Differential mechanisms of resistance to sublethal systemic Aspergillus fumigatus infection in immunocompetent BALB/c and C57BL/6 mice

ArticleinImmunobiology 216(1-2):234-42 · January 2010with78 Reads
Impact Factor: 3.04 · DOI: 10.1016/j.imbio.2010.04.007 · Source: PubMed
Abstract

Studies of systemic and pulmonary Aspergillus fumigatus infection demonstrated differential susceptibility of inbred mice of various genetic background to lethal outcome, with an opposite pattern of Th1 cytokine interferon-γ (IFN-γ) and Th2 cytokine interleukin-4 (IL-4) in susceptible vs resistant mice. We have shown recently reciprocal IFN-γ and IL-4 expression in spleens of Th1-prone C57BL/6 mice in sublethal systemic aspergillosis. In this study, resistance to systemic (i.v.) A. fumigatus infection was investigated in Th2-prone BALB/c mice by survival rate at different fungal inocula, efficiency of reduction of visceral organ and spleen fungal burden at sublethal conidia dose and splenic immune response to this dose and compared to C57BL/6 mice. No strain differences in survival were noted at three A. fumigatus doses, with similar extent and dynamics of fungal eradication from all organs following sublethal conidia dose injection. Progressive decrease in spleen fungal burden was associated with different dynamics and quality of changes in spleen activity of BALB/c and C57BL/6 mice. Increased spleen mass and cellularity was noted in both strains, with higher values in BALB/c mice at some time points what might be ascribed to peripheral blood cell recruitment, as well as hematopoietic activity and red pulp upgrowth. Infection tipped the balance towards pro-inflammatory antifungal splenic response by a highly increasing IFN-γ and without changing the IL-4 expression in BALB/c mice, in contrast to down-regulating anti-inflammatory (IL-4) and a moderately increasing IFN-γ response in C57BL/6 mice. Jointly, stimulation of IL-17 expression noted in both strains provided an optimal inflammatory milieu in the spleen of infected mice that might have contributed to efficient removal of conidia.

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Immunobiology 216 (2011) 234–242
Contents lists available at ScienceDirect
Immunobiology
journal homepage: www.elsevier.de/imbio
Differential mechanisms of resistance to sublethal systemic Aspergillus fumigatus
infection in immunocompetent BALB/c and C57BL/6 mice
Ivana Mirkov
a
, Ivana Stojanovic
b
, Jasmina Glamoclija
a
, Stanislava Stosic-Grujicic
b
,
Lidija Zolotarevski
c
, Dragan Kataranovski
a,d
, Milena Kataranovski
a,e,
a
University of Belgrade, Institute for Biological Research “Sinisa Stankovic”, Department of Ecology, Bulevar despota Stefana 142, 11000 Belgrade, Serbia
b
University of Belgrade, Institute for Biological Research “Sinisa Stankovic”, Department of Immunology, Bulevar despota Stefana 142, 11000 Belgrade, Serbia
c
Military Medical Academy, Institute for Pathology, Crnotravska 11, 11000 Belgrade, Serbia
d
University of Belgrade, Faculty of Biology, Institute of Zoology, Studentski trg 1-3, 11000 Belgrade, Serbia
e
University of Belgrade, Faculty of Biology, Institute of Physiology and Biochemistry, Studentski trg 1-3, 11000 Belgrade, Serbia
article info
Article history:
Received 23 February 2010
Received in revised form 30 April 2010
Accepted 30 April 2010
Keywords:
Aspergillus fumigatus
BALB/c and C57BL/6 mice
IFN-
IL-4 and IL-17
S systemic infection
Spleen
abstract
Studies of systemic and pulmonary Aspergillus fumigatus infection demonstrated differential suscep-
tibility of inbred mice of various genetic background to lethal outcome, with an opposite pattern of
Th1 cytokine interferon- (IFN-) and Th2 cytokine interleukin-4 (IL-4) in susceptible vs resistant mice.
We have shown recently reciprocal IFN- and IL-4 expression in spleens of Th1-prone C57BL/6 mice
in sublethal systemic aspergillosis. In this study, resistance to systemic (i.v.) A. fumigatus infection was
investigated in Th2-prone BALB/c mice by survival rate at different fungal inocula, efficiency of reduction
of visceral organ and spleen fungal burden at sublethal conidia dose and splenic immune response to this
dose and compared to C57BL/6 mice. No strain differences in survival were noted at three A. fumigatus
doses, with similar extent and dynamics of fungal eradication from all organs following sublethal conidia
dose injection. Progressive decrease in spleen fungal burden was associated with different dynamics and
quality of changes in spleen activity of BALB/c and C57BL/6 mice. Increased spleen mass and cellularity
was noted in both strains, with higher values in BALB/c mice at some time points what might be ascribed
to peripheral blood cell recruitment, as well as hematopoietic activity and red pulp upgrowth. Infec-
tion tipped the balance towards pro-inflammatory antifungal splenic response by a highly increasing
IFN- and without changing the IL-4 expression in BALB/c mice, in contrast to down-regulating anti-
inflammatory (IL-4) and a moderately increasing IFN- response in C57BL/6 mice. Jointly, stimulation of
IL-17 expression noted in both strains provided an optimal inflammatory milieu in the spleen of infected
mice that might have contributed to efficient removal of conidia.
© 2010 Elsevier GmbH. All rights reserved.
Introduction
Aspergillus fumigatus is an ubiquitous saprophytic mould species
with airborne conidia (Latge 1999; Mullins et al. 1984; Nolard
1994). Owing to their small diameter, as well as environmental
factors, conidia easily gain access to the lungs (Goodley et al. 1994;
Hospenthal et al. 1998). Infections occur rarely in healthy, immuno-
Abbreviations: A. fumigatus, Aspergillus fumigatus; CFUs, colony forming units;
ELISA, enzyme-linked immunosorbent assay; H&E, hematoxylin and eosin; IFN-,
interferon-; IL, interleukin; i.v., intravenous; mRNA, messenger RNA; NK cells, nat-
ural killer cells; NO, nitric oxide; p.i., post infection; RT-PCR, real-time polymerase
chain reaction; SMA, Sabouraud’s maltose agar; Th, T helper.
