Splenic and lung response to nonlethal systemic Aspergillus fumigatus infection in C57BL/6 mice

Medical Mycology August 2010, 48, 735–743

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Splenic and lung response to nonlethal systemic

Aspergillus fumigatus infection in C57BL/6 mice

© 2010 ISHAM

IVANA MIRKOV*, IVANA STOJANOVIC†, STANISLAVA STOSIC-GRUJICIC†, JASMINA GLAMOCLIJA*,

LIDIJA ZOLOTAREVSKI‡, DRAGAN KATARANOVSKI*§ & MILENA KATARANOVSKI*#

*Department of Ecology & †Department of Immunology, Institute for Biological Research ‘Sinisa Stankovic’, University of Belgrade,

Belgrade, ‡Institute for Pathology, Military Medical Academy, Belgrade, §Institute of Zoology, Faculty of Biology, University of

Belgrade, Belgrade, and #Institute of Physiology and Biochemistry, Faculty of Biology, University of Belgrade, Belgrade, Serbia

Received 7 July 2009; Received i

Accepted 18 November 2009

Correspondence: Milena Katar

Institute for Biological Researc

Belgrade, Belgrade, Despota Stefa

Fax +381 11 2761433; E-mail: mil

In this study, we investigated splenic and lung cell responses to nonlethal systemic Aspergillus fumigatus infection in mice. Apart from basic indices of spleen and lung cell activity, IL-17 expression by cells from both tissues was determined and compared to the expression of IFN-� and IL-4. Progressive decrease in tissue fungal burden cor-related with increased spleen and lung cell activity. Increased IL-17 message was noted in spleen cells at all time points (3, 7 and 15 days post-infection; p.i.), while a modest increase in IFN-� mRNA expression was noted at day 3 p.i. Increased cytokine produc-tion at days 3 and 7 (IL-17) and throughout the experimental period (IFN-�) was found. In contrast, spleen cell IL-4 expression was considerably lower during infection, result-ing in high IFN-�/IL-4 and IL-17/IL-4 ratios in the spleen. Pro-infl ammatory cytokine response was observed in the lungs as well, but primarily as the result of increased production of IFN-� by lung cells in response to challenge with conidia and the absence of change in IL-4 response. Increased activity of cells from both tissues, as well as the pattern of cytokine production, created an optimal pro-infl ammatory milieu for fungal eradication.

Keywords nonlethal murine systemic aspergillosis, spleen, lungs, IL-17, IFN-�, IL-4

Introduction

Aspergillus fumigatus is a ubiquitous saprophytic mould

that forms airborne conidia [1–3]. The small diameter of

the conidia, as well as environmental factors, make them

easily accessible to the lungs [4,5]. Infections occur rarely

in healthy, immunocompetent individuals, as conidia are

effi ciently eliminated by host immune defenses [1]. Thus,

A. fumigatus is regarded as a weak pathogen, responsible

in immunocompetent individuals for only allergic

aspergillosis or aspergilloma. With the increasing number

of immunocompromised/immunodefi cient individuals,

A. fumigatus has emerged as the second major cause of

invasive fungal infections in such patients [1].

n fi nal revised form 22 October 2009;

anovski, Department of Ecology,

h ‘Sinisa Stankovic’, University of

na 142, Serbia. Tel: +381 11 2078375;

[email protected]

Animal models of aspergillosis have been used exten-

sively to study various aspects of infection caused by this

fungus. Murine models have been used to study host resis-

tance in the four main forms of invasive aspergillosis; pul-

monary aspergillosis (IPA), bronchopulmonary aspergillosis

(ABPA), systemic (disseminated) infection and central ner-

vous system (CNS) infection [7]. The use of these models

has been indispensable in enhancing our current under-

standing of both pathogenesis and host resistance to Asper-gillus infections.

