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The Anatomical Origins of Migratory Dendritic Cells in the Intestine Stephanie Ailsa Houston BSc ... PDF

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Houston, Stephanie Ailsa (2013) The anatomical origins of migratory dendritic cells in the intestine. PhD thesis. http://theses.gla.ac.uk/5265/ Copyright and moral rights for this work are retained by the author A copy can be downloaded for personal non-commercial research or study, without prior permission or charge This work cannot be reproduced or quoted extensively from without first obtaining permission in writing from the author The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the author When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given Enlighten:Theses http://theses.gla.ac.uk/ [email protected] The Anatomical Origins of Migratory Dendritic Cells in the Intestine Stephanie Ailsa Houston BSc, MRes Submitted in fulfilment of the requirement for the degree of Doctor of Philosophy Institute of Infection, Immunity and Inflammation College of Medical Veterinary and Life Sciences University of Glasgow December 2013 Abstract The intestine is exposed to a vast array of harmless dietary antigen as well as an enormous community of commensal bacteria. Alongside this harmless antigen, pathogens can enter the body via the intestinal mucosal surface. The intestinal immune system must discriminate between pathogens and innocuous antigens. Dendritic cells (DCs), present in the small intestine (SI) and colon, are fundamental in controlling intestinal immune responses; they migrate to the mesenteric lymph node (MLN) and prime effector or regulatory T cells. Furthermore, DCs direct the immune response in the gut-associated lymphoid tissues (GALT), the Peyer’s patches (PP) and isolated lymphoid follicles (ILFs). The aim of this work was to determine the anatomical origins of DCs in the MLN. First, the anatomical organisation of lymphatic vessels draining to the MLN from the SI and colon was investigated. Second, DC migration in mice lacking specific GALT was explored. Finally, the migration of DCs from PPs to the MLN was investigated. To achieve these aims, DC migration was studied using a variety of labelling methods, mice that lacked specific GALT were employed, and surgical procedures were used to collect DCs from the thoracic duct. Here, I demonstrate the anatomical segregation of DCs that migrate to the MLN from the SI and colon. This was reflected in differences in DC subset composition and antigen presentation in the SI and colon-draining nodes of the MLN. Separate analysis of MLN nodes will allow a more refined understanding of intestinal immune responses. All but one DC subset, CD103-CD11b- DCs, were still present in pseudo-afferent lymph from mice lacking both PPs and ILFs. This subset is therefore likely to originate from either PPs or ILFs. Surprisingly, CD103+CD8α+ DCs were present in these mice, showing that many CD8α+ DCs were resident in the lamina propria and are not limited to lymphoid tissue. Four DC subsets are able to migrate from the intestine in PP-null mice, suggesting that CD103-CD11b- DCs migrate specifically from ILFs. I then demonstrated that DCs migrate from PPs to the MLN. These migrating PP DCs expanded in response to a DC specific growth factor and their migration depended on CCR7 and S1P. These cells may play an important role in driving immune responses in the MLN and their manipulation could lead to advances in controlling intestinal immune responses. 2 Abstract ...................................................................................... 2 Acknowledgments ........................................................................ 11 Authors Declaration ...................................................................... 13 List of Abbreviations ..................................................................... 14 Chapter 1: Introduction ................................................................. 17 1.1 Dendritic Cells- A Brief Introduction ......................................... 17 1.2 Dendritic Cell Development .................................................... 18 1.2.1 Cytokines Involved In Dendritic Cell Development ........................ 20 1.3 Conventional Dendritic Cell Subsets .......................................... 21 1.4 Plasmacytoid Dendritic Cells ................................................... 23 1.5 Pattern Recognition By Dendritic Cells ...................................... 24 1.6 Dendritic Cell Migration ......................................................... 25 1.7 Antigen Uptake and Presentation ............................................. 28 1.8 Dendritic Cell Interactions with the Adaptive Immune Response ...... 30 1.8.1 CD4+ T Cells ..................................................................... 