Corresponding author at: Institute for Biological Research “Sinisa Stankovic”,
Department of Ecology, Bulevar Despota Stefana 142, 11000 Belgrade, Serbia.
Tel.: +381 11 2078375; fax: +381 11 2761433.
E-mail address: milena@ibiss.bg.ac.rs (M. Kataranovski).
competent individuals due to efficient fungal elimination by host
immune defense (Latge 1999). Increase in numbers of immuno-
compromised individuals has, however, placed A. fumigatus as the
second major cause of invasive fungal infections in humans con-
tributing to morbidity/mortality in these individuals (Latge 1999).
The use of animal models has been indispensable for current
understanding of various aspects of Aspergillus infection (Clemons
and Stevens 2005). Four murine models of aspergillosis that are
most often used to explore the mechanisms of resistance to this
microbe are invasive pulmonary aspergillosis, bronchopulmonary
aspergillosis, systemic (disseminated) infection and central ner-
vous system infection (Clemons and Stevens 2005). By using
intranasal or intratracheal inoculation of A. fumigatus conidia, that
mimic the natural route of infection, the role of innate host defense
and the generation of Th1 cytokines in the lungs were shown to
play a critical role in the resistance of mice to invasive pulmonary
aspergillosis (Cenci et al. 1998; Centeno-Lima et al. 2002). To
0171-2985/$ see front matter © 2010 Elsevier GmbH. All rights reserved.
doi:10.1016/j.imbio.2010.04.007
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I. Mirkov et al. / Immunobiology 216 (2011) 234–242 235
increase the susceptibility to infection in these models (Polak 1998;
Schmidt 2002), immunosuppressive treatments (glucocorticoids
and cyclophosphamide) are required, which were shown to mod-
ulate the host immune response both before as well as during A.
fumigatus infection (Armstrong-James et al. 2009). Administration
of A. fumigatus by a systemic route (by i.v. inoculation) establishes
infection in immunocompetent mice with a relatively uniform
pattern of disease. This model of infection is used for infection
dissemination studies and infection to mortality ratio investiga-
tion and is particularly useful in the determination of mechanisms
involved in host resistance or susceptibility to lethal outcome of A.
fumigatus infection (Cenci et al. 1997; Clemons et al. 2000; Mirkov
et al. 2008; Nagai et al. 1995; Polak 1998).
Studies of both systemic and pulmonary A. fumigatus infection
demonstrated that inbred mice of various genetic background dif-
fer significantly in their degree of susceptibility to lethal outcome
of infection (Cenci et al. 1997; Zaas et al. 2008). An opposite pat-
tern of IFN- and IL-4 in susceptible vs resistant strains was noted,
with higher IFN- production and lower IL-4 levels in individu-
als which survived infection compared to non-survivors (Cenci et
al. 1997, 1998). Interestingly, no differences were noted in sus-
ceptibility to lethal systemic A. fumigatus infection (Cenci et al.
1997) between C57BL/6 and BALB/c mice, identified as prototypic
Th1 and Th2 strains, respectively, according to their predisposition
towards IFN- or IL-4 cytokine response to microbial infections
(Mossman and Coffman 1989). Similarly, in invasive pulmonary
aspergillosis studies, immunosuppressed C57BL/6 and BALB/c mice
exhibited similar pattern of survival following conidia adminis-
tration (Svirshchevskaya et al. 2009; Zaas et al. 2008), although
the survival differed somewhat depending on immunosuppression
regimen (Stephens-Romero et al. 2005). Consequently, these both
strains were considered more “resistant” to A. fumigatus infection
as opposed to “susceptible” mice such as A/J, DBA/2J, C3H/HeJ, CBA
and A/Sn mice (Svirshchevskaya et al. 2009; Zaas et al. 2008). In the
light of Th1/Th2 paradigm and knowing that C57BL/6 and BALB/c
were shown to express disparate cytokine (IFN- or IL-4) responses
in a variety of parasitic and bacterial infections (Gervais et al. 1984;
Guler et al. 1996; Tam et al. 1999; Thach et al. 2000; Wakeham et
al. 2000) it seems worthwhile to compare IFN- and IL-4 expres-
sion in response to A. fumigatus infection in these mice. There are
some data concerning Th1 and Th2 cytokine production in resistant
BALB/c mice (Cenci et al. 1998) which showed increase in IFN-
and low levels of splenic IL-4 production in lethal infection, but
with similar levels of production of both cytokines in sublethal
infection with this fungus. In addition, we have shown recently
reciprocal expression (increased IFN- vs decreased IL-4) of these
two cytokines in Th1-prone C57BL/6 mice in response to dissemi-
nated sublethal A. fumigatus infection (Mirkov et al., 2010). Beside
Th1 and Th2 cytokines, the importance of activation of Th17 cells
and expression of IL-17, a main cytokine produced by these cells in
immune response to fungi was stressed (Curtis and Way 2009). The
role for IL-17 in pulmonary aspergillosis is demonstrated recently
in C57BL/6 mice (Zelante et al. 2009) and our recent investigations
showed the involvement of IL-17 in systemic A. fumigatus infection
in this strain (Mirkov et al., 2010). To our knowledge, there are no
data which explore influences of genetic predisposition on IL-17 in
aspergillosis.
