Browsing by Subject "Neutrophil"
- Results Per Page
- Sort Options
Item Open Access Chemical and Microbial Regulation of Epithelial Homeostasis and Innate Immunity(2019) Espenschied, Scott TedmundThe 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.
Item Open Access Mechanisms Underlying Commensal Microbiota Colonization of the Intestine and Effects on Innate Immunity(2019) Murdoch, Caitlin CynthiaDistinct 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.
Item Open Access Regulating Emergency Granulopoiesis(2010) Cain, Derek WilsonNormally, neutrophil pools are maintained by "steady-state" granulopoiesis. Infections and inflammation, however, trigger neutrophilias that are supported by a hematopoietic program of accelerated granulopoiesis known as "emergency" granulopoiesis. Steady-state and emergency granulopoiesis are thought to depend on distinct members of the CCAAT enhancer binding protein (C/EBP) family of transcription factors, yet the extracellular cues that determine these developmental pathways are unclear. I hypothesize that inflammation elicits IL-1 which acts directly on hematopoietic progenitor cells for the induction of emergency granulopoiesis. Indeed, IL-1RI-/- mice fail to mount reactive neutrophilias in response to adjuvant-induced inflammation. Analysis of this specific impairment revealed an unanticipated role for IL-1RI in supporting increased proliferation by granulocyte/macrophage progenitors (GMP) and, surprisingly, more primitive multipotent progenitors (MPP) and hematopoietic stem cells (HSC). Whereas IL-1 drives HSC proliferation directly in vitro, inflammation induces comparable rates of proliferation in IL-1RI deficient and -sufficient HSC, MPP, and GMP in mixed chimeric mice. Thus, IL-1RI signals play a necessary, but indirect role in the support of alum-induced neutrophilias by expanding both pluripotent and myeloid progenitor compartments to accelerate granulopoiesis.
The lack of alum-induced neutrophilia in IL-1RI-/- mice is due to defective mobilization of bone marrow (BM) neutrophils and impaired proliferation of hematopoietic stem and progenitor cells (HSPC). Coincident defects in neutrophil mobilization and HSPC proliferation suggest that the trigger for emergency granulopoiesis might be the exhaustion of neutrophil compartments rather than inflammatory inductions of growth factors. Consistent with this hypothesis, non-inflammatory reductions in BM neutrophil numbers elicit granulopoietic responses similar to those induced by adjuvant. Alum mobilizes BM neutrophils via G-CSF, but increased HSPC proliferation results from a density-dependent mechanism that is only partially dependent on G-CSF. Notably, C/EBPβ, thought to be necessary for enhanced generative capacity of BM, is dispensable for increased proliferation of HSPC, but plays a role in the terminal differentiation of neutrophils. These observations indicate that the draining of BM neutrophil pools is sufficient to activate a latent, homeostatic mechanism of accelerated granulopoiesis. I propose a common model for the regulation of neutrophil production that explains both steady-state and emergency granulopoiesis through negative feedback.