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Genes and Immunity (2004) 5, 576–587
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FULL PAPER
Genetic analysis of innate immunity in resistance
to Candida albicans
A Tuite1, A Mullick2 and P Gros1
Department of Biochemistry, McGill University, Montréal, Québec, Canada; 2Biotechnology Research Institute, Montréal,
Québec, Canada
1
Systemic candidiasis is a significant cause of nosocomial infections and the mechanisms of defense against Candida albicans
in humans remain poorly understood. Studies in animal models have demonstrated the importance of innate immunity in
controlling the response to infection. Although Th1 cytokines have been shown to direct the overall outcome of infection, the
precise role of the Th1/Th2 response and, more generally, the adaptive immune response as a whole, in systemic candidiasis,
appears to apply mainly to the development of resistance to reinfection. A genetic approach to the identification of host factors
regulating pathogenesis and susceptibility to C. albicans infection has been used in humans and in mouse models of infection.
Mouse mutants bearing experimentally induced mutations in specific genes have provided a systematic tool for directly
assessing the role of individual proteins in C. albicans susceptibility. Inbred mouse strains have been valuable in showcasing
the spectrum of naturally occurring variations in initial susceptibility to infection, and type of disease developed. Crosses
between resistant and susceptible strains have led to the detection of additional gene effects affecting innate immunity. Of
particular interest is the major effect of a naturally occurring loss-of-function mutation in the C5 complement component that
has become fixed in many inbred strains. These and other studies have shown that both a functional complement pathway and
robust inflammatory response are critical for resistance to C. albicans.
Genes and Immunity (2004) 5, 576–587. doi:10.1038/sj.gene.6364130
Published online 16 September 2004
Keywords: candida albicans; infection; genetics
Candida albicans infections in humans
Candida albicans is a fungus that can exist as a harmless
colonizer or as an opportunistic pathogen, depending
on the status of its host. In the majority of humans,
Candida is a component of the residential microbial
flora, where it exists as a commensal in the gastrointestinal and genitourinary tracts.1 However, in some—
often immunocompromised—hosts, it has the capacity
to colonize various tissues and organs, causing a range
of diseases. It is the fungal species most frequently
isolated in hospital-acquired infections2 and incidences
of infection have risen with the increased prevalence
of immunosuppressive therapies and broad-spectrum
antibiotic use.3
C. albicans is a member of the genus Candida, and is the
most frequent causative agent of candidiasis in humans.3
It is a diploid organism that undergoes mitosis and has a
cell cycle with stages similar to other yeasts. It has only
recently been shown that C. albicans undergoes mating,
abolishing the previous view of Candida as an asexual
organism.4,5 It exists in severally phenotypically distinct
Correspondence: Dr P Gros, Department of Biochemistry, 3655 Sir
William Osler Promenade, Room 907, Montreal, Quebec, Canada
H3G1Y6. E-mail: [email protected]
Received 22 June 2004; accepted 14 July 2004; published online
16 September 2004
cellular forms, including yeasts or blastospores, pseudohyphae, and hyphae. The dimorphic switch from yeast to
hypha is thought to be one of the principal virulence
mechanisms used by C. albicans to elude the host
immune system during the switch from commensal to
pathogen.6 Mutant strains of C. albicans that are unable to
form either hyphae or yeasts are generally less virulent
than wild-type strains.7–9 There are a number of additional properties of C. albicans that are thought to be
important in the transition from benign colonizer to
pathogen. The production of adhesion factors is important in the early stages of colonization and invasion of
host tissues.10 Hydrolytic enzyme secretion, including
the secreted aspartyl proteinase (Sap) family, phospholipase B enzymes, and lipases, have been demonstrated
to have various roles in the infection process. These
enzymes may be required for nutrient acquisition,
distortion of host cell membranes to assist adhesion
and tissue damage, and digestion of components of the
host immune system to divert antimicrobial attack.11 All
studies of putative virulence factors in C. albicans must be
analyzed in the context of the immune status of the host,
since in the absence of a compromised immune state,
even the so-called virulent forms are not likely to cause
infection. Interestingly, C. albicans has the ability to
exploit a range of weakness in the host defense system,
as illustrated by the spectrum of infections observed in
humans. Obtaining a better understanding of what these
weaknesses are can, in turn, provide insight into the host
Innate immunity in resistance to Candida albicans
A Tuite et al
defenses that are required to protect against invasive
infections by C. albicans.