In this study, resistance of Th2-prone BALB/c mice to systemic
A. fumigatus infection was explored by measuring survival rate
at different conidia doses and by efficiency of conidia elimina-
tion from visceral organs and spleen at sublethal conidia dose
and compared to Th1-prone C57BL/6 mice. Basic indices of splenic
response (spleen mass/cellularity and proliferation) to sublethal A.
fumigatus infection were compared in these two mouse strains, as
well as quantitative and temporal expression of IFN-, IL-4, and
IL-17, main cytokine representatives of Th subsets. Data were pre-
sented which demonstrated roughly similar survival in both strains
at high conidia inocula, with progressive and similar decrease in
liver, lung, kidney and spleen fungal burden at sublethal infec-
tion. Conidia elimination in spleens was associated with changes
in activity and inflammatory cytokine milieu created differently in
the two strains. Animals of both strains responded to infection by
increase in relative mass, cellularity and proliferative capacity, but
with more pronounced red pulp proportion and hematopoiesis in
spleens of BALB/c mice. While highly pronounced increase in IFN-
and IL-17 expression, but unchanged IL-4 expression characterized
antifungal spleen cytokine response in BALB/c mice, increase in the
IFN- and IL-17 cytokine expression was accompanied by down-
regulation of anti-inflammatory IL-4 response in C57BL/6 mice.
Obtained data confirmed the importance of IFN- in immunity to A.
fumigatus and demonstrated the involvement of IL-17 in resistance
to systemic aspergillosis in both strains.
Materials and methods
Mice
Eight to twelve week old female BALB/c and C57BL/6 mice, four
to six per group, were used for the study. The animals were bred
and conventionally housed at the Institute for Biological Research
“Sinisa Stankovic” (Belgrade, Serbia) in a controlled environment
and provided with standard rodent chow and water ad libitum. All
experiments were approved by the Ethical Committee of our Insti-
tute. Fungal burden assessment was conducted at days 1, 3, 7 and
15 post infection (p.i.) and at days 3, 7 and 15 p.i. for the evaluation
of splenic response.
Fungal culture conditions
The human isolate of A. fumigatus was obtained from the Insti-
tute of Public Health of Serbia “Dr Milan Jovanovic-Batut”. The
microorganisms were grown on Sabouraud’s maltose agar (SMA,
Torlak, Belgrade, Serbia) for 7 days (Booth 1971). Conidia were har-
vested by flooding the surface of agar slants with non-pyrogenic
sterile physiological saline.
Animal infection, determination of survival rate and organ fungal
burden
Anesthetized mice (Ketamidor, Richter Pharma, Austria) were
inoculated with 1 × 10
6
,1× 10
7
or 5 × 10
7
conidia of A. fumigatus
in 0.1 ml of pyrogen-free saline via the lateral tail vein. Control
mice received saline solely. Survival, clinical appearance and body
weight were monitored daily for 15 days and survival percentage
was calculated for each group. Fungal burden in the spleen, liver,
kidneys and lungs was determined by quantitative colony forming
units (CFUs) assay (Sheppard et al. 2006). At selected time points (1,
3, 7 and 15 days) p.i. organs were aseptically removed and their wet
masses weighed using a precision balance (±0.01 g). Tissue spec-
imens were homogenized with IKA T18 basic homogenizer (IKA
Works Inc., Wilmington, NC) in 5 ml of sterile saline, on ice. Primary
homogenate dilutions were quantitatively cultured by serial dilu-
tion, plated on SMA plates supplemented with streptomycin sulfate
(ICN-Galenika, Belgrade, Serbia), incubated at 37
C for 24–48 h, and
the number of CFUs per gram of tissue was determined.
Peripheral blood total leukocyte and differential counts
Total leukocyte counts were determined at days 3 and 7 fol-
lowing conidia administration by Türck staining and by using
an improved Neubauer hemocytometer. Leukocyte differential
counts were performed by differentiating at least 200 cells from
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236 I. Mirkov et al. / Immunobiology 216 (2011) 234–242
air-dried whole blood smears stained according to conventional
May-Grunwald-Giemsa protocol.
Spleen histology
Spleens were immediately fixed in 4% formaldehyde (pH 6.9)
and embedded in paraffin wax for sectioning at 5 m. Hematoxylin
and eosin (H&E)-stained histological slides were subsequently
analyzed by light microscopy and scored for the extent of preva-
lence of red to white pulp and for the intensity of expression of
hematopoiesis. Semi-quantitative numeric values were assigned to
these scores as approximate proportion of red pulp (in percents)
and for the degree of expression of hematopoiesis according to
scale: 0 weakly expressed,1–minimally expressed,2–more
expressed, 3 well expressed and 4 highly expressed.
Spleen cell preparation and culture
Spleens were removed at days 3, 7 and 15 following coni-
dia administration, blotted dry and weighed. The relative spleen
mass was calculated according to the following formula: organ
mass/body mass × 100. Spleen cells were obtained aseptically by
gentle teasing of spleen tissue through a cell strainer (BD Falcon,
BD Bioscience, Bedford, USA). Cells were suspended in RPMI-1640
culture medium (Flow, ICN Pharmaceuticals) supplemented with
2 mM glutamine, 20 g/ml gentamycine (Galenika a.d., Serbia),
5% (v/v) heat-inactivated fetal calf serum (PAA Laboratories, Aus-
tria) (complete medium) and 5 g/ml of voriconazole (Pfizer PGM,
France). The capacity of spleen cells to proliferate was determined
ex vivo by culturing 3 × 10
5
cells/well (96-well plate) for 24 h, with
0.5 Ci of
3
H-thymidine (GE Healthcare, UK) per well during the
last 8 h of culture. Incorporation of
3
H-thymidine into cellular DNA
was measured by liquid scintillation counting (LKB, Wallac).
Cell culture supernatants, used for ex vivo detection of cytokines,
were obtained by culturing the cells for 72 h in 24-well culture
plates in 1 ml of standard medium (5 × 10
6
spleen cells/well). For
fungus-stimulated spleen cell IL-17 production, heat-inactivated
conidia (autoclaved at 121
C for 30 min) were used at a ratio
of 2:1 conidia to spleen cells. Cytokine levels in the conditioned
media were determined by enzyme-linked immunosorbent assay
(ELISA).
