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Modulating Cytokines to Polarize Tumor-Infiltrating Immune Cells in the Tumor Microenvironment by Special
Extracts of Eriobotrya japonica
by
Heba Audai Alshaker
A Thesis submitted in Partial fulfillment of the Requirements for the Degree of
Master of Science
in Pharmaceutical Sciences
at
Petra University
Amman-Jordan
June 2010
ii
Modulating Cytokines to Polarize Tumor-Infiltrating Immune Cells in the Tumor Microenvironment by Special
Extracts of Eriobotrya japonica
by
Heba Audai Alshaker
A Thesis submitted in partial fulfillment of the Requirements for the Degree of
Master of Science
in Pharmaceutical Sciences
at
Petra University
Amman-Jordan
June 2010
Supervisor Name Signature 1. Prof. Khalid Z. Matalka ------------------------- Examination committee Name Signature 1. Dr. Fadi Qa’dan ------------------------- 2. Dr. Nidal Qinna ------------------------- 3. Dr. Mona Bustami -------------------------
4. Dr. Said Ismail -------------------------
iii
Abstract
Modulating Cytokines to Polarize Tumor-Infiltrating Immune Cells in the Tumor Microenvironment by Special
Extracts of Eriobotrya japonica
by Heba Audai Alshaker Petra University, 2010
Under the supervision of Prof. Khalid Matalka
Cytokines play key role in immune responses to developing tumors and hence
regarded as important targets for immunomodulation. Manipulation of cytokine actions
provides innovative strategies for enhancing the activity of dendritic cells, macrophages
and polarizing towards T helper 1 type response within tumor microenvironment. One of
these approaches could be the employment of immunomodulators to participate in
achieving this polarized cellular response via their immunopotentiating capabilities.
Eriobotrya japonica (EJ) folium has been known to possess both immunopotentiating
and antitumor activities in vitro. Given the above, the present study sought the in vivo
immunomodulatory effect on interferon-gamma (IFN-γ), interleukin-17 (IL-17), and
transforming growth factor-beta 1 (TGF-β1) evoked by water-extracts prepared from EJ
leaves in the intra-organ environment of normal mice spleen, lung and blood tissues and
further explored these immunomodulatory potentials in the microenvironment of Meth-
A-fibrosarcoma bearing mice and immune active sites.
Determination of the three cytokines kinetics over 0, 2, 6, 24, and 48 hours in
spleen, lungs, and blood of normal mice induced by EJ hydrophilic leaf extract (EJHE)
and EJHE-water residue (WR), prepared from butanol extraction, at 10 µg/ml showed
that EJHE and EJHE-WR increased IFN-γ production from the spleen and lung tissues
iv
over 6-48 hours and in blood over 6-48 hours upon EJHE-WR administration.
Furthermore, in contrast to the discerned increase of IFN-γ in the spleen, TGF-β1 was
down-regulated in response to EJHE and EJHE-WR administration for as long as 48
hours. The latter induced responses, however, were not seen in Meth-A fibrosarcoma-
bearing mice. In the latter model, triple i.p. injections of EJHE at 10 µg/ml 24 hours
apart, increased significantly spleen IFN-γ production whereas EJHE-WR increased
significantly IFN-γ, TGF-β1 and IL-17 production within the tumor microenvironment of
Meth-A fibrosarcoma. In addition, the present results revealed prolonged survival of mice
inoculated i.p. with Meth-A cells following three times/week of EJHE-WR i.p.
administration. The latter prolonged survival effect was not seen with EJHE. These
results conclude that 10 μg/ml EJHE and EJHE-WR increase the production of IFN-γ and
suppress the production of TGF-β1 levels in normal mice spleen. In the tumor
microenvironment of Meth-A fibrosarcoma, however, triple injections of EJHE and
EJHE-WR at least are needed to modulate cytokines level. Thus, the therapeutic value of
EJHE and EJHE-WR as anticancer agent merits further investigation to promote a more
rationale utilization. This could be performed by understanding of the effect of
immunomodulators’ constituents on the cellular components of the tissue
microenvironment which will provide more useful clues for improving the strategies for
cancer treatment.
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v
ACKNOWLEDGEMENTS
First and foremost, I will be always endlessly thankful to ALLAH assistance and
protection throughout my life. It is an honor for me to thank those who made this thesis a
memorable time. In the first place, I would like to express my deepest reverence and
gratitude to my advisor, Prof. Khalid Matalka, for the wonderful opportunity of
participating in this exciting research endeavour accompanied with enthusiastic dialog
that transformed me into a curious and confident researcher. His truly scientist intuition,
wide knowledge, and logical way of thinking have made him as a constant source of
inspiration which was of great value for me. More heartily thanks for his remarkable
advices, unconditional encouragement, and unwavering moral support during my years as
a master’s student. Actually, success at anything requires hard work, dedication and
careful guidance, and he persistently provided all the three. I was extraordinarily
fortunate in having him as my major supervisor and I am indebted to him more than he
knows. I believe that Prof. Matalka is my role model and mentor that I try always to
follow.
My sincere thanks go to Dr. Nidal Qinna for invaluable assistance, suggestions,
and discussions. Dr. Qinna welcomed me into his laboratory so I could learn new bench
techniques but I have gained infinitely more from him. Working in the atmosphere he
provokes taught me to routinely ask more of myself and to take pride in even the smallest
of achievements. Special thanks are owed to Dr. Fadi Qa’dan, Dr. Mona Bustami, and
Prof. Tawfik Alhussainy for their helpful advices and for increasing my self confidence
constantly. I am grateful to my friends specially Mrs. Maysa Al-Hanbali for her friendly
vi
assistance and support during my research and to Mrs. Rasha Alsa'adi encouragement and
nice words. I would like also to thank Mohammad Albayed fine technical assistance and
help at the animal house.
Lastly but sure not least I wish to thank my entire family. As I print these words I
can not express my feelings toward my mother who made it possible for me by her
unrestricted support, patience, special care and infinite love to finalize this work. I owe a
special debt of gratitude to my brothers, especially Abdullah who was always willing to
listen to me, Omar who has never hesitated to help, my sister and my father who
encouraged me valuably through this.
vii
Table of Contents
Subject Page No.
Chapter 1
Thesis Proposal: Modulating Cytokines to Polarize Tumor-Infiltrating Immune Cells in the Tumor Microenvironment by Special Extracts of Eriobotrya japonica
2
1.1 Specific aims 2
1.2 Outline of specific aims 3
1.3 Hypothesis 4
1.4 Background and significance 4
Chapter 2 Review of the Literature: Crosstalk between Tumor Cells and Immune Cells within Tumor Microenvironment: A Possible Immune System Polarization by Special Extracts of Eriobotrya japonica
12
2.1 The immune system and cancer 12
2.2 Dendritic cells (DCs): key link between innate and adaptive immune responses
15
2.3 Role of T cell subsets in cancer development 19
2.3.1 T helper 1, 2 and 17 cell subsets 20
2.3.2 Regulatory T (Treg) cell subset 24
2.4 Myeloid-derived suppressor cells (MDSCs) 28
2.5 Cytokines regulatory roles 29
2.5.1 Interferon-γ (IFN-γ) 30
2.5.2 Interleukin-17 (IL-17) 34
2.5.3 Transforming growth factor-beta (TGF-β) 1 37
2.6 Eriobotrya japonica (EJ) 41
viii
Subject Page No.
Chapter 3
Materials and Methods
47
3.1 Materials 47
3.1.1 Plant material 47
3.1.1.1 Plant material extraction 47
3.1.2 Materials for cytokines extraction and establishment of tumor cell line 48
3.1.3 Materials for cytokine assays 48
3.2 Mice 48
3.3 Methods 49
3.3.1 Tissues specific modulation of IFN-γ, IL-17 and TGF-β1 in normal mice
49
3.3.2 Kinetics of IFN-γ, IL-17 and TGF-β1 induced by EJHE and EHHE-WR administration in normal mice
49
3.3.3 MCA-induced tumor 50
3.3.4 MCA-induced cell lines 50
3.3.5 Inoculation of tumor cells and experimental tumor model 50
3.3.6 IFN-γ, IL-17, and TGF-β1 modulation within the tumor microenvironment
51
3.3.7 Survival of Meth-A tumor-bearing mice after treatment with EJHE and EJHE-WR
51
3.3.8 Cytokine extraction 52
3.3.9 Cytokine assays 52
3.4 Statistical analysis 54
Chapter 4 Results
56
4.1 Tissues specific modulation of IFN-γ, IL-17 and TGF-β1 by EJHE and EJHE-WR following two hours of administration
56
ix
Subject Page No.
4.1.1 EJHE and EJHE-WR modulate IFN-γ and IL-17 expression in blood, spleen, and lung tissues in a comparable manner
56
4.1.2 EJHE and EJHE-WR ubiquitously inhibit TGF-β1 in mice blood, spleen, and lung tissues
60
4.2 Kinetics of IFN-γ, IL-17 and TGF-β1 in specific tissues induced by EJHE and EJHE-WR
62
4.2.1 Kinetics of IFN-γ, IL-17 and TGF-β1 in the blood 62
4.2.2 Kinetics of IFN-γ, IL-17 and TGF-β1 in the spleen 64
4.2.3 Kinetics of IFN-γ, IL-17 and TGF-β1 in the lungs 67
4.3 MCA-induced tumors 69
4.4 Modulation of IFN-γ, IL-17 and TGF-β1 in the spleen and within the tumor microenvironment of Meth-A-bearing mice following EJHE and EJHE-WR administration
71
4.4.1 Modulation of IFN-γ, IL-17 and TGF-β1 in the spleen of Meth-A-bearing mice
71
4.4.2 Modulation of IFN-γ, IL-17 and TGF-β1 within the tumor microenvironment of Meth-A-bearing mice
73
4.5 Survival of Meth-A tumor-bearing mice after treatment with EJHE and EJHE-WR
75
Chapter 5 Discussion and conclusions
77
References 86
x
List of Figures
Figure Legends Page No.
Figure 2.1: Activation of naїve T cells by DCs 18
Figure 2.2: (a) Tumor-derived suppression of APCs and T cell responses (b) Absence of danger signal from tumor
19
Figure 2.3: Inhibition by CD4+CD25+ T cells at the tumor microenvironment 27
Figure 4.1: Effect of EJHE and EJHE-WR on IFN-γ production in the spleen of mice
57
Figure 4.2: Effect of EJHE and EJHE-WR on IL-17 production in the spleen of mice
57
Figure 4.3: Effect of EJHE and EJHE-WR on IFN-γ production in the lungs of mice
58
Figure 4.4: Effect of EJHE and EJHE-WR on IL-17 production in the lungs of mice
58
Figure 4.5: Effect of EJHE and EJHE-WR on IFN-γ production in the blood of mice
59
Figure 4.6: Effect of EJHE and EJHE-WR on IL-17 production in the blood of mice
59
Figure 4.7: Effect of EJHE and EJHE-WR on TGF-β1 production in the spleen of
60
Figure 4.8: Effect of EJHE and EJHE-WR on TGF-β1 production in the lungs of mice
61
Figure 4.9: Effect of EJHE and EJHE-WR on TGF-β1 production in the blood of mice
61
Figure 4.10: Kinetics of IFN-γ production in the blood of mice induced by EJHE and EJHE-WR
63
Figure 4.11: Kinetics of IL-17 production in the blood of mice induced by EJHE and EJHE-WR
63
Figure 4.12: Kinetics of TGF-β1 production in the blood of mice induced by EJHE and EJHE-WR
64
Figure 4.13: Kinetics of IFN-γ production in the spleen of mice induced by EJHE and EJHE-WR
65
Figure 4.14: Kinetics of IL-17 production in the spleen of mice induced by EJHE and EJHE-WR
66
Figure 4.15: Kinetics of TGF-β1 production in the spleen of mice induced by EJHE and EJHE-WR
66
Figure 4.16: Kinetics of IFN-γ production in the lungs of mice induced by EJHE and EJHE-WR
68
Figure 4.17: Kinetic of IL-17 production in the lungs of mice induced by EJHE and EJHE-WR
68
xi
Figure Legends Page No.
Figure 4.18: Kinetic of TGF-β1 production in the lungs of mice induced by EJHE and EJHE-WR
69
Figure 4.19: Fibrosarcoma development in BALB/c mice inoculated with MCA
70
Figure 4.20: Cumulative tumor incidence after s.c inoculation of two different MCA doses
70
Figure 4.21: Effect of EJHE and EJHE-WR on IFN-γ production in the spleen of Meth-A-bearing mice
71
Figure 4.22: Effect of EJHE and EJHE-WR on IL-17 production in the spleen of Meth-A-bearing mice
72
Figure 4.23: Effect of EJHE and EJHE-WR on TGF-β1 production in the spleen of Meth-A-bearing mice
72
Figure 4.24: Effect of EJHE and EJHE-WR on IFN-γ production in the tumor microenvironment of Meth-A-bearing mice
73
Figure 4.25: Effect of EJHE and EJHE-WR on IL-17 production in the tumor microenvironment of Meth-A-bearing mice
74
Figure 4.26: Effect of EJHE and EJHE-WR on TGF-β1 production in the tumor microenvironment of Meth-A-bearing mice
74
Figure 4.27: Effect of i.p. administration of EJHE and EJHE-WR on mice survival after i.p. inoculation with Meth-A cells
75
List of Tables
Table Captions Page No.
Table (2.1): CD4+ T lymphocytes in tumor progression or regression 27
Table (2.2): Effect of cytokines in the tumor microenvironment 40
Table (2.3): Modeling and simulating cytokines’ effect on immune response to tumor cells in the tumor microenvironment
41
xii
List of Abbreviations Abbreviation Phrasing
APCs Antigen-presenting cells
BSA Bovine serum albumin
CTL Cytotoxic T lymphocytes
CTLA-4 Cytotoxic T-lymphocyte-associated antigen 4
CXCL Chemokine (C-X-C motif) ligand
DCs Dendritic cells
EJ Eriobotrya japonica
EJHE Eriobotrya japonica hydrophilic leaf extract
EJHE-WR Eriobotrya japonica hydrophilic leaf extract-water residue
FBS Fetal bovine serum
Foxp3 Forkhead box protein 3
GATA3 GATA-binding protein 3
δγ Gamma delta
IFN Interferon
IFNGR Interferon-gamma receptor
IL Interleukin
I.p. Intraperitoneally
JAK Janus tyrosine kinase
LPS Lipopolysaccharide
MCA 3-Methylcholanthrene
xiii
Abbreviation Phrasing
MDSCs Myeloid-derived suppressor cells
Meth-A 3-methylcholanthrene-induced fibrosarcoma cell line
MHC Major histocompatibility complex
NF-κB Nuclear factor-kappa B
NK Natural killer
NKT Natural killer T
PAMPs Pathogen associated molecular patterns
PBS Phosphate buffered saline
PRRs Pattern recognition receptors
R Receptor
RORγt Retinoid orphan nuclear receptor
s.c. Subcutaneously
Smad Caenorhabditis elegans Sma and Drosophila Mad
STAT Signal transducer and activator of transcription
TCR T cell receptor
TGF-β Transforming growth factor-beta
Th T helper
TLRs Toll-like receptors
Treg T regulatory cells
TNF-α Tumor necrosis factor-alpha
VEGF Vascular endothelial growth factor
1
Chapter 1
Thesis Proposal
Modulating Cytokines to Polarize Tumor-Infiltrating Immune Cells in the Tumor Microenvironment by Special
Extracts of Eriobotrya japonica
2
Chapter 1
Thesis Proposal
Modulating Cytokines to Polarize Tumor-Infiltrating Immune Cells in the Tumor Microenvironment by Special
Extracts of Eriobotrya japonica 1.1 Specific aims:
Cancer treatments especially of solid tumors are still far from being successful.
The aim of this project is to modulate cytokines which are the coordinators of the
immune system in order to enhance and polarize the immune cells within tumor
microenvironment to kill tumor cells. In an attempt of deeper insight into the pleiotropic
activity of cytokines, which apparently could be considered with paradoxical activities,
three cytokines were chosen; interferon-gamma (IFN-γ), interleukin (IL)-17 and
transforming growth factor-beta (TGF-β) 1. An effort to modulate the latter cytokines’
responses and subsequently achieving an overwhelming polarized immune response
within the tumor microenvironment was investigated for developing molecules or a
mixture of molecules capable of manipulating cytokines actions. This could further be
interpreted as a new path for enhancing macrophages, dendritic cells (DCs) and
tumor infiltrating lymphocytes within solid tumors to kill the cancer cells.
