the role of complement in immunity to nippostrongylus
TRANSCRIPT
The Role of Complement in Immunity to Nippostrongylus brasiliensis
A thesis submitted for the degree of
DOCTOR OF PHILOSOPHY
as a portfolio of publications
by
Paul Robert Giacomin
Discipline of Microbiology and Immunology School of Molecular and Biomedical Science
The University of Adelaide Australia
September, 2007
TABLE OF CONTENTS
ABSTRACT……………………………………………………………………………i
DECLARATION……………………………………………………………………...iii
ACKNOWLEDGEMENT OF ANY HELP………………………………………….. iv
STATEMENT OF AUTHORSHIP-CHAPTER 2……………………………………..v
STATEMENT OF AUTHORSHIP-CHAPTER 3…………………………………....vii
STATEMENT OF AUTHORSHIP-CHAPTER 4……………………………………..x
ACKNOWLEDGEMENTS………………………………………………………….xvi
PUBLICATIONS…………………………………………………………………..xviii
COMMONLY-USED ABBREVIATIONS………………………………………….xix
CHAPTER 1: INTRODUCTION AND REVIEW OF THE LITERATURE
1.1 HEALTH AND ECONOMIC CONSEQUENCES OF PARASITIC HELMINTH INFECTIONS ..........................................................................................1 1.2 IMMUNE RESPONSES TO HELMINTH INFECTION .......................................2
1.2.1 Immune recognition and antigen processing ...................................................3 1.2.2 Cytokine responses during helminth infection .................................................4 1.2.3 Immunological basis of gastrointestinal helminth expulsion ...........................6
1.3 LEUKOCYTE-MEDIATED KILLING OF HELMINTHS....................................9 1.3.1 Neutrophils........................................................................................................9 1.3.2 Macrophages...................................................................................................10 1.3.3 Eosinophils......................................................................................................10
1.3.3.1 IL-5 and eosinophils ................................................................................11 1.3.3.2 Eosinophil recruitment.............................................................................12 1.3.3.3 Eosinophil activation, secretion and degranulation .................................13 1.3.3.4 Role of eosinophils in disease..................................................................14 1.3.3.5 Role of eosinophils is killing helminths ..................................................15 1.3.3.6 Other roles for eosinophils during helminth infections ...........................17
1.4 THE COMPLEMENT SYSTEM ..........................................................................19 1.4.1 Function of the complement system ...............................................................19 1.4.2 Pathways to complement activation ...............................................................20 1.4.3 Complement-dependent immunity to pathogens ............................................21
1.5 ROLE OF COMPLEMENT IN IMMUNITY TO HELMINTHS.........................23 1.5.1 Complement activation by helminths .............................................................25 1.5.2 Complement-dependent leukocyte-mediated killing of helminths.................26
1.5.2.1 Recruitment of effector leukocytes..........................................................26 1.5.2.2 Adherence and activation of effector leukocytes.....................................27
1.5.3 Evasion of complement activation and leukocyte adherence .........................27 1.6 NIPPOSTRONGYLUS BRASILIENSIS AS A MODEL FOR STUDYING IMMUNITY TO HELMINTHS.............................................................29
1.6.1 Parasite life cycle ............................................................................................29 1.6.2 Immune responses to N. brasiliensis ..............................................................30
1.6.2.1 Cellular inflammatory responses .............................................................30 1.6.2.2 Cytokine responses ..................................................................................32
1.6.2.3 Role for eosinophils .................................................................................32
1.7 INTRODUCTION TO THIS STUDY...................................................................34 CHAPTER TWO: Quantitation of complement and leukocyte binding to a parasitic helminth species....…………………………………………… 36 LINKAGE TO CHAPTER TWO AND ARTICLE……………………………… 37 CHAPTER THREE: Loss of complement activation and leukocyte adherence as Nippostrongylus brasiliensis develops within the murine host ………. 39 LINKAGE TO CHAPTER THREE AND ARTICLE............................................40 CHAPTER FOUR: The role of complement in innate, adaptive and eosinophil-dependent immunity to the nematode Nippostrongylus brasiliensis.…….43 LINKAGE TO CHAPTER FOUR AND ARTICLE…………………………….. 44 CHAPTER FIVE: DISCUSSION AND CONCLUSION 5.1 GENERAL DISCUSSION ....................................................................................46
5.1.1 Summary of main findings .............................................................................46 5.1.2 Complement and eosinophil-dependent immunity to helminths ....................47
5.1.2.1 Eosinophil-dependent resistance to helminths.........................................47 5.1.2.2 Role of complement in vivo and in vitro..................................................49 5.1.2.3 Complement-dependent eosinophil recruitment to parasite-infected skin .......................................................................................51 5.1.2.4 Complement-independent eosinophil recruitment...................................51 5.1.2.5 Eosinophil versus neutrophil recruitment ................................................53 5.1.2.6 Complement-independent leukocyte adherence to helminths .................54 5.1.2.7 Eosinophil degranulation .........................................................................55 5.1.2.8 Larval aggregation ...................................................................................57
5.1.3 Evasion of complement activation by helminths............................................58 5.1.4 Pulmonary cellular responses following helminth infection ..........................59
5.1.4.1 Restricted early cellular inflammation in the lungs .................................59 5.1.4.2 Delayed cellular inflammation in the lungs.............................................60
5.1.5 Secondary immune response to helminth infection........................................61 5.1.6 Future directions for studies using complement-deficient/IL-5 Tg mice .......62 5.1.7 Issues for design of anthelmintic vaccines .....................................................62
5.2 CONCLUSION......................................................................................................63 REFERENCES…………………………………………………………………….....65
iABSTRACT
Approximately two billion people are infected with helminths worldwide. In order to develop
a vaccine against these pathogens, more needs to be known about the immune response to
helminths. Eosinophils are important for resistance to some helminth species and their
recruitment to infected tissues, attachment to parasites and degranulation may all be critical
processes for immunity. Complement may contribute to these processes via generation of
chemotactic factors (C3a and C5a) or opsonisation of the parasite with C3b/iC3b. The
importance of complement during helminth infection is unclear, though complement does
promote leukocyte-mediated killing of several helminth species in vitro. The aim of the
present study was to investigate the role of complement in immunity of mice to
Nippostrongylus brasiliensis, with a focus on whether complement facilitates eosinophil-
dependent resistance to this parasite. A new fluorescence-based method for quantifying in
vitro complement deposition and leukocyte adherence on N. brasiliensis was developed. C3
from human serum was deposited on infective-stage L3 via the classical or lectin complement
pathways. In contrast, the alternative complement pathway mediated binding of mouse C3
and eosinophil-rich mouse peritoneal leukocytes to L3. Interestingly, the ability of
complement and leukocytes to bind to the parasite changed as it matured. Larvae recovered
from the skin 30 min post-injection (p.i.) were coated with C3, however those harvested 150
min p.i. exhibited reduced C3 binding capacity. Binding of C3 and eosinophils to larvae
recovered from the lungs 24-48 h p.i. (L4) was also diminished compared to that seen on L3.
Adult intestinal worms bound C3 and leukocytes only when treated ex vivo with serum and
cells. Mice lacking in classical (C1q-deficient), alternative (factor B-deficient) or all
complement pathways (C3-deficient) were then employed to determine if complement was
important for resistance of mice to N. brasiliensis. IL-5 Tg mice deficient in individual
complement genes were generated to assess whether complement contributed to eosinophil-
dependent resistance to the parasite. Factor B-deficient mice exhibited impaired C3
deposition on larvae, eosinophil recruitment, eosinophil degranulation and larval aggregation
iiin the skin 30 min p.i. Eosinophil recruitment was similarly abolished by treatment of mice
with the C5aR inhibitor PMX53. However at 150 min p.i., larval aggregation, eosinophil and
neutrophil recruitment, leukocyte adherence and eosinophil degranulation were largely
complement-independent. Ablation of factor B or C3 caused minor but significant increases
in lung-larval burden during primary, but not in secondary, infections. Critically, a lack of C3
or factor B in IL-5 Tg mice failed to greatly impair the strong innate anti-parasite resistance
typical of these animals, suggesting that eosinophils can provide immunity to N. brasiliensis
infection in the absence of complement. This was unexpected, given the evidence from this
and previous studies which suggested that in vitro, complement is important for promoting
eosinophil-dependent killing of N. brasiliensis and other helminth species. The mechanism(s)
by which eosinophils kill N. brasiliensis remain unknown, but may involve the coordination
of the complement system with complement-independent factors that act in the early stages of
infection. Critically, the influence of complement is limited, because soon after entry into the
host, the parasite develops the ability to resist complement activation.
ivACKNOWLEDGEMENT OF ANY HELP
I acknowledge the help of:
All co-authors named on each of the published journal articles comprised in this thesis, for
evaluating manuscript drafts and suggesting changes during the revision process. In
particular, Dr. Lindsay Dent, who acted as co-author for all manuscripts, co-wrote and revised
drafts with myself before submission and critically read sections of my thesis.
Dr. Hui Wang, who contributed to the early development of techniques for measuring C3
deposition on helminths.
Ms. Michelle Knott, for technical assistance with large-scale animal experiments.
NOTE: Statements of authorship appear in the print copy of the thesis held in
the University of Adelaide Library.
xvi ACKNOWLEDGEMENTS
Firstly, I would like to thank my principal supervisor Dr. Lindsay Dent for his dedicated
support, guidance and encouragement throughout my Ph.D. studies. Your mentorship has
made my experience as a Ph.D. student very enjoyable and rewarding. To my co-supervisor,
Professor David Gordon, I also extend a thankyou for sharing your ideas and supporting my
goals.
I acknowledge the help and friendship of all Honours and Ph.D. students who have been part
of the Dent laboratory throughout my time here. In particular, I thank Michelle Knott and
Damon Tumes who have provided constant support and made the lab an enjoyable place to be
around. Also, I thank Hui Wang for her help with my project and technical assistance when I
first joined the lab.
I thank the student and staff members of the Discipline of Microbiology and Immunology and
the School of Molecular and Biomedical Science for making it such a good place to work. In
particular, I thank Nick Eyre, Francesca Bell, Wendy Parker, Georget Reaiche, The Friday
beer crew and the Wednesday soccer crew for making my time at Uni entertaining and
memorable.
To our national and international collaborators Marina Botto, Alex Loukas, Steve Taylor and
Mohamed Daha, I thank you for your assistance with my project.
Whilst conducting these studies I was supported by a University of Adelaide scholarship. I
also thank the School of Molecular and Biomedical Science for their support financially and
for other resources relating to my project.
xviiI thank all of my non-Uni friends for many years of great friendship and support.
I wholeheartedly thank my parents, for whom I am eternally grateful for supporting me with
whatever decisions I have made over the years, allowing me to achieve my goals. I also thank
the rest of my family (and extended family), especially my sister Amanda and all of my
grandparents.
Lastly, I thank my wife Michelle, who has been a loving and inspiring partner since I began
my Ph.D. studies and who I look forward to a spending long life with.
xviiiPUBLICATIONS
Within thesis:
1. Giacomin PR, Wang H, Gordon DL and Dent LA (2004). Quantitation of
complement and leukocyte binding to a parasitic helminth species. Journal of Immunological Methods 289 (1-2): 201-210
2. Giacomin PR, Wang H, Gordon DL, Botto M and Dent LA (2005). Loss of
complement activation and leukocyte adherence as Nippostrongylus brasiliensis develops within the murine host. Infection and Immunity 73 (11): 7442-7449
3. Giacomin PR, Gordon DL, Botto M, Daha MR, Sanderson SD, Taylor SM and
Dent LA (2007). Molecular Immunology 45 (2): 446-455
Other publication arising from Ph.D. studies:
1. Knott ML, Matthaei KI, Giacomin PR, Wang H, Foster PS, Dent LA (2007). Impaired resistance in early secondary Nippostrongylus brasiliensis infections in mice with defective eosinophilopoeisis. International Journal for Parasitology 37 (12): 1367-1378
Previous publications
1. Keating DJ, Rychkov GY, Giacomin P, Roberts ML (2005). Oxygen-sensing pathway for SK channels in the ovine adrenal medulla. Clinical and Experimental Pharmacology and Physiology, 32 (10): 882-887
2. McKay D, Brooker R, Giacomin P, Ridding M, Miles T (2002). Time course of
induction of increased human motor cortex excitability by nerve stimulation. Neuroreport, 13 (10): 1271-1273
Manuscripts in preparation
1. Giacomin PR, Gauld AD, Cava M, Iddewalla D, Gordon DL and Dent LA. Excretory/secretory proteins from Toxocara canis infective larvae reduce eosinophil-dependent innate resistance to Nippostrongylus brasiliensis infection
xixCOMMONLY-USED ABBREVIATIONS
Abbreviation full definition
AAM alternatively-activated macrophage
ADCC antibody-dependent cellular cytotoxicity
AMCase acidic mammalian chitinase
BAL bronchoalveolar lavage
CCR3 chemokine receptor 3
CR complement receptor
CVF cobra venom factor
DAF decay accelerating factor
ES excretory/secretory
EPO eosinophil peroxidase
Ig immunoglobulin
IL interleukin
i.p. intra-peritoneal
L3 third-stage larvae
L4 fourth-stage larvae
MAC membrane attack complex
MASP MBL-associated serine protease
MBL mannan binding lectin
MBP major basic protein
MPO myeloperoxidase
NK natural-killer
p.i. post-infection
PRR pattern recognition receptor
s.c sub-cutaneous
STAT6 signal transducer and activator of transcription 6
Th T-helper
Tg transgenic
VLA very-late antigen
WT wildtype
CHAPTER ONE
CHAPTER ONE: INTRODUCTION AND REVIEW OF THE
LITERATURE
CHAPTER ONE 1
1.1 HEALTH AND ECONOMIC CONSEQUENCES OF PARASITIC
HELMINTH INFECTIONS
Helminth infections cause enormous global health and economic problems. Recent estimates
indicate that approximately 2 billion people are infected worldwide, along with countless
agricultural and domesticated animals. Helminths are a diverse family of parasitic worms,
comprised of two main classes that are only distantly related, nematodes (roundworms) and
platyhelminths (flatworms). Within these two main groups there are many species of
helminths that exhibit complex life cycles, most commonly beginning with either ingestion of
parasite eggs or infective larva that enter the host. The parasite may undergo extensive
growth and maturation while migrating through the host, or in some cases through several
host species, to ultimately accomplish sexual reproduction. To achieve this, helminths must
acquire all nutrients from the host and this can cause significant host morbidity. This is
especially a problem for young host animals, since helminth infections may result in impaired
growth, development, cognitive function and pre-disposition to infection with other pathogens
(Finkelman et al. 1997). Some species of helminth migrate through multiple tissues as part of
the life cycle, causing mechanical damage to delicate tissues such as lung alveoli, the liver or
the eye. In addition to mechanical damage there is significant immunopathology associated
with inappropriate, excessive or chronic immune responses directed against the parasite
(Meeusen 1999). As the survival of the parasite is dependent on the survival of the host,
helminth infection does not typically result in host mortality. Nevertheless, life-threatening
illness and over 150,000 deaths do occur each year in developing countries (Crompton 1999).
Poor levels of nutrition and sanitation, tropical climate and a lack of availability of
anthelmintic drugs increases both the frequency of exposure to parasitic helminths and the
detrimental effects of infection. In developed countries, helminths are a major problem for
agricultural industries, where large losses in product yield and hence profit are seen due to
helminth infection of livestock.
CHAPTER ONE 2
Currently, anthelmintic drugs are the most effective treatment for helminth infections.
However, these drugs are expensive, do not protect against re-infection and there is evidence
of the emergence of drug-resistance in parasites of livestock (Besier and Love 2003; von
Samson-Himmelstjerna and Blackhall 2005). Hence, more effective preventative treatments
are needed to control helminth infections globally. Effective vaccines against human
helminth infections have not been developed. Recently some efficacious vaccines against
tapeworm species have been developed for use in livestock (Lightowlers et al. 2003), leading
to the possibility that a vaccine for preventing infection of humans with some parasite species
is close to fruition. To achieve this, more needs to be known about the nature of the host
immune response to helminth infection and how some helminths have evolved immune
evasion strategies.
