THE ROLE OF MAST CELLS DURING INFLUENZA A VIRUS INFECTION by Amy Catherine ...

299 Pages·2015·2.64 MB·English

by  Amy Graham

THE ROLE OF MAST CELLS
DURING INFLUENZA A
VIRUS INFECTION

by

Amy Catherine Graham

A dissertation submitted in partial fulfillment
of the requirements for the degree

of

Doctor of Philosophy

in

Immunology and Infectious Diseases

MONTANA STATE UNIVERSITY
Bozeman, Montana

April 2015
©COPYRIGHT

by

Amy Catherine Graham

2015

All Rights Reserved
ii
DEDICATION

For my family and friends who were with me throughout this adventure.
iii
ACKNOWLEDGEMENTS

I would first like to thank my mentor, Dr. Joshua Obar, for his years of guidance,
insight, and support. His knowledge and mentorship have been of great value to me, in
both my graduate career and as well as future endeavors. I would also like to thank my
committee for their leadership and assistance in my project.
The Obar lab has provided a great place to work throughout my years at MSU.
Each member has been very helpful in completing my project and assisting when
required. I would like to especially thank them for making my birthdays in Montana a
great and wonderful experience.
I have made many marvelous friends while at MSU. Without them keeping me
sane throughout years of study, I would be lost. I’m excited to see our friendship continue
as we all move on to our next great adventure.
Finally, I would like to give special thanks my amazing family. My parents, sister,
and brother have been incredibly supportive during my studies, and I never would have
made it this far without them.
iv
TABLE OF CONTENTS

1. INTRODUCTION ........................................................................................................ 1
Influenza A Virus .......................................................................................................... 1
IAV Hemagglutinin, Sialic Acid, And the Viral Replication Cycle ....................... 5
Pulmonary Response to IAV ...................................................................................... 12
Unique Role of Mast Cells .......................................................................................... 21
Mast Cell Activation ............................................................................................. 23
Research Goals ........................................................................................................... 29
References ................................................................................................................... 31
2. MATERIALS AND METHODS ................................................................................ 49
Viral Strains ................................................................................................................ 49
Constructing A/WSN/33 D225G ................................................................................ 49
UV Virus Inactivation ................................................................................................. 50
Mouse Strains and Infection Protocol ......................................................................... 51
IAV Propagation in Eggs ............................................................................................ 52
IAV Plaque Assay ....................................................................................................... 53
Histological Analysis of Lungs ................................................................................... 54
H&E ...................................................................................................................... 54
Toluidine Blue ...................................................................................................... 55
Growth of Bone Marrow Cultured Mast Cells (BMCMC)......................................... 55
Generation of Mast Cell Knock-in Mice .............................................................. 56
In Vitro Mast Cell Activation Assay ........................................................................... 56
Assay for Cytokine and Chemokine Secretion ........................................................... 57
Histamine and Leukotriene B Enzyme Immunoassays (EIA) ................................... 57
4
Annexin V Binding for Mast Cell Degranulation ....................................................... 57
Intracellular Staining for Viral Proteins ...................................................................... 58
Statistical Analysis ...................................................................................................... 58
References ................................................................................................................... 59
3. MAST CELLS AND IAV: ASSOCIATION WITH ALLERGIC
RESPONSES AND BEYOND ................................................................................... 61
Contribution of Authors and Co-Authors ................................................................... 61
Manuscript Information Page ..................................................................................... 62
Abstract ....................................................................................................................... 63
Introduction ................................................................................................................. 64
Mast Cell Biology ....................................................................................................... 67
Phases of the Mast Cell Response ........................................................................ 68
De novo Mast Cell Recruitment during Inflammation ......................................... 71
Expression of Pattern-Recognition Receptors by Mast
Cells for Sensing Invading Microbes .................................................................... 73
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TABLE OF CONTENTS - CONTINUED

