universiti putra malaysia - core.ac.uk · potential as an immunotherapeutic target in malaria, ......

UNIVERSITI PUTRA MALAYSIA

SITI SARAH BINTI FAZALUL RAHIMAN

FPSK(m) 2012 37

ROLE, INVOLVEMENT AND POTENTIAL OF INTERLEUKIN-27 AS AN IMMUNOTHERAPEUTIC TARGET IN MURINE MALARIA

© COPYRIG

HT UPM

ROLE, INVOLVEMENT AND POTENTIAL

OF INTERLEUKIN-27 AS AN

IMMUNOTHERAPEUTIC TARGET

IN MURINE MALARIA


MASTER OF SCIENCE

UNIVERSITI PUTRA MALAYSIA

2012

© COPYRIG

HT UPM

ROLE, INVOLVEMENT AND POTENTIAL OF INTERLEUKIN-27 AS AN

IMMUNOTHERAPEUTIC TARGET IN MURINE MALARIA

By


Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia,

in Fulfillment of the Requirements for the Degree of Master of Science

August 2012

© COPYRIG

HT UPM

ii

In the name of Allah, the most Compassionate and the Most Merciful.

To my beloved husband, thank you for your endless support.

To my supportive parents and parents-in-laws, brothers and sisters,

thank you for your unconditional love.

to Prof Madya Dr Abas Hj Hussin, may Allah ease your burdens

and each day is a little bit brighter.

© COPYRIG

HT UPM

iii

Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfillment

of the requirement for the degree of Master of Science

ROLE, INVOLVEMENT AND POTENTIAL OF INTERLEUKIN-27 AS AN

IMMUNOTHERAPEUTIC TARGET IN MURINE MALARIA

By


August 2012

Chair : Rusliza binti Basir, PhD

Faculty : Faculty of Medicine and Health Sciences

Interleukin-27 (IL-27) has been known to exert pleiotropic role in many

inflammatory-related diseases including parasitic infection. However, its

involvement during malaria infection has yet to be elucidated. In this study, the role

and involvement of IL-27 during malaria infection was investigated and the effects

of modulating its release on the course of the infection, the release of major

inflammatory cytokines and the histopathological consequences in major affected

organs during malaria were evaluated. Plasmodium berghei (P. berghei) ANKA

infection in male ICR mice was used as a model for malaria infection. The mice

were inoculated intraperitoneally with 2 x 107 parasite-infected red blood cells

(PRBCs) whereas the controls received an equivalent dilution of normal RBCs. The

concentration of IL-27 in the plasma of malarial mice measured by means of ELISA,

showed persistent elevation of IL-27 right from the early until the late phase of

infection and its release is independent of the degree of disease severity. The

modulation of IL-27 release was carried out by treatment of malarial mice with

recombinant mouse IL-27 (rmIL-27), WSX-1Fc chimera or anti-WSX-1 monoclonal

© COPYRIG

HT UPM

iv

antibody (WSX-1 mAb) intravenously. Inhibition of IL-27 with WSX-1 Fc chimera

and WSX-1 antibodies delayed the appearance of physical signs of illness and

parasitaemia development and also prolonged the survival of malaria-infected mice.

Augmentation of IL-27 with rmIL-27 significantly elevated the release of anti-

inflammatory cytokine (IL-10) whereas inhibition and neutralisation of IL-27 with

WSX-1 Fc chimera and WSX-1 mAb respectively, showed decreased level of IL-10.

A significant elevation of pro-inflammatory cytokines (IFN-γ and IL-6) was also

observed, both during augmentation and inhibition of IL-27. From the pattern of

cytokines release, it can be suggested that IL-27 exerts an anti-inflammatory activity

in the Th1 type response by signalling the production of IL-10 during malaria.

Histopathological examination performed on internal organs including the brain,

lungs, liver, spleen and kidneys of malarial mice showed significant sequestration of

PRBCs in the microvasculature of all the major organs of malarial mice. Other

significant histopathological changes were also observed in malarial mice such as

hyperplasia and hypertrophy of the Kupffer cells in the liver, hyaline membrane

formation in the lungs, enlargement of the white and red pulp element followed by

the lost of structure of germinal centre in the spleen, and vacuolation of the kidney

tubule cells. Treatment with rmIL-27 and WSX-1 Fc chimera failed to show any

significant improvement on the histopathological conditions of the organs of

malaria-infected mice. Overall, the results suggest that IL-27 is involved during

malaria infection and it may play an important anti-inflammatory role during

immune response against the disease. Modulation of its release produced a positive

impact on inflammatory cytokine production during the infection, suggesting its

potential as an immunotherapeutic target in malaria, in which the host may benefit

from its inhibition.

