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Apr 26, 2007 - incubated at 378C for 72 h and the colonies (cfu) counted. .... P, Durand C, Caillot D, Thiel E, Chandrasekar PH, Hodges. MR, Schlamm HT, Troke PF ... Luther K, Rohde M, Heesemann J, Ebel F. Quantification of phagocytosis ...
Mycopathologia (2007) 163:303–307 DOI 10.1007/s11046-007-9013-z

ORIGINAL PAPER

In vitro determination of phagocytosis and intracellular killing of Aspergillus species by mononuclear phagocytes Susanne Perkhofer Æ Cornelia Speth Æ Manfred P. Dierich Æ Cornelia Lass-Flo¨rl

Received: 18 January 2007 / Accepted: 21 March 2007/Published online: 26 April 2007  Springer Science+Business Media B.V. 2007

Abstract We investigated phagocytosis and intracellular killing of clinical and environmental isolates of Aspergillus spp. by human monocyte-derived macrophages (MDMs). Serial pathogens such as Aspergillus fumigatus, Aspergillus flavus and Aspergillus terreus were examined with a microbiological assay. Phagocytosis for resting conidia of Aspergillus spp. was similar for all isolates tested. During 30 min of incubation phagocytosis ranged from 49.9% to 85.5% for clinical isolates and from 40.3% to 87.1% for environmental isolates. MDMs killed A. fumigatus, A. flavus and A. terreus conidia after ingestion for 120 min, as shown by a decrease in colony forming units (cfu) count of intracellular fungi. The killing index for all isolates of Aspergillus spp., ranged from 12.1 ± 1.1% to 90.3 ± 10.4%; isolate-dependent (P < 0.01) differences against the fungicidal action of MDMs were observed. In conclusion, significant differences were noted for killing indices between several strains of Aspergillus spp. whereas phagocytosis was similar for all isolates tested in vitro. No differences were observed within environmental and clinical isolates.

S. Perkhofer (&)  C. Speth  M. P. Dierich  C. Lass-Flo¨rl Department of Hygiene, Microbiology and Social Medicine, Medical University Innsbruck, Fritz Pregl Strasse 3, 6020 Innsbruck, Austria e-mail: [email protected]

Keywords Aspergillus spp.  Intracellular killing  Mononuclear macrophages  Phagocytosis Abbreviations MDMs Monocyte derived macrophages

Introduction Fungal pathogens are recognized as a major and increasing source of infection in immunocompromised hosts [1–3] with Aspergillus species, Fusarium species, zygomycetes, and other usually non-pathogenic fungi being the main pathogens [4, 5]. Infections with Aspergillus spp. are common causes of nosocomial pneumonia and are associated with an extremely high mortality rate of 40% [6]. Aspergillus fumigatus is most frequently isolated from clinical specimen, but other important species include Aspergillus flavus and Aspergillus terreus [5–8]. Uptake of conidia by the respiratory tract is the initial event in Aspergillus infections with alveolar macrophages as the first line of host defence [9, 10]. The high incidence of aspergillosis in leukopenic and agranulocytic patients clearly indicates that phagocytic cells play an important role [9–11]. Several methods are used to determine the microbiocidal activity of phagocytes against conidia and hyphae of Aspergillus spp. [10]. Most in vitro studies of interactions between macrophages and Aspergillus

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spp. were conducted with A. fumigatus [12–15]. Little is known, however, on the behaviour of other species and various isolates of Aspergillus spp. [16]. We therefore investigated phagocytosis and intracellular killing of a wide range of Aspergillus spp. by human blood macrophages and evaluated whether differences exist between clinical and environmental isolates.

Material and methods Strains The in vitro tests were performed on clinical and environmental isolates of Aspergillus spp. comprising eight isolates each of A. fumigatus, A. flavus and A. terreus. Isolates were maintained as conidial suspensions in water at room temperature, and subcultures were grown on sabouraud glucose agar (Merck, Darmstadt, Germany) and incubated at 358C for 5 days. Conidial suspensions were prepared as described by Roilides et al. [11]. In brief, the plates were washed with a physiological sterile saline solution (0.9% [wt/vol] NaCl), and the conidial suspension was filtered twice through a sterile 40 mm pore-size nylon mesh (Falcon, Heidelberg, Germany). Conidia (1 · 106 colony forming units (cfu)/ml) were prepared with phosphate buffered saline (PAA, Linz, Austria) and stored for 4 days at 48C. Monocyte-derived macrophages Human monocytes were isolated from buffy coats as described previously [17] and dispensed into Teflon tubes (Nalge Co.) at concentrations of 1 · 106 cells/ ml. Monocyte-derived macrophages (MDMs) were obtained by culturing for 5–7 days at 378C in 5% CO2. Viability tests using trypan blue exclusion (Merck, Vienna, Austria) were performed routinely. Phagocytosis Ingestion of conidia was assessed using a modification of the procedure by Van Leijh et al. [18]. A total of 100 ml of MDMs (1 · 106 cells/ml), 100 ml of conidia of Aspergillus spp. (1 · 106 cfu/ml) and 5% human serum (Sigma-Aldrich, Vienna, Austria) were

