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The Role and Effects of CD200R Activation in Murine Models of Allergic Airways Inflammation By: Lindsay N Woo A thesis submitted in conformity with the requirements for the degree Master in Science Institute of Medical Science University of Toronto ©Copyright by Lindsay N Woo 2019
Transcript of The Role and Effects of CD200R Activation in Murine Models ... · DeHaas, Kelsey Yang, Elizabeth...
The Role and Effects of CD200R Activation in Murine Models of
Allergic Airways Inflammation
By:
Lindsay N Woo
A thesis submitted in conformity with the requirements
for the degree Master in Science
Institute of Medical Science
University of Toronto
©Copyright by Lindsay N Woo 2019
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The Role and Effects of CD200R Activation in Murine Models of
Allergic Airways Inflammation
Lindsay Woo
Master in Science
Institute of Medical Science
University of Toronto
2019
Abstract
The CD200/CD200R anti-inflammatory signalling pathway plays an important role in immune
homeostasis and has been reported to be dysregulated in asthma, a chronic inflammatory airways
disease. We hypothesized that activation of the CD200R will initiate an anti-inflammatory
signalling cascade which will reduce features of asthma. We explored the role of the
CD200/CD200R signalling pathway in the pathogenesis of an allergic airways inflammation
model and evaluated the effects of CD200R activation with the agonist aptamer PEG.CCS13 on
the features of asthma. Our results found the CD200/CD200R pathway imbalanced in a chronic
model of allergic airways inflammation, suggesting it plays a role in the pathogenesis of the
disease. We further found that activation of the receptor with PEG.CCS13 resulted in a
reduction in features of asthma including eosinophilic inflammation, airway hyperresponsiveness
and goblet cell hyperplasia. We concluded that activation of the CD200R is a promising
therapeutic candidate for an asthma treatment.
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Acknowledgments
I would like to extend my immense gratitude to all those who supported and aided me in this
endeavor- thank you to you all. Firstly, I would like to express my thanks to my supervisor Dr.
Chow for all her guidance, patience and support over these last years. Thank you for all the
opportunities, mentorship and lessons you gave to me.
I would like to thank Dr. Jeremy Scott for his mentorship and support through all my work.
Also, thank you to the member of my committee, Dr. Hartmut Grasemann and Dr. Jean Gariépy
for their collaboration and support. I also would like to thank all my collaborators over the years:
Aaron Prodeus, Amanda Sparkes, Dr. Arthur Chan,
I would also like to acknowledge Xiaomin Wang and thank her for all her expertise and
assistance. Thank you also to Dave (Wang Yuan) Guo, Dong An, and Vanessa Chu for their
help with this project. Thank you to all the members of the Chow lab during my time there,
especially Ashley Young, Sepehr Salehi, Joyce Wu, Sarina Zhang, Sehrish Mahmood, Emily
DeHaas, Kelsey Yang, Elizabeth Cho, Teddy Xu and Queenie (Qian Wen) Huang.
Finally, and most of all I would like to thank my biggest supporters: my friends and family. I am
so grateful for your never-ending love and support. Thank you to my amazing and supportive
parents for the millions of big and small things they’ve done for me. Thank you to my siblings
Rachel and Brendan and all my extended family. Finally thank you to all my friends, especially
Alina, Allen, Cole, Hannah, Jackson, and Jonny, Karan and Madie.
Statement of Contributions
Lindsay Woo worked on study design, experiment completion, figure creation and writing. Dr.
Chow and Dr. Scott worked on experimental design, troubleshooting, supervision and
interpretations. Dr. Jean Gariépy provided the aptamer, aptamer controls. Aaron Prodeus and
Amanda Sparkes provided aptamer expertise and Amanda Sparkes conducted preliminary FACS
experiments. Ashley Young, Sepehr Salehi, Sarina Zhang, Angela Hin, Vanessa Chu and Dave
Guo assisted with the Flexivent Experiments. Xiaomin Wang provided training and assistance
conducting, ELISA, qPCR, FACS, slide preparation, and collagen assay. Dave Guo conducted
FACS experiments.
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Table of Contents
Abstract………………………………………………………………………………………......ii
Acknowledgements……………………………………………………………………………...iii
Table of Contents..........................................................................................................................iv
List of Abbreviations....................................................................................................................vi
List of Tables...............................................................................................................................viii
List of Figures...............................................................................................................................ix
Chapter 1: Literature Review......................................................................................................1
1.1 Asthma......................................................................................................................................1
1.1.1 Prevalence and Burden of Asthma.............................................................................1
1.1.2 Features of Ashtma....................................................................................................2
1.1.3 Asthma Endotypes and Phenotypes..........................................................................10
1.1.4 Current Treatments...................................................................................................12
1.2 Animal Models of Allergic Airways Inflammation................................................................15
1.2.1 Inducing Allergic Airways inflammation in Animals..............................................15
1.2.2 Acute and Chronic Models of Allergic Airways Inflammation...............................18
1.2.3 Asthma Clusters in Mouse Models...........................................................................21
1.2.4 Limitations of HDM Models of Allergic Inflammation...........................................22
1.3 CD200/CD200 Receptor Pathway...........................................................................................23
1.3.1 CD200 and CD200R: Structure, Distribution and Mechanism................................23
1.3.2 CD200/CD20R in Disease Pathogenesis..................................................................25
1.3.3 CD200/CD200R Regulation as a Therapeutic..........................................................27
1.3.4 CD200/CD200R in Allergic Disease........................................................................29
1.4 Aptamers..................................................................................................................................30
1.4.1 Definition and Development.....................................................................................30
1.4.2 Aptamers as Therapeutics.........................................................................................31
1.4.3 Aptamer Modification...............................................................................................33
1.4.4 CD200 Receptor Aptamers.......................................................................................35
Chapter 2: Hypothesis and Objectives.......................................................................................37
2.1 Hypothesis................................................................................................................................37
2.2 Research Objectives.................................................................................................................37
Chapter 3: Materials and Methods............................................................................................38
3.1 Animals....................................................................................................................................38
3.2 Models of Allergic Airways Inflammation..............................................................................38
3.2.1 Acute Model of Allergic Airways Inflammation......................................................38
3.2.2 Four Week Chronic Model of Allergic Airways Inflammation................................39
3.2.3 Eight Week Model of Allergic Airways Inflammation............................................39
3.3 Treatment Regimen with PEG.CCS13....................................................................................41
3.4 Assessment of Lung Function Mechanics and Methacholine Responsiveness.......................42
3.5 Tissue Collection.....................................................................................................................42
3.5.1 Serum Collection......................................................................................................42
3.5.2 Bronchoalveolar Lavage (BAL) and Cell Counts.....................................................43
3.5.3 Lung Tissue...............................................................................................................43
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3.6 Histological Analysis...............................................................................................................43
3.7 Quantification of Serum HDM-Specific IgE...........................................................................44
3.8 Quantification of Total Lung Collagen....................................................................................45
3.9 qPCR Gene Expression Profiling.............................................................................................45
3.10 FACS Analysis of Whole Lungs............................................................................................47
3.11 Statistical Analysis.................................................................................................................50
Chapter 4: Results........................................................................................................................51
4.1 Models of Allergic Airways Inflammation..............................................................................51
4.1.1 All Models Demonstrate AHR with HDM Challenge..............................................51
4.1.2 Differences in Inflammation Between HDM Models...............................................54
4.1.3 Evidence of Remodelling in Models of Allergic Airways Inflammation.................59
4.2 Activation of CD200R in an Acute Model of Allergic Airways Inflammation......................63
4.3 Expression and Localization of CD200 and CD200R.............................................................66
4.4 Changes in Whole Lung Cellular Inflammation in Allergic Airways Inflammation Following
CD200R Activation.......................................................................................................................69
4.5 Changes in Inflammatory Mediators Following CD200R Activation.....................................73
4.6 CD200R Activation Reduces AHR in Established Chronic Allergic Airways Inflammation.75
4.7 Evaluation of Histological and Total Collagen Content with PEG.CCS13 Treatment...........78
Chapter 5: Discussion..................................................................................................................83
5.1 Establishing the Role of CD200/CD200R in Allergic Airways Inflammation........................83
5.1.1 Gene Expression of CD200/CD200R in Allergic Airways Inflammation................83
5.1.2 FACS Analysis of CD200R Expression...................................................................85
5.2 Effect of CD200R Activation on Features of Allergic Airways Inflammation.......................87
5.2.1 Effects on Cellular Inflammation..............................................................................87
5.2.2 Effects of CD200R Activation on HDM-Specific IgE.............................................90
5.2.3 Effects on Inflammatory Gene Expression...............................................................91
5.2.4 Effects on Airway Hyperresponsiveness..................................................................93
5.2.5 Effects on Remodelling............................................................................................95
5.3 Immunogenic Effects from PEG.CCS13.................................................................................96
5.3.1 Immunogenic Effects Seen in Respiratory Responsiveness.....................................96
5.3.2 Immunogenic Effects on Cellular Inflammation......................................................98
Chapter 6: Conclusions.............................................................................................................101
Chapter 7: Future Directions....................................................................................................104
7.1 Exploring Delivery Methods..................................................................................................104
7.2 Other Aptamer Delivery Molecules.......................................................................................105
7.3 Determining How Sex Differences Affect Inflammation and CD200R Activation..............105
7.4 Increasing Treatment Duration or Adding Other Therapeutics.............................................105
Chapter 8: References...............................................................................................................107
Chapter 9: Appendix.................................................................................................................114
9.1 Sex Differences in the Lung Mechanics of BALB/c Mice....................................................116
9.2 VISTA Activation in Acute Model of Allergic Airways Inflammation................................122
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Abbreviations
ABPM Allergic Bronchopulmonary mycosis
AHR Airway Hyperresponsiveness
API Asthma Predictive Indices
ASA Aspirin-Sensitive Asthma
ARG Arginase
BAL Bronchoalveolar Lavage
BALF Bronchoalveolar Lavage Fluid
CCAC Canadian Council on Animal Care
CCL Chemokine
CD200R CD200 Receptor
CST Quasi-Static Compliance
DC Dendritic Cell
ELISA Enzyme-Linked Immunosorbent Assay
FACS Fluorescence Activated Cell Sorting
FDA US Food and Drug Administration
G Peripheral Tissue Dampening
GINA Global Initiative for Asthma
HDM House Dust Mite
H&E Hematoxylin and Eosin
ICS Inhaled Corticosteroids
IFN Interferon
IgE Immunoglobulin E
IL Interleukin
IP Intraperitoneal
IV Intravenous
M1 Classically Activated Macrophages
M2 Alternatively Activated Macrophages
MASS Masson’s Trichrome Stain
MBP Major Basic Protein
MCh Methacholine
MIF Macrophage Migration Inhibitory Factor
MMPs Matrix Metalloproteinases
MUC Mucin
OVA Ovalbumin
PAS Periodic Acid-Schiff
PBMC Peripheral Blood Mononuclear Cell
PBS Phosphate Buffered Saline
PCR Polymerase Chain Reaction
PEG Polyethylene Glycol
PEG.SCRAM PEGylated Scrambled Control Aptamer
PPIA-PS8 Peptidylprolyl Isomerase A Pseudogene 8
Rn Central Newtonian Resistance
Rrs Total Respiratory Resistance
SELEX Systematic Evolution of Ligands by Exponential Enrichment
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SC Subcutaneous
SYK Spleen Tyrosine Kinase
Th1 T Helper 1
Th2 T Helper 2
TLR Toll-Like Receptor
TNF Tumor Necrosis Factor
To Naïve T cells
Treg Regulatory T Cell
VEGF Vascular Endothelial Growth Factor
WHO World Health Organization
WHS World Health Survey
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Table List
Table 1- Established Chronic Models of Allergic Airways Inflammation....................................20
Table 2: TaqMan® Gene Expression Primers...............................................................................46
Table 3: FACs Antibodies.............................................................................................................50
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Figure List
Figure 1: Allergic Asthma Inflammatory Pathway........................................................................10
Figure 2: CD200/CD200R Signalling Pathway.............................................................................25
Figure 3: Allergen Exposure Timelines for HDM Models............................................................40
Figure 4: Allergic Airways Inflammation and Treatment Regimen..............................................41
Figure 5: FACS Gating Strategy....................................................................................................48
Figure 6: FACS Sample FMO Controls........................................................................................49
Figure 7: Enhanced Methacholine Responsiveness Occurs After 2,4, and 8 weeks of D.
pteronyssinus HDM Exposure.......................................................................................................52
Figure 8: Similar Responses to Methacholine Challenge Between Mice Exposed to 10µg and
25µg for Four Weeks.....................................................................................................................53
Figure 9: Quasi-static compliance reduced in all HDM models....................................................53
Figure 10: HDM-Specific IgE Elevated After Four or More Weeks of HDM Exposure..............54
Figure 11: Cellular Inflammation Increases with Duration of HDM Exposure............................56
Figure 12: Higher HDM Dose Leads to Increases in Cellular Inflammation in Four
Week Models.................................................................................................................................57
Figure 13: Length of Model Affects Gene Expression..................................................................58
Figure 14: Airway Remodelling Including Collagen Deposition Observed in Four-Week
HDM Mice.....................................................................................................................................60
Figure 15: 10µg HDM Model Displays Similar Histological Analysis as 25µg HDM Mice.......61
Figure 16: Total Collagen Content Elevated in the 25µg Four-Week and Eight
Week Models.................................................................................................................................62
Figure 17: One Treatment with PEG.CCS13 Reduces Total Respiratory Resistance
in an Acute Model of Allergic Airways Inflammation..................................................................63
Figure 18: PEG.CCS13 Does not Reduce BAL Total Leukocyte Counts in Acute
Model of Allergic Airways Inflammation.....................................................................................64
Figure 19: IV Treatment with PEG.CCS13 Reduces Inflammatory Mediator Gene
Expression in an Acute Model of Allergic Airways Inflammation...............................................65
Figure 20: CD200R Gene Expression is Downregulated in Allergic Airways Inflammation
and Restored with PEG.CCS13 Treatment....................................................................................66
Figure 21: PEG.CCS13 Reduces HDM-Elevated CD200R Expression in Dendritic Cells..........68
Figure 22: PEG.CCS13 Treatment Reduces BAL Eosinophils.....................................................70
Figure 23: CD200R Activation Reduces HDM-Induced Eosinophilia in Chronic Allergic
Airways Inflammation...................................................................................................................72
Figure 24: HDM-Specific IgE was Elevated in all HDM-Exposed Mice.....................................73
Figure 25: Inflammatory Mediator Expression Following Treatment with PEG.CCS13.............74
Figure 26: PEG.CCS13 Reduces AHR Including Rrs, RN, and G..................................................76
Figure 27: Treatment with CD200FC or CCS13 Did Not Reduce Respiratory Resistance..........77
Figure 28: PEG.CCS13 Treatment Results in Histological Evidence of Reduced Cell
Recruitment and Remodelling.......................................................................................................79
Figure 29: Representative Histology of Saline mice Treated with PEG.CCS13...........................80
Figure 30: Representative Histology Images.................................................................................81
Figure 31: Treatment with PEG.CCS13 Did Not Result in A Reduction in Total Collagen
Content...........................................................................................................................................82
Figure 32: CD200R Activation With PEG.CCS13 in Allergic Airway Inflammation................102
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Chapter 1: Literature Review
1.1 Asthma
1.1.1 Prevalence and Burden of Asthma
Asthma is a chronic inflammatory disease of the airways that results in respiratory
symptoms including wheezing, chest tightness, cough, and shortness of breath(GINA 2015). It
comprises a major international public health concern, with doctor diagnosed asthma reaching a
global prevalence rate of 4.3% or approximately 300 million according to the World Health
Survey (WHS)(To, Stanojevic et al. 2012) and the Global Initiative for Asthma (GINA)(GINA
2015) respectively. Prevalence of asthma is currently highest in developed countries. In Canada
8.1% of people 12 years and older reporting an asthma diagnosis in 2014(Canada 2014
). In the United States this number has been reported as high as 13% or 40 million
people((NHIS) 2012).
Although asthma prevalence remains highest in developed countries, increases in asthma
diagnosis has recently been reported in developing countries. This finding is likely related to
increased urbanization and pollution as well as limitations in the ability to access effective
care(Braman 2006). Additionally it is likely that asthma incidence in these areas is underreported
given these aforementioned healthcare limitations faced by developing countries(Braman 2006).
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The pervasiveness of asthma is associated with a high mortality and economic cost. It
has been estimated that asthma accounts for around 180,000(Braman 2006) or 1 in every 250
deaths worldwide each year(Masoli, Fabian et al. 2004). Although the indirect cost of asthma
related to missed school/work days is difficult to estimate, asthma has an undeniably large
economic and social impact(Nunes, Pereira et al. 2017). Uncontrolled asthmatics have been
reported to have 4.6-fold more hospitalizations and 1.8-fold more emergency room visits
compared to non-asthmatics(Sullivan, Slejko et al. 2014). In the year 2011, the total indirect cost
of asthma in just the United States was estimated to be around 56 billion US dollars(Nunes,
Pereira et al. 2017).
Although several therapies such as bronchodilators, anti-inflammatory drugs and steroids
are available to treat asthma symptoms, incidence of asthma continues to grow with no current
therapy able to reverse all symptoms(Masoli, Fabian et al. 2004). This is likely related to the fact
that the pathways and pathogenesis contributing to asthma are not fully understood. Furthermore,
asthma is recognized to be a highly heterogenous disease with multiple factors contributing to its
pathogenesis including genetics, and environmental factors such as allergen exposure, respiratory
viruses and pollutants(Kim, DeKruyff et al. 2010, Hirota and Martin 2013).
1.1.2 Features of Asthma
Asthma is characterized by chronic airway inflammation, airway hyperresponsiveness
(AHR), remodelling and reversible airflow obstruction(Kim, DeKruyff et al. 2010). Asthma can
manifest itself through multiple symptoms including, shortness of breath, coughing, wheezing,
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and chest tightness which may vary in frequency and severity(WHO 2018). Despite the
heterogeneity of asthma three salient features which are present in all forms namely AHR,
airways remodelling and airways inflammation.
Airway Hyper-Responsiveness
Airway hyper-responsiveness is defined as the increased responsiveness or ease of airway
narrowing in the conducting airways to a constrictor agonist(Hargreave, Dolovich et al. 1986). In
asthma this feature can be induced by an allergen or a chemical compound in the cases of
allergic asthma or occupational asthma respectively(Murdoch and Lloyd 2010). AHR can also
be brought about indirectly by cold or exercise(Cockcroft and Davis 2006). In a clinical setting
the presence of AHR is considered a critical feature of asthma and it is frequently used to
diagnose asthma. AHR is measured using spirometry with increasing inhaled doses of
constrictor agonists such as methacholine (MCh), histamine, mannitol or carbachol(O'Byrne and
Inman 2003). Asthmatics have a characteristically steeper dose-response curve than non-
asthmatics reaching the critical >20% drop in FEV1 (Forced Expiratory Volume at 1 second) at a
lower dose(Hargreave, Dolovich et al. 1986).
The exact mechanism of AHR is not fully understood and a wide variety of mechanisms
are believed to be contributing factors. Genetics has long been implicated in playing a role in
AHR(Chapman and Irvin 2015). A mouse study identified a single recessive gene in some strains
of mice which can abrogate AHR(Levitt and Mitzner 1988). Genome wide studies in humans
are not as clear-cut and have identified several genes related to AHR including IL13, IL6R,
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DENND1B and LRRC21(Portelli, Hodge et al. 2015). Several of the genes associated with AHR
are related to inflammation which has also been suggested to play a role in AHR. Eosinophils
and their markers ie. nitric oxide, have also been associated with the severity of AHR(Payne,
Adcock et al. 2001). Other factors affecting AHR include abnormal airway smooth muscle and
structural changes such as airway wall thickening(Chapman and Irvin 2015). Other inflammatory
cells including neutrophils have also been shown to be correlated with AHR(Radermecker, Louis
et al. 2018).
Airway Remodelling
Airway remodelling refers to the structural changes that occur in the lungs of
asthmatics(James and Wenzel 2007, Bergeron, Al-Ramli et al. 2008, Bergeron, Tulic et al. 2010,
Shifren, Witt et al. 2012, Fehrenbach, Wagner et al. 2017). One characteristic features of
remodelling is subepithelial fibrosis, which causes thickening of the basement membrane under
the lamina reticularis(Roche 1998). Degree of fibrosis has been found to be correlated with the
severity of asthma and the presence of fibrosis is associated with all severities and types of
asthma(Chu, Halliday et al. 1998). It is caused by the deposition of both extracellular matrix
proteins and collagen by fibroblasts(Huang, Olivenstein et al. 1999). Some features of
subepithelial fibrosis are known to be further exacerbated by inflammation. For example
collagen deposition is influenced by the presence of matrix metalloproteinases (MMPs),
especially MMP-9 which is regulated by eosinophils(Humbles, Lloyd et al. 2004, Kay, Phipps et
al. 2004, Hirota and Martin 2013).
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Epithelial injury is one other feature of remodelling characteristic of asthma(James and
Wenzel 2007). This type of injury includes the destruction of ciliated cells and epithelial
shedding. Epithelial injury is characterized by the overexpression of epidermal growth factor
receptors and an increase in mucus secreting goblet cells. Studies have found that severity of
epithelial injury corelates with asthma severity(Boulet, Laviolette et al. 1997).
Remodelling is also characterized by changes to both the vasculature and musculature of
the airways(Tanaka, Yamada et al. 2003, Johnson and Burgess 2004, Barbato, Turato et al.
2006). Asthmatics have an increase in smooth muscle in their lungs, caused by both an increase
in the existing smooth muscle and the migration of muscle to the sub epithelium(Joubert, Lajoie-
Kadoch et al. 2005). Angiogenesis of the airway vessels also occurs in asthmatic lungs due to an
increase in the expression of vascular endothelial growth factor (VEGF)(Hoshino, Takahashi et
al. 2001). This subsequent increase in vessel size can result in airway wall edema(Li and Wilson
1997, Johnson and Burgess 2004).
Overall features of airway remodelling are highly correlated with asthma severity. These
features are not exclusive to the airways as aspects of remodelling can be found in the nasal
mucosa(Bergeron, Al-Ramli et al. 2008). While current therapies for asthma are able to treat
acute symptoms there is currently no therapeutic that is able to reverse many features of
remodelling and current research is focused on determining relevant targets to reverse these
features(Beckett and Howarth 2003, Hirota and Martin 2013).
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Airway Inflammation
Chronic airway inflammation is a hallmark feature of asthma, which can also contribute
to other features including AHR and airway remodelling(Louis, Lau et al. 2000, Payne, Adcock
et al. 2001, Kay, Phipps et al. 2004). An association between the severity of both inflammation
and disease has been reported, particularly regarding sputum eosinophils(Louis, Lau et al. 2000).
