University of Pennsylvania ScholarlyCommons Dental Theses Penn Dental Medicine 8-4-2017 P2X7 Receptor Primes IL-1β and the NLRP3 Inflammasome in Astrocytes Subjected to Mechanical Strain Farraj S. Albalawi University of Pennsylvania, [email protected] Wennan Lu Jonarhan M. Beckel Jason C. Lim Stuart A. McCaughey See next page for additional authors Follow this and additional works at: https://repository.upenn.edu/dental_theses Part of the Dentistry Commons Recommended Citation Albalawi, Farraj S.; Lu, Wennan; Beckel, Jonarhan M.; Lim, Jason C.; McCaughey, Stuart A.; and Mitchell, Claire H., "P2X7 Receptor Primes IL-1β and the NLRP3 Inflammasome in Astrocytes Subjected to Mechanical Strain" (2017). Dental Theses. 25. https://repository.upenn.edu/dental_theses/25 This paper is posted at ScholarlyCommons. https://repository.upenn.edu/dental_theses/25 For more information, please contact [email protected]. P2X7 Receptor Primes IL-1β and the NLRP3 Inflammasome in Astrocytes Subjected to Mechanical Strain Abstract Inflammatory responses play a key role in many neural pathologies, with localized signaling from non- immune cells making critical contributions. The NLRP3 inflammasome is an important component of innate immune signaling and can link neural insult to chronic inflammation. Stimulation of the NLRP3 inflammasome is a two-stage process. The priming stage involves upregulation of the biosynthesis of the structural components while activation results in their assembly into the actual inflammasome complex and subsequent activation. The priming step can be rate limiting and can connect insult to chronic inflammation but our knowledge of the signals that regulate NLRP3 inflammasome priming in sterile inflammatory conditions is limited. This study examined the link between mechanical strain and inflammasome priming in neural systems. Transient non-ischemic elevation of intraocular pressure (IOP) increased mRNA for inflammasome components IL-1β, NLRP3, ASC, CASP1 and IL-6 in rat and mouse retinas. The P2X7 receptor was implicated in the in vivo mechanosensitive priming of IL-1β and IL-6 transcription and translation. In vitro experiments with optic nerve head astrocytes demonstrated enhanced expression of the IL-1β and IL-6 genes following stretching or swelling. The increase in IL-1β expression was inhibited by degradation of extracellular ATP with apyrase, blocking pannexin hemichannels with carbenoxolone, probenecid or 10Panx1 peptide, P2X7 receptor antagonists (BBG, A839977 or A740003) as well inhibition of the NFκB transcription factor with Bay 11-7082. The swelling- dependent fall in expression of the NFκB inhibitor IκB-α was reduced by treatment of cells with A839977 and in P2X7 knockout mice. In summary, our data suggest that mechanical trauma to the retina results in priming of the NLRP3 inflammasome components and upregulated IL-6 expression and release. This was dependent upon ATP release through pannexin hemichannels and autostimulation of the P2X7 receptor. Since the P2X7 receptor can also trigger inflammasome activation it appears to have a central role in linking mechanical strain to neuroinflammation. Degree Type Dissertation Degree Name DScD (Doctor of Science in Dentistry) Primary Advisor Dr. Claire Mitchell Keywords IL-1β, astrocytes, glaucoma, pannexin, ATP release, NFκB, IL-18, caspase 1, NLRP3 Subject Categories Dentistry Author Farraj S. Albalawi, Wennan Lu, Jonarhan M. Beckel, Jason C. Lim, Stuart A. McCaughey, and Claire H. Mitchell This dissertation is available at ScholarlyCommons: https://repository.upenn.edu/dental_theses/25 P2X7 receptor primes IL-1β and the NLRP3 inflammasome in astrocytes subjected to mechanical strain Farraj Albalawi A Dissertation Presented to the Faculties of the University of Pennsylvania in Partial Fulfillment of the Requirements for the Degree of Doctor of Science in Dentistry 2017 Supervisor of Dissertation ________________________ Claire H. Mitchell, Ph.D. Professor of Anatomy and Cell Biology Dissertation Committee ________________________ Chair: Jonathan Korostoff, DMD, Ph.D. Professor of Periodontics and Director of Master of Science in Oral Biology Program Member: Kelly Jordan-Sciutto, Ph.D. Chair and Professor, Department of Pathology Member: Kenneth S. Shindler, MD, Ph.D. Associate Professor of Ophthalmology and Neurology P2X7 receptor primes IL-1β and the NLRP3 inflammasome in astrocytes subjected to mechanical strain COPYRIGHT 2017 Farraj Albalawi ACKNOWLEDGMENT First and foremost, I would like to thank my mentor, Dr. Mitchell, for all her help throughout this journey. Without her, this project would not have been possible. Coming from a clinical background where I am used to answering the question, she has taught me how to ask the question and think like a scientist. I would like