Foot-and-mouth disease virus interaction with dendritic cells activates ERK1/2 pathway inducing class I presentation and apoptosis.

CECILIA LANGELLOTTI; JULIETA ALCAIN; IVANA SORIA; MARIELA GAMMELLA; VALERIA QUATTROCCHI; PATRICIA ZAMORANO; MÓNICA VERMEULEN

Medellín

Congreso; Inmunocolombia; 2015

ALAI

Foot-and-mouth disease (FMD) is a highly contagious viral disease of cloven-hoofed animals. This pathology is caused by foot-and-mouth disease virus (FMDV). Previously, we demonstrated immune differences between inactivated and infectious viral particles. Here, we investigated whether these differences are related to intracellular mechanisms on murine dendritic cells (DCs). Attending that activation of PI3K/Akt and MAPK exerts a central role in DCs function as during interaction of RNA virus with host cells. We decided to study signaling differences between inactivated or infective FMDV virus. To evaluate this point, lysates from DCs untreated (Control) or treated (with inactivated (iFMDV) or infective FMDV (FMDV); moi 10), were assayed by western blot with specific antibodies against MAPK. FMDV triggered the activation of ERK1/2; while no differences were observed in Akt phosphorylation and p38 phosphorylation with any viral particle. Since FMDV induces apoptosis of DCs and that ERK1/2 is associated to the activation of caspase-9, we decided to analyze this fact. We show, by cytometry, that FMDV activated caspase-9 at 40 min and maintained until at least 24 h, contrary to observed in the presence of inactivated particles (CT:0.1%±0.01; iFMDV:0.2%±0.03; FMDV:3.7%±0.3, p 0.05). Interestingly, when DCs were incubated with PD98059, an ERK inhibitor, the activation of caspase-9 was prevented. Previously, we demonstrated that infected DCs with FMDV induce production of IFN-gamma by syngeneic lymphocytes. It is known that IFN-gamma is mainly responsible for sustaining the presentation through MHC class I. We decided to evaluate the membrane colocalization of viral particles and class I molecules at different times of DCs culture. The percentages of MHC class I increased in infected DCs by cytometry, being major the effect at 24 h post-infection. On the contrary, inactivated virus showed a moderate increase in the expression of MHC class I at DCs membrane only after 2 h of their co- culture. Besides, in confocal assays, we observed the colocalization of FMDV at membrane level and also in intracellular compartment of DCs 4 h after their co-culture. Next, to study whether ERK1/ERK2 pathway was involved in this modulation, the presentation of MHC class I in presence of PD was analyzed. DCs infected with FMDV in presence of the inhibitor abrogated the presentation of MHC class I, both in their expression by cell as in the percentage of positive cells by cytometry. Taking into account, that after FMDV infection their presentation on MHC class I was induced, we evaluated the modulation of a specific cytotoxicity (CTL). Splenocytes treated in vitro with or without FMDV and stained with CFSE at different concentrations, were injected to infected or naïve BALB/c mice. Finally, lysis of target cells was analyzed by cytometry. As a result, FMDV induced a more vigorous specific CTL response in vivo compared to control mice. So, FMDV enables its presentation by DCs in MHC class I context which induces cytotoxicity against infected cells.As a result, we demonstrated that the internalization of infective virus triggered the phosphorylation of ERK1/2, which was involved in the activation of caspase-9, the intrinsic pathway of apoptosis and the delivery of viral peptides on MHC class I molecules. While, inactivated virus (iFMDV) did not affect this pathway or any function mediated by its activation. Finally, this increased presentation promotes a specific cytotoxic response against infectious virus.