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Neutrophil Reverse Migration Becomes Transparent with Zebrafish  [PDF]
Taylor W. Starnes,Anna Huttenlocher
Advances in Hematology , 2012, DOI: 10.1155/2012/398640
Abstract: The precise control of neutrophil-mediated inflammation is critical for both host defense and the prevention of immunopathology. In vivo imaging studies in zebrafish, and more recently in mice, have made the novel observation that neutrophils leave a site of inflammation through a process called neutrophil reverse migration. The application of advanced imaging techniques to the genetically tractable, optically transparent zebrafish larvae was critical for these advances. Still, the mechanisms underlying neutrophil reverse migration and its effects on the resolution or priming of immune responses remain unclear. Here, we review the current knowledge of neutrophil reverse migration, its potential roles in host immunity, and the live imaging tools that make zebrafish a valuable model for increasing our knowledge of neutrophil behavior in vivo. 1. Introduction “Certain of the lower animals, transparent enough to be observed alive, clearly show in their midst a host of small cells with moving extensions. In these animals the smallest lesion brings an accumulation of these elements at the point of damage. In small transparent larvae, it can easily be shown that the moving cells, reunited at the damage point do often close over foreign bodies [1].” Ilya Mechnikov, one of the fathers of immunology, spoke these words at his Nobel Prize lecture in 1908. More than one hundred years after his seminal studies using transparent starfish larvae to illuminate a role for phagocytosis in immunity, we are again exploiting the power of transparent larvae for research on the immune system. Studies of neutrophils in both humans and mammalian model systems have brought great advances in our knowledge of their functions; however, zebrafish, a small tropical fish with transparent larvae, have demonstrated that direct observation of neutrophils in live animals can provide important insights that would have otherwise faced significant technical challenges using mice. Neutrophils are the most abundant leukocytes in both humans and zebrafish, and they are critical for defending the host against microbial infection [2]. In response to wounding, infection, or other inflammatory stimuli, neutrophils are rapidly recruited to perform their well-known effector functions: degranulation, phagocytosis, production of reactive oxygen species (ROS), secretion of proinflammatory cytokines, and extrusion of neutrophil extracellular traps (NETs) [3, 4]. These responses are acknowledged to kill and sequester microorganisms at their site of entry and promote the activation of the adaptive immune
Mediator Subunit 12 Is Required for Neutrophil Development in Zebrafish  [PDF]
Maria-Cristina Keightley, Judith E. Layton, John W. Hayman, Joan K. Heath, Graham J. Lieschke
PLOS ONE , 2011, DOI: 10.1371/journal.pone.0023845
Abstract: Hematopoiesis requires the spatiotemporal organization of regulatory factors to successfully orchestrate diverse lineage specificity from stem and progenitor cells. Med12 is a regulatory component of the large Mediator complex that enables contact between the general RNA polymerase II transcriptional machinery and enhancer bound regulatory factors. We have identified a new zebrafish med12 allele, syr, with a single missense mutation causing a valine to aspartic acid change at position 1046. Syr shows defects in hematopoiesis, which predominantly affect the myeloid lineage. Syr has identified a hematopoietic cell-specific requirement for Med12, suggesting a new role for this transcriptional regulator.
PhagoSight: An Open-Source MATLAB? Package for the Analysis of Fluorescent Neutrophil and Macrophage Migration in a Zebrafish Model  [PDF]
Katherine M. Henry, Luke Pase, Carlos Fernando Ramos-Lopez, Graham J. Lieschke, Stephen A. Renshaw, Constantino Carlos Reyes-Aldasoro
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0072636
Abstract: Neutrophil migration in zebrafish larvae is increasingly used as a model to study the response of these leukocytes to different determinants of the cellular inflammatory response. However, it remains challenging to extract comprehensive information describing the behaviour of neutrophils from the multi-dimensional data sets acquired with widefield or confocal microscopes. Here, we describe PhagoSight, an open-source software package for the segmentation, tracking and visualisation of migrating phagocytes in three dimensions. The algorithms in PhagoSight extract a large number of measurements that summarise the behaviour of neutrophils, but that could potentially be applied to any moving fluorescent cells. To derive a useful panel of variables quantifying aspects of neutrophil migratory behaviour, and to demonstrate the utility of PhagoSight, we evaluated changes in the volume of migrating neutrophils. Cell volume increased as neutrophils migrated towards the wound region of injured zebrafish. PhagoSight is openly available as MATLAB? m-files under the GNU General Public License. Synthetic data sets and a comprehensive user manual are available from http://www.phagosight.org.
