How do intestinal microbiota interact with host genome-encoded
processes to impact vertebrate health and disease
?
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Microbial ecology in the intestine Microbial regulation of intestinal physiology Adipose tissue physiology and metabolic disease

Overview

Animal physiology is directed by complex interactions between factors encoded in the animal’s genome and those encountered in their environment. The impact of these interactions on animal health is most evident in the intestine, where digestion and absorption of dietary nutrients occur in the presence of complex communities of microorganisms (microbiota). Interactions between diet, microbiota, and animal hosts regulate immune and metabolic homeostasis and also contribute to a spectrum of human diseases, including the inflammatory bowel diseases, obesity, and malnutrition. Our research interests are focused on understanding how environmental factors such as the intestinal microbiota and diet interact with host genome-encoded processes to influence host physiology and pathophysiology. We are using the zebrafish as a vertebrate model system for this research. The small size and optical transparency of the zebrafish facilitate high-resolution in vivo imaging as well as genetic and chemical manipulations that complement the technical limitations of mammalian models. Extensive anatomic, physiologic, and genomic homologies between zebrafish and mammals permit translation of insights gained in zebrafish into advances in human medicine. To facilitate our research, we have developed methods for rearing zebrafish under germ-free conditions and for introducing selected microbial communities and sterilized diets into germ-free fish. We are currently using zebrafish and mouse models to investigate how microbial communities are assembled in the intestine and how microbes and dietary nutrients regulate host metabolism and immunity. We have also established methods for in vivo analysis of adipose tissues in zebrafish, and we are using that experimental platform to elucidate the mechanisms underlying adipose tissue physiology and obesity-associated metabolic disease. The overall objective of our work is to improve our understanding of vertebrate physiology as a complex and dynamic integration of genome-encoded and environmental factors, which is expected to yield new strategies for promoting health in humans and other animals.



Microbial ecology in the intestine Top        

Beginning at birth, the vertebrate intestine is colonized by dynamic microbial communities that contribute significantly to host physiology and disease. Recent advances in high-throughput DNA sequencing have fueled a marked expansion in our understanding of gut microbial community membership[1,2]. However, the ecological and physiologic principles that govern the assembly and persistence of gut microbial communities remain poorly understood. To address these gaps, we are using zebrafish as vertebrate host models because their transparency permits high resolution in vivo imaging and their small size facilitates scaling of sample number. We have used 16S rRNA gene sequencing to define the membership of the zebrafish gut microbiota. We found that the same bacterial phyla dominate the gut microbiota of zebrafish, humans, and mice, although relative phylum abundance and the specific bacteria in those phyla differ[3]. By transplanting gut microbiotas from zebrafish or mouse donors into germ-free mouse or zebrafish recipients, we discovered that differences in community structure between zebrafish and mice arise in part from distinct selective pressures imposed within the gut habitat of each host[4]. In accord, we found striking similarities between gut bacterial communities from zebrafish collected recently from their natural habitat and those domesticated for generations in lab facilities, including a shared “core” gut microbiota. These results suggest that lab-reared domesticated zebrafish can serve as a valid model for investigating coevolved host-microbe relationships that occur in their natural environment[5]. We have also found that feeding increases the abundance of the bacterial phylum Firmicutes in the zebrafish intestine[6]. Diet-dependent enrichment of Firmicutes has also been observed in humans and mice in the context of obesity[7-11], suggesting this might be a conserved ecological theme in the vertebrate intestine. To facilitate direct observation of gut microbial communities, we exploited the transparency of the zebrafish to develop techniques for real-time in vivo imaging of fluorescently labeled bacteria within the intact intestine[12]. Our current research in this area seeks to understand the physiologic and nutritional mechanisms underlying host selection of microbial community assembly in the intestine. This work is expected to provide an improved understanding of the principles underlying the assembly and maintenance of vertebrate gut microbial communities, which will be essential to the design of therapeutic strategies to safely and effectively promote beneficial gut microbial communities and prevent or correct pathogenic ones.



