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UCSF UC San Francisco Electronic Theses and Dissertations Title Immune regulation by the endocannabinoid N-arachidonoyl dopamine (NADA) Permalink https://escholarship.org/uc/item/7wj3d52v Author Lawton, Samira Khakpour Publication Date 2017 Peer reviewed|Thesis/dissertation eScholarship.org Powered by the California Digital Library University of California Immune regulation by the endocannabinoid rV-ar: dopamine by DISSERTATION Submitted in partial satisfaction of the requirements for the degree of Copyright 2017 by Samira Khakpour Lawton ii To Kipp Lawton, my best friend iii ACKNOWLEDGEMENTS First, I would like to thank my graduate advisor, Judith Hellman, for her unconditional support and scientific guidance. Through creativity and determination, we set forth into new frontiers, and I am a better scientist for it. She taught me to follow my heart and never fear the unknown. Judith allowed me to be independent while never feeling alone. I am deeply grateful to have had the chance to experience graduate school with her by my side and will miss her immensely. I also truly appreciate having been able to do science with the rest of the Hellman lab. Fengyun Xu and Erika Wong spent uncountable hours repeating complex experiments, and Alphonso Tran sacrificed endless nights and weekends to help move this project along. I sincerely could not have done this without all of their help and support. Finally, I greatly appreciate my undergraduate research advisor, Will DePaolo, who gave me the foundation in research that has been crucial to my success at UCSF. I would also like to thank my thesis committee, Matt Springer, Shaun Coughlin, and Mehrdad Matloubian, for their consistent guidance and inspiration. Their perspectives have been critical for helping this project transform from an idea into impactful science. Thank you to everyone in the Biomedical Sciences program, especially Lisa Magargal, Demian Sainz, and my academic advisor, Mark Ansel. They have always been there for me with open arms when I needed them, and even when I did not. They made the decision to come to UCSF such an easy one, and I thank them for everything that they have done for me. iv I would like to thank my classmates and friends who have been my family throughout these five years. You have made moving across the country such a seamless transition, and have been there through all of the good and bad. Thank you Brittany Gianetti, Arshiya Fazal, Olga Karagoz, Anju Goyal, Jeewon Lee, Sarah Moshary, and so many others. Thank you Juliet Goldsmith, who never failed to show up on my doorstep with food and wine, thus creating unrealistic expectations for all future friendships. Thank you to my family for their unbelievable love and support through all these years, especially my parents, Bagher and Soraya. They have always encouraged my curiosity and pretended to understand what I was talking about. I am who I am because of them. Thank you to my in-laws, Cathy and Patrick, who opened up their hearts to me when I was only twelve years old and have been family ever since. They have done everything in their power to help and support me, even when I hauled their youngest son 2000 miles away. Thank you to my now official siblings, Erin and Brendan. Not everyone is quite as lucky to inherit such a loving and fun family. And finally, I would like to thank Kipp Lawton, for putting up with me and believing in me through all of this. It is so difficult to find the right way to thank someone who has given me unrelenting and unconditional support for over twelve years. None of this would have been possible without him. While research could be lonely, he showed me that home never had to be. I strive to become the person that he believes me to be. v Contributions to presented work The work in this dissertation was done under the direct supervision and guidance of Dr. Judith Hellman. Chapters 1 and 2 contain material previously submitted for publication: S Khakpour, K Wilhelmsen, and J Hellman. Vascular endothelial cell Toll-like receptor pathways in sepsis. Innate Immunity. 2015 Nov;21(8):827-46. K Wilhelmsen, S Khakpour, A Tran, K Sheehan, M Schumacher, F Xu, and J Hellman. The endocannabinoid/endovanilloid N-arachidonoyl dopamine (NADA) and synthetic cannabinoid WIN55,212-2 abate the inflammatory activation of human endothelial cells. Journal of Biological Chemistry. 