A Novel Aminosaccharide Compound Blocks Immune Responses by Toll-like Receptors and Nucleotide-binding Domain, Leucine-rich Repeat Proteins

Kyoung-Hee Lee,Yuen-Joyce Liu,Amlan Biswas,Chikako Ogawa,Koichi S Kobayashi

doi:10.1074/jbc.m110.108001

Abstract

Toll-like receptors (TLRs) and nucleotide-binding domain, leucine-rich repeat (NLR) proteins are two major forms of innate immune receptors that trigger inflammatory responses by various biological mechanisms such as cytokine production, recruitment of inflammatory cells, or activation of adaptive immunity. Although the innate immune system is designed to fight against infectious pathogens, excessive activation of TLR or NLR signaling pathways may lead to unwarranted inflammation with hazardous outcomes, including septic shock or inflammatory diseases. As part of the search for effective therapeutics to regulate these responses, here we show that a novel aminosaccharide compound, named DFK1012, inhibits immune responses caused by TLR and NLR activation. Treatment with DFK1012, but not its derivatives DFK845 or DFK846, strongly inhibited pro-inflammatory cytokine production upon stimulation via either TLR or NLR proteins in macrophages. Importantly, we have not observed cytotoxicity in any range of its working concentration. Treatment with DFK1012 did not interfere with TLR- or NLR-induced activation of p38 and JNK, phosphorylation/degradation of IκB, and subsequent nuclear translocation of NF-κB subunit p65, suggesting that the inhibitory activity of DFK1012 is not due to the suppression of downstream signaling. Indeed, DFK1012 did not impair transcription of pro-inflammatory cytokine genes but rather promoted post-translational degradation of pro-inflammatory cytokines. Therefore, DFK1012 is a novel anti-inflammatory compound that drives proteolysis of proinflammatory cytokines induced by TLR and NLR stimulation. DFK1012 may represent a novel class of potential therapeutic agents aimed at the treatment of inflammatory disorders.

Highlights

(TLRs)[2] as well as cytoplasmic nucleotide-binding domain and leucine-rich repeat (NBD-LRRs or NLRs) protein families are important groups of pattern recognition receptors that provide immediate immune responses by recognizing different but overlapping microbial components, frequently referred to as pathogen-associated molecular patterns (PAMPs) (2)
DFK1012 Inhibits the Production of Inflammatory Cytokines Induced by Toll-like receptors (TLRs) and NLR Activation—Using muramyl dipeptide (MDP) as a lead compound, we generated various biotinylated derivatives, including DFK1012 (Fig. 1). To determine whether these compounds influence the expression of pro-inflammatory cytokines, we stimulated a murine macrophage cell line (RAW264.7) with LPS (TLR4 ligand) or CpG-B oligodeoxynucleotide (TLR9 ligand) in the presence or absence of DFK1012 and determined levels of IL-6 and TNF-␣ in culture medium by ELISA
The parent molecule MDP enhanced the production of IL-6, TNF-␣, and IL-12 p40 following LPS stimulation (Fig. 2, B and C), which is consistent with an earlier report on the synergism between MDP and TLR signaling (27)

Summary

Introduction

(TLRs)[2] as well as cytoplasmic nucleotide-binding domain and leucine-rich repeat (NBD-LRRs or NLRs) protein families are important groups of pattern recognition receptors that provide immediate immune responses by recognizing different but overlapping microbial components, frequently referred to as pathogen-associated molecular patterns (PAMPs) (2). DFK1012 Inhibits the Production of Inflammatory Cytokines Induced by TLR and NLR Activation—Using MDP as a lead compound, we generated various biotinylated derivatives, including DFK1012 (Fig. 1).

Results

Conclusion