#35 Elucidating key interactions between macrophages and Coccidioides

Abstract

Abstract Background Coccidioidomycosis or Valley Fever is a fungal infection caused by Coccidioides spp. with potentially life-threatening sequelae. Infection is caused after inhalation of spores (arthroconidia) from the environment, which develop into spherules that contain internal cells called endospores. Spherules release their endospores, which in turn can differentiate into new spherules. Clinically, there are a wide range of outcomes of infection, from asymptomatic infection to meningitis, yet the host and fungal factors that underlie these differences remain largely unknown. We are investigating the role of innate immune cells in the early host response to infection, specifically the role of macrophages in host response to Coccidioides arthroconidia. Macrophages are key innate immune cells in the host response to many other fungal infections in the lung and can be subverted as a niche by some of those pathogens, yet little is known about the interaction between Coccidioides and macrophages. We chose to use a facile cell culture model (infection of murine bone marrow derived macrophages) to define key interactions between Coccidioides arthroconidia and macrophages. Method Bone marrow derived macrophages were isolated from wildtype (C57BL/6) or C3aR1-/- mice. Coccidioides posadasii Silveira arthroconidia were used in all experiments. Macrophage phagocytosis of arthroconidia was examined by confocal microscopy using a dual staining approach, incubating macrophages with FITC-labelled arthroconidia at a multiplicity of infection (MOI) of 1 (1 arthroconidia for every macrophage) and at each time point labeling extracellular arthroconidia with calcofluor white. To evaluate the ability of arthroconidia to transition to the host parasitic form, the spherule, in the presence of macrophages, we incubated arthroconidia with macrophages at MOI 0.1 (1 arthroconidia for every 10 macrophages) or MOI 1 and examined fungal morphology by light microscopy over 72hrs. All experiments were conducted at 37C and 5% CO2. Results We show that by 1 hr of infection, half of all macrophages have intracellular arthroconidia, with phagocytosis occurring as quickly at 15 min. The host receptor for C3a, complement 3a receptor (C3aR1), is important for the early phagocytosis of arthroconidia. In macrophages lacking C3aR1, 10% of macrophages have phagocytosed arthroconidia compared to 45% in wildtype cells. We next observed that the presence of macrophages strongly promotes the ability of arthroconidia to transition to spherules at temperatures that would not normally promote significant spherulation in vitro. Small spherules were observed within macrophages, in addition to larger extracellular spherules. Conclusion This work shows that macrophages phagocytose arthroconidia, yet in the presence of macrophages, some arthroconidia are able to develop into the pathogenic host form of Coccidioides. We have created a foundation for better understanding the initial interactions between key host immune cells and the inhaled form of the Coccidioides. We are currently using transcriptional profiling to identify and characterize host pathways that play key roles in the macrophage response to Coccidioides in vitro as arthroconidia develop into spherules.