLDA, validatory qPCR and in situ hybridisation with specific primers and DIG or FITC labelled probes, respectively.&lt;p&gt;&lt;/p&gt; Results: Prior TLDA elicited several dysregulated miRs in RA SF compared to PB CD14+ cells, including miR-34a. Fold change and copy number PCR assays confirmed elevated miR-34a in RA SF cells (n=10; p &lt;0.01). Crucially miR-34a was also elevated in PB CD14+ cells from biologic-resistant RA patients compared to cDMARD good responders or matched healthy controls (n=19–30), associating miR-34a expression with chronic, treatment-resistance. miR-34a expression correlated with disease activity assessed by swollen joint count. In situ hybridisation demonstrated elevated miR-34a expression in RA compared with noninflammatory and inflammatory OA synovial tissues (n=6); double fluorescent staining confirmed expression in lining and sub-lining layer CD68+ macrophages, plus adjacent cells of FLS morphology. miR-34a was rapidly upregulated during monocyte maturation induced by adherence, M-CSF or GM-CSF, but was not further enhanced by addition of TLR ligands (LPS, CLO97, PAM3, PolyIC, CpG). Similarly co-culture with RA derived SF (10%) enhanced miR-34a expression in monocytes. Finally, cognate interactions between CD4+ T cells and macrophages further enhanced miR-34a expression in the latter cells.&lt;p&gt;&lt;/p&gt; Conclusion: miR-34a expression is elevated in RA SF macrophages and in PB monocytes of treatment resistant RA patients where it correlates with clinical disease activity measures. miR-34a is upregulated by the maturation rich synovial microenvironment and by interactions with a activated T cells. Putative miR-34a molecular targets include NOTCH1 rendering it a plausible novel immune regulator in synovitis.&lt;p&gt;&lt;/p&gt

ILDR2 is a novel B7-like protein that negatively regulates T cell responses

The B7-like protein family members play critical immunomodulatory roles and constitute attractive targets for the development of novel therapies for human diseases. We identified Ig-like domain-containing receptor (ILDR)2 as a novel B7-like protein with robust T cell inhibitory activity, expressed in immune cells and in immune-privileged and inflamed tissues. A fusion protein, consisting of ILDR2 extracellular domain with an Fc fragment, that binds to a putative counterpart on activated T cells showed a beneficial effect in the collagen-induced arthritis model and abrogated the production of proinflammatory cytokines and chemokines in autologous synovial-like cocultures of macrophages and cytokine-stimulated T cells. Collectively, these findings point to ILDR2 as a novel negative regulator for T cells, with potential roles in the development of immune-related diseases, including autoimmunity and cancer