Enhancement of antifungal chemotherapy by interferon-gamma in experimental systemic cryptococcosis

Jon E. Lutz, Karl V. Clemons and David A. Stevens*

Department of Medicine, Santa Clara Valley Medical Center and California Institute for Medical Research, San Jose, California and the Division of Infectious Diseases and Geographic Medicine, Department of Medicine, Stanford University School of Medicine, Stanford, CA, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The possible enhancement, using immunotherapy with interferon-gamma (IFN-{gamma}), combined with conventional antifungal therapy, was studied in a murine model of systemic cryptococcosis. Four weeks after intravenous challenge, infection was quantified in brains and livers of survivors. Groups received IFN-{gamma} every other day beginning 7 days before (prophylaxis), or after infection (14 doses), or amphotericin B post-infection, or combinations of these regimens. IFN-{gamma} alone was modestly effective, but impressively and significantly potentiated amphotericin in reducing infection in the most important site of infection, the brain. The efficacy was seen after lethal and non-lethal challenges, and when IFN-{gamma} was given by the intravenous or subcutaneous routes. In non-lethal infection, only the combination amphotericin–IFN-{gamma} resulted in sterilization of the central nervous system. Potentiation of fluconazole was less impressive. Adding prophylactic IFN-{gamma} doses to IFN-{gamma} therapy did not consistently enhance the therapeutic effect. These results suggest IFN-{gamma} may have a role in potentiating conventional antifungal therapy of cryptococcosis.


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Infection due to Cryptococcus neoformans, especially meningitis, is particularly a problem in patients with depressed cellular immunity, and this problem has been sharply exacerbated by the AIDS epidemic.1 Standard antifungal therapy is commonly ineffective in the most severely immunosuppressed.2

Evidence for an important role of interferon-gamma (IFN-{gamma}) in the cell-mediated immune response includes development of anti-cryptococcal activity by IFN-{gamma} treatment of effector cells in vitro,37 augmentation of cryptococcal infection in vivo by administration of antibody to IFN-{gamma}811 and reduction of cryptococcal infection by administration of IFN-{gamma}.9,10,12

We explored the effect of IFN-{gamma} alone, and in combination with different forms of antifungal therapy, in a murine model of systemic cryptococcosis that mimics the human disease. The treatments were prolonged, and were also designed to give insight into the effects of clinical treatment schedules.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Mice

The animal experiments were approved by the institutional animal care and use committee. Male BALB/c mice 6–8 weeks old were infected and divided into groups of 9–12 per treatment regimen.

Isolate

C. neoformans 9759, a typical, encapsulated serotype A isolate, was prepared and quantified for intravenous (iv) challenge as previously described.1315 The challenge was 2.8 x 104–4.3 x 104 colony-forming units (cfu) depending on the experiment. The MIC of amphotericin B and fluconazole by broth macrodilution testing was 1.56 and 6.25 mg/mL, respectively.16

Treatment regimens

Treatments commenced the day following infection. Amphotericin B deoxycholate suspension was given by the intraperitoneal (ip) route, 3 mg/kg thrice weekly for 2 weeks. Fluconazole by gavage in water and recombinant murine IFN-{gamma} (Genentech, South San Francisco, CA), 9.4 x 106 U/mg, in saline, were given as detailed in Results.

IFN-{gamma} every other day (qod) dosing is consistent with pharmacodynamic data.17 Earlier experience in the model or preliminary experiments revealed ip deoxycholate alone or subcutaneous (sc) or iv saline produced the same results as no treatment. Mice were given sterilized food and acidified water ad libitum.

