Project Summary/Abstract The co-evolution of humans with bacteria has resulted in our bodies being colonized by numerous different species of microbes, collectively called the microbiome. Our intimate association with microbes, which begins even before birth, is especially important for immune system development. Changes in the gut microbiome have recently been linked to disease onset and progression for multiple autoimmune disorders ? including type I diabetes (T1D), inflammatory bowel disease, and autism, but the molecular basis for how specific bacterial strains contribute to disease is unknown. The lack of mechanistic understanding of host-microbe interactions in disease prevents development of targeted microbiome-based therapies. The proposed research will identify bacterially produced molecules, and the mechanisms by which they promote or prevent autoimmune disease, with a particular focus on type I diabetes. First, I will generate a library of partially fractionated extracts from cultures of bacterial strains that were identified in a longitudinal T1D study. Strains will be selected based on their association with either disease onset and progression, or non-progression. The strains in this proposal are clinically derived and linked directly to patient outcomes. We predict that bacteria that promote T1D will produce inflammatory molecules, and bacteria that prevent T1D will produce anti-inflammatory molecules. We will test our molecular library using an IL-10/TNF? assay in murine bone marrow-derived macrophages (BMDMs), under both normal and inflammatory conditions. Fractions that either induce IL-10 or suppress TNF? under inflammatory conditions will be considered as anti-inflammatory leads; fractions that suppress IL-10 or induce TNF? will be inflammatory leads. We will conduct activity-guided fractionation to purify active immunomodulators, followed by structure determination using NMR, MS, and X-ray diffraction. Next, we will determine the biosynthetic pathways responsible for immunomodulators by sequencing and analyzing the genomes of the producers. This will enable manipulation of these genes to increase or decrease levels of immunomodulators in vivo. Determination of immunomodulator biosynthetic genes will allow for identification of similar genes in other organisms, and will drive discovery of related compounds with potentially increased or divergent bioactivity. Finally, we will determine immunomodulator modes of action in human cells using a two main functional assays: 1) cytokine profiling and 2) transcription factor-based RNA-seq (TF-seq). This work will expand our understanding of how the microbiome modulates host processes related to autoimmune disease, and provide a basis for therapeutic intervention.