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Genome-wide transcriptional response of bone marrow derived macrophages (BMDM) to tunicamycin (TM).

ABSTRACT: Genome-wide transcriptional response of bone marrow derived macrophages (BMDM) to tunicamycin (TM).

PROVIDER: PRJNA480898 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR7516610.fastq.gz	Fastqsanger.gz
	SRR7516620.fastq.gz	Fastqsanger.gz
	SRR7516630.fastq.gz	Fastqsanger.gz
	SRR7516640.fastq.gz	Fastqsanger.gz
	SRR7516650.fastq.gz	Fastqsanger.gz

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Project description:We analyzed global genome-wide transcriptional response of bone marrow derived macrophages (BMDM) to tunicamycin (TM). This analysis demonstrated that TM treatment changed the expression of 1,426 genes in WT BMDM and 1,075 genes in 4KO BMDM ( Casp1-/-Casp8-/-Casp11-/-Ripk3-/- ) (2-fold cut-off, p<0.05). Among differentially-expressed genes, all downregulated 759 genes were downregulated to similar levels in both WT and 4KO BMDM after TM treatment. However, 667 genes activated by TM treatment were globally upregulated to significantly higher levels in WT, compared to 4KO BMDM which is consistent with previous findings of a muted macrophage responses to triggers in cells lacking CASP8. Next we used gene set enrichment analysis to annotate pathways deregulated by TM in BMDM and compared down-regulated and upregulated gene sets in BMDM to their expression in liver resident Mf, Kupffer cells, at homeostatic conditions in vivo. The most prominently down-regulated pathways in BMDM after TM treatment were cell cycle, DNA replication, and metabolism pathways (normalized enrichment score (NES) =-8.5, -7.9, -7.1, respectively, FDR<1e-4,). Importantly, the gene sets constituting downregulated pathways in TM-treated BMDM were highly expressed in KC in vivo at homeostatic levels (NES= 8.7, 10.5, 6.6 , FDR<1e-4), suggesting the lack of phenotypic similarity between TM-treated BMDM and KC for this set of pathways. However, this same analysis showed that the most prominently enriched pathways in BMDM after TM treatment were pathways related to innate and adaptive immune signaling, cytokine signaling and mitogen-activated protein kinase signaling (NES =8.7, 6.4, 6.9, 3.2, respectively , FDR<1e-4). The gene set enrichment analysis for this set of genes demonstrated that in KC at homeostatic conditions, these same genes are also highly enriched (NES=12.6, 9.8, 7.1, 4.5 , FDR<1e-4), suggesting that for upregulated gene sets, TM treatment drives BMDM in vitro to transcriptomic phenotype similar to that observed in KC in vivo at homeostatic conditions.

Project description:Investigation of whole genome gene expression level changes in BMDM adhered for 4 or 18hrs to Lab Tek Chamber slides pre-coated with 4ug/ml of human serum albumin (HSA) (control protein) or purified human C1q and treated with lipopolysaccharide (LPS: 20ng/ml) Mycobacterium avium 101 (M avium) or apoptotic Jurkat T cells. BMDM were obtained from C57Bl/6 and generated as previously described in Bohlson, SS, Strasser, JA, Bower JJ, Schorey J S 2001 Role of complement in Mycobacterium avium pathogenesis: in vivo and in vitro analyses of the host response to infection in the absence of complement component C3 Infec Immun 69: 7729-7735. Mybacterium avium 101 was obtained from Dr Jeff Schorey (University of Notre Dame). The human Jurkat T cell line was obtained from ATCC (Manassas, VA) and induced to undergo apoptosis with dexamethasone as described in Lillis, AP, Greenlee, M C, Mikhailenko I, Pizzo, S V, Tenner, A J, Strickland, D K, Bohlson, S S 2008 Murine Low-Density Lipoprotein Receptor-Related Protein 1 (LRP) Is Required for Phagocytosis of Targets Bearing LRP Ligands but Is Not Required for C1q-triggered Enhancement of Phagocytosis J Immunol 181: 364: 373. A six chip study using total RNA from 5 separate cultures of BMDM adhered to HSA for 4 hrs, 5 separate cultures of BMDM adhered to C1q for 4 hrs, 5 separate cultures of BMDM adhered to HSA for 4 hrs and infected with M avium at a 1:500 ratio of BMDM to M avium, 5 separate cultures of BMDM adhered to C1q for 4hrs and infected with M avium at a 1:500 ratio ofBMDM to M avium, 5 separate cultures of BMDM adhered to HSA for 4 hrs and infected with M avium at a 1:1000 ratio of BMDM to M avium, 5 separate cultures of BMDM adhered to HSA for 4 hrs and treated with 20ng/ml LPS, 5 separate cultures of BMDM adhered to C1q for 4 hrs and treated with 20ng/ml LPS, 5 separate cultures of BMDM adhered to HSA for 4 hrs and treated with 20ng/ml LPS and co-cultured with apoptotic Jurkat cells at a 1:3 ratio of BMDM to apoptotic cells, 5 separate cultures of BMDM adhered to C1q for 4 hrs and treated with 20ng/ml LPS and co-cultured with apoptotic Jurkat cells at a 1:3 ratio of BMDM to apoptotic cells, 5 separate cultures of BMDM adhered to HSA for 18 hrs, 5 separate cultures of BMDM adhered to C1q for 18 hrs, 5 separate cultures of BMDM adhered to HSA for 18 hrs and infected with M avium at a 1:500 ratio of BMDM to M avium, 5 separate cultures of BMDM adhered to C1q for 18hrs and infected with M avium at a 1:500 ratio of BMDM to M avium, 5 separate cultures of BMDM adhered to HSA for 18 hrs and infected with M avium at a 1:1000 ratio of BMDM to M avium. The chip utilized for these studies is a mouse whole-genome 12-plex expression microarray design by NimbleGen designed from the MM9 genome Candidate probe sequences were verified to have no cross-hybridization to human (HG19) or Mycobacterium avium (NC_008595) targets Note: Study sample BMDM_HSA & M avium (1:500)_18hr_rep2 was not included in this submission due to quality control concerns.

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Genome-wide transcriptional response of bone marrow derived macrophages (BMDM) to tunicamycin (TM).

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