Download PROBING IMMUNE FUNCTION DURING AGING IN ADULT DROSOPHILA MELANOGASTER

PROBING IMMUNE FUNCTION DURING AGING IN ADULT
DROSOPHILA MELANOGASTER
by
Sean Ramsden
A thesis submitted to the Department of Biology
In conformity with the requirements for
the degree of Master of Science
Queen’s University
Kingston, Ontario, Canada
(September 2007)
Copyright ©Sean Ramsden, 2007
Abstract
Virtually all multicellular organisms rely on a highly conserved innate immune
system for defense against foreign microorganisms. Innate immunity consists primarily
of a humeral response that culminates in the expression of antimicrobial peptides. In
contrast to adaptive immunity seen in high order organisms, the innate immune response
is not specific to the invader.
In aging organisms, some of the most dramatic transcriptional changes take place
within the innate immune system. In aging mammals, innate immune reorganization
coincides with declining immune function, which often manifests itself as chronic
inflammation. Similar to this state of chronic inflammation in mammals, Drosophila
exhibit a marked upregulation of many innate immunity related genes. However, it
remains unclear if this upregulation results in a similar decrease in immune function to
that seen in mammals. If Drosophila is to be considered as a model organism in which to
study the relationship between immunity and aging, it must first be determined whether it
too undergoes declining immune function with age.
By examining the response to quantifiable injections of bacteria, we were able to
deduce that adult Drosophila do indeed undergo immune senescence. Elderly wildtype
flies infected with various doses of bacteria showed a decreased ability to survive
infection. Moreover, because the ability to clear the infection remains intact despite
decreased survival following infection, it is believed that a bacterially produced factor is
responsible for immune senescence in adult Drosophila.
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Acknowledgements
I would first like that thank Dr Seroude for the opportunity to work in his lab. Your
guidance and advice have made this an adventure I’ll never forget. I consider myself not only a
better scientist, but also a better person because of this experience.
I would also like to thank all the members of the Seroude lab for their support and help in
the lab. In particular I would like to thank Frederique Seroude and Rhonda Kristensen for their
help maintaining fly stocks and everything else they do to keep the lab running.
I also extend my thanks to two undergraduate students who have helped tremendously
with my work. Both Yeuk Yu Cheung and Lianne Yu proved to be a huge help, especially in
completing some of the more tedious experiments.
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Table of Contents
Abstract ..................................................................................................................................................ii
Acknowledgements ..............................................................................................................................iii
Table of Contents..................................................................................................................................iv
List of Figures ........................................................................................................................................ v
List of Tables ........................................................................................................................................vi
Chapter 1 Introduction and Literature Review .................................................................................... 1
Chapter 2 Experimental Procedures ...................................................................................................19
Chapter 3 Experimental Results .........................................................................................................26
Chapter 4 Discussion...........................................................................................................................58
Appendix A Replicate Survival Curves .............................................................................................73
Appendix B Replicate Clearance Curves ...........................................................................................84
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List of Figures
Figure 1. Imd and Toll pathways
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Figure 2. The relative size of three needles tested
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Figure 3. Percent survival of kenny mutants after infection
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Figure 4. Percent survival of imd mutants after infection
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Figure 5. Internal bacterial titre of imd mutants after infection
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Figure 6. Percent survival across age of w1118 flies in response to infection
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Figure 7. Average difference in survival between injected and non-injected w1118 flies 48
Figure 8. Percent survival across age of CS flies in response to infection
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Figure 9. Average difference in survival between injected and non-injected CS flies
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Figure 10. Bacterial clearance across age following infection of w1118
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Figure 11. Bacterial clearance across age following infection of CS
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Figure 12. Percent survival and internal bacterial titre of fast and slow climbers
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Figure 13. Percent mortality resulting from the infection protocol
60
Figure 14. Percent survival of imd mutants in response to infection with bacterial
supernatant
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Figure 15. Average difference in survival of replicate supernatant infections of imd
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List of Tables
Table 1. Mean internal bacterial titre after infection with three types of needles
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Table 2. A summary of the mean number of bacteria injected into imd mutants in two
independent experiments
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Table 3. Viability of concentrated bacteria cells at time points after preparation
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Chapter 1
Introduction and Literature Review
1
1.1 The free-radical theory of aging
While easy to identify, aging is infinitely more difficult to accurately define, in
large part because of its many forms and manifestations. However, when further
dissected, a common feature of aging in virtually all multicellular organisms is the
progressive decline in the efficiency of various physiological processes once reproductive
maturity is reached. One of the most accepted theories of the underlying mechanisms of
aging postulates that the age related decline of physiological processes associated with
aging is due to the accumulation of molecular oxidative damage. While quite
controversial when first proposed (Harman, 1956), the free radical theory of aging has
garnered increasing support in recent years. This theory holds that because oxygen is a
toxic molecule, and is used by aerobes, it may be hazardous for their long-term survival,
despite being necessary for their immediate survival. This seeming paradox is inherent to
the structure of the oxygen atom. Molecular oxygen, O2, upon single electron additions
can sequentially generate the partially reduced molecules O2.-, H2O2, and OH., which can
in turn generate many other reactive oxygen species (ROS) causing wide spread
biological damage. This damage can manifest itself in many forms. Firstly, peroxidation
of lipids specifically polyunsaturated fatty acid chains, leads to cellular membrane
inflexibility. Protein carbonylation enhances the likelihood of proteolysis (Stadtman,
1992) as well as cross-linking, both rendering the protein non-functional. DNA damage
can take the form of double strand breaks, cross-linking, base hydroxylation and base
excision. Finally, carbohydrate depolymerization, glycooxidation, and glycoaldehyde
production are common outcomes of ROS damage (Masoro, 2006).
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Numerous lines of evidence suggest that the mitochondria are the primary source
of reactive oxygen species. The site of oxidative phosphorylation, the mitochondria are
the major source of energy for the cell. Contained within the inner mitochondrial
membrane is the four complex electron transport chain (ETC), into which the electrons
from food digestion are fed. As the electrons are passed sequentially through the four
complexes of the ETC, an electrochemical gradient is formed, which is used to drive ATP
synthesis. It is hypothesized that age-related oxidative damage induced by ROS to the
mitochondria leads to further damage, thereby producing more ROS. The result is a
vicious cycle of escalating ROS production with age. This escalating damage may be
evidenced in the appearance of mitochondrial swirls in Drosophila that have been aged or
exposed to oxidative stress (Walker and Benzer, 2004).
It is estimated that approximately 2-3% of oxygen consumed by aerobic cells is
eventually converted to ROS. An estimated ten percent of this production is by design,
as ROS play important roles in host defense and signaling pathways (Balaban et al.,
2005). The remaining 90% is able to cause cellular damage. Ames et al (1993) recorded
that a typical cell in rats may undergo as many as 100,000 ROS attacks on its DNA per
day. Moreover, under steady state conditions, approximately 10% of protein molecules
may exhibit carbonyl modifications (Stadtman, 1992).
As with any causal theory of aging, three stipulations must be met in order for the
free radical theory to be validated: 1. The level of molecular oxidative damage increases
with age, 2. Longer life expectancy is associated with lower levels of oxidative damage,
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3. Prolongations of life span by means such as caloric restriction are associated with a
decrease in oxidative damage.
1.1.1 Molecular oxidative damage increases with age
Age-related increases in oxidative damage as well as mtDNA mutational load
have been described in many organisms and organ systems. Specifically, large scale
mtDNA deletions have been described in C. elegans (Melov et al., 1994), Drosophila
(Yui et al., 2003), mouse (Tanhauser and Laipis, 1995), rat (Filburn et al., 1996), monkey
(Lee et al., 1993), and humans (Cortopassi and Arnheim, 1990). Concomitant with these
increases in mtDNA mutational load are increases in indicators of oxidative damage.
However, because of the low steady-state levels of ROS and their transient nature, it is
technically impossible to directly measure ROS levels in vivo. In light of this, the
measurement of ROS byproducts must be measured. In both mammalian and insect
tissues, ratios of the redox couples, such as NADPH:NADP+, glutathione:oxidized
glutathione, NADH:NAD+ tend to shift more towards the pro-oxidant values with
increasing age (McCord, 1995),(Noy et al., 1985). Exponential increases in the levels of
exhaled alkanes and n-pentanes, products of ROS induced peroxidation of membrane
lipids, indirectly indicate an in vivo increase in oxidative damage with age (Sohal and
Weindruch, 1996). Finally, there is a two to three fold increase in the concentration of
oxidatively damaged protein as indicated by a loss of protein –SH groups, protein
carbonylation, and loss of catalytic activity of several enzymes such as glutamine
synthase and alcohol dehydrogenase (Stadtman, 1992).
