Perforin-dependent cryptococcal microbicidal activity in NK cells requires PI3K-dependent ERK1/2 signaling

Previously, NK cells have been reported to kill the opportunistic fungal pathogen Cryptococcus neoformans through a perforin-dependent mechanism; however, the receptor and signaling involved are unknown. In this report we sought to identify the signaling pathways activated and required for direct perforin-mediated killing of microbes. In this study, using the NK-like cell line YT and primary peripheral blood NK cells, it is demonstrated that YT cells kill C neoformans and that the killing is accompanied by the activation of PI3K. We demonstrate that inhibition of either the catalytic subunit (using a pharmacological inhibitor) or the a-regulatory subunit (using small interfering RNA knockdown) of PI3K significantly inhibited the killing of C neoformans. Downstream of PI3K, ERK1/2 was activated in a PI3K-dependent fashion and was required for cryptococcal killing. Furthermore, we demonstrate that perforin release from YT cells can be detected by 4 h after contact of the YT cells with C neoformans and that the release of perforin is blocked by pharmacological inhibition of either PI3K or ERK1/2. Defective degranulation is rooted in the inability to polarize perforin-containing granules toward the target. Finally, we demonstrate that PI3K-ERK1/2-dependent signaling is activated and required for the killing of C neoformans by primary NK cells. Taken together, these data identify a conserved PI3K-ERK1/2 pathway that is used by NK cells during the direct killing of C neoformans and demonstrate that the pathway is essential in the formation and activation of the microbicidal mechanism. The Journal of Immunology, 2007,178: 6456-6464.