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-gamma. The maximum inhibitory effect was observed with one MIC of amphotericin B and 100 or 1000 IU/mL of IFN-gamma. amphotericin B alone (at 1 x MIC), or either 1 x or 50 x MIC of fluconazole in normal or IFN-gamma activated macrophages, did not eradicate the ingested yeast. A potential underlying mechanism of the synergy of amphotericin B in IFN-gamma primed macrophages was investigated by measurement of nitrite level and by use of the NO synthase competitive inhibitor, NG-monomethyl L-arginine (NMMA). One MIC of amphotericin B was able to activate the synthesis of nitrogen reactive intermediates in IFN gamma-primed macrophages. NMMA treated infected macrophages responded less well to IFN-gamma priming, resulting in a moderate inhibition in subsequent amphotericin B exposure.

Reduction of NO Synthase Expression and Tumor Necrosis Factor Alpha Production in Macrophages by Amphotericin B Lipid Carriers

“…Our results indicating that AMB-induced NO production only in the presence of IFN-␥ as a costimulator are in accord with those obtained by several other authors in vitro (21,31,37) as well as in vivo (25). The dose of IFN-␥ necessary for costimulation of mouse peritoneal macrophages (20 IU ml) (Fig.…”

“…These products have been shown to reinforce the antimicrobial activity of AMB by their cytotoxic effects (41,50,51). On the other hand, AMB has been found to induce nitric oxide (NO) production as a result of NO synthase (NOS) induction only in the presence of a costimulator such as gamma interferon (IFN-␥), lipopolysaccharide (LPS), and M-CSF (16,21,37). This NO production may also increase the antimicrobial activity of AMB (29,49).…”

A Repurposing Approach Identifies Off-Patent Drugs with Fungicidal Cryptococcal Activity, a Common Structural Chemotype, and Pharmacological Properties Relevant to the Treatment of Cryptococcosis

“…Previous work indicates that neither FLU nor AMB has activity against intracellular C. neoformans (24). From our set of lysosomotropic anticryptococcal drugs, we selected two agents known to accumulate within macrophages (amiodarone and thioridazine) and found that both were active against C. neoformans within J774 mouse phagocytes.…”

“…neoformans traffics to the phagolysosome of macrophages, replicates, and then escapes as part of its pathogenic life cycle in humans (23). Unfortunately, the two most widely used anticryptococcal drugs, AMB and FLU, do not appear to have activity toward intraphagocytic C. neoformans (24). Thus, molecules able to target C. neoformans within this important niche could serve as either useful stand-alone agents or, possibly, adjuvants to agents currently in use.…”

“…Consequently, anticryptococcal drugs that are able to penetrate and have activity within the phagolysosome are potentially useful (25). Previous studies have shown that neither AMB nor FLU has significant antifungal activity toward C. neoformans once the organism is within the phagocyte (24). The canonical lysosomotropic drug chloroquine has been shown to have activity against C. neoformans within phagocytes (25).…”