The Spatiotemporal Accumulation of Myeloid Lineage Cells Is Mapping Out Fibrosis in the Liver ...

114 Pages·2017·21.49 MB·English

The Spatiotemporal Accumulation of Myeloid Lineage
Cells Is Mapping Out Fibrosis in the Liver after Acute
and Chronic injury

by
Ali-Reza Sadri

A thesis submitted in conformity with the requirements
for the degree of Master of Science
Institute of Medical Science
University of Toronto
© Copyright by Ali-Reza Sadri 2017
The Spatiotemporal Accumulation of Myeloid Lineage Cells Is Mapping Out
Fibrosis in the Liver after Acute and Chronic injury

Ali-Reza Sadri

Master of Science

Institute of Medical Science
University of Toronto

2017

Abstract
Liver fibrosis is known to become problematic after persistent injury. However, little is
known about its response after an acute insult. Myeloid lineage cells, specifically, macrophages
have been implicated in the promotion and resolution of inflammation and fibrosis. We
investigated the role of myeloid lineage cells in the liver after acute injury. Our data show that
thermal injury in mice induces fibrosis predominantly around portal venules whereas myeloid
cells are enriched throughout the liver. The fibrosis peaks 1-2 weeks post injury and resolves by
week 3. Depletion of myeloid cells led to lower fibrosis. Microarray analysis revealed an
inflammatory signature and modulation of components of the serotonin (5-HT) pathway in
myeloid cells. Antagonizing the 5HT receptor decreased fibrosis in thermally injured mice,
2A/2C
likely through promoting an anti-fibrotic phenotype in macrophages. Treating mice with a
5HT receptor antagonist promotes an anti-fibrotic effect, through modulating the phenotype
2A/2C
of macrophages.

  ii
Acknowledgments
“If I have seen further it is by standing on the shoulders of giants”
– Isaac Newton
I am very greatful for the opportunity I was given by Drs. Marc G. Jeschke and Saeid
Amini-Nik to work under their supervision. I have learned many skills and lessons of what it
takes to be a scientist and the impact ones work can have on the lives of patients. Both mentors
are very passionate about finding therpaeutic solutions for patients with thermal injuries, which
is very inspirational to me and highlighted the importance of what we do. I was constantly
challenged and pushed to find solutions to our research problems and to think critically on my
own while having their support when I needed it. Thanks to my mentors, I have numerous skills
that are applicable to any field I decide to enter and I am very appreciative and thankful for that.
I’d also like to thank my committee members Drs. Slava Epelman and Calvin Law for their
ongoing support during my masters.
To Cassandra Belo, thank you for your patience and support as you taught me many of
the lab skills I needed to do the majority of experiments in this thesis. You provided me with
moral and scientific guidance that I am very greatful for.
To the Jeschke lab, thank you for all the good times we shared. In particular, I would like
to thank Andrea Datu for all the help with experiments and editing. Also, the support of Genenve
Awong, the manager of our flow cytometry facility, was invaluable.
I dedicate this work to my dear mother. She has made extradinary scarfices for me to be
where I am today. I am forever greatful for her love and support.

  iii
Table of Contents

ACKNOWLEDGMENTS............................................................................................................iii

TABLE OF CONTENTS.............................................................................................................iv

LIST OF TABLES.......................................................................................................................vii

LIST OF FIGURES....................................................................................................................viii

LIST OF ABBREVIATIONS.......................................................................................................x

- CHAPTER 1 – LITERATURE REVIEW.....................................................................................1

1.1 THE LIVER..............................................................................................................................................2
1.1.1 Overview................................................................................................................................2
1.1.2 Liver Anatomy.......................................................................................................................3
1.1.3 Liver Function and Zonation….............................................................................................7

1.2 SEVERE THERMAL INJURIES.................................................................................................................10
1.2.1 The systemic response post-thermal injury............................................................................10
1.2.2 The hepatic response post-thermal injury and the essential role of macrophages.................11

