Research Article: An Acidic Microenvironment Increases NK Cell Killing of Cryptococcus neoformans and Cryptococcus gattii by Enhancing Perforin Degranulation

Date Published: July 11, 2013

Publisher: Public Library of Science

Author(s): Anowara Islam, Shu Shun Li, Paul Oykhman, Martina Timm-McCann, Shaunna M. Huston, Danuta Stack, Richard F. Xiang, Margaret M. Kelly, Christopher H. Mody, Bruce S. Klein.


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.

Partial Text

The yeast, Cryptococcus causes potentially life threatening pneumonia and meningitis. While C. neoformans causes infections more commonly in immunosuppressed individuals such as those with AIDS or hematologic malignancies [1], the tropical fungus C. gattii has recently emerged on Vancouver Island and the pacific northwest of the United States, where it causes respiratory and meningeal disease in otherwise healthy individuals resulting in disability and even death [2]. Both species produce solid tumor-like lesions called cryptococcomas, although they are somewhat more common in C. gattii disease [3], [4]. Cryptococcomas are large focal collections of organisms with infiltrating macrophages and lymphocytes, among other cells [5]. One study reported the presence of lung and brain cryptococcoma in 48% and 18% of cryptococcosis patients, respectively [3]. Unfortunately, the management of cryptococcoma is difficult as they respond poorly to antifungal therapy and sometimes requires surgery to remove the mass due to a space occupying effect in the brain or other tissue [3]. It is not understood why these patients fail to clear these lesions despite possessing a competent immune system; however, the speculation is that unique environmental factors within the cryptococcoma impair the immune response against this fungus. These observations have led us to explore the influence of microenvironmental factors on immune recognition and killing of this pathogen.

In this study, we made the following observations. 1) Anti-cryptococcal activity of YT cells and primary NK cells is enhanced in acidic pH, which is in contrast to their decreased antitumor activity. 2) Anti-cryptococcal activity of YT cells is mediated via killing. 3) Acidic pH does not increase conjugates with Cryptococcus or the active perforin content of YT cells. 4) Acidic pH enhances the Cryptococcus-induced ERK1/2 signaling as well as perforin degranulation in YT cells, suggesting that these are the mechanisms of greater killing in acidic pH. 5) Acidic pH enhances the cryptococcal replication, and within the same pH, YT cells kill the faster replicating Cryptococcus more efficiently than the slower replicating organisms, indicating that faster cryptococcal replication also plays a role in greater killing in acidic pH. 6) NK cells were found within a cerebral and a lung cryptococcoma. While numerous granzyme B positive cells were found intimately associated with the organism, very few perforin positive cells were identified within the cryptococcoma.




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