Types of candidiasis
Infections caused by C. albicans can be broadly classified
into two major categories: superficial and invasive
candidiasis, with the major differences between these
classes being the type of immune response elicited and
the nature of the ensuing pathology. Superficial candidiasis encompasses a range of infections, including
thrush, chronic atrophic stomatitis, chronic mucocutaneous candidiasis, and vulvovaginitis, and involve
colonization of the skin and mucosal surfaces.12 These
infections tend to be quite specific and self-limited in
non-immunocompromised hosts, with the remedy involving local treatment and basic hygiene measures.12 Tlymphocyte-mediated immunity is the major player in
the control of superficial infections. Among the forms of
superficial candidiasis, chronic mucocutaneous candidiasis (CMC) is the syndrome with the strongest
evidence for the involvement of host genetic factors in
disease manifestation and outcome.13 CMC is characterized by persistent and recurrent infections of the skin,
nails, and mucous membranes by Candida spp., especially C. albicans, and tends to be limited to patients with
impaired T-cell function. CMC is often accompanied by
endocrine and inflammatory disorders, and distinct
syndromes have been identified based on the disorders
that occur in conjunction with CMC.14 The AIRE
(autoimmune regulator) gene has been associated with
the autosomal recessive syndrome autoimmune polyendocrinopathy candidiasis ectodermal dystrophy
(APECED).15,16 This gene was identified by positional
cloning after mapping to chromosome 21q by linkage
analyses in 14 Finnish families17 and subsequent confirmation in an Iranian Jewish population, giving a
maximum LOD score of 10.2.18 An additional gene effect
was identified in a large consanguineous family with
CMC associated with thyroid disease that segregated as
an autosomal dominant trait. A whole genome scan,
followed by fine-mapping in 34 family members identified a 15-cM region on chromosome 2p with a maximum
LOD score of 3.8.19
In contrast to cutaneous infections, which are heavily
dependent on acquired immunity, innate immunity
appears to be more important in determining disease
outcome during invasive candidiasis. This dichotomy
between the two major classes of infections is perhaps
best exemplified by the prevalence of oropharyngeal
candidiasis in HIV-positive and AIDS patients, who have
suppressed T-cell function compared to the relative
dearth of invasive infections in the same patients, who
have relatively intact phagocytic function.20
Invasive candidiasis refers to Candida infections that
occur at sites other than the skin or mucous membranes,
with most cases caused by bloodstream invasion and
subsequent spread of the organisms.21 These infections
occur most frequently in immunocompromised hosts,
although invasive candidiasis in critically ill, nonimmunosuppressed patients is becoming a growing
concern.12 Chronic disseminated candidiasis (CDC) or
hepatosplenic candidiasis occurs in patients undergoing
intensive therapy for acute leukemia, where various
aspects of their treatment put them at increased risk for
infection.22 Patients develop signs of invasive candidiasis
in multiple organs over a period of several weeks to
months.21 Choi et al22 have identified a common
haplotype in the promoter region of IL-4 that is overrepresented among acute leukemia patients with CDC,
which they propose contributes to increased susceptibility to infection due to decreased IL-4 production. This
haplotype was identified in a case/control study.
Patients with the haplotype 1098T/589C/33C are
at a greater risk of developing CDC, with an odds ratio of
2.16. Similarly, the protective 589T polymorphism is
under-represented among CDC patients and this polymorphism is functional for enhanced IL-4 transcriptional
activity.23
Disseminated or systemic candidiasis, another form of
invasive infection, is characterized by the hematogenous
spread of Candida to one or multiple organs.21 Systemic
candidiasis is a life-threatening infection, with the overall
prognosis comparable to septic shock with multiple
organ failure.12 Although it is well established that
systemic infection is associated with insufficiencies in
host defenses,3 to date, no specific genes have been
shown to cause a predisposition to infection. However,
there is some anecdotal evidence that patients with
congenital defects affecting phagocyte function, such as
myeloperoxidase (MPO) deficiency, have a higher occurrence of systemic candidiasis than healthy individuals.24
Among the major risk factors for candidiasis are:
neutropenia, broad-spectrum antimicrobial treatment,
damage to oropharyngeal mucosa through use of
cytostatic drugs, use of central venous catheters, parenteral nutrition, and breaches of skin integrity due to
injury or burns.1,3
577
Epidemiology
The incidence of hospital-acquired infections caused by
Candida has increased steadily since the 1970s; this is
likely due to both an increase in the patients who are
rendered susceptible to infection due to immunosuppressive procedures and improved clinical procedures to
identify yeasts as the causative agents of nosocomial
infections.25 Candidiasis is the fourth most common
cause of nosocomial bloodstream infections.1 Although
invasive candidiasis occurs in 1–8 percent of patients
admitted to hospitals, its occurrence is higher in
intensive care units, with infection rates of approximately 10 percent.12 In intensive care units, candidiasis is
accountable for up to 15 percent of all nosocomial
infections.12 The mortality attributed with candidemia
has been reported to range from 25 to 60 percent.26 There
is also a considerable morbidity associated with Candida
infections, with a median 30 day increase in hospital stay
seen in nonfatal cases compared to uninfected controls.27
Thus, although the incidence of candidiasis in the
general population is not a major problem, the cost in
terms of attributable morbidity and mortality among
hospitalized patients is significant.
Disease pathology
Systemic candidiasis is of particular concern due to the
severity of these infections in already hospitalized, and
often critically ill, patients and the difficultly of treatment
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Innate immunity in resistance to Candida albicans
A Tuite et al
578
with antifungal agents. Although disease manifestation
varies with respect to the target organs and severity of
colonization, some generalizations about infection initiation, progression, and outcome can be made. Systemic
candidiasis usually begins with the penetration of
mucosal barriers, giving the pathogen access to the
bloodstream, where it is subsequently disseminated
throughout the body,26 although external introduction
through central venous catheters and other surgically
implanted devices has also been reported.28,29 Candida
lesions have been found in virtually every tissue of the
body, although the major targets of infection are the
digestive tract, lungs, kidney, and brain.30 The heart is
colonized nearly as often, while the liver and spleen are
less frequently infected. Emboli with yeasts and pseudohyphae are often found in the blood vessels of
systemic candidiasis patients.
C. albicans causes the formation of small, white lesions
in the parenchyma of deep organs.30 These lesions are
multiple discrete abscesses or microabscesses that superficially resemble those formed in miliary tuberculosis or
by diffuse cancer metastases. Candida yeasts and filamentous forms tend to be profuse within the abscesses,
and are usually, but not always, associated with
neutrophil infiltrates.
The study of systemic candidiasis in humans, especially concerning factors which predispose to infection,
has provided some insight into the contribution of the
host in regulating disease susceptibility. It is clear that
the innate immune response, especially neutrophilmediated phagocytosis or killing, is of particular
importance in preventing systemic candidiasis. However, in the absence of an obvious genetic link to
susceptibility to candidiasis, further elucidation of the
contribution of the host in determining disease susceptibility, progression, and outcome has necessitated the use
of animal models.
