Elucidating the Role of HDAC8 in Anthrax Lethal Toxin-Induced Pyroptosis and Cytokine Gene Silencing in Macrophages

ii ABBREVIATIONS . iii CO-AUTHORSHIP STATEMENT . v ACKNOWLEDGEMENTS . vi TABLE OF CONTENTS . vii LIST OF TABLES . xi LIST OF FIGURES . xii CHAPTER 1 . 1 1 INTRODUCTION . 1 1.1 Anthrax . 1 1.1.1 Types of Anthrax . 1 1.1.2 Treatment and Prevention . 2 1.2 B. anthracis Virulence Factors . 3 1.2.1 LeTx . 3 1.3 MAPK Signaling Cascade . 4 1.3.1 ERK1/2 . 5 1.3.2 JNKs . 5 1.3.3 p38 . 6 1.4 TLR signaling . 6 1.5 LeTx and Immune Suppression . 7 1.6 Pyroptosis . 11 1.6. 1 NLRs and Pyroptosis . 11 1.6. 2 LeTx and Pyroptosis . 12 1.6. 3 Mitochondrial Death Genes . 13 viii 1.7 Cellular Adaptation . 15 1.8 Epigenetics . 19 1.8. 1 Histone Modifications and Gene Expression . 19 1.9 Rationale, Hypothesis, and Objectives . 26 CHAPTER 2 . 27 2 MATERIALS AND METHODS . 27 2.1 Cell culture and reagents . 27 2.1.1 Stable isotope labeling by amino acids in cell culture . 28 2.1.2 Inhibitor treatment . 29 2.2 Cell transfection . 30 2.3 Measurement of cell viability . 30 2.4 Lysate preparation and Western blot analysis . 31 2.5 Histone purification . 32 2.6 Digestion of histone H3 . 32 2.7 Selected reaction monitoring mass spectrometry . 33 2.8 Chromatin immunoprecipitation . 34 2.9 Quantitative PCR . 35 2.10 Enzyme-linked immunosorbent assay . 36 2.11 Statistical analysis . 36 CHAPTER 3 . 39 3 RESULTS . 39 3.1 Generating TIR macrophages. 39 3.1.1 TIR macrophages are resistant to LeTx-induced pyroptosis. 39 3.2 TIR is mediated in an HDAC8-dependent manner. 42 3.2.1 Increased HDAC8 expression is correlated with TIR. 42 3.2.2 Inhibiting HDAC8 expression sensitizes TIR macrophages to LeTx. 47 ix 3.2.3 Knocking-down HDAC8 increases expression of the mitochondrial death genes in TIR-BMDMs. 50 3.3 HDAC8-dependent deacetylation at histone H3 lysine 27 (H3K27) mediates TIR. 53 3.3.1 H3K27Ac is reduced in TIR macrophages. 53 3.3.2 HDAC8-dependent H3K27 deacetylation is involved in RAW 264.7 TIR macrophages. 68 3.3.3 HDAC8-dependent H3K27 deacetylation is involved in the downregulation of the mitochondrial death genes in RAW 264.7 TIR macrophages. 73 3.3.4 HDAC8 limits RNA polymerase II accessibility to the mitochondrial death gene promoters. 76 3.4 LeTx suppresses cytokine production in an HDAC8-dependent manner. 81 3.4.1 LeTx-treated RAW 264.7 macrophages are defective in producing IL-1β in an HDAC8-dependent manner in response to LPS. 81 3.4.2 Inhibiting HDAC8 restores the expression and production of IL-1β and TNF-α in LeTx-treated macrophages. 84 3.4.3 Inhibiting HDAC8 in LeTx-treated macrophages increases H3K27Ac and Pol II association in the genomic regions of IL-1β in response to LPS. 91 CHAPTER 4 . 95 4 DISCUSSION . 95 4.1 Resistance to LeTx-induced pyroptosis is HDAC8-mediated . 95 4.2 TIR is mediated by an HDAC8-H3K27-dependent epigenetic mechanism . 96 4.3 LeTx-induced cytokine suppression is mediated by an HDAC8-H3K27-dependent epigenetic mechanism . 103 4.4 Future Studies . 105 4.4.1 Examining the mechanism of LeTx-mediated HDAC8 up-regulation . 105 4.4.2 Examining LeTx-mediated H3K27 methylation . 106 4.4.3 Examining H3K9Ac and H3K14Ac in IL-1β and mitochondrial death gene expression . 107