Involvement of Th17 Pathway in Adverse Drug Reactions - T-Space

130 Pages·2012·3 MB·English

Involvement of Th17 Pathway in Adverse Drug Reactions:
Mechanistic Investigation of Drug-Induced Autoimmunity and
Drug-induced Liver Injury

By

(Ervin) Xu Zhu

A thesis submitted in conformity with the requirements for the degree of
DOCTOR OF PHILOSOPHY
Graduate Department of Pharmaceutical Sciences
University of Toronto
©Copyright by (Ervin) Xu Zhu 2012
ABSTRACT
Involvement of Th17 Pathway in Adverse Drug Reactions: Mechanistic
Investigation of Drug-Induced Autoimmunity and Drug-induced Liver
Injury

By (Ervin) Xu Zhu

Faculty of Pharmacy, University of Toronto 2012

DOCTOR OF PHILOSOPHY

Clinical characteristics of idiosyncratic drug reactions (IDRs) suggest that they are
immune mediated. Penicillamine-induced autoimmunity in Brown Norway rats was used as a
tool for mechanistic studies of this type of IDR. It has been shown that T helper 17 (Th17)
cells play a central role in many types of autoimmune diseases. This study was designed to
test whether Th17 cells are involved in the pathogenesis of penicillamine-induced
autoimmunity. In sick animals, interleukin (IL) 6 and transforming growth factor-β1, known
to be driving forces of Th17 differentiation, were consistently increased following
penicillamine treatment. IL-17 and IL-22, characteristic cytokines produced by Th17 cells,
were increased in sick animals. Furthermore, the percentage of IL-17-producing CD4 T cells
was significantly increased, but only in sick animals. Retinoic acid, which has been reported
to inhibit Th17 cell development, made the autoimmunity worse, increased IL-6 production,
and did not decrease the number of Th17 cells. An infiltration of CD8 cytotoxic T cells in the
liver suggests that they may be the key player in causing liver toxicity induced by D-
penicillamine.
Drug-induced liver injury (DILI) is one of the major causes of morbidity, mortality,
and drug candidate failure. Recently, it has been suggested that Th17 cells may play an active
role in inflammatory human liver diseases. In a study of patients being treated with isoniazid,
II
some patients developed mild liver injury. The percentage of Th17 cells in the blood of these
patients significantly increased when the ALT increased, and this suggests that they play a
role in the mechanism of this liver injury. Furthermore, IL-10-producing T cells also
increased and this may have prevented the development of severe liver injury. In another
study, two hours after treatment of mice with acetaminophen there was a significant increase
in Th17 cells in the liver. This rapid response suggests that Th17 cells can be part of the
innate immune response to liver injury.
Our data provided evidence that Th17 cells are involved in both “toxic” and
idiosyncratic liver toxicity. This pathway could be a new target for the therapeutic
interventions to treat DILI.

III
ACKNOWLEDGEMENT
First of all, I would like to thank and express my deepest gratitude to my supervisor,
Dr. Jack Uetrecht for his great mentorship and continuous support throughout my Ph.D
training in his lab. When I think how much I have learned and gained in the past five years, I
become very emotional. I realized that I have progressed from an insecure student to a fairly
confident one under his supervision. It is a complete privilege and a great honor for me to be
a member of his research group. He is a role model in my life.
I would like to extend my thanks to my advisory committee, Dr. Carolyn Cummins,
Dr. Jack Hay, Dr. Peter J. O’Brien, the internal examiner, Dr. Denis Grant , and the external
examiner of my thesis, Dr. Lance Pohl from NIH. I appreciate very much for their
suggestions and support for my research and the preparation of this thesis.
I would also like to thank my lab mates Jie, Tharsika, Julia, Baskar, Robert, Ping,
Xiao Chu, Maria, Xin, Feng, Winnie, Imir, Amy, and Alex who have always been
encouraging and supportive. They are not only colleagues, but also loyal friends. They made
the life more enjoyable.
Finally, I would like to specially thank my parents, Mr. Ningguo Zhu and Ms.
Songling Yu. Without your selfless support, I would never go this far. I would also like to
dedicate this work to my wife, Ms. Runhua Yang, for her constant care, encouragement and
support from every aspect. I appreciate in deed all she has done for me.

