Fig 1.
Overview of Coccidioides spp. and human neutrophils.
A. Endemic areas of Coccidioides spp. B. Life cycle of Coccidioides spp. Vegetative mycelia exist in the soil and produce arthrospores during periods of low precipitation. Following aerosolization and inhalation of arthrospores, immature spherules develop and transition into large spherules containing hundreds of endospores. The mature spherules eventually rupture and release the endospores, reinitiating the spherule/endospore phase. (Adapted from [12] with permission.). C. Composite videomicrographs of typical C. posadasii endospores (top) and spherules (bottom). D. Composite brightfield videomicrographs of quiescent human neutrophils as used in the experiments. E. H&E-stained human neutrophils after neutrophil enrichment. All images in C-E are shown at the same magnification (some cell shrinkage occurred during H&E-staining). The common scale bar denotes 10 μm.
Fig 2.
Overview of single-cell experiments.
A. Schematic of our dual-micropipette manipulation system. The chamber volume is created by trapping buffer solution between two horizontal microscope coverslips. Facing pipettes access this volume through the chamber's two open sides. Vertically movable water reservoirs allow us to control the pipette-aspiration pressure with high resolution. The aspiration pressure of the right pipette is monitored in real time by measuring the height difference between the main reservoir (which is connected to the pipette) and a pre-zeroed reference reservoir. The included example videomicrographs demonstrate how micropipettes are used to pick up individual targets (B) and neutrophils (C) with gentle suction. After lifting these objects above the chamber bottom, they can be maneuvered in 3D to set up experiments that assess target recognition either from a distance (D) or upon direct physical contact (E). All scale bars denote 10 μm.
Fig 3.
Pure chemotaxis of non-adherent neutrophils toward C. posadasii.
Using micropipettes, endospores (A) (see also S1 Video) and spherules (B) are maneuvered to different positions relative to the cell without touching the cell. In this configuration, chemotaxis takes the form of a directional, protrusive pseudopod extended by the neutrophil toward the target. The relative times of all video images are included. Table 1 summarizes the number of experiments in which this behavior was observed. All scale bars denote 10 μm.
Table 1.
Recognition of endospores and spherules of Coccidioides posadasii by human neutrophils.
Fig 4.
Phagocytosis of C. posadasii endospores by initially passive human neutrophils.
(See also S2 Video.) Four example experiments are presented as vertical filmstrips. The experiment buffer contained 10% heat-treated autologous serum, which prevented chemotaxis (such as shown in Fig 3) but not the engulfment of the endospore after direct contact with the neutrophil surface. The relative times of all video images are included. Table 1 summarizes the number of experiments in which this behavior was observed. All scale bars denote 10 μm.
Fig 5.
Phagocytosis of C. posadasii spherules by initially passive human neutrophils.
(See also S3 Video and S4 Video.) Four example experiments are presented as vertical filmstrips. (The spherule in the first panel is immature [71].) The experimental conditions were the same as in Fig 4. The relative times of all video images are included. Table 1 summarizes the number of experiments in which this behavior was observed. All scale bars denote 10 μm.
Fig 6.
Frustrated phagocytosis of C. posadasii spherules by multiple neutrophils.
A. Shortly after manipulating an immature spherule and a first neutrophil (#1) into contact, a second neutrophil (#2) touches and adheres to the spherule by chance. Both cells proceed to spread over the surface of the spherule. (See also S5 Video.) B. Using micropipettes, three neutrophils are sequentially brought into contact with the same spherule and proceed to attack it. (See also S6 Video.) The relative times of all video images are included. C,D. Bulk assay to verify the recognition of live C. posadasii spherules by human neutrophils. The spherules were incubated with neutrophils for 5 min. (C) or for 20 min. (D) in suspension with gentle mixing on a rotator, then fixed and H&E-stained. Arrows point to particularly spread-out leukocytes. (Some cell shrinkage occurred during H&E-staining.) All scale bars denote 10 μm.
Fig 7.
Analysis of the positional trajectory of a target particle (here: a C. posadasii endospore) during phagocytosis by a pipette-held neutrophil.
The annotated videomicrographs at the top demonstrate our measurement of the distance between the center of the target particle and the opposite side of the main cell body (red straight line). This distance (relative to its initial value) is plotted as a function of time in the bottom graph (red curve). Numbered circles correspond to the time points at which the respective example images were taken. This type of graph allowed us to determine the maximum push-out distance as well as the pull-in speed of the target as shown. The engulfment time (defined in the text) was found by inspection of the recorded video images.
Fig 8.
Quantitative analysis of the time course of phagocytosis of C. posadasii endospores (left) and spherules (right) by human neutrophils.
Representative timelines of the target position (aligned for best overlap of the trajectories during the pull-in phase) (A), the cell-surface area (B), and the cortical tension (C) are shown for three particles of each type. For each target type, a given color indicates the same cell-target pair throughout parts A, B, and C. The three phases identified at the bottom of the figure were determined by inspection of the time-dependent neutrophil morphologies in the recorded image sequences. Positive values of the target position shown in part A reflect a push-out of the particle. A monotonous decrease of the position values characterizes the pull-in phase. The end of the pull-in phase marks the start of the final phase. The inset in the right panels depicts the cell behavior over an extended period of time (~33 minutes). Common axis titles are shown only once at the left and bottom of the figure.
Fig 9.
Side-by-side comparison of the aptitude of passive human neutrophils to recognize various fungal and model targets.
Example videomicrographs show typical outcomes of single-cell experiments assessing chemotaxis (in buffer containing 10% autologous serum) as well as adhesion and phagocytosis (where in the experiments with C. posadasii, C. albicans, and zymosan, the buffer contained 10% of either normal or 52°-C-treated autologous serum). The number of positive responses and the total number of inspected cell-target contacts (in parentheses) are included. In the case of C. posadasii, the numbers comprise the cell responses to both endospores as well as spherules. The outcomes of experiments with zymosan and IgG-coated beads agree with, and include, results that were reported previously [34, 37, 43]. Unambiguous positive (>80% positive response) and negative (never observed) responses are marked by checkmarks and crosses, respectively. All scale bars denote 10 μm.
Fig 10.
Comparison of the behavior of neutrophils from patients with chronic coccidioidomycosis and from healthy donors.
A. Filmstrips illustrate the responses of neutrophils from the two donor groups to contact with C. posadasii endospores and antibody-coated beads, respectively (in the presence of autologous serum). All scale bars denote 10 μm. B. Results of the quantitative analysis of the positional trajectories (cf. Fig 7) of C. posadasii endospores and antibody-coated beads during phagocytosis by neutrophils from the two donor groups. (Error bars denote standard deviations, and asterisks mark differences that are statistically significant. The number N of analyzed single-cell experiments is included in the figure.).