Project 4: Role of Extracellular Remodeling in Epithelial DefenseInfectious agents, allergens and other noxious agents enter through exposed mucosal surfaces, such as therespiratory, gastrointestinal and genitourinary tracts. In response to these patholological agentsinflammatory and immune cells are recruited and cross the epithelial barrier in response to a chemotacticgradient. To maintain their function as a barrier to infection, an adequate number of inflammatory cells mustcross into the luminal spaces. In addition, if the mucosal epithelium is compromised, the defense againstinfection is lost. The epithelial barrier must therefore be restored as quickly as possible, to minimize theopportunity for entry of infectious agents. Matrix metalloproteinases (MMPs) are upregulated during lunginjury, repair and inflammation. Transgenic mouse models with altered proteolytic potential present uniqueopportunities to elucidate important cellular and genetic pathways by which extracellular proteolysisparticipates in these epithelial defense mechanisms. The overall goal of this proposal is to identify thepathways impacting epithelial defense (inflammatory cell recruitment, epithelial repair) regulated by by theseenzymes. We will address the characteristics and functional significance of MMP interactions by combinedin vivo and in vitro approaches: allergic inflammation and infection and the attending epithelial damage. As amucosal tissue, we will primarily focus on the lung, but, as needed, will also will interrogate other mucosaltissues. We propose to determine the role of these genes in regulating inflammatory cell behavior duringtransendothelial and transepithelial egress. We hypothesize that dysregulated airway MMP activity modifiesthe behavior of inflammatory cells by altering extracellular remodeling that impacts on synthesis or release ofchemoattractants and/or antagonists of chemoattractant action, into the airspaces, and thus chemoattractantgradients needed to attract the cells out of the parenchymal space into the airspaces are not formed ormaintained. Moreover we hypothesize that these enzymes regulate the processes of epithelial repairnecessary as a consequence of inflammation. We will study this in vivo using micro-organisms or theirproducts in models of infection (with Project 2) or allergic sensitization in mice that have geneticmodifications in MMP expression or activity, or treat mice with MMP inhibitors. We will evaluate cellrecruitment in the spaces. We will use optical imaging (with Core C) to study the reactions to microorganismsor their products in models of sterile inflammation, infection or allergic sensitization in vivo, ex vivoand in epithelial cell culture models. We will use mice that have genetic modifications in expression of thesegenes, concentrating on MMP2, MMP9 and MMP13. We will use mutant mice, specific inhibitors and normalas well as genetically modified epithelial cells in culture to collaborate with Projects 1, 2, 3 and Cores B andC, to investigate the role of of these genes in epithelial wound healing. The immediate implications of thiswork are in its applications to use of instilled chemoattractant agents and other molecules that modifyepithelial defense and will provide proof-of-principle that these critical MMP substrates are potentiallyefficacious in intervention in epithelial defense mechanisms. Only a thorough understanding of the actionsand effects of these enzymes and their major substrates will help mitigate the defense against infection sothat recruitment of adequate numbers of inflammatory cells into the spaces beyond the epithelial barrier andthe ability of the epithelial to repair itself can be accelerated.
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