HDAC3 Mediates the Inflammatory Response and LPS Tolerance in Human Monocytes and Macrophages.

Mohammed Ghiboub,Theodorus B M Hakvoort,Jing Zhao,Iris Admiraal,Menno P J De Winther,Andrew Y F Li Yim,Peter Henneman,Ronald Schilderink,Patricia H P Van Hamersveld,Caroline Verseijden,Wouter J De Jonge,Jose M Duarte,David F Tough,Nicola R Harker

doi:10.3389/fimmu.2020.550769

Abstract

Histone deacetylases (HDACs) are a group of enzymes that control histone deacetylation and bear potential to direct expression of large gene sets. We determined the effect of HDAC inhibitors (HDACi) on human monocytes and macrophages, with respect to their polarization, activation, and their capabilities of inducing endotoxin tolerance. To address the role for HDACs in macrophage polarization, we treated monocytes with HDAC3i, HDAC6i or pan-HDACi prior to polarization into M1 or M2 macrophages using IFNγ or IL-4 respectively. To study the HDAC inhibition effect on cytokine expression, macrophages were treated with HDACi prior to LPS-stimulation. TNFα, IL-6, and p40 were measured with ELISA, whereas modifications of Histone 3 and STAT1 were assessed using western blot. To address the role for HDAC3 in repeated LPS challenge induction, HDAC3i or HDAC3 siRNA was added to monocytes prior to incubation with IFNγ, which were then repeatedly challenged with LPS and analyzed by means of protein analyses and transcriptional profiling. Pan-HDACi and HDAC3i reduced cytokine secretion in monocytes and M1 macrophages, whereas HDAC6i yielded no such effect. Notably, neither pan-HDACi nor HDAC3i reduced cytokine secretion in M2 macrophages. In contrast to previous reports in mouse macrophages, HDAC3i did not affect macrophage polarization in human cells. Likewise, HDAC3 was not required for IFNγ signaling or IFNβ secretion. Cytokine and gene expression analyses confirmed that IFNγ-treated macrophages consistently develop a cytokine response after LPS repeated challenge, but pretreatment with HDAC3i or HDAC3 siRNA reinstates a state of tolerance reflected by general suppression of tolerizable genes, possibly through decreasing TLRs expression, and particularly TLR4/CD14. The development of endotoxin tolerance in macrophages is important to reduce exacerbated immune response and limit tissue damage. We conclude that HDAC3 is an attractive protein target to mediate macrophage reactivity and tolerance induction in inflammatory macrophages.

Highlights

Histone acetylation controls chromatin remodeling, which in turn is thought to regulate gene transcription
We showed that HDAC3i resulted in the reduced expression of LPS-induced pro-inflammatory cytokines after a second exposure to LPS in human monocytes and M1 macrophages, but not in M2 macrophages, implying that the M1 macrophages have developed some degree of tolerance towards LPS
Through RNA-sequencing, we found that HDAC3i-pretreated macrophages without any LPS exposure upregulate most of the Hallmark gene sets, inflammatory pathways included

Summary

Introduction

Histone acetylation controls chromatin remodeling, which in turn is thought to regulate gene transcription. Where histone acetyltransferases (HAT) add acetyl groups to lysine residues, thereby enabling transcription factor binding and subsequent gene expression, histone deacetylases (HDACs) remove histone residues leading to chromatin compaction, which generally results in gene repression [1, 2]. Such epigenetic mechanisms involving HDACs have gained interest in immunology, as they were found to mediate innate immune-cell memory processes, as well as development of immune training and tolerance towards endotoxins [3,4,5]. To reduce such side effects, HDAC-subtype-specific inhibitors with increased sensitivity and specificity have been developed

Methods

Results

Conclusion