Interferon beta-1a-induced changes in gene expression in PBMCs derived from CIS patients
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE14386 | GEO | 2009/01/13
SECONDARY ACCESSION(S): PRJNA111357
REPOSITORIES: GEO
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Project description:IFNβ, an effective therapy against relapsing-remitting (RR) multiple sclerosis (MS) is naturally secreted during the innate immune response against viral pathogens. The objective of this study was to characterize the immunomodulatory mechanisms of IFNβ targeting innate immune response and their effects on DC-mediated regulation of T-cell differentiation. We found that IFNβ−1a in-vitro treatment of human monocyte-derived dendritic cells (DCs) induced the expression of TLR7 and the members of its downstream signaling pathway, including myeloid differentiation factor 88 (MyD88), IL-1R-associated kinase (IRAK)4, and TNF receptor-associated factor (TRAF)6, while it inhibited the expression of IL-1R. Using siRNA TLR7 gene silencing, we confirmed that IFNβ-1a-induced changes in MyD88, IRAK4 and IL-1R expression were dependent on TLR7. TLR7 expression was also necessary for the IFNβ-1a-induced inhibition of IL-1β and IL-23, and the induction of IL-27 secretion by DCs. Supernatant (SN) transfer experiments confirmed that IFNβ-1a-induced changes in DCs’ cytokine secretion inhibit Th17 cell differentiation as evidenced by the inhibition of retinoic acid-related orphan nuclear hormone receptor C (RORC) and IL-17A gene expression and IL-17A secretion. Our study has identified a novel therapeutic mechanism of IFNβ−1a, that selectively targets the autoimmune response in MS. Overall design: Gene expression changes induced by IFNβ−1a were tested using Affymetrix Human Genome U133 (HG-U133) arrays (Affymetrix) that contain 45,000 probe sets representing 39,000 transcripts derived from approximately 33,000 human genes. 107 PBMCs per condition derived from 15 CIS patients were stimulated with plate-immobilized αCD3 (1 μg/ml) and αCD28 (5 μg/ml) mAb (BD Biosciences) in the absence or presence of IFNβ-1a (1000 U/ml) (EMD Serono Inc) for 24 h in serum-free medium (Gibco). Cells were harvested and the total RNA was isolated using a Rneasy kit (Quiagen). Arrays were hybridized for 16 hours at 45oC in the GeneChip® Hybridization Oven 640 (Affymetrix). The arrays were washed and stained with R-phycoerythrin streptavidin in the GeneChip® Fluidics Station 400 (Affymetrix). The arrays were scanned with a Hewlett Packard GeneArray Scanner. Affymetrix GeneChip® Microarray Suite 5.0 software was used for washing, scanning and basic analysis.
2009-01-17 | E-GEOD-14386 | biostudies-arrayexpress
Project description:IFNb has been used as a first line therapy for relapsing remitting multiple sclerosis (RRMS). Since only a few studies have addressed the role of endogenous IFNb in the pathogenesis of the disease, our objective was to characterize its role in the transcriptional regulation of pathogenic Th17 cytokines in patients with RRMS. In-vitro studies have demonstrated that IFNb inhibited IL-17A, IL-17F, IL-21, IL-22 and IFN-b secretion in CD4+ lymphocytes through the induction of suppressor of cytokine secretion (SOCS)1 and 3. We found that patients with RRMS have increased serum and cerebrospinal fluid (CSF) Th17 (IL-17A and IL-17F) cytokine levels in comparison to the control subjects, suggesting that deficient endogenous IFNbeta secretion and/or signaling may contribute to the dysregulation of those pathogenic cytokines in CD4+ cells. We identified that the endogenous IFNb from serum of RRMS patients induced a