Development of Animal Models to Study Idiosyncratic Drug-Induced Liver Injury

Transcription

1 Development of Animal Models to Study Idiosyncratic Drug-Induced Liver Injury By Feng Liu A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy in Pharmaceutical Science Graduate Department of Pharmaceutical Science Faculty of Pharmacy University of Toronto Copyright by Feng Liu, 2015

2 Development of Animal Models to Study Idiosyncratic Drug- Induced Liver Injury Feng Liu Doctor of Philosophy Department of Pharmaceutical Sciences University of Toronto 2015 Abstract Idiosyncratic drug reactions (IDRs) represent a special problem because of their unpredictable nature and are a major reason for drug withdrawal. The objective of this research was to study the mechanism of DILI caused be amodiaquine (AQ) and D-penicillamine (D-pen). Amodiaquine (AQ) is associated with a relatively high incidence of idiosyncratic drug-induced liver injury (IDILI) and agranulocytosis. In this study, treatment of rats with AQ resulted in a mild delayed-onset liver injury that resolved despite continued treatment with the drug. Immunohistochemistry indicated the presence of Kupffer cell activation, apoptosis, and hepatocyte proliferation in the liver. There was also an increase in serum IL-2, IL-5, IL-9, IL-12, MCP-1, and TGF-β, but a decrease in leptin. Co-treatment with cyclosporin prevented AQinduced liver injury, while Poly I:C co-treatment caused an earlier increase in alanine aminotransferase (ALT), which suggested that AQ-induced liver injury is immune-mediated. Coincident with the elevated serum ALT, liver CD4 + T cells, IL-17 secreting cells, and Th17/Treg cells were increased at week 3 and decreased during continued treatment. An increase in CD161+ cells and activated M2 macrophages were also observed during the liver II

3 injury. These results suggest that the outcome of the liver injury is determined by a balance between effector cells and regulatory cells. Similar results were also observed in AQ-treated C57BL/6 mice. Covalent binding of AQ was detected in the liver, and although AQ forms a glutathione conjugate, depletion of glutathione did not increase covalent binding and paradoxically prevented AQ-induced liver injury. Retinoic acid (RA), which has been reported to enhance natural killer (NK) cell activity, exacerbated AQ-induced liver injury. These characteristics indicate that we have established a rat model of AQ-induced liver injury that mimics mild AQ-induced liver injury in humans. The results suggest that AQ-induced IDILI is immune mediated, and the subsequent adaptation appears to represent immune-tolerance.. III

4 Acknowledgments First and foremost, I would like to express my special appreciation and sincere thanks to my supervisor, Dr. Jack Uetrecht for his great mentorship and continuous support throughout my PhD program. I appreciate all his contributions of time, ideas, and funding to make my Ph.D. experience productive and stimulating. Jack has been a tremendous mentor for me, and I would like to thank him for encouraging my research and for allowing me to grow as a research scientist. It is a great honor and pleasure to work with him. In my personal life, Jack has also been a great friend and a father figure to me: When I was in the darkest time of my life, he was the one who encouraged me, helped me and taught me to make the right decision, to carry on my dream, which I will never forget. I would also like to thank my advisory committee, Dr. Peter McPherson, Dr. Jonathan Rast, Dr. Peter O Brien, the internal examiner, Dr. Denis Grant, and the external examiner of my thesis, Dr. Michael Rieder from University of Western Ontario. I am extremely thankful and indebted to all of you for sharing expertise, and sincere and valuable guidance and encouragement extended to me. Meanwhile, I would like to take this opportunity to express my regards and gratitude to all of my lab mates in the Uetrecht lab, particularly Jie Chen, Julia IP, Ping Cai, Jingze Li, Xiaochu Zhang, Ervin Zhu, Xin Chen, Maria Novalen, Winnie Ng, Amy Sharma, Imir Metushi, and Alexandra Lobach for their help and support. This team has been a source of friendships as well as good advice and collaboration, which made the last few years one of the happiest time in my life. Thank you as well to the other graduate students and friends from Faculty of Pharmacy. I would like to thank Jenny and my parents for their love, encouragement, and attention. Word can t express how grateful I am for the all of the sacrifice and support. Meanwhile, I want IV

5 to give my special thanks to my beloved dogs, Whisky & Tequila, for the unconditional love and cheerfulness. Last, I would like to thank my fiancée, Jenny Lu, for always being there, no matter rich or poor, no matter how hard the life was, I couldn t have come this far without your love and encouragement. Feng Liu V

6 Table of Contents Title... I Abstract... II Acknowledgments...IV Table of Contents...VI List of Tables... X List of Figures...XI List of Abbreviation... XVI List of Appendices... XIX Chapter 1: Introduction Idiosyncratic drug reactions Mechanistic hypotheses Animal models Amodiaquine D-penicillamine Brown Norway rat model Immune system and IDRs Th17 cells and autoimmune disease NK/NKT cells and autoimmune disease Regulatory T cells and immune tolerance Macrophages and drug-induced liver injury Toll-like receptors and autoimmune disease Specific hypotheses and objectives Chapter 2: Establishing an Amodiaquine-induced Hepatotoxicity Model in Rats Abstract Introduction VI

7 3. Materials and methods Animals and sample preparation Detection of liver injury and cytokines/chemokines Histopathology and immunohistochemistry Apoptosis and cell proliferation Detection of serum IgG anti-aq antibody Measurement of serum concentrations of AQ Modulation of the immune response Phenotyping of macrophages and lymphocytes NK cell activation/stimulation Western blotting GSH depletion Regulatory T cell (Treg) depletion Statistical analyses Results Serum ALT and AQ concentrations in AQ-treated rats Liver histological changes induced by AQ treatment in Wistar, BN, and Lewis rats Apoptosis and cell proliferation in AQ-treated male BN rats Cytokine and chemokine changes in AQ-treated male BN rats Serum ALT, anti-aq antibody, cytokines, and chemokines in rechallenged male BN rats Co-treatment of cyclosporin and Poly I:C in BN rats Lymphocyte phenotyping Lymphocyte phenotyping in C57BL/6 mice Macrophage phenotyping in AQ-treated BN rats Effect of temozolomide on Tregs Covalent binding of AQ to hepatic proteins in BN rats treated with AQ GSH depletion by co-treatment with BSO and DEM Effects of RA and DMSO co-treatment Discussion Acknowledgement Declaration of interest Chapter 3: Development of a Novel Mouse Model of Amodiaquine-induced Liver Injury with a Delayed Onset VII

8 1. Abstract Introduction Materials and methods a) Mice and treatments b) Serum concentration of AQ c) Histology and immunohistochemistry d) Western blotting e) Flow cytometry f) Natural killer cell depletion g) Serum cytokines h) Statistical analysis Results a) Treatment of female C57BL/6 mice with AQ results in mild liver injury with delayed onset97 b) Covalent binding of AQ in the liver, spleen, gut, and kidney c) Increase in ALT is associated with lymphocyte infiltration in the liver d) Increase in ALT is associated with activation of T-cells e) Treatment of Rag1 -/- mice and depletion of NK cells Discussion Acknowledgement Declaration of interest Chapter 4: Involvement of T Helper 17 Cells in D-Penicillamine Induced Autoimmune Disease in Brown Norway Rats Abstract Introduction Materials and methods a) Animals b) Chemicals, kits, and solutions c) D-penicillamine treatment Determination of serum IL Phenotyping splenic CD4 + T cells by qrt-pcr Profiling cytokines/chemokines Determination of IL-17 production by ELISPOT Intracellular cytokine staining and flow cytometry VIII

9 3.9. Statistical analysis Serum levels of IL-6 during penicillamine treatment Serum cytokine/chemokine pattern during penicillamine treatment Serum IL-22 during penicillamine treatment Serum cytokines at early time points of penicillamine treatment Th17 cell phenotyping Discussion Funding Acknowledgement Chapter 5: Summary, Discussion and Future Directions Summary of findings and discussion Implications and future directions References IX

10 List of Tables Table 1. Characteristics of activated macrophage subpopulations Table 2. Antibodies used for macrophage phenotyping and activation Table 3. Primer sequences for qrt-pcr X

11 List of Figures Figure 1. Hapten hypothesis by Landsteiner... 3 Figure 2. Danger hypothesis by Polly Matzinger... 5 Figure 3. Pharmacological interaction (PI) hypothesis by Pichler... 6 Figure 4. The chemical structure of amodiaquine... 8 Figure 5. The putative mechanism of amodiaquine-induced liver injury... 9 Figure 6. The chemical structure of D-penicillamine Figure 7. Representation of the signalling pathway between antigen presenting cells (APCs) and other macrophages and T cells involving reaction of an aldehyde on macrophages with an amine on T cells leading to a reversible imine linkage Figure 8. The chemical structure of ARP Figure 9. The schematics of aldehydes binding to D-pen, hydralazine, and isoniazid Figure 10. The dose-response relationship of D-pen-induced autoimmunity in BN rats Figure 11. The differentiation of naive CD4+ T cells into Th1, Th2, and Th17 cells Figure 12. Characterization of M1 and M2 macrophages Figure 13. Postulated mechanisms of AQ-induced liver injury Figure 14. Liver injury induced by AQ treatment in different strains of rats Figure 15. Serum concentration of AQ in male BN rats and male Wistar rats Figure 16. Histological and immunohistochemical staining of the liver from a representative male Wistar rat treated with AQ (62.5 mg/kg/day) for 5 weeks XI

12 Figure 17. Histological changes in the liver of a representative male BN rat treated with AQ for 5 weeks at a dose of 62.5 mg/kg/day Figure 18. Treatment of male BN rats with AQ for 5 weeks is associated with an increase in activated hepatic macrophages and lymphocytes Figure 19. Cell apoptosis and proliferation in the liver and spleen in AQ-treated BN rats Figure 20. Serum levels of cytochrome C and osteopontin in AQ-treated male BN rats after 5 weeks Figure 21. Serum cytokine changes in AQ-treated male BN rats after 5 weeks Figure 22. Liver cytokine and chemokine changes in AQ-treated male BN rats after 5 weeks Figure 23. Serum ALT and anti-aq antibody in male BN rats during primary and re-challenge AQ treatment Figure 24. Serum cytokine changes in male BN rats during the primary and re-challenge AQ treatment Figure 25. The time course of serum ALT during treatment with AQ and cyclosporine in BN rats Figure 26. The time course of serum ALT during treatment with AQ and Poly I:C in BN rats.. 51 Figure 27. The time course curve of serum ALT during AQ treatment in BN rats Figure 28. Time course of changes induced by AQ treatment Figure 29. CD4 + T cells and Th17 cells in different organs during 4 weeks AQ treatment in BN rats Figure 30. IL-17 secreting cells and NK/NKT cells in different organs during AQ treatment in BN rats XII

13 Figure 31. Treg cells in different organs during AQ treatment in BN rats Figure 32. The immune balance of Treg and Th17 in different organs during AQ treatment in BN rats Figure 33. Serum ALT level in C57BL/6 mice treated with AQ Figure 34. Numbers of CD3, CD4, and CD8 cells in in lymph nodes, liver, and peripheral blood of C57BL/6 mice treated with AQ Figure 35. Numbers of CD161, NK, NKT, and NK17 cells in lymph nodes, liver, and peripheral blood of C57BL/6 mice treated with AQ Figure 36. Numbers of IL17 producing, Th17, and Tc17 cells in lymph nodes, liver, and peripheral blood of C57BL/6 mice treated with AQ Figure 37. Differences in serum ALT and various lymphocyte types between mice that developed liver injury after 1 week of AQ treatment and those that did not Figure 38. Changes in Treg and PD-1 + /CD4 + cells in lymph nodes, liver and peripheral blood in C57BL/6 mice treated with AQ Figure 39. The time course for macrophage numbers in different organs of BN rats during AQ treatment Figure 40. The M1 macrophage phenotyping in peripheral blood, lymph nodes, liver, and spleen of BN rats during AQ treatment Figure 41. The M2 macrophage phenotyping in different organs of BN rats during AQ treatment66 Figure 42. Subtypes of M2a macrophages in different organs of BN rats during AQ treatment. 68 Figure 43. Subtypes of M2b macrophages in different organs of BN rats during AQ treatment. 69 Figure 44. Subtypes of M2c macrophages in different organs of BN rats during AQ treatment. 70 XIII

14 Figure 45. The time course of serum ALT during AMQ + temozolomide (TMZ) co-treatment in male BN rats Figure 46. Time course of Treg cell % in PBMC during AMQ + temozolomide (TMZ) cotreatment in male BN rats Figure 47. The total lymphocyte count in blood during AMQ + temozolomide (TMZ) cotreatment in male BN rats Figure 48. Serum ALT and corresponding covalent binding of AQ to hepatic proteins from BN rats treated with AQ Figure 49. Serum ALT change and corresponding covalent binding of AQ to hepatic proteins from BN rats treated with AQ Figure 50. Liver GSH level in BN rats after 1 week of AQ and/or BSO treatment Figure 51. BSO treatment prevents the increase in ALT caused by AQ in BN rats Figure 52. Effects of GSH depletion on covalent binding of AQ to hepatic proteins from AQtreated BN rats Figure 53. Effects of RA co-treatment on serum ALT in AQ-treated BN rats Figure 54. Change of serum ALT in BN rats treated with AQ and/or DMSO for 4 weeks Figure 55. Treatment of C57BL/6 mice with AQ results in mild liver injury Figure 56. Covalent binding of AQ Figure 57. Comparison of AQ covalent binding Figure 58. ALT, liver and spleen weight, and total lymphocyte numbers in female C57BL/6 mice treated with AQ Figure 59. Immunohistochemical staining of livers from female C57BL/6 mice XIV

15 Figure 60. Lymphocyte phenotyping for CD4, CD8, and NK Figure 61. Lymphocyte activation in AQ-treated mice Figure 62. Expression of CD62L + and PD-1 + (CD279) on T-cells Figure 63. Serum cytokine concentrations Figure 64. Liver injury in AQ-treated Rag -/- mice or mice treated with an anti-nk cell antibody113 Figure 65. Serum concentration of IL-6: penicillamine Figure 66. Changes in body weight and cumulative incidence of autoimmunity Figure 67. Serum cytokine/chemokine profiles: sick versus nonsick Figure 68. Serum IL-22 during the development of penicillamine-induced autoimmunity Figure 69. IL-6 and IL-22 serum levels in the first week of penicillamine treatment Figure 70. IL-6, IL-7, and IL-17 production at the end of penicillamine treatment Figure 71. Flow cytometry analysis for intracellular IL-17A in CD4 lymphocytes from control, treated nonsick, and sick animals XV

16 List of Abbreviation ADR ALT APAP APC AQ AQQI ARP BN BSO CYC D-pen DC DEM DMEM DILI EAE ELISA FICZ GSH H&E adverse drug reaction alanine transaminase acetaminophen antigen presenting cell amodiaquine amodiaquine quinine imine aldehyde reactive probe Brown Norway buthionine sulfoximine cyclosporin D-penicillamine dendritic cell diethylmaleate Dulbecco's Modified Eagle's Medium drug-induced liver injury autoimmune encephalomyelitis enzyme-linked immunosorbent assay 6-formylindolo [3,2-b] carbazole glutathione hematoxylin and eosin HMGB1 high mobility group protein 1 HSC hepatic stellate cell XVI

17 HSP IDILI IDR IFN-γ IHM LC-MS/MS LPS MHC MT NK NKT PBS PCNA PI Poly I:C RA RANTES ROS TBST TCR TGF-β TLR TMZ TRAIL heat shock protein idiosyncratic drug-induced liver injury idiosyncratic drug reaction interferon-gamma immunohistochemistry liquid chromatography-tandem mass spectrometry lipopolysaccharides major histocompatibility complex metallothionein natural killer natural killer T cells phosphate buffered saline proliferation cell nuclear antigen pharmacological interaction polyinosinic:polycytidylic acid retinoic acid regulated and normal T cell expressed and secreted reactive oxygen species Tris-buffered saline with Tween T cell receptor transforming growth factor beta toll-like receptor temozolomide TNF-related apoptosis-inducing ligand XVII

18 Treg cells TUNEL regulator T cells terminal deoxynucleotidyl transferase dutp nick end labelling XVIII

19 List of Appendices Table S1. Serum concentrations of AQ and DEAQ in female C57BL/6 mice Figure S1. Covalent binding of AQ in the liver of female C57BL/6 mice Figure S2. Immunohistochemical grading for CD4, CD8, CD11b, KI67, F4/80, and CD45R in the livers of control or AQ-treated female C57BL/6 mice Figure S3. Immunohistochemical staining for CD4, CD8, and CD11b in the spleens of control or AQ-treated female C57BL/6 mice Figure S4. Immunohistochemical staining for KI67, F4/80, and CD45R in the spleens of control or AQ-treated female C57BL/6 mice Figure S5. Lymphocyte activation in AQ-treated female C57BL/6 mice Figure S6. Treatment of Rag-/- mice with AQ XIX

20 Chapter 1: Introduction 1. Idiosyncratic drug reactions Idiosyncratic drug reactions (IDRs) are also referred to as Type B adverse drug reactions, which can be defined as adverse reactions that do not occur in most people at any dose and do not involve the therapeutic activities of the drug. IDRs represent a significant problem; they are often serious, even life threatening, and their unpredictable nature makes them virtually impossible to prevent (Uetrecht, 2007). For example, in the United Kingdom, it was recently reported that adverse drug reactions are responsible for more than 6% of hospital admissions, and the mortality rate was approximately 2% (Pirmohamed et al., 2004). Although IDRs make up only about 5% of adverse drug reactions (ADRs), given the large variety of drugs that cause IDRs and the number of people who take drugs, the number of cases is significant (Uetrecht, 2008). In addition, they also represent a major problem for drug development, and the unpredictability of IDRs makes it very unlikely that they will be discovered in the clinical trials. From 1975 to 2000, about 10% of new drugs approved in the US were either withdrawn or received a black box warning due to unexpected IDRs (Lasser et al., 2002). This uncertainty significantly increases the overall cost of drug development. Therefore, there are major efforts to predict or detect IDR risk early, but presently no reliable methods exist. It is likely that progress toward better prediction and early detection will depend on a better mechanistic understanding Mechanistic hypotheses A major problem in dealing with IDRs is the limited understanding of the mechanisms involved. However, the characteristics of IDRs, such as the delay between starting the drug treatment and 1

21 the onset of adverse reaction suggest that IDRs are immune-mediated (Uetrecht, 2007). In addition, there is a large amount of evidence to suggest that most IDRs are induced by reactive metabolites (Masson et al., 2004a). In the past decades, several hypotheses have been proposed to explain the mechanism of IDRs: the Hapten Hypothesis, the Danger Hypothesis, and the Pharmacological Interaction Hypothesis (Dawson et al., 1993; Fuchs et al., 1996; Seguin et al., 2003b; Uetrecht, 2007). An immune-mediated IDR may also be induced by direct activation of antigen-presenting cells, by alternation in immune balance, or by epigenetic effects Hapten hypothesis In 1935, Landsteiner reported that some small molecules such as 2,4-dinitrochlorobenzene and p-nitrosodimethylaniline induced skin rashes in guinea pigs, but only if they covalently bound to proteins (Landsteiner et al., 1935). A hypothesis was proposed on the basis of this finding that small molecules with a molecular mass less than 1,000 Da are unable to induce an immune response unless these small molecules or their reactive metabolites are bound to a macromolecule such as a protein. This kind of small molecule is called a hapten. A good example of this hypothesis is allergic reactions caused by penicillin and other β-lactam antibiotics. The β-lactam ring of penicillin is chemically reactive and binds to amino and sulfhydryl groups on proteins, which can lead to IgE antibody formation and allergic reactions. For other drugs that are not chemically reactive, reactive metabolites can be formed during metabolism, and these reactive metabolites can act as haptens. The hapten hypothesis applied to IDRs is illustrated in Figure 1 (Uetrecht, 2007). 2

22 Figure 1. Hapten hypothesis by Landsteiner (Uetrecht, 2007). The drug or reactive metabolite binds to proteins making them foreign, the modified proteins are taken up by antigen presenting cells (APCs), processed, and drug-modified peptides are presented in the context of MHC-II to helper (CD4 + ) T cells. Recognition of processed antigen by the T cell receptor (TCR) is referred to as signal 1 and can lead to an immune response Danger hypothesis In 1994, Matzinger challenged the classic Hapten Hypothesis and proposed the famous Danger Hypothesis (Matzinger, 1994). She reasoned that it would be inefficient to respond to 3

23 something unless it was causing injury or was dangerous to the survival of an organism. Damaged cells produce danger signals that activate APCs, which can lead to an immune response. The interaction between the MHC-antigen complex on APCs and TCRs on T cells is referred as signal 1. The co-stimulation of T cells by interactions between activated APCs and T cells, such as between B7 and CD28, is referred to as signal 2. An immune response is initiated when both signal 1 and signal 2 are present, and tolerance will be induced if only signal 1 is present. Lately, several danger signals have been identified, including high mobility group protein 1 (HMGB1), IL-1α, cytosolic calcium binding proteins of the S100 family, heat shock proteins (HSPs), and uric acid crystals (Harris et al., 2006). The danger hypothesis applied to IDRs is illustrated in Figure 2. 4

24 Figure 2. Danger hypothesis by Polly Matzinger (Uetrecht, 2007) Pharmacological interaction (PI) hypothesis Another hypothesis is the pharmacological interaction (PI) hypothesis proposed by Pichler (Pichler, 2002). In this hypothesis, the parent drug acts as a superantigen and binds reversibly to the complex formed by MHC II on APCs and the T cell receptor on T cells to initiate an immune response (Pichler, 2002). The PI hypothesis applied to IDRs is illustrated in Figure 3. 5

25 Figure 3. Pharmacological interaction (PI) hypothesis by Pichler (Uetrecht, 2007). The drug binds directly to the MHC-TCR complex leading to signal 1 and an immune response to the parent drug. This hypothesis does not address the issue of signal Animal models As discussed above, most IDRs are thought to be immune-mediated, and there are many different hypotheses as to how drugs could induce an immune reaction. We need some way to test these mechanistic hypotheses. But given their unpredictable nature, it is impossible to perform controlled experiments in humans. An animal model is the only way to test what 6

26 chemical species is responsible for the IDR and to determine the sequence of events leading up to the IDR. One such model that we have developed is nevirapine-induced skin rash in rats (Shenton et al., 2003). It is immune-mediated and has very similar characteristics to the rash that nevirapine causes in humans. It has been used in the study of the metabolic pathway responsible for rash and the specificity of the T cells involved (Chen et al., 2008; Chen et al., 2009). However, although animals can also have IDRs, they are also unpredictable in animals, and this hampers the development of practical models. If we had a clear understanding of why it is difficult to develop animal models we would know a lot more about their mechanisms. 2. Amodiaquine Amodiaquine (AQ), a 4-aminoquinoline derivative used to treat malaria, is associated with a relatively high incidence of agranulocytosis and hepatotoxicity. This led to its withdrawn from the market in the USA; however, it is still the first-line anti-malaria drug used at lower dose in combination with other drugs in many parts of Africa due to its cheap price and effectiveness against chloroquine-resistant malaria (Clarke et al., 1991; Phillips-Howard et al., 1990; Watkins et al., 1984). Figure 4. The chemical structure of amodiaquine. 7

