The interaction of human monocytes, monocyte-derived macrophages, and polymorphonuclear neutrophils with caspofungin (MK-0991), an echinocandin, for antifungal activity against Aspergillus fumigatus

The interaction of human monocytes, monocyte-derived macrophages, and polymorphonuclear neutrophils with caspofungin (MK-0991), an echinocandin, for antifungal activity against Aspergillus fumigatus

Diagnostic Microbiology and Infectious Disease 39 (2001) 99 –103 www.elsevier.com/locate/diagmicrobio Mycology The interaction of human monocytes, ...

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Diagnostic Microbiology and Infectious Disease 39 (2001) 99 –103

www.elsevier.com/locate/diagmicrobio

Mycology

The interaction of human monocytes, monocyte-derived macrophages, and polymorphonuclear neutrophils with caspofungin (MK-0991), an echinocandin, for antifungal activity against Aspergillus fumigatus Tom Chiller,a,b,c,* Kouros Farrokhshad,a,b Elmer Brummer,a,b,c David A. Stevensa,b,c a

Division of Infectious Diseases, Department of Medicine, Santa Clara Valley Medical Center, San Jose, CA b California Institute for Medical Research, San Jose, CA c Stanford University School of Medicine, Stanford, CA Received 11 September 2000; accepted 30 November 2000

Abstract The collaboration between human effector cells and caspofungin (MK-0991), a 1,3-␤-D glucan synthase inhibitor, was studied for antifungal activity against Aspergillus fumigatus. Caspofungin was co-cultured for 24h with either human monocytes (Monos), monocytederived macrophages (MDM), or polymorphonuclear neutrophils (PMN) against germlings of A. fumigatus and antifungal activity assessed using the XTT metabolic assay. Caspofungin at 0.1 ␮g/ml and 0.05 ␮g/ml or Monos alone against germlings caused significant inhibition. Microscopically this was correlated with less growth and stunted malformed hyphae. The addition of caspofungin at 0.1 ␮g/ml and 0.05 ␮g/ml to the monocyte cultures increased antifungal activity. The inhibition of the combination was significantly greater than drug alone (P ⬍ .01) and Monos alone (P ⬍ .01). MDM against Aspergillus germlings inhibited hyphal growth. The combination of caspofungin at 0.1 ␮g/ml and 0.05 ␮g/ml to the macrophage cultures increased antifungal activity. The growth inhibition by the combination was significantly greater than drug alone (P ⬍ .01) and MDM alone (P ⬍ .01). There was no significant interference with or enhancement of PMNs and caspofungin. These data support the activity of caspofungin against A. fumigatus in vitro, and indicates a cooperative activity with human effector cells. This suggests caspofungin in vivo would have increased efficacy as it combines with host defenses against A. fumigatus. © 2001 Elsevier Science Inc. All rights reserved. Keywords: Aspergillus, caspofungin, antifungals, macrophages, neutrophils, monocytes

1. Introduction A new generation of antifungal agents, known as echinocandins, have consistently demonstrated activity against a variety of fungi (Bartizal et al., 1997; Stevens et al., 1996). The antifungal activity of these drugs is achieved by inhibiting the enzyme 1,3-␤-D glucan synthase. This action inhibits the synthesis of 1,3-␤-D glucan, which is an important component of the fungal cell wall. Caspofungin (MK0991) (Merck Research Labs, Rahway, NJ) is a semisynthetic derivative of the natural product pneumocandin B0

* Corresponding author. Tel.: 1-(408)-885-4307; fax: 1-(408)-8854306. E-mail address: [email protected] (T. Chiller).

and is water-soluble. It has been shown in the case of Aspergillus that these drugs do not give classic MICs in vitro using broth dilution techniques (Kurtz et al., 1994). They do, however, demonstrate clear morphological inhibition in vitro. In addition, several recent reports have shown that caspofungin has good activity against Aspergillus infections in animal models (Abruzzo et al., 1997; Kurtz et al., 1995). Furthermore, using an XTT assay, inhibition of Aspergillus hyphae could be quantified with the echinocandins as well as other antifungals (Brummer et al., 1999). Antifungal agents have been shown to interact with human effector cells against a variety of fungi, including Aspergillus (Brummer et al., 1999; Vora et al., 1998). In this study, we examined the interaction between human monocytes (Monos), monocyte-derived macrophages (MDM), or polymorphonuclear neutrophils (PMN) with caspofungin to inhibit A. fumigatus in vitro in an XTT assay.

