Oligomeric procyanidins stimulate innate antiviral immunity in dengue virus infected human PBMCs

Abstract

Oligomeric procyanidins (OPCs) have been shown to have antiviral and immunostimulatory effects. OPCs isolated from non-ripe apple peel were tested for capacity to reduce dengue virus (DENV) titers. Similar to published accounts, OPCs exhibited direct antiviral activity. The possibility of enhanced innate immune protection was also tested by measuring and characterizing gene and protein expression induced by OPCs during DENV infection. Treatment of DENV-infected human PBMCs with OPCs decreased viral titers and affected the expression of critical innate antiviral immune products. OPCs enhanced expression of MXI and IFNB transcripts in high MOI DENV infected PBMC cultures, and phosphorylation of STAT2 in response to recombinant type I IFN (IFN I). During low MOI infection, addition of OPCs increased expression of STAT1 transcripts, MHC I and TNFα protein production. Thus, OPCs exhibited innate immune stimulation of cells in DENV-infected cultures and uninfected cells treated with IFN I. While OPCs from a number of sources are known to exhibit antiviral effects, their mechanisms are not precisely defined. The capacity of OPCs to increase sensitivity to IFN I could be broadly applicable to many viral infections and two separate antiviral mechanisms suggest that OPCs may represent a novel, robust antiviral therapy.

Introduction

Dengue virus (DENV) is an NIAID Category A pathogen and DENV infection represents one of the most important emerging threats to human health worldwide. Over the past 30 years infection rates have dramatically increased, in part due to population urbanization. Four related viruses of different serotypes, transmitted by mosquitoes, are the cause of DENV infection. Vaccine development is complicated by difficulties in developing a small animal model relevant to human disease as well as the phenomenon of antibody-dependent enhancement (ADE). ADE involves antibodies specific for one DENV serotype that may not neutralize other DENV serotypes. Rather, non-neutralizing, cross reactive antibodies can enhance disease severity upon secondary infection with a different serotype (Halstead and O’Rourke, 1977). There are virtually no antiviral treatments for DENV and mosquito control measures have largely failed to curb DENV incidence in most parts of the world, necessitating novel approaches for protection.

The primary cellular target for DENV infection in humans is the monocyte/macrophage (Chunhakan et al., 2009, Kou et al., 2008). Although details of DENV infection are not completely understood, the early innate response appears to be critical in directing the subsequent infection to either a self-limiting dengue fever, or a severe and potentially lethal dengue hemorrhagic fever (Chen et al., 2007, Shresta et al., 2004). As with many viral infections, type I IFN (IFN I) responses are critical to DENV protection, exemplified by a commonly used mouse model, which utilizes IFN receptor-deficient mice (Shresta et al., 2004).

Polyphenols are a large and diverse group of aromatic-rich plant metabolites that have been identified in a number of food sources and dietary supplements such as cranberry juice, grape seeds, pomegranate and unripe apple peels (Holderness et al., 2008, Terra et al., 2007). Large, oligomeric procyanidins (OPCs) bind to and precipitate basic proline-rich proteins found in saliva (Charlton et al., 2002, Holderness et al., 2008). Antiviral effects of many different types of polyphenols, including monomeric and hydrolysable tannins are described (Haidari et al., 2009). Greater evidence suggests that the robust anti-viral activity is contained in larger order or oligomeric fractions [OPCs; also referred to as condensed tannins or oligomeric proanthocyanidins (Feng et al., 2008, Hauber et al., 2009, Su et al., 2010, Takeshita et al., 2009, Zhuang et al., 2009)]. For example, large-sized OPCs from blueberry leaves block the replication of hepatitis C virus (HCV) RNA in a subgenomic replicon expression system (Takeshita et al., 2009). Additionally, a procyanidin-rich extract from French Maritime Pine blocks HIV-1 intracellular replication and binding to host cells, although the precise mechanisms were not discovered (Feng et al., 2008). Although direct antiviral interaction between OPCs and virus is demonstrated for a variety of viral agents, the descriptions of precise effects are largely limited to blocking early steps in viral entry in cell lines, which is not necessarily relevant to their natural infection in vivo.

We recently identified OPCs derived from unripe apple peels, found in the dietary supplement Applepoly^®, as stimulatory to innate lymphocytes. This response is specific to the oligomeric and not monomeric procyanidins, indicating a unique, non-antioxidant response (Holderness et al., 2007, Holderness et al., 2008). We hypothesized that OPCs would also demonstrate antiviral effects. This potential was tested during DENV infection of human PBMCs. Addition of OPCs to DENV-infected cells consistently decreased viral titers in vitro. As expected, this effect was due, in part, to reduction of infectious titers by direct interaction of OPCs with DENV. The potential for enhanced innate responses to virus infection in the presence of OPCs was also characterized. Results indicated that OPCs enhanced innate immune responses during DENV infection in part by altering expression of genes and proteins in the type I IFN (IFN I) pathway. During high MOI infections, addition of OPCs appeared to circumvent DENV-specific mechanisms that block these pathways. OPCs also enhanced phosphorylation of STAT2 in response to recombinant IFN I and affected cells in low MOI infected cultures, by increasing expression of STAT1 transcripts, MHC I and TNFα protein. Thus, OPCs induced innate antiviral responses that may be broadly applicable to other viral infections. To our knowledge, this study is the first to identify stimulation of critical antiviral innate responses by OPCs in target cells that are highly relevant to viral infection in vivo.