The Effect of Enzymatically Polymerised Polyphenols on CD4 Binding and Cytokine Production in Murine Splenocytes

Abstract

High-molecular weight polymerised polyphenols have been shown to exhibit anti-influenza virus, anti-HIV, and anti-cancer activities. The purpose of this study was to evaluate the immunomodulating activities of enzymatically polymerised polyphenols, and to clarify the underlying mechanisms of their effects. The cytokine-inducing activity of the enzymatically polymerised polyphenols derived from caffeic acid (CA), ferulic acid (FA), and p-coumaric acid (CoA) was investigated using murine splenocytes. Polymerised polyphenols, but not non-polymerised polyphenols, induced cytokine synthesis in murine splenocytes. Polymerised polyphenols induced several cytokines in murine splenocytes, with interferon-γ (IFN-γ) and granulocyte-macrophage colony-stimulating factor (GM-CSF) being the most prominent. The underlying mechanisms of the effects of the polymerised polyphenols were then studied using neutralising antibodies and fluorescent-activated cell sorting (FACS) analysis. Our results show that polymerised polyphenols increased IFN-γ and GM-CSF production in splenocytes. In addition, the anti-CD4 neutralised monoclonal antibody (mAb) inhibited polymerised polyphenol-induced IFN-γ and GM-CSF secretion. Moreover, polymerised polyphenols bound directly to a recombinant CD4 protein, and FACS analysis confirmed that interaction occurs between polymerised polyphenols and CD4 molecules expressed on the cell surface. In this study, we clearly demonstrated that enzymatic polymerisation confers immunoactivating potential to phenylpropanoic acids, and CD4 plays a key role in their cytokine-inducing activity.

Agaricus brasiliensis-derived β-glucans exert immunoenhancing effects via a dectin-1-dependent pathway

“…Because dectin-1 has been reported to synergize with TLR2 or TLR4 [33], these receptors may contribute to the bioactivity associated with the intake of A. brasiliensis fruiting body as food. Furthermore, polymerized substances may also contribute to the immunomodulating activity of this mushroom because of the following reasons: (i) in the present study, we have demonstrated that GM-CSF is a key factor for the induction of cytokine synthesis in murine splenocytes by Agaricus-derived β-glucans, (ii) we have previously reported that A. brasiliensis possesses potent polyphenol oxidase and peroxidase activities in the fruiting body [34][35][36], and (iii) highmolecular-weight polyphenols synthesized by peroxidase induce GM-CSF production in murine leukocytes [37]. GM-CSF exhibits a broad range of biological activities, such as cell differentiation, activation, and inflammatory responses.…”

Species difference in reactivity to lignin-like enzymatically polymerized polyphenols on interferon-γ synthesis and involvement of interleukin-2 production in mice

“…The receptor, which critically modulates the immunoenhancing activity of polymerized polyphenols, remains to be identified. We have previously reported on the interaction between polyphenols and cell surface CD4 [9], also demonstrated that polymerized polyphenols required both T cells and APCs for cytokine synthesis [16]. This suggested that CD4 signal alone could not induce sufficient stimulus to leukocytes.…”

“…Polymerized caffeic (pCA), trans-ferulic (pFA) and trans-p-coumaric (pCoA) acids were synthesized as described previously [9]. Briefly, 200 mg of precursor phenylpropanoic acid was neutralized with 1 N NaOH and diluted to 10 mL with phosphate-buffered saline (PBS) containing 1 mg of HRP.…”

“…After centrifugation, the supernatant was collected and extensively dialyzed (MWCO: 50,000) against deionized water for 2 d, and then lyophilized. The amount of endotoxin (231.5, 93.7 pg/mg, and no detection in pCA, pFA, and pCoA, respectively) was evaluated by a quantitative limulus amoebocyte lysate assay as described previously [9]. All samples were dissolved in saline (1 mg/mL) for stock, and further diluted with saline before use in cell culture.…”

“…These low-molecular weight polyphenols have been considered as anti-inflammatory substances and used as alternative medicines for the symptomatic therapy of inflammatory diseases [6,7]. Our previous study has demonstrated that orally administered polymers, but not monomers, of phenylpropanoids exhibited immunoenhancing activities such as natural killer (NK) cell activation in C3H/HeN mice [8], and induced cytokine production in vitro in C57BL/6 mice [9]. These polymerized phenolic compounds exist in plant cell walls and are easily synthesized by several enzymes such as peroxidases, laccases, and polyphenol oxidases in fresh foods [10][11][12].…”