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Developing Caspase-1 Biosensors to Monitor Inflammation in Vitro and in Vivo

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Developing Caspase-1 Biosensors to Monitor Inflammation in Vitro and in Vivo Loyola University Chicago Loyola eCommons Dissertations Theses and Dissertations 2020 Developing Caspase-1 Biosensors to Monitor Inflammation in Vitro and in Vivo Sarah Talley Follow this and additional works at: https://ecommons.luc.edu/luc_diss Part of the Immunology and Infectious Disease Commons Recommended Citation Talley, Sarah, "Developing Caspase-1 Biosensors to Monitor Inflammation in Vitro and in Vivo" (2020). Dissertations. 3827. https://ecommons.luc.edu/luc_diss/3827 This Dissertation is brought to you for free and open access by the Theses and Dissertations at Loyola eCommons. It has been accepted for inclusion in Dissertations by an authorized administrator of Loyola eCommons. For more information, please contact [email protected]. This work is licensed under a Creative Commons Attribution-Noncommercial-No Derivative Works 3.0 License. Copyright © 2020 Sarah Talley LOYOLA UNIVERSITY CHICAGO DEVELOPING CASPASE-1 BIOSENSORS TO MONITOR INFLAMMATION IN VITRO AND IN VIVO A DISSERTATION SUBMITTED TO THE FACULTY OF THE GRADUATE SCHOOL IN CANDIDACY FOR THE DEGREE OF DOCTOR OF PHILOSOPHY PROGRAM IN INTEGRATIVE CELL BIOLOGY BY SARAH TALLEY CHICAGO, IL AUGUST 2020 TABLE OF CONTENTS LIST OF FIGURES v CHAPTER ONE: INTRODUCTION 1 CHAPTER TWO: REVIEW OF THE LITERATURE 5 Overview 5 Structure of Inflammasomes 6 Function of Inflammasomes 8 NLRP1 8 NLRP3 14 NLRC4 21 AIM2 24 PYRIN 28 Noncanonical Inflammasome Activation and Pyroptosis 31 Inflammatory Caspases 36 Caspase-1 36 Other Inflammatory Caspases 40 Biosensors and Novel Tools to Monitor Inflammation 42 CHAPTER THREE: MONITORING CASPASE-1 BIOSENSOR ACTIVATION IN VITRO 45 Materials and Methods 45 Cell Culture 45 293T Transfections and Luciferase Reporter Assay 45 Generation of Stable Cell Lines 45 Monocyte Derived Macrophages 46 In vitro Inflammasome Activation Assays 46 Salmonella Infection 46 Statistical Analysis 47 Results 47 Caspase-1 Biosensor Design 47 Screen to Identify Putative Biosensors 48 Monitoring Biosensor Activation in THP-1 Cells 49 Monitoring Biosensor Activation in MDMs 52 IQAD biosensors are activated in response to inflammatory and apoptotic stimuli 53 CHAPTER FOUR: GENERATION OF CASPASE-1 REPORTER MICE 55 Materials and Methods 55 Generation of Caspase-1 Reporter Mice 55 In vitro Inflammasome Activation Assays 55 Western Blotting 56 Lactate Dehydrogenase (LDH) Measurements 56 IVIS Imaging 57 Statistical Analysis 57 Results 57 Generation of Caspase-1 Biosensor Transgenic Mice 57 Monitoring Biosensor Activation in BMDMs 59 IQAD Biosensors are Not Activated in Response to Some Inflammatory Stimuli 60 CHAPTER FIVE: MONITORING CASPASE-1 BIOSENSOR ACTIVAITON IN VIVO 64 Materials and Methods 64 S. aureus Culture and Murine Systemic Infection Model 64 DSS-Induced Colitis Model 64 Graft-Versus-Host Disease Model 65 Statistical Analysis 65 Results 66 Staphylococcus aureus Bacterial Infection Model 66 DSS-Induced Colitis Model 72 Graft-Versus-Host Disease Model 79 EPS Treatment Reduces T cell Activation in GVHD Mice 86 CHAPTER SIX: SUMMARY AND DISCUSSION 89 REFERENCE LIST 95 VITA 113 LIST OF FIGURES Figure Page 1. Composition of Canonical and Noncanonical Inflammasomes 7 2. Mechanism of NLRP1 Inflammasome Activation 12 3. Model of NLRP3 Activation on Dispersed trans-Golgi Network 17 4. Schematic of Post-Translation Modifications Regulating NLRP3 Activity 18 5. Schematic of Pyrin Inflammasome Activation. 30 6. Mechanism of Noncanonical Inflammasome Activation and Pyroptosis. 34 7. Caspase-1 Structure, Activation and Inactivation 38 8. Caspase-1 Biosensor Design 48 9. Caspase-1 Biosensors are Activated in 293T Cells 49 10. Caspase-1 Biosensors are Activated in THP-1s 50 11. Caspase-1 Biosensors are Activated in THP-1s in Response to NLRP3 and AIM2 Agonists 50 12. Biosensors are Activated during Bacterial Infection in THP-1 Cells 52 13. Caspase-1 Biosensors are Activated in MDMs 53 14. IQAD Biosensors are Activated in Cells in Response to Inflammatory Stimuli but Secreted from Apoptotic Cells 54 15. Generation of Caspase Reporter Mice 58 16. Biosensor Signal ex vivo IQAD Mice 59 17. Caspase-1 Biosensors are Activated in BMDMs 60 18. Caspase-1 Biosensors are Activated in BMDMs in Response to Some Stimuli 62 19. Caspase-1 Biosensors are Not Activated by Nigericin in BMDMs 63 v 20. Subcutaneous S. aureus Infection and Cytodex-3 Beads Cause Biosensor Activation in the Skin 67 21. Caspase-1 Biosensors are Activated in S. aureus–Infected Mice 69 22. Biosensor Signal, Bacterial Burden and Inflammatory Cytokines and Chemokines Detected in Tissues Isolated from S. aureus-Infected Mice 71 23. Biosensor Signal and IL-1β Correlate with Bacterial CFU in the Kidneys 72 