Organ infectious burden

One day after cessation of therapy, surviving mice were killed by CO2 asphyxiation, and the livers and brains were removed, weighed and homogenized. Dilutions of the homogenates were placed on Sabouraud's agar plates with chloramphenicol, incubated at 35°C, and cfu/organ determined. The lower limit of sensitivity in this assay is 5–7 cfu/ organ.14

Statistics

Comparisons of infectious burdens between groups were performed using the Mann–Whitney U test. Mice that died of progressive infection during experiments were assigned for rank method significance testing a cfu value for infected organs, based on prior experiments in untreated mice dying of infection, higher than that in any surviving animal, to ensure that death was always scored as a worse outcome than survival with any burden of organisms.13,14 Fisher's exact test was performed to compare numbers of animals rendered infection-free in the groups. Significance was set at P < 0.05.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Amphotericin and interferon

Experiment 1. IFN-{gamma} was given iv qod for 14 doses as therapy, or given prophylactically on days –7, –5, –3 and –1 before infection, or both.

Three of 10 infected untreated mice died before the end of the study period. Earlier experiments had demonstrated that residual infection in this model, with challenges of this size and this study duration, were <=100 cfu in the kidney, lung or spleen, so subsequent studies focused on the heavily infected organs, the brain and the liver. The residual infection data are shown in Table IGo and the sterilization of infection in Table IIGo. Amphotericin alone achieved a mean 23000-fold reduction in cfu of C. neoformans from the brains of treated compared with untreated animals. IFN-{gamma} alone produced a 1.7-fold reduction of cfu in brain tissue, but this reduction was not significant. Amphotericin + therapeutic IFN-{gamma} eliminated all brain infection, thus reducing brain infection >830000-fold compared with controls, and also significantly (36-fold) compared with amphotericin alone (Table IGo). Amphotericin + prophylactic and therapeutic IFN-{gamma} eliminated brain infection in all but one animal, thus reducing the cfu of C. neoformans from brain tissue 350000-fold compared with controls and also significantly (15-fold) compared with amphotericin alone. The cfu results obtained with amphotericin + therapeutic IFN-{gamma} versus amphotericin + prophylactic and therapeutic IFN-{gamma} were not significantly different. The number of mice whose brains were cleared of residual infection was significantly greater for those receiving amphotericin + either IFN-{gamma} regimen (18/19 animals receiving any amphotericin–IFN-{gamma}-containing regimen) compared with amphotericin alone (2/10) (Table IIGo).


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Table I. Experiment 1: recovery of Cryptococcus neoformans from the organs of surviving mice treated with IFN-{gamma} and amphotericin B (AmB), alone or in combination
 

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Table II. Experiment 1: number of mice cured (no detectable organisms in either organ) and number of organs free of infection in mice treated with IFN-{gamma} and amphotericin B (AmB) alone or in combination
 
Amphotericin produced a significant, 2700-fold, reduction of liver infection compared with controls (Table IGo). IFN-{gamma} significantly reduced infection, 0.05 log10. Compared with amphotericin alone, amphotericin + therapeutic IFN-{gamma} reduced infection 2.3-fold, and amphotericin + therapeutic and prophylactic IFN-{gamma} reduced it 2.8-fold, but neither improvement was significant. More livers were rendered free of detectable infection in both amphotericin– IFN-{gamma} groups compared with amphotericin alone, the trend was toward significance (P < 0.06 to <0.09).

The number of mice rendered free of detectable cryptococci in both organs, i.e. cured, was significantly greater for either amphotericin–IFN-{gamma} group compared with amphotericin alone (Table IIGo). The two combination groups were not significantly different from each other in comparing cure rates in either or both organs.

Experiment 2, high inoculum. In a similar experiment, the inoculum was 25% higher, and the infection followed a more lethal course. There were only 2/12 (17%) survivors amongst the untreated controls, compared with 3/10 of those receiving therapeutic IFN-{gamma} and 5/10 of those receiving therapeutic + prophylactic IFN-{gamma}. All amphotericincontaining regimens produced 100% survival. Amphotericin alone, again significantly reduced infection in the brain (nine-fold) compared with the surviving controls (Table IIIGo). Amphotericin + therapeutic IFN-{gamma} produced a >100-fold reduction, and amphotericin + therapeutic and prophylactic IFN-{gamma} an even greater 390-fold reduction. Both the combination regimens produced significantly better results than amphotericin alone, an 11-fold improvement for amphotericin + therapeutic IFN-{gamma}, and 43-fold improvement for amphotericin + prophylactic and therapeutic IFN-{gamma}. The latter combined regimen produced 3.8-fold better results than the former. IFN-{gamma} alone given prophylactically followed by a therapeutic dose regimen reduced infection 1.8-fold compared with controls, but therapeutic IFN-{gamma} alone was essentially the same as no treatment.


