Pro-inflammatory cytokine induction by in vitro Dengue Virus-infection was selectively inhibited by patient serum rich in IgG1 subclass antibodies.

Abstract

Event Abstract Back to Event Pro-inflammatory cytokine induction by in vitro Dengue Virus-infection was selectively inhibited by patient serum rich in IgG1 subclass antibodies. Judith Gonzalez Christen1*, Karina R. Flores Avila1, Rosa M. Medina Julian2 and José-Luis Montiel-Hernandez1 1 Facultad de Farmacia Universidad Autónoma del Estado de Morelos, Mexico 2 Laboratorio Estatal de Salud Publica Morelos, Mexico Introduction: Human infection with Dengue Virus (DV) is characterized by fever associated to an inflammatory response, which can progress as Dengue fever (DF) or as severe disease, Dengue Hemorrhagic fever (DHF), where higher levels of TNF, IL-1beta, Il-8, IFN-gamma, and IL-6 are associated with more severe manifestations (1,2,3). Antibodies have been also implicated on the severity of disease. Studies by Koraka and Thein have observed higher levels of anti-dengue lgG1 subclass antibodies in association with DHF(4,5,6). Additionally, results from our group found that total IgG1subclass antibodies were higher in DHF patients than those with DF. In the same context, cross-reactive antibodies from previous DV infections have been suggested to predispose to more severe secondary disease by a mechanism called ADE (antibody-enhanced infection of dengue target cells bearing Fc receptors). Some studies have suggested that immunoglobulin-mediated DV entry could modify the secretory activity of macrophages and induce NO or type I Interferon inhibition (7,8). But since the pre-exiting enhancing antibodies capable of inducing ADE’s effect were found at very low concentrations, it remains to be confirmed if these molecules are the only molecules to induce the severe symptoms of the disease. In this respect, a previous study by our group showed a positive correlation between serum levels of IgG1 and IL-1beta, but not with IL-6 levels, suggesting that even non-specific antibodies could have a role in the pro-inflammatory conditions. The aim of the present study was to characterize the role of neutralizing antibodies in the course of the in vitro DV-infection of macrophages and the secretion of pro-inflammatory cytokines. Additionally, to compare the effects of serum with low levels of IgG1/IL-1beta with those of patients with high concentrations of IgG1/IL-1beta in this mechanism. Material and Methods. THP-1 cell line (ATCC Tib202), differentiated with PMA 10 nM for 72 hrs, was employed as a macrophage model. In vitro DV-Infection assays were done at 1 MOI of Den-2 (New Guinea C). For the opsonized-assays, sera from 5 healthy donors (Ctr) was employed, 5 patient provided sera with low IgG1titers (LI) and 5 patient sera with higher IgG1 titers (HI); virus were pre-incubated for 1 hour with diluted serum (1:1000, in PBS) before infection. 2 hours after infection, cells were washed in PBS and cultured for 12, 24 or 48 hours in RPMI-1640/10% fetal bovine serum. TNF, IL-6 or IL-1beta were quantified using ELISA on cell culture supernatants and infection was evaluated by fluorescence microscopy. Results. DV-infection of macrophages induced the secretion of all cytokines evaluated but showed an outdated profile where peak TNF occurred at 12hrs, peak IL-1beta at 24 hrs and peak IL-6 at 48 hrs. Pre-incubation of DV with serum reduces the rate of infection, even with Ctr serum (17%); but inhibition was significantly higher with patient’s sera (LI, 36%; HI, 59%). Supernatant TNF secretion was decreased with all types of serum evaluated, also showing significant higher inhibitory effect by HI sera (Ctr, 17%; LI, 26% and HI, 38%). IL-6 secretion was also decreased by serum pre-incubation, but the maximum inhibitory effect was observed at 48 hours, where LI and HI showed a similar effect (Ctr, 17%; LI, 31%; HI, 34%). Finally, for IL-1beta we observed an inhibition at 12 hours of 60% with Ctr serum and 68% with LI or HI sera, but at 24hours, the inhibition obtained was of 75%, 67% and 50% for Ctr, LI and HI, respectively. At 48 hrs of DV infection, all sera induced a similar inhibitory effect (56%). Conclusions. Our results lead us to suggest that although the rate of infection is important to promote the pro-inflammatory cytokine secretion, other factors also contribute to modulate this effect since patient’s sera with high IgG1 levels showed increased inhibitory effect mediated by TNF, but this was not the case for IL-6 or IL-1beta. It should be noted that only the TNF’ inhibitory effect correlated with the rate of infection. Paradoxically, IL-1beta inhibition was higher employing sera from healthy donors, in comparison to DV patients, suggesting a modulation by a non-specific and IgG1-independent mechanism. Acknowledgements KFA was suppoted by a scholarship from CONACyT (350115). This work was supported by grant PROMEP-SEP 103.5/13/5259 and Facultad de Farmacia-UAEM.