RNA isolation and RT-PCR
Total RNA from splenocytes (5 × 10
6
) was isolated with mi-Total
RNA Isolation Kit (Metabion, Martinsried, Germany) according to
manufacturer’s instructions. RNA (1 g) was reverse transcribed
using Moloney leukemia virus reverse transcriptase and random
hexamers (both from Fermentas, Vilnius, Lithuania). PCR amplifi-
cation of cDNA (1 l/20 l of PCR reaction) was carried out by a
real-time PCR machine (Applied Biosystems, UK) using SYBRGreen
PCR master mix (Applied Biosystems) as follows: 10 min at 50
C for
dUTP activation, 10 min at 95
C for initial denaturation of cDNA fol-
lowed by 40 cycles (15 s of denaturation at 95
C and 60 s for primer
annealing and chain extension step). Primer pairs for IL-17 were
5
-GGGAGAGCTTCATCTGT-3
and 5
-GACCCTGAAAGTGAAGGG-3
(GenBank accession no. NM 010552.3), IFN- 5
-CATCAGCAAC-
AACATAAGCGTCA-3
and 5
-CTCCTTTTCCGCTTCCTGA-3
(GenBank
accession no. NM 008337.2), IL-4 5
-ATCCTGCTCTTCTTTCTCG-
3
and 5
-GATGCTCTTTAGGCTTTCC-3
(GenBank accession no.
NM 021283.1) and -actin 5
-GGACCTGACAGACTACC-3
and 5
-
GGCATAGAGGTCTTTACGG-3
(NM 007393.2). The expression of
these genes was calculated according to the formula: 2
(C
ti
C
ta
)
where C
ti
is the cycle threshold of the gene of interest and C
ta
is
the cycle threshold value of -actin.
ELISA
Cytokine production was determined in the medium condi-
tioned by spleen cells using commercially available ELISA sets for
mouse IFN- (eBioscience Inc., San Diego, CA, USA), mouse IL-4
(R&D Systems, Minneapolis, USA) and mouse IL-17 (BD Pharmin-
gen, San Diego, CA, USA). Tests were performed according to
manufacturer’s recommendations. Cytokine concentration was cal-
culated from a standard curve constructed by known amounts of
recombinant IFN-, IL-4 and IL-17.
Statistics
Results were pooled from at least three experiments and
expressed as mean values ± S.D. The statistical package used was
Statistica 7.0 (StatSoft Inc., Tulsa, OK, USA). Survival data were ana-
lyzed by means of log-rank comparisons of Kaplan–Meier survival
curves, followed by the Wilcoxon test. Other data were statistically
processed by Mann–Whitney U test (fungal burden, cell culture
data) or t-test (gene expression). p-values less than 0.05 were con-
sidered significant.
Results
Survival of C57BL/6 and BALB/c mice following injection of
different A. fumigatus conidia doses
Examination of survival over a 15-day time period revealed 100%
mortality at 5 × 10
7
conidia injected in both strains, with median
survival time (2 days) in C57BL/6 vs 4 days in BALB/c mice (Fig. 1).
Similar mortality rate (100% and 67%) and median survival time (7
and 10 days) was noted in C57BL/6 and BALB/c mice, respectively, at
lower 1 × 10
7
conidia dose. Injection of 1 × 10
6
conidia resulted in
low mortality (1.9% in C57BL/6 and 2% in BALB/c mice, respectively)
at day 3.
Fungal burden in spleen, liver, lungs and kidneys following i.v.
injection of sublethal dose of A. fumigatus conidia
Determination of fungal burden in visceral organs and spleen of
mice injected with 1 × 10
6
conidia revealed highest levels of conidia
in spleen (7395 ± 42 and 7405 ± 48, BALB/c and C57BL/6, respec-
tively), followed by liver (1167 ± 26 and 1145 ± 40), lungs (352 ± 13
and 351 ± 11) and kidneys (122 ± 8 and 121 ± 7) (Fig. 2), demon-
strating systemic character of infection. Progressive decrease in the
number of CFUs in these organs was noted in BALB/c mice and was
similar to C57BL/6 mice, with conidial elimination at day 15 in both
strains. Histological analysis of liver, lungs and kidneys revealed
Fig. 1. Survival following injection of different numbers of A. fumigatus conidia to
C57BL/6 and BALB/c mice. Results are expressed as percentage of survival.
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I. Mirkov et al. / Immunobiology 216 (2011) 234–242 237
Fig. 2. Time course of fungal burden decrease in spleens, liver, lungs and kidneys of BALB/c and C57BL/6 mice inoculated with Aspergillus fumigatus conidia. Results are
expressed as log
10
of CFU from average values of triplicate determinations. Significantly different at p < 0.05 (*) and p < 0.001 (***) from respective strain day 1 post infection.
presence of microabscesses (liver), mixed leukocyte infiltration
(lungs) in all individuals of both strains, with signs of tubuloint-
erstitial inflammation in some animals of both strains, implying
immune/inflammatory response in these organs (not shown). In
some of the mice prostration, hunchbacked appearance, shiver-
ing and disorientation were noted, implying infection of central
nervous system as well.
Peripheral blood leukocyte counts following i.v. injection of A.
fumigatus
Injection of conidia resulted in a significant decrease in numbers
of peripheral blood leukocytes at day 3 in BALB/c mice, but with-
out statistical significance (p = 0.09) at this time point in C57BL/6
mice (Table 1). Drop in lymphocytes accounted for the observed
leukocyte decrease.
Spleen mass, cell number and proliferation following i.v. injection
of A. fumigatus
Determination of basic parameters relevant for antifungal
response in the spleen revealed an increase (compared to respec-
Table 1
Total and differential peripheral blood leukocyte counts in BALB/c and C57BL/6 mice
following i.v. injection of A. fumigatus conidia.