27 The mechanism of this drug-induced toxicity is still unknown; however, both direct inhibition of cell function and indirect immunological mechanisms have been proposed. In vivo, amodiaquine is oxidized to a reactive iminoquinone by human liver microsome and peroxidases. Formation of such a reactive chemical in vivo and subsequent covalent binding to cellular macromolecules could affect cell function either directly or indirectly via an immunological mechanism (See Figure 5) (Harrison et al., 1992; Jewell et al., 1995; Shimizu et al., 2009). Clinical manifestations of amodiaquine-induced hepatitis include a delay (weeks to months) in the onset of clinical symptoms and the presence of anti-drug IgG antibodies. There is usually prompt recovery upon the discontinuation of treatment, but on rechallenge there is usually a rapid increase in serum ALT, which is consistent with an immune-mediated reaction (Neftel et al., 1986; Uetrecht, 2005). Intriguingly, many patients simultaneously develop agranulocytosis and hepatitis (Neftel et al., 1986). However, due to the low incidence and lack of animal models, the mechanism is hard to study, and it is impossible to predict which patients will have such an adverse reaction. 8

28 Figure 5. The putative mechanism of amodiaquine-induced liver injury. A study of amodiaquine in rats found an elevation of serum ALT and anti-amodiaquine antibodies after treatment at a daily dose of 191 mg/kg for 4 days; however, there were no histological changes in the liver (Clarke et al., 1990). In another published study, a single dose of amodiaquine (180 mg/kg) did not cause any liver injury in mice, but cotreatment with a glutathione synthesis inhibitor, L-buthionine-S,R-sulfoxinine (BSO), induced centrilobular necrosis 6 hours post dose (Shimizu et al., 2009). However, this is very different from the delayed onset hepatotoxicity observed in humans, and if the characteristics are different, it is unlikely that the mechanism will be the same. Although amodiaquine-induced DILI appears to be immune-mediated, amodiaquine also appears to cause direct cytotoxicity that precedes the increase in ALT by weeks; this may be important for the initiation of an immune response. The observation that the increase in ALT in mice treated with amodiaquine resolved despite continued treatment is similar to the pattern observed in humans with drugs that cause 9

29 idiosyncratic DILI (Metushi et al., 2014a). In humans this is referred to as adaptation, and this is much more common than severe DILI. If the delayed onset increase in ALT induced by amodiaquine is immune-mediated, then almost certainly this adaptation represents immune tolerance. If we understood the mechanism of immune tolerance, it might lead to ways to predict in which patients immune tolerance would fail and who would go on to develop liver failure. In addition to idiosyncratic liver injury, amodiaquine is also associated with agranulocytosis in approximately 1 in 2,000 patients (Ng et al., 2012). Amodiaquine is also metabolized by activated human neutrophils to the same reactive quinoneimine, which is suspected to be responsible for this adverse reaction (Tingle et al., 1995). Further in vitro studies have shown that this reactive metabolite is directly toxic to human neutrophils and can react with cell surface proteins leading to hapten formation (Naisbitt et al., 1997). 3. D-penicillamine Figure 6. The chemical structure of D-penicillamine. D-penicillamine (D-pen, see Figure 6) is used clinically to treat rheumatoid arthritis, Wilson's disease, and cystinuria. However, a number of IDRs are associated with its clinical use including a lupus-like syndrome and myasthenia gravis (Uetrecht, 2007). D-pen is well known for two properties: 1) the formation of disulfide links due to the thiol group; 2) the ability to react with aldehydes to form a thiazolidine ring because it also has an amino group (Masson et al., 2004a; 10

30 Willemsen et al., 1990). D-pen is chemically reactive without metabolism; it can react with protein thiols to form mixed disulfides, and it reacts with aldehydes to form a thiazolidine ring (Howard-Lock et al., 1986). One of the signalling pathways between macrophages and T cells involves reaction of an aldehyde on macrophages with an amine on T cells, forming a reversible imine linkage (Figure 7) (Rhodes, 1989). Previous research in our lab demonstrated that D-pen is able to bind to the surface aldehyde groups on macrophages, and it can also activate macrophages both in vivo and in vitro. Robert Li used an aldehyde reactive probe (ARP, see Figure 8) to study D-penmacrophage binding due to its aldehyde-reactive property, and its binding to several proteins was also identified. Furthermore, a microarray study showed that after a 6 hour incubation with D-Pen, several known macrophage activation markers were upregulated (Li et al., 2009). These data suggest that the irreversible reaction of D-pen with the aldehyde groups on macrophages leads to their activation, and in some cases, this may lead to a generalized autoimmune syndrome. This mechanism may not only apply to D-pen but also to other drugs. Hydralazine and isoniazid can also induce a lupus-like syndrome in humans, similar to D-pen (Diamanti et al., 2007). This could be because they both have a hydrazine group that can react with aldehydes to form a hydrazone (see Figure 9). The binding of a drug with aldehyde-containing proteins on antigen presenting cells may represent a mechanism by which drugs can induce IDRs. 11

31 Figure 7. Representation of the signalling pathway between antigen presenting cells (APCs) and other macrophages and T cells involving reaction of an aldehyde on macrophages with an amine on T cells leading to a reversible imine linkage. Figure 8. The chemical structure of ARP 12

32 Figure 9. The schematics of aldehydes binding to D-pen, hydralazine, and isoniazid Brown Norway rat model IDRs are very difficult to study because they are very complex, and they are just as idiosyncratic in animals as they are in humans; therefore, animal models that are directly relevant to humans are quite rare (Uetrecht, 2008). In our lab, BN rats are used as an animal model to study D-peninduced autoimmunity because the syndrome induced on BN rats is similar to the autoimmunity caused by D-Pen in humans. It includes a skin rash, elevated IgE, proteinuria, immune complex glomerulonephritis, vasculitis, production of antinuclear antibodies, hepatic necrosis, arthritis, and weight loss (Donker et al., 1984; Tournade et al., 1990). Therefore the mechanism of autoimmunity in BN rats is very likely similar to that in humans. D-pen-induced autoimmunity in rats is also idiosyncratic because it is strain specific: Lewis and Sprague-Dawley rats do not develop any symptoms when given D-Pen at a dose of 20 mg/day (Donker et al., 1984). 13

33 Moreover, even though BN rats are highly inbred and syngeneic, it only occurs in a little over 50% of male BN rats (Uetrecht, 2008). In addition, there is a delay of about 3 weeks between starting treatment and the onset of the autoimmune syndrome, which is typical of an idiosyncratic reaction. The dose response curve in the BN rat model is unusual: a dose of 20 mg/day is required to induce the syndrome and an increase to 50 mg/day does not significantly increase the incidence, but at 10 mg/day the incidence is zero, and in fact, this low dose leads to immune tolerance, and subsequent treatment with 20 mg/day does not lead to autoimmunity (See Figure 10) (Masson et al., 2004b). Even though it is an immune-mediated reaction, the time to onset is not shortened on rechallenge. Therefore, D-pen-induced autoimmunity in BN rats provides a very important model for mechanistic studies of IDRs. Figure 10. The dose-response relationship of D-pen-induced autoimmunity in BN rats. The incidence of D-pen-induced autoimmunity can be influenced by manipulation of the immune system. The incidence and severity of autoimmunity in the BN rat can be increased by 14

34 a single dose of poly (I:C), which mimics viral RNA and stimulates macrophages through tolllike receptor 3 (Sayeh et al., 2001). Lipopolysaccharide, a toll-like receptor 4 agonist, shares a similar, but smaller effect as poly (I:C) (Masson et al., 2004a). As mentioned above, 2 weeks of low dose D-pen treatment (5 10 mg/day) prior to a dose of 20 mg/day leads to tolerance in 100 percent of BN rats (Masson et al., 2004b). Adoptive transfer of spleen cells from a tolerant animal led to tolerance in naïve animals, thus indicating that it is immune tolerance (Seguin et al., 2004). CD4+ T cells appear to be the major cell responsible for this tolerance; when tolerized animals are treated with 20 mg/day their CD4 + T cells express increased levels of IL- 10 and transforming growth factor-beta (TGF-ß) mrna (Masson et al., 2004b). One dose of misoprostol (a prostaglandin E analog) prevents penicillamine-induced autoimmunity (Seguin et al., 2003a). Treatment of tolerized animals with a combination of poly (I:C) and D-pen partially overcomes tolerance, and it also appears that depletion of macrophages during tolerance induction partially prevents the induction of tolerance (Masson et al., 2004b; Phillips-Howard et al., 1990). This animal model appears to be a valid model of the autoimmune reactions induced by D- pen in humans. Previous studies indicate that the basic mechanism appears to involve direct activation of macrophages with the production of IL-6, but it is not clear in this highly inbred strain of animals why only some of the animals produce an early spike in IL-6, which appears to be essential for the later development of autoimmunity. The activation of macrophages may be an essential step in the initiation of IDRs in general. 4. Immune system and IDRs 4.1. Th17 cells and autoimmune disease 15

35 Figure 11. The differentiation of naive CD4+ T cells into Th1, Th2, and Th17 cells. Ever since it was proposed in 1986, the Th1-Th2 hypothesis has been a major aspect of mechanistic theories of T cell-mediated diseases. For example, organ-specific autoimmune diseases were thought to be driven by Th1 cells (Mosmann, 1992; Mosmann et al., 2005; Singh et al., 1999; Zamvil et al., 1990). A major part of the evidence supporting the role of Th1 cells in autoimmune diseases was obtained from studies of IL-12 (an essential cytokine in Th1 cell development) in several animal models of autoimmune diseases such as experimental autoimmune encephalomyelitis (EAE). However, the Th1 theory of organ specific autoimmunity was challenged because Th1 cytokines were often found to be protective. As a result, much of the attention has switched from IL-12 to IL-23, which contains a unique p19 subunit while sharing a p40 subunit with IL-12. Additional studies of the involvement of IL-23 in autoimmune diseases led to the discovery of a new helper T cell subset characterized by the production of a proinflammatory cytokine, IL-17, which were therefore called Th17 cells (Langrish et al., 2005; McKenzie et al., 2006; Murphy et al., 2003; Weaver et al., 2006). Since its discovery, the signature cytokine pattern of Th17 cells has been expanded with addition of several other key inflammatory cytokines such as IL-21 and IL-22. In spite of many unknowns 16

36 in the function of Th17 cells, significant progress has been made in characterizing this new T cell population. A large number of studies have found that a combination of TGF- and IL-6 is required for the initial commitment of naïve T cells to produce Th17 cells (Bettelli et al., 2006; Mangan et al., 2006; Zhou et al., 2007); exposure to TGF- in the absence of IL-6 leads to T regulatory cells believed to play an important role in immune tolerance (Zhou et al., 2008). In contrast, IL-23 was found to play a very important role in maintaining the growth and expansion of Th17 cells. In addition, the role of transcription factors or signalling molecules such as STAT3, RORγt, and RORα in regulating the expression of IL-17 was discovered (Harris et al., 2007; Ivanov et al., 2006; Yang et al., 2008; Zhou et al., 2009). Numerous studies in both humans and mice strongly suggest that the Th17 cell is a major determinant of the development of many kinds of autoimmune diseases (Fossiez et al., 1996; Ouyang et al., 2008) NK/NKT cells and autoimmune disease Natural killer (NK) cells are a type of cytotoxic lymphocyte that constitutes a major component of the innate immune system, which can be defined as CD161 + /CD3 - cells (Empson et al., 2010). NK cells play a major role in the rejection of tumours and cells infected by viruses. They kill cells by releasing small cytoplasmic granules of proteins called perforin and granzyme that cause the target cell to die by apoptosis (Vivier et al., 2011). They were named "natural killers" because of the initial notion that they do not require activation in order to kill cells that are missing "self" markers of major histocompatibility complex (MHC) class I (Vivier et al., 2011). Cytokines play a crucial role in NK cell activation. Because cytokines often represent stress molecules released by cells upon viral infection, they serve to signal to the NK cell the presence of viral pathogens. 17

37 Natural killer T (NKT) cells are a heterogeneous group of T cells that share properties of both T cells and NK cells, defined as CD161 + /CD3 + cells (Godfrey et al., 2004). NKT cells share other features with NK cells as well, such as CD16 and CD56 expression and granzyme production (Vivier et al., 2004). Upon activation, NKT cells are able to produce large quantities of interferon-gamma, IL-4, and granulocyte-macrophage colony-stimulating factor, as well as multiple other cytokines and chemokines (such as IL-2, IL-13, IL-17, IL-21 and TNF- ) Regulatory T cells and immune tolerance Regulatory T cells, also called Treg cells, are a specialized subpopulation of T cells that act to suppress activation of the immune system and thereby maintain immune system homeostasis and tolerance to self-antigens (Sakaguchi et al., 2010). Natural Treg cells can be identified as CD4 + /CD25 + /FoxP3 +. One interesting phenomena of D-pen-induced autoimmunity is tolerance. In our BN rat model, the incidence is only 50-80% at the dose of 20 mg/day. However, at a dose of 5 to 10 mg/day, the incidence is 0%, and in fact, the lower dose induces tolerance to the 20 mg/day dose (Masson et al., 2004b). This is clearly immune tolerance because it can be transferred to naive animals with spleen cells or T cells from a tolerized animal. These data suggest that Treg cells might play an important role in this immune tolerance. In order to clarify the mechanism of this tolerance, peripheral Treg cells were studied during D-pen and AQ treatment. Temozolomide (TMZ) is one of the most effective chemotherapeutic agents against glioblastoma; however, Banissi reported that it was able to deplete Treg cells in rats (Banissi et al., 2009). 6-Formylindolo[3,2-b]carbazole (FICZ) is a chemical with significant structural similarity to a high affinity natural aryl hydrocarbon receptor, which has been reported to enhance Th17 development and down regulate Treg cells (Ho et al., 2008). To clarify the involvement of Treg cells, TMZ and FICZ were used as co-treatments to regulate Treg cells in 18

38 vivo. If Treg cells are really involved or play an important role in this drug-induced tolerance, then after depletion or regulation of Treg cells, a significant change of severity and/or incidence of this autoimmunity should be noticed Macrophages and drug-induced liver injury Macrophages are a critical activator of the immune response. Dendritic cells (DC) are a special kind of macrophage, which is the primary sensor of the innate immune system and detects the presence of pathogens through highly conserved pattern recognition receptors, including the toll-like receptors. Once DCs initiate contact with the naïve T-cells expressing the appropriate T-cell receptor, DC costimulatory markers interact with CD28 on the T-cell surface, culminating in the initiation of naïve T-cell activation. The cytokines produced by DCs play a critical role for naïve T-cell differentiation into effector Th1, Th2, or Th17 cells. Figure 12. Characterization of M1 and M2 macrophages. 19

39 Analogous to the Th1 and Th2 dichotomy of T cell polarization, macrophages can be polarized by the microenvironment to mount specific M1 or M2 functional programs (Gordon et al., 2005; Mantovani et al., 2005; Mantovani et al., 2002). Classic or M1 macrophage activation in response to microbial products or interferon (IFN)-γ is characterized by a high capacity to present antigen; high IL-12 and IL-23 production and consequent activation of a polarized type I response, and high production of nitric oxide (NO) and reactive oxygen intermediates (Table 1). Thus, M1 macrophages are generally considered potent effector cells that kill intracellular microorganisms and tumor cells and also produce copious amounts of pro-inflammatory cytokines. In contrast, alternative activation of macrophages is promoted by various signals [e.g., IL-4, IL-13, glucocorticoids, IL-10, immune complexes, and toll-like receptor (TLR) ligands] that elicit different M2 forms, which are able to tune inflammatory responses and adaptive Th2 immunity, scavenge debris, and promote angiogenesis, tissue remodelling, and repair (Table 1) (Mantovani et al., 2002; Pulendran et al., 2010). Activation of the classic pathway is stimulated by proinflammatory cytokines, such as TNF-α and IL-1, as well as by recognition of pathogenassociated molecular patterns, and is mostly involved in innate immunity (Bonizzi et al., 2004). M2 macrophage polarization is induced by antiinflammatory cytokines and growth factors, including IL-4, IL-10, and TGF-alpha (Mantovani et al., 2004). 20

40 Table 1. Characteristics of activated macrophage subpopulations (Benoit et al., 2008; Fairweather et al., 2009; Laskin, 2009). There is now growing evidence that the different subtypes of macrophages induce different type of subpopulations in T cells, and thus, control the fate of the immune response from the very beginning of it. It has been suggested that the programmed cell death (apoptosis) of selfcells maintains immune tolerance, and that an increase in inflammatory cell death (necrosis) induces immune responses that could lead to autoimmune disease (Viorritto et al., 2007). Thus, how the antigens are taken up by macrophages may determine the nature of the immune response. TNF- is involved in triggering apoptosis in many immune cells and also in macrophages (Raza et al., 2010). The activation of Langerhans cells by weak antigens can induce Th2 responses and even Treg cells (Pulendran et al., 2010). Additionally, this type of antigen can modify the differentiation pathways of macrophages and induce M2 preferentially over M1 type macrophages (Mantovani et al., 2002). It has been reported that macrophage 21

41 migration inhibitory factor (probably from activated macrophages) can induce the production of Th17 cells, and that the induction of Fas can lead to activation of M1 macrophages (Brown et al., 2004; Stojanovic et al., 2009). This evidence supports the idea that the initial differentiation of macrophages is important in determining whether an immune response or tolerance will be induced Toll-like receptors and autoimmune disease Toll-like receptors (TLRs) are a class of proteins that play a key role in the innate immune system (Rahman et al., 2006). Growing evidence suggests that TLRs may also play an important role in autoimmune diseases (for example systemic lupus erythematosus), including the activation of B cells, production of autoantibodies, and in the subsequent disease progression after immune complex formation. Our previous research indicated that B cells and NK cells may play important roles in D-pen-induced autoimmunity. In addition, TLR ligands such as Poly I:C and LPS can regulate the onset and incidence of D-Pen-induced autoimmunity. This suggests involvement of immune system. In this research, additional TLR ligands were used as immunoregulators to clarify the involvement of innate immune system, more specifically, TLRs (Waltenbaugh C, 2008). 5. Specific hypotheses and objectives Hypotheses: 1) AQ-induced IDILI is immune-mediated. 2) The dominant response to drugs that cause IDILI is immune tolerance. 3) A reactive iminoquinone metabolite is responsible for AQ-induced liver injury. 4) Th17 cells are involved in the pathogenesis of D-pen-induced autoimmunity. 22

42 Specific Objectives: 1) To establish an animal model of AQ-induced liver injury that mimics the mechanism of IDILI in humans. 2) To investigate the mechanism of IDILI in the AQ animal model; specifically, to determine the involvement of different immune components. 3) To further investigate the involvement of NK cells by co-treatment with DMSO, retinoic acid, and NK1.1 antibodies; 4) To investigate the involvement of adaptive immune system in AQ-induced IDILI with Rag1 -/- mice; 5) To develop a model of severe AQ induced IDILI by increasing covalent binding with BSO and diethylmaleate to deplete glutathione. 6) To develop a model of severe AQ-induced IDILI model by co-treatment with anti- CTLA-4 antibodies in PD-1 -/- mice; To determine the involvement of Th17 cells in D-pen-induced autoimmunity by flow cytometry, cytokine profiling, and co-treatment of retinoic acid. 23

43 Chapter 2: Establishing an Amodiaquine-induced Hepatotoxicity Model in Rats Feng Liu 1, Ping Cai 1, Imir Meushi 1, Jinze Li 2, Tetsuya Nakayawa 1, Jack Uetrecht 1* 1 Leslie Dan Faculty of Pharmacy, University of Toronto, Toronto, Ontario, Canada M5S 3M2 2 Department of Immunotoxicology, R&D, Drug Safety Evaluation, Bristol-Myers Squibb Company, New Brunswick, New Jersey, USA *To whom correspondence should be addressed. Tel: Fax: jack.uetrecht@utoronto.ca. Feng Liu and Ping Cai contributed equally to this study and share first authorship, and this chapter will be submitted to Journal of Immunotoxicology. All work was completed by Feng Liu in this chapter. 24

44 1. Abstract Amodiaquine (AQ) is associated with a relatively high incidence of idiosyncratic drug-induced liver injury (IDILI) and agranulocytosis. In this study, treatment of rats resulted in a mild delayed onset liver injury that resolved despite continued treatment with AQ. Immunohistochemistry indicated the presence of Kupffer cell activation, apoptosis, and hepatocyte proliferation in the liver. There was also an increase in serum IL-2, IL-5, IL-9, IL-12, MCP-1, and TGF-β, but a decrease in leptin. Co-treatment with cyclosporin prevented AQinduced liver injury, while Poly I:C co-treatment caused an earlier increase in alanine aminotransferase (ALT), which suggested that AQ-induced liver injury is immune-mediated. Coincident with the elevated serum ALT, liver CD4 + T cells, IL-17 secreting cells, and Th17/Treg cells were increased at week 3 and decreased during continued treatment. An increase in CD161+ cells and activated M2 macrophages were also observed during the liver injury. These results suggest that the outcome of the liver injury is determined by the balance between effector cells and regulatory cells. Similar results were also observed in AQ-treated C57BL/6 mice. Covalent binding of AQ was detected in the liver, and AQ forms a glutathione conjugate; however, depletion of glutathione did not increase covalent binding and paradoxically prevented AQ-induced liver injury. Retinoic acid (RA), which has been reported to enhance natural killer (NK) cell activity, exacerbated AQ-induced liver injury. These characteristics indicate that we have established a rat model of AQ-induced liver injury that mimics mild AQ-induced liver injury in humans. The results suggest that AQ-induced IDILI is immune mediated, and the subsequent adaptation appears to represent immune-tolerance. 25

45 2. Introduction Drug-induced liver injury (DILI) is a common reason for withdrawing drugs from the market (Kaplowitz, 2001). Approximately 50% of liver failure cases are drug-induced, and 13% of DILI is idiosyncratic drug-induced liver injury (IDILI), which makes up approximately 20% of severe liver injury requiring hospitalization in the US (Lee, 2003). The prediction and prevention of IDILI has been impossible due to the low absolute incidence, a lack of screening methods, and limited knowledge of the underlying mechanisms. Valid animal models are essential to rigorously test mechanistic hypotheses (Adams et al., 2010; Lee et al., 2005). A better understand of the mechanism of IDILI will have significant impact on drug development and clinical application. Amodiaquine (AQ) is effective for both prophylaxis and treatment of malaria. It was withdrawn from the market in the US due to agranulocytosis and hepatotoxicity with an incidence of 1: 2,000 (Clarke et al., 1991; Phillips-Howard et al., 1990; Taylor et al., 2004; Watkins et al., 1984). However, it is still the first-line antimalarial drug in many parts of Africa due to its low price and effectiveness against chloroquine-resistant malaria. The first case of AQ-associated agranulocytosis was reported in 1957; since then there have been many reports of similar cases of agranulocytosis and hepatotoxicity (Akindele et al., 1976; Akpalu et al., 2005; Amouretti et al., 1986; Bernuau et al., 1988; Charmot et al., 1987; Glick, 1957; Guevart et al., 2009; Kamagate et al., 2004; Larrey et al., 1986; Markham et al., 2007; Neftel et al., 1986; Sturchler et al., 1987; Woodtli et al., 1986). The hepatitis can be severe, and several fatal cases or cases requiring liver transplantation have been reported (Amouretti et al., 1986; Bernuau et al., 1988; Glick, 1957; Guevart et al., 2009; Markham et al., 2007). As documented in a postmarketing adverse events report in the UK, the incidence of AQ-induced serious hepatic injury is estimated to be ~1 in 15,000 (Hirschel, 2003). 26