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2. Materials and methods 2.1. Aspergillus fumigatus An isolate of A. fumigatus, referred to as AF-10, which was a patient isolate stored at the California Institute for Medical Research, San Jose, CA, was used for all experiments. This was incubated on Sabouraud’s dextrose agar slants at 35°C for 24h and then allowed to form conidia at room temperature over 48 –72h. The conidia were extracted in distilled water and filtered through sterile gauze. The conidia were then washed, diluted in saline, and counted. Conidia suspensions consisted primarily of single conidia (95%) or small groups of conidia with two or three per group (5%). For the Mono and MDM assays, two ml suspensions with 106 conidia/ml in RPMI 1640 medium (Gibco, Grand Island, NY, USA) were placed into 10 ml plastic culture tubes. Over 95% of the conidia germinated when incubated at 37°C for two hours and then at room temperature (26°C) for 16 –20 hrs in order to form single filaments without septa, referred to as germlings. Germlings were harvested and counted. 0.1 ml of a suspension of 104 germlings/ml was added to Monos or MDMs in 96-well microtiter plates for coculture. In the PMN assay, 0.1 ml of a suspension of 105 conidia/ml were placed into wells of 96-well microtiter plates and germinated as described above. PMNs were added to the germinated conidia for coculture. 2.2. Caspofungin Caspofungin powder was supplied by Merck Research Laboratories. The powder was stored at ⫺70°C. The desired amount was diluted in sterile distilled water to a concentration of 1 mg/ml. Portions of this solution were stored at ⫺70°C and used only once per experiment. Final concentrations were made on the day of the experiment in RPMI 1640 for incubation with A. fumigatus. 2.3. Monocyte assay Peripheral blood mononuclear cells (PBMC) were isolated from heparinized human blood by 6% dextran-70 sedimentation followed by density gradient centrifugation of the buffy coat diluted 1:1 with RPMI 1640 on Histopaque 1077 (Sigma). The PBMC layers were harvested, washed, counted, and suspended in RPMI-1640 plus 10% autologous human serum (referred to as complete tissue culture medium, CTCM). 0.1 ml of a suspension of 106 cells/ml was added to wells of 96-well microtiter plates and incubated for 1 hour at 37°C in 5% CO2 ⫹ 95% air. The supernatants from these wells were discarded and nonadherent cells were washed away with two washings of RPMI-1640. There were approximately 10 –20% adherent cells remaining in the wells. Aspergillus germlings were then added to each well as described above and caspofungin

was added to specific wells at desired concentrations. Cultures were incubated at 37°C in 5% CO2 for 24 hrs and the amount of Aspergillus hyphal growth was determined using the XTT assay. 2.4. Monocyte-derived macrophage assay PBMC were isolated from human donors as described above. 0.1 ml of PBMC at 5 ⫻ 106 cells/ml were put into individual wells of 96-well microtiter plates and incubated in RPMI-1640 with 5% human serum for a total of five days. The media was changed after 48 hrs. The supernatants were then removed and the non-adherent cells were washed away twice with RPMI-1640. The remaining adherent cells (approximately 105/well) were used as MDM in coculture with A. fumigatus germlings as described above for the monocyte assay. Caspofungin was added to specific wells and after 24 hrs of coculture antifungal activity determined with the XTT assay. 2.5. PMN assay PMN were isolated from heparinized human blood by 6% dextran-70 sedimentation followed by density gradient centrifugation of the buffy coat diluted 1:1 with RPMI 1640 on Histopaque 1077 (Sigma). The pelleted cells (PMN and some red blood cells) were collected in 0.85% NH4Cl to lyse RBC. PMN were washed, counted, and suspended in CTCM. PMN were added to microtest plate wells containing washed A. fumigatus germlings and incubated with and without caspofungin at 37°C for 24 hrs. The XTT assay was performed to measure antifungal activity. 2.6. XTT assay Cultures in microtest plate wells were centrifuged (2000 rpm, 10 min) and supernatants were carefully aspirated. Wells were washed twice with 0.2 ml of distilled water. Human cells were lysed or killed as shown by debris and deformed cells, observed by microscopy. Inhibition of hyphal growth was measured by the colorimetric XTT-coenzyme Q method (Meshulam et al., 1995) (2,3)-Bis-(2-methoxy-4-nitro-5-sulphenyl(2H)-tetrazolium-5-carboxanilide) sodium salt (XTT) at 5 mg/ml plus 2,3-dimethoxy-5-methyl-1,4-benzoquinone (coenzyme Q) at 0.04 mg/ml in phosphate buffered saline (PBS) pH 7.4 (Sigma Chemical Co., St Louis, MO, USA) constituted the test solution. XTT test solution (0.2 ml) was added to each well and cultures were incubated at 37°C for 1 h. An aliquot of 0.1 ml was transferred to corresponding wells of another plate and the absorbance at 450 nm was recorded with a microtest plate reader (Dynatech, Richmond, VA). Each sample was studied in quadruplicate.