Animal models
Animal models have been used extensively as a tool to
better understand systemic C. albicans infections, from
the perspective of both the host and the pathogen.
Although the mouse is by far the most commonly used
animal rats, rabbits, and guinea-pigs are all approximately equally as susceptible to infection as the mouse
and display similar disease pathology.30 Systemic infections in these animals resemble human candidiasis, with
the kidney being the major target of infection.31 The
major routes used to establish infection are intravenous
(i.v.) and intraperitoneal (i.p.) injection. I.v. inoculation
leads to the establishment of an acute systemic infection
involving multiple deep organs. I.p. infection also leads
to disseminated infection, but the required inoculum to
establish a pathology similar to that observed in i.v.
infection is approximately 10-fold higher.30 An alternate
model uses gastrointestinal (g.i.) candidiasis as a model
for systemic candidiasis of endogenous origin, but
establishing an infection using this method is more
variable, since it relies on dissemination of Candida from
the g.i. tract into the bloodstream, prior to organ
colonization.
An extensive review of the progression of systemic
candidiasis in the mouse has been conducted by Odds.30
In brief, systemic infections in the mouse are characterized by a rapid clearance from the blood, with the
Genes and Immunity
highest fungal loads isolated from the lungs and liver in
the first hours after C. albicans injection. By 1 to 2 days
postinfection, the kidneys have the highest fungal
burden, and this increases with time, due to active
replication. In contrast, fungal loads in the lung, liver,
and spleen decline over time. Depending on the dose
and strain of mouse used, animals may begin dying from
overwhelming infection within the first 24 h, presumably
from kidney failure caused by acute pyelonephritis.31
After the first week of infection, the kidney is the most
obviously affected organ in all surviving animals. Its
surface is studded with macroscopic white abscesses.
These abscesses are focal collections of Candida yeast,
hyphae, and pseudohyphae. The appearance of lesions
varies with the strain of mouse used. They can show little
evidence of an inflammatory reaction, contain large
infiltrates of polymorphs and/or macrophages, or form
granulomas. A similar pattern of tissue damage has been
observed in other affected organs, although the extent
and number of lesions tends to be reduced, compared to
the kidney.
Phenotypes measured
Doses and strains of C. albicans used to study susceptibility in the mouse tend to vary across experiments, with
inocula ranging from 103 to 108 yeasts having been
tested.30 The LD50 for most C. albicans isolates tested in
immunocompetent mice is between 104 and 106 yeasts.
Both the strain of C. albicans and the growth conditions
used have been shown to influence overall lethality
during infection.32 The three phenotypes most frequently
measured to ascertain host response to C. albicans
infection are tissue damage, fungal burden, and mortality, with each representing a different aspect of the host–
fungus interaction.33 Tissue damage is assessed microscopically and is a somewhat difficult phenotype to
measure quantitatively. It represents the interaction
between Candida and the host effector cells at a given
point in time and gives an idea of the type of
inflammatory response elicited and the major cell types
recruited during this response. In the mouse, the organs
most involved in disease pathology are the kidney, brain,
and heart. Measurements of total fungal burden in
affected organs at a given point in time give a more
quantitative measure of infection and reflect the candidacidal or candidastatic efficiency of the host response.
Mortality indicates the overall susceptibility to infection.
A correlation between kidney fungal load and mortality
has been observed.34–36 Cytokine levels at various time
points throughout infection provide an additional
measure of the differences in the host response to
systemic candidiasis. Early in infection, susceptible mice
have elevated levels of TNF-a and IL-637 (Mullick et al,
in press) and these cytokines are useful predictors
of disease outcome. Similarly, mRNA levels of IFN-g,
TNF-a, and IL-6 in the brains of susceptible mice with
systemic candidiasis were found to correlate with the
total infectious burden.38
Mouse models of systemic candidiasis provide a wellestablished foundation upon which to base studies into
the mechanisms of the host response to infection.
Patterns of tissue damage in targeted organs have been
described, as has overall disease pathology and progression. Selection of phenotypes that show clear differences
between resistant and susceptible animals enables the
Innate immunity in resistance to Candida albicans
A Tuite et al
characterization of the effects of various cellular and
gene deficiencies on susceptibility. As a result, mouse
models have been used with great success to elucidate
the involvement of various cell populations in the
defense against systemic infection.
Effector cells
In an attempt to characterize the contribution of specific
cell types of the immune system, the response to
infection has been studied in immunodeficient strains,
which have defects or deficiencies in one or more cell
populations. In addition, the specific depletion of a given
population through antibody neutralization or chemical
means has been used.
Neutrophils are the major effector cells early in
systemic infection. They are rapidly recruited to the sites
of infection. Among the mechanisms employed by
neutrophils to control infection are: phagocytosis of C.
albicans,39,40 production of reactive oxygen species,41 and
the release of mediators, including cytokines and
antimicrobial substances.42 In animals that are pretreated
with agents to reduce the number of neutrophils, there is
a significantly increased susceptibility to systemic C.
