Oxoeicosanoid signaling mediates early antimicrobial defense in zebrafish.

5-oxoETE is a bioactive lipid derived from arachidonic acid generated when phospholipase A2 activation coincides with oxidative stress. Through its G protein-coupled receptor OXER1, pure 5-oxoETE is a potent leukocyte chemoattractant. Yet, its physiological function has remained elusive owing to the unusual OXER1 conservation pattern. OXER1 is conserved from fish to primates but not in rodents, precluding genetic loss-of-function studies in mouse. To determine its physiological role, we combine transcriptomic, lipidomic, and intravital imaging assays with genetic perturbations of the OXER1 ortholog hcar1-4 in zebrafish. Pseudomonas aeruginosa infection induces the synthesis of 5-oxoETE and its receptor, along with other inflammatory pathways. Hcar1-4 deletion attenuates neutrophil recruitment and decreases post-infection survival, which could be rescued by ectopic expression of hcar1-4 or human OXER1. By revealing 5-oxoETE as dominant lipid regulator of the early antimicrobial response in a non-rodent vertebrate, our work expands the current, rodent-centric view of early inflammation.

The yellow brick road to nuclear membrane mechanotransduction.

The nuclear membrane may function as a mechanosensory surface alongside the plasma membrane. In this Review, we discuss how this idea emerged, where it currently stands, and point out possible implications, without any claim of comprehensiveness.

A synergy between mechanosensitive calcium- and membrane-binding mediates tension-sensing by C2-like domains.

When nuclear membranes are stretched, the peripheral membrane enzyme cytosolic phospholipase A2 (cPLA2) binds via its calcium-dependent C2 domain (cPLA2-C2) and initiates bioactive lipid signaling and tissue inflammation. More than 150 C2-like domains are encoded in vertebrate genomes. How many of them are mechanosensors and quantitative relationships between tension and membrane recruitment remain unexplored, leaving a knowledge gap in the mechanotransduction field. In this study, we imaged the mechanosensitive adsorption of cPLA2 and its C2 domain to nuclear membranes and artificial lipid bilayers, comparing it to related C2-like motifs. Stretch increased the Ca2+ sensitivity of all tested domains, promoting half-maximal binding of cPLA2 at cytoplasmic resting-Ca2+ concentrations. cPLA2-C2 bound up to 50 times tighter to stretched than to unstretched membranes. Our data suggest that a synergy of mechanosensitive Ca2+ interactions and deep, hydrophobic membrane insertion enables cPLA2-C2 to detect stretched membranes with antibody-like affinity, providing a quantitative basis for understanding mechanotransduction by C2-like domains.

Components and Mechanisms of Nuclear Mechanotransduction.

The cell nucleus is best known as the container of the genome. Its envelope provides a barrier for passive macromolecule diffusion, which enhances the control of gene expression. As its largest and stiffest organelle, the nucleus also defines the minimal space requirements of a cell. Internal or external pressures that deform a cell to its physical limits cause a corresponding nuclear deformation. Evidence is consolidating that the nucleus, in addition to its genetic functions, serves as a physical sensing device for critical cell body deformation. Nuclear mechanotransduction allows cells to adapt their acute behaviors, mechanical stability, paracrine signaling, and fate to their physical surroundings. This review summarizes the basic chemical and mechanical properties of nuclear components, and how these properties are thought to be utilized for mechanosensing.

Lipid peroxidation regulates long-range wound detection through 5-lipoxygenase in zebrafish.

Rapid wound detection by distant leukocytes is essential for antimicrobial defence and post-infection survival1. The reactive oxygen species hydrogen peroxide and the polyunsaturated fatty acid arachidonic acid are among the earliest known mediators of this process2-4. It is unknown whether or how these highly conserved cues collaborate to achieve wound detection over distances of several hundreds of micrometres within a few minutes. To investigate this, we locally applied arachidonic acid and skin-permeable peroxide by micropipette perfusion to unwounded zebrafish tail fins. As in wounds, arachidonic acid rapidly attracted leukocytes through dual oxidase (Duox) and 5-lipoxygenase (Alox5a). Peroxide promoted chemotaxis to arachidonic acid without being chemotactic on its own. Intravital biosensor imaging showed that wound peroxide and arachidonic acid converged on half-millimetre-long lipid peroxidation gradients that promoted leukocyte attraction. Our data suggest that lipid peroxidation functions as a spatial redox relay that enables long-range detection of early wound cues by immune cells, outlining a beneficial role for this otherwise toxic process.

