functional characterization of annexin a1 … · cell staining with cfse and proliferation analysis...
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FUNCTIONAL CHARACTERIZATION OF ANNEXIN A1 IN TOLL LIKE RECEPTOR 7
SIGNALLING & INFLUENZA VIRUS INFECTION
Suruchi Arora B. Tech , JIIT
A THESIS SUBMITTED FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
DEPARTMENT OF PHYSIOLOGY
NATIONAL UNIVERSITY OF SINGAPORE
2014
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DECLARATION I hereby declare that this thesis is my original work and it has been written by
me in its entirety. I have duly acknowledged all the sources of information, which have been used in the thesis.
This thesis has also not been submitted for any degree in any university previously.
_________________________________________ Suruchi Arora
22nd Januray, 2014
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Publications
Pradeep Bist, Shinla Shu, Huiyin Lee, Suruchi Arora, Sunitha Nair, Jyue Yuen
Lim, Jivanaah Dayalan, Stephan Gasser ,Subhra K. Biswas, Anna-Marie
Fairhurst and Lina H. K. Lim . Annexin-A1 regulates TLR-mediated IFN-beta
production through an interaction with TANK-binding kinase 1. J Immunol.
2013, 191: 4375-4382.
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Contents
Acknowledgements ....................................................................................................... 8
Summary ..................................................................................................................... 12
List of Figures .............................................................................................................. 13
List of Tables ............................................................................................................... 16
List of Abbreviations ................................................................................................... 17
Chapter 1 Introduction ................................................................................................. 20
1.1. Annexin 1 .................................................................................................... 20
1.1.1. Structure of ANXA1 ............................................................................ 20
1.1.2. Functions of ANXA1 ........................................................................... 21
1.1.2.1. Interaction with Phopholipase -2 (PLA2) ........................................ 21
1.1.2.2. Mediation of Glucocorticoid (GC) Actions ..................................... 22
1.1.2.3. Proliferation and Apoptosis ............................................................. 23
1.1.2.4. Annexin 1 Null mouse ..................................................................... 24
1.1.2.5. Pro-inflammatory actions ................................................................ 25
1.1.2.6. Phagocytosis “EAT me” signals ...................................................... 25
1.1.3. The ANXA1 receptor .......................................................................... 28
1.1.4. ANXA1 and APCs............................................................................... 28
1.2. Toll like Receptors (TLRs) .......................................................................... 29
1.2.1. MyD88 dependent pathway ................................................................. 31
1.2.2. TRIF-dependent pathway .................................................................... 31
1.2.3. Intracellular TLRs................................................................................ 32
1.2.4. ANXA1 and TLRs ............................................................................... 34
1.3. Influenza Virus ............................................................................................ 34
1.3.1. Health threat and Global Impact of Influenza Viruses ........................ 35
1.3.2. Signs and Symptoms ........................................................................... 37
1.3.3. Structure of Influenza virus ................................................................. 37
1.3.4. Viral Life Cycle ................................................................................... 43
1.3.5. Influenza Tropism................................................................................ 45
1.3.6. Immune responses against Influenza virus .......................................... 46
1.3.7. Humoral Response ............................................................................... 51
1.3.8. T cell Response .................................................................................... 51
1.3.9. Annexins and viruses ........................................................................... 52
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1.4. Apoptosis ..................................................................................................... 53
1.4.1. The Receptor mediated/ Extrinsic Pathway ......................................... 54
1.4.2. The Intrinsic pathway .......................................................................... 55
1.4.3. The Execution Pathway ....................................................................... 60
1.4.4. Influenza and Apoptosis ...................................................................... 61
1.5. Aims of the study ......................................................................................... 63
1.6 Hypothesis ......................................................................................................... 63
Chapter 2 Materials and Methods ................................................................................ 64
2.1 Media and buffers .............................................................................................. 64
2.1.1. PBS ............................................................................................................ 64
2.1.2. FACS buffer .............................................................................................. 64
2.1.3. Red Blood Cell (RBC) Lysis Buffer .......................................................... 64
2.1.4. Wash buffer for Western Blotting (TBST) ................................................ 65
2.1.5. Buffer for ELISA ....................................................................................... 65
2.1.6. Complete DMEM for cell culture .............................................................. 65
2.1.7. Complete F-12K media for cell culture ..................................................... 65
2.1.8 Plain DMEM at 2X concentration .............................................................. 66
2.2. Cell Culture ...................................................................................................... 66
2.2.1. Cell Lines ................................................................................................... 66
2.2.2. Preparation of L929 Conditioned Media ................................................... 66
2.2.3. Generation of Bone Marrow Derived Macrophages (BMMOs) ................ 67
2.2.4. Generation of Bone Marrow Dendritic Cells (BMDCs) ............................ 67
2.2.5. Isolation of splenic cells ............................................................................ 68
2.2.6. Lung cell culture ........................................................................................ 68
2.2.7. Cell staining with CFSE and proliferation analysis ................................... 68
2.2.8. Thymidine Assay ....................................................................................... 69
2.2.9. Transfection ............................................................................................... 69
2.3. Mice .................................................................................................................. 69
2.3.1. Infection of mice with Influenza virus....................................................... 70
2.3.2. Cellular Infiltration .................................................................................... 70
2.3.3. Lung Histology .......................................................................................... 71
2.4 Influenza Virus Culture and Infection ............................................................... 72
2.4.1. Virus culture in chicken Embryos ............................................................. 72
2.4.2 Concentration of Influenza virus ................................................................ 73
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2.4.3 Plaque forming assay .................................................................................. 73
2.4.4 Infection of cells with Influenza virus ........................................................ 74
2.5 Measurement of Cytokines ................................................................................ 74
2.6 Western Blot ...................................................................................................... 75
2.6.1. Protein lysis ............................................................................................... 75
2.6.2. Protein Concentration Quantification ........................................................ 76
2.6.3 Blotting ....................................................................................................... 76
2.7. Real Time PCR ................................................................................................ 78
2.7.1. RNA Isolation from cells ........................................................................... 78
2.7.2. RNA isolation from lungs ......................................................................... 78
2.7.3. Viral RNA (vRNA) isolation from cell supernatant .................................. 79
2.7.4. cDNA Synthesis ....................................................................................... 79
2.7.5. Real time PCR .......................................................................................... 80
2.8. Luciferase Assay ............................................................................................. 82
2.9. Caspase 3/7 assay ............................................................................................ 82
2.10. Statistics .......................................................................................................... 83
Chapter 3 Results ......................................................................................................... 84
3.1. Endogenous levels of TLR7 in WT and ANXA1-/- are similar in both BMMOs and DCs ................................................................................................ 85
3.2. R848 efficiently activates TLR7 pathway ................................................... 86
3.3. ANXA1 is selectively required for the production of pro-inflammatory cytokines .............................................................................................................. 88
3.4. TLR dependent inflammasome activation is higher in ANXA1-/- cells ....... 93
3.5. ANXA1-deficient splenic B cells exhibit increased activation and proliferation. ........................................................................................................ 95
3.6. ANXA1 deficient dendritic cells exhibit higher production of pro-inflammatory cytokines upon virus infection. ................................................... 102
3.7. ANXA1 expression levels and its promoter activity increase upon virus infection in the epithelial cells in vitro. ............................................................. 104
3.8. Influenza virus replication is inhibited in ANXA1 deficient epithelial cells. ........................................................................................................................... 106
3.9. ANXA1 deficient mice are more protected against Influenza virus infection. ........................................................................................................................... 111
3.10. ANXA1 is a virus inducible protein and is cleaved upon infection ........ 113
3.11. Viral loads in ANXA1 deficient mice are lower compared to the WT mice ........................................................................................................................... 115
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3.12. The cytokine and type-I interferon responses are similar in WT and ANXA1-/- mice. ................................................................................................ 117
3.13. ANXA1-/- mice display higher leukocyte infiltration in the BAL fluid . 120
3.14. ANXA1-/- mice display higher lung inflammation ................................. 123
3.15. ANXA1-/- mice display similar vascular integrity as WT mice ............. 126
3.16. WT mice exhibit more apoptosis in their lungs ........................................ 127
3.17. ANXA1 is required for virus induced apoptosis ...................................... 129
3.18. ANXA1 inhibits NF-κB activation during influenza infection ............... 131
Chapter 4 Discussion ................................................................................................. 133
4.1. Summary of key findings .......................................................................... 133
4.2. Regulation of Cytokines in BMMOs and BMDCs .................................... 134
4.3. Enhanced B cell proliferation in ANXA1-/- cells ..................................... 137
4.4. Regulation of ANXA1 during influenza virus infection ........................... 139
4.5. ANXA1 positively regulates virus propagation ........................................ 140
4.6. Better survival of ANXA1-/- mice upon infection .................................... 141
4.7. Inhibition of NF-κB in ANXA1 overexpressing cells ............................... 146
4.8. Limitations of the Study ............................................................................ 146
4.9. Clinical Significance .................................................................................. 147
4.10. Future Directions ................................................................................... 148
4.11. Conclusion ............................................................................................. 149
References ................................................................................................................. 150
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Acknowledgements
This PhD thesis is like a dream come true…
I would like to thank the following people who have made this possible.
A/P Lina Lim, for your constant guidance and support. Thanks for being so patient even when I made so many mistakes. You have always trusted me and helped me in every way possible way. The constant encouragement from you kept me going and helped to overcome all obstacles. You have always motivated me to do difficult tasks to bring out the best in me.
Dr. Pradeep Bist, for being the best mentor in the world. You have not only taught me everything I know today but also taught me how to live life and smile inspite of all the pains. I have learnt never to give up from you. Thanks for being my family in Singapore. “Master Shifu” you are an ocean of knowledge and I wish you were in lab a bit longer.
Sunitha, I thank God for making you join this lab. Thanks for sharing all the happiness and sorrows. Taking mind off the failed experiments with constant pressure of the deadlines would not have been possible without you. Thanks for always being there for me.
Yuan Yi, you have lived upto your new name “Khushi”. Thanks for always spreading joy and happiness around especially in those tough moments. Having you around during the last two years was wonderful .Thanks for helping with my experiments during the writing period and cooking wonderful tofu and potato for me. And lastly, thanks for teaching me how to use chopsticks and for your constant attempts to teach me mandarin.
Durkesh, your advice always works wonder. Your ability to judge situations and take correct measures always amazes me. Thanks for always guiding and helping me take right decisions.
Claire, you are ray of hope in the darkness, when nothing seems to be working you always come up with a solution or new method. Thanks for a lot for always being ready to help.
Kuan yau and Lay Hoon, Thanks for always arranging the reagents on time for me and sharing your long invaluable experience with me
Neha and Nayana, Both of you have been my agony aunts. Thanks for being so patient and listening to all my stories. I don’t think I could have finished if it had not been for your constant advices. You have always made me stand
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whenever I fell and made sure I never feel lonely. Words will not be enough to express my gratitude. Love you both.
Benson Thanks a lot for putting in so much effort to provide me mice even when you had so many more things to do.
Johan, Thanks for always being so supportive. I have learnt a lot from you.
Dr. Sylvie Alonso and Dr. Anna-marie Fairhurst, Thanks for guidance and support. Your suggestions definitely made my project better. Special Thanks to Dr. Alonso for helping me with the BAL harvests.
Dr. Benedict Yan Thanks for helping and teaching me with the inflammation scoring of the slides.
Paul, Fei Chuin and Guo hui, Thanks for teaching and always being ready for helping me with flow cytometry.
Anjana, I have spent almost 4 years of my PhD life with you. Thanks for always cheering me up and being there for me. You have always guided me towards the right solution and helped me overcome all the troubles I got into. Facing all the non-lab problems wasn’t easy without you. Finally we have sailed through. Cheers to the great times and our friendship.
Indu and Suresh, thanks for all the good times and memories. Times spent with you really de-stresses me. Both of you have always taken time to listen to my lab stories and helped me cope up with all the music lessons I bunked.
Animesh, Thanks for being so supportive and caring. You have always made helped me focus on my goals and made sure I am not lost. Thanks for standing by my side in the worst times and guiding me through it. You have helped me every way you could, so that I could finish on time. Toast to our success.
Kushagra, inspite of hating biology and lab work you have always lent a patient ear to me. I am blessed to have a friend like you. Thanks for all the motivation and chocolates in the writing phase.
Prema Di, Thanks for helping me grow spiritually, which was essential for being peaceful and staying calm in the last four turbulent years.
Anita di, Prachi di and Goutam dada, you guys are my inspiration for doing PhD and my very first mentors since my undergrad years. Thanks for guiding me throughout these years and for all the fun we had. It’s a pleasure to be youngest and get all the attention and love.
Gu Tong, Gracemary, Irene and Gerry, thanks for spreading all the joy around in the midst of the serious lab experiments. Lab has been much more
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fun since you all joined. Gu tong, special thanks for the creatively made lovely cards for all the occasions. Science is fun, enjoy it.
Mumma ,Papa and Bhai, thanks for being my pillars of strength. You gave me wings to follow my dreams. This journey would not have been possible without your love and care. Thanks for always understanding me. I am blessed to have you all.
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Dedicated to my Parents…..
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Summary
ANXA1 is an immune-modulatory protein and is known to play important role
in cell differentiation, adhesion, migration, proliferation and apoptosis. Our lab
has already shown its involvement in TLR3 signalling. To follow up this study
with the other RNA sensing receptors we chose to investigate the effects of
ANXA1 in TLR7 signalling by using an artificial agonist, R848 and a natural
ligand Influenza virus. In this project, cytokine production in various cell types
with TLR7 agonist R848 was observed which indicated the cell-specificity
exhibited by ANXA1, due to which bone marrow macrophages require
ANXA1 for cytokine production while bone marrow dendritic cells can
produce high amounts of cytokines in its absence. We also established that
ANXA1 plays a role in Influenza virus infection and positively regulates virus
titers due to which ANXA1-/- mice have greater survival rates compared to
WT. No differences are observed in cytokine levels or lung damage. Invitro
analysis of viral titers in human alveolar type II epithelial cell line A549, show
similar results as observed in vivo. Overexpression of ANXA1 leads to higher
viral loads whereas the knockdown reduces viral loads. Apoptosis favours viral
propagation and hence higher apoptosis may lead to higher viral loads in the
lungs of infected mice. In all, our study shows that ANXA1 plays a
detrimental role in the influenza infection and better targeting of the molecule
might bring out more efficient therapies against influenza virus.
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List of Figures
Figure 1.1 Schematic representation of ANXA1 protein ................................. 21
Figure 1.2 The diverse biological actions of ANXA1 ...................................... 27
Figure 1.3 TLR signalling pathway .................................................................. 33
Figure 1.4 Structure and genome organization of influenza virus ................... 39
Figure 1.5 Replication and transcription of Influenza virus. ............................ 45
Figure 1.6 RIG-I and MDA-5 Signalling ......................................................... 49
Figure 1.7 Structure of Bcl-2 family proteins .................................................. 57
Figure 1.8 The two main pathways of apoptosis .............................................. 59
Figure 1.9 Model of Influenza induced Apoptosis. .......................................... 62
Figure 3.1 Endogenous TLR7 levels are similar in both WT and ANXA1-/- . 86
Figure 3.2 R848 stimulates maximum production of cytokines. ...................... 87
Figure 3.3 Pro-inflammatory cytokines and Type I interferon expression is
downregulated in ANXA1-/- BMMOs. ............................................................ 89
Figure 3.4 Pro-inflammatory cytokine production is downregulated in
ANXA1-/- BMMOs. ......................................................................................... 90
Figure 3.5 Pro-inflammatory cytokines and Type I interferon expression is
upregulated in ANXA1-/- BMDCs. ................................................................. 91
Figure 3.6 Pro-inflammatory cytokine production is upegulated in ANXA1-/-
BMDCs. ............................................................................................................ 92
Figure 3.7 Increased IL-1β production in ANXA1-/- BMDCs and BMMOs. . 94
Figure 3.8 ANXA1 deficient B cells exhibit increased proliferation compared
to WT. ............................................................................................................... 97
Figure 3.9 ANXA1 deficient B cells exhibit increased activation. .................. 99
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Figure 3.10 ANXA1-/- B cells display enhanced PI3K/Akt signalling in
response to R848. ........................................................................................... 101
Figure 3.11 ANXA1 deficient dendritic cells exhibit higher production of pro-
inflammatory cytokines upon virus infection ................................................. 103
Figure 3.12 ANXA1 is upregulated and cleaved upon viral infection in vitro.
........................................................................................................................ 105
Figure 3.13 Standard curves for viral load quantification. ............................. 107
Figure 3.14 ANXA1 deficient epithelial cell exhibit lower viral titers. ......... 108
Figure 3.15 ANXA1 overexpressing epithelial cell exhibit higher viral titers.
........................................................................................................................ 110
Figure 3.16 ANXA1 defecient mice are protected against influenza A PR8
infection when challenged with 500pfu dose. ................................................ 112
Figure 3.17 ANXA1 is a virus inducible protein and is cleaved upon infection
........................................................................................................................ 114
Figure 3.18 ANXA1 deficient mice exhibit lower viral titers in lungs. ......... 116
Figure 3.19 Cytokine levels are similar in both wild type and ANXA1 deficient
mice. ............................................................................................................... 119
Figure 3.20 Gating Strategy for cell counts in BAL. ..................................... 121
Figure 3.21 Influenza infection leads to higher cellular infiltration in the BAL
from ANXA1-/- mice. .................................................................................... 122
Figure 3.22 Higher Inflammation is observed in infected lungs. ................... 125
Figure 3.23 Vascular Integrity is compromised upon infection. .................... 126
Figure 3.24 Wild type infected mice experience more apoptosis. ................. 128
Figure 3.25 ANXA1 is required for virus induced apoptosis. ........................ 130
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Figure 3.26 NF-κB activity is decreased in presence of ANXA1 upon infection.
........................................................................................................................ 132
Figure 4.1 Putative mechanism for ANXA1 action upon infection ............... 145
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List of Tables
Table 1.1 TLRs and their ligands ..................................................................... 30
Table 2.1 Antibodies used for flow cytometry. ................................................ 70
Table 2.2 Antibodies used for Western Blotting .............................................. 77
Table 2.3 cDNA synthesis initial reaction mix ................................................ 79
Table 2.4 cDNA synthesis final reaction mix .................................................. 79
Table 2.5 qPCR reaction mix ........................................................................... 80
Table 2.6 List of primers .................................................................................. 82
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List of Abbreviations
ANXA1 Annexin A1
Apaf1 Apoptotic protease activating factor
BAD Bcl-2 associated death promoter
BH Bcl-2 homology
BMMOs Bone marrow macrophages
BMDCs Bone Marrow derived dendritic cells
CAD Caspase activated DNAse
COX-2 Cyclooxygenase 2
CARD Caspase recruitment domain
CPSF Cleavage and polyadenylation specificity factor
DEDs Death effector domains
DCs Dendritic cells
DISC Death inducing signalling complex
EGF Epidermal Growth Factor
eIF2α Eukaryotic initiation factor 2α
ERK Extracellular signal-regulated kinases
FADD Fas-associated death doamin
FPR Formyl peptide receptor
GC Glucocorticoid
HA Hemaglutinnin
ICAM-1 Intracellular adhesion molecule – 1
IKK IκB Kinase
IAP Inhibitor of apoptosis proteins
IL-1β Interleukin 1β
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ISGF3 Interferon stimulated gene factor 3
MAPK Mitogen-activated protein kinases
M1 Matrix protein
M2 Ion channel protein
MDA-5 Melanoma differentiation-associated gene 5
MMR Macrophage mannose receptor
MPT Mitochondrial permeability transition
MOMP Mitochondria outer membrane permeabilisation
NA Neuraminidase
NF-κB Nuclear factor κ B
NP Nucleoprotein
NS Non-structural protein
PA Polymerase acid
PAMP Pathogen associated molecular patterns
PB Polymerase basic
PDGF Platelet derived Growth Factor
PI3K Phosphatidylinositide 3-kinases
PKC Protein kinase C
PLA-2 Phospholipase 2
Poly(I:C) Polyinosinic-polycytidylic acid
PRRs Pathogen recognition receptor
PS Phosphotidyl serine
RASF Rheumatoid arthiritis synovial fibroblasts
RIG-I Retinoic acid inducible gene I
RIP 1 Receptor inducing protein kinase 1
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RNP Ribonucleoprotein
SA Sialic acid
TBK-1 TANK binding kinase-1
TGF-β Transforming growth factor β
TLRs Toll-like receptors
TNF α Tumour necrosis factor α
TRAF TNF-receptor associated factor
TRAIL TNF-related apoptosis inducing ligand
UTR Upper respiratory tract
VDAC Voltage dependant anion channel
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Chapter 1 Introduction
1.1. Annexin 1
Annexin 1 (ANXA1) is the first characterized member of the annexin
superfamily which comprises of 13 calcium or phospholipid binding proteins.
It is a 37kDa protein and was identified as an inhibitory protein that mimics the
inhibitory action of glucocorticoids (Flower, 1988). Since the time of its
discovery in late 1970s it has changed various names from macrocortin
(Blackwell et al., 1980) to renocortin (Rothhut, Russo-Marie, Wood, DiRosa,
& Flower, 1983), lipomodulin (Hirata et al., 1981), lipocortin-1 and finally
ANXA1 since ANXA1 encoding gene is located on chromosome 19q24 (Lim
& Pervaiz, 2007).
1.1.1. Structure of ANXA1
The annexins are ubiquitous and are found throughout the animal and plant
kingdom. The structure comprises of tightly packed core domain made of α-
helices. The center of the core is hydrophilic so as to function as a Ca2+
channel. The C terminal of Annexins is highly conserved but N terminal is
unique for a given member. The N terminus is the regulatory region and
contains the sites for phophorylation and proteolysis (Raynal & Pollard, 1994).
Annexins must be able to bind the negatively charged phospholipids in a
calcium dependant manner. All Annexins except Annexin 6 have a core
domain containing four repeating units comprising of approximately 70 amino
acids. The binding is reversible and removal of calcium leads to the disruption
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of interactions between annexins and phospholipids (Gerke & Moss, 2002).