albicans challenge.30,43 Similarly, in mice that are selenium
deficient, a condition where the candidacidal activity of
neutrophils is impaired, there is a high susceptibility to
candidiasis.44 Beige mice, which have impaired natural
killer (NK) cell and neutrophil function, have a reduced
ability to kill C. albicans blastospores and hyphae
compared to immunocompetent controls.45,46 On average, two yeasts are ingested per neutrophil, although up
to ten yeasts have been observed per cell.47,48 The
proportion of ingested yeasts that are killed is between
20 and 30 percent, regardless of the number ingested,
with unkilled yeasts capable of germinating and growing
out through the cell wall.49 Neutrophils kill hyphal and
yeast forms of Candida at similar rates, although yeasts
tend to be killed intracellularly and hyphae are predominantly killed extracellularly.39,40 In addition to
phagocytosis and killing, neutrophils are capable of
directing the development of the host immune response
through cytokine secretion.42
More contentious is the role of macrophages in the
host response against C. albicans. In vitro studies have
shown that the candidacidal capacity of macrophages is
worse, similar to, or better than neutrophils, depending
on the experimental conditions.50 One study showed that
mouse peripheral blood leukocytes and peritoneal
macrophages ingest live C. albicans at the same rate,
but leukocytes more effectively killed intracellular
yeasts.37 Nramp1 is a divalent cation transporter at the
phagosomal membrane of macrophages and neutrophils,
with mutations in this protein causing an increased
susceptibility to several intracellular pathogens.51 A role
for Nramp1 in the killing of Candida yeasts and hyphae
has been shown, despite the fact that they are killed by
intracellular and extracellular mechanisms, respectively.52 Interestingly, NRAMP1 is upregulated in neutrophils and is recruited to the membrane of C. albicanscontaining phagosomes.53 To date, no in vivo studies in
mice have unambiguously demonstrated an absolute
requirement for macrophages in the resolution of C.
albicans infections.43,54
Depletion of NK cells was found to have no effect on
resistance to primary C. albicans infections or in
conferring protection against reinfection55 and it is
generally accepted that NK cells do not play an essential
role in host response against candidiasis.55,56
A role has been proposed for dendritic cells (DCs) as
initiators of adaptive immunity. They are capable of
phagocytosing both the yeast and hyphal forms of C.
albicans, albeit by different mechanisms.57 Depending on
the form of Candida engulfed, different downstream
cellular events are initiated. Since DCs initiate responses
in naı̈ve T cells and participate in T helper (Th) cell
education, they are expected to have a role in regulating
the response to infection.58 Although the relative
contribution of DCs to the control of systemic candidiasis
has not yet been clearly demonstrated, it is believed that
detection by DCs through Toll-like receptors (TLRs) is
involved in pathogen discrimination and initiation of the
appropriate adaptive immune response.59,60
The involvement of T- and B-lymphocytes in host
defense against systemic candidiasis has been studied in
nude (thymus deficient)60–62 and SCID (absence of T and
B cells)43,63 mice. These cell populations are not essential
for protection against primary infection and, in some
instances, deficient strains are actually more resistant.43,47,63,64 Similarly, thymectomized, irradiated, and
bone marrow reconstituted (TXB) mice showed no
difference in disease outcome when compared to
immunocompetent controls.65 However, TXB mice developed no resistance to rechallenge. Even at the later stages
of infection, when acquired immunity is expected to be
operative (days 10–21), no demonstrable role was
observed for T or B cells.63 The major role of T cells
in protection against systemic candidiasis appears to
be in the development of resistance to reinfection after
initial inoculation with a vaccine strain of C. albicans.36,65
CBA/N mice have X-chromosome-linked immune deficiency (XID), which manifests primarily as a defect in
humoral immunity, specifically a reduced serum antibody production. These mice have been used as a B-cell
defective mouse model.66 In both primary and secondary
infections, no difference was seen between CBA/N
mice and B-cell-competent CBA/J mice in response to
i.v. C. albicans infection.66 Conversely, Wagner et al67
observed that JHD B-cell knockout mice, which are
generated by the targeted disruption of the gene segment
that encodes the joining region of the Ig H chain,68 were
more susceptible to systemic infection than wild-type
controls. Although antibodies directed against C. albicans
are produced upon exposure to the pathogen, the
presence of both protective and nonprotective antibodies
complicates any attempt to determine the overall
importance of humoral immune mechanisms in conferring protection.69
Efforts to determine the relative contributions of
various cells of the immune system to the host response
to infection have benefited from the study of immunodeficient mouse models. It is clear that neutrophils are
the major effector cells involved in controlling the spread
and replication of C. albicans during infection. However,
it is likely that other phagocytic cells, such as macrophages are also important in the host response, although
a redundancy in their function may preclude their
detection in depletion studies. Less obvious is the role
of T and B cells in infection resolution, with most studies
suggesting that these cell populations are relatively
dispensable in primary infection resolution.
579
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Innate immunity in resistance to Candida albicans
A Tuite et al
580
Knockout mice have been used to further clarify the
role of both the innate and acquired arms of the immune
system in progression of systemic candidiasis (Table 1).
The effect of a deficiency of a specific component of the
immune system provides a more refined view of the
pathways and networks activated by the host in response
to infectious challenge.
