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Ontogeny of the Intestinal Circadian Clock and Its Role in the Response to Clostridium Difficile Toxin B

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Ontogeny of the Intestinal Circadian Clock and Its Role in the Response to Clostridium Difficile Toxin B Ontogeny of the intestinal circadian clock and its role in the response to Clostridium difficile toxin B A dissertation submitted to the Graduate School of the University of Cincinnati in partial fulfilment of the requirements for the degree of Doctor of Philosophy In the Department of Pharmacology & Systems Physiology of the College of Medicine by Andrew Rosselot B.S. Biology, Wittenberg University October 2019 Committee Chair: Christian I. Hong Ph.D. Abstract: The endogenous clock of the intestine regulates physiological processes ranging from nutrient absorption to the pathogenic response. The developmental timepoint when the human intestinal clock becomes active is unknown. We investigated intestinal circadian clock ontogeny using in vitro samples that are representative of distinct developmental timepoints. Induced pluripotent stem cells (iPSCs) were differentiated into 3D human intestinal organoids (HIOs) to mimic intestinal embryonic development in vitro. HIOs were then matured beyond their early fetal state via kidney capsule transplantation. Differentiation of iPSCs into HIOs did not activate robust circadian clock activity. Enteroids isolated from kidney capsule matured HIOs possessed a functional circadian clock, similar to adult biopsy derived human intestinal enteroids (bHIEs). Samples were challenged with toxin B (TcdB) from Clostridium difficile to provide functional insights on intestinal clock activity. The necrotic cell death response to TcdB was clock phase- dependent in samples that possessed an active clock and anti-phasic between mouse enteroids and bHIEs. RNA-seq analysis of mouse enteroids and bHIEs showed both possess robust rhythmic gene expression with up to 20% and 8% of their transcriptome oscillating, respectively. The phase and identity of rhythmic genes was however species-dependent. Interestingly, we found Rac1 to be the only TcdB target rhythmically expressed in both mouse enteroids and bHIEs. Further, Rac1 expression was anti-phasic between mouse and human samples. In this thesis we have characterized intestinal circadian clock ontogeny using novel 3D in vitro intestinal models. We utilized the characterization to show for the first time that a functional clock is required for a circadian phase-dependent response to Clostridium difficile toxin B. Rhythmic Rac1 expression was anti-phasic between mouse and human enteroids which correlated with their anti-phasic necrotic cell death response to TcdB. These findings underscore the use of the human intestinal organoids/enteroids for understanding fundamental human intestinal circadian biology and its translatable role in promoting circadian phase-dependent human fitness. ii iii Acknowledgements: Thank you to my wife, Hilary. Your support for me throughout my time in graduate school has been ceaseless. During the many peaks and troughs of grad school you were always there to keep me pushing towards this moment. I may have been able to accomplish this goal without your support but luckily didn’t have to nor would I have wanted to. I’m excited to start my “big kid” life with you. Thank you to my family, immediate, extended and non-blood/law. Doing my doctorate at UC allowed me to be close to you all while working towards this accomplishment. Although I don’t have a control to compare against, I have highly valued your proximity while working towards this goal. The respite and memories made during this past 5-years are something I will always be grateful for. Thank you, Chris Hong. As my advisor you always challenged me to think critically and independently. Your high expectations shaped an excellent, valuable training environment, and I am a better scientist for it. Thank you, Toru Matsu-ura. This work would not have been possible without you. I will forever be grateful for your patience and diligence while training me in the many cell biology tasks I didn’t know at the beginning of my grad career. Thank you to all my committee members, Sean Moore, Alison Weiss, Jim Wells and Yana Zavros. From my qualifying exam to final committee meeting, each member showed a great amount of interest and support for my work. Your feedback was crucial in helping me create the project detailed in the following chapters. iv Thank you to the following lab’s and lab members for your assistance, both scientific and technical. The lab of Chris Hong – University of Cincinnati: Miri Park Suengwon Lee Krithika Ramasamy-Subramanian Kaoru Matsu-ura Mokryun Baek The lab of Jim Wells – Cincinnati Children’s Hospital Medical Center: Taylor Broda Heather McCauley Lauren Haines Jorge Munera The lab of Michael Helmrath – Cincinnati Children’s Hospital Medical Center: Nambirajan Sundaram Jennifer Hawkins The lab of Alison Weiss – University of Cincinnati Suman Pradhan The lab of John Hogenesch – Cincinnati Children’s Hospital Medical Center: Lauren Francey Gang Wu v Danilo Flores Robert Schmidt The lab of Yana Zavros – University of Cincinnati Jayati Chakrabati The lab of Garret FitzGerald – University of Pennsylvania Guangrui Yang The lab of Noah Shroyer – Baylor University University of Cincinnati Live Microscopy Core Chet Closson vi Table of Contents: Abstract………………………………………………………………………………………………….