© COPYRIG

HT UPM

v

Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai

memenuhi keperluan untuk Ijazah Master Sains

PERANAN, PENGLIBATAN DAN POTENSI INTERLEUKIN-27 SEBAGAI

SASARAN IMMUNOTERAPEUTIK DALAM MALARIA

Oleh


Ogos 2012

Pengerusi : Rusliza binti Basir, PhD

Fakulti : Fakulti Perubatan dan Sains Kesihatan

Interleukin-27 (IL-27) telah diketahui mempunyai peranan pleiotropik dalam banyak

penyakit berkaitan inflamasi termasuk jangkitan parasit. Walau bagaimanapun,

penglibatannya semasa jangkitan malaria masih belum diperjelaskan. Dalam kajian

ini, peranan dan penglibatan IL-27 dalam jangkitan malaria telah diselidiki dan

kesan modulasi penghasilannya ke atas keadaan keseluruhan jangkitan malaria,

pembebasan sitokin inflamasi utama dan perubahan histopatologi organ-organ utama

yang terjejas semasa jangkitan malaria telah dinilai. Jangkitan Plasmodium berghei

(P. berghei) ANKA dalam mencit ICR jantan telah digunakan sebagai model

jangkitan malaria. Mencit disuntik secara intraperitoneal dengan 2 x 107 sel darah

merah berparasit. Kepekatan IL-27 dalam plasma mencit yang dijangkiti malaria

yang diukur melalui kaedah ELISA, menunjukkan peningkatan berterusan IL-27

bermula dari awal hingga ke akhir fasa jangkitan dan pembebasannya didapati tidak

bergantung kepada tahap ketenatan jangkitan. Modulasi ke atas pembebasan IL-27

dijalankan dengan merawat mencit yang dijangkiti malaria dengan rekombinan

mencit IL-27 (rmIL-27), WSX-1Fc chimera dan antibodi monoklonal anti-WSX-1

© COPYRIG

HT UPM

vi

secara intravena ke atas mencit yang dijangkiti malaria. Perencatan pembebasan IL-

27 oleh WSX-1 Fc chimera dan antibodi WSX-1 memperlahankan kemunculan

tanda-tanda fizikal jangkitan dan perkembangan parasitemia seterusnya melanjutkan

tempoh hayat mencit yang dijangkiti malaria. Penambahan IL-27 dengan rmIL-27

meningkatkan pembebasan sitokin anti-inflamasi (IL-10) secara signifikan manakala

perencatan dan peneutralan IL-27 masing-masing dengan WSX-1 Fc chimera dan

antibodi WSX-1, menunjukkan penurunan tahap IL-10. Peningkatan signifikan

sitokin proinflammasi (IFN-γ dan IL-6) juga diperhatikan semasa penambahan dan

perencatan IL-27. Daripada corak pembebasan sitokin-sitokin, boleh diusulkan

bahawa IL-27 menunjukkan aktiviti anti-inflamasi dalam gerakbalas jenis Th1

dengan mengisyaratkan penghasilan IL-10 semasa malaria. Pemeriksaan

histopatologi yang dilakukan ke atas organ-organ dalaman termasuk otak, paru-paru,

hati, limpa dan ginjal menunjukkan sekuestrasi sel-sel darah merah berparasit dalam

mikrovaskulatur kesemua organ-organ utama mencit yang dijangkiti malaria.

Perubahan histopatologi lain yang signifikan juga diperhatikan dalam mencit yang

dijangkiti malaria termasuk hiperplasia dan hipertrofi sel-sel Kupffer dalam hati,

pembentukan membran hyalin dalam paru-paru, pembesaran elemen pulpa merah

dan putih diikuti dengan kehilangan struktur pusat germinal dalam limpa, dan

vakuolasi sel-sel tubul ginjal. Rawatan dengan rmIL-27 dan WSX-1 Fc chimera

gagal menunjukkan sebarang kesan pembaikan yang signifikan ke atas keadaan

histopatologi organ-organ mencit yang dijangkiti malaria. Secara keseluruhannya,

keputusan kajian mencadangkan penglibatan IL-27 semasa jangkitan malaria dan

ianya mungkin memainkan peranan antiinflamasi yang penting semasa gerakbalas

imun melawan jangkitan malaria. Modulasi pembebasannya menghasilkan impak

yang positif ke atas pembebasan sitokin inflamasi semasa jangkitan, dan ini

© COPYRIG

HT UPM

vii

mencadangkan potensinya sebagai sasaran imunoterapeutik di mana hos boleh

memperolehi manfaat dari perencatannya.