30 1.8.2 CD8+ T cells ..................................................................... 33 1.8.3 B Cells ............................................................................ 34 1.9 Lymphoid Tissue Development ................................................ 35 1.9.1 Lymph Node Development .................................................... 35 1.10 Splenic Development ........................................................... 39 1.11 Mesenteric Lymph Node Development ..................................... 39 1.12 Peyer’s Patch Development .................................................. 39 1.13 Development of Mucosal Associated Lymphoid Tissues ................. 41 1.14 Formation of Tertiary Lymphoid Organs ................................... 44 1.15 The Intestinal Immune System ............................................... 45 1.15.1 Antigen Uptake in the Intestine ............................................ 45 1.15.2 M Cell Mediated Antigen Uptake ........................................... 46 1.15.3 Dendritic Cell Uptake of Luminal Antigen ................................ 47 1.15.4 Antigen Uptake Via the Neonatal Fc Receptor ........................... 51 1.16 Dendritic Cell Subsets in the Intestine ..................................... 52 1.16.1 Dendritic Cell Subsets in the Lamina Propria ............................. 52 1.16.2 Functions of Dendritic Cell Subsets in the Lamina Propria ............. 53 1.16.3 Peyer’s Patch Dendritic Cells ............................................... 54 1.16.4 Functions of Peyer’s Patch Dendritic Cell Subsets ...................... 56 3 1.16.5 Dendritic Cell Subsets in Isolated Lymphoid Follicles ................... 57 1.16.6 Functions of Dendritic Cells Within Isolated Lymphoid Follicles ...... 57 1.17 Functions of Dendritic Cells in the Mesenteric Lymph Nodes ......... 58 1.17.1 Oral Tolerance ................................................................ 58 1.17.2 Functions of Retinoic Acid in Dendritic Cells ............................. 60 1.17.3 The Generation of Foxp3+ Regulatory T Cells in the Mesenteric Lymph Node ..................................................................................... 60 1.17.4 The Induction of Gut Homing Receptors on T Cells by Dendritic Cells 61 1.17.5 Initiation of an Adaptive Immune Response By Dendritic Cells ........ 62 1.18 Hypothesis and Aims ........................................................... 64 Chapter 2: Materials and Methods ..................................................... 66 2.1 Animals ............................................................................. 66 2.2 Surgical Procedures .............................................................. 69 2.2.1 Mesenteric Lymphadenectomy ............................................... 69 2.2.2 Thoracic Duct Cannulation .................................................... 69 2.2.3 Photoconversion of Kaede Mice .............................................. 70 2.2.4 FITC Injections .................................................................. 73 2.2.5 Subserosal Injections of Evan’s Blue ........................................ 73 2.2.6 Swiss Roll Sections of Intestine .............................................. 73 2.3 Reagents ........................................................................... 74 2.4 Cell Isolation ....................................................................... 74 2.4.1 Isolation of Thoracic Duct Leukocytes ...................................... 74 2.4.2 Isolation of Small Intestine Lamina Propria Cells ......................... 74 2.4.3 Isolation of Colonic Lamina Propria Cells ................................... 75 2.4.4 Isolation of Lymph Node and Spleen Cells .................................. 75 2.5 Flow Cytometry ................................................................... 76 2.6 Induction of DSS Colitis .......................................................... 78 2.7 Adoptive Transfers ............................................................... 78 2.7.1 Proliferation Assay ............................................................. 78 2.8 Generation of Bone Marrow Dendritic Cells ................................ 79 2.9 Preparation of Blood and Tissue Samples ................................... 79 2.10 Polymerase Chain Reaction ................................................... 80 2.11 Immunohistochemical Labelling of Slides .................................. 82 2.12 Statistical Analysis .............................................................. 82 4 Chapter 3: Comparison of Dendritic Cell Migration in the Small Intestine and Colon ........................................................................................ 83 3.1 Results .............................................................................. 