Activation of RAW264.7 macrophages by the polysaccharide from the roots of Actinidia eriantha and its molecular mechanisms

Highlights

• We investigated the activation of macrophages by AEPS and its molecular mechanisms.

• AEPS induce the expression of accessory and costimulatory molecules on macrophages.

• AEPS promote the production of proinflammatory factors from macrophages.

• AEPS activate macrophages and elicit a balanced M1/M2 response via TLR/NF-κB pathway.

• This study further expands our knowledge on mechanism how AEPS acts as a potent adjuvant.

Abstract

The polysaccharide from the roots of Actinidia eriantha (AEPS), a potent antitumor agent and immunological adjuvant, was investigated for the immunomodulatory effects on RAW264.7 macrophages and its molecular mechanisms. AEPS could significantly enhance the pinocytic and phagocytic activity, induce the production of NO, TNF-α, IL-10, IL-1β and IL-6, and promote the expression of accessory and costimulatory molecules in RAW264.7 cells. PCR array assay revealed that AEPS up-regulated 28 genes including TLRs (TLR2, TLR8, TLR9), proinflammatory factors (IL-1β, G-CSF, IL-1α, GM-CSF, IL-6, COX-2, TNF-α, IFN-β, CXCL10, CCL2, TNF-β, IL-10), and the genes involved in NF-κB signaling pathway, and down-regulated 6 genes such as TLR3, TLR4, PGLYRP1, EIF2αK2, MAP3K1 and IRF1. AEPS was further showed to promote cytoplasmic IκB-α degradation and increase nuclear NF-κB p65 levels in RAW264.7 cells. These results suggested that AEPS activated RAW264.7 macrophages and elicited a M1 and M2 response through TLRs/NF-κB signaling pathway.

Introduction

Polysaccharides obtained from natural sources represent a structurally diverse class of macromolecules, and are known to affect a variety of biological responses, especially the immune response. Most polysaccharides derived from higher plants are relatively nontoxic and do not cause significant side effects, which is a major problem associated with bacterial polysaccharides and synthetic compounds. The plant polysaccharides are recognized as an effective biological response modifier with low toxicity (Petrovsky & Cooper, 2011). Recently, many polysaccharides have been shown to possess adjuvant potential on specific cellular and humoral immune responses against antigen, and be excellent adjuvant candidates for many vaccines (Licciardi & Underwood, 2011). Thus, the plant polysaccharides have enormous potential for use as an adjuvants in vaccines against both pathogens and cancer (Granell, Fernández-del-Carmen, & Orzáez, 2010).

The roots of Actinidia eriantha Benth (Actinidiaceae) have been used for gastric carcinoma, nasopharyngeal carcinoma, breast carcinoma, and hepatitis in traditional Chinese medicine. The modern pharmacological experiments proved that the water extracts of this drug possessed antitumor and immunopotentiating activities. In our previous works, the polysaccharide from the roots of A. eriantha (AEPS) has been shown to be the main active principles responsible for the antitumor and immunomodulatory effect of this drug (Xu, Yao, Sun, & Wu, 2009). AEPS was also proved to possess the immunological adjuvant activity on specific cellular and humoral immune responses to ovalbumin (OVA) in mice, and elicit a Th1 and Th2 immune responses (Sun, Wang, Xu, & Ni, 2009). Moreover, its low toxicity, no side effects and availability all make it as a safe and efficacious adjuvant candidate suitable for a wide spectrum of prophylactic and therapeutic vaccines. However, the underlying mechanisms of AEPS in the regulation of immune response need to be investigated.

Macrophages occupy a unique niche in the immune system, in that they can not only initiate innate immune responses, but also be effector cells that contribute to fight infection and inflammation. Macrophages can kill pathogens directly by phagocytosis and indirectly via the secretion of pro-inflammatory factors. Macrophages also exert an important role as an interface between innate and adaptive immunity. They are responsible for processes such as antigen processing and presentation to antigen-specific T cells. Following activation, macrophages can induce expression of accessory and costimulatory molecules that promote sustained stimulatory interactions with T cells and the generation of adaptive immunity. Indeed, the basic mechanisms of the immunostimulatory, anti-tumor, bactericidal and other therapeutic effects of plant polysaccharides are thought to occur via activation of immune cells resulting in the induction of immune responses (Beutler, 2004). Macrophages were thought to be the important target cells of some antitumor and immunomodulatory drug (Cheng, Wan, Wang, Jin, & Xu, 2008).

RAW264.7 cells are commonly accepted as a tool to investigate the molecular mechanisms of macrophages involved in regulating immunity (Hartley et al., 2008). The current experiments were designed to investigate the immunomodulatory effects of AEPS on RAW264.7 macrophages by determining the effect on the production and expression of nitric oxide, cytokines, chemokines, accessory and costimulatory molecules, and explore its molecular mechanisms using a high-throughput mouse Toll-like receptor (TLR) signaling pathway PCR array.