Utilizing the intranasal or intratracheal route of inocu-

lating mice with Aspergillus conidia (to simulate the natu-

ral route of infection), the role of innate host defense and

the generation of Th1 cytokines by interstitial lymphocytes

was shown to play a critical role in the resistance to inva-

sive pulmonary aspergillosis [6,8,9]. Immunosuppressive

treatments (e.g., glucocorticoids, cyclophosphamide, etc),

are required to increase the mouse’s susceptibility to infec-

tion [10,11]. Inoculation of A. fumigatus by a systemic route (i.e., intravenous inoculation) leads to infection in

DOI: 10.3109/13693780903496591

© 2010 ISHAM, Medical Mycology, 48, 735–743

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immunocompetent mice with a relatively uniform pattern

of disease. This model of infection is used for disseminated

infection studies, infection-mortality ratio investigations

and is particularly useful in the determination of mecha-

nisms involved in host resistance or susceptibility to lethal

A. fumigatus infection [10,12–15]. Differential production

of cytokines that favor Th1 response (e.g., IFN-�) and Th2

cytokines (e.g., IL-4 and IL-10) by splenic CD4+ cells was

demonstrated in mice found to be resistant or susceptible

to lethal A. fumigatus infections. Although IFN-� was

largely responsible for the resistance to lethal dose of the

fungus, the production of Th2 cytokines was associated

with lethal outcome [13]. Relative resistance to lethal sys-

temic infection could be induced by IFN-� administration

[14] or IL-4 neutralization [13], as well as being found in

mice lacking a functional IL-10 [15].

Although these cited studies clearly demonstrated the

signifi cance of splenic Th1 and Th2 cytokines in the resis-

tance or susceptibility to lethal systemic A. fumigatus infec-

tion, there are few data regarding the relevance of these

cytokines in nonlethal infections. Although these infections

have some of the characteristics of systemic disease, little

attention has been paid to other target tissues. Additionally,

there is, to our knowledge, no data concerning the relevance

of the principal cytokine of Th17 subset, interleukin-17

(IL-17) during systemic infection with A. fumigatus. The

importance of activation of Th17 cells in immune response

to fungi was stressed recently [16,17] and the involvement

of IL-17 in innate host defense to pulmonary aspergillosis

with A. fumigatus has been demonstrated [18,19].

Our previous study demonstrated histopathological

changes in lungs and kidneys of mice that received lethal

or sublethal intravenous doses of A. fumigatus conidia [12].

Using this model of disseminated aspergillosis, we exam-

ined spleen and pulmonary cell responses to nonlethal

doses of A. fumigatus. Basic indices of spleen and lung

activity including changes in mass, cellularity and cell pro-

liferation, as well as leukocyte infi ltration and cell activa-

tion, were determined. Quantitative and temporal expression

of IL-17 in spleen and lung cells were compared to the

expression of IFN-� and IL-4. These data indicated that

resistance to systemic aspergillosis is associated with a

proinfl ammatory response, both in the spleen and the lungs.

Such a response coincided with a progressive decrease in

conidial burden, suggesting its contribution in effi cient

elimination of fungi from these tissues.

Materials and methods

Mice

Eight- to 12-week- old female C57BL/6 mice, four to six

per group, were used for the study. Animals were bred and

housed conventionally in a controlled environment at the

Institute for Biological Research ‘Sinisa Stankovic’

(Belgrade, Serbia) and provided with standard rodent chow

and water ad libitum. All experiments were approved by

the Ethical Committee of our Institute. Mice were eutha-

nized on days 1, 3, 7 and 15 post-infection (p.i.) for fungal

burden assessment, and at days 3, 7 and 15 for the other

analyses.

Fungal culture conditions and infection

A human isolate of A. fumigatus was obtained from the

Institute of Public Health of Serbia ‘Dr Milan Jovanovic-

Batut’. The fungus was grown on Sabouraud maltose agar

(SMA, Torlak, Belgrade, Serbia) for 7 days [20]. Conidia

were harvested by fl ooding the surface of agar slants with

nonpyrogenic sterile physiological saline and the suspen-

sion adjusted to a concentration of 107 conidia/ml. Anes-

thetized mice (Ketamidor, Richter Pharma, Austria) were

inoculated intravenously with 106 conidia in 0.1 ml of

pyrogen-free saline via the lateral tail vein. Control mice

received saline only. In restimulation experiments, conidia

that had been autoclaved at 121°C for 30 min

(heat-inactivated) were used.