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Table III. Experiment 2: recovery of Cryptococcus neoformans from the organs of surviving mice treated with INF-{gamma} and amphotericin B (AmB) alone or in combination
 
In the liver, amphotericin significantly reduced infection (150-fold) compared with controls (Table IIIGo). While neither IFN-{gamma} regimen alone influenced the infection, they decreased the beneficial effect of amphotericin when administered in combination. In the case of therapeutic and prophylactic IFN-{gamma}, this was a 10-fold difference and significant compared with amphotericin alone, or amphotericin + therapeutic IFN-{gamma} (P = 0.003). This IFN-{gamma} interference in the liver was opposite to the trend seen in the earlier experiment.

Experiment 3, route of IFN-{gamma}. Amphotericin was given alone or with IFN-{gamma} administered iv as previously or as sc doses. The infection was again highly lethal, with 90% mortality in control mice. All treatment groups survived (P < 0.01, compared with controls). The residual infection data are shown in Table IVGo. All three treatment regimens markedly reduced infection in both organs compared with controls (P < 0.001 for all six comparisons). Moreover, both combination regimens reduced (>100-fold) brain infection significantly compared with amphotericin alone (P < 0.05 for both comparisons). There was no evidence of IFN-{gamma} interference with amphotericin in the liver as seen in the second (but not the first) experiment.


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Table IV. Experiment 3: recovery of Cryptococcus neoformans from the brain and liver of treated mice, according to route of IFN administration and interaction with amphotericin B (AmB)
 
Fluconazole and interferon

Initial IFN dose-finding experiment (data not shown). IFN was given sc qod for six doses of 2000, 10000 or 50000 U, alone or with 2.5 mg/kg fluconazole bid for 11 days. Fluconazole alone was effective in reducing geometric mean brain cfu from 5.58 to 3.65 log10 (P < 0.01), but the regimens of IFN-{gamma} alone had no effect, and IFN-{gamma} did not potentiate fluconazole. Of interest, examining the liver cfu data, fluconazole treatment, with or without IFN-{gamma} (any dose), was worse than no treatment (P < 0.01 for all four comparisons), as was the lowest IFN-{gamma} dose alone, but IFN-{gamma} at the other two doses did not show this effect, and IFN-{gamma} at all three doses did not affect fluconazole.

Low dose fluconazole (data not shown). A second experiment studied the same dose and number of treatments of IFN-{gamma}, either iv or sc, as used in the amphotericin studies, and fluconazole 7.5 mg/kg/day for 17 days. The inoculum was 50% greater, and 70% of control animals died. Fluconazole + IFN-{gamma} sc significantly prolonged survival (P < 0.05) compared with untreated control, and fluconazole alone (P = 0.06), and fluconazole + IFN-{gamma} iv (P = 0.05) also prolonged survival to a lesser extent. Fluconazole + IFN-{gamma} sc produced the greatest reduction in infection of the brain and liver (P = 0.06 for both organs) compared with controls; this is in contrast to the adverse effect of the combination on the liver data in the previous experiment.