The hydrophilic leaf extract of Eriobotrya japonica (EJ) (EJHE) has been shown
to induce and modulate in vitro and in vivo proinflammatory cytokines production, IL-12,
IFN-γ and tumor necrosis factor-alpha (TNF-α) more than an anti-inflammatory cytokine,
IL-10. The present study extended the latter observation on IFN-γ but with two other
different cytokines; TGF-β1 as a representative of T regulatory (Treg) cells along with
IL-17 as an indicator of T helper (Th) 17 cells. Taken into account the extraction method
3
which provokes key role in natural products and ultimately may or may not modulate the
immune function EJHE was further extracted with n-butanol. The non-extracted material
was referred to as water residue (EJHE-WR). EJHE-WR has been shown to induce more
of IFN-γ than EJHE.
Altogether, this study main objective is to examine EJHE and EJHE-WR possible
treatment of Meth-A, 3-methylcholanthrene (MCA)-induced fibrosarcoma, through the
manipulation of the cytokine triad mentioned previously along with deeper exploration of
this cytokine trilogy synergistic or antagonistic interactions with respect to tumor
immunity at the tumor microenvironment. Furthermore, through this cytokines trilogy;
IFN-γ, IL-17 and TGF-β1 one might speculate type of tumor infiltrating lymphocytes
Th1, Th17 and Treg, respectively. Thus, modulation of the latter cytokines could have
implications for the physiologic regulation as well as manipulation of immune responses
within the local tumor microenvironment.
1.2 Outline of specific aims:
1. Induction and assaying of mouse tissue-extracted protein cytokines (IFN-γ, IL-17 and
TGF-β1) in local mouse tissues/organs; blood, lungs, and spleen following intra-
peritoneal (i.p) administration of EJHE and EJHE-WR at three different concentrations.
2. Determination of the three cytokines kinetics over 0, 2, 6, 24, and 48 hours in chosen
organs/tissues induced by the selected doses.
3. Induction of tumor in the flank region of mice following the administration of
MCA.
4. Developing a mouse tumor model for the exploration of IFN-γ, IL-17, and TGF-β1
role and their correlation to variety of cellular infiltrates within the local tumor
4
microenvironment along with investigation of their amenability to be modulated in the
local microenvironment by the selected dose.
5. Evaluation of the selected dose efficacy in possible treatment of i.p. induced Meth-A
fibrosarcoma.
1.3 Hypothesis:
To accomplish the above aims the following hypothesis will be considered; IL-17
and IFN-γ are reciprocally regulated meanwhile TGF-β1 secreted by Treg cells have the
capacity to down regulate the immune response and subsequently provokes either
protective properties or deleterious effects through the induction of IL-17 by supporting
Th17 differentiation and consequently inhibition of IFN-γ secretion from Th1 cells. At
this point the importance of EJ arises in achieving a potential cytokines modulation by
the induction of protective IFN-γ which outweigh TGF-β1 deleterious effects and
subsequently inhibition of IL-17 within the local tumor microenvironment.
1.4 Background and significance:
Multiple innate and adaptive immune effector cells and molecules partake in the
recognition and destruction of cancer cells to protect against growing tumors, a concept
that is known as cancer immune surveillance. Unfortunately, cancer cells are capable of
avoiding this process by immunoselection of poorly immunogenic tumor cells variants
along with subversion of the immune system and thus shaping both the tumor and its
microenvironment (Zitvogel et al., 2006). Cytokines represent part of the complex pattern
of the immune response which can assist the development of cancer as well as to
eliminate it. Simultaneously, a large number of cytokines may be involved in the
5
complex interactions between host and tumor cells where this dynamic crosstalk, between
tumors and the immune system, can regulate tumor growth and metastasis (Smyth et al.,
2004).
The failure of the immune system to recognize and eradicate cancer cells
may partly be a result of insufficient immunological activation (Adam et al., 2003). It is
now increasingly recognized that the microenvironment plays a critical role in the
progression of tumors where immune-resistant tumor variants are selected initiating the
process of cancer immunoediting (Zou, 2005; Zitvogel et al., 2006). One variable that
might prove conclusive in modulating the host reaction is the mixture of cytokines
produced within tumor microenvironment (Blankenstein et al., 1991; Smyth et al., 2004).
For instance, the aberrant cytokine formation demonstrated in cutaneous T cell
lymphomas is believed to contribute to the tumor development since they might be
responsible for a depressed local antitumor immune response and systemic immune
abnormalities observed in these patients (Asadullah et al., 2000). Meanwhile, local
activation of the immune system at the tumor microenvironment site can be
achieved by experimental transfer and expression of cytokine genes in tumor cells
(Blankenstein et al., 1991; Smyth et al., 2004). Appealingly, cytokines in cancer can
function in a bidirectional way as both; causative agents and potential treatments thusly
necessitating in-depth knowledge of the role of all the various cytokines which in turn
provides new opportunities for improving cancer immunotherapy.
Several medicinal plants are considered immunomodulatory as they display
antitumoral effects. Concurrently, present treatments of cancer display many drawbacks
due to their toxicities as in suppression of the immune system (Sunila & Kuttan, 2004;
6
Davicino et al., 2007). Therefore, harnessing of the cellular immune system represented
by T cell mediated immune responses seems to play a critical role against tumors (Maher
& Davies, 2004) with even prevention rather than treatment of cancer. More effectively
to this endeavour is the manipulation of the cytokines balance within tumor
microenvironment by the administration of natural products as in the case of EJHE or
EJHE-WR providing possible way to reestablish antitumor immune responses which can
be further exploited for cancer therapy and thereby reducing the complications due to
toxicity associated with cytokines systemic administration (Podhajcer et al., 2007).
Customarily, the leaves of EJ Lindl. (Rosaceae) have been used as traditional
medicines for lung, stomach diseases and diabetes and have been found to be effective in
chronic bronchitis, inflammation, and tumors (Ito et al., 2000, 2002; Banno et al., 2005;
Al-Hanbali et al., 2009; Qa’dan et al., 2009). A number of biologically active compounds
have been reported in the leaves of this plant such as polyphenols, tannins, flavonoids,
triterpenes, megastigmane glycosides, and sesquiterpenes (Ito et al., 2000, 2002; Banno
et al., 2005; Lü et al., 2009). Many of these biologically active compounds were
correlated to cytotoxic, antimutagenic and antitumor effects in mouse tumor models and
human oral tumor cell lines (Ito et al., 2000, 2002; Banno et al., 2005). Thus, this study is
concerned in yet deeper exploration of these effects, more precisely, within the tumor
microenvironment of Meth-A fibrosarcoma.
The immune system is diagramed to recognize and destroy foreign pathogens
while preserving immune tolerance to self. Treg cells play a critical role in maintaining
immunological tolerance. The discovery of forkhead box protein (Foxp)3 as a key Treg
transcription factor has enabled new insights into Treg biology and moreover revealed
7
paradoxical features of this lineage (Gavin et al., 2007). There are two major categories
of Foxp3+ Tregs; the naturally occurring CD4+CD25+ Tregs that arise in the thymus and
the TGF-β-induced Tregs produced in the periphery. Both types are likely to be important
in maintaining peripheral tolerance but their individual contributions in vivo still not
established yet. Conversion of CD4+CD25- naїve T cells toward a suppressor T cell
phenotype, similar to that of Treg, and induction of transcription factor Foxp3 expression
in CD25- naїve T cells to enforce transition to Treg cells is achieved by TGF-β (Chen et
al., 2003).
Seemingly, TGF-β is an important immunoregulatory cytokine that in addition to
Treg cell can affect T cell homeostasis and effector cell function (Bommireddy &
Doetschman, 2007). TGF-β is conventionally regarded as an anti-inflammatory agent
(Veldhoen & Stockinger, 2006) but now unequivocally demonstrates both tumor
suppressor and oncogenic activities. In the current paradigm, the suppressor activities
dominate in normal tissue. On the other hand, changes in TGF-β expression and cellular
responses during tumorigenesis tip the balance in favor of its oncogenic activities to drive
malignant progression, invasion and metastasis both in vitro and in vivo (Akhurst &
Derynck, 2001). In fact, understanding the roles of TGF-β in the tumor
microenvironment requires insight into the changing response patterns of many
interacting cell types. Therein, differentiation of recently discovered Th17 cells, a novel
highly inflammatory T cell subset that produces IL-17, has been shown to also require
this cytokine in both humans and murine. However, TGF-β alone was not sufficient for
the induction of Th17 in murine. IL-17 producing murine T helper cells in vitro requires
8
IL-6 plus TGF-β (Mangan et al., 2006; Veldhoen et al., 2006; Manel et al., 2008; Volpe
et al., 2008).
Actually, the discovery that TGF-β is a crucial cytokine for Th17 cell
development suggested that Th17 and Treg cell subsets share reciprocal developmental
pathways from uncommitted CD4+ precursors (Bettelli et al., 2006). More strikingly;
there is a direct lineage relationship of Th17 cells with Treg cells. Indeed, these two
subsets require TFG-β for lineage commitment and it is further observed that there are
direct interactions between the lineage specific transcription factor Foxp3 of Treg and
retinoid orphan nuclear receptor (RORγt) of Th17. Further work showed that TGF-β
signaled in a concentration dependent manner to promote the expression of both Foxp3
and RORγt. Foxp3 directly bound to RORγt preventing Th17 differentiation; an effect
relieved by IL-6, IL-21 and IL-23 therefore confirming the suppressive function of Foxp3
on RORγt (Zhou et al., 2008). Practically, such interactions can not be disregarded within
local tumor microenvironment but even still need to be fully further investigated. A study
has revealed that in parallel to high levels of CD4+Foxp3+ Treg cells, CD4+IL-17+ T cells
were kinetically induced in the tumor microenvironment in multiple mouse and human
tumors. Furthermore, IL-17+ T cells demonstrated a dynamic differentiation in the tumor
microenvironment thus providing new insight of IL-17+ T cells potential role in tumor
immune pathology and therapy (Kryczek et al., 2007).
Concerning tumor development, some human tumor cells could express IL-17
that represents an early event in the development of the inflammatory reaction within the
tumor microenvironment which may successively influence tumor phenotype and growth
(Cirée et al., 2004). Ostensibly, IL-17 seems to be a pleiotropic cytokine with possible
9
protumor or antitumor effects which often depends on the immunogenicity of tumor
models (Benchetrit et al., 2002). Meth-A cells transfected with the human IL-17 gene can
induce tumor-specific antitumor immunity by augmenting the expression of major
histocompatibility complex (MHC) class I and II antigens; this antitumor immunity may
be mediated by CD4+ and CD8+ T cells (Hirahara et al., 2001). Contradictory, IL-17 up-
regulated elaboration of a variety of proangiogenic factors by fibroblasts as well as tumor
cells revealing a novel role for IL-17 as a CD4 T cell-derived mediator of angiogenesis
and tumor growth (Numasaki et al., 2003). A recent work showed that G-197A allele of
the IL-17A gene promoter was significantly associated with an increased risk of
subsequent development of intestinal-type gastric cancer upon its association with the
progression of gastric mucosal inflammation and the development of gastric mucosal
atrophy (Shibata et al., 2009).
IFN-γ is secreted by thymus-derived T cells under certain conditions of activation
and by natural killer (NK) cells, which are targets as well as producers of IFN-γ. IFN-γ
regulates several aspects of the immune response from merely antiviral factor to a broad
spectrum antimicrobial agent to finally be an important player in overall inflammatory
responses to exogenous as well as endogenous agents (Boehm et al., 1997; Billiau &
Matthys, 2009). Furthermore, the finding that IFN-γ receptor knockout mice exhibit
severely impaired antitumor capability illustrated the importance of IFN-γ in tumor
immunity (Qin & Blankenstein, 2000). Induction of IFN-γ is correlated with several
direct and indirect antitumor properties (Qin et al. 2003). Tumor responsiveness to IFN-γ
is necessary for IFN-γ-dependent inhibition of tumor angiogenesis by CD4+ T cells
(Beatty & Paterson, 2001a).
10
Altogether, it is clear that IFN-γ, IL-17 and TGF-β1 are capable of exerting
multiple effects within tumor microenvironment site. Controversially, many of the
exerted effects are contextual hence, compelling further investigations in that regard
along with any attempt of their immunomodulation that should be approached with a
definite knowledge of the initial level of cytokines and their complex interaction with
particular type of tumors and the immune cells.
11
Chapter 2
Review of the Literature: Crosstalk between Tumor Cells and Immune Cells within
Tumor Microenvironment: A Possible Immune System Polarization by Special Extracts of Eriobotrya japonica
12
Chapter 2
Review of the Literature: Crosstalk between Tumor Cells and Immune Cells within Tumor
Microenvironment: A Possible Immune System Polarization by Special Extracts of Eriobotrya japonica
2.1 The immune system and cancer:
The immune system can efficiently protect the body from invading pathogens
including bacteria, viruses, and parasites as well as aberrantly transformed cells. It is a
system with high distribution, self organization and adaptation, which additionally has
the features of diversity, learning, recognition and memory (Adam et al., 2003; Gennery
& Cant, 2006; Li et al., 2009). Protection from infection and disease is provided by the
collaborative efforts of the immune system two major arms; innate and adaptive
(acquired) immunity. Innate immunity is the first line of defense and senses abnormalities
both outside and inside of host cells thus conferring prompt self protection by clearing
pathogens or at least reducing their spread (Shi et al., 2001; Gennery & Cant, 2006). The
innate immune system is composed of two elements: firstly, cells with harmonious
antimicrobial functions, including epithelial cells, neutrophils, NK cells, macrophages,
and DCs. Secondly, proteins such as cytokines or complement factors which are
produced by the cells of immune system or by non-immune cells (Song et al., 2000;
Beutler, 2004; Ulevitch et al., 2004; Gennery & Cant, 2006).
Although innate immunity lacks the ability to specifically recognize pathogens
and provide specific immunity, it instructs the adaptive immune response. The adaptive
immune system is mobilized by cues from the innate response (Zhao et al., 2009). In that,
signals processed and released from innate immune cells can subsequently regulate T
13
cell- and B cell-mediated adaptive immune responses (Fritz et al., 2008). The induction
of specific adaptive immunity requires the participation of professional antigen-
presenting cells (APCs), which include DCs, macrophages, and B lymphocytes. These
cells play pivotal roles in the induction of an adaptive immune response because of their
capacity to phagocytize or endocytose protein antigens and enzymatically digest them to
generate peptides which are then loaded onto class I or class II MHC proteins and
presented to the cell surface T cell receptor (TCR) (Knight & Stagg, 1993; Sprent, 1995;
Rodríguez-Pinto, 2005).
Broadly, T cells can be divided into two classes depending on their effector
functions and accessory molecules; CD8 and CD4. T cells that bear CD8 recognize MHC
class I molecules on the surface of most nucleated cells and are called cytotoxic T
lymphocytes (CTL). Th cells, which have CD4, recognize MHC class II molecules on a
more restricted set of APCs. CD4+ T cells are mainly cytokine-secreting helper cells
which can elicit both immune stimulatory or immune inhibitory effects (Wilson &
Garcia, 1997; Harty et al., 2000; Klein et al. 2003). CD4+ T cells help the activation,
expansion, and persistence of tissue-destructive CD8+ T cells (Zhang et al., 2009b). CD8+
T cells are mainly cytotoxic killer cells that induce antigen-expressing cell death and lyse
tumor cells (Wilson & Garcia, 1997; Harty et al., 2000; Klein et al. 2003).
At least two signals are necessary for the activation of T cells (Fig. 2.1). The first
signal is delivered following engagement of the TCR with peptide-bound MHC
molecules. In the absence of a second signal, the T cells become unresponsive (anergic)
or undergo apoptosis. The second signal involves ligation of costimulatory molecules
CD40, CD80 (B7-1) and CD86 (B7-2) on the APCs to their respective ligands CD40L for
14
CD40, CD28 for CD80/CD86. Activation of T cells is regulated via a negative feedback
loop involving cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4). Following
engagement of CD28 with CD80/CD86, CTLA-4 in the cytoplasm is mobilized to the
cell surface where, because of its higher affinity of binding to CD80/CD86, it displaces
CD28 and subsequently results in suppression of T cell activation and proliferation (Liu
& Linsley, 1992; Sprent, 1995; Alegre et al., 2001).
