The molecular mechanisms underlying the in vitro immunostimulatory and toxic effects of the antifungal drug, amphotericin B: Role of Toll-like receptor 2 and CD14

The primary clinical limitation of the broad-spectrum antifungal compound, amphotericin B (AmB), is its significant toxicity. Hence AmB is no longer the first line of defense against many deep-seated fungal infections, though it is inexpensive and efficacious. We hypothesized that AmB utilizes receptors highly represented in the innate immune cell repertoire, specifically Toll-like receptors (TLR) and the cluster of differentiation-14 antigen (CD14) co-receptor, to elicit toxic effects. We based this hypothesis principally on 3 underlying facts: (1) AmB's toxic effects are inflammatory in nature, (2) AmB is a product of the Gram-positive bacterium, Streptomyces nodosus , and (3) TLR are widely accepted as pro-inflammatory receptors for microbial products. To investigate our hypothesis we examined primary murine macrophages for TLR- and CD14-dependent inflammatory cytokine production and transfected cell lines for TLR signaling following AmB stimulation. We found that macrophages expressing TLR2 and CD14 responded to AmB stimulation with pro-inflammatory cytokine release, while cells deficient in CD14, TLR2 or the TLR signaling adapter, Myeloid Differentiation protein 88 (MyD88), were unable to respond. Furthermore, AmB stimulated NF-kappaB-driven reporter activity in the THP-1 human monocytic cell line transfected with CD14, and in differentiated THP-1 cells expressing up-regulated levels of CD14, but not in undifferentiated THP-1 cells transfected with vector alone. Moreover, transfection of HEK293 cells with TLR2 and CD14 conferred responsiveness to AmB stimulation, resulting in NF-kappaB-driven reporter activity and chemokine release. In conclusion, we discovered that TLR2 and CD14 are required for AmB inflammatory signaling in mammalian cells in vitro and ex vivo. These data offer a receptor-mediated mechanism for the infusion-related toxicity of AmB and specify targets for therapeutic strategies to eliminate these untoward effects. Elucidation of this mechanism for the AmB-mediated promotion of inflammatory toxicity may facilitate development of therapies to prevent these effects.