M cells are specialized epithelial cells that are induced to differentiate over mucosal lymphoid follicles such as Nasal Associated Lymphoid Tissue (NALT) in the upper airways and Peyer's Patches (PP) in the intestine. These cells provide an important immune surveillance function by selectively trapping microparticles and transporting them across the epithelium to underlying antigen presenting cells and lymphocytes. Using a number of closely related cell culture models of M cell differentiation induced by lymphotoxin/TNF family ligands and comparisons to M cell function in vivo, we have found a novel mechanism of particle transcytosis involving the tight junction protein claudin 4. Interestingly, this mechanism shows a distinct hierarchy in the preferential uptake of microbes and synthetic microbeads with preference for S. aureus > E. coli >> S. cerevisiae = Dextran = styrene beads. In the present proposal, we will refine the cell culture model to identify critical components of the transcytosis mechanism within three specific aims: (1) We will establish the importance of specific candidate protein components of the endocytosis mechanism using genetic knockdown and dominant negative constructs in transfected cells. (2) We will develop quantitative and qualitative assays for particle uptake and transcytosis to establish clear relative selectivity in the uptake of particles. This assay will be applied to testing a limited selection of microbes and synthetic beads presenting pathogen associated molecular patterns including peptidoglycan, lipoteichoic acid, and endotoxin extracted from different microbes. (3) We will use phage display libraries to identify peptides showing selective uptake by the endocytosis mechanism. These display peptides will be used as reagents for initial attempts to confirm the role of known receptors used by the cells for selective particle endocytosis, and identify new receptors not yet identified by previous approaches. The studies will provide an important foundation for continuing studies on the molecular basis of M cell specificity in mucosal immune surveillance, and also provide useful information and materials for developing therapeutic strategies for mucosal immunization by targeting selective uptake of vaccines by M cells. A great number of major infectious diseases such as influenza, cholera, and anthrax enter the body through mucosal surfaces, so it is important to understand how the immune system is able to capture and identify such organisms at mucosal surfaces and trigger immune responses. Mucosal M cells are considered the major specialized epithelial cell for trapping and transporting infectious agents across the mucosal barrier to the cells of the immune system, and the proposed studies are expected to provide insights to the function of these cells. Such insights will not only help understand the biology of mucosal immunity, but also provide potential strategies for developing effective mucosal vaccines. ? ? ? ?