to thank my committee members, Dr. Korostoff, Dr. Jordan-Sciutto and Dr. Shindler for taking the time to meet with me and guiding me. Your questions and comments encouraged me to think critically and have inspired me to pursue this research in the dental field. I would also like to thank Dr. Graves and Dr. Jordan-Sciutto who granted me acceptance into the DSCD program. I would like to thank all of the orthodontic department especially Dr. Chung, Dr. Vanarsdall, Dr. Greco, Dr. Coby and Dr. Guan for all their support and guidance. I would like to thank Dr. Mitchell’s lab members, past and present, for being patient and for offering your help along every step of the way. Last but not least I would like to thank my family and friends, I couldn’t have done this without you. iii ABSTRACT P2X7 receptor primes IL-1β and the NLRP3 inflammasome in astrocytes subjected to mechanical strain Farraj Albalawi Claire H. Mitchell, Ph.D. Inflammatory responses play a key role in many neural pathologies, with localized signaling from non-immune cells making critical contributions. The NLRP3 inflammasome is an important component of innate immune signaling and can link neural insult to chronic inflammation. Stimulation of the NLRP3 inflammasome is a two-stage process. The priming stage involves upregulation of the biosynthesis of the structural components while activation results in their assembly into the actual inflammasome complex and subsequent activation. The priming step can be rate limiting and can connect insult to chronic inflammation but our knowledge of the signals that regulate NLRP3 inflammasome priming in sterile inflammatory conditions is limited. This study examined the link between mechanical strain and inflammasome priming in neural systems. Transient non- ischemic elevation of intraocular pressure (IOP) increased mRNA for inflammasome components IL-1β, NLRP3, ASC, CASP1 and IL-6 in rat and iv mouse retinas. The P2X7 receptor was implicated in the in vivo mechanosensitive priming of IL-1β and IL-6 transcription and translation. In vitro experiments with optic nerve head astrocytes demonstrated enhanced expression of the IL-1β and IL-6 genes following stretching or swelling. The increase in IL-1β expression was inhibited by degradation of extracellular ATP with apyrase, blocking pannexin hemichannels with carbenoxolone, probenecid or 10Panx1 peptide, P2X7 receptor antagonists (BBG, A839977 or A740003) as well inhibition of the NFκB transcription factor with Bay 11-7082. The swelling- dependent fall in expression of the NFκB inhibitor IκB-α was reduced by treatment of cells with A839977 and in P2X7 knockout mice. In summary, our data suggest that mechanical trauma to the retina results in priming of the NLRP3 inflammasome components and upregulated IL-6 expression and release. This was dependent upon ATP release through pannexin hemichannels and autostimulation of the P2X7 receptor. Since the P2X7 receptor can also trigger inflammasome activation it appears to have a central role in linking mechanical strain to neuroinflammation. v TABLE OF CONTENTS ACKNOWLEDGMENT .............................................................................................. iii ABSTRACT .............................................................................................................. iv TABLE OF CONTENTS .............................................................................................. vi LIST OF TABLES ....................................................................................................... ix LIST OF ILLUSTRATIONS ........................................................................................... x LIST OF ABBREVIATIONS ......................................................................................... xi Chapter 1 : Introduction........................................................................................... 1 Inflammasome structure: ..............................................................................................4 Inflammasome priming and activation: .........................................................................7 Mechanical strain and inflammation: .......................................................................... 12 Purinergic signaling: .................................................................................................... 15 Hypothesis .................................................................................................................. 21 Chapter 2 : The P2X7 receptor primes IL-1β and the NLRP3 inflammasome in astrocytes exposed to mechanical strain ................................................................ 23 Introduction ...............................................................................................................