High-Throughput Screening for CEBPD-Modulating Compounds in THP-1-Derived Reporter Macrophages Identifies Anti-Inflammatory HDAC and BET Inhibitors

Abstract

This study aimed to identify alternative anti-inflammatory compounds that modulate the activity of a relevant transcription factor, CCAAT/enhancer binding protein delta (C/EBPδ). C/EBPδ is a master regulator of inflammatory responses in macrophages (Mϕ) and is mainly regulated at the level of CEBPD gene transcription initiation. To screen for CEBPD-modulating compounds, we generated a THP-1-derived reporter cell line stably expressing secreted alkaline phosphatase (SEAP) under control of the defined CEBPD promoter (CEBPD::SEAP). A high-throughput screening of LOPAC®1280 and ENZO®774 libraries on LPS- and IFN-γ-activated THP-1 reporter Mϕ identified four epigenetically active hits: two bromodomain and extraterminal domain (BET) inhibitors, I-BET151 and Ro 11-1464, as well as two histone deacetylase (HDAC) inhibitors, SAHA and TSA. All four hits markedly and reproducibly upregulated SEAP secretion and CEBPD::SEAP mRNA expression, confirming screening assay reliability. Whereas BET inhibitors also upregulated the mRNA expression of the endogenous CEBPD, HDAC inhibitors completely abolished it. All hits displayed anti-inflammatory activity through the suppression of IL-6 and CCL2 gene expression. However, I-BET151 and HDAC inhibitors simultaneously upregulated the mRNA expression of pro-inflammatory IL-1ß. The modulation of CEBPD gene expression shown in this study contributes to our understanding of inflammatory responses in Mϕ and may offer an approach to therapy for inflammation-driven disorders.

Epigenetic Regulation of Inflammatory Signaling and Inflammation-Induced Cancer

“…Notably, these epigenetic drugs also harbor anti-inflammatory characteristics, e.g., I-BET151 hampers the expression of the proinflammatory genes IL-1β and TNFα in rheumatoid arthritis synovial fibroblasts, leading to a decreased ability in recruiting immune cells and their lowered proliferative capacity ( Klein et al, 2016 ). A recent report by Ullmann et al (2021) demonstrated that treatment with the BET inhibitors I-BET151 and Ro 11–1,464 in cultured macrophages not only increases endogenous levels of the tumor suppressor protein CEBPD, but also downregulates key cytokine genes like CCL2 and IL-6, buttressing their anti-inflammatory functions. Furthermore, beyond the realm of drug therapeutics, natural dietary supplements like Vitamins C, D and E can also enact both anti-inflammatory and epigenetic effects ( Saccone et al, 2015 ; Gerecke et al, 2018 ; Zappe et al, 2018 ; Yang et al, 2019 ).…”

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

“…In particular, patients with high C/EBPδ positive macrophage numbers are likely to benefit from combination therapy, but this hypothesis needs to be addressed in preclinical PDAC models before one could pursue to clinical studies. Although no C/EBPδ inhibitor has yet been clinically approved, a recent study showed that two histone deacetylase (HDAC) inhibitors, suberoylanilide hydroxamic acid (SAHA) and trichostatin A (TSA), abolished endogenous C/EBPδ mRNA expression levels in THP-1 macrophages [ 44 ]. Whether these HDAC inhibitors do reduce C/EBPδ levels in tumor associated macrophages and subsequently potentiate gemcitabine efficacy in PDAC remains to be established in future experiments.…”

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

“…C/EBPδ is a member of the C/EBP family of transcription factors that, according to the current paradigm, potentiates cytokine production and modulates macrophage function, thereby enhancing the inflammatory response [ 5 , 22 ]. In the current manuscript, we show, however, that cytokine levels are not consistently reduced in LPS-stimulated C/EBPδ deficient macrophages.…”

“…According to the current paradigm, C/EBPδ acts as a pro-inflammatory transcription factor enhancing the expression of pro-inflammatory mediators [ 4 , 5 ]. Indeed, C/EBPδ is shown to regulate IL-1β or collagen-induced cyclo-oxygenase-2 (COX-2) expression [ 6 , 7 , 8 ]; to induce toll-like receptor (TLR)-4 expression and subsequent signalling [ 9 ]; to potentiate Fcγ receptor-mediated tumor necrosis factor alpha (TNFα), macrophage inflammatory protein (MIP)-2 and MIP-1α expression [ 10 ]; to mediate IgG immune complex-induced macrophages inflammatory cytokine and chemokine production in macrophages [ 11 , 12 ]; and to enhance lipopolysaccharide (LPS)-induced IL-6, monocyte chemoattractant protein-1 (MCP-1) and endothelin-1 levels [ 13 ].…”