The crystal structure of ANXA1 consisting of 346 amino acids is described as
a “doughnut”. The N-terminal remains hidden in the pore at low Ca2+
concentrations but an increase in the Ca2+ concentration exposes this region
thereby influencing its biological activity (Rosengarth, Gerke, & Luecke, 2001;
Rosengarth, Rosgen, Hinz, & Gerke, 2001).
Figure 1.1 Schematic representation of ANXA1 protein
The N terminal is the biologically active part while the four repeats represent the conserved C-terminal domain (adapted from Atlas of Genetics and Cytogenetics in Oncology and Haematology).
1.1.2. Functions of ANXA1
ANXA1 is mainly identified as an anti-inflammatory protein that mimics the
effects of glucocorticoids. Its reactions with various inflammatory mediators to
control inflammation are described below.
1.1.2.1. Interaction with Phopholipase -2 (PLA2)
ANXA1 was originally identified as the PLA2 inhibitor. There are two
isoforms of PLA2 namely cytosolic PLA-2 (cPLA2) and secretory PLA2
(sPLA2). cPLA2 is highly specific for the release of arachidonic acid from the
sn2 position of phospholipids (Mukherjee, Miele, & Pattabiraman, 1994). The
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second carbon of the glycerol molecule in phospholipids is described as the sn2
position. The direct interaction between cPLA2 and ANXA1 was determined
by immunoprecipitation and mammalian two hybrid studies. cPLA2 activity
was inhibited by both the full length and C-terminal region but not by the N-
terminal protein (S. W. Kim et al., 2001). The enzyme kinetic studies done by
measuring the degree of inhibition at various substrate and calcium
concentrations revealed that the inhibition is a function of the enzyme activity
and is independent of substrate concentration (K. M. Kim, Kim, Park, Kim, &
Na, 1994) . However, this interaction between ANXA1 and cPLA2 is cell type
specific. A clearer picture of the functional link between these two has been
shown in hepatocytic cell lines (de Coupade et al., 2000).
1.1.2.2. Mediation of Glucocorticoid (GC) Actions
ANXA1 mediates the anti-inflammatory reactions of GCs was revealed when
cyclooxygenase 2 (COX-2) mRNA and protein levels were constitutively
increased in ANXA1-/- mice and GCs were ineffective in bringing out any
effects in these mice (Hannon et al., 2003). ANXA1 can be induced by GCs
and is rapidly mobilized and secreted by target cells to mimic their anti-
inflammatory effects (J. D. Croxtall, van Hal, Choudhury, Gilroy, & Flower,
2002; McLeod, Goodall, Jelic, & Bolton, 1995; Peers, Smillie, Elderfield, &
Flower, 1993; Solito, Nuti, & Parente, 1994). GCs can also induce the
phosphorylation and translocation of ANXA1 to membrane via protein kinase
C (PKC), Phosphatidylinositide 3-kinases (PI3K) and Mitogen-activated
protein kinases (MAPK) dependent pathways (Solito, Mulla, et al., 2003). In
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rheumatoid arthritis ANXA1 was found to be present in arthritic synovium and
mediated the effects of GCs on the migration of neutrophils in carrageenan
induced acute arthritis. The condition aggravated on injection of anti-ANXA1
antibodies and neutrophil activation was inhibited (Y. Yang, Leech,
Hutchinson, Holdsworth, & Morand, 1997).
1.1.2.3. Proliferation and Apoptosis
The role of ANXA1 in proliferation and apoptosis is still unclear. Opposing
phenotypes have been observed based on cell types. ANXA1 is shown to be
anti-proliferative in case of A549 lung cancer cells (J. Croxtall, Flower, &
Perretti, 1993) and macrophages. ANXA1 is believed to exert this anti-
proliferative property in macrophages by being phophorylated by epidermal
growth factor (EGF) and hence keeping MAPK/ERK pathway constitutively
active (Alldridge, Harris, Plevin, Hannon, & Bryant, 1999). It can bind to Grb-
2 adaptor protein, which is upstream of MAPK pathway and bring about
disruption of actin cytoskeleton and inhibition of cyclin D1 (Alldridge &
Bryant, 2003). ANXA1 acts like a substrate or target for various signal
transducing kinases associated with platelet-derieved growth factor (PDGF)
receptor, hepatocyte growth factor receptor (Skouteris & Schroder, 1996) ,
protein kinase C (Varticovski et al., 1988) and TRPM7 channel kinase
(Dorovkov & Ryazanov, 2004) thus being an important player in proliferation.
All the above mentioned data places ANXA1 as an anti-proliferative protein;
however in hepatocytes its role is completely reversed. Its phosphorylation by
hepatocyte growth factor leads to it acting as a signal amplifier to enhance
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proliferation, chemotaxis and differentiation (Skouteris & Schroder, 1996)
ANXA1 also sigmals mitogen-stimulated breast tumour cell proliferation by
activation of frormyl peptide receptors 1&2 (Khau et al.,2010).
The cell-dependant differences shown by ANXA1 in proliferation are also
exhibited in apoptosis. It is believed that ANXA1 mediates the proapoptotic
effects of glucocorticoids. The involvement of ANXA 1 in apoptosis was first
in alveolar cells of mammary duct (McKanna, 1995). Following this
observation a few more papers discussed the role of ANXA 1 in the induction
of apoptosis. ANXA1 is shown to facilitate H2O2 induced apoptosis
(Sakamoto, Repasky, Uchida, Hirata, & Hirata, 1996), promotes caspase-3
activation (Solito, de Coupade, Canaider, Goulding, & Perretti, 2001) and
stimulate transient increase in the intracellular calcium concentrations (Solito,
Kamal, et al., 2003). It also induces apoptosis in neutrophils by intracellular
release of calcium which leads to dephorphorylation of Bcl-2 associated death
promoter (BAD) allowing it to associate with mitochondria (Solito, Kamal, et
al., 2003). Apart from the effects on caspase 3 activation and increase in
calcium concentrations other proposed mechanisms of action for ANXA1 are
inhibition of eicosanoid (Goetzl, An, & Zeng, 1995) and nitric oxide synthesis
(Genaro, Hortelano, Alvarez, Martinez, & Bosca, 1995).
1.1.2.4. Annexin 1 Null mouse
The availability of mice lacking ANXA 1 has given a boost to the research in
this area. It was generated by disruption of ANXA1 gene by homologous
25
recombination with lacZ gene. COX-2 and PLA2 expressions are up regulated
in lungs and thymus. Annexins 2, 4 and 6 are downregulated in ovary and liver
and upregulated in a few other organs like stomach, kidney and spleen
(Hannon, et al., 2003) Annexin 1 knockout mice exhibited responses like
increase in leukocyte transmigration (Chatterjee et al., 2005), delayed repair in
colitis (Babbin et al., 2008) and partial resistance to dexamethasone (Hannon,
et al., 2003).
1.1.2.5. Pro-inflammatory actions
ANXA1 is widely accepted as an anti-inflammatory protein but a few recent
studies have indicated that it can act opposite of its usual role upon cleavage.
The 3kDa cleavage product of ANXA1 is shown to promote neutrophil
transmigration via its effects on endothelial intracellular adhesion molecule 1
(ICAM-1) (Williams et al., 2010) and leukocyte chemotaxis via formyl peptide
receptor (FPR) family members (Ernst et al., 2004). ANXA1 is also secreted
from the rheumatoid arthiritis synovial fibriblasts (RASF) following Tumour
necrosis factor (TNF)-mediated activation to promote RASF matrix
metellaproteinase -1 secretion (Tagoe et al., 2008). The pro-inflammatory
effects can also be observed in the increased phosphorylation of PLA2 and
eicosanoid production in mast cells (Kwon et al., 2012, Chung et al., 2004).
1.1.2.6. Phagocytosis “EAT me” signals
Specific ‘eat me’ signals on the apoptotic cells serve as markers for phagocytes
so that they can recognize and ingest the apoptotic cells. One of the
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characteristics of apoptotic cells is the exposure of phophotidyl serine (PS) on
the outer leaflet of plasma membrane (Lim & Pervaiz, 2007). PS dependant
clearance of cells inhibits the release of pro inflammatory cytokines like
interleukin-1β (IL-1β) and TNF-α but stimulates the release of anti-
inflammatory cytokines like transforming growth factor (TGF-β). It is
suggested that ANXA1 might be an endogenous ligand facilitating the
clearance of apoptotic cells in a caspase or calcium dependant manner by
carrying out experiments where ANXA 1 gene has been silenced and a
defective engulfment of apoptotic cells was observed (Arur et al., 2003).
Monoclonal antibodies to ANXA1 have been shown to inhibit PS dependant
phagocytosis in lymphocytes (Fan, Krahling, Smith, Williamson, & Schlegel,
2004). The PS molecules are present on both phagocytes and apoptotic cells
whereas the PS receptor is present only on the surface of phagocytic cells. It
nay therefore be reasoned that ANXA 1 might act like a receptor to recognize
exposed PS molecules on the surface of apoptotic cells (Lim & Pervaiz, 2007).
27
Figure 1.2 The diverse biological actions of ANXA1
A) ANXA1 was found to inhibit the activity of cytosolic phospholipase A2 (cPLA2) and cyclooxygenase 2 (COX-2), thus exhibiting antiinflammatory, antipyretic, and antihyperalgesic activity. B) Exogenous ANXA1 acts on its receptor, recently identified as the formyl peptide receptor (FPR) and the formyl peptide receptor like-1 (FPRL1) to inhibit cell adhesion and migration, and induce detachment of adherent cells. When cells (particularly neutrophils) adhere onto the endothelium, ANXA1 is released together with the gelatinase granules and can act like exogenous ANXA1. C) ANXA1 expression can be up-regulated with GC treatment through the glucocorticoid receptor (GR), which contributes to its antiinflammatory activity. GCs can also induce rapid ANXA1 phosphorylation and membrane translocation. D) ANXA1 is recruited to the cell surface, where it binds to PS and mediates the engulfment of apoptotic cells. E) ANXA1 can be phosphorylated by a number of kinases, including EGF-R tyrosine kinase, protein kinase C (PKC), platelet-derived growth factor receptor tyrosine kinase (PDGFR-TK), and hepatocyte growth factor receptor tyrosine kinase (HGFR-TK) to mediate proliferation. F) Overexpression of ANXA1 induces apoptosis. ANXA1 can mediate apoptosis by inducing the dephosphorylation of BAD, allowing BAD to translocate to the mitochondria. During apoptosis, ANXA1 itself translocates to the nucleus, which can be inhibited by BCL2 (Lim & Pervaiz, 2007).
28
1.1.3. The ANXA1 receptor
Formyl peptide receptor 2 (FPR2) (or ALXR in humans) is a G-protein coupled
receptor (GPCR) through which ANXA1 signals (Perretti et al., 2002). It
belongs to a family of three receptors FPR1, ALXR/FPR2 and FPR3 which are
expressed by several cell types like monocytes, neutrophils, epithelial cells,
endothelial cells and macrophages (Babbin et al., 2006; Chiang et al., 2006).
The interaction between ANXA1 and its receptor was investigated by
immunoprecipitating ANXA1 from neutrophils with FPR2 when the
leukocytes were adhered to endothelium monolayers (Solito, Kamal, et al.,
2003). FPR2 is also the receptor for another anti-inflammatory molecule
lipoxin A4 (Perretti & D'Acquisto, 2009). Binding of ANXA1 to its receptor
leads to the transient phosphorylation of extracellular–regulated kinase 1 and 2
(ERK-1and ERK-2) (Hayhoe et al., 2006; He, Shepard, Chen, Pan, & Ye,
2006) followed by rapid increase in the levels of intracellular Ca2+ (Babbin, et
al., 2006; Solito, Kamal, et al., 2003). Blocking the receptor with monoclonal
antibodies prevented ANXA1 induced effects on neutrophil transmigration
(Babbin, et al., 2006).
1.1.4. ANXA1 and APCs
Dendritic cells (DC) and macrophages are the key APCs that connect innate
and adaptive immunity. ANXA1 plays a major role DC maturation. ANXA1-/-
BMDCs acquire a mature phenotype during differentiation and lose their
antigen uptake capacity compared to wild-type BMDCs (Huggins, Paschalidis,
Flower, Perretti, & D'Acquisto, 2009). This observation is supported by
29
another study where lack of DC maturation in presence of ANXA1 suppresses
T cell immunity (Weyd et al., 2013). ANXA1 is expressed by different tissue-
specific macrophages such as alveolar (Ambrose et al., 1992), peritoneal (Peers
et al., 1993), synovial (Yang et al., 1998) and microglial cells (Minghetti et al.,
1999) It exhibits a wide variety of roles in these cells such as proliferation
(Alldridge et al., 1999), cytokine production (Y. H. Yang et al., 2006) and
phagocytosis (Yona et al., 2006) making it an important molecule in the cell
functioning. The activation of these APCs is through the cell surface receptors
one of which are toll like receptors which we will discuss in next section.
1.2. Toll like Receptors (TLRs)
TLRS are type 1 transmembrane proteins that detect Pathogen Associated
molecular patterns (PAMPS). They were first identified in drosophila. They
comprise of leucine rich repeats that sense the incoming PAMP and
intracellular Toll-interleukin 1 receptor (TIR) domain that initiates the
downstream signalling. There are 13 TLRs known so far and each one
recognizes a specific PAMP. All TLRs and their ligands are given in the table
below (Table 1.1).
Receptor Ligand(PAMPs) Origin of Ligand
TLR1 Triacyllipopeptides Soluble factors
Bacteria and mycobacteria Neisseria meningitidis
TLR2 Heat Shock protein 70 Peptidogycan Lipoprotein HCV core and NS3 protein
Host Gram-positive bacteria Hepatitis C virus
TLR3 dsRNA Viruses
30
TLR4 Lipopolysaccharides Envelope protein Taxol
Gram-negative bacteria Mouse mammary-tumour virus Plants
TLR5 Flagellin Bacteria
TLR6 Zymosan Lipoteichoic acid Diacyl lipopeptides
Fungi Gram-positive bacteria Mycoplasma
TLR7 ssRNA Viruses
TLR8 ssRNA Viruses
TLR9 CpG-containing DNA Bacteria Plasmodium Viruses
TLR10 Not determined Not determined
TLR11 Profillin-like molecule Taxoplasma gondii
Table 1.1 TLRs and their ligands
TLRs 1, 2,4,5,6 and 11 are localised on the plasma membrane and mainly
recognize microbial membrane components such as lipids and proteins whereas
TLRs 3, 7, 8 and 9 are expressed in intracellular vesicles such as endosomes,
lysosomes and endolysosomes where they recognize the microbial nucleic
acids. TLRs require TIR-domain containing adapters to trigger the downstream
signalling pathways. These adapters are MyD88, Mal, TIR-domain-containing
adapter-inducing interferon-β (TRIF) and TRIF-related adaptor molecule
(TRAM). Each TLR uses one or combination of these adapters which in turn
determines the signalling pathway to be activated. The first discovered adapter,
MyD88 is used by all TLRs except TLR3 to activate NF-κB and MAPKs for
the induction of pro-inflammatory cytokines. TLRs 3 and 4 use TRIF to
activate IRFs and NF- κB for the induction of pro-inflammatory cytokines and
type I interferons. TRAM and Mal act as bridges to recruit TRIF to TLR4 and
31
MyD88 to TLR2 and 4. Therefore, TLR signalling can be classified to MYD88
dependent or TRIF dependent pathways. TLR4 is the only receptor that uses all
four adapter molecules (Kawai and Akira, 2011).
1.2.1. MyD88 dependent pathway
Upon activation of TLRs via MyD88 dependent pathway IL-1 receptor
associated kinase-4 (IRAK-4) is stimulated, which then mediates the activation
of IRAK-1 and IRAK-2. Activated IRAK-1 then interacts with Tumor necrosis
factor receptor-associated factor-6 (TRAF-6) which is an E3 ubiquitin ligase
and catalyzes the synthesis of polyubiquitin chains linked to Lys63. This
activates Transforming Growth Factor β-activated Kinase 1 (TAK1) complex
which simultaneously leads to the activation of two downstream pathways. The
first one results in the activation of activator protein 1 (AP-1) through MAPKs
and the second one activates NF-κB through IκB kinase β (IKKβ) which
phosphorylates IκB thus leading to its degradation and translocation of NF-κB
to the nucleus (Akira & Takeda, 2004; Kawai & Akira, 2010). The intracellular
TLRs 3, 7 and 9 also depend on MyD88-IRAK-TRAF6 for downstream
signalling and activate IRFs mainly IRF7. The above mentioned complex
interacts with IRF7 causing its phosphorylation and translocation to the nucleus
(Honda et al., 2004).
1.2.2. TRIF-dependent pathway
TRIF dependent pathway culminates in the activation of both IRF3 and NF-κB.
TRAF6 is recruited to the N-terminus of TRIF for the activation of NF-κB in a
mechanism similar to that of MyD88 dependent pathway. In addition to this
RIP1 is recruited to the C terminus for the formation of the multiprotein
32
complex comprising of TRIF, TRAF6 and RIP-1 thus activating TAK 1 and
NF-κB (L. A. O'Neill & Bowie, 2007). For the activation of IRF3 and IFN-β
transcription TRIF activates the non-cannonical kinases IKKε and TBK-1
through TRAF3. These kinases phosphorylate IRF-3 and drive its translocation
to the nucleus (H. Hacker & Karin, 2006).
1.2.3. Intracellular TLRs
TLRs 3, 7, 8 and 9 are the intracellular TLRs that recognize the nucleic acids
from microbial origin. Since Influenza is a ssRNA virus and forms a dsRNA
intermediate during replication TLRs 3 and 7 play a major role in its
recognition and activation of downstream signalling. They are localized in ER
in the unstimulated cells and rapidly translocate to the endolysosomes upon
stimulation (Y. M. Kim, Brinkmann, Paquet, & Ploegh, 2008). TLR 3
recognizes double stranded (ds) RNA or its synthetic analog polyionosinic-
polycytidylic acid (poly (I: C)), which mimics viral infection and induces the
production of both pro-inflammatory cytokines and Type I interferons (Akira,
Uematsu, & Takeuchi, 2006). Single stranded (ss) RNA , imidazoquinoline
derivatives and guanine analogs are the ligands for TLR7 (Kawai & Akira,
2006). TLR7 is highly expressed on plasmacytoid dendritic cells (pDCs) that
are able to produce large amounts of type I interferons during a viral infection.
Viruses are internalized and fused to the endolysosomes where TLR7
recognizes ssRNA and antiviral responses are triggered. TLR7 on cDCs also
recognize RNA from bacterial species like Streptococcus and induce type I
interferons (Mancuso et al., 2009). TLR8 is homologous to TLR7. Human
TLR8 also recognizes ssRNA and imidazoquinolines. It is not functional in
33
mice and is expressed on various cells types, the highest expression being on
monocytes. TLR9 recognizes unmethylated 2’-deoxyribo (cytidine-phosphate
guanosine) (CpG) that are present in microbes but uncommon in mammalian
cells. TLR9 ligands activate DCs, macrophages and B cells and drive strong
TH1 responses (Akira, et al., 2006).
Figure 1.3 TLR signalling pathway
TLR signalling begins with recognition of the ligands by TLR receptors. The adaptor, Myd88 associates with TLRs and recruits IRAK. IRAK then activates IKK complex to phosphorylate IκB, resulting in the translocation of NF-κB to the nucleus which induces expression of pro-inflammatory cytokines. TIRAP is another adaptor involved in TLR 2 and 4 signalling.TLR3 and 4 signal through TRIF in a MyD88-independent manner, leading to the activation of IRF-3 and production of IFN-β. Non-cannonical IKKs, IKKi and TBK-1 also activate IRF-3. Another adaptor TRAM is specific to MyD88 independent TLR4 signalling (Akira et al. 2005). .
34
1.2.4. ANXA1 and TLRs
As discussed above TLR signaling pathways culminates in the activation of
MAPKs, NF-κB or IRFs. It has been shown that ANXA1 regulates the
activation of ERK1/2 in response to LPS (Alldridge & Bryant, 2003). ANXA1
also regulates the expression of IL-6 by affecting p38 MAPK and MAPK
phosphatase-1(Y. H. Yang, et al., 2006). GCs have also been shown to interfere
with IRF-3 phosphorylation and promoter complex formation (McCoy et
al.,2008, Ogawa et al.,2005). ANXA1 can constitutively activate NF-κB in
breast cancer cells through the interaction with the IKK complex (Bist et al.,
2011) and ANXA1 deficient macrophages exhibit an aberrant expression of
TLR4 (Damazo et al., 2005). All the studies above indicate that ANXA1 plays
a role in TLR signaling. To understand the mechanism better our lab studied
ANXA1 in context of TLR 3 and 4 and found that ANXA1 C terminus of
ANXA1 directly associates with TANK-binding kinase 1 to regulate IRF3
translocation and phosphorylation (Bist et al., 2013). Influenza virus, being a
ssRNA virus is a natural ligand for TLR7 and hence it will be interesting to
study role of ANXA1 in context of Influenza virus.
1.3. Influenza Virus
Influenza virus is a single stranded RNA virus and belongs to the
orthomyxoviridae family. These are enveloped viruses usually spherical in
shape measuring around 80-120 nm in diameter. Influenza viruses can be
divided into three subtypes A, B and C based on the antigenic differences
35
between the matrix and nucleoproteins (M and NP) that exist in these viruses.
They also differ in morphology, host range surface glycoproteins and genome
organization. The subtype A is the most virulent type and is responsible for all
the pandemics of influenza in the past. It can be further divided based on the
surface glycoproteins haemagglutinin (HA) and neuraminidase (NA). There are
16 HAs and 9 NAs known so far which combine to form distinct serotypes i.e
antibodies to one serotype do not cross-react with others (Lamb and Krug,
2001).
1.3.1. Health threat and Global Impact of Influenza Viruses
The 20th century has seen three influenza pandemics. A pandemic is an
epidemic that spreads on a worldwide scale and infects a large part of human
population. The “H1N1 Spanish flu” in 1918 after the First World War took
approximately 50 million lives, more than the deaths in the war. The following
two pandemics the “H2N2 Asian flu” in 1957 and “H3N2 Hong Kong flu” in
1968 resulted in about two million deaths (Johnson & Mueller, 2002;
Kilbourne, 2006). The current information supports that wildbirds are the
reservoirs for the new evolving strains since they are the natural hosts and
contain all subtypes of HA and NA glycoproteins (Webster, 1997; Zambon,
2001). The first case of direct transmission from birds to human was observed
in 1997 during the avian influenza H5N1 outbreak (Subbarao et al., 1998).