Fungal burden assessment

Fungal burden in lungs, spleen and kidneys was deter-

mined by quantitative colony forming units (CFU) [21]

assay. At selected time points (i.e., 1, 3, 7 and 15 days) p.i., organs were removed aseptically and their wet masses

were determined by weighing them using a precision bal-

ance (� 0.01 g). Tissue specimens were homogenized

using an IKA T18 basic homogenizer (IKA Works INC.,

Wilmington, NC) in 5 ml of sterile saline, on ice. Serial

dilutions of the primary homogenates were inoculated on

SMA plates supplemented with streptomycin sulfate (ICN-

Galenika, Belgrade, Serbia), and incubated at 37°C for

24 to 48 h. The number of CFU per gram of tissue was

determined as described.

Spleen cell preparation and culture

Spleens were removed, blotted dry, and weighed. The rel-

ative spleen mass was calculated according to the follow-

ing formula: organ mass/body mass � 100. Spleen cells

were obtained aseptically by gentle teasing the spleen tis-

sue through a cell strainer (BD Falcon, BD Bioscience,

Bedford, USA). Cells were suspended in RPMI-1640 cul-

ture medium (Flow, ICN Pharmaceuticals) supplemented

with 2 mM glutamine, 20 μg/ml gentamicin (Galenika a.d.,

Serbia) with 5 % (v/v) heat-inactivated fetal calf serum

Proinfl ammatory response in systemic murine aspergillosis 737

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(PAA laboratories, Austria) (complete medium) and

voriconazole (5 μg/ml) (Pfi zer PGM, France) and adjusted

to 3 � 106/ml cells per cell culture well.

The capacity of spleen cells to proliferate was deter-

mined ex vivo or following restimulation with

heat-inactivated conidia of A. fumigatus at a ratio of conidia

to spleen cells of 0.06:1. Briefl y, 0.3 � 106 cells/well

(96-well plate) were cultivated for 24 h (ex vivo prolifera-

tion) or 72 h (restimulated proliferation), with 0.5 μCi of

[3H]-thymidine (GE Healthcare, UK) per well during the

last 8 hours of culture. Incorporation of [3H]-thymidine

into cellular DNA was measured by liquid scintillation

counting (LKB, Wallac).

For cytokine production, 5 � 106/ml spleen cells/well

(24-well plate) were cultured for 72 h in medium without

conidia to assess spontaneous production or with

heat-inactivated conidia of A. fumigatus at a conidia:spleen

cell ratio of 2:1 (restimulation). Cytokine level in the con-

ditioned medium was determined by enzyme-linked immu-

nosorbent assay (ELISA). Cytokine production after in vitro restimulation is expressed as the index of stimulation,

calculated according to the formula of restimulated pro-

duction/spontaneous production.

RNA isolation and RT-PCR

Total RNA from splenocytes (5 � 106 cells) was isolated

with a mi-Total RNA Isolation Kit (Metabion, Martinsried,

Germany) according to the manufacturer’s instructions.

RNA (1 μg) was reverse transcribed using Moloney leuke-

mia virus reverse transcriptase and random hexamers (both

from Fermentas, Vilnius, Lithuania). PCR amplifi cation of

cDNA (1 μl per 20 μl of PCR reaction) was carried out in

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 followed 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′-CATCAGCAACAA-

CATAAGCGTCA-3′ and 5′-CTCCTTTTCCGCTTC-

CTGA-3′ (GenBank accession no. NM 008337.2), IL-4

5′-ATCCTGC TCTTCTTTCTCG-3′ and 5′-GATGCTC

TTTAGGCTTTCC-3′ (GenBank accession no. NM

021283.1) and β-actin 5′-GGACCTGACAGACTACC-3′ and 5′-GGCATAGAGGTCTTTACGG-3′ (NM 007393.2).

The expression of these genes was calculated according

to the formula 2-(Cti-Cta) where Cti is the cycle threshold

of the gene of interest and Cta is the cycle threshold value

of β-actin.

