Synergic inhibitory activity of amphotericin-B and γ interferon against intracellular Cryptococcus neoformans in murine macrophages

Abstract

Cryptococcus neoformans is responsible for pulmonary and meningal infections in HIV patients. The lack of effective cellular cooperation caused by the low level of CD4⁺ cells, and the resistance of C. neoformans to phagocytosis allows growth and persistence of the yeast in the host. We describe here an in-vitro model of intracellular replication of C. neoformans inside J774-A.1 macrophages, and the determination of the intracellular antifungal activity of amphotericin B and fluconazole alone or in association with IFN-γ. The maximum inhibitory effect was observed with one MIC of amphotericin B and 100 or 1000 1U/mL of IFN-γ. amphotericin B alone (at 1× MIC), or either 1 × or 50 × MIC of fluconazole in normal or IFN-γ activated macrophages, did not eradicate the ingested yeast. A potential underlying mechanism of the synergy of amphotericin B in IFN-γ primed macrophages was investigated by measurement of nitrite level and by use of the NO synthase competitive inhibitor, N^G-monomethyl L-arginine (NMMA). One MIC of amphotericin B was able to activate the synthesis of nitrogen reactive intermediates in IFNγ -primed macro-phages. NMMA treated infected macrophages responded less well to IFN-γ priming, resulting in a moderate inhibition in subsequent amphotericin B exposure.