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Analysis of monocyte-derived dendritic cell transcriptome upon HIV-1 challenge and effect of MDA5 knockdown

ABSTRACT: The purpose of this study is to characterize the changes in global transcriptome of monocyte-derived dendritic cells (MDDCs) upon HIV-1 challenge, and examine the effect of MDA5 knockdown on MDDC transcriptome in the presence and absence of HIV-1 challenge. For this study, human CD14+ monocytes were enriched from peripheral blood mononuclear cells (PBMCs) and transduced with lentivectors encoding puromycin acetyltransferase and a short hairpin RNA (shRNA) targeting either control luciferase (Luc) or MDA5. Immediately following challenge with the knockdown vector, monocytes were placed in culture with IL-4 and GM-CSF to promote differentiation into MDDCs. Three days after transduction, puromycin was added to the medium to select for cells that had been transduced. On day seven, the resulting immature MDDCs were either challenged with a single-cycle HIV-1 vector or left untreated for 48 hours. RNA isolated from these samples was used to generate poly(A)-selected RNA-seq libraries. Analyses of these samples revealed that HIV-1 challenge in MDDCs induces a robust type-1 interferon response, whereas MDA5 suppression significantly limits this innate immune response to HIV-1.

ORGANISM(S): Homo sapiens

PROVIDER: GSE201250 | GEO | 2023/12/14

REPOSITORIES: GEO

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Differential gene expression in human THP-1 monocytes expressing the RdRPâ??cat mutant transgene compared to THP-1 empty vector control cells

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2015-11-08 | E-GEOD-62754 | biostudies-arrayexpress

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Project description:Previously, we reported that mice made transgenic for a picornaviral RdRP â the 3Dpol protein of Theilerâs murine encephalomyelitis virus (TMEV) â suppress infection by diverse viruses. Using mouse genetic studies, we determined that uninfected RdRP transgenic mice inherently induce an arsenel of prominent antiviral effectors and that this phenotype is MDA5-, MAVS- and IFNÎ±Î²R-dependent. To determine the mechanism underlying MDA5 activation and induction of constitutive antiviral signaling by the picornaviral RdRP, we constructed mutant RdRP transgenes. First, we introduced pervasive, coding-neutral point mutations into the RdRP cDNA to maximally disrupt primary and secondary RNA structure (RdRPârna). Another mutant, RdRPâcat, lacks catalytic activity due to alanine substitution of the key catalytic center triad aspartate residues (D233, D328, and D329), but is otherwise intact at the nucleotide and amino acid levels. The WT, RdRPârna, and RdRPâcat versions of the RdRP transgenes were transduced with lentiviral vectors into human THP-1 monocytes, with RdRP mRNA transcription controlled by the Spleen Focus Forming Virus (SFFV) promoter. In parallel a control cell line transduced with a vector lacking any RdRP transgene (null THP-1) was generated. RdRP cDNAs and a GFP-puromycin fusion protein selection marker were cloned into transfer constructs containing HIV LTRs, a small portion of gag, an RRE, and a cPPT. Transfer constructs were transfected into HEK 293T cells along with packaging construct Râ8.9 and envelope pseudotyping construct expressing VSV-G. RT-normalized amounts of vector were used to transduce THP-1 cells. After selection in puromycin, whole-genome expression profiling was performed on total RNA extracted from the different THP-1 lines.

2015-11-08 | E-GEOD-62753 | biostudies-arrayexpress

Differential gene expression in human THP-1 monocytes expressing the RdRPârna mutant transgene compared to THP-1 empty vector control cells

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Project description:mRNA array was conducted to identify relevant pathological pathways in activated monocytes in anti-MDA5-positive patients in comparison of those in healthy controls.

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The effects of mutant ASXL1 and BAP1 expression on hematopoietic cells

Project description:To identify target genes of mutant ASXL1 and BAP1 in hematopoietic cells, we performed RNA-seq using murine c-kit positive cells transduced with ASXL1-MT (MT) or ASXL1-MT-K351R (KR) together with vector or BAP1. Method：Murine c-kit positive bone marrow cells were transduced with ASXL1-MT (MT) or ASXL1-MT-K351R (KR) (coexpressing blastcidin resistant gene) together with vector or BAP1 (coexpressing puromycin resistant gene). After the selection with blasticidin and puromycin for three days, colony-forming cells were collected to extract RNA for RNA-seq analysis.

