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

Abstract

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.

Author Summary

Immune responses that protect from infection must occur in a variety of unique and potentially hostile environments. Within these environments, acidosis causes profound affects on protective responses. Low pH can occur in focal tumor-like infections, such as in a cryptococcoma produced by the fungal pathogen Cryptococcus. Similarly, low pH occurs in focal malignant tumors. It follows that Cryptococcus and malignant cells can both be killed by NK cells, which provide an important mechanism of host defense. Thus, we asked whether low pH, which impairs tumor killing, might also affect NK cell killing of Cryptococcus. Surprisingly, despite impaired tumor killing, NK cells possess enhanced killing of Cryptococcus at low pH. The mechanism involved a gain in intracellular signal transduction that led to enhanced perforin degranulation. This led us to examine NK cells in persistent cryptococcoma of a fatal brain infection and lung. We found that NK cells associate with Cryptococcus within the cryptococcoma, but perforin is reduced. These studies suggest NK cell cytotoxicity need not be impaired at low pH, and that enhanced signal transduction and degranulation at low pH might be used to enhance host defense.