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Table 1 Activity of monocytes (Monos) in RPMI-1640 with or without caspofungin against A. fumigatus.

Table 2 Activity of monocytes (Monos) in CTCM with or without caspofungin against A. fumigatus

Treatment (24 hrs)a

Treatment (24 hrs)a

RPMI Monos in RPMI MK (0.05 ␮g/ml) in RPMI MK (0.05 ␮g/ml) ⫹ Monos MK (0.1 ␮g/ml) in RPMI MK (0.1 ␮g/ml) ⫹ Monos a

% inhibition mean ⫾ sd, n⫽4

P vs.

0

Ref b ⬍.01 ⬍.01 ⬍.01 ⬍.01 ⬍.01

31 ⫾ 5.7 11.7 ⫾ 3.6 45.5 ⫾ 5.3 24.9 ⫾ 10.2 53.9 ⫾ 10.7

RPMI

Drug

Monos

Ref ⬍.01 Ref ⬍.01

Ref ⬍.01 ⬍.01 NS ⬍.01

3

The treatment group represents 10 conidia of A. fumigatus per well cocultured with either Monos or MK-0991 or both for 24 hrs. b The term “Ref” refers to the value that is being used as the comparator.

2.7. Quantitation of growth inhibition The absorbance at 450 nm of each well using a 96 well microtest plate reader (Dynatech, Richmond, VA) was used to determine the change in absorbance (⌬A) as compared to control wells with XTT alone. The percentage of inhibition was calculated by the formula (⌬Acontrol⫺⌬Aexperiment/ ⌬Acontrol) ⫻ 100. It has been previously shown (Brummer et al., 1999; Vora et al., 1998) that there is a linear relationship between inoculum and metabolism of XTT as measured by change in absorbance, and that drug induced growth inhibition verified microscopically correlates with inhibition of XTT metabolism. Therefore, decreased change in absorbance (⌬A) of XTT supernatants from caspofungin treated cultures represents inhibition of growth. 2.8. Statistical analysis Student’s t-test was used for statistical analysis of the data and significance set at P ⬍ 0.05. The GB-STAT program (Microsoft, Richmond, VA) was used for Bonferroni’s adjustment to the t-test where appropriate. All values for percentage of inhibition are presented as means from n experiments with their standard deviations.

3. Results

CTCMb Monos in CTCM MK (0.05 ␮g/ml) in CTCM MK (0.05 ␮g/ml) ⫹ Monos in CTCM MK (0.1 ␮g/ml) in CTCM MK (0.1 ␮g/ml) ⫹ Monos in CTCM

P vs. CTCM

Drug

Monos

5.1 ⫾ 4.1 76.6 ⫾ 6.7 58.3 ⫾ 19.2 93.7 ⫾ 6.5

Ref c ⬍.01 ⬍.01 ⬍.01

Ref ⬍.01

Ref NS ⬍.01

78.6 ⫾ 5.8 97.9 ⫾ 2.8

⬍.01 ⬍.01

Ref ⬍.01

NS ⬍.01

a

The treatment group represents 103 conidia of A. fumigatus per well cocultured with either Monos or MK-0991 or both for 24 hrs. b CTCM refers to RPMI-1640 media plus 10% human serum. c The term “Ref” refers to the value that is being used as the comparator.

the cocultures are performed in CTCM instead of RPMI1640. The combination of caspofungin and monocytes in CTCM significantly (P ⬍ .01) enhanced the inhibition of Aspergillus hyphal growth compared with either caspofungin or monocytes alone. The use of CTCM in cocultures served to enhance the inhibitory effect of all groups compared with RPMI-1640 (Table 1, 2). These results were also obtained with human monocytes from three different donors (data not shown). 3.2. Antifungal activity of monocyte-derived macrophages (MDMs) and caspofungin Compared to monocytes, MDMs had similar antifungal activity against A. fumigatus. Table 3 shows the ability of MDMs to inhibit hyphal growth after 24 h coculture. The ability of MDMs and caspofungin to collaborate in vitro is also shown in Table 3. MDMs plus caspofungin produced significantly (P ⬍ .01) more inhibition of Aspergillus Table 3 Activity of monocyte-derived macrophages with or without caspofungin against A. fumigatus. Treatment (24 hrs)a

