Effects of fluvastatin treatment on monocyte gene expression in SLE patients
ABSTRACT: The present gene expression array study of fluvastatin effects on monocytes from SLE patients show that fluvastatin has a global anti-inflammatory effect on monocytes, which includes attenuated expression of several proinflammatory cytokines, and regulated expression of molecules mediating lipoprotein signaling and cholesterol metabolism, as well as atherosclerosis and inflammatory signaling. Twenty seven SLE patients were given 20mg/day fluvastatin for one month. Blood samples were obtained before the starting of the treatment and at the end of treatment. Total RNA from monocytes was extracted using TRIzol reagent. RNA quality control was performed in a 2100 Bioanalyzer. Complementary RNAs from six SLE patients (four before treatment and two after treatment) were prepared for hybridization in an Agilent G4112F platform (Whole human Genome microarray 44K) using the One-color gene expression system (Agilent technologies).
Project description:The present gene expression array study of comparative gene profile in monocytes from patients with primary Antiphospholipid Syndrome, Systemic Lupus Erythematosus and Lupus with Antiphospholipid Syndrome demonstrates that the gene expression profiling allows the segregation of these highly related autoimmune diseases, with specific signatures explaining the pro-atherosclerotic, pro-thrombotic and inflammatory changes. One hundred and twenty six patients, forty one with APS, thirty one with SAPS and fifty four with SLE, as well as sixty one healthy donors were included in the study. Monocytes were purified from peripheral blood samples (non-monocytes depleting kit, Miltenyi Biotech, Bergisch Galdbach, Germany). Total RNA from monocytes was extracted using TRIzol reagent. RNA quality control was performed in a 2100 Bioanalyzer. Complementary RNAs from 3 APS patients, 3 SAPS patients, 3 SLE patients, and 3 healthy donors were prepared for hybridization in an Agilent G4112F platform (Whole human Genome microarray 44K) using the One-color gene expression system (Agilent technologies).
Project description:Many cytokines are involved in the pathogenesis of autoimmune diseases and are recognized as relevant therapeutic targets to attenuate inflammation, such as TNFα in RA and IFNα/γ in SLE. To relate the transcriptional imprinting of cytokines in a cell type-specific and disease-specific manner, we generated gene-expression profiles from peripheral monocytes of SLE and RA patients and compared them to in vitro-generated signatures induced by TNFα, IFNα2a and IFNγ. Monocytes from SLE and RA patients revealed disease-specific gene-expression profiles. In vitro-generated signatures induced by IFNα2a and IFNγ showed similar profiles that only partially overlapped with those induced by TNFα. Comparisons between disease-specific and in vitro-generated signatures identified cytokine-regulated genes in SLE and RA with qualitative and quantitative differences. The IFN-responses in SLE and RA were found to be regulated in a STAT1-dependent and STAT1-independent manner, respectively. Similarly, genes recognized as TNFα-regulated were clearly distinguishable between RA and SLE patients. While the activity of SLE monocytes was mainly driven by IFN, the activity from RA monocytes showed a dominance of TNFα that was characterized by STAT1 down-regulation. The responses to specific cytokines were revealed to be disease-dependent and reflected the interplay of cytokines within various inflammatory milieus. This study has demonstrated that monocytes from RA and SLE patients exhibit disease-specific gene-expression profiles, which can be molecularly dissected when compared to in vitro-generated cytokine signatures. The results suggest that an assessment of cytokine-response status in monocytes may be helpful for improvement of diagnosis and selection of the best cytokine target for therapeutic intervention. Expression profiles of human peripheral blood monocytes activated in vivo and stimulated in vitro. Monocytes from patients with SLE and RA and from healthy donors were used for generating disease-specific gene-expression profiles, where these profiles represent in vivo activation of monocytes. In addition, monocytes from healthy donors were stimulated in vitro by cytokines: TNFα, IFNα2a and IFNγ. Cytokine-specific gene-expression profiles were generated by comparing stimulated monocytes with unstimulated ones. TNFα-, IFNα2a- and IFNγ as cytokine-specific gene-expression profiles were compared with RA and SLE, as disease-specific gene-expression profiles.
Project description:Monocytes from 3 healthy donors were cultured for 6 hours in the presence of 20% serum from three newly diagnosed, untreated SLE patients. Microarray analysis was then performed upon normalizing the gene expression levels of samples incubated with SLE sera to those incubated with autologous serum. Monocytes from 3 healthy donors were cultured for 6 hours in the presence of 20% serum from three newly diagnosed, untreated SLE patients. Microarray analysis was then performed upon normalizing the gene expression levels of samples incubated with SLE sera to those incubated with autologous serum.
