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444. Dendritic Cells Perform Vomocytosis of Cryptococcus

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444. Dendritic Cells Perform Vomocytosis of Cryptococcus Dendritic Cells Perform Vomocytosis of Cryptococcus neoformans Noah Pacifici, Melissa Cruz-Acuña, Neeraj Senthil, and Jamal Lewis University of California, Davis, Davis, CA Statement of Purpose Pathogens have evolved over time to evade the host immune system in various ways. For instance, the fungal species Cryptococcus neoformans (CN), following engulfment by phagocytes, has been observed to stay alive within the acidic phagolysosome and escape through a process called vomocytosis1. Using this phenomenon, CN utilizes host immune cells to disseminate infection throughout the body. One condition due to infection of this microbe is cryptococcal meningitis (CM). Primarily affecting immunocompromised individuals, including an estimated 220,000 HIV/AIDS patients, CM causes ~181,000 deaths/ year worldwide2. Greater understanding of vomocytosis could lead to the development of new CM treatments for patients, as well as novel biomaterial Figure 1. (A) An example image of a vomocytosis event of particulate vaccines. Current studies have mainly focused CN escaping from a DC. (B) Measured expulsions and cell-to- on vomocytosis occurrences in macrophages, as alveolar cell transfer events of live and heat-killed CN from DCs. macrophages are the first immune cell to phagocytose CN Timing of vomocytosis events were also measured for live (C) and ultimately expel this fungal pathogen. However, CN and heat-killed (D) groups. (n=1) may have the ability to induce vomocytosis in other phagocytes due to conserved molecular machinery. windows per condition. Expulsion events were counted as Recently, Yang et al.3 observed vomocytosis-like any time one or more CN escaped from a DC (Figure 1A). expulsion of CN from neutrophils, the most abundant phagocyte found in the body. This finding shows how CN Results and Discussion can use vomocytosis to continually evade the innate Vomocytosis expulsion events and cell-to-cell transfer immune system (macrophages and neutrophils) as it events were recorded for DCs that had phagocytosed live disseminates throughout the body. Another important or heat-killed CN (Figure 1B). 200 DCs containing live phagocyte is the dendritic cell (DC), an antigen presenting fungal cells were observed; of these, 97 expulsions (48.5%) cell that links the innate and adaptive immune system. This and 16 cell-to-cell transfer (8%) events occurred. For the specialized cell scavenges foreign material from around the heat-killed control, 150 DCs were monitored, showing 11 body and shuttles to the lymph node to present pathogen- expulsions (7.3%) and 1 cell-to-cell transfer (0.67%). In specific antigens and help mount an adaptive immune the live fungal condition, vomocytosis events occurred 3- response. This study aims to investigate the potential 13 hrs with an average time of expulsion at 7.5 hrs (Figure occurrence of vomocytosis of CN in DCs. Expulsion from 1C). In the heat-killed control, few vomocytosis events DCs could suggest a new method by which this fungal occurred 3-5 hrs and 8-13 hrs with an average time of pathogen evades the innate and adaptive immune system. expulsion at 8.2 hrs (Figure 1D). The live fungal group displayed high levels of vomocytosis from DCs. Materials and Methods Interestingly, the heat-killed control group should not have Bone marrow derived DCs were harvested from 8 to 12- had any expulsion events; however, 11 events were week-old C57BL/6 mice and differentiated using DC observed. This may be due to inefficient killing of CN media containing 20ng/ml GM-CSF. 10-day old DCs were using heat or inaccuracy of the time lapse method of used for vomocytosis experiments. Cryptococcus quantification. The vomocytosis timing is in line with neoformans var. grubii H99 were cultured by inoculating a literature reported values of 5-15 hrs. Notably, the rate single colony into yeast peptone dextrose media and left to measured in this experiment (48.5%) is higher than shake overnight at 30°C until cloudy. Prior to previously published rates of 5-20%, suggesting that DCs phagocytosis, CN were opsonized by incubation in may be even more likely to perform vomocytosis than 10ug/ml 18B7 mAb and 50% human serum for 1 hr. As a macrophages. control, CN were heat-killed at 60°C for 30 min. Fungal cells were fed to DCs at a 5:1 ratio in the presence of 10% Conclusions human serum in media and incubated for 2 hrs to allow This preliminary study confirmed that DCs have the ability phagocytosis to occur. Afterward, unphagocytosed CN to perform vomocytosis of CN. Future work includes were removed by washing 3 times with PBS. Time lapse validation of DCs purity using cell sorting to remove microscopy was performed using a BZ-X all-in-one contaminating macrophage populations and investigation Keyence microscope from 3 hrs until 17 hrs after of DC trafficking in vivo after phagocytosis of CN. phagocytosis at 4 min intervals in 6 different viewing Acknowledgements: NIH R35 Grant Funding, NIGMS-Funded Pharmacology Training Program (T32GM099608), NSF Graduate Research Fellowship References: 1Alvarez M. Curr Biol. 2006;16:2161-2165. 2CDC 3Yang X. Cell Comm and Signaling. 2019;17(1), 1-9. .
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