Until 2009 few outbreaks of avian influenza were observed throughout the
world. Since this strain was highly pathogenic, had high mortality rate (Komar
& Olsen, 2008) it was suspected to cause another pandemic and had the
36
potential to kill around 5-150 million people. Following this was the 2009
H1N1 pandemic which spread rapidly around the globe. From the
announcement of its first case, it spread to 43 countries within a span of one
month. The most recent are the H7N9 outbreaks in China in April, 2013. The
cases have drastically reduced within a month due to the containment measures
but it is suspected to re-emerge as the weather becomes cooler and has a high
pandemic potential (CDC report, 2013).
Influenza pandemics are the result of antigenic shift or the reassortment of the
genome of influenza virus. Since the vRNA is segmented and present on 8
different segements it allows mixing of RNA if more than one virus particle
infects a cell. It can also take place in wild fowls where all 16 types of HAs and
9 NAs are present. This reassortment allows exchange of large proportions of
the virus genome creating a novel progeny virus that leads to the change of the
host it can infect. Due to changes in the HA and NA the virus overcomes host
immunity and spreads uncontrollably leading to pandemics (Parrish &
Kawaoka, 2005). Influenza A viruses also mutate due to antigenic drift.
Because of the absence of RNA proofreading enzymes, RNA dependant RNA
polymerase makes errors which leads to minor changes in the antigens. This
may lead to epidemics and therefore it necessitates the update of influenza
vaccines annually. Influenza B and C undergo antigenic shifts only compared
to influenza A making them less virulent and posing reduced risk of a
pandemic.
37
1.3.2. Signs and Symptoms
Symptoms of influenza include fever and chills, nasal congestion, cough,
running nose, body aches, fatigue, headache and watering eyes under non-
pandemic conditions. Approximately 33% of the people infected with influenza
virus are asymptomatic (Carrat et al., 2008). The incubation period of the virus
is about 2 days after which the symptoms appear. The infection is usually
cleared by the host immune system unless it develops complications which are
generally observed in either very young (less than 5 years) or old people (more
than 65 years) due to weakened immune system. It is difficult to diagnose
influenza only on the basis of its symptoms as these are similar to the
symptoms caused other respiratory viruses like rhinovirus or parainfluenza
virus but sudden high fever and extreme fatigue indicate a flu infection (Eccles,
2005). Influenza infection can induce hemorrhagic-bronchitis and pneumonia
the above mentioned risk groups (Taubenberger & Morens, 2008) .Secondary
bacterial infections adds to the risk and lead to more pulmonary complications.
It is therefore advisable to treat patients with antibiotics along with antivirals in
case of severe infections (Morens, Taubenberger, & Fauci, 2008).
1.3.3. Structure of Influenza virus
Influenza viruses are usually spherical 80-100nm in size (Horne, Waterson,
Wildy, & Farnham, 1960). It consists of a segmented genome encapsulated by
the envelope. The surface glycoproteins hemagglutinin (HA) and
neuraminidase (NA) spike out from the envelope and the ribonucleoprotein
consisting of the viral polymerase and non-structural proteins reside within the
38
core (Laver & Valentine, 1969). The virus obtains its lipid bilayer from the
plasma membrane during budding (Klenk & Choppin, 1970). Below the lipid
bilayer is another layer of viral membrane Matrix protein (M1). The core
containing the ribonucleoprotein (RNP) is divided into eight segments. Each
RNP contains four proteins namely the nucleoprotein (NP), polymerase basic 1
(PB1), polymerase basic (PB2), polymerase acid (PA) and a segment of RNA.
The RNP complex has RNA-dependant RNA polymerase activity (Chow &
Simpson, 1971; Inglis, Carroll, Lamb, & Mahy, 1976). The eight segments
therefore encode seven structural (PB1, PB2, PA, HA, NA, NP and M1) and
three non-structural (NS1, NS2 and M2) proteins. NS2 and M2 are splice
variations of NS1 and M1 and a recently discovered protein PB1-F2 is the
result of an alternate open reading frame of PB1 (W. Chen et al., 2001; Lamb
& Choppin, 1979).
1.3.3.1. RNA segments 1, 2 and 3: PB1, PB2 and PA
These three proteins are the transcriptase associated proteins. They derive their
nomenclature from isoelectric focussing and polyacrylamide electrophoresis.
The faster migrating basic protein coded by segment 1 is PB2, the slower
migrating protein in segment 2 is PB1 and acidic protein in segment 3 is the
PA (Horisberger, 1980). PB1 is responsible for the addition of nucleotides
during RNA chain elongation. It also possesses the endonuclease activity for
snatching the 5’ cap from host mRNA to provide primer for mRNA synthesis
(Braam, Ulmanen, & Krug, 1983) (M. L. Li, Rao, & Krug, 2001). PB2 plays a
role in replication and transcription initiation by binding to the host cap
structure (Blaas, Patzelt, & Kuechler, 1982) while PA plays a role in vRNA
39
synthesis (Palese, Ritchey, & Schulman, 1977). A few known inhibitors of the
polymerse are 2,4-dioxobutanoic acid and 2,6-diketopiperazine derivatives,
that inhibit the endonuclease activity (PB1) of the influenza virus polymerase.
Ribavirin, another antiviral, and several other nucleotide analogs inhibit viral
polymerase complex, but toxicity remains a problem (Tomassini et al., 1994).
Figure 1.4 Structure and genome organization of influenza virus
(A) HA NA and M2 are the three proteins embedded in host derived plasma membrane. Beneath the lipid bilayer is a layer of M1 which is associated with both RNP and envelope. The core consists of 8 segments or RNA with RNP proteins PB1, PB2 and PA. (B) The figure shows the 8 RNA segments and their splice variants in order of their length. (A) (Horimoto & Kawaoka, 2005) (B)Martinez-Sobrido Laboratory Resources.
1.3.3.2. RNA segment 4: Hemaglutinnin
HA is a glycoprotein responsible for viral adsorption. It is so named because of
its ability to agglutinate red blood cells (RBCs). It is the most important viral
antigens against which the antibodies are directed. Variations in HA is the
A B
40
major cause of influenza epidemics and pandemics. One of the functions of HA
include viral attachment to the host cell (Lazarowitz & Choppin, 1975). The
viral pathogenicity depends on the proteolytic cleavage of HA0 to HA1 and
HA2. Due to this cleavage the hydrophobic region of HA2 containing a fusion
peptide is exposed. Low pH drastically changes the structure of HA and the
fusion peptide inserts itself into endosomal membrane to bring it closer to viral
membrane and hence cause fusion. Presence of more than one HA forms a pore
through which the RNPs can enter the cytoplasm (Skehel et al., 1982). Quinone
derivatives inhibit the conformational changes in HA and thus prevent
infection (Bodian et al., 1993).
1.3.3.3. RNA segment 5: The Nucleocapsid protein
The NP protein is an important constituent of the RNP complex that coats the
RNA. It also interacts with the Polymerase complex comprising of PB1, PB2
and PA. It aids in the process of nuclear import of the vRNA (Cros, Garcia-
Sastre, & Palese, 2005)
1.3.3.4. RNA segment 6: The Neuraminidase
NA is a surface glycoprotein which is mushroom shaped with a stalk and a
head (Wrigley, Skehel, Charlwood, & Brand, 1973). It has a role to play in the
budding of the virus from host membrane as it hydrolytically cleaves
glycosidic bonds of the neuraminic acid (sialic acid) and frees the virus from
any sialic acid containing structures (Palese & Schulman, 1974). It is also
believed to play a role early in infection facilitating the entry of virus (M. N.
Matrosovich, Matrosovich, Gray, Roberts, & Klenk, 2004) or late
41
endosomal/lysosomal trafficking (Suzuki et al., 2005). NA inhibitors are potent
antivirals. Zanamivir (von Itzstein et al., 1993) and oseltamivir (W. Li et al.,
1998) are the two more successful ones.
1.3.3.5. RNA segment 7: The Matrix (M1) and Ion channel (M2) proteins
Segment 7 produces two viral proteins M1 and M2 due to alternative splicing
of the mRNA. The M1 is the most abundant protein of the virus. It forms a
shell beneath the lipid bilayer (Schulze, 1972). It interacts with both the surface
glycoproteins and the RNPs and form a kind of bridge between the two
(Nayak, Hui, & Barman, 2004). It is known to play an important role in virus
assembly by bringing all the viral components to the assembly site and is
indispensable for the formation of viral particles (Gomez-Puertas, Albo, Perez-
Pastrana, Vivo, & Portela, 2000). The M1 protein is synthesized on free
ribosomes and it lacks a signal peptide making its route to the plasma
membrane different from HA and NA.
M2 protein is an ion channel protein that is acid gated and is selective for H+
ions (Mould et al., 2000). It plays a role in the uncoating and release of RNP
into the cytoplasm. When the HA mediated fusion occurs between the viral and
the endosomal membrane it allows the influx of H+ into the virus particle to
cause acidification of the viral core that breaks the protein-protein interactions
to free the RNP from the M1 protein into the cytoplasm (Matlin, Reggio,
Helenius, & Simons, 1981). Amantadine and rimantadine are the antivirals and
inhibits the activity of M2 protein thus affecting the uncoating process (Sugrue
& Hay, 1991; C. Wang, Takeuchi, Pinto, & Lamb, 1993).
42
1.3.3.6. RNA segment 8: NS1 and NS2
Segment 8 is the smallest of the 8 segments and codes for two proteins that are
found only in infected cells, the non-structural proteins 1 and 2. The NS2
mRNA is translated in +1 reading frame both splice variants overlap by 70
amino acids (Inglis & Brown, 1981). NS1 inhibits interferon synthesis by
binding to the intracellular dsRNA. Production of IFN-β depends upon the
transcription of NF-κB, AP-1 and IRF-3. All these transcription factors are
reported to be strongly activated in NS1 mutants (Ludwig et al., 2002; Talon et
al., 2000; X. Wang et al., 2000). It is also involved in the inhibition of PKR, a
host protein that phosphorylates elF2α and ceases protein synthesis thus
preventing viral replication. This function of NS1 is also due to its dsRNA
binding ability. It sequesters the dsRNA which is required for the activation of
PKR (Hatada, Saito, & Fukuda, 1999). NS1 interacts with cleavage and
polyadenylation specificity factor (CPSF) and poly (A)-binding protein II. This
interaction inhibits the processing of pre-mRNAs and thus they are retained in
the nucleus. Since the host antiviral response depends a lot on transcriptional
activation of gene this inhibition delays or in some cases aborts the immune
response (Noah, Twu, & Krug, 2003)
NS2 is found to interact with the nuclear export protein crm1 and also with
M1. The RNP-M1-NS2 complex is formed in the nucleus and is responsible for
the export of RNP from nucleus via interaction with the host cellular export
machinery (R. E. O'Neill, Talon, & Palese, 1998).
43
1.3.4. Viral Life Cycle
The virus life cycle begins with the entry of virus into the host cell. The HA
protein present on the surface of virus form spikes and attaches to the sialic
acid residues on the surface of cells (Skehel & Wiley, 2000). HA is composed
of HA1, the receptor domain and HA2, the fusion peptide linked by disulphide
bonds (Q. Huang et al., 2003). Upon binding, the virus enters the cell by
receptor mediated endocytosis in an endosome (Skehel & Wiley, 2000). The
low pH in endosomes brings conformational changes in HA and exposes the
HA2 subunit which inserts itself into the endosomal membrane bringing about
fusion of viral and endosomal membranes. The acidic environment also opens
the M2 channel and acidifies the core resulting in the release of vRNP in the
cell cytoplasm (Pinto & Lamb, 2006a, 2006b).
The viral transcription and replication happens in the nucleus therefore vRNP
have to be imported into the nucleus. This is done with the nuclear localization
signals present on all the proteins that constitute the vRNP (Boulo, Akarsu,
Ruigrok, & Baudin, 2007). The import takes place with the help of cell’s active
import machinery involving the Crm1 dependent pathway.
Once inside the nucleus the negative strand is transcribed to positive sense to
serve as the template for the production of vRNA. Viral RNA dependant RNA
(RdRp) polymerase replicates the genome. No primer is required for this
process because of the sequence complementarity between the extreme 5’ and
3’ ends which results in base-pairing between the ends and formation of
various cork screw configurations (Crow, Deng, Addley, & Brownlee, 2004).
The mature mRNA contains a 5’ cap and poly (A) tail. During transcription
the RdRp snatches the cap with 10-15 extra nucleotides from the host RNA and
44
uses it to prime the viral transcription (Plotch, Bouloy, & Krug, 1979). Each
viral segment contains five to seven U residues about 17 nucleotides before the
5’ end. The RdRp stutters onto this sequence due to steric hindrance as it nears
the end and moves back and forth over this stretch forming the poly(A) tail
(Poon, Pritlove, Fodor, & Brownlee, 1999).
The negative sense vRNPs are exported out of the nucleus (Shapiro, Gurney, &
Krug, 1987). M1 binds to both vRNPs and NS2. NS2 can then export the
complex out of the nucleus by binding to Crm1 and causing GTP hydrolysis
that occurs normally in Crm1 dependant export pathway (Boulo, et al.,
2007).The exit of vRNPs from the nucleus is from the apical side of the
infected nuclei (Elton, Amorim, Medcalf, & Digard, 2005).
After vRNPs are exported out of the nucleus they are packaged into the viral
envelope and the newly formed virions bud out from the apical side of the
membrane. The virus uses host cell’s plasma membrane to form the lipid
bilayer of the envelope and HA, NA and M2 must be present for the formation
of the virions (Nayak, et al., 2004). Packaging sequences are present in the
coding and non-coding regions of viral segments (Liang, Hong, & Parslow,
2005). The last step before the virus exits the cell is the cleavage of sialic acid
residues from glycoproteins by the NA protein so that the virus can be released
from the membrane (Palese, Tobita, Ueda, & Compans, 1974).
45
Figure 1.5 Replication and transcription of Influenza virus.
The virus is adsorbed into the cell via receptor mediated endocytosis and fuses with the endosomes. The low pH in the endosome results in the release of RNPs into the cytoplasm. The RNP then moves into the nucleus and serves as the template for transcription. The viral proteins are synthesized from mRNA. The replication of the vRNA is through a positive sense intermediate cRNA. The viral proteins then move to the apical plasma membrane where they are packaged and released (Fields virology 5th Edition, 2006).
1.3.5. Influenza Tropism
Most influenza virus strains have restricted tissue tropism but certain strains
can circulate in more than one species. Though the host factors restrict virus
infection in a new species the previous pandemics reiterate the fact that due to
gene assortment the virus is able to overcome these host barriers (Medina &
Garcia-Sastre, 2011). In general the specificity and affinity of HA is one of the
determining steps in host tropism and transmission. The virus infects the cell
46
by binding to the cell surface glycoproteins through HA. The tropism of
influenza virus is based on the differences in sialic acid glycosylation. The
human viruses (containing H1-H3) recognize receptors with terminal α-2,6-
Silaic acid moieties which are usually present on the bronchial epithelial cells
of the human upper respiratory tract (URT) (Stevens et al., 2006). In contrast to
this the avian influenza viruses bind to a slightly different moiety i.e galactose
linked α-2,3-Sialic acid which is abundant on the epithelial cells present in the
intestine of the birds and lower respiratory tract (LRT) of the humans (M.
Matrosovich, Zhou, Kawaoka, & Webster, 1999). Though α-2,3-SA is also
present in non-ciliated cells of the lower tracheal epithelium these cells are in
minority and the density of receptors on these cells is very low , which explains
the low transmissibility of avian viruses in humans (Maines et al., 2006).
However, pigs contain both α-2,6-SA and α-2,3-SA in their trachea and hence
they acts as “mixing vessels” for the reassortment of human and avian strains
resulting in the production of a new pandemic potent strain (Ito et al., 1998).
Turkeys, pheasants, quail and guinea fowl also contain both receptor types in
their intestines and respiratory tract and can thus act as “mixing vessels”.
1.3.6. Immune responses against Influenza virus
The first line of defence against the influenza virus is innate immune system.
Viral nucleic acids act as pathogen associated molecular patterns (PAMPS) and
are recognized by pathogen recognition receptors (PRRs) to activate the
downstream signalling pathways for the antiviral response. There are three
classes of viral nucleic acids namely ssRNA, dsRNA and DNA that can elicit
an immune response. These nucleic acids in the cytoplasm of infected cells can
47
be sensed by ubiquitously expressed dsRNA-dependent protein kinase R
(PKR) , retinoic acid inducible gene I (RIG-I), melanoma differentiation-
associated gene 5 (MDA-5) or cell specific TLRs (described earlier). Each of
them will be discussed in detail in the following sections.
1.3.6.1. PKR
PKR is serine/threonine kinase that recognizes dsRNA. It is an IFN inducible
gene. Upon RNA binding the two RNA binding domains dimerize and causes
autophosphorylation and thus activation of its enzymatic activity (Garcia-
Sastre & Biron, 2006). PKR inhibits cellular and viral protein translation by
phosphorylating eukaryotic initiation factor 2α (eIF2α). This impairs the
GDP/GTP exchange leading to reduced production of GTP-eIF2α which is
essential for translation (Samuel, 2001). PKR can regulate NF-κB and IFN-
stimulated genes to amplify the IFN response.
1.3.6.2. RIG-I and MDA-5
RIG-I and MDA-5 are RNA helicases that recognize the viral RNA in the
cytoplasm. Both helicases belong to the DExD/H box-containing RNA helicase
family with a conserved domain structure. They contain two N-terminal
caspase recruitment domains (CARD) and one C-terminal DExD/H box
helicase domain (Unterholzner & Bowie, 2008). The IFN-β promoter
stimulator-1 (IPS-1) also called mitochondrial anti-viral signalling (MAVS) or
virus induced signalling adapter (VISA), is the adapter molecule that also
contains a CARD domain homologous to that of RIG-I and MDA-5. The
CARD-CARD interaction between these helicases and their adapter leads to
the activation of NF-κB, IRF3 and IRF7 (Kawai et al., 2005; Seth, Sun, Ea, &
48
Chen, 2005; Sun et al., 2006; Xu et al., 2005). IPS-1 is located in the outer
mitochondrial membrane by a transmembrane domain which is essential for
activation of downstream pathways.
IPS-1 makes use of IκB Kinase α (IKKα) , IKKβ , IKKε and Tank binding
kinase (TBK-1) to activate the downstream signalling pathways. IKKγ
facilitates the recruitment of TBK-1 and IKKε to IPS-1-TRAF3 complex.
TRAF3 serves as a bridge for the interaction of these kinases with IPS-1 (Saha
et al., 2006). IPS-1 also interacts with TRAF2, TRAF6, FADD and RIP1 which
are involved in NF-κB activation (Xu, et al., 2005).
Originally, both RIG-I and MDA-5 were thought to be the sensors for dsRNA
but generation of RIG-I and MDA-5 Knockout mice revealed that the function
of the two helicases is not redundant. MDA5 recognizes picornaviruses while
RIG-I recongnizes viruses like Sendai virus, Influenza virus etc. (Kato et al.,
2006). It was observed that viruses that produce large amounts of dsRNA
during replication were detected by MDA-5 while those producing small
amounts of dsRNA only as an intermediate were detected by RIG-I. More
experiments lead to the understanding that MDA-5 recognizes a dsRNA
structure while RIG-I recognizes ss or ds RNAs with a triphosphate at the 5’
end (Pichlmair et al., 2006).
Another intriguing topic that is studied in detail is how self RNA molecules are
distinguished from viruses RNAs. Ribosomal RNA, transfer RNA and pre-
micro RNAs possess a 5’ monophosphate, the mRNAs and small nuclear
RNAs possess methylguanosine cap and some other RNAs possess
pseudouridine or 2’-O-methylation that keep them from detection by RIG-I
(Marques et al., 2006).
49
Figure 1.6 RIG-I and MDA-5 Signalling RIG-I and MDA-5 are cytoplasmic sensors that recognize 5’ tri-phosphate RNA or dsRNA respectively. Both these sensors and their Adaptor protein MAVS contain CARD domains. Due to CARD-CARD interaction between these helicases and their adapter leads to the activation of NF-κB, IRF3 and IRF7 (http://en.wikipedia.org/wiki/File:Simon_%28RIG-I_and_Mda5%29.jpg).
1.3.6.3 Influenza virus Induced signalling cascades
Influenza infection triggers various signalling pathways and activates several
transcription factors like NF-κB, AP-1, IRFs, signal transducers and activators
of transcription (STATs) and nuclear factor IL-6 (NF-IL-6) (Julkunen et al.,
2001). Several cytokines possess NF-κB binding sites in their promoters;
therefore for the production of cytokines, activation of NF-κB is important. It
has also been shown that NF-κB signalling is important for efficient virus
50
propagation (Nimmerjahn et al., 2004). Upon infection, NF-κB is activated in a
biphasic manner. It is first detectable 1hr post infection and the later activation
depends on viral replication and protein synthesis (Ronni et al., 1997). Its
activation is dependent upon activation of IKK in response to viruses.
Another important pathway that is activated is the MAPK pathway. Influenza
activates several members of the MAPK family such as ERK1/2, p38 and JNK
(Kujime, Hashimoto, Gon, Shimizu, & Horie, 2000) resulting in the activation
of AP-1. Unlike NF- κB, AP-1 requires viral replication for activation and is
not affected by over expression of viral proteins (Flory et al., 2000).
TLRs 3, 7 and 9 signalling lead to the activation of IRFs 1, 3, 5 and 7, IRFs 3
and 7 being the most critical. IRF3 and IRF 7 are present in the cytoplasm and
translocate to the nucleus upon phosphorylation of the C-terminus serine
residues (Marie, Durbin, & Levy, 1998). Both these transcription factors
undergo dimerization to activate their targets. The activation of IRF7 depends
on the STAT1-STAT2-IRF9 complex called the Interferon stimulated gene
factor 3 (ISGF3). IRF3 mainly leads to the production of IFN β (and IFN α4)
whereas IRF7 can lead to the production of both IFN β and all forms of IFN α.