© 2010 ISHAM, Medical Mycology, 48, 735–743

Pulmonary cell isolation and activity assays

Lungs were removed aseptically and their wet masses

determined. The relative lung mass was calculated by the

following formula, lung mass/body mass � 100. Lung leu-

kocytes were obtained by digestion with 1 mg/ml type IV

collagenase (Worthington Biochemical Corporation, Lake-

wood, USA) and 30 μg/ml Deoxyribonuclease I (DNase I,

Sigma, Sigma Chemical Co., St Louis, MO, USA) for half

an hour at 37°C. Cells were resuspended in the complete

medium and adjusted to 4 � 106 cells/ml.

The activation of lung cells was evaluated by a quanti-

tative cytochemical assay for the respiratory burst [22],

based upon the spontaneous or phorbol-12-myristate

13-acetate (PMA, Sigma, Sigma Chemical Co., St Louis,

MO, USA) stimulated capacity of the cells to reduce

nitroblue tetrazolium (NBT, ICN Pharmaceutical, Costa

Mesa, CA, USA). Briefl y, 0.2 � 106 cells/well (96-well

plate) were cultured with 500 μg/ml NBT, with or without

(spontaneous) 100 ng/ml PMA (PMA-stimulated NBT

reduction) for 30 min. Formazan produced by cells was

extracted overnight in 10% SDS-0.01N HCl, and optical

density (OD) measured by microplate spectrophotometer

(GRD, Rome, Italy) at 540 and 650 nm. For the measure-

ment of cytokine production, 0.2 � 106 cells/well (96-well

plate) were cultured for 72 h with heat-inactivated conidia

of A. fumigatus at a ratio of 2:1 conidia to cells (restimu-

lated production) or with medium only (spontaneous

production).

ELISA

Cytokine production was determined in the medium con-

ditioned by spleen or lung cells using commercially avail-

able ELISA sets for mouse IL-17 (BD Pharmingen, San

Diego, CA, USA), mouse IFN-� (eBioscience Inc, San

Diego, CA, USA) and mouse IL-4 (R&D Systems,

Minneapolis, USA). Tests were done according to the

manufacturers’ recommendations. Cytokine titer was cal-

culated by reference to a standard curve constructed using

known amounts of recombinant IFN-�, IL-4 or IL-17.

Lung histology

Lung tissue was excised, immediately fi xed in 4% formal-

dehyde (pH 6.9), and embedded in paraffi n wax for sec-

tioning at 5 μm. Hematoxylin and eosin (H&E)-stained

histological slides were analyzed subsequently.

Statistics

Results were pooled from at least three experiments and

expressed as mean values � SD. Results were analyzed

738 Mirkov et al.

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statistically using a Mann-Whitney U test for fungal bur-

den and cell culture data, and a t-test for gene expression.

P-values less than 0.05 were considered signifi cant.

Results

Conidial burden in spleen and lungs following i.v. A. fumigatus

injection

Determination of tissue fungal burden at day 1 postinfec-

tion revealed the highest number of CFU/g in the spleen

(7400 � 5), followed by lungs (352 � 19) and kidneys

(123 � 10). A progressive decrease in the number of CFUs

in these tissues was noted, with conidial elimination at

days 7 and 15 in lungs and spleen, respectively (Fig. 1 A,

B and C). From day 7 onward, studies of tissue homoge-

nates of all animals indicated that they were free of infec-

tion. At day 15, no fungal growth was detected in any

spleen homogenates or in two of four kidney homogenates.

Given the more pronounced, as well as sharper, drop in

conidial burden in spleen and lungs compared to kidneys,

changes in splenocyte and pulmonary cell activity were

investigated further.

Spleen response to conidia application

Basic parameters relevant for antifungal response in the

spleen were examined and included changes in organ mass,

cellularity, cell proliferation rate and cytokine expression.

