Detection and Quantification of Cryptococcus Uptake by Phagocytic Cells Using Imaging Flow Cytometry

Abstract

Cryptococcus neoformans, the predominant etiological agent of cryptococcosis, is an encapsulated fungal pathogen found ubiquitously in the environment that causes pneumonia and life-threatening infections of the central nervous system. Following inhalation of yeasts or desiccated basidiospores into the lung alveoli, resident pulmonary phagocytic cells aid in the identification and eradication of Cryptococcus yeast through their arsenal of pattern recognition receptors (PRRs). PRRs recognize conserved pathogen-associated molecular patterns (PAMPs), such as branched mannans, β-glucans, and chitins that are the major components of the fungal cell wall. However, the key receptors/ligand interactions required for cryptococcal recognition and eventual fungal clearance have yet to be elucidated. Here we present an imaging flow cytometer (IFC) method that offers a novel quantitative cellular imaging and population statistics tool to accurately measure phagocytosis of fungal cells. It has the capacity to measure two distinct steps of phagocytosis: association/attachment and internalization in a high-throughput and quantitative manner that is difficult to achieve with other technologies. Results from these IFC studies allow for the potential to identify PRRs required for recognition, uptake, and subsequent activation of cytokine production, as well as other effector cell responses required for fungal clearance.

Keywords: Cryptococcus; Imaging flow cytometer; Innate immunity; Macrophages; Phagocytosis.