1.3 Liver Fibrosis…..............................................................................................…………………….…..11
1.3.1 Macrophages and The Inflammatory Response Post Acute Liver Injury……..…...11
1.3.2 Macrophages in Wound Healing..............................................................................13
1.3.3 Resident and Recruited Macrophages.......................................................................14
1.3.4 Therapeutic Manipulation of Macrophages..............................................................17
1.3.5 Targeting the Serotonin Pathway as an Anti-Fibrotic Remedy................................20
1.4 THE WNT/β-CATENIN SIGNALING PATHWAY........................................................................................22
1.4.1 The Role of Wnt/β-catenin signalling in Fibrosis...........................................................22
1.5 RATIONALE AND AIMS...........................................................................................................................24
1.6 HYPOTHESIS...........................................................................................................................................27

  iv
- CHAPTER 2 -………….………………………………………………………………….…..28
THE ACCUMULATION OF MYELOID LINEAGE CELLS IN THE LIVER MAPS OUT HEPATIC FIBROSIS
POST ACUTE INJURY: A TARGETABLE LESION USING A 5HT ANTAGONIST
INTRODUCTION…….....................................................................................................................................29
2.1 EXPERIMENTAL METHODS…….............................................................................................................30
2.1.1 Mice.......................................................................................................................................30
2.1.2 Experimental Protocol...........................................................................................................32
2.1.3 Histological Examination......................................................................................................33
2.1.4 Image Acquisition and Analysis............................................................................................33
2.1.5 Fibrosis Index........................................................................................................................33
2.1.6 Clodronate.............................................................................................................................34
2.1.7 Flow Cytometry and Microarray Analysis............................................................................34
2.1.8 Ketanserin Treatment............................................................................................................35
2.1.9 Cell Culture...........................................................................................................................35
2.1.10 Phagocytosis Assay.............................................................................................................36
2.1.11 RNA Isolation and Real-Time Quantitative Polymerase Chain Reaction..........................36
2.1.12 Co-culture of Macrophages and BM-DMSCs....................................................................37
2.1.13 Statistical Analysis.............................................................................................................37
2.2 RESULTS…............................................................................................................................................38
2.2.1 Thermal Injury Results in Liver Fibrosis with a Unique Spatiotemporal Pattern................38
2.2.2 Myeloid Lineage Cells Accumulate in The Liver Post-Thermal Injury...............................41
2.2.3 Ablating Macrophages Prevents Fibrosis in The Liver Post-Thermal Injury.......................43

2.2.4 Liver Myeloid Lineage Cells Show a Gene Expression Profile Favoring Inflammation and
Fibrosis...........................................................................................................................................47
2.2.5 Treating Mice with The 5HT Antagonist Ketanserin Results in Less Liver Fibrosis....52
2A/2C
2.3 DISCUSSION...........................................................................................................................................55
2.4 LIMITATIONS.........................................................................................................................................59
2.5 CONCLUSION.........................................................................................................................................60
2.6 FUTURE DIRECTIONS.............................................................................................................................61
2.7 SUPPLEMENTARY..................................................................................................................................63

  v
- CHAPTER 3 -………………………….…………………………………………………………….....75
β-CATENIN EXPRESION IN MYELOID CELLS HAS AN ESSENTIAL ROLE IN LIVER FIBROSIS POST
THERMAL INJURY
INTRODUCTION.............................................................................................................................................76
3.1 RATIONALE............................................................................................................................................77
3.2 HYPOTHESIS...........................................................................................................................................78
3.3 EXPERIMENTAL METHODS…….............................................................................................................78
3.3.1 Mice.......................................................................................................................................78
3.3.2 Experimental Injury Protocol…………………………………..……………………….…78
3.3.3 Flow cytometry…................................…..............................................................................79
3.4 RESULTS…...…................................................….....................................….........................................79
3.4.1 β-Catenin in myeloid cells is required for fibrogenesis and migration to the wound site in
the liver post-thermal injury...........................................................................................................81
3.4.2 β-Catenin is required in myeloid cells to promote fibrogenesis after chronic CCl induced
4
hepatic injury..................................................................................................................................81
3.4.3 There is a greater accumulation of macrophages in the livers of Ctnnb1 KO mice relative to
wild-type mice................................................................................................................................83
3.4.4 Recruitment of pro-fibrotic Ly6C+ myeloid cells is impaired in Ctnnb1 KO
mice.................................................................................................................................................84
3.4.5 Mice lacking Ctnnb1 expression in their myeloid cells lack the integrin
CD11b......................................................….................................…..............................................86
3.5 DISCUSSION...............................................….........................................................................................89
3.6 LIMITATIONS..........................................................................................................................................90
3.7 CONCLUSIONS.............................................….....................................…..............................................91
3.8 FUTURE DIRECTIONS...............................................….....................................…..................................92

