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

Tatjana Ullmann,Sonja Luckhardt,Michael J Parnham,Eduard Resch,Markus Wolf

doi:10.3390/ijms22063022

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.

Highlights

Inflammation is a protective host response to pathogens and cell or tissue damage that maintains tissue homeostasis and enables the organism to survive during injury or infection
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)
Secreted alkaline phosphatase (SEAP) is a well-established gene reporter, secreted levels in the conditioned medium of which are proportional to the changes in its intracellular mRNA, protein levels, and cell number [46,47]

Summary

Introduction

Inflammation is a protective host response to pathogens and cell or tissue damage that maintains tissue homeostasis and enables the organism to survive during injury or infection. Inflammatory responses are mediated by multiple interactions between immune cells and regulated by molecular mediators and their corresponding signaling pathways. Inflammation is believed to be regulated mainly at the level of gene transcription [1,2,3]. Human CCAAT/enhancer binding protein delta (CEBPD) gene encodes C/EBPδ transcription factor (TF), which belongs to a family of basic-leucine zipper (bZIP) domain TFs [4,5,6]. C/EBPδ functions as a key regulator of inflammatory responses [7,8], which can be activated by various stimuli, such as lipopolysaccharide (LPS) [9,10,11,12,13,14], interferon-gamma (IFN-γ) [9,14], other cytokines [4,9,13,14,15,16,17,18,19], glucocorticoids [20,21], or prostaglandins [22,23]

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