Role of Mast Cells in Allergies and Asthma ........................................................ 74
Is there a Role for Mast Cell Activation and Mediators
during Pathological Viral Infections? ......................................................................... 75
Influenza Virus ..................................................................................................... 76
Dengue Virus ........................................................................................................ 77
Hantavirus ............................................................................................................. 79
Sendai Virus .......................................................................................................... 79
Infectious Bursal Disease Virus (IBDV) .............................................................. 80
Newcastle Disease Virus (NDV) .......................................................................... 81
Porcine Reproductive and Respiratory Syndrome Virus (PRRSV)...................... 81
How Are Mast Cells Activated by Viruses? ......................................................... 82
Is Virus Entry and Replication in Mast Cells Required for Activation? ......... 82
How Are Viral Particles Recognized by Mast Cells? ...................................... 83
Mast Cells as Drug Targets for Limiting Virus-Induced Immunopathology ............. 86
Concluding Remarks ................................................................................................... 89
Acknowledgements ..................................................................................................... 90
References ................................................................................................................... 91
4. INFLAMMATORY RESPONSE OF MAST CELLS DURING
INFLUENZA A VIRUS INFECTION IS MEDIATED BY
ACTIVE INFECTION AND RIG-I SIGNALING ................................................... 106
Contribution of Authors and Co-Authors ................................................................. 106
Manuscript Information Page ................................................................................... 107
Abstract ..................................................................................................................... 108
Introduction ............................................................................................................... 109
Materials and Methods .............................................................................................. 112
Viral Strains ........................................................................................................ 112
Mouse Strains and Infectious Protocol ............................................................... 112
IAV Plaque Assay ............................................................................................... 113
Histological Analysis of Lungs ........................................................................... 113
Growth of Bone Marrow Cultured Mast Cells (BMCMC) And
Generation of Mast Cell Knock-in Mice ............................................................ 114
In vitro Mast Cell Activation Assay ................................................................... 114
Luminex Assay for Cytokine and Chemokine Secretion .................................... 115
Histamine and Leukotriene B Enzyme Immunoassays (EIA) ........................... 115
4
Intracellular Staining for Viral Proteins .............................................................. 115
Statistical Analysis .............................................................................................. 116
Results ....................................................................................................................... 116
Mast Cells are Critical for Inducing the Pulmonary and Systemic
Inflammatory Disease Induced by A/WSN/33 Infection of Mice ...................... 116
Altered Inflammatory Milieu in B6.Cg-KitW-sh Mice during
A/WSN/33 Infection ........................................................................................... 121
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TABLE OF CONTENTS - CONTINUED

In vitro Activation of Mast Cells In Response A/WSN/33 Treatment ............... 122
Mast Cells can be Activated by Human Influenza Virus Isolates ...................... 125
Mast Cells are Infected by A/WSN/33 ............................................................... 127
RIG-I Detection of IAV is Involved In Mast Cell Cytokine, Chemokine,
And Leukotriene Production, but Not Degranulation ......................................... 128
Cytokine and Chemokine Expression by Mast Cells in Response to
A/WSN/33 Treatment Is Partially STING and STAT6 Dependent .................... 129
Discussion ................................................................................................................. 131
Acknowledgements ................................................................................................... 137
Footnotes ................................................................................................................... 138
References ................................................................................................................. 142
5. DIFFERENTIAL ROLE OF INFLUENZA A VIRUS BINDING
PREFERENCE IN MAST CELL ACTIVATION ................................................... 149
Contribution of Authors and Co-Authors ................................................................. 149
Manuscript Information Page ................................................................................... 150
Abstract ..................................................................................................................... 151
Introduction ............................................................................................................... 152
Materials and Methods .............................................................................................. 156
Viral Strains ........................................................................................................ 156
Mouse Strains and Infectious Protocol ............................................................... 157
UV Virus Inactivation ......................................................................................... 158
IAV Plaque Assay ............................................................................................... 158
Growth of Bone Marrow Cultured Mast Cells (BMCMC) And
Generation of Mast Cell Knock-in Mice ............................................................ 159
In vitro Mast Cell Activation Assay ................................................................... 159
Detection Assays for Cytokine and Chemokine Secretion ................................. 160
Histamine Enzyme Immunoassays (EIA) ........................................................... 160
Annexin V Expression For Mast Cell Degranulation ......................................... 161
Intracellular Staining for Viral Proteins .............................................................. 161
Statistical Analysis .............................................................................................. 162
Results ....................................................................................................................... 162
Secondary Mast Cell Mediators are not Responsible for Mast Cell
Induced Disease with A/WSN/33 ....................................................................... 162
BMCMC Degranulation Occurs Early during IAV Replication Cycle .............. 164
BMCMC Degranulation is not Triggered by Entry of A/WSN/33
Into the Cell ....................................................................................................... 170
Sialic Acid Binding is Important for BMCMC Degranulation ........................... 173
A/WSN/33 Binding Specificity is Important for BMCMC Activation .............. 174
A/WSN/33 NA Contributes to BMCMC Degranulation .................................... 176
Sialic Acid Binding Preference Leads To Differential Immune
Response in vivo ................................................................................................. 178
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TABLE OF CONTENTS - CONTINUED