significantly lower IFN-inducible gene expression in comparison to healthy controls (HCs). In addition, in-vitro studies have revealed a deficient endogenous and exogenous IFNb signaling in CD4+ cells derived from MS patients. Interestingly, upon inhibition of the endogenous IFNb signaling by silencing interferon regulatory factor (IRF)7 gene expression, the resting CD4+ T cells secreted significantly higher level of IL-17A, IL-17F, IL-21, IL-22 and IL-9, suggesting that endogenous IFNb suppresses the secretion of these pathogenic cytokines. In-vivo recombinant IFNb-1a treatment induced IFNAR1 and its downstream signaling moleculesM-bM-^@M-^Y gene expression, suggesting that treatment may reconstitute a deficient endogenous IFNbeta regulation of the CD4+ T-cellsM-bM-^@M-^Y pathogenic cytokine production in MS patients. Gene expression changes induced by IFNM-NM-2M-bM-^HM-^R1a were tested using Affymetrix Human Genome U133 (HG-U133) arrays (Affymetrix) that contain 45,000 probe sets representing 39,000 transcripts derived from approximately 33,000 human genes. 107 PBMCs per condition derived from 15 CIS patients were stimulated with plate-immobilized M-NM-1CD3 (1 M-NM-<g/ml) and M-NM-1CD28 (5 M-NM-<g/ml) mAb (BD Biosciences) in the absence or presence of IFNM-NM-2-1a (1000 U/ml) (EMD Serono Inc) for 24 h in serum-free medium (Gibco). Cells were harvested and the total RNA was isolated using a Rneasy kit (Quiagen). Arrays were hybridized for 16 hours at 45oC in the GeneChipM-BM-. Hybridization Oven 640 (Affymetrix). The arrays were washed and stained with R-phycoerythrin streptavidin in the GeneChipM-BM-. Fluidics Station 400 (Affymetrix). The arrays were scanned with a Hewlett Packard GeneArray Scanner. Affymetrix GeneChipM-BM-. Microarray Suite 5.0 software was used for washing, scanning and basic analysis.
2014-05-15 | E-GEOD-53716 | biostudies-arrayexpress
Project description:IFNb has been used as a first line therapy for relapsing remitting multiple sclerosis (RRMS). Since only a few studies have addressed the role of endogenous IFNb in the pathogenesis of the disease, our objective was to characterize its role in the transcriptional regulation of pathogenic Th17 cytokines in patients with RRMS. In-vitro studies have demonstrated that IFNb inhibited IL-17A, IL-17F, IL-21, IL-22 and IFN-b secretion in CD4+ lymphocytes through the induction of suppressor of cytokine secretion (SOCS)1 and 3. We found that patients with RRMS have increased serum and cerebrospinal fluid (CSF) Th17 (IL-17A and IL-17F) cytokine levels in comparison to the control subjects, suggesting that deficient endogenous IFNbeta secretion and/or signaling may contribute to the dysregulation of those pathogenic cytokines in CD4+ cells. We identified that the endogenous IFNb from serum of RRMS patients induced a significantly lower IFN-inducible gene expression in comparison to healthy controls (HCs). In addition, in-vitro studies have revealed a deficient endogenous and exogenous IFNb signaling in CD4+ cells derived from MS patients. Interestingly, upon inhibition of the endogenous IFNb signaling by silencing interferon regulatory factor (IRF)7 gene expression, the resting CD4+ T cells secreted significantly higher level of IL-17A, IL-17F, IL-21, IL-22 and IL-9, suggesting that endogenous IFNb suppresses the secretion of these pathogenic cytokines. In-vivo recombinant IFNb-1a treatment induced IFNAR1 and its downstream signaling molecules’ gene expression, suggesting that treatment may reconstitute a deficient endogenous IFNbeta regulation of the CD4+ T-cells’ pathogenic cytokine production in MS patients.
