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Migratory cells use distinct motility modes to navigate different microenvironments, but it is unclear whether these modes rely on the same core set of polarity components. To investigate this, we disrupted actin-related protein 2/3 (Arp2/3) and the WASP-family verprolin homologous protein (WAVE) complex, which assemble branched actin networks that are essential for neutrophil polarity and motility in standard adherent conditions. Surprisingly, confinement rescues polarity and movement of neutrophils lacking these components, revealing a processive bleb-based protrusion program that is mechanistically distinct from the branched actin-based protrusion program but shares some of the same core components and underlying molecular logic. We further find that the restriction of protrusion growth to one site does not always respond to membrane tension directly, as previously thought, but may rely on closely linked properties such as local membrane curvature. Our work reveals a hidden circuit for neutrophil polarity and indicates that cells have distinct molecular mechanisms for polarization that dominate in different microenvironments., Cells display a high degree of plasticity in migration, but how polarity is organized in different microenvironments has remained unclear. This study uses mechanical perturbations to reveal that migration using actin-rich or bleb-based protrusions are both organized around Rac GTPase.

A major question in research on immune responses in the brain is how the timing and nature of these responses influence physiology, pathogenesis or recovery from pathogenic processes. Proper understanding of the immune regulation of the human brain requires a detailed description of the function and activities of the immune cells in the brain. Zebrafish larvae allow long-term, noninvasive imaging inside the brain at high-spatiotemporal resolution using fluorescent transgenic reporters labeling specific cell populations. Together with recent additional technical advances this allows an unprecedented versatility and scope of future studies. Modeling of human physiology and pathology in zebrafish has already yielded relevant insights into cellular dynamics and function that can be translated to the human clinical situation. For instance, in vivo studies in the zebrafish have provided new insight into immune cell dynamics in granuloma formation in tuberculosis and the mechanisms involving treatment resistance. In this review, we highlight recent findings and novel tools paving the way for basic neuroimmunology research in the zebrafish. GLIA 2015;63:719-735

A major question in research on immune responses in the brain is how the timing and nature of these responses influence physiology, pathogenesis or recovery from pathogenic processes. Proper understanding of the immune regulation of the human brain requires a detailed description of the function and activities of the immune cells in the brain. Zebrafish larvae allow long-term, noninvasive imaging inside the brain at high-spatiotemporal resolution using fluorescent transgenic reporters labeling specific cell populations. Together with recent additional technical advances this allows an unprecedented versatility and scope of future studies. Modeling of human physiology and pathology in zebrafish has already yielded relevant insights into cellular dynamics and function that can be translated to the human clinical situation. For instance, in vivo studies in the zebrafish have provided new insight into immune cell dynamics in granuloma formation in tuberculosis and the mechanisms involving treatment resistance. In this review, we highlight recent findings and novel tools paving the way for basic neuroimmunology research in the zebrafish. GLIA 2015;63:719-735.

Establishing intake recommendations for vitamin C remains a challenge, as no suitable functional parameter has yet been agreed upon. In this report, we review the emerging evidence on neutrophil motility as a possible marker of vitamin C requirements and put the results in perspective with other approaches. A recent in vitro study showed that adequate levels of vitamin C were needed for this function to work optimally when measured as chemotaxis and chemokinesis. In a human study, neutrophil motility was optimal at intakes ≥250 mg/day. Interestingly, a Cochrane review showed a significant reduction in the duration of episodes of common cold with regular vitamin C intakes in a similar range. Additionally, it was shown that at a plasma level of 75 µmol/L, which is reached with vitamin C intakes ≥200 mg/day, incidences of cardiovascular disease were lowest. This evidence would suggest that daily intakes of 200 mg vitamin C might be advisable for the general adult population, which can be achieved by means of a diverse diet. However, additional studies are warranted to investigate the usefulness of neutrophil motility as a marker of vitamin C requirements.

