Lectinas vegetais de Cratylia argentea e Canavalia brasiliensis como adjuvantes terapêuticos contra infecções por Salmonella

Abstract

Plant lectins represent a class of proteins that perform various biological activities, including an immunomodulatory actions. In particular, lectins ConBr (Canavalia brasiliensis) and CFL (Cratylia argentea) have demonstrated an ability to modulate cytokines of the proinflammatory cascade as well as the production of nitric oxide in the murine model of Salmonella infection. Immunomodulatory molecules represent a promising alternative in the prevention and treatment of infections and can enhance immunological and adaptive mechanisms of the host improving the efficacy of antibiotics and vaccines against pathogens. This study evaluated the biotechnological potential of the ConBr and CFL as therapeutic and vaccine adjuvants against the antigen Vi from S. typhi, which has been used with limited success for immunization against typhoid fever. Initially, cytotoxicity assays with lectins were performed with cultured murine peritoneal macrophages to define non-toxic proteins concentrations for assays. Then, macrophages were treated with lectins and infected with a virulent strain of Salmonella Typhimurium. For this purpose, macrophages were incubated with ConBr and CFL lectins (1 and 10 μg / mL) before (preventive treatment) or after (curative treatment) the onset of infection. The following analyses were conducted: macrophage cell viability, intracellular bacterial quantification, iNOS plus pro-and anti-inflammatory cytokines and Toll-like receptor´s (TLR2, TLR4 and TLR9) gene expression. Both preventive and curative treatments significantly reduced the intracellular bacterial load and increased cell viability of infected macrophages. In the preventive treatment with CFL there was an increase in the level of transcripts for IL-6 and TNF-α, whereas ConBr induced an increase of IL-12 (p40). In the curative treatment, CFL induced significant expression of IL-12 (p40), whereas ConBr increased gene expression of IL-1β and TNF-α. In addition, the curative treatment with CFL increased approximately 130 fold the expression of TLR-4 and 24 fold expression of TLR2. In view of the importance of the TLR2 and TLR4 for the control of Salmonella infection, we evaluated the participation of these receptors in the immunomodulatory action of the CFL. Thus, bone marrow-derived macrophages (BMDM) knockout for TLR2/TLR4 were treated with the lectin and gene expression for IL-1β, TNF-α and IL-6 cytokines was evaluated. Naïve BMDM demonstrated high levels of gene expression, increasing 258-fold the number of transcripts for IL-1β, 233-fold for IL-6, and 25-fold for TNF-α. Conversely, double knockout BMDM for TLR2/TLR4 produced low levels of transcripts. Finally, ConBr and CFL were tested as vaccine adjuvants in Swiss mice immunized with the purified Vi antigen through evaluation of anti-Vi IgG antibodies. The results showed that as CFL and ConBr lectins were not able to induce an increase of specific IgG anti-Vi antibodies. Although the lectins did not demonstrate a potential as vaccine adjuvant against the Vi antigen, they showed therapeutic properties beneficial to the control of Salmonella-infected macrophages and were capable to interact with Toll-like receptors, which play a key role on activation of innate immune response and development of adaptive immune response. Such results represent a promising prospect for the development of new drugs against Salmonella.