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Susceptibility to oxidative damage has been shown to be negatively correlated to
maximum lifespan. It has been shown that relatively short lived species such as mouse
and rat are more vulnerable to acute oxidative stress induced by X-rays than relatively
longer lived species such as rabbit, pig and pigeon (Agarwal and Sohal, 1996).
Specifically, brain homogenates from 22 month-old rats were twice as susceptible to
induced oxidative stress as were three month-old rat brain homogenates. These results
are consistent with studies performed on flies indicating that older individuals had
increased levels of oxidative damage (Orr and Sohal, 1994).
1.1.2 Experimental extension of lifespan is associated with less oxidative damage.
The second branch of support for the free radical theory of aging is the many
experimental manipulations that extend lifespan by reducing oxidative damage. One of
the most convincing studies simultaneously over-expressed Cu,Zn-superoxide dismutase
(SOD) and catalase in adult Drosophila (Orr and Sohal, 1994). Cu,Zn-SOD converts
superoxide anion radical to H2O2 and catalase breaks down H2O2 into water and
molecular oxygen. Together, their job is to eliminate the possibility of the production of
the highly reactive hydroxyl free radical. Transgenic flies carrying three copies of each
of these two genes showed an increase in both average and maximum lifespan of up to
one third. Also, these flies exhibited less age accumulated oxidative damage as well as
an increased resistance to induced oxidative stress. Finally, a decrease in mitochondrial
H2O2 production with age as well as a 30% increase in the metabolic potential (total
amount of oxygen consumed during adult life per unit body weight) was seen. Similarly,
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when a mitochondria specific MnSOD is overexpressed, lifespan is increased in
proportion to the amount of enzyme over-expression achieved by a given line (Sun et al.,
2004). Moreover, when Cu,Zn-SOD and MnSOD are simultaneously over-expressed an
additive relationship was revealed, where lifespan extension was greater when coexpressed than when either gene was over-expressed at the same levels (Sun et al., 2004).
In contrast to over expressing specific genes, knocking out genes has also
provided support for the free radical theory of aging. Drosophila with a null mutation in
the G-protein coupled receptor (GPCR) methuselah (mth), which is believed to be
involved in stress response and biological aging, are long-lived (Lin et al., 1998). When
mth flies are exposed to oxidative stress, males were approximately seven times more
resistant than are their wild-type counterparts, whereas females we approximately twice
as resistant. Also, when they are exposed to high temperature, mth flies survive better
than do wild-type flies. Similar to the findings in mth mutants, age-1 Drosophila
mutants, deficient in PI3 kinase, are also long-lived and resistant to a number of
environmental stresses, especially oxidative stress (Larsen, 1993).
1.1.3 Experimental manipulation of free radicals affects lifespan.
Free radicals can be generated by various means. Perhaps the most popular
method is the use of methyl viologen or paraquat. Upon ingestion, paraquat generates in
vivo oxidative stress when it undergoes NADPH-dependent reduction to yield a Pq+
radical. The Pq+ radical in turn rapidly reacts with molecular oxygen to form the super
oxide radical. It has been found on numerous occasions that resistance to paraquat can be
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positively correlated to lifespan. Specifically, it was found that Drosophila strains that
had been previously shown to be long-lived were more resistant to persistent paraquat
stress than were strains previously shown to be short lived (Arking et al., 1991). Shortlived strains reached zero percent survival after only 48 hours while long-lived strains
took approximately one hundred hours to reach the same point. Moreover, when exposed
to varying concentrations of paraquat, long-lived strains consistently showed a higher
percent survival at 48 hours of exposure than did short lived flies. A 2001 study found
similar results when exposing various long- and short-lived strains to a battery of stresses
that included paraquat (Mockett et al., 2001). In addition to measuring lifespan, the
authors measured catalase activity and found that at 10 days old, it was significantly
lower in long-lived lines than in short-lived lines.
Finally, a common technique used for prolongation of lifespan is restricting the
caloric intake of organisms. Although it has been known for approximately sixty years
that caloric restriction without malnutrition extends maximum lifespan, it remained
largely unexplored until the 1970s. However, since then, it has been found that rodents
subjected to caloric restriction show attenuation of age-associated increases in superoxide
and hydrogen peroxide generation, slower accrual of oxidative damage, decreased alkane
production and delayed loss of membrane fluidity (Sohal et al., 1996).
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1.2 Some of the most dramatic transcriptional changes with age occur within the
immune system
Aging has been shown to affect many systems within the body. Using a variety of
experimental techniques, changes in the regulation of immunity related genes have been
shown in a variety or organisms. A series of microarray studies have recently shown the
age-dependent changes of gene expression in rhesus monkeys. Specifically, pathways for
energy metabolism such as the citric acid pathway, and pathways for growth, such as cell
cycling, are down regulated in skeletal muscle, while components of the immune system
such as B cell function, are up-regulated. (Kayo et al., 2001) In a series of similar studies
performed on mice, age dependent changes were also seen in various tissues. Immune
related genes were up-regulated in the brain, whereas pathways related to stress responses
were up-regulated in the skeletal muscle (Lee et al., 1999; Lee et al., 2000). Together,
these studies reveal a species specific difference in age dependent gene regulation. An
additional 2002 microarray study on Drosophila also showed an age-dependent increase
in the regulation of immune related genes. The up-regulation was not restricted to a
specific portion of the immune response, but instead included components from all levels
of the immune response (Pletcher et al., 2002). Consistent with these findings, it was
found using the enhancer trap technique, that Drosophila showed an age-dependent
tissue-specific increase in immunity related genes (Seroude et al., 2002).
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1.3 Innate immunity
An immune system is universal and necessary for all organisms to defend
themselves from foreign invaders. Two different forms of immunity exist, adaptive
immunity, and innate immunity. However, it is only in higher organisms, such as
mammals, that an adaptive, antigen specific immune system has evolved. Study of the
immune system in higher organisms is made difficult by the intimate interaction between
the adaptive and innate immune system. This complexity, however, can be circumvented
by studying invertebrate model organisms, which lack an adaptive immune system, thus
eliminating the complicated interplay between the adaptive and innate immune systems.
One such model organism is Drosophila melanogaster. Because it has a sequenced
genome, is small and easily kept and has a short life cycle, it is ideal for the study of
immune function during aging.
Because the underlying mechanisms of innate immunity have been highly
conserved across evolution, the Drosophila model has been particularly invaluable to
elucidate not only the molecular components and signaling pathways involved in innate
immunity, but also its regulation with age. A recent study suggested that immune
function declines with age in Drosophila because older females failed to terminate the
expression of an antimicrobial peptide as quickly as did younger flies (Zerofsky et al.,
2005). However, this study is not conclusive as it focuses solely on female flies, and
does not report on flies older than middle age. Moreover, it relies on measuring mRNA
transcripts encoding the antimicrobial peptide diptericin as an indirect indicator of
immune function. It remains possible that the persistent expression of antimicrobial
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peptides do not affect the functionality of the immune system. In other words, while older
flies may not terminate the expression of antimicrobial peptides as quickly as younger
flies, they may still clear and survive the infection. Finally, the Drosophila immune
response relies on the coordinated expression of at least 15 antimicrobial peptides (Imler
and Bulet, 2005; Royet et al., 2005). The alteration of the expression of a single peptide is
likely to not affect the functionality of the immune system since the constitutive
expression of a single antimicrobial peptide is sufficient to restore the ability to survive
infection in mutants unable to express any antimicrobial peptides (Tzou et al., 2002).
Recently, however, more evidence has arisen in support of the idea of immune
senescence in adult Drosophila.
Over-expression of a single receptor of the innate
immune system conferred resistance to bacterial infection, regardless of whether the
over-expression was induced in young or old flies. However, the extent of the resistance
conferred differed greatly, with young flies gaining more resistance than old flies. Flies
over-expressing the receptor were also found to be shorter lived, also indicating that
while beneficial early in life, the immune system has the potential to be harmful in old
age (Libert et al., 2006).