…ii Acknowledgements………………………………………………………………………………………iv Figures/Tables...………………………………………………………………………………………..…x Abbreviations……………………………………………….…………………………………………….xi Chapter 1: Introduction…………………………………………………………………………………..1 1.1 Conservation of the molecular clock across eukaryotes……………………………………1 1.2 Circadian misalignment due to modern lifestyle and its implications in disease pathogenesis………………………………………………………………………………….…6 1.3 The mammalian central and peripheral circadian clocks: same clock different story...…7 1.4 Human intestinal organoids and enteroids as models for extending our understanding of the endogenous clock of the intestine...…………………………………………...………..12 1.5 General introduction to Clostridium difficile pathogenesis………………………...……...17 1.5.1 Spore formation and transmission…………………………………………………...17 1.5.2 The Clostridium difficile pathogenicity locus………………………………...…...…18 1.5.3 Toxin B cell receptors…………………………………………………………………18 1.5.4 Mechanism of action for TcdA-/TcdB-mediated cell death………………………..20 Chapter 2: Ontogeny of the intestinal circadian clock……………………………………………….25 2.1 Abstract……....……………………………………………………………………………..….25 2.2 Introduction……………...……………………………………………………………….....….25 2.3 Methods…………………………………………………………………………………..…….31 2.3.1 HIO generation………………………………………………………………………...31 2.3.2 Generation and maintenance of kidney capsule-matured- and biopsy derived- human intestinal enteroids…………………………………….................................32 2.3.3 Bmal1-luciferase lentiviral transduction and monitoring of Bmal1 activity……….33 vii 2.3.4 Timecourse sample collection………………………………………………………..34 2.3.5 Statistical analysis……………………………………………………………………..37 2.3.6 Tables for reagents and primers……………………………………..………………37 2.4 Results………………………………………………………………………………………….40 2.4.1 Bmal1-luc rhythms are not robust in HIOs………………….……………………....40 2.4.2 In vivo maturation of HIOs prompts GI circadian clock development…………....43 2.5 Conclusions and discussion………………………………………………………………….44 Chapter 3: The intestinal circadian clock regulates a phase-dependent response to toxin B from Clostridium difficile………………………………………………………………………….………...…46 3.1 Abstract…………………………………………………………………………………………46 3.2 Introduction………………………………………………………………………………….…47 3.3 Methods………………………………………………………….……………………………..50 3.3.1 Animals……………….………………………………………………………………...50 3.3.2 Isolation and maintenance of mouse enteroids…….………………………………51 3.3.3 Experiment design to test for a circadian phase-dependent response to TcdB...52 3.3.4 RNA-sequencing analysis…………………………………………………………….54 3.4 Results……………………………………………………………………………………….…56 3.4.1 Intestinal 3D cultures with a functional clock have a circadian phase-dependent response to C. diff Toxin B……..……………………………………………………..56 3.4.2 Mouse and human enteroids possess transcriptome wide rhythmic gene expression and out-of-phase expression of core clock genes and Rac1…………62 3.5 Conclusions and discussion………………………………………………………………….65 Chapter 4: General discussion and future directions…………………………………………………68 4.1 Identification of circulating circadian signals that establish circadian clock function in the developing intestine………………………………………………………………………...…69 4.1.1 In vitro activation of the HIO circadian clock….……………………………………...69 viii 4.1.2 Evaluation of host circadian input on clock activation in transplanted HIOs …....71 4.2 Characterization of intestinal clock aging and its influence on the response to Clostridium difficile toxin B…………..………………………………………………………………….…..75 4.3 Summary……………………………………………………………………………………….78 References……………………………………………………………………………………………….82 Appendix…………………………………………………………………………………………………94 ix Figures/Tables: Figure 2.1 Robust circadian clock activity is not present throughout PSC differentiation into HIOs. Figure 2.2 HIOs have low, non-circadian, expression of core clock genes. Figure 2.3 Overview of the source and method to establish in vitro 3D intestinal organoids and enteroids. Figure 2.4 HIO transplantation activates the circadian clock. Figure 2.5 A fully functional TTFL is not present in kcHIEs. Table 2.1 Key resources table. Table 2.2 List of primers. Figure 3.1 Bi-directional interactions between the circadian clock, microbiota and immune system. Figure 3.2 TcdB phase-response experiment design. Figure 3.3 Synchronization of PER2::LUCIFERASE mouse enteroids 12-hours apart drives
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