| Popis: |
The innate immune response is the first barrier against external stimuli arising from disease-causing pathogens. The primary membrane associated Toll-Like receptors (TLRs) play an important role in the innate immune response by recognizing pathogen-associated molecular patterns (PAMPs). Recently nucleotide-binding oligomerization domain (NOD) proteins have been shown to serve as intracellular receptors that are also involved in the innate immune response. Caveolae are small plasma membrane invaginations that exist in a wide range of cell types. The overall goals of this proposal are to examine the molecular determinants of the interaction of the intracellular pathway (NOD2) with plasma membrane (TLR2) mediated events in the response of epithelial cells to bacterial pathogens and to identify the role of caveolae in the signaling events that may underlie this association. Revealing the mechanisms of interaction between NOD2 and pathogens has significant importance to control or prevent Crohn's disease and benefits the public health issues. Accordingly, the specific aims are: Aim 1: To reveal the molecular mechanism for the interaction between NOD2 and MDP. We hypothesize that a cognitive sequence in the LRR of NOD2 recognizes MDP and the cognitive sequence accounts for the interaction of PGN from Gram-positive microorganism in polarized epithelial cells. Aim 2: to determine the requisite role of NOD2/MDP pathway in response to Gram-positive infection. We hypothesize that constitutive levels of NOD2 are low in polarized epithelial cells. Gram-positive infection results in synthesis of TNFa that increases NOD2 expression and TNFa in an autocrine fashion and enables the cells to participate in host defense via synthesis of IL-8. Aim 3: to identify an interaction between NOD2 and TLR2 pathways in mediating Gram-positive bacterium Staphylococcus aureus activation (IL-8 synthesis) in polarized MDCK cells. IL-8 synthesis in response to gram-positive infection will be contrasted in wildtype MDCK cells vs. cells in which NOD2, TLR2 or both have been silenced by siRNA. We also hypothesize that cross talk between NOD2 and TLR2 pathways occurs at a MAP kinase step and accordingly, experiments will be repeated in the presence of pharmacological (or genetic, e.g. dominant negative) inihibitors of p38, JNK and MAPK. |