Specific characteristics of airway inflammation can vary between different asthma types (further
discussed in section 1.1.3). This review will focus on the inflammation which characterizes the
most common subtype: allergic asthma.
Allergic asthma is characterized by T helper 2 (Th2) inflammation(Berger 2000, Barnes
2001). Although recent work has suggested the picture is more complicated, for many years it
has been believed that asthma is a result of an imbalance between two subtypes of CD4+ T cells:
Th2 and T helper 1 (Th1) inflammation. Both inflammatory states produce characteristic
proinflammatory cytokines(Berger 2000). Th1 cytokines include interferon gamma (IFN-
gamma) and interleukin (IL)-2 and which are typically responsible for autoimmune responses
and defense against intracellular parasites and microbial infection(Berger 2000). In contrast Th2
is related to allergic response and acts through IL-4, IL-5, and IL-13(Berger 2000).
Antigen exposure induces a characteristic Th2 response that typifies allergic
asthma(Murdoch and Lloyd 2010). Antigens are detected by dendritic cells (DCs), the main
antigen-presenting cell in allergic asthma(Veres, Zoltán Veres et al. 2011, Gill 2012, Gaurav and
Agrawal 2013). DCs are found in the epithelium and mucosa of the lung and their main function
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is to activate T cells(Veres, Zoltán Veres et al. 2011). There are two main subtypes of DCs,
myeloid DCs (CD11c+) and plasmacytoid DCs (CD11c-), with myeloid DCs playing an
important role in determining the type of T-cell activation(Bharadwaj, Bewtra et al. 2007).
Immature myeloid DCS are thought to promote Th1 and regulatory T cell (Treg) responses while
mature myeloid DCs induce Th2 expansion(Bharadwaj, Bewtra et al. 2007). Epithelial cells also
play a role in early antigen response and DC activation by releasing cytokines including IL-33
via activation of toll-like receptors (TLRs). IL-33 is then detected by DCs and induces the
expression of OX40 ligand (also TNFSF4) on DCs(Fahy 2015). The presence of the OX40
ligand induces DCs to migrate to lymph nodes and activate T cells(Fahy 2015).
Naïve T cells (To) are activated to become Th2 cells via the binding of the OX440 ligand
on migrated DCs to OX40 on the To(Sugita, Kuribayashi et al. 2003). These mature Th2 cells
move into the airway epithelium where they secrete cytokines, particularly IL-4, IL-13 and IL-5,
which induce cellular recruitment and an inflammatory response(Leόn 2017). Secreted IL-4 and
IL-13 interact with B cells to induce IgE class-switching(Fahy 2015). IL-4 secretion is also
crucial to continue the influx of Th2 cells(Kaiko, Horvat et al. 2008). Differentiation into Th2
cells requires the presence of IL-4 which is produced by Th2 cells in addition to other cells
activated by Th2 cell activity including mast cells and basophils(Kaiko, Horvat et al. 2008). Th2
secretion of IL-5 induces the differentiation and maturation of eosinophils, the key effector cell
in allergic asthma, within the bone marrow(Pelaia, Vatrella et al. 2015). IL-13 can bind to the IL-
4 receptor and thus shares several functions with IL-4 but it also functions to induce goblet cell
hyperplasia and AHR(Finkelman, Hogan et al. 2010).
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B cells or B lymphocytes are activated to secrete IgE by a combination of IL-4 and IL-13
and CD40 engagement(Broide 2001). The IgE antibodies then bind IgE surface receptors on B
lymphocytes and eosinophils(Platts-Mills 2001). When these IgE-bearing cells come in contact
with the antigen they trigger a proinflammatory cascade including the release of histamine and
prostaglandins(Platts-Mills 2001).
Eosinophils are the main effector cells in allergic asthma(Fahy 2015). Recruitment of
eosinophils is primarily caused by IL-5 but is also induced IL-4 and IL-13(McBrien and
Menzies-Gow 2017). These cells have been implicated as playing a major role in manifesting
the symptoms of asthma; and eosinophilia, often measured in the sputum, is associated with
severity of asthma and poor outcomes(Louis, Lau et al. 2000). Eosinophils are known to induce
features of airway remodelling(Humbles, Lloyd et al. 2004, Kay, Phipps et al. 2004). They
secrete IL-13 which induces goblet cell hyperplasia and B cell class switching. Eosinophils are
also involved in the secretion of MMP-9 which plays a key role in collagen deposition(Humbles,
Lloyd et al. 2004, Kay, Phipps et al. 2004). Furthermore, they affect airway smooth muscle,
angiogenesis, and the extracellular matrix by secreting TGF-beta. They additionally induce
increased airway smooth muscle with leukotrienes(McBrien and Menzies-Gow 2017) andmay
also play a role in AHR through the secretion of major basic protein (MBP)(McBrien and
Menzies-Gow 2017).
Neutrophils are an effector cell that has research has primarily associated with non-
allergic asthma, however they are also present in allergic asthma and current research is focused
on understanding their role in Th2 inflammation. The presence of neutrophilia in the airways of
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asthmatics is correlated with severity of asthma and exacerbations(Radermecker, Louis et al.
2018). Neutrophils are among the earliest cells recruited to the airways(Panettieri 2018,
Radermecker, Louis et al. 2018). Recruitment of neutrophils in allergy asthma is suggested to be
partially controlled by IL-4(Woytschak, Keller et al. 2016). Although the exact role of
neutrophils in allergic asthma is not fully understood, studies in mice have shown that decreasing
neutrophil recruitment was associated with a subsequent decrease in eosinophilic
inflammation(Hosoki, Aguilera-Aguirre et al. 2015).
Macrophages are the largest population of resident leukocytes in the non-asthmatic lung.
Macrophages generally function as phagocytes to destroy infectious agents and promote tissue
repair(Jiang and Zhu 2016). They function as antigen-presenting cells and although they are not
considered a main factor in the pathway of allergic asthma, recent research suggests that they do
play a role in the disease(Moreira and Hogaboam 2011). There are two main subtypes of
macrophages which are differentially activated when exposed to different cytokines. M1 or
classically activated macrophages are activated by IFN-gamma and LPS. M1 macrophages
release TNF-alpha and IL-12 and they are associated with non-allergic immune responses. There
are two types of M2, or alternatively activated macrophages: M2a and M2c. M2a macrophages
are the subtype associated with allergic asthma(Saradna, Do et al. 2018). They are activated by
the Th2 cytokines, IL-4, IL-13 and IL-33, and secrete IL-4 and IL-13. The final subtype M2c is
associated with Tregs and is activated by IDO and IL-10 and releases IL-10, TGF-beta and
CCL24(Jiang and Zhu 2016).
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Figure 1: Allergic Asthma Inflammatory Pathway- Th2 immune response in allergic asthma is
triggered by allergens which induce OX40L expression on dendritic cells (DCs). The OXL on
DCs then binds to OX 40 on naive CD4+ T cells to trigger their maturation into Th2 cells.
Mature Th2 cells then induce the recruitment of eosinophils, neutrophils, M2 macrophages and
IgE class switching in B cells. These cells further induce inflammatory signals which trigger the
symptoms and features of allergic asthma.
1.1.3 Asthma Endotypes and Phenotypes
Asthma has long been recognized as a heterogeneous disease and recent work has
indicated that it is more likely to be a collection of diseases that can be sub-categorized into
multiple phenotypes and endotypes. An asthma phenotype refers to the clinically observable
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characteristics of the disease but does not take into account the molecular pathways
involved(Bellanti and Settipane 2018).
Early asthma phenotyping identified only two categories: allergic and non-allergic
asthma. Allergic asthma represents a large proportion of the overall asthma population and is
especially high in children(Wenzel 2012). The allergic asthma phenotype is characterized by
early onset as well as allergen-specific IgE and triggers. This is in contrast with non-allergic
asthma which typically develops later in life. However, recently asthma phenotypes have
expanded with studies grouping phenotypes based on cluster analysis of asthma features(Wenzel
2012). Currently clinical phenotypes are defined based on several categories including severity,
frequency of exacerbations, response to therapy and age of onset(Wenzel 2006). Additionally,
phenotypes can be classified based on their triggers as in the case of allergic asthma where
symptoms are triggered by exposure to an allergen. Other examples of trigger-related phenotypes
include: occupational asthma, where the trigger is a workplace chemical, aspirin-induced asthma,
menses-related asthma, and exercise-induced asthma(Wenzel 2006).
Asthma endotypes refer to subtypes of asthma defined based on their cellular and
molecular mechanisms(Bellanti and Settipane 2018). In contrast to phenotypes which focus on
the characteristics of the disease, endotypes are defined by the underlying biological processes
and classified based on parameters including clinical characteristics, biomarkers, physiology,
genetics, histopathology, epidemiology and treatment response(Lötvall, Akdis et al. 2011). As
endotypes are relatively new and tests to establish presence of some biomarkers are less common
there currently is no consensus on endotype definitions(Svenningsen and Nair 2017).
12
Early studies into the mechanisms of asthma suggested that severe, corticosteroid-
dependent asthma can be categorized can be categorized into two groups, the eosinophilic Th2
high endotype and the usually neutrophilic Th2 low endotype(Wenzel, Schwartz et al. 1999).
Several years later, Lötvall et al. expanded on this concept and proposed six distinct endotypes.
The first, aspirin-sensitive asthma (ASA) is characterized by adult onset and eosinophil and
urinary leukotriene inflammation and is triggered by non-steroidal anti-inflammatory medication.
The next endotype- allergic bronchopulmonary mycosis (ABPM) is triggered by molds, often
Aspergillus fumigatus, colonizing the airways inducing eosinophilic and neutrophilic
inflammation as well as mold-specific IgE and IgG(Lötvall, Akdis et al. 2011). Allergic asthma
is one of the most common endotypes and is characterized by childhood onset and eosinophilic
inflammation and allergen-specific IgE and Th2 dominant cytokines/cells. Asthma predictive
indices (API) preschool wheeze endotype occurs in children with more than 3 wheezing episodes
by the age of 3. The severe late-onset hyper-eosinophilic endotype is characterized by adult
onset, hyper-eosinophilia in the blood and sputum and severe exacerbations. The asthma in
cross-country skiers endotype, is categorized by lymphocyte, neutrophil, and macrophage
inflammation but not eosinophilia(Lötvall, Akdis et al. 2011). It has been suggested that these
endotypes are distinct subgroups and that effective treatment for asthma should be personalized
to a patient’s specific endotype(Wenzel, Schwartz et al. 1999).
1.1.4 Current Treatments
Current treatments of asthma remains focused on controlling symptoms and minimizing
risk of future exacerbations(GINA 2015). As such a strong focus is placed on education and
13
communication between patients and care providers. Medications to control symptoms can be
classified as either controller/ maintenance medications, reliever/ rescue medications or
additional therapies for severe asthma(GINA 2015).
Controller medications are used to reduce the likelihood of future exacerbations and often
focus on reducing airway inflammation(GINA 2015). A common controller medication is
inhaled corticosteroids (ICS) or glucocorticosteroids(Barnes 2011). ICSs are viewed as one of
the most effective first-line treatment options for asthma. ICSs bind to glucocorticoid receptors
in the cytoplasm resulting in the downregulation of inflammatory genes that code for cytokines,
chemokines and other inflammatory enzymes, receptors and molecules(Barnes 2011). Although
ICS are highly effective in many patients, patients with severe asthma can develop a resistance to
ICS even at a high dose. Although ICS is considered the gold standard for front-line treatment,
other controller medications including anti-leukotriene modifiers are also used to treat asthma.
These drugs work by blocking cysteinyl leukotrienes which induce bronchoconstriction. They
additionally control mucus secretion and may be involved in eosinophilic inflammation(Barnes
2011).
Rescue medication such as bronchodilators are used to manage asthma attacks as they are
happening. The most effective and common type of bronchodilator used are the β2-adrenergic
agonists. Activation of the β2-adrenergic G-protein coupled receptors results in the relaxation of
smooth muscle cells in the lungs regardless of constricting stimuli(Barnes 2011). Although they
are considered “rescue medications” some agonists such as salmeterol can work over longer
periods, up to 12 hours. Additionally, beta agonists have an additional anti-inflammatory effect
14
as they inhibit mediator release from mast cells. The most common combination asthma therapy
includes an ICS for control and a beta agonist bronchodilator for rescue. The other class of
rescue medication is anticholinergics which are antagonists of muscarinic receptors. They act by
blocking the effect of acetylcholine which increases muscle tone. Although anticholinergics are
not as commonly used as beta agonists, they are still used in cases of severe asthma(Barnes
2010).
Add-on therapies such as theophylline are mostly used in cases of severe asthma.
Theophylline is an inexpensive molecule related to caffeine which results in an anti-
inflammatory effect in the symptoms of asthma through the inhibition of
phosphodiesterases(Barnes 2011). Recently, usage of theophylline is greatly reduced in
developed countries as it is not as effective as a combination of ICS and bronchodilators and can
have side effects. Despite this, theophylline is still used as an add-on therapy in cases of severe
asthma and remains a popular therapy in developing countries(Barnes 2011).
In recent years asthma therapy has become focused on regulation of the immune system
as treatment, particularly for Th2-high allergic asthma(Barnes 2010). Various forms of
immunotherapy have been proposed with a wide variety of molecular targets. Inflammatory
cytokines and chemokines have become promising targets, including IL-4 and Il-13 that have
been documented to play an important role in Th2 allergic response. The drug pitrakinra blocks
the function of IL-4 and IL-13 by binding to and blocking IL-4Rα(Svenningsen and Nair 2017).
Other cytokine blockers such as mepolizumab focus on Il-5, for its crucial role in eosinophilia.
While mepolizumab is able to reduce circulating and sputum eosinophils, it doesn’t reduce
15
allergen response or AHR. Anti-IgE therapy is an option for treatment of patients with an
allergic component to their disease. Omalizumab is an anti-IgE monocolonal antibody used in
patients with severe asthma. In addition to these drugs further research is being done on other
promising targets such as TNF- α, IL-9, IL-17A, GM-CSF, CCL11, CCR3, Peroxisome
proliferator-activated receptor, mast cell inhibitors, and Syk. Although there has been some
success with these immune-targeted therapies, most of them are only effective for a subset of
asthmatics further reinforcing the utility of using endotypes in effective asthma
management(Barnes 2010).
1.2 Animal Models of Allergic Airways Inflammation
1.2.1 Inducing Allergic Airways Inflammation in Animals
One highly useful tool for studying pathogenesis of asthma and for exploring and testing
potential therapeutic targets is animal models(Simeone-Penney, Severgnini et al. 2007). Well-
defined animal models can be used to recapitulate the salient features of asthma including AHR,
airways inflammation and airway remodelling. The most common animal to use is mice
considering their small size and relative ease to handle. However, the use of several other
animals have reported to model features of asthma including Drosophila to study asthma-
associated genes(Roeder, Isermann et al. 2009). Other early animal models involved guinea pigs
which were used to test the efficacy of bronchodilators, but dogs, cats, rats and pigs have all also
been reported(Aun, Bonamichi-Santos et al. 2017).
16
Not all mouse strains develop the same airways inflammation phenotype in response the
allergen sensitization(Leme, Berndt et al. 2010). BALB/c mice are the most commonly used
strain given that they mount a strong and consistent Th2 response however other strains
including C57BL/6 and A/J have also been used(Nials and Uddin 2008). Similarly, sex can also
account for differences in the inflammatory response. It is well recognized that there are
differences in the clinical course of asthma between males and females in both adulthood and
adolescence(Schatz, Clark et al. 2006). Similarly, studies have found that male mice
demonstrate greater AHR, higher total leukocyte cell counts and TNF-α levels in
bronchoalveolar lavage (BAL) in models of allergic airways inflammation(Card, Carey et al.
2006). Despite this finding, most studies only use female mice primarily for economic reasons
as female mice are easier to house together in contrast to male mice which often need to be
housed separately.
Since mice do not develop asthma spontaneously, the salient features of asthma must be
induced. Administration of allergens such as ovalbumin (OVA), house dust mite (HDM),
ragweed or cockroach extracts can induce the features of allergic asthma either individually or in
combination. OVA derived from chicken eggs is the traditionally used allergen(Cates, Fattouh et
al. 2004, Nials and Uddin 2008, Aun, Bonamichi-Santos et al. 2017, Haspeslagh, Debeuf et al.
2017), although it may not be the most clinically relevant allergen choice. Humans do not
develop airway inflammation in response to OVA and tolerance can be developed over long
periods of time(Cates, Fattouh et al. 2004). Additionally, OVA must be administered with an
adjuvant such as aluminum hydroxide or aluminum sulfate to boost its immunogenicity(Swirski,
Sajic et al. 2002, Cates, Fattouh et al. 2004).
17
Given the downsides of OVA as an allergen, other more clinically relevant allergens have
become more common including HDM, molds and cockroach extract(Kim, Merry et al. 2001,
Sarpong, Zhang et al. 2003, Cates, Fattouh et al. 2004, Havaux, Zeine et al. 2005). Of these
HDM is the most common and clinically relevant allergen. Exposure to HDM is a common
occurrence in homes and an estimated 85% of asthmatics demonstrate an allergic response to
HDM(Gregory and Lloyd 2011). Studies of mice have shown that prolonged HDM exposure can
induce the three salient features of asthma(Nials and Uddin 2008)
Administration of these allergens can be delivered in several ways. Some shorter models
that utilize minimal allergen exposure choose to use injection for at least part of the model. This
is frequently an intraperitoneal (IP) injection but can also be given through subcutaneous (SC)
injection. While it has been reported that SC injection is superior to IP when inducing allergic
airways inflammation, most models opt a less invasive to deliver the allergen directly to the
airways(Aun, Saraiva-Romanholo et al. 2015). Models that deliver the allergen directly to the
airways can use either intranasal, intratracheal or aerosolized administration. Intratracheal is less
used as it is the most invasive of these options and more difficult to conduct(Sanbongi, Takano et
al. 2004, Ryu, Yoo et al. 2013, Norimoto, Hirose et al. 2014). Intranasal administration is done
by applying a liquid form of the allergen to the nares to a lightly anesthetized mouse and
allowing it to breathe it in. The volume of instillate has been shown to be an important factor
with 35µl being described as the optimal volume for even distribution in the lungs while
minimizing the volume of fluids delivered(Southam, Dolovich et al. 2002). Aerosolized delivery
is the least invasive of the options as it does not require anesthesia, however it requires a large
18
amount of allergen and it is difficult to control or estimate the amount of allergen that reaches the
airways.
1.2.2 Acute and Chronic Models of Allergic Airways Inflammation
Length of allergen exposure is the most important factor in the development of a model
of allergic airways inflammation. Acute models of exposure typically last up to 3 weeks and
have been reported to result in both airway hyperresponsiveness and aspects of airway
inflammation. Aspects of inflammation that are elevated in some acute models include
increased serum levels of allergen specific IgE and cell counts. Some studies have also reported
aspects of remodelling such as goblet cell hyperplasia and increased epithelial thickness,
however key features of remodelling such as collagen deposition are absent in these shorter
models(Lloyd 2007, Nials and Uddin 2008). Although they lack the three salient features of
asthma, acute models are easy, fast and relatively inexpensive, thus they are useful in proof-of-
concept studies. Despite this, acute models in isolation have limited use in interpreting their
findings given the absence of remodelling as studies of children have found that airway
remodelling occurs early in the development of asthma even before the onset of
symptoms(Roche 1998, Pohunek, Warner et al. 2005).
Chronic models of HDM-induced allergic airways inflammation offer the most clinical
utility as they recapitulate the three salient features of asthma. Models that assess airway
remodelling with collagen deposition require between 5-12 weeks of allergen exposure as well as
significant resources. As can be seen in Table 1, there is significant variation in the duration and
19
dosing of allergen exposure and some novel model classify themselves as “chronic” but fail to
assess remodelling or fully evaluate their biomarkers.
20
Table 1- Established Chronic Models of Allergic Airways Inflammation(Johnson, Wiley et al.
2004, Ulrich, Hincks et al. 2008, Huang, Dong et al. 2016, Vroman, Bergen et al. 2016, Salehi,
Wang et al. 2017)
Authors Strain HDM Exposure
Protocol
AHR Inflammation Remodelling
Johnson et
al., 2004
Female
Balb/c
25µg in
10µl
5 days a
week for
up to
seven
weeks
MCh test
Total and
differential BAL
cell counts,
splenocyte culture,
total IGE and
HDM-specific
IgG1 analysis, flow
cytometry
Present after
five weeks:
Histological
analysis
Vroman,
et al.,
2016
Male
and
female
C57/BL
6,
Cd401-
/- and
Mb1-1-
25µg/40
µl
3 times a
week for
5 weeks
MCh test Flow cytometry, Ig
analysis
Histological
analysis
Huang et
al., 2016
Male,
Balb/c
5mg/kg
body
weight in
10µl
PBS
5 days
for 8
weeks
MCh
test
Protein analysis, Histological
analysis,
immunohisto
chemistry
Salehi et
al., 2016
Female
Balb/c
25µg in
35µl
8 weeks
week 1: 5
consecuti
ve days,
weeks 2-
8: 3 days
a week
MCh test
Gene expression,
IgE analysis,
FACS, total and
differential BAL
cell counts
Present:
Histological
analysis, total
collagen
assay
Ulrich et
al., 2008
Female,
Balb/c
25µgin
10µl
saline
5 days
for 5
weeks
Not
assessed
Total and
differential BAL
cell counts, flow
cytometry,
chemokine and
cytokine analysis
Not assessed
21
Chronic models demonstrate several advantages over acute models, asthma is a chronic
disease and several studies have suggested that remodelling may occur prior to the onset of
symptoms and thus is a key feature of a truly useful model of asthma requires
remodelling(Roche 1998, Pohunek, Warner et al. 2005, Nials and Uddin 2008). Acute models
also require a higher dose of HDM to develop AHR and airway inflammation in a short time
period, and such a high dose is not as clinically relevant as the lower dose that can be used with
chronic models(Nials and Uddin 2008).
1.2.3 Asthma Clusters in Mouse Models
Given the heterogeneity of asthma, a good model will accurately mimic the endotype of
interest. In the case of HDM models the molecular mechanisms and features associated with it
most closely resemble the allergic asthma endotype. In this case, exposure to the HDM antigen
is what induces the symptoms, and HDM-specific IgE production(Johnson, Wiley et al. 2004,
Salehi, Wang et al. 2017). Additionally, key features of allergic asthma such as eosinophilia and
Th2 cytokines and chemokines have been reported in HDM-induced models of allergic airways
inflammation(Brown, Farmer et al. 2003, Johnson, Wiley et al. 2004, Hammad, Chieppa et al.
