The role of integrin αvβ8 on human monocytes and macrophages in intestinal immune homeostasis A thesis submitted to The University of Manchester for the degree of Doctor of Philosophy in the Faculty of Biology, Medicine and Health 2018 Elinor Elizabeth Shuttleworth School of Biological Sciences TABLE OF CONTENTS Chapter 1: Introduction .................................................................................. 15 1.1. Introduction ..................................................................................................... 15 1.2. Intestinal barrier function ............................................................................... 15 1.2.1. Intestinal epithelial layer ............................................................................. 17 1.2.2. PRR function in IECs .................................................................................. 19 1.2.3. Intestinal mucus layer ................................................................................. 20 1.3. Intestinal innate immunity .............................................................................. 20 1.3.1. Monocytes and Macrophages .................................................................... 21 1.3.2. Dendritic cells ............................................................................................. 35 1.4. Intestinal adaptive immunity .......................................................................... 41 1.4.1. Intestinal T cells .......................................................................................... 42 1.4.2. Intestinal B cells .......................................................................................... 46 1.4.3. Homing and localisation of intestinal T and B cells .................................... 47 1.5. Control of TGF-β mediated intestinal immune tolerance ............................ 48 1.5.1. The role of αvβ8 mediated TGF-β activation in immune homeostasis ....... 53 Chapter 2 Materials and Methods .................................................................. 58 2.1. Quantifying expression of integrin αvβ8 on peripheral blood mononuclear cells (PBMCs) ......................................................................................................... 58 2.1.1. Peripheral blood collection ......................................................................... 58 2.1.2. PBMC isolation ........................................................................................... 58 2.1.3. PBMC staining for flow cytometry ............................................................... 58 2.1.4. β8 binding antibody-fluorochrome conjugation ........................................... 60 2.1.5. CD14+ monocyte isolation ......................................................................... 61 2.1.6. RNA synthesis for ITGB8 qPCR ................................................................. 61 2.1.7. cDNA synthesis for ITGB8 qPCR ............................................................... 62 2.1.8. ITGB8 qPCR ............................................................................................... 62 2.2. Investigating factors influencing expression of integrin αvβ8 on peripheral blood mononuclear cells (PBMCs) ....................................................................... 63 2.2.1. PBMC and monocyte cytokine treatment ................................................... 63 2.2.2. PBMC and monocyte TLR ligand treatment ............................................... 63 2.3. Investigating function of αvβ8 on peripheral blood mononuclear cells (PBMCs) .................................................................................................................. 64 2.3.1. TGFβ activation reporter cell co-culture assay ........................................... 64 2.3.2. PBMC and monocyte treatment with anti-TGF-β antibody or anti-αvβ8 antibody ................................................................................................................ 65 2.3.3. Flow cytometry to assess treated CD14+ monocyte phenotype ................ 65 2.4. Quantifying expression and function of integrin αvβ8 on MDM ................. 65 2.4.1. MDM culture from CD14+ monocytes ........................................................ 65 2.4.2. MDM staining for flow cytometry ................................................................ 66 2.4.3. IL-10 ELISA ................................................................................................ 66 2.4.4. Phagocytosis assay .................................................................................... 66 2.4.5. MDM treatment with neutralising antibodies or TLR ligands ...................... 67 2.5. Bioenergetic analysis of MDMs ..................................................................... 67 2.5.1. MDM culture for bioenergetic analysis ....................................................... 67 2.5.2. MDM treatment for bioenergetic analysis ................................................... 68 2.5.3. Bioenergetic analysis using the Seahorse XF96 analyser ......................... 68 2.5.3. Protein quantification using the Bicinchoninic Acid (BCA) assay ............... 69 2.5.4. Seahorse data analysis .............................................................................. 69 2.6. General data analysis ..................................................................................... 70 Chapter 3: Integrin αvβ8 mediated TGF-β activation in human monocytes .......................................................................................................................... 71 2 3.1. Introduction ..................................................................................................... 71 3.2. Results ............................................................................................................. 71 3.2.1. Flow cytometry gating protocols to identify integrin αvβ8 on human PBMC .............................................................................................................................. 71 3.2.2. Expression of integrin αvβ8 on peripheral leucocyte populations .............. 74 3.2.3. Validation of results utilising other integrin β8 staining strategies .............. 77 3.2.4. qPCR analysis of PBMC ITGB8 expression .............................................. 82 3.2.5. Effects of cytokine treatment on PBMC integrin αvβ8 expression ............. 84 3.2.6. Effects of TLR ligand treatment on PBMC integrin αvβ8 expression ........ 89 3.2.7. Integrin αvβ8-dependent TGF-β activation by monocytes .......................... 90 3.2.8. Alterations in CD16 expression in TGF-β, TGF-β blocking and integrin αvβ8 blocking antibody-treated monocytes ................................................................... 94 3.3. Discussion ....................................................................................................... 98 Chapter 4: Integrin αvβ8 mediated TGF-β activation in human macrophages ................................................................................................. 105 4.1. Introduction ................................................................................................... 105 4.2. Results ........................................................................................................... 105 4.2.1. Characterisation of human GM-CSF and M-CSF MDM phenotype ......... 105 4.2.2. Expression of integrin αvβ8 on human MDMs .......................................... 106 4.2.3. Mechanisms governing integrin αvβ8 expression on human MDMs ........ 111 4.2.4. Investigating the role of integrin αvβ8 expression in human MDM phenotype ............................................................................................................................ 112 4.3. Discussion ..................................................................................................... 120 Chapter 5: The role of metabolism in determining macrophage phenotype ........................................................................................................................ 126 5.1. Introduction ................................................................................................... 126 5.2. Results ........................................................................................................... 127 5.2.1 Establishing a protocol for bioenergetics analysis of human MDMs .......... 127 5.2.2. Establishing the bioenergetics profile of human GM-CSF and M-CSF MDMs ................................................................................................................................... 131 5.2.3. Investigating factors which alter the bioenergetics profile of human MDMs ................................................................................................................................... 133 5.3. Discussion ..................................................................................................... 145 Chapter 6. Discussion .................................................................................. 152 6.1. TGF-β activation in inflammation and intestinal immune homeostasis .. 152 6.2 TGF-β in fibrosis ............................................................................................. 154 6.3. TGF-β in cancer ............................................................................................. 156 6.4. Personalised therapy in IBD ......................................................................... 159 References ....................................................................................................