The Lipid Kinase PIKfyve Coordinates the Neutrophil Immune Response through the Activation of the Rac GTPase.

Neutrophils rapidly arrive at an infection site because of their unparalleled chemotactic ability, after which they unleash numerous attacks on pathogens through degranulation and reactive oxygen species (ROS) production, as well as by phagocytosis, which sequesters pathogens within phagosomes. Phagosomes then fuse with lysosomes and granules to kill the enclosed pathogens. A complex signaling network composed of kinases, GTPases, and lipids, such as phosphoinositides, helps to coordinate all of these processes. There are seven species of phosphoinositides that are interconverted by lipid kinases and phosphatases. PIKfyve is a lipid kinase that generates phosphatidylinositol-3,5-bisphosphate and, directly or indirectly, phosphatidylinositol-5-phosphate [PtdIns(5)P]. PIKfyve inactivation causes massive lysosome swelling, disrupts membrane recycling, and, in macrophages, blocks phagosome maturation. In this study, we explored for the first time, to our knowledge, the role of PIKfyve in human and mouse neutrophils. We show that PIKfyve inhibition in neutrophils does not affect granule morphology or degranulation, but it causes LAMP1+ lysosomes to engorge. Additionally, PIKfyve inactivation blocks phagosome-lysosome fusion in a manner that can be rescued, in part, with Ca2+ ionophores or agonists of TRPML1, a lysosomal Ca2+ channel. Strikingly, PIKfyve is necessary for chemotaxis, ROS production, and stimulation of the Rac GTPases, which control chemotaxis and ROS. This is consistent with observations in nonleukocytes that showed that PIKfyve and PtdIns(5)P control Rac and cell migration. Overall, we demonstrate that PIKfyve has a robust role in neutrophils and propose a model in which PIKfyve modulates phagosome maturation through phosphatidylinositol-3,5-bisphosphate-dependent activation of TRPML1, whereas chemotaxis and ROS are regulated by PtdIns(5)P-dependent activation of Rac.

Neutrophils Increase Oral Squamous Cell Carcinoma Invasion through an Invadopodia-Dependent Pathway.

Neutrophils have recently been shown to promote invasion and correlate with a poor prognosis in different cancers, including head and neck squamous cell carcinomas. In this study, we analyze the effects of neutrophils in the invasion of oral squamous cell carcinoma (OSCC) using a combination of conditioned media, direct and indirect coculture of human peripheral blood neutrophils, and UMSCC47 cells (OSCC cell line). Invasion and matrix degradation were determined using a modified in vitro invasion assay and an invadopodia assay, respectively. UMSCC47 and neutrophil cocultures or conditioned media from cocultures increased UMSCC47 invasion, invadopodia formation, and matrix degradation. Further analysis revealed an increase in TNFα and IL8 in supernatants of cocultures compared with neutrophil or UMSCC47 cultures alone and that inhibition of TNFα and IL8 significantly decreased OSCC invasion. Our results show that neutrophils increase the invasiveness of OSCC through the activation of invadopodia and matrix degradation, suggesting a paracrine activation loop between the two cells. Importantly, the presence of neutrophils in the oral environment may modulate the clinical behavior of OSCC.