This application seeks to elucidate how the cell surface receptors Slamf1 and Slamf8 regulate microbicidal innate immune responses of macrophages and monocytes to infections by bacteria and the parasite Trypanosoma cruzi. Surprisingly, the co-stimulatory molecule Slamf1 is also a microbial sensor that enters the phagosome for instance through an interaction with E.coli or S.typhimurium. In the lipid bilayer of the phagosome Slamf1 recruits the intracellular Class III PI-3'kinase Vps34/15 together with several proteins that partake in autophagy to its cytoplasmic tail. Vps34/15 converts phosphatidylinositol into phosphatidylinositol-3-phosphate, PI3P, which regulates two microbicidal processes: generation of reactive oxygen species by Nox2 and phagosomal fusion. By contrast, we find that Slamf8 in the phagosome suppresses Nox2 activity and phagosome maturation. Slamf1 is also a key contributor to murine colitis and peritonitis by affecting monocyte migration to sites of inflammation. Furthermore, upon infection with the parasite Trypanosoma cruzi Slamf1-/- mice do not develop cardiomyopathy, because the replication of parasites in Slamf1-/- macrophages is reduced. Our overall hypothesis is that signaling networks induced by the positive regulator Slamf1 and the negative control element Slamf8 during innate immune responses are in a homeostatic balance. The experiments that are designed to test this hypothesis are grouped in the following Specific Aims: SA#1 Test the hypothesis that Slamf1 specifically recruits an active Vps34/15>Beclin1>UVRAG enzyme complex to Slamf1+ phagolysosomes and Slamf1+ non-lysosomal storage compartments in macrophages and dendritic cells. SA#2: Test the hypothesis that Slamf8 directly counteracts Slamf1 controlled macrophage/monocyte innate immune responses. SA#3: Test the hypothesis that Slamf1 and Slamf8 differentially regulate macrophage and dendritic cell responses to Trypanosoma cruzi.
Upon entering the Gram-negative bacterial phagosome the cell surface receptor Slamf1 recruits an active PI3'kinase enzyme associated with three ubiquitous autophagy proteins to control innate immune responses. o The cell surface receptor Slamf8, which is expressed late in the activation of macrophages, inhibits Slamf1-related microbicidal functions. o The parasite T.cruzi utilizes the mechanisms controlled by Slamf1/8, because they are requisite for the control of phagocyte responses to bacteria. o This should open a novel line of translational research with well-defined targets, which could extend to infections with Measles virus, T.cruzi and possibly other pathogens. o We generated valuable engineered mouse strains [Slamf1-/- Slamf1fl/fl, Slamf8-/- [BALB/c and B6}, monoclonal antibodies, transfectant cells and novel assays, some of which can be applied to other aspects of the innate and adaptive immune responses
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