2009, Ryu, Yoo et al. 2013, Norimoto, Hirose et al. 2014, Dullaers, Schuijs et al. 2016, Hoffman,
Qian et al. 2016, Vroman, Bergen et al. 2016, Ito, Hirose et al. 2017, Woo, Guo et al. 2018).
22
1.2.4 Limitations of HDM Models of Allergic Inflammation
There are several advantages to preclinical HDM mouse models, including that
therapeutic interventions used in human asthma such as dexamethasone, prednisolone,
fluticasone, and roflumilast demonstrate similar effects in HDM mice(Ulrich, Hincks et al.
2008). However, there are limitations to these models that need to be considered in the
translation of this work from animals to humans. Since mice do not develop asthma, HDM
models cannot be considered “true asthma” and instead allergic airway inflammation which
mimics the phenotype of asthma(Nials and Uddin 2008). Additionally, inconsistencies in
methodologies within the literature including the use of naive mice over saline-treated mice as
controls(Cates, Fattouh et al. 2004). Although a study did find there was no significant
difference between naive and saline mice with respect to AHR and eosinophilic inflammation,
frequent handling and anesthesia does lead to stress in mice and thus saline-treated mice are a
more appropriate control over naive mice(Cates, Fattouh et al. 2004, Hurst and West 2010).
Additionally, although chronic exposure to HDM may be an appropriate model for
allergic asthma, this is a limited representation of the clinical spectrum of asthma phenotypes and
endotypes and models representing other non-allergic types of asthma are sorely needed.
Finally, asthma is a chronic disease with flare-ups across prolonged periods of time quiescence,
however it known that in mice some of these features will revert in the absence of ongoing
allergen exposure. Studies have found that leukocyte allergic airways inflammation peaks 24-48
hours after the final HDM dose with a sharp decline overtime(Johnson, Wiley et al. 2004,
Piyadasa, Altieri et al. 2016). Additionally, while airway remodelling has been shown to persist
23
for 2 weeks after 8-week HDM sensitization it is not known if this feature would be maintained
in the long-term absence of allergen exposure(Salehi, Wang et al. 2017). Finally, in these
models, airway remodelling takes weeks of antigen exposure to develop and features of AHR
and of airway inflammation appear prior to the establishment of remodelling. However,
histological analysis of infants has shown that remodelling can precede the development of any
clinical symptoms(Roche 1998, Pohunek, Warner et al. 2005).
1.3 CD200/CD200 Receptor Pathway
1.3.1 CD200 and CD200R: Structure, Distribution, and Mechanism
The CD200 (Cluster of Designation 200) ligand, also called OX2, was initially identified
in 1982 as a membrane glycoprotein found on rat thymocytes, brain, DCs smooth muscle and B-
lymphocytes(Barclay and Ward 1982). The CD200 ligand is roughly 41-47 kDa and is
comprised of two IgSF) domains, a small cytoplasmic domain and a single transmembrane
domain. This ligand is expressed in a wide variety of tissues including the kidney, bone marrow,
retina, hair follicles, lymphoid cells, lung tissue, epithelium and neurons(Gorczynski 2012).
The CD200 receptor (CD200R) is structurally similar to the CD200 ligand and the gene
is located on the same chromosome(Jenmalm, Cherwinski et al. 2006, Gorczynski 2012). The
CD200R also comprises two IgSF domains and a single transmembrane segment. However, the
cytoplasmic domain is much larger in the CD200R suggesting that CD200R but not CD200 is
involved in signaling. Members of the CD200R receptors (CD200R1-R4) family are closely
24
related although only CD200R1 harbors a long cytoplasmic signaling domain(Gorczynski, Chen
et al. 2004, Gorczynski 2012). Structural similarity between the CD200Rs in both mouse and
humans suggests a gene duplication event and made the receptor a promising target for
therapeutic testing(Wright, Cherwinski et al. 2003). In contrast to its ligand, the CD200R is
more narrowly expressed, and is predominantly found on myeloid cells and to a lesser extent on
a subset of lymphoid cells. Although there is some variation across tissues types, the receptor has
been found to be strongly expressed in macrophages and neutrophils but can also be found on
DCs, eosinophils, monocytes, mast cells and CD4+ T-cells(Wright, Cherwinski et al. 2003,
Akkaya, Aknin et al. 2013).
CD200 binds to the CD200 receptor through cell-cell surface interactions and initiates a
downstream signaling cascade: events that lead to a dampening of immune response (Figure
2)(Jenmalm, Cherwinski et al. 2006, Vaine and Soberman 2014). CD200 and CD200R bind via
their N terminal domains. When the ligand and receptor bind, the cytoplasmic NPXY motif on
the CD200R is phosphorylated at three tyrosine residues by SRC kinases(Gorczynski 2012).
After the phosphorylation DOK 1 and DOK 2 are recruited initiating a RAS-GTPase to enact
further downstream changes(Zhang, Cherwinski et al. 2004). Studies of the CD200/CD200R
pathway have shown that the phosphorylation of CD200R leads to changes in cytokine
production including mediators such as TNF-alpha, IFN-gamma and changes in myeloid cell
function(Cherwinski, Murphy et al. 2005, Jenmalm, Cherwinski et al. 2006, Gorczynski 2012).
25
Figure 2: CD200/CD200R Signaling Pathway- The widely expressed CD200 ligand binds to the
CD200R expression on myeloid and to a lesser extent on lymphoid cells. Upon CD200 binding,
the cytoplasmic domain of CD200R is phosphorylated which recruits DOK and RAS leading to
downstream anti-inflammatory signaling resulting in changes to cytokine production and
myeloid activation.
1.3.2 CD200/CD200R in Disease Pathogenesis
The CD200/CD200R axis has been implicated as having a role in multiple disease
states(Minas and Liversidge 2006). Studies of transgenic mice lacking the CD200 ligand have
found that these mice appeared to have a normal life span and breeding capacity but had
26
increased CD200R expressing CD11b+ myeloid cells in the spleen(Hoek, Ruuls et al. 2000).
These knockout mice also showed inflammatory responses in response to stimuli such as injury
or infection(Hoek, Ruuls et al. 2000, Rygiel, Rijkers et al. 2009). Further studies have explored
the role of CD200/CD200R signaling in mouse models of disease including breast cancer and
skin grafts. Transgenic mice overexpressing the CD200 ligand were found to more readily
develop tumor metastasis in comparison to healthy controls when breast tumor cell lines (EMT6)
were grown in the mice(Gorczynski, Clark et al. 2011). In the same model, CD200 ligand
neutralization with an antibody led to a decrease in metastasis and an increase in tumor
suppressing cells(Gorczynski, Clark et al. 2011). Further work showed that mice without CD200
were resistant to developing tumors(Rygiel, Karnam et al. 2012). Studies involving skin grafts
indicate that overexpression of the CD200 ligand led to an increase in allograft survival
compared to control mice. The same study found that blocking the CD200/CD200R pathway
with a CD200 antibody led to graft rejection(Gorczynski, Chen et al. 2009).
Studies of the CD200/CD200 pathway have also found evidence for dysregulation of the
pathway in human studies. Immunohistochemical analysis of tumors from patients with rectal
cancer found that the tumors had high levels of CD200(Bisgin, Meng et al. 2019). Additionally,
CD200R1 was overexpressed in the tissue of patients with metastasis(Bisgin, Meng et al. 2019).
A study that found that individuals endemically infected with Schistosoma haematobium had
elevated CD200R expression on CD4+ T cells correlating with the intensity of their
schistosomiasis(Caserta, Nausch et al. 2012). These CD4+ T cells were also associated with the
Th2 cytokine, IL-4.(Caserta, Nausch et al. 2012) This same study was able to show similar
increases in CD200R on CD4+ T cells in mice infected with chronic exposure to either
27
Salmonella enterica or Schistosoma mansoni. Another study found that CD83+ monocyte-
derived dendritic cells generated from patients with laryngeal cancer had expressed higher levels
of both the CD200 ligand and CD200R than healthy controls(Klatka, Grywalska et al. 2013,
Klatka, Grywalska et al. 2013). Overall these findings suggest that this pathway plays an
important role in immune homeostasis and dysregulation of this pathway may be implicated in
several disease states.
1.3.3 CD200/ CD200R Regulation as a Therapeutic Target
Given the evidence that the CD200R is dysregulated in multiple disease states, several
research studies have explored the activation of the CD200R as a potential target for therapeutic
agents. In a mouse model of collagen-induced arthritis, mice prophylactically treated with
CD200Fc at 3-day intervals didn’t develop features of collagen-induced arthritis(Gorczynski,
Chen et al. 2001). Mice treated with CD200FC had lower arthritic scores, anti-collagen
antibodies and TNF-alpha and IFN-gamma cytokine levels than mice treated with PBS alone.
However in studies where the CD200FC was given in a treatment model after prolonged
exposure to collagen, CD200R activation did not reduce anti collagen IgG levels or cytokines
despite clinical and histological reduction in disease(Gorczynski, Chen et al. 2002, Simelyte,
Criado et al. 2008). These studies suggested that CD200FC could prophylactically prevent
collagen deposition but that the treatment regimen was insufficient to reverse previous collagen
deposition.
28
Further studies have explored the CD200/CD200R pathway in neurobiology as the
CD200 ligand is highly expressed on neurons and some astrocytes(Walker and Lue 2013). A
rodent model of Parkinson’s disease found that blocking the CD200R with an antibody resulted
increased microglia activation and severe disease characteristics(Zhang, Wang et al. 2011).
Similarly, increased disease severity with CD200R blocking was seen in a model of multiple
sclerosis(Walker and Lue 2013).
Other studies have explored activation of the CD200R in models of allogenic skin grafts.
Early studies with of the CD200/CD200R pathway showed that CD200 overexpression increased
graft survival mouse allograft models(Gorczynski, Chen et al. 2009, Gorczynski, Chen et al.
2011). Since then studies have shown that activation of the CD200R with various molecules
prolongs graft survival compared to mice treated with a control(Prodeus, Cydzik et al. 2014).
Although most models are interested in the therapeutic potential of CD200/CD200R
pathway activation; some studies have explored blocking the pathway. Mouse tumour models
have shown that removing the CD200 ligand led to a decrease in tumour burden while activation
of the CD200R had no change in tumour growth(Pilch, Tonecka et al. 2019). Further studies
treated mice with an anti-CD200 antibody and found these mice had reduced tumour growth
compared to control-treated mice(Kretz-Rommel, Qin et al. 2007).
29
1.3.4 CD200/ CD200R in Allergic Disease
Studies of CD200 ligand have found that it is expressed on the epithelial cells of the
lung(Wright, Jones et al. 2001, Snelgrove, Goulding et al. 2008). Additionally the CD200R has
been reported on alveolar macrophages and dendritic cells(Snelgrove, Goulding et al. 2008). A
study of the dysregulated pathways in asthmatics found that gene expression of the CD200
ligand was decreased in the peripheral blood mononuclear cells (PBMCs) of asthmatic children
and associated with asthma exacerbations, suggesting that the CD200/CD200R pathway plays a
role in the pathogenesis of asthma(Aoki, Matsumoto et al. 2009). Another study of adult
asthmatics found that blood serum levels of the CD200 ligand in patients with severe asthma
were elevated relative to healthy controls and patients with controlled asthma(Tural Onur, Yalcin
et al. 2015). It has been suggested that the CD200 ligand is downregulated in resident lung cells
in asthma but ultimately increased in the serum levels(Lauzon-Joset, Marsolais et al. 2019). A
further study of PBMCs reported that expression of the CD200 ligand is regulated by vitamin D
in healthy individuals but not in asthmatics(Dimeloe, Richards et al. 2012). These studies found
that the CD200/CD200R pathway is dysregulated in asthmatics and suggest the pathway may
play a role in asthma pathogenesis and thus may be a potential target for developing future
asthma therapeutics.
A study in rats by Lauzon-Joset et al. explored CD200R activation as a therapeutic target
in a rat model of allergic airways inflammation(Lauzon-Joset, Langlois et al. 2015). Brown
Norway rats were exposed to OVA by intraperitoneal injection and prophylactically treated with
intratracheally delivered CD200Fc fusion protein on day 20 followed by intranasal OVA
30
challenge on day 21. This group was able to show that CD200R activation reduced AHR and
BAL cytokine gene expression of IL-13 while increasing levels of IL-10. BAL cell recruitment
was found to be unaffected by treatment but FACS analysis found myeloid dendritic cells and
Th2 cells were decreased with CD200FC treatment(Lauzon-Joset, Langlois et al. 2015).
Although limited due to the acute and prophylactic nature of the model, these results further
support the suggestion that the CD200/CD200R pathway plays a role in allergic airways
inflammation, and is a logical pathway to target for designing new therapies.
1.4 Aptamers
1.4.1 Definition and Development
Aptamers are short single-stranded DNA, RNA or XNA (nucleic acid analog)-based
oligonucleotides which uniquely fold to bind to specific targets(Keefe, Pai et al. 2010, Lakhin,
Tarantul et al. 2013),(Dunn, Jimenez et al. 2017). Aptamers have garnered interest as a
therapeutic molecule as they bind with high specificity and affinity and are relatively easy to
develop and chemically modify. Additionally, they can be simply and rapidly blocked with
antidotes (antisense oligonucleotides). DNA and chemically-stabilized RNA aptamers also have
a longer shelf life. They can be stored over long periods at ambient temperature and while they
may unfold as a lyophilized product, they refold when resuspended in an appropriate
buffer(Dunn, Jimenez et al. 2017). Additionally, they reportedly display low immunogenicity;
an advantage relative to antibodies and protein therapeutics(Dunn, Jimenez et al. 2017).
31
Aptamers are developed using a process known as Systematic Evolution of Ligands by
Exponential Enrichment (SELEX)(Tuerk and Gold 1990, Dunn, Jimenez et al. 2017). A starting
library of random oligonucleotides is synthesized and incubated with the target (often a protein)
immobilized on a solid support such as a bead or a plate. Unbound oligonucleotides are removed
by repeated washing steps. Bound aptamers are eluted and amplified via polymerase chain
reaction (PCR). A sub-library of oligonucleotide binders is generated by performing up to 20
cycles of amplification. The SELEX process is repeated 10-15 times to enrich for strong target
concentration, increasing the number of wash steps and decreasing the binding time of
oligonucleotides interacting with its target. Final target binding motifs are subsequently
identifies by next generation sequencing.
Delivery of aptamers is usually done systemically (IV or IP) in most published animal
studies. However an aptamer suspended in PBS was delivered intranasally in a study modelling
influenza infection PBS(Jeon, Kayhan et al. 2004). This aptamer targeted the viral surface
glycoprotein hemagglutinin HA and the prophylactic intranasal delivery of the aptamer was able
to successfully block the binding of the virus to the HA antigen(Jeon, Kayhan et al. 2004).
1.4.2 Aptamers as Therapeutic Agents
The use of aptamers as therapeutics has gained much research interested in recent years.
As of 2017, 11 aptamer compounds had undergone clinical trial(Dunn, Jimenez et al. 2017). In
2004, the FDA approved Pegaptanib (brand name Macugen), as a treatment for neovascular age-
related macular degeneration. Pegaptanib is a PEGylated (bound to the compound Polyethylene
32
Glycol) RNA aptamer that acts as an antagonist for vascular endothelial growth factor
(VEGF)(Ng, Shima et al. 2006). Activation of VEGF induces angiogenesis, which plays a role in
vision loss in macular degeneration thus blocking VEGF reduces vision loss. The safety and
efficacy of Pegaptanib was first evaluated in rhesus monkeys followed by two randomized
crossover multicenter trials where Pegaptanib was given by intravitreous injection every 6 weeks
for 48 weeks. These studies found that Pegaptanib reduced vision loss by approximately 50% of
patients in the first year. This and subsequent clinical trials found that Pegaptanib was both safe
and effective after two years leading to its approval by the FDA(Ng, Shima et al. 2006).
Like Pegaptanib, most other aptamer therapeutics have been focused on antagonistic
blocking of biomolecules. ARC1905 is another aptamer which entered clinical trials as a
therapeutic to treat age-related macular degeneration by blocking the activation of complement
factor 5(Majumder, Gomes et al. 2009). Another aptamer ARC1779 was developed against the
von Willebrand factor which initiates platelet adherence to vessels(Majumder, Gomes et al.
2009). Excess activity by the von-Willebrand factor is implicated in blood clots leading to stroke
and heart attack(Majumder, Gomes et al. 2009). Other aptamers have been devised for aptamer-
antidote systems. The REG-1 system combines the RB007 aptamer which binds to the factor
IXa and the RB008 complementary oligonucleotide. REG-1 is in trials to reduce bleeding risk
with anticoagulation targeting therapy. The RB0006 functions as an anticoagulant while the
RB007 is used to control the activity of RB006. The aptamer-antidote system functions together
to reduce the risk of blood clots while minimizing the risk of bleeding(Majumder, Gomes et al.
2009). However there are potential limitations for this as one late stage clinical trial for was
prematurely halted due to potential issues with the PEGylation compound.
33
Although aptamers represent a promising and exciting new therapeutic option, there are
some limitations to their use. The primary disadvantage of aptamers is their short half-life in the
body(Keefe, Pai et al. 2010). Aptamers are very quickly degraded from nuclease enzymes and
removed through renal filtration and thus have a very short circulatory half-life(Keefe, Pai et al.
2010). Aptamers with no modification have a circulatory half-life of only minutes(Healy, Lewis
et al. 2004). Thus, aptamers must be modified to improve their pharmacokinetics if they are to
be used as therapeutics.
1.4.3 Aptamer Modification
Several modification options exist to improve the half-life of aptamers in the body. Since
RNA aptamers in particular are quickly degraded by nucleases, one option is to use nuclease-
resistant nucleotide analogues or to simply switch to generating more stable end-capped DNA
aptamers(Keefe, Pai et al. 2010). High weight polymers such as PEG cahins conjugated to
aptamers can also be used to extend their circulation times by decreasing their renal filtration
rates(Keefe, Pai et al. 2010) .
The most commonly used conjugating compound for aptamers is the FDA approved
polymer polyethylene glycol (PEG)(Keefe, Pai et al. 2010). The acronym PEG is used here as a
generic term to define polyethylene glycol polymers that are specified by their mass range and as
being either linear or branched polymers. PEG is a commonly used modifier of biologics for
drug delivery to increase tissue distribution and reduce excretion(Harris and Chess 2003). PEG
34
offers additional advantages as it is non-ionic and soluble in water. These features can be
advantageous for hydrophobic drug compounds as PEGylation can increase their
solubility(Kolate, Baradia et al. 2014). It is also thought to have low immunogenicity(Kolate,
Baradia et al. 2014). The effectiveness and safety of PEG has been shown in several studies of
animals including monkeys, mice and dogs(Knop, Hoogenboom et al. 2010). Studies of PEG
toxicity toxicity increases with molar mass, the overall remains quite low(Knop, Hoogenboom et
al. 2010). Since 1990, several PEGylated compounds have received approval from various drug
regulatory bodies including the US Food and Drug Administration (FDA)(Knop, Hoogenboom et
al. 2010).
Although PEG is a commonly used compound, recent studies have suggested that PEG
may induce an immunogenic response(Schellekens, Hennink et al. 2013). Early studies with
PEG showed that non-specific interactions of PEG with the blood could lead to blood clotting
and embolism(Keefe, Pai et al. 2010). Additionally, some studies have suggested that
hypersensitivity to PEG can occur and result in anaphylactic reactions(Schellekens, Hennink et
al. 2013). Furthermore, several studies of both animals and humans have reported reassuring
anti-PEG antibodies following exposure to PEG(Richter and Akerblom 1983). However, this
finding is somewhat controversial as the ELISA technique used to identify the anti-PEG
antibodies has not been well defined or validated(Sulkowski, Cooper et al. 2011, Schellekens,
Hennink et al. 2013, Zhang, Liu et al. 2014). Overall PEG has many advantages as a drug
delivery method, however new research suggests that further research is needed to understand its
limitations.
35
Given the disadvantage of PEG several alternative delivery molecules have been
proposed. Natural polymers including heparin, dextran and chitosan are options which have been
previously used in drug delivery(Knop, Hoogenboom et al. 2010). Other options include other
synthetic compounds including polyamino acids or poly(2-oxazoline)s.(Knop, Hoogenboom et
al. 2010) However, despite the availability of alternatives, very few compounds have the same
properties including being water soluble and resisting degradation, thus PEG remains the
standard(Kolate, Baradia et al. 2014).
1.4.4 CD200 Receptor Aptamers
Given the recognized potential for CD200R activation in immune the laboratory group of
Dr. Jean Gariépy at Sunnybrook Health Sciences developed aptamers to target the CD200R.
Unlike most previous aptamers which function as antagonists, these aptamers function as
agonists to activate this receptor. They developed two PEGylated DNA aptamers: M49 and M52
which binds to the murine CD200R(Prodeus, Cydzik et al. 2014). These aptamers were shown to
suppress immune responses both in vitro and in vivo. In vitro analysis showed a suppression of
cytotoxic T-lymphocytes in allogenic-mixed lymphocyte cultures. Furthermore in a murine skin
allograft model mice with skin grafts received intravenous injections of these compound every
three days up to day 15(Prodeus, Cydzik et al. 2014). Further monitoring of the mice found that
mice that had been treated with CD200R1-specific DNA aptamers had longer graft survival
relative to mice treated with PBS alone. The aptamer-treated mice also displayed similar graft
survival to mice treated with the CD200Fc fusion protein which had previously been established
as binding to the CD200R(Prodeus, Cydzik et al. 2014).
36
Dr. Gariépy’s group recently developed another CD200R aptamer, CCS13 (PEGylated:
PEG.CCS13), which is capable of binding to both mouse and human CD200R(Prodeus, Sparkes
et al. 2018). This aptamer was also able to reduce cytotoxic T-lymphocytes in both human and
mouse mixed lymphocyte cultures(Prodeus, Sparkes et al. 2018). In the same murine skin graft
model, mice treated with the PEG.CCS13 similarly to M49 and M52 had longer graft survival
than mice treated with either a control PEGylated aptamer or PBS alone(Prodeus, Sparkes et al.
2018).
37
Chapter 2: Hypothesis and Objectives
2.1 Hypothesis
We hypothesize that activation of the CD200 receptor with the PEG.CCS13 agonist aptamer will
reduce salient features of asthma in a chronic treatment model of allergic airways inflammation.
