Src family kinases are required for nk cell microbicidal activity to cryptococcus neoformans

Abstract

NK cells are well known for their ability to recognize and kill tumor and virally infected cells. Numerous studies have now demonstrated that NK cells also possess the ability to directly recognize and kill bacteria, parasites and fungi such as the pathogenic fungus Cryptococcus neoformans. However, the precise signalling pathways governing NK cell microbicidal activity are still unknown. Previously, it was reported that activation of a conserved PI3K -ERK l /2 pathway is necessary for NK cell anticryptococcal activity. Using YT (NK-like cell line) and primary human NK cells, we sought to identify the upstream signalling elements that lead to the activation of this pathway. We demonstrate that Src family kinases, in particular Fyn, were activated in response to C. neoformans. Furthermore, pharmacologic inhibition with a Src family kinase inhibitor (dasatinib) was found to block C. neoformans induced activation of PI3K and ERKI/2 and abrogate cryptococcal killing. At the same time, dasatinib was observed to disrupt the polarization of perforin-containing granules towards the NK cell-cryptococcal synapse but had no effect on conjugate fom1ation between NK cells and C. neoformans. Finally, double (but not single) siRNA knockdown of two specific Src family kinases, Fyn and Lyn, was found to completely block cryptococcal killing. \Ve also demonstrate that neither Syk nor ZAP-70 were activated in response to C. neoformans. Moreover, anticryptococcal activity was found to be insensitive to both pham1acologic inhibition with a Syk inhibitor (Syk inhibitor II) and siRNA knockdown of ZAP-70. Together these data demonstrate a mechanism whereby the Src family kinases, Fyn and Lyn, redundantly mediate anticryptococcal activity through the IT AM (Syk/ZAP-70) independent activation of PBK-ERKI/2, which in turn facilitates killing by inducing the polarization of perforin-containing granules to the NK cell-cryptococcal synapse.