Toxoplasma gondii infection occurs through the oral route, but we lack important information about how the parasite interacts with the host immune system in the intestine. We used two-photon laser-scanning microscopy in conjunction with a mouse model of oral T. gondii infection to address this issue. T. gondii established discrete foci of infection in the small intestine, eliciting the recruitment and transepithelial migration of neutrophils and inflammatory monocytes. Neutrophils accounted for a high proportion of actively invaded cells, and we provide evidence for a role for transmigrating neutrophils and other immune cells in the spread of T. gondii infection through the lumen of the intestine. Our data identify neutrophils as motile reservoirs of T. gondii infection and suggest a surprising retrograde pathway for parasite spread in the intestine.

The ability of human polymorphonuclear leukocytes (PMNL; neutrophils), to sense and move to sites of infection is essential for our defense against pathogens. Cell motility is critically dependent on a dynamic remodeling of morphology. The morphological polarization toward chemoattractants, such as N-formyl-Met-Leu-Phe (fMLF), is associated with temporary extension and stabilization of lamellipodia in the direction of movement. The underlying mechanisms of cell motility are, however, still not entirely elucidated. It is therefore an urgent task to extend the present experimental evidence to give solid basis for a comprehensive model. Here it is shown that nitric oxide (NO) stimulates the morphological response of neutrophils, most likely due to transient increases in [Ca2+]i, following addition of NO-donors. This will, hypothetically, activate gelsolin and other actin filament severing proteins, leading to a subsequent decrease in filamentous actin. The incapability to efficiently turnover the actin filament network then blocks all motile activity. It is also shown that N-formyl peptide receptors on polarized neutrophils accumulate non-uniformly towards regions involved in motility. It is suggested that neutrophils use the asymmetric receptor distribution for directional sensing and sustained migration. A model for lamellipodium extension, where water fluxes play a pivotal role is presented. It is suggested that water fluxes through water-selective aquaporin (AQP) channels, contribute to the propulsive force for formation of various membrane protrusions and, thus, cell motility. It is well known that small G proteins of the Rho family GTPases play important roles in the intracellular signaling underlying cell motility. In morphologically polarized neutrophils it is shown that Cdc42, Rac2 and RhoA display spatially distinct distributions, which allows for sequential chemoattractant stimulation of neutrophil motility. The specific localizations of Rac2, Cdc42 and RhoA relative to each other and filamentous actin and fMLF receptors support the hypothesized order of activation and regulation of neutrophil cell motility. In conclusion, the detailed analysis of motility-related issues presented here provide new data allowing further refinement of previous models of neutrophil motility.

In previous studies, we reported that sphingosine 1-phosphate (Sph-1-P) inhibits the chemotactic motility of some cancer cell lines such as mouse melanoma cells, as well as human smooth muscle cells, at a very low concentration, as demonstrated by a transwell migration assay method (Proc. Natl. Acad. Sci. USA 89, 9698, 1992; J. Cell Biol. 130, 193, 1995). In this study, we investigated the effect of Sph-1-P on the chemotactic motility and invasiveness of human neutrophils, utilizing three different assay systems: (a) a transwell migration assay where IL-8 or fLMP was added as a chemotactic factor, (b) a phagokinetic assay with gold colloids, and (c) a trans-endothelial migration assay with human umbilical vein endothelial cells (HUVECs) plated on collagen layers. We found that among various sphingosine derivatives, Sph-1-P specifically inhibited the IL-8- or fLMP-induced chemotactic migration of neutrophils at concentrations below 1 μM. Phagokinetic activity of neutrophils was also suppressed by Sph-1-P, but more moderately than by the PKC inhibitory sphingosine analog, trimethylsphingosine. Finally, Sph-1-P inhibited trans-endothelial migration and invasiveness of neutrophils into HUVEC-covered collagen layers, whereas no effect on their adhesion to HUVECs was observed. These observations strongly suggest that Sph-1-P can act as a specific and effective motility regulator of human neutrophils, raising the possibility of future applications of Sph-1-P, or its analogs, as anti-inflammatory agents regulating invasive migration of neutrophils through endothelial layers at injured vascular sites.