Mechanisms by Which Interleukin-12 Corrects Defective NK Cell Anticryptococcal Activity in HIV-Infected Patients

ABSTRACTCryptococcus neoformansis a pathogenic yeast and a leading cause of life-threatening meningitis in AIDS patients. Natural killer (NK) cells are important immune effector cells that directly recognize and killC. neoformansvia a perforin-dependent cytotoxic mechanism. We previously showed that NK cells from HIV-infected patients have aberrant anticryptococcal killing and that interleukin-12 (IL-12) restores the activity at least partially through restoration of NKp30. However, the mechanisms causing this defect or how IL-12 restores the function was unknown. By examining the sequential steps in NK cell killing ofCryptococcus, we found that NK cells from HIV-infected patients had defective binding of NK cells toC. neoformans. Moreover, those NK cells that bound toC. neoformansfailed to polarize perforin-containing granules to the microbial synapse compared to healthy controls, suggesting that binding was insufficient to restore a defect in perforin polarization. We also identified lower expression of intracellular perforin and defective perforin release from NK cells of HIV-infected patients in response toC. neoformans. Importantly, treatment of NK cells from HIV-infected patients with IL-12 reversed the multiple defects in binding, granule polarization, perforin content, and perforin release and restored anticryptococcal activity. Thus, there are multiple defects in the cytolytic machinery of NK cells from HIV-infected patients, which cumulatively result in defective NK cell anticryptococcal activity, and each of these defects can be reversed with IL-12.IMPORTANCEThe mechanisms by which NK cells bind directly to pathogens and deploy their deadly cytolytic machinery during microbial host defense are only beginning to be elucidated. With the goal of understanding this process, we used NK cells from HIV-infected patients, which were known to have a defect in killing ofCryptococcus neoformans. Taking advantage of previous studies that had shown that IL-12 restored killing, we used the cytokine as a gain-of-function approach to define the relevance of multiple steps in the recognition and cytolytic pathway. We demonstrated that NK cells from HIV-infected patients failed to killCryptococcusdue to defects in perforin expression, granule polarization, and release of perforin. Additionally, IL-12 restored recognition ofC. neoformansthrough binding of the NK-activating receptor NKp30. These observations identify important mechanisms used by NK cells to kill microbes and determine that defects in NK cells from HIV-infected patients are reversible.

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

ABSTRACTNatural killer (NK) cells directly recognize and kill fungi, such as the pathogenic fungusCryptococcus 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 toC. neoformans. Furthermore, pharmacologic inhibition with an Src kinase inhibitor blockedC. 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.

Differential Activation of Peritoneal Cells by Subcutaneous Treatment of Rats with Cryptococcal Antigens

ABSTRACT Previous studies in our laboratory have shown that the subcutaneous pretreatment of rats with heat-killed cells (HKC) of Cryptococcus neoformans emulsified in complete Freund adjuvant (CFA) promotes protective immunity against an intraperitoneal challenge with C. neoformans. In contrast, subcutaneous treatment with the capsular polysaccharide (PSC) emulsified in CFA exacerbates the cryptococcal infection. The purpose of this study was to analyze the mechanisms involved in these phenomena. Adherent peritoneal cells from rats treated with HKC-CFA showed upregulated ED2, CD80, and CD86 expression; an increase in the level of production of anticryptococcal metabolites; and the enhanced production of interleukin-12 (IL-12) in comparison with the findings for cells from rats treated with CFA-phosphate-buffered saline (PBS). Adherent peritoneal cells from rats treated with PSC-CFA, however, also presented upregulated ED2, CD80, and CD86 expression compared to the level of expression for peritoneal cells from controls, but these cells showed an increase in arginase activity and decreased levels of production of IL-12 and tumor necrosis factor (TNF) compared with the activity and levels of production by peritoneal cells from CFA-PBS-treated rats. In addition, treatment with HKC-CFA resulted in a rise in the phagocytic and anticryptococcal activities of adherent peritoneal cells compared to those for control rats. However, adherent peritoneal cells from rats treated with PSC-CFA presented a reduction in anticryptococcal activity in comparison with that for cells from animals treated with CFA-PBS. These results show the differential activation between adherent peritoneal cells from HKC-CFA- and PSC-CFA-treated rats, with this differential activation at the primary site of infection possibly being responsible, at least in part, for the phenomena of protection and exacerbation observed in our model.

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

ABSTRACT NK cells, in addition to possessing antitumor and antiviral activity, exhibit perforin-dependent microbicidal activity against the opportunistic pathogen Cryptococcus neoformans. However, the factors controlling this response, particularly whether the pathogen itself provides an activation or rearming signal, are largely unknown. The current studies were performed to determine whether exposure to this fungus alters subsequent NK cell anticryptococcal activity. NK cells lost perforin and mobilized lysosome-associated membrane protein 1 to the cell surface following incubation with the fungus, indicating that degranulation had occurred. Despite a reduced perforin content during killing, NK cells acquired an enhanced ability to kill C. neoformans, as demonstrated using auxotrophs that allowed independent assessment of the killing of two strains. De novo protein synthesis was required for optimal killing; however, there was no evidence that a soluble factor contributed to the enhanced anticryptococcal activity. Exposure of NK cells to C. neoformans caused the cells to rearm, as demonstrated by increased perforin mRNA levels and enhanced loss of perforin when transcription was blocked. Degranulation alone was insufficient to provide the activation signal as NK cells lost anticryptococcal activity following treatment with strontium chloride. However, NK cells regained the activity upon prolonged exposure to C. neoformans, which is consistent with activation by the microbe. The enhanced cytotoxicity did not extend to tumor killing since NK cells exposed to C. neoformans failed to kill NK-sensitive tumor targets (K562 cells). These studies demonstrate that there is contact-mediated microbe-specific rearming and activation of microbicidal activity that are necessary for optimal killing of C. neoformans.