1.2 IMMUNE RESPONSES TO HELMINTH INFECTION
Despite the diverse nature of the species of organisms that we classify as parasitic helminths,
in general, the immune responses directed toward elimination of these organisms are
surprisingly similar. The nature of these inflammatory responses are often similar to those
elicited by allergen challenge, resulting in the release of arrays of particular cytokines
(Finkelman et al. 1997), induction of polyclonal immunoglobulin E (IgE) and activation of
leukocytes such as eosinophils, neutrophils, basophils and mast cells (Kay et al. 1985).
Immune responses directed against helminths are complex by necessity, as diverse responses
must be elicited to parasites that have tissue-invasive and/or gastrointestinal lumen-dwelling
life cycles. Hence, the nature and magnitude of the immune response may change in different
infected tissues. Furthermore, most helminths possess the ability to actively modulate
immune responses to prevent immune-mediated destruction, allowing residence within a
single host for months, or even years as seen for some cestode and trematode species
CHAPTER ONE 3(Sandground 1936).
1.2.1 Immune recognition and antigen processing
In order for a host to elicit an effective innate or adaptive immune response against a specific
parasite, the parasite must first be recognised as non-self. This is made more complicated by
the fact that during the course of an infection with a helminth, the nature of the antigens
expressed by the parasite may change as it matures (Preston et al. 1986; Lightowlers and
Rickard 1988; Tkalcevic et al. 1996). Innate mechanisms of parasite recognition may aid in
elimination of the helminth upon initial exposure and also trigger adaptive immune responses
to protect against re-infection (Abraham et al. 2004; Knight et al. 2004; Pemberton et al.
2004; de Veer et al. 2007). Many species of helminth have been shown to activate the innate
immune system shortly after infection, but the mechanisms involved are not fully understood.
Expression of pattern-recognition receptors (PRRs) enable leukocytes to recognise surface
molecules that are highly conserved amongst pathogens of a given class (Applequist et al.
2002). Such molecules are termed pathogen-associated molecular patterns (PAMPs) and can
include carbohydrates, proteins, lipids and nucleic acids. While much research has
investigated the role of the Toll-like receptor (TLR) family in immunity to bacterial or fungal
pathogens (Netea et al. 2004), limited research has investigated the role of TLRs in eliciting
immune responses to helminths. There is some evidence that antigens produced by
schistosomes can activate TLR signalling (van der Kleij et al. 2002), though bacteria that
reside on or within some helminths may also be responsible for this activation (Brattig et al.
2004). Binding of host C-type lectins (CTLs) to the carbohydrate-rich parasite surface may
also be a mechanism of initiation of immune responses (McGuinness et al. 2003). Mannan-
binding lectin (MBL) is a collectin that has been shown to bind to helminths Schistosoma
mansoni (Klabunde et al. 2000) and Trichinella spiralis (Gruden-Movsesijan et al. 2003).
MBL binding can initiate complement activation on the parasite via the lectin pathway, which
may help leukocytes kill helminths (see section 1.5.2). Since complement can be rapidly and
CHAPTER ONE 4non-specifically activated by the presence of PAMPs, it may be a very important factor in the
recognition of helminths in the very early stages of infection. Many different host cell types
express receptors for complement factors (Gasque 2004), which can recognise pathogens
opsonised with complement molecules such as C3b, or alternatively act directly as PRRs for
lipopolysaccharide (LPS) and β-glycan on microbial surfaces (Ehlers 2000). A more detailed
description of the role of complement in immunity to parasitic helminths is provided in
section 1.5.
Dendritic cells are important for the development of adaptive immune responses to pathogens,
by processing and presenting foreign antigens to T-lymphocytes (Reis e Sousa 2001). The
ways in which helminth antigens are processed and presented are not completely understood.
As most helminths are too large to be internalised, the antigens presented by dendritic cells
may initially consist mostly of those factors excreted or secreted by the helminths
(Lightowlers and Rickard 1988; Balic et al. 2004). Presentation of parasite antigens to naïve
lymphocytes can result in the induction of a cytokine bias (see section 1.2.2) and can result in
the generation of parasite-specific and non-specific antibodies (Whelan et al. 2000).
Antibody that arises after previous exposure to a helminth can allow swift and specific
recognition of the parasite, enabling the rapid instigation of protective adaptive immune
responses.
1.2.2 Cytokine responses during helminth infection
Upon contact with a potentially harmful foreign body, the immune system must be able to
induce a specific set of effector mechanisms to eliminate the pathogen. Some of this
specificity is achieved by inducing the secretion of specific arrays of cytokines. CD4+ T-
lymphocytes are a major cytokine-producing and regulatory cell of the immune system,
though other leukocytes such as macrophages, natural killer (NK)-cells, basophils, mast cells,
eosinophils and CD8+ T cells also readily secrete cytokines upon stimulation. Mice that have
CHAPTER ONE 5impaired CD4-dependent cell function, after treatment with anti-CD4 antibody are less
resistant to infection with Heligomosomoides polygyrus (Urban et al. 1991) and Trichuris
muris (Koyama et al. 1995), with increased worm fecundity and impaired worm expulsion,
respectively. CD4+ T-cells differ in the types of cytokines that are expressed and at least 4
dominant cytokine patterns are commonly described. T-helper 1 (Th1) cells secrete
interleukin (IL)-2, interferon (IFN)-γ and tumour necrosis factor (TNF)-β (commonly termed
as Type-1 cytokines) (Mosmann and Coffman 1989). Th2 cells secrete IL-4, IL-5, IL-6, IL-9,
IL-10 and IL-13 (Type-2 cytokines) (Romagnani 2000). Both Th1 and Th2 cells may secrete
IL-3 and granulocyte-macrophage colony stimulating factor (GM-CSF). A third subset of
cytokine-producing CD4+ T-cells, the Th3 or Type 3 class, produce a more heterogeneous
cytokine profile, most notably transforming growth factor β (TGFβ) that functions mainly to
inhibit the actions of T-cells, macrophages and counteract the actions of other pro-
inflammatory cytokines. Cells within the Type 3 group are commonly known as regulatory
T-cells (T-regs) (Belkaid and Rouse 2005). Lastly, the recently identified Th17 cell lineage
represents a distinct group of CD4+ T-cells that produce, among other factors, the cytokine
IL-17 (Aggarwal et al. 2003; Harrington et al. 2006). Th17 cells are believed to be
particularly important for protection against extracellular bacteria through their ability to
stimulate inflammatory cell recruitment (Happel et al. 2005), but they have also been
implicated in a number of autoimmune diseases (Chen et al. 2006; Komiyama et al. 2006).
Some cytokines possess the ability to enhance production of other cytokines of the same
category, creating a positive feedback loop. This, along with the existence of cross
regulation, i.e. Type 1 cytokines blocking activity or production of Type 2 cytokines and vice
versa, leads to a pronounced cytokine bias in some disease states (Mosmann and Coffman
1989). Infection with several parasitic helminth species has been shown to be a potent
inducer of Type 2-cytokine biased immune responses (Grencis 1997). Exposing mice to
either proteins that are excreted or secreted (ES proteins) by the nematode Nippostrongylus
CHAPTER ONE 6brasiliensis (Holland et al. 2000) or the whole parasite (McKenzie et al. 1998; Min et al.
2004) is sufficient to cause a marked Type 2 cytokine response in mice. A Type 2-cytokine
bias has various effects on the immune status of the host, including IL-5-dependent
eosinophilia (Dent et al. 1990; Urban et al. 1992), IL-4-dependent class switching to promote
IgE production (Finkelman et al. 1988) and mastocytosis (Madden et al. 1991). In contrast,
induction of a Type 1 cytokine response in mice by administration of IL-12 has been shown
to delay clearance of N. brasiliensis and T. muris (Finkelman et al. 1994; Bancroft et al.
1997). The exact mechanism by which a Type 2 cytokine response is triggered during a
parasitic helminth infection is not completely understood. Early production of IL-4 is an
important step in the establishment and amplification of Type 2 immune responses, but how
cells are activated to secrete IL-4 is unclear. Recently, an IL-25-dependent non-B non T-cell
has been shown to be a major mediator of rapid IL-4, IL-5 and IL-13 production during N.
brasiliensis infection (Fallon et al. 2006). Additionally, basophils are an important early
source of IL-4 or IL-13 (Falcone et al. 2001; Min et al. 2004; Voehringer et al. 2004) and
these cells rapidly increase in number in the peripheral blood and infected tissues following
exposure to some helminths (Falcone et al. 2001). In this respect, basophils may be critical
for skewing the immune response in the early stages of helminth infection, in cooperation
with other cytokine-producing leukocytes such as eosinophils and the recently identified IL-
25-dependent non-B non T-cell population.
1.2.3 Immunological basis of gastrointestinal helminth expulsion
Efficient expulsion of intestine-dwelling helminths requires the instigation of physiological
changes to the gastrointestinal environment that act to “force” the parasite out of the intestine.
In particular, helminth infection is associated with increased intestinal smooth muscle cell
contractility. Mouse strains that produce more pronounced smooth muscle contractility (e.g.
NIH Swiss mice) expel T. muris more efficiently than other strains that exhibit lesser changes
in smooth muscle contractility (e.g. B10.BR mice) (Vallance et al. 1997). Helminth
CHAPTER ONE 7infections also result in increases in ion and water secretion, which act to “flush” the parasites
from the intestine (Mettrick et al. 1979). Goblet cells within the intestinal epithelium undergo
rapid proliferation (hyperplasia) at the time of expulsion of several species of helminth (Nawa
et al. 1994; Onah and Nawa 2000). The secretion of mucus by these cells may exclude and
trap worms in the gastrointestinal mucosa and promote expulsion by preventing their
attachment and feeding. Epithelial cells within the intestinal microenvironment have been
shown to produce an array of proteins that may be potent effector molecules against
helminths, such as intelectins and resistin-like molecules (RELMs) (Datta et al. 2005; Nair et
al. 2005), however the functions of these molecules during helminth infection are not fully
understood. An increase in the rate of intestinal epithelial turnover (Symons 1978) and rate of
migration from the base of the crypts to the extrusion zone have been associated with
elimination of T. muris (Cliffe et al. 2005). This may restrict the ability of helminths to attach
to the gut wall and hence aid in worm expulsion.
Mast cells have long been implicated as important for immunity to helminths. Their primary
role may be to promote the expulsion of gastrointestinal parasites, however they are also
prominent in the skin and lungs during infections with some helminth species (Bentley et al.
1981; Matsuda et al. 2001). Intestinal mastocytosis and an increase in tissue mast cell
protease levels coincide with expulsion of N. brasiliensis and T. spiralis (Miller and Jarrett
1971; Woodbury et al. 1984). Mice deficient in mast cells (W/Wv mice) or mice treated with
anti-c-kit antibody are slow to expel T. spiralis and Strongyloides ratti (Abe and Nawa 1987;
Donaldson et al. 1996). However, there is evidence that expulsion of some helminths, such as
N. brasiliensis, proceeds normally in the absence of mast cells (Uber et al. 1980). Mast cells
release an as yet unidentified factor by degranulation that may stimulate parasite expulsion
(Onah and Nawa 2000). In summary, the process of expulsion of gastrointestinal helminths is
complex and involves elements of both the innate and adaptive immune system, co-ordinated
with physiological changes to intestinal tissues and secretions.
CHAPTER ONE 8
It is now commonly believed that changes in intestinal physiology after helminth infection are
a direct result of immune activation. Several Type-2 cytokines have been implicated in the
instigation of mechanisms that promote expulsion of gastrointestinal helminths. In particular,
activation of signal transducer and activation of transcription-6 (STAT6), a transcription
factor activated after ligation of the IL-4R (Takeda et al. 1996), is important. Worm
expulsion is delayed in STAT6-deficient mice infected with N. brasiliensis or Strongyloides
venezuelensis (Urban et al. 1998; Negrao-Correa et al. 2006). Blockade of the IL-4R using
an anti-IL-4R antibody, which inhibits both IL-4 and IL-13 signalling, leads to chronic T.
muris infections in normally resistant mice (Else et al. 1994). Subsequent experiments with
IL-4 or IL-13-deficient mice supported this observation and suggest that both cytokines play a
role in expulsion of this parasite (Bancroft et al. 1997). The type-2 cytokine IL-9 is also
involved in the process of T. muris expulsion, since neutralisation of IL-9 in mice infected
with this parasite delays worm expulsion (Khan et al. 2003). Intestinal smooth muscle
contractility ex vivo is inhibited by anti-IL-9 antibody treatment, suggesting IL-9 may act to
stimulate physiological changes in the small intestine that are necessary for expulsion of
parasites. Elimination of N. brasiliensis is also dependent on IL-4R and STAT6 expression,
however IL-13 may be more important than IL-4 for expulsion this parasite (Urban et al.
1998). The IL-4R must be expressed by non bone marrow-derived cells (i.e. non immune
cells) for timely expulsion of N. brasiliensis (Urban et al. 2001). On the other hand, whilst T.
spiralis expulsion still requires IL-4R signalling, in this model it is essential that the receptor
is expressed by bone marrow-derived immune cells and that mast cells are present in order for
the parasite to be expelled from the host (Urban et al. 2001). In summary, Type 2 cytokines
have been shown to be important for promoting expulsion of worms from the gut. The exact
mechanism of expulsion varies depending on which parasite is present, however activation of
the IL-4/IL-13/STAT6 system appears to be a common element required for expulsion of
gastrointestinal worms.
CHAPTER ONE 9 1.3 LEUKOCYTE-MEDIATED KILLING OF HELMINTHS
While PRRs, complement and antibody may be important for recognition of tissue-invasive
helminths and cytokines are effective at initiating protective immune responses and causing
parasite expulsion, active killing of helminths is typically dependent on cell-mediated
immunity. Macrophages and neutrophils have the capacity to directly inflict damage on
helminths. However it is eosinophils, which are often generated in large numbers during
helminth infections, which may have evolved as key effectors of protective immunity against
some parasite species.
1.3.1 Neutrophils
Neutrophils are effective at killing bacterial or unicellular organisms but there is some
evidence that they can also be cytotoxic for larger helminthic parasites (Incani and McLaren
1981; Shaio et al. 1990). Recruitment of these cells is rapid and is stimulated by many factors
including IL-8, prostaglandins, platelet-activating factor (PAF), leukotriene (LT)B4, N-
formyl methyonyl-leucyl-phenylalanine (fMLP) and complement factors such as C3a and C5a
(Jagels and Hugli 1992). Neutrophils are often observed to be amongst the first leukocytes to
arrive in an infected tissue. Neutrophils express Fc receptors that allow specific recognition
of antibody bound to the parasite surface, which promotes antibody-dependent cell-mediated
cytotoxicity (ADCC). Neutrophils express complement receptors CR1, CR3 and CR4 that
also facilitate their attachment to the surface of a pathogen. Complement-mediated ADCC
has been shown to be effective in the killing of S. mansoni schistosomula (Incani and
McLaren 1981) and Angiostrongylus cantonensis (Shaio et al. 1990) in vitro. Neutrophils can
also kill Strongyloides stercoralis larvae in vivo (Ligas et al. 2003; Galioto et al. 2006). The
mechanism by which neutrophils kill helminths is not clear, but is likely to include the actions
of the enzyme myeloperoxidase (MPO) that generate reactive oxygen intermediates and
CHAPTER ONE 10hypochlorite ions that could damage parasites. Purified MPO can efficiently kill newborn T.
spiralis larvae in vitro (Buys et al. 1984) and co-culture of neutrophils with T. spiralis or N.
brasiliensis results in deposition of superoxide on parasites (MacKenzie et al. 1981). Despite
this, neutrophils are not effective at killing other helminth species such as Toxocara canis
(Huwer et al. 1989). Furthermore, neutrophil responses are typically transient and with time
these cells are often replaced by eosinophils or macrophages in parasite-infected tissues.
1.3.2 Macrophages
As with neutrophils, macrophages are also effective at phagocytosing small microorganisms
and have been reported to be able to kill several helminth species (Sher et al. 1982; Egwang
et al. 1984). Macrophages express Fc receptors that can facilitate ADCC, and CR1, CR3 and
CR4 that allow recognition of complement factors bound to helminths. Macrophages adhere
to and degranulate on N. brasiliensis larvae in the presence of complement or antibody
(MacKenzie et al. 1981) and can kill N. brasiliensis (Egwang et al. 1984) and S. mansoni
(Sher et al. 1982) larvae in vitro. Macrophages also produce MPO and express the enzyme
nitric oxide synthase (NOS), which can generate toxic nitric oxide or peroxynitrite, both of
which may damage helminths (Buys et al. 1984; James et al. 1998; Gupta et al. 2004).