84 3.1.1 Identification of Small Intestinal and Colonic Draining Mesenteric Lymph Nodes ........................................................................... 84 3.1.2 Evaluation of Transgenic Kaede Mice to Monitor Dendritic Cell Migration ........................................................................................... 92 3.1.3 Comparison of Levels of MHCII Expression Upon Migratory and Resident Dendritic Cell Subsets in the Mesenteric Lymph Nodes ......................... 94 3.1.4 Comparison of Dendritic Cell Subsets in the Small Intestine, Colon, Small Intestinal and Colonic Draining Mesenteric Lymph Nodes ............... 96 3.1.5 Comparison of Antigen Presentation by Small Intestinal and Colonic Draining Mesenteric Lymph Node Dendritic Cells in Vitro ..................... 107 3.1.6 Dendritic Cell Subsets in the Small Intestinal and Colonic Draining Mesenteric Lymph Nodes Following Experimental Colitis ...................... 111 3.1.7 Antigen Presentation by Small Intestinal and Colonic Draining Mesenteric Lymph Node Dendritic Cells in Vivo ................................. 118 3.2 Discussion ........................................................................ 124 3.2.1 Identification of Draining Lymph Nodes Draining the Small Intestine and Colon ................................................................................... 124 3.2.2 Validation of Kaede Mice as a Tool to Observe DC Migration .......... 126 3.2.3 Identification of DC subsets in the Small Intestine, Colon and Draining Lymph Nodes .......................................................................... 128 3.2.4 Thoracic Duct Cannulation of Partially Mesenteric Lymphadenectomised Animals ................................................................................ 133 3.2.5 Effects of Experimental Colitis Upon Dendritic Cell Subsets in the Small Intestinal or Colonic Draining Mesenteric Lymph Nodes ....................... 135 3.2.6 Comparison of Antigen Presentation By Dendritic Cell Populations from the Small Intestinal and Colonic Draining Mesenteric Lymph Nodes ......... 136 3.3 Conclusions ...................................................................... 138 Chapter 4: Dendritic Cell Migration in Mice Lacking Peyer’s Patches and Isolated Lymphoid Follicles ........................................................... 140 4.1 Results ............................................................................ 141 4.1.1 Genotyping of RORγtCD3ε and CD3ε Deficient Mice ..................... 141 5 4.1.2 Lymphoid Tissue Development in the Small Intestine of RORγt Deficient Animals ................................................................................ 145 4.1.3 Dendritic Cell Subsets in Pseudo-Afferent Lymph, Small Intestine and Colon of RORγt Deficient Mice ..................................................... 147 4.1.4 Dendritic Cell Development in the Spleen and Bone Marrow of RORγt Deficient Mice ........................................................................ 155 4.1.5 Generation of Lymphotoxin-β Receptor Fusion Protein ................. 159 4.1.6 Lymphoid Tissue in the Intestine of Lymphotoxin-β Receptor Fusion Protein Treated Progeny ............................................................ 161 4.1.7 Dendritic Cell Subsets in the Mesenteric Lymph Nodes, Small Intestine, Colon and Migrating in Pseudo -Afferent Lymph of Lymphotoxin-β Receptor Fusion Protein Treated Progeny ................................................... 164 4.1.8 Development of Lymph Nodes, Spleen and Dendritic Cells in Lymphotoxin-β Receptor Fusion Protein Treated Progeny ..................... 173 4.2 Discussion ........................................................................ 181 4.2.1 Abrogated Lymphoid Tissue But Normal Dendritic Cell Development in RORγtCD3ε Deficient Mice .......................................................... 181 4.2.2 Abrogated Peyer’s Patch But Normal Lymph Node and Dendritic Cell Development in LTβR-Ig Treated Progeny ........................................ 184 4.2.3 Dendritic Cell Subsets In The Intestine of Mice Lacking Peyer’s Patches or Peyer’s Patches and Isolated Lymphoid Follicles ............................ 186 4.3 Conclusions ...................................................................... 190 Chapter 5: Dendritic Cell Migration From Peyer’s Patches to the Mesenteric Lymph Nodes ............................................................................ 193 5.1 Results ............................................................................ 194 5.1.1 Dendritic Cells from the Peyer’s Patches Migrate to the Mesenteric Lymph Nodes in Kaede Mice ........................................................ 194 5.1.2 Dendritic Cells Migrating From Peyer’s Patches to the Mesenteric Lymph Nodes Expand In Response to Flt3 Ligand ............................... 