2.2 Research Objectives
Given that previous research has established the CD200R as a promising target for inflammatory
disease including asthma we aimed to:
1. Evaluate the role of CD200/CD200R signaling in the allergic airways inflammation
phenotype and,
2. Evaluate the effectiveness of the PEG.CCS13 aptamer in reducing or reversing the
features of asthma in a murine model of allergic airways inflammation
38
Chapter 3: Materials and Methods
3.1 Animals
All animal protocols were approved the University Health Network Animal Care
Committee and conducted in compliance with guidelines by the Canadian Council on Animal
Care (CCAC). Female BALB/c mice (aged 6-12 weeks old) were ordered from Charles River
Laboratories, Montreal, Canada or Jackson Laboratories, Bar Harbor, ME, USA. Animals were
housed in a pathogen-free environment with a 12:12 hour light:dark cycle, and acclimatized for
one week before the start of the experiment. Mice were provided with standard chow and water
ad libitum. The health status of animals was monitored daily. No animals became ill or died
before the study-end point.
3.2 Models of Allergic Airways Inflammation
3.2.1 Acute Model of Allergic Airways Inflammation
100µg HDM extract (Stallergenes Greer, Boston, MA, USA) (D. pteronyssinus) was re-
suspended in normal saline. 50µl was introduced to the nares with a 20-200µl pipette tip by
intranasal instillation with the mouse lightly anesthetized with 5% isofluorane. Control mice
were treated with 50µl saline and handled identically. Mice were given intranasal instillation on
days 0-4 and day 11 (Figure 3).
39
3.2.2 Four Week Chronic Model of Allergic Airways Inflammation
For the chronic four-week model, mice were either intranasally exposed to either HDM
or saline (control) five consecutive days/week (ie. Monday to Friday) for four weeks. Mice were
tested at two HDM doses of either 10µg or 25µg D. pteroyssinus in 35µl saline (Figure 3).
3.2.3 Eight Week Model of Allergic Airways Inflammation
For the eight-week chronic model mice were intranasally exposed to 25µg D.
pteronyssinus in 35µl saline for five consecutive days on week one and every other weekday
(Monday, Wednesday and Friday) on weeks 2-8 (Figure 3).
40
Figure 3: Allergen Exposure Timelines for HDM models- (A) The 2-week acute model
intranasally exposed mice to 100µg/50µl HDM or saline control on days 0-4 and day 11. (B)
The 4-week chronic model exposed mice to 25µg/35µl HDM or saline for 5 consecutive days for
4 weeks. (C) The 8-week chronic model exposed mice to 25µg/35µl HDM or saline for 5
consecutive days on week one followed by 3 alternating days during weeks 2-8. All mice were
sacrificed 24 hours following final HDM or Saline challenge.
41
3.3 Treatment Regimen with PEG.CCS13
After exposure to HDM or saline for four weeks according to the 4-week model, HDM
challenge was continued twice a week for weeks 5&6. Treatment with the PEG.CCS13 aptamer,
PEG.Scrambled aptamer (negative control), CD200FC (CD200R fusion protein), the naked
(unPEGylated) aptamer CCS13, (30µg/35µl) or vehicle (saline control) was given intranasally
alternating days three days a week for weeks five and six (Figure 4). For the acute model mice
were intranasally exposed to the HDM or Saline as described in 3.2.1 followed by one tail vein
IV treatment with PEG.CCS13, CD200FC or Vehicle (Saline).
Figure 4: Allergic Airways Inflammation and Treatment Regimen- Mice were exposed to HDM
(25µg/35µl) or saline five days a week for weeks 1-4. On week’s 5-6 mice were given HDM or
saline twice a week and treated with either PEG.CCS13, PEG.Scram, CD200FC, CCS13 or
Vehicle (35µg/35µl) three days a week. Sacrifice occurred 24 hours following final treatment.
42
3.4 Assessment of Lung Function Mechanics and Methacholine Responsiveness
24 hours after the final exposure or treatment, mice were anesthetized with ketamine
(50mg/kg via intraperitoneal injection (i.p.), Wyeth Animal Health, Guelph, ON, Canada)
and xylazine (10mg/kg i.p., Bayern Inc, Toronto, ON, Canada). After toe-pinch reflex was gone
the trachea was exposed and intubated with an 18-gage stainless steel cannula (BD Biosciences
Canada, Mississauga, ON, Canada). Following intubation mice were ventilated on the Flexi-
vent® system (SciReq Inc., Montreal, QC, Canada). Rocuronium (1.8-4.8mg/kg i.p., Sandoz
Canada Inc., Boucherville, QC, Canada) was given to prevent artifacts from respiratory drive.
After waiting five minutes in vivo respiratory mechanics were measured using the Flexi-Vent®,
baseline respiratory mechanics were assessed prior to challenge with increasing concentrations
of nebulized methacholine (MCh; Sigma-Aldrich, Mississauga, ON). Anesthesia was maintained
during pulmonary function tests with ketamine-xylazine i.p injections every twenty minutes.
3.5 Tissue Collection
3.5.1 Serum Collection
Following assessment of lung function mechanics mice were euthanized with a
ketamine/xylazine overdose. The thoracic cavity was opened, and blood was collected via
cardiac puncture. Blood was kept at 4 ̊C overnight and then serum supernatant was collected
after centrifugation.
43
3.5.2 Bronchoalveolar Lavage (BAL) and Cell Counts
In a subset of mice (n= 7-8/group) bronchoalveolar lavage (BAL) was performed by
lavaging the lungs via the canula with 1mL of Phosphate Balanced Saline (PBS) followed by two
washes of 0.5mL. BAL was then centrifuged for 10 minutes at 400g at 4 ̊C. The bronchoalveolar
lavage fluid (BALF) supernatant was collected and stored at -80 ̊C. The cell pellet was
resuspended in 500mL of PBS and total leukocyte counts were counted using a hemocytometer
and trypan blue (Sigma-Aldrich, Oakville, ON, Canada). 70µl of cell pellet suspension were
spun onto slides using StatSpin Cytofuge 2 (Beckman Coulter Inc, CA, USA) and stained using
Diff-Quick (Invitrogen Life Technology, Carlsbad, CA, USA). Differential cell counts were
done under a light microscope.
3.5.3 Lung Tissue
Lungs were excised and the left lung was stored at -80 ̊C for future protein isolation. The
right lung was collected and stored in RNAlater (ThermoFisher Scientific, Mississauga, ON,
Canada) at 4 ̊C overnight before being transferred to -20 ̊C for storage.
3.6 Histological Analysis
In a subset of mice (n=3-4/group) lung tissues was excised and inflation-fixed to 25cm
H2O in 4% paraformaldehyde in accordance with American Thoracic Society Guidelines(Hsia,
44
Hyde et al. 2010). Lung were stored for twenty-four hours at room temperature before being
transferred to 70% ethanol and stored at -20 ̊C. Lung were dehydrated using ethanol and xylene
and embedded in paraffin. Hematoxylin and eosin (H&E), periodic acid-Schiff (PAS), and
Masson Trichrome staining were conducted at the Toronto Centre for Phenogenomics (Toronto,
ON, Canada).
3.7 Quantification of Serum HDM-Specific IgE
Serum HDM-specific Ige was assessed using antigen-capture ELISA. Serum samples
were precleared by incubation at a 1:1 ratio with Protein G Sepharose beads (GE Healthcare,
Mississauga, ON, Canada) overnight at 4 ̊C. Plates were coated using 5µg HDM in 100µl
coating buffer and incubated overnight at 4 ̊C. 200µl of assay diluent was used to block non-
specific binding. After washing, 50µl of undiluted serum was added to the wells and incubated at
4 ̊C overnight. After washing 100µl of biotin anti-mouse IgE (Biolegend, CA, USA) was added
and incubated for one hour followed by 30 minutes of incubation with avidin-horse radish
peroxidase (Biolegend, CA, USA). 100µl of TMB substrate solution was added and incubated in
the dark for 30 minutes. Optical densities were read at 450nm with reference at 570nm using the
Titertek Multiskan® Ascent Spectrophotometer (Titertek Instruments Inc., Huntsville, AL,
USA). Data was normalized to the respective Vehicle controls.
45
3.8 Quantification of Total Lung Collagen
A Quickzyme Total Collagen Assay Kit (Quickzyme Biosciences Inc, Leiden,
Netherlands) was used to determine total collagen content. 50mg of lung tissue was hydrolyzed
in 6M HCl at 95 ̊C for 20h and then diluted 15-fold for the four-week model and 20-fold for the
eight-week model. 35µl of tissue hydrolysate was incubated on a microtiter plate with 75µl
assay buffer for 20 min at room temperature. 75µl of detection reagents A and B (2:3 ratio) were
added and the plate was incubated at 60 ̊C for an hour. After the plate was cooled to room
temperature it was read at 570nm. Standard curves to the collagen standard were prepared via
serial dilutions according to the manufacturer’s instructions. To standardize to the total collagen
content, collagen content was normalized to the weight of the lung.
3.9 qPCR Gene Expression Profiling
An RNAeasy Mini Kit (Qiagen Inc, Toronto, ON, Canada) was used to extract total RNA. SYBR
green real time Quantitative RT-PCR kit (Roche Bioscience, Mississauga, ON, Canada) was
used for qPCR on a CFX384 Touch Real-Time PCR Detection System (BioRad, ON, Canada).
TaqMan® Gene primer sets can be found in Table 2.
46
Table 2: TaqMan® Gene Expression Primers
Gene Assay ID
Peptidylprolyl isomerase A pseudogene 8 (Ppia-ps8) Mm02342429_g1
Chemokine (C-C motif) ligand 11 (Eotaxin, CCL-11) Mm00441238_m1
Chemokine (C-X-C motif) ligand 1 (CXCL-1) Mm04307460_m1
Interleukin 4 (IL-4) Mm00445259_m1
Interleukin 6 (IL-6) Mm00446190_m1
Interleukin 10 (IL-10) Mm01288386_m1
interleukin 13 (IL-13) Mm00434204_m1
Interleukin 17A (IL-17A) Mm00439618_m1
Spleen tyrosine kinase (Syk) Mm01333032_m1
C-type lectin domain family 7, member a (CLEC7A, Dectin-1) Mm01183349_m1
Arginase, liver (Arg-1) Mm00475988_m1
Tumor Necrosis Factor-Alpha (TNF-Alpha) Mm00443258_m1
Interferon-Gamma (IFN-Gamma) Mm01168134_m1
CD200 ligand Mm00487740_m1
CD200 receptor Mm00491164_m1
Macrophage Migration Inhibitory Factor (MIF) Mm01611157_gH
Muc-5AC Mm01276718_m1
Interleukin-1B (IL-1B) Mm00434228_m1
47
3.10 FACS Analysis of Whole Lung Cells
A subset of mice (n=6/group) were used for fluorescence-activated cell sorting (FACS).
Twenty-four hours following final treatment mice were sacrificed and whole lungs were excised
and digested with collagenase A and DNase I using a gentleMACSTM Octo Dissociator (Miltenyi
Biotec, Cambridge MA, USA). The tissue homogenate was then passed through a cell strainer
and incubated with red blood cell lysis buffer to remove erythrocytes. Sample gating strategy can
be found in Figure 5. Antibodies and Live/Dead Fix stain are found in Table 3. Live dead cells
and doublets were removed using PI stain and FSC/SSC. CD45+ stain was used to identify
leukocytes. Neutrophils were isolated using Ly6G+ stain. Siglec F+, CD11C+ macrophages and
Siglec F+, CD11C- eosinophils were subsequently isolated followed by MHCII+, CD11C+ DCs
and LycC+ monocytes.
48
Figure 5: FACS Gating Strategy- Sample gating strategies from both Saline and HDM mice.
Representative of N=5-6 mice/group.
49
Figure 6: FACS Sample FMO Controls- Images of neutrophils (A), eosinophils (B), dendritic
cells (C) and CD4+ T cells (D) gating for each group along with the accompanying Fluorescence
Minus One (FMO) images. Representative of N=4-6/group
50
Table 3: FACs Antibodies
Antibody Fluorochrome/Colour
Live/Dead Fix Red (Dead Cells Staining) PE-TexRed
Brilliant Violet 785™ anti-mouse CD45 Antibody BV786
Pacific BlueLy anti-mouse Ly-6G Antibody Pacific Blue
BV480 Rat Anti-Mouse Siglec-F BV480
PerCP/Cyainine5.5 anti-mouse I-A/I-E (MHC-II) Antibody PerCPCy5.5
Brilliant Violet 605™ anti-mouse/human CD11b Antibody BV605
Brilliant Violet 650™ anti-mouse CD11c Antibody BV650
APC/Cyainine 7 anti-mouse Ly-6C Antibody APC-Cy7
Alexa Fluor 700 anti-mouse CD19 Antibody Alexa Fluor 700
PE/Cy7 anti-mouse CD3ɛ PE-Cy7
APC anti-mouse CD8a Antibody APC
FITC anti-mouse CD4 FITC
PE anti-mouse CD200R (OX2R) Antibody PE
3.11 Statistical Analysis
All data is expressed as mean ± SEM. A two-way ANOVA test was used to compare
MCh dose-response curves and FACS analysis. Group comparisons were made using one-way
ANOVA using post hoc comparison via Tukey’s test or Kruskal-Wallis Test with Dunn’s post
hoc test as appropriate. Statistical analyses were conducted using GraphPad Prism (version 7.0;
GraphPad Software, La Jolla, CA, USA).
51
Chapter 4: Results
4.1 Models of Allergic Airways Inflammation
4.1.1 All Models Demonstrate AHR with HDM Challenge
Mice intranasally exposed to D. pteronyssinus HDM for two weeks developed MCh
responsiveness demonstrated by elevated total respiratory resistance (Rrs), central Newtonian
resistance (RN) and peripheral tissue dampening (G) (Figure 7).
52
Figure 7: Enhanced Methacholine Responsiveness Occurs After 2, 4, and 8 weeks of D.
pteronyssinus HDM Exposure- Rrs (A,B,C,D), RN (E,F,G,H) and G (I, J, K, L) were significantly
elevated following MCh challenge relative to saline controls. All models showed similar
increases in these respiratory parameters. N=7-10/group, *p<0.05.
Mice exposed to HDM (D. pteronyssinus) for four weeks developed AHR with elevated
Rrs, RN and G (Figure 6B,F,J). Mice were exposed to two doses of HDM, a low dose of
10µg/35µl, or a high dose of 25µg/35µl. We found all parameters were similarly elevated from
MCh challenge relative to saline mice between the two HDM doses (Figure 8). Mice exposed to
D. pteronyssinus for 8 weeks also developed increased respiratory resistance parameters (Figure
53
7). Additionally, all models demonstrated reduced quasi-static compliance (Cst) relative to saline
mice (Figure 9). Overall all models demonstrated similar changes to their respiratory parameters
in comparison to saline mice.
Figure 8: Similar Responses to Methacholine Challenge Between Mice exposed to 10µg and
25µg for Four Weeks- Respiratory resistance parameters (Rrs, RN, G) were elevated in mice
treated with HDM for four weeks regardless of dose (10µg or 25µg)
Figure 9: Quasi-static compliance reduced in all HDM models- Cst was reduced in all HDM
models relative to saline controls. There was no difference between the HDM groups (n= 7-
10/group, *p<0.05).
54
4.1.2 Differences in Inflammation Between HDM Models
Establishing an inflammatory profile which closely mimics the asthma cluster being
modeled is necessary for a good model. To evaluate allergic response HDM-specific IgE levels
in serum were evaluated. HDM-specific IgE levels were elevated only in the four-week and
eight-week models with the highest levels found at 4-weeks (Figure 10). There were no
differences between the 10µg and 25µg doses in the 4-week model.
Figure 10: HDM-Specific IgE Elevated After Four or More Weeks of HDM Exposure- Serum
levels of HDM-specific IgE were most elevated relative to saline after four weeks of HDM
exposure regardless of whether they were given 10µg or 25µg of HDM. Mice treated with HDM
for eight weeks also had elevated IgE. (n=6/group *p<0.05 vs. saline, #p<0.05 vs. 4-week HDM
models).
55
Cellular inflammation of the models was measured with BAL cell counts. Here we found
that total leukocyte counts increased with duration of HDM exposure with the highest total
leukocyte counts found after eight weeks of HDM exposure (Figure 11A). The four-week and
eight-week models were significantly elevated relative to saline mice however the two-week
model was not significantly elevated.
Differential cell counts revealed neutrophils were elevated in all models (Figure 11B)
with the highest increase in the eight-week model. Both leukocytes and eosinophils were
elevated only in the two longer models (Figure 11C,D). Macrophages were significantly elevated
only in the eight-week model (Figure 11E).
56
Figure 11: Cellular Inflammation Increases with Duration of HDM Exposure- Total leukocyte
counts were elevated in the four and eight-week models (A). Neutrophils were elevated in all
models and highest in the eight-week model (B). Lymphocytes and eosinophils were only
elevated in the four and eight-week models (C,D). Macrophages were only elevated in the eight-
week model (E). N=6-7/group *p<0.05 vs saline mice #p<0.05 vs. 4-week HDM.
57
When comparing the 10µg and 25µg four-week models, we found no differences in
overall leukocyte counts between the two doses (Figure 12A). Both neutrophils and lymphocytes
were similarly elevated between the two doses (Figure 12B,C). Macrophages were elevated
compared to saline mice only in the higher dose (Figure 12D). Eosinophils were elevated with
both HDM doses relative to saline mice and the 25µg mice had higher eosinophil levels when
compared to 10µg mice (Figure 12E).
Figure 12: Higher HDM Dose Leads to Increases in Cellular Inflammation in Four Week
Models- Overall total leukocyte counts (A) were not significantly different between the two
HDM doses or in differential neutrophil (B) or lymphocyte (C) counts. Macrophage counts were
only elevated in the higher 25µg model (D). Eosinophils were elevated in both 4-week models
but were further increased in the 25µg model. N=6-7/group *p<0.05 vs saline #p<0.05 vs 10µg
HDM.
58
Gene expression analysis using quantitative polymerase chain reaction (qPCR) found
gene expression patterns varied with the length of HDM exposure. Eotaxin-1 aka CCL-11 levels
were elevated in all models but increased with increased duration of HDM exposure (Figure
13A). Levels of CXCL-1, IL-17A, IL-6, and IL-10 (Figure 13B-E) were elevated only in the
two-week model. Il-4 and IL-13 both showed the greatest increase in the 8-week model (Figure
13F,G).
Figure 13: Length of Model Affects Gene Expression- Differential gene expression was observed
based on the length of the model. Eotaxin-1 levels increased with model length (A). CXCL-1
(B), IL-17A (C), IL-6 (D), IL-10 (E) had the highest increases in the shorter, higher dose
(100µg) two-week model. IL-4 (F) and IL-13 (G) had the highest levels in the eight-week model
followed by the two-week model. N=6-7/group *p<0.05 Vs. saline, #p<0.05 Vs. HDM
59
4.1.3 Evidence of Remodelling in Models of Allergic Airways Inflammation
Airway remodelling was assessed with histological analysis and collagen assay.
Histological analysis of inflation-fixed lungs found that inflammatory infiltrates, measured with
a hematoxylin and eosin (H&E) stain, were present in all HDM models relative to saline mice
(Figure 14A,D,G,J). Collagen deposition as measured with a Trichrome Masson stain and was
only found in the four-week model (Figure 14B,E,H,K). Goblet cells were stained with a PAS
stain and were found in all models (Figure 14C,F,I,L). Inflammatory infiltrates, collagen and
goblet cell hyperplasia were also observed in the 10µg four-week HDM dose (Figure 15A-C).
Histological data of the eight-week model has previously been published(Salehi, Wang et al.
2017).
60
Figure 14: Airway Remodelling Including Collagen Deposition Observed in Four-Week HDM
Mice- No difference was observed between saline mice (A-F). Inflammatory infiltrates (G,J) and
goblet cell hyperplasia (I,L) were observed in both two and four-week models. Collagen
deposition (H,K) was observed only in the longer 4-week model. 8- week model images were
previously published(Salehi, Wang et al. 2017). Images representative of N=3/group
61
Figure 15: 10µg HDM Model Displays Similar Histological Analysis as the 25µg HDM Mice-
Inflammatory infiltrates (A), collagen deposition (B), and goblet cell hyperplasia (C) were
similar to those found in the higher 25µg dose. Images representative of N=3/group.
Total lung collagen content was assessed with a hydroxyproline kit. We found that both
the 4 and 8-week HDM-exposed mice had elevated levels of collagen content relative to saline-
exposed mice with the highest collagen found in the 8-week mice (Figure 16A). When
comparing the two doses of four-week models, only the higher 25µg dose model had elevated
collagen content relative to saline (Figure 16B).
62
Figure 16: Total Collagen Content Elevated in the 25µg Four-Week and Eight Week Models-
Collagen content was significantly elevated relative to saline mice in the 25µg four-week and
eight-week models but not in the lower dose 10µg four-week model. N=6-8/group *p<0.05 vs
saline, #p<0.05 VS 8 wk.
63
4.2 Activation of CD200R in an Acute Model of Allergic Airways Inflammation
Methacholine responsiveness was measured in HDM and saline mice treated with
PEG.CCS13, the fusion protein CD200FC or control Vehicle (Saline). We observed that mice
acutely exposed to HDM and treated with a Vehicle developed increased Rrs compared to saline
exposed and treated controls. HDM-mice treated with either PEG.CCS13 or CD200FC both
demonstrated reduced Rrs compared to HDM mice treated with Vehicle (Figure 17A). Rn was
elevated in all HDM-exposed mice and was not significantly reduced by either PEG.CCS13 or
CD200FC treatment (Figure 17B).
Figure 17: One Treatment with PEG.CCS13 Reduces Total Respiratory Resistance in an Acute
Model of Allergic Airways Inflammation- Mice exposed to five doses of HDM over two weeks
developed increased Rrs relative to saline mice. HDM mice treated with either PEG.CCS13 or
CD200FC had reduced Rrs relative to HDM mice treated with Vehicle. Rn was elevated in all
HDM mice relative to saline mice. N=6-7/group *p<0.05.
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Figure 18: PEG.CCS13 Does Not Reduce BAL Total Leukocyte Counts in Acute Model of
Allergic Airways Inflammation- HDM mice treated with either Vehicle or PEG.CCS13 had
elevated BAL leukocyte counts compared to saline Mice. HDM mice treated with CD200FC did
not have elevated leukocyte counts. N=5-15/group *p<0.05.
Gene expression analysis with qPCR found that inflammatory mediators IL-10, IL-13 and
Eotaxin-1 were elevated in HDM- exposed mice relative to saline exposed mice (Figure 19A-C).
Gene expression of these inflammatory mediators were reduced in mice treated with one IV dose
of PEG.CCS13 relative to mice treated with Vehicle. Levels of CXCL-1 and MIF were elevated
in all mice exposed to HDM (Figure 19D,E). IL-17A levels were higher in HDM-Vehicle mice
compared to Saline-Vehicle (Figure 19F). No changes between any groups were seen in gene
expression levels of IL-6, TNF-alpha, or IFN-gamma (Figure 19G-I).
