Download Acute susceptibility of aged mice to infection with Candida albicans

J. Med. Microbiol. - Vol. 48 (1999), 1095-1 102
0 1999 The Pathological Society of Great Britain and Ireland
ISSN 0022-26 15
MYCOLOGY
Acute susceptibility of aged mice to infection with
Candida albicans
ROBERT B. ASHMAN, JOHN M. PAPADIMITRIOU and A L M A FULURIJA
School of Dentistry, University of Queensland, Brisbane, Queensland 4072 and Pathology Department,
University of Western Australia, Nedlands, WA 6009, Australia
The effect of aging on host resistance to systemic candidosis was assessed by monitoring
the course of infection in 16-month-old CBA/CaH mice (aged non-immune) and in a
comparable group that had been infected with a sublethal dose of Candida albicans at 6
weeks of age (aged immune). Aged non-immune mice showed rapid progression of the
disease, with a marked increase in the number of mycelia in the brain and kidney, and
early morbidity. Foci of myocardial necrosis were evident, but inflammatory cells were
sparse. The histological picture in the aged immune mice was similar to that in the aged
non-immune group, although fewer mycelial aggregates were seen. Both groups of aged
mice showed a significantly lower fungal burden in the brain on day 1 of infection, but
on day 4, colony counts increased significantly in the aged non-immune mice.
Comparison of cytokine gene expression in the infected brains showed that the relative
amount of interferon-y and tumour necrosis factor-a cDNA were similar in all three
groups. Interleukin-6 was elevated in both infected non-immune and uninfected aged
mice. Aged immune mice showed no morbidity after challenge, and both colonisation
and tissue damage were reduced in comparison with the aged non-immune animals.
Introduction
Immunocompetence declines with advancing age [ 11
and this is associated with a general increase in
susceptibility to infection. Epidemiological investigations have shown that the elderly are at higher risk for
pneumococcal and other respiratory infections [2], and
experimental studies have demonstrated an increased
susceptibility of aging mice to organisms as diverse as
Leishmania major [3], Trypanosoma musculi [4],
Legionella pneumophila [5] and Sendai virus [6].
Candida albicans is an opportunist yeast that is carried
as a commensal by the majority of the human
population. Mucosal candidosis (oral and vaginal
thrush) is common in the general population, although
chronic infections usually occur in conjunction with
deficiencies or abnormalities in the cell-mediated
immune response [7, 81. Systemic, or disseminated
candidosis is rare outside the hospital environment and
Received 20 Oct. 1998; revised version received 26 April
1999; accepted 30 April 1999.
Corresponding author: Professor R. B. Ashman (e-mail:
[email protected]).
is typically associated with dysfbnctional neutrophils
[9] or neutropenia [lo, 111. Oral infections with C.
albicans are common in the elderly [ 121, but although
advanced age increases susceptibility of mice to
intravenous challenge with Cryptococcus neoformans
[13], it is not a recognised factor for disseminated
candidosis.
This paper reports the effect of aging in CBA/CaH
mice on susceptibility to systemic challenge with C.
albicans and the effect of immunity induced as a
consequence of infection with the yeast early in adult
life on the severity of infection induced by re-challenge
in aged animals.
Materials and methods
Mice
Specific-pathogen-free female CBA/CaH mice were
purchased from the Animal Resources Centre, Perth,
Australia. Animal experiments were approved by the
Animal Experimentation Ethics Committee of the
University of Western Australia and performed in
accordance with the NH&MRC/CSIRO/Australian
Agricultural Council’s Code of Practice for the Care
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R.B. ASHMAN, J.M. PAPADIMITRIOU AND A. FULURIJA.
and Use of Animals for Experimental Purposes in
Australia, 1985.
Yeast preparation
C. albicans isolate 3630 was obtained from the
Mycology Reference Laboratory at the Royal North
Shore Hospital, Sydney, Australia, and stored at -70°C
in Sabouraud's broth with glycerol 15%. Yeasts were
grown in Sabouraud's broth for 2 days at room
temperature, with constant agitation. Blastospores were
washed in phosphate-buffered saline (PBS) and adjusted to the appropriate concentration for inoculation.