46 Figure 13. Postulated mechanisms of AQ-induced liver injury. AQ is metabolized by CYP2C8 in the liver to N-desethylamodiaquine, bisdesethylamodiaquine, and 2-hydroxyamodiaquine (Adjei et al., 2009; Gil, 2008; Li et al., 2002). The primary metabolite, N-desethylamodiaquine, which concentrates in the blood cells, is the major species responsible for AQ s antimalarial effect (Adjei et al., 2009; Churchill et al., 1985; Li et al., 2002). The mechanism by which AQ causes hepatotoxicity is unknown; both direct toxicity and indirect immune-mediated hypersensitivity have been postulated (Clarke et al., 1991; Lind et al., 1973). AQ is oxidized to a reactive iminoquinone by human liver microsomes and peroxidases; this chemically reactive iminoquinone covalently binds to glutathione (GSH) and cellular macromolecules, and this binding is presumably responsible for the liver injury, either directly or indirectly via an immunological mechanism (See Figure 13) (Harrison et al., 1992; Jewell et al., 1995; Shimizu et al., 2009). 27

47 Clinical characteristics of AQ-induced hepatotoxicity suggest an immune-mediated mechanism: a delayed onset, the presence of anti-drug IgG antibodies, and a prompt increase of ALT on rechallenge with AQ (Clarke et al., 1991; Neftel et al., 1986; Uetrecht, 2005). However, due to the low incidence, unpredictability, and lack of a valid animal model, it has been impossible to study the mechanism in detail. Therefore, the development of a valid animal model with characteristics similar to the IDILI that occurs in humans would greatly increase our understanding of this IDILI, and by extension, other IDILI caused by other drugs. Recently, our lab developed a new model of AQ-induced liver injury in C57BL/6 mice with characteristics similar to IDILI in humans (Metushi et al., 2014a). Studies in this model suggest that NK cells play an important role in mild injury, and that immune tolerance limits the injury, which is similar to the adaptation that occurs in most patients. This is the first animal model of IDILI that mimics the clinical characteristics of mild IDILI in humans, and the injury appears to be immune mediated. Further study indicated that co-treatment of PD-1 -/- mice with anti-ctl-4 antibody and AQ resulted in a more severe liver injury with piece-meal necrosis that did not resolve with continued treatment (Metushi et al., 2014b). This is the first animal model of IDILI similar to more severe IDILI in humans, and it was accomplished by breaking immune tolerance. In the present study, the model of AQ-induced liver injury was extended to several strains of rats, including Brown Norway (BN), Lewis, and Wistar rats to determine the generality of the model. In addition, pathological changes, apoptosis, and cell proliferation were determined. We also examined cytokines/chemokines and anti-aq antibodies. To further test the effects of immune system perturbation, the effects of co-treatment with cyclosporine (CYC), polyinosinic:polycytidylic acid (Poly I:C), and retinoic acid were studied. The nature of the immune response was studied by phenotyping of lymphocytes and macrophages in different organs. To better understand the mechanism of AQ-induced hepatotoxicity, we investigated the 28

48 covalent binding of AQ to hepatic proteins. In addition, given the fact that AQ forms a GSH conjugate, the effects of GSH depletion were studied. 3. Materials and methods 3.1. Animals and sample preparation Male BN, male and female Lewis, or Wistar rats (150~175 g) were purchased from Charles River (Montreal, QC, Canada) and housed in standard cages (2 per cage) in a hour lightdark cycle at 22 ºC. The rats were given free access to standard rat chow and tap water for a week-long acclimatization period, and then the rats were given AQ suspended in saline, 6 days/week by oral gavage at a dose of 62.5 mg/kg/day. Male C57BL/6 mice (6-8 weeks) were obtained from Charles River (Montreal, Quebec). The mice were kept in standard cages (4 per cage) in a hour light-dark cycle at 22 ºC. The mice were given free access to standard mice chow and tap water for a week-long acclimatization period. AQ was mixed thoroughly with food and given to mice at a dose of 0.2% of AQ by weight. Based on food consumption, the dose of AQ to mice is about 200 mg/kg/day. The blood was collected from the tail veins of rats or from the saphenous veins of mice. The harvested blood was allowed to clot for 30 min at room temperature, and then centrifuged at 5000 X g for 5 min at 4 ºC to isolate the serum. The serum was aliquoted and stored at -80 ºC for future biochemical assays. All animal protocols used in this study were approved by the University of Toronto Animal Care Committee and conducted in an animal facility accredited by the Canadian Council on Animal Care Detection of liver injury and cytokines/chemokines The liver injury marker, ALT (Infinity kit, Pittsburgh, PA) was measured according to the manufacture s instructions. Twenty-three cytokines or chemokines (Eotaxin, GM-CSF, G-CSF, 29

49 GRO/KC, IFNγ, IL-1α, IL-2, IL-4, IL-5, IL-6, IL-9, IL-10, IL-13, IL-17, IL-18, IP-10, leptin, MIP-1α, MCP-1, RANTES, TNFα and VEGF) were measured in serum and liver homogenates using a MILLIPlex kit from Millipore (Billerica, MA). The livers were homogenized in 0.9% saline (20 ml/g liver) and then centrifuged at X g. Supernatants from the liver homogenates or serum (12.5 µl) were used for cytokine quantification. The fluorescence was measured by a Luminex-100 luminometer (Bio-Rad, ON). Rat IL-12 +p40 ELISA kits were purchased from Invitrogen Corporation (Camarillo, CA), rat TGF-β1 ELISA kits were purchased from R&D Systems (Minneapolis, MN), and the serum levels were measured according to manufacturer s instructions Histopathology and immunohistochemistry At the end of each experiment, animals were euthanized by CO2, followed by the removal of livers, spleens, cervical lymph nodes, and lungs. The organs were then fixed in 10% neutralbuffered formalin (Sigma-Aldrich; Oakville, ON) followed by paraffin-embedment and haematoxylin and eosin (H&E) staining, which were performed by the Department of Pathology at the Hospital of Sick Children (Toronto, ON). Macrophages in the spleen and liver of rats were detected using mouse anti-rat ED1 (CD68), and ED2 (CD163), and lymphocytes with anti-cd-45ra (OX33) monoclonal antibodies (Santa Cruz Biotechnology Inc.; Santa Cruz, CA). The formalin-fixed paraffin-embedded sections of liver and spleen were deparaffinised with xylene (Sigma-Aldrich) for 5 min X 2 and rehydrated with 100% ethanol (Sigma-Aldrich)X 2, followed by 95%, 80%, and 70% ethanol once for 3 min. After washing, the sections were rinsed with phosphate buffered saline (PBS; Sigma- Aldrich) twice and then immersed in 3.0% H2O2 (Sigma-Aldrich) in methanol to block endogenous peroxidases. Antigens were retrieved by using 10 µg/ml proteinase K (Thermo 30

50 Fisher Scientific Inc., Rockford, IL) at 37 o C for 10 min. After washing, the samples were blocked with 1% bovine serum albumin (BSA; Fisher Scientific, Toronto, ON) for 1 h and then the samples were covered with the primary antibody in 1% BSA for 2 h. After washing, they were incubated with secondary antibody conjugated with horseradish peroxidase (HRP; Bethyl Laboratories Inc., TX) for 1 h. After the final washing in PBS, antibody binding was detected using Vector NovaRED (Vector Laboratories, Inc., Burlingame, CA) as the enzyme substrate. Sections were counterstained with hematoxylin (Sigma-Aldrich), dehydrated, and mounted Apoptosis and cell proliferation Formalin-fixed paraffin-embedded sections of the liver and spleen were stained for the presence of proliferating cell nuclear antigen (PCNA) by using a biotinylated anti-pcna antibody kit (ZYMED Laboratories Inc.; San Francisco, CA) according to the manufacturer s instructions. Formalin-fixed paraffin-embedded sections of the liver were stained for the presence of internucleasomal DNA fragments by using Apo-Brdu-IHC kit from BioVision (Milpitas, CA), which was a two-color terminal deoxynucleotidyl transferase dutp nick end labelling (TUNEL) assay for labelling DNA breaks in apoptotic cells. Meanwhile, serum cytochrome C and osteopontin were also measured as possible markers of apoptosis and cell proliferation, respectively. The Quantikine rat/mouse cytochrome C ELISA kit and the Quantikine mouse osteopontin kit were purchased from R&D Systems Inc. (Minneapolis, MN) Detection of serum IgG anti-aq antibody To determine if AQ-induced liver injury had immune memory, male BN rats were treated with AQ at 62.5 mg/kg for 3 weeks, the AQ withdrawn for 4 weeks, and then the rats were rechallenged with AQ for 4 weeks. Serum ALT, cytokines, chemokines, and anti-aq antibodies were measured. An AQ-metallothionein conjugate was prepared according to the protocol from 31

51 Kevin Park s lab except manganese dioxide (MnO2, Sigma-Aldrich) was used as the oxidant instead of silver oxide (Clarke et al., 1990). Briefly, AQ (0.148 mmol) in 2 ml of chloroform was stirred with MnO2 (115 mg) in the presence of anhydrous sodium sulphate (50 mg) for 30 min at room temperature. The reaction mixture was filtered and the solvent evaporated under nitrogen. The quinone imine of amodiaquine (AQQI) precipitated and was dissolved in 6 ml of dioxane (Sigma-Aldrich), which was added dropwise to a stirred solution of 10 mg rabbit metallothionein (rmt) in phosphate buffer (5 ml, 50 mm, ph 7.2) over a period of 30 min. Ascorbic acid (50 mg, Sigma-Aldrich) was added to reduce unreacted AQQI, and the solution was dialyzed for 24 h against phosphate buffer (20 mm, ph 7.2). An ELISA Starter Accessory Package was bought from Bethyl (Montgomery, TX). The anti-rat IgG-HRP was purchased from Cell Signaling Technology (Danvers, MA). The microwell strips were coated with AQrMT (4 µg/well) in coating buffer. The serum was diluted 1:400 in saline, 100 µl samples were added, and the strips were incubated for 2 h. The 3,3,5,5 -tetramethylbenzidinesubstrate solution (100 µl) was added and incubated for 10 min at room temperature. Serum IgG anti-aq antibody was measured according to the manufacturer s instructions Measurement of serum concentrations of AQ Methanol (80 µl; VWR; Mississauga, ON) containing 0.1 µm internal standard (4- dimethylaminoantipyrene; Sigma-Aldrich) was added to 10 µl of serum sample to precipitate the protein. The mixture was vortexed and stored at -20 o C for 30 min. Thawed samples were centrifuged at X g for 10 min. The supernatant was evaporated to dryness under a gentle stream of nitrogen. Residues were dissolved in 100 µl of the HPLC mobile phase, and 20 µl of the aliquot was injected to the LC MS/MS system consisting of Shimadzu LC10 HPLC and a API3000 mass spectrometer (PE Sciex, Concord, ON, Canada). Separation was done on a 32

52 mm Luna C18 3µm analytical column from Phenomenex (Torrance, CA) and the flowrate was 0.2 ml/min. The mobile phases used for chromatography were 0.1% formic acid (solvent A; Sigma-Aldrich) and methanol (solvent B; Sigma-Aldrich). The mobile phase was delivered using a linear gradient elution program: 5% methanol (solvent B) at 0 min and increased to 90% solvent B at 2.5min. Multiple reaction monitoring transitions were m/z 356 m/z 283 for AQ, m/z232 m/z 56 for internal standard, respectively. Data were collected and processed using Analyst software (Applied Biosystems, Burlington, ON) Modulation of the immune response An immunosuppressant, cyclosporine (20 mg/kg/day), was administrated by oral gavage to male BN rats for 5 weeks during treatment with AQ at the dose of 62.5 mg/kg/day. Another group of BN rats were treated with same dose of AQ for 5 weeks. Control rats were treated with water only. Blood samples were drawn from the tail vein on Days 0, 7, 14, 21, 28, and 35. Liver injury was monitored by serum ALT. In another experiment, Poly I:C was used to stimulate the immune system. Poly I:C was intraperitoneally injected at a dose of 10 mg/kg in male BN rats 1 day before the start of AQ treatment. Another group of BN rats was treated with same dose of AQ for 6 weeks. Control rats were intraperitoneally injected with PBS only Phenotyping of macrophages and lymphocytes BN rats treated with AQ were monitored for liver injury by serum ALT, and groups of animals were sacrificed weekly for evaluation of the phenotype of mononuclear cells in the blood, liver, spleen, and cervical lymph nodes by flow cytometry. Paraffin slices of liver and spleen were also prepared for histological studies. Lymphocyte isolation from liver: Rats were injected with 0.2 ml heparin (1000 U/ml, i.p.; Fisher Scientific) 10 min before sacrifice. Right after the sacrifice, a needle was carefully 33

53 inserted into the portal vein. The liver was perfused with 50 ml PBS (ph 7.0; Sigma-Aldrich) with heparin (100 U/ml) and then kept in cold PBS. The liver was dissected and gently passed through a 200-gauge stainless steel mesh and then suspended in RPMI 1640 medium (Sigma- Aldrich) containing 100 ml/l fetal calf serum (FCS; Sigma-Aldrich). The above cell suspension was centrifuged at 350 X g for 5 min. The pellet was resuspended in 35% Percoll solution (Sigma-Aldrich) containing 10 U/ml heparin, and then loaded on the layer of 50% Percoll solution followed by centrifugation at 500 X g for 15 min at 4 C. The cells were aspirated from the Percoll interface and harvested by centrifugation. The pellet was resuspended in 2 ml RBC lysis buffer (ebioscience, San Diego, CA) for 10 min. The cells were washed twice and suspended with Hanks Balanced Salt Solution (HBSS; Sigma-Aldrich) containing 1% FCS. Macrophage isolation from liver: the cell pellet from liver was prepared as described in last paragraph. The pellet was resuspended in 100 ml digestion buffer and incubated at 37 C water bath for 30 min. The digestion buffer refers to Dulbecco's Modified Eagle's Medium (DMEM; Sigma-Aldrich) containing 250 µg/ml collagenase IV (Life Technology, Burlington, ON) and MgSO4, 15 mg/100 ml (VWR, Mississauga, Canada). The cell suspension was centrifuged at 30 X g for 2 min to remove the precipitate. The supernatant were centrifuged again at 350 X g for 5 min. The pellet was suspended in ml of 11% Nycdenz (Sigma-Aldrich) in DMEM and loaded on 3 ml 18% Nycodenz in HBSS, and then centrifuged at 800 X g for 15 min. The white interface was collected and washed 3-4 times with DMEM, followed by centrifugation for 15 min at 800 X g. The pellet (liver macrophages) was suspended in DMEM containing 1% FCS for further analysis. Flow cytometry: single cell suspensions from spleen, lymph nodes (5 nodes), and blood (1 ml) as previously described (Metushi et al., 2014a). Lymphocytes and macrophages were isolated from livers according to the protocol above. Anti-rat CD32 antibody was used to block 34

54 non-specific binding. Anti-rat CD161 (NK1.1) and CD4 antibodies were used to stain CD4 T cells, NK cells, and NKT cells, respectively. Th17 cells were studied by intracellular staining with anti-rat CD4 and anti-mouse IL-17 antibodies in the presence of phorbol 12-myristate 13- acetate (20 ng/ml) + ionomycin (1 µg/ml) for 5 h. Treg cells were characterized as CD4 + /CD25 + /Foxp3 +, Th17 cells as CD4 + /IL-17 +, Tc17 cells as CD8 + /IL-17 +, IL-17 producing cells as IL-17 +, NK cells as CD4 - /NK1.1 +, NKT cells as CD4 + /NK1.1 +, NK17 cells as CD4 - /NK1.1 + /IL Macrophages were stained by anti-rat CD68, CD163, MHC II, TNFα, CCL2, and IL-10 antibodies. As shown in Table 2, M1 macrophages were characterized as MHC II + /CD163 + /CD68 -, activated M1 macrophages as MHC II + /CD163 + /CD68 - /CCL2 + /TNFα + /IL10 -, M2a macrophages as MHC II + /CD163 - /CD68 +, activated M2a macrophages as MHC II + /CD163 - /CD68 + /CCL2 - /TNFα - /IL10 +, M2b macrophages as MHC II + /CD163 + /CD68 +, activated M2b macrophages as MHC II + /CD163 + /CD68 + /CCL2 - /TNFα + /IL10 +, M2c macrophages as MHC II - /CD163 - /CD68 +, activated M2c macrophages as MHC II - /CD163 - /CD68 + /CCL2 - /TNFα - /IL10 +. All antibodies above were purchased from ebioscience (San Diego, CA). 4',6-Diamidino-2- phenylindole (DAPI, Molecular Probes, Burlington, ON) was used as a live cell marker. Cells were counted with a LSRII cytometer (Becton Dickinson Immunocytometry System, San Jose, CA) and analyzed using FlowJo 10 software (Tree Star, Inc., Ashland, OR). 35

55 Table 2. Antibodies used for macrophage phenotyping and activation (Benoit et al., 2008; Fairweather et al., 2009; Laskin, 2009) NK cell activation/stimulation It was recently reported that retinoic acid (RA) is able to activate liver NK cell killing of hepatocytes and hepatic stellate cells (Lee et al., 2012; Ochi et al., 2004; Radaeva et al., 2007; Taimr et al., 2003). RA (Sigma-Aldrich) was dissolved in saline at the concentration of 300 mg/ml. Male BN rats were treated with AQ by gavage at the dose of 62.5 mg/kg/day for 4 weeks. Meanwhile, RA was also given by oral gavage at the dose of 30 mg/kg/day as co-treatment for 4 weeks. Dimethyl sulfoxide (DMSO) was reported to be able to activate hepatic NK and NKT cells in vivo as evidenced by increased NK/NKT cell numbers and higher intracellular levels of the cytotoxic effector molecules such as IFN- and granzyme B in both cell types (Masson et al., 2008). In another study, DMSO was also employed to activate NK/NKT cells. A 10% DMSO (Sigma-Aldrich) solution was prepared in PBS (Sigma-Aldrich). Male BN rats were treated with AQ by gavage at a dose of 62.5 mg/kg/day for 5 weeks. The low dose DMSO group of animals 36

56 were intraperitoneally injected with 2 ml 10% DMSO on Day -1 (1 day before the AQ treatment); high dose DMSO group of animals were intraperitoneally injected with 2 ml 10% DMSO on Day -1, Day 7, and Day 14; Control animals were intraperitoneally injected with 2 ml of PBS Western blotting Rats were euthanized and a large part of liver was removed from at least 2 different lobes and placed in 30 ml of PBS (ph7.4; Sigma-Aldrich). Liver (0.5 g) was placed in a 15 ml tube, and 5 ml Cell Lysis Buffer (Fisher Scientific) with protease inhibitor was added. Liver was homogenized for at least 2 min, followed by centrifugation at 1000 X g for 10 min at 4 C. Two ml of the middle layer was collected and centrifuged at 9000 X g for 30 min at 4 C. The middle layer was collected and stored in 100 µl aliquots at -80 C. Liver proteins (20 µg) were loaded onto a 8% SDS gel and run for approximate 1 h (5 W, 150 V). Protein transfer was performed for 1.5 h at 250 ma (approximately 25 W, 100 V) in the cold room, followed by 1 h incubation with 5% non-fat milk (Bio-Rad, Hercules, CA) at room temperature. The membrane was incubated with primary antibody (anti-aq antibody, 1:10 000) overnight at 4 C. It was washed 3 times for 15 min with tris-buffered saline Tween-20 (TBST; Bioshop, Burlinton, ON) followed by incubation with secondary antibody (goat anti-rabbit conjugated with horseradish peroxidase, 1:20,000; Sigma Aldrich) for 1.5 h at room temperature. After 3 TBST washes, the membrane was stained with Pierce Western Blotting Detection System (Fisher Scientific) for 5 min before imaging by a FluorChem FC2 imager (Alpha Innotech, Toronto, ON) GSH depletion Buthionine sulfoximine (BSO; Sigma, Oakville, Canada) and diethyl maleate (DEM; Sigma) were administrated as co-treatments with AQ to deplete GSH. Male C57/BL6 mice were treated 37

57 with AQ in food for 4 weeks at a dose of 200 mg/kg/day as described above. One week prior to AQ treatment, BSO was given in drinking water (4.4 g/l) and this treatment lasted for 5 weeks. One day before AQ treatment, a dose of DEM (4 mmol/kg) was administrated to mice by i.p. injection. Liver protein was isolated as described above. Liver GSH levels were measured by a GSH assay kit (Cayman Chemical, Ann Arbor, MI) according to manufacturer s instructions Regulatory T cell (Treg) depletion In order to decrease immune tolerance, temozolomide (Sigma) was administrated as a cotreatment because it was reported to be able to deplete Treg cells in rats (Banissi et al., 2009). Male BN rats were treated with AQ at the dose of 62.5 mg/kg/day for 3 weeks. Temozolomide was administered orally with schedules designed to mimic the temozolomide regimens currently used in humans: High dose group with 10 mg/kg per day for 21 days and low-dose temozolomide group with 2 mg/kg per day for 21 days. Peripheral blood Treg cells were analysed by flow cytometry using CD3, CD4, CD25, and Foxp3 mabs. 4. Statistical analyses GraphPad Prism (GraphPad Software, San Diego, CA) was used to perform the statistical analyses. When appropriate, data were analyzed by two-way analysis of variance (ANOVA), Mann-Whitney U-test, or two-tailed Student s t-test. All p values <0.05 were considered significant (*p<0.05; **p<0.01; ***p<0.001). 5. Results 5.1. Serum ALT and AQ concentrations in AQ-treated rats In male BN rats, AQ treatment increased ALT by a factor of 1.7 times after 3 weeks of AQ treatment, and the peak was 2.9 times at week 4 (Figure 14A). The increase in ALT in male 38