1.3.3 Eosinophils
There is strong evidence to support a role for eosinophils in protection against some helminth
species. Eosinophils are terminally differentiated granulocytic leukocytes produced from
myeloid CD34+ precursors in the bone marrow (Warren and Moore 1988). The early stage of
their differentiation from these precursors is controlled primarily by the cytokines GM-CSF
and IL-3 (Warren and Moore 1988). In the later stage, IL-5 is the critical cytokine involved
in the terminal differentiation and maturation of eosinophils (Lopez et al. 1986; Dent et al.
1990).
CHAPTER ONE 11Eosinophil granules store proteins that are both toxic to invading pathogens but also
potentially damaging to host tissues in some disease states. In humans there are four main
eosinophil granule proteins; major basic protein (MBP), eosinophil peroxidase (EPO),
eosinophil-derived neurotoxin (EDN) and eosinophil cationic protein (ECP) (Hamann et al.
1991). MBP and EPO are also expressed by mouse eosinophils; however the ribonucleases
EDN and ECP are not. Instead, mouse eosinophils express at least 11 genes that encode
proteins with ribonuclease activity, termed eosinophil-associated ribonucleases (EARs)
(Cormier et al. 2001). MBP is located within the electron-dense crystalline core of eosinophil
granules, while the other proteins are located within the surrounding matrix (Gleich et al.
1976; Dvorak et al. 1994; Egesten et al. 1997). In addition to these granule proteins,
eosinophils also express an array of other proteins, including cytokines, chemokines, growth
factors and lipid mediators (Rothenberg and Hogan 2006).
1.3.3.1 IL-5 and eosinophils
IL-5 controls the differentiation, maturation and survival of eosinophils in many species
including humans (Warren and Sanderson 1985; Lopez et al. 1986) and the maturation of B-1
lymphocytes in mice (Kinashi et al. 1986). Raised levels of IL-5 are commonly seen in
animals infected with parasitic helminths (Coffman et al. 1989), under allergic and asthmatic
conditions (Sur et al. 1995; Nagai et al. 1996) and in some autoimmune diseases (Hellmich et
al. 2005). Basal eosinophil numbers in the peripheral blood are typically low in naïve
animals (<5% of total leukocytes) with most residing within the gastrointestinal tract and
more variably in the female reproductive tract (Weller 1991; Mishra et al. 1999). However,
raised levels of IL-5 can stimulate eosinophilopoiesis in the bone marrow and vast increases
in blood and tissue eosinophil numbers (Dent et al. 1990). Treatment of humans or animals
with anti-IL-5 antibody reduces peripheral blood eosinophilia during experimental helminth
infections (Coffman et al. 1989; Sher et al. 1990) and in asthmatic and hyper-eosinophilia
syndrome (HES) patients (Leckie et al. 2000; Garrett et al. 2004; Simon et al. 2005).
CHAPTER ONE 12
The relationship between IL-5 and eosinophils in vivo has been explored using mice over-
expressing IL-5 transgenes (IL-5 Tg mice) and mice genetically deficient in IL-5 (IL-5-/-). IL-
5 Tg mice have been developed by several strategies and in each case the mice express
constitutive eosinophilia (Dent et al. 1990; Vaux et al. 1990; Tominaga et al. 1991), however
the distribution of eosinophils in various tissues differs depending on the nature of the
transgene used. IL-5 Tg lines developed by ligation of the dominant controlling region of the
human CD2 gene to a murine genomic DNA IL-5 gene and flanking regions yields IL-5 Tg
expression that is largely T-cell specific (Dent et al. 1990). Eosinophilia in these mice is
most evident in the peripheral blood, bone marrow, spleen, intestinal wall and lungs. The
degree of eosinophilia is dependent on transgene copy number, such that the Tg(0IL5)C2 line
(abbreviated Tg5C2) with approximately 49 copies of the IL-5 transgene express more
pronounced eosinophilia that the Tg(0IL5)C1 line (abbreviated Tg5C1), which has
approximately 8 copies of the IL-5 transgene. Conversely, IL-5-/- mice fail to develop
eosinophilia when challenged with aeroallergens in a mouse model of asthma or when
infected with helminths (Foster et al. 1996; Kopf et al. 1996; Matthaei et al. 1997; Ovington
et al. 1998). However these mice do exhibit very low numbers of eosinophils that are
functionally and morphologically similar to those in wild type (WT) mice. Although
eosinophils can be generated in small numbers in the absence of IL-5, presumably due to the
actions of GM-CSF and IL-3 in early differentiation (Warren and Moore 1988), eosinophilia
is dependent on IL-5 (Dent et al. 1990; Foster et al. 1996; Yu et al. 2002).
1.3.3.2 Eosinophil recruitment
Rapid and specific recruitment of eosinophils to sites of infection may be critical for
immunity to pathogens and can be mediated by many different factors (Resnick and Weller
1993). After maturation in the bone marrow, eosinophils circulate in the blood and traffic to
the gastrointestinal tract, thymus, mammary glands or uterus under normal, uninfected
CHAPTER ONE 13conditions. This trafficking is dependent on the chemokine eotaxin-1 (Gouon-Evans et al.
2000; Rothenberg et al. 2001; Rothenberg and Hogan 2006), which is a major chemotactic
factor for eosinophils (Mould et al. 1997; Mishra et al. 1999). However, other cells have
been shown to express the eotaxin-1 receptor chemokine receptor 3 (CCR3) and eotaxin can
be chemotactic for other leukocytes (Menzies-Gow et al. 2002). Many different factors can
mediate eosinophil recruitment aside from eotaxin-1, including the chemokines regulated on
activation, normal T-cell expressed and secreted (RANTES), some cytokines, complement
factors C3a and C5a, PAF, leukotrienes, integrins and acidic mammalian chitinase
(AMCase)(reviewed in Resnick and Weller 1993; Rothenberg and Hogan 2006). Upon
stimulation, circulating eosinophils adhere to the endothelial wall via expression of leukocyte
function-associated antigen (LFA)-1, very late antigen (VLA)-4, P-selectin glycoprotein
ligand (PSGL)-1 and Mac-1, which interact with their corresponding ligands on endothelial
cells (Rothenberg and Hogan 2006). The cells then migrate through the endothelium,
localising at the source of the inflammatory signal.
1.3.3.3 Eosinophil activation, secretion and degranulation
Once recruited to the site of infection, tissue injury or allergen exposure, eosinophils can
respond to a variety of stimuli, becoming activated and selectively releasing a range of
products. Typically, eosinophils circulating in the peripheral blood are far less active than
those present in tissues (Nonaka et al. 1999), but become more activated during recruitment
and extravasation. Eosinophil activation, manifested as the ability to express and release
granule proteins, is enhanced by the presence of IL-5 (Fujisawa et al. 1990). However under
various conditions, eosinophil degranulation can be triggered by cytokines such as IL-3 and
GM-CSF, chemokines, complement factors (C3b, C3a, C5a), PAF, ligation of FcγR and
factors released by tissue-invasive pathogens (Abu-Ghazaleh et al. 1992; Rothenberg and
Hogan 2006). The exact mechanism by which eosinophils release their granule contents is
not fully understood. Regulated exocytosis of cytoplasmic vesicles is the most commonly
CHAPTER ONE 14described mechanism, where eosinophils selectively release specific granules in a piecemeal
fashion (Henderson and Chi 1985; Dvorak et al. 1992; Logan et al. 2003; Clark et al. 2004).
Selective release of granule proteins in response to varying stimuli may explain the diversity
of functions for eosinophils. Eosinophils have also been demonstrated to degranulate by
cytolysis, where entire cytoplasmic contents are released non-specifically and this may cause
significant tissue pathology (Erjefalt et al. 1998).
1.3.3.4 Role of eosinophils in disease
Eosinophils can be destructive to the host, particularly in allergic diseases. Eosinophils are
commonly detected in elevated numbers in asthmatic humans and levels of the toxic granule
protein MBP are raised in bronchoalveolar lavage (BAL) fluid from such patients (Gleich et
al. 1979). IL-5 has been thought of as a useful target molecule to alleviate the symptoms of
asthma. Administering antibodies that inhibit IL-5 to guinea pigs (Mauser et al. 1993) and
mice (Corry et al. 1996) prevents the establishment of allergen-induced eosinophilia but was
not observed to greatly affect airways hyper-reactivity. However, in studies with IL-5-/- mice
(Foster et al. 1996) or mice with a complete lack of eosinophils (Lee et al. 2004), more
encouraging results were obtained, with animals failing to develop airways pathology and
hyper-reactivity after allergen challenge. In humans, trials have used anti-IL-5 antibodies to
target eosinophilia in an attempt to alleviate symptoms for asthmatic patients (Leckie et al.
2000). This study yielded discouraging results, where peripheral blood eosinophilia was
successfully reduced but symptoms were not significantly improved. Anti-IL-5 therapy may
not completely prevent eosinophilopoeisis or recruitment into tissues (Flood-Page et al. 2003)
and sufficient numbers remain after therapy, contributing to lung pathology and loss of
function. There remains some controversy as to the role of eosinophils in allergic asthma, but
it is now becoming apparent that eosinophils also contribute to other diseases, notably several
diseases affecting the gastrointestinal tract (Rothenberg 2004).
CHAPTER ONE 151.3.3.5 Role of eosinophils in killing helminths
Evidence that eosinophils protect against helminth infection was initially derived from
observations that these cells were found located around dead larvae from tissues and that the
magnitude of eosinophilia was positively correlated with the degree of resistance to infection
(Taliaferro and Sarles 1939; Butterworth 1984; Hagan et al. 1985). The precise mechanisms
by which eosinophils kill helminths remain unknown, though adherence to the organism and
degranulation may be critical. Early in vitro studies demonstrated that eosinophils kill S.
mansoni schistosomula in the presence of antibody and a heat-labile component of serum
(Butterworth et al. 1975; David et al. 1980). Eosinophils were observed to adhere to the
schistosomula and degranulate, releasing MBP directly onto the surface of the parasite.
Eosinophils are also effective at killing larval stages of T. spiralis (MacKenzie et al. 1980;
MacKenzie et al. 1981; Venturiello et al. 1995), N. brasiliensis (McLaren et al. 1977;
MacKenzie et al. 1981; Shin et al. 2001), Haemonchus contortus (Rainbird et al. 1998), H.
polygyrus (Penttila et al. 1983) and some filarial nematodes (Chandrashekar et al. 1990;
Brattig et al. 1991). In these studies, antibody appears to be important for eosinophil
adherence, degranulation and killing, but other serum factors also contribute to these
processes. In particular, the complement system is postulated to play an important role in
both the presence and absence of antibody (Butterworth 1984).
Infecting mice that display constitutive eosinophilia (IL-5 Tg mice) with helminths has
provided important insight into the role of eosinophils in resistance to helminth infection. IL-
5 Tg mice infected with N. brasiliensis (Dent et al. 1997a; Dent et al. 1997b; Shin et al. 1997;
Daly et al. 1999), A. cantonensis (Sugaya et al. 1997), Angiostrongylus costaricensis (Sugaya
et al. 2002), Litomosoides sigmodontis (Martin et al. 2000) and S. stercoralis (Herbert et al.
2000) have much lower parasite burdens than similarly treated WT mice. This is associated
with rapid eosinophil recruitment to sites of infection and restriction of parasite migration and
development. These results demonstrate that if eosinophils are sufficiently numerous and/or
CHAPTER ONE 16activated at the time of the initial infection, robust resistance can be mounted against some
helminths. In contrast, IL-5 Tg mice infected with parasites such as T. canis (Sugane et al.
1996; Dent et al. 1997a; Dent et al. 1999), S. mansoni (Dent et al. 1997b) or T. spiralis (Dent
et al. 1997a; Hokibara et al. 1997) were either no more resistant than WT animals or carried
high parasite loads, suggesting these parasites may have evolved strategies to evade attack by
eosinophils and that tissue eosinophilia may interfere with the function of other elements of
the immune response.
Studies using anti-IL-5 antibodies or IL-5-/- mice to diminish eosinophilia during helminth
infection have yielded mixed results in terms of the effect on parasite load. IL-5-/- mice
infected with Brugia malayi and S. ratti display higher worm burdens than WT mice during a
primary infection, however a lack of IL-5 does not impair resistance to re-infection with the
same parasites (Ovington et al. 1998; Ramalingam et al. 2003; Simons et al. 2005). Similar
results were observed for S. stercoralis, where IL-5-/- mice and mice treated with anti-CCR3
antibody had impaired killing of larvae during primary but not secondary infections (Herbert
et al. 2000; Galioto et al. 2006). Transfer of eosinophils or neutrophils to IL-5-/- mice
restored their ability to kill this parasite during primary infections (Galioto et al. 2006).
Significantly increased parasite burdens after anti-IL-5 antibody treatment were also observed
for infections with A. cantonensis (Sasaki et al. 1993), Onchocerca lienalis (Folkard et al.
1996) and S. venezuelensis (Korenaga et al. 1991). Therefore, for these helminths,
eosinophils are important for parasite killing during primary infections but may not be
required during secondary infections.
IL-5 and eosinophils do not appear to play a significant role in resistance to other helminth
species. IL-5 plays only a very minor role in immunity to T. spiralis, where anti-IL-5
antibody treatment of mice or infection of IL-5-/- mice had no or little effect on parasite
persistence in the intestine (Herndon and Kayes 1992; Vallance et al. 2000). IL-5-/- mice, or
CHAPTER ONE 17mice treated with anti-IL-5 antibodies exhibit similar parasite burdens to WT mice infected
with S. mansoni (Sher et al. 1990), N. brasiliensis (Coffman et al. 1989), T. muris (Betts and
Else 1999) or T. canis (Takamoto et al. 1997) despite effective blockade of the development
of eosinophilia. However, IL-5-/- or eosinophil-deficient ∆dblGATA mice exhibit higher
parasite fecundity than in WT mice during primary infections with N. brasiliensis (Knott et
al. 2007).
Eosinophils do degranulate in parasite-infected tissues but whether this contributes to parasite
killing is not clear (Hsu et al. 1974; von Lichtenberg et al. 1977; Kephart et al. 1984; Daly et
al. 1999). Preparations of eosinophil granule proteins can kill or immobilise parasite larvae in
vitro (Butterworth et al. 1979; Buys et al. 1981; Jong et al. 1981; Hamann et al. 1990), hence
degranulation may be important for eosinophil-dependent parasite killing. Whether
adherence to the parasite is essential for triggering degranulation is unclear. Mice deficient in
the granule proteins MBP and EPO develop higher worm burdens than WT animals infected
with L. sigmodontis (Specht et al. 2006), but the role of the eosinophil granule proteins in
killing other helminths in vivo is unclear. Resistance of mice to S. stercoralis (Abraham et al.
2004) and B. malayi (Ramalingam et al. 2005) is eosinophil-dependent, but not affected by an
absence of EPO and for B. malayi, resistance is also unaffected by the absence of MBP. This
suggests that for these parasites, EPO or MBP alone are not essential for parasite killing and
eosinophils possess other factors in their arsenal that are capable of damaging helminths.
1.3.3.6 Other roles for eosinophils during helminth infections
While the primary function of eosinophils during helminth infection may be active killing via
the release of toxic mediators, they may also contribute in other ways. There is evidence that
eosinophils can present antigens to T-cells (Weller et al. 1993; MacKenzie et al. 2001),
including helminth antigens that induce a Type-2 cytokine response (Padigel et al. 2006).
Eosinophils can also be a major source of cytokines during helminth infection, in particular
CHAPTER ONE 18by producing IL-4, which is important for induction of a Type-2 cytokine bias (Shinkai et al.
2002; Voehringer et al. 2004). While not directly demonstrated during helminth infection,
eosinophils are thought to be associated with tissue remodelling and wound healing in other
models (Bassett et al. 1977; Gouon-Evans et al. 2000; Sferruzzi-Perri et al. 2003), potentially
by the release of TGF-β (Wong et al. 1991) and hence could conceivably aid in repairing
tissues damaged by helminths after resolution of infection.
