Perforin-Dependent Cryptococcal Microbicidal Activity in NK Cells Requires PI3K-Dependent ERK1/2 Signaling

Abstract

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 α-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.

Enhanced Innate Immune Responsiveness to PulmonaryCryptococcus neoformansInfection Is Associated with Resistance to Progressive Infection

“…Both NF-B and PI3K/Akt are well-recognized elements of the innate immune response against infection (14,22). TLR4 stimulation by cryptococcal GXM has been shown to activate NF-B (60), and PI3K/Akt has been implicated in the killing of C. neoformans organisms by human NK cells (66). Therefore, pharmacologic inhibitors and immu-noblotting were used to study and confirm the differential activation of NF-B and PI3K/Akt in SJL/J macrophages following cryptococcal stimulation.…”

“…volvement of NF-B and PI3K in C. neoformans Fc␥R signaling has been shown in human microglial cells (61) and NK cells (66), yet the role of these pathways in C. neoformans infectionassociated macrophage signaling has not been well studied. Therefore, as a first step in defining the role of these intermediates in the production of TNF-␣, we treated resident peritoneal macrophages with pharmacologic inhibitors of NF-B (BAY11-7082) and PI3K (LY294002) prior to stimulation with C. neoformans infection.…”

Cryptococcal Interactions with the Host Immune System

“…Although NK cells express both proteins, perforin is the main mediator of anticryptococcal killing via a PI3K-dependent ERK1/2 signaling pathway (120,126,195). In contrast, CD4 ϩ and CD8…”

Cryptococcus neoformansDirectly Stimulates Perforin Production and Rearms NK Cells for Enhanced Anticryptococcal Microbicidal Activity

“…In particular, at least some of the signaling molecules that NK cells require to elicit responses to these two targets are conserved. NK cells require phosphoinositol 3-kinase and extracellular signal-regulated kinase 1/2 phosphorylation for both tumor lysis and killing of C. neoformans (4,6,21,58,61,62). However, NK cells bind maximally to tumor targets after 20 min of exposure through formation of an intimate synapse involving LFA-1, and subsequent killing is detected 10 to 15 min following binding (43).…”