Publications by authors named "John F Rawls"

76 Publications

Age-related changes in the upper respiratory microbiome are associated with SARS-CoV-2 susceptibility and illness severity.

medRxiv 2021 Mar 23. Epub 2021 Mar 23.

Children are less susceptible to SARS-CoV-2 and typically have milder illness courses than adults. We studied the nasopharyngeal microbiomes of 274 children, adolescents, and young adults with SARS-CoV-2 exposure using 16S rRNA gene sequencing. We find that higher abundances of species are associated with SARS-CoV-2 infection and SARS-CoV-2-associated respiratory symptoms, while higher abundances of are present in SARS-CoV-2-infected individuals respiratory symptoms. We also demonstrate that the abundances of these bacteria are strongly, and independently, associated with age, suggesting that the nasopharyngeal microbiome may be a potentially modifiable mechanism by which age influences SARS-CoV-2 susceptibility and severity.

Summary: Evaluation of nasopharyngeal microbiome profiles in children, adolescents, and young adults with a SARS-CoV-2-infected close contact identified specific bacterial species that vary in abundance with age and are associated with SARS-CoV-2 susceptibility and the presence of SARS-CoV-2-associated respiratory symptoms.
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http://dx.doi.org/10.1101/2021.03.20.21252680DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8010748PMC
March 2021

The Pediatric Obesity Microbiome and Metabolism Study (POMMS): Methods, Baseline Data, and Early Insights.

Obesity (Silver Spring) 2021 Mar;29(3):569-578

Department of Pediatrics, Duke University, Durham, North Carolina, USA.

Objective: The purpose of this study was to establish a biorepository of clinical, metabolomic, and microbiome samples from adolescents with obesity as they undergo lifestyle modification.

Methods: A total of 223 adolescents aged 10 to 18 years with BMI ≥95th percentile were enrolled, along with 71 healthy weight participants. Clinical data, fasting serum, and fecal samples were collected at repeated intervals over 6 months. Herein, the study design, data collection methods, and interim analysis-including targeted serum metabolite measurements and fecal 16S ribosomal RNA gene amplicon sequencing among adolescents with obesity (n = 27) and healthy weight controls (n = 27)-are presented.

Results: Adolescents with obesity have higher serum alanine aminotransferase, C-reactive protein, and glycated hemoglobin, and they have lower high-density lipoprotein cholesterol when compared with healthy weight controls. Metabolomics revealed differences in branched-chain amino acid-related metabolites. Also observed was a differential abundance of specific microbial taxa and lower species diversity among adolescents with obesity when compared with the healthy weight group.

Conclusions: The Pediatric Metabolism and Microbiome Study (POMMS) biorepository is available as a shared resource. Early findings suggest evidence of a metabolic signature of obesity unique to adolescents, along with confirmation of previously reported findings that describe metabolic and microbiome markers of obesity.
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http://dx.doi.org/10.1002/oby.23081DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7927749PMC
March 2021

Acoustofluidic rotational tweezing enables high-speed contactless morphological phenotyping of zebrafish larvae.

Nat Commun 2021 02 18;12(1):1118. Epub 2021 Feb 18.

Department of Mechanical Engineering and Material Science, Duke University, Durham, NC, USA.

Modern biomedical research and preclinical pharmaceutical development rely heavily on the phenotyping of small vertebrate models for various diseases prior to human testing. In this article, we demonstrate an acoustofluidic rotational tweezing platform that enables contactless, high-speed, 3D multispectral imaging and digital reconstruction of zebrafish larvae for quantitative phenotypic analysis. The acoustic-induced polarized vortex streaming achieves contactless and rapid (~1 s/rotation) rotation of zebrafish larvae. This enables multispectral imaging of the zebrafish body and internal organs from different viewing perspectives. Moreover, we develop a 3D reconstruction pipeline that yields accurate 3D models based on the multi-view images for quantitative evaluation of basic morphological characteristics and advanced combinations of metrics. With its contactless nature and advantages in speed and automation, our acoustofluidic rotational tweezing system has the potential to be a valuable asset in numerous fields, especially for developmental biology, small molecule screening in biochemistry, and pre-clinical drug development in pharmacology.
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http://dx.doi.org/10.1038/s41467-021-21373-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7892888PMC
February 2021

Enteroendocrine cells sense bacterial tryptophan catabolites to activate enteric and vagal neuronal pathways.

Cell Host Microbe 2021 02 21;29(2):179-196.e9. Epub 2020 Dec 21.

Department of Molecular Genetics and Microbiology, Duke Microbiome Center, Duke University School of Medicine, Durham, NC 27710, USA; Division of Gastroenterology, Department of Medicine, Duke University School of Medicine, Durham, NC 27710, USA. Electronic address:

The intestinal epithelium senses nutritional and microbial stimuli using epithelial sensory enteroendocrine cells (EEC). EECs communicate nutritional information to the nervous system, but whether they also relay signals from intestinal microbes remains unknown. Using in vivo real-time measurements of EEC and nervous system activity in zebrafish, we discovered that the bacteria Edwardsiella tarda activate EECs through the receptor transient receptor potential ankyrin A1 (Trpa1) and increase intestinal motility. Microbial, pharmacological, or optogenetic activation of Trpa1EECs directly stimulates vagal sensory ganglia and activates cholinergic enteric neurons by secreting the neurotransmitter 5-hydroxytryptamine (5-HT). A subset of indole derivatives of tryptophan catabolism produced by E. tarda and other gut microbes activates zebrafish EEC Trpa1 signaling. These catabolites also directly stimulate human and mouse Trpa1 and intestinal 5-HT secretion. These results establish a molecular pathway by which EECs regulate enteric and vagal neuronal pathways in response to microbial signals.
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http://dx.doi.org/10.1016/j.chom.2020.11.011DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7997396PMC
February 2021

Conserved anti-inflammatory effects and sensing of butyrate in zebrafish.