To summarise, while the role of eosinophils in killing parasitic helminths remains
controversial, there is considerable evidence to suggest that these cells can be effective at
killing or damaging some species of parasite if present in adequate numbers and at the
appropriate time during the course of an infection. Eosinophils appear to be most effective at
killing tissue-invasive helminths, though there are notable exceptions including T. canis, T.
spiralis and S. mansoni. For those studies where eosinophils were not demonstrated to play a
major role, it is possible that eosinophils were either not available in sufficient numbers to
exert an effect, or may only be effective against a particular and perhaps immature stage of
maturation of the parasite. It is also likely that some helminth species have evolved immune
evasion mechanisms that suppress eosinophil function. Certainly, parasites capable of
residing in host tissues for long periods would have been under strong pressure to evolve
strategies to evade damage induced by eosinophils. Equally, parasites such as N. brasiliensis
that migrate rapidly through tissues and normally have a relatively short period of residence
in the host are inadequately protected if eosinophils are present in large numbers at the onset
of infection. Eosinophils may be most effective against helminths soon after entry into the
host, particularly if the portal of entry is the skin. Understanding how eosinophils kill
helminths at these times may be critical for the development of effective vaccines that can
target these infective larval stages and prevent progression of the infection. The mechanism
by which eosinophils can be recruited to sites of infection, adhere to the target and kill by
degranulation (or by other mechanisms) is an area that requires further investigation.
CHAPTER ONE 19 1.4 THE COMPLEMENT SYSTEM
1.4.1 Function of the complement system
The complement system is important for both innate and adaptive immune responses to
infection with a variety of pathogens, mediating processes such as immune cell recruitment,
adhesion to the target, host cell activation, phagocytosis and direct lysis of small microbial
pathogens. The complement system consists of more than 35 fluid phase and cell-bound
proteins, most of which circulate as inactive enzymes until initiation events trigger activation
by proteolytic cleavage. Ultimately, the complement cascade can result in formation of the
membrane attack complex (MAC) which creates pores in cell membranes and may lead to cell
lysis. This is of importance for some smaller membrane-bound pathogens such as bacteria
and viruses, however due to their size and structure, the MAC is unlikely to cause significant
damage to most tissue-invasive parasitic helminths. However, certain events and factors
generated prior to the formation of the MAC may be advantageous for triggering immune
responses to helminths. Activation of complement occurs by three distinct routes (see section
1.4.2), but in all cases results in cleavage of the C3 molecule. Figure 1.1a details the
consequences of C3 activation schematically. C3 cleavage leads to generation of the
anaphylotoxin C3a, which can act as a chemotactic factor for leukocytes, and C3b which can
covalently bind to the surface of the pathogen or remain soluble (Rother and Till 1988).
Surface-bound C3b can act to cleave the C5 molecule into the anaphylotoxin C5a, another
chemoattractant molecule, and C5b which forms the first component of the MAC (C5b-9).
This covalent tagging of foreign molecules with C3b has other important functions, such as
opsonisation that facilitates recognition by leukocytes via CR1 (CD35). CR1 is found on
lymphocytes, erythrocytes and granulocytes such as eosinophils and neutrophils, where it
promotes phagocytosis (Gasque 2004). C3b can be further converted into iC3b by factor I-
mediated cleavage, in the presence of cofactors such as C4b and factor H (Rother and Till
Fi
gure
1.1
a: C
onse
quen
ces o
f C3
clea
vage
Figu
re 1
.1b:
Pat
hway
s to
com
plem
ent a
ctiv
atio
n
CHAPTER ONE 201988). The iC3b molecule is an “inactive” form of C3 in that it does not cause amplification
of the complement cascade. However, CR3 (CD11c/CD18) found on monocytes, neutrophils,
eosinophils and NK cells has high affinity for iC3b, which is also important for cell adhesion
and phagocytosis (Fischer et al. 1986; Rother and Till 1988; Gasque 2004). Further cleavage
of iC3b yields the fragments C3c, C3dg and C3d, the latter two of which can be recognised by
CR2 (Cooper et al. 1990). Although the complement system may be important in providing
protection against invading pathogens, when activated inappropriately it can be extremely
damaging to host tissues, as is seen in age-related macular degeneration, rheumatoid arthritis,
asthma, inflammatory bowel disease, ischemia-reperfusion injury and some infectious
diseases. Host cells are normally protected from the potentially damaging effects of
complement activation by various inhibitory molecules such as factor H, C1-inhibitors, C4-
binding protein, decay accelerating factor (DAF) and factor I (Gasque 2004).
1.4.2 Pathways to complement activation
There are three main pathways of initiation of the complement cascade, the classical, lectin
and alternative pathways, detailed schematically in Figure 1.1b. Cleavage of the C3 molecule
is central to all pathways. The classical pathway of complement activation begins primarily
with C1q recognition of pathogen-bound antibody, though C1q can recognise other pathogen-
associated molecules such as bacterial lipopolysaccharide and C-reactive protein (Gewurz et
al. 1993). C1q complexes with C1r and C1s in a Ca2+-dependent manner to form an esterase
that cleaves both C4 and C2 in the presence of Mg2+ (Rother and Till 1988). The resultant
cleavage factors C4b and C2a join to form the membrane-bound classical pathway C3
convertase (C4b2a) which then cleaves C3. The lectin pathway is similar to the classical
pathway, but does not require C1q and involves Ca2+-dependent recognition of microbial
carbohydrates by fluid-phase pattern recognition molecules such as MBL or ficolins (Endo et
al. 2006). This recognition triggers activation of the MBL-associated serine proteases
(MASP-1, MASP-2 and MASP-3), where MASP-2 cleaves and activates C2 and C4 to
CHAPTER ONE 21produce the same C3 convertase (C4b2a) found in the classical pathway (Wallis et al. 2007).
In addition, MASP-1 and MASP-2 have the ability to cleave C3 directly (Ambrus et al.
2003). The alternative pathway of complement activation is initiated by interactions with any
surface, including host cells, as well as carbohydrate-rich structures such as bacterial, yeast or
parasite cell walls. Activation is triggered by spontaneous hydrolysis of C3, resulting in
formation of a “C3b-like” molecule C3(H2O) (Rother and Till 1988). This combines with
factor B, which is cleaved by factor D to form C3(H2O)Bb, a C3 convertase that cleaves C3 to
form C3b, which then binds to the surface of the target. Metastable C3b then interacts with
factor B in the presence of Mg2+ to form C3bBb which is a C3 convertase that acts to generate
more C3b, hence amplifying C3b opsonisation of the microbial surface (Rother and Till
1988). If this process remained unimpeded it would result in consumption of all serum C3,
but it is normally controlled by negative regulators such as DAF, which blocks the interaction
between factor B and C3b (Kinoshita et al. 1986), and by factors I and H, which accelerate
conversion of C3b to inactive iC3b (Brown et al. 1983).
1.4.3 Complement-dependent immunity to pathogens
The complement system is typically the first line of defence against a wide range of
pathogenic organisms. Because microbial activation of the classical pathway usually requires
the presence of antibody, it is the alternative or lectin pathways of complement that would
typically be most important for innate immunity to bacteria and viruses in humans and other
animals (Kolble and Reid 1993; Petersen et al. 2001). However, naturally-occurring
antibodies reactive with micro-organisms are common and could activate the classical
pathway even without prior exposure of the host to a specific pathogen (Kozel et al. 1996;
Mold et al. 2002). Antibody-independent activation of the classical pathway may also play a
role, as has been demonstrated for both Gram negative (Loos et al. 1978) and Gram positive
(Eads et al. 1982) bacteria and for some viruses (Bartholomew and Esser 1980).
CHAPTER ONE 22Many species of bacteria, fungi, parasites and viruses have been shown to activate
complement in vitro; though the role of complement in vivo for immunity to these pathogens
is less well defined. Several methods of studying the role of complement in vivo are used.
One is the pharmacological depletion of complement components (typically C3) by treatment
of animals with cobra venom factor (CVF) which acts to generate an alternative complement
pathway convertase that is resistant to factor I-dependent inhibition (Shin et al. 1969). CVF-
treated animals are more susceptible to infection with pathogens such as Streptococcus
pneumoniae (Winkelstein et al. 1975), Candida albicans (Gelfand et al. 1978) and
Leishmania amazonensis (Laurenti et al. 2004). Similarly, CVF-treated mice immunised with
Escherichia coli are as susceptible to secondary infection as naïve mice (Ahlstedt 1981) and
CVF-treated chickens are highly vulnerable to infection with fowlpox virus (Ohta et al.
1986). While CVF-treatment of animals is a useful tool, C3 depletion is not absolute, with
levels of C3 reduced to 5-15% of those found in untreated animals (Jones and Ogilvie 1971).
More complete ablation of complement activity can be seen in animals with natural genetic
deficiencies in particular complement components. The mechanisms of complement-
dependent resistance can be probed in greater detail through the study of the consequences of
genetic mutations in C2, C4, C5 and C6 molecules. Humans deficient in C5 have been shown
to be extremely susceptibile to Neisseria gonorrhoeae and Neisseria meningitidis infections
(reviewed in Guenther 1983; Gianella-Borradori et al. 1990) and C3-deficient humans are
highly susceptible to Haemophilus influenzae type B (Winkelstein and Moxon 1992) and
various pyogenic bacterial infections. Studies with spontaneous-mutant C5-deficient mice
have demonstrated that the terminal arm of the complement cascade has an important function
during infection and the influence of complement is not just a consequence of C3 deposition
(Lovchik and Lipscomb 1993; Ferreira et al. 2000; Mullick et al. 2004).
Lastly and most recently, more comprehensive analysis of the role of complement has been
achieved using mice genetically engineered to be deficient in specific complement factors.
CHAPTER ONE 23This has the advantage that the roles of multiple factors can be analysed in the same host
species and strain. A large range of complement-deficient mice are now available and these
have been used in many studies to illustrate the in vivo role of various complement proteins in
immunity to bacterial, fungal, parasitic and viral infections (Wessels et al. 1995; Fischer et al.
1996; Matsumoto et al. 1997; Botto et al. 1998; Taylor et al. 2001; Pickering et al. 2002;
Mehlhop and Diamond 2006; Yuste et al. 2006).
Complement can also contribute to other elements of the immune response. Complement
receptors have been identified on lymphocytes and follicular dendritic cells (Carroll 2004)
and deficiency in C3 induced by CVF treatment (Pepys 1974) or genetic C3 or C4 deficiency
(Fischer et al. 1996) leads to impaired antibody responses to T lymphocyte-dependent
antigens. Complement is important for the activation of naïve B cells via complement
receptors CD21/CD35 and for persistence of antibody secretion (Carroll 2004). Elements of
the complement system can also modulate the function of T lymphocytes (Carroll 2004),
though its relative importance for T cell biology is unclear. Hence, complement can function
at several levels to provide resistance to infection with micro-organisms, from inducing the
activation of innate effector leukocytes to modulating adaptive, lymphocyte-dependent
responses during infection.
1.5 ROLE OF COMPLEMENT IN IMMUNITY TO HELMINTHS
Relatively little is known about the role of complement in vivo in providing resistance to
helminth infections. S. mansoni has been the most widely studied helminth with regards to
the role of complement, with varying outcomes between studies. CVF-treatment of mice
impairs both primary (Santoro et al. 1982) and secondary (Tavares et al. 1978) resistance to
S. mansoni in some studies, but not in others (Sher et al. 1982; Vignali et al. 1988). These
discrepancies can be attributed to differences in the timing and efficacy of the de-
CHAPTER ONE 24complementation treatment, as susceptibility to attack by complement can change as the
parasite matures. Skin-stage schistosomula are vulnerable to lysis by normal rat serum (NRS)
in vitro, however lung-stage schistosomula, which are resistant to complement activation in
vitro, are highly susceptible to complement in vivo (Vignali et al. 1988). In contrast, C5 or
C3 deficiency in mice has no impact on liver or intestinal parasite loads or egg production in
either primary or secondary S. mansoni infections (Ruppel et al. 1982; Sher et al. 1982; La
Flamme et al. 2003), though the development of a long-term Type-2 cytokine response is
compromised in C3-deficient animals (La Flamme et al. 2003).
The role of complement in host resistance to other helminth species can be even more
uncertain. CVF treatment of naturally-resistant strains of mice reduces the extent of killing of
T. taeniaeformis in the liver (Davis and Hammerberg 1988) and comparable outcomes are
observed when C5-deficient are used (Davis and Hammerberg 1990). Similarly, C5-deficient
mice are more susceptible to the development of large cysts when chronically infected with E.
granulosus and this correlates with reduced eosinophil recruitment to infected sites,
implicating C5a as a chemotactic factor (Ferreira et al. 2000). In contrast, C5 does not play a
role in innate or adaptive resistance to S. stercoralis, as C5-deficient mice efficiently recruit
granulocytes and larval killing is similar to that seen in WT mice (Kerepesi et al. 2006). For
S. stercoralis, killing of larvae by neutrophils during primary and secondary infections is C3-
dependent, suggesting that larval killing requires C3-mediated neutrophil adherence rather
than C5a-mediated cell recruitment (Kerepesi et al. 2006). Killing of S. stercoralis larvae
during secondary infections is IgG-dependent and sensitive to CVF treatment, suggesting that
the classical complement pathway is required (Ligas et al. 2003). Together, these studies
suggest that the relative importance and role of complement varies depending on the species
and life cycle of the parasite, the tissue infected and the nature of the humoral or cellular
inflammatory response. The following sections address the mechanism(s) through which the
complement system may protect against helminth infection.
CHAPTER ONE 25 1.5.1 Complement activation by helminths
Complement activation by helminths in vitro has been described for a number of species.
Deposition of the central molecule of the complement system (C3) on the parasite surface is
most often studied but there is also evidence that proteins excreted or secreted by some
parasites also activate complement (Baeza et al. 1994; Irigoin et al. 1996). Binding of other
molecules such as C1q, C4 (Linder and Huldt 1983) and MBL (Klabunde et al. 2000) to
parasites has also been examined. The generation of soluble factors such as C5a has not been
explored in vitro for parasitic helminths. Components of the terminal end of the complement
cascade (e.g. C5-C9) are also deposited on some helminth species (Ruppel et al. 1984;
Kennedy and Kuo 1988), though few studies have demonstrated that the MAC directly
damages helminths. For example, the early stages of some helminths such as cercariae and
newly-transformed schistosomula of S. mansoni (Marikovsky et al. 1986) and Echinococcus
granulosus oncospheres (Heath et al. 1994) are susceptible to complement-mediated lysis.
The pathway to initiation of in vitro complement activation has been described for some
helminths. It is clear that the relative importance of each pathway varies for different parasite
species and the nature of the host serum used. In the presence of serum from naïve animals,
the alternative pathway dominates for various life stages of T. spiralis (Hong et al. 1992), H.
polygyrus (Prowse et al. 1979) and Dirofilaria immitis (Abraham et al. 1988). Different
stages of S. mansoni have been shown to activate all three pathways, with schistosomula and
adult worms capable of triggering the alternative (Santoro et al. 1979; Marikovsky et al.
1986) and classical pathways (Santoro et al. 1979; Linder and Huldt 1983) and cercariae and
adult worms activating the lectin pathway (Klabunde et al. 2000). T. spiralis larvae can also
activate the lectin pathway (Gruden-Movsesijan et al. 2003). These studies are based on
methods such as the determination of sensitivity to Ca2+ depletion and Mg2+ chelation, C4
binding, consumption of complement components, serum C4-depletion and the use of C4 or
CHAPTER ONE 26C1q-deficient serum. As yet, no study has comprehensively investigated the role of all three
complement pathways for complement activation on any helminth species. This is now
feasible given the recent availability of mice genetically engineered to be deficient in various
complement factors.
Very few studies have established that complement is activated in vivo by helminths during
the course of an infection, the first study to do so was by Befus 1977. S. stercoralis L3
recovered from diffusion chambers implanted in skin of naïve and immunised mice were
coated with C3 and the extent of complement activation increased over a 6-24 hr period
(Brigandi et al. 1996). Furthermore, immune mice had higher levels of C3 deposition,
consistent with classical pathway activation. Activation of complement during E. granulosus
infection has been indirectly examined by analysing complement consumption, i.e. observing
a reduction of biologically active serum C3 after infection (Marikovsky et al. 1990; Diaz et
al. 1995).
