Modulación de los mecanismos de activación del macrófago durante la infección por "Trypanosoma cruzi": invasión celular e inhibición de la maduración del macrófago
Author
Maganto García, ElenaAdvisor
Fresno Escudero, Manuel
Entity
UAM. Departamento de Biología Molecular; Centro de Biología Molecular Severo Ochoa (CBM)Date
2006-02-19Subjects
Trypanosoma cruzi-Inmunología-Tesis doctorales; Biología y Biomedicina / BiologíaNote
Tesis Doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura 19-02-2006Abstract
The 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.
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