Mechanisms Underlying Commensal Microbiota Colonization of the Intestine and Effects on Innate Immunity
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Distinct microbial communities colonize diverse vertebrate mucosal surfaces including the intestinal tract. These microorganisms, termed the intestinal microbiota, impact many aspects of host physiology including metabolism, behavior, and immune development. A majority of gut associated microbes are genetically intractable and thus the mechanisms that mediate their colonization and influence on the host remain undefined. To define genetic mechanisms of bacterial colonization and their subsequent impact on host innate immune development and function, we utilized a gnotobiotic zebrafish model. Using germ free zebrafish, devoid of microbiota, compared to zebrafish colonized with either large isogenic mutant pools or complex microbial communities, we identify bacterial mechanisms of colonization (Chapter 2) and mechanisms of microbial control of host innate immune function (Chapter 3).
In Chapter 1, I introduce the intestinal microbiota and experimental strategies to interrogate their influence on host immunity. I highlight mechanisms by which animals recognize microbiota derived signals and products. Further I detail the cellular responses that lead to phenotypic alterations of host epithelial and innate immune cells following microbiota colonization. In Chapter 2 we use gnotobiotic zebrafish to investigate the mechanisms of intestinal colonization of a gut bacterial commensal, Exiguobacterium acetylicum. We performed parallel in vitro and in vivo competitions of large pools of E. acetylicum mutants. These experiments identified several mutations that are differentially enriched specifically in vivo. We also elucidated the ability of different E. acetylicum strains to colonize the zebrafish intestine utilizing high resolution live imaging of labeled strains. Our data indicate that traits, such as motility, are not always the major drivers of successful colonization.
The intestinal microbiota influences the development and function of myeloid lineages such as neutrophils, but the underlying molecular mechanisms are unresolved. In Chapter 3, we identified the immune effector Serum amyloid A (Saa) as one of the most highly induced transcripts in digestive tissues following microbiota colonization in gnotobiotic zebrafish. Saa is a conserved secreted protein produced in the intestine and liver with enigmatic in vivo functions. We engineered saa mutant zebrafish to test requirements for Saa on innate immunity in vivo. Zebrafish mutant for saa displayed impaired neutrophil responses to wounding but augmented clearance of pathogenic bacteria. Saa’s effects on neutrophils further depend on microbiota colonization, suggesting this protein mediates the microbiota’s effects on host innate immunity. To test tissue-specific roles of Saa on neutrophil function, we over-expressed saa in the intestine or liver and found that sufficient to partially complement neutrophil phenotypes observed in saa mutants. These results indicate Saa produced by the intestine in response to microbiota serves as a systemic signal to neutrophils to restrict aberrant activation, decreasing inflammatory tone and bacterial killing potential while simultaneously enhancing their ability to migrate to wounds. These findings identify a host molecular mechanism that promotes innate immune tolerance to the commensal microbiota. In Chapter 4, I offer perspectives as to how Saa may function at the crossroads between host metabolism and immunity. I posit that Saa, and other microbiota-induced host factors from IECs, are the molecular underpinnings of animal-microbiota symbiosis, orchestrating systemic alterations in host metabolism and immune function.
serum amyloid a
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