Increased relative spleen mass and cell numbers compared

to spleens of control animals were noted at all time points

following infection (Fig. 2A and 2B). An increase in

ex vivo proliferation of spleen cells was found at days

3 and 7 postinfection with a tendency (P � 0.07) towards

increases in cell proliferation at day 15 (Fig. 2C). Restim-

ulation with A. fumigatus conidia resulted in increased

spleen cell proliferation at days 3 and 7. However, this did

not reach statistical signifi cance at day 3 (Fig. 2D). Increase

in spleen cellularity correlated with a decrease in spleen

fungal burden (r � −0.84; P � 0.001; y � 229.8197 −

0.0792*x). A signifi cant positive correlation was noted

between spleen cellularity and spleen mass (r � 0.74; P �

0.001; y � 0.02 + 2.9365E-5*x) and between spontaneous

splenocyte proliferation and spleen cellularity (r � 0.61;

P � 0.001; y � 145.5 + 0.0019*x).

Examination of the kinetics of spleen cytokine gene-

expression after infection with conidia (Fig. 3) revealed

increased IL-17 mRNA levels in spleen cells throughout

the p.i. period, but did not reach statistical signifi cance at

day 7. Signifi cantly, more IFN-� message was noted on

day 3 only. In contrast, a decrease in IL-4 mRNA expres-

sion was found for all time points examined. High IL-17/

IL-4 mRNA ratio of 1.7 � 1.1 (P � 0.05), 2.9 � 2.5 and

1.8 � 1.1, (P � 0.05) at days 3, 7 and 15, respectively,

compared to 0.2 � 0.1 in controls was noted. In addition,

IFN-�/IL-4 ratios of 1473.7 � 812.4, (P � 0.05), 1258.9

� 805.0, (P � 0.05) and 933.1 � 306.5, (P � 0.05) at

days 3, 7 and 15, respectively, compared to 348.8 � 221.0

in controls, were also observed.

Determination of cytokine protein content in media

conditioned by spleen cells from mice infected with

A. fumigatus conidia revealed an increase in the spontane-

ous production of IL-17 at days 3 and 7 p.i. and increased

IFN-� production at all time points (Fig. 4A). Reduced

spontaneous production of IL-4 was found throughout the

period of examination. Calculation of pro-infl ammatory

cytokine versus IL-4 content demonstrated high IL-17/

Fig. 1 Time course of fungal burden in spleen, lungs and kidneys of animals

inoculated with Aspergillus fumigatus conidia. Fungal burdens were

determined by a colony forming unit (CFU) assay in homogenates of spleen

(A), lung (B) and kidney (C) tissue of four mice per time point. Results are

expressed as average values of CFU from triplicate determinations.

Signifi cance at **P � 0.01 and ***P � 0.001) from day 1 postinfection.

© 2010 ISHAM, Medical Mycology, 48, 735–743

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IL-4 ratios of 96.0 � 90 (P � 0.05) or 17.0 � 6.0

(P � 0.05) at days 3 and 7, respectively, as compared to 6.0 � 2.0 in controls, as well as IFN-�/IL-4 at all time

points, i.e., 338.1 � 361.4 (P � 0.05), 190.8 � 229.1 (P

� 0.05) and 151.2 � 74.7 (P � 0.01) versus 10.0 � 7.0

in controls.

Restimulation of splenocytes from mice infected with A. fumigatus conidia resulted in increased production of pro-

infl ammatory cytokines, with the highest stimulation index

for IFN-� at the beginning of infection (compared to later

time points), and at later time points for IL-17. Interest-

ingly, some responsiveness to secondary stimulation with

the conidia was noted regarding IL-4 production, although

without difference among time points p.i. (Fig. 4B).

Pulmonary cell activity

No changes in relative lung mass were noted between con-

trol and infected animals (not shown). Application of

A. fumigatus conidia resulted in histologically evident pul-

monary infl ammation expressed as perivascular leukocyte

infi ltration, with vascular changes including vessel wall

edema and narrowing of vascular lumen (Fig. 5A).