2014-05-15 | GSE53716 | GEO
Project description:Constitutively active RAS plays a central role in the development of skin cancer in the classical two stage skin carcinogenesis in mice and in a number of human cancers. Ras-mediated tumor formation is commonly associated with upregulation of cytokines and chemokines that mediate an inflammatory response considered relevant to oncogenesis. MyD88 is a crucial intermediate in the expression of multiple innate immune responders through signaling from the Toll-like/IL-1R family. We report that mice ablated for MyD88 or the IL-1R are resistant to topical skin carcinogenesis, and cultured MyD88-/- keratinocytes transduced with an oncogenic ras vector form only a few small tumors in orthotopic grafts. Initiated keratinocytes arising from oncogenic activation of ras are hyperproliferative but also resist signals for induced differentiation and upregulate a host of pro-inflammatory genes. Ras-transduced MyD88-/- keratinocytes are also hyperproliferative but the differentiation response is intact and pro-inflammatory genes are not upregulated. Using both genetic and pharmacological approaches, we find that in keratinocytes, the differentiation and immune regulation functions mediated by oncogenic ras require the establishment of an autocrine loop through IL-1α and its receptor leading to NF-κB activation. In the absence of MyD88 or IL-1R, this loop cannot be established. Further, blocking the IL-1a mediated NF-κB activation in ras-transduced wildtype keratinocytes corrects the defect in both differentiation response and proinflammatory gene expression. Collectively, these results demonstrate that ras activation converts normal keratinocytes to an initiated phenotype through a series of potentially reversible feedback signals that provide therapeutic opportunities through inhibition of IL-1 signaling. Gene expression comparison of wild type ras-transformed keratinocytes untreated (n=3) or treated (n=3) in vitro with the IL1R antagonist Anakinra.
2011-08-28 | E-GEOD-22777 | biostudies-arrayexpress
Project description:Constitutively active RAS plays a central role in the development of skin cancer in the classical two stage skin carcinogenesis in mice and in a number of human cancers. Ras-mediated tumor formation is commonly associated with upregulation of cytokines and chemokines that mediate an inflammatory response considered relevant to oncogenesis. MyD88 is a crucial intermediate in the expression of multiple innate immune responders through signaling from the Toll-like/IL-1R family. We report that mice ablated for MyD88 or the IL-1R are resistant to topical skin carcinogenesis, and cultured MyD88-/- keratinocytes transduced with an oncogenic ras vector form only a few small tumors in orthotopic grafts. Initiated keratinocytes arising from oncogenic activation of ras are hyperproliferative but also resist signals for induced differentiation and upregulate a host of pro-inflammatory genes. Ras-transduced MyD88-/- keratinocytes are also hyperproliferative but the differentiation response is intact and pro-inflammatory genes are not upregulated. Using both genetic and pharmacological approaches, we find that in keratinocytes, the differentiation and immune regulation functions mediated by oncogenic ras require the establishment of an autocrine loop through IL-1α and its receptor leading to NF-κB activation. In the absence of MyD88 or IL-1R, this loop cannot be established. Further, blocking the IL-1a mediated NF-κB activation in ras-transduced wildtype keratinocytes corrects the defect in both differentiation response and proinflammatory gene expression. Collectively, these results demonstrate that ras activation converts normal keratinocytes to an initiated phenotype through a series of potentially reversible feedback signals that provide therapeutic opportunities through inhibition of IL-1 signaling.
2011-08-29 | GSE22777 | GEO
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2008-10-23 | GSE13300 | GEO
Project description:Progressive dysfunction and death of photoreceptors occurs in blinding diseases such as age-related macular degeneration and retinitis pigmentosa. The MyD88 protein is a central adaptor molecule for innate immune system Toll-like receptors (TLR) and interleukin-1 receptor (IL-1R), which are active in retinal disease and induce cytokine secretion from inflammatory cells. We recently demonstrated that inhibiting MyD88 in mouse models of retinal degeneration led to increased rod photoreceptor survival, which was associated with altered cytokine expression and increased neuroprotective microglia. However, additional molecular changes associated with MyD88 inhibitor-mediated neuroprotection are not known. Quantitative proteomics using iTRAQ LC-MS/MS is a high-throughput method ideal for providing new information about the molecular mechanisms contributing to photoreceptor protection. In this study, we used isobaric tags for relative and absolute quantification (iTRAQ) labeling followed by liquid chromatography–tandem mass spectrometry (LC-MS/MS) for quantitative proteomic analysis on the rd10 mouse model of retinal degeneration to identify protein pathways changed by MyD88 inhibition. A total of 42 proteins were differentially expressed in retinas from mice treated with MyD88 inhibitor compared with control. Notably, increased expression of multiple crystallins and chaperones that respond to cellular stress and have anti-apoptotic properties were identified in the MyD88 inhibited mice. Additional differentially expressed biological processes included pyrophosphatase activity and peptide biosynthesis. These data suggest that inhibiting MyD88 may enhance chaperone-mediated tissue protective pathways. Therefore, this study provides new insight into molecular events that contribute to photoreceptor protection from modulating inflammation.
