Atividade citotóxica e pró-apoptótica de antígenos de ancylostoma ceylanicum: Implicações na modulação da resposta imune na ancilostomíase

Abstract

Hookworm infection is one of the most prevalent parasitic diseases, infecting an estimated 740 million people in tropical and subtropical regions of the world. A robust but ineffective immune response, characterized by the ablation of parasite-specific T cell proliferative response (hyporesponsiveness), is often observed in the hookworm infection resulting in a long term parasite survival in the host lumen. While several mechanisms of modulation have been demonstrated for hookworm and other neglected tropical infections, the influence of apoptosis into the immunomodulation of hookworm infection is still poorly understood. In the current study, MTT assay for cell viability, of Jurkat T cells and mesenteric lymph nodes cells from infected and non-infected hamsters was performed in the presence of different concentrations of adult Ancylostoma ceylanicum excreted/secreted products (ES) and crude extract (HEX) (100, 50, 25, 12.5, 6.25, 3.125 g/mL). Besides that we evaluated the DNA fragmentation, using a hypotonic fluorocromic solution (HFS), in Jurkat T cells and mesenteric lymph nodes cells from infected and non-infected hamsters, after ES and HEX stimulation. Moreover, the phenotypic profile of circulating lymphocytes from Necator americanus infected and healthy non-infected donors were evaluated by flow cytometry using Annexin V-FITC and Propidium Iodide (PI) staining. Finally, we evaluated by Real Time PCR, the expression of 84 genes related with different apoptotic pathways, after hookworms antigens stimulation. Our results demonstrated that stimulation of Jurkat T cells with ES and HEX antigen induced a decrease of cell viability in a dose-dependent manner when compared with control non-stimulated cells. Similarly, decrease of cell viability of lymph nodes cells after antigenic stimulation was also observed in both infected and non-infected groups. The analysis of HFS assay showed that the stimulation with hookworm ES antigen induced an increase of DNA fragmentation in Jurkat T cells (p=0,0001) as well as in mesenteric lymph nodes cells from infected (p=0,0073) and non-infected hamsters (p<0.0001), when compared with non-stimulated cells. Flow cytometric analysis demonstrated that hookworm infected patients presented a significant increase of early apoptotic CD4+ , CD8+ , and CD19+ lymphocytes (Annexin V-FITC+ ) (p=0.0052, p=0.0269 and p< 0.0001, respectively) and also in the late apoptotic CD8+ and CD19+ lymphocytes (Annexin V-FITC+ /PI+ ) (p=0.0248 and p=0.0065, respectively) when compared with non-infected individuals. The down-modulation of the members of the TNF receptor superfamily (death receptors), as well as the up-regulation of the pro-apoptotic genes belonging the BCL-2 and P53 family, observed by qPCR analysis, suggest that hookworms antigens induce apoptosis by intrinsic mitochondrial pathway. In conclusion, this study demonstrated the potential cytotoxic and pro-apoptotic activity of the hookworms` antigens for T and B cells, which contribute to the downmodulation of immune response and the survival of the hookworms in their hosts.