Accordingly, the operating of innate and of specific T cell immunity is interlinked
via DCs, major APCs (Ghiringhelli et al., 2007). However, the importance of the innate
immunity lies in its instantaneous promptness to recognize even a small number of
abnormally transformed cells (Deichman, 2002). Tumor immune surveillance is a process
in which the immune system recognizes precancerous or/and cancerous cells and
eradicates them before they can elicit any harm. This process is an important host defense
process to prevent carcinogenesis and to maintain cellular homeostasis (Dunn et al.,
2002). The relationship, however, between the immune system and cancer is dynamic,
multiple and far more complicated than was previously thought (Sengupta et al., 2010).
In virtue of the interaction of host and tumor cells, three essential phases were proposed:
elimination, equilibrium and escape, which are designated as the three Es (Dunn et al.,
2004b).
Elimination is equivalent to immune surveillance, in which innate and adaptive
components of the immune system act to eradicate cancers that arise in the organism
(Dunn et al., 2004a; Zitvogel et al., 2006). Sometimes, throughout the course of
elimination a selective pressure is provided for immune sculpting that generates immune-
resistant cells (Smalley et al., 2005). Equilibrium, the second stage of immunoediting, is
15
turned up when the immune system can still control tumors but no longer eradicate them
(Koebel et al., 2007; Teng et al., 2008). However, dormant tumors in equilibrium, with
continued selection, may evolve into the third stage of immune escape, wherein cells can
defeat or evade the immune system (Aguirre-Ghiso, 2007).
Tumor-derived soluble factors can impel various mechanisms for escape from
immune attack in the tumor microenvironment (Kim et al., 2006). The tumor
microenvironment is a pivotal factor in the course of carcinogenesis and is largely
dependent on its interactions with microenvironmental components in a bidirectional way
and consequently tumor progression or regression (Witz, 2009). The tumor
microenvironment was lately recognized as the product of a developing crosstalk
between different cells types. In addition to tumor cells, the tumor microenvironment is
comprised of immune cells, fibroblasts, endothelial cells and the extracellular matrix
(Zou, 2005). Normal cellular microenvironment can inhibit tumor cell proliferation and
cancer formation (Hu & Polyak, 2008). Contrariwise, as tissue becomes cancerous
pathological interactions between cancer cells and host immune cells in the tumor
microenvironment create an immunosuppressive network that protects the tumor from
immune attack (Zou, 2005) leading to tumor growth, progression, invasion and metastasis
which is basically due to a deranged relationship between tumor and stromal cells
(Mbeunkui & Johann, 2009).
2.2 Dendritic cells (DCs): key link between innate and adaptive immune responses:
DCs are migratory population of specialized APCs that are bone-marrow-derived
leukocytes capable of either initiating or preventing the stimulation of primary antigen-
specific T cell responses (Banchereau et al., 2000). Based on surface marker expression
16
and function, several different states of DCs maturation exist (Lutz & Schuler, 2002).
Immature DCs are very efficient at endocytosing antigen and processing the antigenic
proteins into short peptides. However, the latter lack the expression of high levels of
MHC molecules and costimulatory molecules required for optimal T cell priming
(Steinman, 2003). Matured DCs are not so efficient at capturing and processing antigen,
but have high levels of surface MHC molecules and costimulatory molecules (eg, CD80
(B7-1) or CD86 (B7-2)), and secrete cytokines that stimulate T cell function (Steinman,
2003). DCs can be matured by a variety of agents, including cytokines and other stimuli
(Guermonprez et al., 2002). Signals that induce cytokine production by DCs are
associated with pathogen recognition by the pathogen associated molecular patterns
(PAMPs) through pattern recognition receptors (PRRs) on or inside immature DCs
represented via Toll-like receptors (TLRs), which consequently can promote the
functional maturation of DCs (Iwasaki & Medzhitov, 2004; Reis e Sousa, 2004).
Lymphoid organs as lymph nodes, spleen, and thymus contain immature DCs
under steady state conditions that play a role in maintaining peripheral self-tolerance
(Wilson et al., 2003). Ordinarily, surveillance DCs are found in a majority of non-
lymphoid tissues and organs including skin, liver, lung, and mucosal surfaces wherein
tissue microenvironments appear to have major impact on the function of the residential
DCs (Hart, 1997; Ardavín, 2003). In the periphery, before entry into the secondary
lymphoid tissues, DCs might interact with NK and natural killer T (NKT) cells (Fig. 2.1).
Activated NK cells are a substantial source of IFN-γ and have been implicated in the
activation of DCs (Gerosa et al., 2002). The production of IFN-γ or IL-4 by NKT cells
can also modulate DCs function before T cell activation (Stober et al., 2003). B cells also
17
have a remarkable propensity to form synapses with APCs from which they can acquire
antigen and subsequently might progressively interact with T cells (Batista et al., 2001;
Lund, 2008). Naϊve T lymphocytes are not situated within peripheral tissues but localize
within specialized lymphoid organs. Immature DCs capture antigens in the periphery,
migrate with environmental self and antigens to areas more commonly associated with
adaptive immunity such as the draining lymph nodes, and cause the aggregation and
stimulation of naϊve lymphocytes (Knight & Stagg, 1993).
The capture of antigens by immature DCs in the periphery promotes maturation of
DCs and results in the local production of cytokines, such as IL-12, which in turn can
amplify the innate immune response and stimulate T cell function (Ferrantini, 2008).
Production of IL-12 is an important sign of DCs activation (Mosca et al., 2000). IL-12
produced by activated APCs is a major inducer of Th1 cell and its cytokines (Matalka,
2003a, 2003b). Remarkably, the cytokines produced in the local microenvironment
modulate the type of response that will be generated. For instance, in murine spleen DCs
which secrete IL-12 induce Th1 responses whereas DCs which do not secrete IL-12
induce Th2 responses (Maldonado-López et al., 1999; Pulendran et al., 1999).
18
Fig. 2.1: Activation of naїve T cells by DCs. Optimally, antigen-specific activation of CD4+ or CD8+ T cells occurs in secondary lymphoid tissue where naїve T cells interact with DCs. Efficient presentation of MHC class I or peptide complexes to CD8+ T cells or MHC class II or peptide complexes to CD4+T cells is achieved by mature DCs that express high levels of MHC molecules and costimulatory molecules. In the periphery, production of IFN-γ by NK or NKT cells can modulate DCs function before T cell activation.
As surveillance cells in the tumor microenvironment, DCs have been shown to
infiltrate tumors and monitor the presence of novel antigen derived from tumors (Preynat-
Seauve et al., 2006). DCs may be activated by danger signals released from stressed or
necrotic tumor cells which may include cytokines, heat shock proteins, intracellular
nucleotides, and intact double-stranded DNA (Sauter et al., 2000; Gallucci & Matzinger,
2001; Matzinger, 2002). Activation and migration of DCs from the tumor site of antigen
capture to secondary lymphoid organs is crucial in the initiation and amplification of
immune response thereby, triggering a maturation program that includes expression of
multiple costimulatory molecules and cytokines that result in efficient priming of effector
T cell responses and beneficial antitumor immunity (Smyth et al., 2001; Matzinger, 2002;
Preynat-Seauve et al., 2006; Lin et al., 2010).
Conversely, DCs at the tumor microenvironment, which possess an immature
phenotype, is not necessarily conducive to the activation of antitumor immune responses
19
(Fig. 2.2) due to tumor-derived local immunosuppressive cytokines milieu as TGF-β, and
IL-10 and growth factors as vascular endothelial growth factor (VEGF) that may instead
suppress DCs function or condition DCs to secrete TGF-β to induce dampening of T cells
and the augmented Treg cells function, and ultimately inducing anergy thus favoring
tumor evasion (Gabrilovich, 2004; Bennaceur et al., 2009; Lin et al., 2010). Inevitably,
efficient maturation of DCs at the tumor microenvironment is pivotal for priming T cells
responses and mounting effective antitumor immunity (Ghiringhelli et al., 2007).
Fig. 2.2: (a) Tumor-derived suppression of APCs and T cell responses. Production of immunosuppressive cytokines (TGF-β and IL-10) in the local microenvironment by tumor themselves can suppress the antitumor response by acting directly on CD4+ or CD8+ T cells or indirectly via APCs. (b) Absence of danger signal from tumor. If the tumor present does not send out danger signals, APCs may not be sufficiently activated and may not express sufficient costimulatory molecules to activate specific T cells. Consequently, these T cells will fail to respond to the tumor. Adapted from (Smyth et al., 2001).
2.3 Role of T cell subsets in cancer development:
From early stages of disease, a ceaseless dynamic battle exists between immune
cells and tumor cells which, in some circumstances, can manifestly favor the growth of
the latter. Cancer cells evolve from normal cells as a consequence of accumulating
numerous genetic alterations. Characteristically, cancer cells provide their own growth
signals, disregard growth inhibitory signals, escape cell death, replicate endlessly, sustain
20
angiogenesis, and invade tissues through basement membranes and capillary walls
(Hanahan & Weinberg, 2000). In addition to these six hallmarks of cancer that ultimately
dictate the course of malignant growth, leukocyte infiltration into developing tumors is
now considered one of the hallmarks of cancer development (Colotta et al., 2009).
CD4+ and CD8+ T cells are the principal helper and effector cells, respectively, of
adaptive cellular immunity. Unsurprisingly, most efforts to detect a role of T cells in
tumor immunity have been placed on CD8+ T lymphocytes since tumor cells are MHC
class I positive but MHC class II negative (Wang, 2001; Gerloni & Zanetti, 2005).
Nonetheless, growing body of evidence indicates that tumor-specific CD4+ T cells can be
directly involved in mediating in vivo tumor protection and for priming efficient and long
lasting immunity against tumors (Wang, 2001; Corthay et al., 2005; Gerloni & Zanetti,
2005). Th cells can be assorted into various subsets, able of evoking a variety of
conflicting effects sometimes, leading either to the immune rejection of cancer or to
suppression of the immune responses and tumor growth (Muranski & Restifo, 2009a).
2.3.1 T helper 1, 2 and 17 cell subsets:
Upon receiving signals through the binding of antigen to the TCR, in the presence
of polarizing cytokines IL-12 or IL-4, naїve Th cells differentiate into distinctive CD4+ T
cell subsets; Th1 or Th2 effector cells, respectively. Th1 and Th2 secrete unique
repertoires of cytokines that mediate their responses (Table 2.1). Th1 cells secrete IL-2,
TNF-α, and IFN-γ and therefore direct cell-mediated immunity responses whereas Th2
cells secrete IL-4, IL-5, and IL-13 facilitating local humoral immunity responses (Janson
et al., 2009; Wilson et al., 2009). While IL-12 and IFN-γ are endowed with a propensity
of inhibiting Th2 response, Th2 cytokines such as, IL-10 and IL-4, can alternately inhibit
21
Th1 activity. Therefore, Th1 and Th2 responses are mutually inhibitory (Steinman,
2007).
The classical classification of Th cells into the binary system of Th1 and Th2 had
been updated by including a third distinct member denoted as Th17 (Table 2.1). This
lineage is called in light of their large quantities production of IL-17, also called IL-17A,
in addition to IL-17F, IL-21 and IL-22 (Harrington et al., 2005; Bettelli et al., 2008). A
novel transcription factor, RORγt, has emerged as a central protein for Th17
development. RORγt is regulated by signal transducer and activator of transcription
(STAT) 3, and overexpression of either STAT3 or RORγt promotes IL-17 transcription
(Ivanov et al., 2006). Interestingly, another orphan nuclear receptor, RORα, also
promotes Th17 differentiation in a similar STAT3 dependent manner (Yang et al.,
2008b). The reciprocal relation between Th1 and Th2 extends to play role in regulating
Th17 in which their cytokines inhibit Th17 suggesting a cross regulation in Th1, Th2 and
Th17 cell development (Harrington et al., 2005, 2006). Both Th1 and Th2 cell specific
transcriptional regulators (STAT1 and T-bet, STAT6 and GATA-binding protein 3
(GATA3), respectively) inhibited Th17 cell differentiation (Harrington et al., 2005).
Despite the need for IL-23 for the expansion and activation of Th17, it is not
essential for the initiation of Th17 development. Alternately, a combination of IL-6 and
TGF-β seems to be the proper formula for mice Th17 induction (Veldhoen et al., 2006).
However, this is not the case for human Th17 development wherein IL-23 and IL-1β
induces IL-17 production from naїve human CD4+ cells (Wilson et al., 2007; Manel et
al., 2008). Other present works reinstate TGF-β as a critical factor in inducing both
human and mouse Th17 differentiation from naїve CD4+ T cells, in concert with
22
inflammatory mediators including IL-1, IL-21, IL-6 and IL-23 (Manel et al., 2008; Volpe
et al., 2008; Yang et al., 2008a). Thus, decidedly at least two cytokines are needed to
differentiate Th17 cells.
CD4+ T cells can steer immune responses in different directions which could be
beneficial or detrimental. Th1 cells recognize cognate tumor antigen peptides presented
by MHC class II molecules on APCs, promote tissue destruction, and tumor rejection by
providing cytokine to activate cytotoxic CD8+ T cells thus amplifying their activation and
clonal expansion (Nishimura et al., 1999; Assudani et al., 2007). On the other hand, Th2
cells promote antibody production by B cells and skew immunity away from a beneficial
cell-mediated antitumor response (Nishimura et al., 1999; Assudani et al., 2007). Cross
regulation of Th1 and Th2 subsets through cytokine network has also been observed in
peripheral blood of patients with bladder cancer. Proportions of Th1 cells identified on
the basis of intracellular production of IFN-γ are markedly reduced, whereas proportions
of Th2 cells producing IL-4 are significantly elevated (Agarwal et al., 2006). Likewise, T
cells infiltrating human cervical carcinomas exhibit enhanced Th2 cytokine profiles,
particularly increased IL-4 and decreased IFN-γ production (Sheu et al., 2001). Th2
polarization is dependent upon, and leads to, production of IL-4 which might have direct
immunosuppressive effects on CD8+ T cells at the tumor site. Furthermore, favoring Th2
development promotes tumor immune evasion leading to detrimental antitumor response
(Kemp & Ronchese, 2001; Ziegler et al., 2009). Meanwhile, STAT1-deficient mice
displayed an increase in Th2 polarization due to the block in IFN-γ signaling, and were
more susceptible to tumor development (Fallarino & Gajewski, 1999).
23
Physiologically, Th17 responses are likely to emerge as an early response to a
number of pathogens not handled well by Th1- or Th2-type immunity and which require
robust tissue inflammation to be cleared (Bettelli et al., 2008). Moreover, Th17 cells
might facilitate the migration of other Th cells (such as Th1 cells) into the target tissue,
which could further propagate and modulate inflammation and tissue damage (Dardalhon
et al., 2008). Currently, Th17 cells have been shown to play important roles in
inflammation and autoimmune diseases, but relatively little is known about their exact
roles in tumor immunity (Ji & Zhang, 2010) since the information about the relevance of
Th17 in cancer biology is meager and contradictory (Bronte, 2008). Some studies,
however, detected low number of Th17 cells in several types of tumor. The levels of
tumor-infiltrating Th17 cells were reduced in a group of ovarian cancer patients with
more advanced disease and seemed to positively anticipate the outcome (Kryczek et al.,
2009a). Th17 cells were even able to contribute to protective human tumor immunity
through recruiting effector cells to the tumor microenvironment (Kryczek et al., 2009a).
Tumor-specific Th17-polarized cells can eradicate large established melanoma in mice
(Muranski et al., 2008). A possible explanation lies in that Th17 CD4+ T cells can be
unstable and evolve into IFN-γ-producing Th1-like cells capable of tumor destruction.
This effect also seems to be contingent on IFN-γ, as blocking antibodies prevent Th17
cell transfer from causing tumor regression (Muranski et al., 2008).
Contrarily, Th17 cells are potent inducers of autoimmunity through the promotion
of tissue inflammation and the mobilization of the innate immune system (Dardalhon et
al., 2008). The resulting inflammatory mediators may contribute to tumor progression by
upregulating immune suppressive cells of the adaptive and innate immune systems.
24
Substantial evidence indicates that the inflammatory reaction at a tumor site can promote
tumor growth and progression (Dougan & Dranoff, 2008; Colotta et al., 2009). Tumor-
associated inflammatory cytokines like IL-6 and TNF-α probably regulate Th17 cells in
the tumor microenvironment of ovarian cancer mouse model (Charles et al., 2009). Also,
tumor-activated monocytes promote expansion of Th17 cells through secreting a set of
key proinflammatory cytokines in the peritumoral stroma of hepatocellular carcinoma
tissues (Kuang et al., 2010).
Notably, Kryczek et al. (2007) reported the prevalence of Th17 cells in peripheral
blood and tumor microenvironment in both human and mice (Kryczek et al., 2007).