1.4 The Drosophila immune response
The Drosophila immune system consists of three lines of defense. The first is a
phenoloxidase reaction that deposits black melanin around wounds and foreign objects,
forming a scar tissue of sorts. The second is a cellular response resulting in the
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phagocytosis or encapsulation of the intruder and the third is a humeral response
culminating in the generation of antimicrobial peptides.
1.4.1 Wound Repair
The physical barrier the cuticle provides is the first obstacle potential pathogens
must circumvent in order to colonize their host. Therefore, it is essential that any
breeches of the cuticle be repaired efficiently. This is achieved through the clotting of
haemolymph and melanization. Currently, much of what is known about the wound
healing process in Drosophila has been garnered from work on larvae rather than adults.
Upon injury, a clot made primarily from fibers that trap haemocytes is quickly generated
at the wound. Upon entrapment, the haemocytes release several proteins. Hemolectin is
released in the largest quantities and is a large protein with several domains that are also
found in other clotting factors (Goto et al., 2003),(Scherfer et al., 2004). Following the
formation of the plug, the outermost portion melanizes and the surrounding epidermal
cells orient towards the plug and fuse to form a syncytium (Galko and Krasnow, 2004).
Reestablishment of the epithelium is mediated though Jun N-terminal kinase (JNK)
signaling (Ramet et al., 2002), (Galko and Krasnow, 2004).
1.4.2 Cellular response
The cellular response is generated by circulating blood cells called haemocytes
(Carton and Nappi, 1997). Haemocytes fall into three categories based on structural and
functional characteristics: plasmatocytes, lamellocytes and crystal cells. Plasmatocytes
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are the main class of haemocytes, making up 90-95% of all mature larval haemocytes.
They are responsible for phagocytozing bacteria and other foreign particles as well as
dead host cells. Lamellocytes are large, flat and adherent cells produced in a specialized
reaction after infection by larger parasites, and are involved in forming a cellular capsule
around particles too large for phagocytosis. Lamellocytes are not found in embryos and
rarely in larvae. Crystal cells make up approximately 5% of all haemocytes and are nonphagocytic cells containing phenoloxidase. Crystal cells play an important role in the
melanization process (Lemaitre and Hoffmann, 2007).
As can be predicted by their abundance, phagocytosis by plasmatocytes is the
primary cellular defense mechanism against foreign antigens. Plasmatocytes, shown to
phagocytoze a variety of particles from Sephadex beads to dsRNA, recognize their
targets via a wide array of surface receptors such as members of the scavenger receptor
family, the EGF-domain protein Eater and the Ig superfamily domain protein Dscam
(Lemaitre and Hoffmann, 2007). Upon recognition, antigens are internalized and
destroyed as part of the phagosome.
Some antigens may also be opsonized by three thioester containing proteins;
TEP1, TEP2 and TEP4, as well as secreted forms of Dscam (Lagueux et al., 2000),
(Lemaitre and Hoffmann, 2007) before destruction. Once opsonized, antigens are
recognized by lamellocytes released from the lymph gland. The lamellocytes form a
multi-layered capsule around the invader, a process accompanied by melanization (Nappi
et al., 1995). Within the capsule, the pathogen is eventually killed by a mechanism that is
as yet largely unknown.
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The responsibility of crystal cells, activation of the phenoloxidase is essential for
melanization. The process is rapid, and takes place in the haemolymph of wounded or
infected animals (Ashida, 1998), (Soderhall and Cerenius, 1998). A cascade of serine
proteases generates active phenoloxidase from an inactive proenzyme. Active
phenoloxidase oxidizes phenols such as tyrosine into reactive species that cross-link and
polymerize into melanin, which is later deposited around wounds.
1.4.3 Humeral response
Arguably the most important part of the Drosophila immune system, the humeral
response ultimately culminates in the expression of seven classes of antimicrobial
effector molecules. The seven classes of antimicrobial peptides (AMPs) are small
molecules (<10kDa), with the exception of Attacin, cationic and exhibit a broad range of
specificities (Imler and Bulet, 2005). Diptericin, Drosocin and Attacin are most effective
against, and are induced in response to Gram-negative bacteria (Wicker et al., 1990),
(Asling et al., 1995), (Bulet et al., 1993). Defensin is active against gram-positive
bacteria (Dimarcq et al., 1994), while Drosomycin and Metchnikowin are antifungal in
nature (Fehlbaum et al., 1994), (Levashina et al., 1995). Finally, Cecropin acts against
both bacteria and fungi (Kylsten et al., 1990). Because of their specificity, it is
advantageous for Drosophila to limit the expression of the AMPs to those effective at
fighting the class of pathogen that has been detected. This is achieved through the
activation of two primary pathways, the Toll pathway and the Imd pathway. A third,
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lesser pathway, the Jak/STAT-pathway, is known to be involved in the immune response,
but its exact nature and contribution is not yet fully known.
1.4.3.1 The Toll pathway
The Toll signaling pathway responds to and defends against fungal and grampositive bacterial attacks (Lemaitre et al., 1995a),(Lemaitre et al., 1996),(Lemaitre et al.,
1997) in addition to its roles in dorso-ventral patterning during development. The Toll
pathway can be initiated by one of five circulating recognition proteins. Persephone is
responsible for recognition of fungal invaders (Ligoxygakis et al., 2002). Peptidoglycan
recognition protein PGRP-SA and the Gram-negative binding protein GNB1 encoded by
the semmelweis and osiris genes respectively work together to detect Lysine-type
peptidoglycans (Michel et al., 2001),(Gobert et al., 2003). PGRP-SD also detects the
Lysine-type peptidoglycans that activate the PGRP-SA/GNBP1 complex (Bischoff et al.,
2004). Gram-negative binding protein GNBP3, unlike its name would have you believe,
detects fungal glucan (Bangham et al., 2006). Upon detection of an invader, the
recognition proteins induce a proteolytic cascade that activates spaetzle processing
enzyme (SPE) (Jang et al., 2006). Active SPE cleaves the polypeptide Spaetzle into an
active ligand for the Toll membrane receptor (Weber et al., 2003). Once activated, Toll
dimerizes and interacts with at least three cytoplasmic proteins: MyD88, Pelle and Tube.
This interaction results in the phosphorylation of I- κB factor Cactus by Pelle, resulting in
its dissociation from the dorsal-related immune factor (DIF) and degradation by the
proteosome (Nicolas et al., 1998). In the absence of Toll activation, Cactus bound DIF,
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an NF-κB transcription factor, is inactive. Once activated, DIF translocates from the
cytoplasm to the nucleus where it acts as a transcription factor to activate hundreds of
genes (Ip et al., 1993), (De Gregorio et al., 2001; De Gregorio et al., 2002), (Irving et al.,
2001). These genes are involved in the production of anti-microbial peptides.
1.4.3.2 The Imd pathway
The Imd pathway is initiated by membrane bound recognition proteins, unlike the
Toll pathway which is triggered by circulating recognition proteins (Choe et al., 2002),
(Gottar et al., 2002). PGRP-LC has several spliceoforms, two of which are required for
the activation of the Imd pathway. As dimers, these spliceoforms can detect either
monomeric peptidoglycan (PGN) or polymeric PGN. Dimeric PGRP-LCx is required for
recognition of polymeric PGN, whereas PGRP-LCa/PGRP-LCx is required for
monomeric PGN recognition (Kaneko et al., 2004). A second recognition protein,
PGRP-LE, has an affinity for diaminopimelic (DAP)-type PGN, which is primarily found
in Gram-negative bacteria (Takehana et al., 2002), (Takehana et al., 2004).
Once activated, the recognition proteins activate membrane bound Imd (Kaneko
et al., 2006), (Choe et al., 2005), which in turn binds dFADD which in turn binds Dredd
(Hu and Yang, 2000). Dredd, an apical caspase, has been suggested to bind and cleave
relish, the NF-κB transcription factor in the Imd pathway (Stoven et al., 2000). Unlike
DIF, relish is not inhibited by Cactus in the cytoplasm. Instead, Relish carries its own
ankyrin repeat inhibitory region (Dushay et al., 1996) and its activation and nuclear
translocation are dependent on its endoproteolytic cleavage by Dredd to remove the
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inhibitory domain (Stoven et al., 2000). However, before cleavage, relish must first be
phosphorylated by the IKK signaling complex (Silverman et al., 2000). The IKK
complex is proposed to be activated by TAK1 and TAB2 in an Imd dependent manner.
Once active, relish has been shown by microarray to activate the transcription of several
hundred genes, including the antimicrobial peptide genes (De Gregorio et al., 2002),
(Irving, 2001).