Deficiencies of innate immunity and involvement in
early infection
Innate immunity is a major component of the response to
systemic C. albicans infection, since it is the first arm of
the immune system to detect and react to the invading
pathogen. C. albicans is cleared rapidly from the bloodstream and becomes lodged in various organs. Among
the first cells at the site of infection are professional
phagocytes, and deficiencies that affect the ability of
these cells to ingest and kill C. albicans are implicated in
increased susceptibility to infection. Detection of Candida
occurs mainly through TLRs and the complement
system. Uptake of unopsonized C. albicans occurs
through pattern recognition receptors,70 whereas serum-opsonized Candida is phagocytosed by complement
receptors on phagocytic cells.49
TLRs have the ability to recognize the invading fungi
and initiate signaling cascades to recruit and activate
effector cell populations.70 TLR2 and TLR4 are the most
well characterized of the TLRs with respect to their
involvement in fungal recognition. TLR4, which is
expressed on the surface of macrophages, DCs, and
endothelial cells,71,72 recognizes C. albicans-derived mannan,73 possibly in conjunction with the CD14 receptor. It
is involved in the control of fungal replication in the
kidneys, likely by inducing the secretion of CXC
cytokines such as KC and MIP-2, which subsequently
recruit neutrophils to the site of infection.74 Conflicting
results exist for the mechanism of TLR2’s involvement in
the regulation of infection outcome. TLR2 expression is
detected on neutrophils, DCs, and monocytes,72 and it is
thought to detect phospholipomannan from the cell-wall
surface of C. albicans.75 Villamon et al76 have proposed
that TLR2 is essential in murine defense against C.
albicans infection, and in the absence of TLR2, mice have
a decreased survival time after infection. Conversely,
Netea et al77 showed that signaling through TLR2
appears to be detrimental to host survival, since the
Table 1 Involvement of genes in resistance to primary infection
with C. albicans
Gene
Phenotype measured
Fungal load
Mortality
Fungal load
Mannose receptor Mortality and
NADPH oxidase Mortality and
MPO
Mortality and
TNF-a
Mortality and
IL-6
Mortality and
IFN-g
Fungal load
IL-12
Mortality and
IL-10
Fungal load
IL-4
Fungal load
Role in primary
infection
TLR4
TLR2
Genes and Immunity
fungal
fungal
fungal
fungal
fungal
load
load
load
load
load
fungal load
kSusceptibility
Uncertain
mSusceptibility
No effect
kSusceptibility
kSusceptibility
kSusceptibility
kSusceptibility
kSusceptibility
No effect
mSusceptibility
kSusceptibility
absence of TLR2-mediated signaling results in an
increased resistance to systemic candidiasis. They attribute this to an expansion of the CD4 þ CD25 þ regulatory
T-cell population and a decreased macrophage candidacidal capacity in the presence of TLR2. These findings
were supported by Bellocchio et al,78 who found a
reduced fungal load in the kidneys of TLR2-deficient
mice but no difference in mortality. The reason for the
apparently opposing role of TLR2 in controlling the
response to systemic candidiasis is unclear, but may
reflect differences in the strain of C. albicans used, since
TLRs have been shown to discriminate between fungal
morphotypes.32,78
The mannose receptor is another pattern recognition
receptor that was thought to recognize C. albicans
through the binding of specific cell wall glycans, based
on in vitro studies.79,80 However, a recent study in
mannose receptor knockout mice showed no significant
difference in overall susceptibility compared to wildtype controls,81 suggesting that this is not a major
receptor for C. albicans detection, or that a redundancy
exists with the TLR4/CD14 system.
The complement system, which is composed of plasma
proteins and membrane proteins, is important in the
opsonization, phagocytosis, and killing of microorganisms.82 C. albicans activates the complement system
through the alternative pathway.83 Guinea-pigs deficient
in complement component 4 were unaffected in their
ability to respond to infectious challenge with C. albicans,
suggesting that the classical pathway is not essential in
pathogen recognition and clearance.83 The involvement
of the membrane attack complex in direct killing of the
pathogen has not been demonstrated.84 The chemotactic
activity of the complement is likely to be important in
recruiting effector cells to infected tissues. Complement
enhances phagocytosis of C. albicans by murine blood
leukocytes, but it does not affect killing by these cells.85
MPO and NADPH oxidase generate reactive oxygen
species, which are an integral part of the microbicidal
system used by neutrophils against Candida.86 Defects in
these complexes have been implicated in enhanced
susceptibility to candidiasis.24,41 NADPH oxidase generates superoxide anion during the phagocytosis-associated respiratory burst. Mice with X-linked chronic
granulomatous disease (X-CGD), which have deficiencies in NADPH oxidase, have an increased susceptibility
to disseminated candidiasis, as determined by survival
time and fungal load.86 This correlates with the observed
decreased in vitro candidacidal activity of neutrophils
from human CGD patients, despite the normal uptake of
C. albicans.49
MPO is mainly expressed in neutrophils and monocytes and leads to the production of hypochlorous acid.87
In vivo studies in MPO knockout mice demonstrated that
these mice are increasingly susceptible to systemic
candidiasis,86,87 in agreement with the finding in humans
of an elevated rate of C. albicans infections in patients
with congenital MPO deficiencies.24 Neutrophils isolated
from a patient with disseminated candidiasis were able
to ingest but not kill C. albicans in vitro, and these
leukocytes were subsequently found to be completely
devoid of MPO.49
The innate immune system is a major player in the
detection, uptake, and killing of C. albicans during
systemic infection. Studies in knockout mice have
Innate immunity in resistance to Candida albicans
A Tuite et al
revealed the importance of the TLR pathway and the
complement cascade in the initial interaction between
the host and pathogen. Following detection and possible
phagocytosis, depending on the morphogenic form of
Candida, the microbicidal system is key in controlling the
replication and spread of the invading organism.