58 Wistar rats (Figure 14C) was similar to that in BN rats although more sustained, and the increase in ALT was greatest in male Lewis rats (Figure 14D). The ALT increase was less in female Wistar (Figure 14B) and female Lewis rats (Figure 14E), although female Lewis rats lost about 20% of their body weight. The rats could not be treated with AQ for longer periods because in all cases the intake of food in AQ-treated rats was only 1/2 to 2/3 that of the control rats, and this led to a lack of weight gain or weight loss. The experiments were repeated and the results were similar (data not shown). The serum concentration of AQ in Wistar rats increased over the first week to a level of 0.1 µm and then decreased despite the continued treatment (Figure 15). In contrast, the serum concentration of AQ in BN rats gradually increased and reached a plateau of ~2 µm at day 14, which is within the range of therapeutic concentration of AQ ( nm) in humans (White et al., 1987). 39

59 Figure 14. Liver injury induced by AQ treatment in different strains of rats. AQ (62.5 mg/kg/day) was administrated to different strains of rats for 2 to 6 weeks by oral gavage. (A) Male BN rats. (B) Female Wistar rats. (C) Male Wistar rats. (D) Male Lewis rats; (E) Female Lewis rats. Each bar represents the mean ± SD for 4 animals. The data were analyzed for statistical significance by two-way ANOVA. (Significantly different from the control group *p<0.05, **p<0.01, ***p<0.001). 40

60 Figure 15. Serum concentration of AQ in male BN rats and male Wistar rats. Rats were treated with 62.5 mg/kg/day AQ by gavage for 4 weeks Liver histological changes induced by AQ treatment in Wistar, BN, and Lewis rats In male Wistar rats treated with AQ for 5 weeks, some hyperplastic cells near the central vein of liver were observed on H&E stained sections, and activated Kupffer cells were observed using anti-ed1 (CD 68) staining (Figure 16). Similarly, hypertrophy of Kupffer cells was found in AQ-treated male BN rat liver (Figure 17B). Some hypertrophic cells were also noticed in lung alveolar cells (Figure 17D). However, there were no pathological changes observed in the liver of AQ-treated male or female Lewis rats (data not shown), despite the fact that male Lewis rats had the greatest increase in ALT. These results suggested that AQ is able to induce mild liver injury in rats with possible involvement of Kupffer cells. To determine the involvement of Kupffer cells, male BN rats were treated with AQ. After 5 weeks of treatment, the expression of ED1, ED2, and CD45RA surface markers were increased in the liver (Figure 18), which indicated that AQ treatment resulted in an inflammatory response in the liver. 41

61 Figure 16. Histological and immunohistochemical staining of the liver from a representative male Wistar rat treated with AQ (62.5 mg/kg/day) for 5 weeks. (A) Liver of control (H&E). (B) Liver of AQ-treated rat; arrow shows hyperplastic cells (H&E). (C) Liver of control stained with an anti-ed1 antibody (450X). (D) Liver of AQ-treated rat; the ED1 + cells are stained brown (450X). 42

62 Figure 17. Histological changes in the liver of a representative male BN rat treated with AQ for 5 weeks at a dose of 62.5 mg/kg/day (H&E staining, 450X). (A) Control liver. (B) Liver of AQtreated rat, arrows show hypertrophic Kupffer cells. (C) Control lung. (D). Lung of AQ-treated rat; arrows show some hypertrophic cells. 43

63 Figure 18. Treatment of male BN rats with AQ for 5 weeks is associated with an increase in activated hepatic macrophages and lymphocytes (ED1, 450X; ED2 and CD45, 250X) Apoptosis and cell proliferation in AQ-treated male BN rats In another experiment, TUNEL and PCNA assays were employed to study apoptosis and cell proliferation, respectively. Apoptosis was found to be significantly increased in the liver of AQtreated male BN rats compared to control rats (Figure 19A, 19B). No significant difference in cell proliferation was observed in the liver of AQ-treated male BN rats (Figure 19C, 7D); however, cell proliferation in the germinal center of spleen was found to be significantly increased (Figure 19E, 19F). Serum cytochrome C, which was not detectable in control animals, started to increase from week 3 and reached a peak at week 4 followed by a reduction at week 5 (Figure 20A). This correlates with the serum ALT pattern during AQ treatment (Figure 14A) and suggests a significant increase in cell apoptosis. The serum osteopontin level was significantly increased at day 6 (1.5 times that of control) and reached a peak at 12 days (2.3 times that of control, Figure 20B), which is consistent with lymphocyte activation/cell proliferation. 44

64 Figure 19. Cell apoptosis and proliferation in the liver and spleen in AQ-treated BN rats. (A) TUNEL staining in the liver of control rats (400X). (B) TUNEL staining in the liver of AQtreated rats (400X). (C) PCNA staining in the liver of control rats (400X). (D) PCNA staining in the liver of AQ-treated rats (400X). (E) PCNA staining of the spleen of control rats (250X). (F) PCNA staining of the spleen of AQ-treated rats (250X). Figure 20. Serum levels of cytochrome C and osteopontin in AQ-treated male BN rats after 5 weeks. Values represent the mean ± SE from 4 animals per group. The data were analyzed for statistical significance by two-way ANOVA. (Significantly different from the control group *p<0.05, ***p<0.001). 45

65 5.4. Cytokine and chemokine changes in AQ-treated male BN rats Changes in serum cytokines and chemokines in BN rats induced by AQ treatment are shown in Figure 21: Serum IL-12 was increased on day 3. Serum TGF-β1 and IL-2 were increased on Day 14. Serum IL-5, IL-9, and chemokine MCP-1were increased on Day 28 although the pattern of MCP-1 with respect to time is unusual. However, leptin was significantly decreased after 35 days of the treatment. No significant changes in eotaxin, G-CSF, GRO/KC, IFNγ, IL-1α, IL-4, IL-6, IL-10, IL-13, IL-17, IP-10, MIP-1α, or RANTES were observed (data not shown). Changes in liver cytokines and chemokines were also studied by ELISA and the Luminex assays. RANTES and IL-18 were significantly increased in the AQ-treated group at 5 weeks compared to the control group (Figure 22). However, IL-2, IL-5, IL-6, IL-12 were significantly decreased in the AQ-treated group compared to the control group (Figure 22). No significant changes were observed for eotaxin, G-CSF, GRO/KC, IFNγ, IL-1α, IL-4, IL-9, IL-10, IL-13, IL-17, leptin, TGFβ, IP-10, MIP-1α, or MCP-1 (data not shown). 46

66 Figure 21. Serum cytokine changes in AQ-treated male BN rats after 5 weeks. Values represent the mean ± SE from 4 animals per group. The data were analyzed for statistical significance by two-way ANOVA. (Significantly different from the control group *p<0.05, **p<0.001, ***p<0.001). 47

67 Figure 22. Liver cytokine and chemokine changes in AQ-treated male BN rats after 5 weeks. Values represent the mean ± SE from 4 animals per group. The data were analyzed for statistical significance by student t-test. (Significantly different from the control group *p<0.05, **p<0.001, ***p<0.001) Serum ALT, anti-aq antibody, cytokines, and chemokines in rechallenged male BN rats In order to determine if AQ-induced liver injury had immune memory, BN rats were treated with AQ for 3 weeks, withdrawn for 4 weeks, and then they were rechallenged with AQ for 4 more weeks. The serum ALT started to increase at the 2 nd Week of primary treatment and reached a peak at Week 3, followed by a reduction at Week 4, which was not significantly different from the rechallenge treatment; however, the ALT appeared to be higher on rechallenge (Figure 23). The anti-aq antibody had increased by Week 1 and plateaued by Week 2 (Figure 23). The Luminex assay for cytokines and chemokines showed that IFN-γ (at day 21), IL-13 (at day 21), IL-4 (at day 28) and IL-12p70 (day 28) were increased after rechallenge (Figure 24). No significant changes were observed for eotaxin, G-CSF, GRO/KC, IFNϒ, IL-1α, IL-2, IL-5, IL-6, IL-9, IL-10, IL-17, IL-18, leptin, TGFβ, IP-10, MIP-1α, MCP-1 or RANTES (data not shown). 48

68 Figure 23. Serum ALT and anti-aq antibody in male BN rats during primary and re-challenge AQ treatment. Values represent the mean ± SE from 4 animals per group. The data were analyzed for statistical significance by Mann-Whitney U-test. 49

69 Figure 24. Serum cytokine changes in male BN rats during the primary and rechallenge AQ treatment. Values represent the mean ± SE from 4 animals per group. The data were analyzed for statistical significance by Mann-Whitney U-test Co-treatment of cyclosporin and Poly I:C in BN rats Various co-treatments were used to determine the effect of perturbation of the immune response. Co-treatment with cyclosporine prevented the ALT increase induced by AQ (Figure 25). As shown in Figure 26, co-treatment with the toll-like receptor agonist, Poly I:C, led to an earlier onset of ALT elevation, and the animals appeared quite ill so the animals had to be sacrificed, but Poly-I:C did not appear to significantly increase the severity of the injury and the ALT was decreasing at the time of sacrifice. 50

70 Figure 25. The time course of serum ALT during treatment with AQ and cyclosporine in BN rats. Values represent the mean ± SE. The data were analyzed for statistical significance by twoway ANOVA. (Significantly different from the control group *p<0.05, **p<0.001, ***p<0.001). Figure 26. The time course of serum ALT during treatment with AQ and Poly I:C in BN rats. Values represent the mean ± SE from 4 animals per group. The data were analyzed for statistical significance by two-way ANOVA. (Significantly different from the control group *p<0.05, **p<0.001, ***p<0.001). 51

71 5.7. Lymphocyte phenotyping As shown in Figure 27, a significant ALT increase was observed in AQ-treated BN rats by the 3rd week, and then it decreased by the 4th week. No ALT increase was observed in the control group. However, well before the liver injury was observed, the liver weight significantly decreased (Figure 28). The total number of lymphocytes in the liver slightly increased from the first week and reached the peak at the 3 rd week, then appeared to decrease by the 4 th week, which is coincident with the time course of the ALT (Figures 27 and 28). For the secondary lymphoid organs, both spleen and lymph nodes (data not shown) were enlarged from the first week; an increase in total lymphocyte number in cervical lymph nodes was also noticed during the treatment (Figure 28). Figure 27. The time course curve of serum ALT during AQ treatment in BN rats. Values represent the mean ± SE from 8 animals per group. The data were analyzed for statistical significance by two-way ANOVA. (Significantly different from the control group *p<0.05, **p<0.001, ***p<0.001). 52

72 Figure 28. Time course of changes induced by AQ treatment. Values represent the mean ± SE from 8 BN rats per group. The data were analyzed for statistical significance by Mann-Whitney U-test. (Significantly different from the control group *p<0.05, **p<0.001, ***p<0.001). Phenotyping of lymphocytes in the liver revealed a small increase in CD4 + T cells at Week 3 and then they appeared to decrease by Week 4, which was consistent with the time course of the ALT (Figure 29). Lymph node CD4 + T cells appeared to increase from the first week, but the change did not reach statistical significance, and no significant change was observed in spleen and blood CD4 + T cells. The phenotyping results of Th17 cells and IL-17 secreting cells (Figure 29 and 30) indicate that Th17 and IL-17 secreting cells shared a similar tendency as the CD4 + T cells in liver. In addition, it appears that there may be a reciprocal relationship between the Th17 cells in the liver and spleen. As shown in Figure 30, NK1.1 + cells (NK/NKT cells) started to 53

73 increase in the liver and lymph nodes, and to a lesser extent in the peripheral blood from the 2 nd week, and kept on increasing as the treatment continued. However, no significant change was observed in the spleen. As shown in Figure 31, peripheral blood Treg cells decreased by Week 1 followed by a return towards normal. The decrease of Treg cells was also noticed in livers at Week 1, lymph nodes and spleens at Week 2; however, none of these changes is statistically significant. The ratio of Th17/Treg was calculated as an indication of immune-balance and is depicted in Figure 32. There was a significant increase in the ratio in the spleen and lymph nodes at Week 2 and an increase in the liver at Week 3 the time of maximal ALT - followed by a return toward normal after that. 54

74 Figure 29. CD4 + T cells and Th17 cells in different organs during 4 weeks AQ treatment in BN rats. Th17 cells were characterized as CD4 + /IL Values represent the mean ± SE from 8 animals per group. The data were analyzed for statistical significance by Mann-Whitney U-test. (Significantly different from the control group *p<0.05, **p<0.001, ***p<0.001). 55

75 Figure 30. IL-17 secreting cells and NK/NKT cells in different organs during AQ treatment in BN rats. IL-17 secreting cells were characterized as IL-17 +, NK/NKT cells as NK Values represent the mean ± SE from 8 animals per group. The data were analyzed for statistical significance by Mann-Whitney U-test. (Significantly different from the control group *p<0.05, **p<0.001, ***p<0.001). 56

76 Figure 31. Treg cells in different organs during AQ treatment in BN rats. Treg cells were characterized as CD4 + /CD25 + /Foxp3 +. Values represent the mean ± SE from 8 animals per group. The data were analyzed for statistical significance by Mann-Whitney U-test. (Significantly different from the control group *p<0.05, **p<0.001, ***p<0.001). 57

77 Figure 32. The immune balance of Treg and Th17 in different organs during AQ treatment in BN rats. Treg cells were characterized as CD4 + /CD25 + /Foxp3 +, and Th17 cells as CD4 + /IL Values represent the mean ± SE from 8 animals per group. The data were analyzed for statistical significance by Mann-Whitney U-test. (Significantly different from the control group *p<0.05, **p<0.001, ***p<0.001) Lymphocyte phenotyping in C57BL/6 mice As shown in Figure 33, there was a small, but significant, increase in ALT in mice after one week of AQ treatment, which appeared to slowly decrease after that. The size of lymph nodes (data not shown) and the number of lymphocytes in lymph nodes were increased after 1 week of AQ treatment, which suggested an immune response as shown in Figure 33. Although there was not a significant change in lymphocytes in the liver, there was a significant increase in CD3 +, CD4 +, and CD8 + T cells in lymph nodes (Figure 34). Further lymphocyte phenotyping showed that there was a significant increase of NK and NKT cells in liver, lymph nodes, and PBMC 58

78 after 1 week of AQ treatment, which was followed by a return towards baseline as the treatment continued (Figure 35). This change also applied to CD161 + cells, Th17 cells, Tc17 cells, and NK17 cells (Figure 35 and 36). Serum ALT and lymphocyte phenotyping results were compared between sick and non-sick animals after 1 week of AQ treatment (Not all of the animals had an increase in ALT after one week of AQ treatment), as shown in Figure 37. Compared to control and non-sick animals, sick animals always had a significantly higher level of ALT and more cervical node lymphocytes, including CD161 + cells, NK cells, IL-17 secreting cells, Th17 cells, and NK17 cells, which suggests possible critical roles for NK cells and/or IL-17 related cells at an early stage of this liver injury. Figure 38 shows the phenotyping of Treg cells and PD- 1 + /CD4 + T cells in different organs during the AQ treatment. For both Treg cells and PD- 1 + /CD4 + T cells, the total cell number in the liver did not change significantly during AQ treatment; however, the total cell number in lymph nodes significantly increased after 1 week. Figure 33. Serum ALT level in C57BL/6 mice treated with AQ. Values represent the mean ± SE from 8 animals per group. The data were analyzed for statistical significance by Mann-Whitney U-test. (Significantly different from the control group *=P<0.05; **= P<0.01;***=P<0.001). 59

79 Figure 34. Numbers of CD3, CD4, and CD8 cells in in lymph nodes, liver, and peripheral blood of C57BL/6 mice treated with AQ. Values represent the mean ± SE from 8 animals per group. The data were analyzed for statistical significance by Mann-Whitney U-test. (Significantly different from the control group *p<0.05, **p<0.001, ***p<0.001). 60

80 Figure 35. Numbers of CD161, NK, NKT, and NK17 cells in lymph nodes, liver, and peripheral blood of C57BL/6 mice treated with AQ. NK cells were characterized as CD4 - /NK1.1 +, NKT cells as CD4 + /NK1.1 +, NK17 cells as CD4 - /NK1.1 + /IL Values represent the mean ± SE from 8 animals per group. The data were analyzed for statistical significance by Mann-Whitney U-test. (Significantly different from the control group *p<0.05, **p<0.001, ***p<0.001). 61

81 Figure 36. Numbers of IL17 producing, Th17, and Tc17 cells in lymph nodes, liver, and peripheral blood of C57BL/6 mice treated with AQ. Th17 cells were characterized as CD4 + /IL- 17 +, Tc17 cells as CD8 + /IL-17 +, IL-17 producing cells as IL Values represent the mean ± SE from 8 animals per group. The data were analyzed for statistical significance by Mann- Whitney U-test. Significantly different from the control group (*p<0.05, **p<0.001, ***p<0.001). 62

82 Figure 37. Differences in serum ALT and various lymphocyte types between mice that developed liver injury after 1 week of AQ treatment and those that did not. Th17 cells were characterized as CD4 + /IL-17 +, NK cells as CD4 - /NK1.1 +, NK17 cells as CD4 - /NK1.1 + /IL Values represent the mean ± SE from 4 animals per group. The data were analyzed for statistical significance by student t-test. (Significantly different from the control group *p<0.05). 63

83 Figure 38. Changes in Treg and PD-1 + /CD4 + cells in lymph nodes, liver and peripheral blood in C57BL/6 mice treated with AQ. Treg cells were characterized as CD4 + /CD25 + /Foxp3 +. Values represent the mean ± SE from 8 animals per group. The data were analyzed for statistical significance by Mann-Whitney U-test. (Significantly different from the control group *p<0.05, **p<0.001, ***p<0.001) Macrophage phenotyping in AQ-treated BN rats Macrophage phenotyping was performed in AQ-treated BN rats to investigate the involvement of macrophages in AQ-induced liver injury. Macrophages in the peripheral blood decreased from the first week and then gradually went back to normal by the third week (Figure 39). Lymph node macrophage number decreased in the first week and then increased to above baseline by the third week. There was no significant change in the total number of liver macrophages, and spleen macrophages appeared to increase slightly by the 4 th week. 64

84 Further phenotyping of the macrophages indicated that blood M1 macrophages decreased continuously over the first 4 weeks of AQ treatment (Figure 40). In contrast, there was a decrease, then an increase, and finally a decrease in M1 macrophages in lymph nodes. In the liver there was a small but significant increase in M1 macrophages at weeks 3 and 4, which corresponds to the liver injury while in the spleen there was a corresponding decrease in M1 macrophages (Figure 40). The pattern of activated M1 macrophages was similar but less distinct. In contrast, in general there was an increase in M2 macrophages of all subtypes and in most locations at later time points, and we suspect that is, at least in part, what leads to the resolution of liver injury (Figure 41-44). Figure 39. The time course for macrophage numbers in different organs of BN rats during AQ treatment. Values represent the mean ± SE from 8 animals per group. The data were analyzed for statistical significance by Mann-Whitney U-test. (Significantly different from the control group *p<0.05, **p<0.001, ***p<0.001). 65

85 Figure 40. The M1 macrophage phenotyping in peripheral blood, lymph nodes, liver, and spleen of BN rats during AQ treatment. Values represent the mean ± SE from 8 animals per group. M1 macrophages were characterized as MHC II + /CD163 + /CD68 -, activated M1 macrophages as MHC II + /CD163 + /CD68 - /CCL2 + /TNFα + /IL10 -. The data were analyzed for statistical significance by Mann-Whitney U-test. (Significantly different from the control group *p<0.05, **p<0.001, ***p<0.001). 66

86 Figure 41. The M2 macrophage phenotyping in different organs of BN rats during AQ treatment. (M2=M2a+M2b+M2c). Values represent the mean ± SE from 8 animals per group. The data were analyzed for statistical significance by Mann-Whitney U-test. (Significantly different from the control group *p<0.05, **p<0.001, ***p<0.001). 67

87 Figure 42. Subtypes of M2a macrophages in different organs of BN rats during AQ treatment. M2a macrophages were characterized as MHC II + /CD163 - /CD68 +, activated M2a macrophages as MHC II + /CD163 - /CD68 + /CCL2 - /TNFα - /IL10 +. Values represent the mean ± SE from 8 animals per group. The data were analyzed for statistical significance by Mann-Whitney U-test. (Significantly different from the control group *p<0.05, **p<0.001, ***p<0.001). 68

88 Figure 43. Subtypes of M2b macrophages in different organs of BN rats during AQ treatment. M2b macrophages were characterized as MHC II + /CD163 + /CD68 +, activated M2b macrophages as MHC II + /CD163 + /CD68 + /CCL2 - /TNFα + /IL10 +. Values represent the mean ± SE from 8 animals per group. The data were analyzed for statistical significance by Mann-Whitney U-test. (Significantly different from the control group *p<0.05, **p<0.001, ***p<0.001). 69

89 Figure 44. Subtypes of M2c macrophages in different organs of BN rats during AQ treatment. M2c macrophages were characterized as MHC II - /CD163 - /CD68 +, activated M2c macrophages as MHC II - /CD163 - /CD68 + /CCL2 - /TNFα - /IL10 +. Values represent the mean ± SE from 8 animals per group. The data were analyzed for statistical significance by Mann-Whitney U-test. (Significantly different from the control group *p<0.05, **p<0.001, ***p<0.001). 70

90 5.10. Effect of temozolomide on Tregs Instead of decreasing Tregs and increasing liver injury, co-treatment with the alkylating agent, temozolomide, did not significantly affect liver injury (Figure 45), and it increased the % Tregs (Figure 46), although there was a general lymphopenia (Figure 47). Figure 45. The time course of serum ALT during AMQ + temozolomide (TMZ) co-treatment in male BN rats. Values represent the mean ± SE. The data were analyzed for statistical significance by two-way ANOVA. (Significantly different from the control group *p<0.05, **p<0.001, ***p<0.001). 71

91 Figure 46. Time course of Treg cell % in PBMC during AMQ + temozolomide (TMZ) cotreatment in male BN rats. Treg cells were characterized as CD4 + /CD25 + /Foxp3 +. Values represent the mean ± SE. The data were analyzed for statistical significance by two-way ANOVA. (Significantly different from the control group *p<0.05, **p<0.001, ***p<0.001). Figure 47. The total lymphocyte count in blood during AMQ + temozolomide (TMZ) cotreatment in male BN rats. Values represent the mean ± SE. The data were analyzed for statistical significance by two-way ANOVA. (Significantly different from the control group *p<0.05, **p<0.001, ***p<0.001). 72

92 5.11. Covalent binding of AQ to hepatic proteins in BN rats treated with AQ As in previous experiments, treatment of BN rats with AQ led to an increase in ALT as shown in Figure 48A. There was significant covalent binding of ALT at 1 week, and this did not significantly increase at Week 2 (Figure 48B). There was covalent binding to proteins at a wide range of molecular mass, and there was no difference in covalent binding between sick and unsick animals (Figure 49). Figure 48. (A) Serum ALT in BN rats treated with AQ. Values represent the mean ± SE from 4 animals per group. The data was analyzed for statistical significance by two-way ANOVA. (Significantly different from the control group *p<0.05). (B) Corresponding covalent binding of AQ to hepatic proteins from BN rats treated with AQ. W1, animals treated for 1 week; W2, 73

