Summary Macrophages are among the earliest immune cells present during fetal development. Different populations of macrophages have distinct functions, with early yolk sac derived macrophages having an immune tolerant function and later liver derived macrophages having a more robust inflammatory profile. In the mouse lung, the inflammatory profile of liver derived macrophages increases late in development and peaks around the time of birth. This pro-inflammatory phenotype of fetal macrophages conflicts with previous notions that the fetal immune system resides solely in a tolerant state. Inflammatory macrophages may function to protect the developing fetus and newborn from pathogens encountered right at birth, but might also contribute to inflammatory disease pathogenesis, particularly in infants born preterm. We hypothesize that newly formed macrophages arising from the fetal liver are programmed for inflammation via the IKKb/NF-kB pathway. However, once macrophages travel to various organs in the developing fetus, they may adopt tissue-specific features. This proposal will use state of the art, complementary approaches to measure developmental changes in the immune signature of fetal macrophages within developing mouse tissues. Experiments using knockout mice will test if canonical IKKb/NF-kB signaling is required for the pro-inflammatory phenotype. While the myelopoietic cytokine GM-CSF is known to promote alveolar macrophage differentiation in the lung after birth, our preliminary data suggest it may also regulate the inflammatory phenotype in fetal lung macrophages. Studies using Csf2 (the gene encoding GM-CSF) knockout mice will test the requirement of GM-CSF on the developing lung immune system and specifically macrophages. The project will bring together experts in developmental immunology and computational modeling and employ novel, cutting edge approaches to complex systems immunology. The results generated by this proposal will fill a significant gap in our understanding of the fetal immune system and the unique functional properties of macrophages protecting newborns.