© COPYRIG

HT UPM

viii

ACKNOWLEDGEMENTS

I would like to express my deepest gratitude and sincerest appreciation to my

supervisor Dr. Rusliza Basir for her outstanding supervision and support for this

research. Special thanks to my co-supervisors, Dr Herni Talib and Dr Norshariza

Nordin for excellently guiding me through with their expertise. This research would

not have been successful without their valuable guidance, enthusiastic help as well

as constructive criticisms throughout the research.

I would like to express my sincere thanks to Hazirah Azman who provided me with

an endless support for this project and my laboratory colleagues for their assistance

during my laboratory work. Not forgetting, many thanks to the laboratory staffs for

guiding me through all the technical difficulties during the project. They helped me

out far more than they ever realised, their supports are very much appreciated.

I would like to express my heartiest and sincerest appreciation to my husband and

my daughter for their endless support and encouragement throughout the research

period. Special thanks to my beloved parents and family members who had backed,

inspired and paved me to succeed in my project.

Finally, thank you to Universiti Sains Malaysia and Ministry of Higher Education

for providing me the scholarships. Thank you as well to Universiti Putra Malaysia

for funding this project under Research University Grant Scheme (RUGS).

© COPYRIG

HT UPM

x

This thesis was submitted to the Senate of Universiti Putra Malaysia and has been

accepted as fulfillment of the requirement for the degree of Master of Science. The

members of the Supervisory Committee were as follows:

Rusliza binti Basir, PhD

Senior Lecturer

Faculty of Medicine and Health Sciences

Universiti Putra Malaysia

(Chairman)

Norshariza binti Nordin, PhD

Senior Lecturer



(Member)

Herni binti Talib, PhD

Senior Medical Lecturer



(Member)

_______________________________

BUJANG BIN KIM HUAT, PhD

Professor and Dean

School of Graduate Studies


Date:

© COPYRIG

HT UPM

xi

DECLARATION

I declare that the thesis is my original work except for quotations and citations

which have been duly acknowledged. I also declare that it has not been previously,

and is not concurrently, submitted for any other degree at Universiti Putra Malaysia

or at any other institution.

______________________________________


Date: 7 August 2012

© COPYRIG

HT UPM

xii

TABLE OF CONTENTS

Page

ABSTRACT iii

ABSTRAK v

ACKNOWLEDGEMENTS viii

APPROVAL ix

DECLARATION xi

LIST OF TABLES xvi

LIST OF FIGURES xvii

LIST OF APPENDICES xx

LIST OF ABBREVIATIONS xxi

CHAPTER

1 INTRODUCTION 1

2 LITERATURE REVIEW 8

2.1 Human malarial parasites 8

2.2 Life cycle of Plasmodium 10

2.3 Animal models of malaria infection 13

2.4 Clinico-pathology of malaria 15

2.4.1 Fever 15

2.4.2 Anaemia 16

2.4.3 Cerebral malaria (CM) 18

2.4.4 Metabolic acidosis 20

2.4.5 Splenomegaly 20

2.4.6 Hepatomegaly 21

2.4.7 Pulmonary oedema 22

2.4.8 Renal damage 23

2.5 Immune response in malaria 24

2.5.1 Innate immunity 24

2.5.2 Adaptive immunity 26

2.5.3 Th1 and Th2 responses during malaria 28

2.6 Pro-inflammatory cytokines in malaria 30

2.6.1 Tumor necrosis factor alpha (TNF-α) 30

2.6.2 Interferon-gamma (IFN-γ) 31

2.6.3 Interleukin-12 (IL-12) 32





2.7 Anti-inflammatory cytokines in malaria 36



2.7.3 Transforming growth factor beta (TGF-β) 38

2.7.4 Granulocyte-macrophage colony-stimulating

factor (GM-CSF)

