JAC Advance Access originally published online on February 13, 2008
Journal of Antimicrobial Chemotherapy 2008 61(4):810-817; doi:10.1093/jac/dkn036
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Original research |
Amphotericin B formulations variably enhance antifungal activity of human neutrophils and monocytes against Fusarium solani: comparison with Aspergillus fumigatus
1 Laboratory of Infectious Diseases, 3rd Department of Paediatrics, School of Medicine, Aristotle University, Hippokration Hospital, Thessaloniki 54642, Greece 2 Laboratory of Medical Biotechnology, Department of Medical Laboratories, Technological Educational Institute of Thessaloniki, Thessaloniki 57400, Greece 3 Immunocompromised Host Section, National Cancer Institute, Bethesda, MD 20892, USA
* Correspondence address. 3rd Department of Paediatrics, Hippokration Hospital, Konstantinoupoleos 49, GR-54642 Thessaloniki, Greece. Tel: +30-2310-892444; Fax: +30-2310-992981; E-mail: roilides{at}med.auth.gr
Received 2 August 2007; returned 9 October 2007; revised 27 December 2007; accepted 13 January 2008
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Abstract |
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Objectives: Lipid formulations of amphotericin B (AMBF) are widely used in the treatment of life-threatening infections caused by Aspergillus fumigatus and Fusarium solani. We aimed to compare the immunomodulatory effects of four AMBF, deoxycholate (DAMB), liposomal (LAMB), lipid complex (ABLC) and colloidal dispersion (ABCD), on the oxidative antifungal activities of human neutrophils (PMNs) and monocytes (MNCs) against hyphae of A. fumigatus and F. solani.
Methods: Human PMNs and MNCs were pre-incubated with 1 or 5 mg/L DAMB and 5 or 25 mg/L for each of LAMB, ABLC and ABCD. Hyphal damage was then assessed by XTT assay, and O2– production was assessed by cytochrome c assay.
Results: All agents resulted in increased hyphal damage induced by phagocytes against both A. fumigatus and F. solani (P < 0.05). The high concentrations of AMBF elicited higher phagocyte-induced hyphal damage of both fungi than the low concentrations. There was, however, no consistent superiority of any of the AMBF or substantial effector cell:target ratio-dependent differences in the degree of hyphal damage enhancement. By comparison, O2– produced by PMNs or MNCs upon hyphal challenge was not generally affected by any of the AMBF. F. solani hyphae were significantly more resistant to H2O2 than A. fumigatus.
Conclusions: These findings suggest that AMBF have enhancing effects of variable degree on phagocyte-induced hyphal damage of A. fumigatus and F. solani. Other fungicidal mechanisms, perhaps non-oxidative, are more likely to mediate these immunomodulatory effects of AMBF on host defence against the two medically important filamentous fungi.
Keywords: filamentous fungi , phagocytes , hyphal damage , oxidative burst
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Introduction |
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Invasive aspergillosis and fusariosis cause excessive morbidity and mortality in immunocompromised patients. Invasive aspergillosis caused by Aspergillus fumigatus is the most common cause of fungal pneumonia in immunocompromised patients.1,2 Fusarium spp., the most common of which is Fusarium solani, are responsible for life-threatening local or disseminated infections in severely immunocompromised patients.3,4 Indeed, among immunocompromised patients, mainly patients with haematological malignancies, Fusarium spp. are the second most common pathogenic filamentous fungi.5 Aspergillus spp. and especially Fusarium spp. may be refractory to current antifungal therapies making treatment of infections caused by these fungal pathogens extremely difficult.2,6 The refractoriness of these infections to antifungal therapies underscores the importance of innate host defences against them.
The immune response varies with respect to the species and growth form encountered. Circulating phagocytes, particularly neutrophils (PMNs) and monocytes (MNCs), constitute the main innate host defence against fungal pathogens that cause invasive infections, including invasive aspergillosis and fusariosis.7,8 Oxidative pathways are among the antifungal mechanisms of PMNs and MNCs that are involved in hyphal damage. During the oxidative burst, activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase results in superoxide anion (O2–) production, a reactive oxygen intermediate released.9
Amphotericin B is an important antifungal agent in the treatment of mycoses caused by filamentous fungi. However, due to excessive nephrotoxicity and infusion-related adverse reactions of the conventional deoxycholate amphotericin B (DAMB),10 lipid formulations of amphotericin B, such as liposomal (LAMB), lipid complex (ABLC) and colloidal dispersion (ABCD), have been developed. These formulations have been recently shown to have immunomodulatory effects on immune response-related genes of human phagocytes11 and on various antifungal functions resulting in inhibition of A. fumigatus in vitro.12,13
Little is known, however, about the effects of lipid formulations of amphotericin B on antifungal host defences against filamentous fungi and in particular against non-Aspergillus filamentous fungi. To better understand the interaction between amphotericin B formulations (AMBF) and human phagocytes in treatment of invasive aspergillosis and fusariosis, we investigated the antifungal activities of human PMNs to induce damage on A. fumigatus hyphae, and also the antifungal activities of human PMNs and MNCs to elicit a respiratory burst and to induce damage on F. solani hyphae after exposure to AMBF.