1.5.2 Complement-dependent leukocyte-mediated killing of helminths
1.5.2.1 Recruitment of effector leukocytes
Little work has been done on the role of complement in cellular inflammatory responses
during helminth infections. The rapid generation of chemotactic factors C3a and C5a makes
this system a prime candidate for mediating early cellular inflammatory responses during an
acute helminth infection. Generation of C3a and C5a can also mediate mast cell activation
(Johnson et al. 1975), which can stimulate the recruitment of other inflammatory leukocytes
by releasing leukotrienes and chemokines (Lukacs et al. 1998; Malaviya and Abraham 2000).
Natural deficiency of C5 impairs eosinophil, but not neutrophil recruitment to the peritoneal
cavity three days post-secondary infection with E. granulosus (Ferreira et al. 2000) but does
not affect eosinophil recruitment to liver six days post primary infection with Taenia
taeniaeformis (Davis and Hammerberg 1990). Similarly, absence of C3, C3aR or C5 does not
CHAPTER ONE 27impair neutrophil or eosinophil recruitment into sub-cutaneous diffusion chambers 1-3 days
after primary or secondary S. stercoralis infection (Kerepesi et al. 2006).
1.5.2.2 Adherence and activation of effector leukocytes
Complement may allow attachment and activation of effector leukocytes, by opsonising the
surface of the parasite with C3 and its proteolytic products. Leukocyte adherence to parasitic
helminths in vitro is mostly dependent on complement and antibody. In the absence of
specific antibody (i.e. in the presence of naïve serum), complement is most important
(MacKenzie et al. 1980; MacKenzie et al. 1981; Butterworth 1984; Badley et al. 1987;
Desakorn et al. 1987; Venturiello et al. 1995; Shin et al. 2001), though other mediators such
as adhesion molecules can also play a role (Brattig et al. 1995; Shin et al. 2001). Eosinophil
and neutrophil adherence to H. polygyrus (Prowse et al. 1979; Penttila et al. 1983) and A.
cantonensis (Shaio et al. 1990) is complement-dependent in the presence of normal serum,
but antibody plays a greater role in immune serum. Whereas newborn T. spiralis larvae are
killed by eosinophils in the presence of antibody (Venturiello et al. 1995), eosinophil
adherence to and killing of infective-stage larvae is partially complement-dependent
(MacKenzie et al. 1980). Adherence of eosinophils and macrophages to N. brasiliensis
(MacKenzie et al. 1980; Egwang et al. 1985; Shin et al. 2001) and eosinophils to H. contortus
(Rainbird et al. 1998) promotes larval killing and this is similarly complement-dependent.
The pathway of complement activation that promotes cell adherence and killing of helminths
is unknown. Cell adherence to helminths does occur in vivo (Wang and Bell 1988; Melo et
al. 1990), and eosinophil degranulation can be detected in close proximity to parasite larvae
(Daly et al. 1999), however the mechanism by which these processes occur has not been
described.
1.5.3 Evasion of complement activation and leukocyte adherence
The complement system has ancient origins. Homologues of complement proteins such as C3
CHAPTER ONE 28have been isolated from the horseshoe crab (Limulus polyphemus), a “living fossil” that has
existed for at least 550 million years (Zhu et al. 2005). Hence, to enhance the chance of
survival within a host, some parasites have needed to evolve strategies to either avoid or limit
complement activation. S. mansoni recovered from either murine skin (Ruppel et al. 1984) or
lungs (Pearce et al. 1990) do not have C3 bound on their surface even though non-injected
infective-stage schistosomula are strong activators of complement in vitro. Similarly, T.
spiralis larvae recovered from muscle fix little C3 in vivo, but strongly activate complement
in vitro (Stankiewicz et al. 1989). There is evidence that the ability of complement and
leukocytes to bind to helminths changes as parasites mature to different life stages. Newborn
T. spiralis larvae or S. mansoni schistosomula activate complement more than their respective
adult parasitic stages (Marikovsky et al. 1990; Hong et al. 1992). Complement-dependent
leukocyte adherence to fourth-stage larvae of D. immitis (Abraham et al. 1988) and
Onchocerca volvulus (Brattig et al. 1991) is less pronounced than on third-stage larvae. The
mechanisms through which these parasites evade complement after only a short time within a
host is not fully understood, but may involve surface expression (Deng et al. 2003) or active
secretion (Badley et al. 1987; Marikovsky et al. 1988) of complement-activating or inhibitory
proteins, or by acquisition of host complement inhibitory factors (Meri et al. 2002).
In conclusion, the role of complement in immunity to parasitic helminths requires further
investigation. Importantly, there is very little information on the relative roles of individual
pathways of complement activation during infection with any helminth species. Considering
the large amount of in vitro evidence that complement contributes to killing of helminths by
facilitating the adherence of eosinophils and other leukocytes, as well as its potential for
promoting inflammatory cell recruitment to sites of infection, there is a significant gap in the
knowledge as to whether complement contributes to eosinophil-dependent killing of
helminths in vivo.
CHAPTER ONE 29
1.6 NIPPOSTRONGYLUS BRASILIENSIS AS A MODEL FOR
STUDYING IMMUNITY TO HELMINTHS
1.6.1 Parasite life cycle
The nematode N. brasiliensis is a parasite of rodents and has been widely used in
experimental studies because the life cycle of the parasite and the immune responses elicited
resemble those seen with some of the tissue-invasive gastrointestinal helminths that infect
humans and domesticated animals (Ogilvie and Jones 1971). N. brasiliensis infections in
mice and rats are convenient because the parasite is not pathogenic for humans, can be easily
maintained in the laboratory, does not require an intermediate host and has a short life-cycle
in these species. Figure 1.2 details the life cycle of N. brasiliensis in mice. In the natural
scenario, the life cycle of the parasite begins with the infective third-stage larvae (L3)
entering the host by penetrating through intact skin. Larvae then migrate from the skin to the
alveoli of the lungs over a period of 1-4 days where they moult and mature into fourth-stage
larvae (L4) (Kassai 1982). Larvae then migrate to the small intestine via the trachea,
oesophagus and stomach. In the small intestine, the parasite undergoes its final moult to
develop to the adult stage, a process that begins approximately five days post-infection (p.i.).
Eggs begin to appear in the faeces by day 7 p.i. and can persist until final parasite expulsion,
9-14 days after infection. Eggs hatch in the external environment and mature through two
moults from first-stage larvae (L1) to L3 (Kassai 1982).
Generally, the life cycle of the parasite in rats and mice is similar, however the total length of
the infection is usually shorter in mice (Kassai 1982). As gene knockout technology is far
easier to achieve and more advanced in mice than in rats, more recently much of the work
investigating immune responses to N. brasiliensis has been done in mice. For experimental
purposes, rodents are usually infected by subcutaneous injection of L3 in a minimal volume
Figu
re 1
.2:
Sche
mat
ic re
pres
enta
tion
of th
e lif
e cy
cle
of N
. bra
silie
nsis
CHAPTER ONE 30of liquid, allowing for more accurate control of delivery and dose. The kinetics of parasite
migration can be monitored experimentally by assessing escape from the skin, determining
lung larval burden 1-2 days p.i., with peak L4 numbers at day 2. Parasite migration to the
small intestine can be examined from day 3 to 7 p.i. and mature egg-producing adult worms
are recoverable in peak numbers from days 6-8 p.i.
The precise route that N. brasiliensis larvae take from the site of inoculation to reach the
lungs is unknown, but may involve transportation through the lymphatic and circulatory
systems (Clarke 1967). Larvae that reach the lungs are passively trapped in arterioles of the
pulmonary parenchyma before actively passing through into the terminal bronchioles and in
the process, cause significant haemorrhage and oedema of lung tissue. This is associated with
increased lung weight, mucus production and tissue damage (Ramaswamy and Befus 1993).
Upon reaching the small intestine, the parasite resides within the gastrointestinal mucosa
where it feeds off nutrients from the gut wall (Ogilvie and Jones 1971; Kassai 1982), growing
very rapidly and undergoing a final moult before reaching sexual maturity. The intestinal
epithelium is damaged during feeding by digestive enzymes released from the parasite’s
oesophageal and excretory glands and cellular debris is taken up via the parasite’s mouth
(Bottjer and Bone 1985). Worms preferentially inhabit the anterior third of the small
intestine, aggregating with other worms (Brambell 1965), presumably to facilitate mating.
Significant changes in the distribution pattern of intestinal worms has been attributed to
immune-mediated damage to the parasite prior to or during the intestinal stage of infection
(Kassai 1982; Dent et al. 1999).
1.6.2 Immune responses to N. brasiliensis
1.6.2.1 Cellular inflammatory responses
The cellular inflammatory response to N. brasiliensis begins very soon after injection into
CHAPTER ONE 31skin. Subcutaneous injection of naïve WT mice typically results in a cellular inflammatory
response that peaks 2-8 h p.i. and is comprised mostly of neutrophils (Taliaferro and Sarles
1939; Daly et al. 1999). This cellular infiltrate subsides two days after infection, by which
time most larvae have migrated from the skin. In fact, in WT mice, relatively few larvae are
recoverable from skin as little as two hours after inoculation (Daly et al. 1999). Hence,
neutrophils that are recruited to the site of infection are largely ineffective at damaging or
killing N. brasiliensis larvae. Eosinophils, lymphocytes and macrophages are also recruited
to N. brasiliensis-infected skin during primary infections of WT mice, albeit in smaller
numbers. The factors which promote this early inflammatory response to N. brasiliensis
infection have not been examined.
Within 4 days of infection, eosinophil numbers in the bone marrow increase significantly
(Rennick et al. 1990) and substantial peripheral blood eosinophilia develops within 10-13
days p.i. (Coffman et al. 1989; Urban et al. 1993). An acute inflammatory response in the
lung peaks 6-9 days after initial infection (Ramaswamy and Befus 1993; Arizono et al. 1996;
Daly 1999; Voehringer et al. 2004), 2-5 days after the larvae had left this site, en route to the
small intestine. There is also evidence that this belated inflammatory response is biphasic,
such that a second substantial peak response occurs approximately 16 days after initial
infection (Ramaswamy and Befus 1989). Curiously, during the short period in which N.
brasiliensis resides in the lungs (1-2 days), the inflammatory response is very mild relative to
that in the skin (Daly 1999; McNeil et al. 2002). The reason for this is unclear and there is
also no obvious explanation for the development of a substantial but delayed and biphasic
inflammatory response after the parasite has left the lungs. Eosinophils are the most prevalent
cell type in this late inflammatory response (Voehringer et al. 2004), though there has been
much interest in IL-4-producing CD4+ T-cells found at 8-12 days p.i. (Shinkai et al. 2002).
The chemoattractant molecules promoting cell recruitment to skin may therefore not be
generated in the lung, or alternatively the process of cell recruitment may be inactivated by
CHAPTER ONE 32the parasite. The cellular immune response in the intestine following N. brasiliensis infection
involves the participation of mast cells and goblet cells, as described in section 1.2.3.
The rapid nature of the life cycle of the N. brasiliensis parasite in naïve mice ensures that it
typically escapes a tissue before the cellular inflammatory response mounted against it is able
to trap the parasite. This is certainly of benefit during primary infections as it means the
parasite is not extensively damaged in transition to the small intestine. However if sufficient
effector cells of the right type can be recruited, for example during secondary infections with
the parasite or perhaps if the host has a concurrent infection with another pathogen, a more
effective immune response can be elicited, resulting in reduced parasite migration, maturation
and egg production (Taliaferro and Sarles 1939; Kassai 1982).
1.6.2.2 Cytokine responses
The cytokine response to N. brasiliensis, along with a number of other helminthic parasites, is
a predominately Type-2 biased response (Kopf et al. 1993; Grencis 1997; McKenzie et al.
1998; Min et al. 2004). The type-2 cytokine response following N. brasiliensis infection is
described in detail in earlier sections, in particular the rapid generation of IL-4-producing
cells (see section 1.2.3), the importance of STAT6 and IL-13 in worm expulsion (see section
1.2.4) and the role of IL-5 in stimulating eosinophil responses (see section 1.3.3.1).
1.6.2.3 Role for eosinophils
While the role of eosinophils in killing some species of helminth remains controversial, the
evidence for these cells as effectors in immunity to N. brasiliensis is strong. Eosinophils have
been shown to adhere to L3 in vitro and release their granule contents (McLaren et al. 1977;
MacKenzie et al. 1981) and can damage the parasite (Shin et al. 2001), such that the larvae
have impaired ability to migrate when subsequently injected into a naïve murine host (Daly et
al. 2004). As discussed in section 1.3.3, the exact mechanisms by which eosinophils kill
CHAPTER ONE 33helminths, including N. brasiliensis in vitro are not completely understood, though adherence
of eosinophils to the parasite has been shown to be antibody and/or complement-dependent
(MacKenzie et al. 1980; Shin et al. 2001). It is not known whether adherence and/or
degranulation of these cells are absolutely required for parasite killing. Other leukocytes such
as macrophages, mast cells and neutrophils have been shown to adhere to N. brasiliensis in
vitro (MacKenzie et al. 1980; Egwang et al. 1984; Egwang et al. 1985), but only
macrophages and eosinophils have been demonstrated to be able to damage the parasite.
Studies with N. brasiliensis infections in IL-5 Tg mice clearly demonstrate that pre-existing
eosinophilia is associated with potent and early resistance, even in naïve animals (Dent et al.
1997a; Shin et al. 1997). These mice exhibit drastically reduced numbers of N. brasiliensis
lung larvae, intestinal worms and faecal eggs when compared to similarly-treated WT mice.
This resistance develops extremely rapidly (within hours) and occurs prior to the lung stage
and independently of adaptive immune mechanisms. Larvae are attacked by eosinophils in
the skin and may be trapped for up to 24-48 h (Daly et al. 1999). Eosinophils also provide
potent resistance to N. brasiliensis during secondary infections, as IL-5-/- and eosinophil-
deficient ∆dblGATA mice exhibit higher lung larval burdens than WT mice 48 h post-
secondary challenge (Knott et al. 2007).
The mechanism of eosinophil-dependent killing of N. brasiliensis in vivo is unknown. Rapid
recruitment of these cells to infected skin, recognition of the parasite and attachment followed
by release of toxic granule proteins may be critical processes. While multiple factors have
been shown to be important for eosinophil recruitment (see section 1.3.3.2) and adherence
(see section 1.3.3.5) in other models, the complement system may be most critical as it can
mediate both processes and potentially trigger degranulation (Takafuji et al. 1996; Egesten
and Malm 1998). Furthermore, the complement cascade can be triggered very rapidly and
independent of adaptive immunity and so it satisfies the criteria as a rapid innate effector
CHAPTER ONE 34mechanism. Complement activation in the very early stages of infection may therefore be
critical for eosinophil-dependent resistance to N. brasiliensis. Until recently, our knowledge
of interactions between the complement system and N. brasiliensis has largely been limited to
in vitro studies (MacKenzie et al. 1980; MacKenzie et al. 1981; Egwang et al. 1984; Shin et
al. 2001). However, in one study of CVF-treated rats, the rate of worm expulsion 15 days p.i.
was unaffected (Jones and Ogilvie 1971). The impact of complement depletion on earlier
stages of N. brasiliensis migration and inflammatory responses in the host has not been
reported.
1.7 INTRODUCTION TO THIS STUDY
The present studies will address the role of complement in the immune response of mice to
infection with the nematode N. brasiliensis. The N. brasiliensis model is a useful model in
which to study the role of complement. Despite the fact that naïve WT mice possess only
modest resistance to the parasite, potent innate immunity is seen in eosinophilic IL-5 Tg mice
and WT mice develop robust resistance to re-infection. The hypotheses underpinning this
thesis are that, during N. brasiliensis infection, complement:
1. stimulates recruitment of effector cells, such as eosinophils;
2. promotes interactions of these cells with the parasite surface;
3. enhances primary and/or secondary resistance of WT mice and
4. facilitates eosinophil-dependent primary resistance of IL-5 Tg mice.
The first aim was to comprehensively characterise the nature of the interactions between N.
brasiliensis, complement proteins and eosinophils. In particular, we were interested in the
specific pathway(s) that mediate complement activation on N. brasiliensis in vitro and if this
mediated the adherence of eosinophils to the parasite surface. To do this we first needed to
develop a new method for analysing and quantifying interactions between complement
CHAPTER ONE 35proteins, eosinophils and parasitic worms. Secondly, we aimed to establish the role of
complement in providing resistance of mice to N. brasiliensis infections. Our focus was on
the role of complement in eosinophil-dependent anti-parasite resistance in the early stages of
infection.
