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Transcriptional effects of siRNA-mediated MAFB knockdown on SARS-CoV-2-infected human M-CSF and GM-derived macrophages.

ABSTRACT: Transcriptional effects of siRNA-mediated MAFB knockdown on SARS-CoV-2-infected human M-CSF and GM-derived macrophages.

PROVIDER: PRJNA929954 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR23287076.fastq.gz	Fastqsanger.gz
	SRR23287077.fastq.gz	Fastqsanger.gz
	SRR23287078.fastq.gz	Fastqsanger.gz
	SRR23287079.fastq.gz	Fastqsanger.gz
	SRR23287080.fastq.gz	Fastqsanger.gz

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Transcriptional effects of siRNA-mediated MAFB knockdown on SARS-CoV-2-infected human M-CSF and GM-derived macrophages.

Project description:To investigate the MAFB-dependent genes in the SARS-COV-2 infection on human macrophages, we generated M-CSF and GM-CSF-dependent monocyte-derived macrophages in which MAFB has been knocked down by siRNA and then infected with SARS-CoV-2. Monocytes from three independent donors (0.5 x 106 cells/ml, >95% CD14+ cells) were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) at 37°C in a humidified atmosphere with 5% CO2 for 6 days in the presence of GM-CSF or M-CSF, with cytokine addition every two days. Then, monocyte-derived macrophages were transfected with MAFB-specific siRNA or control siRNA, kept in culture for 6 hours, and then were exposed to SARS-CoV-2 (SARS-CoV-2 clinical isolate Gisaid EPI_ISL_1120962, corresponding to ancestral S D614G, at an MOI=1) or uninfected. After 36 hours, cells were lysed and RNA isolated for transcriptional analysis. We performed gene expression profiling analysis using data obtained from RNA-seq of 3 different donors.

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Transcriptional effects of MAF- and MAFB-siRNAs on M-CSF-derived macrophages

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Transcriptional effects of siRNA-mediated MAF and MAFB knockdown on IVIg treatment and TLR7 activation of M-CSF-derived macrophages

Project description:Analysis of the role of MAF and MAFB on the transcriptional signature of TLR7-activated human M-CSF-dependent monocyte-derived macrophages. Methods: Human Peripheral Blood Mononuclear Cells (PBMC) were isolated from buffy coats from donors over a Lymphoprep gradient according to standard procedures. Monocytes were purified from PBMC by magnetic cell sorting using anti-CD14 microbeads (>95% CD14+ cells). Monocytes from three independent donors (0.5 x 106 cells/ml, >95% CD14+ cells) were cultured in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) at 37°C in a humidified atmosphere with 5% CO2 and 21% O2 for 6 days in the presence of 1000 U/ml M-CSF, with cytokine addition every two days. Then, monocyte-derived macrophages were transfected with MAF and MAFB-specific siRNA or control siRNA, kept in culture for 24 hours, and then stimulated (or not) with IVIg (10mg/ml) and the TLR7 ligand CL264 (1 microgram/ml). After 4 and 12 hours, cells were lysed and RNA isolated for transcriptional analysis.

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Project description:GM-CSF is involved in immune complex (IC)-mediated arthritis. However, little is known about what is the cellular source of GM-CSF and how it is regulated during IC-mediated inflammation. Using novel GM-CSF reporter mice, we show that NK cells produce GM-CSF during an IC-mediated model of inflammatory arthritis. NK cells promoted STIA in a GM-CSF-dependent manner, as deletion of NK cells and selective removal of GM-CSF production by NK cells abrogated disease. Furthermore, we show that myeloid cell activation by GM-CSF is restrained by induction of JAK/STAT checkpoint inhibitor cytokine-inducible SH2-containing protein, CIS. Myeloid cells from CIS-deficient mice had exaggerated responses to GM-CSF, and these mice develop exacerbated STIA. Our data suggest that tissue NK cells may amplify joint inflammation in arthritis via GM-CSF production and thus represent a novel target in IC-mediated pathology. Endogenous CIS provides a key brake on signaling through the GM-CSF receptor and strategies that boost its function may provide an alternative anti-inflammatory approach.

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