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Drugs and reagents
Deoxycholate amphotericin B was purchased from Bristol–Myers Squibb (La Grande Nord, Paris, France), LAMB from Gilead Sciences (San Dimas, CA, USA), ABLC from Enzon Pharmaceuticals (Piscataway, NJ, USA) and ABCD from Sequus Pharmaceuticals (Menlo Park, CA, USA). Stock solutions and final dilutions were prepared as previously described.12 RPMI 1640 medium, fetal calf serum (FCM), penicillin, streptomycin, Hanks balanced salt solution without Ca2+ and Mg2+ (HBSS–) or with Ca2+ and Mg2+ (HBSS+) and Ficoll (Lymphocyte Separation Medium) were obtained from Gibco BRL, Life Technologies Ltd (Paisley Scotland, UK). Dextran T500, phorbol myristate acetate (PMA), cytochrome c, 2,3-bis [2-methoxy-4-nitro-5-sulfophenyl]2H-tetrazolium-S-carboxantide (XTT) and coenzyme Q were purchased from Sigma Chemical Co. (St Louis, MO, USA).
In the assays performed (described below), the final concentrations of DAMB were 1 and 5 mg/L and those of LAMB, ABLC and ABCD were 5 and 25 mg/L. The drug concentrations used were selected to be relevant to the therapeutically achievable concentrations in plasma and tissues14,15 as well as by preliminary experiments with a range of drug concentrations. Culture medium consisted of RPMI 1640 supplemented with 10% heat inactivated fetal calf serum, 100 U/mL penicillin and 100 mg/L streptomycin (FCM). PMA at a final concentration of 2 mg/L was used as a control stimulus of oxidative burst of MNCs and PMNs.
Aspergillus fumigatus. Strain AF 4215 of A. fumigatus [American Type Culture Collection (ATCC, Rockville, MD, USA) MYA 1163] isolated from a cancer patient with invasive pulmonary aspergillosis was used. This strain was preserved on potato dextrose agar slants at –30°C. From suspensions containing 105 [for hyphal damage and assay of hyphal susceptibility to hydrogen peroxide (H2O2)] or 2 x 106 (for O2– production) conidia/mL in yeast nitrogen base (YNB) broth supplemented with 2% glucose, 200 µL was plated in 96-flat-bottomed-well plates (Corning, Inc., New York, NY, USA) and incubated at 32°C with 5% CO2 for 18 h to become hyphae.
Fusarium solani. Strain F. solani NIH#26 isolated from a cancer patient with invasive fusariosis was also used. The preparation of F. solani hyphae was the same with that of A. fumigatus with the exception of a 16 h incubation for F. solani conidia to become hyphae.
PMNs were purified from healthy adult donors by dextran sedimentation and Ficoll centrifugation. Whole blood was mixed with dextran to a final concentration of 1%, and the red blood cells (RBCs) were allowed to sediment for 20 min. The top layer of the biphasic solution was placed above Ficoll and centrifuged at 400 g for 20 min. The supernatant was removed, and the remaining RBCs were lysed by hypotonic shock. The PMNs were washed with HBSS–, resuspended in HBSS– and counted with a haemocytometer. The PMN concentration was adjusted to 106 cells/mL, and immediately after their preparation, they were incubated with 1 and 5 mg/L DAMB or 5 and 25 mg/L LAMB, ABLC and ABCD, respectively, at 37°C for 2 h. PMNs were then washed and used for the functional assays.