Elevated proportion of Th17 cells were also detected both in the peripheral blood and in
the tumor-draining lymph nodes of patients with gastric cancer, which were associated
with clinical stage (Zhang et al., 2008). Th17 cells were also suggested as a prognostic
marker in hepatocellular carcinoma (Zhang et al., 2009a) suggesting that Th17 cells can
also play an active role in tumor pathogenesis. Nonetheless, whether Th17 cells play the
same roles in the different types and stages of cancers remains to be determined (Ji &
Zhang, 2010).
2.3.2 Regulatory T (Treg) cell subset:
The fourth subset of CD4+ T lymphocyte lineage is naturally arising CD4+CD25+
Treg cells (Table 2.1). CD4+CD25+ Treg cells characterized as cells expressing CTLA-4
(Tang & Bluestone, 2008), and uniquely, the transcription factor Foxp3, which acts as a
master switch gene for their development and function (Fontenot et al., 2003; Gavin et
al., 2007). CTLA-4 is important for the maintenance of the Treg cells pool in the
25
periphery since it is involved in the induction of Foxp3 and is required for Treg
suppressor function (Sansom & Walker, 2006).
CD4+CD25+ Treg cells produce TGF-β and IL-10, two cytokines endowed with
immunosuppressive functions that play crucial functions in Treg cell biology (Chatila,
2005). Despite the disparate functions of Treg and Th17 subsets, both have been shown
to be dependent on TGF-β for their development (Burgler et al., 2009). Therein, TGF-β
induces the Treg specific transcription factor Foxp3. However, addition of IL-6 to TGF-β
inhibits the generation of Treg cells and induces Th17 cells (Bettelli et al., 2006). Thus,
TGF-β in the absence of inflammatory cytokines will induce Foxp3+ Treg cells
development (Korn et al., 2007; Zhou et al., 2008). Moreover, the cytokines that promote
Th17 responses significantly counteract the activation and functionality of the Tregs (La
Cava, 2008). Reciprocal relationship between Treg and Th17 cells is further supported by
recent finding that Treg cell transcription factor, Foxp3, and Th17 cell transcription
factor, RORγt, may counteract each other’s actions (Zhou et al., 2008).
Normally, CD4+CD25+ Treg cells have emerged as being particularly critical for
the maintenance of immunologic tolerance, suppressing inflammatory and allergen-
driven responses, and preventing autoimmune diseases by regulating self reactive T cells
(Choileain & Redmond, 2006; D'Ambrosio, 2006). The cross-talk between Treg cells and
targeted cells, such as APCs and T cells, is crucial for ensuring suppression by Treg cells
in the appropriate microenvironment by soluble factors and direct cell-cell contact (Wei
et al., 2006). For instance, Treg cells inhibit DCs function through binding of CTLA-4 to
CD80/86 (Vignali et al., 2008). Furthermore, Treg cells suppression of CD4+ and CD8+ T
26
cell responses is mediated both in the secondary lymphoid organs, where T cell activation
occurs, and in the tissues (Rudensky & Campbell, 2006).
Treg cells comprise 6%-10% of CD4+ T cells in human and mouse blood, thymus,
lymph nodes, and spleen (Wei et al., 2006). Notably, elevated proportions of CD4+CD25+
Treg in the total CD4+ T cell populations are present in the tumor microenvironment of
various types of cancer where they mediate immune suppression (Wang & Wang, 2007).
Tregs at tumor sites potently suppress CD4+ and CD8+ T cell responses, and suppress
immune responses to tumor antigens as well thereby promoting tumor growth (Chaput et
al., 2007) (Fig. 2.3) along with down-regulation of NK and NKT reactivity (Nishikawa et
al., 2005). Additionally, Treg cells can mediate suppression in the tumor
microenvironment through the actions of IL-10 and TGF-β in vivo in mouse tumor
models which inhibit expression of MHC molecules, CD80, CD86 and IL-12 and thus
inhibition of APCs function and T cell activation (Chen et al., 2005; Ghiringhelli et al.,
2005; Zou, 2006). Suppression within the tumor microenvironment might also be
mediated locally by APC-derived IL-10 as Treg cells might condition APCs and enable
them to express functional IL-10 (Kryczek et al., 2006). CD4+CD25+ Treg cells enhance
susceptibility to MCA-induced-tumorigenesis (Nishikawa et al., 2005). However,
depletion of CD4+CD25+ Treg cells reduced tumor growth of MCA-induced
fibrosarcomas (Betts et al., 2007).
27
Fig 2.3: Inhibition by CD4+CD25+ T cells at the tumor microenvironment. CD4+CD25+ T cells have the potential to depress tumor-specific immune responses, by interfering with the ability of APC to activate CD4+ and CD8+ T cells. Adapted from (Smyth et al., 2001).
Table 2.1: CD4+ T lymphocytes in tumor progression or regression
Th1 cells Th2 cells Th17 cells CD4+CD25+ Treg References
Priming cytokines
IL-12 IFN-γ IL-4
TGF-β and inflammatory
cytokine (IL-1, IL-21, IL-6 and
IL-23)
TGF-β1
(Veldhoen et al., 2006); (Bettelli et al., 2008); (Wilson et al., 2009)
Transcription factors
T-bet STAT-1
GATA-3 STAT-6
RORγt RORα Foxp3
(Fontenot et al., 2003); (Yang et al., 2008b); (Wilson et al., 2009)
Major secreted effector
cytokines
IL-2 IFN-γ TNF-α
IL-4 IL-5
IL-13
IL-17A (IL-17) IL-17F IL-21 IL-22
TGF-β1 IL-10
(Chatila, 2005); (Bettelli et al., 2008); (Janson et al., 2009)
Role in tumor micro-
environment
Help CD8+ T
cells
Suppress CD8+ T
cells
Recruit Th1 effector T cells/ Suppress CD8+
T cells and promote early growth in the inflammatory environment?
Suppress CD4+ and
CD8+ T cells
Outcome Tumor regression
Tumor progression
Tumor regression/ progression?
Tumor progression
(Ronchese, 2001); (Zou, 2006); (Bronte, 2008); (Kemp &; (Kryczek et al., 2009a); (Ji & Zhang, 2010)
28
The beneficial effect of stimulated tumor-specific T cell response can be
highlighted in T cells ability to move on into to the antigen-expressing tumor regardless
of their location in the host’s tissues. Besides, T cells can proliferate in response to
immunogenic proteins expressed in tumors till extermination of all the tumor cells is
achieved (Disis et al., 2009). Increasing evidence suggests that tumor-infiltrating immune
cells may have dual functions: inhibiting and promoting tumor growth and progression.
Among the host cells infiltrating the tumor, some can create a microenvironment that is
highly unfavorable to lymphocyte activity (Zou, 2005).
2.4 Myeloid-derived suppressor cells (MDSCs):
Tumor progression in many patients and experimental animals with cancer is
commonly associated with the expansion of a cell population of myeloid origin (Murdoch
et al., 2008; Peranzoni et al., 2010). As facilitators of tumor progression, myeloid-derived
suppressor cells (MDSCs) are a recently described population of myeloid cells
comprising immature macrophages, granulocytes, DCs and myeloid cells at earlier stages
of differentiation (Gabrilovich & Nagaraj, 2009). MDSCs can be found in the blood,
bone marrow, spleen, lymph nodes and immune-activated environment where they exert
immunosuppressive activity (Serafini et al., 2006; Dolcetti et al., 2008). Once recruited
to tumors, MDSCs have potent immunosuppressive activity by preventing the activation
of CD4+ and CD8+ T cells, reducing the number of mature DCs, swerving macrophage
activity, and suppressing NK cell cytotoxicity leading to immunosuppression of
antitumor effectors (Serafiini et al., 2006; Dolcetti et al., 2008; Ostrand-Rosenberg,
2010). MDSCs mediate their suppressive activity principally through multiple
29
mechanisms, including the production of arginase and/or inducible nitric oxide synthase
(Serafini et al., 2006), the promotion of tumor angiogenesis (McLean & Buckanovich,
2008), and the induction of Treg cells (Huang et al., 2006a).
2.5 Cytokines regulatory roles:
Cytokines are key proteins or glycoproteins of low molecular weight that can be
produced in all tissues and by most cells (e.g. tissue fibroblasts, endothelial cells and
enterocytes) (Hopkins, 2003; Matalka et al., 2005). Cytokines are intracellular,
extracellular or membrane-bound proteins that regulate the growth, differentiation and
activation of immune cells (Dranoff, 2004; Matalka et al., 2005). They act on many
different adjacent target cells (pleiotropism) often in an additive, synergistic, or
antagonistic manner (Nicolini et al., 2006). These cytokines are significant arbitrators of
host defense as they can act as first danger signals in alerting the immune system by
promoting antigen presentation, the differentiation and activation of DCs, and T cell-
mediated immune responses and consequently shaping both innate and adaptive immune
responses (Belardelli & Ferrantini, 2002).
Tumor immunity involves a concerted interplay between cytokines and effector
cells. Classically, tumor microenvironment encompasses tumor cells, fibroblasts,
endothelial cells, and infiltrating leukocytes (Ariztia et al., 2006). However, this
interaction between various cell types in the tumor microenvironment culminates in the
production of cytokines that control autocrine and paracrine communication within and
between these cells resulting in reciprocal influences which can either stimulate or inhibit
tumor development and progression (Dranoff, 2004; Salazar-Onfray, 2007). On the other
hand, cancer cells themselves produced cytokines that depict a complex network with a
30
spacious diversity of molecularly and functionally different members (Nicolini et al.,
2006). The cytokine repertoire present at the tumor site can steers the type of host
response directed towards the tumor (Belardelli & Ferrantini, 2002). Therefore, it is not
surprising that cytokines significantly affect the growth of tumors in vivo.
Cytokines are also probably involved in the mechanism from tumor cell detection
as well as evasion of the immune surveillance system (Salazar-Onfray, 2007). They
manipulate the immune response, impact growth and survival of the tumor cells, manage
the leukocytes infiltrating the tumor mass, regulate remodelling of the extracellular
matrix, stimulate neovascularisation, and may contribute to metastasis (Wilson &
Balkwill, 2002). One of the strategies to improve tumor local environment is to provide
appropriate cytokines systemically or locally (Joyce, 2005). An adequate balance of the
cytokine environment is critical to achieve protective immunity. Thus, extensive efforts
have focused on understanding the roles of cytokines and their interactions with effector
cells for the production of effective antitumor immunity (Chabalgoity et al., 2007).
2.5.1 Interferon-γ (IFN-γ):
IFNs are a family of cytokines that evoke a pleiotropic biological effect having
antiviral, immunomodulatory, and antiproliferative effects (Jonasch & Haluska, 2001).
They are sorted as type I (IFN-α, and β) which are induced in response to viral infection
of cells, and type II (IFN-γ) which are produced following activation with immune and
inflammatory stimuli rather than viral infection (Jonasch & Haluska, 2001;
Theofilopoulos et al., 2005; Billiau, 2006). IFN-γ is the signature cytokine produced by
Th1 cells but is also derived from CD8+ T cells, NKT, and NK cells (Boehm et al., 1997).
31
It is now well recognized that IFN-γ exerts its biologic effects by interacting with
an IFN-γ receptor (IFNGR) that is ubiquitously expressed on nearly all cells and
consisting of IFNGR1 and IFNGR2 (Pestka et al., 1997). IFN-γ responses in cells result
from the ligand-induced binding of the activated IFN-γ receptor complex to particular
components of the Janus tyrosine kinase (JAK)-STAT signaling pathway (Kalvakolanu,
2003). JAK1 and JAK2 protein tyrosine kinases of the Janus family constitutively
associate with IFNGR1 and IFNGR2, respectively, and binding ultimately leads to
phosphorylation of two STAT1 molecules followed by their dimerization and nuclear
translocation (Schindler & Plumlee, 2008).
IFN-γ is an inhibitor of Th2-mediated responses through its capacity to suppress
many IL-4 mediated effects thus, favoring the development of Th1 over Th2 cells
(Schroder et al., 2004). IFN-γ produced by Th1 cells provides help from Th1 cells to
macrophages promoting their activation and provides help from Th1 cells to B cells
(Billiau & Matthys, 2009). IFN-γ also upregulates MHC class I and II expression and
stimulates antigen presentation and cytokine production by APCs (Schroder et al., 2004).
IFN-γ released by NK cells has recently been studied intensively due to its
importance in molding the innate and adaptive immunity (Lion et al., 2009). NK cells are
not only producers of IFN-γ but also are targets of IFN-γ action. The crosstalk between
NK cells, DCs and T cells initiates and sustains immune responses against pathogens and
tumors (Fig 2.1) (Cooper et al., 2004). In this process NK cells provide early and
important sources of IFN-γ production as they might be the first to recognize developing
tumors and thus producing the initial levels of IFN-γ (Ikeda et al., 2002; Martin-Fontecha
et al., 2004). IFN-γ secreted at the tumor site by NK cells augments MHC expression on
32
tumor cells increasing their immunogenicity which in turn can induce tumor cell death
(Sengupta et al., 2010). NK cells not only operate in early stage of tumor development
but also act as a helper in priming process of CD8+ and Th1 cells by producing IFN-γ
(Seo et al., 2002). Likewise, NK and NKT cells are regarded as major sources of IFN-γ
during the early phase of tumor development, whereas CD4+ and CD8+ T cells may
become additional sources as adaptive immunity evolves (Li et al., 2007c).
NK cells are endowed with remarkable ability of recognizing potential target cells
without the need of being firstly educated by APCs and then activated, as in T cells.
Importantly, NK cell lineage is capable of killing a variety of tumor cells instinctively
while sparing normal cells (Smyth et al., 2002). NK cells are even capable of identifying
tumors with aberrant human leukocyte antigen that may evade T cell killing (Smyth et
al., 1998, 1999) through their special receptors (Moretta et al., 2006). In the course of
tumor mass, infiltration of NK cells into human neoplasms appears to correlates with a
better prognosis whereas low numbers of NK cells in advanced human neoplasms
indicate that NK cells do not normally home efficiently to malignant tissues (Albertsson
et al., 2003).
IFN-γ is a cytokine that is well recognized to play a central role in coordinating
tumor immune responses (Table 2.2). Myriad of studies have been performed to dissect
the mechanisms which IFN-γ employs to reject tumors (Dunn et al., 2006). An
unequivocal demonstration of cancer immune surveillance is that tumor suppressor
function of the immune system is critically dependent on the actions of IFN-γ which
along with lymphocytes collaborate to protect against development of carcinogen-
induced sarcomas and spontaneous epithelial carcinomas (Shankaran et al., 2001). In
33
tumor cells, IFN-γ upregulates the expression of the MHC class I antigen-processing and
-presentation pathway, thereby enhancing tumor cell immunogenicity and facilitating
tumor recognition and elimination by lymphocytes (Dunn et al., 2005). Actually, IFN-γ
displays an essential role for tumor responsiveness by regulating the migration of T cells
into tumor tissue (Beatty & Paterson, 2001b). The IFN-γ produced by tumor-infiltrating T
cells might play two distinct roles in antitumor activity: activation of antitumor T cells
and direct tumoricidal activity by generating inducible nitric oxide synthetase (Yu et al.,
1997). MCA-induced fibrosarcoma (Meth-A) is an immunogenic fibrosarcoma that arises
in BALB/c mice and grows progressively when transplanted subcutaneously (s.c.) into
naїve syngeneic mice (Ikeda et al., 2002). The loss of IFN-γ sensitivity is correlated with
an increased tumor incidence in animals treated with the chemical carcinogen MCA
(Beatty & Paterson, 2001b).
Studies of the benefaction of IL-12 to antitumor immunity provide further insight
into the physiologically relevant stimuli for IFN-γ production to enhance antitumor
immunity (Yang et al., 1999; Berenson et al., 2004). Cytokines as IL-12 can stimulate
IFN-γ production from T cells, NK cells, NKT cells (Sinlgaglia et al., 1999). IFN-γ
produced by IL-12-activated tumor-infiltrating CD8+T cells directly induced apoptosis of
mouse hepatocellular carcinoma cells (Komita et al., 2006). Moreover, IFN-γ and TGF-
β1 show reciprocal antagonistic effects (Li & Flavell, 2008). IFN-γ can antagonize the
production of immunosuppressive cytokines such as TGF-β, which can promote the
development of otherwise highly immunogenic tumors, improves the establishment of an
effective antitumor memory immune response, and thus control ongoing tumor growth
(Martini et al., 2010).
34
IFN-γ has a profound impact on solid tumors growth and metastasis (duPre' et al.,
2008) and appeared to play an early role in protection from metastasis (Street et al.,
2001). Developing solid tumors demand new blood vessel creation in order to grow.