It is expected that this study will provide a detailed analysis of the function of the
complement system during infections with tissue-invasive parasitic helminths.
CHAPTER TWO 36
CHAPTER TWO: QUANTITATION OF COMPLEMENT AND
LEUKOCYTE BINDING TO A PARASITIC HELMINTH SPECIES
1Paul R. Giacomin, 1Hui Wang, 2David L. Gordon and 1Lindsay A. Dent
1School of Molecular and Biomedical Science, University of Adelaide, North Tce, South
Australia, Australia
2Department of Microbiology and Infectious Diseases, Flinders Medical Centre, University of
South Australia, Bedford Park, Adelaide, Australia.
Journal of Immunological Methods-2004 Jun, volume 289, issue 1-2, pages 201-210 doi:10.1016/j.jim.2004.04.024
CHAPTER TWO 37
LINKAGE TO CHAPTER TWO
Complement is the immune system’s first line of defense against invading pathogens and the
notion that it may promote protective immunity to parasitic helminths has been long-held.
The primary importance of complement in this context may be to aid in leukocyte-mediated
immunity, by mediating recruitment of these cells to infected tissues and their adherence to
the parasite. Helminths are strong activators of complement in vitro (Santoro et al. 1979) and
several leukocyte subsets can adhere to these parasites in both complement-dependent and
independent manners (MacKenzie et al. 1981). The importance of the complement system
may vary depending on the species of the helminth, the stage of parasite maturation and the
species of the host. Much of our understanding of the nature of the interactions between
complement proteins, helminths and leukocytes has been derived from in vitro studies. While
these studies gained valuable information regarding the importance of complement, the
methodologies employed had some major limitations, where they were often purely
qualitative, subjective and inefficient. Previously used methods for quantifying complement
activation and cell adherence on helminths are outlined in greater detail in the introduction to
Chapter Two.
The broad aim of this research project was to further our understanding of how complement
may promote resistance to parasitic helminth infection. Determining the specific pathway of
complement activation involved was a major focus, which initially required the development
of improved in vitro assays that allowed sensitive, automated and objective quantification of
complement deposition and cell adherence to helminths.
In summary, Chapter Two describes novel fluorescence-based methods for analysing and
quantifying both the activation of complement on a species of helminth (N. brasiliensis) and
the adherence of eosinophils to this same parasite. Importantly, the new techniques were used
CHAPTER TWO 38to establish the pathway involved in activation of human and mouse complement on N.
brasiliensis infective larvae and the role of complement in eosinophil adherence to the
nematode.
Giacomin, P.R., Wang, H., Gordon, D.L. and Dent, L.A. (2004) Quantitation of complement and leukocyte binding to a parasitic helminth species. Journal of Immunological Methods, v. 289, (1-2), pp. 201-210, June 2004
NOTE: This publication is included in the print copy of the thesis
held in the University of Adelaide Library.
It is also available online to authorised users at:
http://dx.doi.org/10.1016/j.jim.2004.04.024
CHAPTER THREE 39
CHAPTER THREE: LOSS OF COMPLEMENT ACTIVATION AND
LEUKOCYTE ADHERENCE AS NIPPOSTRONGYLUS BRASILIENSIS
DEVELOPS WITHIN THE MURINE HOST
1Paul R. Giacomin, 1Hui Wang, 2David L. Gordon, 3Marina Botto and 1Lindsay A. Dent
1School of Molecular and Biomedical Science, University of Adelaide, North Tce, South
Australia, Australia
2Department of Microbiology and Infectious Diseases, Flinders Medical Centre, University of
South Australia, Bedford Park, Adelaide, Australia.
3Molecular Genetics and Rheumatology Section, Faculty of Medicine, Imperial College,
London W12 0NN, England, UK.
Infection and Immunity-2005 Nov, volume 73, issue 11, pages 7442-7449 doi:10.1128/IAI.73.11.7442-7449.2005
CHAPTER THREE 40
LINKAGE TO CHAPTER THREE
Chapter Two outlined the development of new and improved methods for quantification of
the binding of complement proteins and leukocytes to parasitic helminths. Development of
these methodologies was critical for the studies comprised within Chapter Three.
Implementation of these methods generated important experimental data regarding the in vitro
interactions between the complement system, leukocytes and helminths. The mechanism of
deposition of both human and mouse C3 was determined and interestingly, the pathway by
which complement was activated differed depending on the species of serum used. Human
C3 was deposited either via the Ca2+-dependent classical or lectin pathways, we could not
conclusively determine the relative contribution of each individual pathway due to lack of
reagents. Activation of the classical pathway in naïve human serum would be unlikely to
have occurred due to the absence of parasite-specific antibody, whether antibody-independent
classical pathway activation occurred was not resolved. The fact that the Ca2+-independent
alternative pathway dominated for mouse C3 deposition was expected, as the alternative
pathway has been shown to be important for complement activation on a number of species of
helminth (Prowse et al. 1979; Santoro et al. 1979; Abraham et al. 1988; Hong et al. 1992).
As N. brasiliensis is a parasite that infects rodents but not humans, the primary focus for the
remainder of this study was on the role of the mouse complement system in immunity to the
parasite. While methods of Ca2+/Mg2+ depletion are useful for some purposes, they are not
very specific as EGTA can chelate Mg2+ at high concentrations. Hence, a more conclusive
demonstration of the relative roles of each complement pathway is required and this will
involve the use of serum taken from mice genetically engineered to be deficient in various
complement factors. The outcomes of these studies are included in the following chapter.
Chapter Two demonstrated that complement is the major contributor to the adherence of
eosinophil-rich mouse peritoneal leukocytes to infective N. brasiliensis larvae. This was in
CHAPTER THREE 41agreement with previous studies using the N. brasiliensis model as well as some other
helminths (MacKenzie et al. 1980; MacKenzie et al. 1981; Butterworth 1984; Badley et al.
1987; Desakorn et al. 1987; Venturiello et al. 1995; Shin et al. 2001). Nevertheless, some
degree of cell adherence did occur in the presence of serum depleted of C3 activity by heat
treatment, suggesting complement-independent factors can contribute, though this may be a
minor pathway. Methods for heat-inactivation of serum may not be 100% effective, hence
using serum deficient in various complement factors will clarify the influence of complement
on cell adherence to N. brasiliensis L3, while also determining the precise pathway involved.
This will represent the most definitive demonstration of the role of each individual
complement pathway in mediating eosinophil adherence to a parasitic helminth species; the
results of this study are included in Chapter Three.
As N. brasiliensis is a parasite that undergoes several stages of maturation as it migrates
through different tissues, the ability of the complement system and leukocytes to interact with
the parasite may change. This may be part of an immune evasion mechanism used by the
parasite to avoid complement recognition, inflammatory responses, eosinophil adherence and
eosinophil degranulation. The results from Chapter Two clearly indicate that infective-stage
L3 are sensitive to activation of the mouse alternative complement pathway and adherence of
eosinophils. The ability of complement and leukocytes to bind to other life stages of N.
brasiliensis, e.g. skin-recovered larvae, lung-stage L4 or adult intestinal worms is unknown.
Hence, the capacity for in vivo binding of host-derived complement as well binding of
exogenous C3 from NMS will be assessed for each of the aforementioned life-stages. The
specific pathway to complement activation will also be compared. Determining if and when
complement and eosinophils are effective at recognising N. brasiliensis will be important for
addressing our final goal of examining the role of complement in vivo.
CHAPTER THREE 42In summary, the following chapter compares the ability of mouse complement and
eosinophil-rich leukocytes to bind to infective-stage L3, skin-recovered larvae, lung-stage L4
and adult intestinal worms, with a particular focus on determining the most important
complement pathway involved using various complement-deficient sera.
Giacomin, P.R., Wang, H., Gordon, D.L., Botto, M. and Dent, L.A. (2005) Loss of complement activation and leukocyte adherence as Nippostrongylus brasiliensis develops within the murine host.Infection and Immunity, v. 73 (11), pp. 7442-7449, November 2005
NOTE: This publication is included in the print copy of the thesis
held in the University of Adelaide Library.
It is also available online to authorised users at:
http://dx.doi.org/10.1128/IAI.73.11.7442-7449.2005
CHAPTER FOUR 43
CHAPTER FOUR: THE ROLE OF COMPLEMENT IN INNATE,
ADAPTIVE AND EOSINOPHIL-DEPENDENT IMMUNITY TO THE
NEMATODE NIPPOSTRONGYLUS BRASILIENSIS
1Paul R. Giacomin, 2David L. Gordon, 3Marina Botto, 4Mohamed R Daha, 5Sam D.
Sanderson, 6Stephen M. Taylor and 1Lindsay A. Dent
1School of Molecular and Biomedical Science, University of Adelaide, North Tce, South
Australia, Australia
2Department of Microbiology and Infectious Diseases, Flinders Medical Centre, University of
South Australia, Bedford Park, Adelaide, Australia.
3Molecular Genetics and Rheumatology Section, Faculty of Medicine, Imperial College,
London, UK.
4Leiden University Medical Center, Department of Nephrology, Albinusdreef 2, Leiden, The
Netherlands
5University of Nebraska Medical Center, Omaha, Nebraska, USA
6School of Biomedical Sciences, University of Queensland, Brisbane, Queensland, Australia
Published online: Molecular Immunology doi:10.1016/j.molimm.2007.05.029
Full citation: Molecular Immunology-2008 Jan, volume 45, issue 2, pages 446-
455
CHAPTER FOUR 44
LINKAGE TO CHAPTER FOUR
Chapter Three described that the ability of complement and eosinophils to recognise N.
brasiliensis changes as the parasite matures. Infective-stage larvae were the most susceptible
to complement activation by the alternative pathway and cell adherence, though the lectin
pathway played a minor role in causing C3 deposition. Complement was activated in vivo on
larvae injected into skin air pouches; however parasites recovered from lungs or adult worms
taken from the intestine did not have complement bound on their surface. Furthermore, while
adult worms did bind C3 and leukocytes via alternative pathway activation after treatment
with exogenous serum and peritoneal cells, lung-stage larvae resisted complement activation
and cell adherence and the small amount of C3 that did bind was via lectin pathway
activation. These findings have important implications for the next part of the study which
aims to investigate the role of complement in immunity to N. brasiliensis in vivo. This will be
addressed using the various lines of complement-deficient mice whose sera we used in the
studies described in Chapter Three. Specifically, mice deficient in either the classical
pathway (C1qa-/-), alternative pathway (Bf-/-) or all complement pathways (C3-/-) were used.
To address the question of whether complement mediates eosinophil-dependent resistance to
N. brasiliensis, eosinophilic IL-5 Tg mouse strains deficient in C1q, factor B or C3 were
generated. We then used these mouse strains in the well-established N. brasiliensis model,
analysing immunity during both primary and secondary parasite infections.
Complement and eosinophils may be most effective in the very early stages of innate N.
brasiliensis infection, soon after parasite entry into the skin, as once the parasite reaches the
lungs their impact may be reduced (see Chapter Three). Hence, much of Chapter Four
focused on the early events of N. brasiliensis infection (0-150 min p.i.) using a skin air pouch
model. Parameters such as in vivo C3 deposition on the parasite, leukocyte adherence,
inflammatory cell recruitment and eosinophil degranulation were monitored. In addition, rate
CHAPTER FOUR 45of parasite migration from the skin to the lungs and the small intestine in normal and
complement-deficient mice with or without IL-5 transgene expression were assessed. Our
hypothesis, based on our findings from Chapter Three, was that the alternative pathway would
be most important for immunity to N. brasiliensis. Hence, absence of factor B in mice may
result in reduced inflammatory leukocyte recruitment to the skin, attachment of these cells to
the parasite and consequently the rate of parasite migration would be enhanced compared to
WT or single-mutant IL-5 Tg control mice.
In summary, the following chapter describes the role of various pathways of complement
activation in immunity of mice to N. brasiliensis infection with a focus on their role in
promoting eosinophil-dependent resistance to the parasite.
Paul R. Giacomin, P.R., Gordon, D.L., Botto, M., Daha, M.R., Sanderson, S.D., Taylor, S.M. and Dent, L.A. (2008) The role of complement in innate, adaptive and eosinophil-dependent immunity to the nematode Nippostrongylus brasiliensis. Molecular Immunology, v. 45 (2), pp. 446-455, January 2008
NOTE: This publication is included in the print copy of the thesis
held in the University of Adelaide Library.
It is also available online to authorised users at:
http://dx.doi.org/10.1016/j.molimm.2007.05.029
CHAPTER FIVE
CHAPTER FIVE: DISCUSSION AND CONCLUSION
CHAPTER FIVE 46
5.1 GENERAL DISCUSSION
Eosinophils equip the host immune system with a potent weapon against infection with
parasitic helminths. The precise mechanisms used by these cells to damage helminths are
unknown, though previous in vitro research has suggested that complement-mediated
adherence to the surface of the parasite may be important (MacKenzie et al. 1980;
Butterworth 1984; Shin et al. 2001). Furthermore, complement activation may be critical for
leukocyte recruitment to parasite-infected tissues. The aim of the present study was to
investigate the pathways to complement activation important for the in vitro interaction of
eosinophils with N. brasiliensis and also to determine if complement was important to provide
eosinophil-dependent resistance to helminth infections.
5.1.1 Summary of main findings
N. brasiliensis L3 activate human and mouse complement when exposed to serum in vitro and
we designed a novel assay that allowed rapid, automated and objective quantification of
complement activation and cell adherence to N. brasiliensis (Giacomin et al. 2004). The
assay was used to demonstrate that N. brasiliensis L3 activate human complement via the
classical or lectin pathways. In contrast, the alternative pathway mediated mouse C3
deposition on L3 and this was the primary mediator of the adherence of eosinophil-rich mouse
leukocytes in vitro (Giacomin et al. 2005). However, the roles of complement in vivo are
more complex. N. brasiliensis larvae did activate the alternative pathway of complement 30
min after injection into the skin of mice and this correlated with aggregation of larvae.
Unexpectedly, cell adherence to the parasite and larval aggregation at this site also occurred in
complement-deficient mice over a longer time frame of up to 2.5 h (Giacomin et al. 2008). In
the absence of the alternative complement pathway, eosinophil degranulation in the skin 30
min post-parasite injection was reduced, but eosinophils efficiently degranulated in the
absence of complement at a later time. The presence of the alternative complement pathway
CHAPTER FIVE 47 and the C5aR was critical for eosinophil recruitment to the skin 30 min p.i., though other
factors recruited eosinophils at later times. Interestingly, parasites recovered from the lungs
resisted complement activation and leukocyte adherence when exposed to serum and
eosinophil-rich leukocytes ex vivo and the small amount of complement that did bind was via
the lectin pathway of activation. Evasion of complement activation by the parasite began in
the first 2.5 h of infection, while the parasite resided within the skin and this may explain in
part why complement was of most significance in the first 30 min of infection. Intestinal
worms bound complement and leukocytes ex vivo via the alternative pathway of activation,
but not in vivo. Absence of the alternative complement pathway caused slight but significant
increases in lung larval burdens in normal and IL-5 Tg mice during a primary infection.
However, IL-5 Tg mice remained highly resistant to primary N. brasiliensis infection even in
the complete absence of complement, suggesting that eosinophils can provide resistance in a
complement-independent manner. Similarly, robust resistance of mice to secondary N.
brasiliensis infection was complement-independent. In conclusion, the alternative
complement pathway is important for mediating interactions of eosinophils with N.
brasiliensis in vitro and in the very early stages of a parasite infection. However evasion of
the complement system by the parasite and the participation of complement-independent
factors over time means its influence is reduced and that overall, complement does not
contribute greatly to immunity of mice to N. brasiliensis infection.
5.1.2 Complement and eosinophil-dependent immunity to helminths
5.1.2.1 Eosinophil-dependent resistance to helminths
While the role of eosinophils during helminth infection remains somewhat controversial
(Behm and Ovington 2000; Meeusen and Balic 2000), eosinophils are undoubtedly effective
mediators of resistance to N. brasiliensis in mice. One of the most conclusive pieces of
evidence for this was obtained using eosinophilic IL-5 Tg mice, where larvae were trapped at
the skin injection site within the first 2 h, with fewer larvae migrating to the lungs and small
CHAPTER FIVE 48 intestine than in WT mice (Dent et al. 1997a; Daly 1999; Daly et al. 1999). The results from
the present study similarly support a role for eosinophils in resistance to helminths. We
hypothesised that complement would be particularly important for mediating innate killing of
the parasite by eosinophils at the site of initial infection, in the skin. We therefore generated
IL-5 Tg mouse strains deficient in various complement factors. Our evidence clearly
demonstrates that in this model, and contrary to our initial hypothesis, complement-deficient
IL-5 Tg mice are almost as resistant to primary N. brasiliensis infection as IL-5 Tg animals.
