YB-1 regulates mesothelioma cell migration via snail but not EGFR, MMP1, EPHA5 or PARK2.

Pleural mesothelioma (PM) is characterized by rapid growth, local invasion, and limited therapeutic options. The multifunctional oncoprotein Y-box-binding protein-1 (YB-1) is frequently overexpressed in cancer and its inhibition reduces aggressive behavior in multiple tumor types. Here, we investigated the effects of YB-1 on target gene regulation and PM cell behavior. Whereas siRNA-mediated YB-1 knockdown reduced cell motility, YB-1 overexpression resulted in scattering, increased migration, and intravasation in vitro. Furthermore, YB-1 stimulated PM cell spreading in zebrafish. Combined knockdown and inducible overexpression of YB-1 allowed bidirectional control and rescue of cell migration, the pattern of which was closely followed by the mRNA and protein levels of EGFR and the protein level of snail, whereas the mRNA levels of MMP1, EPHA5, and PARK2 showed partial regulation by YB-1. Finally, we identified snail as a critical regulator of YB-1-mediated cell motility in PM. This study provides insights into the mechanism underlying the aggressive nature of PM and highlights the important role of YB-1 in this cancer. In this context, we found that YB-1 closely regulates EGFR and snail, and, moreover, that YB-1-induced cell migration depends on snail.

A two-step search and run response to gradients shapes leukocyte navigation in vivo.

Migrating cells must interpret chemical gradients to guide themselves within tissues. A long-held principle is that gradients guide cells via reorientation of leading-edge protrusions. However, recent evidence indicates that protrusions can be dispensable for locomotion in some contexts, raising questions about how cells interpret endogenous gradients in vivo and whether other mechanisms are involved. Using laser wound assays in zebrafish to elicit acute endogenous gradients and quantitative analyses, we demonstrate a two-stage process for leukocyte chemotaxis in vivo: first a "search" phase, with stimulation of actin networks at the leading edge, cell deceleration, and turning. This is followed by a "run" phase, with fast actin flows, cell acceleration, and persistence. When actin dynamics are perturbed, cells fail to resolve the gradient, suggesting that pure spatial sensing of the gradient is insufficient for navigation. Our data suggest that cell contractility and actin flows provide memory for temporal sensing, while expansion of the leading edge serves to enhance gradient sampling.

Live Imaging of Chemokine Receptors in Zebrafish Neutrophils During Wound Responses.

Leukocyte guidance by chemical gradients is essential for immune responses. Neutrophils are the first cells to be recruited to sites of tissue damage where they execute crucial antimicrobial functions. Their trafficking to these loci is orchestrated by several inflammatory chemoattractants, including chemokines. At the molecular level, chemoattractant signaling is regulated by the intracellular trafficking of the corresponding receptors. However, it remains unclear how subcellular changes in chemokine receptors affect leukocyte migration dynamics at the cell and tissue level. Here we describe a methodology for live imaging and quantitative analysis of chemokine receptor dynamics in neutrophils during inflammatory responses to tissue damage. These tools have revealed that differential chemokine receptor trafficking in zebrafish neutrophils coordinates neutrophil clustering and dispersal at sites of tissue damage. This has implications for our understanding of how inflammatory responses are self-resolved. The described tools could be used to understand neutrophil migration patterns in a variety of physiological and pathological settings and the methodology could be expanded to other signaling receptors.

Neutrophil Swarming in Damaged Tissue Is Orchestrated by Connexins and Cooperative Calcium Alarm Signals.