IRF3 homodimer or IRF3/IRF7 heterodimer can produce small amount of
interferons which bind to its receptor IFNAR resulting in robust production of
type I interferons forming a positive feedback loop (Honda, Yanai, Takaoka, &
Taniguchi, 2005).
51
1.3.7. Humoral Response
Humoral immunity provides long term protective effects through neutralizing
antibodies against HA and NA proteins of the virus. They prevent reinfection
by an antigenically similar or identical strain. Neutralizing antibodies are the
basis of seasonal vaccinations and have to be periodically updated due to rapid
mutations in the viral coat. They also play a role in viral clearance during the
primary challenge since mice deficient in B cells have compromised clearance
of virus from the respiratory tract (Kris, Asofsky, Evans, & Small, 1985). The
main influenza-specific antibody isotypes are IgA, IgG and IgM. Mucosal or
secretory IgA antibodies are produced locally in the infection site and
transported along the mucus of the respiratory tract thus, conferring local
protection from infection of airway epithelial cells. These antibodies are also
able to neutralize influenza virus intracellularly (Mazanec, Coudret, &
Fletcher, 1995). IgAs are produced rapidly after influenza virus infection and
the presence of these antibodies indicates a recent infection (Rothbarth, Groen,
Bohnen, de Groot, & Osterhaus, 1999). IgG antibodies predominantly
transudate into the respiratory tract and provide long-lived protection (Murphy
et al., 1982). IgM antibodies initiate complement mediated neutralization of
influenza virus and are a hallmark of primary infection (Fernandez Gonzalez,
Jayasekera, & Carroll, 2008).
1.3.8. T cell Response
Influenza infection leads to the proliferation of both CD4+ and CD8+ T cells.
CD4+ T cells mainly secrete cytokines like IL-4 and IL-13 to promote B cell
52
responses and IFN-γ and IL-2 to aid cellular responses. CD8+ T cells are
responsible for the lysis of the infected cells and thus prevent the production of
progeny virus. Upon infection these cells are activated in the lymphoid tissues
and recruited to the site of infection. CD8 T-cells recognise infected cells by
interaction of the T cell receptor (TCR) with viral antigens loaded onto major
histocompatibility complex I (MHC I) on the cell surface. The cytolytic
activity is due to the perforins and granzymes. Perforins permeabilize the
membrane of the infected cells and granzymes enter these to induce apoptosis.
They can also induce apoptosis through Fas and TRAIL interactions (Brincks,
Katewa, Kucaba, Griffith, & Legge, 2008; Topham, Tripp, & Doherty, 1997).
Effector CD8 T cells can secrete large amounts of cytokines like IFN-γ, TNF-α
and GM-CSF. Human CTL induced by influenza virus infection are mainly
directed against NP, M1 and PA proteins (Gotch, McMichael, Smith, & Moss,
1987). These proteins are highly conserved and therefore CTL responses
display a high degree of cross-reactivity unlike humoral immunity, even
between different subtypes of influenza A virus
1.3.9. Annexins and viruses
As discussed above the one of the important host defence mechanism against
Influenza is triggering the signalling through TLR pathway and a novel player
in this pathway is ANXA1. Whether and how ANXA1 is involved in
virus‐triggered signalling is unknown. Mass spectrometry data obtained by our
lab shows that ANXA1 might be involved in anti‐viral immunity. Annexins
have been known to be involved in infectious diseases such as Tuberculosis
53
(Gan et al., 2008), HIV (Ryzhova et al., 2006) and Hepatitis C (Backes et al.,
2010). Importantly, Annexin V was identified as a protein binding to the
proteins of Influenza virus (Otto, Gunther, Fan, Rick, & Huang, 1994).
Annexin V helps the entry of influenza virus into the cell. In addition, it has
also been proposed that annexins might be serving as the receptor for the
viruses (R. T. Huang, Lichtenberg, & Rick, 1996). Annexin II has been shown
to support viral replication when incorporated in Influenza virus by converting
plasminogen into plasmin (LeBouder et al., 2008). Annexin 1 were also found
to inhibit Cytomagalovirus infection (Derry, Sutherland, Restall, Waisman, &
Pryzdial, 2007). Furthermore it was reported that ANXA1 levels were
upregulated in porcine monocytes infected with swine flu virus (Katoh, 2000).
Another report shows human Annexin 6 binds to cytoplasmic tail of the ion
channel protein (M2) of the Influenza virus and negatively modulates infection
(Ma et al., 2012). Recently published data from lab shows the ANXA1
regulates TLR-mediated production of IFN-β by interacting with (TBK-1) in
macrophages upon activation with LPS or Poly(I:C) (Bist, et al., 2013). This
led us to postulate that ANXA1 could be involved in anti‐viral immunity. It is
known that Influenza virus induces apoptosis and ANXA1 also plays a role in
apoptosis however it is not known if ANXA1 plays a role in influenza induced
apoptosis. Apoptosis is discussed in detail in the next section.
1.4. Apoptosis
The term Apoptosis is was coined by John Kerr in 1972 referring to the
formation of small apoptotic bodies formed during the disintegration of the cell
(Kerr, Wyllie, & Currie, 1972). It also also known as Programmed cell death
54
and is important for the development, tissue homeostasis and effective immune
sytem. Due to its importance any disturbance in its regulation brings out
numerous pathological conditions like autoimmune disorders and cancers
(Cory & Adams, 2002). Once the apoptotic switch is triggered it results in the
activation of a family of cysteine proteases known as caspases. This switch
however can be activated either by transmembrane receptor-mediated
interactions, also known as extrinsic pathway (Strasser, O'Connor, & Dixit,
2000) or by intracellular damage, known as the Intrinsic pathway (Gross,
McDonnell, & Korsmeyer, 1999).
1.4.1. The Receptor mediated/ Extrinsic Pathway
The receptor mediated pathway involves death receptors that are members of
TNF receptor gene family like Fas (CD95) and TNF-R1 (Locksley, Killeen, &
Lenardo, 2001). These receptors contain a cysteine-rich extracellular domain
and a cytoplasmic death-domain of about 80 amino acids (Ashkenazi & Dixit,
1998) . This pathway activates caspas-8 and caspase 10 in humans (initiator
caspases) via Fas-associated death domain (FADD). FADD associates with
pro-caspase 8 due to dimerization of homologous death effector domains
(DEDs) which leads to the formation of Death inducing signalling complex
(DISC). DISC brings zymogens in close proximity resulting in auto-catalytic
activation of pro-caspase 8 (Kischkel et al., 1995). After the activation of
caspase-8 the execution pathway if triggered which involve the downstream
caspases. The extrinsic pathway can be inhibited by a protein called c-FLIP, a
dominant negative caspase-8 which binds to FADD and pro-caspase 8 leading
55
to the formation of an inactive DISC (Kataoka et al., 1998; Scaffidi, Schmitz,
Krammer, & Peter, 1999). Apart from Fas there are other death receptors as
well like DR4, DR5, CAR1, TNF-R1 and p75 which are activated by TNF-
related apoptosis inducing ligand (TRAIL). These receptors are present on
most cell types and recruit FADD and induce DISC in a similar fashion as Fas
(Lawen, 2003). TRAIL also plays a role in the apoptosis of autoreactive T cells
(Lamhamedi-Cherradi, Zheng, Maguschak, Peschon, & Chen, 2003). TNF-R1
unlike Fas can also signal for proliferation depending upon the downstream
molecules recruited to DISC. TNF-R associated death domain protein is the
one that signals either via FADD to induce apoptosis or RIP1 and TRAF to
promote survival (MacEwan, 2002).
1.4.2. The Intrinsic pathway
The intrinsic pathway leads to the initiation of apoptosis due to the intracellular
stimuli which directly acts on mitochondria. This leads to the release of
mitochondrial proteins into cytoplasm due to opening of the mitochondria
permeability transition (MPT) pore and loss of mitochondria transmembrane
potential, a process known as mitochondria outer membrane permeabilisation
(MOMP) (Ekert & Vaux, 2005). The released proteins can be divided into 2
main groups. The first one consists of cytochrome c, Smac/DIABLO and the
serine protease HtrA2/Omi (Du, Fang, Li, Li, & Wang, 2000; Elmore, 2007;
Garrido et al., 2006). Cytochrome c induces a conformational change in
Apoptotic protease activating factor (Apaf-1) which allows it to recruit
procaspase-9 into the resulting giant complex known as “apoptosome” (Hill,
56
Adrain, Duriez, Creagh, & Martin, 2004). Mature caspase-9 is formed from its
precursor zymogen due to allosteric interactions and dimerization rather than
cleavage (Rodriguez & Lazebnik, 1999). Smac/DIABLO and HtrA2/Omi
promote apoptosis by inhibiting the inhibitors of apoptosis proteins (IAPs). The
second group of mitochondrial proteins consists of caspase dependent
Apoptosis inducing factor (AIF) or independent Caspase activated DNase
(CAD) endonucleases which translocates to the nucleus and leads to DNA
fragmentation (Susin et al., 2000).
The release of cytochrome c is a crucial event in apoptosis and hence must be
tightly regulated. This task is carried out by Bcl-2 family of proteins. This
family is divided into 3 subgroups depending on the structural and functional
characteristics. The Bcl-2 like proteins Bcl-2, Bcl-XL, Bcl-w, Mcl-1 and A1 are
inhibitors of apoptosis, while Bax, Bak and BH3-only proteins are pro-
apoptotic. BH3 only proteins trigger the cytochrome release by activating Bax
and/or Bak (G. Hacker & Weber, 2007).
57
Figure 1.7 Structure of Bcl-2 family proteins The proteins are divided based on the Bcl-2 homology (BH) domains. Most Bcl-2 like proteins have all 4 BH domains, the “multidomain” proteins share 3 (BH1, BH2 and BH3) and the BH3-only proteins just have 1 BH domain (G. Hacker & Weber, 2007). Localization of Bcl-2 family members is important for their function. Bcl-2 is a
membrane protein even in healthy cells, Mcl-1 is cytosolic and the others Bcl-
w and Bcl-XL bind to membranes only upon the apoptotic stimuli. (Hsu,
Wolter, & Youle, 1997). Bax is a cytosolic protein while Bak is bound to the
membrane in healthy cells (Griffiths et al., 1999; Mikhailov et al., 2001). Upon
stimulation Bax migrates to the mitochondrial membrane and undergoes
conformational changes. Bak also undergoes conformational change and forms
oligomers. Bax or Bak oligomers coalesce to from larger complexes
58
(Mikhailov et al., 2003) that can form pores in the mitochondria leading to
cytochrome c release (Antonsson, Montessuit, Lauper, Eskes, & Martinou,
2000).
A few mechanisms have been proposed for the exact apoptotic pathway but
none has been proven fully. An example of a cross-talk between extrinsic and
intrinsic pathway is mitochondrial damage by Fas pathway and caspase-8
cleavage of Bid (Igney & Krammer, 2002). Bad is regulated by serine
phosphorylation and once phosphorylated it is trapped by 14-3-3 (phophoserine
binding molecules). Upon unphosphorylation it moves to mitochondria and
releases cytochrome c (Zha, Harada, Yang, Jockel, & Korsmeyer, 1996). Bad
can also associate with Bcl-2 and Bcl-XL neutralizing their anti-apoptotic
effects (E. Yang et al., 1995). Bcl-2 and Bcl-XL when free inhibit the release of
cytochrome c and control the activation of caspases (Newmeyer et al., 2000). A
few other members of bcl-2 family like Puma and Noxa induce apoptosis in p-
53 dependant manner. Puma can activate Bax and lead to lowering to
mitochondrial potential resulting in cytochrome c release (Liu, Newland, & Jia,
2003). Noxa on the other hand migrates to the mitochondria and sequesters
anti-apoptotic proteins resulting in caspase 9 activation (Oda et al., 2000).
Mechanism for activation of Bax and Bak is still unclear, however it is
believed that BH3-only protein Bid plays an important role in their activation
(Desagher et al., 1999). Three models have been proposed for the interplay
between the Bcl-2 family members. (A) Bcl-2 proteins inhibit the activation of
Bax/Bid by controlling direct interactions of BH3-only proteins with Bax/bak.
(B) BH3-only proteins indirectly activate Bax/BAk by inhibiting Bcl-2 protein
59
activities. (C) A few BH3-only proteins activate Bax/Bak while others inhibit
Bcl-2 proteins (Adams, 2003).
Figure 1.8 The two main pathways of apoptosis The receptor mediated pathway (on the left) is triggered by Fas/Cd95 binding to its receptor and acativating caspase-8 via the adapter molecule FADD. The intrinsic pathway is activated by the intracellular insults like DNA damage in this case and leads to the release of cytochrome c, AIF and Smac/DIABLO due to the interplay between Bcl-2 family members. The released cytochrome c aids the formation of apoptosome comprising of itself, procaspase-9 and Apaf-1. Both receptor mediated and intrinsic pathways culminates at the point of activation of executioner caspases like caspase 3, 6 or 7 resulting in the downstream execution pathway (Hengartner, 2000).
60
1.4.3. The Execution Pathway
Both extrinsic and intrinsic pathways culminate in the execution phase, which
is considered as the final step of apoptosis. Activation of the caspases bring
about the initiation of this step. There are 14 capases known so far and they can
be divided into three classes (1) the initiator caspases that are described earlier
in the extrinsic and intrinsic pathway, characterised by long prodomains
containing either DED domains (eg. Caspase 8 or 10) or a caspase recruitment
domain (CARD) (eg. Caspase 2 and 9). (2) The executioner caspases
containing short prodomains (caspase 3, 6 and 7) and (3) Caspases playing a
role in cytokine production rather than apoptosis (Grutter, 2000) . Initiator
caspases cleave executioner caspases and activate them. Execution caspases
then activate endonucleases and proteases that degrade nuclear material and
cytoplasmic and nuclear proteins. The executioner caspases have various
substrates like Poly (ADP-ribose) polymerase (PARP), cytoskeletal protein α-
fodrin, NuMA etc. which bring about the morphological and biochemical
changes in the dyeing cells (Slee, Adrain, & Martin, 2001).
Caspase-3 is considered as the most important execution caspase. It realeases
the endonuclease CAD from its inhibitor ICAD and brings about the degration
of the chromosomal DNA (Sakahira, Enari, & Nagata, 1998). It also cleaves
Gelsolin, an actin binding protein resulting in the disruption of cytoskeleton,
cell division and signal transduction (Kothakota et al., 1997). Apoptosis ends
with the phagocytocis of apoptotic cells due to the “eat me” signals like
flipping of the PS molecule towards the outer leaflet of the cell, relayed from
the cell (Bratton et al., 1997).
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1.4.4. Influenza and Apoptosis
Apoptosis is a powerful strategy used by the host to inhibit viral propagation
but over time many viruses have evolved to control the apoptotic response in
the host (Galluzzi, Brenner, Morselli, Touat, & Kroemer, 2008). Three out of
eleven influenza virus proteins namely NS1, PB1-F2 and M2 have been
implicated in manipulation of apoptosis (Herold, Ludwig, Pleschka, & Wolff,
2012). NS1 is an important factor that promotes viral gene expression by
inhibiting the Type 1 interferon activity of the host (Hale, Randall, Ortin, &
Jackson, 2008; Wolff & Ludwig, 2009). It was observed that delNS1 mutants
cause more apoptosis compared to the WT suggesting that NS1 extends viral
lifetime in the cell (Zhirnov, Konakova, Wolff, & Klenk, 2002). These effects
were partially attributed due to the anti-interferon activity of the protein
because they were not observed in IFN deficient cells (Schultz-Cherry,
Dybdahl-Sissoko, Neumann, Kawaoka, & Hinshaw, 2001). NS1 also interacts
with the p85 subunit of the PI3K to activate the pro-survival pathways
(Ehrhardt, Wolff, & Ludwig, 2007; Ehrhardt et al., 2007) and suppresses
protein kinase R (PKR) activity to inhibit DISC formation and hence apoptosis
(Balachandran et al., 2000).
PB1-F2, contrary to NS1 is a pro-apoptotic protein contributing to viral
pathogenicity (W. Chen, et al., 2001). It interacts with mitochondrial proteins
like adenine nucleotide translocase 3 and voltage-dependent anion channel
(VDAC) leading to MOMP (McAuley et al., 2010; Zamarin, Garcia-Sastre,
Xiao, Wang, & Palese, 2005). Strain dependant differences have been observed
in case of PB1-F2 with respect to viral replication (Le Goffic et al., 2010),
62
polymerase modulation (Mazur et al., 2008), and mitochondrial localization (C.
J. Chen et al., 2010).
The ion channel protein M2 inhibits macroautophagy which is important for
cell survival and also binds to beclin-1 suggesting that it contributes to the
induction of apoptosis by influenza virus (Gannage et al., 2009).
Figure 1.9 Model of Influenza induced Apoptosis. (A) In the early stages of infection NS1 interacts with p85 subunit of PI3K and activates Akt pathway by phosphorylation which regulates the downstream pro-apoptotic factors like caspases, BAD and GSK3β and suppress apoptosis in the early stages.(B and C) In the later stages the virus induces NF-κB expression by activating IKK which phosphorylaties the inhibitor of κB alpha (IκBα) and promotes its localisation into the nucleus.NF-κB activation leads to the expression of pro-apoptotic factors like TRAIL and FasLwhich induce caspases leading to the increased RNP transport (Herold, et al., 2012) As shown in the figure, the apoptotic pathways in the later stages are beneficial
for the viruses. These pathways promote viral replication and propagation. In
caspase deficient cell line MCF-7 the replication efficiency of IV was very low
and was boosted upon ectopic expression of caspase-3 (Wurzer et al., 2003).
63
Overexpressions of anti-apoptotic protein Bcl-2 also lead to decreased virus
titers (Olsen, Kehren, Dybdahl-Sissoko, & Hinshaw, 1996). Mechanistically,
this observation was due to the retention of vRNP complexes in the nucleus.
The transport of RNPs can be either via the caspase dependent passive
transport machinery or Crm-1 dependant active transport as observed by using
caspase and Crm-1 inhibitors (Elton et al., 2001; Faleiro & Lazebnik, 2000).
These findings can be merged to develop a model where the vRNP is
transported via the active machinery in the initial stages but once the caspases
are activated they cleave the active transport proteins and then the transport is
through the widened nuclear pores created by caspase due to the degradation of
nuclear pore proteins (Faleiro & Lazebnik, 2000; Herold, et al., 2012; Kramer
et al., 2008). Also, upon infection the death ligands TRAIL and FasL are
upregulated in a NF-κB dependant manner and hence regulate virus
propagation through apoptosis (Wurzer et al., 2004).
1.5. Aims of the study
This project aims to characterize the role of ANXA1 in TLR7 signalling and
influenza virus pathogenesis by conducting both in vitro and in vivo studies.
1.6 Hypothesis
ANXA1 might plays a role in TLR 7 signalling and influence influenza virus
infection since it is a natural TLR7 ligand.
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Chapter 2 Materials and Methods 2.1 Media and buffers
2.1.1. PBS
10X PBS buffer was obtained from 1st base (Singapore). The stock comprised
of 137mM NaCl, 2.7mM KCl and 10mM phosphate buffer. It was diluted to
1X using 9 parts of sterile water. pH was adjusted to 7.2-7.4 and the solution
was autoclaved.
2.1.2. FACS buffer
FACS buffer consists of 1X PBS supplemented with 1% FBS and 2mM
EDTA. 0.05% sodium azide was added as a preservative to allow usage under
non sterile conditions.
2.1.3. Red Blood Cell (RBC) Lysis Buffer
10X RBC lysis buffer was prepared by dissolving 8.3g Ammonium Chloride,
0.84g Sodium Bicarbonate and 0.03g EDTA in water, the pH adjusted to 7.4
the solution was then filter sterilized using 0.22um filter (Sartorius,Singapore)
and volume was made up to 100ml. For use as a 1X solution, the 10X stock
solution was diluted with water.
65
2.1.4. Wash buffer for Western Blotting (TBST)
Wash buffer for Western Blotting was 1X Tris-Buffered Saline (TBS)
supplemented with 0.5% v/v Tween-20, pH adjusted to 7.2 to 7.4.
2.1.5. Buffer for ELISA
Wash buffer for ELISA was 1X PBS with 0.5% v/v Tween-20, pH adjusted to
7.2 to 7.4. Blocking buffer was 1X PBS with 1% w/v BSA, unless or otherwise
stated by the ELISA kit.
2.1.6. Complete DMEM for cell culture
Complete DMEM was prepared using Dulbecco’s Modified Eagle Medium with L-
Glutamine, 4.5g/L D-glucose and sodium bicarbonate (Sigma,Singapore),
supplemented with 10% heat-inactivated Fetal Bovine Serum (Biowest, France)
and 1% Pencillin-Streptomycin solution ( Hyclone, USA).
2.1.7. Complete F-12K media for cell culture
Nutrient Mixture F-12 Ham Kaighn's Modification (Sigma, USA) with L-
Glutamine and sodium bicarbonate was supplemented with 10% heat-
inactivated Fetal Bovine Serum (Biowest, France) and 1% Pencillin-
Streptomycin solution (Hyclone, USA).
66
2.1.8 Plain DMEM at 2X concentration
DMEM powder with L-Glutamine and 4.5g/L D-Glucose (Sigma, Singapore)
was dissolved in half the recommended volume of sterile water. 3.7g/L of
Sodium bicarbonate (Sigma, Singapore) was added. The pH of the solution was
adjusted to 7.4 and supplemented with 2% v/v Non-essential amino acids
(Sigma, Singapore), as well as 1% Penicillin-Streptomycin (Hylone,
Singapore) before filter sterilization through a 0.22μm filter (Nalgene, USA).
2.2. Cell Culture
2.2.1. Cell Lines
The human epithelial lung cancer cell line A549 (CCL-185, ATCC, USA) was
cultured in complete F-12K media (Sigma, Singapore). Macrophage Colony
Stimulating Factor (M-CSF) producing L929 cells were generously provided
by Dr. Sylvie Alonso and were cultured in complete DMEM
(Sigma,Singapore) media. All cell lines and primary cells were grown at 37ºC
in a humidified 5% CO2 incubator (Thermo Scientific, Singapore).
