? The project has three aims: 1) To determine whether or not glycolipids from Leishmania sp (L.sp.) and Trypanosoma cruzi (TC) activate mast cells (MC) through Toll-like receptors (TLR); 2) identify the TLR activated (most likely TLR2), the signaling pathways activated, and the biological responses of MC to these glycolipids; and 3) examine the role of MC in the immunological/pathological reactions to infection from the above organisms in a mouse model. A subsidiary theme is to evaluate the MC as a model for a) studies of the effect of drugs on TLR-mediated responses and b) for identifying other parasite components that act through TLRs. MC are an important component of the innate immune system and are now thought to be essential for elimination of parasite infestation but the mechanism is undefined. Recent reports indicate that MC express TLR2, 4, 6, and 8 which can be activated by bacterial glycolipids via TLR2 to induce production of inflammatory cytokines and degranulation: and via TLR4 to induce production of cytokines but not degranulation. Glycolipids from T.C. also activate TLR2 on macrophages but have yet to be tested on MC. The question we address is whether TLRs are the """"""""recognition"""""""" receptors that allow MC to participate in parasite elimination through release of inflammatory mediators. The core signaling pathway activated via TLR2 and TLR4 has been identified but many questions remain, especially so for MC, on how TLR activation leads to gene expression for cytokine production and other biological responses.
For aims 1 & 2, studies will be conducted with MC lines as well as cultured bone marrow-derived MC from TLR2- and TLR4- knockout mice. Purified glycolipid components from L.sp. and T.C. will be evaluated for potency along with bacterial glycolipids that activate specifically TLR2 or TLR4. Known TLR-signaling pathways will be examined along with pathways that are responsible for degranulation and cytokine production in antigen-stimulated MC. Dexamethasone will be used as a prototype drug to examine effects on TLR-mediated signals as this drug disrupts several key signaling events in antigen-stimulated MC.
For aim 3, normal, mast cell-deficient and TLR-deficient mice will be used to evaluate host responses to L.sp. and T.C. infection using experimental models that are well established in our institution. ? ?