Infection and immunisation
Mice were immunised with 3 X lo5 blastoconidia
administered in 0.2 ml of PBS via the lateral tail vein
at 6 weeks of age. The same yeast isolate, dose and
route of administration were used both for infection of
the aged non-immune mice and re-challenge of the
aged immune animals.
Histology
Tissue samples were fixed in formalin, sectioned and
stained with haematoxylin and eosin (H&E), or
periodic acid-Schiff (PAS). The sections were coded,
examined blind and re-evaluated when the code had
been broken.
Clearance of C. albicans
Mice were killed at 1,4, 8 and 14 days after infection. A
transverse slice 2-3 mm thick was cut from the central
portion of the brain and placed in 1 ml of PBS.
Additional brain tissue was stored in liquid nitrogen
for mRNA extraction and analysis of cytokine gene
expression. Each tissue sampled was weighed and
disrupted with an Ultra Turrax T-25 homogeniser (IKA
Labortechnik, Staufen, Germany) running at 13 500 rpm
at room temperature. The samples were diluted appropriately and 100-pl volumes were plated on Sabouraud's
agar containing chloramphenicol. The plates were
incubated at 37°C for 2 days and the colonies were
counted. Each determination was performed in duplicate
in a minimum of five mice. The results were calculated
as loglo c h / g of tissue.
RNA preparation and reverse transcription
Total cellular RNA was prepared from infected brain
tissue with 'Ultraspec' RNA Isolation Reagent (Biotecx
Laboratories, Houston, TX, USA) according to the
manufacturer's instructions. The concentration and
purity of the RNA samples were determined by
spectrophotometry at 260 and 280nm. cDNA was
prepared by reverse transcription of 2 pg of each
RNA, with an oligo d(T)15 primer and AMV reverse
transcriptase, according to the manufacturer's instruc-
tions (Promega Corporation, Madison, WI, USA).
Briefly, 5 m~ MgClz, 1 X reverse transcription buffer,
1 m~ each dNTP, 0.5 URNAasin, 15 UAMV reverse
transcriptase and 0.5 pg oligo (dT)15 primer were
incubated in a 20-pl reaction mix at 42°C for 1 h,
heated to 99°C for 5 min, then cooled on ice. The cDNA
was stored at -20°C until used.
PCR
Primer sequences for interferon-y (IFN-y), interleukin4 (IL-4), interleukin-6 (IL-6) and tumour necrosis
factor-a (TNF-a) were obtained from published data
[14], and the PCR was performed as described
previously [ 151. The amplification mix consisted of
50 ng DNA, 200 m d N T P s , 0.5 U Taq polymerase
(Biotech International, Perth, Western Australia), 1X
reaction buffer, either 1 m~ or 2 m~MgC12and either
1 ml or 2 ml of primers in a total volume of 25 pl. The
mixture was overlaid with paraffin oil, then amplified
with a PTC-100 thermal cycler (MJ Research, Waltham, MA, USA). The amplification protocol was 94"C,
1 min; 60"C, 2 min; 72"C, 2 min; for 35-40 cycles.
Following amplification, 10 pl of product were analysed by electrophoresis through agarose 3% gels. The
gels were stained with ethidium bromide and the bands
were visualised with a W transilluminator.
Competitive PCR for comparison of cytokine
cDNA
Plasmid MCQ (pMCQ) was generously donated by Dr
Cornelia Platzer, Humboldt University, Berlin, Germany. This plasmid (control fragment) contains the
primer sequences for interleukins 1-6, IFN-y, TNF,
lymphotoxin and p-actin, arranged so that the products
amplified by cytokine-specific primers differ in size
from those amplified from the target DNA, and thus
can be distinguished by electrophoresis through an
agarose gel [ 161. Samples showing reactivity with
cytokine primers were subjected to semi-quantitative
analysis as described previously [ 151. Briefly, the
cDNA was adjusted to equal concentrations by coamplification of a fixed amount of the cDNA with 10fold, and then two-fold, dilutions of the control
fragment @MCQ), with primers for P-actin. For
quantification and comparison of cytokine mRNA
expression, equal amounts of cDNA were amplified in
the presence of 10-fold and then three-fold dilutions of
the control fragment. The linearised plasmid was used
at dilutions ranging from lo3 to lo5 for the
standardisation of the cDNA and from lo5 to lo8 for
comparison of cytokine concentrations. The experiments were repeated three times. The end-points for the
aged mice, expressed in logarithms, were subtracted
from those for the young controls, and the resultant
value gave a factor by which the cDNA concentrations
in the aged mice were greater or less than those in the
controls. A positive value indicated that the concentra-
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CANDIDIASIS IN AGING MICE
tions in the aged mice were greater than in the young
animals and vice versa.