Project description:To better characterize the molecules that could potentially confer antigen presenting capacity to SLE monocytes, we assessed their gene expression profile. Blood monocytes from five healthy controls and five pediatric SLE patients were isolated using CD14+ selection. Because drugs used to treat SLE could induce considerable transcriptional changes, we selected active, newly diagnosed patients who had never received oral or intravenous (IV) medications.
Project description:To determine if proinflammatory and prothrombotic biomarkers are differentially upregulated in persistently antiphospholipid antibody (aPL)-positive patients, and to examine the effects of fluvastatin on these biomarkers.Four groups of patients (age 18-65) were recruited: (a) primary antiphospholipid syndrome; (b) systemic lupus erythematosus (SLE) with antiphospholipid syndrome (APS) (SLE/APS); (c) persistent aPL positivity without SLE or APS (Primary aPL); and (d) persistent aPL positivity with SLE but no APS (SLE/aPL). The frequency-matched control group, used for baseline data comparison, was identified from a databank of healthy persons. Patients received fluvastatin 40 mg daily for 3 months. At 3 months, patients stopped the study medication and they were followed for another 3 months. Blood samples for 12 proinflammatory and prothrombotic biomarkers were collected monthly for 6 months.Based on the comparison of the baseline samples of 41 aPL-positive patients with 30 healthy controls, 9/12 (75%) biomarkers (interleukin (IL)-6, IL1?, vascular endothelial growth factor (VEGF), tumour necrosis factor (TNF)-?, interferon (IFN)-?, inducible protein-10 (IP10), soluble CD40 ligand (sCD40L), soluble tissue factor (sTF) and intracellular cellular adhesion molecule (ICAM)-1) were significantly elevated. Twenty-four patients completed the study; fluvastatin significantly and reversibly reduced the levels of 6/12 (50%) biomarkers (IL1?, VEGF, TNF?, IP10, sCD40L and sTF).Our prospective mechanistic study demonstrates that proinflammatory and prothrombotic biomarkers, which are differentially upregulated in persistently aPL-positive patients, can be reversibly reduced by fluvastatin. Thus, statin-induced modulation of the aPL effects on target cells can be a valuable future approach in the management of aPL-positive patients.
Project description:Previous studies showed that bone marrow derived-mesenchymal stem cells (BMMSCs) from patients with systemic lupus erythematosus (SLE) and lupus animal models have deficiency in their capacities of proliferation, differentiation, secretion of cytokines and other functions. In this study, we aim to investigate the different gene patterns of BMMSCs between normal and SLE individuals using genome-scale DNA microarrays. We found that among all the genes investigated in microarray slides, a total of 1, 905 genes were differentially expressed by BMMSCs SLE patients, in which 652 genes were up-regulated and 1253 genes were down-regulated. Gene ontology analysis showed that the majority of these genes were related to cell cycle and protein binding. Pathway analysis showed that differentially regulated signal pathways involved actin cytoskeleton, focal adhesion, tight junction and TGF-β pathway. Our data suggested that there were differential gene expression patterns of BMMSCs between normal and SLE individuals, and further studies are needed to reveal the mechanisms for such differences. Bone marrow (BM) was obtained from 4 patients with SLE and 4 normal controls. BMMSCs were cultured and mRNA was extracted. Standard human reference RNA were purchased. Every slide: SLE or normal vs control, then SLE vs normals.
Project description:Neutrophil Extracellular Traps (NETs) are chromatin-derived extracellular structures that are expelled from neutrophils in response to infectious or inflammatory stimuli. NET DNA structures are decorated with proteins including histones, myeloperoxidase and neutrophil elastase. NETs are implicated in the development of auto-immunity in diseases including rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE) through the externalisation of intracellular neoepitopes e.g. dsDNA and nuclear proteins in SLE and citrullinated peptides in RA. The aim of this work was to use quantitative proteomics to identify and measure NET proteins produced by neutrophils from healthy individuals, and from patients with RA and SLE.