2014 May 9;289(19):13079- 100. Epub 2014 Mar 18. Chapter 2 data was collected in collaboration with Kevin Wilhelmsen, who performed qPCR, western blot, neutrophil adhesion, and COMT assays, and helped perform in vitro TLR agonist challenge experiments. Alphonso Tran provided assistance with ELISAs. All other experiments were performed by Samira Khakpour Lawton. For data presented in Chapter 3, Fengyun Xu and Alphonso Tran conducted TLR agonist challenge experiments. Erika Wong and Fengyun Xu performed CLP surgeries. Alphonso Tran and Erika Wong helped with the collection and processing of murine plasma samples and assisted with ELISAs. Kevin Wilhelmsen and Arun Prakash helped perform multiplex experiments. All other experiments were performed by Samira Khakpour Lawton. For data presented in Chapter 4, Fengyun Xu performed in vivo TLR vi agonist challenge experiments. The Roth Laboratory at the University of North Carolina Chapel Hill performed the GPCR-ome screening assay. All other experiments were performed by Samira Khakpour Lawton. I would like to acknowledge the following funding sources: UCSF Department of Anesthesia and Perioperative Care, UCSF Biomedical Sciences Graduate Program, NSF Graduate Research Fellowship Program (1144247), and the UCSF REAC Program (Huntington Fund). vii Immune regulation by the endocannabinoid N-arachidonoyl dopamine (NADA) Samira Khakpour Lawton ABSTRACT Arachidonic acid-based lipids, or eicosanoids, are well-known regulators of the inflammatory response. While there are additional classes of eicosanoids that have also been shown to possess immunomodulatory activity, such as the cannabinoids, they remain largely unstudied. We have found that the endocannabinoid N-arachidonoyl dopamine (NADA) decreases inflammatory responses in vitro in endothelial cells after challenge with TLR agonists, and that this activity is dependent upon the cannabinoid receptors (CB1R and CB2R). Furthermore, NADA dramatically reduces systemic inflammation in murine models of bacterial lipopeptide, endotoxemia, and polymicrobial intraabdominal sepsis. Consistent with its classification as a potent endovanilloid, the activity of NADA in vivo depends upon the cation channel transient receptor potential vanilloid 1 (TRPV1) expressed in non-hematopoietic cells and may be mediated through neuronal TRPV1. Furthermore, the activity of NADA systemically, in endothelial cells, and in the hematopoietic compartment is dependent upon CB2R. Finally, we show that the immunomodulatory activity of NADA is mediated in part by its ability to regulate prostanoid production, and its metabolism into the novel lipid prostaglandin D2- dopamine. These data indicate that NADA can modulate inflammatory activation as viii both an endocannabinoid and endovanilloid, and suggests that these pathways may be targeted therapeutically for the treatment of acute inflammatory disorders, such as sepsis. ix TABLE OF CONTENTS Chapter 1: Background .................................................................................................... 1 Chapter 2: The endocannabinoid/endovanilloid N-arachidonoyl dopamine (NADA) and synthetic cannabinoid WIN55,212-2 abate the inflammatory activation of human endothelial cells ......................................................................................................... 7 Chapter 3: N-arachidonoyl dopamine (NADA) modulates acute systemic inflammation via non-hematopoietic TRPV1 ................................................................................. 80 Chapter 4: N-arachidonoyl dopamine (NADA) affects inflammation by regulating endothelial and systemic prostanoid metabolism .................................................. 126 Chapter 5: Conclusions ................................................................................................ 177 References ................................................................................................................... 182 x LIST OF TABLES Chapter 2 Table 2.1. Mean ΔCt for cannabinoid receptor qPCR data ............................................ 66 Table 2.2. Mean ΔCt for metabolic enzymes qPCR data ............................................... 67 Table 2.3. Cannabinoid receptor agonists/antagonists used in experiments ................. 68 Table 2.4. Mean inflammatory and viability indices, SEM, and significance for cannabinoid receptor agonists/antagonists ............................................................. 69 Chapter 4 Table 4.1. The effects of endocannabinoids on arachidonic acid metabolic enzymes in endothelial cells ..................................................................................................... 165 Table 4.2. Binding affinity of PGD2-DA to GPCRs ........................................................ 167 Table 4.3. List of qPCR primers used .......................................................................... 170 xi LIST OF FIGURES Chapter 1 1.1: Major outputs after endothelial cell TLR pathways are activated .............................. 5 Chapter 2 2.1: ECs express a similar subset of CBR mRNA transcripts ........................................ 31 2.2: ECs express CBR and TRPV1