An acidic microenvironment increases NK cell killing of Cryptococcus neoformans and Cryptococcus gattii by enhancing perforin degranulation.

Cryptococcus gattii and Cryptococcus neoformans are encapsulated yeasts that can produce a solid tumor-like mass or cryptococcoma. Analogous to malignant tumors, the microenvironment deep within a cryptococcoma is acidic, which presents unique challenges to host defense. Analogous to malignant cells, NK cells kill Cryptococcus. Thus, as in tumor defense, NK cells must kill yeast cells across a gradient from physiologic pH to less than 6 in the center of the cryptococcoma. As acidic pH inhibits anti-tumor activities of NK cells, we sought to determine if there was a similar reduction in the anticryptococcal activity of NK cells. Surprisingly, we found that both primary human NK cells and the human NK cell line, YT, have preserved or even enhanced killing of Cryptococcus in acidic, compared to physiological, pH. Studies to explore the mechanism of enhanced killing revealed that acidic pH does not increase the effector to target ratio, binding of cytolytic cells to Cryptococcus, or the active perforin content in effector cells. By contrast, perforin degranulation was greater at acidic pH, and increased degranulation was preceded by enhanced ERK1/2 phosphorylation, which is essential for killing. Moreover, using a replication defective ras1 knockout strain of Cryptococcus increased degranulation occurred during more rapid replication of the organisms. Finally, NK cells were found intimately associated with C. gattii within the cryptococcoma of a fatal infection. These results suggest that NK cells have amplified signaling, degranulation, and greater killing at low pH and when the organisms are replicating quickly, which would help maintain microbicidal host defense despite an acidic microenvironment

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

Cryptococcus neoformans is 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 kill C. neoformans via 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 of Cryptococcus, we found that NK cells from HIV-infected patients had defective binding of NK cells to C. neoformans. Moreover, those NK cells that bound to C. neoformans failed 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 to C. 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

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

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

NK cell mediated cryptococcal killing is proportional to the rate of replication of the organisms.

<p>(A&B) H99<i>ras1Δ</i> and H99<i>ras1Δ</i>+<i>RAS1</i> were cultured alone at 37°C for 20 h in pH 7.4 (A) and pH 6.6 (B). Fold increase in CFU was determined by dividing the CFU_{t = 20 h} by CFU_t = 0. (C&D) H99<i>ras1Δ</i> and H99<i>ras1Δ</i>+<i>RAS1</i> were cultured with or without YT cells at 37°C for 20 h in pH 7.4 (C) and pH 6.6 (D). The CFU reduction was calculated as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003439#ppat-1003439-g001" target="_blank">figure 1</a>. Each bar represents the mean of four replicates. Data are representative of three experiments. (E&F) H99<i>ras1Δ</i> and H99<i>ras1Δ</i>+<i>RAS1</i> were either cultured alone or with YT cells at 37°C for 20 h at pH 7.4 (E) and pH 6.6 (F) and the CFUs for each time point (t = 0 and t = 20 h) are shown. For H99<i>ras1Δ</i>, two different starting inocula are shown. Each bar represents the mean of four replicates. Data are representative of two experiments.</p

Anti-cryptococcal activity of NK cells is greater in pH 6.6 compared to pH 7.4.

<p>YT cells were co-cultured with different strains of <i>Cryptococcus</i>; <i>C. gattii</i> strain R265 (A), <i>C. neoformans</i> strain H99 (B), or <i>C. neoformans</i> strain B3501 (C). Primary human NK cells were co-cultured with B3501 (D) in different pH for 20 h. The reduction in CFU was calculated by deducting the number of experimental CFU_{t = 20 h} (<i>Cryptococcus</i> cultured with NK cells) from the number of control CFU_{t = 20 h}(<i>Cryptococcus</i> cultured alone). Each bar represents the mean of four replicates. ns, not significant, *, p value≤0.05, **, p value≤0.01 ***, p value≤0.001 compared to pH 7.4. Data are representative of three (A, C and D) or two (B) experiments. (E) CFSE-labeled K562 cells (tumor cells) were cultured either alone or with YT cells at 50∶1 E∶T ratio for 4 h. The number of K562 cells killed by YT cells was calculated as described in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003439#s4" target="_blank">materials and methods</a>. Each bar represents the mean of three replicates *, p value≤0.05 compared to pH 7.2. Data are representative of three experiments.</p

<i>Cryptococcus</i>-induced perforin loss in YT cells is greater in pH 6.6 compared to pH 7.4.