1.4.3.3 The JAK/STAT pathway
The JAK/STAT pathway remains largely a mystery in terms of function. It
consists of three main cellular components: the receptor Domeless, the Janus Kinase
(JAK) Hopscotch and the STAT transcription factor (Agaisse et al., 2004). JAK/STATdeficient flies show no differential response to bacterial and fungal infections, similar to
wild type flies and they express a normal profile of AMPs. However, they are sensitive
to the Drosophila C virus (Dostert et al., 2005). It is also thought that the pathway could
respond to tissue damage. Haemocyte released Unpaired-3 (Upd-3) activates the
pathway by binding Domeless (Agaisse et al., 2003). Together, these findings suggest
that the JAK-STAT pathway plays a role in wound healing and viral defense.
In addition to its role in immunity, the JAK/STAT pathway has been proven to
play significant roles in germ cell sex determination as well or morphogenesis
(Wawersik, 2006)
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Figure 1. A schematic of the humeral response in adult Drosophila. Adapted from
Lemaitre and Hoffmann, 2007.
17
1.5 Experimental Aims
Similar to the upregulation of immune components in mammals, Drosophila exhibit a
marked increase in the expression of immunity related genes with age. However, what is
not known is whether this upregulation coincides with a decrease in overall immune
functionality, as it does in mammalian systems. Because study of overall immune
function is made difficult in mammals because of the complex interplay between adaptive
and innate immunity, insects such as Drosophila are ideal model organisms in which to
study immune senescence. However, if Drosophila is to be used as a model organism to
study immune senescence, it must first be established whether they undergo this age
related decrease in immune function. In order to test this we administered known doses
of bacteria to adult Drosophila of varying age and followed their ability to survive the
infection as well as their ability to clear the infection. It was predicted that because of the
high degree of homology in innate immunity between mammals and insects that adult
Drosophila would indeed exhibit immune senescence with age.
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Chapter 2
Experimental Procedures
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2.1 Drosophila strains and culture
Two different strains were used to control for the influence of the genetic
background. The Canton-S strain is a wild-type strain whereas the w1118 strain is a
laboratory strain derived from an Oregon-R wild-type strain in which a recessive
mutation conferring white eyed has been introduced. This strain was included since it is
the background used in aging experiments involving transgenes. The w1118 strain has a
shorter lifespan than the Canton-S strain. The w;imd immune deficiency strain is a w1118
background with a P-element-induced recessive mutation in the imd gene (Lemaitre et al.,
1995b). All fly lines were treated with tetracycline before initiating any experiments to
ensure none would carry Wolbachia bacteria, which is known to influence lifespan and
can cause neurodegeneration in some fly strains (Min and Benzer, 1997).
Flies were maintained and aged at 25°C on standard fly media containing 0.01%
molasses, 8.2% cornmeal, 3.4% killed yeast, 0.94% agar, 0.18% benzoic acid, 0.66%
propionic acid. Newly emerged flies (0-48h) were anaesthetized with nitrogen for a
maximum of 2 minutes, and were segregated by sex into vials (20-30 individuals per
vial). Until reaching the appropriate age, flies were transferred to fresh food every 3–4
days.
2.2 Infections
Bacteria were grown to exponential growth phase (OD600nm=0.50±0.05), and the
desired amount of bacteria was centrifuged for 5 minutes at 4000RCF. The resulting
bacterial pellet was re-suspended in the appropriate amount of growth media to obtain the
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desired bacterial concentration. The concentration was independently confirmed by
plating. The bacterial solution was mixed periodically to prevent sedimentation of the
bacterial cells. The bacteria used were a lab strain of E. coli DH5α containing the plasmid
pHC60 conferring tetracycline resistance and GFP expression (Cheng and Walker, 1998;
Elrod-Erickson et al., 2000). During infection, flies were anaesthetized with nitrogen for
no more than 5 minutes. Flies were injected in the pteropleura of the thorax, in the axial
plane. To verify that each fly was injected with comparable amounts of bacteria, five to
eight single flies from the infected population were homogenized immediately after
infection and a 1/100 dilution of the extract was plated.
The growth media used was 2TY. Liquid media consisted of 10g/L of yeast
extract (BioShop, cat#YEX401), 16g/L of Bio-Tryptone (BioShop, cat#TRP402) and
5g/L of Sodium Chloride (BioShop, cat#SOD001.1). For solid media, follow same
recipe, but in addition add 15g/L of bacteriological grade agar (BioShop,
cat#AGR001.1).
2.2.1 Needle tests
The first needles tested were glass needles, produced as commonly described in
the literature. These were made with a Sutter Instrument Co. Micropipette puller (set for
1 mm diameter) and glass capillary tubes from World Precision Instruments
(cat#1B100F-4).
The needles were dipped into a bacterial solution, prepared as
previously stated, and inserted into the thorax through the pteropleura.
21
The second needles tested were from a Cereus species cactus. The needle was
glued into the end of a glass capillary tube to be used as a handle. Flies were infected
using the same protocol as was followed in the glass needle trials.
The final needle used for injections was a custom ordered 33 gauge, 30° tip and
1.75-inch length needle (Hamilton, cat#780305) mounted onto a Hamilton 5µl syringe
with plunger (Hamilton, cat# 87930). Unlike microinjection systems using capillary glass
needles, this setup can accommodate the viscosity of the bacterial mixture allowing
higher bacterial concentrations without clogging. It was determined that a 0.1µL volume
was the maximum volume that could be injected into each fly. Flies were always infected
at the same time of the day to avoid any influences of circadian rhythms.
2.3 Survival experiments
2.3.1 Immune deficient mutant survival experiments
Kenny mutants were infected as per the above infection protocol using a glass
needle dipped into a 40 OD/ml bacteria solution. Flies were infected as described above.
The flies dead within an hour following infection were excluded since those animals
succumbed from needle injury. Every 1-3 days thereafter, the number of dead flies was
counted and the surviving flies were transferred to new vials with fresh food media. As
these were the first, early experiments, instead of having a sterile injection control
population of the same genotype, a wildtype control infected with the same bacterial
solution was used for comparison.
22
Imd mutants were infected as per the protocol outlined in section 2.2. Three
different concentrations were used for the infection. 0.04 OD/ml, 4 OD/ml and 40
OD/ml populations were injected with 0.1µL of their respective concentrated bacterial
solutions. To verify how much bacteria was injected, five randomly sampled flies from
each population were homogenized and a dilution was plated. The number of colonies
for these five flies was averaged and used to calculate an estimate of the number of
bacteria injected into each population. All populations were treated within three hours of
each other. Cox regression and log-rank statistics were performed with Prism (GraphPad
Software Inc.) to identify statistically significant differences (P<0.05) in survival among
treatments.
2.3.2 Wildtype Populations
In each experiment, four populations were treated. The no-injection control was
anaesthetized under nitrogen and put to recover on new food, without injection. The
negative control population was anaesthetized under nitrogen and injected with 0.1µL of
sterile 2TY. 4 OD/ml and 40 OD/ml populations were injected with 0.1µL of their
respective concentrated bacterial solutions. To verify how much bacteria was injected
into the 4 OD/ml and 40 OD/ml populations, five randomly sampled flies from each
population were homogenized and a dilution was plated. The number of colonies for the
five flies was averaged and used to calculate an estimated number of bacteria injected
into each population. All populations were treated within three hours of each other.
23
Flies were infected as described above in section 2.2. The flies dead within an
hour following infection were excluded since those animals succumbed from needle
injury. Two controls were used. The negative controls are injected with sterile 2TY
without bacteria. The “no injection” controls were anesthetized like the injected animals
but not injected in order to discern death from natural aging from death from infection.
Every 1-3 days thereafter, the number of dead flies was counted and the surviving flies
were transferred to new vials with fresh food media.
Cox regression and log-rank statistics were performed with Prism (GraphPad
Software Inc.) to identify statistically significant differences (P<0.05) in survival among
treatments. At least two independent experiments were performed for each genotype and
age.
Clearance
Flies were infected as described above in section 2.2. At set time points after
infection, five to eight single flies from the infected cohort were homogenized and a
dilution of the homogenate was plated on 2TY media containing tetracycline (10ug/ml).
After 24 hours incubation at 37°C the number of colonies was counted. The colonies
were also confirmed to be expressing GFP to exclude any tetracycline-resistant bacteria
that may be present naturally inside the fly. This number and the dilution factor were
used to calculated the actual number of bacteria within each fly at a given time point. At
least two independent experiments were performed for each genotype and age. The same
24
protocol was used for wildtype and imd mutants.