Given that at 30 min p.i., eosinophil recruitment and degranulation, as well as larval
aggregation were reduced in the absence of factor B, we expected that eosinophils would have
impaired ability to restrict the migration of N. brasiliensis larvae. There was some evidence
that trapping of larvae in the skin of IL-5 Tg mice 150 min p.i. was partially dependent on the
alternative pathway. In the present study, the entrapment of larvae in the skin was not as
extensive as seen previously (Daly et al. 1999), perhaps due to differences in the strain of the
mice used. Nevertheless, overall resistance to N. brasiliensis was not impaired in the absence
of C3 or factor B, as parasite burdens in the lungs or small intestine were not greatly enhanced
compared to IL-5 Tg. This implies that eosinophils can efficiently limit N. brasiliensis
migration in the absence of complement and in our IL-5 Tg model, without specific antibody.
Damage to the parasite may be inflicted at the site of initial infection and possibly at other
sites in the pre-lung stage. The precise pathway that N. brasiliensis undertakes to get to the
lungs has not been conclusively established, though the parasite may pass through the
peripheral blood and lymphatic systems (Clarke 1967). Alternatively, damage incurred in the
skin may impair the ability of the parasite to colonise the small intestine, in particular. This
highlights one of the limitations of our “snapshot” method for analysing parasite migration
through the host, where mice are sacrificed at specific time points and only a limited number
of organs are examined for parasites. In the future, parasite migration in vivo should be
analysed using techniques such as whole-body imaging, where larvae labeled with a
fluorochrome or similar indicator might be tracked through a living rodent host, as has been
CHAPTER FIVE 49 done for green-fluorescent protein (GFP)-expressing bacteria (Zhao et al. 2001). Purified
eosinophils could then be labeled with a different colour dye and injected into the rodent so
that these cells could be tracked simultaneously to establish where they interact and co-
localise with the parasite.
5.1.2.2 Role of complement in vivo and in vitro
The present study included a comprehensive examination of the role of complement during
infection with a species of parasitic helminth, taking it from an in vitro setting through to its
role in vivo under various levels of immunity. These findings highlight that care must be
taken when trying to extrapolate in vitro observations to the in vivo situation. Our in vitro
work, and that done by others (MacKenzie et al. 1980; Shin et al. 2001), suggest that
eosinophils adhere to N. brasiliensis L3 via activated complement proteins and this is
important for parasite killing. However, while the alternative complement pathway did
mediate several processes in vivo that may be important for inflicting damage to the parasite
(C3 deposition, eosinophil and neutrophil recruitment, EPO release, larval aggregation), other
mechanisms compensated for a lack of complement and these may not be present in vitro.
Our results echo those from previous investigations on the role of complement in immunity to
other helminth species. For example, complement may contribute to some important immune
responses such as leukocyte recruitment (Ferreira et al. 2000; Giacomin et al. 2008), cytokine
responses (La Flamme et al. 2003) and cellular cytotoxicity against a particular larval stage or
within an individual infected tissue (Giacomin et al. 2004; Giacomin et al. 2005; Kerepesi et
al. 2006), but ultimately ablation of complement may not greatly affect parasite infection
kinetics or worm burdens. Complement can and probably does play an important role during
N. brasiliensis infection of normal mice, but there are compensating factors that may be
accentuated in complement-deficient mice. This is an inherent problem when working with
genetically modified mice. Mice surviving a gene deletion may only be able to do so if
capable of compensating for the deletion by up-regulating what may otherwise be relatively
minor homeostatic or defensive mechanisms. This, compounded with evasion mechanisms
CHAPTER FIVE 50 used by the parasite in vivo, may limit the efficacy of the complement system in anti-parasite
resistance.
We have classified the relative importance of the three pathways of complement activation
and established that in the mouse, the alternative pathway plays the greatest role in vivo and in
vitro. The lectin pathway does contribute to in vitro C3 deposition on L3 and L4 but we were
unable to directly determine the relative importance of the lectin pathway in vivo in this
infection model. This might be addressed by using C4-deficient mice, but these animals are
also deficient in the classical pathway (Fischer et al. 1996). Similarly, MBL-deficient lines of
mice have been produced, but the lectin pathway can be initiated by binding of a variety of
lectins other than MBL (Chan et al. 2006). In any case, the absence of C3 in mice caused no
greater defect than ablation of factor B for any of the parameters studied and so the lectin
pathway would seem to play little or no role in the overall immunity to N. brasiliensis.
Interestingly, the pathway to complement activation on L3 differed depending on whether
human or mouse sera were used (Giacomin et al. 2004), leading to the possibility that the role
of complement may vary depending on the species of the host. It is very common for the
immune response to helminth infection to be studied in mice using non-natural parasite
species, hence care must be taken when making conclusions from such studies. N.
brasiliensis is a parasite that naturally infects both rats and mice (Kassai 1982), but there is
evidence that immune responses to non-natural parasites can include enhanced non-specific
inflammation and parasite rejection (Meeusen and Balic 2000). Perhaps complement
activation is enhanced, or alternatively immune evasion mechanisms used by the parasite are
less effective during infection with non-natural helminth species. Indeed, studies with non-
natural parasites of mice have yielded more positive results regarding the role of complement
during helminth infection than we have in the present study (Santoro et al. 1982; Ferreira et
al. 2000; Kerepesi et al. 2006). Humans are accidental hosts for some species of helminth
CHAPTER FIVE 51 (e.g. T. canis) and can cause significant pathology, so understanding the immune responses to
both natural and non-natural helminths is important.
5.1.2.3 Complement-dependent eosinophil recruitment to parasite-infected skin
The present study has advanced our knowledge of how eosinophils are recruited to sites of
helminth infection. Activation of the alternative complement pathway played an important
role in the early C5aR-dependent recruitment of eosinophils to the skin during N. brasiliensis
infections. We did not directly test whether C5a was generated in the skin of mice during
infection, however there are ELISAs available that could be used to measure mouse C5a in air
pouch supernatants. One point to consider is that C5a (and C5a des-Arg) also bind to the
C5L2 receptor (Okinaga et al. 2003), which is not blocked by the C5aR inhibitor we used.
Hence it is conceivable that C5a could react with leukocytes via different pathways, though
C5L2 is considered to be a non-signalling C5a receptor with little biological role (Okinaga et
al. 2003). While the role of C3a has not been directly elucidated, C5a is clearly more
important in this model since C5aR inhibition completely blocked early eosinophil
recruitment to the same degree as in C3-/- mice, which are unable to generate C3a. Hence,
during the early stages of N. brasiliensis infection, C5a is generated by activation of the
alternative complement pathway and eosinophils that are exquisitely sensitive to C5a are
rapidly and preferentially recruited to the inoculation site.
5.1.2.4 Complement-independent eosinophil recruitment
Eosinophil recruitment to the skin was complement-independent within 2.5 h of infection.
Many factors may have mediated this recruitment, including eotaxin, PAF and AMCase
(Resnick and Weller 1993; Rothenberg and Hogan 2006). Data from our laboratory suggest
that eotaxin-1-deficient/IL-5 Tg mice recruit less eosinophils than single-mutant IL-5 Tg mice
2-4 h post-N. brasiliensis infection (Knott et al, unpublished data), consistent with the time
frame where complement-independent eosinophil recruitment is apparent. Similarly,
blockade of CCR3 with mAbs impairs blood and tissue eosinophil responses in N.
CHAPTER FIVE 52 brasiliensis-infected mice (Grimaldi et al. 1999). Eotaxin is a prominent eosinophil chemo-
attractant molecule during infections with other species of helminth (Simons et al. 2005;
Dixon et al. 2006). However, as with our observations in complement-deficient/IL-5 Tg
mice, absence of eotaxin in IL-5 Tg mice does not impair resistance to N. brasiliensis (Knott
et al, unpublished data). Factor B- and eotaxin-deficient/ IL-5 Tg triple-mutant mice could be
generated to test whether further reductions in eosinophil recruitment impair resistance to this
parasite, but the genetic backgrounds of the mutant animals currently available to our
laboratory are not compatible, hence extensive backcrossing may be required. A less time-
and labour-expensive strategy would be to treat eotaxin-deficient mice with the C5aR
inhibitor PMX53 and then monitor parasite infection kinetics and inflammatory responses.
Chitinase-like proteins, such as Ym1 and AMCase have been shown to be up-regulated during
helminth infection (Nair et al. 2005; Pesce et al. 2006) and these may directly or indirectly
promote inflammatory responses such as leukocyte recruitment (Owhashi et al. 2000; Zhu et
al. 2004). Recently, intranasal chitin administration was shown to induce recruitment of IL-4-
expressing eosinophils and neutrophils into the lungs of mice and also to activate AAMs
(Reese et al. 2007). Some species of helminth express chitin synthase (Harris et al. 2000),
though this has not been described for N. brasiliensis. Since the functional importance of
parasite-derived chitin and mammalian chitinase proteins during helminth infection is yet to
be fully defined, this represents an exciting new direction for the study of immunity to
helminths. Lastly, the number of eosinophils in the skin of IL-5 Tg mice under basal
conditions may have been sufficient to cause killing of N. brasiliensis, rendering the large
degree of eosinophil recruitment unnecessary. However, co-injection of peritoneal
eosinophils with N. brasiliensis L3 does not affect kinetics of parasite migration (Daly 1999).
Since those peritoneal cells may have been in a different state of activation to those residing
in the skin of IL-5 Tg mice, this issue remains unresolved. Further experiments could be
conducted to determine if leukocytes recruited into air pouches provide protection when
CHAPTER FIVE 53 transferred with larvae into WT or eosinophil-deficient ΔdblGATA mouse recipients. This
would address whether the process of recruitment activated eosinophils to a level sufficient
for anti-parasite immunity.
5.1.2.5 Eosinophil versus neutrophil recruitment
The recruitment of eosinophils in response to N. brasiliensis infection was extremely rapid
and consistent with that observed in experimental models of nasal and pulmonary allergic
inflammation (Tedeschi et al. 1994; Tiberio et al. 2003). To our knowledge, this is the most
rapid recruitment of eosinophils into parasite-infected tissues yet reported. In some infectious
diseases, neutrophils are the leukocytes most rapidly recruited and these cells are seen in large
numbers in the first 2 h of N. brasiliensis infection of WT mice (Daly et al. 1999). We have
shown that neutrophil recruitment to the skin following N. brasiliensis infection is partially
dependent on the alternative complement pathway and C5aR, however other factors also play
a role. Potential alternative neutrophil recruitment factors include PAF, LTB4, prostaglandins
and fMLP (Jagels and Hugli 1992). The role that neutrophils play in the skin is unclear
though they do not appear to greatly restrict larval migration. WT mice exhibit only a modest
level of resistance to infection, despite an extensive influx of neutrophils into the skin soon
after injection of larvae. Neutrophils have been shown to adhere to N. brasiliensis larvae both
in vitro (MacKenzie et al. 1981) and in the current study in the skin, however there is little
evidence of significant damage to L3. Although neutrophils may be non-specific and rapidly
recruited effector cells that specialize in phagocytosis, these cells have been shown to be able
to damage some helminths (Incani and McLaren 1981; Shaio et al. 1990). In particular, the
nematode S. stercoralis is susceptible to attack by neutrophils in the skin, though in these
studies larvae were mechanically trapped in diffusion chambers for days and hence exposed to
an ongoing barrage of neutrophils (Ligas et al. 2003; Galioto et al. 2006; Kerepesi et al.
2006). This is in contrast to what happens in the N. brasiliensis model, where larvae are free
to undergo their natural route of infection, limiting the opportunity of neutrophils to interact
CHAPTER FIVE 54 with the parasite.
5.1.2.6 Complement-independent leukocyte adherence to helminths
So, if complement activation is not essential, how do eosinophils recognize and kill N.
brasiliensis in vivo? Logically, eosinophils would need to make close contact with the
surface of the parasite to inflict damage, hence adherence may be a critical step. It is possible
that the same factors that mediate the relatively minor degree of complement-independent cell
adherence in vitro (Giacomin et al. 2004), also operate in vivo. The level of the fibronectin
receptor VLA-4 on eosinophils increases during infections with some helminth species
(Brattig et al. 1995). Both fibronectin and complement deposition on N. brasiliensis L3 have
been shown to occur in vitro (Shin et al. 2001). Each of these factors was demonstrated to be
important for the adherence of eosinophils, as treatment with either anti-VLA-4 or anti-CR3
antibodies both abolished cell adherence and the larval immobilization that followed. The
fact that blocking either fibronectin receptors or complement receptors completely inhibited
adherence was curious and the authors suggested, but did not prove, that simultaneous
engagement of both receptors may be essential. The in vitro data gained from the current
study does not refute the involvement of such a mechanism, as absence of C3 or heat-
treatment of serum almost completely blocked eosinophil adherence. However, our in vivo
data does not support such an hypothesis, as cells adhered effectively in vivo in C3-deficient
mice. Other factors may be mediating cell adherence in vivo, or perhaps the involvement of
the fibronectin pathway is enhanced in complement-deficient mice as a compensatory
mechanism
Clearly, more work needs to be done to establish the role of fibronectin and other adhesion
molecules in eosinophil-dependent killing of helminths. Firstly, deposition of fibronectin on
the surface of N. brasiliensis larvae in the skin of mice could be detected by
immunofluorescence. Secondly, anti-VLA-4 antibodies could be administered locally in skin
air pouches to attempt to block cell adherence in both WT and complement-deficient mice.
CHAPTER FIVE 55 There is evidence that other adhesion molecules can contribute to eosinophil-parasite
interactions, as anti-L-selectin antibodies reduce in vitro cytotoxicity against S. mansoni
schistosomula (Nutten et al. 1999). Curiously, the authors did not observe any differences in
cell adherence to larvae, hence this ligand may have other roles in leukocyte effector cell
function, such as activation or degranulation. Other factors such as natural antibodies may
also promote eosinophil adherence to helminths. TLRs are expressed by eosinophils (Plotz et
al. 2001) and can facilitate eosinophil recognition and activation during viral infections
(Phipps et al. 2007), though very little is known regarding the role of TLRs during infections
with helminths. Alternatively, eosinophils may not need to adhere to the parasite in order to
inflict damage. Other cell types, such as platelets, have been shown to be toxic to
schistosomula without adhering, most probably via the release of toxic oxygen free radicals
on the parasite surface at a distance (Joseph et al. 1985). Exploring such complement-
independent factors was beyond the scope of the current study. Future research should further
investigate how eosinophils adhere to parasites and focus on determining if it is necessary for
the immobilisation and/or killing of these pathogens.
5.1.2.7 Eosinophil degranulation
The precise role of eosinophil degranulation in killing parasitic helminths is unclear.
Furthermore, there is also debate as to whether mouse eosinophils readily degranulate in
models of asthma-like pulmonary eosinophilic inflammation (Persson et al. 1997; Lee and
Lee 2005). The present study has demonstrated that mouse eosinophils do degranulate under
physiological conditions during helminth infections and this is consistent with previous
studies with N. brasiliensis and other parasites (Daly et al. 1999; Herbert et al. 2000; Shinkai
et al. 2002; Simons et al. 2005). Furthermore, we demonstrated that EPO levels in the skin
30 min after N. brasiliensis infection were reduced in the absence of the alternative
complement pathway. We did not establish whether this reduction in EPO activity was
because fewer eosinophils were recruited into the skin at this time, or if complement
activation is important for triggering eosinophil degranulation. Certainly, eosinophil
CHAPTER FIVE 56 degranulation was restored to normal levels at a later time point (150 min p.i.) when
eosinophil recruitment was more pronounced. It is therefore possible that the process of
recruitment is sufficient to activate eosinophils, as tissue-dwelling eosinophils generally have
a greater ability to degranulate than those in the peripheral blood (Dvorak and Ishizaka 1994).