Controls Day 3 Day 7
BALB/c
Total leukocytes (×10
9
/L) 6.8 ± 1.2 4.8 ± 0.9
*
7.3 ± 1.4
Lymphocytes 5.6 ± 1.0 3.9 ± 0.8
***
5.9 ± 1.1
Neutrophils 0.9 ± 0.3 0.7 ± 0.1 1.1 ± 0.3
Monocytes 0.3 ± 0.1 0.2 ± 0.1 0.3 ± 0.1
C57BL/6
Total leukocytes (×10
9
/L) 8.1 ± 2.2 6.2 ± 1.0 10.0 ± 2.3
Lymphocytes 6.2 ± 2.5 5.0 ± 0.9 8.1 ± 2.1
Neutrophils 1.0 ± 0.6 0.9 ± 0.2 1.5 ± 0.5
Monocytes 0.3 ± 0.1 0.3 ± 0.1 0.4 ± 0.1
*
Significantly different at p < 0.05 from controls.
***
Significantly different at p < 0.001 from controls.
tive uninfected controls) of relative spleen mass and cell number in
individuals of both strains, that was greater at days 3 and 7 (spleen
mass) and day 3 (cell number) following conidia administration in
BALB/c mice than in C57BL/6 mice (Table 2). Increase of ex vivo
spleen cell proliferation was noted nearly throughout infection,
with roughly similar levels and dynamics in two strains.
Table 2
Basic parameters of spleen response of BALB/c and C57BL/6 mice to i.v. injection of A. fumigatus conidia.
Controls Day 3 Day 7 Day 15
Relative spleen mass
BALB/c 0.51 ± 0.02 0.82 ± 0.13
***,##
0.83 ± 0.13
***,##
0.76 ± 0.09
***
C57BL/6 0.48 ± 0.04 0.63 ± 0.15
***
0.69 ± 0.12
***
0.77 ± 0.21
**
Cell number (×10
6
)
BALB/c 133.7 ± 12.4 220.3 ± 35.2
***,###
187.6 ± 25.9
***
173.2 ± 35.5
***
C57BL/6 112.2 ± 25.3 156.7 ± 30.3
*
167.2 ± 35.2
***
183.2 ± 39.6
***
Proliferation (×10
3
c.p.m.)
BALB/c 6.0 ± 3.3 7.8 ± 0.4 9.5 ± 6.8
*
15.5 ± 10.6
***
C57BL/6 4.9 ± 3.3 9.7 ± 5.9
**
7.0 ± 3.5
*
23.9 ± 14.4
Results were pooled from at least three experiments with four to six mice per group and expressed as mean values ± S.D.
*
Significantly different at p < 0.05 vs respective control animals.
**
Significantly different at p < 0.01 vs respective control animals.
***
Significantly different at and p < 0.001 vs respective control animals.
##
Significantly different at p < 0.01 vs C57BL/6 mice.
###
Significantly different at and p < 0.001 vs C57BL/6 mice.
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238 I. Mirkov et al. / Immunobiology 216 (2011) 234–242
Fig. 3. Histological evaluation of spleen following i.v. injection of A. fumigatus coni-
dia. Semi-quantitative analysis of red to white pulp proportion (a), hematopoiesis
intensity score (b), (c)1–minimally expressed hematopoiesis (H&E 20×), (d) 3-
well expressed hematopoiesis (H&E 20×). Significantly different at p < 0.05 (*) vs
respective strain control animals.
Fig. 4. IFN- and IL-4 gene expression in spleen cells of BALB/c and C57BL/6 mice
after inoculation of mice with A. fumigatus. Data are presented as means ± S.D. of
samples pooled from three independent experiments with three to four animals
per group for each time point and expressed as percentages of mRNA expression
in spleen cells of infected mice relative to cells from respective control noninfected
mice. BALB/c mice (a) and C57BL/6 mice (b). Significantly different at p < 0.05 (*) vs
respective strain control animals.
Spleen histology following i.v. injection of A. fumigatus
Histological analysis of spleens of infected mice revealed no
significant differences in the spleen activity in terms of germi-
nal centre and marginal zone appearance (not shown). However,
higher proportion of red pulp (Fig. 3a) with more expressed
hematopoietic activity (Fig. 3b) was noted in infected BALB/c mice,
while even a tendency of decrease in this spleen compartment was
noted in C57BL/6 mice early in infection.
Interferon- (IFN-) and interleukin-4 (IL-4) expression and
secretion by spleen cells following i.v. injection of A. fumigatus
The examination of kinetics of spleen IFN- gene expression
after infection of mice by conidia revealed differences both in the
level and dynamics of cytokine message expression in spleen cells
of two strains (Fig. 4). In spleen cells of infected BALB/c mice an
increased IFN- mRNA levels were noted during the first week fol-
lowing conidia administration (statistically significant) and late in
infection (solely numerical), with unchanged IL-4 mRNA expres-
sion throughout the infection (Fig. 4a). In contrast, moderate,
although significantly higher (compared to uninfected pairs) IFN-
message was detected in C57BL/6 mice at day 3 p.i. only. In
contrast to BALB/c mice, a decrease in IL-4 mRNA expression in
spleens of infected C57BL/6 mice was noted at all time points exam-
ined (Fig. 4b). Consequently, a high IFN-/IL-4 mRNA ratio was
present throughout infection in spleens of animals of both strains
(2833 ± 613, 2277 ± 302 and 2131 ± 161 in BALB/c vs 1474 ± 812,
1259 ± 805 and 933 ± 306 in C57BL/6 at days 3, 7 and 15, respec-
tively) but higher in BALB/c mice (p < 0.001 and p < 0.01) at days 3
and 15 p.i.
