Down-regulation of IL-6 production by astaxanthin via ERK-, MSK-, and NF-κB-mediated signals in activated microglia

https://doi.org/10.1016/j.intimp.2010.09.007Get rights and content

Abstract

In this study, we investigated the effect of astaxanthin on IL-6 in activated microglial cells because excessive interleukin-6 (IL-6) production by activated brain microglia has been linked to many neurological disorders and proper regulation of IL-6 is critical for maintaining brain homeostasis. Astaxanthin inhibited lipopolysaccharide (LPS)-stimulated IL-6 mRNA and protein in BV-2 microglial cells. Moreover, LPS-induced p-IKKα, p-IκBα, and p-NF-κB p65 levels were all suppressed by astaxanthin. The translocation of p-NF-κB p65 from the cytosol into the nucleus and transcriptional activity were inhibited by astaxanthin. IL-6 expression and NF-κB transcriptional activation were inhibited by astaxanthin, as well as inhibitors of NF-κB and MAPK in LPS-stimulated BV-2 microglial cells. Consistent with these findings, astaxanthin down-regulated the activation of p-extracellular signal-regulated kinase 1/2 (p-ERK1/2) and p-mitogen- and stress-activated protein kinase 1(p-MSK1), but not of p-c-jun N-terminal kinase (p-JNK). Astaxathin also decreased IL-6 mRNA and protein levels in LPS-stimulated primary microglial cells, RAW264.7 macrophages, and peritoneal macrophages. In addition, IL-6 suppression through astaxanthin-induced down-regulation of p-ERK1/2, p-MSK1, and p-NF-κB p65 occurred in microglial cells stimulated with LPS or stromal derived factor (SDF)-1α. Astaxathin also inhibited the secretion and mRNA expression of IL-6 in SDF-1α-stimulated microglial cells. SDF-1α-stimulated ERK1/2, MSK1, and NF-κB p65 phosphorylation were reduced by astaxanthin. Therefore, our results suggest that astaxanthin regulates IL-6 production through a p-ERK1/2-MSK-1- and p-NF-κB p65-dependent pathway in activated microglial cells.

Research highlights

► Astaxanthin suppresses IL-6 level in activated microglial cells and macrophages. ► The inhibitory effect of astaxanthin on IL-6 expression occur through NF-κB-mediated signaling pathway. ► MAPK pathway mediates the inhibitory effect of astaxanthin on IL-6 expression.

Introduction

The cytokine interleukin-6 (IL-6) is one of the major pro-inflammatory cytokines produced by monocytes and macrophages [1]. This cytokine is involved in autoimmune disorders and chronic inflammation and mediates the innate-adaptive immunity interface [1], [2], [3], [4], [5], [6]. IL-6 has both beneficial and destructive effects depending on the physiological conditions in the central nervous system (CNS) [1]. An elevation of IL-6 levels is closely associated with many neurological disorders, including Alzheimer's disease, multiple sclerosis, and viral and bacterial infections [7]. Over-expression of IL-6 leads to neuropathology, including neurodegeneration, breakdown of the blood-brain barrier, angiogenesis, impaired learning, and influences glial cells as messengers between the immune system and the CNS [7], [8], [9].

Microglia, the resident macrophages of the CNS, play important roles as cellular mediators of neuroinflammatory processes associated with neurodegeneration, toxicology, and immunity [10]. Many signals that activate microglial cells can trigger IL-6 synthesis via pathological processes in the brain [11]. Moreover, IL-6 contributes to inflammation-associated carcinogenesis in cancers of the colon and liver, and is elevated in the cystic fluids of malignant glioblastomas and astrocytomas of the CNS, affecting both tumor growth and malignancy [12], [13], [14]. Thus, IL-6 is not just a pro-inflammatory cytokine that regulates immune responses and potently mediates cell–cell interactions and cellular growth in the brain. Proper regulation of IL-6 concentrations may be critical to maintaining brain homeostasis. Within this context, identification of therapeutic molecules and detailed signaling pathways that modulate IL-6 activity will be useful for treating neurodegenerative disorders, malignant brain tumors, and gliomas. Currently, some clinical trials involving the blockade or modulation of IL-6 are under way with the aim of treating lymphoma, myeloma, renal cancer, rheumatoid arthritis, Crohn's disease, and hematological malignancy [15].

Astaxanthin is a naturally occurring carotenoid pigment that belongs to a phytochemical class known as terpenes and has significant antioxidant capacity [16], [17]. It is found in many kinds of marine organisms, such as microalgae, yeast, salmon, trout, krill, and shrimp [18]. Astaxanthin inhibits tumor invasion and carcinogenesis by decreasing extracellular matrix production [19]. Additionally, astaxanthin inhibits low density lipoprotein oxidation, scavenger receptor expression, and matrix metalloproteinase activity in macrophages [20]. Astaxanthin has been reported to possess a wide variety of biological functions, including antioxidant and anti-tumor activities [21], [22]. However, the mechanisms and signaling pathways underlying these properties are not fully understood. Moreover, it has not yet been reported whether astaxanthin can modulate the expression and production of IL-6 in microglial cells.

Therefore, we investigated whether astaxanthin could modulate levels of IL-6 produced from activated microglia. The present study was performed using the immortalized murine microglial cell line BV-2, which has been used as a valid substitute for primary microglia in many experiments [23]. In activated BV-2 microglial cells, we demonstrated that astaxanthin down-regulates IL-6 expression and production via the p-ERK1/2- MSK-1- and p-NF-κB p65-dependent pathway.

Section snippets

Materials

The primary antibodies used for Western blotting were rabbit anti-IKKα/β, anti-p-IKKα/β, anti-IκBα, anti-p-IκBα, anti-ERK1/2, anti-pERK1/2, anti-NF-κB p65, anti-p-NF-κB p65, p-MSK1, anti-JNK1/2, anti-p-JNK1/2, and anti-GAPDH. All antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). PDTC (pyrrolidine-1-carbodithioic acid), SP600125, U0126, and stromal-derived factor (SDF)-1α were all obtained from Calbiochem (Gibbstown, NJ, USA).

Cell culture

The mouse microglial cell line BV-2 was

The inhibitory effect of astaxanthin on IL-6 expression

Initially, we ensured that our experiments were all performed under conditions in which astaxanthin did not affect cell viability. Next, to know whether astaxanthin could modulate IL-6 expression in activated microglial cells, we examined IL-6 production at both the mRNA and protein level in LPS-stimulated BV-2 microglial cells. LPS induced IL-6 mRNA expression (Fig. 1A). When BV2 cells were pretreated with various concentrations of astaxanthin (0, 6.25, 12.5, 25, and 50 μM) for 24 h and then

Discussion

IL-6 has detrimental effects in various chronic inflammation-associated neuronal diseases and cancers [1]. Increased levels of IL-6 in the brain have been linked to inflammation-related neuronal diseases and gliomas [8], [9]. IL-6 expression in the brain increases after LPS injection and during the development of neurodegenerative disease [28]. In our studies, we demonstrated that astaxanthin could inhibit increases in IL-6 mRNA and protein in microglial cells after LPS stimulation. The

Acknowledgements

We thank the Korean Basic Science Institute Chuncheon Center for assistance with confocal microscopy imaging. We thank Dr. S.Y. Choi of Hallym University for providing BV-2 microglial cells. This research was supported by Future–based Technology Development Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education, Science and Technology (2009-0081760).

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