2.2.2. Preparation of L929 Conditioned Media
L929 cells were split from one T-75 flask into five T-75 flasks when it was
almost 90% confluent, using trypsin (Hycone ,USA). When these five T-75
flasks were confluent in about approximately 60hrs, they were trypsinized and
seeded equally into thirteen T-175 flasks, each with 50 ml of complete DMEM.
After these flasks became confluent they were incubated for additional two
67
days to condition the media. The supernatant of all 13 T-175 flasks was filtered
and stored at -20oC for further use.
2.2.3. Generation of Bone Marrow Derived Macrophages (BMMOs)
Six to ten week old Balb/c mice were used for the extraction of Bone marrow.
Mice were sacrificed and their hind limbs were obtained. The bones were
cleared of the flesh using a gauze cloth/ tissue and were disinfected by dipping
in 70% ethanol .They were then transferred to a dish containing DMEM media.
The two ends of each bone were cut so that a 27G needle can pass through it,
flushing the bone marrow out into a 50 ml Falcon tube through a 70 μm cell
strainer (BD, U.S.A.) placed on top of the tube. Tubes were then spun at 280g
for 10 mins at 4°C. Cell pellet was resuspended in 1 ml of red blood cell lysis
buffer and incubated on ice for 5 mins. 5 ml of full DMEM was added to stop
the lysis and the cells were then spun at 2000 rpm for 5 mins. They were then
counted and seeded with 20% L929 conditioned media (L cell media), and
incubated for seven days. The media was topped up on day 3 to replenish the
MCSF required for differentiation.
2.2.4. Generation of Bone Marrow Dendritic Cells (BMDCs)
Bone marrow cells were obtained as described above for BMMOs. After RBC
lysis cells cells were resuspended in complete RPMI media supplemented with
non-essential amino acids (1X) (Sigma, Singapore), vitamins (1X) (Sigma,
Singapore) and GM-CSF derieved from supernatant of J558L cell line (a kind
gift from Dr. Veronique Angeli) at a concentration of 2x106/ml. On day two,
the dish was topped up with equal amount of media already present in the dish.
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Cells were pelleted and media was completely replaced on day four and
replenished with fresh media. Cells were plated again at the same concentration
as mentioned above and were used on day seven.
2.2.5. Isolation of splenic cells
Spleens were isolated from the mice and homogenized. The cell suspension
was passed through a 70µm strainer to obtain a single cell suspension. This cell
suspension was used for the CFSE staining. For western blot B cells were
purified using Easysep mouse B cell enrichment kit (STEMCELL
technologies, Singapore). All cells were cultured in complete DMEM.
2.2.6. Lung cell culture
Lungs were harvested from euthanized mice and excised into small pieces in
1mg/ml Collagenase Type A (Roche Diagnostics, Singapore) and digested for
45 min at 370C. The suspension was homogenized using a Pasteur pipette after
20 mins of digestion. The digested lungs were physically disrupted into a
single cell suspension by passing through a 70µm cell strainer (Fischer
Scientific, Singapore). Single cell suspensions were washed with F-12K media
to remove collagenase and were plated into 10 cm diameter plate. The media
was changed after three days. The cells were ready to use after 7 days.
2.2.7. Cell staining with CFSE and proliferation analysis
Splenic cells were obtained as described earlier. The cells were then stained
with 5µM CFSE dye (Sigma, Singapore) for 30 mins at 37ºC. The cells were
washed twice and plated in complete DMEM. They was then treated with
1µg/ml of R848 (Invivogen, USA). Cells were harvested after 72 hrs, data was
69
acquired using CyAn analyzer (Beckman Coulter, Singapore) and analysis was
done using FlowJo software (TreeStar, USA).
2.2.8. Thymidine Assay
Splenocytes (2x105) were treated with various concentrations of R848 for 72
hrs and thymidine at a concentration of 0.5milliCurie was added 18 hrs before
harvesting cells. The readings were taken using a scintillation counter.
2.2.9. Transfection Over-expression and knock down of ANXA1 in A549 cells was done using
Turbofect transfection reagent (Fermentas, USA). DNA to reagent ratio was
maintained at 1:2. 100,000 cells were seeded in a 24 well plate the day before
transfection antibiotic free media. Transfection was performed according to
manufacturer’s instructions. 500ng of DNA was transfected per well. Media
containing the transfection complexes was changed to complete media 6 hrs
after transfection. Cells were used for subsequent experiments 24-36hrs post
transfection.
2.3. Mice
Balb/c mice 6-10 weeks old were purchased from Centre for Animal Resources
at the National University of Singapore. ANXA1-/- mice were a kind gift from
Prof. Roderick Flower. Mice were housed and bred in pathogen free conditions
.in compliance with the Institutional Animal Care and Use Committee
guidelines at the national University of Singapore.
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2.3.1. Infection of mice with Influenza virus
6-8 weeks old Balb/c mice were anesthetized with intraperitoneal injections of
Ketamine, 100mg/kg and Medetomidine, 15mg/kg cocktail. (Comparative
Medicine, NUS). Intratracheal challenge with influenza was carried out with
500pfu of the virus in 20µl of PBS. Atipamezole (Comparative Medicine,
NUS), 5mg/kg was administered by intraperitoneal injection to reverse the
effects of the anaesthetic.
2.3.2. Cellular Infiltration
Broncheoalveolar lavage (BAL) was obtained from the uninfected and
infected mice 1,3 and 7 days post infection and was spun to separate the cells
and lavage. The RBCs were removed using a RBC lysis buffer and cells were
counted using a hemacytometer. These cells were then stained with CD45,
CD11c, CD3/ CD19, Ly6-G, CD11b, CD14, F4/80 and Siglec F to determine
the cellular composition.
Target Host Conjugation Source
CD3 Rat PerCp Cy5.5 ebioscience
CD11b Rat APC Cy7 eBioscience
CD11c Hamster PE Texas Red eBioscience
CD14 Rat FITC eBioscience
CD19 Rat PerCp Cy 5.5 eBioscience
CD45 Rat PE Cy7 BD Pharmingen
F4/80 Hamster APC BD Pharmingen
Ly6G Rat Pacific Blue Biolegend
Siglec F Rat PE BD Pharmingen
Table 2.1 Antibodies used for flow cytometry.
71
2.3.3. Lung Histology
To observe the cellular infiltration and lung damage Haematoxylin and Eosin
(H&E) staining was performed on the lung sections from naive, 5 and 10 days
infected mice. Essentially, lungs were isolated from the thoracic cavity after
the mouse was euthanized, fixed in 10% neutral buffered formalin solution for
at least 48 hours, and processed in a tissue processor (Leica Microsystems,
Wetzler, Germany). Briefly, lungs were dehydrated in a series of ethanol
mixtures (70% to 80% to 90% to 100%, 30 minutes each and 2 hours for
100%), and immersed in xylene for 10.5 hours. Lungs were perfused with hot
paraffin for 3 hours and embedded in paraffin wax. The specimens were then
cut into 5 μm sections using a microtome (Leica Microsystems, Wetzler,
Germany) and fixed on slides. For H&E staining, slides were deparaffinized
with HistoClear for 10 min and rehydrated in a series of ethanol to water
mixtures (100% to 90% to 70% to water and each for 2 minutes). The sections
were then stained with Harris haematoxylin for 5 minutes, washed in the
distilled water, and differentiated in 0.1% acid alcohol solution for 30 seconds.
The sections were washed with tap water for 5 minutes and counter stained
with Eosin for 1 minute. Subsequently, the sections were dehydrated in a series
of ethanol solutions (70 % to 90% to 100% and each for 30 seconds) and
immersed in HistoClear for 10 minutes. Evaluation of inflammation around
peribronchial and perivascular areas was semi-quantitatively performed in a
blind manner. A subjective scale (0-3) was assigned as follows:
0, no inflammation
72
1. mild, inflammatory cell infiltrate of the perivascular/peribronchiolar
compartment
2. moderate, inflammatory cell infiltrate of the perivascular/peribronchiolar
space with modest extension into the alveolar parenchyma
3. severe, inflammatory cell infiltrate of the perivascular/peribronchiolar space
with a greater magnitude of inflammatory foci found in the alveolar
parenchyma. The blinded scoring was kindly done by Dr. Ben Yan,
Pathologist, KK Hospital, Singapore.
2.4 Influenza Virus Culture and Infection
2.4.1. Virus culture in chicken Embryos
Nine to ten day old embryonated chicken eggs were purchased from Bestar
Laboratories (Singapore). After locating the air cell and marking out the large
blood vessels, a 21G needle was used to puncture the egg shell. Using a fine
needle, 10 HAU of Influenza A/PR8/34 (H1N1) stock kindly provided by Prof.
Vincent Chow, diluted in PBS buffer was carefully injected into the allantoic
cavity through the hole. The hole was then sealed with melted wax to preserve
sterility. Eggs were then kept in a 37oC incubator without CO2. They were
candled every day for the viability of the embryos. After 2 to 3 days, the
embryos were culled by placing the eggs at 4oC for 4 hours after which the egg
was broken through the air cell and the allatoic fluid was harvested. The
allantoic fluid was clarified by spinning at 3000 g before it was concentrated
and stored at -80oC.
73
2.4.2 Concentration of Influenza virus
The clarified allantoic fluid containing the virus obtained from the eggs was
poured into a Vivaspin 20 centrifugal concentrator with a membrane pore size
of 100,000kDa molecular weight cut-off (Sartorius AG, Germany). The
concentrator was centrifuged at 3000g at 4oC, stopping at intervals of 30 mins
to resuspend the top fractions to permit a better flow rate and to discard the
flowthrough in the bottom fraction. The top fractions were then pooled and
quantified using plaque assays on MDCK cells.
2.4.3 Plaque forming assay
MDCK cells were seeded at a density of 2.5 x 105 cells/well in a 24-well plate
and cultured in complete DMEM until fully confluent and the spaces between
cells are barely distinguishable. Immediately before infection, the media was
withdrawn and the cells were washed once with PBS to remove any traces of
serum which may affect the ability of the virus to bind to the cells. The virus
was then serially diluted 10-folds in serum-free DMEM and overlaid on
MDCK cells for 1 hour at 37oC to allow the virus to adsorb onto the cells. The
volume of the inoculum was kept small, typically 250μl per well so as to
ensure maximal adsorption of the virus onto the cells. During the incubation
period, a 2% agarose solution in sterile water was prepared and kept in molten
state by incubating in a water bath at 47oC. At the same time, 2X DMEM was
warmed to 37oC and TPCK Trypsin was added to a final concentration of
4μg/ml. After one hour the molten 2% agarose was mixed with an equal
volume of 2X DMEM to obtain a 1X DMEM agarose solution. The virus
inoculums were quickly removed and cells were overlaid with the DMEM-
74
agarose. Plates were incubated for 3 days at 37oC to allow the formation of
plaques. To visualise plaques on the third day, 0.5 to 1ml of PBS was added on
top of the agarose layer to facilitate removal of the agarose overlay and a bent
needle was used to gently dig out the agarose layer above the cells. Care must
be taken to avoid scratching the bottom of the plate. Plaques were visualised by
adding crystal violet solution and incubating for at least 5 minutes. Excess stain
was removed by washing the plates with water.
2.4.4 Infection of cells with Influenza virus
For infection A549 cells were plated in complete F-12K media one day before.
The media was aspirated and the monolayer was washed with PBS. The
inoculum containing virus at specified MOI in serum free medium was
incubated with cells for 1hr following which it was aspirated and the cells were
washed with PBS. For viral titer assay F-12K+ 2% FBS + 1µg/ml TPCK
trypsin was added. For all other experiments complete F-12K was added.
2.5 Measurement of Cytokines
Levels of cytokines : IL-6, IL-12p70, IL-10, IL-1β, TNFα, TGF-β IFNα , IFNβ
and IFN-g were detected using the commercially available Ebioscience Ready-
Set-go® kits according to manufacturer’s instructions. Briefly, the capture
antibody was diluted to the working concentration in the coating buffer and a
96-well maxisorp plate (NUNC) was coated with 50µL per well of the diluted
capture antibody. The plate was then sealed and incubated at 4ºC overnight.
Next day, each well was aspirated and then washed with the ELISA wash
buffer three times. After the last wash, inverting the plate and blotting it on
75
clean paper towels removed any remaining wash buffer. The plate was then
blocked by adding 100µl of blocking buffer and incubating for 1h at room
temperature. It was then washed as mentioned earlier. 50µL of sample or
standard in the blocking buffer was added to the wells. The plates were then
sealed and incubated for 2h at room temperature or O/N at 4ºC. After the
incubation plate was washed and detection antibody was added for 1 hr. Plate
was then washed and a working dilution of Streptavidin-HRP (50µL) was
added to each well. The plate was covered and incubated for 30mins at room
temperature after which it was washed and 50µl of TMB substrate was added
to each well. After incubation for color development of 15 mins 50µl of stop
solution 2N H2SO4 was added to each well. The absorbance was then measured
at 450nm.
2.6 Western Blot
2.6.1. Protein lysis
The culture medium was aspirated and cell monolayer was washed once with
1X PBS. Cells were scraped in ice cold PBS and pelleted by spinning at
2000rpm for 10mins. They were then lysed using the 1X RIPA buffer
consisting of 50mM sodium chloride (NaCl), 0.1% NP-40, 0.5% sodium
deoxycholate (C24H39O4Na), 0.1% SDS and 50mM Tris-HCl supplemented
with protease inhibitor (Thermo Scientific Halt Protease inhibitor).Cells were
left to stand on ice for 30 minutes and tapped intermittently before being
centrifuged at 12000 rpm for 15 minutes. The supernatant was carefully
removed and stored at -80°C.
76
2.6.2. Protein Concentration Quantification
Protein concentrations were estimated using 1X Bradford’s reagent (BioRad
Protein Assay) and BSA standards (Thermo Scientific Pierce). Absorbance was
measured using a BioRad spectrophotometer at 595nm.
2.6.3 Blotting 30-50 μg of proteins per samples were loaded onto a 10% or 12% SDS-PAGE
gel which was run in 1X Running buffer (0.025 M Tris, 0.192 M Glycine and
0.1% SDS) at 80 V for 20 minutes (stacking phase) and 125 V for 90 minutes
(resolving phase).Wet transfer was performed using 1X Transfer buffer (20%
Methanol, 25 mM Tris and 192 mM Glycine) onto a nitrocellulose membrane
(BioRad) for 60 minutes at 0.35 mA on ice. Membranes were stained with
Ponceau S (0.1% w/v in 1% acetic acid) to check for successful transfer.
Membranes were blocked using 5% skimmed milk (Anlene, Fonterra) for 30
minutes at room temperature on an orbital shaker (80 rpm). They were then
washed twice with 1X TBST (1X TBS + 0.1% Tween-20) for 2-3 minutes
each. Incubation with primary antibodies was performed overnight at 4°C on
an orbital shaker (table of antibodies used is given below). Membranes were
washed twice with 1X TBST for 5 minutes each followed by incubation with
secondary antibodies for 60 minutes at room temperature on the shaker.
Membranes were washed thrice with 1X TBST for 3-5 minutes each.
Chemiluminescent detection was performed by exposure to SuperSignal West
Pico Chemiluminescent Subtrate (Thermo Scientific). This was followed by
exposure to CL-Xposure (Thermo Scientific) films in an autoradiography
77
cassette (Amersham Hypercassette™) until protein bands could be visualised
using Konica Minolta SRX-101A tabletop processor.
Primary Antibody Dilution Company
Rabbit Annexin A1 1:000 Cell Signaling Techonlogy Inc (Danvers, MA, U.S.A)
Rabbit p-Akt (s473) 1:1000 Cell Signaling Techonlogy Inc (Danvers, MA, U.S.A)
Rabbit total Akt 1:1000 Cell Signaling Techonlogy Inc (Danvers, MA, U.S.A)
Rabbit Cleaved Caspase 3
1:1000 Cell Signaling Techonlogy Inc (Danvers, MA, U.S.A)
Rabbit Caspase 3 1:1000 Cell Signaling Techonlogy Inc (Danvers, MA, U.S.A)
Rabbit Cleaved Caspase 7
1:1000 Cell Signaling Techonlogy Inc (Danvers, MA, U.S.A)
Rabbit Caspase 7 1:1000 Cell Signaling Techonlogy Inc (Danvers, MA, U.S.A)
Rabbit Cleaved PARP 1:1000 Cell Signaling Techonlogy Inc (Danvers, MA, U.S.A)
Rabbit PARP 1:1000 Cell Signaling Techonlogy Inc (Danvers, MA, U.S.A)
Mouse Tubulin 1:2000 Sigma-Aldrich Co (St. Louis, MO, U.S.A.)
Table 2.2 Antibodies used for Western Blotting
78
2.7. Real Time PCR
2.7.1. RNA Isolation from cells Total RNA was isolated using the phenol-chloroform extraction method. Cells
were lysed using Omics MaestroZolTM RNA plus extraction reagent (Omics
Biotechnology). The homogenised samples were incubated at room
temperature for 5 minutes followed by addition of 0.2 ml of chloroform
(Sigma-Aldrich,) per ml of MaestroZol reagent. Samples were mixed by
inverting a few times and incubated at room temperature for 10 minutes. They
were then centrifuged at 12,000 g for 15 minutes at 4°C. The clear aqueous
layer was transferred to a new, clean 1.5 ml Eppendorf tube. 0.5 ml
isopropanol per ml MaestroZol was added to the clear aqueous layer and mixed
well. The samples were incubated at room temperature for 10 minutes and
centrifuged at 12,000 g for 10 minutes at 4°C. The RNA pellet was washed
with at least 1 ml of 75% ethanol. The samples were mixed by vortexing and
centrifuged at 7500 g for 5 minutes at 4°C. Supernatant was removed and RNA
pellets were air dried for 10 minutes at room temperature before being
dissolved in nuclease free water. The samples were quantitated using a
nanodrop spectrophotometer (BioFrontier Technology). Only RNA samples
with A260/A280 ratio of values equal to or greater than 1.8 were used and
RNA was stored at -80°C.
2.7.2. RNA isolation from lungs
Infected and naive mice were euthanized using CO2 and lungs were harvested
which were then cut into small pieces and stored in RNA later (Qiagen). RNA
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was isolated using RNAeasy kit according to manufacturer’s instructions
(Qiagen).
2.7.3. Viral RNA (vRNA) isolation from cell supernatant
Viral RNA from culture media of infected cells was harvested using viral RNA
extraction kit (Geneaid) according to the manufacturer’s protocol.
2.7.4. cDNA Synthesis
For cDNA synthesis following mix was prepared in each of the sample tubes
and incubated at 65°C for 5 minutes after which the tubes were immediately
put on ice.
Total RNA 1 µg
Random Primers 200ng
10mM dNTPs 1µl
Nuclease free water Upto 12µl
Table 2.3 cDNA synthesis initial reaction mix
After the initial reaction the following master mix was prepared and added to
each sample. Tubes were spun and incubated at 25°C for 2 mins.
5X M-MLV RT buffer 4µl
RNAsin(40U/ml) 0.25µl
Nuclease free water To make
Final volume
19µl
Table 2.4 cDNA synthesis final reaction mix
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Finally 1ul of reverse transcriptase enzyme was added, and the mixture was
incubated at 25°C for 10mins , followed by 42°C for 60 mins and 70°C for 15
mins. cDNA was stored at -20°C.
2.7.5. Real time PCR
GoTaq® qpCR master mix (Promega) was used and a master reaction was
prepared. For each reaction
2X GoTaq® qpCR master mix 10 μl
10uM forward primer 0.4 μl
10uM reverse primer 0.4 μl
cDNA template (5 ng) 1.0 μl
Nuclease-free water 7.4 μl
Table 2.5 qPCR reaction mix
The following PCR protocol was used:
50°C, 2 minutes,
95°C, 10 minutes,
40 cycles of 95°C, 15 seconds and 60°C, 1 minute
50°C, 15 seconds
60°C, 1 minute,
95°C, 30 seconds,
60°C, 15 seconds
Melting curve analysis was performed after that at 95°C for 1 minute and 55°C
for 1 minute. The ABI 7500 real-time PCR system (Applied Biosystems) was
used for analysis. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or
81
beta-actin was used as the endogenous control. Relative expression was
computed by ΔΔCt a method. Primer sequence list is provided below.
Primer Sequence Company
Mouse IL-6 (Forward)
5’-GGG ACT GAT GCT GGT GAC AA-3’ 1st Base (Singapore)
Mouse IL-6 (Reverse)
5’-TCC ACG ATT TCC CAG AGA ACA-3’ 1st Base (Singapore)
Mouse IL-12 p40 (Reverse)
5’-AAC TTG AGG GAG AAG TAG GAA TGG-3’
1st Base (Singapore)
Mouse β-actin (Forward)
5’-CTT AGG AA TGC CTC TGG GAG GTC C-3’
1st Base (Singapore)
Mouse β-actin (Reverse)
5’-GCA GAC GCG AGG AAG GAG-3’ 1st Base (Singapore)
Mouse TNF-α (Forward)
5’-GGC AAG GAT GAG CCT TTT AGG-3’ 1st Base (Singapore)
Mouse TNF-α (Reverse)
5’-TTG GTT TGG GAG GAA AGG G-3’ 1st Base (Singapore)
Mouse IFN α (Forward)
5’-TGTCTGATGCAGCAGGTG-3’ 1st Base (Singapore)
Mouse IFN α (Reverse)
5’-AAGACAGGGCTCTCCAGA-3’ 1st Base (Singapore)
Mouse IFN β (Forward)
5’-GGCCGACTTCAAGATCCCTAT-3’ 1st Base (Singapore)
Mouse IFN β (Reverse)
5’-GGATGGCAAAGGCAGTGTAAC-3’ 1st Base (Singapore)
Mouse ANXA1 (Forward)
5’-AAGGTGTGGATGAAGCAACC-3’ 1st Base (Singapore)
Mouse ANXA1 (Reverse)
5’-TGCATCAAACTGAGCTGGAG-3’ 1st Base (Singapore)
Viral M gene (Forward)
5’-GGACTGCAGCGTTAGACGCTT-3’ 1st Base (Singapore)
Viral M gene (Reverse)
5’-ATCCTGTTGTATATGAGGCCCAT-3’ 1st Base (Singapore)
82
Viral NS1 gene (Forward)
5’-TTCACCATTGCCTTCTCTTC-3’ 1st Base (Singapore)
Viral M gene (Reverse)
5’-CCCATTCTCATTACTGCTTC-3’ 1st Base (Singapore)
Human GAPDH (Forward)
5’-CACTTTGTTTTTGGACATAGCTG-3’ 1st Base (Singapore)
Human GAPDH (Reverse)
5’-CCACACCTAGCAACCAGAAGTTAG-3’ 1st Base (Singapore)
Table 2.6 List of primers
2.8. Luciferase Assay
For the luciferase assay 100,000 A549 cells were plated in a 24 well plate one
day before transfection. The following day 200 ng of NF-κB luciferase reporter
plasmid and 10 pg of Renilla plasmid along with scrambled or ANXA1 shRNA
or ANXA1-V5 tagged plasmids were transfected. 24hrs later cells were
infected with Influenza virus at a MOI of 1 for 12hrs. Cells were then lysed in
120µl of passive lysis buffer and Luciferase activity was determined using
Dual-Luciferase® Reporter Assay System (Promega) as per manufacturer’s
protocol. Luciferase activity was measured using a spectrophotometer (Perkin
Elmer VICTOR3™ V Multilabel Counter Model 1420).