Increased lung cell recovery was noted at all time points

p.i. (Fig. 5B), mainly due to lymphocytes, and possibly

neutrophils at day 7, when a tendency to increase

© 2010 ISHAM, Medical Mycology, 48, 735–743

(P � 0.07) was noted. On day 15 p.i. a decrease in numbers

of recovered monocytes/macrophages and neutrophils was

found, whereas numbers of lymphocytes still remained

elevated. Increased activation (spontaneous NBT reduction

capacity), as well as PMA-induced reduction of NBT were

Fig. 2 Basic parameters of antifungal spleen response after inoculation of mice with Aspergillus fumigatus conidia. Spleens were isolated from infected

mice or from control uninfected mice (control) at indicated time points p.i. (A) Relative spleen mass. (B) Number of cells isolated from the spleen. (C)

Spontaneous (ex vivo) proliferation of spleen cells. (D) Spleen cell proliferation after restimulation with Aspergillus fumigatus conidia in vitro. Results

were pooled from at least three experiments with 4–6 mice per group and expressed as mean values � SD. Signifi cance at *P � 0.05, **P � 0.01 and

***P � 0.001 vs control animals.

Fig. 3 Cytokine gene expression kinetics in spleen cells after inoculation

of mice with Aspergillus fumigatus conidia. Cell RNA from spleens of

infected or control mice was extracted, reverse transcribed and a relative

expression of IL-17, IFN-� and IL-4 genes was analyzed usig real-time

(RT)-PCR. The expression was normalized to β-actin to account for

differences in total RNA content of each sample. Data are presented as

mean � SD 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 control noninfected mice. Signifi cance at *P � 0.05 vs control

animals.

© 2010 ISHAM, Medical Mycology, 48, 735–743

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noted in cultures of lung cells of mice exposed to A. fumig-atus conidia at almost all time points examined (except day

3 p.i. for spontaneous activation) (Fig. 5C). A signifi cant

correlation was noted between the stimulated lung cell

activity and decrease in conidial burden (r � −0.82; P �

0.05; y � 3.57 − 15.6*x).

Examination of cytokine production by lung cells

revealed no changes in spontaneous cytokine production.

However, increased proinfl ammatory cytokine production

in response to restimulation with A. fumigatus conidia was

found in cultures of lung cells at day 15 p.i (IL-17) and at

all time points (IFN-�) (Fig. 6A & B). Neither spontaneous

nor stimulated IL-4 production by lung cells was noted

throughout the period of examination, remanining at low

levels, ranging from 10–20 pg/ml (data not shown).

Increased ratio of stimulated IFN-� to IL-4 production was

noted at day 7 (217.4 � 210.7, P � 0.01) and day 15 (271.1

� 417.6, P � 0.01), compared to the ratio calculated in

controls (32.2 � 25.6), while numerically increased IL-17/

IL-4 ratio of stimulated cytokine production was observed

only on day 15 p.i. (119.2 � 96.9 vs 79.9 � 20.5 in con-

trols; P � 0.05).

Discussion

Spleen and lung cell activity in systemic nonlethal

A. fumigatus infection were evaluated in this study. Both

Fig. 4 Production of cytokines by spleen cells after inoculation of mice

with Aspergillus fumigatus conidia. Cells from spleens of infected or control

mice (5 � 106/well) were cultured for 72 h in medium only (spontaneous

production) (A) or with heat-inactivated conidia of Aspergillus fumigatus

(restimulated production) (B). The production of IL-17, IFN-� and IL-4 was

measured in cell culture supernatants by using ELISA. Data are presented

as mean � SD of samples pooled from three independent experiments

with four to six animals per group for each time point. Results are

expressed as cytokine protein concentration (pg/ml) (A) or index of

stimulation (restimulated/spontaneous cytokine production) in infected

mice (B). Signifi cance at *P � 0.05 and **P � 0.01 vs control animals (A)

or vs stimulation index at day 3 postinfection (IFN-� B).

Fig. 5 Pulmonary infl ammation in mice after inoculation with Aspergillus fumigatus conidia. Lungs were isolated from immunized mice or from

control uninfected mice (control) at indicated time points. (A) Histological

appearance of lungs of infected mice by day 7 p.i. Multifocal perivascular

leukocyte infi ltration. Blood vessel wall edema and narrowing of vascular

lumen (insert). (B,C) Activity of leukocytes obtained by collagenase/

DNase digestion from lungs of infected mice or control mice. Total and

differential number of recovered leukocytes (B). Spontaneous and PMA-

induced respiratory burst of lung leukocytes evaluated by quantitative

NBT reduction assay (C). Signifi cance at *P � 0.05, **P � 0.01 and

***P � 0.001 vs control animals.

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spleen cell and stimulated lung cell activity correlated sig-

nifi cantly with a decrease in tissue CFUs, suggesting the

importance of these cells in the elimination of fungi.

