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BACKGROUNDHealthy animals and humans co-exist with a dense microbiota in the lower intestine, so there are many more microbial than host cells in the body. There is an extensive and diverse molecular exchange between the microbiota and their host, although live microbes are mainly confined to the lumen of the intestine. The mucosal immune system is extensively adapted to the presence of the microbiota: we have shown that small numbers of commensal intestinal bacteria can be sampled by dendritic cells at the mucosal surface (Science 2004; 303, 1662) with the induction of compartmentalised mucosal immune responses of anti-commensal IgA (Science 2000; 288, 2222) and regulatory T cells (Immunity 2011; 34, 794). A serious problem in studying how mucosal immunity functions to maintain mutualism with live commensal intestinal microbes, was that induction of host mucosal immunity involved irreversible microbial colonisation of the intestine. We have solved this by engineering a bacterial strain of Escherichia coli with auxotrophic deletions that prevents it surviving in the germ-free intestine; so we can experimentally uncouple specific mucosal immune induction and intestinal microbial colonisation (Science 2010; 328, 1705) for functional gnotobiotic experiments.OBJECTIVESIn this grant we will address three facets of host microbial interaction, with a focus on the function of secretory IgA. 1. The way in which specific secretory IgA (SIgA) influences dynamics of bacterial proliferation, death, retention and shedding from the different intestinal segments. 2. How specific SIgA determines the ‘handshake’ between live intestinal microbes and their molecular products at the mucosal surface and above the inner mucus layer of the small and large intestines. 3. How the mucosal IgA plasma cell compartment size may be critical to determine the functional repertoire of SIgA against commensals, and whether any systemic antibody tolerance to commensals occurs during intestinal IgA induction in the face of mucosal-systemic immune compartmentalisation.METHODSOur method of reversible colonisation permits mucosal immune priming of specific antibacterial IgA secretion in germ-free animals: we will compare bacterial colonisation, replication, metabolism and shedding dynamics in different intestinal segments after challenge with replication-competent and incompetent bacteria. Using different germ-free mouse strains (e.g. C57BL/6 wild-type, JH-/- or IgA-/- antibody deficient, somatic hypermutation deficient, T cell deficient) we will determine the in vivo functional performance of the immune system in these experiments. Bacterial replication will be measured with IPTG-dependent plasmids and by metabolic labelling. Mass spectroscopy will assess penetration of 13C-labelled bacterial metabolites at the interface with the host.IMPACTMutualism between mammalian hosts and their commensal microbiota is critical for health, but poorly understood. In this grant we will use powerful axenic and gnotobiotic methodology to independently manipulate intestinal bacteria and host mucosal immunity to address this critical interface. Epidemiological studies show an importance of the microbiota in inflammatory bowel disease, obesity, autoimmunity, dyslipidaemia, endotoxaemia and malignancy. An improved understanding of the host-microbial handshake is fundamental to advances in human health.