39



© COPYRIG

HT UPM

xiii

2.8 Interleukin-27 (IL-27) 42

2.9 IL-27 receptor (IL-27R) 43

2.10 IL-27 signalling 44

2.11 IL-27 and pro-inflammatory responses 45

2.12 IL-27 and anti-inflammatory responses 47

2.13 Potential therapies of IL-27 48

2.14 Histopathological changes during malaria 52

2.14.1 Brain 52

2.14.2 Liver 53

2.14.3 Spleen 54

2.14.4 Kidneys 55

2.14.5 Lungs 55

3 MATERIALS AND METHODOLOGY 56

3.1 Animals 56

3.2 Measurement of body weight and body temperature 56

3.3 Murine malaria parasite 56

3.4 Induction and maintenance of malaria 57

3.5 Parasitaemia measurement 57

3.6 Plasma preparation 58

3.7 Preparation of stock solutions and buffers 59

3.7.1 Alsever’s buffer solution 59

3.7.2 Sodium chloride (NaCl) 0.85% solution 59

3.7.3 Phosphate buffer saline (PBS) solution 59

3.7.4 Block buffer solution 59

3.7.5 Tris-buffered saline (TBS) solution 60

3.7.6 0.05% Tween 20 in TBS solution 60

3.7.7 Wash buffer solution 60

3.7.8 Reagent diluent solution 60

3.7.9 Mouse IL-27 standard solution 60

3.7.10 Mouse IL-27 kit control solution 61

3.7.11 IFN-γ capture antibody solution 61

3.7.12 IFN-γ detection antibody solution 61

3.7.13 IFN-γ standard solution 62

3.7.14 IL-6 capture antibody solution 62

3.7.15 IL-6 detection antibody solution 62

3.7.16 IL-6 standard solution 63

3.7.17 IL-10 capture antibody solution 63

3.7.18 IL-10 detection antibody solution 63

3.7.19 IL-10 standard solution 64

3.7.20 Streptavidin-horseradish peroxidase (HRP)

solution

64

3.8 Preparation of Leishman’s stain (0.02%w/v) 64

3.9 Preparation of chemicals and reagents for

histopathological study

65

3.9.1 Formalin 10% solution 65

3.9.2 Ethanol 95% solution 65



3.9.5 Acid alcohol 1% solution 65

© COPYRIG

HT UPM

xiv

3.9.6 Weak ammonia 3% solution 66

3.10 Preparation of drugs 66

3.10.1 Recombinant mouse IL-27 (rmIL-27) 66

3.10.2 Recombinant mouse WSX-1 Fc chimera

(WSX-1 Fc chimera)

66

3.10.3 Monoclonal anti-mouse WSX-1 antibody

(WSX-1 mAb)

66

3.11 Cytokine assays 67

3.11.1 IL-27 ELISA 67

3.11.2 IFN-γ ELISA 68

3.11.3 IL-6 ELISA 70

3.11.4 IL-10 ELISA 71

3.12 Experimental procedures 74

3.12.1 Malaria model establishment 74

3.12.2 Post- mortem examination on internal

organs in the control and malaria-infected

mice

75

3.12.3 Determination of IL-27 systemic

concentrations in control and malaria-

infected mice

75

3.12.4 The effects of treatment with rmIL-27,

WSX-1 Fc chimera and WSX-1 mAb on

the course of malaria infection

76

3.12.5 The effects of treatment with rmIL-27,

WSX-1 Fc chimera and WSX-1 mAb on

pro- and anti-inflammatory cytokines

systematic release

77

3.12.6 The effects of treatment with rmIL-27 and

WSX-1 Fc chimera on the

histopathological changes of malaria

78

3.13 Statistical analysis 81

4 RESULTS AND DISCUSSION 82

4.1 Establishment of malaria model 82

4.1.1 Visual observation on the physical signs of

illness

82

4.1.2 Post-mortem examinations on the internal

organs of control and malaria-infected mice

84

4.1.3 Effect of malaria on body weight 86

4.1.4 Effect of malaria on body temperature 88

4.1.5 Parasitaemia levels of malarial mice 90

4.1.6 Percentage of red blood cells (RBCs) in

malarial mice

91

4.1.7 Survival of malarial mice 94

4.2 IL-27 involvement during malaria 96

4.2.1 IL-27 release during malaria infection 96

4.2.2 Correlation of IL-27 release and

parasitaemia development

96

4.3 The effects of modulating IL-27 release on

morbimortality during murine malaria

100

© COPYRIG

HT UPM

xv

4.3.1 The effects of modulating IL-27 release

on physical signs of illness of malaria-

infected mice

100

4.3.2 The effects of modulating IL-27 release on

parasitaemia level during murine malaria

102

4.3.3 The effects of modulating IL-27 release on

the survival of malaria-infected mice

111

4.4 The effects of modulating IL-27 release on pro-

and anti-inflammatory cytokines during rodent

malaria

116

4.5 The effects of modulating IL-27 release on

histopathological changes of internal organs during

murine malaria

124

5 SUMMARY, CONCLUSION AND

RECOMMENDATION FOR FUTURE RESEARCH

155

5.1 Research summary 155

5.2 Limitations and recommendations 160

5.3 Conclusion 162

REFERENCES 163

APPENDICES 201

BIODATA OF STUDENT 206

LIST OF PUBLICATIONS 207

universiti putra malaysia - core.ac.uk · potential as an immunotherapeutic target in malaria, ......

Documents