Partially, this occurs through both: induced outgrowth of the preexisting vasculature and
de novo recruitment of vascular cell precursors from the circulation (Gupta & Massagué,
2006). Th1 cells can impair tumor angiogenesis either directly by inhibiting endothelial
cell proliferation through IFN-γ or indirectly through induction of antiangiogenic
chemokines such as chemokine (C-X-C motif) ligand (CXCL)9 and CXCL10 leading to
both angiostasis in and around the tumor and chemoattraction of immune effector cells
into the tumor site (Ikeda et al., 2002; Muller-Hermelink et al., 2008).
2.5.2 Interleukin-17 (IL-17):
IL-17 or (IL-17A) is the original member of IL-17 family which further includes
IL17B, IL17C, IL17D, IL17E (also called IL-25), and IL17F which displays the highest
degree of homology with IL-17A (Gaffen, 2008). IL-17 is the signature cytokine of a
unique lineage of CD4+ T cells termed Th17 (Bettelli et al., 2008). Additional cellular
sources of IL-17A include gamma delta (δγ) T cells, CD8+ T cells, neutrophils and
eosinophils (Kawaguchi et al., 2004; Roark et al., 2008). IL-17 is also produced by a
distinct population of activated NKT cells upon TCR stimulation (Lee et al., 2008b). IL-
17 binds to and signals through IL-17 receptor A (IL-17RA), a member of the IL-17R
family, which is widely expressed by epithelial cells, endothelial cells and fibroblasts. IL-
17R is detected also ubiquitously in tissues including lung, kidney, liver, spleen, and
brain (Moseley et al., 2003; Pappu et al., 2008).
35
Production of IL-17 by variety of cellular sources of both innate and adaptive T
cells could be correlated to a number of its exerted effects. Therein, whereas IFN-γ is
crucial in orchestrating the cellular immune response to intracellular pathogens, IL-17
generates T cell-mediated immune responses to extracellular pathogens and fungal
pathogens such as Klebsiella pneumoniae and Candida albicans, respectively
(Kawaguchi et al., 2004; Matsuzaki & Umemura, 2007). On the other hand, IL-17A is an
inflammatory cytokine that plays critical roles in the pathogenesis of multiple
autoimmune diseases including rheumatoid arthritis, psoriasis, inflammatory bowel
disease, and asthma (Kolls & Lindĕn, 2004; Kikly et al., 2006; Toy et al., 2006). IL-17
evokes various proinflammatory cytokines including induction of TNF-α, IL-1β, IL-6,
and IL-23 (Lin & Karin, 2007; Muranski & Restifo, 2009b).
IL-17 secreting cells have been identified in many types of human cancers and in
murine models as well, however, there are discrepant data (Table 2.2) on a possible role
for IL-17 in carcinogenesis (Muranski & Restifo, 2009b). Antitumor activity of IL-17
could be achieved by means of a T cell-dependent mechanism as in an increased
generation of specific CTLs (Benchetrit et al., 2002). Transfection of IL-17 into Meth-A
fibrosarcoma cell lines was shown to augment the expression of both MHC class I and II
antigens, therefore inducing tumor specific antitumor immunity (Hirahara et al., 2001).
Tumor growth and lung metastasis were intensified in IL-17 deficient mice (Kryczek et
al., 2009b).
It is generally accepted that IL-17 acts as an angiogenic factor in vitro as well as
in vivo. IL-17 stimulates the migration of vascular endothelial cells in vitro whereas it
elicits vessel formation in vivo (Numasaki et al., 2003; Takahashi et al., 2005).
36
Additionally, IL-17 promoted the growth rate and tumorigenicity of human cervical
tumors transplanted into nude mice (Tartour et al., 1999), knockdown of the IL-17
receptor in 4T1 mouse mammary cancer cells decreased tumor growth in vivo (Nam et
al., 2008) and IL-17 depletion delays chemically induced papillomas development (Xiao
et al., 2009). Remarkably, this effect seems to be driven by a pathway of IL-17 inducing
IL-6 production by both neoplastic and stromal cells, which successively leads to
activation of Stat3 (Wang et al., 2009). Seemingly, not merely Stat3 is involved but also
a number of inflammatory cytokines does play critical roles in this process.
In the course of the tumor microenvironment, TNF-α enhanced tumor growth via
the inflammatory cytokine IL-17 in a mouse model of ovarian cancer and in patients with
advanced cancer (Charles et al., 2009). Moreover, a set of key cytokines profile (IL-1β,
IL-6, TNF-α, and TGF-β) was detected in ovarian tumor cells, tumor-derived fibroblasts,
and APCs, which formed a cytokine milieu that regulated and expanded human IL-17-
producing Th17 cells (Miyahara et al., 2008) suggesting that these inflammatory
cytokines within the tumor microenvironment can provide a favorable niche for Th17
cells generation and potentially directing into a dangerous feedback loop.
Supplementary, proinflammatory cytokine as IL-23 is an important cytokine for
the expansion and survival of Th17 cells (Langrish et al., 2005). IL-23 demonstrated to
be an important link between tumor-promoting and proinflammatory processes. IL-23p19
mRNA was upregulated in significant manner in the majority of cancer samples from
various organ types, comprising colon, ovarian, lung, breast, and stomach cancers as well
as melanoma (Langowski et al., 2006). Stat3 signaling within the tumor
microenvironment induces the protumor cytokine, IL-23, while inhibiting a central
37
antitumor cytokine, IL-12, thereby shifting the balance of tumor immunity toward
carcinogenesis (Kortylewski et al., 2009).
2.5.3 Transforming growth factor-beta (TGF-β) 1:
Consisting of a family with highly pleiotropic properties TGF-β is a member of a
large family of developmental conserved proteins. Three closely related isoforms have
been identified of this family including TGF-β1, TGF-β2 and TGF-β3 (Gorelik & Flavell,
2002). TGF-β can be produced by both non-immune and immune cells and function in an
autocrine and paracrine pattern (Letterio & Roberts, 1998; Zhang et al., 2006). TGF-β1 is
the predominant isoform expressed in the immune system and lymphoid organs and is
essential for maintaining T cell homeostasis, Treg cells and effector cells function and
carcinogenesis (Gorelik & Flavell, 2002; Bommireddy & Doetschman, 2007). TGF-β1 is
produced by every leukocyte lineage including lymphocytes, macrophages, and DCs, and
its autocrine and paracrine expression serves to control the differentiation, proliferation,
and these immune cells activation (Letterio & Roberts, 1998).
TGF-β1 is generally secreted in a biologically latent form and cannot interact with
its receptor. Following activation by the cell releasing TGF-β1 or the target cell (Khalil,
1999), TGF-β1 provokes its biological effects via hetero-oligomerization of membrane-
bound type I and type II TGF-β serine/threonine kinase receptors, resulting in subsequent
activation of downstream signaling cascades (Itoh & Dijke, 2007). Caenorhabditis
elegans Sma and Drosophila Mad (Smad) proteins play a particularly important role in
the intracellular signaling of TGF-β1. Upon TGF-β receptor activation, Smad2 and
Smad3 are phosphorylated, oligomerize with Smad4, and translocate into the nucleus
where they participate in regulating gene expression (Xu, 2006).
38
TGF-β1 and IFN-γ are antagonistic cytokines that regulate Th1 development in
opposite directions. Therein, TGF-β1 inhibits Th1 development by inhibiting IFN-γ’s
induction of T-bet and by inhibiting receptor-proximal IFN-γ-Jak-Stat signaling
responses (Park et al., 2007). TGF-β1 inhibits the differentiation of both Th1 and Th2
cells (Li & Flavell, 2008). By contrast, TGF-β1 is a crucial factor for the differentiation
of Th17 cells from naïve CD4+ T cells, in concert with an inflammatory cytokine as
mentioned earlier (Veldhoen & Stockinger, 2006). However, the balance of an
inflammatory cytokine, as IL-6, and TGF-β1 determines whether a T cell become a Treg
or Th17 (Korn et al., 2007; Zhou et al., 2008).
TGF-β signaling is contextual, depends on the cell type, and has both positive and
negative effects on cancer. However, changes in TGF-β signaling often correlate with
tumor stage and rate of progression (Bierie & Moses, 2006). Despite TGF-β capacity in
suppressing immune cells via an autocrine and paracrine pattern this is not the case with
cancer cells. TGF-β has the capacity to inhibit the growth of early tumor cells but also
might enhance tumor progression (Pardali & Moustakas, 2007). Seemingly, cancer cells
protect themselves and tend to acquire increasing resistance to ignore TGF-β growth
inhibitory signals. Subsequently, cancer cells start secreting non-physiological levels of
TGF-β in an autocrine and paracrine manner which may affect the differentiation of the
tumor cells and the surrounding cellular environment, respectively, leading to
development of the tumor and metastasis in an immunosuppressive environment that is
rich in TGF-β (Moustakas et al., 2002).
A great number of cancerous cell lines and tumor-associated T cells are known to
produce TGF-β directly (Smyth et al., 2004). Treg cells are regarded as a primary source
39
of TGF-β1 (Bommireddy & Doetschman, 2007). Accumulation of CD4+CD25+Foxp3+
Treg cells in both the peripheral blood and the tumor may maintain the
immunosuppressive environment, which can be aggravated by the considerable quantities
of TGF-β that some tumors can produce (Liu et al., 2007; Sengupta et al., 2010).
Paradoxically, both Th17 and Foxp3+ Treg cells were at their maximal levels in advanced
tumors. Despite that elevated Treg cells foretell low Th17 cell levels, a potentiation of
TGF-β levels by the high Treg cells may resolve this paradox in the inflammatory tumor
microenvironment (Moutsopoulos et al., 2008). The high levels of TGF-β available
within the tumor microenvironment can even induce the conversion of normal
CD4+CD25- T cells into CD4+CD25+Foxp3+ Treg cells (Zou, 2006).
TGF-β is an immunosuppressive cytokine that function at several levels in the
tumor microenvironment (Table 2.2) which also encompasses reduction in the amount of
antigen presentation by DCs (Bennaceur et al., 2008), reduction in the proliferation of T
cells (Th1, Th2 and CTL), suppression of NK cells cytotoxic activity, and stimulation of
Treg cells proliferation thus impeding immune surveillance of the developing tumor
(Massagué, 2008). In the context of malignant niche, TGF-β induces epithelial transition
that in turn converts cancer cells into invasive cells. Tumor-derived TGF-β causes
remodeling of the tumor matrix: by acting on stromal fibroblasts; inducing the expression
of mitogenic signals towards the carcinoma cells; and by promoting angiogenesis through
stimulating VEGF and other angiogenic factors production (Bierie & Moses, 2006;
Pardali & Moustakas, 2007).
40
Table 2.2: Effect of cytokines in the tumor microenvironment
Cytokine Secreting cells Cytokine effects References
IFN-γ
CD4+ Th1 CD8+ T
NK NKT
Positive effects on tumor immune surveillance: Direct anti-proliferative and cytotoxic effects Upregulates MHC expression on tumor cells Inhibit angiogenesis Antagonize suppression by tumor-derived TGF-β
(Ikeda et al., 2002); (Dunn et al., 2005); (Martini et al., 2010)
IL-17 Th17
Positive and negative effects on tumor immune surveillance, depending on tumor model: Suppress tumor progression by enhancing antitumor immunity, or promote tumor progression by an increase in inflammatory angiogenesis?
(Benchetrit et al., 2002); (Kryczek et al., 2009b); (Muranski & Restifo, 2009b)
TGF-β1 CD4+CD25+
Foxp3+
Treg
Negative effects on tumor immune surveillance: Remodeling of tumor matrix and stromal cells Promotes angiogenesis Induction of Treg cells development Inhibits development, proliferation, and function of both the innate and the adaptive immunity
(Bierie & Moses, 2006); (Pardali & Moustakas, 2007); (Massagué, 2008)
The targeted cells react in a microenvironmental context where different stimuli
can impact their behavior. Hence, care should be taken in the creation of therapeutic
interventions utilizing compounds that could stable the delicately reharmonized
equilibrium that exists at the base of cytokine network. Additional evidence for the
criticality of cytokines in cancer is their antagonistic efficacy as aforementioned.
Modeling and simulating cytokines’ effect on immune response to tumor cells in the
tumor microenvironment in a potential favorable manner is listed in (Table 2.3).
41
Table 2.3: Modeling and simulating cytokines’ effect on immune response to tumor cells in the tumor microenvironment
Cytokine Rationale Favorable outcome
IFN-γ
Tumor growth inhibition: Stimulation of cellular immunity; Antagonize TGF-β1 deleterious effects; Inhibit IL-17 production
↑ STAT1 activation
IL-12 Tumor growth inhibition: Acts via IFN-γ and activates NK, Th1,CD8+ T cell and promote DCs maturation
↑ STAT4 activation
IL-17
Tumor growth promotion: TGF-β1 with an inflammatory cytokine supports IL-17 production via STAT3 activation; Inhibit IL-12 favorable effects by STAT3 activation
↓ STAT3 activation
TGF-β1
Tumor growth promotion: Inhibits IFN-γ favorable effects; Supports Treg cells differentiation; Supports Th17 cells differentiation with an inflammatory cytokine
↓ Smad3 activation
2.6 Eriobotrya japonica (EJ):
Loquat (EJ Lindl.) is a renowned member of the Rosaceae family with high
medicinal values. Presumably, EJ is indigenous in south-eastern China and has been
cultivated in China and Japan since ancient times. Presently, EJ is known to many
countries and is cultivated in other regions as in the Mediterranean area (Martínez-Calvo
et al., 1999; Louati et al., 2003; Freihat et al., 2008). This evergreen shrub or small tree
has white flowers that give rise to tasty pale-yellow or deep-orange fruits which can be
either eaten fresh or processed into jam and jelly (Zhou et al., 2007b; Ferreres et al.,
2009). The tree narrow leaves are dark green on the upper surface with a lighter woolly
under surface (Ferreres et al., 2009).
42
Practically, these plant different organs (flowers, fruits, seeds, and leaves) were
utilized following treatment with different extraction methods that ultimately led to the
isolation of several compounds with distinctive biological activities (Nishioka et al.,
2002; Zhou et al., 2007a, 2007b). Most momentously, the extraction solvent utilized on
every occasion crucially affected the terminal biological activity obtained (Nishioka et
al., 2002; Matalka et al., 2007). For instance, certain experimental assays implied that the
favorable effect of the extracts on the liver functions fluctuates according to the
extracting solvent employed. Ethylic- and methylic-extracts of EJ seeds were found to
inhibit the development of liver fibrosis in hepatopathic rats (Nishioka et al., 2002).
Likewise, the solvent used for extraction could also significantly affect the type and level
of components recovered. Ethylic-extract of EJ seeds provides a richer source of
polyphenolic antioxidants with high ability to scavenge free radicals and suppress the low
density lipoprotein oxidation than the seeds water-extract (Koba et al., 2007).
Additionally, EJ seeds exerted a hypoglycemic property and improved glucose tolerance.
Theses effects are also attributable to the polyphonlic components extracted by ethanol as
in several kinds of flavonoids e.g., (+)-catechin, epicatechin, epigallocatechin,
epicatechingallate, and chlorogenic acid (Tanaka et al., 2008). Furthermore, in addition to
polyphenols, ethylic EJ seeds extract contains various contents such as phytosterols, fatty
acids, and amino acids that were probably involved in inhibiting formation of gastric
mucosal injury (Yokota et al., 2008).
Traditionally, the flowers of this plant were valuable for the treatment of cough,
cold and sputum and its ethylic-extract contained triterpenoidal saponins, amygdalin, and
triterpene acids such as oleanolic and ursolic acids (Zhou et al., 2007a). The mature fruits
43
are rich in carotenoids, as principal pigments responsible for the color of loquat fruits
(Zhou et al., 2007b). Organic acids, majorly malic and citric acids are one of the
important factors influencing fruit flavour (Chen et al., 2009). The ethylic-extract of fruit
flesh contains significant levels of polyphenolic compounds such as chlorogenic acid,
cyanidine glycoside, and epicatechin which exhibited free radical scavenging capabilities
(Koba et al., 2007). Moreover, the antioxidant defensive action of EJ fruit alcoholic-
extract on leukocytes and erythrocytes proposes that the latter may be used as an
antioxidant agent complementing the administration of medicinal treatments to
counteract the oxidative stress generated in blood and other tissues by oxidant agents
(Eraso & Albesa, 2007).