Statistical analysis
The statistical significance of differences between
groups in the number of yeast cells recovered from
infected brains was determined by Scheffe’s test on the
one-way analysis of variance. Cytokines were compared as follows. The mean and standard deviation of
the factors by which the aged mice differed fiom the
controls were calculated for each data set, and the
hypothesis that the cDNA concentrations in young and
aged mice were not significantly different was tested by
determining whether the 95% confidence intervals
included zero.
Results
Long-term effects of systemic infection
Female CBA/CaH mice infected with 3 X lo5 C.
albicans yeast cells at 6-8 weeks of age and a group
of uninfected, age-matched control mice were maintained in conventional animal facilities for 16 months.
The mean weight (30.2 SD 6.1 g) of the aged immune
mice was significantly less (p<O.Ol) than that of the
uninfected controls (35.2 SD 3.9 g), but they were
otherwise active and healthy. Within 3 days after
intravenous challenge with 3 X lo5 C. albicans yeast
cells, the aged non-immune mice displayed visible
signs of distress and this entire group was killed on day
4 for ethical reasons. In contrast, the old immune mice
and a group of 6-week-old control mice given a similar
challenge remained outwardly healthy and showed no
unusual symptomatology at any time during the 14-day
period of observation.
Histology of the lesions
The histological features of disseminated C. albicans
infection in 6-8-week-old CBA/CaH mice were as
described previously [ 171. Briefly, mycelia were readily
detected in the brains of the young control mice on the
first day after infection. They were present to a lesser
extent in the kidney, but were rarely found in the heart,
although a focal myocarditis had already developed. By
day 4, the encephalitis, pyelonephritis and myocarditis
had increased in severity. The brain exhibited abscesses
(Fig. 1A) containing predominantly yeasts, with some
mycelial growth forms, the latter being present in
greater numbers than on day 1. At day 14, mycelia
were difficult to find. The myocarditis had abated, but
the encephalitis and pyelonephritis had increased in
severity.
On day 1 after infection of the aged non-immune mice,
mycelia were seen in brain, kidney and heart, in
numbers similar to those in the young controls. In the
heart, scattered foci of myocardial necrosis were
1097
surrounded by a few inflammatory cells. By the fourth
day of infection, the number of mycelia in the brain
and kidney had increased markedly in comparison with
those in the young mice, the brain being the most
severely affected organ (Fig. 1B). Necrotic debris was
seen in the vicinity of the mycelia, but only a few
inflammatory cells were detected. Mycelia were not
seen in the heart, but foci of myocardial necrosis,
including calcified cardiac myocytes, were readily
found (Fig. 2). Again, inflammatory cells were sparse.
The aged immune mice were similar to the other two
groups in terms of the numbers of mycelia in the brain
and kidney at 24 h after infection. By the fourth day,
the mycelial population in the brain (Fig. 1C) and
kidney had increased, but to a lesser extent than in the
aged non-immune mice. Polymorphonuclear leucocytes
(PMNLs) were easily detected at the periphery of the
mycelial aggregates in the brain (Fig. 3), while in the
heart they were present as focal infiltrates in the
interstitium. Abscesses had formed in both brain and
kidney at this time point, but the myocarditis remained
unchanged. Calcified myocytes were detected, but at a
much lower fiequency than in the non-immune aged
mice. By day 14, mycelia were only detectable in the
kidney. The focal encephalitis, pyelonephritis and
myocarditis were reduced in intensity compared with
the aged non-immune mice, but were more severe than
lesions in the young controls at the same time point.
Kinetics of fungal clearance
The fungal burden in the brain of both the aged nonimmune and the aged immune mice was significantly
less than in the young mice on the first day after
infection ( p < O . O l ) , but by day 4 the number of
colonies recovered from the brains of aged nonimmune mice was significantly greater (p < 0.01) than
that in the other two groups (Fig. 4). After this time,
the number of colonies recovered from the brains of
the aged immune mice declined to levels similar to
those in the young controls.
