Poster U14 - Investigating the Effects of Parenteral Delivery on Cryptococcus neoformans Biodistribution in Healthy and Immunosuppressed Murine Models

Introduction: In Greek mythology, the Greeks used a ‘Trojan Horse’ to infiltrate the walls of the fortress of Troy. In microbiology, the fungal species Cryptococcus neoformans (Cn) is notorious for circumventing the innate immune system upon inhalation and escaping into circulation as a free cell or, even while engulfed by a phagocyte like a Trojan Horse. Prior literature suggests that Cn infiltrates the central nervous system via three mechanisms: 1) hitchhiking monocytes after phagocytosis across the blood-brain barrier followed by vomocytosis (non-lytic exocytosis), 2) transcytosis upon adhering to the blood-brain barrier and, 3) degradation of the tight junctions of the blood-brain barrier. These mechanisms are exacerbated in immunosuppressed populations due to their inability to combat infection. Despite 220,000 infections, 180,000 deaths annually, and clinical manifestation of cryptococcosis in various organs, Cn dissemination has yet to be elucidated. Therefore, there is a need for a comprehensive study that examines the effects of immunosuppression and the route of infection on Cn dissemination in vivo. We hypothesize that Cn dissemination patterns will differ based on the route of injection and the state of the host’s immune system

Materials and

Methods: Briefly, 6- to 8-week-old healthy BALB/C mice were inoculated with 5E6 colony-forming units of a bioluminescent, luciferase-expressing strain of Cn (NE1269) via intranasal (IN), intravenous (IV), intramuscular (IM), intraperitoneal (IP), and subcutaneous (SubQ) delivery. On days 0, 1, and 3, the mice were injected with 150mg/kg of luciferin and sacrificed. The brain, lungs, liver, spleen, kidneys, popliteal and inguinal lymph nodes were excised and imaged via in vivo imaging system (IVIS) to characterize differential biodistribution of Cn based on the route of administration. Upcoming work will include inoculating mice treated with tofacitinib (30mg/kg/day), a JAK inhibitor, to model immunosuppressed mice to compare Cn dissemination in healthy and immunosuppressed mice.

Results, Conclusions, and Discussions: Our preliminary IVIS data supports our hypothesis that the route of administration does result in differential biodistribution of Cryptococcus neoformans in healthy mice (Figure 1). Interestingly, after IM injection, a bioluminescent signal was observed in the brain as early as Day 0 and Day 1 after IV injection. However, no signal was detected in the brain after IN administration, which models the clinical manifestation of cryptococcal infection. The trends in Cn dissemination after IN or IV inoculation have been observed throughout the literature, with IV injection having more rapid Cn dissemination to the central nervous system compared to IN inoculation. This could be attributed to the fact that the IV-administered can directly interact with the BBB to facilitate neurotropism while IN-administered Cn must interact with the pulmonary innate immune system. Since these mice were otherwise healthy, the intact immune system may prevent Cn escape from the lungs, thus reducing its overall dissemination to other tissues at these early timepoints. As such, we may observe more clinically reflective trends in the immunosuppressed models where the Cn may be able to thwart the efforts of the pulmonary immune system. In the future, we plan to complement our IVIS data with flow cytometry and immunofluorescent histology to determine Cn spatial distribution within the specified organs. So far, our data indicate that depending on the route of infection, there may be a risk to develop cryptococcosis in unsuspecting regions of the body.

Acknowledgements (Optional): Reference: Cruz-Acuña, M., Pacifici, N., & Lewis, J. S. (2019). Vomocytosis: Too much booze, base, or calcium? mBio, 10(6). https://doi.org/10.1128/mbio.02526-19