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combined and incubated at 378C under continuous rotation (4 rev./min). At several time points (0, 30 min, 60 min and 120 min), 50 ml aliquot of the suspension was removed and added to 150 ml ice-cold gelatin-HBSS (PAA Laboratories, Linz, Austria) to stop phagocytosis. This sample was centrifuged for 4 min at 110 · g. The non-ingested conidia remained in the supernatant fluid; dilutions were made and 100 ml was pipetted on sabauroud glucose agar, incubated at 378C for 72 h and the colonies (cfu) counted. The number of viable conidia/ml was calculated from the means of the colony counts of duplicate plates. Tests were performed twice in triplets. Intracellular killing of conidia Phagocytosis of conidia was performed as described above. On the basis of conidia:MDMs cell ratio of one, 150 ml of a cell suspension was added to an equal volume of pre-opsonized conidia (1 · 106 cfu/ml). Phagocytosis was stopped after 30 min by placing the tube in crushed ice and shaking it for 1 min. Extracellular conidia were removed by differential centrifugation (4 min at 100 · g) and two washes with gelatin-HBSS. The phagocytes were resuspended in HBSS with 5% serum and incubated at 378C. At various time points (0, 120 min) 50 ml samples of the suspensions were removed and added to 50 ml ice-cold HBSS to stop the intracellular killing. Lysis of macrophages was performed with 2.5% deoxycholate (Sigma, Vienna, Austria). The number of viable conidia was determined as described above; assays were performed twice in triplets. Calculations Phagocytosis at a given time point is expressed as the percentage decrease in the initial number of viable extracellular conidia according to the following formula: P(t) = (1 Nt/N0) · 100, whereby P(t) is the phagocytic index at times t = t; N0 and Nt are the number of viable extracellular conidia at time t = 0 and t = t, respectively. The killing index K(t) is calculated using the formula K(t) = (1 N t / N0) · 100, where N0 and Nt are the numbers of viable intracellular conidia at times t = 0 and t = t, respectively.

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Statistics

pathogens [9–11]. Phagocytic functions, such as migration, ingestion and release of reactive oxygen intermediates, which appear to be responsible for the killing activity, are important in host defence against Aspergillus spp. [12, 19]. The mechanisms of elimination of conidia of Aspergillus spp., however, are not fully understood [11, 19, 20]. The second line of defence are circulating polymorphic neutrophils mediating damage of escaping hyphae [21]. Neutropenia has long been recognized as a significant risk factor for invasive aspergillosis [21]. The results of the phagocytosis assay for clinical isolates of Aspergillus spp. revealed that conidia were ingested during the first 30 min, with minor differences over the remaining 90 min. The range of phagocytosis was similar for clinical isolates (49.9%– 85.5%) and environmental isolates (40.3–87.1%) as shown in Fig. 1a–f. No significant differences within and among the strains tested. Phagocytosis of the various isolates was fast, and uptake was comparable to human polymorphonuclear cells [22]. Our data are also in agreement with previous reports [11, 14] showing phagocytosis of 40–82% of A. fumigatus by

Each experiment was performed with cells of one donor and by use of triplicate testing for each condition. The average values were taken as the value for this particular donor/experiment. Experiments were repeated twice with different donors. Statistical significance was determined by using the Student’s t-test. P-values less than 0.05 were considered significant.

Results and discussion We studied phagocytosis and intracellular killing of various isolates of Aspergillus spp. by MDMs. No differences between clinical and environmental isolates were observed, yet isolate-dependent differences of susceptibility (P < 0.01) against the fungicidal action of MDMs were noted. Pulmonary aspergillosis results from inhalation, and deposition of conidia with alveolar macrophages forming the first line of defence against fungal

d)

A. fumigatus

100 80 60 40 20 0

% P ha g o cy t o sis

% P ha g o cy t o sis

a)

30

60

A. fumigatus

100 80 60 40 20 0 30

120

min CAf1

CAf2

CAf3

EAf1

CAf4

e)

A. terreus

100 80 60 40 20 0 30

60

c)