93 animals treated for 2 weeks; W3, animals treated for 3 weeks. The ALT level highlighted in the black-box represents liver injury. Figure 49. (A) Serum ALT change in BN rats during AQ treatment. (B) Corresponding covalent binding of AQ to the hepatic proteins from BN rats treated with AQ. 2 BN rats were treated with AQ and sacrificed at Weeks 3 and Week 4 as they developed liver injury and immune tolerance, respectively. W3S, the rat was treated for 3 weeks and developed liver injury. W4T, the rat was treated for 4 weeks and developed immune tolerance GSH depletion by co-treatment with BSO and DEM BN rats were treated with AQ at the dose of 62.5 mg/kg/day for 1 week, and BSO was used as co-treatment to deplete GSH. After the isolation of liver protein, the total liver GSH level was determined by an ELISA kit as shown in Figure 50. There was a slight and insignificant decrease of total liver GSH after 1 week of AQ treatment, but marked depletion of liver GSH in both BSO- and AQ+BSO-treated animals, and the average depletions were 65% and 82%, respectively compared to the liver GSH in the control animals. GSH depletion by AQ+BSO was greater and more consistent compared to BSO alone. 74

94 Figure 50. Liver GSH level in BN rats mice after 1 week of AQ and/or BSO treatment. Values represent the mean ± SE from 4 animals per group. Values represent the mean ± SE from 4 animals per group. The data were analyzed for statistical significance by Mann-Whitney U-test. BSO and DEM were administrated to AQ-treated male BN rats to study the effect of GSH depletion on covalent binding and liver injury. Paradoxically, co-treatment with BSO or BSO+DEM prevented AQ-induced liver injury (Figure 51). This phenomenon was observed in C57/BL6 and Wistar rats as well (data not shown). Co-treatment with BSO or BSO+DEM appeared to decrease instead of increase AQ covalent binding; however, the difference was not significant (Figure 52). 75

95 Figure 51. BSO treatment prevents the increase in ALT caused by AQ in BN rats. Values represent the mean ± SE from 3 animals per group. The data were analyzed for statistical significance by two-way ANOVA. (Significantly different from the control group *p<0.05, **p<0.001, ***p<0.001). 76

96 Figure 52. Effects of GSH depletion on covalent binding of AQ to hepatic proteins from AQtreated BN rats. BSO and DEM were used to deplete GSH and hepatic proteins were isolated after 4 weeks of AQ treatment Effects of RA and DMSO co-treatment RA was orally administrated to AQ-treated male BN rats as described above. Co-treatment with RA exacerbated AQ-induced liver injury resulting in a greater increase in ALT; however, tolerance was still observed at week 4 (Figure 53). DMSO did not appear to have any significant effect on AQ-induced liver injury (Figure 54). 77

97 Figure 53. Effects of RA co-treatment on serum ALT in AQ-treated BN rats. Values represent the mean ± SE from 6 or 8 animals per group. The data was analyzed for statistical significance by two-way ANOVA. 78

98 Figure 54. Change of serum ALT in BN rats treated with AQ and/or DMSO for 4 weeks. Values represent the mean ± SE from 3 or 4 animals per group. The data was analyzed for statistical significance by two-way ANOVA. 6. Discussion The characteristics of AQ-induced IDILI and agranulocytosis suggest that they are immunemediated; however, it is very difficult to perform studies in humans that would conclusively test this hypothesis (Clarke et al., 1991; Neftel et al., 1986; Uetrecht, 2005). The reactive iminoquinone metabolite of AQ is presumed to be responsible for these adverse reactions, but again there has been no way to conclusively test this hypothesis (Harrison et al., 1992; Jewell et al., 1995). Others have performed studies in rats and mice; specifically, treatment of rats led to an increase in ALT and anti-drug antibodies, and in mice, co-treatment with buthionine sulfoximine to deplete glutathione increased the hepatotoxicity (Clarke et al., 1990; Shimizu et 79

99 al., 2009). However, these were acute studies with high doses, and the characteristics of the toxicity were different from the liver injury observed in humans. Recently, our lab developed a new model of drug-induced liver injury in C57BL/6 mice: treatment of AQ in female C57BL/6 mice lead to mild liver injury with a delay in onset and resolution of the injury despite continued treatment (Metushi et al., 2014a). Evidence pointed to the involvement of NK cells in this injury. Further studies found that co-treatment of PD-1 -/- mice with anti-ctl-4 antibody and AQ resulted in more severe liver injury that did not resolve with continued treatment, and it was characterized by piecemeal necrosis similar to IDILI in humans (Metushi et al., 2014b). This is the first animal model of IDILI similar to IDILI of humans, and it was achieved by breaking immune tolerance. In this study, we extended the AQ model to rats and used different co-treatments to explore the mechanism and possible risk factors for AQ-induced liver injury. The dose chosen was 62.5 mg/kg/day, which results in a serum AQ concentration of about 2 µm, which is within the therapeutic range in humans ( µm) (White et al., 1987). All of the rat strains tested sustained liver injury when treated with AQ, but female rats were relatively resistant. The histology of AQ-induced liver injury in rats demonstrated a mild infiltrate of mononuclear cells (CD45RA positive), suggesting that AQ induced an immune response. The increased number of cells expressing ED1 (CD68) and ED2 (CD163) implied activation of macrophages in response to AQ treatment. CD68 is a pan-macrophage marker whose expression is correlated with the phagocytic activity of macrophages while CD163 is expressed on resident macrophages and has been used as a Kupffer cell marker in the liver (Dijkstra et al., 1985a, b). AQ induced a small amount of apoptosis in liver as indicated by both the TUNEL assay and detection of cytochrome C in serum. The apoptosis in the liver after AQ-treatment suggests the existence of cell/tissue damage, which has the potential to initiate an immune response by 80

100 activating macrophages, neutrophils, or lymphocytes. Apoptosis is a common mechanism of cell death in DILI including hepatotoxicity induced by acetaminophen (APAP), carbamazepine, and phenytoin (Gujral et al., 2002; Kon et al., 2007; Santos et al., 2008). Cell proliferation is another common phenomenon in DILI, which was detected after AQ-treatment by the PCNA assay, and there was also an increase in osteopontin. The significant increase of PCNA expression in the spleen of AQ-treated animals suggests activation of immune system. Proliferation of hepatocytes is a common reaction of the liver to injury (Apte et al., 2004; Baier et al., 2006; Cardin et al., 2002; Trautwein et al., 1998). Recent studies have shown that osteopontin is not only able to enhance cell proliferation in many tissues such as muscle, kidney, and prostate (Angelucci et al., 2004; Liu et al., 2009; Phillips et al., 2012; Sodhi et al., 2001; Xiao et al., 2012), but it also plays an important role in mediating hepatic inflammation and the ensuing liver toxicity (Ramaiah et al., 2007). Osteopontin was found to enhance acetaminophen toxicity by recruitment of inflammatory cells, activation of macrophages and T cells, and production of proinflammatory cytokines in the liver (He et al., 2012; Ramaiah et al., 2008). Meanwhile, osteopontin was also reported to be closely related to production of IL-17, a proinflammatory cytokine, during the pathogenesis of chronic hepatitis B and concanavalin A- induced hepatitis (Diao et al., 2012). The dominant hypothesis of the mechanism of AQ-induced hepatotoxicity is that it is immune-mediated, which is supported by the results of this study. First, a mild delayed liver injury and the presence of anti-drug antibody were observed in both AQ-treated and rechallenged animals. Second, significant changes in serum cytokines were observed during AQ-treatment; both proinflammatory and antiinflammatory cytokines were produced in response to AQ treatment. Although we did not see an elevation of ALT until Day 21, IL-12 started to increase on Day 3, followed by the elevation of IL-2 and IL-5 on Day 14. IL-12 is a 81

101 proinflammatory cytokine, which enhances the cytotoxicity of NK cells and CD8 + T lymphocytes by stimulating the production of IFN-γ and TNF-α from T cells and NK cells (Papadakis et al., 2004; Robertson et al., 1996; Trinchieri, 2003). IL-2 is also generally considered a proinflammatory cytokine, which is able to activate Kupffer cells, induce leukocyte adhesion, and could lead to liver injury (Nakagawa et al., 1996). Clinically, many patients undergoing therapy with IL-2 often experience elevations of ALT, aspartate aminotransferase, and alkaline phosphatise (King et al., 2001). Recently, several studies reported that MCP-1 is closely related to hepatotoxicity, possibly by recruiting leukocytes and increasing proinflammatory cytokines. Increased expression of MCP-1 was observed in HCV-related liver disease and liver injury induced by carbon tetrachloride and galactosamine, and deficiency of MCP-1 protects mice against alcoholic liver injury (Czaja et al., 1994; Mandrekar et al., 2011; Marra et al., 1999; Muhlbauer et al., 2003). This could also explain the macrophage and lymphocyte infiltration we observed. The significant increase in antiinflammatory cytokines such as TGF-β suggests the presence of hepatocyte damage, which appears to be involved in wound repair as well as the regulation of proinflammatory cytokines, such as IL-6 and IL-17 (Amento et al., 1991; Letterio et al., 1998; Yoshimura et al., 2011). As the AQ-induced liver injury resolved, the concentration of several proinflammatory cytokines also decreased, including IL-2, IL-5, IL-6, and IL-12. This decrease in proinflammatory cytokines matches the decrease of ALT. However, enhanced IL-18 and RANTES (regulated and normal T cell expressed and secreted, or CCL5) were observed after 5 weeks of treatment, both of which appear to be involved in NK cell proliferation and activation. Significant elevation of several cytokines was also found in AQ rechallenged animals, including IL-4, IL-12, IL-13, and INF-γ. IFN-γ is a proinflammatory cytokine mainly produced by NK cells and T cells, which plays important roles in innate and adaptive immunity, promoting Th1 differentiation, suppressing 82

102 Th2 differentiation, stimulating NK cells and macrophages activities, and inducing the production of IgG from B cells (Schoenborn et al., 2007; Schroder et al., 2004). IL-4 and IL-13 were recently shown to be hepatoprotective in acetaminophen-induced liver injury by suppressing Th1 differentiation, promoting Th2 differentiation, and activating M2 macrophages (Ryan et al., 2012; Van Dyken et al., 2013; Yee et al., 2007). All of these results suggest that the immune system is activated in AQ-induced hepatotoxicity. Co-treatment with cyclosporine prevented AQ-induced liver injury, while Poly I:C caused an earlier increase in ALT. These results suggest AQ-induced liver injury is immune-mediated. A few findings were observed in the first week in AQ-treated BN rats, including liver lymphocyte infiltration, and enlarged spleen and lymph nodes. These observations suggested the involvement of the immune system and also an early immune response during the first week. The trend of liver lymphocyte infiltration, especially CD4 + T cells and Th17 cells, matched the changes in ALT, which suggested that lymphocytes play a role in both liver injury and the tolerance that followed. The increase of CD4 + T cells, Th17 cells, and NK/NKT in the first week implied: 1) the activation of both innate and adaptive immune systems; 2) those lymphocytes might be the effector cells in the early immune response. A continuous and constant NK/NKT cell accumulation was observed in the liver. The covalent binding of AQ reactive metabolites to cellular macromolecules could affect cell function such as impairing mitochondria and leading to further cell damage or apoptosis. This cell damage or apoptosis could release chemokines or danger signals to activate and recruit NK/NKT cells to clean it up. The possible critical role of NK cells in AQ-induced liver injury was also previously reported by Metushi et al. (Metushi et al., 2014a). The decrease and rebound of macrophages in PBMC and lymph nodes suggests the involvement of macrophages. The M1 and M2 changes in the liver during the 1 st week suggest 83

103 that at the early stage of the immune-response (1 st week or even earlier), local M2 macrophages had been activated and play a protective role. As discussed before, AQ could induce direct or indirect toxicity in the liver followed by the recruitment of lymphocytes (CD4 + T cells, Th17 cells, NK/NKT cells), which would lead to the activation of M2a macrophages that are involved in repair of tissue damage and regulating the immune response. As the liver injury progresses, at Week 3 more M1 macrophages were observed in the liver; however, the total activated M1 macrophages did not change during the entire treatment. The total number of activated M2 macrophages was found to gradually but significantly increase during the entire AQ treatment, which suggested the possible function of M2 macrophages in liver is to decrease the pathogenic effect, such as cytotoxic effect of NK/NKT cells whose number also gradually increased during the AQ treatment. This accumulation of M2 macrophages and NK/NKT cells might be important to the liver injury and subsequent tolerance. For example, M2 macrophages could upregulate Treg cells or secret IL-10, which will further down-regulate the immune response from effect cells, such as Th1, Th2, Th17 and cytotoxic T cells, and lead to the immune tolerance. Assuming that covalent binding is responsible for the injury, one strategy to enhance the liver injury caused by AQ is to increase the covalent binding. The reactive metabolite of AQ is a soft electrophile, which binds preferentially to thiols such as glutathione. GSH-depleted animals have been used to study the mechanism of toxicity caused by reactive metabolites of drugs. Watanabe found that GSH depletion by BSO enhanced mouse sensitivity to APAP (Watanabe et al., 2003). Mizutani employed GSH deficient model to investigate the hepatotoxicity induced by styrene, eugenol, and methimazole (Mizutani et al., 1994; Mizutani et al., 1999; Mizutani et al., 1991). In the present work, the effect of GSH depletion on covalent binding and the AQ-induced hepatotoxicity was investigated. Our hypothesis was that GSH depletion would result in decreased detoxication of AQQI by GSH, an increased level of 84

104 covalent binding to proteins, and increased liver injury. Surprisingly, BSO prevented the AQinduced liver injury, and this effect was true for both BN rats and C57BL/6 mice. Covalent binding analysis showed that there was no significant increase in protein binding after GSH depletion. One plausible explanation is that the major detoxication mechanism for the reactive metabolite of AQ is reduction back to the parent drug. That does not explain why GSH depletion paradoxically protected against AQ-induced liver injury. It has been reported that NK function is dependent on normal GSH levels, and therefore it is possible that the mechanism by which GSH depletion led protection against AQ-induced liver injury was by impairing NK cell function. Our results conflict with those of Shimizu who reported that one single dose of AQ (180 mg/kg by gavage) did not cause liver injury in mice unless the AQ administration was combined with BSO-induced GSH depletion. However, the liver injury in the Shimizu model is very different from the liver injury in our model as well as the liver injury that occurs in humans; specifically, it is acute toxicity caused by a significantly higher dose of AQ rather than the delayed injury in our model and in humans (Shimizu et al., 2009). If GSH depletion does not significantly increase covalent binding, it may potentiate direct cytotoxicity through a lack of some other protective effect of GSH, but it is difficult to speculate because the mechanism of this acute toxicity is unknown. It has been reported that NK cells are functionally suppressed and undergo apoptosis induced by ROS such as O2 - and H2O2 (Betten et al., 2004; Hansson et al., 1996; Hellstrand et al., 1994). Peraldi s work revealed that oxidative stress results in a reduced expression of the NK cell activating receptor NKG2D (Peraldi et al., 2009). It is not surprising that GSH, as a ROS scavenger, is able to regulate NK cells activity: GSH is a key component to enhance NK cell function (Millman et al., 2008). Viora reported that decreased levels of intracellular GSH severely impairs NK cell activity, and on the other hand, the antioxidant activity of N-acetyl cysteine can lead to an increased NK cell-mediated cytotoxic binding and 85

105 lysis of target cells (Viora et al., 2001). The fact that GSH depletion prevents AQ-induce hepatotoxicity reinforces the hypothesis that NK cells play a critical role in mild AQ-induced liver injury. This hypothesis is further supported by the finding of NK cell accumulation in the liver and elevated expression of NK cell-related cytokines/chemokines, such as IL-2, IL-12, IL- 18 and CCL5. The presence of IFN-γ, IL-2, IL-6, IL-12. and IL-18 in the inflamed liver has been reported to contribute to an activated phenotype of NK cells, whereas IL-2 may sustain the survival and activation of these cells while IL-12 may be responsible for the NK cell recruitment (Shi et al., 2011). Millman reported that the combination of IL-2 and IL-12 in combination with glutathione augments NK cells functions, such as cytolytic activity, activating receptor expression, induction of apoptosis, and cytokine synthesis (Millman et al., 2008). Earlier research from our lab has already shown the critical role of NK cells in AQ-treated C57BL/6 mice: depletion of NK cells significantly attenuated the AQ-induced liver injury (Metushi et al., 2014a). To investigate the involvement of NK cells in AQ-induced hepatotoxicity, RA was administrated as co-treatment in AQ-treated BN rats. Recently, several studies found that RA is able to activate liver NK cell killing of hepatocytes and hepatic stellate cells (Lee et al., 2012; Ochi et al., 2004; Radaeva et al., 2007; Taimr et al., 2003). Taimr found that RA induces expression of RAE-1, an activating ligand for NK cells, and further enhance NK cells cytotoxicity by releasing TNF-related apoptosis-inducing ligand (TRAIL) (Taimr et al., 2003). The co-treatment with RA exacerbated AQ-induced liver injury with an earlier onset (Day 7) and increased severity. This result of RA co-treatment again supports the plausible pathogenic role of NK cells in AQ-induced hepatotoxicity. NK cells have been reported to play a role in DILI. For example, Dugan reported that NK cells mediated severe liver injury in a murine model of halothane hepatitis in an IFN-γ- and perforin-dependent manner (Dugan et al., 2011). NK cells contribute to liver injury in several ways (Sharma et al., 2012): 1) NK cells can 86

106 be activated by poly I:C or cytokines and then kill hepatocytes by producing TRAIL, perforin, and granzyme B (Kahraman et al., 2008; Stout-Delgado et al., 2007). 2) NK cells can be activated by RAE-1 and further induce hepatocyte damage (Chen et al., 2007b). 3) NK cells are able to produce pro-inflammatory cytokines, which can also contribute to liver injury (Chen et al., 2007a). This study provides new insights into the mechanism of AQ-induced liver injury. In this research we found that liver CD4 + T cells and IL-17 secreting cells increased in the liver and may be involved in AQ-induced liver injury and subsequent immune-tolerance. AQ also induces an accumulation of NK cells and activated M2 macrophages in the liver. These cells are also likely to play a role in the balance between liver injury and repair/immune tolerance induced by AQ reactive metabolites. However, a recent study indicated that AQ caused even greater liver injury in a T cell deficient mice model (Metushi et al., 2014b). These results imply that the adaptive immune system is more important in the ultimate tolerance that occurs in this model than in the injury. However, when immune tolerance was impaired, then the injury appeared to be mediated by CD8 T cells. If this liver injury is mediated by NK cells, it can also explain the lack of immune memory we observed during the AQ rechallenge in BN rats. Our results presented in this study suggest that the covalent binding of AQ to hepatic proteins is a necessary but not sufficient component in AQ-induced liver injury, and NK cells play a pathogenic role in AQ-induced hepatotoxicity. In conclusion, although we have not yet been able to develop an animal model of severe AQ-induced IDILI, we have found that AQ causes a delayed onset of mild IDILI in BN rats that appears to be immune-mediated and resolves with what appears to be immune tolerance. Therefore, this may be an excellent model to study the phenomena of adaptation; we postulate that it is only when immune tolerance fails that severe liver injury results. The outcome of liver 87

107 injury and tolerance depends on the complicated immune balance between effector cells and regulatory cells, which suggests that it may be possible to develop an AQ-induced server IDILI model in rats by manipulating with the immune system. 7. Acknowledgement Dr. Jack Uetrecht is the recipient of the Canada Research Chair in Adverse Drug Reactions. This work was supported by a grant received from the Canadian Institutes of Health Research. Part of this work was present by Ping Cai, Feng Liu and Jack Uetrecht at the Society of Toxicology Annual Meeting in Washington, D.C, USA, in Declaration of interest The authors declare no competing financial interest. The authors alone are responsible for the content and writing of the paper. 88

108 Chapter 3: Development of a Novel Mouse Model of Amodiaquineinduced Liver Injury with a Delayed Onset This work has been published in the following journal and is reproduced with permission: Metushi IG, Cai P, Dervovic D, Liu F, Lobach A, Nakagawa T, Uetrecht J. Development of a novel mouse model of amodiaquine-induced liver injury with a delayed onset. Journal of Immunotoxicolgy Jul;12(3): Epub 2014 Jul 21. In this chapter, all the work was designed and written by Metushi IG; lymphocytes phenotyping and the measurement of serum cytokines were completed by Liu F and Metushi IG; immunohistochemistry and serum concentration were completed by Cai P. 89

109 1. Abstract Amodiaquine (AQ) treatment is associated with a high incidence of idiosyncratic drug-induced liver injury (IDILI) and agranulocytosis. Evidence suggests that AQ-induced IDILI is immune mediated. A significant impediment to mechanistic studies of IDILI is the lack of valid animal models. This study reports the first animal model of IDILI with characteristics similar to mild IDILI in humans. Treatment of female C57BL/6 mice with AQ led to liver injury with delayed onset, which resolved despite continued treatment. Covalent binding of AQ was detected in the liver, which was greater in female than in male mice, and higher in the liver than in other organs. Covalent binding in the liver was maximal by Day 3, which did not explain the delayed onset of alanine aminotransferase (ALT) elevation. However, coincident with the elevated serum ALT, infiltration of liver and splenic mononuclear cells and activation of CD8 T-cells within the liver were identified. By Week 7, when ALT levels had returned close to normal, down-regulation of several inflammatory cytokines and up-regulation of PD-1 on T-cells suggested induction of immune tolerance. Treatment of Rag1 -/- mice with AQ resulted in higher ALT activities than C57BL/6 mice, which suggested that the adaptive immune response was responsible for immune tolerance. In contrast, depletion of NK cells significantly attenuated the increase in ALT, which implied a role for NK cells in mild AQ-induced IDILI. This is the first example of a delayed-onset animal model of IDILI that appears to be immune-mediated. Keywords Covalent binding, drug-induced liver injury, immune-mediated, immune tolerance, reactive metabolite 2. Introduction 90

110 Amodiaquine (AQ), a 4-aminoquinoline, was approved to treat malaria because of the development of resistance to chloroquine. Although it is still used in some countries, AQ was withdrawn from the market because of severe idiosyncratic drug reactions (IDR) that included hepatotoxicity, agranulocytosis, and aplastic anemia (Hatton et al., 1986; Larrey et al., 1986; Neftel et al., 1986; Rouveix et al., 1989). The mechanism of AQ-induced adverse reactions is currently not well understood, but AQ is metabolized to N-desethylamodiaquine (DEAQ) by CYP2C8, and both AQ or DEAQ can be oxidized to a reactive quinoneimine, which reacts with proteins to form covalent adducts (Li et al., 2002; Maggs et al., 1987; Maggs et al., 1988). As with most drugs that cause idiosyncratic drug-induced liver injury (IDILI), the clinical characteristics of AQ-induced liver injury involve a delayed onset of several weeks, and the injury usually resolves despite continued treatment (Neftel et al., 1986). There is a prompt recurrence of liver injury upon rechallenge, which is consistent with immune memory. Histopathology shows necrosis and the presence of inflammatory cells including Kupffer cells (Larrey et al., 1986; Neftel et al., 1986). It has also been reported that there are circulating anti- AQ IgG antibodies in humans who developed a severe adverse reaction and anti-aq antibodies were also observed in rats (Clarke et al., 1990; Clarke et al., 1991). These observations suggest that the mechanism of AQ-induced IDILI is an immune-mediated reaction. Although the clinical characteristics of AQ-induced liver injury and other drugs that are associated with IDILI suggest an immune mechanism, the lack of a valid animal model has hampered progress in understanding the molecular mechanism and factors that lead to the initiation of liver injury and liver failure. Recently, a model of AQ-induced hepatotoxicity has been reported (Shimizu et al., 2009). However, as with most animal models, this is an acute model of hepatotoxicity in which mice were treated with buthionine sulfoximine and AQ for up to 24 hours, and this resulted in an increase in alanine transaminase (ALT) levels. A similar 91

