Microarrays of human pDCs derived from Rheumatoid Arthritis (RA) patients in remission (responding to anti-TNF agents) versus pDCs from healthy donors
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ABSTRACT: In Rheumatoid Arthritis, restoration of immune self-tolerance represents an unmet therapeutic need, since a significant proportion of RA patients show inadequate clinical response. Based on the previously described tolerogenic phenotype of pDCs in RA responding to anti-TNF agents, we aimed to explore the molecular mechanisms involved in the tolerogenic reprogramming of pDCs in RA.
Project description:Transcriptome analysis was conducted to investigate the gene expression profiles of pDCs using bulk RNA-sequencing (RNA-seq). Peripheral blood mononuclear cells (PBMCs) from healthy donors (n=3) were stained with lineage markers (CD3, CD14, CD16, CD19, and CD56), and pDCs were identified via flow cytometry (fluorescence-activated cell sorting [FACS]) based on the co-expression of IL-3R (CD123) and BDCA-2 (CD303). mDCs were identified using CD11c and sorted from the same PBMC donor as a control. After sorting, mRNA was extracted from the sorted cells, including mDCs and pDCs. The whole transcriptome profile was analyzed via RNA-seq.
Project description:Two well-characterized blood dendritic cell populations, conventional (cDC) and plasmacytoid (pDC), exhibit multiple phenotypic, migratory and functional differences that suggest specialized and may be complementary and coordinated functions. To study this possible coordination, cDCs and pDCs from healthy blood donors were sorted and cDCs were stimulated either with LPS or R848 whereas pDCs were CFSE-labelled and maintained in IL3. Then, CFSE labelled-pDCs and stimulated-cDCs were mixed and co-cultured. Following this "conditioning", CFSE-pDCs were sorted again and further analyzed. Conditioned pDCs showed moderate phenotypic maturation and acquired allostimulatory capacity. Microarray and RT-PCR analyses showed the induction of different genes including chemokines and proinflammatory cytokines that were dependent on the stimulation received from the "conditioner" cDC. Additionally, the differential pattern of conditioning was confirmed by protein secretion analyses with the production of specific chemokines and cytokines by conditioned pDCs. Importantly, conditioning of pDCs by activated cDCs required cell-cell contact.
Project description:Multiple Sclerosis (MS) is an immune-mediated chronic inflammatory disease affecting the central nervous system. The cause of MS is not known and the mechanism of IFN-beta, a disease-modifying treatment (DMT) approved for MS, is not well-understood. Oligonucleotide microarrays were used to study gene expression in plasmacytoid denditic cells (pDCs) which are antigen-presenting cells implicated in MS pathogenesis. We analyzed gene expression in pDCs of healthy controls, untreated and IFN-beta treated MS patients. We were able to identify 60 genes which were abnormally expressed in untreated MS patients and were corrected after treatment with IFN-beta. PDCs were separated from healthy donors and MS patients at two time points: before and 3 months after initiation of treatment with IFN-beta for RNA extraction and hybridization on Affymetrix microarrays. Gene expression data analysis of was done by GeneSpring software (Agilent Technologies). An unpaired t-test was applied to select genes with significant difference in expression between healthy donors and untreated MS patients. A paired t-test was applied to select genes with significant difference in expression in MS patients before and after IFN-beta treatment. To select differentially expressed/regulated genes, the cut-off criteria consisted of a P value < 0.05 and fold change >1.5.
Project description:Plasmacytoid dendritic cells (pDCs) rapidly produce type I interferon (IFN-I) in response to viruses and are essential for antiviral immune responses. Although related to classical dendritic cells (cDCs) in their development and expression profile, pDCs possess many distinct features. Unlike cDCs, pDCs develop in the bone marrow (BM) and emerge into peripheral lymphoid organs and tissues as fully differentiated cells. We now report that pDCs specifically express Runx2, a Runt family transcription factor that is essential for bone development. Runx2-deficient murine pDCs developed normally in the BM but were greatly reduced in the periphery. The defect was cell-intrinsic and was associated with the retention of mature Ly49Q+ pDCs in the BM. Runx2 was required for the expression of several pDC-enriched genes including chemokine receptors Ccr2 and Ccr5. Mature pDCs expressed high levels of Ccr5 at the surface, and Ccr5-deficient pDCs in a competitive setting were reduced in the periphery relative to the BM. Thus, Runx2 is required for the emergence of mature BM pDCs into the periphery, in a process that is partially dependent on Ccr5. These results establish Runx2 as a lineage-specific regulator of immune system development. Total BM cells were isolated from Runx2-/- and wildtype fetal liver chimeras 2 months post-reconstitution. BM cells were stained with antibodies against surface markers CD11b, CD11c, BST2, and B220. CD11b- BST2+ B220+ pDCs were purified by flow cytometry on a BD FACS Aria. RNA was purified immediately and prepared for microarray analysis using the Ambion WT labeling kit. Expression was analyzed using Affymetrix Mouse Gene 1.0 ST.