Human mononuclear cells were obtained from the blood of healthy adult volunteers and separated by centrifugation over Ficoll, as previously described.16 Briefly, the cells were washed and resuspended in HBSS–. They were counted on a haemocytometer by Trypan Blue staining, and percentages of MNCs over total number of mononuclear leucocytes were calculated after staining with May-Grunwald-Giemsa. MNCs were adjusted to 106 cells/mL in FCM. MNC-enriched cell populations were obtained from mononuclear leucocytes by adherence on plastic surfaces in 12-well plates during incubation in a humidified CO2 incubator at 37°C for 2 h. Following adherence, the cells were washed with warm HBSS– and incubated in fresh FCM at 37°C for 24 h prior to incubation with the drugs in order to avoid cell activation due to handling. MNCs were then incubated with 1 or 5 mg/L DAMB and 5 or 25 mg/L LAMB, ABLC and ABCD, respectively, at 37°C for 22 h. They were then washed and used for the functional assays.
Viability of PMNs or MNCs, either untreated or amphotericin B-treated, was tested using the Trypan Blue exclusion method by counting stained (dead) versus unstained (alive) cells on a haemocytometer. Viability was >95% in all experiments.
Hyphal damage was assessed by a modified method of the XTT colorimetric assay.17 During this assay, tetrazolium salts are taken up by viable hyphae and are reduced by mitochondrial dehydrogenase of the fungi to coloured tetrazolium products that are determined spectrophotometrically. Pre-treated PMNs were added to 104 hyphae in HBSS– in each well at effector cell:target (E:T) ratios of 5:1 and 10:1 and incubated for 2 h. Similarly, pre-treated MNCs were added to 104 hyphae at E:T ratios of 10:1 and 20:1 and incubated for 2 h. After the 2 h of incubation at 37°C with 5% CO2, PMNs or MNCs were lysed by washing with H2O three times before adding 150 µL of a solution containing 0.25 g/L XTT plus 40 mg/L coenzyme Q. After incubation at 37°C with 5% CO2 for 1 h, hyphal damage was assessed at 450 nm with a reference wavelength of 690 nm. Antihyphal activity was calculated according to the following formula: percent hyphal damage = (1 – X/C) x 100, where X is the absorbance of experimental wells and C is the absorbance of control wells with hyphae only.18
Superoxide anion (O2–) release was assessed by a modification of a previously described cytochrome c reduction method.19 YNB broth was replaced by 100 µL of HBSS+ in all wells. Cytochrome c was added to all wells at a final concentration of 65 µM, and PMNs or MNCs that had been pre-treated with drugs for 2 or 22 h, respectively, were added to wells containing 2 x 105 hyphae at an E:T ratio of 1:1. Control (untreated) PMNs or MNCs were also added to wells containing 2 x 105 hyphae at an E:T ratio of 1:1. PMA was added to certain wells without hyphae together with untreated PMNs or MNCs as a positive control of O2– production. Plates were incubated at 37°C with 5% CO2 for 1 h. Aliquots of 200 µL were assessed for cytochrome c reduction spectrophotometrically at 550 nm with a reference wavelength of 690 nm.
Hyphal susceptibility to hydrogen peroxide
Susceptibility of A. fumigatus and F. solani hyphae to H2O2 was assessed by a modification of a previously described method.20 Suspensions of both fungi containing 105 conidia/mL in YNB were incubated to become hyphae. They were then incubated with different H2O2 concentrations of 0, 50, 100 and 200 mM H2O2 solutions for 10 min. Each hyphal suspension was then washed with H2O twice before adding 150 µL of a solution containing 0.25 mg/mL XTT plus 40 mg/L coenzyme Q. After incubation at 37°C with 5% CO2 for 30 min, hyphal damage was assessed at 450 nm with a reference wavelength of 690 nm. Antihyphal activity was calculated according to the following formula: percent hyphal damage = (1 – X/C) x 100, where X is the absorbance of experimental wells and C is the absorbance of control wells with hyphae only.18
Each experiment investigating hyphal damage and O2– production was performed with PMNs or MNCs of one blood donor using duplicate wells for each condition. For each set of functional assays (hyphal damage by PMNs and MNCs at each E:T ratio and O2– production), five experiments were performed. The average value of these replicate wells was taken as the value for a blood donor/experiment. These values were then used in the data analysis to calculate the mean ± SE of all the donors at the same condition. Comparisons between drug-treated and untreated PMNs or MNCs and also between high and low AMBF concentrations as well as other conditions were performed using one-way analysis of variance (ANOVA) with Dunnett as the post-test for multiple comparisons. A P value of <0.05 indicated statistical significance.
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Hyphal damage induced by PMNs on A. fumigatus
Pre-treatment of PMNs with both low and high concentrations of all AMBF for 2 h resulted in significantly increased PMN-induced hyphal damages as compared with hyphal damage induced by PMNs alone at both E:T ratios of 5:1 and 10:1 (P < 0.01; Figure 1a and b).