High-dose fluconazole. A third experiment studied the same IFN regimens, and fluconazole at a 10 mg/kg/day dose for 17 days. All animals survived, and the residual infection data are shown in Table VGo. No animals were cleared of infection. Fluconazole, IFN-{gamma} sc, or IFN-{gamma} iv reduced brain infection significantly, and the fluconazole–IFN-{gamma} sc and fluconazole–IFN-{gamma} iv regimens did so to an even more marked degree. The combination regimens were superior to fluconazole alone, and to IFN-{gamma} alone (either combination P < 0.01 versus IFN-{gamma} alone sc, and P < 0.05 versus IFN alone iv). IFN-{gamma} alone, either route, produced a significant reduction in cfu from the liver as compared with the controls. For a third time, fluconazole was ineffective in reducing the burden of infection in the liver, and was also inferior to IFN-{gamma} alone (either route; IFN-{gamma} sc and IFN-{gamma} iv versus fluconazole, P < 0.05 and <0.01, respectively). Fluconazole did not enhance the effect of IFN-{gamma} (combination fluconazole–IFN-{gamma} was not superior to IFN-{gamma} alone), neither did it demonstrate an antagonistic effect (both fluconazole–IFN-{gamma} combination regimens were significantly superior to fluconazole alone).


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Table V. Recovery of Cryptococcus neoformans from the brain and liver of fluconazole (FCZ)-treated mice: interaction with INF-{gamma}
 

    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
These studies show a modest efficacy of IFN-{gamma} given therapeutically alone, and an impressive potentiation of amphotericin therapy, in reducing infection in the most important site of infection in this murine model (and in the human disease), the central nervous system. The efficacy was seen in lethal and non-lethal challenges, and when IFN-{gamma} was given by the iv or sc route. In a non-lethal infection, only the combination of amphotericin + IFN-{gamma} (not either component alone) produced the most desirable and profound result of all, sterilization of the central nervous system. Potentiation of fluconazole was less impressive. Adding prophylactic doses of IFN-{gamma} to IFN-{gamma} therapy did not consistently enhance the therapeutic effect. IFN-{gamma} often showed efficacy in reducing liver infection, but effects in this organ were not consistent.

These results corroborate and extend the observations of Joly et al.12 in mice, who studied short course regimens of rat IFN-{gamma} (three doses, two given prophylactically) and amphotericin (one dose). They showed prolongation of survival by IFN-{gamma} alone and potentiation of amphotericin therapy, reduction of brain infection by the combination regimen, but not sterilization. Our sub-acute model and prolonged therapy more closely mimics the human disease and its therapy.

Murine macrophages stimulated in vitro with rIFN-{gamma} suppress growth of extracellular cryptococci,3 apparently by an extracellular stimulated macrophage product. IFN-{gamma} alone had no growth-inhibitory effect. Opsonized cryptococci can be killed intracellularly by murine macrophages activated by rIFN-{gamma} in vitro or in vivo.6 Differences in the capacity to kill encapsulated and non-encapsulated cryptococci have been noted.6 Rat rIFN-{gamma}-treated rat alveolar macrophages inhibit growth of cryptococci by a mechanism involving increased adhesion or uptake.7 IFN-{gamma} is involved in activating human astrocytes,5 but not microglia,18 to kill cryptococci and in enhancing anti-cryptococcal activity of mixed lymphocyte populations from HIV-infected persons.4

Treatment of mice with a monoclonal antibody against IFN-{gamma} enhanced their susceptibility to pulmonary9,10 or systemic11 challenge, and this also occurs in immune animals.8

Amphotericin can prime macrophages for a second signal from IFN-{gamma}, producing an enhanced oxidative burst.19,20 IFN-{gamma} and amphotericin interacted cooperatively in murine macrophages to eradicate ingested cryptococci, whereas fluconazole and IFN-{gamma} did not.21

Intraperitoneal rIFN-{gamma} significantly prolonged survival of mice challenged intra-tracheally with C. neoformans.9,10 Interleukin-12, a potent stimulator of IFN-{gamma} production, enhances host resistance against systemic cryptococcal challenge and potentiates fluconazole therapy.22 Others have corroborated this,23 and shown that the effect is mediated by IFN-{gamma}.

