CEBPD Potentiates the Macrophage Inflammatory Response but CEBPD Knock-Out Macrophages Fail to Identify CEBPD-Dependent Pro-Inflammatory Transcriptional Programs

Abstract

CCAAT/enhancer-binding protein delta (C/EBPδ) is a member of the C/EBP family of transcription factors. According to the current paradigm, C/EBPδ potentiates cytokine production and modulates macrophage function thereby enhancing the inflammatory response. Remarkably, however, C/EBPδ deficiency does not consistently lead to a reduction in Lipopolysaccharide (LPS)-induced cytokine production by macrophages. Here, we address this apparent discrepancy and show that the effect of C/EBPδ on cytokine production and macrophage function depends on both the macrophage subtype and the LPS concentration used. Using CRISPR-Cas generated macrophages in which the transactivation domain of C/EBPδ was deleted from the endogenous locus (ΔTAD macrophages), we next show that the context-dependent role of C/EBPδ in macrophage biology relies on compensatory transcriptional activity in the absence of C/EBPδ. We extend these findings by revealing a large discrepancy between transcriptional programs in C/EBPδ knock-out and C/EBPδ transactivation dead (ΔTAD) macrophages implying that compensatory mechanisms do not specifically modify C/EBPδ-dependent inflammatory responses but affect overall macrophage biology. Overall, these data imply that knock-out approaches are not suited for identifying the genuine transcriptional program regulated by C/EBPδ, and we suggest that this phenomenon applies for transcription factor families in general.

Heterogeneity in quiescent Müller glia in the uninjured zebrafish retina drive differential responses following photoreceptor ablation

“…Expression of cebpd was also upregulated at this initial phase. cebpd (CCAAT enhancer binding protein delta) while being associated with a reactive phenotype of mammalian astrocytes, Müller glia and immune cells (Roesch et al, 2012;Spek et al, 2021) (Wang et al, 2016), is also involved in proliferation (Ko et al, 2015) and may be crucial in the zebrafish Müller glia proliferative response.…”

Macrophage C/EBPδ Drives Gemcitabine, but Not 5-FU or Paclitaxel, Resistance of Pancreatic Cancer Cells in a Deoxycytidine-Dependent Manner

“…Macrophage-secreted chemokines and growth factors are suggested to induce drug resistance [ 18 ], suggesting that macrophage C/EBPδ potentiates gemcitabine resistance by enhancing chemokine/cytokine secretion. Analysis of recently obtained RNA-Seq data to determine transcriptional changes induced by C/EBPδ deletion in macrophages (GSE173552 [ 31 ]) revealed, however, no apparent changes in the expression of chemokines/cytokines. KEGG pathway enrichment analyses of differentially expressed genes between wild type and C/EBPδ-deficient macrophages, in fact, identified the pyrimidine metabolism pathway amongst the most significant pathways down-regulated by C/EBPδ deficiency ( Figure 2 A).…”

“…PANC-1, MIA PaCa-2 and BxPc3 PDAC cells (ATCC, Manassas, VA, USA) were cultured in DMEM (Lonza, Basel, Switzerland), whereas wild type and C/EBPδ knock out RAW264.7 macrophages, generated and characterized as described previously [ 31 ], were cultured in IMDM medium (Gibco, Thermo Fischer Scientific, Waltham, MA, USA). All media were supplemented with 10% fetal calf serum (FCS), 2 mM L-glutamine, 100 units/mL penicillin, and 500 μg/mL streptomycin (all Lonza, Basel, Switzerland) according to routine cell culture procedures.…”

“…Expression levels were normalized to the expression of the reference genes TBP, B2M and UBC using the primers listed in Supplementary Table S1 . RNA-Seq results and the corresponding experimental design were described previously [ 31 ] and represent transcriptional differences between wild type and C/EBPδ-deficient RAW264.7 cells. The complete sequence libraries are publicly available through the National Center for Biotechnology Information gene expression omnibus under the following accession number: GSE173552.…”

Allele-specific Effects of Three-dimensional Genome Architecture in Hybrid Pigs

“…Of these, VEGFA reportedly plays essential roles in promoting angiogenesis and hypoxiaresponse under in ammation 68 , while MAP4K4 is a mediator of oxidative stress-induced cell death 69 . In addition, three typical in ammatory markers also displayed greater RPS and expression levels in the Tibetan allele compared to that of the Berkshire allele, as well as greater sequence divergence between alleles, including ABI3 and ITGA6 (two hypoxia-induced regulators) 70,71 , and CEBPD (a hepatic lipogenic transcription factor that potentiates macrophage in ammatory response) 72 (Supplementary Fig. 20).…”