Th1 and Th2 cytokines in experimental systemic
candidiasis
Although no definitive role for T cells has been observed
during infection, it is clear that cytokine production by
Th cells can influence infection outcome. The Th cell
response during systemic candidiasis in the mouse has
been well studied.88 The profile of Th cell differentiation
in response to candidiasis is directed by cytokines
produced during the initial phase of infection. Various
effector cells have been shown to secrete cytokines,
thereby influencing the adaptive immune response by
directing CD4 þ Th cell differentiation. These cytokines
prime the activation of naı̈ve T cells into either Th1 or
Th2 cells. The Th1 response is associated with cellular
immunity, leading to the activation of macrophages and
neutrophils to a candidacidal state. The Th2 response is
involved in the development of humoral immunity,
resulting in the activation of B cells. Since the cytokines
produced by one T-cell subset tend to inhibit activation
of the other set, the overall effect is the dominance of
either humoral or cell-mediated immunity, although
mixed Th1/Th2 responses do occur.89 In the case of
systemic candidiasis, the development of a Th1 response
is associated with protection against infection, while a
Th2 response is more frequently observed in susceptible
hosts.90 The overall importance of the Th1/Th2 paradigm
in disease outcome is mainly applicable to the development of protective immunity after initial infection. The
development of protective immunity has been mainly
studied by evaluating the effects of various cytokine
deficiencies on resistance to reinfection, following primary infection with an avirulent vaccine strain.36 In this
system, Th1 cytokines have been shown to be important
in protective immunity development, while Th2 cytokines tend to prevent the development of resistance to
reinfection.57 The roles of these various cytokines in
primary infection have also been studied, and individual
cytokines that can be classified as either Th1 or Th2 do
influence disease progression and outcome. Thus, they
likely have a dual role during infection, regulating both
effector cell functions and T-cell development.
The proinflammatory cytokine TNF-a is important in
innate and adaptive immunity against C. albicans. It
recruits and activates effector phagocytes, induces
costimulatory molecules, and is required for Th1
response induction.90 TNF-a production is initiated in
the first few hours after infection, primarily in monocytes
and macrophages,91 although neutrophils also secrete
this cytokine.92 It does not appear to be a mediator of
early death, since neutralization does not alter the time of
death, and administration of TNF-a modestly protects
against infection.93 TNF-a knockout mice have a
decreased survival time and higher fungal loads in the
kidney, spleen, and liver.94 In the absence of TNF-a, there
is a reduced recruitment of neutrophils to the sites of
infection, which likely explains the increased susceptibility of knockout mice.95 Similar findings are seen in IL6 knockout mice,96 with IL-6 deficiency leading to a more
severe pathology. These cytokines act very early in
infection, arguing against Th1 response induction as
their primary role during infection. Since the TNF-a and
IL-6 knockout mice die early in infection, even upon
injection of a low virulence strain of C. albicans, their
effect on the development of protective immunity has
not been determined.36 However, the in vitro analysis of
cytokine production of activated spenocytes from infected mice showed the high levels of production of Th2
cytokines.96,97
IFN-g is another Th1 cytokine that is important in
primary infection. IFN-g knockout mice are more
susceptible than wild-type controls to systemic candidiasis.98 Experiments investigating the roles of IL-12 and
IFN-g in systemic infection99 highlight the complexity of
the balance of cytokines during infection progression.
Despite its importance in protection against reinfection,100 native IL-12 is not required during primary
systemic candidiasis,36,101 and exogenous IL-12 administration actually exacerbates disease severity, apparently
by inducing IFN-g overproduction. The IFN-g receptor is
required for protection against reinfection with C.
albicans.102
The Th2 cytokines IL-10 and IL-4 have disparate effects
on the control of infection progression. IL-10 inhibits
cytokine production by Th1 cells, and has a detrimental
effect on resistance to systemic infection, as demonstrated by both neutralization103 and knockout studies.104
In contrast, IL-4 appears to be important in protection
against the early stages of fungal infection, with knockout mice having an increase in fungal replication in
target organs.104 Interestingly, this Th2 cytokine is
required for the development of resistance to re-infection.105
Based on these and other studies on the effects of
various cytokines on disease outcome, it is apparent that
they are an integral part of the host response to
candidiasis. However, the relatively simplistic view of
the protective Th1 vs the nonprotective Th2 response,
while providing a framework by which to better understand the nature of the response to infection, is not able
to fully encapsulate the complexity of this response. In
particular, the timing of cytokine production can have a
major influence on the effect on infection progression. A
number of protective Th1 cytokines have their effects
very early during infection, before acquired immunity is
active. However, in the context of protection from reinfection, Th1 response development is important.
Numerous studies in knockout mice have clarified the
importance of individual genes in disease pathogenesis.
However, these analyses are limited to the exploration
pathways and molecules that are predicted to be relevant
to the host defense against invading pathogens. Additionally, these studies in mice may be confounded by
unforeseen genetic background effects. A parallel approach to identify novel or not previously implicated
mechanisms of host defense is gene discovery through
the use of inbred mouse strains.
581
Studies in inbred mouse strains
From studies in knockout mice, it is clear that the host
response to candidiasis is complex, involving a number
host–pathogen interactions, as well as interactions
between various components of the immune system.
An alternative approach to identifying host genetic
Genes and Immunity
Innate immunity in resistance to Candida albicans
A Tuite et al
582
factors involved in disease outcome is through the use of
inbred mouse strains, which possess a diversity of
genetic backgrounds that have been fixed through
generations of inbreeding. Over the years, the response
of a number of different inbred strains to systemic
candidiasis has been assessed, with fungal load and
mortality being the two most commonly measured
phenotypes (Table 2).33,35,74,106–111 For the more frequently
tested strains, results between experiments are fairly
consistent, despite differences in experimental conditions, such as C. albicans strain and dose used and the
time of assessment of fungal load (fixed time point or at
time of death). Interstrain differences are apparent with
respect to susceptibility to infection with C. albicans,
although correspondence between mortality and fungal
burden is not always seen, suggesting the possibility of
alternate genetic control for these traits. Exploitation of
differences between strains may allow for the mapping
of loci that affect the host response to infection, which in
turn may lead to the identification of their counterparts
in humans. Eventual cloning of any identified genes
would provide insight into factors affecting susceptibility
to systemic infection and potentially improve treatment
of these infections.