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Figure 19: IV Treatment with PEG.CCS13 Reduces Inflammatory Mediator Gene Expression in
an Acute Model of Allergic Airways Inflammation- Gene expression levels of IL-10 (A), IL-13
(B) and Eotaxin-1 (C) were elevated in HDM mice relative to saline. These inflammatory
mediators were significantly reduced in HDM-mice treated with PEG.CCS13. CXCL-1 (D) and
MIF (E) levels were elevated in both HDM groups and IL-17A (F) was only elevated in HDM-
Vehicle mice. No changes in any group were found in levels of IL-6 (G), TNF-alpha (H), or IFN-
gamma (I). N= 6-7/group *p<0.05.
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4.3 Expression and Localization of CD200 and CD200R
QPCR analysis of the CD200 ligand gene expression found that there was no difference
between any groups regardless of exposure or treatment (Figure 20A). Levels of the CD200R
receptor expression were significantly reduced in HDM mice treated with Vehicle (saline)
compared to saline mice but not in HDM mice treated with PEG.CCS13 (Figure 20B).
Figure 20: CD200R Gene Expression is Downregulated in Allergic Airways Inflammation and
Restored with PEG.CCS13 Treatment- CD200R expression was reduced in HDM mice treated
with Vehicle but not in HDM mice treated with PEG.CCS13 (B). CD200 ligand expression was
unchanged in all groups (A) N=6-7/group *p<0.05
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CD200R expression on CD45+ leukocytes, neutrophils, DCs, CD4+ T cells and
monocytes was assessed with FACS. Under basal conditions (saline-treated mice) the receptor
was found to be most strongly expressed on DC with it being found on around 5% of DCs
(Figure 21A). After HDM exposure the percentage of DCs and CD4+ T cells expressing
CD200R increased (Figure 21B). The percentage of CD4+ T cells expressing CD200R was
similarly elevated in HDM-PEG.CCS13 mice while the percentage of DCs expressing CD200R
were reduced similar to saline mice. The percentage of CD200R-expressing monocytes were
elevated only in HDM mice treated with PEG.CCS13 and no change was found between any
groups in CD200R expression on either neutrophils or leukocytes (Figure 21B).
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Figure 21: PEG.CCS13 Reduces HDM-Elevated CD200R Expression in Dendritic Cells-
CD200R expression was unchanged by HDM exposure (A). The percentage of cells expressing
CD200R was elevated expression was elevated in DCs and CD4+ T cells following HDM
Exposure. Treatment with PEG.CCS13 reduced CD200R expression on DCs but not on CD4+ T
cells. Monocytes were elevated relative to saline mice in following PEG.CCS13 treatment. No
changes in CD200R expression were found in leukocytes or neutrophils (B). N=5-6/group,
*p<0.05.
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4.4 Changes in Whole Lung Cellular Inflammation in Allergic Airways Inflammation
Following CD200R Activation
BAL leukocyte counts found that total leukocyte counts were elevated in all mice treated
with HDM (Figure 22A). Neutrophils and lymphocytes were elevated in all HDM-exposed mice
(Figure 22B,C). Macrophages were increased from saline-treated mice in HDM mice treated
with aptamer compounds (Figure 22D). BAL eosinophils were elevated in HDM mice, however
HDM mice treated with PEG.CCS13 had reduced eosinophils counts compared to HDM mice
treated with Vehicle (Figure 22E).
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Figure 22: PEG.CCS13 Treatment Reduces BAL Eosinophils- HDM mice had elevated
leukocytes (A), neutrophils (B), and lymphocytes (C) relative to saline mice. Macrophages were
elevated in HDM mice treated with aptamer compounds (D). Eosinophils were elevated in all
HDM mice but mice treated with PEG.CCS13 had lower counts compared to HDM mice treated
with Vehicle (E). N=6/7/group *p<0.05 Vs saline mice, #p<0.05 Vs HDM. PEG.CCS13 mice.
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FACS analysis identified changes in cellular recruitment following HDM exposure and
with PEG.CCS13 analysis (Figure 24). HDM exposure resulted in an increase in total neutrophils
per lung compared to saline-exposure. Eosinophils per lung were also highly increased in the
HDM model. No significant changes in other cell types (leukocytes, DCs, macrophages, B cells,
T cells or monocytes) were found between saline and HDM-treated mice.
HDM mice treated with PEG.CCS13 had an increase in total DC and total macrophage
per lung recruitment compared to both saline and HDM mice (Figure 23). Additionally, the
increase in total neutrophils and eosinophils per lung seen in HDM-Vehicle mice was not seen in
the HDM-PEG.CCS13 treated mice. Total leukocytes, B cells, T cells and monocytes were all
unaffected by PEG.CCS13 compared to both saline and HDM mice.
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Figure 23: CD200R Activation Reduces HDM-Induced Eosinophilia in Chronic Allergic
Airways Inflammation- FACS analysis found that HDM exposure resulted in increased
eosinophils and neutrophils which was not found in HDM mice treated with PEG.CCS13.
Dendritic cells (DCs) and macrophages (Mfs) were increased in HDM-PEG.CCS13 mice relative
to saline-Vehicle and HDM-Vehicle mice. No changes were observed in total leukocytes, B
cells, T cells or monocytes between any groups. N=5-6/group, *p<0.05.
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4.5 Changes in Inflammatory Mediators Following CD200R Activation
Evaluation of HDM-specific IgE with antigen capture ELISA found that all HDM-
exposed mice had elevated levels of HDM-specific IgE. Treatment with PEG.CCS13 or
PEG.Scram did not reduce HDM-specific IgE levels in HDM mice (Figure 24).
Figure 24: HDM-Specific IgE was Elevated in all HDM-Exposed Mice- All HDM-exposed mice
had elevated HDM-Specific IgE levels which were not reduced with PEG.CCS13 treatment.
N=7-8/group *p<0.05.
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Inflammatory mediators assessed with qPCR found that mediators including Muc-5,
Eotaxin-1, IL-13, IL-10 were elevated in all HDM mice (Figure 25A-D). Eotaxin-1, IL-13, and
IL-10 had no difference between HDM-Vehicle mice and HDM-PEG.CCS13 mice while HDM-
PEG.CCS13 mice had elevated levels of Muc-5 compared to HDM-Vehicle mice. Levels of
Interferon gamma (IFN-γ) levels were unchanged in all groups (Figure 25E). Tumor necrosis
factor alpha (TNF-α) was downregulated in HDM mice treated with Vehicle but not HDM mice
treated with PEG.CCS13 (Figure 25F).
Figure 25: Inflammatory Mediator Expression Following Treatment with PEG.CCS13- Mice
exposed to HDM had elevated levels Muc-5 (A), Eotaxin-1 (B), IL-13 (C), IL-10 (D) relative to
saline mice. HDM mice treated with PEG.CCS13 had increased levels of Muc-5 relative to
HDM mice treated with Vehicle (A). There was no difference between the HDM groups in
Eotaxin-1 (B), IL-13 (C), IL-10 (D). Gene expression of IFN- γ (E) was unchanged in all
groups. HDM-Vehicle mice had reduced levels of TNF-α (F). N=6-7/group, *p<0.05.
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4.6 CD200R Activation Reduces AHR in Established Chronic Allergic Airways
Inflammation
HDM mice treated with Vehicle had exhibited increases in Rrs, RN, and G in response to
MCh responsiveness relative to saline-Vehicle mice (Figure 26). HDM mice treated with
PEG.CC13 reduced had significantly reduced Rrs (Figure 26A), RN (Figure 26C), G (Figure 26E)
from HDM-Vehicle mice. HDM mice treated with the PEG.Scrambled aptamer did not
demonstrate reduced respiratory parameters (Figure 26B,D,F). HDM mice treated with
CD200FC or with the naked (unPEGylated) CCS13 aptamer did not have decreased Rrs relative
to HDM-Vehicle mice (Figure27A,B).
Saline mice treated with aptamer compounds (CCS13, PEG.CCS13 or PEG.Scram) had
elevated Rrs following MCh challenge compared to saline-Vehicle mice, there was no increase in
RN or G (Figure 26 & Figure 27A).
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Figure 26: PEG.CCS13 Reduces AHR Including Rrs, RN, and G- HDM-exposed, Vehicle-treated
mice had elevated Rrs, RN, and G compared to saline mice (A-F). HDM mice treated with
PEG.CCS13 had reduced Rrs, RN, and G relative to HDM-Vehicle mice (A,C,E). Saline mice
treated with aptamers had elevated Rrs relative to saline mice treated with Vehicle (A,B).
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Figure 27: Treatment with CD200FC or CCS13 Did Not Reduce Respiratory Resistance- HDM
mice treated with CD200FC or CCS13 did not have reduced Rrs relative to HDM-Vehicle mice.
Saline mice treated with the CCS13 aptamer had elevated Rrs relative to saline-Vehicle mice.
N=9-10/group *p<0.05 Vs. saline-Vehicle #p<0.05 Vs. HDM-Vehicle
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4.7 Evaluation of Histological and Total Collagen Content with PEG.CCS13 Treatment
Histological analysis of inflammatory infiltrates with an H&E stain found that
inflammatory infiltrates (*) were elevated in HDM-Vehicle mice compared to saline-Vehicle
mice while HDM-PEG.CCS13 treated mice had reduced inflammatory infiltrates relative to
HDM-Vehicle mice (Figure 28A,D,G).
Collagen deposition was also elevated in HDM-Vehicle mice and showed reduced
staining in HDM-PEG.CCS13 mice (Figure 28B,E,H). Similarly, goblet cell hyperplasia
measured with a PAS stain was elevated in HDM-Vehicle mice compared to saline-Vehicle mice
and HDM-PEG.CCS13 mice exhibited reduced goblet cells (Figure 28C,F,I). Saline mice treated
with PEG.CCS13 did not have any evidence of goblet cells although they too have increased
inflammatory infiltrates relative to saline mice (Figure 29). Representative images of saline-
Vehicle, HDM-Vehicle and HDM-PEG.CCS13 mice can be found in Figure 30.
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Figure 28: PEG.CCS13 Treatment Results in Histological Evidence of Reduced Cell
Recruitment and Remodelling- HDM mice treated with Vehicle (Veh) demonstrated increased
inflammatory infiltrates (D), collagen deposition (E), and goblet cell hyperplasia (F) compared to
saline-Vehicle mice (A-C). HDM-PEG.CCS13 mice had reduced histological evidence of
inflammation (G), collagen (H), and goblet cells (I) relative to HDM-Veh mice. Images
representative of N=3-4/group.
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Figure 29: Representative Histology of Saline mice Treated with PEG.CCS13- Saline exposed
mice treated with PEG.CCS13 had no evidence of goblet cell hyperplasia from PAS stains
(C,F,I). However, H&E stains (A,D,G) found that they appeared to have elevated inflammatory
infiltrates relative to saline-Vehicle Mice (Figure 29A and Figure 30). Collagen deposition was
mainly localized around veins (B,E,H).
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Figure 30- Representative Histology Images- Representative Images of Saline-Vehicle (A-F),
HDM-Vehicle (G-L) and HDM-PEG.CCS13 (M-R). Images from N=3-4/group
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Hydroxyproline assay for total collagen content found that total collagen was elevated in
all HDM-exposed mice. Despite the histology suggesting a reduction in collagen content with
PEG.CCS13 treatment, the assay found no treatment resulted in a reduction in collagen (Figure
29).
Figure 31: Treatment with PEG.CCS13 Did Not Result in A Reduction in Total Collagen
Content- Hydroxyproline assay found that collagen levels were elevated in all HDM-exposed
mice regardless of treatment. N=6-7/group, *p<0.05.
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Chapter 5: Discussion
5.1 Establishing the Role of CD200/CD200R in Allergic Airways Inflammation
5.1.1 Gene Expression of CD200/CD200R in Allergic Airways Inflammation
The CD200/CD200R pathway has been found to be dysregulated in several diseases
including asthma(Aoki, Matsumoto et al. 2009). In this study we aimed to evaluate the role of
CD200/CD200R pathway in a model of chronic allergic airways inflammation which exhibits the
features of asthma(Woo, Guo et al. 2018). To achieve this, we used both qPCR and FACS
analysis to measure both the gene and receptor expression of the whole lung and determine if the
pathway is dysregulated in allergic airways inflammation.
Our results found that the gene expression of the CD200 ligand was unaffected by HDM
exposure. This seemed to contrast with a study of asthmatic children which found the CD200
ligand was downregulated in asthmatic cells and a study of adult asthmatics which found serum
levels of the CD200 ligand were elevated(Aoki, Matsumoto et al. 2009, Tural Onur, Yalcin et al.
2015). These varying results from both our study and the literature are likely due to the different
tissues/ cell types assessed. The studies in children asthmatics evaluated the gene expression of
CD200 only in peripheral blood mononuclear cells while the adult study evaluated CD200 in
serum (Aoki, Matsumoto et al. 2009, Tural Onur, Yalcin et al. 2015). In contrast, our study
looked at the gene expression of the whole lung tissue. The CD200 ligand has been reported to
be widely distributed across a multitude of cell types and our analysis of the whole lung would
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have included a multitude of cell types expressing CD200 including epithelial cells (Gorczynski
2012, Lauzon-Joset, Marsolais et al. 2019). Given the findings of the two previously mentioned
experiments, this suggests that regulation of the CD200 ligand in asthmatics varies between
different cells/tissues. Additionally these differences may be attributable to differences in the
immune system between humans and mice.
In our study, we evaluated gene expression of the whole lung which includes multiple
cell types including a large number of CD200-expressing epithelial cells. Thus, it is somewhat
unsurprising that expression of the CD200 ligand within the whole lung is unaffected by HDM-
exposure, especially as only myeloid cells have been reported to demonstrate CD200
dysregulation in asthma and these cells make up only small proportion of lung tissue(Aoki,
Matsumoto et al. 2009, Tural Onur, Yalcin et al. 2015). Future studies should be focused on
evaluating the regulation of the CD200 ligand within specific cell types in asthmatics.
Our evaluation of the gene expression of the CD200R in the whole lung found that the
receptor was downregulated in HDM-exposed mice but not in HDM mice treated with
PEG.CCS13. The CD200R is much more narrowly expressed than the ligand, being mostly
localized on myeloid cells and some lymphoid cells(Gorczynski 2012). While few studies have
looked at the expression of the CD200R in asthmatics, our findings suggest that the receptor and
ligand are imbalanced in mice exhibiting HDM-induced allergic airways inflammation. We
further suggest that this imbalance may contribute to the pro-inflammatory features of asthma
which characterize the model. We suggest a pro-inflammatory state may arise as a result of
decreased receptor activation due to decreased ligand availability. However, it should be noted
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that the gene expression analysis of the whole lung is not equivalent to cellular receptor
expression or activity. Furthermore, we conducted qPCR on whole lung tissue and did not
distinguish between RNA expression in different cell types.
5.1.2 FACS Analysis of CD200R Expression
FACS analysis of the CD200R was used to determine the expression of CD200R on
individual cell types. We were able to evaluate the expression of the receptor on CD45+
leukocytes, neutrophils, DCs, CD4+ T cells and monocytes. Other cell types could not be
evaluated due to either low receptor expression or low levels of the cell type under basal/saline-
exposed conditions (ie. eosinophils). Furthermore, we evaluated the percentage of cells which
express the CD200R not the overall receptor expression and are unable to assess whether the
subpopulations strongly or weakly express the receptor.
Our results showed that overall expression of the CD200R was unchanged by HDM
exposure in leukocytes. However, when we evaluated individual leukocyte populations, we
found that DC and CD4+ T cells both had an increased percentage of cells expressing the
CD200R. CD200R has previously been reported to be expressed on lymphoid subpopulations,
particularly CD4+ T cells(Caserta, Nausch et al. 2012). Although the receptor expression has not
been reported in a similar model, studies of CD200R expression in a Th2 driven infection model
have found that receptor expression is increased on CD4+ T cells following chronic
infection(Caserta, Nausch et al. 2012). The increase we reported in a primarily anti-
inflammatory receptor under inflammatory conditions, may suggest that in allergic airways
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inflammation the receptor is not being sufficiently activated despite its increased expression. It
is possible that the CD200R is upregulated in a compensatory mechanism in response the
inflammatory phenotype.
Previous studies have reported that CD200R is an indicator molecule of a specific
subtype of CD4+ cell which is more prevalent in allergic airways inflammation. Studies of
CD200R distribution have found that the receptor is mostly expressed on Th2 cells over Th1
cells(Wright, Cherwinski et al. 2003, Caserta, Nausch et al. 2012). Given that Th2 cells
characterize our model and the allergic asthma endotype(Murdoch and Lloyd 2010, Barnes 2011,
Woo, Guo et al. 2018) it is likely that the increase in CD200R expression is related to an influx
of Th2 cells and a shift away from Th1 cells. HDM mice treated with PEG.CCS13 were found
to have similarly elevated levels of CD200R expression compared to HDM-Vehicle mice
suggesting that PEG.CCS13 treatment did not decreased the proportion of Th2 cells.
CD200R expression has also been previously reported to be expressed on DCs. It is not
known if CD200R expression on DCs is associated with a specific population of DCs. However
previous studies have highlighted the role DCs in determining T cell response(Bharadwaj,
Bewtra et al. 2007). Specific T cell responses have been associated with different DC
populations. Of the two types of DCs, myeloid and plasmacytoid DC, myeloid DCs are
associated with allergic response. Immune myeloid DCs are associated with Th1 and Treg
responses, while mature myeloid DCs are associated with Th2 response(Bharadwaj, Bewtra et al.
2007). The increase in CD200R expression following HDM exposure may suggest that similar
to what was found with T cells the presence of CD200R on DCs may be indicative of a Th2
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response. Further research would be needed to confirm if the CD200R is also an indicator of
mature myeloid DCs.
HDM mice treated with PEG.CCS13 had fewer DCs expressing the CD200R than those
treated with Vehicle. If as we suggest, the CD200R is primarily expressed on mature myeloid
DCs this would indicate a shift away from Th2-related DCs, however future work would be need
to confirm this.
Overall, we were able to show that both the gene expression and cellular expression of
CD200R was dysregulated in our model of allergic airways inflammation which we suggest is
indicative of a shift towards Th2 inflammation. These findings further support the hypothesis
that the CD200/CD200R pathway plays a role in the pathogenesis of asthma and may be a
promising therapeutic candidate.
5.2 Effect of CD200R Activation on Features of Allergic Airways Inflammation
5.2.1 Effects on Cellular Inflammation
Both allergic asthma and our model of allergic airways inflammation are characterized by
cellular inflammation. In our model we found an increase in eosinophil and neutrophil
recruitment in both BAL cell counts and FACS analysis(Woo, Guo et al. 2018). These findings
are expected given that neutrophils are associated with asthma severity and eosinophils are the
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main effector cells in allergic asthma and previous studies of HDM models which have reported
eosinophilic and neutrophilic inflammation(Broide 2001).
In general, our analysis of cell recruitment corresponded between FACS and BAL cell
counts. Both techniques found a that the eosinophilic inflammation induced by HDM exposure
was not present in HDM mice treated with PEG.CCS13. As previously discussed, eosinophilia
plays an important role in the pathogenesis of asthma, influencing other factors including
remodelling and AHR(McBrien and Menzies-Gow 2017). Although we were not able to assess
it, previous findings have reported that the CD200R is expressed on eosinophils and future
studies may focus on the presence of the CD200R on eosinophils under different treatment
conditions(Vaine and Soberman 2014).
FACS analysis of neutrophils found they followed a similar pattern as eosinophils with
HDM-PEG.CCS13 mice not elevated compared to saline mice. This is significant because
although neutrophils are traditionally associated with non-allergic asthma, recent research has
suggested that they do still play a role in allergic asthma(Radermecker, Louis et al. 2018). The
decrease in neutrophils with CD200R activation was not seen in the BAL cell counts, despite a
non-significant trend. This difference between the FACS and BAL cell counts can likely be
attributed to the difference in samples. Our FACS analysis sampled the whole lung while the
BAL samples a 2mL lavage, thus some differences between the two are not unexpected.
DC recruitment assessed with FACS analysis was not significantly different between
HDM-Vehicle and saline-Vehicle mice, however HDM-PEG.CCS13 mice had elevated levels of
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DCs. This raises the possibility that the DCs are still detecting the HDM antigen even with
CD200R activation and continue to try and initiate an inflammatory response due to the
continued HDM exposure and the blocked downstream inflammatory response. Alternatively
the increased DC may be due to the ability of the CD200R family to alter the differentiation of
DC(Gorczynski 2005, Chen, Chen et al. 2008). A previous study found that activation of the
CD200R2 induced a shift to TLR-activated DCs which are involved in the initiation of
Treg(Martín-Orozco, Norte-Muñoz et al. 2017). Tregs function to prevent autoimmune disease
and have been hypothesized to have potential to suppress allergic asthma, although further work
on the subject is required(Martín-Orozco, Norte-Muñoz et al. 2017). Thus, the increased DCs in
PEG.CCS13 treated mice may be representative of a shift towards TLR-activated DCs and
further Treg activity(Martín-Orozco, Norte-Muñoz et al. 2017). Alternatively, it may be due to
an immunogenic effect from the aptamer, this will be further discussed in section 5.3.
Macrophages populations were recorded as elevated in HDM-PEG.CCS13 mice in both
BAL and FACs analysis. This may a similar phenomenon as is seen in DCs related to their
activation. Studies have shown that macrophage expression of CD200R is associated with the
alternatively activated macrophage subtype M2a the macrophage subtype associated with
allergic asthma(Koning, van Eijk et al. 2010). Although we were unable to assess the expression
of CD200R on macrophages due to their low numbers, the increased recruitment with
PEG.CCS13 could suggest we assessed cell population as they were shifting away from the M2a
population associated with allergic asthma. However, BAL leukocyte counts found that mice
treated with the PEGylated scrambled control aptamer also had elevated macrophages. It is
unclear if the elevated macrophages are elevated due to the treatment compounds, the receptor
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activation or both, this will be further discussed in section 5.3. Regardless, further quantification
of the macrophage populations with and without treatment over multiple treatment lengths and
timepoints would further illuminate the role of CD200R activation in macrophage activation.