202 5.1.3 Dendritic Cells From Peyer’s Patches Migrate in Pseudo-Afferent Lymph .......................................................................................... 205 5.1.4 Subsets of DCs Migrating From the Peyer’s Patches to the Mesenteric Lymph Node ........................................................................... 205 6 5.1.5 Dendritic Cells from the Peyer’s Patches Migrate to the Mesenteric Lymph Nodes Following FITC Injection ........................................... 209 5.1.6 Dendritic Cells Migrating From Peyer’s Patches to the Mesenteric Lymph Nodes Do Not Expand In Response to R848 or Cholera Toxin ......... 212 5.1.7 Dendritic Cells Migrating From Peyer’s Patches to the Mesenteric Lymph Nodes Are Dependent Upon CCR7 and S1PR ............................. 214 5.1.8 Subsets of DCs Migrating From the Peyer’s Patches to the Mesenteric Lymph Node ........................................................................... 219 5.2 Discussion ........................................................................ 221 5.2.1 Dendritic Cells from the Peyer’s Patches Migrate to the Mesenteric Lymph Nodes .......................................................................... 222 5.2.2 Dendritic Cells Migrating From Peyer’s Patches to the Mesenteric Lymph Nodes Expand In Response to Flt3 Ligand ............................... 224 5.2.3 Proportions of Dendritic Cells Migrating From Peyer’s Patches to the Mesenteric Lymph Nodes Do Not Increase in Response to R848 or Cholera Toxin ................................................................................... 225 5.2.4 Dendritic Cells Migrating From Peyer’s Patches to the Mesenteric Lymph Nodes Are Dependent Upon CCR7 and S1PR ............................. 227 5.2.5 Subsets of DCs Migrating From the Peyer’s Patches to the Mesenteric Lymph Node ........................................................................... 229 5.3 Conclusions ...................................................................... 231 Chapter 6: Final Discussion ........................................................... 233 6.1 Conclusions ...................................................................... 239 Chapter 7: References ................................................................. 240 7 Figure 1-1 Dendritic Cell Development. ............................................... 19   Figure 1-2 Functions of Dendritic Cell Subsets ....................................... 23   Figure 1-3 Development of T helper Subsets ......................................... 32   Figure 1-4 Timeline of the Sequential Development of Secondary Lymphoid Organs. .................................................................................. 36   Figure 1-5 Lymph Node Development. ................................................. 38   Figure 1-6 The Development of Cryptopatches and their Expansion into Isolated Lymphoid Follicles. ................................................................... 43   Figure 2-1 Peyer’s Patch Photoconversion ............................................ 71   Figure 2-2 Small Intestine Photoconversion ........................................... 72   Figure 3-1 Subserosal Injection of Evans Blue Distinguishes Distinct Parts of the MLN Drain the Small Intestine and Colon. ......................................... 86   Figure 3-2 DCs Originating in the Small Intestine Drain to Specific Parts of the MLN. ..................................................................................... 89   Figure 3-3 Dendritic Cells Originating in the Colon Drain to Specific Nodes of the MLN. ..................................................................................... 91   Figure 3-4 Photoconversion of kaede bone marrow derived DCs does not upregulate the expression of CD80 or CD86. ...................................... 93   Figure 3-5 Migratory Photoconverted kaede Red+ DCs in the Small intestine and Colon Express Higher Levels of MHCII in the MLN. ............................... 95   Figure 3-6 Four Distinct Subsets of DCs are Present in the Small Intestinal Lamina Propria. ........................................................................ 98   Figure 3-7 Three Distinct Subsets of DCs are Present in the Colonic Lamina Propria. ................................................................................. 99   Figure 3-8 Dendritic Cell Subsets in Small Intestinal Draining MLN and Colonic Draining MLN. ......................................................................... 103   Figure 3-9 Expression of CX3CR1 on Migratory and Resident Dendritic Cell Subsets in siMLN and coMLN. ....................................................... 104   Figure 3-10 Small Intestine and Colon with Draining Mesenteric Lymph Nodes Normally Removed Following Mesenteric Lymphadenectomy. ................ 