<p>(A) YT cells were cultured alone or with live <i>Cryptococcus</i> (B3501 in upper panel and R265 in lower panel, E∶T, 1∶10) in pH 7.4 and 6.6 for 20 hours. Cells were harvested, made permeable and immunolabeled with either anti-perforin or isotype control antibodies. Data are representative of three experiments. (B) Reduction in perforin MFI was calculated by deducting the MFI of <i>Cryptococcus</i> stimulated YT from the MFI of unstimulated YT. (C) Same as A except for heat killed B3501. Data are representative of three experiments. (D) YT cells were pretreated for 2 h with 10 nM concanamycin A (B3501+YT+CMA) and anticryptococcal activity was compared with untreated group (B3501+YT). ***, p<0.0001. Data are representative of three experiments.</p

Acidic pH does not increase the amount of active perforin within YT cells.

<p>YT cells alone were cultured in complete media at pH 7.4 and 6.6 for 20 h. Cells were harvested at the beginning and end of incubation, made permeable and immunolabeled with either anti-perforin or isotype control antibodies and then analyzed by flow cytometry. Data are representative of three experiments.</p

Acidic pH does not increase the effector to target (E∶T) ratio.

<p>(A) YT cells were cultured for 20 h in different pH (7.4, 7.2, 7.0, 6.8, and 6.6) and the numbers of viable YT cells were counted by light microscopy using trypan blue staining at the beginning and end of the incubation. Fold increase in the number of viable YT cells was calculated by dividing the number of viable YT cells at 20 h by the number of viable YT cells at t = 0. Each bar represents the mean of three replicates. Data are representative of three experiments. <i>C. gattii</i> strain R265 (B), <i>C. neoformans</i> strains H99 (C), or B3501 (D) were cultured in different pH (7.4, 7.2, 7.0, 6.8, 6.6) for 20 h. The number of viable organisms was detected by counting the CFU on agar plates. Each bar represents the mean of four replicates. *, p value≤0.05, ***, p value≤0.001 compared to the fold increase at pH 7.4. Data are representative of three experiments.</p

Cryptococcomas contain numerous granzyme B positive, but very few perforin positive NK cells.

<p>Case 1: The brain was removed at autopsy and fixed in formalin with subsequent sectioning and examination. Sections were taken from the cryptococcoma in the cerebral cortex and stained with mucicarmine (200×) (A); or stained with PAS and labeled with either anti-CD56 (400×) (B); anti-CD57 (600×) (C); anti-granzyme B (1000×) (D); or anti-perforin (E, 600×) antibody. <i>Cryptococcus</i> (open arrow) in association with labeled cells (solid arrows) are shown. Perforin positive cells (brown) in NK cell lymphoma in lung, shown as a positive control for perforin (F). Case 2: Wedge resection of lung cryptococcoma, fixed in formalin with subsequent sectioning, PAS staining and labeled with either anti-CD57 (G, 400×), anti-granzyme B (H, 1000×) or anti-perforin (I, 600×). No organisms are present in figure G. The organisms stain bright pink with PAS, whilst positive staining for antigens is dark brown.</p

Acidic pH does not increase the number of conjugates between YT cells and <i>C. neoformans</i>.

<p>TRITC-labeled YT cells were cultured with CFSE-labeled B3501 in pH 7.4 and pH 6.6 for up to 300 minutes. At the indicated times, samples were isolated and fixed in ice-cold PBS containing 0.5% formalin and data acquired immediately using flow cytometry. Cells were identified based on their labeling and the percentage of events that simultaneously contained both TRITC-labeled YT cells and CFSE-labeled B3501 was determined. At least 5000 NK cells and 300 conjugates were counted at each time point. Data are representative of three experiments.</p