Our results found that there was no change in any groups in B cells, CD4+ T cells or
monocytes. Although we did not evaluate the proportions of Th2 and Th1 cells within each
group, given previous studies of HDM models and our characterization of the model as having
Th2 cellular and mediator inflammation, it is likely there is an increase in the proportion of Th2
T cells. This supports previous studies of asthma pathogenesis which suggest that allergic
asthma is caused by an imbalance between Th2 and Th1 responses. Thus, although we do not see
an overall change in CD4+ T cells, there is likely due to an increase in Th2 cells and a
subsequent decrease in Th1 cells, which is also suggested by the increase in CD200R expression
under HDM conditions (previously discussed in section 5.1).
5.2.2 Effects of CD200R Activation on HDM-Specific IgE
For this study we evaluated the effects of CD200R activation on inflammatory mediators
including cytokines and HDM-specific IgE. Our results showed that HDM-specific IgE was
elevated in all HDM-exposed mice and not reduced with PEG.CCS13 treatment. Few studies
have studied the effect of CD200R activation on antigen-specific IgE, however models of
collagen-induced arthritis have evaluated anti-collagen IgE(Gorczynski, Chen et al. 2001,
Gorczynski, Chen et al. 2002). These studies found that prophylactic studies were able to
prevent the development of anti-collagen IgE while treatment with CD200FC after inducing the
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disease did not reduce anti-collagen IgE(Gorczynski, Chen et al. 2001, Gorczynski, Chen et al.
2002, Simelyte, Criado et al. 2008). Additionally, our model continued HDM exposure for only
a two-week treatment phase, but our previous work showed HDM-specific IgE takes four weeks
of HDM exposure to be established(Woo, Guo et al. 2018). Thus increasing the duration and/or
frequency of the treatment could also be explored to determine if long term blockage of the
CD200R would reduce HDM-specific IgE.
5.2.3 Effects on Inflammatory Gene Expression
RNA expression of inflammatory meditators measured with qPCR found that few
mediators showed differences between HDM-Vehicle and HDM-PEG.CCS13 mice. The
mediator eotaxin-1, which is associated with eosinophil recruitment, was found to be unchanged
in the chronic model despite a reduction in overall eosinophils with PEG.CCS13 treatment(Kim,
Merry et al. 2001). Notably PEG.CCS13 treatment did downregulate eotaxin-1 RNA in the acute
model, which may be related to differences in inflammatory response between the acute and
chronic models(Woo, Guo et al. 2018). In particular, the acute HDM model is characterized by
an exuberant gene response in several cytokines which is not as pronounced in the chronic
models, this is likely related to the high dose of HDM used (Woo, Guo et al. 2018). Thus, these
findings suggest that the CD200/CD200R pathway regulates eosinophils differently in acute and
chronic models of allergic airways inflammation.
When we looked at the gene expression of other inflammatory mediators, we found that
there were few differences between the HDM-Vehicle treated mice and HDM-PEG.CCSS13
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mice although both were elevated in IL-13 and IL-10. Both these mediators were decreased with
PEG.CCS13 treatment in the acute model(Prodeus, Sparkes et al. 2018). This further
demonstrates the differences in inflammatory regulation between the chronic and acute models
and the potential for future work to explore the regulation of the models in more depth.
Muc-5 is associated with mucus secretion in airways disease, particularly in chronic
obstructive pulmonary disease(Bonser and Erle 2017). Our results did find that this mediator
elevated in HDM-Vehicle mice which corresponded with histological increase in goblet cells.
Surprisingly although HDM mice treated with PEG.CCS13 had reduced goblet cells, gene
expression of Muc-5 was further increased. This may suggest CD200R expression results in a
decrease in goblet cells via a mechanism that is downstream of gene expression, thus future work
may wish to conduct ELISA assays or western blots to determine protein expression of some
inflammatory mediators. Alternatively, the elevated Muc-5 may be due to an effect from the
treatment compounds and contributing to elevated respiratory mechanics. This will be further
discussed in Section 5.3.
Previous studies exploring the therapeutic effects of CD200R activation have also found
that the effects of CD200R activation on gene expression have greatly varied depending on the
model, despite resolution of other disease symptoms. For example, IL-10 gene expression was
previously recorded as being decreased in an acute model of OVA-induced allergic airways
inflammation(Lauzon-Joset, Langlois et al. 2015). This corresponds with our findings in the
acute model, but not the chronic model further highlighting the differences between the chronic
and acute models. Previous studies of CD200R activation effect on the gene expression of
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inflammatory mediators has been inconsistent, varying with the model used. Although the acute
models of allergic airways inflammation found a decrease in IL-10, other studies including a
murine skin grafts model found elevated IL-10 levels(Gorczynski, Cattral et al. 1999). Still a
further study of collagen induced arthritis found that although gene expression of IL-10 was
reduced, cytokine levels were not(Simelyte, Criado et al. 2008). Similarly, contradictory results
were found in the literature regarding TNF-alpha expression. Two studies, one of collagen-
induced arthritis(Simelyte, Criado et al. 2008) and an in vivo study of human mesenchymal cells
found that CD200R activation reduced TNF-alpha(Pietilä, Lehtonen et al. 2012) but our model
and a model of OVA-induced acute allergic airways inflammation found no change with receptor
activation(Lauzon-Joset, Langlois et al. 2015). Overall the contradictory literature can most
plausibly be explained with the suggestions that the effects of CD200R activation on
inflammatory gene expression are highly model dependant. In general, we found most mediators
were unchanged with PEG.CCS13 treatment suggesting that in our chronic model the pathway
regulates inflammation downstream of gene expression.
5.2.4 Effects on Airway Hyperresponsiveness
Our results showed that PEG.CCS13 treatment reduced AHR in HDM mice when
assessed with the Flexivent®. The reduction in AHR was seen in both the acute and chronic
model. Importantly CD200FC was only found to reduce AHR in the acute model when the
treatment was delivered by IV as opposed to the intranasal delivery given in the chronic model.
This suggests that the PEG.CCS13 aptamer has advantages over the protein CD200FC when
delivered intranasally. We also found that the unPEGylated CCS13 aptamer did not have any
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effects on AHR. This indicates that the addition of the PEG moiety, is essential for the
effectiveness of the drug as the CCS13 aptamer is likely degraded and/or removed from the
system too quickly.
Although there is no firm consensus on the mechanisms causing AHR, several
contributing factors include genetics, inflammation and smooth muscle(Cockcroft and Davis
2006, Chapman and Irvin 2015). In our model it is likely that inflammation is the largest
contributor to AHR given that all our mice are genetically identical. Additionally, an acute OVA
model determined that abrogation of antigen induced AHR was independent of smooth muscle
contractility, this is likely to be the case in our model too given the similarities of animal model
and antigen exposure(Lauzon-Joset, Langlois et al. 2015). Given that eosinophils had the
greatest proportional numerical increase in our chronic model and have been implicated in
causing AHR it is likely that their subsequent reduction with PEG.CCS13 treatment contributes
to the reduction in AHR.
Importantly we also noted an increase in AHR in our control (saline) mice treated with
both PEG.CCS13 and PEG.Scram and CCS13. This may be caused by an immunogenic effect of
either the aptamer compound or the method of delivery. This will be further discussed in section
5.3.
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5.2.5 Effects on Remodelling
In evaluating the structural features of remodelling we found that the inflammatory
infiltrates, collagen deposition and goblet cell hyperplasia all appeared to be reduced in
histological analysis. The reduction in inflammatory infiltrates corresponded with our BAL and
FACS results which showed decreased eosinophils. However, our quantitative collagen assay
found that all HDM treated mice had elevated collagen regardless of treatment. Although the
histological evidence that CD200R activation influences remodelling is promising, it should be
noted that histological analysis is not a quantitative measure.
Previous studies of the CD200/CD200R pathway on collagen remodelling have been
focused on murine models of collagen induced arthritis. These studies used H&E stained tissue
and a semi-quantitative histological scoring system to conclude that there were reductions in
features of remodelling. However, when given in a treatment model these studies found that
CD200FC was not able to reduce anti-collagen IgG. It should be noted that none of these studies
ran the same collagen assay as we did or were able to find quantitative reductions in collagen in
non-prophylactic models despite histological changes or amelioration of other disease
characteristics.
Although it was disappointing that we were not able to show a reduction in collagen
content it was not entirely surprising as no therapeutic has been shown to completely reverse
collagen deposition. However, eosinophils, which were reduced with CD200R activation, play a
role in remodelling including collagen deposition through MMPs. Other studies have shown that
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knockout of the Th2 cytokines IL-4 and IL-13 can protect against remodelling. Thus, a longer,
increasing the duration and frequency of the treatment regimen could be explored in future
studies.
5.3 Immunogenic Effects from PEG.CCS13
5.3.1 Immunogenic Effects Seen in Respiratory Responsiveness
We have demonstrated the effectiveness of PEG.CCS13 at reducing features of asthma
including eosinophils and AHR. However, our results have suggested that there may be some
unintended, likely immunogenic effects from our treatment.
When assessing AHR with the Flexivent® device, we observed saline mice treated with
PEG.CCS13 mice had elevated total respiratory resistance relative to saline mice treated with
Vehicle. This same effect was observed in the PEGylated control aptamer. This effect was only
seen in Rrs and not in any other measured respiratory mechanics. This was particularly
interesting because the levels of Rrs between HDM-PEG.CCS13 mice and saline-PEG.CCS13
mice were very similar and it suggests the possibility that without the immunogenic effect of the
treatment, CD200R activation may result in even further resolution of AHR.
Given the elevated Rrs noted in the PEGylated aptamers and the previous literature which
has implicated immunogenic effects in PEGylated compounds, we added an aptamer group
which did not include the PEG compound to determine if the AHR from treatment was
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exclusively due to the PEG moiety. We found that the CCS13 “naked” aptamer also had
elevated Rrs relative to saline-Vehicle mice. This suggested that the effect was not exclusively
caused by the PEG moiety and may be cause by the aptamer compounds themselves or our
method of intranasal administration.
We further observed an increase in the levels of Muc-5 in HDM-mice treated with
PEG.CCS13 over and above what was found in HDM-Vehicle mice. This was despite a
histological reduction, but no complete amelioration, of goblet cells within the airways. Muc-5
is associated with mucus secretion which plays a role in inducing AHR. Thus, the increase in
AHR may be due to an increase in mucus secretion in the lungs of mice treated with the aptamer
compounds. IL-13 is also associated with the production of mucus(Cohn 2006). Notably IL-13
was found to be decreased with PEG.CCS13 in the acute models of allergic airways
inflammation but not in our chronic model. Future work should be done to determine if the
increase in gene expression of Muc-5 is related to an increase in mucus secretion.
It is also possible that the increased AHR is due to our method of administration. Few
studies have explored the use of aptamers via intranasal administration and none to our
knowledge have studied the effect of aptamers given intranasally in the absence of a disease
model. Thus, further work is needed to determine if this increase in AHR is unique to intranasal
instillation of compounds.
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5.3.2 Immunogenic Effects on Cellular Inflammation
Our results from BAL cell counts showed that mice treated with both HDM mice treated
with PEG.CCS13 and PEG.Scrambled had elevated macrophages relative to saline mice. Further
examination of BAL cell counts also showed that HDM-CD200FC mice and HDM-CCS13 mice
both had elevated macrophages recruitment relative to the saline-Vehicle mice (Appendix).
Interestingly this effect was only seen in mice which were treated with HDM and a non-
vehicle compound, this effect was not seen in saline mice treated with these compounds. This
does suggest that HDM exposure does play a role. This is further supported by the presence of
elevated macrophages seen in our eight-week model. However, this increase in macrophages is
unlikely to be exclusively caused HDM exposure as HDM-Vehicle mice do not have elevated
macrophages. Thus, there is likely due to additive effects from both the HDM and the treatment
compounds.
Given that this elevation was seen in the BAL cell counts of all mice treated with all
treatment compounds, this suggests that the immunogenic responses observed are non-specific
responses from intranasal administration of compounds rather than a specific response to any of
the compounds. Given that we have shown this in both aptamer and protein compounds this
effect is likely not due to the aptamer compound. Similarly, we have tested aptamer compounds
with and without the PEG moiety and compounds which do not bind to the CD200R, indicating
the increase in macrophages is not solely attributable either the PEG moiety or CD200R
activation.
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FACS analysis found that, similarly to the BAL cell counts, macrophages were elevated
in HDM mice treated with PEG.CCS13. This is likely due to the same non-specific effect of
treatment that was described earlier, however in FACS analysis we did not assess the effects of
CCS13, PEG.Scram or CD200FC. Additionally, macrophages do not play a strong role in the
pathways of allergic airways inflammation which suggests that the effect is due to the treatment
administration as can be concluded with the same findings in the BAL.
FACS analysis of DCs found they demonstrated a similar pattern to macrophages with
the greatest recruitment seen in HDM-PEG.CCS13 treated mice. There could be either due to a
change in DC activation as previously discussed or due to a similar non-specific effect as was
seen in the macrophages. Since the scrambled aptamer was not evaluated in FACS we are not
able to make a determination as to whether the effect is due to the pathway or the delivery
without further experimentation.
Finally, in observing the histology images, saline mice treated with PEG.CCS13 appeared
to show more evidence of inflammatory infiltrates in the H&E stain. As previously mentioned,
histological analysis is qualitative, but these findings align with the previously discussed
macrophage inflammation present in HDM mice treated with PEG.CCS13. This also
corresponded with the BAL leukocyte counts of saline-PEG.CCS13 mice which had a trend
towards increased leukocyte counts.
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Overall, we found that there were effects from the intranasal administration of the
treatment. Evaluation of the effect of different compounds on the BAL leukocyte counts suggests
that this is unlikely completely due to either the PEG moiety or the aptamer compounds but
rather a result of compounds other than saline being introduced intranasally. Although not all
these compounds were evaluated at all endpoints, the constancy across the endpoints tested
suggest this immune response is likely due to the intranasal administration.
These findings suggest that intranasal administration may not be the best delivery method
for therapeutics in mouse models, particularly those involving BALB/c mice. Other methods of
administration such as IV or subcutaneous injection or aerosol inhalation may prevent these non-
specific immunogenic effects. In our acute model the use of one IV dose of both CD200FC and
PEG.CCS13 led to a reduction in AHR similar to what was seen with intranasal instillation,
however the effect of these compounds was not explored in saline mice. If these effects are
found to be absent this may suggest IV as a better delivery option, however this not be a very
clinically relevant method of delivery as most asthma patients would likely find it very
unappealing to take their medication by injection. Additionally, although previous models using
a form of systemic delivery have not reported any adverse effects, there are potential as yet
unknown effects on other organ systems to be taken into account. Aerosol delivery is an option
for which would also results in only local delivery to the airways similar to intranasal. However,
this also has disadvantages is it would require a large amount of compound and it is difficult to
measure the exact dose that would be delivered to the animal’s lungs. Furthermore, a precise
dose would be very difficult to deliver so further innovations and research in this form of
delivery would be needed.
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Chapter 6: Conclusions
Over the course of thesis, we have explored the role of the CD200/CD200R pathway in
allergic airways inflammation and the effects of CD200R activation across two models.
We showed that one IV treatment with the CD200R agonist aptamer, PEG.CCS13 was
able to reduce features of AHR and inflammatory gene expression in an acute model of allergic
airways inflammation. These findings demonstrated the potential of both PEG.CCS13 as a
therapeutic agent and validated the potential of CD200R activation as therapeutic pathway in
allergic airways inflammation.
Building on the promising results of the acute model we have described the development
and characterization of a chronic model of allergic airways inflammation which recapitulates the
three salient features of asthma in a shorter timeframe than previous models and with a lower
antigen dose than acute models. This offers advantages as a faster, lower cost model which has
been extensively characterized. This model can be used for the development and testing of
therapeutics and also for further characterizing and exploring the pathways behind the allergic
airways inflammation phenotype.
We further described the role of the CD200/CD200R pathway in the pathogenesis of
chronic allergic airways inflammation. We found that CD200R gene expression was reduced in
the overall lung tissue of HDM-exposed mice. We also demonstrated an elevated percentage of
DC and CD4+ T cells expressed CD200R in allergic airways inflammation likely indicating the
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presence of Th2 specific cells. These findings support the role of the dysregulated
CD200/CD200R pathway in the pathogenesis of allergic airways inflammation
Finally, we evaluated the effects of PEG.CCS13 CD200R activation on the features of
asthma in the chronic model of allergic airways inflammation. We concluded that activation of
the receptor reduced eosinophilic inflammation, AHR and goblet cell hyperplasia (Figure 33).
These findings suggest that the CD200R is a promising therapeutic target for asthma treatment
and demonstrate the promise of the PEG.CCS13 aptamer as a therapeutic agent.
Figure 32: Effects of CD200R Activation With PEG.CCS13 in Allergic Airway Inflammation-
Activation of the CD200R with the PEG.CCS13 aptamer resulted in downstream reduction in
eosinophilia, AHR and goblet cell hyperplasia.
Recent research in the pathogenesis of asthma has focused on treating the various
subtypes and endotypes of the disease with allergic asthma taking the forefront as one of the
103
most common. Given the known inflammatory profile of allergic asthma, much research has
focused on the potential of targeted immunotherapies. This has led to the development of
immune targeting therapies which show a lot of promise in the treatment of asthma as well as
other inflammatory diseases. This body of work has advanced our understanding of the role of
CD200/CD200R pathway in inflammation and the potential for immunoregulation of this
pathway in allergic asthma.
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Chapter 7: Future Directions
7.1 Exploring Delivery Methods
In this work we reported an immunogenic effect that appeared with all treatment
compounds except our Vehicle (saline) treatment. We suggest that these effects may be due to
our intranasal administration of compounds. For future studies we propose the further
exploration of this non-specific effect from intranasal delivery. Our work did not fully explore
these effects in all endpoints and further quantifying these effects and determining if there are
differences between the intranasal administration of proteins and aptamer may have important
implications for drug development.
We further suggest that other methods of delivery including IV or subcutaneous injection
may be alternatives which prevent the immunogenic effect we reported despite its undesirability
to patients. Aerosol administration is also a promising future study that can be used for aptamer
delivery and as previously mentioned will require some development to ensure the delivery of a
consistent dose. It is important to establish the systemic vs local effects of CD200R activation
and the role delivery method may play in these. A previous study did establish differences in
response between mice with hyperactivated CD200 and mice receiving CD200FC by IV
injection, highlight possible differences in both method of delivery and activation ability
between the cellular CD200 ligand and the CD200FC protein which have yet to be fully
explored(Chen, Chen et al. 2008).
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7.2 Other Aptamer Delivery Molecules
Although our results suggest that the immunogenic effects we reported are likely due to
the intranasal delivery method, it remains that there have been several adverse effect associated
with the PEG moiety including anaphylactic reactions, including a case of a halted phase II
trial(Zhou and Rossi 2017). Given this, it is reasonable to explore alternative molecules which
can serve the same function without the potential downsides associated with the PEG moiety.
Substitute conjugating molecules such as heparin, dextran or polyamino acids should be explored
as potential alternatives to the PEG moiety(Zhang, Liu et al. 2014).
7.3 Determining Sex Differences in Inflammation and CD200R Activation
As work in the appendix of this thesis will demonstrate, there are known differences in
lung function mechanics between male and female mice, studies have also shown this to be the
case with immune responses as well. These differences are also evident between the human
sexes so a most accurate animal model should utilize both sexes to account for this. Thus, a
future study should be conducted to determine if the effects of CD200R activation and the role of
the CD200R are identical in both sexes.
7.4 Increasing Treatment Duration/ Adding other Therapeutics
In this work we were able to show that two weeks of PEG.CCS13 treatment given 3
times/week was able to reduce some features of asthma while others showed no effect. Follow
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up studies should focus on increasing treatment duration and frequency. Since all features of
remodelling take a minimum of 4 weeks of HDM exposure to develop, it may not be surprising
that after only 2 weeks of treatment we did not see changes in some features such as quantitative
collagen deposition, similarly the HDM models we presents showed that two week of HDM
exposure did not lead to the development of HDM-specific IgE. However, with the two weeks
of treatment, we did see reduction in features that are quicker to develop such as eosinophils
which are known to play a role in remodelling. Thus, it is worth investigating to determine if a
longer, and more rigorous treatment regimen could lead to a reduction in features of remodelling.
Another possibility for future work in the administration of the PEG.CCS13 aptamer in
conjunction with other existing therapies. Given that not all features of inflammation were
affected by the CD200R activation it is possible that other inflammatory pathways continue to be
activated that are not influenced by the CD200R. As it has been shown that certain members of
the CD200R family cause downstream effects independent of IgE we propose the use of
PEG.CCS13 in conjunction with the IgE blocking drug omalizumab. Together these two
therapies may lead to a better coverage of the inflammatory pathways that contribute to allergic
airways inflammation than either alone.
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Chapter 8: References
(NHIS), N. H. I. S. (2012). "Data, Statistics, and Surveillance." from http://www.cdc.gov/asthma/nhis/2012/data.htm. Akkaya, M., M. L. Aknin, B. Akkaya and A. N. Barclay (2013). "Dissection of agonistic and blocking effects of CD200 receptor antibodies." PLoS One 8(5): e63325. Aoki, T., Y. Matsumoto, K. Hirata, K. Ochiai, M. Okada, K. Ichikawa, M. Shibasaki, T. Arinami, R. Sumazaki and E. Noguchi (2009). "Expression profiling of genes related to asthma exacerbations." Clin Exp Allergy 39(2): 213-221. Aun, M. V., R. Bonamichi-Santos, F. M. Arantes-Costa, J. Kalil and P. Giavina-Bianchi (2017). "Animal models of asthma: utility and limitations." J Asthma Allergy 10: 293-301. Aun, M. V., B. M. Saraiva-Romanholo, F. M. Almeida, T. R. Brüggemann, J. Kalil, M. e. A. Martins, F. M. Arantes-Costa and P. Giavina-Bianchi (2015). "Sensitization by subcutaneous route is superior to intraperitoneal route in induction of asthma by house dust mite in a murine mode." Einstein (Sao Paulo) 13(4): 560-566. Barbato, A., G. Turato, S. Baraldo, E. Bazzan, F. Calabrese, C. Panizzolo, M. E. Zanin, R. Zuin, P. Maestrelli, L. M. Fabbri and M. Saetta (2006). "Epithelial damage and angiogenesis in the airways of children with asthma." Am J Respir Crit Care Med 174(9): 975-981. Barclay, A. N. and H. A. Ward (1982). "Purification and chemical characterisation of membrane glycoproteins from rat thymocytes and brain, recognised by monoclonal antibody MRC OX 2." Eur J Biochem 129(2): 447-458. Barnes, P. J. (2001). "Th2 cytokines and asthma: an introduction." Respir Res 2(2): 64-65. Barnes, P. J. (2010). "New therapies for asthma: is there any progress?" Trends Pharmacol Sci 31(7): 335-343. Barnes, P. J. (2011). "Biochemical basis of asthma therapy." J Biol Chem 286(38): 32899-32905. Beckett, P. A. and P. H. Howarth (2003). "Pharmacotherapy and airway remodelling in asthma?" Thorax 58(2): 163-174. Bellanti, J. A. and R. A. Settipane (2018). "Unraveling the conundrum of asthma phenotypes and endotypes." Allergy Asthma Proc 39(1): 1-2. Berger, A. (2000). "Th1 and Th2 responses: what are they?" BMJ 321(7258): 424. Bergeron, C., W. Al-Ramli and Q. Hamid (2008). "Remodeling in Asthma." Proceedings of the American Thoracic Society 6(3): 301-305. Bergeron, C., M. K. Tulic and Q. Hamid (2010). "Airway remodelling in asthma: from benchside to clinical practice." Can Respir J 17(4): e85-93. Bharadwaj, A. S., A. K. Bewtra and D. K. Agrawal (2007). "Dendritic cells in allergic airway inflammation." Can J Physiol Pharmacol 85(7): 686-699. Bisgin, A., W. J. Meng, G. Adell and X. F. Sun (2019). "Interaction of CD200 Overexpression on Tumor Cells with CD200R1 Overexpression on Stromal Cells: An Escape from the Host Immune Response in Rectal Cancer Patients." J Oncol 2019: 5689464. Bonser, L. R. and D. J. Erle (2017). "Airway Mucus and Asthma: The Role of MUC5AC and MUC5B." J Clin Med 6(12). Boulet, L. P., M. Laviolette, H. Turcotte, A. Cartier, M. Dugas, J. L. Malo and M. Boutet (1997). "Bronchial subepithelial fibrosis correlates with airway responsiveness to methacholine." Chest 112(1): 45-52. Braman, S. S. (2006). "The global burden of asthma." Chest 130(1 Suppl): 4S-12S. Broide, D. H. (2001). "Molecular and cellular mechanisms of allergic disease." J Allergy Clin Immunol 108(2 Suppl): S65-71.