Gut Microbes 2020 11;12(1):1-11

Tuberculosis Research Program at the Centenary Institute, The University of Sydney , Camperdown, Australia.

Short-chain fatty acids (SCFAs) are produced by microbial fermentation of dietary fiber in the gut. Butyrate is a particularly important SCFA with anti-inflammatory properties and is generally present at lower levels in inflammatory diseases associated with gut microbiota dysbiosis in mammals. We aimed to determine if SCFAs are produced by the zebrafish microbiome and if SCFAs exert conserved effects on zebrafish immunity as an example of the non-mammalian vertebrate immune system. We demonstrate that bacterial communities from adult zebrafish intestines synthesize all three main SCFA , although SCFA were below our detectable limits in zebrafish intestines . Immersion in butyrate, but not acetate or propionate, reduced the recruitment of neutrophils and M1-type pro-inflammatory macrophages to wounds. We found conservation of butyrate sensing by neutrophils via orthologs of the () gene. Neutrophils from Hcar1-depleted embryos were no longer responsive to the anti-inflammatory effects of butyrate, while macrophage sensitivity to butyrate was independent of Hcar1. Our data demonstrate conservation of anti-inflammatory butyrate effects and identify the presence of a conserved molecular receptor in fish.
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http://dx.doi.org/10.1080/19490976.2020.1824563DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7575005PMC
November 2020

Transcriptional programmes underlying cellular identity and microbial responsiveness in the intestinal epithelium.

Nat Rev Gastroenterol Hepatol 2021 01 6;18(1):7-23. Epub 2020 Oct 6.

Department of Molecular Genetics and Microbiology, Duke Microbiome Center, Duke University School of Medicine, Durham, NC, USA.

The intestinal epithelium serves the unique and critical function of harvesting dietary nutrients, while simultaneously acting as a cellular barrier separating tissues from the luminal environment and gut microbial ecosystem. Two salient features of the intestinal epithelium enable it to perform these complex functions. First, cells within the intestinal epithelium achieve a wide range of specialized identities, including different cell types and distinct anterior-posterior patterning along the intestine. Second, intestinal epithelial cells are sensitive and responsive to the dynamic milieu of dietary nutrients, xenobiotics and microorganisms encountered in the intestinal luminal environment. These diverse identities and responsiveness of intestinal epithelial cells are achieved in part through the differential transcription of genes encoded in their shared genome. Here, we review insights from mice and other vertebrate models into the transcriptional regulatory mechanisms underlying intestinal epithelial identity and microbial responsiveness, including DNA methylation, chromatin accessibility, histone modifications and transcription factors. These studies are revealing that most transcription factors involved in intestinal epithelial identity also respond to changes in the microbiota, raising both opportunities and challenges to discern the underlying integrative transcriptional regulatory networks.
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http://dx.doi.org/10.1038/s41575-020-00357-6DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7997278PMC
January 2021

Single-cell imaging of human cancer xenografts using adult immunodeficient zebrafish.

Nat Protoc 2020 09 21;15(9):3105-3128. Epub 2020 Aug 21.

Molecular Pathology Unit, Mass General Research Institute, Charlestown, MA, USA.

Zebrafish are an ideal cell transplantation model. They are highly fecund, optically clear and an excellent platform for preclinical drug discovery studies. Traditionally, xenotransplantation has been carried out using larval zebrafish that have not yet developed adaptive immunity. Larval engraftment is a powerful short-term transplant platform amenable to high-throughput drug screening studies, yet animals eventually reject tumors and cannot be raised at 37 °C. To address these limitations, we have recently developed adult casper-strain prkdc, il2rgα immunocompromised zebrafish that robustly engraft human cancer cells for in excess of 28 d. Because the adult zebrafish can be administered drugs by oral gavage or i.p. injection, our model is suitable for achieving accurate, preclinical drug dosing. Our platform also allows facile visualization of drug effects in vivo at single-cell resolution over days. Here, we describe the procedures for xenograft cell transplantation into the prkdc, il2rgα model, including refined husbandry protocols for optimal growth and rearing of immunosuppressed zebrafish at 37 °C; optimized intraperitoneal and periocular muscle cell transplantation; and epifluorescence and confocal imaging approaches to visualize the effects of administering clinically relevant drug dosing at single-cell resolution in vivo. After identification of adult homozygous animals, this procedure takes 35 d to complete. 7 days are required to acclimate adult fish to 37 °C, and 28 d are required for engraftment studies. Our protocol provides a comprehensive guide for using immunocompromised zebrafish for xenograft cell transplantation and credentials the model as a new preclinical drug discovery platform.
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http://dx.doi.org/10.1038/s41596-020-0372-yDOI Listing
September 2020

Short-Chain Fatty Acid Production by Gut Microbiota from Children with Obesity Differs According to Prebiotic Choice and Bacterial Community Composition.

mBio 2020 08 11;11(4). Epub 2020 Aug 11.

Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, North Carolina, USA

Pediatric obesity remains a public health burden and continues to increase in prevalence. The gut microbiota plays a causal role in obesity and is a promising therapeutic target. Specifically, the microbial production of short-chain fatty acids (SCFA) from the fermentation of otherwise indigestible dietary carbohydrates may protect against pediatric obesity and metabolic syndrome. Still, it has not been demonstrated that therapies involving microbiota-targeting carbohydrates, known as prebiotics, will enhance gut bacterial SCFA production in children and adolescents with obesity (age, 10 to 18 years old). Here, we used an system to examine the SCFA production by fecal microbiota from 17 children with obesity when exposed to five different commercially available over-the-counter (OTC) prebiotic supplements. We found microbiota from all 17 patients actively metabolized most prebiotics. Still, supplements varied in their acidogenic potential. Significant interdonor variation also existed in SCFA production, which 16S rRNA sequencing supported as being associated with differences in the host microbiota composition. Last, we found that neither fecal SCFA concentration, microbiota SCFA production capacity, nor markers of obesity positively correlated with one another. Together, these findings suggest the hypothesis that OTC prebiotic supplements may be unequal in their ability to stimulate SCFA production in children and adolescents with obesity and that the most acidogenic prebiotic may differ across individuals. Pediatric obesity remains a major public health problem in the United States, where 17% of children and adolescents are obese, and rates of pediatric "severe obesity" are increasing. Children and adolescents with obesity face higher health risks, and noninvasive therapies for pediatric obesity often have limited success. The human gut microbiome has been implicated in adult obesity, and microbiota-directed therapies can aid weight loss in adults with obesity. However, less is known about the microbiome in pediatric obesity, and microbiota-directed therapies are understudied in children and adolescents. Our research has two important findings: (i) dietary prebiotics (fiber) result in the microbiota from adolescents with obesity producing more SCFA, and (ii) the effectiveness of each prebiotic is donor dependent. Together, these findings suggest that prebiotic supplements could help children and adolescents with obesity, but that these therapies may not be "one size fits all."
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http://dx.doi.org/10.1128/mBio.00914-20DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7439474PMC
August 2020

Rationale and design of "Hearts & Parks": study protocol for a pragmatic randomized clinical trial of an integrated clinic-community intervention to treat pediatric obesity.

BMC Pediatr 2020 06 26;20(1):308. Epub 2020 Jun 26.

Duke Clinical Research Institute, Duke University, Durham, NC, 27710, USA.

Background: The prevalence of child and adolescent obesity and severe obesity continues to increase despite decades of policy and research aimed at prevention. Obesity strongly predicts cardiovascular and metabolic disease risk; both begin in childhood. Children who receive intensive behavioral interventions can reduce body mass index (BMI) and reverse disease risk. However, delivering these interventions with fidelity at scale remains a challenge. Clinic-community partnerships offer a promising strategy to provide high-quality clinical care and deliver behavioral treatment in local park and recreation settings. The Hearts & Parks study has three broad objectives: (1) evaluate the effectiveness of the clinic-community model for the treatment of child obesity, (2) define microbiome and metabolomic signatures of obesity and response to lifestyle change, and (3) inform the implementation of similar models in clinical systems.

Methods: Methods are designed for a pragmatic randomized, controlled clinical trial (n = 270) to test the effectiveness of an integrated clinic-community child obesity intervention as compared with usual care. We are powered to detect a difference in body mass index (BMI) between groups at 6 months, with follow up to 12 months. Secondary outcomes include changes in biomarkers for cardiovascular disease, psychosocial risk, and quality of life. Through collection of biospecimens (serum and stool), additional exploratory outcomes include microbiome and metabolomics biomarkers of response to lifestyle modification.

Discussion: We present the study design, enrollment strategy, and intervention details for a randomized clinical trial to measure the effectiveness of a clinic-community child obesity treatment intervention. This study will inform a critical area in child obesity and cardiovascular risk research-defining outcomes, implementation feasibility, and identifying potential molecular mechanisms of treatment response.

Clinical Trial Registration: NCT03339440 .
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http://dx.doi.org/10.1186/s12887-020-02190-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7318397PMC
June 2020

RSPO3 impacts body fat distribution and regulates adipose cell biology in vitro.

Nat Commun 2020 06 3;11(1):2797. Epub 2020 Jun 3.

Oxford Centre for Diabetes, Endocrinology and Metabolism, Radcliffe Department of Medicine, University of Oxford, Oxford, OX3 7LE, UK.

Fat distribution is an independent cardiometabolic risk factor. However, its molecular and cellular underpinnings remain obscure. Here we demonstrate that two independent GWAS signals at RSPO3, which are associated with increased body mass index-adjusted waist-to-hip ratio, act to specifically increase RSPO3 expression in subcutaneous adipocytes. These variants are also associated with reduced lower-body fat, enlarged gluteal adipocytes and insulin resistance. Based on human cellular studies RSPO3 may limit gluteofemoral adipose tissue (AT) expansion by suppressing adipogenesis and increasing gluteal adipocyte susceptibility to apoptosis. RSPO3 may also promote upper-body fat distribution by stimulating abdominal adipose progenitor (AP) proliferation. The distinct biological responses elicited by RSPO3 in abdominal versus gluteal APs in vitro are associated with differential changes in WNT signalling. Zebrafish carrying a nonsense rspo3 mutation display altered fat distribution. Our study identifies RSPO3 as an important determinant of peripheral AT storage capacity.
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http://dx.doi.org/10.1038/s41467-020-16592-zDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7271210PMC
June 2020

Feeling the Burn: Intestinal Epithelial Cells Modify Their Lipid Metabolism in Response to Bacterial Fermentation Products.

Cell Host Microbe 2020 03;27(3):314-316

Department of Molecular Genetics and Microbiology, Duke Microbiome Center, Duke University School of Medicine, Durham, NC 27710, USA. Electronic address:

Intestinal epithelial absorption of dietary lipids is a major determinant of animal energy balance and metabolic health. Recent studies uncovered significant roles for intestinal microbiota in this process, but underlying mechanisms remain unresolved. Araújo et al. (2020) identify two end-products of bacterial fermentation that regulate intestinal lipid absorption and metabolism.
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http://dx.doi.org/10.1016/j.chom.2020.02.009DOI Listing
March 2020

High fat diet induces microbiota-dependent silencing of enteroendocrine cells.

Elife 2019 12 3;8. Epub 2019 Dec 3.

Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, United States.

