Interleukin-21 is increased in active systemic lupus erythematosus patients and contributes to the generation of plasma B cells.

OBJECTIVES: Excessive interleukin- (IL-) 21 production by T cells has been implicated in the pathogenesis of systemic lupus erythematosus (SLE). We explored the expression and function of IL-21 in human SLE. METHODS: IL-21 and IL-21 receptor (IL-21R) expression was assessed by real-time PCR and flow cytometry in peripheral blood mononuclear cells (PBMCs) of SLE patients and healthy controls. PBMCs, purified CD19+CD27- naïve and CD19+CD27+ memory B cells were stimulated with IL-21 and CpG-ODN2006 (TLR-9 agonist) to examine generation of memory and plasma (CD19+CD38highIgD-) B cells. Apoptosis was assessed by 7AAD staining. RESULTS: Active SLE patients had 4-fold higher IL-21 mRNA and increased levels of intracellular IL-21 in peripheral blood CD4+ T cells (mean±SD fluorescence intensity, 1.7±0.1 in active versus 0.9±0.3 in inactive SLE and controls, p=0.035). IL-21R mRNA was comparable between SLE and healthy controls. Stimulation of PBMCs with IL-21 increased the proportion of memory and plasma cells; addition of CpG-ODN2006 enhanced these effects. Both naïve and memory B cells responded to IL-21/TLR-9 by increased generation of memory and plasma B cells, respectively; an anti-apoptotic effect was observed. In active SLE, PBMCs stimulation with IL-21 and/or CpG-ODN increased memory and plasma B cells, comparable to healthy controls. Addition of IL-21 to lupus autologous mixed lymphocyte cultures induced significant IgG production, and treatment with IL-21R.Fc to block IL-21/IL-21R interaction reduced the proportion of plasma cells. CONCLUSIONS: Increased IL-21 may synergise with TLR-9 signalling and contributes to generation of plasma cells in active SLE patients.

Identification of novel microRNA signatures linked to human lupus disease activity and pathogenesis: miR-21 regulates aberrant T cell responses through regulation of PDCD4 expression.

OBJECTIVE: MicroRNAs (miRNAs) regulate the expression of genes involved in immune activation. A study was undertaken to characterise the miRNA signature and identify novel genes involved in the regulation of immune responses in systemic lupus erythematosus (SLE). METHODS: The expression of 365 miRNAs in peripheral blood mononuclear cells of patients with SLE and healthy controls was analysed using TaqMan Low Density Arrays. The results were validated by quantitative real-time PCR and potential target genes were identified using prediction analysis software. The effect of miR-21 on T cell function was assessed by transfection with antago-miR-21 or pre-miR-21. RESULTS: A 27-miRNA signature was identified in patients with SLE; 19 miRNAs correlated with disease activity. Eight miRNAs were deregulated specifically in T cells and four miRNAs in B cells. miR-21 was upregulated and strongly correlated with SLE disease activity (r(2)=0.92). Compared with controls, CD4 T lymphocytes from patients with SLE had higher basal and activation-induced miR-21 expression. Silencing of miR-21 reversed the activated phenotype of T cells from patients with SLE--namely, enhanced proliferation, interleukin 10 production, CD40L expression and their capacity to drive B cell maturation into Ig-secreting CD19+CD38(hi)IgD-(plasma cells. Overexpression of mMiR-21 in normal T cells led to acquisition of an activated phenotype. Investigation of putative gene- targets showed that PDCD4 (a selective protein translation inhibitor) was suppressed by miR-21 and its expression was decreased in active SLE. CONCLUSIONS: miRNAs represent potential biomarkers in SLE as their expression reflects underlying pathogenic processes and correlates with disease activity. Upregulated miR-21 affects PDCD4 expression and regulates aberrant T cell responses in human SLE.

Gene network analysis of bone marrow mononuclear cells reveals activation of multiple kinase pathways in human systemic lupus erythematosus.

BACKGROUND: Gene profiling studies provide important information for key molecules relevant to a disease but are less informative of protein-protein interactions, post-translational modifications and regulation by targeted subcellular localization. Integration of genomic data and construction of functional gene networks may provide additional insights into complex diseases such as systemic lupus erythematosus (SLE). METHODOLOGY/PRINCIPAL FINDINGS: We analyzed gene expression microarray data of bone marrow mononuclear cells (BMMCs) from 20 SLE patients (11 with active disease) and 10 controls. Gene networks were constructed using the bioinformatic tool Ingenuity Gene Network Analysis. In SLE patients, comparative analysis of BMMCs genes revealed a network with 19 central nodes as major gene regulators including ERK, JNK, and p38 MAP kinases, insulin, Ca(2+) and STAT3. Comparison between active versus inactive SLE identified 30 central nodes associated with immune response, protein synthesis, and post-transcriptional modification. A high degree of identity between networks in active SLE and non-Hodgkin's lymphoma (NHL) patients was found, with overlapping central nodes including kinases (MAPK, ERK, JNK, PKC), transcription factors (NF-kappaB, STAT3), and insulin. In validation studies, western blot analysis in splenic B cells from 5-month-old NZB/NZW F1 lupus mice showed activation of STAT3, ITGB2, HSPB1, ERK, JNK, p38, and p32 kinases, and downregulation of FOXO3 and VDR compared to normal C57Bl/6 mice. CONCLUSIONS/SIGNIFICANCE: Gene network analysis of lupus BMMCs identified central gene regulators implicated in disease pathogenesis which could represent targets of novel therapies in human SLE. The high similarity between active SLE and NHL networks provides a molecular basis for the reported association of the former with lymphoid malignancies.

Gene expression in systemic lupus erythematosus: bone marrow analysis differentiates active from inactive disease and reveals apoptosis and granulopoiesis signatures.

OBJECTIVE: The cells of the immune system originate from the bone marrow, where many of them also mature. This study was undertaken to examine gene expression in the bone marrow of patients with systemic lupus erythematosus (SLE), in order to better understand the aberrant immune response in this disease. METHODS: Bone marrow mononuclear cells (BMMCs) from 20 SLE patients (11 with active disease and 9 with inactive disease) and peripheral blood mononuclear cells (PBMCs) from 27 patients (16 with active disease and 11 with inactive disease) were studied; BMMCs and PBMCs from 7 healthy individuals and 3 osteoarthritis patients were studied as controls. Samples were analyzed on genome-scale DNA microarrays, with 21,329 genes represented. RESULTS: We identified 102 genes involved in various biologic processes that were differentially expressed between patient and control BMMCs; 53 of them are genes that are involved in major networks, including cell death, growth, signaling, and proliferation. Comparative analysis revealed 88 genes that were differentially expressed between bone marrow and blood, the majority of which are involved in cell growth and differentiation, cellular movement and morphology, immune response, and other hematopoietic cell functions. Unsupervised clustering of highly expressed genes revealed 2 major SLE patient clusters (active disease and inactive disease) based on gene expression in bone marrow, but not in peripheral blood. The up-regulated genes in the bone marrow of patients with active disease included genes involved in cell death and granulopoiesis. CONCLUSION: Microarray analysis of the bone marrow differentiated active from inactive SLE and provided further evidence of the role of apoptosis and granulocytes in the pathogenesis of the disease.