CAt2

120

EAf4

A. terreus

CAt3

CAt4

EAt1

60

120

f) % P h a g o c y t o si s

60

120

CAfl3

CAfl4

EAt2

EAt3

EAt4

A. flavus

100 80 60 40 20 0 30

min CAfl2

EAf3

min

A. flavus

CAfl1

EAf2

30

100 80 60 40 20 0 30

120

100 80 60 40 20 0

min CAt1

60 min

% Pha g o cy t o sis

% P ha g o cy t o sis

b)

% Pha g o cy t o sis

Fig. 1 Phagocytosis of clinical isolates of Aspergillus fumigatus (a), A. terreus (b) and A. flavus (c) and environmental isolates of Aspergillus fumigatus (d), A. terreus (e) and A. flavus (f) by monocyte derived macrophages (MDMs) in the presence of 5% human serum at a fungus:cell ratio of 1:1. The results are presented as mean ± SD. C = clinical isolates. E = environmental isolates

EAfl1

60 min

EAfl2

120

EAfl3

EAfl4

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Table 1 Intracellular killing (%) of clinical and environmental isolates of Aspergillus spp. by monocyte derived macrophages (MDMs)a Aspergillus strains

Clinical isolates

Killing index (%) at 120 min

Environmental isolates

Killing index (%) at 120 min

A. fumigatus

CA.f 1

50.7 ± 4.2

EA.f 1

90.3 ± 10.4

CA.f 2

77.8 ± 5.4

EA.f 2

51.8 ± 4.7

CA.f 3

13.7 ± 1.2b

EA.f 3

63.7 ± 5.4

CA.f 4

35.9 ± 3.3

EA.f 4

46.9 ± 5.6

CA.fl 1

42.2 ± 2.1

EA.fl 1

17.2 ± 2.1b

EA.fl 2

54.1 ± 3.8

EA.fl 3

37.8 ± 3.8b

A. flavus

A. terreus

a

CA.fl 2

27.5 ± 1.3

CA.fl 3

38.8 ± 2.5

b

b

CA.fl 4

14.2 ± 1

EA.fl 4

65.4 ± 6.2

CA.t 1

37.7 ± 2.3

EA.t 1

26.7 ± 2.5b

CA.t 2

63.2 ± 4.6

EA.t 2

57.2 ± 5.1

CA.t 3 CA.t 4

23.1 ± 3.2b 12.1 ± 1.1b

EA.t 3 EA.t 4

31.1 ± 2.8b 65.9 ± 5.8

Intracellular killing was measured at a conidia:MDMs ratio of 1:1 at 378C. Values represent the means of at least two experiments

b

Statistical significance in comparison to killing index of highest value is indicated as P < 0.01. C = clinical isolates. E = environmental isolates

alveolar macrophages. By contrast, phagocytosis did not significantly increase during a prolonged incubation period, as found by others [19, 23]. So far, species dependent differences in phagocytosis were found by others showing A. fumigatus conidia more phagocytosed in comparison to non-fumigatus Aspergillus strains [24, 25]. It has been shown that the antifungal activities of macrophages depend on the anatomical location of the macrophages [12]. Alveolar macrophages killed 90% of A. fumigatus in 30 h, whereas peritoneal macrophages were even able to inhibit germination of ingested conidia. MDMs used in our study killed A. fumigatus, A. flavus and A. terreus conidia after ingestion during 120 min, as shown by a decrease in cfu count of intracellular fungi. The killing index for clinical isolates ranged from 12.1 ± 1.1% to 77.8 ± 5.4% and was comparable for environmental isolates with 17.2 ± 2.1–90.3 ± 10.4% as shown in Table 1. However, significant isolatedependent differences (P < 0.01) were observed among the fungi tested in vitro and was given for A. fumigatus, A. flavus and A. terreus (Table 1), respectively. The reason for the differences of susceptibility against the fungicidal action of MDMs is not known yet. Various fungus related factors e.g., surface properties may play an important role in the attempt

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to survive in phagocytes [14]. So far, MDMs used in our tests were obtained from healthy volunteers, meaning that the qualitative and quantitative efficiency of phagocytes in patients at risk remains an open question. It has been reported, however, that resting conidia are resistant to oxygen-dependent metabolites and non-oxidative antimicrobial products of phagocytes [26, 27]. These data only partly agree with our in vitro observations since the killing index was >50% in some isolates. Several factors, such as the assay [18] or the reisolation method of conidia [15] could account for the diversity of these results. In conclusion, significant differences were noted for killing indices between several-strains whereas phagocytosis was similar for all isolates tested in vitro. Several studies are necessary to evaluate whether intracellular survival of conidia in MDMs is of clinical relevance.

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