Chemical and Microbial Regulation of Epithelial Homeostasis and Innate Immunity

The intestine is a multifunctional organ that must perform dichotomous roles in order to maintain health. While it is the primary site of absorption of dietary nutrients, it must also serve as a barrier to both the multitude of microorganisms which reside in the intestinal lumen (the microbiota) and foreign compounds (xenobiotics) which can be toxic to the host. Moreover, the microbiota are required for normal physiology, regulating immunological development, metabolism and behavior. Understanding how the intestine maintains homeostasis and responds to insult in the face of a chemically and microbially complex and dynamic environment is not only a fundamental question of biology, but has important implications for human health. We used zebrafish in order to better understand how the intestine responds to xenobiotics (Chapter 2) and transduces signals from the microbiota to the immune system (Chapter 3).

In Chapter 1, I introduce the complex and reciprocal interactions between xenobiotics, the microbiota, and the host. I highlight examples whereby the microbiota modulates the activity and toxicity of pharmaceuticals, with relevance to diseases of different organ systems. I also describe mechanisms by which the intestine responds to xenobiotic toxicity, and finally advocate for the use of novel model organisms to improve our understanding of these complex interactions.

In Chapter 2, I present our work using the NSAID Glafenine to explore how the intestine responds to xenobiotic challenge. Using transgenic zebrafish and high resolution in vivo imaging, we demonstrate loss epithelial cells in a live animal following xenobiotic challenge. Moreover, Glafenine causes intestinal inflammation, which is potentiated by microbial dysbiosis. We also show that Glafenine can directly alter microbiota composition. Glafenine treatment resulted in activation of the unfolded protein response (UPR), and while pharmacological inhibition of the UPR sensor Ire1a suppressed Glafenine-induced IEC loss, this was associated with increased inflammation and mortality. Ultimately, we demonstrate that Glafenine-induced intestinal toxicity is likely due to off-target inhibition of multidrug resistance (MDR) efflux pumps, as other MDR inhibitors were able to elicit similar phenotypes. Collectively, our findings revealed that (i) MDRs serve an evolutionarily conserved role in maintenance of intestinal homeostasis and (ii) IEC delamination is a protective mechanism which serves to limit inflammation and promote animal survival.

While studies in gnotobiotic mice and zebrafish have demonstrated that the microbiota are required for normal development of the innate immune system, the underlying host and microbial signals which mediate these effects remain largely unknown. We had previously demonstrated that motility of gut commensal bacteria in zebrafish was important for successful colonization of some strains and stimulation of the normal host innate immune response to colonization. In Chapter 3, we describe how microbiota colonization is associated with changes in the PMN transcriptome in addition to promoting systemic abundance and distribution of myeloid cells. Intriguingly, the only pattern recognition receptors found to be differentially expressed in PMNs were the Flagellin receptors tlr5a and tlr5b. Colonization of zebrafish larvae with bacteria lacking Flagellin resulted in attenuated PMN transcriptional activation compared to larvae colonized with isogenic wild type (WT) bacteria. We subsequently demonstrated that direct exposure to purified Flagellin can potently induce transcriptional activation in zebrafish PMNs. These findings identify how the presence of the microbe associated molecular pattern (MAMP) Flagellin serves as a bacterial cue from the microbiota which promotes PMN activation. In Chapter 4, I offer perspectives as to how the Glafenine-zebrafish model system can be used to more deeply investigate host-microbiota-xenobiotic interactions, and genetic, biochemical and computational analyses can help delineate mechanisms by which MDR efflux pumps function in the maintenance of intestinal homeostasis. Moreover, I propose the use of bacterial screens as well as inflammatory and infectious challenge assays in order to better understand the functional outcomes of PMN transcriptional activation elicited by microbiota-derived signals such as Flagellin.