Melatonin suppresses TLR9-triggered proinflammatory cytokine production in macrophages by inhibiting ERK1/2 and AKT activation

Xiongfei Xu,Lingling Ai,Guoquan Wang,Jing Zhang,Yu-Xia Chen,Jianhui Shi

doi:10.1038/s41598-018-34011-8

Abstract

Toll-like receptor (TLR) signaling plays major roles in innate immune response in macrophages. Melatonin regulates TLR3- and TLR4-mediated innate immune responses in macrophages. However, it remains unknown whether melatonin regulates TLR9-mediated innate immune responses in macrophages. Here we demonstrated that melatonin suppressed TLR9 ligand-induced proinflammatory cytokines mRNA and protein production in peritoneal macrophages without interrupting the viability of peritoneal macrophages. Using a melatonin membrane receptors MT1/MT2 antagonist luzindole, we found that MT1 and MT2 were dispensable for melatonin’s inhibitory effects on TLR9-mediated proinflammatory cytokines production, even though melatonin upregulated mRNA expression of MT1 and MT2 in macrophages. Furthermore, melatonin did not affect mRNA expressions of TLR9 and MyD88 but attenuated TLR9 ligand-induced ERK1/2 and AKT phosphorylation without affecting p38 and NF-κB p65 phosphorylation. Also, melatonin inhibited TLR9-mediated proinflammatory cytokines production in vivo. Taken together, our results demonstrate that melatonin suppresses TLR9-triggered proinflammatory cytokines production in macrophages via melatonin membrane receptor-independent manners and probably through inhibiting ERK1/2 and AKT activation, which further elucidates the roles of melatonin in regulating TLR-mediated innate immune responses in macrophages.

Highlights

Toll-like receptor (TLR), an important pattern-recognition receptor (PRR), is critical for host defence against invading pathogens[1,2]
To investigate whether melatonin could regulate TLR9-mediated inflammatory responses in macrophages, primary peritoneal macrophages were pre-treated with melatonin and stimulated with TLR9 ligand CpG-ODN
Melatonin effectively suppressed the production of TNF-α, IL-6 and IL-12 p70 induced by CpG-ODN in a dose-dependent manner (Fig. 2A–C), whereas it exerted no notable suppression on anti-inflammatory IL-10 production (Fig. 2D)

Summary

Introduction

Toll-like receptor (TLR), an important pattern-recognition receptor (PRR), is critical for host defence against invading pathogens[1,2]. Ligation of TLRs by PAMPs induces the activation of intracellular signaling molecules such as MyD88 and TRIF, which in turn lead to the production of nuclear factor-κB (NF-κB) and mitogen-activated protein kinases (MAPKs)-dependent pro-inflammatory cytokines, or the production of interferon regulatory factor (IRF)-dependent type I interferons (IFNs)[1,2,3]. More and more studies show that melatonin modulates the functions of innate immune cells such as macrophages, monocytes, natural killer cells and neutrophils[13,17]. Its suppressive effects on TLR9 signaling did not depend on melatonin membrane receptors MT1 and MT2, but probably through inhibiting ERK1/2 and AKT activation without affecting mRNA expressions of TLR9 and MyD88. The results of our study clarify the inhibitory effects of melatonin on TLR9-mediated innate immune responses in macrophages, probably through suppressing ERK1/2 and AKT activation

Methods

Results

Conclusion