In line with the gene expression data, an increase of sponta-
neous production of IFN- (Fig. 5a) with unchanged IL-4 production
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I. Mirkov et al. / Immunobiology 216 (2011) 234–242 239
Fig. 5. IFN- and IL-4 production by spleen cells from spleens of BALB/c and C57BL/6
mice injected with A. fumigatus. Results are expressed as cytokine protein concentra-
tion (pg/ml) for BALB/c (a), C57BL/6 (b), and IFN- to IL-4 ratio (c). Data are presented
as means ± S.D. of samples pooled from three independent experiments with four
to six animals per group for each time point. Significantly different at p < 0.05 (*), at
p < 0.01 (**) and p < 0.001 (***) vs respective strain control animals, and at p < 0.01
(
##
) vs C57BL/6 mice.
(Fig. 5b) by spleen cells was noted in infected BALB/C mice. In com-
parison to this mouse strain, much lower levels of IFN- production
were noted in C57BL/6 mice at all time points (p < 0.01, at days 3
and 15, and p < 0.001 at day 7 p.i.), with reduced spontaneous pro-
duction of IL-4 (Fig. 5b). Observed pattern of spleen cell cytokine
production resulted in high ratio of IFN-/IL-4 in infected mice
of both strains at all time points, but significantly much higher
(p < 0.01) during the last week p.i. in BALB/c mice (Fig. 5c).
Interelukin-17 (IL-17) expression and secretion by spleen cells
following i.v. injection of A. fumigatus
The IL-17 mRNA levels were increased in the spleen cells of
both strains throughout the examined period following infection
(although without reaching statistical significance at days 15 and
7 p.i. in BALB/c and C57BL/6 mice, respectively) (Fig. 6a). However,
unchanged (day 3) and even significantly lower protein secretion
later in infection (days 7 and 15) was noted by splenocytes of
infected BALB/c mice, while spleen cells of C57BL/6 mice responded
to A. fumigatus infection by an increase in IL-17 production dur-
ing first week of infection (Fig. 6b). When splenocytes from non
infected (naïve) mice were stimulated in vitro with A. fumigatus
conidia, higher production was observed in BALB/c mice compared
to C57BL/6 mice (Fig. 6c), thus contrasting reduced IL-17 secretion
seen in infected animals (Fig. 6b).
Discussion
In this study, resistance to systemic A. fumigatus infection was
evaluated in two mouse strains, BALB/c and C57BL/6 known to dif-
fer in resistance/susceptibility to various microbial infections and
in the type of cytokine response to these insults (Gervais et al. 1984;
Guler et al. 1996; Tam et al. 1999; Thach et al. 2000; Wakeham et
al. 2000). Survival rate at different conidia doses and efficacy of
conidia elimination from visceral organs and spleen at sublethal
conidia dose were examined and compared in these two strains.
Basic indices of spleen activity, as well as the production of sig-
nature pro-inflammatory (IFN- and IL-17) and anti-inflammatory
(IL-4) cytokines shown to be relevant for the response to lethal
systemic A. fumigatus infection (IFN- and IL-4) and in pulmonary
aspergillosis (IL-17) (Cenci et al. 1997; Zelante et al. 2007) were
determined at sublethal conidia dose.
Survival study revealed 100% mortality in 6 days (BALB/c) and
2 days (C57BL/6) at high (5 × 10
7
) dose in both strains, in line with
data of Takemoto et al. (2004) which demonstrated 100% mortality
of outbred ddY mice in 4 days at 3 × 10
7
conidia. In agreement with
the study of lethal invasive aspergillosis in mice of several differ-
ent strains (Cenci et al. 1997), no differences in survival between
BALB/c and C57BL/6 mice were noted when infection was induced
by 1 × 10
7
A. fumigatus conidia, although 100% mortality (67% in our
study) was noted in BALB/c mice. While no lethal outcome follow-
ing administration of 1 × 10
6
conidia was noted in that study, fatal
outcome at this conidia dose was detected in 2% BALB/c mice (1/49)
and 1.9% (1/49) of C57BL/6 mice in our study. Variance in colonies
of mice might account for minor differences in survival data in that
and our study.
In line with data obtained in systemic sublethal A. fumigatus
infection in CBA mice (Corbel and Eades 1977), the highest numbers
of CFUs were noted in the spleen and liver of infected animals. Sim-
ilar dynamics of reduction in fungal burden in spleen, liver, lungs
and kidneys following administration of 1 × 10
6
demonstrated sim-
ilar capacity to resist infection in these two strains as well. However,
while similar dynamics of conidial removal from spleens of infected
mice of both strains was noted, the magnitude, dynamics and the
quality of spleen immune responses differed in some aspects in
these two strains. Although measurements of IFN- and IL-4 pro-
duction in lethal systemic A. fumigatus infection have been already
published in several mouse strains (Cenci et al. 1997), data pre-
sented in this study are novel in terms of comparative evaluation
of expression of these cytokines in sublethal Aspergillus infection in
C57BL/6 and BALB/c mice, mouse strains most often used as Th1-
and Th2-prone mice, respectively (Thach et al. 2000; Wakeham
et al. 2000). In particular, the expression of IL-17, the principal
cytokine of Th17 T cells was evaluated for the first time in BALB/c
mice with systemic aspergillus infection and was compared in these
two strains.
