Table Of ContentLung Complications in Adenosine Deaminase (ADA)
Deficiency: A Mouse Model for the Human Disease
by
Rupreet Dhanju
A thesis submitted in conformity with the requirements
for the degree of Master of Science
Institute of Medical Science
University of Toronto
© Copyright by Rupreet Dhanju 2012
Lung Complications in Adenosine Deaminase (ADA)
Deficiency: A Mouse Model for the Human Disease
Rupreet Dhanju
Master of Science
Institute of Medical Science
University of Toronto
2012
Abstract
Recently, we discovered patients with inherited adenosine deaminase (ADA) deficiency are
predisposed to pulmonary alveolar proteinosis (PAP). PAP is characterized by the
accumulation of surfactant in the alveoli. To overcome ethical issues and limited patient
samples, animal models are often utilized. Here, I investigated the lung abnormalities in
ADA deficient (ADA -/-) mice, which suffer from severe hypoxia, till their death at 3 weeks.
I hypothesized that, similar to ADA-deficient patients, ADA -/- mice demonstrate evidence
of PAP. Indeed, electron microscopy showed thickening of type I cells, accumulation of
apoptotic foamy alveolar macrophages, cholesterol and lipoproteinaceous material that is
periodic-acid Schiff (PAS) positive and diagnostic of PAP. Moreover, the pulmonary
abnormalities were corrected with supplementation of ADA. In conclusion, we demonstrated
evidence of PAP in ADA -/- mice for the first time and their suitability to study pathogenesis
of PAP in ADA deficiency.
ii
Acknowledgments and Contributions
First and foremost, I would like to thank Dr. Eyal Grunebaum for his guidance, patience
and support over the two years. I have learned a great deal from Dr. Grunebaum and I am
truly grateful for the experience. I would like to extend my gratitude to Dr. Nades Palaniyar
for his continuous encouragement. I sincerely appreciate Dr. Palaniyar for welcoming me to
his lab and for his efforts to make my goals become a reality. I couldn’t have made it this far
without the mentorship of both Dr. Grunebaum and Dr. Palaniyar.
My program advisory committee members, Dr. Yigal Dror, Dr. Nicola Jones and Dr.
Chetankumar Tailor have always been supporting and encouraging. I would like to thank
them for always making helpful suggestions and their continued kindness.
A special thanks goes to Dr. Cameron Ackerley for conducting the electron microscopy
for this study. Weixian Min for animal husbandry and for his eagerness to always lend a
helping hand. Huimin Wang for her help with sectioning and staining some tissues. Pascal
Djiadeu for helping me with the apoptosis studies and Ron Flannagan for showing me the
phagocytosis technique. I would also like to thank Casey and Francis at the PMH flow
facility for introducing me to flow cytometry and Michael Woodside from the SickKids
imaging facility for answering my microscope related questions.
Thank you to all my friends in Dr. Dror’s and Dr. Palaniyar’s lab for always making
everyday an adventure. Moreover, this could not have been possible without the support of
my loving family and amazing friends. Lastly, I would like to acknowledge CIHR for
providing me with a Frederick Banting and Charles Best Master’s Award.
iii
Table of Contents
Acknowledgments
and
Contributions
...................................................................................
iii
Table
of
Contents
..........................................................................................................................
iv
List
of
Figures
.................................................................................................................................
vi
List
of
Tables
.................................................................................................................................
xii
List
of
Appendices
......................................................................................................................
xiii
Chapter 1: Introduction
.................................................................................................................
1
1
Introduction
.............................................................................................................................
2
1.1
Adenosine
Deaminase
Deficiency
............................................................................................
2
1.1.1
History
..........................................................................................................................................................
3
1.1.2
Metabolism
.................................................................................................................................................
3
1.1.3
ADA
mutations
..........................................................................................................................................
8
1.1.4
Clinical
Aspects
.........................................................................................................................................
9
1.2
Importance
of
ADA
Deficiency
and
Broader
Relevance
................................................
10
1.3
Management
of
ADA
Deficiency
.............................................................................................
11
1.3.1
Bone
Marrow
Transplant
..................................................................................................................
11
1.3.2
Enzyme
Replacement
Therapy
with
PEG-‐ADA
(Adagen®)
.................................................
11
1.3.3
Gene
Therapy
..........................................................................................................................................
12
1.4
Pulmonary
Abnormalities
in
patients
with
ADA
deficiency
........................................
13
1.5
Pulmonary
Alveolar
Proteinosis
...........................................................................................
17
1.6
Surfactant
......................................................................................................................................
21
1.6.1
Surfactant
Homeostasis
.....................................................................................................................
22
1.7
Lung
Structure
and
Development
.........................................................................................
23
1.8
Mouse
Models
..............................................................................................................................
26
1.8.1
Development
of
ADA
-‐/-‐
mice
..........................................................................................................