IV
TABLE OF CONTENTS
ABSTRACT ................................................................................................................................ II
ACKNOWLEDGEMENT ....................................................................................................... IV
TABLE OF CONTENTS ........................................................................................................... V
LIST OF THESIS PUBLICATIONS .................................................................................. VIII
LIST OF ABBREVIATIONS ................................................................................................. IX
LIST OF FIGURES ................................................................................................................. XII
LIST OF TABLES ................................................................................................................. XIV
CHAPTER 1 ................................................................................................................................ 1
GENERAL INTRODUCTION .................................................................................................. 1
1.1. ADVERSE DRUG REACTIONS ....................................................................................... 2
1.2. IDIOSYNCRATIC DRUG REACTIONS .......................................................................... 5
1.3. PROPOSED HYPOTHESIS OF MECHANISMS OF IDRS ......................................... 11
1.3.1. THE HAPTEN HYPOTHESIS .................................................................................. 11
1.3.2. THE DANGER HYPOTHESIS ................................................................................ 12
1.3.3. THE PHARMACOLOGICAL INTERACTION HYPOTHESIS .............................. 13
1.4. D-PENICILLAMINE-INDUCED IDR ANIMAL MODEL ........................................... 14
1.5. IL-17 AND TH17 CELLS .................................................................................................. 17
1.5.1. IL-17 AND TH17 CELLS IN HOST DEFENSE ...................................................... 18
1.5.2. IL-17 AND TH17 CELLS IN AUTOIMMUNITY ................................................... 19
1.5.3. IL-17 AND TH17 CELLS IN LIVER DISEASES .................................................... 20
CHAPTER 2 .............................................................................................................................. 24
INVOLVEMENT OF T HELPER 17 (TH17) CELLS IN D-PENICILLAMINE-INDUCED
AUTOIMMUNE DISEASE IN BROWN NORWAY RATS ................................................ 24
2.1. INTRODUCTION .............................................................................................................. 25
V
2.2. MATERIALS AND METHODS ....................................................................................... 28
2.3. RESULTS ............................................................................................................................ 32
2.4. DISCUSSION ...................................................................................................................... 40
CHAPTER 3 ............................................................................................................................. 43
MODULATION OF D-PENICILLAMINE-INDUCED AUTOIMMUNITY IN THE BROWN
NORWAY RATS WITH PHARMACOLOGICAL AGENTS THAT INTERFERE WITH THE
TH17 PATHWAY: THE EFFECTS OF RETINOIC ACID ................................................. 43
3.1. INTRODUCTION ............................................................................................................. 45
3.2. MATERIALS AND METHODS ....................................................................................... 47
3.3. RESULTS ............................................................................................................................ 49
3.4. DISCUSSION ...................................................................................................................... 52
CHAPTER 4 ............................................................................................................................. 54
CHARACTERIZATION OF THE ROLE OF THE IMMUNE SYSTEM IN D-
PENICILLAMINE-INDUCED LIVER INJURY. ................................................................. 54
4.1. INTRODUCTION .............................................................................................................. 55
4.2. MATERIALS AND METHODS ....................................................................................... 57
4.3. RESULTS ............................................................................................................................ 59
4.4. DISCUSSION ...................................................................................................................... 67
CHAPTER 5 ............................................................................................................................. 70
INVESTIGATION OF THE ROLE OF TH17 CELLS IN ISONIAZID-INDUCED LIVER
INJURY ...................................................................................................................................... 71
5.1. INTRODUCTION .............................................................................................................. 72
5.2. MATERIALS AND METHODS ....................................................................................... 74
5.3. RESULTS ............................................................................................................................ 76
5.4. DISCUSSION ...................................................................................................................... 83
VI
CHAPTER 6 ............................................................................................................................. 86
TH17 CELLS ARE INCREASED IN THE LIVER FOLLOWING ACETAMINOPHEN
TREATMENT OF MICE: TH17 CELLS AND THE INNATE IMMUNE SYSTEM ....... 86
6.1. INTRODUCTION .............................................................................................................. 88
6.2. MATERIALS AND METHODS ....................................................................................... 90
6.3. RESULTS AND DISCUSSION ......................................................................................... 93
CHAPTER 7 ............................................................................................................................. 98
OVERALL CONCLUSIONS AND FUTURE DIRECTIONS ............................................. 98
7.1. INTRODUCTION .............................................................................................................. 99
7.2. SUMMARY AND CONCLUSIONS ............................................................................... 100
7.3. IMPLICATIONS AND FUTURE DIRECTIONS ........................................................ 101
REFERENCES ........................................................................................................................ 105

