Mecanismos de resistência à infecção por Leishmania major e Trypanosoma cruzi mediados por IFN- e independentes de Óxido Nítrico: do reconhecimento à imunidade efetora

Abstract

An adequate immune response is essential for optimal host resistance against invading microorganisms. Here we show that a combination of different Toll Like Receptors (TLRs) are required for adequate recognition of pathogen and optimal immune response with high production of IFN- and nitric oxide (NO). NO is considered the major IFN--induced killing agent against L major and T. cruzi and its production depends on adequate immune response activation through a combination of different Toll Like Receptors. Recently, new data challenged such assertion. Cummings et al (2004) showed that while IFN- deficient (KO) mice are extremely susceptible to T. cruzi, NOS2 deficient KO mice can be resistant. Similar results can be also observed in leishmaniasis, we found that IFN- KO animals died 11 weeks after infection with L. major, while NOS2 KO mice survived up to 30 weeks. In addition, pathology and visceralization of parasites were dramatic in IFN- KO mice, with high levels of tissue parasitism in liver and spleen, sites barely affected in NOS2 KO. Such results clearly demonstrate that, similarly to infection with T. cruzi, additional IFN--induced mechanisms are important to control L. major. ROS was investigated as a possible mechanism associated to NO in inducing host resistance to L. major or T. cruzi, but in none of these parasites, ROS was found to play major role in containment of parasite replication. However, our results suggest that ROS may have a role in restricting systemic effects of high levels of NO induced by T. cruzi infection since phox KO animals displayed 100% of mortality till day 21 post-infection. Such mortality was found to be associated to a good control of parasite in tissues and blood, and augmented levels of NO in serum. The mechanisms involved in enhanced susceptibility of phox KO mice to T. cruzi infection are still under investigation. Our further investigation demonstrates that recently described IFN-induced p47GTPases might be the connecting key between IFN--dependent and NO-independent effector mechanisms. Infected wild-type (WT) and NOS2 KO mice were found to express higher levels of LRG-47, IRG and IGTP in the footpad, lymph node, spleen and liver than IFN- KO animals. Coherently, unpublished data by Belkaid et al. show that both LRG-47 KO and IGTP KO mice are highly susceptible to L. major. We also found LRG-47 was up regulated in the spleen during T. cruzi infection. To better investigate the role of p47GTPase LRG-47 in response this pathogen, we infected WT and LRG-47 KO animals with 100 blood trypomastigotes and evaluated parasitemia, survival and immune-pathologic responses. LRG-47 KO mice displayed almost similar parasitemia to WT control till 12 days of infection. Thereafter, control of parasites in the blood and tissues was lost in LRG-47 KO mice. This enhanced parasitism was associated with 100% of mortality at day 19 of infection. LRG-47 KO mice exhibited unimpaired inflammatory responses throughout the infection. In vitro studies revealed that LRG-47-deficient macrophages present impaired killing of T. cruzi even when stimulated with IFN- and that such deficiency was not associated with reduced TNF or NOS2 expression. Inhibition of NOS2 in LRG-47 KO macrophages and gene silencing LRG-47 expression in NOS2 deficient macrophages revealed that both mechanisms are independent of each other. We suggest that both mechanisms cooperate for optimal IFN--induced control of parasite replication.