Discussion ................................................................................................................. 183
References ................................................................................................................. 193
6. DISCUSSION AND FUTURE DIRECTIONS ........................................................ 202
Discussion ................................................................................................................. 202
Future studies ............................................................................................................ 214
Understanding the Cellular Mechanism Driving Mast Cell Degranulation
Sialic Acid Expression on Mast Cells................................................................. 217
Further Explore the Role of the IAV NA in Driving Mast Cell
Degranulation ...................................................................................................... 218
Role of Mast Cells in the Innate Immune Response to IAV .............................. 222
Explore the Different Inflammatory Milieus Induced by α2,3- and α2,6-
Sialic Acid Binding Influenza A Viruses ........................................................... 224
Understanding the Link Between Asthma, Mast Cells, and IAV ....................... 225
Conclusion ................................................................................................................ 226
References ................................................................................................................. 230

REFERENCES CITED ....................................................................................................243

APPENDIX A: Supporting Information ..........................................................................277
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LIST OF TABLES

Table Page

4.1 Cytokine and Chemokine Produced by BMCMC after
A/WSN/33 Treatment ....................................................................................140
ix
LIST OF FIGURES

Figure Page

1.1 IAV Replication Cycle .....................................................................................10

1.2 IAV Binds to α2,3- or α2,6- Linked Sialic Acid Linkages to
Initiate Entry into Host Cells ...........................................................................11

1.3 Biology of IAV Infection .................................................................................20

1.4 Mast Cells Response to Stimuli Includes Degranulating or
Producing de novo Mediators ..........................................................................28

3.1 Mast Cell Activation in Response to Viral Replication ...................................70

3.2 The Effect of Mast Cell Activation on the Inflammatory
Induced by Virus ..............................................................................................71

3.3 Mast Cell Inhibitors .........................................................................................89

4.1 B6.Cg-KitW-sh is Less Sensitive to A/WSN/33 ...............................................119

4.2 B6.Cg-KitW-sh Mice Reconstituted with BMCMC Were
Susceptible to WSN .......................................................................................120

4.3 B6.Cg-KitW-sh Mice Had Lower Levels of Cytokines in the
Lung during A/WSN/33 Infection .................................................................122

4.4 In vitro BMCMC Activation with A/WSN/33 Results in
Mast Cell Activation ......................................................................................124

4.5 Human Influenza Virus Isolates can Activate Mast Cells .............................126

4.6 Mast Cells are Infected by A/WSN/33 ..........................................................127

4.7 Mast Cell Activation by A/WSN/33 is Dependent on RIG-I ........................129

4.8 Mast Cell Activation is Partially Dependent on STING and
STAT6............................................................................................................131

4.9 Naïve Allantoic Fluid does not Cause BMCMC Activation .........................139

4.10 Cytokine and Chemokine Expression from BMCMC .................................141