105   Figure 3-11 Dendritic Cell Subsets in the Lymph of siMLNx and coMLNx Mice. 106   Figure 3-12 Migratory Dendritic Cells From the Small Intestine and Colon are Able to Present Antigen to Naïve T Cells. ........................................ 108   Figure 3-13 Migratory Dendritic Cells From the Small Intestine and Colon are Able to Induce CCR9 Expression in Responding T Cells. ........................ 110   Figure 3-14 Dendritic Cell Populations are Comparable in Small Intestinal and Colonic Draining MLN During and After Experimental Colitis. ................. 113   Figure 3-15 Migratory Dendritic Cell Subsets are Comparable in Small Intestinal and Colonic Draining MLN During and After Experimental Colitis ............ 114   Figure 3-16 Resident Dendritic Cell Subsets are Comparable in Small Intestinal and Colonic Draining MLN During and After Experimental Colitis ............ 115   8 Figure 3-17 CD64+CD11b+CX3CR1+ Cells are Unchanged in Small Intestinal and Colonic Draining MLN During and After Experimental Colitis. ................. 117   Figure 3-18 Orally Administered Antigen Preferentially Causes Proliferation of CD4+ T Cells in Small Intestinal Draining Lymph Nodes. ........................ 120   Figure 3-19 Orally Administered Antigen Causes Proliferation of CD8α+ T Cells Specifically in Small Intestinal Draining Lymph Nodes. ........................ 121   Figure 3-20 Small Intestinal Derived Ovalbumin is Only Presented to T cells in Small intestinal Draining Lymph Nodes ........................................... 123   Figure 4-1 The Absence of CD3+ Lymphocytes in blood samples from RORγtCD3ε and CD3ε deficient mice. ........................................................... 142   Figure 4-2 The Presence of Neomycin and RORγt Protein in RORγCD3ε and CD3ε Deficient mice. ....................................................................... 144   Figure 4-3 Organised Lymphoid Structures Are Absent From the Small Intestine of RORγt Deficient Mice. ............................................................ 146   Figure 4-4 CD103-CD11b- DCs Are Missing From The Lymph of RORγtCD3ε- Deficient Mice ........................................................................ 148   Figure 4-5 Expression of CD8α on Dendritic Cell Subsets in Lymph. ............. 149   Figure 4-6 CD103-CD11b- DCs Are Missing From Small Intestinal Lamina Propria of RORγt Deficient Mice. ............................................................... 151   Figure 4-7 Expression of CD8α on Dendritic Cell Subsets in Small Intestinal Lamina Propria. ....................................................................... 152   Figure 4-8 CD103-CD11b- DCs Are Fewer In The Colonic Lamina Propria of WT, CD3ε and RORγtCD3ε Deficient Mice. ............................................. 154   Figure 4-9 Characterisation of Cell Subsets in the Spleen of WT, CD3ε and RORγtCD3ε Deficient Mice. ......................................................... 157   Figure 4-10 Characterisation of Cell Subsets Generated from Bone Marrow of WT, CD3ε and RORγtCD3ε Deficient Mice After Culture with Flt3L. .......... 158   Figure 4-11 SDS-PAGE Gel of Purified LTβR-Ig. ...................................... 160   Figure 4-12 The Concentration of Purifed LTβR-Ig by Bradford Assay. .......... 160   Figure 4-13 Progeny of Mice Injected With LT-βR-Ig at Embryonic Day 14 and 16 Lack Peyer’s Patches But Retain Mesenteric Lymph Nodes. ................... 162   Figure 4-14 Peyer’s Patches Are Absent From the Small Intestine of LTβR-Ig Treated Progeny. ..................................................................... 163   Figure 4-15 Four Subsets of Dendritic Cells DCs Are Present In The Lymph of Mice Treated in Utero With LTβR-Ig. .............................................. 166   Figure 4-16 Characterisation of Cell Subsets in the MLN of WT mice and Mice Treated with LTβR-Ig in Utero. .................................................... 167   Figure 4-17 Characterisation of Migratory and Blood Derived Dendritic Cell Subsets in the MLN of WT and LTβR-Ig Progeny. ................................ 168   Figure 4-18 Characterisation of Dendritic Cells in Small Intestinal Draining and Colonic Draining MLN of WT and LTβR-Ig-treated Progeny. ................... 169   9

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Institute of Infection, Immunity and Inflammation. College of Medical Veterinary and Life Sciences. University of Glasgow. December 2013 increased dermal DC migration from the skin (Johnson and Jackson, 2013). investigate these cells other mucosal adjuvants such as heat-liable enterotoxin.
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Most books are stored in the elastic cloud where traffic is expensive. For this reason, we have a limit on daily download.