Project description:OBJECTIVE:The inflammasome complex is a driver of organ damage in patients with systemic lupus erythematosus (SLE). Although type I interferons (IFNs) are well established as mediators of SLE pathogenesis, their role in inflammasome activation in SLE has not been assessed. The aim of this study was to examine type I IFNs as regulators of the inflammasome. METHODS:SLE patients fulfilled ?4 American College of Rheumatology criteria and were recruited from the University of Michigan Lupus Cohort. Primary monocytes were isolated from SLE patients or healthy controls by negative selection, treated with inflammasome activators in the presence or absence of IFN?, and IL-1? secretion was measured by enzyme-linked immunosorbent assay. Expression levels of IFN and inflammasome-related molecules were assessed by real-time polymerase chain reaction and Western blotting. IFN regulatory factor 1 (IRF-1) expression was specifically down-regulated by small interfering RNA (siRNA) transfection and a chemical inhibitor. RESULTS:Monocytes from patients with SLE exhibited increased expression and enhanced activation of the inflammasome by ATP when compared with control monocytes. Expression of inflammasome and IFN-regulated genes was significantly correlated in monocytes from SLE patients but not in control monocytes. Inflammasome activity was increased after prolonged exposure to IFN?. Reduction of IRF-1 expression via siRNA blocked caspase 1 up-regulation after treatment with IFN?. Importantly, hyperactivity of the inflammasome in the monocytes of SLE patients was significantly reduced after knockdown or inhibition of IRF-1. CONCLUSION:Prolonged type I IFN exposure, as seen in SLE patients, primes monocytes for robust inflammasome activation in an IRF-1-dependent manner. IRF-1 inhibition may serve as a novel target for treatment of SLE-associated inflammation and organ damage.
Project description:Factor (F) Xa reactive IgG isolated from patients with antiphospholipid syndrome (APS) display higher avidity binding to FXa with greater coagulant effects compared to systemic lupus erythematosus (SLE) non APS IgG. FXa signalling via activation of protease-activated receptors (PAR) leads to increased intracellular calcium (Ca2+). Therefore, we measured alterations in Ca2+ levels in human umbilical vein endothelial cells (HUVEC) following FXa-mediated PAR activation and investigated whether FXa reactive IgG from patients with APS or SLE/APS- alter these responses. We observed concentration-dependent induction of Ca2+ release by FXa that was potentiated by APS-IgG and SLE/APS- IgG compared to healthy control subjects' IgG, and FXa alone. APS-IgG and SLE/APS- IgG increased FXa mediated NF?B signalling and this effect was fully-retained in the affinity purified anti-FXa IgG sub-fraction. Antagonism of PAR-1 and PAR-2 reduced FXa-induced Ca2+ release. Treatment with a specific FXa inhibitor, hydroxychloroquine or fluvastatin significantly reduced FXa-induced and IgG-potentiated Ca2+ release. In conclusion, PAR-1 and PAR-2 are involved in FXa-mediated intracellular Ca2+ release in HUVEC and FXa reactive IgG from patients with APS and/or SLE potentiate this effect. Further work is required to explore the potential use of IgG FXa reactivity as a novel biomarker to stratify treatment with FXa inhibitors in these patients.
Project description:Gene expression studies of peripheral blood mononuclear cells from patients with systemic lupus erythematosus (SLE) have demonstrated a type I interferon signature and increased expression of inflammatory cytokine genes. Studies of patients with Aicardi Goutières syndrome, commonly cited as a single gene model for SLE, have suggested that accumulation of non-coding RNAs may drive some of the pathologic gene expression, however, no RNA sequencing studies of SLE patients have been performed. This study was designed to define altered expression of coding and non-coding RNAs and to detect globally altered RNA processing in SLE.Purified monocytes from eight healthy age/gender matched controls and nine SLE patients (with low-moderate disease activity and lack of biologic drug use or immune suppressive treatment) were studied using RNA-seq. Quantitative RT-PCR was used to validate findings. Serum levels of endotoxin were measured by ELISA.We found that SLE patients had diminished expression of most endogenous retroviruses and small nucleolar RNAs, but exhibited increased expression of pri-miRNAs. Splicing patterns and polyadenylation were significantly altered. In addition, SLE monocytes expressed novel transcripts, an effect that was replicated by LPS treatment of control monocytes. We further identified increased circulating endotoxin in SLE patients.Monocytes from SLE patients exhibit globally dysregulated gene expression. The transcriptome is not simply altered by the transcriptional activation of a set of genes, but is qualitatively different in SLE. The identification of novel loci, inducible by LPS, suggests that chronic microbial translocation could contribute to the immunologic dysregulation in SLE, a new potential disease mechanism.