Enteroendocrine cells (EECs) are specialized sensory cells in the intestinal epithelium that sense and transduce nutrient information. Consumption of dietary fat contributes to metabolic disorders, but EEC adaptations to high fat feeding were unknown. Here, we established a new experimental system to directly investigate EEC activity in vivo using a zebrafish reporter of EEC calcium signaling. Our results reveal that high fat feeding alters EEC morphology and converts them into a nutrient insensitive state that is coupled to endoplasmic reticulum (ER) stress. We called this novel adaptation 'EEC silencing'. Gnotobiotic studies revealed that germ-free zebrafish are resistant to high fat diet induced EEC silencing. High fat feeding altered gut microbiota composition including enrichment of bacteria, and we identified an strain sufficient to induce EEC silencing. These results establish a new mechanism by which dietary fat and gut microbiota modulate EEC nutrient sensing and signaling.
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http://dx.doi.org/10.7554/eLife.48479DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6937151PMC
December 2019

Commensal Microbiota Regulate Vertebrate Innate Immunity-Insights From the Zebrafish.

Front Immunol 2019 6;10:2100. Epub 2019 Sep 6.

Department of Molecular Genetics and Microbiology, Duke Microbiome Center, Duke University School of Medicine, Durham, NC, United States.

Microbial communities populate the mucosal surfaces of all animals. Metazoans have co-evolved with these microorganisms, forming symbioses that affect the molecular and cellular underpinnings of animal physiology. These microorganisms, collectively referred to as the microbiota, are found on many distinct body sites (including the skin, nasal cavity, and urogenital tract), however the most densely colonized host tissue is the intestinal tract. Although spatially confined within the intestinal lumen, the microbiota and associated products shape the development and function of the host immune system. Studies comparing gnotobiotic animals devoid of any microbes (germ free) with counterparts colonized with selected microbial communities have demonstrated that commensal microorganisms are required for the proper development and function of the immune system at homeostasis and following infectious challenge or injury. Animal model systems have been essential for defining microbiota-dependent shifts in innate immune cell function and intestinal physiology during infection and disease. In particular, the zebrafish has emerged as a powerful vertebrate model organism with unparalleled capacity for imaging, a full complement of genetic approaches, and facile methods to experimentally manipulate microbial communities. Here we review key insights afforded by the zebrafish into the impact of microbiota on innate immunity, including evidence that the perception of and response to the microbiota is evolutionarily conserved. We also highlight opportunities to strengthen the zebrafish model system, and to gain new insights into microbiota-innate immune interactions that would be difficult to achieve in mammalian models.
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http://dx.doi.org/10.3389/fimmu.2019.02100DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6742977PMC
October 2020

Lysosome-Rich Enterocytes Mediate Protein Absorption in the Vertebrate Gut.

Dev Cell 2019 10 29;51(1):7-20.e6. Epub 2019 Aug 29.

Department of Cell Biology, Duke University, Durham, NC 27710, USA; Regeneration Next Initiative, Duke University, Durham, NC 27710, United States. Electronic address:

The guts of neonatal mammals and stomachless fish have a limited capacity for luminal protein digestion, which allows oral acquisition of antibodies and antigens. However, how dietary protein is absorbed during critical developmental stages when the gut is still immature is unknown. Here, we show that specialized intestinal cells, which we call lysosome-rich enterocytes (LREs), internalize dietary protein via receptor-mediated and fluid-phase endocytosis for intracellular digestion and trans-cellular transport. In LREs, we identify a conserved endocytic machinery, composed of the scavenger receptor complex Cubilin/Amnionless and Dab2, that is required for protein uptake by LREs and for growth and survival of larval zebrafish. Moreover, impairing LRE function in suckling mice, via conditional deletion of Dab2, leads to stunted growth and severe protein malnutrition reminiscent of kwashiorkor, a devastating human malnutrition syndrome. These findings identify digestive functions and conserved molecular mechanisms in LREs that are crucial for vertebrate growth and survival.
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http://dx.doi.org/10.1016/j.devcel.2019.08.001DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6783362PMC
October 2019

Commensal Bacteria Regulate Gene Expression and Differentiation in Vertebrate Olfactory Systems Through Transcription Factor REST.

Chem Senses 2019 10;44(8):615-630

Center for Evolutionary and Theoretical Immunology (CETI), Department of Biology, MSC03 2020, 1 University of New Mexico, Albuquerque, NM, USA.

Sensory systems such as the olfactory system detect chemical stimuli and thereby determine the relationships between the animal and its surroundings. Olfaction is one of the most conserved and ancient sensory systems in vertebrates. The vertebrate olfactory epithelium is colonized by complex microbial communities, but microbial contribution to host olfactory gene expression remains unknown. In this study, we show that colonization of germ-free zebrafish and mice with microbiota leads to widespread transcriptional responses in olfactory organs as measured in bulk tissue transcriptomics and RT-qPCR. Germ-free zebrafish olfactory epithelium showed defects in pseudostratification; however, the size of the olfactory pit and the length of the cilia were not different from that of colonized zebrafish. One of the mechanisms by which microbiota control host transcriptional programs is by differential expression and activity of specific transcription factors (TFs). REST (RE1 silencing transcription factor, also called NRSF) is a zinc finger TF that binds to the conserved motif repressor element 1 found in the promoter regions of many neuronal genes with functions in neuronal development and differentiation. Colonized zebrafish and mice showed increased nasal expression of REST, and genes with reduced expression in colonized animals were strongly enriched in REST-binding motifs. Nasal commensal bacteria promoted in vitro differentiation of Odora cells by regulating the kinetics of REST expression. REST knockdown resulted in decreased Odora cell differentiation in vitro. Our results identify a conserved mechanism by which microbiota regulate vertebrate olfactory transcriptional programs and reveal a new role for REST in sensory organs.
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http://dx.doi.org/10.1093/chemse/bjz050DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6796929PMC
October 2019

Epithelial delamination is protective during pharmaceutical-induced enteropathy.

Proc Natl Acad Sci U S A 2019 08 7;116(34):16961-16970. Epub 2019 Aug 7.

Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, NC 27710;

Intestinal epithelial cell (IEC) shedding is a fundamental response to intestinal damage, yet underlying mechanisms and functions have been difficult to define. Here we model chronic intestinal damage in zebrafish larvae using the nonsteroidal antiinflammatory drug (NSAID) Glafenine. Glafenine induced the unfolded protein response (UPR) and inflammatory pathways in IECs, leading to delamination. Glafenine-induced inflammation was augmented by microbial colonization and associated with changes in intestinal and environmental microbiotas. IEC shedding was a UPR-dependent protective response to Glafenine that restricts inflammation and promotes animal survival. Other NSAIDs did not induce IEC delamination; however, Glafenine also displays off-target inhibition of multidrug resistance (MDR) efflux pumps. We found a subset of MDR inhibitors also induced IEC delamination, implicating MDR efflux pumps as cellular targets underlying Glafenine-induced enteropathy. These results implicate IEC delamination as a protective UPR-mediated response to chemical injury, and uncover an essential role for MDR efflux pumps in intestinal homeostasis.
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http://dx.doi.org/10.1073/pnas.1902596116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6708343PMC
August 2019

Disrupted Maturation of the Microbiota and Metabolome among Extremely Preterm Infants with Postnatal Growth Failure.

Sci Rep 2019 06 3;9(1):8167. Epub 2019 Jun 3.

Division of Neonatology, Department of Pediatrics, Duke University, Durham, NC, 27710, USA.

Growth failure during infancy is a major global problem that has adverse effects on long-term health and neurodevelopment. Preterm infants are disproportionately affected by growth failure and its effects. Herein we found that extremely preterm infants with postnatal growth failure have disrupted maturation of the intestinal microbiota, characterized by persistently low diversity, dominance of pathogenic bacteria within the Enterobacteriaceae family, and a paucity of strictly anaerobic taxa including Veillonella relative to infants with appropriate postnatal growth. Metabolomic profiling of infants with growth failure demonstrated elevated serum acylcarnitines, fatty acids, and other byproducts of lipolysis and fatty acid oxidation. Machine learning algorithms for normal maturation of the microbiota and metabolome among infants with appropriate growth revealed a pattern of delayed maturation of the microbiota and metabolome among infants with growth failure. Collectively, we identified novel microbial and metabolic features of growth failure in preterm infants and potentially modifiable targets for intervention.
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http://dx.doi.org/10.1038/s41598-019-44547-yDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6546715PMC
June 2019

Visualizing Engrafted Human Cancer and Therapy Responses in Immunodeficient Zebrafish.

Cell 2019 06 25;177(7):1903-1914.e14. Epub 2019 Apr 25.

Molecular Pathology Unit, Massachusetts General Hospital Research Institute, Charlestown, MA 02129, USA; Massachusetts General Hospital Cancer Center, Harvard Medical School, Charlestown, MA 02129, USA; Center for Regenerative Medicine, Massachusetts General Hospital, Boston, MA 02114, USA; Harvard Stem Cell Institute, Cambridge, MA 02139, USA. Electronic address:

Xenograft cell transplantation into immunodeficient mice has become the gold standard for assessing pre-clinical efficacy of cancer drugs, yet direct visualization of single-cell phenotypes is difficult. Here, we report an optically-clear prkdc, il2rga zebrafish that lacks adaptive and natural killer immune cells, can engraft a wide array of human cancers at 37°C, and permits the dynamic visualization of single engrafted cells. For example, photoconversion cell-lineage tracing identified migratory and proliferative cell states in human rhabdomyosarcoma, a pediatric cancer of muscle. Additional experiments identified the preclinical efficacy of combination olaparib PARP inhibitor and temozolomide DNA-damaging agent as an effective therapy for rhabdomyosarcoma and visualized therapeutic responses using a four-color FUCCI cell-cycle fluorescent reporter. These experiments identified that combination treatment arrested rhabdomyosarcoma cells in the G2 cell cycle prior to induction of apoptosis. Finally, patient-derived xenografts could be engrafted into our model, opening new avenues for developing personalized therapeutic approaches in the future.
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http://dx.doi.org/10.1016/j.cell.2019.04.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6570580PMC
June 2019

Intestinal Serum amyloid A suppresses systemic neutrophil activation and bactericidal activity in response to microbiota colonization.

PLoS Pathog 2019 03 7;15(3):e1007381. Epub 2019 Mar 7.

Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, North Carolina, United States of America.

The intestinal microbiota influences the development and function of myeloid lineages such as neutrophils, but the underlying molecular mechanisms are unresolved. Using gnotobiotic zebrafish, we identified the immune effector Serum amyloid A (Saa) as one of the most highly induced transcripts in digestive tissues following microbiota colonization. Saa is a conserved secreted protein produced in the intestine and liver with described effects on neutrophils in vitro, however its in vivo functions remain poorly defined. 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. At baseline, saa mutants exhibited moderate neutrophilia and altered neutrophil tissue distribution. Molecular and functional analyses of isolated neutrophils revealed that Saa suppresses expression of pro-inflammatory markers and bactericidal activity. Saa's effects on neutrophils depended 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.
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http://dx.doi.org/10.1371/journal.ppat.1007381DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6405052PMC
March 2019

An explant technique for high-resolution imaging and manipulation of mycobacterial granulomas.

Nat Methods 2018 12 30;15(12):1098-1107. Epub 2018 Nov 30.

Department of Molecular Genetics and Microbiology, Duke University School of Medicine, Durham, NC, USA.

