Elsevier

Molecular Immunology

Volume 114, October 2019, Pages 179-188
Molecular Immunology

The novel methyltransferase SETD4 regulates TLR agonist-induced expression of cytokines through methylation of lysine 4 at histone 3 in macrophages

https://doi.org/10.1016/j.molimm.2019.07.011Get rights and content

Highlights

  • LPS stimulation leads to enhanced expression of SETD4.

  • SETD4−/− macrophages display diminished IL-6 and TNF-α upon TLR-agonist stimulation.

  • SETD4 translocates from the cytosol to the nucleus in response to LPS stimulation.

  • SETD4 substantially enhances H3K4me1 and H3K4me2 levels.

  • SETD4 deficiency impairs the enrichment of H3K4me1 and H3K4me2 at the TNF-α and IL-6 promoters in LPS-stimulated BMDMs.

Abstract

The production of inflammatory cytokines is closely related to pathogen-associated molecular pattern (PAMP)-triggered activation of the Toll-like receptor (TLR), intracellular signal transduction pathways such as MAPK and NF-κB, and histone modifications. Histone methylation, a type of histone modifications, is mainly accomplished by a class of SET family proteins containing highly conserved SET domains. In the present study, we found that SET domain-containing protein 4 (SETD4) regulated inflammatory cytokines in response to TLR agonists. LPS stimulation led to the enhanced SETD4 expression, while the increased IL-6 and TNF-α release from LPS-stimulated RAW264.7 cells was attenuated by depletion of SETD4 using RNA interference. The results were further confirmed in BMDMs and pMφ isolated from SETD4-deficient mice where SETD4−/− macrophages treated with LPS, BLP or Poly(I:C) showed down-regulated IL-6 and TNF-α mRNA and protein levels when compared with SETD4+/+ macrophages. Moreover, the mRNA levels of all NF-κB-dependent genes including IL-1β, IL-10, NFKBA, DUSP1, CCL2, CCL5, and CXCL10 in SETD4−/− macrophages were substantially reduced. To further clarify the regulatory mechanism(s) by which SETD4 modulates inflammatory cytokines, we examined the effect of SETD4 on the activation of MAPK and NF-κB signalling pathways, and found that knockout of SETD4 had no effect on phosphorylation of p38, ERK, JNK, p65, and IκBα. Notably, SETD4 translocated quickly from the cytosol to the nucleus upon LPS stimulation, suggesting that SETD4 may exert its regulatory function downstream of the MAPK and NF-κB pathways. To characterize this, we performed an in vitro HMTase assay to measure histone methyltransferase (HMTase) activity of SETD4. H3K4me1 and H3K4me2 levels were enhanced dramatically with the supplementation of SETD4, whereas both H3K4me1 and H3K4me2 were strongly attenuated in SETD4−/− BMDMs. Moreover, the LPS-stimulated recruitment of H3K4me1 and H3K4me2 at both TNF-α and IL-6 promoters was severely impaired in SETD4−/− BMDMs. Collectively, these results demonstrate that SETD4 positively regulates IL-6 and TNF-α expression in TLR agonist-stimulated macrophages by directly activating H3K4 methylation.

Introduction

The host response to infection involves the detection of pathogen-associated molecular patterns (PAMPs) by innate immune cells via pattern recognition receptors (PRRs), such as Toll-like receptors (TLRs) (Vijay, 2018). TLRs recognize PAMPs such as lipopolysaccharide (LPS), bacterial lipoprotein (BLP) and polyinosinic:polycytidylic acid (poly (I:C), trigger multiple signalling pathways including the MAPK and NF-κB pathways, and induce the expression of inflammatory cytokines (Kaukonen et al., 2015). Uncontrolled and excessive expression of inflammatory cytokines leads to severe tissue injury and a variety of life-threatening pathological conditions including septic shock and multiple organ dysfunction syndrome (MODS) (Lakshmikanth et al., 2016; Liu and Yao, 2010). Therefore, the tight control and modulation of transcriptional expression of inflammatory cytokines during the pathological process are an important area of research.