Taken together, these in vitro and in vivo results, and our results, indicate that IFN-{gamma} potentiates conventional antifungal therapy, particularly amphotericin. This argues for further studies of the possible mechanisms of this potentiation and for exploration of this beneficial effect in the clinical setting. This enhancement may be most critical in the patient with a marginal endogenous response to infection, the immunocompromised patient, who is so susceptible to cryptococcal disease. However, more experimental evidence addressing this particular question would be of interest; one must always also be cautious in extrapolating conclusions from experimental systems into the clinic.24 If adjunctive IFN-{gamma} therapy could allow shorter courses or lower doses of amphotericin, reducing the potential for amphotericin toxicity and/or shortening the duration of the patient's hospital stay, this would also be an important benefit.


    Acknowledgments
 
Presented in part to the 34th Infectious Disease Society of America Annual Meeting, New Orleans. Supported in part by a grant from Genentech, Inc., South San Francisco, CA, USA.


    Notes
 
* Correspondence address. Division of Infectious Diseases, Department of Medicine, Santa Clara Valley Medical Center, 751 S. Bascom Avenue, San Jose, CA 95128-2699, USA. Tel: +1-408-885-4303; Fax: +1-408-885-4306; E-mail: stevens{at}leland.stanford.edu Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
1 . Mitchell, T. G. & Perfect, J. R. (1995). Cryptococcosis in the era of AIDS—100 years after the discovery of Cryptococcus neoformans. Clinical Microbiology Reviews 8, 515–48.[Abstract]

2 . Powderly, W. G. (1993). Cryptococcal meningitis and AIDS. Clinical Infectious Diseases 17, 837–42.[ISI][Medline]

3 . Flesch, I. E., Schwamberger, G. & Kaufmann, S. H. (1989). Fungicidal activity of interferon-gamma-activated macrophages. Extracellular killing of Cryptococcus neoformans. Journal of Immunology 142, 3219–24.[Abstract/Free Full Text]

4 . Horn, C. A. & Washburn, R. G. (1995). Anticryptococcal activity of NK cell-enriched peripheral blood lymphocytes from human immunodeficiency virus-infected subjects: responses to interleukin-2, interferon-gamma, and interleukin-12. Journal of Infectious Diseases 172, 1023–7.[ISI][Medline]

5 . Lee, S. C., Dickson, D. W., Brosnan, C. F. & Casadevall, A. (1994). Human astrocytes inhibit Cryptococcus neoformans growth by a nitric oxide-mediated mechanism. Journal of Experimental Medicine 180, 365–9.[Abstract]

6 . Levitz, S. M. & DiBenedetto, D. J. (1988). Differential stimulation of murine resident peritoneal cells by selectively opsonized encapsulated and acapsular Cryptococcus neoformans. Infection and Immunity 56, 2544–51.[ISI][Medline]

7 . Mody, C. H., Tyler, C. L., Sitrin, R. G., Jackson, C. & Toews, G. B. (1991). Interferon-gamma activates rat alveolar macrophages for anticryptococcal activity. American Journal of Respiratory Cell and Molecular Biology 5, 19–26.[ISI][Medline]

8 . Aguirre, K., Havell, E. A., Gibson, G. W. & Johnson, L. L. (1995). Role of tumor necrosis factor and gamma interferon in acquired resistance to Cryptococcus neoformans in the central nervous system of mice. Infection and Immunity 83, 1725–31.

9 . Kawakami, K., Kohno, S., Kadota, J. I., Tohyama, M., Teruya, K., Kudeken, N. et al. (1995). T cell-dependent activation of macrophages and enhancement of their phagocytic activity in the lungs of mice inoculated with heat-killed Cryptococcus neoformans: involvement of IFN-gamma and its protective effect against cryptococcal infection. Microbiology and Immunology 39, 135–43.[ISI][Medline]

10 . Kawakami, K., Tohyama, M., Teruya, K., Kudeken, N., Xie, Q. & Saito, A. (1996). Contribution of interferon-gamma in protecting mice during pulmonary and disseminated infection with Cryptococcus neoformans. FEMS Immunology and Medical Microbiology 13, 123–30.[ISI][Medline]

11 . Salkowski, C. A. & Balish, E. (1991). A monoclonal antibody to gamma interferon blocks augmentation of natural killer cell activity induced during systemic cryptococcosis. Infection and Immunity 58, 486–93.

