The in vitro effects of interferon-gamma, alone or in combination with amphotericin B, tested against the pathogenic fungi Candida albicans and Aspergillus fumigatus

Abstract

Objective: Recent studies into the antifungal activity of NK-cells against the Aspergillus fumigatus have presented differing accounts on their mode of antifungal activity. One of these mechanisms proposed that NK-cells may kill the fungus via the direct effects of exposure to Interferon gamma (IFN-γ).

Results: In this study we investigated the direct antifungal effects of recombinant human IFN-γ against a range of pathogenic fungi by measuring cellular damage using an XTT-based assay and cell viability through plate counts. It was found that 32 pg/ml of IFN-γ exhibited a significant but small antifungal effect on A. fumigatus (p = 0.02), Aspergillus flavus (p = 0.04) and Saccharomyces cerevisiae (p = 0.03), inhibiting growth by 6, 11 and 17% respectively. No significant inhibitory effects were observed in Candida species (p > 0.05 for all species tested) or Cryptococus neoformans (p = 0.98). Short term exposure (3 h) to a combination of amphotericin B (1 µg/ml) and IFN-γ (32 pg/ml) increased the effectiveness of amphotericin B against A. fumigatus and S. cerevisiae but not Candida albicans. These data suggest that IFN-γ does not possess strong antifungal activity but can enhance the effect of amphotericin B under some testing conditions against Aspergillus species.

Keywords: Amphotericin B; Antifungals; Aspergillosis; Interferon-gamma.

Fig. 1

A Measurement of inhibition caused by treatment with IFN-γ on several pathogenic fungi using the XTT assay. The results were expressed as the percentage of the metabolic activity of treated cells compared to untreated cells. The absorbance reading at OD₄₉₂ for the untreated control for each species was taken as 100%; the mean OD₄₉₂ value was 0.44 (±0.07). The data shown are means and standard errors of the growth inhibition (treated/untreated control) from three replicate experiments. Data were analysed by t-test to compare low to high doses of IFN-γ (*p < 0.05, **p < 0.01, ***p < 0.001), p-values for each test are shown in the figure. B Measurement of inhibition caused by incubation of several pathogenic fungi with IFN-γ using the XTT assay over an extended dose range. No significant differences in fungal survival were found when treatment with 32 pg/ml to treatment with 50 or 100 pg/ml of IFN-γ were compared. Data in 1B were analysed by one-way ANOVA (p-value shown in the figure) and Dunn’s post-test (p-values shown as asterisks) to compare the range of doses of IFN-γ (*p < 0.05, **p < 0.01, ***p < 0.001). Asterisks indicate that doses are significantly different to the lowest dose

Fig. 2

The effect of short term exposure to IFN-γ alone or in combination with amphotericin B on selected fungi. The addition of (I+; 32 pg/ml) and amphotericin B (A+; 1.0 µg/ml. A++; 2.0 µg/ml) were tested on C. albicans, S. cerevisiae and A. fumigatus. The effect was measured as CFU following a three hour incubation relative to the untreated control samples (100%) were used to calculate the growth inhibition a S. cerevisiae 3.1 × 10⁴/ml (p < 0.0001); b C. albicans 3.5 × 10⁴/ml (p = 0.0011) and c A. fumigatus 4.1 × 10⁴/ml (p = 0.002). The data presented are means and standard errors of growth inhibition (treated/untreated) from, three replicate experiments. The data was analysed by one-way ANOVA and Dunn’s multiple comparison test to compare all treatments (*p < 0.05, **p < 0.01, ***p < 0.001). Three independent experiments were conducted for each treatment