No clearance experiments were
performed on kenny mutants.
Climbing Assay
Ten-day-old flies were infected with 40 OD/ml as per section 2.2. After six
hours, flies were separated based on climbing ability. Following separation, the survival
of the flies was monitored. Concurrently, the clearance of the infections was followed as
previously mentioned.
Confirming bacterial solution characteristics
To determine the viability of the cells in the 4 OD/ml and 40 OD/ml bacterial
solution, 100ul of a 1/100 dilution of each paste was plated at 0, 1 and 2 hours after
preparation. The solutions were prepared as described above in section 2.2.
Supernatant infection of Imd
Imd mutants were infected with either sterile 2TY or the supernatant from an
overnight culture of E. coli. To prepare the supernatant, the culture was spun at 4000
RCF for 5 minutes. After centrifugation, the pellet was discarded and the resulting
supernatant was filtered four times through a filter with a filter size of 0.22um. It was
found that after being filtered four times, no colonies would grow when the supernatant
was plated (data not shown). Imd mutants were infected with the resulting supernatant or
sterile 2TY as per section 2.2.
25
Chapter 3
Results
26
3.1 Delivery method
In order to sufficiently test the functionality of the immune system, it was first
necessary to be able to introduce a consistent amount of bacteria into each fly, and follow
the response to the infection. The first technique tested is common in the literature. A
fine needle is dipped into a concentrated bacteria solution then pricked into either the
abdomen or thorax of the fly, thereby depositing some of the bacteria solution into which
the needle was dipped. We first attempted this technique using pulled capillary tubes,
pricking the flies in the thorax. To measure if each fly was injected with comparable
amounts of bacteria, three to five flies from the infected population were homogenized
immediately after infection and a 1/10 dilution of the extract was plated on a tetracycline
media. After 24 hours incubation at 37°C, the number of colonies was counted. These
colony counts were used to calculate an approximation of the average number of bacteria
injected into each fly in the population. This protocol did not yield reproducible results
from individual to individual and between experiments (Table 1). In experiment one, the
number of bacteria that was introduced into each fly is highly variable when comparing
within each gender and between the genders. This is highlighted by the extremely high
standard deviation in the average number of bacteria inside the three individuals.
Moreover, when experiment one is compared to experiment two, the number of bacteria
in each fly is over one hundred times higher for males and ten times higher in females.
27
28
The second needles tested were from a Cereus species cactus. The needle was glued into
the end of a glass capillary tube to be used as a handle. The same protocol as the glass
needle infections was followed with similar results (Table 1). In experiment three, males
received almost three times the amount of bacteria as did the females despite being
infected by the same bacteria solution. This is highlighted in females, where the standard
deviation of the mean number of bacteria injected was almost as large as the mean itself.
When compared to experiment four, the average number of bacteria introduced to each
fly is dramatically reduced. Both males and females received at least one hundred times
less bacteria than they did in experiment three.
The third technique attempted was to use the microinjection system, commonly
used for creating transgenic fly lines. This system offered a way in which to consistently
inject a set amount of volume of bacterial solution into each fly. It was believed because
of this that the system would be ideal for our goals. However, it was found that the
Eppendorf® tips used for injecting each fly not only clogged with the bacterial solution,
but also with the cuticle of the fly. Because of these technical difficulties, this protocol
was also abandoned.
The final needle tested was a custom ordered 33 gauge, mounted onto a microliter
syringe. This set-up, unlike the microinjection system, can accommodate the viscosity of
the bacterial mixture. This set-up was the most consistent at injecting each fly with the
same amount of bacteria both within a given experiment and between independent
experiments (Table 1). In experiment five, both males and females were injected with
comparable amounts of bacteria. Moreover, the standard deviations for the average
29
number of bacteria are significantly lower than in previous experiments with other
injections techniques. When experiment five is compared to experiment six, the average
number of bacteria injected is comparable. Finally, the averages seen in both males and
females in experiment six are almost identical, further indicating that this is by far the
most consistent technique.
A visual comparison of all three needles tested relative to a male and female fly
can be seen in figure 2.
Figure 2. The relative diameter of the glass needle, cactus needle and Hamilton syringe.
For size comparison, a male (bottom) and female (top) fly are shown.
30
3.2 Infection protocol
Two critical outcomes of a successful immune response, the elimination of
invaders and the ability to survive them, need to be tested in order to investigate subtle
changes in immune function because aging may affect the ability to clear infections
without translating into reduced survival. It is also necessary to test various levels of
infection since aging may also affect the capacity of the immune system. We took
advantage of the immune-deficient mutants available in Drosophila to establish the
infection protocol. Different amounts of bacteria were injected into kenny and imd
mutants and their survival was monitored.
When kenny mutants were tested, it was seen that their survival was significantly
decreased in response to infection with the gram negative bacteria E. coli (Figure 3.).
Kenny mutants were infected with glass needles as a preliminary test. Infected males
showed a survival percentage of less than twenty percent after only four days after
infection, reaching less than ten percent survival after five days. Females declined
slightly later than males, reaching approximately seventy percent survival after four days,
and continuing to zero percent survival after six days post infection. Both males and
females show a significantly decreased survival in response to infection from the w1118
controls. Although the tests with kenny mutants proved that this protocol was able to
introduce bacteria into the flies, as previously discussed, it was not possible to
consistently introduce the same amount into each fly.
31
A
B
Figure 3. Percent survival of male (A) and female (B) w1118 and kenny flies. Figure
legends are indicated in the bottom left corner of each graph. Sample size of each
population is indicated in brackets. Both male and female kenny mutants showed a
decreased ability to survive after infection with E. coli.
32
When imd flies were tested using the Hamilton syringe, similar results were
obtained. Males showed 50% mortality in less than one day and 100% mortality in just
two days when infected with 40 OD/ml of bacteria (Figure 4A). When infected with 4
and 0.04 OD/ml of bacteria it took them three and five days, respectively, to reach 50%
mortality, while it took eight days for both to reach 100% mortality. All three male
populations are significantly different (P<0.05). Similarly, females infected with 40
OD/ml of bacteria reached 50% and 100% mortality in less than one day and two days,
respectively.
Females infected with 4 and 0.04 OD/ml of bacteria displayed 50%
mortality after 6 and 7 days, respectively, and 100% mortality 8 days after infection
(Figure 4B). The 0.04 and 4 OD/ml treatments are significantly different from the 40
OD/ml treatment (P<0.0001), but not from each other.
Simultaneously, the internal bacterial titre was measured to confirm that the
severity of the effect on survival is indeed due to the introduction of more bacteria
resulting in faster proliferation inside the animal (Figure 5). Males infected with 40
OD/ml and 4 OD/ml of bacteria showed increases in internal bacterial titre. However,
males infected with the lowest concentration of bacteria, 0.04 OD/ml, showed no
indication of bacterial proliferation despite the decrease in survival seen in this
population. Similar to males, females infected with 40 OD/ml and 4 OD/ml of bacteria
showed an increase in internal bacterial titre, indicating bacterial proliferation. The
extent of which, however, was far less than that seen in males. Moreover, females
infected with 0.04 OD/ml of bacteria showed little to no bacterial proliferation, despite
significant mortality observed in this population.
33
A
B
Figure 4. Percent survival of imd mutants after infection with three different
concentrations of bacteria. Males are shown in (A) and females are shown in (B). Figure
legends are depicted in the top right corner of each graph. In brackets, the sample size
and average number of bacteria injected into each population is indicated.
34
A
B
Figure 5. Percentage of bacteria remaining within populations of imd mutants infected
with three concentrations of bacteria. Males are shown in the top graph (A), and females
are shown in the bottom graph (B). Figure legends are indicated at the top of each graph.
In brackets, the sample size and average number of bacteria injected into each population
is indicated. Flies from figure 3 and 4 were treated together as part of a single
experiment.
35
3.3 Comparing actual and expected values in infection protocol
Expected numbers of bacteria were calculated based of colony counts after plating
0.1mL of the injection solutions. When the ratio of mean number of bacteria to expected
number of bacteria is observed, it can be seen that as the concentration of the injection
solution increases, the ratio decreases, indicating a lower proportion of the injection
solution is entering the fly (Table 2). This is a likely source of variability between flies.
