Coccidioides Endospores and Spherules Draw Strong Chemotactic, Adhesive, and Phagocytic Responses by Individual Human Neutrophils

Abstract

Coccidioides spp. are dimorphic pathogenic fungi whose parasitic forms cause coccidioidomycosis (Valley fever) in mammalian hosts. We use an innovative interdisciplinary approach to analyze one-on-one encounters between human neutrophils and two forms of Coccidioides posadasii. To examine the mechanisms by which the innate immune system coordinates different stages of the host response to fungal pathogens, we dissect the immune-cell response into chemotaxis, adhesion, and phagocytosis. Our single-cell technique reveals a surprisingly strong response by initially quiescent neutrophils to close encounters with C. posadasii, both from a distance (by complement-mediated chemotaxis) as well as upon contact (by serum-dependent adhesion and phagocytosis). This response closely resembles neutrophil interactions with Candida albicans and zymosan particles, and is significantly stronger than the neutrophil responses to Cryptococcus neoformans, Aspergillus fumigatus, and Rhizopus oryzae under identical conditions. The vigorous in vitro neutrophil response suggests that C. posadasii evades in vivo recognition by neutrophils through suppression of long-range mobilization and recruitment of the immune cells. This observation elucidates an important paradigm of the recognition of microbes, i.e., that intact immunotaxis comprises an intricate spatiotemporal hierarchy of distinct chemotactic processes. Moreover, in contrast to earlier reports, human neutrophils exhibit vigorous chemotaxis toward, and frustrated phagocytosis of, the large spherules of C. posadasii under physiological-like conditions. Finally, neutrophils from healthy donors and patients with chronic coccidioidomycosis display subtle differences in their responses to antibody-coated beads, even though the patient cells appear to interact normally with C. posadasii endospores.

Controlled One-on-One Encounters between Immune Cells and Microbes Reveal Mechanisms of Phagocytosis

“…A local stimulus acting at the cell surface leads to reorganization of the adjacent cytoskeleton, which in turn creates a pushing force that displaces a membrane patch outward. This basic mechanism is consistent with the cell morphology observed during pure (i.e., cell-substrate-adhesion-free) chemotaxis (9)(10)(11)33). But it also implies that immediately after cell-target contact, a phagocyte should always form a protrusive pseudopod directly underneath the region of contact (where the phagocytosis-triggering stimulus is strongest).…”

“…1 B), and have already revealed insight into cellular behavior that had been inaccessible to traditional techniques. (For movies of representative single-live-cell experiments see https://www.youtube.com/user/HeinrichLab or the supplemental videos of (7,(9)(10)(11)(12)). …”

Quantifying the Sensitivity of Human Immune Cells to Chemoattractant

“…For example, the typical time lag from placing a zymosan particle near a neutrophil to the first sign of a newly forming chemotactic pseudopod was found to be on the order of~60 s (9). Similar results were obtained for bacterial and fungal pathogens (6,8). On the other hand, for the typical cell-target distances (Dr z 5 mm) and target sizes (R z 2.5 mm) used in those experiments, Eq.…”

“…How does the cell decide on a particular response? Such questions touch on the core of our mechanistic understanding of immune-cell behavior, and have inspired the paradigm that immunotaxis comprises an intricate spatiotemporal hierarchy of distinct chemotactic processes (1)(2)(3)(4)(5)(6). The systematic dissection of this hierarchy is an enormous interdisciplinary challenge that requires, among others, quantitative analyses of the stimulus-specific sensitivity of the responding cells.…”

“…Recent single-cell experiments have validated human neutrophils as uniquely capable biodetectors of minuscule amounts of complement-derived anaphylatoxins in the proximity of microbial and model pathogens ( Fig. 1) (6)(7)(8). But the question just how sensitive these immune cells are was not addressed by earlier studies.…”