Browsing by Subject "Intestine"

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.

Foxp3-expressing regulatory T cells (Tregs) are essential mediators of immunological tolerance in both mice and humans. Although Tregs have traditionally been considered a relatively homogeneous population, they are now thought to comprise numerous cellular subtypes each with distinct roles in maintaining organismal homeostasis. In this regard, Tregs undergo functional specialization leading to diverse phenotypes and effector functions depending on (1) the inflammatory milieu encountered during an immune response, and (2) the specific anatomical location in which they reside. This process of context-dependent adaptation of effector function is facilitated by changes in transcriptional programming resulting from the coordinated action of numerous transcription factors (TFs). However, aside from the demonstrated roles for classical lineage-defining transcription factors – such as RORt, GATA-3, T-bet and Bcl-6 – the transcriptional regulatory circuity underlying Treg specialization has remained largely unexplored.

The activator protein-1 (AP-1) superfamily consists of a large number of TFs which bind similar DNA sequences as either homo- or hetero-dimers to influence transcription. Transcriptomic profiling studies have demonstrated that numerous AP-1 TFs exhibit subset- or tissue-specific expression patterns in Tregs, suggesting that these factors might regulate Treg specialization. Specifically, AP-1 TFs c-Maf and JunB were shown to exhibit preferential expression in specialized Tregs and have established regulatory roles in the differentiation of helper T cells. Therefore, I hypothesized that c-Maf and JunB might play undiscovered roles controlling the process of Treg specialization.

Using mice with Treg-specific ablation of Maf (encoding c-Maf), I evaluated the role of c-Maf in Treg specialization within the intestinal immune compartment that hosts a variety of specialized Treg subsets. Mice with Tregs lacking c-Maf appeared largely normal, with no apparent health problems, indicating that c-Maf was dispensable for general Treg function. However, using multicolor flow cytometry to perform comprehensive immunophenotyping of these mice, I found that c-Maf played a critical role in the generation of RORt+ Tregs, which constitute a major subset of specialized Tregs in the intestine but are rarely found in other organs. Additionally, these analyses showed that c-Maf was important for the generation of a second specialized Treg subset, T follicular regulatory (Tfr) cells, which are found in secondary lymphoid organs and are important for controlling antibody responses. However, in contrast to RORt+ Tregs and Tfr cells, c-Maf was not required for differentiation of other types of specialized Tregs, indicating a novel and specific role for c-Maf in Treg specialization. Mechanistically, in vitro culture experiments showed that upregulation of c-Maf and RORt in Tregs could be driven by exposure to IL-6 signaling in the presence of TGF, suggesting that c-Maf functions to link environmental signals to Treg specialization in the intestine. This work demonstrated that c-Maf is a novel and critical regulator of subset-specific Treg specialization for RORt+ Tregs and Tfr cells.

Separately, I investigated the role of JunB in Treg specialization by employing a similar approach as for c-Maf. Surprisingly, I found that mice with Treg-specific ablation of JunB developed a spontaneous phenotype of immune dysregulation which lead to weight loss and colonic inflammation, indicating that JunB was critical for normal Treg functioning. JunB expression was greatly elevated in intestinal Tregs relative to other populations and inflammation was most pronounced within the colons of animals with JunB-deficient Tregs, suggesting that JunB played a specific role in the function of colon-resident Tregs. Like c-Maf, JunB was absolutely required for the development of Tfr cells; however, JunB was otherwise dispensable for the development of all other specialized Tregs examined. Unexpectedly, I found that the loss of Tfr cells in mice with Tregs lacking JunB stemmed from impaired maintenance of the CD25- Treg population, which may be due to novel JunB-dependent metabolic requirements in these cells. Examining the colon-specific effects of JunB ablation using RNA-sequencing of intestinal Tregs, I found that JunB was required for expression of a tissue-specific set of Treg effector genes, such as granzyme B, which may represent a novel suppressive mechanism employed by multiple subtypes of colonic Tregs. Therefore, JunB plays a novel, organ-specific role in Treg specialization of both Tfr cells and all colonic Tregs.

Taken together, my work has revealed novel functions for two AP-1 family TFs – c-Maf and JunB – in the control of Treg specialization. Although c-Maf and JunB are both important for Treg specialization, their contributions to this process are distinct. c-Maf predominantly plays a subset-specific role in RORt+ Tregs and Tfr cells, whereas JunB plays both subset-specific and organ-specific roles depending on anatomical location. Of note, both c-Maf and JunB are essential for differentiation of Tfr cells, although the underlying molecular mechanisms appear to be different. These findings highlight the importance of AP-1 TFs in the control of Treg diversification and functional specialization and suggest that AP-1-mediated transcriptional regulatory circuits are an important mechanism controlling subset- and tissue-specific gene expression in Tregs.

There are fundamental differences in the way that neonatal and adult intestines absorb nutrients. In adults, macromolecules are efficiently broken down into simpler molecular components in the lumen of the small intestine, then absorbed. In contrast, neonates are thought to rely more on bulk intake of nutrients and subsequent degradation in the lysosome. Here, we identify the Maf family transcription factors, MafB and cMaf, as markers of terminally-differentiated intestinal enterocytes throughout life. The expression of these factors is regulated by HNF4a/g, master regulators of the enterocyte cell fate. Loss of Maf factors results in a neonatal-specific failure to thrive and loss of bulk uptake of nutrients. RNA-Seq and CUT&RUN analyses defined an endo-lysosomal program as being downstream of these transcription factors. We demonstrate major transcriptional changes in metabolic pathways, including fatty acid oxidation and increases in peroxisome number in response to loss of Mafs. Additionally, we show that deletion of Blimp1, which represses adult enterocyte genes in the neonatal gut, shows highly overlapping changes in gene expression and similar defects in nutrient uptake. This work defines transcriptional regulators that are necessary for bulk uptake in neonatal enterocytes.