2.9. Caspase 3/7 assay
10,000 cells were plated in a 96 well plate one day before transfection with
control or ANXA1 siRNA . 24 hours post transfection, cells were infected with
Influenza virus at a MOI of 1 and were grown for 48 hrs before apoptosis was
assessed by Caspase-Glo® 3/7 Assay Systems (Promega). Substrate was added
to the well containing media in the ratio 1:1. Luminiscence was measured
83
using luminometer (Perkin Elmer VICTOR3™ V Multilabel Counter Model
1420).
2.10. Statistics
Unpaired sets of data were compared using unpaired Student’s t-test (two-
tailed). In all cases, p<0.05 was accepted as statistically significant. Survival
curve was compaired using Kaplan-Meier survival curve analysis.
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Chapter 3 Results
Annexins have been known to be involved in infectious diseases such as
Tuberculosis (Gan, et al., 2008), HIV (Ryzhova, et al., 2006) and Hepatitis C
(Backes, et al., 2010). The presence of ANXA1 on the phagosomal membrane
seems to be functionally important in macrophages and neutrophils. Rapid
translocation of ANXA1 to the phagosome membrane has been observed
during the phagocytosis of non-pathogenic or killed bacteria (Harricane, Caron,
Porte, & Liautard, 1996; Kaufman, Leto, & Levy, 1996), and macrophages
from ANXA1-knockout mice decreases phagocytosis of non-opsonised
zymosan (Yona, et al., 2006). ANXA1 is also involved in TLR-mediated
production of pro- or anti-inflammatory cytokines (Y. H. Yang, Aeberli,
Dacumos, Xue, & Morand, 2009; Y. H. Yang, et al., 2006; Yona, et al., 2006)
indicating that ANXA1 may be crucial regulator for the balance between pro-
and anti-inflammatory cytokines. Dendritic cells obtained from ANXA1 KO
mice exhibit higher maturation and hence lower phagocytocis and migratory
potential. The activation of NF-κB, ERK1/2 and Akt is also decreased in
ANXA1 KO dendritic cells (Huggins, et al., 2009). It also regulates T-cell
activation and differentiation (D'Acquisto et al., 2007). More recently, we
have shown that ANXA1 is a binding partner of IKK complex and keeps NF-
κB constitutively active (Bist, et al., 2011). Our recent publication also
revealed that ANXA1 plays a critical role in TLR3-mediated IFN regulation by
binding to TBK-1 and regulating IRF3 phosphorylation and its translocation to
the nucleus. Having observed an important role of ANXA1 in TLR3 signalling,
85
in this project we wished to characterize its role in TLR7 signalling using
artificial ligands and in influenza virus infection since single stranded (ss)RNA
viruses are the natural ligands of TLR7.
3.1. Endogenous levels of TLR7 in WT and ANXA1-/- are similar in both BMMOs and DCs
TLRs act as the first line defense mechanism against microbial infections by
playing a pivotal role in the recognition of bacteria and viruses. Aberrant
expression of these pathways has serious consequences in autoimmune
diseases and cancer. As mentioned above, ANXA1 plays an important role in
TLR3 mediated production of IFN-β and CXCL10 by associating with TBK-1
through its C-terminal (Bist, et al., 2013). TLR7 is one of the most important
PRRs for ssRNA viruses. Efficiency of detection of ssRNA or
imidazoquinoline compounds depend on TLR7 gene expression (S. Huang,
Wei, & Yun, 2009). In order to investigate if antigen presenting cells from
ANXA1-/- have an inherent advantage in recognition of ligands we determined
the endogenous levels of TLR7 in bone marrow macrophages (BMMOs)
(Figure 3.1A) and bone marrow dendritic cells (BMDCs) (Figure 3.1B).
However the levels were comparable and no significant differences were
observed.
86
Figure 3.1 Endogenous TLR7 levels are similar in both WT and ANXA1-/-
RNA from WT and ANXA1-/- (A) BMMOs and (B) DCs was extracted and endogenous levels of TLR7 were determined using qPCR. Results are representative of two independent experiments expressed as mean±SD.
3.2. R848 efficiently activates TLR7 pathway
TLR7 recognizes ssRNAs as their natural ligand and also small synthetic
molecules such as imidazoquinolines and nucleoside. BMMOs from WT mice
were treated with CL264 (1µg/ml), Gardiquimod (1µg/ml), Imiquinod (R837)
(1µg/ml) and Resiquimod (R848) (300ng/ml) and activation was assessed by
production of pro-inflammatory cytokines IL-6 and IL-12p40 (Figure 3.2).
The maximum production for both cytokines was observed in the case of R848
compared to the other three agonists, even at a very low dose. Use of R848 has
also been widely reported in the literature therefore we used R848 for all
subsequent experiments. R848 is a low molecular weight molecule that
A B
ns ns
87
exhibits potent antiviral activity and stimulates cells in a MyD88-dependant
manner.
Figure 3.2 R848 stimulates maximum production of cytokines.
BMMOs were treated with different TLR7 ligands, CL264 (1µg/ml), Gardiquimod (1µg/ml), Imiquinod (R837) (1µg/ml) and Resiquimod (R848) (300ng/ml) for 24 hrs and pro-inflammatory cytokines (A) IL-6 and (B) IL-12p40 were measured to detect the efficacy of the ligands. Results are representative of two independent experiments and are expressed as mean±SD.
88
3.3. ANXA1 is selectively required for the production of pro-inflammatory cytokines
TLR activation triggers the production of pro and anti-inflammatory cytokines,
and these cytokines play an important role in shaping the adaptive immune
response. We therefore compared the expression of the proinflammatory
cytokines, type I interferons and the transcription factors that regulate them
from WT and ANXA1-/- mice after stimulation with R848 in BMMOs and
BMDC. Since the regulation of these cytokines begin at transcriptional level
post treatment, we first determined the RNA levels for IL-6, IL-12p40, TNF-α,
IFNα, IFNβ and the regulators of type I interferons IRF7 and IRF5 for both
cell types, followed by ELISA for IL-6, IL-12p40 and TNF-α to verify if the
differences observed at RNA levels can be seen post-translationally. In case of
BMMOs, IL-6, IL-12p40, IFNβ and IRF7 RNA expression was impaired in
ANXA1-/- cells while TNF-α, IFNα and IRF5 were not affected (Figure 3.3).
These differences in IL-6 and IL-12p40 were observed in protein levels as
well, as shown by ELISA (Figure 3.4).
Surprisingly, cytokine expression in ANXA1-/- BMDCs was different to that of
BMMO. All the genes except IRF5 (Figures 3.5 and 3.6) were upregulated in
ANXA1-/- cells. A discrepancy was observed in the levels of IL-12p40 where
the RNA expression levels were higher in ANXA1-/- BMDC (Figure 3.5B)
whereas the protein levels were lower in these cells. These results indicate a
selective requirement of ANXA1 in TLR7-mediated signalling pathway and it
differentially regulates the production of inflammatory cytokines depending
upon the cell type.
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Figure 3.3 Pro-inflammatory cytokines and Type I interferon expression is downregulated in ANXA1-/- BMMOs.
BMMOs were treated with 300ng/ml of R848 and were harvested 2,4 and 8 hrs later. RNA was collected and qPCR was done for (A) IL-6, (B) IL-12p40, (C) TNFα, (D) IFNα, (E) IFNβ, (F) IRF7 and (G) IRF5. Data is represented as mean ±SEM from three independent experiments and analysed using two-way ANOVA. *p<0.05, **p<0.01 , #0.05<p<0.1.
90
Figure 3.4 Pro-inflammatory cytokine production is downregulated in ANXA1-/- BMMOs.
BMMOs were treated with 300ng/ml of R848 and supernatants were harvested 24hrs later. ELISA was performed for (A) IL-6, (B) IL-12p40 and (C) TNFα. Data is representative of three independent experiments expressed as mean±S.D. *p<0.05, **p<0.01.
C
BA
91
Figure 3.5 Pro-inflammatory cytokines and Type I interferon expression is upregulated in ANXA1-/- BMDCs.
BMDCs were treated with 300ng/ml of R848 and were harvested 2, 4 and 8 hrs later. RNA was collected and qPCR was done for (A) IL-6, (B) IL-12p40, (C) TNFα, (D) IFNα, (E) IFNβ, (F) IRF7 and (G) IRF5. Data is represented as mean ±SEM from three independent experiments. *p<0.05, **p<0.01.
92
Figure 3.6 Pro-inflammatory cytokine production is upregulated in ANXA1-/- BMDCs.
BMDCs were treated with 300ng/ml of R848 and supernatants were harvested 24hrs later. ELISA was performed for (A) IL-6, (B) IL-12p40 and (C) TNFα. Data is representative of three independent experiments expressed as mean±S.D. *p<0.05, **p<0.01 .
C
BA
93
3.4. TLR dependent inflammasome activation is higher in ANXA1-/- cells
Another important pro-inflammatory cytokine is IL-1β. However, its
production through TLRs is not direct. TLRs only act as a primary signal for
the production of proIL-1 β and activation of few inflammasome components,
which is cleaved by capsase 1 inflammasome upon activation by another signal
from danger associated molecular patterns (DAMPS) leading to the production
of mature IL-1β (Mariathasan & Monack, 2007). Though any TLR agonist can
provide the first signal for the production of IL-1β, RNA viruses such as
influenza virus trigger this host response via TLR7 (Ichinohe, Pang, & Iwasaki,
2010). Therefore, we next used R848 to activate the inflammasome in WT and
ANXA1-/- BMDC.
To assess the effects of ANXA1 on inflammasome activation, we primed
ANXA1-/- and WT BMMOs and BMDCs with lipopolysaccharide (LPS) (used
as a positive control) or R848 for 16 hr in order to induce pro-IL-1β synthesis
and stimulated with ATP to activate IL-1β maturation via inflammasome
activation. Extracellular adenosine triphosphate (ATP) is a DAMP that is
released at sites of cellular injury or necrosis. LPS or R848 alone was not able
to induce IL-1β production, however cotreatment of ATP with either LPS or
R848 resulted in IL-1β production in both WT and ANXA1-/-. Interestingly,
ANXA1-/- cells produced more IL-1β in both cell types, indicating that
ANXA1 deficiency amplifies ATP-driven IL-1β production with R848 or LPS
(Figure 3.7). The results obtained for BMDCs are in conjunction with previous
cytokine production results that seem to suggest that TLR7 signalling is more
activated in ANXA1-/- dendritic cells.
94
Figure 3.7 Increased IL-1β production in ANXA1-/- BMDCs and BMMOs.
BMDCs and BMMOs were isolated from ANXA1-/-and WT mice, and subsequently primed either with LPS or R848 for 16 h. Thereafter, the cells were stimulated with ATP for another 4 h. IL-1β release was assessed by ELISA. Data is representative of three independent experiments expressed as mean±S.D. *p<0.05, ***p<0.001.
A B
95
3.5. ANXA1-deficient splenic B cells exhibit increased activation and proliferation.
Having established a phenotype for involvement of ANXA1 in cytokine
production involving TLR7 in innate immune cells, we next studied its role in
adaptive immune cells. In addition to the cytokine secretion by macrophages
and dendritic cells, imidazoquinolines also possess activation and proliferation
potential for B cells (Bishop et al., 2000). To demonstrate the involvement of
ANXA1 in proliferative responses, splenocytes were obtained from 6-8 wk-old
ANXA1-/- and WT mice. These were then stimulated with R848 and CpGB (a
very potent proliferation inducer of B cells) followed by staining with cell
specific markers and CFSE to determine proliferating cells. Proliferation was
observed in B220+ cells in both WT and ANXA1-/- cells, suggesting that B cells
proliferated in response to TLR stimulation. Interestingly, more proliferation
was observed in ANXA1-/- as compared to WT in both R848 (Figure 3.8B)
and CpGB (Figure 3.8C). Each peak represents one cell division. ANXA1-/-
cells undergo 3 and 5 cell divisions compared to WT which undergo 2 and 3
cell divisions with R848 and CpGB respectively. Quantification was done by
calculating % proliferating cells starting from the second peak (Figure
3.8D&E). For both the agonists used ANXA1-/- cells exhibited higher
proliferation compared to the WT. To confirm this finding, thymidine
incorporation assay was performed in isolated splenic B cells which again
demonstrated higher proliferation in the ANXA1-/- cells when treated with
various doses of R848 (Figure 3.8F). These results indicate that ANXA1 may
inhibit B cell proliferation upon TLR7 activation.
96
A
B220 +ve cells unstimulatedWT KO
B220 +ve cells stimulated with 1ug of R848
CFSE
0
2
10
12
3
B
B220 +ve cells unstimulated
WT KO
B220 +ve cells stimulated with 1uM of CpG-B
CFSE
Cou
nts
01
23
4
53
21
0
C
97
Figure 3.8 ANXA1 deficient B cells exhibit increased proliferation compared to WT.
Spenocytes were isolated and stained with CFSE followed by treatment with 1µg/ml of R848 or 1μM CpGB. (A) B cell were identified by FSC/SSC and B220. (B&C) CFSE analysis shows multiple peaks upon stimulation with R848 and CpGB, representing proliferation. (D&E) Quantification of proliferation was done was analyzing %proliferation after treatment. (F) Splenocytes were treated with various doses of R848. 3
1H was added 16hrs prior to the measurement at 72 hrs. Data is represented as mean ±SEM from three independent experiments. *p<0.05, **p<0.01.
F
D E
98
Since B cells are converted to antibody producing plasma cells during
infection, we investigated the numbers of plasma cells induced during
proliferation by staining the cells with CD138. No significant differences were
detected in response to the R848 in WT and ANXA1-/- cells (data not shown).
We next investigated R848-induced activation of splenic B cells from WT and
ANXA1-/- mice using flow cytometry. CD86 and CD69 are the early activation
markers present on a wide variety of cell types including B cells. Expression
levels of these two co-stimulatory molecules were higher in ANXA1-/- cells as
compared to WT (Figure 3.9B & C).
99
Figure 3.9 ANXA1 deficient B cells exhibit increased activation.
Spenocytes were isolated and treated with various doses of R848 for 24hrs (A) B cells were gated based on lymphocyte forward and side scatter, followed by B220 positivity. (B) B cells were analysed for activation markers CD69 and CD86. Red and green lines represent WT control and treated respectively while orange and blue represent ANXA1-/- control and treated. (C) Activation was quantified using MFI. Data represents mean±SEM from three independent experiments. *p<0.05,**p<0.01.
A
CD69CD86
% o
f max
B
C
100
Upon encountering pathogens, stimulation of TLRs induce signaling events
that culminate in the activation of transcription factors needed for cytokine
production and up-regulation of costimulatory molecules (Beutler, 2009). The
serine-threonine protein kinase Akt mediates many of the downstream effects
of PI3K and consequently plays a central role in cell proliferation and survival.
Akt kinase is activated by phosphorylation of Ser 473. Thus, we evaluated Akt
activation in splenic B cells. WT and ANXA1-/- B cells were stimulated with
300 ng/ml R848 at different time points, and western blot was conducted.
Notably, high levels of basal Akt phosphoylation was detected in the ANXA1-/-
B cells as compared to WT which increased upon stimulation (Figure 3.10).
Akt phosphorylation was time dependent in WT, however sustained activation
was observed in ANXA1-/- cells. This suggests that ANXA1 might regulate
proliferation through sustained Akt phosphorylation.
101
Figure 3.10 ANXA1-/- B cells display enhanced PI3K/Akt signalling in response to R848.
(A) B cells were stimulated with 300 ng/ml R848 for the indicated time points, followed by Western blot analysis using phospho-Akt (p) antibody. Blots were stripped and reprobed with anti-Akt1. (B) Quantification of pAKT with respect to total in WT and ANXA1-/- cells.
0 30 60 120 0 30 60 120 (min)
WT KO
T-AKT
pAKT(ser473)
0
1
2
3
4
5
6
7
8
0 30 60 120
fold
ch
ange
Mins post treatment
pAKT
WT
ANXA1-/-
ANXA1-/- A
B
102
3.6. ANXA1 deficient dendritic cells exhibit higher production of pro-inflammatory cytokines upon virus infection.
Our data so far with TLR7 ligand R848 indicates that ANXA1 affects TLR7
signalling pathway in both innate and adaptive immune cells. To advance into
a more clinical scenario we next infected WT and ANXA1-/- BMMOs and
BMDCs with live and UV inactivated Influenza A/PR8/34 (H1N1) influenza
virus (PR8) at an MOI=10, which being a single stranded virus, is a natural
ligand for TLR7. UV inactivation preserves the virus structure but makes it
unable to replicate. Live virus induced high levels of pro-inflammatory
cytokines compared to UV inactivated virus in BMDCs (Figure 3.11). As
observed with R848, ANXA1 deficient DCs produced higher levels of IL-6,
IL-12p40 compared to WT upon treatment with both live and UV inactivated
virus. Levels of cytokines observed in BMMOs were almost negligible (data
not shown).
These results thus far obtained clearly show a difference in cytokine expression
between WT and ANXA1-/- cells in response to TLR7 stimulation and
influenza virus infection. However, due to the reverse trends observed between
the innate immune cells (macrophages and DCs), it is difficult to predict
whether ANXA1 will be a positive or negative player in influenza virus
pathogenesis. To clarify this, we shifted our focus from the immune cells to
epithelial cells which are the sites for virus replication and infected them with
influenza virus to observe gene activation and expression of ANXA1.
103
Figure 3.11 ANXA1 deficient dendritic cells exhibit higher production of pro-inflammatory cytokines upon virus infection
BMDCs were treated with UV inactivated and live virus at a MOI=10 and supernatants were harvested 24hrs later. ELISA was performed for IL-6 (A), IL-12p40 (B) and TNFα (C). Data is representative of two independent experiments expressed as mean±S.D. **p<0.01, ***p<0.001 .
A B
C
104
3.7. ANXA1 expression levels and its promoter activity increase upon virus infection in the epithelial cells in vitro.
Alveolar type II epithelial cells are the major sites for viral replication therefore
we assessed the levels of ANXA1 in A549 cell line. A549 cells were infected
with H1N1 PR8 at a MOI of 1 for various time points. ANXA1 increases in a
time dependent manner with maximum levels obtained 48hrs p.i. (Figure
3.12A). Upon infection ANXA1 is cleaved into a smaller 33kDa fragment from
24 hrs. The viability of the cells 48hrs post infection is around 40%. The
promoter activity of ANXA1 as determined by luciferase reporter assay also
increases three folds upon induction with influenza virus (Figure 3.12B). The
ANXA1 luciferase plasmid was obtained from Prof. Lezarbe (Lecona et. al.,
2008). This data suggests that ANXA1 might be a virus inducible protein.
Annexins 5 and 6 have been implicated to be playing a role influenza virus
infection. While Annexin 5 is suggested to be involved in the entry of the virus
(R. T. Huang, et al., 1996), Annexin 6 disrupts viral budding (Ma, et al., 2012).
The following experiments were performed to assess any effects of ANXA1 on
viral titers.
105
Figure 3.12 ANXA1 is upregulated and cleaved upon viral infection in vitro.
(A) ANXA1 is cleaved and upregulated 24hrs onwards, post infection. Tubulin is used as loading control. (B) Quantification of ANXA1 western blot with respect to tubulin. (C) Promoter activity of ANXA1 as determined by luciferase reporter assay increases upon infection. Data is represented as mean±SEM from 3 independent experiments. *p<0.05
0 4 8 12 24 36 48 (hrs p.i)
ANXA1
Tubulin
A
B
0
1
2
3
4
5
6
0 4 8 12 24 36 48
Fold
Cha
nge
Hours post infection
ANXA1
C
106
3.8. Influenza virus replication is inhibited in ANXA1 deficient epithelial cells.
In an attempt to decipher at which step of viral propagation ANXA1 plays a
role, A549 cells were transfected with scrambled or ANXA1-shRNA and
infected with Influenza virus at a MOI=1 and virus titers were monitored 24
and 48 hrs p.i. in the cells and the supernatant. This was done to determine
whether ANXA1 plays a role in early (cells) or later (supernatant) stages of
viral propagation. To quantify the titers in the supernatant, standard curves for
the serial dilutions of known virus concentrations against the Ct value were
plotted for M1 (Figure 3.13A) and NS1 (Figure 3.13B) genes. The Ct values
obtained were fitted in the equation and corresponding virus concentrations
were obtained. The knock down efficiency was tested using qPCR (Figure
3.13C) and western blot (Figure 3.13D). Viral titers in the cells increased in a
time dependant manner but the levels in ANXA1-shRNA transfected cells were
significantly lower compared to the scrambled-shRNA transfected cells
(Figure 3.14 A). The titers were quantified by measuring the expression of
viral M1 and NS1 genes by qPCR. Similar data was obtained in the
supernatants of infected cells (Figure 3.14B) which is indicative of the viral
titers post-budding. In addition to qPCR from supernatant, plaque assay was
also performed to assess the viral load (Figure 3.14C) and similar trend was
observed again, where silencing of ANXA1 inhibited virus replication or
propagation. Lower viral titers in both cells and supernatant lead us to conclude
that ANXA1 may play a role early in virus life cycle rather than later.