3.1. Antifungal activity of monocytes and caspofungin As shown in Table 1, monocytes alone in RPMI-1640 significantly inhibited Aspergillus hyphal growth. Inhibition of Aspergillus by monocytes increased in CTCM (Table 2). Results in Table 1 also demonstrate collaboration between caspofungin and monocytes in inhibiting Aspergillus. The ability of monocytes in RPMI-1640 plus caspofungin to inhibit A. fumigatus hyphae was significantly higher than either caspofungin or monocytes alone. This collaboration of monocytes and caspofungin was additive. The results shown in Table 2 demonstrate a similar additive effect when

% inhibition mean ⫾ sd, n⫽4

CTCMb MDM in CTCM MK (0.05 ␮g/ml) in CTCM MK (0.05 ␮g/ml) ⫹ MDM in CTCM MK (0.1 ␮g/ml) in CTCM MK (0.1 ␮g/ml) ⫹ MDM in CTCM a

% inhibition mean ⫾ sd, n⫽4

P vs. CTCM

Drug

MDM

0 47.5 ⫾ 5.3 58.3 ⫾ 19.2 77.6 ⫾ 12.4

Ref c ⬍.01 ⬍.01 ⬍.01

Ref ⬍.05

Ref NS ⬍.01

78.6 ⫾ 5.8 90.4 ⫾ 3.7

⬍.01 ⬍.01

Ref ⬍.01

⬍.01 ⬍.01

The treatment group represents 103 conidia of A. fumigatus per well cocultured with either MDMs or MK-0991 or both for 24 hrs. b CTCM refers to RPMI-1640 media plus 10% human serum. c The term “Ref” refers to the value that is being used as the comparator.

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Table 4 Activity of PMNs with and without caspofungin against A. fumigatus. Treatment (24 hrs)a

CTCMb PMN ⫹ CTCM MK (0.05 ␮g/ml) ⫹ CTCM MK (0.05 ␮g/ml) ⫹ PMN ⫹ CTCM MK (0.1 ␮g/ml) ⫹ CTCM MK (0.1 ␮g/ml) ⫹ PMN ⫹ CTCM

% inhibition mean ⫾ sd, n⫽6

P vs. CTCM

Drug

PMN

0 45.1 ⫾ 13 35.8 ⫾ 10.8 55.9 ⫾ 15.1

Ref c ⬍.01 ⬍.01 ⬍.01

Ref NS

Ref NS NS

57.0 ⫾ 11.2 47.5 ⫾ 21.7

⬍.01 ⬍.01

Ref NS

NS NS

a The treatment group represents 103 conidia per well of A. fumigatus cocultured with either PMNs or MK-0991 or both for 24 hrs. b CTCM refers to RPMI-1640 media plus 10% human serum. c The term “Ref” refers to the value that is being used as the comparator.

hyphal growth than either caspofungin or MDMs alone. This was repeated with human MDMs from three donors with similar results. 3.3. Activity of PMNs with and without caspofungin against A. fumigatus As is shown in Table 4, PMNs alone with effector:target (E:T) ratios of 20:1 were capable of significant inhibition of A. fumigatus growth. Similar results were obtained with E:T ratios as low as 1:1 and increased inhibition was observed with increasing E:T ratios. No significant collaboration between PMNs and caspofungin could be measured. For example, the activity of drug alone and the activity of PMN alone were similar to the activity of both together, e.g. there was not a significant additive effect. There was, however, a trend toward collaboration.

4. Discussion Several investigators have demonstrated the inhibition of hyphal growth of Aspergillus fumigatus by monocytes, monocyte-derived macrophages, and PMNs (Brummer et al., 1999; Roilides et al., 1994; Vora et al., 1998). In our present study, we demonstrate that monocytes and monocyte-derived macrophages cooperate with the echinocandin caspofungin for inhibition of hyphal growth of Aspergillus fumigatus. As the echinocandins do not produce classic MICs against Aspergillus in vitro (Kurtz et al., 1994), it is useful to know that these compounds can collaborate with host effector cells to combine their ability to inhibit Aspergillus. This would be favorable for possible clinical efficacy and support the fact that caspofungin has been found to have substantial activity in vivo (Abruzzo et al., 1997; Kurtz et al., 1995). Collaboration between the echinocandin LY 303366 and

