PROJECT SUMMARY/ABSTRACT Innate immune factors are critical for the selection and maintenance of regional commensal gut microbiota that provide beneficial functions for the host and deterrence against pathogens and pathobionts. In the previous grant cycle, we discovered that peptide YY (PYY), an anorexigenic or satiety-related gut hormone of intestinal enteroendocrine L-cell origin, is expressed by innate immune intestinal Paneth cells (PC) and functions as an antimicrobial peptide (AMP) in its unmodified PYY1-36 form (henceforth referred to as PC-PYY1-36). PYY's structure differs from other PC-α and β−defensins but is remarkably similar to the amphibian AMP, magainin-2, that has both anti-fungal and anti-bacterial properties. PC-PYY1-36 further differs from other PC-AMPs by selectively targeting and killing the virulent (hyphal), but not commensal (yeast) forms of polymorphic gut fungi like Candida albicans, thereby playing an important role in maintaining fungal (and also bacterial) commensalism of the gut. PC-PYY1-36 also differs from the endocrine PYY3-36 that is derived from DPP-IV conversion of L-cell PYY in structure and function by being vectorally secreted into and retained by intestinal mucus where its activity is optimal and protected from dipeptidyl peptidase IV (DPP-IV) modification that would otherwise convert it to the endocrine PYY3-36 form. We subsequently discovered that PCs also express another anorexigenic peptide highly related to PYY, neuropeptide Y (NPY1-36), that also has AMP properties. However, its regulation, antimicrobial spectrum, and compartmentalization upon secretion in luminal fluid rather than overlying mucus differs from that of PC-PYY. Based on our findings, we hypothesize that PC-PYY and PC-NPY are a novel class of PC- AMPs that play important roles in maintaining gut microbial commensalism and conferring innate immune defense against certain pathogens and pathobionts. Two specific aims are proposed: (1) to define and differentiate the drivers, mediators, and signaling pathways involved in regulation of PC-PYY and PC-NPY gene expression, secretion, and regional compartmentalization under physiological and pathophysiological conditions, and (2) to determine and differentiate the antimicrobial spectra of PC-PYY and -NPY in vitro and in vivo and their impact on gene expression and adaptive responses of targeted bacterial and fungal species. These studies will employ newly established in vivo (PC-PYY and PC-NPY KO and PC-PYY transgenic mice) as well as in vitro and ex vivo approaches. The MPI team combines unique expertise and resources in experimental research, multi'omic technologies, and host-microbe interactions (Chang, UChicago), experimental physiology and pathophysiology (Pierre, UWisc-M), and microbiology, bioinformatics, and genetics (Peters, UTHSC). Thus, the whole is much greater than the sum of the parts. The MPI groups have collaborated productively over the past two years to acquire the preliminary a...