GSE117042
Genome-wide transcriptional response of bone marrow derived macrophages (BMDM) to tunicamycin (TM).
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. Overall design: Bone marrow cells were harvested from femurs of  mice (pooled cells from three mice were used for each genotype) . Marrow cells were re-suspended in DMEM media supplemented with 10% FBS (Life Technologies, Carlsbad, CA), 1×pencillin/ streptomycin (Life Technologies, Carlsbad, CA) and 40ng/ml of macrophage colony–stimulating factor, mouse M-CSF (R&D, Minneapolis, MN). After 6 days differentiation, bone marrow derived macrophages (BMDM) were retrieved and plated on FBS pre-treated 4-well glass bottom slides (Thermo Fisher Scientific Inc, Suwanee, GA) at 2-2.2× 105 cell/well. One day later, culture media were changed with phenol-red free DMEM with 20ng/ml of M-CSF in the presence of DMSO (control) or 0.5µg/ml of tunicamycin (Sigma) for 6 hrs. For each genotype four replicate samples were  analyzed. Kupffer cells were isolated from five C57Bl6 mice and submitted for RNA-sequencing independently.