111 study was reported in Swiss mice (Mishra et al., 2011). Because there is a delayed onset in liver injury in humans, it is likely that the mechanism of AQ-induced liver injury in humans is very different from these models. In this report we describe the development of an animal model of AQ-induced liver injury with a delayed onset that resolves despite continued treatment. This is similar to what often occurs in humans, i.e. the IDILI resolves despite continued treatment with the drug and mild IDILI is more common than severe IDILI. 3. Materials and methods a) Mice and treatments Mice (C57BL/6NCrl; 6 8-weeks-of-age) were purchased from Charles River Laboratories (Montreal, QC, Canada). T- and B-cell immunodeficient Rag -/- (Rag1 tm1mom /J; 6 8-weeks-of-age) mice were purchased from Jackson Laboratories (Bar Harbor, ME). All mice were housed in a specific pathogen-free environment maintained at 22 with a 30 70% relative humidity and a 12-h light/dark cycle. All mice had ad libitum access to filtered water. Upon arrival, all mice were allowed to acclimatize for 1 week before treatment. The University of Toronto animal care committee approved all protocols. AQ was thoroughly mixed with food and given to rodents at a dose of 0.2% [w/w] in food. Food was provided to the animals (four mice/cage) in small jars ad libitum, and the average amount consumed was measured; this resulted in an AQ dose of mg/kg/day. Liver enzyme activities were measured by collecting blood from the saphenous vein at baseline or every week, unless otherwise stated, during the course of the exposures. As biomarker of liver injury, the activity of alanine aminotransferase (ALT, Thermo Scientific, Middletown, VA) was measured (Metushi et al., 2012; Metushi et al., 2014c; Metushi et al., 2014d). b) Serum concentration of AQ 92

112 Methanol (80 ml), including 0.1 mm of the internal standard 4-dimethylaminoantipyrene (Sigma, Oakville, ON), was added to 10 ml serum to precipitate proteins. The mixture was vortexed and stored at -20 for 30 min, then centrifuged at X g for 10 min. The supernatant was evaporated to dryness under a gentle stream of nitrogen. Residues were dissolved in 100 ml of mobile phase, and a 20 ml aliquot was injected into the LC MS/MS system consisting of a Shimadzu LC10 HPLC and a API3000 mass spectrometer (PE Sciex, Concord, ON, Canada). The HPLC column was a 2.1 mm 50 mm Luna C18 3 mm analytical column; mobile phase flow rate was 0.2 ml/min. The mobile phases used were 0.1% formic acid (Solvent A) and methanol (Solvent B) and were delivered using a linear gradient elution of 5% methanol (Solvent B) at 0 min increasing to 90% Solvent B at 2.5 min. Optimization for multiple reaction monitoring transitions were performed using synthetic standards (Toronto Research Chemicals, Toronto, ON) for AQ (Q1/Q3: 356.1/283.2), DEAQ (Q1/Q3: 328.1/283.2) and internal standard (232.1/56.1). Data were collected and processed using Analyst software (Applied Biosystems, Grand Island, NY). c) Histology and immunohistochemistry At various end-points over the course of the exposures, animals were euthanized by placement inside a CO2 chamber, after which their livers were removed, perfused, extracted, and then placed in 10% neutral buffered formalin solution (Sigma; Oakville, ON) overnight. For preparation of frozen sections, the liver tissue was placed in OCT medium (VWR International; Radnor, PA) and immediately frozen using liquid nitrogen. Paraffin and frozen section slides were cut and H&E stained by the Department of Pathology at the Hospital for Sick Children (University of Toronto). 93

113 Rat monoclonal primary antibodies against mouse CD11b (clone M1/70), F4/80 (clone CI:A3-1), CD45R (clone RA3-6B2), and rabbit polyclonal antibody against mouse KI67 were purchased from Abcam (Cambridge, MA). Dr P. Ohashi (Princess Margaret Hospital, University of Toronto) donated monoclonal antibodies against mouse CD4 (clone GK 1.5) and CD8 (clone YTS169). Rabbit anti-aq produced in our laboratory, as previously described was shown to be specific for AQ binding to macromolecules (Lobach et al., 2014). Polyclonal rabbit secondary antibody anti-rat IgG-biotinylated and streptavidin-peroxidase were purchased from Dako (Burlington, ON) and goat anti-rabbit IgG-peroxidase from Sigma. Each experiment was repeated at least twice, and the signal was developed using 3,3 -diaminobenzidine for paraffinembedded slides or NovaRed for frozen slides (Vector; Burlington, ON) with Mayer s hematoxylin (Sigma) as counterstain. Paraffin-embedded slides were stained with antibodies against F4/80, CD45R, and KI-67. Antibodies against CD11b, CD4, and CD8 were used with frozen sections. Immunohistochemical grading was blinded, and the numbers of cells per field of view under a microscope were counted; at least two slices of tissue (3 6 mm 2 ) were mounted on glass slides and cell numbers from five areas of each slice were counted. d) Western blotting The liver, spleen, gut (consisting of small and large intestine), and kidney were removed at necropsy, and each homogenized in the presence of protease inhibitors (Sigma) as previously described and centrifuged at 2500 X g (i.e., S2.5 fraction) (Metushi et al., 2012). Alternatively, in an independent experiment, the liver was removed, homogenized, and centrifuged at 9000 X g (referred to as S9 fraction). Protein concentration in each sample was measured using a bicinchoninic acid kit (Fisher Scientific; Ottawa, ON). From each sample, 10 mg of protein (for S2.5 fractions) and 20 mg protein (for S9 fraction) was loaded into dedicated lanes on an 8% SDS-PAGE gel and the proteins then separated by electrophoresis. Super-signal enhanced 94

114 molecular weight marker was loaded onto one of the lanes (Fisher Scientific) to monitor sample progress. The resolved proteins were then electrotransferred onto a nitrocellulose membrane (BioRad, Mississauga, ON). After blocking to prevent non-specific binding, the membrane was incubated with rabbit anti-aq (1: dilution; primary antibody) for h at 4 ; after washing away unbound primary antibody, the membrane was incubated with goat anti-rabbit IgG-peroxidase secondary antibody (1: dilution; Sigma) for 1 2 h at room temperature. For all studies, mouse monoclonal anti-gapdh (Sigma) was used as loading control and detected using goat anti-mouse IgG-peroxidase (Jackson ImmunoResearch; West Grove, PA). Bound peroxidase was then visualized using Supersignal West Pico Chemiluminescent Substrate (Fisher Scientific). The blots were then scanned with a FluorChem FC2 imager (Alpha Innotech, Toronto, ON). e) Flow cytometry Mouse liver was perfused with cold phosphate-buffered saline (PBS, ph 7.4), removed, and crushed with a syringe plunger. The cells were suspended in 35% Percoll (GE Healthcare, Mississauga, ON) and passed through a 100-mm cell strainer. This material was then centrifuged at 1000 X g for 20 min and the lymphocytes at the bottom collected. The cells were then incubated in red cell lysis buffer for 5 min and washed with FACS buffer (10% fetal calf serum in PBS). Non-specific binding was blocked by incubating the cells with anti-mouse CD32 antibody (ebioscience, San Diego, CA). In addition, half of the spleen and five cervical lymph nodes collected from each mouse at necropsy were also analyzed. Single cell suspensions of the liver, spleen, and lymph nodes were prepared by passing each tissue through a 100-mm cell strainer using a syringe plunger. Following lymphocyte isolation, red cell lysis was performed followed by a second filtration through a 40-mm cell strainer. The remaining cells were resuspended in 10% fetal calf serum in PBS buffer for cell counting and the number of 95

115 lymphocytes counted using a Countless Automated Cell Counter by Invitrogen (Life Technologies, Grand Island, NY). From each suspension, 0.5 X 10 6 cells were stained for surface markers using antibodies against mouse CD4-PECY7, CD69-APC, NKp46-eFluor450, streptavidin-pecy5 (ebioscience), CD8-APCCY7, CD279-APC (BD Biosciences, Mississauga, ON), NK1.1-FITC, CD62L-PE, CD44-Biotin, and CD152-Biotin (Sunnybrook Hospital, Toronto, ON); 4,6 -diamidino-2-phenylindole (DAPI, Molecular Probe, Burlington, ON) was used as a live cell marker. Analyses were performed by gating first on the lymphocyte population; cell aggregates were gated out and live cells used for analysis by using DAPI viability dye by gating on the negative population. Results are reported as percentages and as total cell numbers, which were calculated by multiplying the percentage of each cell type by the total number of lymphocytes from each organ (corrected for organ weight), with the exception of cervical lymph nodes for which a total of five nodes from each host were used for cell counting. f) Natural killer cell depletion Depletion of NK cells was performed by intraperitoneal injection of 200 µg/mouse of anti- NK1.1 antibody (clone PK136) diluted in sterile PBS at -3 and -1 days before drug initiation, as described in Lang et al. (2012), or IgG1 isotype control diluted in PBS (both from Sunnybrook Hospital, Toronto, ON) (Lang et al., 2012). Control mice were injected with PBS only. The drug was started at Day 0, and injection of anti-nk1.1 antibody, IgG1 isotype, or PBS was repeated every week to maintain suppressed NK activity (Koo et al., 1986). Administration of this anti- NK1.1 antibody has been previously shown to deplete NK and NKT cells (Masson et al., 2008). In our laboratory, injection with anti-nk1.1 antibody depletes NK and NKT cells in the liver, spleen, blood, and cervical lymph nodes; depletion of NK cells was also confirmed by staining with anti-nkp46 antibody (another cellular marker for NK cells; data not shown). 96

116 g) Serum cytokines Female C57BL/6 mice were treated with AQ for 1, 3, or 7 weeks. Blood was collected at the end of each timepoint by euthanizing the mice in a CO2 chamber. The harvested blood was allowed to clot for 30 min at room temperature, and then centrifuged at 2000 X g for 10 min at 4 to isolate the serum. Cytokines were ultimately measured in 25 ml serum using a BioRad BioPlex Pro Mouse Cytokine 23-plex, as per manufacturer instructions. The cytokines assessed included: interleukin (IL)-1, -1, -2, -3, -4, -5, -6, -9, -10, -12 (p40), -12 (p70), -13, and-17a, as well as eotaxin, G-CSF, GM-CSF, interferon (IFN)-γ, KC (CXCL1), MCP-1 (MCAF), MIP-1α, MIP-1β, RANTES, and tumor necrosis factor (TNF)-α. h) Statistical analysis Statistical analyses were performed using GraphPad Prism (GraphPad Software, San Diego, CA). Data were analyzed using two-way analysis of variance (ANOVA) or Mann-Whitney U- test. All p values <0.05 were considered significant (*p<0.05, **p<0.01, ***p<0.001). 4. Results a) Treatment of female C57BL/6 mice with AQ results in mild liver injury with delayed onset Treatment of female C57BL/6 mice with AQ resulted in a mild increase in ALT, which had a delayed onset of at least 1 week and resolved despite continued treatment (Figure 55a). The ALT peak was the highest between 3 4 weeks and had returned to almost normal by Week 6. There was no increase in ALT in male C57BL/6 mice (Figure 55b). In general, mice that were treated with AQ in food did not eat as much food as control mice, but their body weight remained stable throughout the treatment (Figures 55c and d). The liver histology of female mice did not show signs of significant abnormalities at the end of 6 weeks (data not shown); it is 97

117 likely that this is because the injury is mild and resolves despite continued treatment. AQ has a long terminal half-life; the serum concentration of AQ in female mice was ~ ng/ml (Table S1), which is within the range of the plasma AQ concentrations found in humans after intravenous dosing (CAQ= ng/ml) (White et al., 1987). Female mice had higher serum concentrations of AQ (2.5-fold higher) and DEAQ (3.7-fold higher) than male mice (Table S1); this corresponded to the greater extent of liver injury in female mice. Serum concentrations of AQ were somewhat higher at Week 1 than at Weeks 3 or 7 (Table S1); thus, the delay in injury cannot be explained by accumulation of AQ. b) Covalent binding of AQ in the liver, spleen, gut, and kidney AQ covalently binds proteins in the liver (Shimizu et al., 2009). An anti-aq antibody was used here to evaluate the extent of covalent binding in different tissues. Covalent binding in the liver was greater in the centrilobular region (Figures 55e and f), an outcome that correlates with the highest concentrations of cytochromes P450; this is similar to what was observed with other drugs such as isoniazid (Metushi et al., 2012). AQ was also found to bind in the red pulp of the spleen that contains antigen-presenting cells (APC; Figures 55g and h). The relationship between ALT elevation and covalent binding in the liver was also examined. Both in female and male mice, AQ covalent binding in the liver was significant at Day 1 and appeared slightly greater on Day 3, but there was no significant increase in binding after that (Figures 56a and b). In an independent experiment we examined covalent binding of AQ in the liver of female mice in the S9 fraction of liver homogenate, and covalent binding peaked after Day 2 (Supplemental Figure S1). Covalent binding in the gut was never very high and did not increase after Day 3 (Figures 56a and b); covalent binding in the spleen and kidney continued to increase past 1 week. A comparison of covalent binding between different tissues revealed that the greatest covalent binding occurred in the liver, followed by the spleen, kidney, and gut (Figures 57a and 98

118 b). AQ covalent binding was greater in the liver and kidney of female compared to male mice, but there did not appear to be any difference in covalent binding in the spleen and gut (Figure 57c). c) Increase in ALT is associated with lymphocyte infiltration in the liver To determine whether the increase in ALT was associated with an immune response, lymphocyte infiltration into the liver, spleen, and cervical lymph nodes was examined. The greatest increase in ALT was observed at Weeks 3 4 (Figure 55a). Consequently, to determine the immune response before, during, and after the timepoint of maximal ALT elevation, mice were sacrificed at Weeks 1, 3, and 7. ALT activity was highest at Week 3, followed by at Weeks 1 and 7 (Figure 58a). Liver weights of the control and AQ-treated mice were similar (Figure 58b), but the total number of lymphocytes isolated from the liver of AQ-treated mice was highest at Week 3 (Figure 58d), which coincides with the greatest increase in ALT. The spleen weight of the AQ-treated mice was greatest at Week 3 but decreased after that time (Figure 58c). The number of lymphocytes associated with the AQ-treated mice was highest at Week 1 in the cervical lymph nodes and at Week 3 in the spleen (Figures 58e and f). 99

119 Figure 55. Treatment of C57BL/6 mice with AQ results in mild liver injury. (a) Serum ALT in female or (b) male mice treated with AQ for up to 6 weeks. (c, d) Body weight of mice in (a) and (b). (e) Covalent binding of AQ in liver of a control female C57BL/6 mouse or (f) treated with AQ for 3 weeks. (g, h) Covalent binding in spleens of the same mice (20X magnification). For covalent binding studies, figures are representative of four animals/group. Values shown are 100

120 mean ± SE. Analyzed for statistical significance by two-way ANOVA; p values <0.05 were considered significant (*p<0.05; **p<0.01; ***p<0.001). Immunohistochemical analysis revealed an increase in immune cells staining positive for CD4, CD8, CD11b, F4/80, and CD45R in livers of mice treated with AQ, as well as increased cell proliferation as determined by cells that stained positive for KI67 (Figure 59a). The increase in cell numbers was greatest at Week 3 except for F4/80 cells that remained elevated at Week 7 (Figure 59b). The grading of immunohistochemistry is shown in Figure S2. In the spleen, an increase in cells staining for CD11b, KI67, F4/80, and CD45R in the red pulp (Figures S3 and S4) was observed. Most of these changes were apparent between Week 1 and 3 (data not shown), with the exception for KI67 that increased only at Weeks 3 and 7 and not on Week 1 (data not shown for all slides). d) Increase in ALT is associated with activation of T-cells In addition to immunohistochemistry, flow cytometry was used to quantify lymphocyte infiltration and activation. No major changes were detected in the number of CD4 + T-cells in the livers of AQ-treated mice, but there was an increase in total number of CD4 + T-cells in cervical lymph nodes at Week 1 and in the spleen at Week 3 (Figure 60). Moreover, an increase in the percentage and total CD8 + and NK1.1 + cells was observed in the liver of these hosts at Weeks 1 and 3 (Figure 60). In addition, an increase in CD8 + and NK1.1 + cells was also apparent in the cervical lymph nodes at Week 1 and in the spleen at Week 3 (Figure 60). The number of activated CD4 + and CD8 + T-cells was determined by staining for CD62L, CD44, and CD69 (Bjorkdahl et al., 2003; Chao et al., 1997; Ziegler et al., 1994). No significant increase in the total number of activated CD4 + cells in the liver was observed in the AQ-treated hosts (Figure 61); in fact, the percentage of activated CD4 + cells decreased (Figure S5). 101

121 However, an increase in the activation of CD4 + cells in the spleen and cervical lymph nodes was noted at Week 1 (Figure 61, Figure S5). A greater increase was seen in the total number of activated CD8 + cells in the liver, followed by trends that suggest activation in the spleen and cervical lymph nodes of the AQ-treated mice (Figure 61). The percentage of CD8 + activated lymphocytes in the liver also increased (Figure S6). Further analysis of CD4 T-cells revealed that there was an increase in CD4 + CD62L + T cells in the liver at Weeks 1 and 3, which could be characterized as central memory T-cells (Figure 62a) with no significant increase in CD8 + CD62L + cells observed (data not shown). In addition, there was an increase in the percentage and total number of cells that stained positive for CD279 (otherwise known as PD-1) in the liver (Figure 62b), suggesting induction of immune tolerance. Of note, no changes in the surface expression of CD152 (known as CTLA-4) were observed (data not shown). Serum cytokines such as IL-1α, IL-12 (p40), and IL-17 increased at Week 1 in the AQtreated hosts (Figure 63). However, a general trend suggested that most serum cytokines followed the pattern of ALT; by Week 7 there was a down-regulation that was significant for a variety of cytokines including IL-3, IL-5, IL-10, IL-12(p70), IL-13, IFNγ, TNFα, and G-CSF (Figure 64). 102

122 Figure 56. Covalent binding of AQ. Binding in the liver, spleen, gut, and kidney of (a) female and (b) male C57BL/6 mice (n=2) as a function of time. Mice were treated for 5 weeks with AQ (0.2% [w/w] in food). The covalent binding to proteins in the S2.5 fraction was determined on Western blots with antibodies that recognize AQ bound to proteins as described in the Methods section. The same amount of protein was loaded in each lane so that the binding could be compared between different treatment durations. 103

123 Figure 57. Comparison of AQ covalent binding. Binding in the liver, spleen, gut, and kidney (n=2) of (a) female or (b) male C57BL/6 mice. (c) Comparison of AQ covalent binding between female and male C57BL/6 mice (under the same conditions and on the same blot). The methods are the same as described in the legend for Figure 56. The timepoint is 5 weeks. e) Treatment of Rag1 -/- mice and depletion of NK cells To determine whether the adaptive immune system was involved in the pathogenesis of liver injury in this model, Rag1 -/- mice (B- and T-cell deficient) were treated with AQ. In preliminary experiments, an increase in ALT was noted in wild-type (WT) and Rag1 -/- mice (Figure S6A); by Week 5, the ALT appeared higher in the Rag1 -/- mice. The covalent binding of AQ in the liver of Rag1 -/- mice was equal to that of WT mice (Figure S6B), an outcome that suggested 104

124 Rag1 -/- mice may have impaired immune tolerance at later timepoints. We then monitored ALT activities in C57BL/6 or Rag1 -/- mice for up to 11 weeks and found ALT was consistently higher in Rag1 -/- mice. However, these mice did not progress to liver failure (Figure 64a). Covalent binding of AQ in the liver at Week 11 was similar between WT and Rag1 -/- mice, suggesting that covalent binding was not the reason for the differences in ALT (Figure 64b). An increase was observed in NK cell numbers (Figure 59) and IL-12 levels (Figure 63), the latter which is produced by activated macrophages and can activate NK cells. Consequently, the role of NK cells in this model was investigated. Upon depletion of NK cells, there was a significant attenuation of AQ-induced ALT elevation compared to the isotype-treated controls, but there was no difference in the covalent binding of AQ in the liver 64c and d). 5. Discussion IDILI is a significant problem because it is associated with significant patient mortality and can lead to the withdrawal of an otherwise useful drug (Bjornsson, 2010; Senior, 2007). It will be difficult to effectively deal with the problem without a better mechanistic understanding. A major impediment to a better mechanistic understanding of IDILI is the lack of valid animal models that could be used to test hypotheses. To be a valid animal model it must mimic the IDILI that occurs in humans. This includes a delay in the onset of liver injury for at least 1 week and a liver histology that consists of mononuclear inflammatory infiltrates (consisting mostly of CD8 + T-cells and macrophages) that ultimately lead to hepatocyte necrosis (Ng et al., 2012; Watkins, 2005). If a drug can cause idiosyncratic liver failure, it is always associated with a higher incidence of mild IDILI, which often resolves despite continued treatment with the drug; this is referred to as adaptation (Watkins, 2005). 105

125 Figure 58. ALT, liver and spleen weight, and total lymphocyte numbers in female C57BL/6 mice treated with AQ. (a) ALT activity after treatment with AQ for 1, 3, or 7 weeks. (b, c) Liver and spleen weights divided by body weights of control or AQ-treated animals from (a). (d f) Total number of lymphocytes of control or AQ-treated animals from (a). The number of lymphocytes was adjusted for liver and spleen weight, and a total of five lymph nodes were isolated/mouse. Values shown are mean ± SE. Analyzed for statistical significance versus control by Mann-Whitney U-test; p values<0.05 were considered significant (*p<0.05; **p<0.01; ***p<0.001). 106

126 Figure 59. Immunohistochemical staining of livers from female C57BL/6 mice. (a) Staining for CD4, CD8, CD11b, KI67, F4/80, or CD45R. (b) Grading of the number of cells in (a); grading is based on the results of statistical analysis from Figure S2. Figures are representative of four animals/group. For illustration purposes, slides from mice treated with AQ for 3 weeks are shown as they showed the greatest changes 107

