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.

Figure 1

OPCs derived from Applepoly^® consistently decreased DENV titers upon addition to infected human PBMCs. A. In DENV-infected human PBMCs from different human subjects (each represented by a different line, open circle denotes MOI>10, closed circle denotes MOI<5), addition of a crude extract from Applepoly^® consistently resulted in lower titers as measured by endpoint titration. B. The putative trimeric OPCs purified from Applepoly^® were slightly more effective and consistent than crude extract at reducing titers in DENV-infected human PBMCs. C. As shown in other viral systems, monomeric procyanidins (catechin or epicatechin) had a minimal and inconsistent effects on viral titers in the same assays.

Figure 2

Addition of OPCs to high MOI DENV-infected human PBMC affects gene expression in the IFN I signaling pathway. A. Transcripts encoding IFNB and MX1 remained unchanged in cells from most subjects in uninfected, OPC-treated and DENV-infected cells, but increased upon addition of OPCs to DENV-infected cultures. Significance was calculated using the Student’s paired t-test, *p<0.05, n=5. B. The expression of CXCL10 and STAT1 were consistently reduced in the presence of OPC during DENV infection. *p<0.05, n=5. D. OAS1 transcript expression in response to DENV-infection and OPC addition is not consistent between human donors. n=5. E. Phosphorylated STAT2 was only barely detectible in IFN only-treated cells and consistently clearly detected in OPC- and IFN I-treated human PBMCs. Normalized densitometric values for each blot (n=5) and a representative blot is shown. Significance was calculated using the Student’s paired t-test *p<0.05, **p<0.01.

Figure 3

OPCs altered gene and protein expression during low MOI DENV infection and stimulation with IFN I. A. When OPCs were added to low MOI DENV infected cells, expression of STAT1 was increased compared to vehicle only controls **p<0.01. B. Human cells (from n=4 donors) were untreated, treated with low rIFN I (100U/ml), or infected with DENV at MOI=1, then stimulated with OPCs or vehicle only and expression of MHC I (HLA-A, B, C) on CD11b⁺ (monocyte/macrophages) was measured by flow cytometry. *p<0.05, **p<0.01. C. Representative histograms from one donor.

Figure 4

OPCs altered production of TNFα during DENV infection in vitro. A. At low MOI (closed circles) infection, addition of OPCs significantly increased production of TNFα in supernatant fluids from infected human PBMCs. *p=0.0156, n=7. At high MOI (open circles), DENV infection alone appeared to increase production of TNFα and the addition of OPCs decreased it. n=3, insufficient for statistical significance.

Figure 5

Model for responses of cells to OPCs. Circles represent cells. Larger font and boldface type denotes intensified expression of genes (in cells) or proteins (on or by cells).