Drift-Diffusion Analysis of Neutrophil Migration during Inflammation Resolution in a Zebrafish Model  [PDF]
Geoffrey R. Holmes,Giles Dixon,Sean R. Anderson,Constantino Carlos Reyes-Aldasoro,Philip M. Elks,Stephen A. Billings,Moira K. B. Whyte,Visakan Kadirkamanathan,Stephen A. Renshaw
Advances in Hematology , 2012, DOI: 10.1155/2012/792163
Abstract: Neutrophils must be removed from inflammatory sites for inflammation to resolve. Recent work in zebrafish has shown neutrophils can migrate away from inflammatory sites, as well as die in situ. The signals regulating the process of reverse migration are of considerable interest, but remain unknown. We wished to study the behaviour of neutrophils during reverse migration, to see whether they moved away from inflamed sites in a directed fashion in the same way as they are recruited or whether the inherent random component of their migration was enough to account for this behaviour. Using neutrophil-driven photoconvertible Kaede protein in transgenic zebrafish larvae, we were able to specifically label neutrophils at an inflammatory site generated by tailfin transection. The locations of these neutrophils over time were observed and fitted using regression methods with two separate models: pure-diffusion and drift-diffusion equations. While a model hypothesis test (the F-test) suggested that the datapoints could be fitted by the drift-diffusion model, implying a fugetaxis process, dynamic simulation of the models suggested that migration of neutrophils away from a wound is better described by a zero-drift, “diffusion” process. This has implications for understanding the mechanisms of reverse migration and, by extension, neutrophil retention at inflammatory sites. 1. Introduction The fate of neutrophils following completion of the inflammatory programme is of critical importance for the outcome of episodes of acute inflammation and can determine whether there is prompt healing of a wound or the development of chronic inflammation and tissue injury. Neutrophils recruited to sites of inflammation may leave the site or die in situ [1]. The most widely accepted mechanism of neutrophil disposal is the programmed cell death or apoptosis, of the neutrophil followed by macrophage uptake and clearance (reviewed in [2]). Recently, other routes have been proposed; neutrophils may move away from the inflamed site into the bloodstream (“reverse transmigration” [3]), by migration through other tissues (“retrograde chemotaxis” or “reverse migration” [4–6]), or be lost into the inflammatory exudate [7, 8]. Current understanding of the process of reverse migration is reviewed elsewhere [9]. The uncertainty as to the in vivo fates of individual cells relates in part to the difficulty in following individual cells during inflammation resolution in vivo. The transgenic zebrafish model is emerging as a key model for the study of vertebrate immunity [10] and allows direct
BMP Signaling Modulates Hepcidin Expression in Zebrafish Embryos Independent of Hemojuvelin  [PDF]
Yann Gibert,Victoria J. Lattanzi,Aileen W. Zhen,Lea Vedder,Frédéric Brunet,Sarah A. Faasse,Jodie L. Babitt,Herbert Y. Lin,Matthias Hammerschmidt,Paula G. Fraenkel
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0014553
Abstract: Hemojuvelin (Hjv), a member of the repulsive-guidance molecule (RGM) family, upregulates transcription of the iron regulatory hormone hepcidin by activating the bone morphogenetic protein (BMP) signaling pathway in mammalian cells. Mammalian models have identified furin, neogenin, and matriptase-2 as modifiers of Hjv's function. Using the zebrafish model, we evaluated the effects of hjv and its interacting proteins on hepcidin expression during embryonic development. We found that hjv is strongly expressed in the notochord and somites of the zebrafish embryo and that morpholino knockdown of hjv impaired the development of these structures. Knockdown of hjv or other hjv-related genes, including zebrafish orthologs of furin or neogenin, however, failed to decrease hepcidin expression relative to liver size. In contrast, overexpression of bmp2b or knockdown of matriptase-2 enhanced the intensity and extent of hepcidin expression in zebrafish embryos, but this occurred in an hjv-independent manner. Furthermore, we demonstrated that zebrafish hjv can activate the human hepcidin promoter and enhance BMP responsive gene expression in vitro, but is expressed at low levels in the zebrafish embryonic liver. Taken together, these data support an alternative mechanism for hepcidin regulation during zebrafish embryonic development, which is independent of hjv.
