Requirement and Redundancy of the Src Family Kinases Fyn and Lyn in Perforin-Dependent Killing of Cryptococcus neoformans by NK Cells

Abstract

Natural killer (NK) cells directly recognize and kill fungi, such as the pathogenic fungus Cryptococcus neoformans, via cytolytic mechanisms. However, the precise signaling pathways governing this NK cell microbicidal activity and the implications for fungal recognition are still unknown. Previously, it was reported that NK cell anticryptococcal activity is mediated through a conserved phosphatidylinositol 3-kinase-extracellular signal-regulated kinase 1/2 (PI3K-ERK1/2) pathway. Using YT (a human NK-like cell line) and primary human NK cells, we sought to identify the upstream, receptor-proximal signaling elements that led to fungal cytolysis. We demonstrate that Src family kinases were activated in response to C. neoformans. Furthermore, pharmacologic inhibition with an Src kinase inhibitor blocked C. neoformans-induced downstream activation of PI3K and ERK1/2 and abrogated cryptococcal killing. At the same time, the inhibitor disrupted the polarization of perforin-containing granules toward the NK cell-cryptococcal synapse but had no effect on conjugate formation between the organism and the NK cell. Finally, small interfering RNA (siRNA) double (but not single) knockdown of two Src family kinases, Fyn and Lyn, blocked cryptococcal killing. Together these data demonstrate a mechanism whereby the Src family kinases, Fyn and Lyn, redundantly mediate anticryptococcal activity through the activation of PI3K and ERK1/2, which in turn facilitates killing by inducing the polarization of perforin-containing granules to the NK cell-cryptococcal synapse.

Ras-related C3 Botulinum Toxin Substrate (Rac) and Src Family Kinases (SFK) Are Proximal and Essential for Phosphatidylinositol 3-Kinase (PI3K) Activation in Natural Killer (NK) Cell-mediated Direct Cytotoxicity against Cryptococcus neoformans

“…We also investigated PLC␥ by pretreating YT cells with the PLC␥ inhibitor, U-73122, at concentrations found to inhibit tumor killing (34). The anti-cryptococcal activity was examined, and we found that the addition of YT cells significantly reduced the number of cryptococcal cfu, which is consistent with our previous observations of killing (1,27,35). Despite using concentrations of U-73122 in excess to inhibit PLC␥, there was no impact on YT anti-cryptococcal killing, suggesting that PLC␥ was not involved in NK cell-mediated cryptococcal killing (Fig.…”

“…We found that the addition of YT cells to C. neoformans cultures reduced the number of cfu for both strain B3501 (Fig. 1, F and G) and strain 145 (data not shown), demonstrating NK cell killing of Crypto- coccus (1,24,27) and demonstrating that the vehicle control did not affect this activity. Treatment of YT cells with EHT1864 or Rac inhibitor II caused a dose-dependent increase in cfu compared with vehicle controls, indicating a loss of anti-cryptococcal activity (Fig.…”

“…Rac and SFK Activate Anti-cryptococcal Activity through Independent Signaling Pathways-Previous studies have established that SFK are required for the activation of the PI3K 3 Erk signaling cascades in NK cell-mediated cryptococcal killing (4,27). Because inhibition of Rac prevented PI3K activity, we hypothesized that Rac would be distal to SFK but proximal to PI3K.…”

“…Although two different pathways were initiated, both pathways converged into a Vav1 3 PLC␥ pathway that led to degranulation (25). In cryptococcal killing, the SFK 3 PI3K 3 Erk cytotoxicity pathway has been identified (4,27). We considered the possibility that multiple anti-cryptococcal signaling pathways converge on to this central pathway.…”

STAT1 Signaling within Macrophages Is Required for Antifungal Activity against Cryptococcus neoformans

“…Furthermore, NO production by STAT1-mediated M1 macrophages is critical for control of the intracellular growth of C. neoformans. Even in the presence of other immune cells with demonstrated anticryptococcal activity, including DCs, neutrophils, and NK cells (61,63,64,76,77), the loss of macrophages capable of polarizing to an M1 phenotype is crippling to the host's ability to mount protective responses. We definitively show that STAT1-mediated M1 macrophage activation is crucial to anticryptococcal responses, providing an effector cell population and mechanism by which the host executes protective responses against C. neoformans.…”