127 Figure 60. Lymphocyte phenotyping for CD4, CD8, and NK1.1. Control mice (n=8) or mice treated with AQ for 1, 3, or 7 weeks (n=8). Values shown are mean ± SE. Analyzed for statistical significance versus control by Mann-Whitney U-test; p values <0.05 were considered significant (*p<0.05; **p<0.01; ***p<0.001). In this paper, we describe the successful development of an animal model (female C57BL/6) of AQ-induced liver injury, which has a delayed onset and resolves despite continued treatment. 108

128 The delayed onset in this animal model is similar to the IDILI caused by most drugs that induce idiosyncratic drug reactions (Bjornsson, 2010; Uetrecht, 2009). Liver injury occurred only in female mice. This is most easily explained by the higher blood levels of AQ and DEAQ in female mice. Greater AQ covalent binding was also detected in the liver of female mice compared to male mice. The greater injury in female mice was consistent with the observation that women appear more susceptible to most forms of IDILI, especially when liver injury is more severe as in the case of fulminant hepatic failure (De Valle et al., 2006; Lucena et al., 2009; Metushi et al., 2014d; Russo et al., 2004; Sgro et al., 2002). Covalent binding of AQ occurred in the liver as well as in the spleen, kidney, and gut; however, the highest amount of covalent binding was observed in the liver, the target organ of the injury. AQ covalent binding in the liver plateaued after 3 days of treatment; however, the liver injury was not evident until after 1 2 weeks of treatment, and peaked at 3 weeks. This disconnect between the rapid covalent binding but delayed onset of liver injury is consistent with a mechanism in which the covalent binding does not directly cause the liver injury, but instead initiates an immune response that results in liver injury. Coincident with an increase in ALT, an infiltration of T- cells, B-cells, macrophages, and dendritic cells in the liver and spleen was observed. In addition, AQ treatment resulted in an increase in the size of the spleen and lymphocyte infiltrates in the liver, spleen, and cervical lymph nodes. Taken together, these data indicated an immune response. Furthermore, the increase in the number of lymphocytes in lymph nodes appeared to precede the increase in the spleen and liver. This was in line with an immune response causing the injury rather than being a response to the injury (Bowen et al., 2005). 109

129 Figure 61. Lymphocyte activation in AQ-treated mice. Cells were from control (n=4) or mice treated with AQ (n=4) for 1, 3, or 7 weeks. Values shown are mean ± SE. Analyzed for statistical significance versus control by Mann-Whitney U-test; p values <0.05 were considered significant (*p<0.05; **p<0.01; ***p<0.001). 110

130 Figure 62. Expression of CD62L + and PD-1 + (CD279) on T-cells. Cells were from control (n=4) or mice treated with AQ (n=4) for 1, 3, or 7 weeks. Values shown are mean ± SE. Analyzed for statistical significance versus control by Mann-Whitney U-test; p values <0.05 were considered significant (*p<0.05; **p<0.01; ***p<0.001). 111

131 Figure 63. Serum cytokine concentrations. Levels in serum of control (n=8) or mice treated with AQ (n=8) for 1, 3, or 7 weeks. Values shown are mean ± SE. Analyzed for statistical significance versus control by Mann-Whitney U-test; p values <0.05 were considered significant (*p<0.05; **p<0.01; ***p<0.001). 112

132 Figure 64. Liver injury in AQ-treated Rag -/- mice or mice treated with an anti-nk cell antibody. (a) ALT activity in C57BL/6 or Rag -/- mice treated with AQ for up to 11 weeks. (b) Western blot showing covalent binding of AQ to the S9 liver fraction from mice in (a). (c) ALT activity in female C57BL/6 mice. Control = untreated mice but injected with PBS on the same schedule as anti-nk1.1 antibody. IgG Isotype + AQ = mice that were injected with IgG isotype control while being treated with AQ at 0.2% [w/w] in food for up to 3 weeks. Anti-NK1.1 + AQ = mice were injected with anti-nk1.1 antibody (as described in Methods) while receiving AQ at 0.2% [w/w] in food for up to 3 weeks. ALT levels were similar to control when mice were injected with anti-nk1.1 antibody alone (data not shown). (d) Western blotting showing covalent binding of AQ in the liver of mice at the end of 3 weeks. Quantification of AQ covalent binding expressed as relative intensity (and corrected for GAPDH) was performed for both (b) and (d), but there were no differences between groups (data not shown). For (a) and (c), values shown 113

133 are mean ± SE. Analyzed for statistical significance using two-way ANOVA; p values <0.05 were considered significant ( # or*p<0.05; **p<0.01; ***p<0.001; *=significantly different from control; #=significantly different from AW-treated groups). The dominant immune cell infiltrating the livers of patients with drug-induced liver failure are CD8 + cells (Foureau et al., 2012b). We determined the phenotype of infiltrating CD4 + and CD8 + T-cells by using a variety of activation markers such as CD62L, CD44, and CD69 (Ziegler et al., 1994). Overall, it was shown that the greatest cell activation in the liver occurred in CD8 + T-cells. An increase in the number and activation of CD4 + cells in the spleen and cervical lymph nodes, but not in the liver, was also detected. These results suggested an immune response in the spleen and lymph nodes involving helper T-cells. In addition, an increase in CD4 + central memory (TCM) T-cells (defined as CD62L + ) and an increase in CD8 + effector memory (TEM) cells (defined as CD62L - ) was observed in the liver that could be involved in the resolution of liver injury (Bowen et al., 2005; Sallusto et al., 2004; Yang et al., 2011). Consistent with these findings we found the increase in CD8 + CD44 + cells in the liver and in the spleen. Although CD44 (a cell-adhesion molecule that binds to hyaluronan) is commonly used as a marker of activation, recently CD44 has been implicated in leukocyte recruitment to inflammatory areas and resolution of inflammation in models of hepatitis (Johnson et al., 2009; Pure et al., 2001; Siegelman et al., 1999). This is consistent with the fact that studies with acetaminophen and carbon tetrachloride revealed that the lack of CD44 gene exacerbated liver injury in animals and humans, as reviewed elsewhere (Tujios et al., 2011). In the present study, the greatest increase in CD44 + liver lymphocytes was observed at Week 3 and could be involved in the resolution of liver injury. The data clearly shows activation of CD8 T-cells in the liver. If the role of these CD8 T-cells were pathogenic, then one would expect that Rag1 -/- mice that are B- and T-cell deficient would not develop an increase in ALT. The fact that Rag1 -/- mice 114

134 showed a greater increase in ALT suggests that the adaptive immune system may play a more important role in induction of immune tolerance than injury in this model. Consistent with induction of tolerance by the adaptive immune system is the fact that the delayed onset of ALT was followed by a resolution in injury and with a decrease in the number of most cell types in the liver except for macrophages, which are classically involved in the repair of injury (Singer et al., 1999). In addition, the resolution of liver injury was preceded by an increase in hepatic T- cells (both CD4 + and CD8 + ) that stained positive for PD-1, a molecule involved in immune tolerance (Pardoll, 2012). Additional data here that support the hypothesis of immune tolerance included decreases detected at Week 7 in levels of pro-inflammatory cytokines such as IL-12, TNFα, IFNγ, IL-5, and IL-1β. This is not a model of idiosyncratic drug-induced liver failure, but it does mimic the mild liver injury that is more common than liver failure and usually has a delayed onset with resolution despite continued treatment with the drug. Although the peak ALT was not very high, in general the ALT is not as high in chronic liver injury as it is in acute injury. Thus, these data suggest that IDILI may be initiated with a sequence of events, starting with bioactivation of AQ and covalent binding. This appears to trigger an immune response by activated helper T-cells migrating to lymph nodes (and to a lesser extent the spleen) within the first week. This is followed by an increase in serum inflammatory cytokines (IL-1α, Il-12 and IL-17) and an early increase in CD8 + T-cells in the liver. Given the observation that the injury is greater in Rag1 -/- mice and that there was an increase in PD-1 + T-cells in the liver (both CD4 + and CD8 + ), with the ultimate result that the injury resolved, this appears to represent the more common response of humans to a drug that can cause IDILI; namely, immune tolerance. The protective effect of anti- NK1.1 antibodies provided clear evidence that NK cells a major cell type in the liver and implicated in other types of liver injury mediated much of the liver injury in this model (Tian 115

135 et al., 2013). However, the major cell type found in the liver of patients with IDILI resulting in liver failure is the CD8 + T-cell (Foureau et al., 2012a). In this model both innate and adaptive immune cells were involved. It is plausible that the initial liver injury in humans is also mediated by NK cells, but this usually resolves with immune tolerance; it is only if immune tolerance fails that cytotoxic CD8 + T-cells become the dominant cell responsible for serious liver injury. In conclusion, this model provides investigators with a unique opportunity to study an immune response to a drug that causes significant IDILI in humans. 6. Acknowledgement Dr. Jack Uetrecht is the recipient of the Canada Research Chair in Adverse Drug Reactions. This work was supported by a grant received from the Canadian Institutes of Health Research. 7. Declaration of interest The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. 116

136 Chapter 4: Involvement of T Helper 17 Cells in D-Penicillamine Induced Autoimmune Disease in Brown Norway Rats T his work has been published in the following journal and is reproduced with permission Zhu X, Li J, Liu F, Uetrecht JP. Involvement of T helper 17 cells in D-penicillamine-induced autoimmune disease in Brown Norway rats. Toxicol Sci Apr; 120(2): In this chapter, all the experiment was completed by Feng Liu, except Fig 65 and Fig

137 1. Abstract Idiosyncratic drug reactions (IDRs) are poorly understood, but their clinical characteristics suggest that they are immune mediated. Penicillamine-induced autoimmunity in Brown Norway rats has been utilized as an animal model for mechanistic studies of one type of IDR because it closely mimics the autoimmune syndromes that it causes in humans. Our previous work suggested that it is T-cell mediated. 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 and to establish an overall serum cytokine/chemokine profile for this IDR. In total, 24 serum cytokines/chemokines were determined and revealed a dynamic process. In sick animals, interleukin (IL) 6 and transforming growth factor-b1, known to be driving forces of Th17 differentiation, were consistently increased at both early and late stages of penicillamine treatment; however, no significant changes in these cytokines were observed in animals that did not develop autoimmunity. IL-17, a characteristic cytokine produced by Th17 cells, was increased in sick animals at both the messenger RNA and serum protein level. In addition, serum concentrations of IL-22, another characteristic cytokine produced by Th17 cells, were found to be elevated. Furthermore, the percentage of IL-17 producing CD4 T cells was significantly increased but only in sick animals. These data strongly suggest that Th17 cells are involved in penicillamine-induced autoimmunity. Such data provide important mechanistic clues that may help to predict which drug candidates will cause a relatively high incidence of such autoimmune IDRs. Key Words: idiosyncratic drug reactions; penicillamine; Th17; autoimmune; animal models. 2. Introduction 118

138 Idiosyncratic drug reactions (IDRs) refer to a group of adverse drug reactions that do not occur in most patients within their therapeutic dose range and cannot be explained by the known pharmacological properties of the drug (Uetrecht, 2009). IDRs can be very severe, even life threatening, thus representing a significant clinical problem. They also present a challenge to the pharmaceutical industry by adding an additional level of uncertainty to new drug development. At present, it is impossible to predict IDRs largely because the mechanisms involved are unknown. Nevertheless, the delay between starting the drug and the onset of the adverse reactions suggests that most are immune mediated (Uetrecht, 2007). Animal models are essential tools for mechanistic studies; unfortunately, IDRs are also idiosyncratic in animals, and so there are very few practical models with characteristics similar to idiosyncratic reactions that occur in humans (Shenton et al., 2004). Penicillamine-induced autoimmunity in Brown Norway (BN) rats represents an important model for the mechanistic study of one type of IDR, drug-induced autoimmunity, because it mirrors the variety of autoimmune reactions that it can cause in humans: both can involve the presence of antinuclear antibodies, a skin rash, deposits of immunoglobulin G (IgG) along the glomerular basement membrane, arthritis, hepatic necrosis, and weight loss (Jaffe, 1981; Sayeh et al., 2001; Tournade et al., 1990). By definition, drug-induced auto-immunity is an immune-mediated IDR. Penicillamine-induced autoimmunity in rats is also idiosyncratic: it is strain specific treatment of Lewis and Sprague-Dawley rats does not induce autoimmunity. Moreover, even though BN rats are highly inbred and syngeneic, autoimmunity only occurs in a little over 50% of male BN rats. Ever since it was proposed in 1986, the T helper (Th1)-Th2 hypothesis has been a significant aspect of mechanistic theories of T-cell mediated diseases (Mosmann, 1992). Based on an elevation of interleukin (IL) 4 and IgE, which are associated with Th2-type responses, it was 119

139 proposed that penicillamine-induced autoimmunity as well as autoimmunity induced by gold salts and graft versus host disease were Th2-driven immune reactions (Goldman et al., 1991). We tested this hypothesis by using a series of agents such as misoprostol that were expected to tip the Th1/Th2 balance; however, the effects were the opposite of those predicted by the Th2 hypothesis (Sayeh et al., 2001). In contrast, it was proposed that organ-specific autoimmune diseases were mediated by Th1 cells, which are driven by IL-12 and produce interferon (IFN)-γ (Singer et al., 1999). However, the Th1 theory of organ-specific autoimmunity was challenged because Th1 cytokines were often found to be protective. For example, IFN-γ knockout mice had a higher mortality in an experimental autoimmune encephalomyelitis model than the wildtype animals (Ferber et al., 1996). In an animal model of arthritis, it was found that it was IL-23, which shares a p40 subunit with IL-12, and not IL-12 that was required for the development of arthritis (Murphy et al., 2003). Additional studies of the involvement of IL-23 in autoimmune diseases led to the discovery of a new helper T-cell sub-set characterized by the production of a proinflammatory cytokine, IL-17, which were therefore named Th17 cells (Langrish et al., 2005; McKenzie et al., 2006). Since its discovery, the signature cytokine pattern of Th17 cells has been expanded with addition of several other key inflammatory cytokines such as IL-21 and IL-22. In spite of many unknowns in the function of Th17 cells, significant progress has been made in characterizing this new T-cell population. A large number of studies have found that a combination of transforming growth factor (TGF)-β and IL-6 are required for the initial commitment of naive T cells to become Th17 cells (Mangan et al., 2006; Zhou et al., 2007); exposure to TGF-β in the absence of IL-6 leads to T regulatory cells believed to play an important role in immune tolerance (Zhou et al., 2008). In contrast, IL-23 was found to play a very important role in maintaining the growth and expansion of Th17 cells. Numerous studies in both humans and mice strongly suggest that the Th17 cell is a major determinant of the 120

140 development of many kinds of autoimmune diseases (Ouyang et al., 2008). Although the exact role of Th17 cells is controversial, there is compelling evidence that Th17 cells are involved in many inflammatory and autoimmune reactions (Tesmer et al., 2008). This study was designed to examine the involvement of Th17 cells in penicillamine-induced autoimmunity as a model of a drug-induced autoimmune IDR. 3. Materials and methods a) Animals Male BN rats ( g) were purchased from Charles River (Montreal, Quebec, Canada) and doubly housed in standard cages in a 12:12 h light:dark cycle at 22LC. The rats were given free access to standard rat chow (Agribrands Purina Canada, Strathroy, Ontario, Canada) and tap water for a week long acclimatization period before starting an experiment. All the animal protocols were approved by the University of Toronto Animal Care Committee. b) Chemicals, kits, and solutions D-Penicillamine was purchased from Richman Chemical Inc. (Lower Gwynedd, PA). MACS anti-rat CD4 magnetic microbeads and magnetic columns were purchased from Miltenyi Biotec (Auburn, CA). ELISA and enzyme-linked immunosorbent spot (ELISPOT) kits were purchased from R&D Systems (Minneapolis, MN). Luminex kits were purchased from Millipore (St Charles, MO). RNeasy Mini kits and OmniScript reverse transcriptase 285 kits were purchased from Qiagen (Mississauga, Ontario, Canada). Oligo (dt15) primers, RNAse inhibitor, and LightCycler SYBR Green I kits for quantitative real-time PCR (qrt-pcr) were all purchased from Roche (Montreal, Quebec, Canada). High performance liquid chromatography-purified primers for qrt-pcr were designed and obtained from Integrated DNA Technologies (Coralville, IA). Phorbol 12-myristate-13-acetate and calcium ionomycin were purchased from 121

141 Sigma (Oakville, Ontario, Canada). Fixation/permeabilization solution was purchased from ebioscience (San Diego, CA). Monensin was purchased from BD Biosciences (San Jose, CA). Conjugated monoclonal antibodies for CD4 (clone W3/25) were purchased from Cedarlane (Burlington, Ontario, Canada), and IL-17 (clone ebio17b7) was purchased from ebioscience. Table 3. Primer sequences for qrt-pcr c) D-penicillamine treatment Rats were given D-penicillamine dissolved in tap water at a concentration of 1.5 mg/ml with an average water intake of 25 ml/day. The D-penicillamine solution was prepared fresh every 2 days because of the slow formation of inactive penicillamine disulfide. Unless otherwise indicated or unless signs of a severe autoimmune syndrome led to sacrifice of the animal, the duration of D-penicillamine treatment was 8 weeks. Red ears were the primary sign used as a surrogate to determine which animals had developed autoimmunity Determination of serum IL-6 Blood samples were drawn via tail vein on day 0 and at the end of each week of penicillamine treatment. Blood samples were allowed to clot for 2 h at room temperature before centrifuging 122

142 for 20 min at approximately 2000 X g. Sera were aliquoted and stored at -80. Serum IL-6 levels were determined by ELISA Phenotyping splenic CD4 + T cells by qrt-pcr At the end of penicillamine treatment, splenic CD4 + T cells were isolated using rat CD4 magnetic microbeads according to the manufacturer s instructions. RNA was isolated from CD4 + T cells using RNeasy mini kits as described by the manufacturer. RNA concentration and purity were determined spectrophoto-metrically. RNA (0.5 µg) from each sample was reverse transcribed to complementary DNA (cdna). The expression level of Th17-related cytokine messenger RNAs (mrnas) was determined using qrt-pcr carried out with a LightCycler instrument (Roche) using the primers listed in Table 3. The basic PCR program for all samples was as follows: 95 for 10 min, 45 cycles of 95 for 5 s, annealing temperature (primer specific, 295 range ) for 5 s, and elongation at 72 for various times (because of difference in PCR product length, range 5 16 s). Melting curve analysis was performed after amplification and carried out at a temperature transition rate of 0.2 /s up to 95. Data were normalized by calculating the absolute concentration of the cdna of interest relative to absolute glyceraldehyde 3-phosphate dehydrogenase concentration in each cdna sample Profiling cytokines/chemokines Male BN rats (n=20) were treated with penicillamine, and blood samples were drawn via tail vein on day 0 and at end of each week of treatment. Serum was isolated as described above. A Luminex assay of 24 cytokines/chemokines (eotaxin, granulocyte macrophage colonystimulating factor, granulocyte colony stimulating factor, monocyte chemotactic protein-1 (MCP), leptin, macrophage inflammatory protein-1α, IFN-γ, IL-1α, IL-1β, IL-2, IL-4, IL-5, IL- 6, IL-9, IL-10, IL-12p70, IL-13, IL-17, IL-18, interferon gamma-induced protein-10, growth 123

143 related oncogene (GRO)/KC, regulated upon activation, normal T cell expressed and secreted (RANTES), tumor necrosis factor-α, and vascular endothelial growth factor) was performed to determine the overall pattern of serum cytokines/chemokines over the course of penicillamine treatment using the protocol provided by the manufacturer. Serum concentration of IL-22 was determined separately by ELISA during penicillamine treatment of male BN rats (n=8) at days 0, 7, 14, 16, 18, 20, 22, 24, and 28. In another study (n=8), IL-6 and IL-22 were measured by ELISA at days 1, 3, 5, and 7 of penicillamine treatment to determine if an early change predicted which animals would develop autoimmunity. In addition, at the end of treatment, serum IL-6 and IL-7 were determined by ELISA and IL-17 production in blood, lymph nodes, and the spleens was determined by ELISPOT according to the protocol provided by manufacturer as below Determination of IL-17 production by ELISPOT The frequency of IL-17 producing cells from different organs was evaluated by ELISPOT using an IL-17A ELISPOT kit. Briefly, single-cell suspensions free of red blood cells were prepared from the spleens, cervical lymph nodes, and peripheral blood mononuclear cells (PBMC). An aliquot of 2.0 X 10 5 lymphocytes was added to each well and stimulated with 50 ng/ml of phorbol 12-myristate-13-acetate and 0.5 µg/ml calcium ionomycin for 16 h at 37 in a 5% CO2 incubator, which was followed by streptavidin conjugation and 5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium chromogen staining according to the manufacturer s instructions. The spots were counted using an ImmnunoSpot Reader Intracellular cytokine staining and flow cytometry The presence of CD4 + IL-17 + cells was evaluated by flow cytometry. Lymphocytes were isolated from the cervical lymph nodes, and after cell surface staining, cells were washed and 124

144 resuspended in fixation/permeabilization solution, and intracellular staining was performed following the manufacturer s instructions. For the detection of IL-17, cells were incubated for 4 h with 50 ng/ml phorbol myristate acetate and 1 µg/ml ionomycin in the presence of monensin in tissue culture incubator at 37LC. Stained cells were all analyzed by a BD FACSAria flow cytometer, and data were analyzed by FlowJo software Statistical analysis Statistical analyses were performed using GraphPad prism (GraphPad Software, San Diego, CA). T-test with Welch s correction was conducted to examine the ELISA results. One-way ANOVA was carried out for flow cytometry analysis. Two-tailed analysis was carried out with significance defined as p < 0.05 with 95% confidence. 125

145 Results 4.1. Serum levels of IL-6 during penicillamine treatment In a preliminary experiment, two out of three penicillamine-treated rats developed an autoimmune syndrome (rat # 2 (P2): day 18; rat # 3 (P3): day 20). Serum IL-6 was significantly increased at about the time that the animals developed clinical signs of autoimmunity, whereas the serum IL-6 level of the nonsick and control animals remained at nondetectable levels throughout the treatment (Figure 65). Figure 65. Serum concentration of IL-6: penicillamine (n=3) versus control (n=3). Out of three penicillamine-treated rats, two developed autoimmunity (P2 and P3). Significant serum IL-6 levels were only detected in the two sick animals, not in nonsick (P1) and control animals (C1, C2, and C3). (P, penicillamine treated; C, Control). At the end of the experiment described in the previous paragraph, the rats were sacrificed and total RNA was isolated from purified splenic CD4 + T cells. IL-21 and IL-4 mrna was increased in all three treated animals (fourfold and twofold, respectively). In contrast, a twofold increase of IL-17 mrna was only observed in the two sick animals, and there was no 126