Increased spleen cellularity may have accounted for the

increase in relative spleen mass following infection, and

may indicate splenocyte proliferation ex vivo, as judged by

the correlation between relational parameters. However,

the contribution of the infl ux of leukocytes into the spleen

to the increased spleen mass/cellularity ratio could not be

ruled out, since the spleen is a site of intense leukocyte

migration in the setting of infections [23].

Exposure to A. fumigatus conidia resulted in increased

expression of pro-infl ammatory (IL-17 and IFN-�) cytok-

ines by splenocytes. Although splenic IFN-� and IL-4 pro-

duction in systemic (lethal) A. fumigatus infection have

been already reported [13], our data are novel concerning

the IL-17 expression in systemic nonlethal A. fumigatus

infection. Increased gene expression, as well as production

of this cytokine nearly throughout the infection indicates

the relevance of this cytokine in systemic nonlethal Asper-gillus infection. Difference in the dynamics of expression,

as well as in the magnitude of the increase of IL-17 message

(2–3 times versus control) compared to the modest 30% for

IFN-� may potentially imply differential importance of

these two cytokines in systemic aspergillosis. However,

increased production of IFN-� by spleen cells in culture

indicates differential regulation of these two cytokines dur-

ing Aspergillus infection. In this regard, recent data demon-

strated importance of IL-17 production by draining lymph

node cells under conditions of relative IFN-� defi ciency in

murine pulmonary Aspergillus infections [18]. A lack of an

increase in IL-17 protein expression at day 15 may have

been related to regulation of its production, as it is subject

to tight control [19]. Splenic IL-17 production occurs mainly

in CD4+ T cells, as demonstrated in host response to pul-

monary Aspergillus infection [18]. However, CD8+ cells, as

well as NK cells, have shown a capacity to produce this

cytokine under certain conditions [24], and thus, may have

contributed to the total amount of the cytokine produced.

Recent data have demonstrated the importance of IL-17

in lethal systemic infections caused by Cryptococcus

neoformans [25] and Candida albicans [26] in mice. Our

results also showed the relevance of IL-17 in hosts that have

successfully recovered from Aspergillus infection. Various

IL-17-related effector activities in pathogen killing [27], as

well as the T-cell activating properties of IL-17 suggested in

experimental hypersensitivity in mice [28], might be rele-

vant for its antifungal activity in the spleen.

Supplementary to the gene expression data, increased

spontaneous IFN-� production demonstrated the relevance

of this cytokine in the spleen in a systemic nonlethal model

of infection with A. fumigatus. Increased IFN-� protein

content, compared to negligible mRNA expression, might

© 2010 ISHAM, Medical Mycology, 48, 735–743

have resulted in the 72-h conditioned medium from various

cell-derived products, such as IL-12 or nitric oxide with

IFN-�-stimulating potential. Responsiveness of spleen

cells to secondary stimulation with A. fumigatus conidia

observed at time points when no increase in IFN-� mRNA

supports such an assumption. Both CD4+ [13] and CD8+ T

cells, as well as NK cells, are well known sources of IFN-�

in settings of various microbial infections [29] and may be

a source of splenic IFN-� during A. fumigatus infection.

Much higher spontaneous (throughout the p.i. period

examined), as well as conidia-restimulated production

(particularly at day 3 p.i.) of IFN-� compared to IL-17 by

spleen cells implies stringent requirements for IFN-� in

nonlethal A. fumigatus infections. In accord with this

assumption are our preliminary data in which signifi cant

IFN-� production (compared to IL-17) was noted in

infected BALB/c mice (known as low IFN-� producers in

lethal parasitic and some bacterial infections; data not

shown). Such considerations are in line with data that dem-

onstrated higher ConA-stimulated IFN-� production by

spleen cells from mice that survived systemic lethal A. fumigatus infection as compared to non-survivors [13].

