Metazoans carry microbial communities on their mucosal surfaces with which they establish complex interactions essential to many aspects of the host physiology. In return, the microbiota derives benefit from the association with its host by inhabiting a protected nutrient rich environment. This mutually beneficial symbiosis relies on a homeostatic host/microbiota relationship which, when uncoupled results in pathological outcomes. For metazoans to tolerate certain amounts of beneficial microbes, it is critical that the activation threshold of the gut immune response is tightly regulated. Indeed, breakdown of gut immune tolerance disturbs reciprocal host/microbes interactions causing chronic inflammatory disease. Despite being instrumental to the control of gut inflammation, we are currently far from having a clear understanding of how innate immune tolerance to the gut microbiota is established and how this mechanism impacts on gut physiology and overall organismal fitness. The present proposal adresses these issues with the global objectives to reveal a new mechanism of immune tolerance to the gut microbiota, analyse the impact of a rupture of this mechanism on gut physiology and host fitness and study the physiopathological mechanisms associated with a rupture of immune tolerance. To this end, we propose integrated approach in an invertebrate host model, Drosophila melanogaster to unravel the function and the consequences of the loss of a new immune modulator, PIMS. PIMS is a novel protein essential to regulate the homeostasis of NF-kB dependent immune responses mediated by the IMD signalling pathway and to establish immune tolerance to the gut microbiota. The first aim of our proposal is to understand the mechanism by which PIMS mediates immune tolerance to the gut microbiota. To this end, we will further elucidate how PIMS inhibits the IMD signalling. Based on our previous results obtained using expression of tagged proteins in Drosophila cultured cells, PIMS impacts on the cellular localisation of its binding partner, the pattern recognition receptor of the IMD pathway, PGRP-LC. Therefore we propose to elucidate through a functional cell biology approach how PIMS regulates the cellular localisation of PGRP-LC. The second aim of our proposal is to study the impact of pims loss of function on host physiology. To this end, we will study the physiological consequences of pims loss of function at three different levels. First, we will reveal the local perturbation of the host microbiota in pims mutant guts. Then, we will apprehend the impact of pims loss of function on the intestinal tissue integrity. Finally, we will reveal if pims mutant present a reduced fitness by monitoring fitness related-traits and if their metabolism is affected by monitoring established metabolic parameters. The results of these experiments will have revealed the local and systemic physiological perturbation associated with pims loss of function. At this point, we will then be in a favorable position to integrate the descriptive data into a more functional approach aiming at revealing the putative mechanisms leading to pims loss of function phenotypes, functionally test them, pinpoint in which tissue they operate and adopt an integrated approach to reveal if the phenotypes observed at the systemic level are a consequence of local perturbations of gut homeostasis or if they result from distinct physiopathological mechanisms. This research program, which takes advantage of the enormous resources and technical potential of the Drosophila model, will provide fresh insights on how host factors manipulate the balance between the microbiota and the host immune response to control gut homeostasis and promote host fitness. Because of its fundamental nature using an animal model with conserved physiological and immunological features, this project is relevant to all animals including humans and insect vectors of human pathogens