108
Brown, A., K. Farmer, L. MacDonald, N. Kalsheker, D. Pritchard, C. Haslett, J. Lamb and J. M. Sallenave (2003). "House dust mite Der p 1 downregulates defenses of the lung by inactivating elastase inhibitors." Am J Respir Cell Mol Biol 29(3 Pt 1): 381-389. Canada, S. (2014 ). "Asthma, 2014." from https://www150.statcan.gc.ca/n1/pub/82-625-x/2015001/article/14179-eng.htm. Card, J. W., M. A. Carey, J. A. Bradbury, L. M. DeGraff, D. L. Morgan, M. P. Moorman, G. P. Flake and D. C. Zeldin (2006). "Gender differences in murine airway responsiveness and lipopolysaccharide-induced inflammation." J Immunol 177(1): 621-630. Caserta, S., N. Nausch, A. Sawtell, R. Drummond, T. Barr, A. S. Macdonald, F. Mutapi and R. Zamoyska (2012). "Chronic infection drives expression of the inhibitory receptor CD200R, and its ligand CD200, by mouse and human CD4 T cells." PLoS One 7(4): e35466. Cates, E. C., R. Fattouh, J. Wattie, M. D. Inman, S. Goncharova, A. J. Coyle, J. C. Gutierrez-Ramos and M. Jordana (2004). "Intranasal exposure of mice to house dust mite elicits allergic airway inflammation via a GM-CSF-mediated mechanism." J Immunol 173(10): 6384-6392. Chapman, D. G. and C. G. Irvin (2015). "Mechanisms of airway hyper-responsiveness in asthma: the past, present and yet to come." Clin Exp Allergy 45(4): 706-719. Chen, Z., D. X. Chen, Y. Kai, I. Khatri, B. Lamptey and R. M. Gorczynski (2008). "Identification of an expressed truncated form of CD200, CD200tr, which is a physiologic antagonist of CD200-induced suppression." Transplantation 86(8): 1116-1124. Cherwinski, H. M., C. A. Murphy, B. L. Joyce, M. E. Bigler, Y. S. Song, S. M. Zurawski, M. M. Moshrefi, D. M. Gorman, K. L. Miller, S. Zhang, J. D. Sedgwick and J. H. Phillips (2005). "The CD200 receptor is a novel and potent regulator of murine and human mast cell function." J Immunol 174(3): 1348-1356. Chu, H. W., J. L. Halliday, R. J. Martin, D. Y. Leung, S. J. Szefler and S. E. Wenzel (1998). "Collagen deposition in large airways may not differentiate severe asthma from milder forms of the disease." Am J Respir Crit Care Med 158(6): 1936-1944. Cockcroft, D. W. and B. E. Davis (2006). "Mechanisms of airway hyperresponsiveness." J Allergy Clin Immunol 118(3): 551-559; quiz 560-551. Cohn, L. (2006). "Mucus in chronic airway diseases: sorting out the sticky details." J Clin Invest 116(2): 306-308. Dimeloe, S., D. F. Richards, Z. L. Urry, A. Gupta, V. Stratigou, S. Farooque, S. Saglani, A. Bush and C. M. Hawrylowicz (2012). "1α,25-dihydroxyvitamin D3 promotes CD200 expression by human peripheral and airway-resident T cells." Thorax 67(7): 574-581. Dullaers, M., M. J. Schuijs, M. Willart, K. Fierens, J. Van Moorleghem, H. Hammad and B. N. Lambrecht (2016). "House dust mite-driven asthma and allergen-specific T cells depend on B cells when the amount of inhaled allergen is limiting." J Allergy Clin Immunol. Dunn, M. R., R. M. Jimenez and J. C. Chaput (2017). "Analysis of aptamer discovery and technology." Nature Reviews Chemistry 1. Fahy, J. V. (2015). "Type 2 inflammation in asthma--present in most, absent in many." Nat Rev Immunol 15(1): 57-65. Fehrenbach, H., C. Wagner and M. Wegmann (2017). "Airway remodeling in asthma: what really matters." Cell Tissue Res 367(3): 551-569. Finkelman, F. D., S. P. Hogan, G. K. Hershey, M. E. Rothenberg and M. Wills-Karp (2010). "Importance of cytokines in murine allergic airway disease and human asthma." J Immunol 184(4): 1663-1674. Gaurav, R. and D. K. Agrawal (2013). "Clinical view on the importance of dendritic cells in asthma." Expert Rev Clin Immunol 9(10): 899-919. Gill, M. A. (2012). "The role of dendritic cells in asthma." J Allergy Clin Immunol 129(4): 889-901.
109
GINA (2015). The Global Strategy for Asthma Management and Prevention, Global Initiative for Asthma (GINA). Gorczynski, R., Z. Chen, Y. Kai, L. Lee, S. Wong and P. A. Marsden (2004). "CD200 is a ligand for all members of the CD200R family of immunoregulatory molecules." J Immunol 172(12): 7744-7749. Gorczynski, R. M. (2005). "CD200 and its receptors as targets for immunoregulation." Curr Opin Investig Drugs 6(5): 483-488. Gorczynski, R. M. (2012). "CD200:CD200R-Mediated Regulation of Immunity." ISRN Immunology 2012: 682168. Gorczynski, R. M., M. S. Cattral, Z. Chen, J. Hu, J. Lei, W. P. Min, G. Yu and J. Ni (1999). "An immunoadhesin incorporating the molecule OX-2 is a potent immunosuppressant that prolongs allo- and xenograft survival." J Immunol 163(3): 1654-1660. Gorczynski, R. M., Z. Chen, W. He, I. Khatri, Y. Sun, K. Yu and I. Boudakov (2009). "Expression of a CD200 transgene is necessary for induction but not maintenance of tolerance to cardiac and skin allografts." J Immunol 183(3): 1560-1568. Gorczynski, R. M., Z. Chen, I. Khatri and K. Yu (2011). "Graft-infiltrating cells expressing a CD200 transgene prolong allogeneic skin graft survival in association with local increases in Foxp3(+)Treg and mast cells." Transpl Immunol 25(4): 187-193. Gorczynski, R. M., Z. Chen, L. Lee, K. Yu and J. Hu (2002). "Anti-CD200R ameliorates collagen-induced arthritis in mice." Clin Immunol 104(3): 256-264. Gorczynski, R. M., Z. Chen, K. Yu and J. Hu (2001). "CD200 immunoadhesin suppresses collagen-induced arthritis in mice." Clin Immunol 101(3): 328-334. Gorczynski, R. M., D. A. Clark, N. Erin and I. Khatri (2011). "Role of CD200 expression in regulation of metastasis of EMT6 tumor cells in mice." Breast Cancer Res Treat 130(1): 49-60. Gregory, L. G. and C. M. Lloyd (2011). "Orchestrating house dust mite-associated allergy in the lung." Trends Immunol 32(9): 402-411. Hammad, H., M. Chieppa, F. Perros, M. A. Willart, R. N. Germain and B. N. Lambrecht (2009). "House dust mite allergen induces asthma via Toll-like receptor 4 triggering of airway structural cells." Nat Med 15(4): 410-416. Hargreave, F. E., J. Dolovich, P. M. O'Byrne, E. H. Ramsdale and E. E. Daniel (1986). "The Origin of Airway Hyperresponsiveness." Allergy and Clinical Immunology 78(5): 825-832. Harris, J. M. and R. B. Chess (2003). "Effect of pegylation on pharmaceuticals." Nat Rev Drug Discov 2(3): 214-221. Haspeslagh, E., N. Debeuf, H. Hammad and B. N. Lambrecht (2017). "Murine Models of Allergic Asthma." Methods Mol Biol 1559: 121-136. Havaux, X., A. Zeine, A. Dits and O. Denis (2005). "A new mouse model of lung allergy induced by the spores of Alternaria alternata and Cladosporium herbarum molds." Clin Exp Immunol 139(2): 179-188. Healy, J. M., S. D. Lewis, M. Kurz, R. M. Boomer, K. M. Thompson, C. Wilson and T. G. McCauley (2004). "Pharmacokinetics and biodistribution of novel aptamer compositions." Pharm Res 21(12): 2234-2246. Hirota, N. and J. G. Martin (2013). "Mechanisms of airway remodeling." Chest 144(3): 1026-1032. Hoek, R. M., S. R. Ruuls, C. A. Murphy, G. J. Wright, R. Goddard, S. M. Zurawski, B. Blom, M. E. Homola, W. J. Streit, M. H. Brown, A. N. Barclay and J. D. Sedgwick (2000). "Down-regulation of the macrophage lineage through interaction with OX2 (CD200)." Science 290(5497): 1768-1771. Hoffman, S. M., X. Qian, J. D. Nolin, D. G. Chapman, S. B. Chia, K. G. Lahue, R. Schneider, J. L. Ather, M. J. Randall, D. H. McMillan, J. T. Jones, D. J. Taatjes, M. Aliyeva, N. Daphtary, S. Abdalla, L. K. Lundblad, Y. S. Ho, V. Anathy, C. G. Irvin, E. F. Wouters, N. L. Reynaert, A. E. Dixon, A. van der Vliet, M. E. Poynter and Y. M. Janssen-Heininger (2016). "Ablation of
110
Glutaredoxin-1 Modulates House Dust Mite-Induced Allergic Airways Disease in Mice." Am J Respir Cell Mol Biol 55(3): 377-386. Hoshino, M., M. Takahashi and N. Aoike (2001). "Expression of vascular endothelial growth factor, basic fibroblast growth factor, and angiogenin immunoreactivity in asthmatic airways and its relationship to angiogenesis." J Allergy Clin Immunol 107(2): 295-301. Hosoki, K., L. Aguilera-Aguirre, A. R. Brasier, A. Kurosky, I. Boldogh and S. Sur (2015). "Facilitation of Allergic Sensitization and Allergic Airway Inflammation by Pollen-Induced Innate Neutrophil Recruitment." Am J Respir Cell Mol Bio 54(1): 81-90. Hsia, C. C., D. M. Hyde, M. Ochs, E. R. Weibel and A. E. J. T. F. o. Q. A. o. L. Structure (2010). "An official research policy statement of the American Thoracic Society/European Respiratory Society: standards for quantitative assessment of lung structure." Am J Respir Crit Care Med 181(4): 394-418. Huang, C., H. Dong, M. Zou, L. Luo, Y. Hu, Z. Xie, Y. Le, L. Liu, F. Zou and S. Cai (2016). "Bevacizumab reduced auto-phosphorylation of VEGFR2 to protect HDM-induced asthma mice." Biochem Biophys Res Commun 478(1): 181-186. Huang, J., R. Olivenstein, R. Taha, Q. Hamid and M. Ludwig (1999). "Enhanced proteoglycan deposition in the airway wall of atopic asthmatics." Am J Respir Crit Care Med 160(2): 725-729. Humbles, A. A., C. M. Lloyd, S. J. McMillan, D. S. Friend, G. Xanthou, E. E. McKenna, S. Ghiran, N. P. Gerard, C. Yu, S. H. Orkin and C. Gerard (2004). "A critical role for eosinophils in allergic airways remodeling." Science 305(5691): 1776-1779. Hurst, J. L. and R. S. West (2010). "Taming anxiety in laboratory mice." Nat Methods 7(10): 825-826. Ito, T., K. Hirose, A. Norimoto, T. Tamachi, M. Yokota, A. Saku, H. Takatori, S. Saijo, Y. Iwakura and H. Nakajima (2017). "Dectin-1 Plays an Important Role in House Dust Mite-Induced Allergic Airway Inflammation through the Activation of CD11b+ Dendritic Cells." J Immunol 198(1): 61-70. James, A. L. and S. Wenzel (2007). "Clinical relevance of airway remodelling in airway diseases." Eur Respir J 30(1): 134-155. Jenmalm, M. C., H. Cherwinski, E. P. Bowman, J. H. Phillips and J. D. Sedgwick (2006). "Regulation of myeloid cell function through the CD200 receptor." J Immunol 176(1): 191-199. Jeon, S. H., B. Kayhan, T. Ben-Yedidia and R. Arnon (2004). "A DNA aptamer prevents influenza infection by blocking the receptor binding region of the viral hemagglutinin." J Biol Chem 279(46): 48410-48419. Jiang, Z. and L. Zhu (2016). "Update on the role of alternatively activated macrophages in asthma." J Asthma Allergy 9: 101-107. Johnson, J. R., R. E. Wiley, R. Fattouh, F. K. Swirski, B. U. Gajewska, A. J. Coyle, J. C. Gutierrez-Ramos, R. Ellis, M. D. Inman and M. Jordana (2004). "Continuous exposure to house dust mite elicits chronic airway inflammation and structural remodeling." Am J Respir Crit Care Med 169(3): 378-385. Johnson, P. R. and J. K. Burgess (2004). "Airway smooth muscle and fibroblasts in the pathogenesis of asthma." Curr Allergy Asthma Rep 4(2): 102-108. Joubert, P., S. Lajoie-Kadoch, I. Labonté, A. S. Gounni, K. Maghni, V. Wellemans, J. Chakir, M. Laviolette, Q. Hamid and B. Lamkhioued (2005). "CCR3 expression and function in asthmatic airway smooth muscle cells." J Immunol 175(4): 2702-2708. Kaiko, G. E., J. C. Horvat, K. W. Beagley and P. M. Hansbro (2008). "Immunological decision-making: how does the immune system decide to mount a helper T-cell response?" Immunology 123(3): 326-338. Kay, A. B., S. Phipps and D. S. Robinson (2004). "A role for eosinophils in airway remodelling in asthma." Trends Immunol 25(9): 477-482. Keefe, A. D., S. Pai and A. Ellington (2010). "Aptamers as therapeutics." Nat Rev Drug Discov 9(7): 537-550.
111
Kim, H. Y., R. H. DeKruyff and D. T. Umetsu (2010). "The many paths to asthma: phenotype shaped by innate and adaptive immunity." Nat Immunol 11(7): 577-584. Kim, J., A. C. Merry, J. A. Nemzek, G. L. Bolgos, J. Siddiqui and D. G. Remick (2001). "Eotaxin represents the principal eosinophil chemoattractant in a novel murine asthma model induced by house dust containing cockroach allergens." J Immunol 167(5): 2808-2815. Klatka, J., E. Grywalska, M. Klatka, M. Rahnama, A. Polak and J. Rolinski (2013). "Expression of CD200 and CD200R regulatory molecules on the CD83+ monocyte-derived dendritic cells generated from patients with laryngeal cancer." Folia Histochem Cytobiol 51(1): 59-65. Klatka, J., E. Grywalska, M. Klatka, M. Wasiak, A. Andrzejczak and J. Rolinski (2013). "Expression of selected regulatory molecules on the CD83+ monocyte-derived dendritic cells generated from patients with laryngeal cancer and their clinical significance." Eur Arch Otorhinolaryngol 270(10): 2683-2693. Knop, K., R. Hoogenboom, D. Fischer and U. S. Schubert (2010). "Poly(ethylene glycol) in drug delivery: pros and cons as well as potential alternatives." Angew Chem Int Ed Engl 49(36): 6288-6308. Kolate, A., D. Baradia, S. Patil, I. Vhora, G. Kore and A. Misra (2014). "PEG - a versatile conjugating ligand for drugs and drug delivery systems." J Control Release 192: 67-81. Koning, N., M. van Eijk, W. Pouwels, M. S. Brouwer, D. Voehringer, I. Huitinga, R. M. Hoek, G. Raes and J. Hamann (2010). "Expression of the inhibitory CD200 receptor is associated with alternative macrophage activation." J Innate Immun 2(2): 195-200. Kretz-Rommel, A., F. Qin, N. Dakappagari, E. P. Ravey, J. McWhirter, D. Oltean, S. Frederickson, T. Maruyama, M. A. Wild, M. J. Nolan, D. Wu, J. Springhorn and K. S. Bowdish (2007). "CD200 expression on tumor cells suppresses antitumor immunity: new approaches to cancer immunotherapy." J Immunol 178(9): 5595-5605. Lakhin, A. V., V. Z. Tarantul and L. V. Gening (2013). "Aptamers: problems, solutions and prospects." Acta Naturae 5(4): 34-43. Lauzon-Joset, J.-F., D. Marsolais, É. Tardif-Pellerin, D. Patoine and E. Bissonnette (2019). "CD200 in Asthma." The International Journal of Biochemistry & Cell Biology. Lauzon-Joset, J. F., A. Langlois, L. J. Lai, K. Santerre, A. Lee-Gosselin, Y. Bossé, D. Marsolais and E. Y. Bissonnette (2015). "Lung CD200 Receptor Activation Abrogates Airway Hyperresponsiveness in Experimental Asthma." Am J Respir Cell Mol Biol 53(2): 276-284. Leme, A. S., A. Berndt, L. K. Williams, S. W. Tsaih, J. P. Szatkiewicz, R. Verdugo, B. Paigen and S. D. Shapiro (2010). "A survey of airway responsiveness in 36 inbred mouse strains facilitates gene mapping studies and identification of quantitative trait loci." Mol Genet Genomics 283(4): 317-326. Levitt, R. C. and W. Mitzner (1988). "Expression of airway hyperreactivity to acetylcholine as a simple autosomal recessive trait in mice." FASEB J 2(10): 2605-2608. Leόn, B. (2017). "T Cells in Allergic Asthma: Key Players Beyond the Th2 Pathway." Curr Allergy Asthma Rep 17(7): 43. Li, X. and J. W. Wilson (1997). "Increased vascularity of the bronchial mucosa in mild asthma." Am J Respir Crit Care Med 156(1): 229-233. Lloyd, C. M. (2007). "Building better mouse models of asthma." Curr Allergy Asthma Rep 7(3): 231-236. Louis, R., L. C. Lau, A. O. Bron, A. C. Roldaan, M. Radermecker and R. Djukanović (2000). "The relationship between airways inflammation and asthma severity." Am J Respir Crit Care Med 161(1): 9-16. Lötvall, J., C. A. Akdis, L. B. Bacharier, L. Bjermer, T. B. Casale, A. Custovic, R. F. Lemanske, A. J. Wardlaw, S. E. Wenzel and P. A. Greenberger (2011). "Asthma endotypes: a new approach to classification of disease entities within the asthma syndrome." J Allergy Clin Immunol 127(2): 355-360.
112
Majumder, P., K. N. Gomes and H. Ulrich (2009). "Aptamers: from bench side research towards patented molecules with therapeutic applications." Expert Opin Ther Pat 19(11): 1603-1613. Martín-Orozco, E., M. Norte-Muñoz and J. Martínez-García (2017). "Regulatory T Cells in Allergy and Asthma." Front Pediatr 5: 117. Masoli, M., D. Fabian, S. Holt, R. Beasley and G. I. f. A. G. Program (2004). "The global burden of asthma: executive summary of the GINA Dissemination Committee report." Allergy 59(5): 469-478. McBrien, C. N. and A. Menzies-Gow (2017). "The Biology of Eosinophils and Their Role in Asthma." Front Med (Lausanne) 4: 93. Minas, K. and J. Liversidge (2006). "Is the CD200/CD200 receptor interaction more than just a myeloid cell inhibitory signal?" Crit Rev Immunol 26(3): 213-230. Moreira, A. P. and C. M. Hogaboam (2011). "Macrophages in allergic asthma: fine-tuning their pro- and anti-inflammatory actions for disease resolution." J Interferon Cytokine Res 31(6): 485-491. Murdoch, J. R. and C. M. Lloyd (2010). "Chronic inflammation and asthma." Mutat Res 690(1-2): 24-39. Ng, E. W., D. T. Shima, P. Calias, E. T. Cunningham, D. R. Guyer and A. P. Adamis (2006). "Pegaptanib, a targeted anti-VEGF aptamer for ocular vascular disease." Nat Rev Drug Discov 5(2): 123-132. Nials, A. T. and S. Uddin (2008). "Mouse models of allergic asthma: acute and chronic allergen challenge." Dis Model Mech 1(4-5): 213-220. Norimoto, A., K. Hirose, A. Iwata, T. Tamachi, M. Yokota, K. Takahashi, S. Saijo, Y. Iwakura and H. Nakajima (2014). "Dectin-2 promotes house dust mite-induced T helper type 2 and type 17 cell differentiation and allergic airway inflammation in mice." Am J Respir Cell Mol Biol 51(2): 201-209. Nunes, C., A. M. Pereira and M. Morais-Almeida (2017). "Asthma costs and social impact." Asthma Res Pract 3: 1. O'Byrne, P. M. and M. D. Inman (2003). "Airway hyperresponsiveness." Chest 123(3 Suppl): 411S-416S. Panettieri, R. A. (2018). "The Role of Neutrophils in Asthma." Immunol Allergy Clin North Am 38(4): 629-638. Payne, D. N., I. M. Adcock, N. M. Wilson, T. Oates, M. Scallan and A. Bush (2001). "Relationship between exhaled nitric oxide and mucosal eosinophilic inflammation in children with difficult asthma, after treatment with oral prednisolone." Am J Respir Crit Care Med 164(8 Pt 1): 1376-1381. Pelaia, G., A. Vatrella, M. T. Busceti, L. Gallelli, C. Calabrese, R. Terracciano and R. Maselli (2015). "Cellular mechanisms underlying eosinophilic and neutrophilic airway inflammation in asthma." Mediators Inflamm 2015: 879783. Pietilä, M., S. Lehtonen, E. Tuovinen, K. Lähteenmäki, S. Laitinen, H. V. Leskelä, A. Nätynki, J. Pesälä, K. Nordström and P. Lehenkari (2012). "CD200 positive human mesenchymal stem cells suppress TNF-alpha secretion from CD200 receptor positive macrophage-like cells." PLoS One 7(2): e31671. Pilch, Z., K. Tonecka, M. Skorzynski, Z. Sas, A. Braniewska, T. Kryczka, L. Boon, J. Golab, L. Meyaard and T. P. Rygiel (2019). "The pro-tumor effect of CD200 expression is not mimicked by agonistic CD200R antibodies." PLoS One 14(1): e0210796. Piyadasa, H., A. Altieri, S. Basu, J. Schwartz, A. J. Halayko and N. Mookherjee (2016). "Biosignature for airway inflammation in a house dust mite-challenged murine model of allergic asthma." Biol Open 5(2): 112-121. Platts-Mills, T. A. (2001). "The role of immunoglobulin E in allergy and asthma." Am J Respir Crit Care Med 164(8 Pt 2): S1-5.