Comparison of cytokine cDNA proJiles
It was of interest to determine whether the increased
susceptibility of the aged mice was associated with a
change in the relative gene expression in the infected
brain of cytokines that are known to play a part in host
defence against C. albicans infection. The cytokine
profiles were assessed by comparison of the end-points
of competitive PCR assays. The relative amounts of
IFN-y and TNF-a cDNA in the aged non-immune mice
were not different fiom those in the young controls
(Fig. 5 ) , but IL-6 cDNA levels were significantly higher
in the aged infected mice. However, comparison of IL6 gene expression in young and aged uninfected mice
showed a 3-5-fold increase in aged animals (data not
shown). There were no significant differences between
aged immune mice and the young controls in the
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R.B. ASHMAN, J.M. PAPADIMITRIOU AND A. FULURIJA.
Fig. 1. (A) Brain of a 6-8-week-old CBA/CaH mouse 4 days after i.v. challenge with 3 X lo5 C. albicans yeast cells. A large
abscess contains fungal elements and inflammatory infiltrates. (B) Brain of aged non-immune CBA/CaH mouse 4 days after
challenge as above. There is extensive fungal proliferation, with prominent mycelial elements. Few inflammatory cells are
present in the lesion. (C) Brain of aged immune CBA/CaH mouse, 4 days after challenge as above. An abscess shows yeast
and mycelial forms, and a dense inflammatory infiltrate. All sections stained with periodic acid-Schiff (X380).
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CANDIDIASIS IN AGING MICE
1099
Fig. 2. Aggregates of calcified myocytes (arrows) in the heart of an aging CBA/CaH mouse, 4 days after intravenous
challenge with 3 X lo5 C. albicans yeast cells. (X160).
Fig. 3. Brain of aged immune mouse 4 days after intravenous challenge with 3 X lo5 C. albicans yeast cells.
Inflammatory cells can be seen scattered around the focus of fungal infection. H&E (X400).
relative expression of IL-6, IFN-y or TNF-a (data not
shown). IL-4 was detected in the brains of all three
control mice on day 4 after infection, but was not
found in either the aged non-immune (Fig. 6) or aged
immune mice (data not shown) at any time point.
Discussion
Systemic infection with C. albicans in aged mice
causes acute disease and early death. The rapid onset
of morbidity, within 2-3 days after challenge,
suggested that the animals may have suffered some
kind of septic shock, a condition that has also been
associated with candidaemia in elderly patients [ 181.
Although there was a quantitative increase in the
fungal burden in the brains of the aged non-immune
mice at 4 days after challenge, the striking feature of
the infection in these animals was the widespread
appearance of mycelial growth forms, which are
potent stimulators of TNF-a production by both
macrophage cell lines [ 191 and macrophages obtained
from different anatomical regions throughout the body
POI
*
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R.B. ASHMAN, J.M. PAPADIMITRIOU AND A. FULURIJA.
Fig.4.Number of C. albicans in the brains of normal (young; o), aged non-immune (FA),and aged immune (m)
CBA/CaH mice, at various times after intravenous challenge with 3 X lo5 yeast cells. Each bar shows the mean and
SEM of loglo cfu/gram of tissue.
Fig. 5. Relative amounts of cytokine cDNA in the brain of aged non-immune CBA/CaH mice: IFN-y (day 1, 0; day 4,
o), IL-6 (day 1, 0;day 4, m), TNF-a (day 1, A; day 4, A). Each point represents the mean and 95% confidence
intervals of the difference in cytokine cDNA concentrations between old and young animals, as estimated by
competitive PCR with equivalent amounts of cDNA. mRNA was prepared separately from each of three mice per group
and each determination was carried out at least twice.
Fig. 6. IL-4 gene expression in brain tissue of young
(lanes 1-3) and aged non-immune (4-6) mice 4 days
after intravenous challenge with 3 x 105 c. albicans
yeast cells.