Customarily, the dried leaves of EJ have been used as a Chinese crude drug to
treat stomachache, restrain vomiting, promote antidiarrheal, diuretic, eliminate phlegm,
regulate cough, and clear away lung-heat (Kawahara et al., 2002; Chen et al., 2008) and a
traditional therapy to treat cancers utilized the leaves in compress (Ito et al., 2002). So
far, the research on EJ different organs is divergent and still ongoing, however, studies of
extraction, identification and pharmacological activities of EJ functional constituents are
chiefly centered on its leaves (Zhou et al., 2007a). The pharmacological tests on anti-
hyperglycemic effects prove that EJ leaves are an outstanding material to develop
medicine for treatment of diabetes mellitus with an astonishing multi-targeted anti-
diabetic profile (Rollinger et al., 2010). An ethylic-extract of the leaves showed a
significant hypoglycaemic effect (Li et al., 2007b). The research on the leaves ethylic-
extract reveals the occurrence of several chemical components like sesquiterpene
glycoside (Chen et al., 2008), flavonoids (Lü et al., 2009a), and triperpene acids (Lü et
44
al., 2009b) wherein all proved to have hypoglycemic effect in vivo. On the other hand,
cinchonain Ib isolated from the water-extract of the leaves has a novel insulinotropic
effect suggesting its potential use for managing type 2 diabetes (Qa’dan et al., 2009).
The isomeric pentacyclic oleanolic and ursolic acids are prevalent triterpene acids
present in EJ leaves (Zhou et al., 2007a). Recently, extensive investigations on the
triterpene acids in the leaves of this plant disclosed significant antiviral (De Tommasi et
al., 1992), anti-inflammatory (Huang et al., 2007), antioxidative (Huang et al., 2006b),
antimutagenic (Young et al., 1994), and antitumor properties (Ito et al., 2000, 2002;
Banno et al., 2005). Collectively, besides triterpene acids, megastigmane glycosides and
polyphenolic constituents encompassing flavonoid glycosides and procyanidins were
isolated from EJ leaves and have been reported to exert antitumor activities as well (Ito et
al., 2000, 2002). In an in vivo two-stage carcinogenesis test of mouse tumor euscaphic
acid and procyanidin B-2, one of triterpene acids and polyphenols, respectively, isolated
from the methylic-extract of EJ leaves, exhibited marked antitumor-promoting activity
(Ito et al., 2002; Banno et al., 2005) whereas roseoside, a megastigmane glycoside,
displayed a potent antitumor-initiating effect providing an evidence that EJ is promising
as a potential source for chemoprevention of cancer (Ito et al., 2002).
A strong cytotoxicity of EJ leaves methanol-extract was documented in vitro
against human cancer cell lines representing different tissues such as breast
adenocarcinoma, as well as cervix epitheloid and lung carcinoma (Kang et al., 2006).
Moreover, the latter extract also inhibited the adhesion, migration and invasion of human
breast cancer cells, partially through the inhibition of matrix metalloproteinase activity
(Kim et al., 2009). Uniquely, among the various individual polyphenols isolated and
45
tested from different fractions of EJ leaves extract, a polyphenol obtained from the water-
soluble fraction, namely procyanidin oligomer, exhibited the highest selective cytotoxic
activity in vitro against human oral tumor cell lines (Ito et al., 2000). The above findings
furnish a comprehensive foundation for the possibility of EJ folium as a potential agent
for the treatment and/or prevention of cancer.
Latterly, Matalka et al. (2007) established EJ leaves water-extract as a rich source
of polysaccharides and polar phenolic compounds that are capable of inducing
proinflammatory cytokines production in vitro as well as in vivo, whereas the ethylic
acetate- and methylic-extracts displayed anti-inflammatory activity (Banno et al. 2005;
Matalka et al. 2007). Correspondingly, EJ leaves water-extract is considered
immunostimulatory as it displays a variety of proinflammatory and antitumoral effects.
Akin to these findings, and yet to be fully exploited in a tumor model whether water-
extract of EJ leaves is capable of exerting these immunostimulating capabilities, more
precisely, at the tumor site in vivo and therefore reducing tumor development (Matalka et
al., 2007), which the following chapters will clarify such hypothesis.
46
Chapter 3
Materials and Methods
47
Chapter 3 Materials and Methods
3.1 Materials: 3.1.1 Plant material:
Fresh leaves of EJ LINDL (Rosaceae) were collected from Jabal Al Hussein area
in Amman-Jordan (06/2009), washed profoundly with tap water, dried for one week at
room temperature, and then grounded into powder. The plant material was identified in
comparison with authentic EJ obtained from the Botanical Institute, University of
Cologne (Germany).
3.1.1.1 Plant material extraction:
Five hundred grams of powdered leaves were thoroughly extracted with boiled
water. In portions, boiled water (300 ml) was added to 50 g powder for five minutes
followed by filtration into a flask. This step was repeated three times. The native liquid
EJHE was combined at the end of this step giving a total volume of 2 liters. A part of the
combined native liquid EJHE (300 ml) was freeze dried to obtain 8.93 g of the EJHE.
The remaining (1700 ml) of the combined native liquid EJHE was successively
partitioned with (5600 ml) of n-butanol (1:1). Each portion of the remaining native EJHE
was repeatedly extracted (three times or until solution became clear) with n-butanol. The
water phase, non-extracted material, was combined at the end of this step and referred to
as EJHE-WR. A part of the combined liquid EJHE-WR (300 ml) was freeze dried to
obtain 10.76 g of the EJHE-WR.
Before utilization in animal testing, each of EJHE and EJHE-WR were dissolved
in endotoxin-free phosphate buffered saline (PBS) and sterilized through filtration with
48
sterile filters (0.2 µm). Then, the required concentrations were subsequently prepared in
endotoxin-free PBS and used fresh for each experiment.
3.1.2 Materials for cytokines extraction and establishment of tumor cell line:
Endotoxin-free Dulbecco's PBS without calcium and magnesium, PRMI-1640,
fetal bovine serum (FBS), and penicillin-streptomycin were purchased from Euroclone
(Siziano, Italy). Trypsin was obtained from PAA Laboratories Gmbh (Linz, Austria)
whereas Igepal CA-630, MCA 98%, and lipopolycaccharide (LPS, L-6143) were
obtained from Sigma (St. Louis, MO, USA). T75 flasks were purchased from Costar
(Cambridge, MA, USA).
3.1.3 Materials for cytokine assays:
The following materials and reagents: standards (recombinant mouse IFN-γ, IL-
17 and TGF-β1), anti-cytokine capture antibodies, anti-cytokine-biotinylated detector
antibodies, and strepavidin-Horseradish peroxidase (Duoset cytokine kits); color reagent
A (stabilized peroxidase) and color reagent B (stabilized chromogen) solutions were
purchased from R&D Systems, UK. HEPES buffer was obtained from PAA Laboratories
Gmbh (Linz, Austria) whereas bovine serum albumin (BSA) was obtained from Research
Organics (Cleveland, OH, USA). Maxisorb 96-well flat bottom plates were purchased
from Nunc International (Denmark).
3.2 Mice:
BALB/c males and females mice were utilized throughout the experiments as
indicated. BALB/c mice were purchased from Yarmouk University (Irbid, Jordan). All
49
mice were housed in a pathogen free environment at 22°C with 12 hours light/dark cycle
with food and tap water ad libitum.
3.3 Methods:
3.3.1 Tissues specific modulation of IFN-γ, IL-17 and TGF-β1 in normal mice:
In test of tissue specific modulation of cytokines, thirty-five male mice of 8-10
weeks old were divided into seven groups each composed of five mice. Groups 1, 2 and 3
were administered i.p. with 1 ml of 1, 10 and 100 µg EJHE, respectively. Groups 4, 5 and
6 were administered i.p. with 1 ml of 1, 10 and 100 µg EJHE-WR, respectively. The
remaining group was administered i.p. with 1 ml of the endotoxin-free PBS alone and
considered as the control group. Following 2 hours, mice were sacrificed by cervical
dislocation and tissues/organs were collected within 5-10 minutes. The collected
tissues/organs were blood, lungs, and spleen. Cytokines were extracted from the latter
tissues/organs in accordance with a method previously described by Matalka et al. (2005)
as mentioned below (3.3.8).
3.3.2 Kinetics of IFN-γ, IL-17 and TGF-β1 induced by EJHE and EHHE-WR administration in normal mice:
The kinetics of IFN-γ, IL-17 and TGF-β1 modulation following the
administration of EJHE and EHHE-WR were performed using fifty-two male mice of 8-
10 weeks old. Mice were divided into three groups each of sixteen mice. Group 1 and 2
were administered i.p. with 1 ml of 10 µg EJHE and EJHE-WR, respectively. Group 3
was administered i.p. with 1 ml containing 2 µg LPS. The remaining four mice received
no treatment, considered as the control group, and were sacrificed at zero time point.
Following single i.p. injection of the latter concentrations four mice were sacrificed from
50
each group by cervical dislocation at specific time points (2, 6, 24 and 48 hours) and
blood, lungs, and spleen were collected as described below (3.3.8).
3.3.3 MCA-induced tumors:
Mice were inoculated s.c. into the right hind flank with MCA dissolved in olive
oil at two different doses. Initially, nineteen male mice of 10-12 weeks old were
inoculated with (0.5 mg/mouse). Then, seven male and seven female mice of 2-4 weeks
old were inoculated with (1 mg/mouse). Mice were inspected weekly for tumor
development. When tumors reached a certain size (1-2 cm in diameter), mice were
sacrificed and the tumor tissue was surgically removed. The tissue was then processed for
the establishment of cell lines.
3.3.4 MCA-induced cell lines:
Murine primary MCA-induced tumors were aseptically excised, cut into small
pieces, minced, treated with trypsin, and cultured in T75 flasks in PRMI-1640 medium
supplemented with 10% FBS, and antibiotics (100 U/ml penicillin and 100 μg/ml
streptomycin), at 37 ◦C in a humidified atmosphere of 5% CO2. The medium was
changed every 2-3 days. The monolayers were split by trypsinization as required to
maintain logarithmic growth.
3.3.5 Inoculation of tumor cells and experimental tumor model:
Established MCA-induced fibrosarcoma (Meth-A cells), were grown in T75
flasks and harvested as described in (3.3.4) in logarithmic growth phase. Meth-A cells
were counted and resuspended in endotoxin-free PBS to the desired concentrations. A
suspension of Meth-A cells was inoculated s.c in the right flank of the mouse to establish
51
a tumor model of Meth-A fibrosarcoma. In this experimental system, twenty-one male
mice and thirty female mice were inoculated with 2×106 and 0.67×106 corresponding
tumor cells in 200 μl endotoxin-free PBS, respectively. Recipient mice were inspected
twice weekly. Tumor-bearing mice were defined by the presence of a progressively
growing tumor with a diameter of ≥7 mm measured using a digital caliper. Noteworthy,
following tumor inoculation the mice behaved normally, no apparent suffering, and no
signs of pain or other disorders were noticed.
3.3.6 IFN-γ, IL-17 and TGF-β1 modulation within the tumor microenvironment:
Tumor-bearing mice with progressively growing transplanted-tumors as
delineated in (3.3.5) were divided into five groups, where each group consists of three to
five mice. Groups 1 and 2 were administered i.p. with 1 ml of 10 µg EJHE and EJHE-
WR, respectively. Group 3 was administered i.p. with 1 ml of endotoxin-free PBS and
considered as the control group. Following 24 hours, mice from groups 1, 2, and 3 were
sacrificed. This experiment was performed twice and the pattern of cytokine change was
the same in the two experiments. Therefore, the data presented here were derived from a
single experiment. Groups 4 and 5 were administered i.p. with 1 ml of 10 µg EJHE and
EJHE-WR, respectively, once every 24 hours for three consecutive days and then
sacrificed on the fourth day. Mice were sacrificed by cervical dislocation then spleen and
tumor were collected as described below (3.3.8).
3.3.7 Survival of Meth-A tumor-bearing mice after treatment with EJHE and EJHE-WR:
Thirty female mice were divided into three groups each of ten mice. Mice were
inoculated i.p. with 2×106 Meth-A cells in 0.5 ml endotoxin-free PBS. Three days after
52
tumor inoculation, group 1 and 2 were administered i.p. with 0.5 ml of 10 µg EJHE and
EJHE-WR, respectively. Group 3 was administered i.p. with 0.5 ml of endotoxin-free
PBS and considered as the control group. The mice in the three groups were repeatedly
administered with the corresponding doses every 48 hours with one day off following
each three consecutive administrations. The weights of the mice were documented before
each administration. The mice in each group were observed daily to monitor their
survival. The survival of the mice was recorded till their death.
3.3.8 Cytokine extraction:
Post sacrificing each mouse blood was directly collected from the cardiac
chamber, placed into prechilled tube, weighed and incubated with 2 ml of ice-cold
endotoxin-free PBS containing 0.1% igepal CA-630 nonionic detergent under ice. After
collecting blood samples, the lungs, spleen, and tumor were removed from mice,
weighed, placed in prechilled tube containing 2 ml of ice-cold endotoxin-free PBS with
0.1% igepal CA-630 nonionic detergent and incubated under ice. Following incubation
period of 10 minutes, the tissues were homogenized with a tissue disrupter (Janke and
Kundle) and were then centrifuged (6000 rpm for 6 minutes). The resulting supernatants
were transferred to labeled microcentrifuge tubes and reserved at –30 °C till cytokine
assays (Matalka et al., 2005).
3.3.9 Cytokine assays:
Measurements of mouse tissue-extracted cytokines (IFN-γ, IL-17, and TGF-β1)
were accomplished by sandwich ELISAs developed in accordance with the
manufacturer's recommendations (Duoset R & D Systems, UK). Initially, 96-well
53
microplates were coated with captured antibodies in PBS (137 mM NaCl, 2.7 mM KCl,
8.1 mM Na2HPO4, 1.5 mM KH2PO4) pH 7.2-7.4, at 4.0 µg/ml for IFN-γ, TGF-β1, and at
2.0 µg/ml for IL-17, sealed and incubated overnight at room temperature. Day after, wells
were washed three times with washing buffer containing 0.05% Tween 20 in PBS pH
7.2-7.4. On the hour, IFN-γ and IL-17 were blocked with the same buffer, TGF-β1 was
blocked by 5% Tween in PBS pH 7.2-7.4 followed by washing the wells three times with
washing buffer. TGF-β1 samples were activated to the immunoreactive form by adding
2.5 N Acetic Acid/ 10 M Urea (100 µl) to each 100 µl of tissues samples, mixed well,
incubated 10 minutes at room temperature and neutralized by adding 2.7 NaOH/ 1 M
HEPES (100 µl). Next, standards in the range of 15.6-2000 pg/ml were used for the
assays (with 7 points standard curves and the zero standard), tissues samples for IFN-γ,
IL-17 and neutralized tissues samples for TGF-β1 diluted (1:4), all run in duplicates, with
reagent diluents for each cytokine being added in the wells to give a final volume of (100
μl/well), incubated for two hours at room temperature and washed three times with
washing buffer. Biotinylated detector antibodies (100μl/well) were added at
concentrations (800 ng/ml, 400 ng/ml, and 200 ng/ml for IFN-γ, IL-17, and TGF-β1,
respectively), incubated for two hours at room temperature and washed three times with
washing buffer. Streptavidin-Horseradish peroxidase conjugate (100 μl/well) diluted
(1:200) in reagent diluents was added to each well, incubated for 20 minutes at room
temperature and washed four times with washing buffer. Color developed upon the
addition of 100μl/well of the substrate solution (1:1 mixture) containing color reagent A
(stabilized peroxidase) and color reagent B (stabilized chromogen) solutions. Color
development was halted by addition of 1M H2SO4 (50 μl/well). Plates were read at 450
54
nm by SCO GmbH (Dingelsadt, Germany) ELISA plate reader and absorbance was
transformed to cytokines concentrations (pg/ml) and then to (pg/g) of tissues using a
standard curve computed on Excel sheet after transforming values to log to construct a
straight line on a log-log graph.
3.4 Statistical analysis:
All data in the figures are depicted as the mean ± standard error and assessed by
using one way ANOVA analysis followed by a Tukey’s test (95% confidence) for
multiple comparisons (SPSS version 17). P value of <0.05 is considered statistically
significant.