Tibetan herbal formula Padma Digestin modulates gastrointestinal motility in vitro  [cached]
Bruno M Balsiger,Magali Krayer,Andreas Rickenbacher,Beatrice Flogerzi
World Journal of Gastrointestinal Pharmacology and Therapeutics , 2013, DOI: 10.4292/wjgpt.v4.i1.9
Abstract: AIM: To examine the effects of Padma Digestin on the smooth muscle motility of different gastrointestinal segments in vitro. METHODS: The effects of the ethanolic extract of Padma Digestin (at 8.16 mg/mL or 81.6 mg/mL) on the contractility and susceptibility to acetylcholine (ACh) of muscle strips from the cardia, antrum, pylorus, duodenum, jejunum, ileum and colon of male Wistar rats were analyzed. RESULTS: Compared with the control treatment, the Padma Digestin extract had a procontractile effect on the antral smooth muscle strips. Padma Digestin decreased ACh sensitivity in cardia muscle strips and increased it in those from the antrum and pylorus. In the intestinal segments, spontaneous contractility was inhibited in both the duodenal and jejunal strips, whereas reactivity to ACh was inhibited in the jejunal strips only. In the colonic samples, Padma Digestin inhibited spontaneous and ACh-stimulated contractility at a low dose but seems to have increasing effects at a high dose. CONCLUSION: Padma Digestin extract has region-specific effects on the contractility and excitability of gastrointestinal smooth muscle. Our results support the traditional use of Padma Digestin for maldigestion and functional gastrointestinal disorders.
A Complex of BBS1 and NPHP7 Is Required for Cilia Motility in Zebrafish  [PDF]
Yun Hee Kim, Daniel Epting, Krasimir Slanchev, Christina Engel, Gerd Walz, Albrecht Kramer-Zucker
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0072549
Abstract: Bardet-Biedl syndrome (BBS) and nephronophthisis (NPH) are hereditary autosomal recessive disorders, encoded by two families of diverse genes. BBS and NPH display several overlapping phenotypes including cystic kidney disease, retinitis pigmentosa, liver fibrosis, situs inversus and cerebellar defects. Since most of the BBS and NPH proteins localize to cilia and/or their appendages, BBS and NPH are considered ciliopathies. In this study, we characterized the function of the transcription factor Nphp7 in zebrafish, and addressed the molecular connection between BBS and NPH. The knockdown of zebrafish bbs1 and nphp7.2 caused similar phenotypic changes including convergent extension defects, curvature of the body axis, hydrocephalus, abnormal heart looping and cystic pronephros, all consistent with an altered ciliary function. Immunoprecipitation assays revealed a physical interaction between BBS1 and NPHP7, and the simultaneous knockdown of zbbs1 and znphp7.2 enhanced the cystic pronephros phenotype synergistically, suggesting a genetic interaction between zbbs1 and znphp7.2 in vivo. Deletion of zBbs1 or zNphp7.2 did not compromise cilia formation, but disrupted cilia motility. Although NPHP7 has been shown to act as transcriptional repressor, our studies suggest a crosstalk between BBS1 and NPHP7 in regulating normal function of the cilium.
Modulation of Neutrophil Function by a Secreted Mucinase of Escherichia coli O157∶H7  [PDF]
Rose L. Szabady,Mary A. Lokuta,Kevin B. Walters,Anna Huttenlocher,Rodney A. Welch
PLOS Pathogens , 2009, DOI: 10.1371/journal.ppat.1000320
Abstract: Escherichia coli O157:H7 is a human enteric pathogen that causes hemorrhagic colitis which can progress to hemolytic uremic syndrome, a severe kidney disease with immune involvement. During infection, E. coli O157:H7 secretes StcE, a metalloprotease that promotes the formation of attaching and effacing lesions and inhibits the complement cascade via cleavage of mucin-type glycoproteins. We found that StcE cleaved the mucin-like, immune cell-restricted glycoproteins CD43 and CD45 on the neutrophil surface and altered neutrophil function. Treatment of human neutrophils with StcE led to increased respiratory burst production and increased cell adhesion. StcE-treated neutrophils exhibited an elongated morphology with defective rear detachment and impaired migration, suggesting that removal of the anti-adhesive capability of CD43 by StcE impairs rear release. Use of zebrafish embryos to model neutrophil migration revealed that StcE induced neutrophil retention in the fin after tissue wounding, suggesting that StcE modulates neutrophil-mediated inflammation in vivo. Neutrophils are crucial innate effectors of the antibacterial immune response and can contribute to severe complications caused by infection with E. coli O157:H7. Our data suggest that the StcE mucinase can play an immunomodulatory role by directly altering neutrophil function during infection. StcE may contribute to inflammation and tissue destruction by mediating inappropriate neutrophil adhesion and activation.