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Among the low concentrations of AMBF at an E:T ratio of 5:1, ABLC (5 mg/L)-pre-treated PMNs tended to exhibit the largest increase in hyphal damage as compared with PMNs alone (P < 0.01). By comparison, among the high concentrations of AMBF, DAMB (5 mg/L)-pre-treated PMNs tended to exhibit the largest increase in hyphal damage as compared with PMNs alone (Figure 1a; P < 0.01). By analogy, at an E:T ratio of 10:1, ABLC (5 mg/L) among the low concentrations and DAMB (5 mg/L) among the high concentrations of AMBF again tended to exhibit the largest increase in hyphal damages as compared with PMNs alone (Figure 1b; P < 0.01).
Of note, significant differences in hyphal damage induced by PMNs pre-treated with all AMBF and then incubated with A. fumigatus at an E:T ratio of 5:1 were observed between high and low concentrations of the drugs (Figure 1a). By comparison, at an E:T ratio of 10:1, significant differences were observed only between high and low concentrations of DAMB and LAMB with LAMB-pre-treated PMNs exhibiting lower hyphal damage than the corresponding DAMB pre-treatment (Figure 1b; P 
0.002).
Hyphal damage induced by PMNs on F. solani
Pre-treatment of PMNs with either low or high concentrations of virtually all AMBF for 2 h resulted in significantly increased PMN-induced hyphal damages as compared with PMNs alone at both E:T ratios of 5:1 and 10:1 (P < 0.05; Figure 2a and b).
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PMNs pre-treated with DAMB at high concentrations and incubated with F. solani hyphae at either 5:1 or 10:1 E:T ratios resulted in the largest increases of hyphal damage among all four AMBF. In particular, PMNs pre-treated with 5 mg/L DAMB at an E:T ratio of 5:1 resulted in 90.7 ± 2.1% hyphal damage as compared with 12.2 ± 2.5% of PMNs alone (Figure 2a; P < 0.001). At an E:T ratio of 10:1, PMNs pre-treated with 5 mg/L DAMB resulted in a relatively very high percentage of hyphal damage as compared with PMNs alone (96 ± 2% versus 22.3 ± 4.1%, respectively, P < 0.001; Figure 2b).
Significant differences were also observed in hyphal damage against F. solani induced by PMNs pre-treated with AMBF and then incubated with F. solani at an E:T ratio of 5:1 between high and low concentrations of DAMB, LAMB and ABCD. At an E:T ratio of 10:1, significant differences were observed between high and low concentrations of DAMB and LAMB only. The largest difference between high and low concentrations of AMBF was noticed with LAMB at an E:T ratio of 10:1 (Figure 2b; P < 0.0001).
Hyphal damage induced by MNCs on F. solani
Pre-treatment of MNCs with the high concentrations of AMBF (5 mg/L DAMB and 25 mg/L of lipid formulations) for 22 h resulted in a significant increase in MNC-induced hyphal damage as compared with MNCs alone at both E:T ratios of 10:1 and 20:1 (Figure 3a and b).
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Among all four AMBF, pre-treatment of MNCs with both concentrations of ABLC and subsequent incubation with F. solani hyphae at both E:T ratios resulted in the largest increases in hyphal damage (Figure 3). Specifically, MNCs pre-treated with 5 or 25 mg/L ABLC resulted in very high percentages of hyphal damage at both E:T ratios of 10:1 and 20:1 as compared with MNCs alone (Figure 3a and b; P < 0.01).
Significant differences in hyphal damage against F. solani induced by pre-treated MNCs at an E:T ratio of 10:1 were also observed between high and low concentrations of DAMB, LAMB and ABCD. In particular, treatment of MNCs with low (1 mg/L) and high (5 mg/L) concentrations of DAMB resulted in significant differences between these concentrations (P = 0.0002). In addition, the same trend was observed when MNCs were pre-treated with 5 and 25 mg/L LAMB (P = 0.014) and ABCD (P = 0.018). At an E:T ratio of 20:1, significant differences also were observed between high and low concentrations of DAMB (P = 0.003) and LAMB (P = 0.046).
Superoxide anion production by PMNs
Aspergillus fumigatus. In general, neither low nor high concentrations of AMBF showed significant changes in the O2– production in response to hyphae of both fungi as compared with untreated PMNs. Interestingly, pre-treatment of PMNs with 1 mg/L DAMB and 25 mg/L ABCD significantly reduced the O2– production in response to hyphae of A. fumigatus as compared with PMNs alone (1.76 ± 0.23 and 1.39 ± 0.38 versus 2.83 ± 0.22 nmol O2–/106 PMN/h, respectively; P < 0.01).