A central and critical structure in tuberculosis, the mycobacterial granuloma consists of highly organized immune cells, including macrophages that drive granuloma formation through a characteristic epithelioid transformation. Difficulties in imaging within intact animals and caveats associated with in vitro assembly models have severely limited the study and experimental manipulation of mature granulomas. Here we describe a new ex vivo culture technique, wherein mature, fully organized zebrafish granulomas are microdissected and maintained in three-dimensional (3D) culture. This approach enables high-resolution microscopy of granuloma macrophage dynamics, including epithelioid macrophage motility and granuloma consolidation, while retaining key bacterial and host characteristics. Using mass spectrometry, we find active production of key phosphotidylinositol species identified previously in human granulomas. We also describe a method to transfect isolated granulomas, enabling genetic manipulation, and provide proof-of-concept for host-directed small-molecule screens, identifying protein kinase C (PKC) signaling as an important regulator of granuloma macrophage organization.
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http://dx.doi.org/10.1038/s41592-018-0215-8DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6312189PMC
December 2018

Pneumococcal Colonization and the Nasopharyngeal Microbiota of Children in Botswana.

Pediatr Infect Dis J 2018 11;37(11):1176-1183

Division of Pediatric Infectious Diseases, Ann and Robert Lurie Children's Hospital, Chicago, IL.

Background: Nasopharyngeal colonization precedes infections caused by Streptococcus pneumoniae. A more detailed understanding of interactions between S. pneumoniae and the nasopharyngeal microbiota of children could inform strategies to prevent pneumococcal infections.

Methods: We collected nasopharyngeal swabs from children 1 to 23 months of age in Botswana between August 2012 and June 2016. We tested samples for S. pneumoniae and common respiratory viruses using polymerase chain reaction. We sequenced the V3 region of the bacterial 16S ribosomal RNA gene and used random forest models to identify clinical variables and bacterial genera that were associated with pneumococcal colonization.

Results: Mean age of the 170 children included in this study was 8.3 months. Ninety-six (56%) children were colonized with S. pneumoniae. Pneumococcal colonization was associated with older age (P = 0.0001), a lack of electricity in the home (P = 0.02) and household use of wood as a cooking fuel (P = 0.002). Upper respiratory symptoms were more frequent in children with S. pneumoniae colonization (60% vs. 32%; P = 0.001). Adjusting for age, nasopharyngeal microbiota composition differed in colonized and noncolonized children (P = 0.001). S. pneumoniae colonization was associated with a higher relative abundance of Moraxella (P = 0.001) and lower relative abundances of Corynebacterium (P = 0.001) and Staphylococcus (P = 0.03). A decision tree model containing the relative abundances of bacterial genera had 81% sensitivity and 85% specificity for the determination of S. pneumoniae colonization status.

Conclusions: S. pneumoniae colonization is associated with characteristic alterations of the nasopharyngeal microbiota of children. Prospective studies should determine if nasopharyngeal microbial composition alters the risk of pneumococcal colonization and thus could be modified as a novel pneumonia prevention strategy.
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http://dx.doi.org/10.1097/INF.0000000000002174DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6181769PMC
November 2018

A High-Throughput Organoid Microinjection Platform to Study Gastrointestinal Microbiota and Luminal Physiology.

Cell Mol Gastroenterol Hepatol 2018 22;6(3):301-319. Epub 2018 May 22.

Joint Department of Biomedical Engineering, University of North Carolina at Chapel Hill/North Carolina State University, Chapel Hill, North Carolina.

Background & Aims: The human gut microbiota is becoming increasingly recognized as a key factor in homeostasis and disease. The lack of physiologically relevant in vitro models to investigate host-microbe interactions is considered a substantial bottleneck for microbiota research. Organoids represent an attractive model system because they are derived from primary tissues and embody key properties of the native gut lumen; however, access to the organoid lumen for experimental perturbation is challenging. Here, we report the development and validation of a high-throughput organoid microinjection system for cargo delivery to the organoid lumen and high-content sampling.

Methods: A microinjection platform was engineered using off-the-shelf and 3-dimensional printed components. Microinjection needles were modified for vertical trajectories and reproducible injection volumes. Computer vision (CVis) and microfabricated CellRaft Arrays (Cell Microsystems, Research Triangle Park, NC) were used to increase throughput and enable high-content sampling of mock bacterial communities. Modeling preformed using the COMSOL Multiphysics platform predicted a hypoxic luminal environment that was functionally validated by transplantation of fecal-derived microbial communities and monocultures of a nonsporulating anaerobe.

Results: CVis identified and logged locations of organoids suitable for injection. Reproducible loads of 0.2 nL could be microinjected into the organoid lumen at approximately 90 organoids/h. CVis analyzed and confirmed retention of injected cargos in approximately 500 organoids over 18 hours and showed the requirement to normalize for organoid growth for accurate assessment of barrier function. CVis analyzed growth dynamics of a mock community of green fluorescent protein- or Discosoma sp. red fluorescent protein-expressing bacteria, which grew within the organoid lumen even in the presence of antibiotics to control media contamination. Complex microbiota communities from fecal samples survived and grew in the colonoid lumen without appreciable changes in complexity.

Conclusions: High-throughput microinjection into organoids represents a next-generation in vitro approach to investigate gastrointestinal luminal physiology and the gastrointestinal microbiota.
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http://dx.doi.org/10.1016/j.jcmgh.2018.05.004DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6092482PMC
July 2019

Deep phenotyping in zebrafish reveals genetic and diet-induced adiposity changes that may inform disease risk.

J Lipid Res 2018 08 23;59(8):1536-1545. Epub 2018 May 23.

Department of Molecular Genetics and Microbiology, Duke University, Durham, NC 27710.

