Non-mouse models can be fertile ground for the discovery of previously unappreciated immunologic paradigms and provide new perspectives of vertebrate immunity. The proposed work addresses a fundamental question of bony fish (teleost) immunity: are melano-macrophage centers (MMC) the sites where fish B cells proliferate and generate high affinity antibody, homologous to germinal centers (GC) in mammals? To answer this long- standing question, we will characterize the threespine stickleback (Gasterosteus aculeatus) MMC response. Clarifying MMC function will yield important insights into the basis of humoral adaptive immunity in fish, and the evolutionary origins of vertebrate adaptive immunity.
The first Aim of this project will investigate MMC function in response to immunization. To determine if MMCs are the site of B cell proliferation, immunized fish will be pulse labeled with BrdU so that proliferating BrdU+ cells can be localized. Next, to clarify whether somatic hypermutation occurs in MMCs, we will quantify immunoglobulin gene mutations in MMC-adjacent and non- adjacent B cells. To resolve if MMC function is indeed GC-like, we will compare the MMC response to immunization with T-dependent (NP-CGG) and T-independent (NP-dextran) antigens. The results of this first Aim will determine the suitability of the MMC as a biomarker of teleost humoral adaptive immunity, and whether the MMC is an ?evolutionarily primitive? GC. Preliminary data strongly support our hypothesis that MMCs are GC-like. Consequently, in our second Aim we will apply our knowledge of MMC function to investigate the immuno-modulatory effects of a helminth parasite, Schistocephalus solidus. By combining experimental infection and immunization we will determine whether this tapeworm suppresses teleost adaptive immunity. We find that some stickleback genotypes are refractory to this immunosuppression. Using a combination of Quantitative Trait Locus (QTL) mapping, population genomics, and RNAseq analysis of gene expression we will locate the host loci responsible for variable parasite-mediated immune-modulation. We will then use the CRISPR/Cas9 system to modify these genes in cultured MMCs and evaluate their role in MMC function in vitro. In the final Aim, we will identify the helminth-derived factors that dampen MMC activity. Through QTL mapping we will identify the tapeworm-specific loci responsible for MMC suppression. To complement this approach, we will assess whether helminth excretory/secretory products directly modulate MMC gene expression. Clarifying the mechanistic basis of helminth-mediated immunomodulation in fish may reveal new approaches to regulate vertebrate immune function. This work will also generate a new and broadly applicable assay of teleost immunity, which has the potential to be used in the study of diverse fish (and other poikilotherm) species. Beyond expanding our view of vertebrate immunobiology, this work has implications for the disciplines of disease ecology, host-pathogen co-evolution, and vaccine development.
? relevance to public health Parasitic worms are a public health concern as infected individuals can exhibit a compromised vaccine response. This project will develop and apply new approaches to study how parasitic worms manipulate their hosts? immune systems. We will employ a novel fish model system to identify host and tapeworm genes that enable tapeworms to suppress host responsiveness.
