Platycodon grandiflorum polysaccharide induces dendritic cell maturation via TLR4 signaling

Highlights

• PG increases the expression of MHC-I/II and co-stimulatory molecules on DC surface.

• PG increases cytokine production and allogeneic T cell stimulation capacity of DCs.

• PG does not induce maturation of TLR4-mutated DCs.

• PG activates MAPK and NF-κB signaling downstream of TLR4 in DCs.

Abstract

Dendritic cell (DC) maturation is critical for initiation of the adoptive immune response. DC maturation is often attenuated in several pathological conditions including cancer. In this study, we report the effect of Platycodon grandiflorum polysaccharide (PG) on DC maturation. PG induced phenotypic maturation of DCs, as proved by the increase in the expression of CD40, CD80, CD86, and major histocompatibility complex (MHC)-I/II on the cell surface. PG also induced functional maturation of DCs, as proved by elevated production of interleukin (IL)-12, tumor necrosis factor-α, IL-1β, IL-6, IL-10, and interferon-β, and by enhanced allogeneic T cell stimulation ability of PG-treated DCs. PG efficiently induced maturation of DCs from C3H/HeN mice, which have normal Toll-like receptor-4 (TLR4), but not that of DCs from C3H/HeJ mice, which have mutated TLR4, suggesting that TLR4 might be one of the PG receptors in DCs. In line with TLR4 activation, PG increased the phosphorylation of ERK, p38, and JNK, and the nuclear translocation of p-c-Jun, p-CREB, and c-Fos. PG also activated NF-κB signaling, as evidenced by degradation of IκBα/β and nuclear translocation of p65 and p50. In summary, our data suggest that PG induces DC maturation by activating MAPK and NF-κB signaling downstream of TLR4.

Introduction

Dendritic cells (DCs) are the most potent antigen-presenting cells that initiate immune responses. Immature DCs differentiate from lymphoid or myeloid progenitors, are located at different sites of the body, and encounter the invading foreign antigens or pathogens (Joffre et al., 2009). After maturation, DCs show enhanced antigen presentation through up-regulation of the major histocompatibility complex (MHC) and co-stimulatory molecule expression, actively migrate to lymph nodes as a consequence of up-regulation of CCR7, and strongly stimulate T cells (Forster et al., 2012). However, DC functions are usually attenuated in tumor tissues, which impairs the induction of potent anti-tumor immune responses (Gallois and Bhardwaj, 2013). Several factors such as vascular endothelial growth factor, transforming growth factor-β, interleukin (IL)-10, prostaglandin E₂, and gangliosides down-regulate DC functions (Gallois and Bhardwaj, 2013, Steinhagen et al., 2011). Actually, several deficiencies in maturation, cytokine production, migration to lymph nodes, and T cell activation ability of DCs were observed in the tumor microenvironment.

Recovery of DC maturation is an important issue in cancer therapy (Kim et al., 2010b). Several studies have identified endogenous inducers of DC maturation, such as prostaglandins and inflammatory cytokines including tumor necrosis factor (TNF)-α, IL-6, and IL-1β (Mittal and Prasadarao, 2008). In addition, there are several exogenous inducers of DC maturation, including agonists of Toll-like receptor (TLR) 3, TLR4, and TLR9 (Mata-Haro et al., 2007). In particular, TLR4 agonists have been studied as the most promising inducers of DC maturation (Mazzoni and Segal, 2004). TLR4 has many exogenous agonists, which include compounds originating from foreign invaders, such as bacterial lipopolysaccharide (LPS), fungal mannan, protozoan glycoinositol phospholipids, and viral F protein (Uematsu and Akira, 2008). Among these compounds, LPS is the strongest natural agonist for TLR4 in DCs; however, its toxicity prevents its use as a DC activator or vaccine adjuvant in humans (Steinhagen et al., 2011). Monophosphoryl lipid A (MPL), a chemical derivative of LPS, was synthesized to maintain immunogenicity with reduced toxicity (Reed et al., 2009). MPL absorbed to alum is used clinically as an adjuvant in vaccines against human hepatitis and papilloma virus (Dubensky and Reed, 2010). MPL increases co-stimulatory molecule expression and cytokine production in DCs and their migration to lymph nodes (Didierlaurent et al., 2009). Glucopyranosyl lipid A, another synthetic analog of LPS, appears to be a TLR4 agonist that allows DC maturation (Pantel et al., 2012). In addition, many natural polysaccharides isolated from fungi such as Cordyceps militaris (Kim et al., 2006a), Coriolus versicolor (Kanazawa et al., 2004), Paecilomyces cicadae (Kim et al., 2012), Phellinus linteus (Kim et al., 2004), and Sparassis crispa (Kim et al., 2010b) and plants such as Angelica dahurica (Kim et al., 2013b), Morus alba (Shin et al., 2013) and Pueraria lobata (Kim et al., 2013a), appear to induce DC maturation by mediating TLR4 binding. DC activator from a fungus Grifola frondosa has been assessed in clinical trials of cancer immunotherapy (Ferreira et al., 2010).

Here, we describe another natural TLR4 agonist that stimulates DC maturation. The roots of Platycodon grandiflorum have been used as a food source and in traditional oriental medicine for the treatment of bronchitis, asthma, hyperlipidemia, hypertension, and diabetes (Xu et al., 2011). A polysaccharide (PG) isolated from P. grandiflorum stimulates B cells and macrophages, but not T cells (Han et al., 2001). PG also activates RAW264.7 cells through activation of MAPK and NF-κB signaling (Yoon et al., 2003, Yoon et al., 2004). However, the effects of PG on DCs have remained elusive, which prompted us to investigate its effect on such DC functions as MHC and co-stimulatory molecule expression, cytokine production, migration, and T cell activation ability. We focused on the identification of the PG receptor and intracellular signaling events in DCs.