The regional distribution of adipose tissues is implicated in a wide range of diseases. For example, proportional increases in visceral adipose tissue increase the risk for insulin resistance, diabetes, and CVD. Zebrafish offer a tractable model system by which to obtain unbiased and quantitative phenotypic information on regional adiposity, and deep phenotyping can explore complex disease-related adiposity traits. To facilitate deep phenotyping of zebrafish adiposity traits, we used pairwise correlations between 67 adiposity traits to generate stage-specific adiposity profiles that describe changing adiposity patterns and relationships during growth. Linear discriminant analysis classified individual fish according to an adiposity profile with 87.5% accuracy. Deep phenotyping of eight previously uncharacterized zebrafish mutants identified as a novel gene that alters adipose distribution. When we applied deep phenotyping to identify changes in adiposity during diet manipulations, zebrafish that underwent food restriction and refeeding had widespread adiposity changes when compared with continuously fed, equivalently sized control animals. In particular, internal adipose tissues (e.g., visceral adipose) exhibited a reduced capacity to replenish lipid following food restriction. Together, these results in zebrafish establish a new deep phenotyping technique as an unbiased and quantitative method to help uncover new relationships between genotype, diet, and adiposity.
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http://dx.doi.org/10.1194/jlr.D084525DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6071777PMC
August 2018

Zebrafish Transcription Factor ORFeome for Gene Discovery and Regulatory Network Elucidation.

Zebrafish 2018 04 27;15(2):202-205. Epub 2017 Nov 27.

1 Department of Integrative Biology and Pharmacology, McGovern Medical School, University of Texas Health Science Center at Houston , Houston, Texas.

The completion of the zebrafish genome sequence and advances in miniaturization and multiplexing were essential to the creation of techniques such as RNA-seq, ChIP-seq, and high-throughput behavioral and chemical screens. Multiplexing was also instrumental in the recent enhancement of the classic yeast one-hybrid interaction techniques to provide unprecedented discovery capabilities for protein-DNA interactions. Unfortunately its use for zebrafish research is currently hampered by the lack of an open reading frame (ORF) clone collection. As a first step toward a complete collection, we describe a small library of transcriptional regulatory proteins comprising 142 ORFs and its potential applications.
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http://dx.doi.org/10.1089/zeb.2017.1486DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6112160PMC
April 2018

Microbial colonization is required for normal neurobehavioral development in zebrafish.

Sci Rep 2017 09 11;7(1):11244. Epub 2017 Sep 11.

U.S. EPA/ORD/NHEERL/ISTD, RTP, NC, USA.

Changes in resident microbiota may have wide-ranging effects on human health. We investigated whether early life microbial disruption alters neurodevelopment and behavior in larval zebrafish. Conventionally colonized, axenic, and axenic larvae colonized at 1 day post fertilization (dpf) were evaluated using a standard locomotor assay. At 10 dpf, axenic zebrafish exhibited hyperactivity compared to conventionalized and conventionally colonized controls. Impairment of host colonization using antibiotics also caused hyperactivity in conventionally colonized larvae. To determine whether there is a developmental requirement for microbial colonization, axenic embryos were serially colonized on 1, 3, 6, or 9 dpf and evaluated on 10 dpf. Normal activity levels were observed in axenic larvae colonized on 1-6 dpf, but not on 9 dpf. Colonization of axenic embryos at 1 dpf with individual bacterial species Aeromonas veronii or Vibrio cholerae was sufficient to block locomotor hyperactivity at 10 dpf. Exposure to heat-killed bacteria or microbe-associated molecular patterns pam3CSK4 or Poly(I:C) was not sufficient to block hyperactivity in axenic larvae. These data show that microbial colonization during early life is required for normal neurobehavioral development and support the concept that antibiotics and other environmental chemicals may exert neurobehavioral effects via disruption of host-associated microbial communities.
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http://dx.doi.org/10.1038/s41598-017-10517-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5593827PMC
September 2017

Genomic dissection of conserved transcriptional regulation in intestinal epithelial cells.

PLoS Biol 2017 Aug 29;15(8):e2002054. Epub 2017 Aug 29.

Department of Molecular Genetics and Microbiology, Center for the Genomics of Microbial Systems, Duke University, Durham, North Carolina, United States of America.

The intestinal epithelium serves critical physiologic functions that are shared among all vertebrates. However, it is unknown how the transcriptional regulatory mechanisms underlying these functions have changed over the course of vertebrate evolution. We generated genome-wide mRNA and accessible chromatin data from adult intestinal epithelial cells (IECs) in zebrafish, stickleback, mouse, and human species to determine if conserved IEC functions are achieved through common transcriptional regulation. We found evidence for substantial common regulation and conservation of gene expression regionally along the length of the intestine from fish to mammals and identified a core set of genes comprising a vertebrate IEC signature. We also identified transcriptional start sites and other putative regulatory regions that are differentially accessible in IECs in all 4 species. Although these sites rarely showed sequence conservation from fish to mammals, surprisingly, they drove highly conserved IEC expression in a zebrafish reporter assay. Common putative transcription factor binding sites (TFBS) found at these sites in multiple species indicate that sequence conservation alone is insufficient to identify much of the functionally conserved IEC regulatory information. Among the rare, highly sequence-conserved, IEC-specific regulatory regions, we discovered an ancient enhancer upstream from her6/HES1 that is active in a distinct population of Notch-positive cells in the intestinal epithelium. Together, these results show how combining accessible chromatin and mRNA datasets with TFBS prediction and in vivo reporter assays can reveal tissue-specific regulatory information conserved across 420 million years of vertebrate evolution. We define an IEC transcriptional regulatory network that is shared between fish and mammals and establish an experimental platform for studying how evolutionarily distilled regulatory information commonly controls IEC development and physiology.
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http://dx.doi.org/10.1371/journal.pbio.2002054DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5574553PMC
August 2017

Elucidating the role of plexin D1 in body fat distribution and susceptibility to metabolic disease using a zebrafish model system.

Adipocyte 2017 Oct 9;6(4):277-283. Epub 2017 Aug 9.

a Department of Molecular Genetics and Microbiology , Duke University , Durham , NC , USA.

