Vomocytosis of Cryptococcus neoformans cells from murine, bone marrow-derived dendritic cells

Abstract

Cryptococcus neoformans (CN) cells survive within the acidic phagolysosome of macrophages (MΦ) for extended times, then escape without impacting the viability of the host cell via a phenomenon that has been coined ‘vomocytosis’. Through this mechanism, CN disseminate throughout the body, sometimes resulting in a potentially fatal condition—Cryptococcal Meningitis (CM). Justifiably, vomocytosis studies have focused primarily on MΦ, as alveolar MΦ within the lung act as first responders that ultimately expel this fungal pathogen. Herein, we hypothesize that dendritic cells (DCs), an innate immune cell with attributes that include phagocytosis and antigen presentation, can also act as ‘vomocytes’. Presciently, this report shows that vomocytosis of CN indeed occurs from murine, bone marrow-derived DCs. Primarily through time-lapse microscopy imaging, we show that rates of vomocytosis events from DCs are comparable to those seen from MΦ and further, are independent of the presence of the CN capsule and infection ratios. Moreover, the phagosome-altering drug bafilomycin A inhibits this phenomenon from DCs. Although DC immunophenotype does not affect the total number of vomocytic events, we observed differences in the numbers of CN per phagosome and expulsion times. Interestingly, these observations were similar in murine, bone marrow-derived MΦ. This work not only demonstrates the vomocytic ability of DCs, but also investigates the complexity of vomocytosis regulation in this cell type and MΦ under multiple modulatory conditions. Understanding the vomocytic behavior of different phagocytes and their phenotypic subtypes is needed to help elucidate the full picture of the dynamic interplay between CN and the immune system. Critically, deeper insight into vomocytosis could reveal novel approaches to treat CM, as well as other immune-related conditions.

A Multi-Fluorophore Staining Scheme for Identification and Quantification of Vomocytosis

“…Interestingly, the finding that CQ reduces MΦ vomocytosis rates conflicts with prior studies investigating J774 MΦ cell lines by Ma et al and Nicola et al but is in agreement with other observations in neutrophils and DCs. ,,, The significant increase in vomocytosis rates in BFA-treated MΦ and DC infections differs from observations by Nicola et al (in J774 MΦ) and Pacifici et al (in primary DCs) that BFA treatment decreases expulsion rates. , All these findings differ from results reported by Yang et al, showing that neutrophil vomocytosis rates are unaffected by BFA treatment . Lastly, the observed increase in vomocytosis rates for CYT hi-treated MΦ and DC groups aligns with previous studies investigating J774 MΦs by Alvarez et al and neutrophils by Yang et al , However, again there exists disagreement within the literature, with decreases in CYT-modulated vomocytosis rates seen in J774 MΦs by Dragotakes et al and DCs by Pacifici et al , This inconsistency in prior studies may be due to the variety of cell types, cell sources, and vomocytosis measurement methodologies. Despite some differences compared to already conflicting literature, these findings indicate that the reporter system can measure differences in vomocytosis rates of drug-treated groups with high resolution and repeatability.…”

“…For DCs infected with CN, the BFA and CYT hi groups also recorded higher incidences of vomocytosis measured by the reporter system (Figure 4C). Interestingly, the finding that CQ reduces MΦ vomocytosis rates conflicts with prior studies investigating J774 MΦ cell lines by Ma et 8,16 This inconsistency in prior studies may be due to the variety of cell types, cell sources, and vomocytosis measurement methodologies. Despite some differences compared to already conflicting literature, these findings indicate that the reporter system can measure differences in vomocytosis rates of drugtreated groups with high resolution and repeatability.…”

“…Cells were treated with 100 nM bafilomycin A1 (BFA, Sigma), 4 μM cytochalasin B (CYT hi, Sigma), 100 nM cytochalasin B (CYT lo), or 10 μM chloroquine (CQ, Thermo Fisher Scientific). These drugs and their concentrations were selected based on relevant reference experiments from vomocytosis studies. ,,,, …”

“…Due to the relevance of MΦs in CM infections, vomocytosis studies thus far have primarily focused on the occurrence of this event in this one cell type. However, other phagocytic cell typesneutrophils and dendritic cells (DCs)have recently been documented to perform vomocytosis of CN during infection. However, the underlying mechanisms of this phenomenon are mostly unknown, with only a small handful of phagosomal properties, proteins, and immunological phenotypes known to affect expulsion rates. , Moreover, in the published literature there is much disagreement, which likely stems from the lack of an objective tool to measure vomocytosis.…”

An Image Processing Algorithm for Facile and Reproducible Quantification of Vomocytosis

“…Vomocytosis is where an internalized fungal pathogen escapes from a phagocyte without compromising the viability of both the pathogen and the host cell . This mechanism has been observed and measured in Cryptococcus neoformans (CN) infections of macrophages, neutrophils, and dendritic cells. − CN infections are known to affect immunocompromised patients, disseminating from the lungs to the central nervous system. Notably, vomocytosis is thought to be one of CN’s core mechanisms of immune evasion that allows for traversal through the blood–brain barrier and eventual dissemination into the brain environment .…”