At the bottom of the table, the ratios between means and expected numbers of bacteria
are shown. By comparing the ratio of the means to the ratio of the expected number of
bacteria, it can be seen that the observed dilution factor is very similar to the expected
dilution factor.
It was also seen that the ratio of the 40 OD/ml bacterial solution to 4 OD/ml paste
was not always the expected 10:1 and it was this hypothesized that because of their
extreme concentration, the bacteria within the 40 OD/ml paste in fact are more prone to
die over the two hours it takes to inject the cohorts of flies. This idea was confirmed by
plating dilutions of several 40 OD/ml and 4 OD/ml solutions over the course of two hours
(Table 3). It was seen that three of the 40 OD/ml pastes show decreases in the number of
cfu/ml over two hours, while two showed increases. In contrast, the 4 OD/ml solutions
always showed an increase in the number of cfu/ml. This finding is reflected in the initial
numbers of bacteria injected into flies in the wildtype experiments in various
experiments. Occasionally a near 10:1 ratio was observed, but more often a ratio less
than 10:1 was seen.
36
Table 2. A summary of the mean number of bacteria injected into imd mutants in two
independent experiments. Also shown are the expected and observed ratios of the
different bacterial solutions.
37
Table 3. The percentage of viable bacteria (as calculated by cfu) at various time points
after bacterial solutions were prepared. A 4 OD/ml and 40 OD/ml solution were prepared
for each trial and aliquots were plated every hour there after. Percentage at a given time
is relative to the number of colonies at zero hours.
Trial 1
Trial 2
Trial 3
Trial 4
Trial 5
0 hours
1 hour
2 hours
4 OD
100.00
157.04
131.89
40 OD
100.00
94.54
120.93
4 OD
100.00
151.54
114.62
40 OD
100.00
124.70
91.69
4 OD
100.00
151.03
164.55
40 OD
100.00
301.74
118.04
4 OD
100.00
105.60
123.92
40 OD
100.00
133.22
72.53
4 OD
100.00
141.26
181.92
40 OD
100.00
80.38
77.08
38
3.4 Surviving an infection is dependent on age.
3.4.1 w1118
When three days old w1118 flies were infected with 4 OD/ml of bacteria, there was
consistently no difference in survival when compared to the negative or no injection
controls (Figure 6A). Less consistent results were observed with 40 OD/ml infections.
Out of three independent experiments, significantly lower survival was obtained in two
female experiments and one male experiment (Figure 6).
At ten days old, a decreased ability to survive 40 OD/ml infections becomes
consistently evident (Figure 6B), with males reaching approximately twenty and ten
percent survival after two and seven days respectively. Ten day old females infected
with 40 OD/ml of bacteria were seemingly less susceptible than similarly treated males,
reaching approximately fifty percent survival after two days post infection, further
decreasing to less than forty percent survival after seven days post infection. Both males
and females injected with 4 OD/ml or with sterile media show no significant difference in
survival. However, both treatments are significantly different from both the non-injected
population as well as the population treated with 40 OD/ml (P<0.02).
At thirty days old, flies become increasingly susceptible to infection. When males
are infected with 40 OD/ml of bacteria, they reach less than fifty percent survival after
just one day, and reach zero percent survival six days after infection (Figure 6C). Males
infected with 4 OD/ml are not significantly different from the negative control, with both
reaching approximately sixty percent survival seven days after infection. Female w1118
infected at thirty days show similar results to males. However, it appears that females are
39
slightly less susceptible to all three treatments. Females treated with 40 OD/ml of
bacteria reached fifty percent survival after two days, while they never reached zero
percent survival within the seven-day assessment. Those treated with 4 OD/ml or the
negative treatment both decreased to approximately seventy percent after seven days.
Forty-day-old males treated with 40 OD/ml showed nearly identical results to
flies infected at thirty days old (Figure 6D). Fifty percent survival was reached one day
after infection, while zero percent survival was reached after approximately six days.
Also similar to the thirty-day-old flies, the 4 OD/ml treatment and the negative control
were not significantly different. However, a much lower survival of thirty percent was
reached after seven days. Moreover, non-injected flies showed susceptibility to infection,
reaching a survival rate of sixty percent after seven days. These results are consistent
across independent trials (Figure 7). Females infected at forty days show similar trends
to thirty-day-old females, if slightly less severe.
While the 4 OD/ml and negative
treatments are again not significantly different, all other comparisons are significantly
different (P<0.05).
40
Figure 6. Percentage survival of male and female w1118 flies infected with varying
amounts of bacteria at different ages. Representative experiments of at least two
independent trials are shown. In all graphs, the Y-axis shows percentage survival and the
X-axis shows days after infection. The sample size is provided in parenthesis. The
average number of bacteria injected into infected populations is also indicated (colony
forming units) (A) Three days old males show no significant differences between
treatments. Females infected with 40 OD/ml are significantly different from all other
treatments (P<0.0001) (B-D) Both male and female ten (B), thirty (C) and forty days old
(D) flies show significant differences (male P<0.02, female P<0.005) between all
treatments except between the 4 OD/ml and negative treatments (P>0.095).
41
A
B
Figure 7. The average difference in percent survival between each treatment and the noninjected w1118 flies two days after infection as a function of the age at which flies were
infected. Error bars represent the range. Males (A) and females (B) are shown.
42
3.4.2 Canton-S
To ensure that these observations do not result from the genetic background, a
different strain was examined and produced mostly similar results.
Three-day-old
Canton-S flies infected with both 4 and 40 OD/ml consistently exhibited no susceptibility
to infection (Figure 8A, 9). Moreover, the negative control flies injected with sterile
media are not yet susceptible to the injection protocol at this age.
At ten days old, males infected with 40 OD/ml showed a significant decrease in
survival, reaching fifty percent by three days and approximately twenty percent by seven
days (Figure 8B). Neither the negative or 4 OD/ml treatments showed any signs of
decreasing survival. Females, like males, show a significant, though smaller, decrease in
survival in response to infection with 40 OD/ml reaching sixty percent survival seven
days after infection. While the 4 OD/ml treatment is not significantly different from the
no-injection control population, the negatively treated flies are significantly different
from both the 4 OD/ml treated and non-injected flies (P<0.02).
At thirty days old, males infected with 40 OD/ml show an almost identical trend
as do ten-day-old males infected with the same titre of bacteria (Figure 8C). At two days
post infection, thirty-day-old males reach forty percent survival, and continue to twenty
percent survival at seven days post infection. However, unlike at ten days, males treated
with either sterile media (negative) or 4 OD/ml of bacteria show decreases in survival.
Negatively treated males decrease to approximately eighty-five percent survival after two
days and remain there consistently for the remainder. 4 OD/ml treated flies decrease to
approximately seventy percent survival after two days and like the negatively treated
43
flies, do not decrease further. In thirty-day-old males, all series are significantly different
from one another (P<0.01).
Thirty-day-old females treated with 40 OD/ml of bacteria decrease to
approximately fifty percent after two days, and continue to decrease to forty percent
survival after seven days. However, unlike males of the same age, thirty-day-old females
treated with 4 OD/ml show no difference from the negatively treated flies. Both the
negative and 4 OD/ml flies decrease to approximately eighty percent survival after two
days and remain largely unchanged after seven days. Similar to thirty-day males, these
two series are significantly different from the non-injected control flies (P<0.0001). In
thirty-day-old females, all series are significantly different from one another (P<0.0004),
except when comparing the 4 OD/ml and negatively treated flies.
Forty-day-old males in all four populations begin to show decreases in survival
(Figure 8D). Those treated with 40 OD/ml show a trend consistent with both ten-day-old
and thirty day old males in reaching approximately sixty percent survival after two days,
and twenty percent survival after seven days. 40 OD/ml males are significantly different
from all other populations. Forty-day-old males treated with 4 OD/ml show slightly less
susceptibility to infection than do thirty-day-old flies infected with the same titre of
bacteria, reaching approximately eighty-five percent survival after two days, and seventy
percent survival after seven days post-infection. Negatively treated flies, like the 4
OD/ml treated flies appear to show an improvement over the thirty day old flies, reaching
ninety percent survival after two days and not decreasing further after seven days.
Finally, the non-injected flies show signs of mortality in decreasing to approximately
44
ninety-five percent survival after seven days post-infection.
All forty-day old male
populations are significantly different from one another (P<0.007) except when
comparing the non-injected and negatively treated populations (P=0.08).
