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Development of Dual Reconstituted Humanized Mice for Studies of HIV-1 Neuropathogenesis

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Development of Dual Reconstituted Humanized Mice for Studies of HIV-1 Neuropathogenesis University of Nebraska Medical Center DigitalCommons@UNMC Theses & Dissertations Graduate Studies Spring 5-6-2017 Development of Dual Reconstituted Humanized Mice for Studies of HIV-1 Neuropathogenesis Weizhe Li University of Nebraska Medical Center Follow this and additional works at: https://digitalcommons.unmc.edu/etd Recommended Citation Li, Weizhe, "Development of Dual Reconstituted Humanized Mice for Studies of HIV-1 Neuropathogenesis" (2017). Theses & Dissertations. 182. https://digitalcommons.unmc.edu/etd/182 This Dissertation is brought to you for free and open access by the Graduate Studies at DigitalCommons@UNMC. It has been accepted for inclusion in Theses & Dissertations by an authorized administrator of DigitalCommons@UNMC. For more information, please contact [email protected]. Development of Dual Reconstituted Humanized Mice for Studies of HIV-1 Neuropathogenesis by Weizhe Li A DISSERTATION Presented to the Faculty of The Graduate College in the University of Nebraska In Partial Fulfillment of the Requirements For the Degree of Doctor of Philosophy Pharmacology and Experimental Neuroscience Under the Supervision of Professors Larisa Y. Poluektova and Howard E. Gendelman University of Nebraska Medical Center Omaha, Nebraska March, 2017 i TABLE OF CONTENTS TABLE OF CONTENTS ......................................................................................... i LIST OF FIGURES ............................................................................................... vi LIST OF TABLES ................................................................................................. vii ACKNOWLEDGEMENTS .................................................................................... viii ABSTRACT .......................................................................................................... xi LIST OF ABBREVATIONS .................................................................................. xiii CHAPTER - 1: Introduction ................................................................................... 1 1.1. HIV-associated neurocognitive disorders (HAND) and the neuropathogenesis of HIV-1 infection ................................................................... 2 1.1.1. Immune activation and neuroinflammation .............................................. 3 1.1.2. Neurotoxic viral products .......................................................................... 5 1.2. Brain microenvironment under homeostatic conditions ................................. 6 1.3. Brain microenvironment altered by HIV-1 and other infectious agents and endogenous damage ............................................................................................. 7 1.4. Adjunctive strategies for restoration of brain homeostasis for HIV infection of the brain .............................................................................................................. 11 1.4.1. Anti-inflammatory therapies ................................................................... 12 1.4.2. Combination adjunctive and antiretroviral strategies ............................. 16 ii 1.4.3. Cell-based gene and immune modulatory therapies .............................. 18 CHAPTER - 2: Animal Models for NeuroAIDS .................................................... 37 2.1. Introduction ................................................................................................... 38 2.2. Brain cells critical for HIV neuropathogenesis .............................................. 39 2.2.1. Brain macrophages and microglia .......................................................... 39 2.2.3. Astrocytes .............................................................................................. 41 2.3. Non-human primate models ......................................................................... 42 2.4. HIV transgenic rodent models ...................................................................... 43 2.5. Humanized models ....................................................................................... 47 2.5.1. The HIV encephalitis mouse model ....................................................... 47 2.5.2. Human immune system reconstituted models ....................................... 49 2.6. Humanization of mouse brain and future direction ....................................... 51 CHAPTER - 3: Combinatorial Brain and Blood Humanized Mouse .................... 55 3.1. Abstract ........................................................................................................ 56 3.2. Introduction ................................................................................................... 56 3.3. Results ......................................................................................................... 58 3.3.1. Human brain cell transplantation ............................................................ 58 3.3.2. Peripheral hematolymphoid humanization ............................................. 60 3.3.3. Transcriptional changes in the humanized mouse brain ........................ 60 iii 3.3.4. Region-specific alterations of mouse transcriptom by engrafted human glial cells ........................................................................................................... 61 3.4. Discussion .................................................................................................... 62 3.5. Materials and Methods ................................................................................. 79 3.5.1. Animals, cell transplantation .................................................................. 79 3.5.2. Neural progenitors isolation and culture ................................................. 79 3.5.3. Human hematopoietic stem cell isolation ............................................... 80 3.5.4. Flow cytometry ....................................................................................... 81 3.5.5. Immunohistochemistry ........................................................................... 81 3.5.6. Immunofluorescence .............................................................................. 83 3.5.7. RNA isolation and next generation sequencing ..................................... 84 3.5.8. Statistical analysis .................................................................................. 85 CHAPTER - 4: HIV-1 Infection and Glial Innate Immune Response In the Combinatorial Humanized Mouse Model ............................................................ 86 4.1. Abstract ........................................................................................................ 87 4.2. Introduction ................................................................................................... 88 4.3. Results ......................................................................................................... 89 4.3.1. HIV-1 infection of dual humanized mice ................................................. 89 4.3.2. Transcriptional changes in the humanized mouse brain ........................ 90 4.3.3. Region-specific alterations of mouse transcriptom by HIV-1 infection in combinatorial humanized mice ......................................................................... 92 iv 4.3.4. Non-overlapping alterations in the human and mouse transcriptomes following viral infection ..................................................................................... 92 4.3.5. Human astrocytes produce type 1 IFN during infected macrophage- astrocyte co-cultivation ..................................................................................... 94 4.3.6. RT-PCR confirmation of affected human genes in humanized mouse brain tissues ................................................................................................... 95 4.4. Discussion .................................................................................................... 96 4.5. Materials and Methods ................................................................................ 124 4.5.1. Animals, cell transplantation, and HIV-1 infection ................................ 124 4.5.2. Flow cytometry ..................................................................................... 124 4.5.3. Viral load in plasma, brain, and spleen ................................................ 125 4.5.4. Immunohistochemistry ......................................................................... 126 4.5.5. Immunofluorescence ............................................................................ 127 4.5.6. RNA isolation and next generation sequencing ................................... 128 4.5.7. Macrophage and human fetal astrocyte co-culture .............................. 130 4.5.8. IFN-β enzyme-linked immunosorbent assay (ELISA) .......................... 131 4.5.9. RT-PCR for selected human and mouse transcripts ............................ 131 CHAPTER - 5: Summary and Future Directions ............................................... 133 5.1. Summary .................................................................................................... 134 5.2. Further needed refinements of the dual humanized mice .......................... 136 v 5.3. Applications of dual humanized mice model in development of diagnostics and therapeutics for NeuroAIDS and infections of other pathogens ................. 138 References ........................................................................................................ 140 vi LIST OF FIGURES Figure 1.1.
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