TNF-a is a known mediator of septic shock
syndromes [21] and accumulates to high levels in
the serum of infected mice [22,23]. Although aged
mice are more susceptible than younger mice to the
lethal toxicity of TNF-a [24], the relative levels of
TNF-a cDNA in the brains of the aging non-immune
mice were not significantly different from those in the
Young control group. This suggested that TNF-a was
not implicated in the early morbidity and mortality of
these animals. A recent study of C. aEbicans septic
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CANDIDIASIS IN AGING MICE
shock syndrome in rats [25] also excluded TNF-a as a
mediator and the authors of the latter study suggested
that the syndrome was mediated either by host
cytokines other than TNF-a, or by circulating fungal
virulence factors.
As mice age, the concentration of leucocytes in the
blood rises [26]. This could account for the transient
reduction in the number of viable yeasts in the brain of
both the aged non-immune and aged immune mice, as
compared with the controls, on the first day after
infection. However, the ability to mobilise PMNLs
from the marrow into blood in the elderly is markedly
reduced when compared with young subjects [27] and
PMNLs from older individuals also exhibit a pronounced impairment of their chemotactic, phagocytic
and killing capacities [28]. PMNLs from aging rats
show a reduction in bactericidal capacity and in the
generation of oxygen radicals, after stimulation with
low levels of IFN-y [29].
Hyphal growth forms of the yeast are readily susceptible to killing by neutrophils [30], and the marked
increase in mycelial elements seen in the aging nonimmune mice might have reflected a quantitative or
qualitative reduction in the contribution of this cell
type to resolution of the lesions [31]. The aging mice
also displayed foci of myocardial calcification and
necrosis, which have otherwise only been observed in
mice depleted of neutrophils [32]. There is increasing
evidence that neutrophils are the major effector
mechanism mediating host responses against the yeast
[33-351, so a gradual decline in neutrophil function
may account both for the extent of tissue invasion in
the aged non-immune animals and the predominance of
mycelial forms throughout the later stages of infection
in the aged immune mice.
Neutrophil activation for killing of C. albicans is
mediated predominantly by IFN-y and TNF-a, acting
either separately or synergically [36]. However, the
relative gene expression of these two cytokines in the
brains of aged non-immune mice was similar to that in
the young controls. This tended to exclude a failure of
cytokine-mediated activation as a basis for the increased susceptibility of the aged non-immune animals.
Nevertheless, cytokine production and function in C.
albicans infection are subject to complex regulation,
particularly by cytokines of the T-helper type 2 (Th2)
subset [37]. Of these, IL-4 was detected in the young
but not in the aged non-immune or aged immune
animals, although the relevance of this host resistance
is unknown. In contrast, IL-6 was expressed in
significantly higher amounts in the brains of aged
non-immune mice when compared with young controls,
but levels in aged uninfected mice were also elevated.
The cellular origin of the increased IL-6 in the aged
mice, and whether its presence is a cause or a
consequence of the increased susceptibility, remain to
be determined.
1101
The induction of protective immunity consequent upon
recovery from an initial sublethal infection is most
readily demonstrated in mice of the CBA/CaH strain
[38, 391. The present data confirm that protective
immunity elicited by systemic infection of young mice
persists for at least 12 months, and probably for the life
of the mouse. Colony counts in the brains of the aged
immune mice were reduced by about 0.5 loglo relative
to the aged non-immune group, which was similar to
the effect achieved by immunisation of young animals
[3 81. Nevertheless, the increased susceptibility demonstrated by the aged non-immune mice was also evident
in the aged immune mice when compared with the
responses of younger animals. It is unclear whether this
reflects an overall decline in the candidacidal potential
of phagocytic effector cells, or a reduction in function
of some of the antibody-dependent mechanisms that
accelerate clearance of the yeast in immune mice.
In summary, these studies in the mouse provide
evidence that the changes in host defence mechanisms
associated with advanced age markedly increase the
severity of disseminated candidosis. It follows that the
increasing prevalence of infections with Candida spp.
in the hospital environment [40] may represent a threat
not only to the debilitated and immunosuppressed, but
also to the elderly patient.
We thank Phillip Williams for photography. R.B.A. was supported in
part by the Arnold Yeldham and Mary Raine Medical Research
Foundation. The research was funded by grants from the Raine
Foundation, the Child Health Research Foundation of Western
Australia, the Sylvia and Charles Viertel Charitable Foundation,
and the Medical Research Fund of Western Australia.
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