Brucella spp. Lumazine Synthase Induces a TLR4-Mediated Protective Response against B16 Melanoma in Mice

Abstract

Brucella Lumazine Synthase (BLS) is a highly immunogenic decameric protein which can accept the fusion of foreign proteins at its ten N-termini. These chimeras are very efficient to elicit systemic and oral immunity without adjuvants. BLS signaling via Toll-Like Receptor 4 (TLR4) regulates innate and adaptive immune responses, inducing dendritic cell maturation and CD8(+) T-cell cytotoxicity. In this work we study the effect induced by BLS in TLR4-expressing B16 melanoma. In order to evaluate the effectiveness of BLS as a preventive vaccine, C57BL/6J mice were immunized with BLS or BLS-OVA, and 35 days later were subcutaneously inoculated with B16-OVA melanoma. BLS or BLS-OVA induced a significant inhibition of tumor growth, and 50% of mice immunized with the highest dose of BLS did not develop visible tumors. This effect was not observed in TLR4-deficient mice. For treatment experiments, mice were injected with BLS or BLS-OVA 2 days after the inoculation of B16 cells. Both treatments induced significant and equal tumor growth delay and increased survival. Moreover, BLS and BLS-OVA stimulation were also effective in TLR4-deficient mice. In order to study whether BLS has a direct effect on tumor cells, B16 cells were preincubated with BLS, and after 48h, cells were inoculated. Tumors induced by BLS-stimulated cells had inhibited growth and survival was increased. In the BLS group, 40% of mice did not develop tumors. This effect was abolished by the addition of TLR4/MD2 blocking antibody to cells before BLS stimulation. Our work demonstrates that BLS immunization induces a preventive antitumor response that depends on mice TLR4. We also show that BLS generates a therapeutic effect in mice inoculated with B16 cells. Our results show that BLS acts directly in cultured tumor cells via TLR4, highly suggesting that BLS elicits its therapeutic effects acting on the TLR4 from B16 melanoma cells.

Fig 1. BLS immunization induces a protective effect against B16 melanoma.

C57Bl/6J (C57, (A) and (B)) and C57BL/10ScNJ mice (SCN, (C) and (D)) were immunized with 100 or 200 μg of BLS or 100 μg of BLS-OVA in PBS or left untreated (control) subcutaneously in the base of the tail. After 35 days, all mice were inoculated with 2.5x10⁵ B16 melanoma or B16-OVA cells subcutaneously in the right flank. Tumor growth was monitored and diameters were measured using a caliper; Tumor volume was estimated as ½ (length × width²). (A) and (C) show tumor growth; (B) and (D) show the survival rate. Data from two independent experiments with B16-OVA cells have been pooled (5 mice per group). n = 10, * p<0.05, ** p<0.01.

Fig 2. BLS stimulation does not protect mice with B16 melanoma at 10 days of tumor inoculation.

C57Bl/6J mice were inoculated with 2.5x10⁵ B16-OVA cells sc in the right flank; 10 days later, mice were injected sc in the base of the tail with 100 μg of BLS or BLS-OVA or left untreated (control). Tumor growth was monitored; curves show survival rate; n = 6.

Fig 3. BLS induces a therapeutic effect in mice with B16 melanoma at 2 days of tumor inoculation.

C57Bl/6J (C57, (A) and (B)) and C57BL/10ScNJ mice (SCN, (C) and (D)) were inoculated with 2.5x10⁵ B16 melanoma or B16-OVA cells and 2 days later were immunized with 100 or 200 μg of BLS or 100 μg of BLS-OVA or left untreated (control). Tumor growth was monitored and diameters were measured using a caliper; Tumor volume = ½ (length × width²). (A) and (C) show tumor growth; (B) and (D) show the survival rate. Data from two independent experiments with B16-OVA cells have been pooled (5 mice per group). n = 10, * p<0.05, ** p<0.01 and *** p< 0.001.

Fig 4. TLR4 expression decreases in B16-OVA tumors.

TLR4/MD2 expression was determined in (A): cultured B16-OVA cells and (B): excised tumors from C57Bl/6J mice at different times post-B16-OVA sc inoculation. Representative histograms from 3 independent experiments are shown (n = 4).

Fig 5. BLS signals B16 cells directly via TLR4.

B16 cells were preincubated in vitro with 100 μg of BLS or 5 ng of LPS. After 48h cells were washed and 2.5x10⁵ cells were sc inoculated in the right flank of C57BL/6J mice (A). To block TLR4, cells were incubated with TLR4/MD2 monoclonal antibody; then they were stimulated with 100 μg of BLS (BLS+TLR4/MD2 Ab) and inoculated into C57BL/10ScNJ (B) and C57Bl/6J (C) mice. Figures show the survival rate; n = 7, * p<0.05, *** p< 0.001.

Fig 6. BLS direct effect on B16 cells.

B16 cells were cultured in a 6-well plate (2.5x10⁵cells/well) in 2 mL standard cell culture medium with 100 μg of BLS or 5 ng of LPS for 48h. (A): Apoptosis was assessed by staining with Annexin V-PE/7-AAD and fluorescence-activated cell sorter analysis was performed. Representative dot plots of unstimulated (control), BLS- and LPS-stimulated B16 cells are shown. (B): Expression of surface TLR4/MD2 was analyzed by FACS in B16 melanoma. Results depict representative overlayed histograms of unstimulated (control) B16 cells, BLS- and LPS-stimulated cells. (C): Expression of CD80 in B16 melanoma was analyzed by FACS. Representative overlayed histograms are shown of unstimulated (control) B16 cells, BLS- and LPS-stimulated cells.