55
Chapter 4
Results
56
Chapter 4
Results
4.1 Tissues specific modulation of IFN-γ, IL-17 and TGF-β1 by EJHE and EJHE-WR following two hours of administration: 4.1.1 EJHE and EJHE-WR modulate IFN-γ and IL-17 expression in blood, spleen, and lung tissues in a comparable manner:
Two hours following i.p. administration of EJHE (1-100 µg/ml) significantly
modulated and increased IFN-γ and IL-17 production in the spleen. This increase was
concentration dependent. IFN-γ and IL-17 levels were increased significantly in the
spleen at 1 and 10 µg/ml of EJHE (P<0.0001 for IFN-γ, 0.001 and 0.0001 for IL-17,
respectively). At higher doses of EJHE (100 µg/ml), however, these modulations in IFN-
γ and IL-17 levels were reversed (P<0.0001 and >0.05, respectively). As for EJHE-WR,
1 µg/ml increased significantly IFN-γ and IL-17 production (P>0.05 and <0.001,
respectively). This increase was not observed at higher doses (Fig. 4.1 & 4.2).
In the lungs a different pattern was noticed in that, increasing concentration of
EJHE (1-100 µg/ml) was inversely related to inhibition of IFN-γ and IL-17 (P<0.0001).
The maximum suppression of IFN-γ and IL-17 was noticed at 1 µg/ml of EJHE
(P<0.0001), whereas the maximum increase was seen with 10 µg/ml of EJHE-WR
(P<0.0001 and =0.0045, respectively) (Fig. 4.3 & 4.4). In blood, levels of IFN-γ were
reduced after (1-100 µg/ml) of EJHE and 1 µg/ml of EJHE-WR administrations
(P=0.0003 and >0.05, respectively), whereas blood IL-17 was not significantly
modulated following either dose of EJHE or EJHE-WR (P>0.05) (Fig. 4.5 & 4.6).
57
Fig 4.1: Effect of EJHE and EJHE-WR on IFN-γ production in the spleen of mice. Two hours following i.p. administration of 1 or 10 µg/ml EJHE increased significantly IFN-γ production as opposed to significant suppression at higher dose of 100 µg/ml (*P<0.0001). EJHE-WR at 1 µg/ml apparently increased IFN-γ production (P>0.05).
Fig 4.2: Effect of EJHE and EJHE-WR on IL-17 production in the spleen of mice. Two hours following i.p. administration of 1 or 10 µg/ml EJHE increased significantly the production of IL-17 (*P<0.001 and 0.0001, respectively). This increase was not observed at higher dose (P>0.05). EJHE-WR at 1 µg/ml increased IL-17 production whereas 10 and 100 µg/ml led to reduction (**P<0.001, =0.014, and =0.029, respectively).
58
Fig. 4.3: Effect of EJHE and EJHE-WR on IFN-γ production in the lungs of mice. Two hours following i.p. administration of 10 µg/ml EJHE or EJHE-WR increased significantly the production of IFN-γ whereas 1 µg/ml EJHE or 100 µg/ml EJHE-WR decreased significantly the production of IFN-γ (*, **P<0.0001).
Fig. 4.4: Effect of EJHE and EJHE-WR on IL-17 production in the lungs of mice. Two hours following i.p. administration of 10 µg/ml EJHE-WR increased significantly the production of IL-17 (P=0.0045), whereas 1 µg/ml EJHE decreased significantly the production of IL-17 (*P<0.0001).
59
Fig 4.5: Effect of EJHE and EJHE-WR on IFN-γ production in the blood of mice. Two hours following i.p. administration of increasing concentrations (1-100 µg/ml) of EJHE reduced significantly the production of IFN-γ (*P=0.0003), whereas 1 µg/ml of EJHE-WR showed a trend towards a decrease in IFN-γ production (P>0.05).
Fig 4.6: Effect of EJHE and EJHE-WR on IL-17 production in the blood of mice. Blood IL-17 was not significantly modulated following either dose of EJHE or EJHE-WR (P>0.05).
60
4.1.2 EJHE and EJHE-WR ubiquitously inhibit TGF-β1 in mice blood, spleen, and lung tissues:
Two hours following i.p. administration of increasing concentration of EJHE and
EJHE-WR (1-100 µg/ml) inhibited significantly TGF-β1 production in the lungs and
blood. The highest inhibition was noticed at high dose of EJHE (100 µg/ml) (P<0.0001)
whilst EJHE-WR achieved similar suppression at low doses (1 and 10 µg/ml) in the
spleen (P=0.003 and <0.001, respectively) and lungs (P<0.0001) (Fig. 4.7 & 4.8). In the
blood, the highest inhibition was observed at low doses of EJHE (1 and 10 µg/ml)
(P<0.0001) (Fig. 4.9).
Fig 4.7: Effect of EJHE and EJHE-WR on TGF-β1 production in the spleen of mice. Two hours post i.p. administration of 100 µg/ml of EJHE inhibited significantly the production of TGF-β1 (*P<0.0001). EJHE-WR at 1 and 10 µg/ml suppressed significantly the production of TGF-β1 (**P=0.003 and <0.001, respectively) with a trend towards a decrease at 100 µg/ml (P>0.05).
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Fig. 4.8: Effect of EJHE and EJHE-WR on TGF-β1 production in the lungs of mice. Two hours following i.p. administration of increasing concentrations of EJHE or EJHE-WR (1-100 µg/ml) suppressed significantly the production of TGF-β1 (*, **P<0.0001).
Fig 4.9: Effect of EJHE and EJHE-WR on TGF-β1 production in the blood of mice. Two hours following i.p. administration of increasing concentrations (1-100 µg/ml) of EJHE or EJHE-WR inhibited significantly the production of TGF-β1 (*, **P<0.0001).
62
4.2 Kinetics of IFN-γ, IL-17 and TGF-β1 in specific tissues induced by EJHE and EJHE-WR: 4.2.1 Kinetics of IFN-γ, IL-17 and TGF-β1 in the blood:
EJHE at 10 µg/ml did not induce any significant change in blood IFN-γ levels 24
hours following i.p. administration (P>0.05). Contrariwise, EJHE-WR at 10 µg/ml
displayed a trend towards an increase in blood IFN-γ levels by 41-98% over 6-48 hours
post i.p. administration (P>0.05). LPS on the other hand, increased significantly IFN-γ
production in blood 6 hours following i.p. injection by 311%. This maximum increase
subsided below basal levels after 24 hours and maintained for as long as 48 hours post
administration (P<0.0001) (Fig 4.10).
EJHE at 10 µg/ml displayed a trend towards a decrease in blood IL-17 production
24-48 hours post i.p. administration by 25-52% (P>0.05). In contrast, EJHE-WR at 10
µg/ml displayed a trend towards an increase in blood IL-17 production 6 hours following
i.p. administration by 39% (P>0.05) while LPS inhibited significantly blood IL-17
production over 24 hours after administration but then ascended back to basal levels
(P=0.0344) (Fig 4.11).
After 2 hours of administration, 10 µg/ml of EJHE demonstrated a trend towards
a transient increase in blood TGF-β1 production as opposed to a trend towards a decrease
in TGF-β1 production by EJHE-WR (P>0.05). Single i.p. administration of LPS
significantly suppressed blood TGF-β1 production over 24 hours post administration but
then went up to slightly above the basal TGF-β1 levels (P<0.0001) (Fig 4.12).
63
Fig 4.10: Kinetics of IFN-γ production in the blood of mice induced by EJHE and EJHE-WR. EJHE at 10 µg/ml did not induce any changes in IFN-γ levels 2-48 hours post i.p. administration (P>0.05). EJHE-WR at 10 µg/ml displayed a trend towards an increase in IFN-γ levels 6-48 hours (P>0.05). LPS increased significantly IFN-γ production 6 hours post i.p. injection. This maximum increase subsided to lower than the basal levels after 24 hours and maintained for as long as 48 hours post administration (*P<0.0001).
Fig 4.11: Kinetics of IL-17 production in the blood of mice induced by EJHE and EJHE-WR. EJHE at 10 µg/ml displayed a trend towards a decrease in IL-17 production for as long as 48 hours post i.p. administration (P>0.05). In contrast, EJHE-WR at 10 µg/ml displayed a trend towards an increase in IL-17 production 6 hours post i.p. administration (P>0.05), however, LPS inhibited significantly IL-17 production all over 24 hours post administration, but then ascended back to basal levels (P=0.0344).
64
Fig 4.12: Kinetics of TGF-β1 production in the blood of mice induced by EJHE and EJHE-WR. 10 µg/ml of EJHE demonstrated a trend towards a transient increase in TGF-β1 levels as opposed to a trend towards a decrease in TGF-β1 production by 10 µg/ml of EJHE-WR post 2 hours of administration (P>0.05). LPS significantly suppressed TGF-β1 production allover 24 hours post i.p. administration but then went up again to basal levels (*P<0.0001).
4.2.2 Kinetics of IFN-γ, IL-17 and TGF-β1 in the spleen:
A single i.p. injection of 10 µg/ml EJHE or EJHE-WR into mice modulated
significantly spleen cytokines level. Each of EJHE and EJHE-WR at 10 µg/ml increased
significantly IFN-γ production in the spleen over 6-48 hours post i.p. administration
(P=0.0239 and 0.0091, respectively). The increase was highest 24 hours post
administration by 351% for EJHE-WR followed by a decline at 48 hours but yet to higher
levels than basal levels by 196%. EJHE at 10 µg/ml displayed a transient suppression in
spleen IFN-γ levels 2 hours after administration but then kept increasing for as long as 48
hours by 197% (Fig. 4.13). On the other hand, i.p. administration of LPS increased
significantly IFN-γ production over the first 24 hours of administration (P<0.0001). A
prominent increase by 738% was discerned 6 hours post i.p. injection, however, this
65
increase declined to even below basal IFN-γ levels over the period 24-48 hours (Fig.
4.13).
In the spleen and following single i.p. injection, EJHE (10 μg/ml) exhibited a
trend toward suppression of IL-17 level at 24 hours post administration (P>0.05). This
EJHE-induced suppression was not observed at 48 hours post administration. However,
IL-17 levels did not change after i.p. administration of EJHE-WR or LPS (P>0.05) (Fig.
4.14).
EJHE and EJHE-WR at 10 µg/ml exhibited a different pattern in spleen TGF-β1
modulation. Single i.p. injection of 10 µg/ml EJHE or EJHE-WR suppressed significantly
spleen TGF-β1 production for as long as 48 hours post administration (P=0.0004 and
0.0017, respectively). The suppression was highest 24 hours post administration and kept
over 24-48 hours by 69-57% and 63-54% for EJHE and EJHE-WR, respectively, and to
levels yet lower than LPS-induced levels (P=0.0156) (Fig. 4.15).
Fig 4.13: Kinetics of IFN-γ production in the spleen of mice induced by EJHE and EJHE-WR. EJHE or EJHE-WR at 10 µg/ml increased significantly IFN-γ production over 6-48 hours post i.p. administration (P=0.0239 and **P=0.0091, respectively). LPS increased significantly IFN-γ production over the first 24 hours of administration (*P<0.0001).
66
Fig 4.14: Kinetics of IL-17 production in the spleen of mice induced by EJHE and EJHE-WR. EJHE (10 µg/ml) exhibited a trend toward suppression at 24 hours post administration. This transient decline went up again to levels comparable to basal levels after 48 hours (P>0.05). However, neither EJHE-WR nor LPS led to significant change in IL-17 production (P>0.05).
Fig 4.15: Kinetics of TGF-β1 production in the spleen of mice induced by EJHE and EJHE-WR. EJHE or EJHE-WR at 10 µg/ml suppressed significantly TGF-β1production for as long as 48 hours post administration (**P=0.0004 and 0.0017, respectively). Similarly, i.p. injection of LPS suppressed significantly TGF-β1production for as long as 48 hours (*P=0.0156).
67
4.2.3 Kinetics of IFN-γ, IL-17 and TGF-β1 in the lungs:
Single i.p. injection of EJHE or EJHE-WR at 10 µg/ml modulated the production
of cytokine levels in mice lung tissues. EJHE-WR at 10 µg/ml increased significantly
IFN-γ production in the lungs all over 24 hours post i.p. administration (P=0.0298). The
increase was highest 6 hours post administration by 87% followed by a decline at 48
hours to even below the basal levels. Similarly, EJHE demonstrated a trend towards an
increase in IFN-γ production for as long as 48 hours after administration with a maximum
increase by 101% noticed 24 hours after i.p. injection (P>0.05). Single i.p. administration
of LPS increased significantly IFN-γ production in the lungs 2 hours after administration
by 101% followed by decline over the next 6-48 hours after administration to lower
levels than the basal IFN-γ levels (P=0.0014) (Fig. 4.16).
Neither EJHE nor EJHE-WR modulated IL-17 or TGF-β levels in the lungs.
Similarly LPS did not induce any significant change in IL-17 levels in the lungs (Fig.
4.17), but demonstrated a trend towards a decrease of TGF-β levels over 2 to 24 hours
post i.p. injection. This LPS-induced suppression of TGF-β was not observed at 48 hours
post administration (Fig. 4.18).
68
Fig 4.16: Kinetics of IFN-γ production in the lungs of mice induced by EJHE and EJHE-WR. EJHE-WR at 10 µg/ml increased significantly IFN-γ production over 24 hours post i.p. administration (P=0.0298). Similarly, EJHE demonstrated a trend towards an increase in IFN-γ production for as long as 48 hours (P>0.05). I.p. administration of LPS increased significantly IFN-γ production 2 hours after administration followed by decline over the next 6-48 hours (*P=0.0014).
Fig 4.17: Kinetic of IL-17 production in the lungs of mice induced by EJHE and EJHE-WR. Neither EJHE nor EJHE-WR modulated IL-17 levels in the lungs.
69
Fig 4.18: Kinetic of TGF-β1 production in the lungs of mice induced by EJHE and EJHE-WR. Neither EJHE nor EJHE-WR modulated TGF-β levels in the lungs. Administration of LPS suppressed TGF-β1 levels 2-24 hours post injection (P>0.05).
4.3 MCA-induced tumors:
The tumors were noted locally at the sites of MCA injection and grow with a
reproducible growth pattern (Fig. 4.19). The tumor incidence reached 26% (5 of 19 mice)
within 21 weeks and 36% (5 of 14 mice) within 15 weeks in the groups inoculated with
0.5 mg and 1 mg MCA, respectively (Fig. 4.20). Notably, the surfaces of tumors were
often ulcerated and areas of hemorrhage surrounding the tumors were sometimes noticed.
The tumors were generally nodular and irregular in shape. The tumors as whole were
firm or fleshy with soft, fragile regions and were uniformly reddish-white or pale grey-
white.
70
Fig 4.19: Fibrosarcoma development in BALB/c mice inoculated with MCA.
Fig 4.20: Cumulative tumor incidence after s.c inoculation of two different MCA doses.
71
4.4 Modulation of IFN-γ, IL-17 and TGF-β1 in the spleen and within the tumor microenvironment of Meth-A-bearing mice following EJHE and EJHE-WR administration: 4.4.1 Modulation of IFN-γ, IL-17 and TGF-β1 in the spleen of Meth-A-bearing mice:
EJHE at 10 µg/ml administered i.p. once every 24 hours for three consecutive
days increased significantly spleen IFN-γ production (P=0.011). This increase, however,
was not seen with triple i.p. injection of EJHE-WR or single i.p. injection of EJHE and
EJHE-WR at 10 µg/ml (P>0.05) (Fig. 4.21). Neither EJHE nor EJHE-WR modulated IL-
17 or TGF-β1 levels in the spleen after either single or triple i.p. injection (P>0.05) (Fig.
4.22 & 4.23).
Fig 4.21: Effect of EJHE and EJHE-WR on IFN-γ production in the spleen of Meth-A-bearing mice. Administration of 10 µg/ml of EJHE once every 24 hours for three consecutive days increased significantly spleen IFN-γ production (*P=0.011).
72
Fig 4.22: Effect of EJHE and EJHE-WR on IL-17 production in the spleen of Meth-A-bearing mice. Neither EJHE nor EJHE-WR modulated IL-17 levels in the spleen following either single or triple i.p. injection (P>0.05).
Fig 4.23: Effect of EJHE and EJHE-WR on TGF-β1 production in the spleen of Meth-A-bearing mice. Neither EJHE nor EJHE-WR modulated TGF-β1levels in the spleen following either single or triple i.p. injection (P>0.05).
73
4.4.2 Modulation of IFN-γ, IL-17 and TGF-β1 within the tumor microenvironment of Meth-A-bearing mice:
EJHE-WR at 10 µg/ml administered i.p. once every 24 hours for three
consecutive days increased significantly IFN-γ and IL-17 (P=0.027) as well as TGF-β1
(P=0.004) production within the tumor microenvironment of Meth-A-bearing mice.
