The therapeutic effect of brucella lumazine synthase correlates with the expression of TLR4 in B16 melanoma
Farias A, Rossi AH, Fernandez JE, Goldbaum FA, Bonomi HR, Berguer PM
Fundación Instituto Leloir, Buenos Aires, Argentina
Brucella lumazine synthase (BLS) is a highly immunogenic decameric protein. By inserting foreign proteins at the 10 N-terminus of BLS chimeras can be generated that elicit immunity without adjuvants. BLS activates dendritic cells via TLR4, induces the cross-presentation of attached peptides and generates a strong long-lasting humoral immune response. Here we investigate the effect induced by the chimera of BLS with an ovalbumin peptide (BLS-OVA) or BLS alone in TLR4 and ovalbumin expressing B16 F1 melanoma. Mice were immunized with 100μg of BLS-OVA or BLS, 2 or 10 days after the inoculation of B16 cells and tumor growth and survival was evaluated. A delay in tumor growth and an increased survival was observed only after 2 days post-inoculation with BLS or BLS-OVA, showing that the presence of a tumor antigen does not increase the effect. Similar results were obtained in TLR4-deficient mice, showing that BLS per se has a protective role in mice with B16 melanoma and that this effect is independent of mice TLR4 signaling. Therefore, we measured the levels of TLR4 by FACS in B16 cells. At the time of the inoculation, TLR4/MD-2 is expressed in 84% of B16 cells and this percentage drops to 5.7% at day 10. To study if BLS has a direct effect on tumor cells, B16 cells were preincubated in vitro with PBS, BLS or LPS. After 48h we determined that cells were viable and not apoptotic. Tumors induced by BLS-stimulated B16 cells had an inhibited growth and survival was significantly increased (p<0.05). Moreover, 40% of mice failed to develop tumors. The effect was abolished by the addition of TLR4/MD2 blocking antibody to cells before BLS stimulation. Furthermore, we observed that the presence of selection antibiotic in culture decreases the levels of surface TLR4 abolishing the direct effect of BLS or BLS-OVA. The TLR4 levels are restored after a week of culture without antibiotics and so is the direct effect. Therefore, these results strongly suggest that BLS has a direct effect in B16 cells that slows or inhibits subsequent tumor growth via TLR4.
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