12 . Joly, V., Saint-Julien, L., Carbon, C. & Yeni, P. (1994). In vivo activity of interferon-gamma in combination with amphotericin B in the treatment of experimental cryptococcosis. Journal of Infectious Diseases 170, 1331–4.[ISI][Medline]

13 . Clemons, K. V. & Stevens, D. A. (1998). Comparison of Fungizone, Amphotec, AmBisome, and Abelcet for treatment of systemic murine cryptococcosis. Antimicrobial Agents and Chemotherapy 42, 899–902.[Abstract/Free Full Text]

14 . Hostetler, J. S., Clemons, K. V., Hanson, L. H. & Stevens, D. A. (1992). Efficacy and safety of amphotericin B colloidal dispersion compared with those of amphotericin B deoxycholate suspension for treatment of disseminated murine cryptococcosis. Antimicrobial Agents and Chemotherapy 36, 2656–60.[Abstract]

15 . Hostetler, J. S., Hanson, L. H. & Stevens, D. A. (1993). Effect of hydroxypropyl-beta-cyclodextrin on efficacy of oral itraconazole in disseminated murine cryptococcosis. Journal of Antimicrobial Chemotherapy 32, 459–63.[Abstract]

16 . Stevens, D. A. & Aristizabal, B. H. (1997). In vitro antifungal activity of novel azole derivatives with a morpholine ring, UR-9746 and UR-9751, and comparison with fluconazole. Diagnostic Microbiology and Infectious Disease, 29, 103–6.[ISI][Medline]

17 . Chen, S. A., Shalaby, M. R., Crase, D. R., Palladino, M. A. & Baughman, R. A. (1988). Pharmacokinetics of recombinant murine interferon-gamma and human interferon-alphaA/D (Bgl). administered in concert and their influence on natural killer cell function in mice. Journal of Interferon Research 8, 597–608.[ISI][Medline]

18 . Lipovsky, M. M., Juliana, A. E., Gekker, G., Hu, S., Hoepelman, A. I. & Peterson, P. K. (1998). Effect of cytokines on anticryptococcal activity of human microglial cells. Clinical and Diagnostic Laboratory Immununology 5, 410–11.

19 . Perfect, J. R., Granger, D. L. & Durack, D. T. (1987). Effects of antifungal agents and gamma interferon on macrophage cytotoxicity for fungi and tumor cells. Journal of Infectious Diseases 156, 316–23.[ISI][Medline]

20 . Wolf, J. E. & Massof, S. E. (1990). In vivo activation of macrophage oxidative burst activity by cytokines and amphotericin B. Infection and Immunity 58, 1296–300.[ISI][Medline]

21 . Herrmann, J. L., Dubois, N., Fourgeaud, M., Basset, D. & Lagrange, P. H. (1994). Synergic inhibitory activity of amphotericin B and gamma interferon against intracellular Cryptococcus neoformans in murine macrophages. Journal of Antimicrobial Chemotherapy 34, 1051–8.[Abstract]

22 . Clemons, K. V., Brummer, E. & Stevens, D. A. (1994). Cytokine treatment of central nervous system infection: efficacy of interleukin-12 alone and synergy with conventional antifungal therapy in experimental cryptococcosis. Antimicrobial Agents and Chemotherapy 38, 460–4.[Abstract]

23 . Kawakami, K., Tohyama, M., Xie, Q. & Saito, A. (1996). IL-12 protects mice against pulmonary and disseminated infection caused by Cryptococcus neoformans. Clinical and Experimental Immunology 104, 208–14.[ISI][Medline]

24 . Reardon, C. C., Kim, S. J., Wagner, R. P. & Kornfeld, H. (1996). Interferon-gamma reduces the capacity of human alveolar macrophages to inhibit growth of Cryptococcus neoformans in vitro. American Journal of Respiratory Cell and Molecular Biology 15, 711–5.[Abstract]

Received 19 November 1999; returned 25 January 2000; revised 9 March 2000; accepted 22 May 2000