monocytes or PMNs has been previously reported (Brummer et al., 1999). In this study, we have demonstrated that MDM as well as monocytes can collaborate with caspofungin to inhibit the hyphal growth of Aspergillus. There are several possible mechanisms involved in this collaboration. The caspofungin could damage the fungal hyphae and render them better targets for human effector cells. The effector cells could similarly damage the fungal hyphae, allowing for better activity of caspofungin, or they could provide enhanced drug delivery and concentration. In data not shown, we could not demonstrate any significant difference in the effect of hyphae preincubated for 6 hrs with MK-0991 to which effector cells were then added compared with the results described in this report where both were added at the same time. This might suggest that the latter hypothesis is true. PMNs alone inhibited growth of Aspergillus fumigatus hyphae, but they did not significantly collaborate with caspofungin. PMN activity, when combined with caspofungin, was different from activity of either Monos or MDMs. On the other hand, previous findings with the echinocandin LY 303366 did show additive collaboration with PMN against A. fumigatus (Brummer et al., 1999). In contrast to the current paper, experiments done with LY303366 involved adding the drug to the conidia prior to the formation of germlings. Therefore the drug was present in the cultures for 24 hours prior to the addition of PMNs. This difference may have influenced the result obtained in those experiments. These two drugs also differ in chemical structure; therefore they may not share the same collaborative properties with each type of human effector cell. In summary, the fact that caspofungin collaborates with monocytes and MDMs suggest that this drug would work better in vivo as it reaches areas of infection where host effector cells are present. Studies done examining in vitro versus in vivo efficacy suggest that this is indeed the case (Bartizal et al., 1997; Abruzzo et al., 1997). The fact that PMNs are not essential for human effector cell collaboration with caspofungin might suggest that this drug would still have good efficacy, even in neutropenic patients, as long as either monocytes of MDMs were not depleted. This effect should be further studied in vivo.

References Abruzzo, G. K., Flattery, A. M., Gill, C. J., Kong, L., Smith, J. G., Pikounis, V. B., Balkovec, J. M., Bouffard, A. F., Dropinski, J. F., Rosen, H., Kropp, H., & Bartizal, K. (1997). Evaluation of the echinocandin antifungal caspofungin (L-743,872): Efficacies in mouse models of disseminated aspergillosis, candidiasis, and cryptococcosis. Antimicrob Agents Chemother 41, 2333–2338. Bartizal, K., Gill, C. J., Abruzzo, G. K., Flattery, A. M., Kong, L., Scott, P. M., Smith, J. G., Leighton, C. E., Bouffard, A., Dropinski, J. F., & Balkovec, J. (1997). In vitro preclinical evaluation studies with the

T. Chiller et al. / Diagnostic Microbiology and Infectious Disease 39 (2001) 99 –103 echinocandin antifungal caspofungin (L 743,872). Antimicrob Agents Chemother 41, 2326 –2332. Brummer, E., Chauhan, S. D., & Stevens, D. A. (1999). Collaboration of human phagocytes with LY 303366 for antifungal activity against Aspergillus fumigatus. J Antimicrob Chemother 43, 491– 496. Kurtz, M. B., Bernard, E. M., Edwards, F. F., Marrinan, J. A., Dropinski, J., Douglas, C. M., & Armstrong, D. (1995). Aerosol and parenteral pneumocandins are effective in a rat model of pulmonary aspergillosis. Antimicrob Agents Chemother 39, 1784 –1789. Kurtz, M. B., Heath, I. B., Marrinan, J., Dreikorn, S., Onishi, J., & Douglas, C. (1994). Morphological effects of lipopeptides against Aspergillus fumigatus correlate with activities against (1,3)-beta-D-glucan synthase. Antimicrob Agents Chemother 38, 1480 –1489. Meshulam, T., Levitz, S. M., Christin, L., & Diamond, R. D. (1995). A simplified new assay for the assessment of fungal cell damage with the tetrazolium dye (2,3)-Bis-(2-methoxy-4-nitro-5-sulphenyl-(2H)-tetra-

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zolium-5-carboxanilide) sodium salt (XTT). J Infect Dis 172, 1153– 1156. Roilides, E., Holmes, C., Blake, C., Venzon, D., Pizzo, P. A., & Walsh, T. J. (1994). Antifungal activity of elutriated human monocytes against Aspergillus fumigatus hyphae; enhancement by granulocyte-macrophage colony stimulating factor and interferon-gamma. J Infect Dis 170, 894 – 899. Stevens, D. A., Martinez, M., & Devine, M. J. (1996). Antifungal activity of LY 303366, an echinocandin beta glucan synthase inhibitor. In Program Abstracts of the Thirty-Sixth ICAAC, New Orleans, LA. Abstract F46, p. 107. ASM, Washington D.C. Vora, A., Chauhan, S., Brummer, E., & Stevens, D. A. (1998). Activity of voriconazole combined with neutrophils or monocytes against Aspergillus fumigatus: effects of Granulocyte colony stimulating factor and Granulocyte-macrophage colony stimulating factor. Antimicrob Agents Chemother 42, 2299 –2303.