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

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.

The Vi capsular polysaccharide enables Salmonella enterica serovar typhi to evade microbe-guided neutrophil chemotaxis.

Salmonella enterica serovar Typhi (S. Typhi) causes typhoid fever, a disseminated infection, while the closely related pathogen S. enterica serovar Typhimurium (S. Typhimurium) is associated with a localized gastroenteritis in humans. Here we investigated whether both pathogens differ in the chemotactic response they induce in neutrophils using a single-cell experimental approach. Surprisingly, neutrophils extended chemotactic pseudopodia toward Escherichia coli and S. Typhimurium, but not toward S. Typhi. Bacterial-guided chemotaxis was dependent on the presence of complement component 5a (C5a) and C5a receptor (C5aR). Deletion of S. Typhi capsule biosynthesis genes markedly enhanced the chemotactic response of neutrophils in vitro. Furthermore, deletion of capsule biosynthesis genes heightened the association of S. Typhi with neutrophils in vivo through a C5aR-dependent mechanism. Collectively, these data suggest that expression of the virulence-associated (Vi) capsular polysaccharide of S. Typhi obstructs bacterial-guided neutrophil chemotaxis.

Differential effects of serum heat treatment on chemotaxis and phagocytosis by human neutrophils.

Neutrophils, in cooperation with serum, are vital gatekeepers of a host's microbiome and frontline defenders against invading microbes. Yet because human neutrophils are not amenable to many biological techniques, the mechanisms governing their immunological functions remain poorly understood. We here combine state-of-the-art single-cell experiments with flow cytometry to examine how temperature-dependent heat treatment of serum affects human neutrophil interactions with "target" particles of the fungal model zymosan. Assessing separately both the chemotactic as well as the phagocytic neutrophil responses to zymosan, we find that serum heat treatment modulates these responses in a differential manner. Whereas serum treatment at 52°C impairs almost all chemotactic activity and reduces cell-target adhesion, neutrophils still readily engulf target particles that are maneuvered into contact with the cell surface under the same conditions. Higher serum-treatment temperatures gradually suppress phagocytosis even after enforced cell-target contact. Using fluorescent staining, we correlate the observed cell behavior with the amounts of C3b and IgG deposited on the zymosan surface in sera treated at the respective temperatures. This comparison not only affirms the critical role of complement in chemotactic and adhesive neutrophil interactions with fungal surfaces, but also unmasks an important participation of IgGs in the phagocytosis of yeast-like fungal particles. In summary, this study presents new insight into fundamental immune mechanisms, including the chemotactic recruitment of immune cells, the adhesive capacity of cell-surface receptors, the role of IgGs in fungal recognition, and the opsonin-dependent phagocytosis morphology of human neutrophils. Moreover, we show how, by fine-tuning the heat treatment of serum, one can selectively study chemotaxis or phagocytosis under otherwise identical conditions. These results not only refine our understanding of a widely used laboratory method, they also establish a basis for new applications of this method.

Blurred line between chemotactic chase and phagocytic consumption: an immunophysical single-cell perspective.

An innate immune cell can sense a pathogen, either from a distance by recognizing chemoattractant stimuli or by direct physical contact. The pathogen is subsequently neutralized, which usually occurs through its phagocytic internalization. By investigating chemotaxis and phagocytosis from an immunophysical single-cell perspective, it now appears that the demarcation between these two processes is less distinct than originally thought. Several lines of evidence support this notion. First, chemotactic stimulation does not cease at the moment of initial contact between the cell and the pathogenic target. Second, even when classical chemotaxis of neutrophils is suppressed, the early cell response to contact with typical chemoattractant targets, such as zymosan, fungal spores or chemokine-coated particles, can still involve morphological attributes of chemotaxis. Recognizing that the changing morphology of motile cells is inextricably linked to physical cell behavior, this Commentary focuses on the mechanical aspects of the early response of innate immune cells to chemotactic and phagocytic stimuli. On the basis of this perspective, we propose that the combined study of chemotaxis and phagocytosis will, potentially, not only advance our grasp of the mechanisms underlying immune-cell motility but also open new lines of research that will promote a deeper understanding of the innate recognition of pathogens.

Target-specific mechanics of phagocytosis: protrusive neutrophil response to zymosan differs from the uptake of antibody-tagged pathogens.

The physical mechanisms that control target-specific responses of human neutrophils to distinct immune threats are poorly understood. Using dual-micropipette manipulation, we have quantified and compared the time courses of neutrophil phagocytosis of two different targets: zymosan (a prominent model of fungal infection), and antibody-coated (Fc) particles. Our single-live-cell/single-target approach exposes surprising differences between these two forms of phagocytosis. Unlike the efficient uptake of 3-µm Fc targets (within ~66 seconds), the engulfment of similarly sized zymosan is slow (~167 seconds), mainly due to the formation of a characteristic pedestal that initially pushes the particle outwards by ~1 µm. Despite a roughly twofold difference in maximum cortical tensions, the top 'pull-in' speeds of zymosan and Fc targets are indistinguishable at ~33 nm/second. Drug inhibition shows that both actin as well as myosin II partake in the regulation of neutrophil cortical tension and cytoplasmic viscosity; other than that, myosin II appears to play a minor role in both forms of phagocytosis. Remarkably, an intact actin cytoskeleton is required to suppress, in antibody-mediated phagocytosis, the initially protrusive deformation that distinguishes the neutrophil response to zymosan.

Protrusive push versus enveloping embrace: computational model of phagocytosis predicts key regulatory role of cytoskeletal membrane anchors.

Encounters between human neutrophils and zymosan elicit an initially protrusive cell response that is distinct from the thin lamella embracing antibody-coated targets. Recent experiments have led us to hypothesize that this behavior has its mechanistic roots in the modulation of interactions between membrane and cytoskeleton. To test and refine this hypothesis, we confront our experimental results with predictions of a computer model of leukocyte mechanical behavior, and establish the minimum set of mechanistic variations of this computational framework that reproduces the differences between zymosan and antibody phagocytosis. We confirm that the structural linkages between the cytoskeleton and the membrane patch adherent to a target form the "switchboard" that controls the target specificity of a neutrophil's mechanical response. These linkages are presumably actin-binding protein complexes associating with the cytoplasmic domains of cell-surface receptors that are engaged in adhesion to zymosan and Fc-domains.