113
Pohunek, P., J. O. Warner, J. Turzíková, J. Kudrmann and W. R. Roche (2005). "Markers of eosinophilic inflammation and tissue re-modelling in children before clinically diagnosed bronchial asthma." Pediatr Allergy Immunol 16(1): 43-51. Portelli, M. A., E. Hodge and I. Sayers (2015). "Genetic risk factors for the development of allergic disease identified by genome-wide association." Clin Exp Allergy 45(1): 21-31. Prodeus, A., A. Abdul-Wahid, A. Sparkes, N. W. Fischer, M. Cydzik, N. Chiang, M. Alwash, A. Ferzoco, N. Vacaresse, M. Julius, R. M. Gorczysnki and J. Gariépy (2017). "VISTA.COMP - an engineered checkpoint receptor agonist that potently suppresses T cell-mediated immune responses." JCI Insight 2(18). Prodeus, A., M. Cydzik, A. Abdul-Wahid, E. Huang, I. Khatri, R. Gorczynski and J. Gariépy (2014). "Agonistic CD200R1 DNA Aptamers Are Potent Immunosuppressants That Prolong Allogeneic Skin Graft Survival." Mol Ther Nucleic Acids 3: e190. Prodeus, A., A. Sparkes, N. W. Fischer, M. Cydzik, E. Huang, I. Khatri, L. Woo, C.-W. Chow, R. Gorczynski and J. Gariépy (2018). "A synthetic cross-species CD200R1 agonist suppresses inflammatory immune responses in vivo." Molecular Therapy- Nucleic Acids 12: 350-358. Radermecker, C., R. Louis, F. Bureau and T. Marichal (2018). "Role of neutrophils in allergic asthma." Curr Opin Immunol 54: 28-34. Raghavan, D. and R. Jain (2016). "Increasing awareness of sex differences in airway diseases." Respirology 21(3): 449-459. Richter, A. W. and E. Akerblom (1983). "Antibodies against polyethylene glycol produced in animals by immunization with monomethoxy polyethylene glycol modified proteins." Int Arch Allergy Appl Immunol 70(2): 124-131. Roche, W. R. (1998). "Inflammatory and structural changes in the small airways in bronchial asthma." Am J Respir Crit Care Med 157(5 Pt 2): S191-194. Roeder, T., K. Isermann and M. Kabesch (2009). "Drosophila in asthma research." Am J Respir Crit Care Med 179(11): 979-983. Rygiel, T. P., G. Karnam, G. Goverse, A. P. van der Marel, M. J. Greuter, R. A. van Schaarenburg, W. F. Visser, A. B. Brenkman, R. Molenaar, R. M. Hoek, R. E. Mebius and L. Meyaard (2012). "CD200-CD200R signaling suppresses anti-tumor responses independently of CD200 expression on the tumor." Oncogene 31(24): 2979-2988. Rygiel, T. P., E. S. Rijkers, T. de Ruiter, E. H. Stolte, M. van der Valk, G. F. Rimmelzwaan, L. Boon, A. M. van Loon, F. E. Coenjaerts, R. M. Hoek, K. Tesselaar and L. Meyaard (2009). "Lack of CD200 enhances pathological T cell responses during influenza infection." J Immunol 183(3): 1990-1996. Ryu, J. H., J. Y. Yoo, M. J. Kim, S. G. Hwang, K. C. Ahn, J. C. Ryu, M. K. Choi, J. H. Joo, C. H. Kim, S. N. Lee, W. J. Lee, J. Kim, D. M. Shin, M. N. Kweon, Y. S. Bae and J. H. Yoon (2013). "Distinct TLR-mediated pathways regulate house dust mite-induced allergic disease in the upper and lower airways." J Allergy Clin Immunol 131(2): 549-561. Salehi, S., X. Wang, S. Juvet, J. A. Scott and C. W. Chow (2017). "Syk Regulates Neutrophilic Airway Hyper-Responsiveness in a Chronic Mouse Model of Allergic Airways Inflammation." PLoS One 12(1): e0163614. Sanbongi, C., H. Takano, N. Osakabe, N. Sasa, M. Natsume, R. Yanagisawa, K. I. Inoue, K. Sadakane, T. Ichinose and T. Yoshikawa (2004). "Rosmarinic acid in perilla extract inhibits allergic inflammation induced by mite allergen, in a mouse model." Clin Exp Allergy 34(6): 971-977. Saradna, A., D. C. Do, S. Kumar, Q. L. Fu and P. Gao (2018). "Macrophage polarization and allergic asthma." Transl Res 191: 1-14. Sarpong, S. B., L. Y. Zhang and S. R. Kleeberger (2003). "A novel mouse model of experimental asthma." Int Arch Allergy Immunol 132(4): 346-354. Schatz, M., S. Clark and C. A. Camargo (2006). "Sex differences in the presentation and course of asthma hospitalizations." Chest 129(1): 50-55.
114
Schellekens, H., W. E. Hennink and V. Brinks (2013). "The immunogenicity of polyethylene glycol: facts and fiction." Pharm Res 30(7): 1729-1734. Schulz, H., C. Johner, G. Eder, A. Ziesenis, P. Reitmeier, J. Heyder and R. Balling (2002). "Respiratory mechanics in mice: strain and sex specific differences." Acta Physiol Scand 174(4): 367-375. Shifren, A., C. Witt, C. Christie and M. Castro (2012). "Mechanisms of remodeling in asthmatic airways." J Allergy (Cairo) 2012: 316049. Simelyte, E., G. Criado, D. Essex, R. A. Uger, M. Feldmann and R. O. Williams (2008). "CD200-Fc, a novel antiarthritic biologic agent that targets proinflammatory cytokine expression in the joints of mice with collagen-induced arthritis." Arthritis Rheum 58(4): 1038-1043. Simeone-Penney, M. C., M. Severgnini, P. Tu, R. J. Homer, T. J. Mariani, L. Cohn and A. R. Simon (2007). "Airway epithelial STAT3 is required for allergic inflammation in a murine model of asthma." J Immunol 178(10): 6191-6199. Snelgrove, R. J., J. Goulding, A. M. Didierlaurent, D. Lyonga, S. Vekaria, L. Edwards, E. Gwyer, J. D. Sedgwick, A. N. Barclay and T. Hussell (2008). "A critical function for CD200 in lung immune homeostasis and the severity of influenza infection." Nat Immunol 9(9): 1074-1083. Southam, D. S., M. Dolovich, P. M. O'Byrne and M. D. Inman (2002). "Distribution of intranasal instillations in mice: effects of volume, time, body position, and anesthesia." Am J Physiol Lung Cell Mol Physiol 282(4): L833-839. Sugita, M., K. Kuribayashi, T. Nakagomi, S. Miyata, T. Matsuyama and O. Kitada (2003). "Allergic bronchial asthma: airway inflammation and hyperresponsiveness." Intern Med 42(8): 636-643. Sulkowski, M. S., C. Cooper, B. Hunyady, J. Jia, P. Ogurtsov, M. Peck-Radosavljevic, M. L. Shiffman, C. Yurdaydin and O. Dalgard (2011). "Management of adverse effects of Peg-IFN and ribavirin therapy for hepatitis C." Nat Rev Gastroenterol Hepatol 8(4): 212-223. Sullivan, P. W., J. F. Slejko, V. H. Ghushchyan, B. Sucher, D. R. Globe, S. L. Lin and G. Globe (2014). "The relationship between asthma, asthma control and economic outcomes in the United States." J Asthma 51(7): 769-778. Svenningsen, S. and P. Nair (2017). "Asthma Endotypes and an Overview of Targeted Therapy for Asthma." Front Med (Lausanne) 4: 158. Swirski, F. K., D. Sajic, C. S. Robbins, B. U. Gajewska, M. Jordana and M. R. Stämpfli (2002). "Chronic exposure to innocuous antigen in sensitized mice leads to suppressed airway eosinophilia that is reversed by granulocyte macrophage colony-stimulating factor." J Immunol 169(7): 3499-3506. Tanaka, H., G. Yamada, T. Saikai, M. Hashimoto, S. Tanaka, K. Suzuki, M. Fujii, H. Takahashi and S. Abe (2003). "Increased airway vascularity in newly diagnosed asthma using a high-magnification bronchovideoscope." Am J Respir Crit Care Med 168(12): 1495-1499. To, T., S. Stanojevic, G. Moores, A. S. Gershon, E. D. Bateman, A. A. Cruz and L. P. Boulet (2012). "Global asthma prevalence in adults: findings from the cross-sectional world health survey." BMC Public Health 12: 204. Tuerk, C. and L. Gold (1990). "Systematic evolution of ligands by exponential enrichment: RNA ligands to bacteriophage T4 DNA polymerase." Science 249(4968): 505-510. Tural Onur, S., A. D. Yalcin, B. Celik and S. Gumuslu (2015). "Evaluation of d-dimer, CXCL8, homocysteine, eosinophil cationic peptide, 25(OH)-vitamin D and immunomodulatory OX-2 levels in allergic patients." J Asthma 52(2): 123-127. Ulrich, K., J. S. Hincks, R. Walsh, E. M. Wetterstrand, M. D. Fidock, S. Sreckovic, D. J. Lamb, G. J. Douglas, M. Yeadon, C. Perros-Huguet and S. M. Evans (2008). "Anti-inflammatory modulation of chronic airway inflammation in the murine house dust mite model." Pulm Pharmacol Ther 21(4): 637-647. Vaine, C. A. and R. J. Soberman (2014). "The CD200-CD200R1 inhibitory signaling pathway: immune regulation and host-pathogen interactions." Adv Immunol 121: 191-211.
115
Veres, T. Z., T. Zoltán Veres, S. Voedisch, E. Spies, T. Tschernig and A. Braun (2011). "Spatiotemporal and functional behavior of airway dendritic cells visualized by two-photon microscopy." Am J Pathol 179(2): 603-609. Vroman, H., I. M. Bergen, B. W. Li, J. A. van Hulst, M. Lukkes, D. van Uden, R. W. Hendriks and M. Kool (2016). "Development of eosinophilic inflammation is independent of B-T cell interaction in a chronic house dust mite-driven asthma model." Clin Exp Allergy. Walker, D. G. and L. F. Lue (2013). "Understanding the neurobiology of CD200 and the CD200 receptor: a therapeutic target for controlling inflammation in human brains?" Future Neurol 8(3). Wenzel, S. E. (2006). "Asthma: defining of the persistent adult phenotypes." Lancet 368(9537): 804-813. Wenzel, S. E. (2012). "Asthma phenotypes: the evolution from clinical to molecular approaches." Nat Med 18(5): 716-725. Wenzel, S. E., L. B. Schwartz, E. L. Langmack, J. L. Halliday, J. B. Trudeau, R. L. Gibbs and H. W. Chu (1999). "Evidence that severe asthma can be divided pathologically into two inflammatory subtypes with distinct physiologic and clinical characteristics." Am J Respir Crit Care Med 160(3): 1001-1008. WHO. (2018). "Chronic Respiratory Diseases, Asthma." from http://www.who.int/respiratory/en/. Woo, L. N., W. Y. Guo, X. Wang, A. Young, S. Salehi, A. Hin, Y. Zhang, J. A. Scott and C. W. Chow (2018). "A 4-Week Model of House Dust Mite (HDM) Induced Allergic Airways Inflammation with Airway Remodeling." Sci Rep 8(1): 6925. Woytschak, J., N. Keller, C. Krieg, D. Impellizzieri, R. W. Thompson, T. A. Wynn, A. S. Zinkernagel and O. Boyman (2016). "Type 2 Interleukin-4 Receptor Signaling in Neutrophils Antagonizes Their Expansion and Migration during Infection and Inflammation." Immunity 45(1): 172-184. Wright, G. J., H. Cherwinski, M. Foster-Cuevas, G. Brooke, M. J. Puklavec, M. Bigler, Y. Song, M. Jenmalm, D. Gorman, T. McClanahan, M. R. Liu, M. H. Brown, J. D. Sedgwick, J. H. Phillips and A. N. Barclay (2003). "Characterization of the CD200 receptor family in mice and humans and their interactions with CD200." J Immunol 171(6): 3034-3046. Wright, G. J., M. Jones, M. J. Puklavec, M. H. Brown and A. N. Barclay (2001). "The unusual distribution of the neuronal/lymphoid cell surface CD200 (OX2) glycoprotein is conserved in humans." Immunology 102(2): 173-179. Zhang, F., M. R. Liu and H. T. Wan (2014). "Discussion about several potential drawbacks of PEGylated therapeutic proteins." Biol Pharm Bull 37(3): 335-339. Zhang, S., H. Cherwinski, J. D. Sedgwick and J. H. Phillips (2004). "Molecular mechanisms of CD200 inhibition of mast cell activation." J Immunol 173(11): 6786-6793. Zhang, S., X. J. Wang, L. P. Tian, J. Pan, G. Q. Lu, Y. J. Zhang, J. Q. Ding and S. D. Chen (2011). "CD200-CD200R dysfunction exacerbates microglial activation and dopaminergic neurodegeneration in a rat model of Parkinson's disease." J Neuroinflammation 8: 154. Zhou, J. and J. Rossi (2017). "Aptamers as targeted therapeutics: current potential and challenges." Nat Rev Drug Discov 16(3): 181-202.
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Chapter 9: Appendix
Supplementary Figure 1: BAL Macrophages Elevated in all HDM Mice Treated with a
Compound- HDM mice treated with non-saline (vehicle) compounds had significantly elevated
BAL macrophages. N=6-7/group p<0.05.
9.1 Sex Differences in the Lung Mechanics of BALB/c Mice
9.1.1 Background
Lung diseases, including asthma constitute a significant healthcare burden affecting
millions of people worldwide(GINA 2015). Thus, understanding the mechanics of lung function
are important to minimizing the burden of lung disease. Reference values compiled using
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thousands of lung function tests have shown that there are many factors that influence lung
function, including sex(Raghavan and Jain 2016).
Murine models are a useful research tool which can be used to study the pathogenesis of
respiratory diseases as well as potential therapeutics. However, many of these models only use
one sex to explore disease pathology(Schulz, Johner et al. 2002). Most studies of respiratory
diseases utilize only female mice as they can be housed together which cuts down on the costs of
the experiment even sex differences in lung mechanics are known to exist in humans. This may
reduce the significance of claims and inter-study comparability. Here we explore the sex-based
differences in respiratory mechanics using the Flexivent® system. We hypothesize that
significant differences will exist in the respiratory mechanics of male and female BALB/c mice.
9.1.2 Methods
Animals- 10 male and 10 female BALB/c mice from Jackson were housed in a pathogen-free
environment with a 12:12 hour light dark cycle and given food and water ad litem. All animal
protocols were approved by the CCAC and were monitored daily. No animals became ill or died
prior to the end of the experiment.
Lung Assessment- Mice were anesthetized and cannulated as described in section 3.1.
Respiratory mechanics and methacholine responsiveness were assessed with the Flexivent®
system. The script assessment included: deep inflation, OSC, NPFE, PV loop perturbations and
MCh challenge.
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Table 1: Respiratory Mechanics
Respiratory Mechanic Measured Definition
Single Compartment Model
Rrs Total Respiratory Resistance
Ers Elastance
Constant Phase Model
Rn Central Newtonian Resistance- resistance in the large
conducting airways
H Tissue Elastance- energy stored within the tissues
following deformation
G Tissue damping- energy lost to heat as a result of
tissues, related to resistance of the peripheral airways
Eta Hysteresivity- ratio fo G/H
Pressure Volume Loop
CST Static Compliance- intrinsic elastic properties of the
respiratory system
K Curvature of PV curve
Hysteresis Area between the ascending and descending portions
of the PV loop
NPFE (Negative Pressure- Driven Forced Expiration)
FEV(0.1, 0.05, 0.2) Forced Expiratory Volume in 0.1 second, 0.05 second,
0.2 second
FEV PEF Forced Expiratory Volume Peak Forced Expired Flow
FVC Forced Vital Capacity
FEF Forced Expiratory Flow
PEF Peak Forced Expiratory Flow
TPEF Total Peak Forced Expiratory Flow
IC Inspiratory Capacity
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9.1.3 Results
Our results showed that at baseline (prior to MCh challenge) there was no difference between
male and female mice in Rrs, Rn, G, H, or K (Supp Figure 2 A,C,D,E,H), however males had
higher baseline eta, Cst and hysteresis (Supp Figure F, G, I) and females had higher Ers (Supp
Figure 2B).
Supplementary Figure 2: Differences in Single Compartment and Constant-Phase Model
Parameters at Baseline- Most baseline respiratory mechanics had no differences between male
and female mice (A,C, D,E,I). Male mice demonstrated higher eta, Cst and hysteresis (FGH) and
female mice had greater Ers. N=8-10/group *p<0.05.
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Most NPFE parameters which had significant differences between the male and female
mice had higher values in the male mice with only FEV0.05/FVC having higher values in the
female mice. Overall male had higher inspiratory capacity, FVC and higher FEV/0.1 values
(Supplementary Figure 3).
Supplementary Figure 3: Differences in NPFE Parameters Between Male and Female Mice- At
baseline male mice demonstrated higher IC, FVC, FEV0.1, FEV0.2, FEV0.1/FVC, FEF0.1,
FEF0.05 and FEF0.2 (A,B,E,G,I,M,N,O) while female mice had higher FEV0.5/FVC (J). N=8-
10/group *p<0.05.
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Following MCh dose response all single compartment (Supp Figure 4A,B) and constant-
phase parameters (Supp Figure 5C-F) were elevated in male mice higher than in females. PV
loop parameters were not different between the sexes following MCh challenge.
Supplementary Figure 4: Dose Response Differences Between Male and Female Mice- All
single compartment (A,B) and constant phase (C-F) model parameters were higher in male mice.
No change was found between male and female mice in the PV parameters (G-I). N=8-10/group
*p<0.05.
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9.1.4 Discussion and Conclusions
Overall, we found differences with both baseline parameters and parameters measured
after MCh challenge between the male and female mice. Parameters that accounted for animal
size such as IC and FVC were unsurprisingly larger in male mice at baseline. We found that
parameters that different were typically higher in male mice, both at baseline and with dose
response. This suggests that male mice have higher respiratory resistance at baseline and greater
sensitivity to MCh challenge. From these findings we conclude that it is insufficient to assess the
lung function of only one sex of mice. Further studies should be done to quantify any differences
in other parameters including inflammation.
9.2 The VISTA Activation in Acute Model of Allergic Airways Inflammation
9.2.1 Introduction
The V-domain Immunoglobulin Suppressor of T-cell Activation (VISTA) is an immune
checkpoint ligand which has been shown to suppress T-cell activity similar to the PD-1 immune
checkpoint regulators where have been implicated in asthma(Prodeus, Abdul-Wahid et al. 2017).
The VISTA domain itself has been implicated in several disease states including autoimmune
diseases and cancer(Prodeus, Abdul-Wahid et al. 2017). Here we aim to evaluate the
effectiveness of VISTA compounds in an acute model of allergic airways inflammation.
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9.2.2 Methods
10 Female BALB/c mice obtained from Jackson were housed as previously described.
Mice were intranasally exposed to 2 weeks of 5 days/week HDM exposure followed by one
week of treatment 3 days a week while continuing exposure 2 days a week. Mice were treated
intranasally with either VISTA-FC, VISTA.COMP a construct or PBS control.
9.2.3 Results
Our results found that treatment with VISTA-FC or VISTA.COMP resulted in increased
AHR in HDM treated mice over and above what was seen from the saline controls.
Supplementary Figure 5: VISTA Compounds Result in Elevated AHR in HDM Mice- HDM mice
treated with either VISTA-FC or VISTA.COMP had elevated Rrs above what was seen with HDM
mice treated with PBS control. Saline mice treated with VISTA.COMP also demonstrated elevated
resistance relative to saline mice. N=3-4/group *p<0.05.
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BAL cell counts found HDM mice treated with PBS had elevated total leukocyte counts
including neutrophils, lymphocytes, macrophages and eosinophils. HDM mice treated with
VISTA compounds had reduced total leukocyte counts and neutrophils. Additionally, eosinophil
counts of mice treated with VISTA compounds were not elevated relative to saline mice.
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Supplementary Figure 6: VISTA Compounds Result in Decreased BAL Cellular Recruitment-
HDM mice treated with VISTA compounds had reduced BAL total cell counts and neutrophils.
All cell counts were elevated relative to controls in HDM mice except eosinophils which were
only elevated in HDM mice treated with PBS. N=3-4/group *p<0.05.
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9.2.4 Discussion and Conclusions
Overall these findings with the VISTA compound are preliminary and further work is
needed to make conclusions regarding their effects. Although our results were promising with
respect to dampening cell recruitment, the increase respiratory resistance in mice treated with the
compounds suggests further work needs to be done to evaluate their safety in intranasal
exposure.
Publication Permissions
Work characterizing the 4-week chronic HDM model and PEG.CCS13 treatment in the acute
model have both been published in (Woo & Guo et al., 2018) Scientific Reports and (Prodeus &
Sparkes et al., 2018) Molecular Therapy-Nucleic Acids respectively. We received permission
from both journals for this work to be included in this thesis.
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