To determine if complement activation on helminths promotes eosinophil degranulation,
purified eosinophils and N. brasiliensis L3 could be co-injected into air pouches of WT or
factor B-deficient mice. Pouches could be lavaged 30 min after co-injection and cell-free
EPO levels could then be measured in the exudates. This experiment would assist in
determining if absence of the alternative complement pathway reduces the level of
degranulation of the transferred eosinophils.
While not demonstrated in this study, it is likely that eosinophil degranulation occurred within
the aggregates of larvae and cells formed in the skin. Closer inspection of these aggregates by
electron microscopy or staining for MBP using antibodies is required to determine if these
cells do indeed degranulate. A reduction in eosinophil degranulation and larval aggregation
in the very early stages of infection may have caused the increased rate of parasite migration
to the lungs that was observed in factor B-deficient animals. In addition, the technique for
measuring cell-free EPO activity may have underestimated the level of eosinophil
degranulation, as it is possible that eosinophil granule proteins were sequestered from the
aqueous phase by binding directly to the surface of the parasite. It is unclear whether
eosinophil degranulation is a mechanism by which eosinophils kill N. brasiliensis. Future
studies should assess N. brasiliensis infection kinetics in MBP or EPO-deficient mouse
strains. These mice have been used in other models of helminth infection, where the granule
proteins were shown either to be protective (Specht et al. 2006) or had no impact on the
progress of parasite infection (Abraham et al. 2004; Ramalingam et al. 2005). Similar studies
could be conducted using MBP-deficient/IL-5 Tg or EPO-deficient/IL-5 Tg mice, which
would have large numbers of eosinophils with more limited granule contents. The present
CHAPTER FIVE 57 study has built on important observations made by our group and by others regarding the
nature of eosinophil degranulation during helminth infection. The exact stimuli for eosinophil
degranulation remains unknown and the roles of individual granule proteins are yet to be
determined.
5.1.2.8 Larval aggregation
The aggregation of larvae after injection into mouse skin was an unexpected phenomenon that
has not been described elsewhere in the literature. It is interesting that the rate at which
aggregation occurred was dependent on the alternative complement pathway. In our in vitro
assay a small degree of larval aggregation did occur (Giacomin et al. 2004), but these weakly-
formed aggregates were easily disaggregated in the washing steps. Aggregates formed in vivo
were more difficult to break apart by pipetting. However, larvae have a startling ability to
escape from aggregates within a few hours, such that relatively few larvae were recoverable
by 2.5 h p.i. Furthermore, the formation of aggregates in WT mice did not greatly impede
migration to the lungs, as lung larval burdens in these mice were only slightly lower than in
factor B-deficient mice where aggregation did not occur until later. Hence, further research is
required to determine how this parasite escapes aggregates. It is feasible that ex-sheathment
and/or the release of proteolytic factors are key to this process.
We were unable to conclusively determine the identity of the fibrous material in the
aggregates that “tethers” larvae together. It is possible that it was fibrin, which is generated
via activation of the coagulation cascade after hydrolysis of fibrinogen by thrombin (Bouma
and Mosnier 2006). Binding of fibrin or prothrombin to the surface of a parasite could not
only facilitate aggregation of larvae but could also promote cell adherence (Kuijper et al.
1997) or the generation of chemotactic factors such as C5a (Huber-Lang et al. 2006). Why
complement activation on larvae would enhance activation of the coagulation system is
unclear, however there is evidence of functional overlap between both pathways, where
complement activation can induce tissue factor, which stimulates thrombin formation (Esmon
CHAPTER FIVE 58 2004). Hence, elements of the clotting system may make significant contributions to the
immune response to N. brasiliensis and may explain the limited role for complement in
resistance of IL-5 Tg mice to infection. Future studies should investigate this by examining
fibrin deposition and thrombin activation in vivo. Inhibitors of coagulation (e.g. antithrombin
III or Hirudin) could be used in experiments designed to assess larval aggregation, cell
adherence, leukocyte recruitment and resistance to parasite infection.
5.1.3 Evasion of complement activation by helminths
N. brasiliensis has the ability to resist complement activation after a short period of time
within the host. We initially reported that lung-stage larvae recovered 24-48 h p.i. do not bind
appreciable levels of C3 or leukocytes in vivo or ex vivo (Giacomin et al. 2005), but further
analysis revealed that the ability to resist C3 deposition begins while the parasite resides in
the skin (Giacomin et al. 2008). C3 deposited on the surface of the parasite within the first 30
min was reduced or was no longer detectable after 2.5 h. This was a surprise, as C3b
deposition is covalent and the molecule is not easily shed, though it may have been degraded
by factor I to a state that was unable to be detected by our anti-C3 antibodies, for example
C3d or C3dg. It is unlikely that reductions in C3 deposition were solely due to parasite ex-
sheathment, as chemically ex-sheathed larvae still bound C3. Acquisition of host complement
inhibitory proteins such as factor H may be one mechanism through which N. brasiliensis
evades complement, as has been described for O. volvulus (Meri et al. 2002). We have
demonstrated that N. brasiliensis L3 (Baker et al. 2004) and L4 (unpublished data) bind
human factor H after incubation with NHS. Immunofluorescence and Western blotting
should be used to determine if mouse factor H is taken up by N. brasiliensis L3 or L4 in vitro
or in vivo. This analysis should focus on the time required for factor H acquisition and the
tissues in which it occurs. Unfortunately, antibodies specific for mouse factor H are not
currently available. There are other host proteins that could be adsorbed by N. brasiliensis to
evade complement, including DAF that binds to schistosomula (Ramalho-Pinto 1987) and
CHAPTER FIVE 59 C4-binding protein. Alternatively, N. brasiliensis may excrete or secrete proteins that can
either directly or indirectly inhibit complement, as has been reported for other helminth
species (Badley et al. 1987; Suchitra and Joshi 2005; Garcia-Hernandez et al. 2007).
Proteomic technology could be used to detect differences in protein expression profiles by L3
and L4 and identify potential immune evasion proteins, some of which may be novel.
Activation of the complement system is a potent stimulator of inflammatory responses in a
variety of models. The present study has demonstrated this clearly for a model of helminth
infection, but excessive or prolonged activation of complement can also be damaging for the
host. Inappropriate complement activation can contribute to the pathogenesis of
inflammatory diseases such as asthma, neurodegenerative disorders and atherosclerosis
(Oksjoki et al. 2003; van Beek et al. 2003; Sarma et al. 2006). The fact that lung-stage larvae
of N. brasiliensis can inhibit complement activation means that it may be possible to identify
parasite-derived factors that can therapeutically limit excessive complement activation in
other diseases. To achieve this, the mechanism of complement evasion must be elucidated.
5.1.4 Pulmonary cellular responses following helminth infection
5.1.4.1 Restricted early cellular inflammation in the lung
In the present study, we focused on the prominent inflammatory response induced in the skin
by injection of N. brasiliensis L3. The nature of the inflammatory response in other tissues
such as the lungs has not been fully characterised. Leukocyte infiltration of the lungs whilst
larvae are in situ is very limited (Daly 1999; Daly et al. 1999; Knott et al. 2007), even though
large numbers of cells are recruited to this site in allergic inflammatory diseases, such as
asthma. The lack of inflammation in the lungs may be a consequence of poor activation of
complement on L4 and/or a failure to generate C5a. To test this, levels of C5a in BAL fluid
following N. brasiliensis infections should be compared with levels seen in lavage fluid from
skin air pouches. Alternatively, this could be assessed by comparing the inflammatory
CHAPTER FIVE 60 response induced by injection of L3 and L4 into the skin. However, recent studies in our
laboratory suggested that L4 induce a similar cellular inflammatory response to L3 when
injected into skin air pouches (Cava 2007). Hence it is possible that the ability of L4 to
inhibit inflammation may be tissue-specific. For example, the presence of anti-inflammatory
cells in the lung may restrict cell recruitment. Such cells may be alternatively-activated
macrophages (AAMs) which are known to be present in the lungs during N. brasiliensis
infection (Reece et al. 2006). The anti-inflammatory properties of these cells could be
determined by co-injecting lung leukocytes into the skin to see if they interfere with the
normally strong cellular inflammatory response induced by injection of L3. Should anti-
inflammatory activity be detected, these cells could be fractionated and their phenotype and
secretory products determined.
5.1.4.2 Delayed cellular inflammation into the lungs
It is not widely acknowledged that the inflammatory response in the lung does not develop
until days after the parasite has left the site. A substantial inflammatory response, including
eosinophils, develops 2-4 days after N. brasiliensis leaves the lungs (Voehringer et al. 2004).
There are several unknowns regarding this delayed inflammatory response, including the
stimulus for its eventual development. It is possible that the response is initiated by parasite
antigens or sheaths shed into the lung environment as the larvae depart for the gut. It could
also be a consequence of processes promoting tissue repair, a role which both AAMs and
eosinophils may play a role (Williams 2004; Reece et al. 2006). However despite the
presence of a strong inflammatory response from days 4-6, there is no evidence that parasite
killing occurs in the lungs during either primary or secondary infections (Knott et al. 2007).
The chemotactic factors that promote cell recruitment into the lungs are also unknown. The
late inflammatory response to N. brasiliensis infection should therefore be examined in mouse
strains deficient in factors that may recruit eosinophils, such as complement factors,
chemokines such as eotaxin-1 and cytokines such as IL-4, -5 and -13.
CHAPTER FIVE 61 5.1.5 Secondary immune response to helminth infection
The present study focused primarily on innate mechanisms of immunity to N. brasiliensis,
since we proposed that complement would play its greatest role upon initial exposure to the
parasite. However, there are still many questions to be answered regarding the nature of the
strong secondary resistance to N. brasiliensis infection. Absence of IL-5 or a complete lack
of eosinophils (in ΔdblGATA mice) is associated with impaired early resistance to N.
brasiliensis infection (Knott et al. 2007). Both of these mutant strains had higher lung larval
loads than WT mice during secondary infections. In the present study we have demonstrated
that complement-deficient mice are highly resistant to secondary infection, suggesting that
early eosinophil-dependent killing of N. brasiliensis during secondary infection is mediated
by other factors. Such factors may be the same as those that promote innate eosinophil-
dependent anti-larval immunity in the pre-lung stage of primary infections, or could be
parasite-specific antibody that mediates ADCC. Based on many other models of helminth
infection, it has long been thought that parasite-specific antibody protects against secondary
infection via neutrophil or eosinophil-dependent ADCC (MacKenzie et al. 1980; Incani and
McLaren 1981; Shaio et al. 1990; Venturiello et al. 1995). However, this study suggests that
the presence of parasite-specific antibody during secondary N. brasiliensis infection does not
promote classical pathway-dependent parasite killing, since C1q-deficient and C3-deficient
mice exhibit similar parasite burdens to WT animals in the lungs and small intestine. Hence,
complement is not required for anti-parasite resistance during secondary N. brasiliensis
infection.
The companion study of Knott et al (2007) indicates that eosinophils are also not essential for
expulsion of the adult intestinal stage of N. brasiliensis during secondary infections. Similar
results are seen during infections with other helminths, including B. malayi (Ramalingam et
al. 2003; Simons et al. 2005) and S. ratti (Ovington et al. 1998), suggesting that while
eosinophils may be protective in the early stages of secondary infections with some species of
CHAPTER FIVE 62 helminth, other elements of the immune response are sufficient to cause elimination of the
parasite from the host. It is likely that such mechanisms are dependent on STAT6 and IL-
4/IL-13, which may invoke physiological changes in the gastrointestinal environment that act
to “flush” or “force” helminths from the intestine (Mettrick et al. 1979; Nawa et al. 1994;
Bancroft et al. 1997; Urban et al. 1998; Cliffe et al. 2005).
5.1.6 Future directions for studies using complement-deficient/IL-5 Tg mice
We have investigated, using a unique mouse model, the relationship between IL-5,
eosinophils and the complement system during infection with a parasitic helminth. The
complement system plays an important role in regulating eosinophil trafficking and activation
during eosinophilic conditions. Hence, it may also play a role in other eosinophilic disease
models. The tools used and the data generated in this study could be applied to other disease
models, including infections with other parasite species. IL-5 Tg mice have been shown to be
resistant to infection with S. stercoralis, L. sigmodontis, A. cantonenesis and A. costaricensis
(Sugaya et al. 1997; Herbert et al. 2000; Martin et al. 2000; Sugaya et al. 2002). The
complement-deficient/IL-5 Tg mouse strains we developed as part of this study could be used
as hosts for these and other parasite species to establish if complement is more important for
eosinophil-dependent anti-parasite resistance. While the use of IL-5 Tg mice is essentially an
artificial system, the resultant eosinophilia is characteristic of a number of diseases. The role
of complement during eosinophilia could also be assessed in murine asthma models to
determine if the absence of specific pathways of complement activation lessens symptoms.
Similar approaches could be used in animal models of allergen-induced eosinophilic
oesophagitis, rhinitis, and gastroenteritis.
5.1.7 Issues for design of anthelmintic vaccines
Ultimately, the major goal for those studying immunity to helminths is the development of
vaccines to protect humans and domesticated animals from infection. The life cycles and
CHAPTER FIVE 63 pathology caused by the human pathogens S. stercoralis and the hookworms Necator
americanus and Ancylostoma duodenale are similar to the tissue-invasive stages of N.
brasiliensis in mice, albeit much longer in duration. Developing a vaccine that promotes
rapid eosinophil recruitment to sites of infection and limits the ability of the parasite to evade
complement may be a useful strategy. Such a vaccine would mirror what might occur in
natural and acquired resistance, restricting parasite migration and hence breaking the lifecycle
at an early stage within the host. Gastrointestinal helminths may become more difficult to
destroy as they mature and migrate through the host to the gut. Therefore, using the early
stages of parasite maturation as vaccine targets may be the most useful strategy, as it would
prevent much of the pathology caused by the helminth and stop parasite reproduction and
spreading of the disease.
Many studies using humans or animal subjects have investigated potential vaccine candidates,
including the use of attenuated larvae, native parasite ES proteins and individual recombinant
ES proteins (Schallig et al. 1997; Lightowlers et al. 2003; Vercauteren et al. 2004; Hewitson
et al. 2005). The present study has not conclusively demonstrated a mechanism by which
eosinophils might provide resistance to N. brasiliensis, but the process appears to be
dependent on multiple factors. Complement activation can rapidly trigger several important
immune responses, including eosinophil and neutrophil recruitment and it may also trigger
eosinophil degranulation. Evasion mechanisms employed by parasites mean that the
influence of complement is limited over time. Discovering the mechanisms used by
helminths to eventually avoid complement activation may identify new target molecules for
vaccination.
5.2 CONCLUSION
The present study has comprehensively examined the role of complement in immunity to the
nematode N. brasiliensis. Previously, most studies of the immune response to gastrointestinal
CHAPTER FIVE 64 helminth infections have focused on responses in the gut that promote parasite expulsion.
Our work is relatively unique in that we have concentrated on earlier events and especially
those that occur in the skin within the first few hours of an infection. Understanding how
parasites are killed at this early stage may be critical for developing strategies, including
vaccines, which will limit the progression an infection. Critically, eosinophils can be
important for anti-parasite resistance if available in sufficient numbers in the early stages of
infection (Daly et al. 1999; Dent et al. 1999). A crucial next step will be to determine if
similar protection is possible with some of the major nematode parasites of humans. In the
current study we have found that the infective stage of this parasite is exquisitely sensitive to
the alternative pathway of complement activation and to eosinophil attachment in vitro. We
have demonstrated that complement contributes to a number of important immune responses
in the first 30 min of sub-cutaneous injection. However the complement system becomes
largely redundant after just 2.5 h, most likely due to evasion strategies instituted by the
parasite and other factors within the immune system that can contribute to anti-parasite
immunity. The parasite can hence migrate through to the lungs and gut largely unaffected by
complement, improving its chances of maturation and reproduction. Hence, while the
interaction between eosinophils, complement and N. brasiliensis appears to be fairly straight-
forward in vitro, the in vivo setting is far more complex. This highlights that the mammalian
immune system is a complicated entity, where functional overlap between different elements
can lead to apparent redundancy of some components. Since parasites usually need to live in
close contact with the host’s immune system, in some cases for years, they have evolved
sophisticated strategies to limit potentially damaging arms of the immune response and
inhibition of the complement system may be a critical first stage in this.
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