Gene transcription is regulated epigenetically through alterations in the patterns of DNA methylation and covalent histone modifications (Berger, 2007; Nishioka et al., 2002). Histone methylation, one of the most characterized posttranslational modifications, regulates the transcription and replication of genes by providing binding platforms for diverse transcription factors that specify whether the promoter of an associated gene is in an active or repressed chromatin state, thereby controlling specific biological outcomes such as the inflammatory response (Falnes et al., 2016). Histone methylation can be associated with either transcriptional activation or transcriptional repression depending on its position and methylation state. For example, tri- or dimethylation at residue lysine 4 of histone H3 (H3K4) correlates with the activation of gene expression, whereas H3K36 dimethylation and H3K27 trimethylation is commonly linked to gene silencing (Kubicek and Jenuwein, 2004; Wei et al., 2009). Histone lysine methylation, which is tightly regulated by specific lysine methyltransferases and lysine demethylases, plays a critical role in the transcriptional expression of inflammatory cytokines (Medzhitov and Horng, 2009). For example, Smyd2, a SET (Su[var]3-9, E[z] and trithorax) domain family protein, can specifically facilitate H3K36 dimethylation at the Tnf and Il6 promoters, which suppresses the production of proinflammatory cytokines induced by macrophage activation (Xu et al., 2015). Ezh1 promotes TLR-triggered production of inflammatory cytokines, including IL-6, TNF-α and IFN-β, by suppressing the TLR negative regulator Tollip through its H3K27me3 methyltransferase activity (Liu et al., 2015). However, the detailed roles of histone methyltransferases, especially in TLR-mediated innate immune responses, remain poorly understood. A better understanding of the function of these enzymes in innate immunity and the pathophysiology of relevant diseases, as well as the underlying mechanisms, will provide new insight into the epigenetic regulation of immune responses.

SETD4 is a member of the SET family, which contains a highly conserved SET domain structure. To date, only a few studies have reported that SETD4 may play a role in the development of breast cancer (Faria et al., 2013), hepatocellular carcinoma (Li et al., 2014) and cell quiescence during diapause formation in Artemia (Dai et al., 2017). However, no report has investigated the role of SETD4 in innate immunity-associated inflammatory responses. Here, we report that SETD4 is a positive regulator of the expression of inflammatory cytokines including IL-6 and TNF-α in TLR agonist-triggered macrophages and SETD4 might exert its effect by enhancing H3K4 methylation directly. We propose that this newly identified SETD4 protein may play an important role in TLR agonist-induced inflammatory cytokine expression.

Section snippets

Reagents and antibodies

LPS (E. coli, serotype O111:B4) and Poly(I:C) were purchased from Sigma-Aldrich (St. Louis, MO, USA). BLP (Pam3CysSKKKK) was purchased from EMC Microcollections (Tubingen, Germany). TRIzol RNA was purchased from Invitrogen (Thermo Fisher Scientific, USA). SYBR® Green Real-time PCR and ReverTra Ace®qPCR RT Kit were obtained from TOYOBO (Osaka, Japan). Small interfering RNAs (siRNA) including specifically targeting SETD4 and its scrambled siRNA were obtained from Gene Pharma (Shanghai, China). A

SETD4 is involved in the inflammatory response

TLR-induced proinflammatory cytokines such as IL-6 and TNF-α are critical mediators in the immune response and inflammation against pathogens. To investigate whether SETD4 is involved in the regulation of inflammation, down-regulation of SETD4 by small RNA interference was used to assess the role of SETD4 in regulating inflammatory cytokines. After determining the efficiency of SETD4 RNA interference (Fig. 1A), IL-6 and TNF-α production upon LPS treatment were examined in SETD4 down-regulated

Discussion

Epigenetic modifications such as histone methylation are involved in the transcriptional regulation of multiple TLR agonist-mediated genes. For example, SMYD4, an SET domain family protein, is a negative regulator of inflammatory response genes. SMYD4 can trimethylate H4K20 as a repression checkpoint that restricts the expression of TLR4 signal pathway-mediated target genes in macrophages through its association with nuclear receptor co-repressor (NCoR) complexes (Stender et al., 2012). Ash1l,

Conclusions

Taken together, SETD4 positively regulates LPS-stimulated and other TLR agonist-stimulated a wild range of inflammatory mediators including both pro- and anti-inflammatory cytokines as well as chemokines, indicating that by up-regulating inflammatory cytokine expression and production, SETD4 plays an important role in host innate immune responses against microbial infection. Results from the present study indicate that SETD4 is essential for initiating an efficient inflammatory response via its

Funding

This work was supported by the National Natural Science Foundation of China (grant numbers 81471901, 81272149, 81873620), the Guangdong Provincial Key Laboratory Construction Project of China (grant number 2014B030301044), the Guangdong Provincial Natural Science Foundation (grant number 2015A030311031), and the South Wisdom Valley Innovative Research Team Program (grant number CXTD-001).

Declaration of Competing Interest

None.

Acknowledgements

We thank the Wellcome Trust Sanger Institute Mouse Genetics Project (Sanger MGP) and its funders for providing the mutant mouse line (Allele: SETD4), and the distribution centre from which we received the mouse line, the KOMP Repository at the University of California, Davis (www.komp.org). Funding and associated primary phenotypic information may be found at www.sanger.ac.uk/mouseportal.

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