Toll-like receptors (TLR) recognize chemically conserved structures on microbes and respond by triggering signalling pathways important in innate host defense. Although individual TLR were originally thought to be designed to distinguish different classes of pathogens, there is a growing consensus that anti-microbial responses involve stimulation of multiple TLR. Previous studies had established that murine host resistance to the bacterial and protozoan pathogens Mycobacterium tuberculosis and Trypanosoma cruzi requires signalling through MyD88, an adaptor molecule used by most TLR receptors. This year we demonstrated that TLR9 plays a role in the control of both microbes and co-operates with TLR2 in mediating host resistance. Dendritic cells from TLR9 deficient mice displayed impaired IL-12 production when stimulated in vitro with either pathogen and these responses were even further impaired in TLR2/TLR9 double knockout (KO) animals. More importantly the double KO mice were more susceptible to in vivo infection than either of the single TLR2 or TLR9 deficient strains. In the case of M. tuberculosis, this decreased survival was reflected in augmented pulmonary pathology and in T. cruzi infection by increases in blood parasitemia equivalent to those seen in infected MyD88 deficient hosts. Together, these findings established a role for TLR9 in resistance to both intracellular pathogens while demonstrating the co-operative effect of multiple TLR signals in host defense. ? ? In a second project, we studied the role of the IFN inducible GTPase LRG47 in host resistance to Trypanosoma cruzi. This molecule had previously been shown by us to be required for IFN-gamma dependent defense against a variety of different bacterial and protozoan pathogens. When assayed on day 12 after parasite inoculation, LRG-47 KO mice, in contrast to IFN-gamma KO mice, controlled early parasitemia almost as effectively as WT animals. However, the infected LRG-47 KO mice displayed a rebound in parasite growth on day 15, and all succumbed to the infection by day 19. Additional analysis indicated that LRG-47-deficient mice exhibit unimpaired proinflammatory responses throughout the infection. Instead, reactivated disease in the KO animals was associated with severe splenic and thymic atrophy, anemia, and thrombocytopenia not observed in their WT counterparts. In addition, in vitro studies revealed that IFN-gamma-stimulated LRG-47 KO macrophages display defective intracellular killing of amastigotes despite normal expression of TNF and NO synthetase type 2 and that both NO synthetase type 2 and LRG-47 are required for optimum IFN-gamma-dependent restriction of parasite growth. Together, these data establish that LRG-47 can influence pathogen control by simultaneously regulating macrophage-microbicidal activity and hemopoietic function.
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