THE FUSION PROTEIN BLS-FLIC HAS THE CAPACITY TO TRIGGERS DENDRITIC CELLS ACTIVATION IN A TLR-4 AND TLR-5 DEPENDENT WAY

AGUSTINA ERREA; GRISELDA MORENO; ANDRÉS HUGO ROSSI; YANINA HIRIART; JEAN CLAUDE SIRARD; FERNANDO A. GOLDBAUM; PAULA M. BERGUER; MARTIN RUMBO

Los Cocos

Congreso; Reunión Anual de la sociedad argentina de inmunología; 2013

SAI

During the last years increasing researchin the field of subunits vaccines has highlighted the need for adjuvantdevelopment. TLR agonists are adjuvant candidates based on the capacity toboost adaptative response. With the aim to generate a new adjuvant system, wegenerated a fusion protein combining Lumazine synthase from Brucella spp.(BLS), which has showed TLR4 dependent properties, and flagellin (FliC), aTLR-5 agonist.Here, we addressed the potential of thefusion protein BLS-FliC to induce DC activation. We also studied thecontribution of TLR5, TLR4 and adaptor proteins involved in signalingtransduction to DC activation.Bone marrow dendritic cells (BMDC) were derivedfrom C57BL/6 mice (wt), TLR4-/-, TLR5-/- or MyD88-/- mice with GM-CSF. Cellswere stimulated during 24hs with 30μg/ml BLS-FliC. Resulting response wascompared with the one elicited by stimulation with 1μg/ml FliC and 9μg/ml ofBLS. LPS and non treated cells were used as control. BMDC activation wasdetermined measuring levels of co-stimulatory molecules by flow citometry andcytokines secretion by ELISA.BLS-FliC induced expression of CD80 andCD86 and secretion of IL12p40 and IL6 on wt dendritic cells (p< 0.001 vs nontreated cells). Moreover, at doses employed, all measured parameters werehigher for BLS-FliC treatment compared to stimulation with BLS or FliC assingle molecules (p<0.001). On the other hand, dendritic cells activationtriggered by BLS-FliC was conserved in absence of TLR5 and the absence of TLR4signaling suggesting TLR4 and TLR5 stimulatory property of chimeric protein dueto BLS and FliC components.Finally, we found that BLS-FliC stimulatorypotential was reduced in MyD88 ko BMDC compared to wt cells but still remainedstatistically different from control treatment for CD86 expression (p<0.05).All together our results demonstrate thatour fusion protein BLS-FliC is able to activate dendritic cells through a TLR-5and TLR-4 pathways and partially dependent on MyD88.