Carg1 and Carg2
To date, two gene effects controlling susceptibility to
systemic candidiasis, as measured by tissue damage,
have been proposed. The discrete differences in tissue
damage seen in BALB/c and CBA/CaH and the
segregation pattern observed in a [BALB/c CBA/
CaH] F2 population led Ashman and Papadimitriou112
to propose the existence of a codominant, Mendelian
gene. The resistance conferred by this gene appeared to
be acting through cells derived from the bone marrow.
They subsequently investigated this trait in AKR and
C57/L mice; the kidneys of AKR mice showed moderate
tissue damage after i.v. infection, whereas the damage in
C57/L kidneys was of mild severity.113 Using a panel of
15 AKXL recombinant inbred strains, this gene, C.
albicans resistance gene 1 (Carg1), was tentatively
assigned to chromosome 14.114 Carg1 is hypothesized to
be involved in overall susceptibility to tissue damage.
However, tissue damage in some of the recombinant
inbred strains was more severe than either of the parental
strains, suggesting that a second locus, designated C.
albicans resistance gene 2 (Carg2), may be involved. This
gene is proposed to influence specifically the level of
susceptibility of the kidney to fungal damage,115 having
relatively minor effects in overall susceptibility. The
genomic location of this putative gene has not yet been
mapped. The characterization of the roles of these loci in
the mechanism of resistance to infection, as well as any
interaction with other components of the host immune
system, awaits further mapping and cloning.
Involvement of complement component 5
In looking at the susceptibility to C. albicans across mouse
strains, the importance of complement component 5
(Hc/C5) in determining disease outcome is striking. The
association of C5 deficiency with increased susceptibility
is clear, although the correlation is not perfect. A similar
relationship is seen for susceptibility to infection with
Cryptococcus neoformans116 and Saccharomyces cerevisiae.117
Up to 40 percent of the commonly used inbred mouse
strains118 have a 2-bp deletion in an exon near the 50 -end
of the mRNA, introducing a premature stop codon 4-bp
downstream of the deletion.119 This leads to the production of a truncated 216 amino-acid translation product
compared to the wild-type 1680 amino-acid protein.
This truncated protein is not secreted.119 The functional
consequences of a C5 deficiency in the response to
systemic candidiasis have been studied extensively.111,120,121 In two histocompatible B10.D2 congenic
strains that are expected to differ primarily in their C5
status, the C5-deficient strain had a higher fungal burden
Table 2 Response of inbred strains to systemic infection with C. albicans
Strain
A/J
AKR/J
B10.D2/oSn
B10.D2/nSn
BALB/c
BALB/cN
C3H/HeN
C3H/HeJ
C57/L
C57BL/10SnJ
C57BL/6J
C57BL/6J beige-J
C57BL/KsJ
CBA/CaH
CBA/J
CBA/N
DBA/1
DBA/2
NZB
RF/J
SJL
a
Hc allelea
0
0
0
1
1
1
1
1
1
1
1
1
1
1
1
1
1
0
0
0
1
0 — C5 deficient; 1 — C5 sufficient.
Genes and Immunity
Survival
Fungal load
Susceptible
Resistant
—
—
Resistant
Susceptible
Resistant
Susceptible
—
—
Resistant
Intermediate
Resistant
Susceptible/Intermediate
—
Intermediate
Resistant
Susceptible
Susceptible
Susceptible
Resistant
Susceptible
Susceptible/Intermediate
Susceptible
Resistant
Resistant
Resistant
Resistant
Susceptible/Resistant
Resistant
Resistant
Resistant
Intermediate
Resistant
Intermediate
Susceptible
Susceptible
Resistant
Susceptible
—
—
—
References
33, 107, 110
33, 35, 107
111
111
33
106
33, 74
74, 106, 108, 110, 111
33
35
33, 106, 107, 110
106
106,107
33, 107
35, 110
106
33, 107
33, 107, 110
109
35
107
Innate immunity in resistance to Candida albicans
A Tuite et al
in the kidney early in infection.111 Although the presence
of C5 did allow for a more rapid initial control of C.
albicans replication after challenge, as the infection
proceeded there was no significant difference between
the C5-sufficient and -deficient strains in terms of kidney
fungal load. This suggests that additional genetic factors
can contribute to the later stages of infection control,
even in the absence of functional C5. The treatment of
C5-deficient DBA/2 mice, which are extremely sensitive
to infection, with C5-containing serum had a protective
effect, reducing fungal load, and increasing survival
time.120 Depletion of neutrophils did not completely
abrogate the protective effects of C5 treatment, indicating
that C5 is not acting exclusively through neutrophils and
supporting the view that other cell populations are also
recruited to control C. albicans replication. We have
provided additional evidence for the important role of
C5 in regulating the response to infection. The recombinant congenic strain BcA70, which derives 87.5 percent
of its genome from the resistant, C5-sufficient C57BL/6J
(B6) strain and 12.5 percent from the susceptible, C5deficient A/J strain,122 is as susceptible to infection as A/
J. Despite being on a resistant background, BcA70 is
deficient for C5, suggesting that C5 is a major determinant in controlling the response to systemic candidiasis.
To map genes controlling the host response to systemic
candidiasis, we have assessed fungal load in the kidney
and brain in a susceptible and a resistant inbred strain.