Fusarium solani. Hyphae of F. solani stimulated O2– production to a smaller degree (Figure 4). However, the AMBF generally did not affect the production of O2– by PMNs in response to hyphae of F. solani. When PMA was used as a stimulus of PMNs (positive control of O2– production), it considerably stimulated O2– production as compared with PMNs alone (3.56 ± 0.48 versus 0.69 ± 0.11 nmol O2–/106 PMN/h, respectively; P = 0.0002; Figure 4).
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Superoxide anion production by MNCs
PMA considerably stimulated O2– production by MNCs (3.5 ± 0.13 versus 0.71 ± 0.18 nmol O2–/106 MNC/h, respectively; P < 0.001). Similarly to previous experiments with PMNs, AMBF generally did not affect the production of O2– by MNCs in response to hyphae of F. solani. However, hyphae of F. solani stimulated O2– production to a smaller degree [from a baseline O2– production of 0.71 ± 0.18 nmol O2–/106 MNC/h by untreated (control) MNCs, there was no significant difference in O2– production by MNCs treated with any AMBF, P > 0.05].
Susceptibility of A. fumigatus and F. solani hyphae to H2O2
Hyphal damage induced by H2O2 at a concentration of 50 mM was 47.1% versus 42.9% for A. fumigatus and F. solani, respectively, as shown in Table 1 (P > 0.05). However, at higher concentrations of 100 and 200 mM, A. fumigatus hyphal damage was statistically significantly higher as compared with F. solani. F. solani seems to be more resistant to H2O2 than A. fumigatus.
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Although A. fumigatus and F. solani are both opportunistic filamentous fungi with great mortality, they have distinct characteristics with regard to their biology, drug susceptibility and interaction with host defences, and may thus have different susceptibilities to PMNs and/or MNCs under the influence of specific or all AMBF. In this study, we have demonstrated that: (i) deoxycholate and lipid formulations of amphotericin B enhance antifungal activities of both types of phagocytes, PMNs and MNCs, against A. fumigatus and F. solani, as evidenced by phagocyte-induced hyphal damage; (ii) the activity of a given AMBF in increasing the hyphal damage varies as a function of phagocyte and organism; (iii) there are no substantial E:T ratio-dependent differences in AMBF enhancement; (iv) there is, however, an AMBF concentration dependency of AMBF effects; and (v) of note, interaction between AMBF and human phagocytes is not accompanied with enhanced oxidative burst as shown by unchanged or reduced O2– production by pre-treated phagocytes in response to hyphae of these fungal species.
In a previous report, we presented data on the effects of the four AMBF on the hyphal damage induced by MNCs and on O2– production elicited by MNCs in response to A. fumigatus.12 In this report, we expand our studies on the effects of AMBF on the hyphal damage induced by PMNs in response to A. fumigatus, and on the effects of AMBF on the hyphal damage induced by PMNs and MNCs and on O2– production elicited by PMNs and MNCs in response to F. solani.
This study indicates that the immune response against A. fumigatus and F. solani, as evidenced by hyphal damage activity, may be species dependent. Taken the results of this study and those of other studies together,12,13 the effects of AMBF on the antihyphal activity of PMNs against F. solani were somewhat lower than the effects on the antihyphal activity of PMNs against A. fumigatus. The strains of Aspergillus and Fusarium that have been used are typical isolates of these organisms and are well characterized in terms of their basic host–pathogen interaction, pathogenicity and drug susceptibility.21 F. solani seems to be more resistant to oxidative antifungal mechanisms as shown in the experiments of hyphal susceptibility to H2O2, as compared with A. fumigatus hyphae. A possible explanation is that F. solani hyphae tend to have decreased susceptibility to antifungal peptides or to other non-oxidative antifungal mechanisms as compared with A. fumigatus hyphae.21
ABLC-pre-treated MNCs exhibited a trend towards the highest degree of antihyphal activity against A. fumigatus and F. solani as shown in this study and our previous study.12 By comparison, PMNs result in more variable degrees of hyphal damage of the two organisms after incubation with the four AMBF. The antifungal activity of ABLC-pre-treated MNCs among the AMBF does not appear to be due to a direct effect on antifungal MNC function by enhanced expression of a combination of immunoenhancing cytokines (i.e. TNF-
or IL-1β), since these cytokines are not enhanced by ABLC.11 Release of lipases by A. fumigatus or F. solani breaking down the lipid complex ribbon structures of intracellular ABLC could cause an enhanced delivery of amphotericin B and fungicidal products of the MNC to the hyphal membrane, which might account for the enhanced antifungal effect observed.12,18 However, it is unclear why ABLC does not consistently have a similarly pronounced effect on PMNs against the two fungi.