The increase in relative spleen mass, cellularity and proliferation
in infected animals of both strains reflects the response to injected
conidia. Increased spleen masses noted in BALB/c mice during infec-
tion might rely on the increase in relative proportion of red pulp in
these mice. Rodent spleen is known hematopoietic organ (Mebius
and Kraal 2005) and pronounced red pulp and hematopoietic activ-
ity might imply higher propensity of spleen of BALB/c mice to
respond to host demands for leukocytes during acute fungal infec-
tion. Cell influx to spleens of BALB/c mice might have contributed
to a greater increase in spleen cell numbers early in infection com-
pared to C57BL/6 mice as spleen is the site of intense leukocyte
trafficking in the settings of various infections (Mebius and Kraal
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240 I. Mirkov et al. / Immunobiology 216 (2011) 234–242
Fig. 6. IL-17 gene expression and cytokine production by spleen cells of BALB/c and C57BL/6 mice after inoculation of mice with A. fumigatus. IL-17 mRNA expression
(a), spontaneous IL-17 production (b), (c) IL-17 production following in vitro stimulation of spleen cells form naive mice with A. fumigatus conidia. Data are presented as
means ± S.D. of samples pooled from three independent experiments with four to six animals per group for each time point. Results are expressed as percentages of mRNA
expression in spleen cells of infected mice relative to cells from respective control noninfected mice (a) and cytokine protein concentration (pg/ml) (b and c). Significantly
different at p < 0.05 (*) and at p < 0.01 (**) vs respective strain controls and at p < 0.01 (
##
) vs C57BL/6 mice.
2005). A decrease in peripheral blood leukocyte numbers, more
pronounced in BALB/c than in C57BL/6 mice, might have accounted
for the observed difference. Proliferative capacity of spleen cells
possibly contributed equally to spleen cellularity, based on sim-
ilar ex vivo proliferation in both strains, although hematopoietic
activity might had a share in spontaneous proliferation in BALB/c
mice.
A. fumigatus conidia application resulted in increased expres-
sion of mRNA for IFN- in splenocytes of both strains, confirming
the importance of this cytokine in infection with the fungus.
Higher production of IFN- by spleen cells of BALB/c mice might
be explained by the need for higher quantities of this cytokine
to combat infection in these mice. Interferon- is potent acti-
vator of antifungal effector activities (phagocytosis, nitric oxide
production) in phagocytic cells (Bernhisel-Broadbent et al. 1991;
Rex et al. 1991; Roilides et al. 1993) of which macrophages were
shown as indispensable for adaptive immunity to aspergillosis (De
Repentigny et al. 1993). Macrophages from BALB/c mice, com-
pared to C57BL/6 macrophages respond weakly by NO production
to IFN- stimulation (Mills et al. 2000) and it is possible that
higher cytokine amounts are needed for production of this effec-
tor molecule. High IFN- levels in spleen of BALB/c mice might be
needed to overcome the effects of locally produced IL-4 (unchanged
in this strain vs decreased in C57BL/6) during A. fumigatus infec-
tion in these mice. Higher NO production and fungicidal efficiency,
noted by macrophages from IL-4 knockout BALB/c mice infected
with Candida albicans (Mencacci et al. 1998), as well as higher
hyphal damage capacity of macrophages from IL-4 knockout BALB/c
infected with A. fumigatus (Cenci et al. 1999) supports such consid-
erations.
Both higher IFN- gene expression and production noted in
splenocytes of infected BALB/c mice point to a greater propensity
of this mouse strain to respond to systemic A. fumigatus infection
by producing IFN-. This is in line with the data that demonstrated
the importance of Th1-mediated immune response to the fungus
Paracoccidioides brasiliensis in BALB/c mice (Taborda et al. 1998),
where protective response to fungal antigens was characterized
by high lymph node cell production of IFN-. High expression of
IFN- compared to unchanged IL-4 in spleen cells of BALB/c mice
upon A. fumigatus infection in our study is in contrast to data which
demonstrated similar production of both cytokines in this mouse
strain exposed to sublethal A. fumigatus infection (Cenci et al. 1998).
However, for stimulation of spleen cells, the authors employed
polyclonal (ConA) stimulation in the presence of exogenous acces-
sory cells that might have equalized cytokine response. In that
study, cytokine production by CD4
+
cells was evaluated, while we
used unseparated spleen cells, and therefore both CD4
+
and CD8
+
T cells (Cenci et al. 1998), as well as NK cells, known sources of
IFN- in the settings of various microbial infections (Abbas et al.
2007), might have contributed to higher splenic IFN- in A. fumi-
gatus infection in BALB/c mice as presented here.
In adherence to data obtained in systemic lethal murine
aspergillosis (Cenci et al. 1999) that showed a lower spleen IL-4
expression in survivors, compared to increased expression of this
cytokine in the spleens of non-survivors, lack of change in produc-
tion of IL-4 by spleen cells of BALB/c mice as well as decreased
expression and production of IL-4 in infected C57BL/6 mice com-
pared to controls demonstrated the relevance of this cytokine in
sublethal fungal infection as well. Unchanged expression of this
cytokine in BALB/c mice might be explained by higher expression
of IFN-, shown to control IL-4 production in murine aspergillosis
(Cenci et al. 1999). Decreased IL-4 expression might be beneficial
for infected C57BL/6 mice, as an increase in susceptibility to inva-
sive pulmonary aspergillosis evoked by IL-4-mediated suppression
of protective type 1 responses was demonstrated in mice (Cenci
et al. 1999). Lack of change in production of IL-4 by spleen cells of
BALB/c mice noted in our study is in line with the finding of low
production of IL-4 (as compared to high IFN-) in experimental
paracoccidiomycosis in this mouse strain (Taborda et al. 1998).