2022-02-17 | PXD024501 | Pride
Project description:Most autoreactive B cells are normally counterselected during early B cell development. To determine whether Toll-like receptors (TLRs) regulate the removal of autoreactive B lymphocytes, we tested the reactivity of recombinant antibodies from single B cells isolated from patients deficient for IL-1R-associated kinase (IRAK)-4, myeloid differentiation factor 88 (MyD88) and UNC-93B. Indeed, all TLRs except TLR3 require IRAK-4 and MyD88 to signal and UNC-93B-deficient cells are unresponsive to TLR3, TLR7, TLR8 and TLR9. All patients suffered from defective central and peripheral B cell tolerance checkpoints resulting in the accumulation of large numbers of autoreactive mature naïve B cells in their blood. Hence, TLR7, TLR8, and TLR9 may prevent the recruitment of developing autoreactive B cells in healthy donors. Paradoxically, IRAK-4-, MyD88- and UNC-93B-deficient patients did not display autoreactive antibodies in their serum nor developed autoimmune diseases, suggesting that IRAK-4, MyD88 and UNC-93B pathway blockade may thwart autoimmunity in humans. Experiment Overall Design: RNA was extracted from 105-3.105 batch sorted new emigrant and mature naïve B cells isolated from donors using the Absolutely RNA microprep kit (Stratagene). 100-200 ng of RNA was obtained per sample, and the quality of the purified RNA was assessed by the Bioanalyzer from Agilent. Using the Ovation biotin system kit from Nugen, 30-50ng of RNA was amplified and labeled to produce cDNA. Labeled cDNA was hybridized on chips containing the whole human genome (Human Genome U133 2.0 from Affymetrix). Raw data from new emigrant (1 healthy donor) and mature naive (4 healthy donors) B cells were analyzed in order to determine the expression of some molecules involved in the TLR pathway in these B cell population in humans.
2008-11-13 | E-GEOD-13300 | biostudies-arrayexpress
Project description:Inflammasomes are multi-protein complexes that control the production of pro-inflammatory cytokines such as IL-1beta. Inflammasomes play an important role in the control of immunity to tumors and infections, and also in autoimmune diseases, but the mechanisms controlling the activation of human inflammasomes are largely unknown. We found that human activated CD4+CD45RO+ memory T-cells specifically suppress P2X7R-mediated NLRP3 inflammasome activation, without affecting P2X7R-independent NLRP3 or NLRP1 inflammasome activation. The concomitant increase in pro-IL-1β production induced by activated memory T-cells concealed this effect. Priming with IFNβ decreased pro-IL-1β production in addition to NLRP3 inflammasome inhibition and thus unmasked the inhibitory effect on NLRP3 inflammasome activation. IFNβ did not suppress NLRP3 inflammasome activation by acting directly on monocytes. The inhibition of pro-IL-1β production and suppression of NLRP3 inflammasome activation by IFNβ-primed human CD4+CD45RO+ memory T-cells is partly mediated by soluble FasL and is associated with down-regulated P2X7R mRNA expression and reduced response to ATP in monocytes. CD4+CD45RO+ memory T-cells from multiple sclerosis (MS) patients showed a reduced ability to suppress NLRP3 inflammasome activation, however their suppressive ability was recovered following in vivo treatment with IFNβ. Thus, our data demonstrate that human P2X7R-mediated NLRP3 inflammasome activation is regulated by activated CD4+CD45RO+ memory T cells, and provide new information on the mechanisms mediating the therapeutic effects of IFNβ in MS. Memory T-cells were cultured in the presence of monocytes with and without Interferon-beta, resorted and expression profile was determined
2012-12-01 | E-GEOD-35601 | biostudies-arrayexpress
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