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Microbiota-released extracellular vesicles (MEVs) have emerged as key players in intercellular signaling in host-microbiome communications. However, their role in gut-brain axis signaling has been poorly investigated. Here, we performed deep multi-omics profiling of MEVs generated ex-vivo and from stool samples to gain insight into their role in gut-brain-axis signaling. Metabolomics unveiled a wide array of metabolites embedded in MEVs, including many neurotransmitter-related compounds such as arachidonyl-dopamine (NADA), gabapentin, glutamate, and N-acylethanolamines. To test the biodistribution of MEVs from the gut to other parts of the body, Caco-2, RIN-14B, and hCMEC/D3 cells showed the capacity to internalize labeled MEVs through an endocytic mechanism. Additionally, MEVs exhibited dose-dependent paracellular transport through Caco-2 intestinal cells and hCMEC/D3 brain endothelial cells. Overall, our results revealed the capabilities of MEVs to cross the intestinal and blood-brain barriers to delivering their cargo to distant parts of the body.

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Research has shown that our gut microbiota confers many beneficial functions, including aiding the development of the immune system, metabolism, modulating stress reactivity and behaviour. The diverse population of the gut microbiota has been shown to be heterogeneous between individuals, with host genetic factors emerging as a contributor to gut microbiota composition. Recent work suggests that microRNA may act as a mediator of communication between the host and resident gut microbiota. Here we explore host genetic differences in gut microbiota composition and fecal miRNA profiles in two inbred mouse strains BALB/C and C57BL/6, in relation to gastrointestinal homeostasis. Furthermore, we evaluate the role of host genetics in response to perturbation of the gut microbiota using broad-spectrum antibiotic treatment. Distinct differences in the gut microbiota composition evaluated by fecal 16s rRNA gene sequencing between BALB/c and C57BL/6 mice were found with notable significant differences in genera Prevotella, Alistipes, Akkermansia and Ruminococcus. Significant host genetic differences were also observed in fecal miRNA profiles evaluated using the nCounter Nanostring platform. A BLASTn analysis was used to identify conserved fecal miRNA target regions in bacterial metagenomes, which identified numerous bacterial gene targets. Of those miRNA targets that were conserved in our dataset, 14 significant correlations were found between fecal miRNA and predicted taxa relative abundance. Treatment with broad-spectrum antibiotics for a period of 2 weeks resulted in BALB/c mice exhibiting a decrease in barrier permeability while C56BL/6 barrier permeability remained unchanged, demonstrating a host-specific physiological response to antibiotics at the gastrointestinal barrier. Differential response to antibiotics was also observed in the expression of barrier regulating genes in both host strains. Individual taxa were found to respond differentially by host strain, with Parabacteroides and Bacteroides associating with changes in barrier function. Together these findings suggest that host genetics play a role in determining the host-microbe relationship in both healthy homeostatic conditions and altered microbial conditions. / Thesis / Master of Science (MSc)

The human gut and brain have a bidirectional communication that has shown to play a pivotal role in our health and disease. Literature has shown that microbiota composition and diversity can be influenced by both genetic and environmental factors, contributing to shaping an individual’s microbial composition. The current work includes analysis of the microbiome of several mouse models to better understand how gene-environmental interactions during early development can influence the composition of microbiota within the gut. Here, male and female mice from several strains (C57BL/6, Balb/C, FVB, CD1) and genetically modified mice including T-cell receptor knock out mice (TCRβ-/-δ-/-) and Fragile-X-mice (FMR1-KO) were exposed to early life stressors including lipopolysaccharide (LPS) injection on postnatal day 3 (P3) and/or overnight maternal separation on P9. Fecal samples were collected at P24 and microbiota composition was determined by amplifying the 16s rRNA gene variable 3(v3) region and sequenced using the MiSeq Illumina platform. DADA2, was used to analyze this data in R software. Among the group, strain was found to be significant among alpha and beta diversity metrics while sex and stress were found to contribute to within strain variation, which demonstrated that both genetic and environmental factors are important in shaping an individual’s microbial composition. Secondly, we also explored the role of gut microbiota on the development of the immune system in TCRβ-/-δ-/- and C57BL/6 mice. Mice that lack T-lymphocytes were found to have a lower alpha diversity, as well as separated from their wild-type controls by beta diversity. Several bacterial taxa were found to be influenced by the immune system, demonstrating a bidirectional communication between the gut and T-cells. Lastly, the influence of litter, an environmental factor on microbial composition was explored within inbred mouse strains, C57BL/6 and Balb/C. Litter was found to influence alpha diversity, in which litters among C57BL/6 exhibited the greatest variation in such diversity. Beta diversity was also found to be influenced by litter, as related litters were found to cluster together. Differences in bacterial taxa between the inbred strains were observed and a subset of those taxa were found to be influenced by litter. Hierarchical clustering and co-occurrence analysis revealed different clusters of co-occurring taxa between both strains. These findings demonstrate that environmental factors can contribute to influence the composition of microbiota. / Thesis / Master of Science (MSc)

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