107
Figure 3.13 Standard curves for viral load quantification.
Serial dilutions of known virus concentrations are plotted against their corresponding Ct values for (A) M1 and (B) NS1 genes to quantify the viral load. The knock down efficiency is shown by (C)qPCR and (D) western blot. GAPDH is used as a loading control.
y = 2E+22x-12.81
R² = 0.9762
1
10
100
1000
10000
100000
1000000
10000000
0 10 20 30 40
pfu
/ml
Ct value
M1
y = 2E+15x-9.118
R² = 0.9875
1
10
100
1000
10000
100000
1000000
10000000
0 10 20 30
pfu
/ml
Ct value
NS1A B
0
0.2
0.4
0.6
0.8
1
1.2
scrambled shRNA ANXA1
Fol
d c
han
ge
ANXA1
GAPDH
C DScrambled ShRNA-
ANXA1
108
Figure 3.14 ANXA1 deficient epithelial cell exhibit lower viral titers.
A549 cells transfected with scrambled or ANXA1-shRNA were infected with influenza virus. Cells (A) and supernatant (B) was harvested 12, 24 and 48 hrs post infection and subjected to qPCR or plaque assay(C) to quantify the viral loads. Results are expressed as representative mean±S.D from three independent experiments. *p<0.05,**p<0.01, ***p<0.001.
A
C
B
109
To verify this data, gain-of-function studies were performed where ANXA1
was overexpressed in A549 cells using V5-tagged ANXA1 cloned in
pcDNA3.1 vector. Figure 3.15 shows that significantly higher titers were
observed in ANXA1 overexpressing cells compared to the cells transfected
with empty vector (EV) both in cells (Figure 3.15A) and supernatant (Figure
3.15B). The overexpression efficiency is shown by qPCR (Figure 3.15C).
Overall, this data shows that ANXA1 acts to negatively regulate virus titers
and plays a role in the early stages of viral infection. To extend our findings
further, we infected mice with influenza virus and investigated the innate
immune reactions in response to virus infection.
110
Figure 3.15 ANXA1 overexpressing epithelial cell exhibit higher viral titers.
A549 cells transfected with empty vector or ANXA1 plasmid were infected with influenza virus. Cells and supernatant was harvested 24 and 48 hrs post infection and subjected to qPCR (A) or plaque assay (B) to quantify the viral loads. (C) Overexpression efficiency determining ANXA1 levels is tested by qPCR Results are expressed as representative mean±S.D from three independent experiments. *p<0.05, **p<0.01.
B
A
0
100
200
300
400
500
600
ev ANXA1-V5
fold
incr
ease
C
111
3.9. ANXA1 deficient mice are more protected against Influenza virus infection.
To assess the involvement of ANXA1 in Influenza virus induced in vivo, we
infected WT and ANXA1-/- mice with a lethal dose (500 pfu) of Influenza
H1N1 PR8 virus intratracheally and weight loss was monitored every day for
20 days. Weight loss is an indirect method to assess the severity of the disease
and is a good non-invasive way to determine infection related morbidity. The
mice start to lose weight about 4 days post infection and the consistently lose
until day 10. After they have lost 25% of the initial body weight, they were
sacrificed in accordance with IACUC guidelines. Interestingly, when WT and
ANXA1-/- mice were compared, it was noted that only 20% of WT mice
survived while 80% of ANXA1-/- survived. (p=0.0065) (Figure 3.16A), as WT
mice experienced drastic weight loss which was not observed in the ANXA1-/-
mice. Mice which lost 25% of body weight were sacrificed by day 10 and the
remaining mice recovered and reached their initial body weight again by day
13 (Figure 3.16B). These results indicate a role of ANXA1 in influenza virus
infection, where the absence of ANXA1 protected mice from the effects of the
virus. We next determined the regulation of ANXA1 post infection and
characterized the infection on the basis of viral titers, cytokine responses,
cellular infiltration and lung damage.
112
Figure 3.16 ANXA1 defecient mice are protected against influenza A PR8 infection when challenged with 500pfu dose.
(A) 10 mice /grp were infected intratracheally with 500pfu and the weight loss was monitored over 20days. Mice were sacrificed when they lost 25% of their body weight. Figure (B) represents the weight loss when infected with 500pfu. The data is representative of two independent experiments, n=10 /grp. Data is analysed using the Kaplan-Meier survival curve analysis.
B
A
113
3.10. ANXA1 is a virus inducible protein and is cleaved upon infection
To elucidate the mechanism on how ANXA1 exerts its effects, we first
determined if ANXA1 is regulated in vivo in the lungs upon infection as
observed in the lung epithelial cells, we harvested the lungs from WT mice 1,
3 and 5 days post infection and qPCR (Figure 3.17A) and western blot (Figure
3.17B) was performed for ANXA1 expression. ANXA1 was observed to be
upregulated both transcriptionally and translationally post infection. RNA
levels rapidly increase by two folds one day p.i. and decrease subsequently.
Increase in protein levels followed soon after and ANXA1 upregulation and
cleavage was evident 3 days p.i. These results are similar to the ones obtained
by Katoh et. al where ANXA1 levels were upregulated in porcine monocytes
infected with swine flu virus (Katoh, 2000). ANXA1 is cleaved at its N-
terminus by serine proteases and the current view in the field is that the 33kDa
cleaved form is pro-inflammatory (Vong et al., 2007; Williams, et al., 2010).
This cleaved form is present in high amounts in broncheo-alveolar lavage
(BAL) from cystic fibrosis patients and correlates with amount of neutrophil
infiltration and neutophil elastase (Tsao, Meyer, Chen, Rosenthal, & Hu,
1998). However, in the influenza infection both the full length and cleaved
forms can be observed and how the balance is maintained is still unclear.
114
Figure 3.17 ANXA1 is a virus inducible protein and is cleaved upon infection
Levels of ANXA1 are increased in lungs of the infected mice as determined by (A) qPCR (B) western blot 1, 3 and 5 days post infection. Data in (A) is expressed as mean ± SEM from three independent experiments and analysed using student’s t test **p<0.01. (B) Tubulin is used as loading control.(C) Quantification of ANXA1 blot with respect to tubulin.
115
3.11. Viral loads in ANXA1 deficient mice are lower compared to the WT mice
To determine the virus load in the lungs of infected mice, mice were sacrificed
5 days p.i and lungs were harvested. Lungs were then either homogenized for
the plaque assay on MDCK cells (Figure 3.18A) or stored in RNA later for
total RNA isolation. qPCR was performed with the isolated RNA and the
amount of viral M (Figure 3.18B) and NS1 (Figure 3.18C) genes was
quantified. Interestingly, we observed that the virus titers in the lungs of
ANXA1-/- mice were lower compared to that of the WT mice by at least one
log. This data complements our in vitro data where we observe less virus titers
upon ANXA1 knock down and higher titers upon overexpression. The lower
viral loads in the ANXA1-/- mice can be attributed either to defective
replication and propagation or to the efficient clearance of the virus. To address
the virus clearance, levels of pro-inflammatory cytokines and Type I
interferons were determined locally in the BAL. The results are described in
the next section.
116
Figure 3.18 ANXA1 deficient mice exhibit lower viral titers in lungs.
Viral load in the lungs of infected mice was measured using (A) plaque assay and qPCR (B) M1 (C) NS1 genes 5 days post infection. Data is representative of two independent experiments where each experiment had at least 5 mice/group. *p<0.05. **p<0.01.
A
CB
117
3.12. The cytokine and type-I interferon responses are similar in WT and ANXA1-/- mice.
Influenza virus up regulates several pro and anti-inflammatory cytokines along
with type I interferons. Macrophages and dendritic cells are the major
producers of these. Previously, in our in vitro assays we observed opposite
trends in cytokine production between WT and ANXA1-/- BMMOs and
BMDCs. when treated with R848. Influenza infection is mainly localized to
lungs in the early stages therefore the maximum amount of cytokines can be
detected in the BAL. To determine the kinetics of production of these
cytokines, BAL from mice 1, 3 and 7 days post infection was harvested and
subjected it to ELISA. We chose a comprehensive panel to include most of the
important cytokines and divded it into three sections pro-inflammatory (IL-6,
IL-12, TNF-α, IL-1β), anti-inflammtory (IL-10 and TGF-β) and Type I/II
interferons (IFNα, IFNβ and IFNγ). Levels of IL-6 increased in a time
dependent manner from day 1 to 7 for WT and ANXA1-/- mice whereas IL-12,
TNF-α and IL-1β peaked at day 1 or 3 and subsided by day 7. The anti-
inflammatory cytokines IL-10 and TGF-β were also at their maximum levels 3
days post-infection and dropped down to the basal levels by day 7. IFN α/β
peaked at day 3 and IFNγ increased in a time dependent manner until day 7 p.i
(Figure 3.19). However, no significant differences were observed between WT
and ANXA1-/- mice.
118
BA
DC
FE
119
Figure 3.19 Cytokine levels are similar in both wild type and ANXA1 deficient mice.
Cytkokine analysis was performed on the bronchioalveolar lavage obtained from mice 1,3,and 7 days post infection by ELISA. Figures A-D represent pro-inflammatory cytokines, E-F anti-inflammatory cytokines and G-I type I & II interferons. Data is representative of two independent experiments where each experiment had atleast 3 mice/group /time point.
HG
I
120
3.13. ANXA1-/- mice display higher leukocyte infiltration in the BAL fluid
To further characterize the inflammatory cell milieu that is present in the
airways during influenza infection, differential cell counts were performed
using multicolour flow cytometry.
Cells were differentiated on the basis of following cell surface markers.
Eosinophils - CD45+ SigF+ Ly6Glow/- F4/80- CD11clow
Neutrophils (PMNs) - CD45+ SigF- Ly6G+
Monocytes - CD45+ SigF- Ly6G- F4/80+ CD14+
Alveolar macrophages – CD45+ Sig F+ Ly6Gint CD11c+ F480+
Lymphocytes – CD45+ SigF- Ly6G- F4/80- CD14- CD3/CD19+
Significant rise in the total number of cells retrieved in the BALF of influenza-
treated mice was observed as compared to the naive mice, and were higher in
ANXA1-/- compared to the WT (Figure 3.20B). The early innate immune
response was marked by significant increases in neutrophils and alveolar
macrophages (Figure 3.21 A&B) by day 3 pi and lowered by day 7 p.i..
Monocytic cells (Figure 3.21C) were significantly elevated between days 3
and 7 p.i. A marked increase was observed in lymphocytes (Figure 3.21D) at
day 7 p.i. Monocytes, neutrophils and lymphocytes were observed to be
significantly higher in ANXA1-/- mice compared to the WT mice. Eosinophils
were not observed until day 7 p.i., and numbers were not significantly different
between WT and ANXA1-/- mice (Figure 3.21E)
121
Figure 3.20 Gating Strategy for cell counts in BAL.
(A) Cells present in BAL were stained with various cell surface markers and categorised into eosinophils, neutrophils, alveolar macrophages, lymphocytes and monocytes.(B) Cells obtained in BAL were pelleted and counted using trypan blue. Data is representative of 2 independent experiments n=5/group/time point and is analysed using two-way ANOVA. Results are represented as mean±SD, *p<0.01.
FSC-A
CD45
SigF
Ly6G
PMNs
CD14
F4/80
Monocytes
CD19/CD3CD11b
Lymphocytes
Alveolar macs
Alveolar macs
Eosinophils
F4/80
CD11c
A
B
122
Figure 3.21 Influenza infection leads to higher cellular infiltration in the BAL from ANXA1-/- mice.
Cells obtained in BAL were stained and characterized 3 and 7 days p.i.using flow cytometry. Data is representative of 2 independent experiments n=5/group/time point. Results are represented as mean±SD and analysed using two-way ANOVA. *p<0.01,***p<0.001
A B
C
E
D
123
3.14. ANXA1-/- mice display higher lung inflammation
Having established the increased infiltrates in the lungs by flow cytometry, we
further performed histopathology using H&E staining to visualize the
infiltration and assess the inflammation. We focused on the large conducting
airways close to the site of bronchus bifurcation as this is the site of viral
replication during early infection and provides clear visualisation of the
bronchial epithelial lining and cellular infiltration into the peribronchial and
alveolar spaces. In the uninfected lungs the characteristic “honeycomb”
structure could be observed. The peribronchiolar spaces were free from
infiltrates and the airways looked clear. By day 5, mild inflammatory cell
infiltrate was observed in the peribronchiolar compartment and traces of red
blood cells were also detected in the conducting airways as well as the alveolar
air spaces, indicative of pulmonary haemorrhage. By day 10, severe
inflammatory cell infiltrate of the peribronchiolar space with a greater
magnitude of inflammatory foci was found in the alveolar parenchyma in
ANXA1-/- mouse lungs while in the WT mouse, the inflammation was still
mild or moderate and did not extend to the alveolar parenchyma. The
inflammation was scored from 0-3 using the following criterion by an
independent blinded pathologist (Figure 3.22).
0 no inflammation 1 mild, inflammatory cell infiltrate of the perivascular/peribronchiolar
compartment; 2 moderate, inflammatory cell infiltrate of the
perivascular/peribronchiolar space with modest extension into the alveolar parenchyma;
3 severe, inflammatory cell infiltrate of the perivascular/peribronchiolar space with a greater magnitude of inflammatory foci found in the alveolar parenchyma.
124
KOWT
UI
Day 5
Day 10
200μm
A
200μm
200μm
200μm
200μm
200μm 200μm
125
Figure 3.22 Higher Inflammation is observed in infected lungs.
(A) Lungs from naïve and mice infected for 5 or 10 days were harvested and fixed with 4% paraformaldehyde. Sections were made and slides were stained with H&E stain to assess the infiltrationg cells. The blue stained cells represent the infiltrate and the arrows point towards them. Slides are representative of sections made from 5 different mice/ group (B) Depending on the amount of infiltration and extent of spread the slides were scored from 0-3. UI stands for Uninfected.
B
126
3.15. ANXA1-/- mice display similar vascular integrity as WT mice
The concentration of serum albumin in BAL fluid reflects vascular integrity in
pulmonary tissues and permeability of the epithelial lining of the airways and
was thus measured as an indicator of lung pathology (Figure 3.23). Albumin
levels increased drastically in the infected mice between days 1-7 p.i, however
no significant difference was observed between WT and ANXA1-/- lungs.
Figure 3.23 Vascular Integrity is compromised upon infection.
Albumin leakage into BAL was determined by ELISA as a measure of lung damage. The data is represented as mean±SD for 5 mice/group/time point.
127
3.16. WT mice exhibit more apoptosis in their lungs
Caspase 3 activation is essential for viral propagation (Wurzer, et al., 2003)
and ANXA1 has been shown to be involved in apoptosis (Lim & Pervaiz,
2007). To determine whether deficiency of ANXA1 effects apoptosis post
influenza infection we harvested the lungs from naive and three days infected
mice. Lysates were prepared from small pieces of lungs and western blot for
cleaved caspase 3 and 7 was performed. Indeed, the levels of cleaved caspases
observed in the infected WT mice were significantly higher than those of
ANXA1-/- (Figure 3.24). This data establishes that apoptosis in epithelial cells
might help virus to replicate to better and make the infection more severe. The
next step would be to find out the exact role of ANXA1 in the pathway and
decipher its mechanism. To carry out these experiments we first evaluated
whether this experiment is reproducible in vitro in lung epithelial cells.
128
Figure 3.24 Wild type infected mice experience more apoptosis.
(A) Lungs from mice infected with 500pfu H1N1 PR8 were harvested 3 days post infection and lysed in RIPA buffer. Protein obtained was run on a SDS-PAGE and probed with apoptotic markers. The blot shows samples from 2 naïve and 2 infected mice.(B) Quantification of Cleaved caspases 3 and 7 with respect to tubulin.
Naive NaiveInfected Infected
WT KO WT KO WT KO WT KO
Cleaved caspase 7
Total caspase 7
Cleaved caspase 3
Total caspase 3
Tubulin
ANXA1
Set1 Set2
0
0.5
1
1.5
2
2.5
WT ANXA1-/-
fold
ch
ange
Cleaved Caspase 7
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
WT ANXA1-/-
fold
ch
ange
Cleaved Caspase 3
A
B
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3.17. ANXA1 is required for virus induced apoptosis
Caspase 3/7 activity is the hallmark for apoptosis. To determine influenza
induced apoptosis and whether there was any difference between WT and
ANXA1 deficient cells we first derived cells from lungs by digesting them and
then culturing the single cell suspensions. The cells were infected with
influenza virus and caspase 3/7 activity was measured. The activity measured
was low and not much apoptosis was seen. However, we still observed a 2 fold
difference between the capase 3/7 activity in WT compared knock down cells
(Figure 3.25A). This might be because the whole lung culture was a mixture of
epithelial and fibroblast cell population and PR8 virus cannot replicate
efficiently in fibroblasts (Stewart & Frickey, 1966). Following this, the
overexpression and knock down studies were carried out in A549 cells. As
expected influenza virus induced severe apoptosis in A549 cells which resulted
in a significant increase in caspase activity. Comparing these values between
WT and ANXA1 silenced cells there was a partial decrease in the ANXA1
knock down cells (Figure 3.25B) which was reversed by ANXA1
overexpression (Figure 3.25C). This observation supports our in vivo data
where higher apoptosis is observed in WT mice and our hypothesis that
inhibition of apoptosis might be responsible of lower viral loads in ANXA1
silenced cells.
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Figure 3.25 ANXA1 is required for virus induced apoptosis.
(A) Single cell suspension obtained from lung after digestion were cultured and infected with Influenza virus. Caspase activity was measured 48hrs p.i. (B) ANXA1 was silenced in A549 cells using shRNA and caspase activity was measured 48hrs p.i (C) The reversal of phenotype is shown by overexpression of ANXA1 48hrs p.i. Data is represented as mean±S.D and is representative of three independent experiments. Cntrl represents control siRNA, KD is ANXA1 siRNA , EV is empty vector and ANXA1-V5 is ANXA1 overexpression plasmid with V5 tag.
C
B
A
131
3.18. ANXA1 inhibits NF-κB activation during influenza infection
ANXA1 has been shown to interact with NF-κB and inhibit its activation (Z.
M. Wang et al., 2011; Zhang, Huang, Zhao, & Rigas, 2010). Zhang et. al also
reported induction of apoptosis with inhibition of NF-κB due to ANXA1. NF-
κB has been reported as a pro-survival gene widely in the literature, therefore
we hypothesized that inactivation of NF-κB upon infection might lead to
higher apoptosis and hence higher viral loads. To put our hypothesis to test we
overexpressed ANXA1 in A549 cells and measured NF-κB promoter activity
by dual luciferase reporter assay. The cells transfected with empty vector
displayed a threefold increase in the activity whereas no increase was detected
in the cells overexpressing ANXA1 (Figure 3.26). A significant difference was
also observed between the EV and ANXA1 transfected cells after infection,
thus confirming that NF-κB activity reduced in presence of ANXA1 and results
in higher apoptosis which may in turn be the cause of higher viral titers
observed in presence of ANXA1. Overall, our data points towards the role of
ANXA1 in apoptosis might be Nf-κB dependant.
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Figure 3.26 NF-κB activity is decreased in presence of ANXA1 upon infection.
EV and V5-tagged ANXA1 were transfected in A549 cells for 36hrs followed by infection for 4 hrs. Promoter activity was determined by dual luciferase reporter assay. Data is representative of 3 independent experiments, expressed as mean±SD. *p<0.05, **p<0.01 as determined by t test.
NF-B luciferase
UI Infected 0
500
1000
1500
2000
2500EVANXA1-V5
*
RL
U
133
Chapter 4 Discussion
4.1. Summary of key findings
This study has established that ANXA1 controls TLR7 signalling in a cell
specific manner and is a positive regulator of Influenza virus infection, both in
vivo and in vitro. We show that ANXA1 plays a crucial role in TLR7
signalling. R848 activated BMMOs from ANXA1-/- mice exhibit low levels
pro-inflammatory cytokines while BMDCs from these mice produce very high
levels of pro-inflammatory cytokines compared to their WT controls,
highlighting the importance of cell-type specificity. B cells from ANXA1-/-
mice also demonstrate an activated phenotype and proliferate faster compared
to the WT. TLR7 dependent inflammasome activation is higher in the
ANXA1-/- cells. We also show that ANXA1-/- mice have a better survival rate
compared to the WT mice. ANXA1 is a virus inducible protein and its presence
supports viral propagation. It is upregulated and cleaved upon virus infection.
Viral titers drop when ANXA1 is knocked out of the system and increase upon
over expression. Higher cellular infiltration was observed in ANXA1-/- mice
however, not much difference was seen in terms of cytokines and interferons.
Lower levels of apoptosis noticed in ANXA1-/- mice and cells, might be
implicated as the cause for reduced virus loads. Overall, our data establishes
importance of ANXA1 in TLR7 signalling and Influenza virus infection.
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4.2. Regulation of Cytokines in BMMOs and BMDCs
Previous studies from our lab demonstrate that ANXA1 plays a role in TLR3
signalling by aiding phosphorylation and translocation of IRF3 to the nucleus
(Bist, et al., 2013). TLR7 and TLR3 can both recognize influenza virus because
of the ssRNA and dsRNA intermediate it forms during replication. However,
TLR3 has detrimental contributions in influenza pathogenesis (Le Goffic et al.,
2006) whereas TLR7 agonists are seen as a promising antivirals (Lund et al.,
2004), and when we started this project, no other study had reported the role of
ANXA1 on TLR7 signalling. Therefore we focused our work on the role of
ANXA1 in TLR7 signalling. In this study R848, a TLR7 agonist was used to
determine the cytokine responses in BMMOs and BMDCs. R848 was observed
to be the most potent out of all TLR7 agonists we used due to the chemical
modifications in its structure. It is derived from R837 (Burns, Ferbel, Tomai,
Miller, & Gaspari, 2000). R848 stimulates the Myd88 dependent pathway to
produce downstream cytokines (Hemmi et al., 2002) . In the case of BMMOs
we observed that cytokines like IL-6 and IL-12p40 are downregulated both at
RNA and protein level. Stimulation with LPS which activates both MyD88 and
TRIF, abrogated production of TRIF dependent cytokines in BMMOs but had
no effect on IL-6 production (Bist, et al., 2013). However, TLR7 dependant
cytokine production is reversed in DCs where IL-6 and IL-12p40 production
was observed to be higher in ANXA1-/- cells. Our data is consistent with Weyd
et. al who showed recently that ANXA1 suppresses DC activation upon R848
treatment (Weyd, et al., 2013). This inhibition in cytokine production by
ANXA1 was observed previously in fibroblasts and peritoneal macrophages,
where ANXA1 negatively regulated IL-6 and TNF-α production by regulation
135
of p38 MAPK and MAPK phosphatase-1 (Y. H. Yang, et al., 2009; Y. H.