Ferroptosis occurs through an osmotic mechanism and propagates independently of cell rupture.

Ferroptosis is a regulated form of necrotic cell death that is caused by the accumulation of oxidized phospholipids, leading to membrane damage and cell lysis1,2. Although other types of necrotic death such as pyroptosis and necroptosis are mediated by active mechanisms of execution3-6, ferroptosis is thought to result from the accumulation of unrepaired cell damage1. Previous studies have suggested that ferroptosis has the ability to spread through cell populations in a wave-like manner, resulting in a distinct spatiotemporal pattern of cell death7,8. Here we investigate the mechanism of ferroptosis execution and discover that ferroptotic cell rupture is mediated by plasma membrane pores, similarly to cell lysis in pyroptosis and necroptosis3,4. We further find that intercellular propagation of death occurs following treatment with some ferroptosis-inducing agents, including erastin2,9 and C' dot nanoparticles8, but not upon direct inhibition of the ferroptosis-inhibiting enzyme glutathione peroxidase 4 (GPX4)10. Propagation of a ferroptosis-inducing signal occurs upstream of cell rupture and involves the spreading of a cell swelling effect through cell populations in a lipid peroxide- and iron-dependent manner.

A Photo-clickable ATP-Mimetic Reveals Nucleotide Interactors in the Membrane Proteome.

ATP is an important energy metabolite and allosteric signal in health and disease. ATP-interacting proteins, such as P2 receptors, control inflammation, cell death, migration, and wound healing. However, identification of allosteric ATP sites remains challenging, and our current inventory of ATP-controlled pathways is likely incomplete. Here, we develop and verify mipATP as a minimally invasive photoaffinity probe for ATP-interacting proteins. Its N6 functionalization allows target enrichment by UV crosslinking and conjugation to reporter tags by "click" chemistry. The additions are compact, allowing mipATP to completely retain the calcium signaling responses of native ATP in vitro and in vivo. mipATP specifically enriched for known nucleotide binders in A549 cell lysates and membrane fractions. In addition, it retrieved unannotated ATP interactors, such as the FAS receptor, CD44, and various SLC transporters. Thus, mipATP is a promising tool to identify allosteric ATP sites in the proteome.

Quantitative Imaging of Endogenous and Exogenous H2O2 Gradients in Live Zebrafish Larvae.

Quantitative aspects of extracellular H2O2 signaling in animals, such as its spatiotemporal dynamics within tissues, remain little understood. Here we detail an optimized, experimental setup for measuring the dynamics and physiological consequences of extracellular H2O2 application to live tissues by intravital biosensor imaging in zebrafish larvae.

Live imaging of leukocyte recruitment in a zebrafish model of chemical liver injury.

Studying early immune responses to organ damage in situ requires animal models amenable to intravital imaging. Here, we used transparent zebrafish larvae, a powerful animal model for innate immunity, to measure leukocyte recruitment to damaged livers. Bath application of metronidazole (Mtz) to fish expressing nitroreductase (NTR) under a liver-specific promoter damaged the organ within 24 hours causing oxidative stress, distorted liver morphology, accumulation of TUNEL-positive cells, and transcriptional upregulation of apoptotic and antioxidant genes. Inflammatory gene transcription in damaged hepatocytes was attenuated. In line with predominant apoptosis, macrophages were massively recruited into Mtz/NTR-damaged livers. By contrast, neutrophil infiltration was more variable and delayed, consistent with less abundant necrosis and an attenuated inflammatory capacity of damaged hepatocytes.

Tissue Damage Signaling Is a Prerequisite for Protective Neutrophil Recruitment to Microbial Infection in Zebrafish.

Tissue damage and infection are deemed likewise triggers of innate immune responses. But whereas neutrophil responses to microbes are generally protective, neutrophil recruitment into damaged tissues without infection is deleterious. Why neutrophils respond to tissue damage and not just to microbes is unknown. Is it a flaw of the innate immune system that persists because evolution did not select against it, or does it provide a selective advantage? Here we dissect the contribution of tissue damage signaling to antimicrobial immune responses in a live vertebrate. By intravital imaging of zebrafish larvae, a powerful model for innate immunity, we show that prevention of tissue damage signaling upon microbial ear infection abrogates leukocyte chemotaxis and reduces animal survival, at least in part, through suppression of cytosolic phospholipase A2 (cPla2), which integrates tissue damage- and microbe-derived cues. Thus, microbial cues are insufficient, and damage signaling is essential for antimicrobial neutrophil responses in zebrafish.