26
1.8.2
Severe
Pulmonary
Abnormalities
in
ADA
-‐/-‐
mice
.................................................................
27
1.8.3
PAP
Models
..............................................................................................................................................
28
1.9
Collectins:
Surfactant
Proteins
(SP)-‐D
and
SP-‐A
..............................................................
29
iv
1.9.1
SP-‐D
and
SP–
A
structure
...................................................................................................................
29
1.9.2
The
role
of
SP-‐D
in
surfactant
homeostasis
and
inflammation
.........................................
31
Chapter 2: Hypothesis and Aims
...............................................................................................
33
2
Hypothesis
and
Aims
...........................................................................................................
34
2.1.1
Rationale
...................................................................................................................................................
34
2.1.2
Hypothesis
...............................................................................................................................................
34
2.1.3
Aims
............................................................................................................................................................
35
Chapter 3: Methods
......................................................................................................................
36
3
Methods
....................................................................................................................................
37
3.1
Animals
..........................................................................................................................................
37
3.2
Anesthesia
.....................................................................................................................................
37
3.3
Enzyme
Activity
...........................................................................................................................
37
3.4
Arterial
blood
oxygen
saturation
..........................................................................................
38
3.5
Electron
Microscopy
..................................................................................................................
38
3.6
Lung
Lavage
..................................................................................................................................
38
3.7
Protein
Quantification
..............................................................................................................
39
3.8
Western
Blot
................................................................................................................................
39
3.9
Immunofluorescence
................................................................................................................
40
3.10
Cholesterol
Assay
.....................................................................................................................
40
3.11
Apoptosis
Detection
................................................................................................................
41
3.12
Histology
and
Oil
Red
O
.........................................................................................................
41
3.13
PEG-‐ADA
treatment
.................................................................................................................
42
3.14
Kaplan-‐Meier
Survival
Curve
...............................................................................................
42
3.15
Phagocytosis
Assay
..................................................................................................................
42
3.16
Viable
cell
counting
.................................................................................................................
43
3.17
Statistical
analysis
...................................................................................................................
44
Chapter 4: Results
.........................................................................................................................
45
4
Results
......................................................................................................................................
46
4.1
Aim
1
-‐
Investigating
Lung
Structure
...................................................................................
48
4.1.1
Abnormal
lung
ultrastructure
in
the
ADA
-‐/-‐
mice
................................................................
48
v
4.1.2
Accumulation
of
PAS
positive
diastase
resistant
lipoproteinaceous
material
in
the
alveolar
spaces
of
ADA
-‐/-‐
mice
.....................................................................................................................
54
4.1.3
Decreased
SP-‐D
and
SP-‐A
in
the
ADA
-‐/-‐
BALF
........................................................................
57
4.1.4
SP-‐D
and
SP-‐A
in
the
lung
homogenate
.......................................................................................
59
4.1.5
Immunofluorescence
of
SP-‐D
and
SP-‐A
in
the
lung
tissue
sections
................................
59
4.1.6
Conclusion
................................................................................................................................................
62
4.2
Aim
2
-‐
Alveolar
macrophage
in
ADA
-‐/-‐
mice
..................................................................
63
4.2.1
Accumulation
of
foamy
AM
in
ADA
-‐/-‐
BALF
............................................................................
63
4.2.2
Accumulation
of
lipids
and
remnants
of
tubular
myelin
in
ADA
-‐/-‐
AM
.......................
66
4.2.3
Increased
cholesterol
in
ADA
-‐/-‐
lungs
.......................................................................................
68
4.2.4
Cell
death
in
ADA
-‐/-‐
AM
....................................................................................................................
70
4.2.5
ADA
-‐/-‐
AM
phagocytose
IgG-‐coated
beads
effectively
........................................................
72
4.2.6
Conclusion
................................................................................................................................................
75
4.3
Aim
3
-‐
Correcting
lung
abnormalities
................................................................................
76
4.3.1
PEG-‐ADA
can
extend
survival
of
ADA
-‐/-‐
mice
.........................................................................
76
4.3.2
Treatment
with
PEG-‐ADA
normalizes
abnormal
lung
architecture
...............................
79
4.3.3
SP-‐D
and
SP-‐A
in
the
BALF
of
PEG-‐ADA
treated
ADA
-‐/-‐
mice
..........................................
79
4.3.4
Cholesterol
accumulation
in
ADA
-‐/-‐
AM
can
be
normalized
............................................
83
4.3.5
Conclusion
................................................................................................................................................
83
Chapter 5: Discussion
...................................................................................................................
85
5
Discussion
...............................................................................................................................
86
Chapter 6: Conclusions and Future Directions
......................................................................
96
6
Conclusions
and
Future
Directions
................................................................................
97
6.1
Conclusions
...................................................................................................................................
97
6.2
Future
Directions
.......................................................................................................................
98
References
...................................................................................................................................