 Microbial regulation of intestinal physiology  

The intestinal microbiota has been identified as an important environmental factor that contributes to many aspects of human health and disease[1,2], which has prompted considerable interest in understanding the underlying microbial signals and responsive host signal transduction mechanisms. One of the most powerful experimental approaches for this work is to rear animals under conditions in which all microbial life forms are either excluded or known (gnotobiotic). We have developed zebrafish as a gnotobiotic host model because its amenability to in vivo imaging plus genetic and gnotobiotic manipulation provides a useful complement to existing mammalian models[13]. We used these methods to conduct functional genomic comparisons of gene expression in digestive tracts of zebrafish reared germ-free (GF) to ex-GF animals colonized with a conventional microbiota (conventionalized of CONVD) or to animals reared from birth with a microbiota (conventionally raised or CONV-R). This study revealed zebrafish genes regulated by the microbiota including many that are conserved in the mouse intestine and involved in stimulation of epithelial proliferation, promotion of nutrient metabolism, and innate immune responses[3]. To test the specificity of these responses, we performed reciprocal transplantations of gut microbiotas from CONV-R zebrafish or mouse donors into GF zebrafish or mouse recipients. In both of zebrafish and mouse recipients, metabolic responses were stimulated by microbiota donated from either species. In contrast, immune responses in each recipient host were induced only by their respective normal microbiota, suggesting that immune and metabolic responses to the microbiota are evoked by distinct bacterial signals. We also identified individual culturable representatives of the zebrafish and mouse gut microbiotas that elicit conserved responses, providing candidates for mechanistic studies[4]. Based on these results, we used Pseudomonas aeruginosa as a genetically manipulatable model gut bacterium to define the mechanisms by which members of the microbiota colonize and elicit conserved responses in zebrafish hosts. Using in vivo imaging in the zebrafish intestine, we found that bacteria display complex patterns of localization and behavior, including flagella-mediated directional motility. Genetic analysis in P. aeruginosa revealed that loss of flagellar function results in attenuation of conserved host innate immune responses but not of conserved metabolic responses[12,14]. Current research in our lab seeks to use gnotobiotic zebrafish and mouse models to understand the mechanisms underlying conserved innate immune responses to members of the microbiota. Once available, this information could be used to develop novel approaches for reducing inflammation in the context of chronic inflammatory diseases such as IBD or for enhancing immunity to prevent opportunistic infections.

     

    There is currently intense interest in understanding the physiologic mechanisms by which the gut microbiota regulates host nutrient metabolism and energy balance. It is known that microbial fermentation of otherwise indigestible polysaccharides into short chain fatty acids improves digestive efficiency and promotes positive energy balance. In contrast, the contributions of the microbiota to metabolism of other dietary nutrient classes such as energy-rich lipids remain unresolved. We used in vivo imaging of fluorescent fatty acid (FA) analogs delivered into gnotobiotic zebrafish hosts to reveal that the microbiota stimulates FA uptake and lipid droplet (LD) formation in the intestinal epithelium and liver. We found that the Firmicutes bacterium Exiguobacterium indicum and its products were sufficient to increase epithelial LD number, whereas LD size was increased by other bacterial types. Therefore, different members of the intestinal microbiota promote lipid absorption via distinct mechanisms[6]. To further explore mechanisms of nutrient absorption, we have developed methods for micro-gavage of zebrafish larvae with selected materials[15]. We are currently investigating the bacterial factors that promote intestinal absorption of lipids and other nutrients, and the responsive host mechanisms that mediate these effects on nutrient absorption. Once identified, these factors would represent promising targets for new therapies designed to address human diseases such as malnutrition, obesity, and associated metabolic diseases. 

           Physiologic responses in eukaryotic cells are often mediated by changes in gene transcription that are specific to the cell type and the stimulus. The specificity of the transcriptional response is determined by targeted alterations in chromatin accessibility and the resulting interactions between specific transcription factors (TFs) and open chromatin at DNA cis-regulatory modules (CRMs)[16]. A fundamental challenge in the study of host-microbiota interactions is to understand how host cells interpret dynamic microbial signals within the context of tissue-specific regulatory networks to evoke appropriate transcriptional responses. The amenability of the zebrafish to in vivo imaging, transgenesis, and gnotobiotic manipulations provides exciting opportunities to address these questions. The NF-kB transcriptional control pathway is known to mediate microbial stimuli that regulate expression of diverse host genes[17]. However, the temporal and spatial activation of NF-kB in response to microbial signals had not been determined in whole living organisms. We generated transgenic zebrafish that express fluorescent protein under the transcriptional control of NF-kB and used them to show that distinct cell types in the digestive tract and other tissues display robust and dynamic responses to variations in the microbial environment[14]. The capacity of the microbiota to promote fat storage is caused in part by the microbial suppression of intestinal epithelial transcription of Angiopoietin-like 4 (Angptl4/Fiaf), a circulating inhibitor of lipoprotein lipase (LPL) that inhibits hydrolysis of serum triglycerides for storage in adipose tissues[18,19].  We used in vivo transgenic reporter assays to identify discrete tissue-specific CRMs at zebrafish angptl4 that drive expression in intestinal enterocytes and other tissues. Strikingly, the microbiota suppressed the transcriptional activity of the intestine-specific CRM similar to the endogenous Angptl4 conservationangptl4 gene. These results suggest that the microbiota might regulate host intestinal Angptl4 protein expression and peripheral fat storage by suppressing the activity of an intestine-specific transcriptional enhancer[20]. We are currently seeking to identify the TFs that mediate the tissue specificity and microbial sensitivity of this and other CRMs. We are also using functional genomic methods in intestinal epithelial cells from gnotobiotic animals to elucidate global transcriptional regulatory networks underlying physiologic responses to the microbiota. The expected outcomes of this work include fundamental new insights into the genomic foundations of host-microbe commensalism and novel molecular targets for the manipulation of intestinal physiology and pathophysiology in humans and other vertebrates.



