The Chemotactic Defect in Wiskott-Aldrich Syndrome Macrophages Is Due to the Reduced Persistence of Directional Protrusions

Wiskott-Aldrich syndrome protein (WASp) is an actin nucleation promoting factor that is required for macrophages to directionally migrate towards various chemoattractants. The chemotaxis defect of WASp-deficient cells and its activation by Cdc42 in vivo suggest that WASp plays a role in directional sensing, however, its precise role in macrophage chemotaxis is still unclear. Using shRNA-mediated downregulation of WASp in the murine monocyte/macrophage cell line RAW/LR5 (shWASp), we found that WASp was responsible for the initial wave of actin polymerization in response to global stimulation with CSF-1, which in Dictyostelium discoideum amoebae and carcinoma cells has been correlated with the ability to migrate towards chemoattractants. Real-time monitoring of shWASp cells, as well as WASp−/− bone marrow-derived macrophages (BMMs), in response to a CSF-1 gradient revealed that the protrusions from WASp-deficient cells were directional, showing intact directional sensing. However, the protrusions from WASp-deficient cells demonstrated reduced persistence compared to their respective control shRNA and wild-type cells. Further examination showed that tyrosine phosphorylation of WASp was required for both the first wave of actin polymerization following global CSF-1 stimulation and proper directional responses towards CSF-1. Importantly, the PI3K, Rac1 and WAVE2 proteins were incorporated normally in CSF-1 – elicited protrusions in the absence of WASp, suggesting that membrane protrusion driven by the WAVE2 complex signaling is intact. Collectively, these results suggest that WASp and its phosphorylation play critical roles in coordinating the actin cytoskeleton rearrangements necessary for the persistence of protrusions required for directional migration of macrophages towards CSF-1

Transcriptomic Analysis Comparing Tumor-Associated Neutrophils with Granulocytic Myeloid-Derived Suppressor Cells and Normal Neutrophils

The role of myeloid cells in supporting cancer growth is well established. Most work has focused on myeloid-derived suppressor cells (MDSC) that accumulate in tumor-bearing animals, but tumor-associated neutrophils (TAN) are also known to be capable of augmenting tumor growth. However, little is known about their evolution, phenotype, and relationship to naïve neutrophils (NN) and to the granulocytic fraction of MDSC (G-MDSC)

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

Cell adhesion and response to synthetic nanopatterned environments by steering receptor clustering and spatial location

During adhesion and spreading, cells form micrometer-sized structures comprising transmembrane and intracellular protein clusters, giving rise to the formation of what is known as focal adhesions. Over the past two decades these structures have been extensively studied to elucidate their organization, assembly, and molecular composition, as well as to determine their functional role. Synthetic materials decorated with biological molecules, such as adhesive peptides, are widely used to induce specific cellular responses dependent on cell adhesion. Here, we focus on how surface patterning of such bioactive materials and organization at the nanoscale level has proven to be a useful strategy for mimicking both physical and chemical cues present in the extracellular space controlling cell adhesion and fate. This strategy for designing synthetic cellular environments makes use of the observation that most cell signaling events are initiated through recruitment and clustering of transmembrane receptors by extracellular-presented signaling molecules. These systems allow for studying protein clustering in cells and characterizing the signaling response induced by, e.g., integrin activation. We review the findings about the regulation of cell adhesion and focal adhesion assembly by micro- and nanopatterns and discuss the possible use of substrate stiffness and patterning in mimicking both physical and chemical cues of the extracellular space