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 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

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

Mir-125a: A novel regulator of IL-6 and TLR driven pathways in RA pathogenesis

Background/Purpose: Molecular mechanisms driving disease initiation and chronicity in RA are incompletely understood. There is increasing interest in the role played therein by microRNAs–small RNA species that mediate post-transcriptional regulation of integrated pathways in mammalian cells. We recently profiled miRs in RA synovial fluid (SF) CD14+ macrophages in comparison to peripheral blood (PB) monocytes and identified dysregulation of miR-125a, which has not previously been associated with RA pathophysiology. Herein we aimed to characterise its expression and functional significance. Methods: Matched PB and SF CD14+ cells were isolated from RA patients (n=10). Additional PB samples were obtained from RA DMARD good responders (n=17), recurrent non-responders (n=11) and matched healthy controls (n=17). Primary human monocytes and THP-1 cells were stimulated with TLR ligands (LPS, PAM3, PolyIC, CLO97) and differentiated using M-CSF or GM-CSF for up to 7 days. Copy number of miR-125a was evaluated using qPCR. Targeted pathways were identified using prediction algorithms (e.g. TargetScan) and transcriptional profiling of SF CD14+ cells. Direct molecular interactions were confirmed using luciferase reporters. miR-125a or control mimic were transfected into THP-1 cells and IL-6R expression was evaluated by flow cytometry. Results: miR-125a was up-regulated in RA SF CD14+ macrophages compared to PB controls (p=0.002). Moreover, miR-125a was up-regulated in RA PB CD14+ monocytes compared with healthy controls, regardless of DMARD response status (p &lt;0.02). Copy number of miR-125a in resting control PB monocytes was low. Extensive activation profiling revealed M-CSF, GM-CSF and 10% RA SF all induced miR125a expression at discrete time-points between 24h and 7ds. Of TLR ligands tested, only TLR4 agonism increased miR125a. Prediction algorithms identified members of the IL-6 signalling pathway (IL-6R, gp130) as potential targets of miR-125a. Luciferase reporter assays thereafter confirmed functional target interactions. Transfection of THP-1 cells with miR-125a but not control mimic reduced IL-6R membrane expression measured by FACS. miR-125a also targeted the negative regulators of TLR signalling, TNFAIP3 and IRF4, assessed by TargetScan and luciferase reporter. Commensurate with this IRF4 was down-regulated in RA PB monocytes (both DMARD good and recurrent non-responders) compared to healthy donors. Conclusion: Inflammatory cytokines, maturation factors and articular DAMPs drive elevated miR-125a expression in monocyte/macrophage lineages. miR-125a, in turn represents a novel molecular pathway that cross regulates IL-6R and TLR pathway activation in RA macrophages. We conclude that miR-125a represents an intriguing molecular marker with therapeutic and biomarker potential

Mir-125a: A novel regulator of IL-6 and TLR driven pathways in RA pathogenesis

Background/Purpose: Molecular mechanisms driving disease initiation and chronicity in RA are incompletely understood. There is increasing interest in the role played therein by microRNAs–small RNA species that mediate post-transcriptional regulation of integrated pathways in mammalian cells. We recently profiled miRs in RA synovial fluid (SF) CD14+ macrophages in comparison to peripheral blood (PB) monocytes and identified dysregulation of miR-125a, which has not previously been associated with RA pathophysiology. Herein we aimed to characterise its expression and functional significance. Methods: Matched PB and SF CD14+ cells were isolated from RA patients (n=10). Additional PB samples were obtained from RA DMARD good responders (n=17), recurrent non-responders (n=11) and matched healthy controls (n=17). Primary human monocytes and THP-1 cells were stimulated with TLR ligands (LPS, PAM3, PolyIC, CLO97) and differentiated using M-CSF or GM-CSF for up to 7 days. Copy number of miR-125a was evaluated using qPCR. Targeted pathways were identified using prediction algorithms (e.g. TargetScan) and transcriptional profiling of SF CD14+ cells. Direct molecular interactions were confirmed using luciferase reporters. miR-125a or control mimic were transfected into THP-1 cells and IL-6R expression was evaluated by flow cytometry. Results: miR-125a was up-regulated in RA SF CD14+ macrophages compared to PB controls (p=0.002). Moreover, miR-125a was up-regulated in RA PB CD14+ monocytes compared with healthy controls, regardless of DMARD response status (p &lt;0.02). Copy number of miR-125a in resting control PB monocytes was low. Extensive activation profiling revealed M-CSF, GM-CSF and 10% RA SF all induced miR125a expression at discrete time-points between 24h and 7ds. Of TLR ligands tested, only TLR4 agonism increased miR125a. Prediction algorithms identified members of the IL-6 signalling pathway (IL-6R, gp130) as potential targets of miR-125a. Luciferase reporter assays thereafter confirmed functional target interactions. Transfection of THP-1 cells with miR-125a but not control mimic reduced IL-6R membrane expression measured by FACS. miR-125a also targeted the negative regulators of TLR signalling, TNFAIP3 and IRF4, assessed by TargetScan and luciferase reporter. Commensurate with this IRF4 was down-regulated in RA PB monocytes (both DMARD good and recurrent non-responders) compared to healthy donors. Conclusion: Inflammatory cytokines, maturation factors and articular DAMPs drive elevated miR-125a expression in monocyte/macrophage lineages. miR-125a, in turn represents a novel molecular pathway that cross regulates IL-6R and TLR pathway activation in RA macrophages. We conclude that miR-125a represents an intriguing molecular marker with therapeutic and biomarker potential

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