Adipose tissue physiology and metabolic disease TopTop  

The current epidemic of obesity and obesity-associated metabolic diseases represents major public health challenges. Obesity is caused by prolonged positive energy balance resulting in storage of excess energy as neutral lipid in adipocytes within white adipose tissues (AT). AT forms in several distinct anatomic regions and expands through addition of new adipocytes from undifferentiated precursors (hyperplasia) or by increase in adipocyte size (hypertrophy). The regional distribution and cellular morphology of AT have been identified as critical factors linking obesity to metabolic disease. Specifically, selective expansion of visceral AT and hypertrophic AT morphology are associated with increased risk for metabolic diseases such as insulin resistance (IR)[21-24]. However, the mechanisms governing regional distribution and cellular morphology of AT remain poorly understood. To address these gaps, we have pioneered the use of the zebrafish model for investigating AT development and physiology. We developed methods for vital labelingWt and gh1 and in vivo imaging of zebrafish AT[25], and used them to reveal extensive molecular, cellular, and physiological homologies between zebrafish and mammalian white adipocytes[26]. Using these methods, we found that zebrafish growth hormone 1 (gh1) mutants exhibit delayed somatic growth, increased AT accumulation, and disrupted adipose plasticity during nutrient deprivation[27]. We are currently exploring evolutionarily conserved mechanisms by which the vascular system and fibrotic programs determine regional AT morphology. This work could lead to new strategies for preventing human obesity and associated metabolic diseases by selectively controlling distinct aspects of AT development and physiology, including AT distribution and cellular morphology.  


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10. Hildebrandt, M. A., Hoffmann, C., Sherrill-Mix, S. A., Keilbaugh, S. A., Hamady, M., Chen, Y. Y., Knight, R., Ahima, R. S., Bushman, F. & Wu, G. D. High-fat diet determines the composition of the murine gut microbiome independently of obesity. Gastroenterology 137, 1716-1724 e1711-1712, (2009).

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15 .Cocchiaro, J. L. & Rawls, J. F. Microgavage of zebrafish larvae. J Vis Exp, e4434, (2013).

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17. Karrasch, T. & Jobin, C. NF-kappaB and the intestine: friend or foe? Inflamm Bowel Dis 14, 114-124, (2008).

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19. Bäckhed, F., Manchester, J. K., Semenkovich, C. F. & Gordon, J. I. Mechanisms underlying the resistance to diet-induced obesity in germ-free mice. Proc Natl Acad Sci U S A 104, 979-984, (2007).

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21. Fox, C. S., Massaro, J. M., Hoffmann, U., Pou, K. M., Maurovich-Horvat, P., Liu, C. Y., Vasan, R. S., Murabito, J. M., Meigs, J. B., Cupples, L. A. et al. Abdominal visceral and subcutaneous adipose tissue compartments: association with metabolic risk factors in the Framingham Heart Study. Circulation 116, 39-48, (2007).

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23. Penders, J., Thijs, C., Vink, C., Stelma, F. F., Snijders, B., Kummeling, I., van den Brandt, P. A. & Stobberingh, E. E. Factors influencing the composition of the intestinal microbiota in early infancy. Pediatrics 118, 511-521, (2006).

24. Hoffstedt, J., Arner, E., Wahrenberg, H., Andersson, D. P., Qvisth, V., Lofgren, P., Ryden, M., Thorne, A., Wiren, M., Palmer, M. et al. Regional impact of adipose tissue morphology on the metabolic profile in morbid obesity. Diabetologia 53, 2496-2503, (2010).

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27. McMenamin, S. K., Minchin, J. E., Gordon, T. N., Rawls, J. F. & Parichy, D. M. Dwarfism and Increased Adiposity in the gh1 Mutant Zebrafish vizzini. Endocrinology 154, 1476-1487, (2013).

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