2018-05-25 | GSE114861 | GEO

Lineage relationship in human dendritic cells

Project description:The lineage relationships and fate of human blood and tissue dendritic cells (DC) has significance for a number of diseases including HIV where both blood and tissue DC may be infected. We used gene expression profiling of monocyte and DC sub-populations sorted directly from blood and skin and compared this to monocyte derived DC (MDDC) and MUTZ3 Langerhans cells (LCs) to define the lineage relationships. Hierarchical clustering analysis showed that plasmacytoid DCs formed the most discrete cluster. The ex vivo derived myeloid cells formed two separate clusters of cells derived from blood, and skin. Separate and specific DC populations could be determined within the sub-clusters. During overnight culture CD14+ dermal DCs (DDC) converted to CD1a+ expressing cells in situ consistent with origin of the CD1a+ DDC from a local precursor rather than from circulating blood DC or monocyte precursors. The in vitro derived MDDC and MUTZ3 populations grouped within the skin DC cluster and MDDCs clustered most closely to CD14+ DDC consistent with the proposed similarity between these two cell types. We identified differential expression of novel genes in particular DC subsets including genes related to DC surface receptors (including C-type lectin receptors, toll-like receptors and galectins). Total RNA was extracted and hybridised to 24 bead arrays. Dendritic cells and monocytes from human blood and skin using magnetic bead and flow cytometry based cell sorting both before and after culture for 24 hours

2011-12-01 | E-GEOD-32400 | biostudies-arrayexpress

Lineage relationship in human dendritic cells - cDNA arrays

Project description:The lineage relationships and fate of human blood and tissue dendritic cells (DC) has significance for a number of diseases including HIV where both blood and tissue DC may be infected. We used gene expression profiling of monocyte and DC sub-populations sorted directly from blood and skin and compared this to monocyte derived DC (MDDC) and MUTZ3 Langerhans cells (LCs) to define the lineage relationships. Hierarchical clustering analysis showed that plasmacytoid DCs formed the most discrete cluster. The ex vivo derived myeloid cells formed two separate clusters of cells derived from blood, and skin. Separate and specific DC populations could be determined within the sub-clusters. During overnight culture CD14+ dermal DCs (DDC) converted to CD1a+ expressing cells in situ consistent with origin of the CD1a+ DDC from a local precursor rather than from circulating blood DC or monocyte precursors. The in vitro derived MDDC and MUTZ3 populations grouped within the skin DC cluster and MDDCs clustered most closely to CD14+ DDC consistent with the proposed similarity between these two cell types. We identified differential expression of novel genes in particular DC subsets including genes related to DC surface receptors (including C-type lectin receptors, toll-like receptors and galectins). Total RNA was extracted and hybridised to 62 cDNA arrays. Dendritic cells and monocytes from human blood and skin using magnetic bead and flow cytometry based cell sorting both before and after culture for 24 hours

2011-12-01 | E-GEOD-32648 | biostudies-arrayexpress

Breaching self-tolerance to Alu duplex RNA underlies MDA5-mediated inflammation [ADAR1KO HEK293T and WT MDA5]

Project description:Aberrant activation of innate immune receptors can cause a spectrum of immune disorders, such as Aicardi-Goutières syndrome (AGS). One such receptor is MDA5, a viral double-stranded RNA (dsRNA) sensor that induces antiviral immune response. We here demonstrate that constitutive activation of MDA5 in AGS results from the loss of tolerance to cellular dsRNAs formed by Alu retroelements. While wild-type MDA5 cannot efficiently recognize Alu-dsRNA because its filament formation on dsRNA is impaired by the imperfect duplex structure, AGS-variants of MDA5 display reduced sensitivity to duplex structural irregularities, assembling signaling-competent filaments on Alu-dsRNA. Moreover, we identified an unexpected role of RNA-rich cellular environment in suppressing aberrant MDA5 oligomerization, highlighting context-dependence of self vs. non-self discrimination. Overall, our work demonstrates that the increased efficiency of MDA5 to recognize dsRNA comes at a cost of self-recognition, and implicates a unique role of Alu RNAs as virus-like elements that shape the primate immune system.

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