100
Appendices
.................................................................................................................................
109
vi
Abbreviations
A2B – adenosine 2 B
ABC – ATP-binding cassette
ADA – adenosine deaminase deficiency
AM – alveolar macrophages
ATP- adenosine triphosphate
BAL – bronchoalveolar lavage
BALF - bronchoalveolar lavage fluid
BCA – bicinchoninic acid
CRD – carbohydrate recognition domain
DAPI – 4’,6-diamidino-2-phenylindole
dATP - deoxyadenosine triphosphate
ELISA – enzyme-linked immunosorbent assay
EM – electron microscope
FITC – fluorescein isothiocyanate
GM-CSF – granulocyte macrophage colony stimulating factor
HLA- human leukocyte antigen
IgG – immunoglobulin G
LAMP – lysosomal-associated membrane protein
LP – lipoproteinaceous
vii
MMP- matrix metalloproteinase
NF- κβ – nuclear factor kappa beta
PAP – pulmonary alveolar proteinosis
PBS – phosphate buffered saline
PCR – polymerase chain reaction
PE – phycoerythrin
PPAR – peroxisome proliferator- activated receptor
SFM – serum free media
SP – surfactant protein
viii
List of Figures
Figure 1: Schematic representative of adenosine deaminase (ADA) deficiency metabolic
pathway………………………………………………………………………………………..6
Figure 2: Biochemical effects of adenosine and deoxyadenosine……………………………7
Figure 3: Biopsy demonstrating pulmonary alveolar proteinosis in adenosine deaminase
deficient patients…………………………………………………………………………..…16
Figure 4: Mechanisms of surfactant homeostasis and pulmonary alveolar proteinosis
(PAP)…………………………………………………………………………………………20
Figure 5: The structure of the alveoli………………………………………………………..30
Figure 6: Collectin structure………………………………………………………………...25
Figure 7: Reduced arterial blood saturation in ADA -/- mice…………………………........47
Figure 8: Electron microscopy shows accumulation of lipoproteinaceous material in the
ADA -/- alveolar spaces………………………………………………………………….......50
Figure 9: Electron microscopy indicates ADA -/- mice have thickened type I cells……......51
Figure 10: Alveolar type II cells in ADA -/- mice lungs show no apparent abnormalities
compared to ADA +/+ mice…………………………………………………………….........52
Figure 11: Accumulation of lipoproteinaceous material and macrophages in the alveoli are
characteristic of ADA -/- mice lungs…………………………………………………….......53
Figure 12: Light microscope show PAS positive and diastase resistant material indicative of
pulmonary alveolar proteinosis in ADA -/- mice……………………………………............55
Figure 13: Increased protein concentration in BALF retrieved from ADA -/- mice……….56
Figure 14: Western blot analyses show decreased SP-D and SP-A in the BALF of ADA -/-
mice lungs…………………………………………………………………………………...58
ix
Figure 15: Western blot show that ADA -/- mouse lung tissues have similar amounts of
SP-D and SP-A in lung homogenate as wild type littermate control………………………...60
Figure 16: SP-D and SP-A immunofluorescence in lung sections of ADA +/+ and ADA -/-
mice…………………………………………………………………………………………..61
Figure 17: Light microscope shows an accumulation of enlarged foamy alveolar
macrophages in ADA -/- mouse BALF……………………………………………….……..65
Figure 18: Electron microscopy shows accumulation of lipids phospholipids and tubular
myelin remnants in ADA -/- alveolar macrophages………………………………….……...67
Figure 19: Increased cholesterol in ADA -/- mice macrophages and BALF………..……...69
Figure 20: Alveolar macrophages from ADA -/- mice are more susceptible to
apoptosis……………………………………………………………………………..……...71
Figure 21: ADA -/- mice alveolar macrophages exhibit effective phagocytosis of IgG-coated
polysterene beads (~3.5um) compared to ADA +/+ mice alveolar macrophages…..……...73
Figure 22: Similar number of beads phagocytosed by ADA +/+ and ADA -/- mice alveolar
macrophages………………………………………………………………………...……...74
Figure 23: Kaplan-Meier survival curve demonstrates reduced survival in ADA -/- mice can
be prolonged with PEG-ADA treatment…………………………………………….……...77
Figure 24: Normal blood oxygen saturation in ADA -/- mice following PEG-ADA
treatment……………………………………………………………………………..……...78
Figure 25: PEG-ADA treatment on day 7 corrects lung structure and accumulation of
macrophages and protein in the alveolar spaces of ADA -/- mice…………………..……...81
Figure 26: PEG-ADA treatment restores SP-D and SP-A in BALF of ADA -/-
mice………………………………………………………………………………………...82
x
Description:Recently, we discovered patients with inherited adenosine deaminase (ADA)
deficiency are predisposed to pulmonary BAL – bronchoalveolar lavage.
