Placental Growth Factor Enhances Toll-like Receptor-Induced Inflammatory Cytokine Gene Expression Transcriptionally in Human Mononuclear Phagocytes

Abstract The production of placental growth factor (PlGF) increases during normal third trimester pregnancy, a point at which women are at an increased risk of morbid viral infections (e.g. an 8-fold increased risk of mortality during the 2009 H1N1 influenza pandemic). Pathologic increases in PlGF have also been detected in sickle cell disease patients and are associated with similarly increased morbidity (hospitalizations and acute chest syndrome). While treatment of normal donor mononuclear cells with exogenous PlGF reportedly increases cytokine transcripts (including tumor necrosis factor-alpha [TNF]), whether TNF mRNA is translated is unclear, as are: (a) the comparative activity of PlGF isoforms, (b) the scope of inflammatory cytokines involved, (c) the TNF-dependency of other induced cytokines, (d) and the potential effects of PlGF when combined with toll-like receptor (TLR) pathway activators. Consequently, we sought to test the hypothesis that PlGF could enhance pro-inflammatory responses of peripheral blood monocytes to TLR-stimulation and focused on endosomal TLRs which would be expected to be activated by the types of viral infections (ssRNA) for which pregnant women and sickle cell anemia patients are at risk. Normal primary peripheral blood CD14+ cells were exposed to the PlGF isoforms (PlGF-1, -2, -3). None induced TNF secretion. However, when CD14+ cells were exposed to both PlGF-1 (250 ng/ml) and the TLR-7/8 ligand R848 (3 μM), production of TNF mRNA (qRT-PCR) at 4-6 hours (mean 2.52-fold, p=0.05, 10 separate experiments) and TNF protein (ELISA) at 24-hours (mean 2.15-fold, p=0.0007, 24 separate experiments) was significantly enhanced. PlGF-2 and -3 had no effect on TLR-7/8 activated cells. PlGF-1 did not prolong TNF mRNA half-life (Actinomycin D chase results) but did enhance TNF gene transcription. Specifically, in kinetic studies we quantified equivalent increases in both spliced and unspliced TNF mRNA and TNF protein (Figure 1). The same experiments indicated that PlGF-1 prolongs the duration of TNF gene transcription compared to cells treated with R848 alone. The synergistic effect of PlGF with TLR-7/8 was not seen with the TLR-4 activator LPS. VEGF-A exhibited no influence on TLR-7/8 induced TNF gene expression. We sought to identify signaling pathways involved in the PlGF/TLR effect and determined by western blotting (total IKK-α and β, and phospho-IKK-α/β) that PlGF markedly enhanced R848-induced phosphorylation of IKK. Moreover, treatment of primary cells with an IKK inhibitor (BMS 345541; 0.5, 1.5, 5 μM) inhibited the enhancing effect of PlGF on R848-induced TNF production. TNF gene expression was not inhibited by small molecule inhibitors of p38 MAPK, PI3K, MEK-1/2, or ERK-1/2. Gene microarray analysis (Affymetrix HTA 2.0) revealed a PlGF/R848-specific increase in RIPK1 mRNA encoding a known IKK activator. To identify other inflammatory cytokines influenced by PlGF/TLR, and their dependency on TNF, we performed Luminex multiplex cytokine arrays of conditioned culture media from CD14+ cells cultured for 6 and 24-hours with PlGF-1 (250 ng/ml) and R848 (3 μM), and found (n=2 biological replicates) that PlGF enhanced R848-induced protein levels of: TNF, interleukin-6 (IL-6), IL-8, IL-1β, macrophage inflammatory protein 1 alpha (MIP-1α), MIP-1β, monocyte chemoattractant protein-1 (MCP-1), interferon-alpha, G-CSF, and monocyte interferon gamma inducing factor (MIG). To test the idea that the production of these factors was TNF-dependent, CD14+ cells were cultured as above in the presence and absence of soluble TNF receptor (sTNFR) (0.1, 1, 10 μg/ml). Complete inhibition of signal transduction through the TNF receptor had no effect on the production of other inflammatory cytokines (i.e. IL-8). We conclude that PlGF directly contributes to an exaggerated pro-inflammatory response in human mononuclear phagocytes incited by TLR-7/8 activation and suggest that it does so by promoting the expression or activation of an IKK kinase (e.g. RIPK1) and/or suppressing an IKK phosphatase. We also conclude that the production of inflammatory cytokines by PlGF and R848 is independent of autocrine TNF production. Clarification of the effect of PlGF on TLR-pathway hyper-reactivity may lead to the identification of key pathways that can be safely suppressed in individuals with high PlGF levels to achieve the goal of reducing virus related morbidity and mortality. Download : Download high-res image (56KB) Download : Download full-size image Figure 1 . Disclosures Tyner: Constellation Pharmaceuticals: Research Funding; Aptose Biosciences: Research Funding; Array Biopharma: Research Funding; Janssen Pharmaceuticals: Research Funding; Incyte: Research Funding.