Yang, et al., 2006). This shows ANXA1 regulates the signalling pathways in a
cell specific manner. ANXA1 displayed cell specific effects with respect to
proliferation. In RAW264.7 cells, a macrophage cell line, ANXA1 inhibited
proliferation while it induced proliferation in hepatocytes (Lim & Pervaiz,
2007). ANXA1 has been shown to interact with NF-κB, the master regulator of
pro-inflammatory cytokines and it is essential for IRF-3 activation and
translocation to the nucleus (Bist, et al., 2013; Zhang, et al., 2010). DNA
binding of NF-κB subunit p65 is reduced in presence of ANXA1 (Weyd, et al.,
2013). Therefore, regulation of TLR7 pathways by ANXA1 through
differential interference with NF-κB and IRF-3 activation might be the reason
behind this selective production of cytokines in different cell types. This cell
specificity might also contribute to the reason of why we do not observe any
differences in the cytokine levels in vivo. Another reason could be the different
expression of the receptor levels of TLR7 in both macrophages and DCs, but in
our study, we found that the levels were similar. We also observed differences
TLR7 dependent IL-1β production through the activation of inflammasome but
no differences were observed in IL-1β in vivo. This might be due to the
redundancy of IL-1β production pathways i.e the involvement of caspase-1
independent pathways that do not require TLR signalling (Mayer-Barber et al.,
2010) .
We next infected BMMOs and BMDCs with influenza virus and evaluated the
pro-inflammatory cytokine response. Levels of IL-6, IL-12 and TNFα in
BMMOs were negligible while DCs produced intermediate levels compared to
what we observed with R848. Infection of both BMMOs and BMDCs is
136
abortive, i.e. these cells can be infected, though at a very low percentage but
virus cannot replicate inside (Ioannidis, Verity, Crawford, Rockman, & Brown,
2012; Wells, Albrecht, Daniel, & Ennis, 1978). Differential cytokine
production between BMMOs and BMDCs has also been observed upon
mycobacterium (Hickman, Chan, & Salgame, 2002) and salmonella infections
(Yrlid, Svensson, Johansson, & Wick, 2000).
There are a variety of other factors which can regulate infection such as type of
DCs or macrophages. Alveolar macrophages are infected better compared to
peritoneal macrophages (B. M. Hartmann et al., 2013; Lee, Dutry, & Peiris,
2012). In addition, the type of influenza virus strain can predict infectivity of
abortive cells. Highly pathogenic strains like H5N1 infects almost all cell
types as compared to H1N1 (Chan, Leung, Nicholls, Peiris, & Chan, 2012) and
the host, (ferrets or human) blood cells are better compared to mouse cells (van
Riel et al., 2007) . The mechanism of infection is not well understood but it
may be through phagocytosis. However, the question that remains to be
answered is why DCs are more susceptible than macrophages. The
conventional sialic acid receptor is absent in both cell types and alternate
receptors such as C-type lectin receptors namely macrophage mannose receptor
(MMR), macrophage galactose-type lectin (MGL) etc. have been shown to aid
influenza virus entry (Londrigan, Tate, Brooks, & Reading, 2012; Upham,
Pickett, Irimura, Anders, & Reading, 2010) Apart from these two receptors,
DCs have been shown to express a few more receptors like DCIR, Langerin,
DC-SIGN, Dectin-1 and Dectin-2 (Denda-Nagai, Kubota, Tsuiji, Kamata, &
Irimura, 2002). Presence of these receptors that aid phagocytosis and
endocytosis might provide DCs an edge over the macrophages, possibly
137
leading to more cytokine production after infection. Another reason might be
the presence of Mx gene that confers resistance to influenza infection is very
highly expressed in macrophages (Arnheiter & Haller, 1983; Horisberger,
Staeheli, & Haller, 1983; Staeheli, Haller, Boll, Lindenmann, & Weissmann,
1986; Staeheli et al., 1986).
4.3. Enhanced B cell proliferation in ANXA1-/- cells
B lymphocytes play an important role in both adaptive and innate immunity.
TLR agonists like LPS and immiquinods activates B cells and stimulates
proliferation and IgM secretion (Yazawa et al., 2003). Exogenous
physiological signals leading to B-cell activation have been classified as
“signal 1,” the stimulation of the B-cell receptor (BCR) by antigen binding;
“signal 2,” the stimulation of CD40 by the CD40 ligand molecule, expressed
on activated helper T cells; and “signal 3,” the stimulation of Toll-like
receptors (TLRs) by microbial components or their mimics. All three signals
together are required for maximal proliferation of naive B cells (Ruprecht &
Lanzavecchia, 2006). However, stimulation with TLR ligands alone, for
example, CpG DNA, is sufficient to cause transient B-cell activation, including
proliferation and induction of immune effector molecules such as CD86, a T-
cell-costimulatory molecule (G. Hartmann & Krieg, 2000). B cells express
several TLRs on their surface mainly TLRs 1,6,7,9, 10 and RP-105 and main
TLR induced responses include proliferation, activation and plasma cell
differentiation (Bohnhorst et al., 2006). R848 induced proliferation is similar to
the effects brought about by CD40-CD40L interactions (Bishop, et al., 2000) .
138
We observed that R848 activates and induces proliferation in both WT and
ANXA1-/- cells however, both these are significantly higher in the ANXA1-/-
cells. On the other hand, effect of TLR ligands on T cells is controversial. Most
studies indicate that T cells express no or very low expression of only some
TLRs such as TLR6, TLR8 and TLR2 (Rich et. al. 2005) while others have
demonstrated direct effects of TLR on T cell with TLR9 ligands like CpG.
However, in our hands we do not observe any T cell proliferation upon
treatment with R848 (data not shown).
Higher phosphorylation in Akt, an important player in cell survival was
observed which might contribute to the higher proliferation in ANXA1-/- cells.
Akt has not been shown to be regulated by ANXA1 before but it is known to
regulate other cell proliferation players such as MAPKs ERK1/2 and p38
(Alldridge & Bryant, 2003; Y. H. Yang, et al., 2006). TLRs play a role in the
early stages of B cell response such as induction of proliferation and fine
tuning of IgG class switching, but are not sufficient alone in the maturation and
secretion of antibodies. Help from other factors like IFNα is required for proper
response. As our in vivo data with influenza virus indicates that there are no
significant differences in the production of IFNα, we might expect with all
other factors remaining constant, proliferation induced in absence of ANXA1
might contribute to a better humoral response. In our previous publication with
respect to asthma we did observe higher allergen-specific and total IgE, IgG2a
and IgG2b levels in ANXA1-/- mice (Ng et al., 2011). Influenza specific serum
IgM levels usually peak after 8 days p.i and IgG even later (Wolf et al., 2011).
However, most of our experiments do not exceed day 7 because that is the time
when most mice lose 25% of the body weight and have to be sacrificed.
139
Therefore, we could not quantify antibody production in this set of
experiments. However we predict that the antibody production will be higher
in the serum of ANXA1-/- mice.
4.4. Regulation of ANXA1 during influenza virus infection
Expression of ANXA1 is detected in various cell types like neutrophils (Oliani,
Paul-Clark, Christian, Flower, & Perretti, 2001), monocytes (Harricane, et al.,
1996), mast cells (Oliani, Christian, Manston, Flower, & Perretti, 2000) and
epithelial cells (Sena et al., 2006). The levels of the protein increase during
some inflammatory reactions however the exact reason of this upregulation is
still unknown. We have observed this increase in the lungs of influenza
infected mice. This is in line with previous studies where increased levels of
ANXA1 was observed in porcine monocytes infected with swine flu virus
(Katoh, 2000). However, this increase in ANXA1 is not specific to viral
infections but also has been shown in bacterial infections. Previously an
increase has been observed in ocular taxoplasmosis (Mimura, Tedesco,
Calabrese, Gil, & Oliani, 2012) and LPS induced endotoxemia (Damazo, et al.,
2005). These observations indicate towards ANXA1 being a virus-inducible or
infection-inducible protein. We have shown in our study that this increase is
due to the enhanced promoter activity of ANXA1.
We also observed that ANXA1 is cleaved into a 34kDa fragment and a small
3kDa fragment upon infection. Similar cleavage has also been shown when
Raw 264.7 cells were treated with LPS and poly (I:C) (data not shown) and in
tuberculosis infection (Gan, et al., 2008). This cleavage is thought be a result of
140
proteolytic action of enzymes such as elastase or calpain and cleaves at the N-
terminal of the protein (Rescher, Goebeler, Wilbers, & Gerke, 2006; Williams,
et al., 2010). The cleaved form is observed at day 3 but absent at day 5. This
might be because the infiltration of neutrophils is highest at day 3 and they
secrete enzymes like elastases which as described earlier can cleave ANXA1.
Most studies have been done in macrophages or neutrophils; however this
phenomenon has not been previously tested in epithelial cells, and we are the
first to show this. The cleaved ANXA1 is implicated to play a role in apoptosis
or is observed to have pro-inflammatory activities against the previous
conventions that ANXA1 is anti-inflammatory (Williams, et al., 2010). WT
cells exhibit higher levels of apoptosis which support the experiments by Peng
et al. where cyclohexamide treatment increases levels of cleaved ANXA1
which is abolished on treatment with calpain inhibitors.
WT and ANXA1-/- mice have similar cytokine levels in their BAL after
infection even though lower levels may be expected in WT due to the presence
of anti-inflammatory ANXA1. However, since ANXA1 is cleaved upon
infection, we can reason that the pro-inflammatory effects may balance the
anti-inflammatory effects.
4.5. ANXA1 positively regulates virus propagation
Our data clearly shows that ANXA1 plays a crucial role in virus propagation.
We have shown in vivo and in vitro that presence of ANXA1 leads to higher
virus titers. By carrying out both loss and gain of function studies, the positive
correlation between ANXA1 and viral titers were observed. To make our
141
assays more sensitive we used both qPCR and plaque assays. Plaque assay
estimates the number of live virions capable of producing cytopathic effects in
the MDCK cell monolayers. Though the plaque assay is a more direct way of
quantifying the virions released in the supernatant, it is less sensitive than
qPCR. Real time PCR detects every single copy of viral RNA whereas the
plaque assay will detect a plaque forming unit that might contain any number
of viruses. Viral propagation can be divided into three main processes entry,
replication and budding. By assessing viral titers in the cells infected by the
virus, we confirmed that ANXA1 plays a role in the early stages of propagation
i.e entry or replication, and these lower viral titers are also reflected after
budding into the supernatant. Two recent reports have been published on
ANXA6 and influenza. Both show a negative correlation between ANXA6 and
influenza virus titers. The mechanisms of regulation revealed by the two papers
are however very different. Musinol et. al show that viral replication and
propagation is inhibited in ANXA-6 deficient cells due to disturbance in
cholesterol levels in the late endosomes (Musiol et al., 2013) whereas the Ma
et. al demonstrated that the early steps in viral life cycle are not affected
ANXA-6 disrupts the budding process. Our mechanism of ANXA1 on
apoptosis is different from the two processes mentioned but it would be
interesting to also study the involvement of ANXA1 in cholesterol balancing or
interaction of ANXA1 with viral proteins.
4.6. Better survival of ANXA1-/- mice upon infection
142
We observed a strong phenotype in the survival of mice when we infected
them with a lethal dose of flu virus where ANXA1-/- mice have better survival
rates. Influenza virus replicates in epithelial cells due to which they produce
chemokines and cytokines and recruit the mononuclear cell population to the
site of infection. The first question that was to be answered was whether it is
the cytokine storm that is the reason for extensive weight loss in WT mice.
Since ANXA1 is an immunomodulatory molecule it would be quite unlikely
that WT mice have higher levels compared to ANXA1-/- but we still needed to
investigate it. To our surprise, the levels of cytokines in both WT and
ANXA1-/- were similar and no significant difference was observed in pro or
anti-inflammatory cytokines and type I interferons. The kinetics of production
of these cytokines over 7 days was also similar in WT and ANXA1-/- mice.
Cytokines and chemokines are the mediators which cause the clinical signs
related to the infection. IL-1, IL-6 and IL-12 have been correlated with the
symptom pathogenesis during acute influenza (Kaiser, Fritz, Straus, Gubareva,
& Hayden, 2001; Kostense et al., 1998; Schmitz, Kurrer, Bachmann, & Kopf,
2005). The viral titers however may not negatively correspond to the cytokines.
In our study the cytokine levels are similar but viral load in ANXA1-/- is lower
and another study shows lower cytokine levels in TLR3-/- mice but higher viral
loads compared to the WT (Le Goffic, et al., 2006). Lesser viral loads in the
early phases of infection and no differences in the cytokines led us to
hypothesize that influenza virus is unable to propagate efficiently in ANXA1-/-
mice rather than it being a case of efficient clearance. The lung damage as
determined by Albumin ELISA was also similar in both WT and ANXA1-/- .
143
Accumulation of inflammatory cell infiltrate is another hallmark of influenza
virus infection. Lymphocytes mainly CD8+ T cells infiltrate from the lymph
nodes later in the infection and APCs infiltrate earlier which provide
costimulatory signals for the activation of effector CD8+ cells following which
effector CD8+ T cells cause cytolysis of infected epithelial cells (Hufford et al.,
2012). We found higher numbers of infiltrating lymphocytes, monocytes and
neutrophils in ANXA1-/- mice. Other studies related to lung fibrosis (Damazo
et al., 2011), intestinal ischemia (Guido, Zanatelli, Tavares-de-Lima, Oliani, &
Damazo, 2013) and zymosan induced inflammation(Getting, Flower, &
Perretti, 1997) also observe higher recruitment of leukocytes in absence of
ANXA1. Inhibition of leukocyte migration is one of the ways by which
ANXA1 brings about its anti-inflammatory effects (Getting, et al., 1997; Lim,
Solito, Russo-Marie, Flower, & Perretti, 1998) . The mechanism of this is
proposed to be the inhibition of transmigration of cells through the
endothelium due to the externalization of ANXA1 on the surface of adherent
cells (Perretti et al., 1996). The same levels of cytokines in BAL and more
cellular infiltration might point towards less cytokine being produced per cell
in the ANXA1-/- mice. The human N-formyl peptide receptor (FPR) is a key
modulator of chemotaxis directing granulocytes toward sites of infections.
Since ANXA1 is a ligand for all FPR receptors, it desensitizes the cells towards
subsequent activation from other stimuli and the inhibits transmigration of
leukocytes (Ernst, et al., 2004).
Our data suggests that ANXA1 affects viral propagation but the mechanism is
not clear. There might be few mechanisms i) ANXA1, like ANXA6 might
interact with M2 which is an ion channel protein (Ma, et al., 2012) , and
144
interfere with the uncoating of the viral envelope ii) ANXA1 might induce
dephosphorylation of BAD upon infection and allow its transportation to the
nucleus for induction of apoptosis through intrinsic pathway as influenza virus
has been shown to induce apoptosis via BAD mediated mitochondria
dysregulation (Tran, Cortens, Du, Wilkins, & Coombs, 2013) iii) ANXA1
interferes with RIP-1 poly- ubiquitination by E3 ligase (unpublished data) and
this might lead to inhibition of NF-κB signalling and hence enhancing
apoptosis.
Apoptosis plays an important role in viral propagation (Wurzer, et al., 2003)
and ANXA1 is a pro-apoptotic molecule (Lim & Pervaiz, 2007; Solito, et al.,
2001; Solito, Kamal, et al., 2003) which might help the virus in its better
propagation and hence lower survival of the mice. In case of complex disease
like Influenza, a combination of multitude of factors determines survival out of
which one might be apoptosis. Our results indeed show that higher apoptosis
corresponds with higher viral loads.
145
Figure 4.1 Putative mechanism for ANXA1 action upon infection
(i) ANXA1 might interact with M2 protein and interfere with the uncoating of the viral envelope ii) ANXA1 might induce dephophorylation of BAD upon infection and allow its transportation to the nucleus for induction of apoptosis through mitochondria iii) ANXA1 interferes with RIP-1 poly-ubiquitination leading to inhibition of NF-κB signalling and hence enhancing apoptosis.
146
4.7. Inhibition of NF-κB in ANXA1 overexpressing cells
Suppression of NF-κB by ANXA1 has been shown previously in cancer studies
(Ouyang et al., 2012; Zhang, et al., 2010). Here, we also observe inhibition of
NF-κB activity upon ANXA1 overexpression. NF-κB inhibits TNF-α induced
apoptosis(Van Antwerp, Martin, Kafri, Green, & Verma, 1996) and Curcumin,
an anti-cancer drug induces apoptosis by inhibiting NF-κB (Shishodia, Amin,
Lai, & Aggarwal, 2005). These and many other evidences indicate that NF-κB
is a pro-survival gene and its inhibition leads to higher degree of apoptosis. As
stated earlier apoptosis is essential for viral propagation, and its inhibition
might interfere with the propagation. However, a few studies reveal the need of
NF-κB activation in the propagation (Ehrhardt et al., 2013; Wurzer, et al.,
2004). As discussed before, apoptosis is inhibited in the early stages of
infection and induced in the later stages and NF-κB activation is also in phases
therefore, further time dependant experiments need to be conducted to answer
this discrepancy.
4.8. Limitations of the Study
There are a few limitations to this work. The first is the virus dose. We observe
the protective phenotype only at 500pfu and no significant difference was
observed at 750pfu (data not shown). This dose however may or may not be
physiological. Also we do not have the range where absence of ANXA1 can
provide the protective effect. The second limitation is the strain of the mice.
147
C57/B6 mice are the best models to study influenza since Balb/c mice show
some resistance to the infection and high doses of virus are required to infect
them. A third limitation would be the use of only one virus strain H1N1 PR8.
Other strains like H3N2 or HIN1 WSN/33 could be used.
4.9. Clinical Significance
According to our findings, ANXA1 deficiency has proven to be beneficial for
the hosts. By restricting the production of progeny virions it helps in the
containment of the disease and decreases morbidity. Influenza is a one of the
major threats today. Due to past pandemics and their deadly potential to kill
millions of people constant surveillance and monitoring is done by health
organizations throughout the world and new vaccines are generated every 6
months based on circulating strains. Vaccines are the most effective preventive
measure against influenza but due to the vast effects of ANXA1 on so cellular
functions it cannot be a component of the vaccine itself. ANXA1 is a
glucocorticoid inducible protein and brings about their anti-inflammatory
effects. Based on this, N-terminal ANXA1 peptides based therapies are being
tested in mice and rats for ailments like colitis, ischaemic injury, allergic
inflammation etc. However, no therapies targeting ANXA1 and lowering its
expression have been studied yet. For the first time detrimental effects of
ANXA1 expression have been shown with respect to infectious disease. A few
possibilities that can possibly be tested would be to administer anti-ANXA1
antibodies or FPR inhibitors along with antivirals to block ANXA1 and in turn
control viral loads.
148
4.10. Future Directions
This study has established that ANXA1 plays an important role in influenza
virus pathogenesis and acts as a positive regulator. However, there are a few
questions that remain to be solved.
The lower viral titers have been associated with lower apoptosis in absence of
ANXA1. The executioner caspases have been shown to be affected in ANXA1
deficient cells. These caspases are common for both extrinsic and intrinsic
pathways but it is yet to be determined which apoptotic pathway
(mitochondrial/intrinsic or receptor mediated/extrinsic) involves ANXA1 upon
infection. The first report that established caspase 3 activation being necessary
for viral propagation (Wurzer, et al., 2003) have linked the vRNP transport
from nucleus to cytoplasm, to apoptotic signalling. In this respect it will be
interesting to observe the movements of ANXA1 in the cell upon infection and
its interaction with vRNP if any.
Ma et. al have shown that human Annexin A6 interacts with the M2 protein of
the virus, however the functional significance of this interaction is not
determined yet. Annexins possess a high structural and sequence homology
except for the N-terminal region. ANXA1 might also interact with M2 or other
proteins like NS1 and PB2-F2, which are known to induce apoptosis upon
infection.
As mentioned earlier ANXA1 has various domains, the C-terminal region is
conserved while the N-teminal is the unique domain for all annexins. C-
terminal interactions with TBK-1 help ANXA1 to execute its role in TRIF
149
dependent signalling (Bist, et al., 2013). Different N and C terminal mutants
can be used to detect the domains important for regulating viral pathogenesis.
ANXA1 on the surface of apoptotic cells suppresses CD8+ T cell immunity by
preventing DC activation (Weyd, et al., 2013). ANXA1 deficiency in CD4+ T
cells also allows for exacerbation of T cell dependent immune responses (Y. H.
Yang et al., 2013). ANXA1 has also been described as the molecular tuner for
TCR signalling (D’Acquisto, et al.,2007). To find the mechanism of better
survival of ANXA1 deficient mice we mainly concentrated on the innate
immune pathways however, considering the above mentioned observations a
few experiments on adaptive immune cells might provide useful insights into
the mechanism governing survival.
4.11. Conclusion
This study has established that ANXA1 plays an important role in TLR7
signalling and Influenza virus pathogenesis. The novel findings include the cell
specific role of ANXA1 in innate immune cells upon activation with TLR7
ligand R848 and its deficiency provides protection against influenza virus in
mice. ANXA1 interferes with the viral propagation in the early stages of
infection. The innate immune responses mounted against influenza virus are
similar in WT and ANXA1-/- mice. Higher apoptosis in the epithelial cells, the
hosts for virus infection, leads to higher viral loads and morbidity in WT mice.
Clinically, it will be challenging to target ANXA1 because of its various roles
in cell function maintenance. Current therapies involve administration of
ANXA1 in the form of recombinant protein or peptide but lowering its
expressions or blocking production might also be a part of future therapies.
150
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