  vi
List of Tables
Table 1. Genes associated with inflammation are up-regulated in microarray analysis.
Table 2. Genes associated with fibrogenesis are up-regulated in microarray analysis.

  vii
List of Figures
Figure 1. Anatomy of the liver.
Figure 2. Portal blood flow.

Figure 3. The gradient of oxygen delivery and metabolic activity.

Figure 4. Investigating the underlying mechanisms of liver repair after injury.

Figure 5. Schematic of the Cre-LoxP system.
Figure 6. Thermal injury primes liver fibrosis in a spatiotemporal pattern.

Figure 7. Myeloid cells are recruited to the liver post-thermal injury.
Figure 8. Myeloid cells have an essential role in induction of fibrosis post-thermal injury.
Figure 9. Isolation of myeloid cells from control and thermally injured mice for
microarray analysis.
Figure 10. EYFP+ cells show a gene expression profile favoring a pro-inflammatory
phenotype post-thermal injury.
Figure 11. Ketanserin decreases liver fibrosis post thermal injury.
Figure 12. Wnt/β-catenin signalling pathway has an essential role in mobilizing myeloid
lineage cells to the wound area post-thermal injury.
Figure 13. Knocking down β-catenin impairs fibrogenesis after chronic hepatic injury in
mice.
Figure 14. Increased concentration of myeloid lineage cells in the livers of Ctnnb1 KO mice
relative to control.
Figure 15. Impaired recruitment of Ly6Chi, EYFP+ myeloid cells to the liver in the KO
group relative to wild-type mice.
Figure 16. Mice that lack Ctnnb1 expression in their myeloid cells do not recruit CD11b
positive cells to the liver after chronic CCl induced hepatic injury.
4

  viii
Figure 17. Expression of CD11b in myeloid cells is regulated through β-catenin signaling.

Supplementary Figures
Supporting Information Figure 1: Majority of EYFP+ve cells in the liver are F4/80+ve as
well as CD11b+ve.
Supporting Information Figure 2: Thermal injury increases the percentage of of EYFP+ve
cells in the bone marrow.
Supporting Information Figure 3: Thermal injury up-regulate several pathways that
associated with liver injury in myeloid cells.
Supporting Information Figure 4: EYFP+ve cells are skewed more towards a

proinflammatory phenotype 1-week post thermal injury in vivo.

Supporting Information Figure 5: Pathway analysis shows deregulation of numerous

pathways associated with inflammation and fibrosis.

  ix
List of Abbreviations

5HT Serotonin, 5-hydroxytryptamine

AALF Acetaminophen-induced acute liver failure
AD Alzheimer’s disease
AMs Alveolar macrophages
Ang2 Angiopoietin 2
APC Adenomatous polyposis coli
BD Bile ducts
BMDM Bone marrow derived macrophages
BMP Bone morphogenic proteins
CBP CREB-binding protein
CK1α Casein kinase 1α
CSF-1 Colony stimulating factor
CV Central veins
CCl2 Chemokine ligand 2
CCR2 Chemokine receptor 2
E Embryonic day
ECM Extracellular matrix
EGFR Epidermal growth factor receptor
ER Endoplasmic reticulum
EYFP Enhanced yellow fluorescent protein

  x