Although a triple consecutive i.p. injections of EJHE showed trend toward increase in the
production of the three cytokines within tumor microenvironment but without reaching
statistical significance. A single i.p. injection of EJHE and EJHE-WR did not lead to any
significant changes in IFN-γ, IL-17 and TGF-β1 levels in the tumor microenvironment of
Meth-A-bearing mice (P>0.05) (Fig. 4.24, 4.25 & 4.26).
Fig 4.24: Effect of EJHE and EJHE-WR on IFN-γ production in the tumor microenvironment of Meth-A-bearing mice. I.p. injection of EJHE-WR at 10 µg/ml once every 24 hours for three consecutive days increased significantly IFN-γ production (**P=0.027).
74
Fig 4.25: Effect of EJHE and EJHE-WR on IL-17 production in the tumor microenvironment of Meth-A-bearing mice. I.p. injection of EJHE-WR at 10 µg/ml once every 24 hours for three consecutive days increased significantly IL-17 production (**P=0.027).
Fig 4.26: Effect of EJHE and EJHE-WR on TGF-β1 production in the tumor microenvironment of Meth-A-bearing mice. I.p. injection of EJHE-WR at 10 µg/ml once every 24 hours for three consecutive days increased significantly TGF-β1 production (**P=0.004).
75
4.5 Survival of Meth-A tumor-bearing mice after treatment with EJHE and EJHE-WR:
Observing from the overall mortality patterns of the Meth-A-bearing mice, it was
found that i.p. administration of EJHE-WR could prolong the life-span of Meth-A-
bearing mice. However, the mortality pattern of the Meth-A-bearing mice were found to
be overlapped between the EJHE treated group and the control group indicating that
EJHE did not affect the long-term survival of Meth-A-bearing mice (Fig. 4.27).
Fig 4.27: Effect of i.p. administration of EJHE and EJHE-WR on mice survival after i.p. inoculation with Meth-A cells. Prolongation of survival following administration of EJHE-WR-treated Meth-A-bearing mice was observed.
76
Chapter 5
Discussion and Conclusions
77
Chapter 5
Discussion and Conclusions
Eriobotrya japonica is a renowned plant for its distinctive constituents that were
identified following treatment with different extraction methods utilizing various arrays
of solvents as ethyl acetate, acetone-water, or alcoholic solvents (Ito et al., 2000, 2002;
Banno et al., 2005, Al-Hanbali et al., 2009). During this study, EJ leaves were first
extracted with water and then fractioned further with n-butanol. The latter extracts were
termed EJHE and EJHE-WR, respectively. Lately, it has been shown that water-extract of
EJ, termed EJHE, provides an affluent source of polysaccharides and polar phenolic
compounds like procyanidins and flavonoid glycosides and likewise other molecules that
may induce and modulate cytokines production providing distinguishable
immunomodulatory (Matalka et al., 2007) and putative anticancer activity (Ito et al.,
2000). The fractionation of EJHE with n-butanol (EJHE-WR) was performed to
concentrate polysaccharides, high molecular weight oligomeric procyanidins and related
polyphenols and to exclude low molecular weight compounds that will be recovered by
n-butanol (Ito et al., 2000).
Accurate measurement of cytokine levels in tissues and their relative changes may
be useful indicators of the effectiveness of local immune responses (Matalka et al., 2005).
A thorough understanding of the cytokine networks that regulate the development of
local immune responses in their local microenvironments (or intra-organ environment) is
a valuable tool in settling the benefaction of immunomodulators as EJHE and EJHE-WR.
Thereby, this study was undertaken to investigate the specific immunomodulative
potential of EJHE and EJHE-WR in tissues of normal mice and Meth-A fibrosarcoma-
78
bearing mice including the tumor microenvironment. In this experimental system, the
method utilized for extraction of cytokines from tissues/organs enabled the detection of
EJHE-, EJHE-WR-, and LPS-induced- extracellular, intracellular, and membrane-bound
cytokines from immune cells and non-immune cells like endothelial cells and fibroblasts
(Matalka et al., 2005). Lymphoid organs such as spleen, and non-lymphoid organ such as
lungs that includes structural cells such as epithelial cells, endothelial cells and
fibroblasts have been selected in the study. The latter include itinerant immune cells that
serve a critical function in the immune defenses such as monocytes/macrophages, DCs
and lymphocytes that are circulating in the blood as well (Rescigno et al., 1999; Mebius
& Kraal, 2005; Suzuki et al., 2008).
Initially, three different concentrations of EJHE and EJHE-WR were chosen (1,
10, and 100 μg/ml) and their immunomodulative activity was sought in mice organs,
including the spleen and lungs following two hours of administration. Collectively, this
experiment demonstrated that the in vivo favorable immunomodulative activities of EJHE
and EJHE-WR in mice tissues as increase of IFN-γ and inhibition of TGF-β1 levels were
best achieved by 10 μg/ml of EJHE and EJHE-WR. Subsequently, the selected
concentrations were further utilized to investigate the direct immunomodulative tissue-
specific effects of EJHE, EJHE-WR, in comparison with LPS on IFN-γ, IL-17, and TGF-
β1 kinetics alterations over 48 hours. The current study demonstrates that EJHE and
EJHE-WR at 10 μg/ml modulate and increase in vivo cytokines production in tissue- and
time-specific manner. The 2 to 24 hour time interval appeared to be pivotal for
occurrence of cytokines modulatory response by EJHE-, EJHE-WR-, and LPS in mice
tissues with respect to cytokine and tissue types.
79
EJHE and EJHE-WR at 10 μg/ml increased IFN-γ production from the spleen and
lung tissues over 6-48 hours and in blood over 6-48 hours upon EJHE-WR
administration. LPS also increased IFN-γ production from the spleen and blood at 6
hours, and at 2 hours in the lungs. Furthermore, the results illustrated that in contrast to
the discerned increase of IFN-γ in the spleen, TGF-β1 was down-regulated in response to
EJHE and EJHE-WR administration for as long as 48 hours. Likewise, blood, spleen, and
lungs TGF-β1 levels were down-regulated upon LPS administration over 2-24, 2-48, and
2-24 hours, respectively.
Earlier studies in vitro showed that low concentrations of EJHE induced the
production of proinflammatory cytokines (IL-12, IFN-γ, and TNF-α) whilst higher
concentrations reduced the latter induction along with an increase in anti-inflammatory
cytokine; IL-10 (Matalka et al., 2007). Similarly, unspecified water-extract of EJ leaves
utilized at high concentrations inhibited LPS- induced proinflammatory cytokines; IL-6,
IL-8, TNF-α; and IL-1β, IL-8, TNF-α from human mast cells and human lung epithelial
cells, respectively, likely by inhibiting Nuclear factor kappa B (NF-κB) activation (Lee et
al., 2008a; Kim & Shin, 2009). The latter coincides with the inhibition of
proinflammatory cytokines and the increase in anti-inflammatory cytokine; IL-10,
observed by Matalka et al. (2007) at higher EJHE concentrations. Correspondingly, the
data in the present study demonstrated a similar action in vivo as higher concentration
(100 μg/ml) inhibited IFN-γ production in the spleen and lung tissues probably in a
likewise manner, i.e. inhibition to NFκB, while on the contrary lower concentration (10
μg/ml) increased IFN-γ production. Howbeit, NF-κB activity is essential for the
80
development of Th1 type responses and it is involved in the production of IFN-γ (Kojima
et al., 1999).
Since EJHE and EJHE-WR induced and modulated cytokines in several organs,
their cytokine modulatory mechanism might be postulated by the compounds they
contain. These extracts contain mainly polysaccharides and polar phenolic compounds
like procyanidins and flavonoid glycosides. Such compounds stimulate APCs; DCs and
macrophages, via the expressed TLRs (Reis e Sousa, 2004). The latter cells induce IL-12
levels through the activation of NF-κB (Matalka et al., 2007). Consequently, APCs-
derived IL-12 may preferentially stimulate T and B cells in vivo (Pulendran et al., 1999;
Schulz et al., 2000; Lund, 2008; Theiner et al., 2008) besides its action on NK cells that
in turn can influence the development of Th1 response and thus supports IFN-γ
production (Lieberman & Hunter, 2002; Trinchieri, 2003). The recognition of the
polysaccharides with procyanidins and flavonoid glycosides by TLRs-expressed on DCs,
macrophages, as well as B and T cell subtypes (Kabelitz et al., 2007; Lund, 2008)
induces subsequent engagement of signaling pathways that explain the timing of cytokine
secretion pattern. This is apparent in IFN-γ highest increase at 2 or 6 hours upon LPS
administration versus 24-48 hours after EJHE and EJHE-WR administration. Ostensibly,
this differential induction time proposes that molecules within EJHE and EJHE-WR
induce cytokine release in more complex pathways involving several intracellular
signaling in comparison with LPS. Still, other non-immune cellular sources could be
implicated in the cytokines responses depicted herein. Apart from immune cells, TLRs
are expressed on tissues such as the lung epithelial cells (Zaas & Schwartz, 2005; Suzuki
et al., 2008). Type 2 alveolar epithelial cells, for instance, have been shown to express
81
mRNA for TLR2 and TLR4 and to have functional response with the release of cytokines
in response to LPS (Armstrong et al., 2004).
Moreover, it cannot be ruled out that the changes observed in the present study
may occur when EJHE-, EJHE-WR-, and LPS-induced IFN-γ bind to T cell populations
in a paracrine fashion probably via employment of the JAK-STAT signaling pathway
(Kalvakolanu, 2003; Chen & Liu, 2009). Generally, within a period of hours, upon the
accumulation of activated STATs in the nucleus the signal decays and the STATs are re-
exported back to the cytoplasm for a new cycle of signaling (Schindler et al., 2007).
IFN-γ inhibits the TGF-β-induced phosphorylation of Smad3, the subsequent binding of
Smad3 with Smad4, and the accumulation of Smad3 in the nucleus. Furthermore, upon
IFN-γ signalling through Jak1 and STAT1 proteins and activation of NFκB, IFN-γ
induces Smad7 expression which prevents the interaction of Smad3 with the TGF-β
receptor leading to inhibition of TGF-β signaling and so reduction in its levels (Ulloa et
al., 1999; Derynck & Zhang, 2003). Probably, in this way, the crosstalk between the
IFN-γ and TGF-β signals lead to the changes depicted herein.
Furthermore, Treg cells expressing Foxp3 are major source of TGF-β1 which
regulates their generation as well (Bettelli et al., 2006; Li et al., 2007a). Mice with a T
cell-specific deletion of the Tgfb1 gene showed enhanced Th1 cells proliferation,
activation, and differentiation, however, TGF-β1 produced by Foxp3+ Treg cells was
required to inhibit Th1 cells differentiation (Li et al., 2007a). Thereby, TGF-β1
suppression upon EJHE, EJHE-WR, and LPS administration in the spleen supported Th1
development and thus the enhanced IFN-γ production. Additionally, taking into account
the delicate balance in the cytokines' network, the information conveyed by an individual
82
cytokine depends on the pattern of regulators to which a cell is exposed, and not on one
single cytokine (Correa et al., 2007). Recent studies have established that TGF-β1 not
only regulates the generation of Foxp3+ Treg cells but also acts as an essential regulator
of Th17 cell differentiation from naїve CD4+ T cells together with IL-6 (Bettelli et al.,
2006; Mangan et al., 2006; Veldhoen et al., 2006). In the current study, the disappearance
of a crucial cytokine for Th17 differentiation as TGF-β1 in the spleen upon EJHE, EJHE-
WR, and LPS administration could foretell the absence of absolute Th17 cells and thus
predict no change in IL-17 levels noticed herein.
Commonly, the immune system may be either compromised or suppressed
overwhelmingly in cancer patients. In view of the antitumor effects of EJHE and EJHE-
WR on the immune system and, more precisely, within the tumor microenvironment it
would be of considerable interest to investigate the immunorestorative activities in Meth-
A tumor-bearing mice. In the present study, Meth-A cells were injected into the s.c. layer
which consists of loose connective tissue permitting the injected cells to diffuse easily
(Qin et al., 2002). The local microenvironment of Meth-A cells administered s.c. is
complex since, besides fibroblasts, the microenvironment can also comprise numerous
types of resident or infiltrating cells, like endothelial cells, muscular cells as well as
granulocytes, macrophages, and lymphocytes (Masuda et al., 1997; Qin et al., 2002). The
results from the present study demonstrated that i.p. administration of EJHE at 10 µg/ml
24 hours apart increased significantly spleen IFN-γ production. Additionally, triple i.p.
injection of EJHE-WR at 10 µg/ml increased significantly IFN-γ, and unexpectedly TGF-
β1 and IL-17 production within the tumor microenvironment of Meth-A-bearing mice.
83
The antitumor cytokine; IFN-γ increased in the tumor microenvironment and
spleen of Meth-A-bearing mice by triple i.p. injection of EJHE-WR and EJHE,
respectively. This is probably connected with the extracts constituents enhancing effect
allowing the intratumoral penetration of antitumor effectors and polarizing towards Th1
response. The latter is mostly achieved by macrophages and DCs produced IL-12 which
in turn can support IFN-γ production noticed within the microenvironment (Takeuchi et
al., 2009). Many studies displayed critical functions for IFN-γ dependent tumor rejection
of transplanted tumors and MCA-mediated tumorigenesis in mice (Shankaran et al.,
2001; Dunn et al., 2005). Immune responses against Meth-A tumor cells appeared to be
closely associated with augmented IFN-γ production and CTL activity against tumor
which can injure tumor-feeding vessels in tumor tissue (Masuda et al., 1997;
Blankenstein & Qin, 2003).
Indeed, to our surprise, this favorable increase in IFN-γ by EJHE-WR within the
tumor microenvironment was accompanied with an increase in TGF-β1 and IL-17. The
presence of TGF-β1 along with an inflammatory cytokine e.g. TNF-α (Matalka et al.,
2007) may further supports Th17 differentiation and thus the increase in IL-17 production
was observed (Nam et al., 2008). Actually, cells within the tumor microenvironment
respond in a microenvironmental context where different stimuli can impact their
behavior. Therefore, the latter observations are worthy of further investigation of the
various cellular populations that were recruited to the tumor microenvironment of Meth-
A-bearing mice upon repeated i.p. administration of EJHE and EJHE-WR and thus led to
these present changes. However, these changes were not provoked by single injection of
84
either EJHE or EJHE-WR in the spleen or in the tumor microenvironment of Meth-A-
bearing mice.
Furthermore, the present results demonstrated prolonged survival of mice
inoculated i.p. with Meth-A cells throughout long period i.p. administration of EJHE-WR
in comparison with Meth-A-bearing mice treated with EJHE (10 µg/ml) and PBS.
Previous work showed that a fractionated water-extract of EJ leaves with butanol
recovered high molecular weight procyanidin oligomer which displayed the highest
cytotoxic activity in vitro against two human oral tumor cell lines (Ito et al., 2000).
Particularly, fractionation of EJHE with n-butanol to obtain EJHE-WR provided an
abundant source of high molecular weight procyanidin oligomer together with
polysaccharides and polar phenolics compounds that possess low anti-inflammatory
activity. Meanwhile, these compounds exert immunostimulating potentials (Matalka et
al., 2007) and can also be involved in antitumor and cytotoxic activities (Ito et al., 2000).
However, further study will be needed to clarify the antitumor activity involved detail
mechanisms of these compounds in Meth-A bearing mice.
In conclusion, EJHE and EJHE-WR cytokine modulatory potentials have been
characterized in tissues from normal and tumor-bearing mice. It is clear that
multidimensional factors partake in depicting the responses noticed herein. In that
respect, (i) the nature of the immunomodulating agent that elicits the immune response in
each occasion, (ii) the type and timing of cytokine secretion either by the stimulating
APCs, T and B lymphocytes through TLRs mediated pathways, or other onlooker cell
populations, and (iii) the impact of tissue/organ/tumor being studied with its burden and
phenotype of residing cell populations that also cannot be excluded.
85
Immunomodulators which can be used for long period without or with minimum
side effects are substantial in the cancer therapy. Ordinarily, targeted cancer mono-
therapy can end up with bypass mechanisms which in turn forced the employment of
either combination therapy or agents that interfere with multiple cell-signaling pathways
as a current paradigm for most treatments. Therefore, as future directions it would be of
particular interest to inject known anti-cancer compounds with EJHE and EJHE-WR to
see if their antitumor effect would be superior. Further understanding of the effects of
immunomodulators’ constituents on the cellular components of the tissue
microenvironment is needed. This will provide more useful clues for improving the
strategies for cancer treatment and thus can open up a new frontier for future studies.
86
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