Neutrophils are major inflammatory cells that rapidly infiltrate wounds to provide antimicrobial functions. Within the damaged tissue, neutrophil migration behavior often switches from exploratory patrolling to coordinated swarming, giving rise to dense clusters that further disrupt tissue architecture. This aggregation response is self-organized by neutrophil paracrine chemoattractant signaling (most notably of the inflammatory mediator leukotriene B4 [LTB4]). The coordination mechanism and possible evolutionary benefits of neutrophil swarms are elusive. Here, we show that neutrophil swarms require mutual reinforcement of damage signaling at the wound core. New biosensors and live imaging in zebrafish revealed that neutrophil chemoattractant synthesis is triggered by a sustained calcium flux upon contact with necrotic tissue that requires sensing of the damage signal ATP. This "calcium alarm" signal rapidly propagates in the nascent neutrophil cluster in a contact-dependent manner via connexin-43 (Cx43) hemichannels, which are mediators of active ATP release. This enhances chemoattractant biosynthesis in the growing cluster, which is instrumental for coordinated motion and swarming. Inhibition of neutrophil Cx43 compromises clearance of wound-colonizing P. aeruginosa bacteria and exacerbates infection-induced morbidity. Thus, cooperative production of alarm signals among pioneer clustering neutrophils fuels the growth of dense antimicrobial cell masses that effectively seal off breached tissue barriers from opportunistic pathogens.

Author Correction: Chemokine receptor trafficking coordinates neutrophil clustering and dispersal at wounds in zebrafish.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Chemokine receptor trafficking coordinates neutrophil clustering and dispersal at wounds in zebrafish.

Immune cells congregate at specific loci to fight infections during inflammatory responses, a process that must be transient and self-resolving. Cell dispersal promotes resolution, but it remains unclear how transition from clustering to dispersal is regulated. Here we show, using quantitative live imaging in zebrafish, that differential ligand-induced trafficking of chemokine receptors such as Cxcr1 and Cxcr2 orchestrates the state of neutrophil congregation at sites of tissue damage. Through receptor mutagenesis and biosensors, we show that Cxcr1 promotes clustering at wound sites, but is promptly desensitized and internalized, which prevents excess congregation. By contrast, Cxcr2 promotes bidirectional motility and is sustained at the plasma membrane. Persistent plasma membrane residence of Cxcr2 prolongs downstream signaling and is required for sustained exploratory motion conducive to dispersal. Thus, differential trafficking of two chemokine receptors allows coordination of antagonistic cell behaviors, promoting a self-resolving migratory response.

Biphasic CD8+ T-Cell Defense in Simian Immunodeficiency Virus Control by Acute-Phase Passive Neutralizing Antibody Immunization.

UNLABELLED: Identifying human immunodeficiency virus type 1 (HIV-1) control mechanisms by neutralizing antibodies (NAbs) is critical for anti-HIV-1 strategies. Recent in vivo studies on animals infected with simian immunodeficiency virus (SIV) and related viruses have shown the efficacy of postinfection NAb passive immunization for viremia reduction, and one suggested mechanism is its occurrence through modulation of cellular immune responses. Here, we describe SIV control in macaques showing biphasic CD8(+) cytotoxic T lymphocyte (CTL) responses following acute-phase NAb passive immunization. Analysis of four SIVmac239-infected rhesus macaque pairs matched with major histocompatibility complex class I haplotypes found that counterparts receiving day 7 anti-SIV polyclonal NAb infusion all suppressed viremia for up to 2 years without accumulating viral CTL escape mutations. In the first phase of primary viremia control attainment, CD8(+) cells had high capacities to suppress SIVs carrying CTL escape mutations. Conversely, in the second, sustained phase of SIV control, CTL responses converged on a pattern of immunodominant CTL preservation. During this sustained phase of viral control, SIV epitope-specific CTLs showed retention of phosphorylated extracellular signal-related kinase (ERK)(hi)/phosphorylated AMP-activated protein kinase (AMPK)(lo) subpopulations, implying their correlation with SIV control. The results suggest that virus-specific CTLs functionally boosted by acute-phase NAbs may drive robust AIDS virus control. IMPORTANCE: In early HIV infection, NAb responses are lacking and CTL responses are insufficient, which leads to viral persistence. Hence, it is important to identify immune responses that can successfully control such HIV replication. Here, we show that monkeys receiving NAb passive immunization in early SIV infection strictly control viral replication for years. Passive infusion of NAbs with CTL cross-priming capacity resulted in induction of functionally boosted early CTL responses showing enhanced suppression of CTL escape mutant virus replication. Accordingly, the NAb-infused animals did not show accumulation of viral CTL escape mutations during sustained SIV control, and immunodominant CTL responses were preserved. This early functional augmentation of CTLs by NAbs provides key insights into the design of lasting and viral escape mutation-free protective immunity against HIV-1 infection.