Burn Injury Reduces Neutrophil Directional Migration Speed in Microfluidic Devices  [PDF]
Kathryn L. Butler,Vijayakrishnan Ambravaneswaran,Nitin Agrawal,Maryelizabeth Bilodeau,Mehmet Toner,Ronald G. Tompkins,Shawn Fagan,Daniel Irimia
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0011921
Abstract: Thermal injury triggers a fulminant inflammatory cascade that heralds shock, end-organ failure, and ultimately sepsis and death. Emerging evidence points to a critical role for the innate immune system, and several studies had documented concurrent impairment in neutrophil chemotaxis with these post-burn inflammatory changes. While a few studies suggest that a link between neutrophil motility and patient mortality might exist, so far, cumbersome assays have prohibited exploration of the prognostic and diagnostic significance of chemotaxis after burn injury. To address this need, we developed a microfluidic device that is simple to operate and allows for precise and robust measurements of chemotaxis speed and persistence characteristics at single-cell resolution. Using this assay, we established a reference set of migration speed values for neutrophils from healthy subjects. Comparisons with samples from burn patients revealed impaired directional migration speed starting as early as 24 hours after burn injury, reaching a minimum at 72–120 hours, correlated to the size of the burn injury and potentially serving as an early indicator for concurrent infections. Further characterization of neutrophil chemotaxis using this new assay may have important diagnostic implications not only for burn patients but also for patients afflicted by other diseases that compromise neutrophil functions.
A Unique Protein Phosphatase with Kelch-Like Domains (PPKL) in Plasmodium Modulates Ookinete Differentiation, Motility and Invasion  [PDF]
David S. Guttery equal contributor,Benoit Poulin equal contributor,David J. P. Ferguson,Balázs Sz??r,Bill Wickstead,Paula L. Carroll,Chandra Ramakrishnan,Declan Brady,Eva-Maria Patzewitz,Ursula Straschil,Lev Solyakov,Judith L. Green,Robert E. Sinden,Andrew B. Tobin,Anthony A. Holder,Rita Tewari
PLOS Pathogens , 2012, DOI: 10.1371/journal.ppat.1002948
Abstract: Protein phosphorylation and dephosphorylation (catalysed by kinases and phosphatases, respectively) are post-translational modifications that play key roles in many eukaryotic signalling pathways, and are often deregulated in a number of pathological conditions in humans. In the malaria parasite Plasmodium, functional insights into its kinome have only recently been achieved, with over half being essential for blood stage development and another 14 kinases being essential for sexual development and mosquito transmission. However, functions for any of the plasmodial protein phosphatases are unknown. Here, we use reverse genetics in the rodent malaria model, Plasmodium berghei, to examine the role of a unique protein phosphatase containing kelch-like domains (termed PPKL) from a family related to Arabidopsis BSU1. Phylogenetic analysis confirmed that the family of BSU1-like proteins including PPKL is encoded in the genomes of land plants, green algae and alveolates, but not in other eukaryotic lineages. Furthermore, PPKL was observed in a distinct family, separate to the most closely-related phosphatase family, PP1. In our genetic approach, C-terminal GFP fusion with PPKL showed an active protein phosphatase preferentially expressed in female gametocytes and ookinetes. Deletion of the endogenous ppkl gene caused abnormal ookinete development and differentiation, and dissociated apical microtubules from the inner-membrane complex, generating an immotile phenotype and failure to invade the mosquito mid-gut epithelium. These observations were substantiated by changes in localisation of cytoskeletal tubulin and actin, and the micronemal protein CTRP in the knockout mutant as assessed by indirect immunofluorescence. Finally, increased mRNA expression of dozi, a RNA helicase vital to zygote development was observed in ppkl? mutants, with global phosphorylation studies of ookinete differentiation from 1.5–24 h post-fertilisation indicating major changes in the first hours of zygote development. Our work demonstrates a stage-specific essentiality of the unique PPKL enzyme, which modulates parasite differentiation, motility and transmission.
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