The nonpathogenic intestinal flora play critical roles in human health and disease, yet very little is known about how commensal microbes communicate with their mammalian hosts to establish mutually beneficial relationships. To study these relationships, we use an in vivo system in which germ-free mice are colonized with Bacteroides thetaiotaomicron, a predominant member of the intestinal flora in mice and humans. Laser capture microdissection and microarray analysis have established that B. thetaiotaomicron regulates the transcription of genes encoding antimicrobial proteins in Paneth cells, a small intestinal epithelial lineage specialized for antimicrobial defense. Moreover, changes in Paneth cell antimicrobial gene expression that are normally associated with the weaning transition can be elicited by bacterial colonization of adult germ-free mice, suggesting that commensal bacteria play an instructive role in the development of the Paneth cell antimicrobial arsenal. These results have led to the hypothesis that commensal gut bacteria shape intestinal antimicrobial defense and mucosal barrier function by modulating Paneth cell gene expression. Three specific aims are proposed to address this hypothesis. Aim 1 will characterize the expression patterns of bacterially-regulated Paneth cell antimicrobial genes during postnatal development and in response to microbial factors. Aim 2 will examine the contributions of host pattern recognition receptors, including Toll-like receptors and Nod proteins, to bacteria-Paneth cell interactions. Aim 3 proposes to investigate an antimicrobial function for a member of the Reg family of proteins which is strongly induced in Paneth cells by interactions with commensal bacteria. Together, these studies will yield novel insights into how commensal bacteria shape gut epithelial innate immunity, and will lead to a better understanding of the molecular mechanisms underlying bacteria-epithelial interactions in vivo. Furthermore, this work will aid in designing novel strategies for strengthening the role of the microflora in enhancing the mucosal barrier.