Increased gene expression of IL-17 nearly throughout the infec-
tion in spleens of both BALB/c and C57BL/6 mice pointed out the
relevance of this cytokine in systemic sublethal Aspergillus infection
in both strains. While in BALB/c mice an increased IL-17 gene tran-
scription was observed, the unchanged (day 3) and even decreased
secretion (during second weak of infection) of IL-17 protein was
detected. This could be explained by the demonstrated relatively
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I. Mirkov et al. / Immunobiology 216 (2011) 234–242 241
high IFN- production by spleen cells from infected BALB/c mice
at these time points, as suppression of IL-17 by IFN- was shown
in some experimental systems (Bi et al. 2007). Higher production
of IL-17 by naïve spleen cells to in vitro stimulation with A. fumiga-
tus conidia at time point when no substantial increase in IFN- is
observed supports such an assumption. Although not directly com-
parable, higher IL-17 production by both naïve as well as infected
BALB/c splenocytes in response to in vitro stimulation and challenge
with conidia is in line with the data which demonstrated higher
frequency of IL-17 producing cells in thymocytes of BALB/c mice
(compared to C57BL/6 mice) stimulated via CD3 in vitro (Hofstetter
et al. 2006). The significance of slightly higher IL-17 production by
spleen cells of BALB/c mice is at present unknown, as there is no, to
our knowledge, systematic study of influence of genetic predisposi-
tion on IL-17 expression. Splenic IL-17 production may rest mainly
on CD4
+
T cells, as demonstrated in host response to pulmonary
Aspergillus infection (Zelante et al. 2007) although CD8
+
cells, as
well as NK cells, shown to have a capacity to produce this cytokine
in certain conditions (Weaver et al. 2007) might also have con-
tributed. Supplementary to data that demonstrated the importance
of IL-17 in lethal systemic Cryptococcus neoformans (Kleinschek et
al. 2006) and C. albicans infection in mice (Huang et al. 2004), our
results showed the relevance of IL-17 in hosts that have success-
fully combated Aspergillus infection. Various IL-17-related effector
activities postulated to influence the capacity of protective T cells
to eliminate pathogens (Matsuzaki and Umemura 2007) might be
relevant for splenic response to A. fumigatus infection. Consistent
with the means of protection against other fungi (Curtis and Way
2009), increased production of IL-17 in Aspergillus infection might
be associated with mobilization of peripheral blood leukocytes to
infected spleen and stimulation of spleen mononuclear cell activity.
In conclusion, similar levels of lethal outcome at high fun-
gal inocula and resistance to sublethal conidia dose were noted
in Th2-prone BALB/c and Th1-prone C57BL/6 mice. Host anti-
fungal response to low conidia dose was associated with similar
progressive decrease in fungal burden from visceral organs and
spleen in both strains. Sublethal systemic aspergillosis in immuno-
competent BALB/c and C57BL/6 mice tips the balance towards
pro-inflammatory antifungal splenic response by an increase in a
Th1 arm of response by pronounced increase in IFN-, but with-
out changing the anti-inflammatory Th2 (IL-4) response in BALB/c
mice and by an increase in the IFN- and down-regulation of IL-4
response in C57BL/6 mice. Triggering of Th17 arm of the immune
response in both strains provided optimal inflammatory milieu that
might have contributed to efficient removal of conidia.
Acknowledgements
This study was supported by the Ministry of Science and Tech-
nological Development of the Republic of Serbia, Grants # 143038
and # 143029. The authors would like to thank Sandra Belij and
Aleksandra Popov for expert technical assistance in some of the
aspects of this investigation.
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    • "In contrast to the increase of IFN-␥ in WT mice, which confirms our previous findings concerning this cytokine in sublethal systemic aspergillosis in immunocompetent C57BL/6 mice (Mirkov et al. 2010, 2011), the lower production and expression of IFN-␥ in MIF −/− compared to WT mice, imply Th1 promoting activity of MIF in systemic Aspergillus infection. This is in line with the pro-inflammatory activity of MIF during protection against bacterial infections (Koebernick et al. 2002) and in a wider context with data that MIF is equally efficiently involved in the adaptive immune response through favouring Th1 activation and differentiation (Cvetkovic and Stosic-Grujicic 2006; Koebernick et al. 2002). "
    [Show abstract] [Hide abstract] ABSTRACT: Inflammation plays an important role in protective immunity against fungi, including the opportunistic pathogen, Aspergillus fumigatus. The balance between pro-inflammatory and anti-inflammatory cytokines is a key determinant of infection outcome. Since macrophage migration inhibitory factor (MIF) is an upstream regulator of many cytokines, we analyzed herein the role of endogenous MIF in the host control of hematogenously disseminated aspergillosis using MIF⁻/⁻ mice. As revealed by their mortality rate, MIF⁻/⁻ mice were more susceptible to disseminated infection than WT mice. Moreover, pharmacologic inhibition of MIF with (S,R)-3-(4-hydroxyphenyl)-4,5-dihydro-5-isoxazole acetic acid methyl ester, (ISO-1) increased the susceptibility of WT mice to lethal infection. The higher tissue fungal burden early in sublethal infection indicated increased susceptibility of MIF⁻/⁻ mice to sublethal infection as well. Substantial down-regulation of innate and acquired antifungal responses, characterized by decreased production of IL-1β, IL-6, TNF-α, IFN-γ and IL-17 in the spleen was noted in sublethally infected MIF⁻/⁻ mice. In contrast, IL-4 was higher in MIF⁻/⁻ than in WT mice. Taken together, our findings show that MIF contributes to host resistance against progressive invasive A. fumigatus infection by controlling downstream pro-inflammatory versus anti-inflammatory cytokine production thus determining the outcome of infection.
    Full-text · Article · Mar 2011 · Immunobiology
  • No preview · Article · Mar 2011 · Journal of Vascular and Interventional Radiology
  • [Show abstract] [Hide abstract] ABSTRACT: Model of systemic Aspergillus fumigatus infection induced by intravenous application of conidia is suitable for studying important aspects of invasive aspergillosis including relationship between infection and mortality, dissemination of infection and immune mechanisms involved in host resistance to this fungus. Use of this model allows the investigation of both innate and adaptive immune response characteristics in resistant/susceptible host, and investigating the contribution of genetic background and cytokine gene deficiency improves the knowledge of the diversity of mechanisms of immune response to Aspergillus infection. Studying of various aspects of systemic aspergillosis contributes to development of antifungal drugs.
    No preview · Article · Mar 2012 · Immunologic Research
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