VII
LIST OF THESIS PUBLICATIONS
Zhu X, Li J, Liu F, Uetrecht JP. Involvement of T helper 17 cells in D-penicillamine-
(cid:122)
induced autoimmune disease in Brown Norway rats. Toxicol Sci. 2011 Apr; 120(2):331-
8.
Jinze Li, Xu Zhu, Feng Liu, Ping Cai, Carron Sanders, William M. Lee, and Jack
(cid:122)
Uetrecht. Cytokine and autoantibody patterns in acute liver failure. J Immunotoxicol.
2010 Jul-Sep; 7(3):157-64.
Metushi IG, Cai P, Zhu X, Nakagawa T, Uetrecht JP. A fresh look at the mechanism of
(cid:122)
isoniazid-induced hepatotoxicity. Clin Pharmacol Ther. 2011 Jun; 89(6):911-4.
Zhang X, Liu F, Chen X, Zhu X, Uetrecht J. Involvement of the immune system in
(cid:122)
idiosyncratic drug reactions. Drug Metab Pharmacokinet. 2011; 26(1):47-59. Review
Dugoua JJ, Machado M, Zhu X, Chen X, Koren G, Einarson TR. Probiotic safety in
(cid:122)
pregnancy: a systematic review and meta-analysis of randomized controlled trials of
Lactobacillus, Bifidobacterium, and Saccharomyces spp J Obstet Gynaecol Can. 2009
Jun;31(6):542-52. Review.
Zhu X, Li J, and Uetrecht JP. Th17 cells are increased in the liver following
(cid:122)
acetaminophen treatment of mice: Th17 cells and the innate immune system (Submitted)
J Immunotoxicol.
Zhu X, Li J, and Uetrecht JP. Modulation of D-penicillamine-induced autoimmunity in
(cid:122)
the Brown Norway rats with pharmacological agents that interfere with the Th17
pathway: The effects of retinoic acid. (Submitted) J Immunotoxicol.

VIII
LIST OF ABBREVIATIONS
Chapter 1
ADR, Adverse drug reaction
ALT, Alanine aminotransferase
APC, Antigen presenting cell
AST, Aspartate aminotransferase
BN, Brown Norway
CD, Cluster of differentiation
ConA, Concanavalin A
DILI, Drug induced liver injury
DNA, Deoxyribonucleic acid
EAE, Experimental autoimmune encephalomyelitis
FOXP3, Forkhead box P3
HBV, Hepatitis B
HCV, Hepatitis C
HLA, Histocompatibility leukocyte antigen
HMGB, High mobility group protein
HSP, Heat shock proteins
IDILI, Idiosyncratic drug induced liver injury
IDR, Idiosyncratic drug reaction
IFN, Interferon
IgE, Immunoglobulin E
IL, Interleukin
IL-17R, IL-17 receptor
G-CSF, Granulocyte colony stimulating factor
GI, Gastrointestinal
LPS, Lipopolysaccharide
MHC, Major histocompatibility
mRNA, Messenger ribonucleic acid
MS, Multiple sclerosis
NK, Natural Killer
NKT, Natural killer T
PI, Pharmacological interaction
Poly I:C, Polyinosinic-Polycytidylic acid
RAGE, Receptors for advanced glycation end products
ROR, Retinoic acid receptor-related orphan receptor
SJS, Steven-Johnson syndrome
IX
STAT, Signal transducer and activator of transcription
TCR, T cell receptor
TEN, toxic epidermal necrolysis
TGF, Transforming growth factor
Th, T helper cell
TNF, Tumor necrosis factor
Treg, Regulatory T cells
Chapter 2
BCIP, 5-bromo-4-chloro-3-indolyl-phosphate
CCL, Chemokine (C-C motif) ligand
cDNA, Complementary DNA
IgG, Immunoglobulin G
ELISA, Enzyme-linked immunosorbent assay
ELISPOT, Enzyme-linked immunosorbent spot
GAPDH, Glyceraldehyde 3-phosphate dehydrogenase
GM-CSF, Granulocyte macrophage colony-stimulating factor
MACS, Magnetic cell separation technology
MCP, Monocyte chemotactic protein
MIP, Macrophage inflammatory protein
NBT, Nitro blue tetrazolium
GRO/KC, Growth-related oncogene
PBMC, Peripheral blood mononuclear cells
PCR, Polymerase chain reaction
qRT-PCR, Quantitative real-time PCR
RANTES, Regulated upon Activation, Normal T-cell Expressed, and Secreted
VEGH, Vascular endothelial growth factor
Chapter 3
PMA, Phorbol myristate acetate
RA, Retinoic acid
Chapter 4
ANOVA, Analysis of variance
APAP, Acetaminophen
D-PBS, Dulbecco's Phosphate-Buffered Saline
H&E, Hematoxylin and eosin
RPMI, Roswell Park Memorial Institute
SDH, Sorbitol dehydrogenase
Chapter 5
INH, Isoniazid
X