Project description:Microarray experiments were performed to compare the gene expression profiles exhibited by immature and activated bone-marrow (BM) derived conventional DCs (BM-cDCs) and plasmacytoid DCs (BM-pDCs) from WT and miR155-/- mice. (time points 0, 4 and 24 hours)
Project description:To identify microRNA changes during plasmacytoid dendritic cell (PDC) activation, we stimulated human primary PDCs with 10ug/ml R837 (Invivogen, San Diego, CA, USA) for 4 hours. Purified human pDCs were divided into two parts: one was cultured with medium alone, another was cultured with R837. 4 hours later, cells were collected and total RNA was extracted for the TaqManM-BM-. Human MicroRNA Arrays. The experiment was duplicated (sample1 and sample2).
Project description:The goal of this study is to determine if pDCs reconstituted from T cell depleted allogeneic STAT1-/- bone marrow express genes that contribute to Graft versus host disease (GVHD) resistance as compared to STAT1+/+ bone marrow Lethally irradiated B6 x C3H.SW mice were transplanted with T cell depleted 129 or 129 STAT1-/- bone marrow. 14 days later, pDCs were isolated from spleens by magnetic cell selection, and pooled from 12 mice/group. cDNA was generated and hybridized to an Affymetrix genechip mouse genome 430 2.0 array. Samples were normalized by RMA algorithm to a log2 intensity value and analyzed by Ingenuity Pathway Analysis v8.5 software.
Project description:The purpose of this experiment was to assess the genes upregulated when pDCs were stimulated with TLR7 agonist imiquimod and TLR9 agonist CpG C. pDCs were isolated from six healthy donors by FACS sorting, and were stimulated with CpG and imiquimod for 18 hours, after which RNA was extracted for next generation sequencing on the Illumina HiSeq platform. Unstimulated samples were included as controls.
Project description:Robust type I interferon (IFN-alpha/beta) production in plasmacytoid dendritic cells (pDCs) is critical for anti-viral immunity. Here we demonstrated a role for the mammalian target of rapamycin (mTOR) pathway in regulating interferon production by pDCs. Inhibition of mTOR or the ‘downstream’ mediators of mTOR p70S6K1,2 kinases during pDC activation via Toll-like receptor 9 (TLR9) blocked the interaction of TLR9 with the adaptor MyD88 and the subsequent activation of interferon response factor 7 (IRF7), resulting in impaired IFN-alpha production. Microarray analysis confirmed that inhibition of mTOR by the immunosuppressive drug rapamycin suppressed anti-viral and anti-inflammatory gene expression. Consistent with this, targeting rapamycin-encapsulated microparticles to antigen-presenting cells in vivo resulted in a diminution of IFN-alpha production in response to CpG DNA or the yellow fever vaccine virus strain 17D. Thus, mTOR signaling plays a critical role in TLR-mediated IFN-alpha responses by pDCs. CpGA is a TLR9 agonist. pDCs were isolated from mouse spleen or human PBMC. The effect of rapamycin on pDCs IFN-alpha production as induced by TLR ligands was studied. The mechanism of rapamycin effect was dissected in RAW cell line.
Project description:Type I IFNs are critical in initiating protective antiviral immune responses and plasmacytoid DCs (pDCs) represent a major source of these cytokines. Here we show that only few pDCs are capable to produce IFN? after virus infection or CpG stimulation. Utilizing IFN?/YFP reporter mice, we identify these IFN?-producing cells in the spleen as a CCR9+CD9- pDC subset exclusively localized within the T/B cell zones. IFN?-producing pDCs exhibit a distinct transcriptome profile with higher expression of genes encoding cytokines and chemokines, facilitating T cell recruitment and activation. These distinctive characteristics of IFN?-producing pDCs are independent of the type I IFN receptor mediated feedback loop. Furthermore, IFN?-producing pDCs exhibit enhanced CCR7-dependent migratory properties in vitro and in a peritoneal inflammation model they effectively recruit T cells in vivo. We define “professional type I IFN-producing cells” as a distinct subset of pDCs specialized in coordinating cellular immune responses. IFN? associated gene expression in ex vivo sorted IFN?/YFPpos vs. IFN?/YFPneg splenic pDCs was measured at 6 hr after i.v. injection of CpG 1668 complexed to DOTAP. Two independent experiments were performed using pooled samples of at least 12 mice per experiment.