Non-communicable diseases (NCDs) such as cardiovascular disease, diabetes and cancer were responsible for 68% of all deaths worldwide in 2012. The regional distribution of lipid deposited within adipose tissue (AT) - so called body fat distribution (BFD) - is a strong risk factor for NCDs. BFD is highly heritable; however, the genetic basis of BFD is almost entirely unknown. Genome-wide association studies have identified several loci associated with BFD, including at Plexin D1 (PLXND1) - a gene known to modulate angiogenesis. We recently demonstrated that zebrafish homozygous for a null mutation in plxnd1 had a reduced capacity to store lipid in visceral AT (VAT) leading to altered BFD. Moreover, we found that type V collagens were upregulated in plxnd1 mutants, and mediated the inhibitory effect of Plxnd1 on VAT growth. These results strengthen evidence that Plxnd1 influences BFD in human populations, and validate zebrafish as a model to study BFD. However, many pertinent questions remain unanswered. Here we outline potential Plxnd1 mechanisms of action in AT, and describe the genetic architecture at human PLXND1 that is associated with BFD and NCD susceptibility.
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http://dx.doi.org/10.1080/21623945.2017.1356504DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5736248PMC
October 2017

The Nasopharyngeal Microbiota of Children With Respiratory Infections in Botswana.

Pediatr Infect Dis J 2017 Sep;36(9):e211-e218

From the *Botswana-UPenn Partnership, Gaborone, Botswana; †Division of Pediatric Infectious Diseases, Duke University Medical Center, Durham, North Carolina; ‡Department of Medicine, McMaster University, §Department of Pathology and Molecular Medicine, McMaster University, ¶St. Joseph's Healthcare, and ‖Department of Pediatrics, McMaster University, Hamilton, Ontario, Canada; **Global Health Center, and ††Division of Pediatric Infectious Diseases, Children's Hospital of Philadelphia, Philadelphia, Pennsylvania; ‡‡Department of Pathology and Laboratory Medicine, BC Children's Hospital, Vancouver, British Columbia, Canada; §§University of Botswana School of Medicine, Gaborone, Botswana; ¶¶Department of Molecular Genetics and Microbiology, Center for the Genomics of Microbial Systems, Duke University Medical Center, Durham, North Carolina; and ‖‖Divisions of Hospital Medicine and Infectious Diseases, Cincinnati Children's Hospital Medical Center, Cincinnati, Ohio.

Background: Nearly half of child pneumonia deaths occur in sub-Saharan Africa. Microbial communities in the nasopharynx are a reservoir for pneumonia pathogens and remain poorly described in African children.

Methods: Nasopharyngeal swabs were collected from children with pneumonia (N = 204), children with upper respiratory infection symptoms (N = 55) and healthy children (N = 60) in Botswana between April 2012 and April 2014. We sequenced the V3 region of the bacterial 16S ribosomal RNA gene and used partitioning around medoids to cluster samples into microbiota biotypes. We then used multivariable logistic regression to examine whether microbiota biotypes were associated with pneumonia and upper respiratory infection symptoms.

Results: Mean ages of children with pneumonia, children with upper respiratory infection symptoms and healthy children were 8.2, 11.4 and 8.0 months, respectively. Clustering of nasopharyngeal microbiota identified 5 distinct biotypes: Corynebacterium/Dolosigranulum-dominant (23%), Haemophilus-dominant (11%), Moraxella-dominant (24%), Staphylococcus-dominant (13%) and Streptococcus-dominant (28%). The Haemophilus-dominant [odds ratio (OR): 13.55; 95% confidence interval (CI): 2.10-87.26], the Staphylococcus-dominant (OR: 8.27; 95% CI: 2.13-32.14) and the Streptococcus-dominant (OR: 39.97; 95% CI: 6.63-241.00) biotypes were associated with pneumonia. The Moraxella-dominant (OR: 3.71; 95% CI: 1.09-12.64) and Streptococcus-dominant (OR: 12.26; 95% CI: 1.81-83.06) biotypes were associated with upper respiratory infection symptoms. In children with pneumonia, HIV infection was associated with a lower relative abundance of Dolosigranulum (P = 0.03).

Conclusions: Pneumonia and upper respiratory infection symptoms are associated with distinct nasopharyngeal microbiota biotypes in African children. A lower abundance of the commensal genus Dolosigranulum may contribute to the higher pneumonia risk of HIV-infected children.
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http://dx.doi.org/10.1097/INF.0000000000001607DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5555803PMC
September 2017

Microbiota regulate intestinal epithelial gene expression by suppressing the transcription factor Hepatocyte nuclear factor 4 alpha.

Genome Res 2017 07 6;27(7):1195-1206. Epub 2017 Apr 6.

Department of Molecular Genetics and Microbiology, Center for the Genomics of Microbial Systems, Duke University, Durham, North Carolina 27710, USA.

Microbiota influence diverse aspects of intestinal physiology and disease in part by controlling tissue-specific transcription of host genes. However, host genomic mechanisms mediating microbial control of intestinal gene expression are poorly understood. Hepatocyte nuclear factor 4 (HNF4) is the most ancient family of nuclear receptor transcription factors with important roles in human metabolic and inflammatory bowel diseases, but a role in host response to microbes is unknown. Using an unbiased screening strategy, we found that zebrafish Hnf4a specifically binds and activates a microbiota-suppressed intestinal epithelial transcriptional enhancer. Genetic analysis revealed that zebrafish activates nearly half of the genes that are suppressed by microbiota, suggesting microbiota negatively regulate Hnf4a. In support, analysis of genomic architecture in mouse intestinal epithelial cells disclosed that microbiota colonization leads to activation or inactivation of hundreds of enhancers along with drastic genome-wide reduction of HNF4A and HNF4G occupancy. Interspecies meta-analysis suggested interactions between HNF4A and microbiota promote gene expression patterns associated with human inflammatory bowel diseases. These results indicate a critical and conserved role for HNF4A in maintaining intestinal homeostasis in response to microbiota.
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http://dx.doi.org/10.1101/gr.220111.116DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5495071PMC
July 2017