Forty-day-old females infected with 40 OD/ml of bacteria appear to do better than
thirty-day-old flies infected with the same titre of bacteria. While thirty-day-old flies
reached forty percent survival after seven days post infection, forty-day-old females
infected with 40 OD/ml of bacteria reached only sixty percent survival. However, like
the thirty-day-old flies, forty days old flies infected with 40 OD/ml are significantly
different from all other series. Females infected with 4 OD/ml decrease to approximately
eighty-five percent survival after two days post infection, further declining to
approximately eighty percent survival after seven days. Negatively treated flies, while
not significantly different from the 4 OD/ml treated flies, decrease to only ninety percent
survival after infection. Non-injected flies appear to show no decrease in survival. All
forty day female treatments are significantly different from one another (P<0.003), except
when comparing the 4 OD/ml and negative treatments (P=0.242).
45
Figure 8. Percentage survival of male and female CS flies infected with varying
amounts of bacteria at different ages. Representative experiments of at least two
independent trials are shown. In all graphs, the Y-axis shows percentage survival and the
X-axis shows days after infection. The sample size is provided in parenthesis. The
average number of bacteria injected into infected populations is also indicated (colony
forming units) (A) Three days old flies show no significant differences between
treatments. (B) Ten days old flies infected with 40 OD/ml of bacteria are the only
populations to show a significant difference from all other treatments (P<0.0001). (C-D)
Both male and female thirty (C) and forty days old (D) flies show significant differences
(male P<0.0008, female P<0.002) between all treatments except between the 4 OD/ml
and negative treatments (P>0.01). In one out of two independent experiments the
negative and no injection treatments in 40 days old males was not significant (P=0.089).
46
A
B
Figure 9. The average difference in percent survival between each treatment and the noninjected Canton-S flies two days after infection as a function of the age at which flies
were infected. Error bars represent the range. Males (A) and females (B) are shown.
47
3.5 Bacterial clearance remains largely unchanged through age.
In w1118 flies, there appears to be little difference between the 4 and 40 OD/ml
treatments in terms of rate of clearance. At 3 days old, both males and females clear the
majority of the injected bacteria within 48 hours post infection (Figure 10A). This trend
is mirrored in ten-day-old males. Female ten-day-old w1118 flies show nearly identical
clearance for both treatments (Figure 10B). For both thirty and forty day old w1118 flies,
the 4 and 40 OD/ml treatments show similar trends.
Three-day-old CS flies show similar clearance patterns between male and female
flies for both the 4 OD/ml and 40 OD/ml treatments, clearing approximately twenty five
percent of bacteria by forty eight hours after infection (Figure 11A). Ten-day-old males
infected with 40 OD/ml of bacteria appear to clear bacteria initially slower than those
infected with 4 OD/ml, however catch up and reach a similar level of clearance by
seventy-two hours post infection. Both treatments in females show similar clearance
rates (Figure 11B). Thirty and forty-day-old CS flies show similar results for males and
females for both sexes, with the 4 OD/ml treated populations clearing to less than twenty
five percent within forty one hours, and the 40 OD/ml treated flies taking approximately
seventy two hours (11C, 11D).
48
Figure 10. Bacterial clearance following infection in male and female w1118 flies across
age. The average number of bacteria injected into infected populations is also indicated
(colony forming units). In all graphs, Y-axis shows percentage remaining of initial
infection and X-axis shows hours after infection. Error bars represent two standard
deviations.
49
Figure 11. Bacterial clearance following infection in male and female CS flies across
age. The average number of bacteria injected into infected populations is also indicated
(colony forming units). In all graphs, Y-axis shows percentage remaining of initial
infection and X-axis shows hours after infection. Error bars represent two standard
deviations.
50
3.6 Cohort effects did not influence clearance experiment results
While performing clearance experiments, potential cohort effects may have arisen
as a result of the protocol where flies alive at a given time point are homogenized. As a
result, it was possible that two subpopulations existed and that the strongest flies were
selected. To address this issue, ten-day-old flies were infected with 40 OD/ml of bacteria
and at six hours post infection, were separated based on climbing ability. The survival of
the two subpopulations, good climbers and poor climbers, was followed. It was found
that the climbing assay was predictive of lifespan, with good climbers living longer than
poor climbers (Figure 12). Moreover, when the clearance of these two populations was
followed, it was found that there was no significant difference.
51
Figure 12. Percent survival of ten-day-old CS and w1118 flies separated based on
climbing ability after infection with 40 OD/ml of bacteria. Fast climbing females survive
significantly better than slow climbers. In all males and females, there is no significant
difference in internal bacterial titre at six hours post infection (counts shown in brackets).
52
3.7 Genotype specific increase in mortality from infection protocol with age
When the number of flies that die as a result of the infection protocol are
quantified and plotted by age at which they were infected, an interesting trend develops
(Figure 13). While w1118 male flies showed no significant change in mortality with age,
female w1118 flies showed a slight, though insignificant decrease in mortality with age. In
an opposite trend, both male and female Canton-S flies showed an increase in mortality.
In male Canton-S flies, a significant increase in mortality occurred between the ten and
thirty days of age (P<0.03). In females, a significant increase in mortality occurs
between thirty and forty days of age (P<0.005).
53
Figure 13. Percent mortality resulting from the injection protocol as a function of age at
infection. While CS populations show a significant increase in mortality with age, w1118
populations show no significant change.
54
2.7 A bacterial toxin, not proliferation, causes increased mortality upon infection
To test whether bacterial toxins were responsible for the mortality in response to
infection that was evident in our survival experiments, we again took advantage of the
immune deficient mutants available in Drosophila. Imd flies were injected with either
sterile 2TY or supernatant from an overnight culture of E. coli. After three repeats it
became evident that females injected with supernatant survived worse than those infected
with sterile 2TY (Figure 14). However, the results were not as clear in males. In two of
three experiments, males showed a decreased ability to survive in response to infection
with supernatant. This variability is evident when the average difference in percent
survival between flies injected with sterile media and those injected with supernatant is
plotted (Figure 15). Males exhibit extremely large error bars as a result of the
discrepancy between experiments.
55
A
B
Figure 14. The percent survival of male (A) and female (B) imd mutants in response to
infection with either sterile 2TY or supernatant from an overnight culture of E. coli. Both
males and females infected with supernatant are significantly different from those
infected with sterile 2TY (male P<0.0001, female P=0.0003)
56
Figure 15. Average percent difference between imd flies injected with sterile bacterial
growth media and flies injected with the supernatant of an overnight culture of bacteria.
Males and females are shown. Error bars depict two standard deviations.
57
Chapter 4
Discussion
58
4.1 Delivery method
The functionality of the immune system in many mammalian systems declines
with age. As such, in order to use Drosophila as a model organism to study such changes
in immune functionality, it was first necessary to determine whether Drosophila
undergoes the same decline in immune function with age. To test this, it was necessary
to find a method to introduce a consistent amount of bacteria into each fly in order to
challenge their immune system.
The first method of introducing bacteria into flies was common amongst the
literature, where a needle was dipped into a concentrated bacterial solution and then used
to prick the fly. However, it was found that this method produced extreme variation in
the amount of bacteria introduced into each fly. This was the case for both types of
needles used for this method. The variation is likely due to several factors. Firstly, the
amount of bacteria introduced is dependent on how far the needle is dipped into the
bacterial mixture, which is extremely difficult and tedious to control from dip to dip.
Secondly, the depending on how far that needle is inserted into the fly, more or less
bacteria will be deposited. This again is very difficult to control from fly to fly. Finally,
especially with the glass needles, the needles are prone to break; therefore the wound
sizes between flies can change in the course of the experiment, leading to variable
amounts of bacteria introduced.
59
4.2 Survival and Clearance experiments
By modifying current protocols for infecting adult flies, we were better able to
quantify and control immune challenges, and thus better able to discern subtle changes in
the immune system with age. Two observations show that immune senescence does occur
in Drosophila. Whereas 3 days old flies can survive any infection, older animals are
clearly sensitive to infection with the highest dose of bacteria indicating that the capacity
of the immune system to survive E. coli decreases with age. We also found that older
animals become susceptible to injections with sterile media likely resulting from airborne
microorganisms that cannot be fully prevented when performing aseptic infections in an
unconfined laboratory. Although it cannot be tested because the nature of these
microorganisms is unknown, the reproducibility of our results between different
experiments and different strains makes it highly unlikely that the reduced survival
resulted from exposing old flies to different kinds of microorganisms.