Wound redox gradients revisited.

Evidence emerges that redox gradients regulate morphogenesis, inflammation, regeneration, and healing of tissues. At the example of redox signaling during the zebrafish wound response, I briefly discuss current ideas on how such patterns might be sensed and spatially regulated to guide physiological processes over distances in animals.

Image-Based Measurement of H2O2 Reaction-Diffusion in Wounded Zebrafish Larvae.

Epithelial injury induces rapid recruitment of antimicrobial leukocytes to the wound site. In zebrafish larvae, activation of the epithelial NADPH oxidase Duox at the wound margin is required early during this response. Before injury, leukocytes are near the vascular region, that is, ∼100-300 µm away from the injury site. How Duox establishes long-range signaling to leukocytes is unclear. We conceived that extracellular hydrogen peroxide (H2O2) generated by Duox diffuses through the tissue to directly regulate chemotactic signaling in these cells. But before it can oxidize cellular proteins, H2O2 must get past the antioxidant barriers that protect the cellular proteome. To test whether, or on which length scales this occurs during physiological wound signaling, we developed a computational method based on reaction-diffusion principles that infers H2O2 degradation rates from intravital H2O2-biosensor imaging data. Our results indicate that at high tissue H2O2 levels the peroxiredoxin-thioredoxin antioxidant chain becomes overwhelmed, and H2O2 degradation stalls or ceases. Although the wound H2O2 gradient reaches deep into the tissue, it likely overcomes antioxidant barriers only within ∼30 µm of the wound margin. Thus, Duox-mediated long-range signaling may require other spatial relay mechanisms besides extracellular H2O2 diffusion.

Nuclear membrane stretch and its role in mechanotransduction.

Most research in nuclear mechanotransduction has focused on the nuclear lamina and lamin binding proteins. These structures provide mechanical stability to the nucleus, establish a link between the cytoskeleton and chromatin, and can transmit mechanical signals. At the same time, mechanical perturbations to the nucleus also affect its phospholipid membranes. In this commentary, we discuss how changes in nuclear membrane tension can mediate mechanotransduction.

Neutrophil mechanotransduction: A GEF to sense fluid shear stress.

Forces deriving from blood flow shear modulate vascular adherence and transendothelial migration of leukocytes into inflamed tissues, but the mechanisms by which shear is sensed are unclear. In this issue, Fine et al. (2016. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201603109) identify the guanosine nucleotide exchange factor GEF-H1 as critical for shear stress-induced transendothelial neutrophil migration.

Illuminating Phagocyte Biology: The View from Zebrafish.

Many phagocyte behaviors, including vascular rolling and adhesion, migration, and oxidative bursting, are better measured in seconds or minutes than hours or days. Zebrafish is ideally suited for imaging such rapid biology within the intact animal. We discuss how this model has revealed unique insights into various aspects of phagocyte physiology.

A Case for the Nuclear Membrane as a Mechanotransducer.

The cell nucleus is becoming increasingly recognized as a mechanosensitive organelle. Most research on nuclear mechanosignaling focuses on the nuclear lamina and coupled actin structures. In this commentary, we discuss the possibility that the nuclear membrane senses and transduces mechanical signals similar to the plasma membrane. We briefly summarize possible (i) pathophysiological sources of nuclear membrane tension, (ii) features that render nuclear membranes particularly suited for mechanotransduction, and (iii) molecular sensing mechanisms.

The early wound signals.

Wounding of tissue barriers, such as epithelia, disrupts homeostasis and allows infection. Within minutes, animals detect injury and respond to it by recruitment of phagocytes and barrier breach closure. The signals that activate these first events are scarcely known. Commonly considered are cytoplasmic factors released into the extracellular space by lysing cells (Damage Associated Molecular Patterns, DAMPs). DAMPs activate inflammatory gene transcription through pattern recognition receptors. But the promptness of wound responses is difficult to explain by transcriptional mechanisms alone. This review highlights the emerging role of nonlytic stress signals in the rapid detection of wounds.