Protective effects of this cytokine in lethal A. fumigatus

infection in mice [14] might be of relevance for antifungal

activity of the spleen demonstrated in this study. IFN-� is

a known stimulator of antifungal activity by phagocytes

[30–32] and activated splenic macrophages were shown to

be responsible for initial defense against invasive aspergil-

losis by their phagocytosis of conidia and conidiacidal

activity [33].

A decrease in IL-4 expression noted in our study is in

accord with data from a study of systemic lethal murine

aspergillosis which showed lower IL-4 expression by

spleens of survivors as compared to increased expression

of this cytokine by spleens of non-survivors [13]. Decreased

IL-4 expression might be benefi cial in nonlethal A. fumig-atus infection as IL-4 has been demonstrated to enhance

susceptibility to invasive pulmonary aspergillosis [34].

Although decreased gene transcription, as well as protein

expression of IL-4 was noted in the spontaneous response,

an increased production was observed in the restimulation

response. This could be explained by the demonstrated

stimulation of IL-4 response with heat-inactivated A. fumigatus conidia [35], which were used in restimulation

experiments in our study.

Progressive elimination of A. fumigatus conidia from

lungs of infected mice was associated with pulmonary

infl ammation. Similar to the pulmonary response to conidia

that have gained access to lungs by the respiratory route

[8,36], we noted increased activation of lung cells by conidia

deposited in the lungs following systemic infection.

In contrast to response in the spleen, IL-17 in the lungs

is produced in recall response only during the later course

742 Mirkov et al.

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of the infection. According to recent data, mucosal sites

are rich in naturally occurring Th17 cells which are thought

to be responsible for resistance to fungi [17]. This implies

a protective role of lung IL-17 in A. fumigatus infection.

However, in analogy to the recent data that indicated

involvement of IL-17 in tissue injury in pulmonary asper-

gillosis [18], lung IL-17 might be deleterious as well.

The capacity of lung cells to respond to conidia by

IFN-� production is in accord with data demonstrating

increased IFN-� response in the lungs to intranasal appli-

cation of A. fumigatus in immunocompetent mice [8,36].

The increased capacity of lung cells to produce IFN-� in

response to secondary stimulation throughout infection

compared to the modest and late production of IL-17,

might imply greater relevance of IFN-� in lung response

to systemic A. fumigatus infection. Interstitial lymphocytes

infi ltrating lungs following systemic infection, might be

responsible for IFN-� production, as shown in murine

invasive pulmonary aspergillosis [8]. Unchanged IL-4

production by mice lung cells with nonlethal A. fumigatus

infections is in line with the data from immunocompetent

mice with pulmonary Aspergillus infection. In the latter,

IL-4 production by interstitial lung cells remained

unchanged, in contrast to immunocompromised mice in

which increased production of this cytokine by lung cells

was noted [8].

In conclusion, nonlethal systemic aspergillosis in immu-

nocompetent mice tips the balance towards Th1 antifungal

spleen response by down-regulating Th2 response and by

triggering the Th17 arm of the immune response. This results

in a high infl ammatory cytokine (IL-17 and IFN-�) to IL-4

ratio in the spleen microenvironment during infection.

A proinfl ammatory cytokine response was noted in the lungs

as well, but was due to the increased capacity of IFN-� pro-

duction by lung cells in response to challenge with conidia

and not to a change in the IL-4 response. The observed

patterns of cytokine expression created an infl ammatory

milieu that might contribute to effi cient conidial removal.

The presented data add new information regarding mecha-

nisms of resistance to invasive A. fumigatus infections.

Acknowledgements

This study was supported by the Ministry of Science and

Technological Development of the Republic of Serbia,

Grants # 143038 and 143029. The authors would like to

thank Sandra Belij and Aleksandra Popov for expert techni-

cal assistance in some of the aspects of this investigation.

Declaration of interest: No confl ict of interest.

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This paper was fi rst published online on Early Online on 25 January 2010.