Early events in macrophage killing of Aspergillus fumigatus conidia: new flow cytometric viability assay

Abstract

Detailed investigations of macrophage phagocytosis and killing of Aspergillus fumigatus conidia have been limited by technical difficulties in quantifying fungal uptake and viability. In order to study early events in cell pathogen ingestion and killing, we developed a new flow cytometry assay that utilizes the fungus-specific viability dye FUN-1. Metabolically active A. fumigatus conidia accumulate orange fluorescence in vacuoles, while dormant or dead conidia stain green. After incubation within THP-1 cells, recovered conidia are costained with propidium iodide (PI) to discriminate between dormant and dead cells. Flow cytometric measurements of FUN-1 metabolism and PI uptake provide indicators of conidial viability, dormancy, and death. Conidial phagocytosis and killing are also assessed by measurement of green and orange FUN-1 fluorescence within the THP-1 cell population. Compared to previously described methods, this assay has less error introduced by membrane permeability changes and serial dilution of filamentous fungal forms. Results suggest that the THP-1 cells kill conidia rapidly (within 6 h) after exposure. Conidia that are preexposed to human serum are ingested and killed more quickly than are nonopsonized conidia.

FIG. 1

CFU recovered from THP-1 cells relative to inoculum (y axis) after 2- and 4-h incubations (“2 hour kill” and “4 hour kill”) and after 2- and 4-h growth in the absence of THP-1 cells (“2 hour growth” and “4 hour growth”). Data shown are from three experiments, each with triplicate plating per well.

FIG. 2

Forward scatter characteristics (FSC-H, y axis) are plotted against PI uptake (x axis) for live, naked A. fumigatus conidia (a); live, PI-exposed A. fumigatus conidia (b); and heat-killed, PI-exposed conidia (c). Large PI-positive cells within the live population are apparent (b) despite the absence of significant autofluorescence (a). Heat killing (verified using serial dilution plating) resulted in a high proportion of PI-positive cells (c).

FIG. 3

(a) FUN-1-stained green and orange A. fumigatus conidia. (b) FUN-1 metabolism of live A. fumigatus conidia over time. After indicated incubations at 30°C, conidial metabolism was halted by rapid exposure to 4°C and cells were fixed, as described in Materials and Methods. The fluorescence-activated cell sorting profiles shown were produced by gating upon the entire cellular population. Log fluorescence intensity is shown on the x axis, and the relative cell number is shown on the y axis, for each time point indicated (5, 30, 60, and 120 min).

FIG. 4

Control experiments for FUN-1 metabolism (top) and PI uptake within the FUN-1 orange-negative population (bottom) in freshly harvested live cells (left) and heat-killed conidia (right).

FIG. 5

(a) Histogram plot of FUN-1 metabolism of live A. fumigatus conidia (dotted line) and conidia harvested from macrophages after 2-h (solid line) and 6-h (bold line) incubations. Log fluorescence intensity is shown on the x axis, and the relative cell number is on the y axis. (b) PI uptake in live conidia and conidia recovered from THP-1 cells after 2 and 6 h. FUN-1 green-to-orange metabolism is measured within the entire population of recovered conidia, while PI uptake is measured only within the FUN-1 orange-negative population. Shown are the results of one experiment, which is representative of at least six different experiments.

FIG. 6

Histogram plots of green fluorescence (a) and orange fluorescence (b) within intact THP-1 cells after 5-min (solid line) and 2-h (bold line) incubations, compared to intracellular staining of sodium azide-treated controls after 2 h (solid gray area).

FIG. 7

(a) Histogram plots of intracellular orange fluorescence 2 h after exposure to nonopsonized A. fumigatus conidia (solid gray area) and conidia preexposed to non-heat-inactivated human serum (solid line). Counts are shown on the y axis. (b) Percent conidia viable (FUN-1)orange) and dead (PI positive) after recovery under the conditions indicated. Data from triplicate measures in three different experiments.

FIG. 8

Percentage of THP-1 cells with internalized conidia (FUN-1 orange, y axis) after indicated incubations (x axis). Percentages of cells with internalized naked A. fumigatus conidia and conidia preexposed to fresh serum are shown. Results were obtained from triplicate measurements in two experiments.