Elsevier

International Immunopharmacology

Volume 40, November 2016, Pages 254-264
International Immunopharmacology

A bis-malonic acid fullerene derivative significantly suppressed IL-33-induced IL-6 expression by inhibiting NF-κB activation

https://doi.org/10.1016/j.intimp.2016.08.031Get rights and content

Highlights

  • A bis-malonic acid fullerene derivative inhibits the IL-33-induced IL-6 expression.

  • A bis-malonic acid fullerene derivative inhibits the IL-33-induced NF-κB activation.

  • A bis-malonic acid fullerene derivative directly inhibits IKK activation.

Abstract

IL-33 functions as a ligand for ST2L, which is mainly expressed in immune cells, including mast cells. IL-33 is a potent inducer of pro-inflammatory cytokines, such as IL-6, and has been implicated in the pathogenesis of allergic inflammatory diseases. Therefore, IL-33 has recently been attracting attention as a new target for the treatment of inflammatory diseases. In the present study, we demonstrated that a water-soluble bis-malonic acid fullerene derivative (C60-dicyclopropane-1,1,1′,1′-tetracarboxylic acid) markedly diminished the IL-33-induced expression of IL-6 in bone marrow-derived mast cells (BMMC). The bis-malonic acid fullerene derivative suppressed the canonical signaling steps required for NF-κB activation such as the degradation of IκBα and nuclear translocation of NF-κB by directly inhibiting the IL-33-induced IKK activation. Although p38 and JNK also contributed to IL-33-induced expression of IL-6, the bis-malonic acid fullerene derivative did not affect their activation. Furthermore, the bis-malonic acid fullerene derivative had no effect on the NF-κB activation pathway induced by TNFα and IL-1. These results suggest that the bis-malonic fullerene derivative has potential as a specific drug for the treatment of IL-33-induced inflammatory diseases by specifically inhibiting the NF-κB activation pathway.

Graphical abstract

The inhibitory mechanism of IL-33signaling pathway by the bis-malonic acid fullerene derivative.

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Introduction

ST2L is a member of the interleukin (IL)-1 receptor (IL-1R)/Toll-like receptor (TLR) superfamily and expressed on several immune cells, including Th2 lymphocytes, and mast cells [1], [2]. Schmitz et al. identified IL-33 as a functional ligand of ST2L in 2005 [3], and the physiological function of IL-33 through ST2L has since been elucidated in detail. The finding of alterations in the mRNA and protein expression of IL-33 in the serum and mucosa of patients with inflammatory diseases was of importance. The expression of IL-33 was previously shown to be enhanced by asthma, and anaphylactic shock [4], [5]. Furthermore, the secretion of chemical mediators from mast cells was augmented in patients with these conditions, and was attributed to the stimulating effects of IL-33, suggesting that IL-33 contributes to the pathogenesis of chronic autoimmune diseases and inflammatory diseases [6]. These findings clearly demonstrate the importance of IL-33 in the onset of inflammatory diseases, and have prompted research with a focus on IL-33 as a target for new therapeutic anti-inflammatory drugs.

Once stimulated with IL-33, ST2L forms a protein complex with the IL-1R accessory protein (IL-1RAcP) that activates downstream signaling molecules such as the mitogen-activated protein (MAP) kinase family and nuclear factor-kappa B (NF-κB), which is a critical transcription factor in inflammation [7], [8]. The ST2L-IL-1RAcP complex has also been shown to recruit signaling molecules including myeloid differentiation factor 88 (MyD88), IL-1R-associated kinase-1 (IRAK-1), and tumor necrosis factor (TNF) receptor-associated factor 6 (TRAF6), similar to other members of the IL-1R/TLR superfamily [3], [9]. The formation of this signaling complex is known to activate the canonical pathway of NF-κB. IκB kinase (IKK), which is downstream of TRAF6, phosphorylates two N-terminal serine residues of the inhibitor of NF-κB (IκBα). Phosphorylated IκBα is recognized by the ubiquitin ligase mechanism, which results in polyubiquitination and proteasomal degradation [10]. The released NF-κB complex has been shown to translocate to the nucleus, in which it binds to specific sequences in the promoter or enhancer regions of target genes involved in inflammatory reactions [11], [12].

Fullerene (C60) is a spherical molecule with a diameter of 0.7 nm and is a new kind of organic compound with a cage-like structure [13]. Chemical modifications with several water-soluble groups into the fullerene core have increased its solubility, and water-soluble fullerene derivatives were previously reported to possess various biological and pharmacological properties [14]. A pyrrolidinium fullerene derivative has been shown to exhibit anti-cell proliferative activities by inducing apoptosis, which was attributed to the generation of reactive oxygen species (ROS), indicating that fullerene derivatives have potential as anti-cancer drugs [15], [16]. A previous study reported that proline-modified fullerene derivative exhibited inhibitory activities against human immunodeficiency virus (HIV)-reverse transcriptase and Hepatitis C virus RNA polymerase, which demonstrated that fullerene derivatives may be candidates for antiviral agents [17].

In the present study, we found that a bis-malonic acid fullerene derivative significantly inhibited the IL-33-induced expression of IL-6 by inhibiting the NF-κB activation pathway. These results clearly show the potential of the bis-malonic acid fullerene derivative as a novel anti-inflammatory drug.

Section snippets

Antibodies and reagents

A bis-malonic acid fullerene derivative was synthesized as previously described [18], [19]. Recombinant murine IL-33, IL-1β, and TNFα were purchased from PEPROTECH (Rocky Hill, NJ, USA). Antibodies against NF-κB (p65), Lamin B, IκBα, IKKγ, and β-actin were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-GST antibody was purchased from Nacalai Tesque (Tokyo, Japan). Antibodies against JNK, phosphorylated JNK at Thr183/Tyr185, p38, phosphorylated p38 at Thr180/Tyr182,

The bis-malonic acid fullerene derivative significantly inhibited IL-33-induced expression of IL-6

In order to investigate the anti-inflammatory effects of the bis-malonic acid fullerene derivative (C60-dicyclopropane-1,1,1′,1′-tetracarboxylic acid) (Fig. 1A), we tested its effects on the IL-33 signaling pathway in BMMC that had differentiated for 4 weeks in the presence of IL-3. The results of a flow cytometry analysis revealed the significant cell-surface expression of ST2L on BMMC (Supplemental Fig. 1). We first analyzed the cytotoxicity of the bis-malonic acid fullerene derivative against

Discussion

Fullerenes are soccer ball-shaped carbon cages that may be functionally derivatized by numerous types of chemical modifications. Due to their unique chemical characteristics, fullerenes are now being investigated as a novel tool to diagnose, monitor, and treat certain conditions [14], [15], [16], [17], [18]. A previous study reported that one of the fullerene derivatives, N-ethyl-polyamino C60, inhibited histamine release from mast cells and prevented the IgE-induced production of ROS in mast

Conflict of interest statement

All the authors declare that they have no conflict of interest.

Acknowledgments

We thank Mr. Shigenobu Yokota and Mr. Kazuhi Okamoto for their technical help. We thank Dr. Kazuo Umezawa (School of Medicine, Aichi Medical University) for the gift of DHMEQ. This work was supported in part by grants (25460073) from MEXT, the Takeda Science Foundation and Uehara Memorial Foundation. This work was also supported by the Platform for Drug Discovery, Informatics, and Structural Life Science from the Ministry of Education, Culture, Sports, Science and Technology, Japan.

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