146 significant change in IFN-γor IL-10 mrna. Given that this was a preliminary experiment with limited numbers of animals, the results could only be used to direct further experiments Serum cytokine/chemokine pattern during penicillamine treatment During 5 weeks of penicillamine treatment, 15 out of 20 rats developed autoimmunity, and in most cases, the time to onset was between 14 and 21 days. The body weight and cumulative incidence are shown in Figure 66. The total splenocytes in sick animals was more than double those in nonsick animals indicating significant splenomegaly. Of the 24 cytokines/chemokines, serum levels of IL-6, TGF-β1, IL-17, IL-2, IL-4, IL-5, IL-9, IL-10, IL-13, IL-18, GRO/KC, MCP-1, leptin, and RANTES were found to be significantly different between sick and nonsick animals at different time points (Figure 67), whereas other analytes were either nondetectable in all treated animals (i.e., IL-1α) or there was no difference between sick and nonsick rats throughout the penicillamine treatment (i.e., IFN-γ). Figure 66. Changes in body weight (A) and cumulative incidence of autoimmunity (B) in 20 animals that were treated with penicillamine for 5 weeks; 15 developed autoimmunity and 5 did not. 127

147 Figure 67. Serum cytokine/chemokine profiles: Sick versus Nonsick. 128

148 4.3. Serum IL-22 during penicillamine treatment Serum concentrations of IL-22 during penicillamine treatment were monitored and compared between sick and nonsick animals. As shown in Figure 68, there was an elevation of serum IL- 22 about 4 days before the onset of autoimmunity in sick rats, whereas no elevation of IL-22 was detected in nonsick rats. Figure 68. Serum IL-22 during the development of penicillamine-induced autoimmunity. The onset of autoimmunity for four sick animals were day 15 for rat 5, day 20 for rat 4, day 22 for rat 6, and day 27 for rat Serum cytokines at early time points of penicillamine treatment If IL-6 drives the formation of Th17 cells, we would expect to see an increase in IL-6 at early time points, but in Figure 67, there is no increase in IL-6 until day 21. It is possible that we missed an early spike in IL-6, and so another experiment was done to look at IL-6 and IL-22 levels during the first week. Out of eight treated rats, four eventually developed autoimmunity. Concentrations of serum IL-6 (day 1) and IL-22 (day 3) were elevated more in animals that developed autoimmunity as shown in Figure

149 Figure 69. IL-6 (panel A) and IL-22 (panel B) serum levels in the first week of penicillamine treatment (n=8: 4 developed autoimmunity and 4 did not by the end of treatment) Th17 cell phenotyping Marked IL-17 production by cells from lymph nodes, the spleen, and PBMC is clearly shown by ELISPOT with few IL-17 producing cells in the wells containing cells from control and nonsick animals (Figure 70). Consistent with previous findings, a dramatically elevated level of IL-6 was detected in sick animals at the end of treatment. In contrast, the serum concentration of IL-7 was lower in sick animals than in nonsick animals. More definitive evidence of Th17 cell involvement in penicillamine-induced autoimmunity is the marked increase in CD4 + IL-17 + cells in sick animals compared with control and nonsick animals (Figure 71). 130

150 Figure 70. IL-6, IL-7, and IL-17 production at the end of penicillamine treatment. Panel A shows the serum IL-6 and IL-7 levels. Panel B shows the number of cells from cervical lymph nodes (ALN), the spleen, and PBMC that produce IL-17 as determined by ELISPOT. The number in bold next to each spot is the number of cells producing IL

151 Figure 71. Flow cytometry analysis for intracellular IL-17A in CD4 + lymphocytes from control, treated nonsick, and sick animals. Panel A shows a representative plot from one animal from each group, and panel B shows the average percentage of CD4 + /IL-17 + cells (n=4 in each group). 132

152 4. Discussion Th17 cells have been implicated in many types of immune-mediated pathology, but there is currently little evidence for their involvement in IDRs. This study provides a very complete cytokine/chemokine profile as a function of time during the development of an autoimmune IDR. An increase in IL-17 producing cells as determined by ELISPOT was observed in sick animals but that does not prove the involvement of Th17 cells. For example, in a previous study, we found an increase in serum IL-17 levels in some patients with idiosyncratic drug-induced liver failure, which suggested that Th17 cells were also involved in this IDR. However, some of the patients with acetaminophen-induced liver failure also had increased serum levels of IL-17, and acute acetaminophen-induced liver injury is very unlikely to be mediated by Th17 cells (Li et al., 2010). It is now known that several other cell types, especially innate immune cells (i.e., γδt cells, invariant natural killer T cells, natural killer cells), can produce IL-17 (Cua et al., 2010). These innate immune cell populations are probably the major cellular sources of IL-17 in response to early cell stress or damage. Thus, the first peak of serum IL-17 around 1 week of penicillamine treatment is likely released from innate cells that can be directly activated by penicillamine. In contrast, later in the course of treatment, animals with evidence of penicillamine-induced autoimmunity had a marked increase in CD4 + / IL-17 + cells, which defines Th17 cells, and this provides strong evidence for their involvement in penicillamineinduced autoimmunity. In addition, the pattern of cytokines is exactly what would be expected for a Th17 response, specifically an early spike of IL-6 (Figure 69) and the presence of increased TGF-β (Figure 67), which are required for the development of Th17 cells as discussed in the Introduction section. This was followed by an increase in the Th17 cytokine IL-22 (Figure 68) just before the animal developed signs of autoimmunity, and there was also a second 133

153 increase in IL-17. It appears that many other types of IDRs may have an autoimmune component (Uetrecht, 2009); therefore, Th17 cells may be involved in other types of IDRs. In addition to the Th17-associated cytokines, i.e., IL-17 and IL-22, there is also a marked increase in IL-2, IL-5, IL-6, IL-9, IL-10, IL-18, and MCP-1 and a marked decrease in RANTES and leptin when the animals develop clinical evidence of autoimmunity (Figure 67). IL-9 can be produced by Th17 cells, but it also appears to be produced by Th2 and Th9 cells and is associated with autoimmunity (Nowak et al., 2010). The major source of IL-6, IL-18, and MCP- 1 (monocyte chemotactic protein also known as chemokine (C-C motif) ligand) is macrophages, and these cytokines/chemokines are associated with inflammation and generally increased in autoimmune reactions. In particular, IL-18 appears to be a good indicator of disease activity in lupus (Favilli et al., 2009). Therefore, although these results are consistent with the hypothesis that penicillamine-induced autoimmunity is mediated by Th17 cells, it is also to be expected that any immune response involves a symphony of different cells interacting over time, and this is demonstrated by these data. The very early spike in IL-6 predicted which animals would develop autoimmunity. Therefore, it is clear that a very early decision is made by the immune system that determines how it will evolve even though the manifestations of autoimmunity occur weeks later. It is possible that such a pattern could be used to predict which patients will develop an IDR; however, there is no evidence at present that most IDRs are mediated by Th17 cells. Although the spike in IL-6 clearly predicted which animals would ultimately develop autoimmunity, it is not clear what factors determine which animals will develop auto-immunity, especially when these animals are syngeneic, thus virtually genetically identical, and they were housed together which should minimize environmental factors. A better understanding of cellular sources of cytokines (i.e., IL-6, IL-13, IL-17, etc.) that significantly changed during 134

154 early drug treatment might be able to explain the response difference and would shed light on the pathogenesis of drug-induced idiosyncratic autoimmunity. In summary, this study provides a very nice picture of cytokine changes that occur during the development of an IDR. It demonstrates the involvement of Th17 cells, but not surprisingly, there are changes in many cytokines produced by other cells. It remains to be determined whether this is a common profile for most IDRs, is specific for autoimmune reactions, or is unique to penicillin-induced autoimmunity. IL-17 intracellular staining determined by flow cytometry represents an efficient way to define the Th17 cell population. However, it would be a challenge to obtain fresh peripheral blood cells from patients early in the course of an IDR for the assessment of Th17-mediated pathology. 5. Funding Canadian Institutes of Health Research. 7. Acknowledgement J.U. holds a Canada Research Chair in Adverse Drug Reactions. 135

155 Chapter 5: Summary, Discussion and Future Directions 1. Summary of findings and discussion IDRs represent a significant problem; they are often serious, even life threatening, and their unpredictable nature makes them virtually impossible to prevent (Uetrecht, 2007). In addition, they also represent a major problem for drug development, and the unpredictability of IDRs makes it unlikely that they will be discovered in the clinical trials. This uncertainty significantly increases the overall cost of drug development. However, due to the low incidence and unpredictable natures in humans, the mechanism is hard to study, and it is impossible to perform controlled experiments in humans, which makes it important to develop animal models that can mimic the clinical characteristics of IDRs in humans. In this thesis, two drugs (AQ & D-pen) were studied to explore their mechanisms of IDRs. In the 1 st and 2 nd projects, we established AQ-induced DILI models in both rats and mice. To date, the mechanism by which AQ causes hepatotoxicity is still unknown. Several characteristics suggest that AQ-induced hepatotoxicity has an immune-mediated mechanism: a delayed onset, the presence of anti-drug IgG antibodies, and a prompt increase of ALT on rechallenge of AQ antibodies (Clarke et al., 1991; Neftel et al., 1986; Uetrecht, 2005). However, due to the low incidence in patients and lack of animal models, it is impossible to predict, and the mechanism is hard to study. Previously there was no valid animal model that mimics the AQ-induced delayed onset hepatotoxicity observed in humans, which is critical for us to understand the mechanism of AQ-induced liver injury. First, AQ-induced liver injury was studied in different strains of rats, including BN, Lewis, and Wistar rats. We found that treatment of rats resulted in a mild delayed onset liver injury that 136

156 resolved despite continued treatment with AQ. In addition, pathological changes, apoptosis, and cell proliferation were determined by immunohistochemistry, TUNEL, and PCNA assays. The results indicated Kupffer cell activation and hepatocyte apoptosis and proliferation in the liver. We also examined cytokines/chemokines and anti-aq antibodies and observed that there was also an increase in serum IL-2, IL-5, IL-9, IL-12, MCP-1, and TGF-β, but a decrease in leptin. The above results imply the involvement of immune system. To further test the effects of immune system perturbation, the effects of co-treatment with cyclosporin and Poly I:C were studied. Co-treatment with cyclosporin prevented AQ-induced liver injury, while Poly I:C cotreatment caused an earlier increase in ALT, which suggested that AQ-induced liver injury is immune-mediated. To further explore the nature of the immune response in AQ-induced liver injury, phenotyping of lymphocytes and macrophages was employed in different organs of AQ-treated BN rats. Coincident with the elevated serum ALT, liver CD4 + T cells, IL-17 secreting cells, and Th17/Treg cells were increased at week 3 and decreased during continued treatment. An increase in CD161 + cells and activated M2 macrophages was also observed during the liver injury. These results suggest that the outcome of the liver injury is determined by the balance between effector cells and regulatory cells. Similar results were also observed in AQ-treated C57BL/6 mice. To better understand the mechanism of AQ-induced hepatotoxicity, we investigated the covalent binding of AQ to hepatic proteins. We investigated the covalent binding of AQ to hepatic proteins by an immunoblot assay. A significant amount of covalent binding was observed in the livers of all AQ-treated animals. To overcome immune tolerance, BSO and DEM were used to deplete GSH in vivo. However, depletion of GSH did not increase covalent binding and paradoxically prevented AQ- 137

157 induced liver injury. This implies that glutathione conjugation is not the major pathway for detoxication of the AQ reactive metabolite. An alternative pathway is reduction back to the parent drug. More puzzling is why BSO prevented AQ-induced hepatotoxicity. One reasonable explanation is the involvement of ROS in NK cell function. If a major pathway of AQ metabolite detoxication is reduction it may generate ROS, and GSH is able to detoxify ROS through the glutathione peroxidase pathway (Tafazoli et al., 2009). GSH depletion could substantially increase ROS accumulation in vivo, which may impair NK cell activity. To test the involvement of NK cells we examined the effect of retinoic acid, which has been reported to enhance NK cell activity. Co-treatment with retinoic acid exacerbated AQ-induced liver injury leading to an earlier onset (Day 7) and increased severity. The fact that GSH depletion prevents AQ-induce hepatotoxicity is consistent with the hypothesis that NK cells play a critical role in AQ-induced liver injury. Earlier studies also provided strong evidence for involvement of NK cells in AQ-induced liver injury. This conclusion is supported by the finding of increased numbers of NK cells in the liver and increased NK cell-related cytokines/chemokines such as IL-2, IL-12, IL-18, and CCL5. The presence of IFN-γ, IL-2, IL-6, IL-12, and IL-18 in the inflamed liver has been reported to be associated with an activated NK cell phenotype. In addition, IL-2 may sustain the survival and activation of these cells, and IL-12 may be responsible for the NK cell recruitment (Shi et al., 2011). Our results suggest that the covalent binding of AQ to hepatic proteins is a necessary but not sufficient component of AQ-induced liver injury, and NK cells play a pathogenic role in this mild AQ-induced hepatotoxicity. Meanwhile, in the 2 nd project (Chapter 3), we described the development of a new model of AQ-induced liver injury in C57BL/6 mice with characteristics similar to IDILI in humans. We found that treatment of female C57BL/6 mice with AQ led to liver injury with delayed onset, 138

158 which resolved despite continued treatment. Covalent binding of AQ was detected in the liver, which was greater in female than in male mice, and higher in the liver than in other organs. Covalent binding in the liver was maximal by Day 3, which did not explain the delayed onset of ALT elevation. However, coincident with the elevated serum ALT, infiltration of liver and splenic mononuclear cells and activation of CD8 T-cells within the liver were observed. By Week 7, when ALT levels had returned close to normal, down-regulation of several inflammatory cytokines and up-regulation of PD-1 on T-cells suggested induction of immune tolerance. Treatment of Rag1 -/- mice with AQ resulted in higher ALT activities than C57BL/6 mice, which suggested that the adaptive immune response was responsible for immune tolerance. In contrast, depletion of NK cells significantly attenuated the increase in ALT, which implied a role for NK cells in mild AQ-induced IDILI. However, the major cell type found in the liver of patients with IDILI resulting in liver failure is the CD8 + T-cell (Foureau et al., 2012a). In this model both innate and adaptive immune cells were involved. It is plausible that the initial liver injury in humans is also mediated by NK cells, but this usually resolves with immune tolerance; it is only if immune tolerance fails that cytotoxic CD8 + T-cells become the dominant cell responsible for serious liver injury. In conclusion, this model provides investigators with a unique opportunity to study an immune response to a drug that causes significant IDILI in humans. Overall, we successfully developed two AQ-induced hepatotoxicity models in both BN rats and C57BL/6 mice, which mimic most of clinical characteristics of mild idiosyncratic druginduced liver injury in humans. Although we have not yet been able to develop an animal model of severe AQ-induced IDILI, we have found that AQ causes a delayed onset of mild IDILI that appears to be immune-mediated and resolves with what appears to be immune tolerance. Therefore, these may be the excellent models to study the phenomena of adaptation. We 139

159 postulate that adaptation represents immune tolerance, and it is only when immune tolerance fails that severe liver injury results. The outcome of liver injury and tolerance depends on a complex immune balance between effector cells and regulatory cells, which suggests that it may be possible to develop an AQ-induced server IDILI model in rats and/or mice by manipulating the immune system. In the 3 rd project (Chapter 4), we studied the mechanism of D-pen-induced autoimmunity as a model of autoimmune IDRs. Penicillamine-induced autoimmunity in BN rats has been utilized as an animal model for mechanistic studies of one type of IDR because it closely mimics the autoimmune syndromes that it causes in humans. Our previous work suggested that it is T-cell mediated. It has been shown that 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 and to establish an overall serum cytokine/chemokine profile for this IDR. In total, 24 serum cytokines/chemokines were determined and revealed a dynamic process. Even though BN rats are highly inbred, only about 50% of the animals develop autoimmunity when treated with D-pen. In sick animals, IL-6 and transforming growth factor-b1, known to be driving forces of Th17 differentiation, were consistently increased at both early and late stages of D-pen treatment; however, no significant changes in these cytokines were observed in animals that did not develop autoimmunity. In fact a transient spike in IL-6 predicted which animals would develop autoimmunity weeks later. IL- 17, a characteristic cytokine produced by Th17 cells, was increased in sick animals at both the messenger RNA and serum protein level. In addition, serum concentrations of IL-22, another characteristic cytokine produced by Th17 cells, were found to be elevated. Furthermore, the percentage of IL-17 producing CD4 T cells was significantly increased, but only in sick animals. These data strongly suggest that Th17 cells are involved in penicillamine-induced 140

160 autoimmunity. Such data provide important mechanistic clues that may help to predict which drug candidates will cause a relatively high incidence of such autoimmune IDRs. Overall, this study provides a very nice picture of cytokine changes that occur during the development of an IDR. It demonstrates the involvement of Th17 cells, but not surprisingly, there are changes in many cytokines produced by other cells. It remains to be determined whether this is a common profile for most IDRs, is specific for autoimmune reactions, or is unique to penicillin-induced autoimmunity. IL-17 intracellular staining determined by flow cytometry represents an efficient way to define the Th17 cell population. However, it would be a challenge to obtain fresh peripheral blood cells from patients early in the course of an IDR for the assessment of Th17-mediated pathology. 2. Implications and future directions Although our research suggests the involvement of immune system, the mechanism of AQinduced DILI is still unclear. This thesis also raised several critical questions that need to be investigated in order to further understand AQ-induced liver injury and IDRs in general. First of all, more and more evidence points to involvement of the immune system in IDRs; however, we still can t be sure if this immune response leads to liver injury or is just a response to liver injury. In addition, we observed the presence of NK cells and IL-17 at the early stage of AQinduced liver injury. The results of serum ALT and lymphocyte phenotyping were compared between sick and non-sick C57BL/6 mice after 1 week of AQ treatment, and sick animals always had a significantly higher level of ALT and more cervical node lymphocytes, including CD161 + cells, NK cells, IL-17 secreting cells, Th17 cells, and NK17 cells, which suggests the possible roles of NK cells and/or IL-17-related cells at early stages of this IDR. However, how NK cells are activated and which cells release IL-17 is still unknown. A study of NK cell 141

161 activation and IL-17 secretion at very early stages of AQ treatment can be carried out to help us understand the sequence of events that lead to AQ-induced liver injury. Specifically, NK cell activation could be studied by flow cytometry within 7 days of treatment in different organs, including liver, blood, spleen, and lymph nodes. In addition, Wu and Kleinewietfeld reported that NaCl is able to markedly stimulate or induce pathogenic Th17 cells (Kleinewietfeld et al., 2013; Wu et al., 2013). We could try to use NaCl to manipulate AQ-induced hepatotoxicity and determine the involvement of Th17 cells. Another important question is what is the real biological function of macrophages in this AQ-induced liver injury : Do they play a pathogenic role or protective role? As we discussed before, one plausible hypothesis is: In vivo, AQ is oxidized to a reactive iminoquinone metabolite, which can act as a hapten by covalent biding to proteins. This haptenization can activate the innate immune system (macrophages). The activated macrophages are likely to evoke an adaptive immune response, which could, with the co-stimulation of danger signals released by cell damage, lead to liver injury. On the other hand, some regulatory cells, specifically M2 macrophages, could also be activated to decrease hepatotoxicity and induce tolerance. However, due to the limited data and highly complicated nature of the immune system, little is known about the precise function of macrophages in this process: pathogenic role, protective role, or even both? To investigate the involvement of macrophages, clodronate liposome could be employed to deplete macrophages, and hopefully it could give us a clue to further understand the possible roles of macrophages in AQ-induced liver injury. Further D-pen studies could be carried out to investigate the involvement of NK cells. A surprising result from the previous D-pen study is that RA treatment increased the incidence and severity of D-pen-induced autoimmune disease. As discussed before, RA is able to stimulate NK cells, and this suggests that NK cells play a pathogenic role in D-pen induced autoimmunity. 142

162 Previous studies of the source of IL-17 indicate that NK1.1 + cells are the major source of IL-17 instead of T cells in both spleen and lymph nodes. As reported by Passos, IL-6 is able to promote NK cell production of IL-17 (Passos et al., 2010). If this is true, then NK cells, instead of Th17 cells, could be the cells stimulated by elevated IL-6 to produce IL-17. This could even explain the lack of immune memory in D-pen-induced autoimmunity. In the present research, due to the limited data and highly complicated nature of the immune system, we still don t know the mechanistic details of AQ- or D-pen-induced IDRs. However, these models provide to tool to further investigate the mechanistic details of IDRs. A better mechanistic understanding could lead to biomarkers that predict IDR risk in drug candidates, which would save millions of dollars in the process of drug development, and it could also lead to better treatments of serious IDRs. 143

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178 Supplemental Tables Table S1: Serum concentrations of AQ and DEAQ in female C57BL/6 mice Values reported as Mean ± S.E. and analysed for statistical significance by Mann Whitney-U test (*p < 0.05). The statistical significance comparison is between male vs. female mice for AQ and DEAQ. 159

179 Supplemental Figures Figure S1: Covalent binding of AQ in the liver of female C57BL/6 mice. AQ was given at 0.2% w/w in food and the S9 fraction was prepared to look at the covalent binding as described in the methods section. 160

180 Figure S2: Immunohistochemical grading (at 40X or 10X magnification) for CD4, CD8, CD11b, KI67, F4/80, and CD45R in the livers of control (n = 4) or AQ-treated (n = 4) female C57BL/6 mice. Values represent the mean ± S.E. Analyzed for statistical significance vs. control by Mann-Whitney U test. 161

181 Figure S3: Immunohistochemical staining for CD4, CD8, and CD11b in the spleens of control or AQ-treated female C57BL/6 mice. Figures are representative of 4 animals per group, and AQ represents mice treated for 3 weeks with AQ. Given the large number of CD4 and CD8 T cells in the white pulp area, it was difficult to grade the staining; an increase in cell numbers was shown by flow cytometry. A clear increase was observed for CD11b. 20X magnification. 162

182 Figure S4: Immunohistochemical staining for KI67, F4/80, and CD45R in the spleens of control or AQ-treated female C57BL/6 mice. Figures are representative of 4 animals per group and AQ represents mice treated for 3 weeks with AQ. An obvious increase in KI67 staining was observed in the red pulp and in the follicle; for F4/80 and CD45R, the greatest increases were observed in the red pulp. 20X magnification. 163

183 Figure S5: Lymphocyte activation in AQ-treated female C57BL/6 mice. The data are from untreated controls (n = 4) or animals treated with AQ (n = 4) for 1, 3, or 7 weeks. Values represented as mean ± S.E. Analyzed for statistical significance vs. control by Mann-Whitney U test. A p value < 0.05 was considered significant (*p < 0.05; **p < 0.01; ***p < 0.001). 164

184 Figure S6: Treatment of Rag-/- mice with AQ. (A) ALT activity in mice treated with AQ for up to 5 weeks. Values represented as mean ± S.E. and analyzed for statistical significance by Mann-Whitney U test. A p value < 0.05 was considered significant (*p < 0.05; **p < 0.01; ***p <0.001). (B) Western blotting showing covalent binding of AQ in the livers of mice from panel A. Quantification of AQ covalent binding is expressed as relative intensity and corrected for GAPDH. 165