The immunomodulatory effect of AMBF on hyphal damage against the two fungi is concentration dependent. We used two concentrations of each drug, a low concentration and a high concentration, that are clinically achievable in plasma and/or tissue. Our data support the notion that a higher concentration may have a better immunomodulatory effect on PMN/MNC antifungal function.
The mechanism underlying the immunomodulatory effects of AMBF may be related to their interaction with Toll-like receptors (TLRs).22,23 It has been previously shown that DAMB as well as LAMB induce signal transduction via TLR2 on the surface of phagocytes. Through an NF-
B-mediated pathway, these formulations modify cytokine expression and antifungal function of phagocytes. AMBF may also affect other molecules on the surface or in the cytoplasm of effector cells ultimately modulating their antifungal function.24,25 In addition, amphotericin B through TLR2 signalling activates oxidative mechanisms of killing, whereas the liposomes through TLR and intracellular pathogen-associated molecular patterns (PAMPs) polarize PMNs to non-oxidative mechanisms of killing, which are more important for hyphal versus conidial damage.24
The reported effects of AMBF on O2– production by human phagocytes have been variable.12,26–28 In the present study, none of the AMBF affected O2– release from pre-treated MNCs and PMNs in response to hyphae of A. fumigatus and F. solani. The exception was pre-treated PMNs with DAMB at 1 mg/L and ABCD at 25 mg/L, which significantly reduced the production of O2– in response to A. fumigatus hyphae. This suppressive effect may be due to the binding of DAMB and ABCD to sterols of MNCs and subsequent cell membrane injury. The same suppressive effect has been found to occur with high concentrations of DAMB in previous studies.28,29 In our previous report,12 MNCs pre-treated with a high concentration (25 mg/L) of ABLC significantly reduced the production of O2– in response to serum-opsonized A. fumigatus hyphae. This finding probably suggests that, in addition to DAMB, lipid amphotericin B formulations, including ABLC and ABCD, although they are better tolerated and less toxic antifungal agents, can also cause subsequent cell membrane injury especially at high concentrations.
The remarkable finding of increased hyphal damage with a suppressed O2– production after treatment of phagocytes with certain AMBF is in agreement with the result of our previous study.12 Although there is no definitive explanation, one can speculate that while O2– constitutes the first reactive oxygen intermediate released during the oxidative burst, it must act within a short period of time, rapidly leading to more powerful oxidizing species production, such as H2O2 and H2O2-dependent intracellular intermediates.30 Therefore, the increased phase during its measurement may be missed. Additionally, a dissociation of O2– from H2O2 levels may occur through differential expression of enzymes catalyzing reactive oxygen intermediates such as dismutase and myeloperoxidase.31 Furthermore, enhanced hyphal damage without a concomitant increase in O2– production may be related to modulation of antimicrobial peptides found within the granules of phagocytes. Phagocyte-induced hyphal damage is mediated by the oxidative burst in combination with complementary non-oxidative mechanisms, the latter consisting of a variety of antimicrobial peptides.32 Some of these antimicrobial peptides, including azurocidin, lactoferrin and lysozyme, exhibit antifungal activity and can be found in high concentrations at the site of infection. Incubation of hyphae with cationic peptides and other granule constituents results in the release of cell wall glycoproteins, thereby indicating hyphal damage.33 This hypothesis may suggest a potential role of amphotericin B-induced modulation of other fungicidal mechanisms, perhaps non-oxidative, on the immune response to fungi.
In conclusion, we have demonstrated that AMBF, at clinically relevant ranges of concentrations, enhance antihyphal activity of human phagocytes, PMNs or MNCs, against A. fumigatus and F. solani. This augmentation of the antifungal function of human phagocytes is not associated with significantly augmented O2– production suggesting a critical role for other innate immune mechanisms, perhaps non-oxidative. Although further studies on the molecular mechanisms of these interactions are required, differential immunomodulatory effects of AMBF may be of clinical significance in the management of invasive aspergillosis and fusariosis.
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This study was supported in part by the Aristotle University and the intramural research programme of the National Cancer Institute, Bethesda, MD, USA.
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None to declare.
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References |
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