Modulación de los mecanismos de activación del macrófago durante la infección por "Trypanosoma cruzi": invasión celular e inhibición de la maduración del macrófago

Abstract

Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura 19-02-2006The mechanism by which T. cruzi modulates macrophage function during the acute phase of Chagas` disease is of great importance to understand the immunopathogenesis of this disease. In this work we have analyzed several aspects of this, such as the regulation of different signal transduction pathways involved in the control of immunomodulatory molecules and the process of entry of T. cruzi into macrophages, both in vivo and in vitro infected cells. Primarily we analyzed the activation status of macrophages in BALB/c mice in response to T. cruzi acute infection with circulating blood tripomastigotes. We analyzed in peritoneal macrophages from acutely infected the expression of different molecules required relevant for the infection such as TNF-α, COX-2 and iNOS. The infection induced the expression of all them with different kinetics being the peak of TNF-α production coincident with the highest parasitemia, while COX-2 and iNOS are expressed in a later times. As this response is normally regulated in macrophages by kinases such as p38-MAPK and PI3K, we studied their activation/phosphorylation during the course of infection. PI3K activity was inhibited upon infection, while p38- MAPK was induced coinciding with COX-2 and iNOS induction during the course of the infection in vivo. A second aim was to determine the effect of T. cruzi infection on antigen presentation in vivo as well as in vitro. Expression of co-stimulatory molecules CD80 and CD86 in peritoneal macrophages from infected BALB/c mice was strongly inhibited at the end of the acute phase. However, the expression of MHCII expression increased transiently during the peak of parasitemia but subsequently returned to basal levels. This effect on the expression co-stimulatory molecules was also observed in our in vitro model of infection, where we found that T. cruzi infection inhibited CD80 and CD86 cell surface expression independently of p38-MAPK activation. Infection experiments in vitro also suggest that the intracellular replicative form of Τ. cruzi is inhibiting the expression of MHCII on the cell surface in response to IFN-γ, interfering this way with the antigen presentation to T CD4+ cells by macrophages. Finally, we have analyzed the molecules that are involved in the endocytic pathway that T. cruzi uses to enter into the macrophage. We chose Rab5 as a candidate marker since it is one of the first molecules implicated in phagosome maturation. We have observed that T. cruzi infection induces the binding of Rab5 to early phagosomes containing live parasite through PI3K activation, confirming that the activation of Rab5 in the PI3K pathway is essential for the entrance of T. cruzi into the cell. Using various knockout mice, we have found that certain cell surface receptors, such as TLR2, TLR4 and SLAM (CD150), are involved in the entry and activation of macrophages by T. cruzi. Activation of Rab5 and fusion of early endosomes by T. cruzi infection involved TLR2 and SLAM receptors but was independent of TLR4. In summary, we have established two phases during T. cruzi infection of the macrophage that occur in vivo and can be further reproduced more detailed in vitro. A first stage involves macrophage interaction with the parasite, followed by infection, and a second stage that involves intracellular replication. Both of these processes are altered by T. cruzi, which interferes with the mechanisms of macrophage activation and likely uses it for its intracellular replication and development