Fungal load in the brain is significantly correlated with
kidney fungal load (Po0.0001), suggesting that they are
under the same genetic control. Based on fungal loads in
these organs measured 48 h after i.v. infection, A/J is
susceptible and B6 is resistant to infection. The distribution pattern for fungal load measured in [A/J B6] F2
progeny was indicative of susceptibility acting as an
autosomal recessive trait under simple genetic control. A
genome-wide scan on the DNA from 128 F2 mice
followed by linkage analysis led to the identification of
a statistically significant linkage on the proximal portion
of chromosome 2, which we determined to be due to the
effect of C5. We also looked at heart fungal load, serum
TNF-a levels, and overall mortality in additional F2 mice.
Segregation analysis verified that these differences
between susceptible and resistant mice were explained
by the effect of C5. Thus, C5 deficiency in mice is
associated with a decreased resistance to infection. This
gene acts in a recessive manner, with heterozygotes
displaying a similar degree of resistance as their wildtype counterparts. Our results provide further support
for the importance of C5 in regulating the response to
systemic candidiasis (Tuite, Mullick, and Gros, unpublished).
We have observed that C5 deficiency leads to elevated
serum TNF-a levels and Ashman and Papadimitriou37
have shown that TNF-a secretion is proportional to the
infectious burden. A clue as to the role of C5 in
controlling TNF-a production is provided by experimental studies of sepsis; in a rat model, C5a has been
shown to inhibit TNF-a production in neutrophils
through induction of IkBa, preventing nuclear translocation of NFkB and thereby leading to reduced transcription of TNF-a.123 This mechanism is specific for
neutrophils and, in fact, the opposite effect is observed
in macrophages, with C5 enhancing TNF-a production.
Thus, the increased serum TNF-a levels in C5-deficient
mice may be due to uncontrolled TNF-a production in
circulating neutrophils induced in response to the
pathogen.
The precise role of C5 in disease pathogenesis is likely
due to C5a, which is generated by cleavage of C5 during
complement system activation.124 The other cleavage
product, C5b, has a role in opsonization and is required
for assembly of the membrane attack complex, but its
role in control of candidiasis is not clear. Specifically,
direct lysis by the membrane attack complex does not
appear to be an essential mechanism for the killing of C.
albicans in vivo.84 Although complement does accelerate
the rate of C. albicans phagocytosis by neutrophils,49,85 the
fact that Candida is cleared equally as rapidly from the
bloodstream of C5-sufficient and -deficient mice argues
against the opsonization activity of C5b as regulating the
efficiency of the host response.
C5a is an anaphylatoxin and a chemoattractant.125 As
an anaphylatoxin, C5a induces various features of acute
inflammation, including increased blood flow, edema,
and smooth muscle contraction.126 However, the profile
of cytokines produced in A/J mice during infection is
characteristic of an extreme inflammatory reaction
suggesting that in the absence of C5, there is actually
an unregulated production of proinflammatory molecules. Contrary to C5’s more well-characterized role as
an inducer of inflammation, in candidiasis C5 may have
a role as a negative regulator of the inflammatory
response. The most defined role for C5a during invasive
candidiasis is as a chemotactic agent for neutrophils. In
the absence of C5a, mice have been shown to have a
reduced recruitment of effector cells to the site of
infection. Neutrophils express high levels of the C5a
receptor (C5aR/CD88),126 explaining their rapid infiltration to sites of infection in response to C5. C5a signaling
pathways in neutrophils lead to the assembly of
catalytically-active NADPH oxidase at the cell membrane, which has an established role in the killing of
phagocytosed C. albicans (discussed above).
C5’s role as a mediator of host response to infection
has been demonstrated in a number of organisms and
against a number of pathogens. In particular, C5
deficiencies in humans are associated with recurrent
Neisserial infections.82 In mice, C5 deficiency has been
linked to susceptibility to Listeria monocytogenes infection.127 Additionally, virulent group A streptococci have
been shown to express a cell-surface factor capable of
inactivating C5a, which they use as a mechanism of
eluding the host immune response.128
The results of the genome-wide scan clearly illustrate
the importance of C5 as a major gene controlling the
differential susceptibility to systemic C. albicans infections seen in A/J and B6 mice. A deficiency in C5
explains increased fungal outgrowth in target organs,
elevated serum TNF-a levels, and an increase in overall
mortality in susceptible mice. The physiological relevance of a C5 deficiency in humans with systemic
candidiasis is unclear, since although this deficiency
renders patients increasingly susceptible to a range of
microbial infections, an increased incidence of candidiasis has not been reported.124 This may simply be due to
the decreased pathogenicity of Candida, relative to other
microorganisms that are better adapted at exploiting
the weakened defense systems of a host that is deficient
in C5.
583
Genes and Immunity
Innate immunity in resistance to Candida albicans
A Tuite et al
584
Conclusion
Recognition of systemic candidiasis as an important
medical concern has been increasing in concert with
the rise in immunosuppressive and broad-spectrum
antimicrobial therapies. The development of candidiasis
is due to the complex interaction between the host
and colonizing C. albicans, in addition to environmental
factors. As resistance to antifungal agents becomes a
growing concern, a better understanding of the host–
pathogen interaction is invaluable for the development
of any new therapies. Animal models provide a useful
tool to study the response to systemic candidiasis
and studies in mice have indicated the importance
of the innate immune system in controlling infection. In
particular, the involvement of neutrophils in the initial
stages of infection parallels the importance of neutrophils in human infections and validates the mouse as a
physiologically relevant model of systemic candidiasis.
Acknowledgements
PG is a distinguished scientist of the Canadian Institutes
of Health Research and a James McGill Professor. AT is
supported by a studentship from the Natural Sciences
and Engineering Research Council of Canada. This work
was supported by the Genomics and Health Initiative of
the National Research Council of Canada. This is an NRC
publication number 37729.
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