Our data does not show any evidence that aging affects the ability to recognize
and eliminate bacteria. It is possible this may result from the experimental protocol. The
flies collected at a given time point during the clearance experiments are a fraction of the
original population that survived to that point. This fraction may constitute a
subpopulation of flies that survived because they maintain the ability to clear bacteria. To
address this issue, we tested whether flies close to death, identifiable by severely reduced
locomotor abilities, show signs of bacterial proliferation. A climbing assay was
performed 6 hours after infection to separate good and poor climbers (Goddeeris et al.,
60
2003). The average internal bacterial titres between good and poor climbers did not show
any significant differences (Figure 12).
Several things are also worth considering in light of our research. If the immune
system is not saturated with the bacterial challenge, it might be difficult to detect and
record any differences that arise after infection. Saturation of the immune system would
ensure that any differences in efficiency that might arise with age would be exploited and
revealed. Without saturation, the level of stress on the immune system may not be
sufficient to elicit a differential response between young and old flies. If, for example, an
old fly had only 80% of the immune capacity as a young fly, but the bacterial challenge
only required 50% of immune capacity to be cleared, then the difference in immune
functionality between young and old flies would not be revealed. However, when the
survival experiments are considered, it is evident that this was not the case, as a decline in
survival in response to infection was seen.
Also, only one pathway of one component of the immune system was tested with
the bacterial challenges. Neither the phenoloxidase, nor cellular response were
monitored and may themselves undergo changes in functionality with age. If a
significant portion of any change in immune functionality occurred in one of these two
alternative forms of immunity, we would not have been able to detect their these changes.
However, it should be noted that the humeral response is the most important component
of the immune system of flies (Hultmark, 2003). Moreover, the Toll pathway was left
unchallenged or monitored as we only challenged flies using E. coli, a Gram-negative
strain of bacteria. Because both pathways initiate the transcription of different anti61
microbial peptide genes, although related, they are distinct pathways. As such, changes
in one would not necessarily correlate to a change in the other. Because of this, it is
possible that a change in the Toll pathway went undetected. However, this seems
unlikely because as discussed before, Tzou et al. (2002) showed that the expression of
only one anti-bacterial peptide is sufficient to restore wild type immune function. Thus,
any change to the immune system large enough to affect its overall functionality would
likely be large enough to be detected using the clearance studies we conducted.
Moreover, decreases in the functionality of the cellular and encapsulation responses
would have been apparent in both survival and clearance experiments.
Finally, the injection process itself could have had an effect on flies that we were
not able to detect. Ha et al. showed that an antioxidant pathway within epithelial lining
of the gut in Drosophila is essential for survival (2005). When this pathway was
blocked, flies showed a significantly reduced survival. This illustrates that a physical
barrier such as the gut epithelium is imperative for proper immune system function and
overall fly health. Injection disrupts the cuticle, the exoskeletal physical barrier of the
fly. While it is unknown whether injury disrupts any immune pathways that may exist
within the cuticle, it does allow the bacteria to circumvent them. For example, immune
senescence might occur as a result of a decreased thickness of the cuticle leading to a
constant over-exposure of the immune system to exogenous bacteria with this over
exposure eventually exhausting the immune system. However, indications of this would
have been apparent in the no injection controls where it would be expected that they
would show a decline in survival with age. This would be further tested in axenic fly
62
lines where the presence of bacteria is eliminated, and it would be expected that a decline
in survival due to persistent exposure to bacteria would not occur.
4.3 A bacteria derived toxin is responsible for immune senescence
Tzou et al. published in 2002 that overexpression of a single anti-bacterial peptide
can restore a wildtype phenotype in immunodeficient mutant flies. Moreover, several
microarray studies indicate that the immune system is up-regulated with age. In light of
these findings, it would seem unlikely that all antimicrobial peptides are down regulated;
therefore immune senescence likely occurred as a result of an event not related to the
expression of antimicrobial peptides. This idea is consistent with several of our findings.
Firstly, in our initial set of experiments in which the infection protocol was established by
infecting imd mutant flies, it was evident that mutants infected with the lowest dose of
bacteria, while succumbing to the infection, did not show any signs of bacterial
proliferation as indicated by the clearance experiments. This implies that the mere
presence of the bacteria is enough to kill the flies.
Our findings in elderly flies are consistent with the idea that the bacteria kill
Drosophila through the production of a bacterial toxin. It became evident that elderly
wild-type flies were especially susceptible to doses of bacteria. However, this
susceptibility did not coincide with a decrease in ability to clear the infection. This
indicates that the flies’ ability to identify and eliminate invaders remains intact with age.
Therefore, the bacteria must be producing a factor that is killing the flies. Moreover, it
also implies that the immune senescence that was detected was not a result of a decreased
63
capacity to eliminate invaders, but instead a result of a decreased ability to cope with the
bacterially produced factors that accompany the infection.
Several preliminary experiments yielded results that were consistent with this
theory. It was observed that flies treated with the supernatant of overnight cultures of
bacteria do indeed survive less well than flies injected with sterile 2TY in all female trials
and two of three male trials. This discrepancy is likely explained by the fact that during
the experiments, the OD of the culture was not controlled. It is possible that in the one
male trial that did not yield the same results as the other two that the overnight culture
was in fact younger than in the other two trials and as such a sufficient concentration of
bacterial factors was not reached. It will be interesting to pursue the identification of the
bacterial products responsible for killing adult Drosophila.
4.4 Research Applications
As previously mentioned, it is well known that mammalian aging is accompanied
by an increase in expression of immunity related genes resulting in a chronic
inflammatory state. Coincident with these changes are a marked decrease in the
functionality of the immune system. Elderly individuals exhibit increased morbidity and
mortality in response to infection and have a reduced ability to generate high affinity
antibodies. Similar to the upregulation seen in mammals, Drosophila exhibit a dramatic
up regulation of many immunity related genes. However, it is not known whether these
changes in gene expression are accompanied by a change in immune function as they are
in mammals. If Drosophila is to be used as a model organism to study changes in
64
immune function with age, it was first necessary to determine if they do indeed undergo
immune senescence.
Because of the complicated interplay between the adaptive and innate immune
systems in mammalian model organisms, study of immune function as a whole in these
organisms can be complicated. For this reason, the use of Drosophila to study immune
function is ideal. In light of our results indicating that immune senescence does occur in
adult Drosophila, it appears that Drosophila would be an ideal model organism in which
to study immune senescence with age.
65
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Appendix A
Replicate Survival Studies
73
Figure 1. Replicate survival studies for three day old male (top) and female (bottom) w1118 flies
after infection.
74
Figure 2. Replicate survival studies for ten day old male (top) and female (bottom) w1118 flies
after infection.
75
Figure 3. Replicate survival studies for thirty day old male (top) and female (bottom) w1118 flies
after infection.
76
Figure 4. Replicate survival studies for forty day old male (top) and female (bottom) w1118 flies
after infection.
77
Figure 5. Replicate survival studies for three day old male (top) and female (bottom) CS flies
after infection.
78
Figure 6. Replicate survival studies for ten day old male (top) and female (bottom) CS flies after
infection.
79
Figure 7. Replicate survival studies for thirty day old male (top) and female (bottom) CS flies
after infection.
80
Figure 8. Replicate survival studies for forty day old male (top) and female (bottom) CS flies
after infection.
81
A
B
Figure 9. The percent survival of male (A) and female (B) imd mutants in response to
infection with either sterile 2TY or supernatant from an overnight culture of E. coli. Both
males and females infected with supernatant are significantly different from those
infected with sterile 2TY (male P<0.0001, female P<0.0001)
82
A
B
Figure 10. The percent survival of male (A) and female (B) imd mutants in response to
infection with either sterile 2TY or supernatant from an overnight culture of E. coli.
While males infected with supernatant are significantly different from those infected with
sterile 2TY (P=0.9849), females show a significant difference (P=0.0006)
83
Appendix B
Replicate Clearance Curves
84
Figure 1. Replicate clearance studies for three day old male (top) and female (bottom) w1118 flies.
85
Figure 2. Replicate clearance studies for ten day old male (top) and female (bottom) w1118 flies.
86
Figure 3. Replicate clearance studies for three day old male (top) and female (bottom) CS flies.
87
Figure 4. Replicate clearance studies for ten day old male (top) and female (bottom) CS flies.
88
Figure 5. Replicate clearance studies for thirty day old male (top) and female (bottom) CS flies.
89