24. Biosensor Signal Correlates with Inflammatory Markers in the Kidneys 73 25. Biosensor Signal and IL-1β do Not Correlate with Bacterial CFU in the Liver and Heart 73 26. Biosensor Signal in the Colons in vivo 74 27. Mice Exhibit Some Clinical Measurements of Colitis on Day 5 and Day 7 75 28. Caspase-1 Biosensors are Activated in Inflamed Areas of the Colon during Colitis – Males 76 29. Caspase-1 Biosensors are Activated in Inflamed Areas of the Colon during Colitis – Females 77 30. Biosensors are Activated in Regions of Caspase-1 Activation and Inflammatory Cytokine / Chemokine Production in the Colon 78 31. Caspase-1 Biosensors are Activated in the Brain during Colitis 79 32. Biosensors are Activated in Alloreactive Donor Cells during GVHD 81 33. Mice Exhibit Clinical Measurements of GVHD 82 34. Increased Inflammatory Cytokines in the Serum of GVHD Mice 82 35. IQAD Biosensor is Activated in Donor T cells in Mice with GVHD 84 36. IQAD Biosensor is Activated in Alloreactive Donor T cells during GVHD 85 37. Caspase-1 Activation in Donor T cells in GVHD Spleens 85 38. Biosensor Activation is Attenuated in EPS-Treated Mice in vivo 87 39. EPS-Treated Mice Exhibit Reduced Biosensor Signal in Tissues and Reduced Clinical Disease 88 vi CHAPTER ONE INTRODUCTION Inflammation is an integral part of our host defense against various pathogens, but when uncontrolled, can drive the pathology of numerous diseases. Many of these conditions are exacerbated with age, and their incidences will only increase as the general population ages and lifespans increase. A key mediator of inflammatory responses is caspase-1. The recognition of pathogen associated molecular patterns (PAMPs) and damage associated molecular patterns (DAMPs) drives the formation of multiprotein complexes termed inflammasomes, which recruit and activate caspase-1. Caspase-1 cleaves numerous downstream targets, including the proinflammatory cytokines proIL-1β and proIL-18, leading to their maturation and release from cells. The processing and release of these cytokines and other inflammatory effectors mediate inflammation and trigger immune responses. Inflammation plays a central role in numerous diseases and novel methods to study the initiation of early inflammatory events and the progression of inflammation over time would provide experimental value. The primary assays used to measure inflammasome activation are the proteolytic cleavage of procaspase-1 to caspase-1 by immunoblot or secretion of IL-1β in the serum by ELISA, both of which have notable limitations. Measuring active caspase-1 in vitro by immunoblot is technically challenging and often only detectable in the supernatant from cells stimulated directly with drugs that potently activate caspase-1. Measuring caspase-1 activation during inflammation in vivo requires tissue removal from sacrificed animals, preventing the dynamic measurement of caspase-1 in individual animals overtime. Furthermore, immunoblot detection of caspase-1 activation is often not sensitive enough to detect modest and/or transient activation of caspase-1 that can drive very consequential levels of inflammation in disease pathogenesis. Other common methods used to detect inflammation involve harvesting tissues for microscopic of qRT-PCR analyses to examine the histopathology or the presence of 1 2 certain inflammatory markers. These techniques also require tissue removal from sacrificed animals. Serum IL-1β levels measured by ELISA can be used as an indirect measurement of inflammasome activation. During systemic inflammation, enough IL-1β can be secreted in the bloodstream to be detected by ELISA, but this is often not the case, especially when inflammation is localized to specific tissues. Even if enough IL-1β is secreted, this assay gives no insight into where the inflammation is coming from. We therefore sought to develop a biosensor of caspase-1 to overcome the technical limitations associated with monitoring inflammasome activation in vivo. Developing biosensors that directly monitor caspase-1 activation in real time would allow for the identification of specific tissues where inflammation is occurring in living animals and the ability to monitor how this inflammation changes in these animals over time. To better understand the role of caspase-1 in various disease settings, we developed a series of bioluminescent sensors that allow us to detect and quantify caspase-1 activation. We employed a circularly permuted form of luciferase in which the N- and C-terminal domains necessary for bioluminescence are physically separated by a linker region. Caspase-1 target sequences were cloned into this linker region, so that upon caspase-1 activation, these sequences are cleaved allowing the two domains of luciferase to come together and luminescence can be quantified. In our initial screen, we identified biosensors that exhibited a significant
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