title: Inhibitors of neutrophil recruitment identified using transgenic zebrafish to screen a natural product library.
abstract: Cell migration is fundamental to the inflammatory response, but uncontrolled cell migration and excess recruitment of neutrophils and other leukocytes can cause damage to the tissue. Here we describe the use of an in vivo model - the Tg(mpx:GFP)(i114) zebrafish line, in which neutrophils are labelled by green fluorescent protein (GFP) - to screen a natural product library for compounds that can affect neutrophil migratory behaviour. Among 1040 fungal extracts screened, two were found to inhibit neutrophil migration completely. Subfractionation of these extracts identified sterigmatocystin and antibiotic PF1052 as the active components. Using the EZ-TAXIScan chemotaxis assay, both compounds were also found to have a dosage-dependent inhibitory effect on murine neutrophil migration. Furthermore, neutrophils treated with PF1052 failed to form pseudopods and appeared round in shape, suggesting a defect in PI3-kinase (PI3K) signalling. We generated a transgenic neutrophil-specific PtdIns(3,4,5)P3 (PIP3) reporter zebrafish line, which revealed that PF1052 does not affect the activation of PI3K at the plasma membrane. In human neutrophils, PF1052 neither induced apoptosis nor blocked AKT phosphorylation. In conclusion, we have identified an antibiotic from a natural product library with potent anti-inflammatory properties, and have established the utility of the mpx:GFP transgenic zebrafish for high-throughput in vivo screens for novel inhibitors of neutrophil migration.
authors.affiliation: Institute of Molecular and Cell Biology, 61 Biopolis Drive, Proteos, 138673 Singapore.
authors.forename: Xingang
authors.initials: X
authors.lastname: Wang
authors.forename: Anne L
authors.initials: AL
authors.lastname: Robertson
authors.forename: Jingyu
authors.initials: J
authors.lastname: Li
authors.forename: Ruth Jinfen
authors.initials: RJ
authors.lastname: Chai
authors.forename: Wang
authors.initials: W
authors.lastname: Haishan
authors.forename: Pranvera
authors.initials: P
authors.lastname: Sadiku
authors.forename: Nikolay V
authors.initials: NV
authors.lastname: Ogryzko
authors.forename: Martin
authors.initials: M
authors.lastname: Everett
authors.forename: Kanagasundaram
authors.initials: K
authors.lastname: Yoganathan
authors.forename: Hongbo Robert
authors.initials: HR
authors.lastname: Luo
authors.forename: Stephen A
authors.initials: SA
authors.lastname: Renshaw
authors.forename: Philip W
authors.initials: PW
authors.lastname: Ingham
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D017209:Apoptosis; D002462:Cell Membrane / Q000378:metabolism; D002633:Chemotaxis / Q000187:drug effects*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006801:Humans; D007249:Inflammation; D008297:Male; D051379:Mice; D008810:Mice, Inbred C57BL; D009281:Naphthalenes / Q000494:pharmacology*; D020556:Neutrophil Infiltration; D009504:Neutrophils / Q000166:cytology* / Q000187:drug effects; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010766:Phosphorylation; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D011760:Pyrrolidinones / Q000494:pharmacology*; D013241:Sterigmatocystin / Q000494:pharmacology*; D013347:Subcellular Fractions; D019076:Transgenes; D015027:Zebrafish
chemical_list: D000900:Anti-Bacterial Agents; D009281:Naphthalenes; C000591384:PF1052; D011760:Pyrrolidinones; D013241:Sterigmatocystin; D049452:Green Fluorescent Proteins; D019869:Phosphatidylinositol 3-Kinases; D051057:Proto-Oncogene Proteins c-akt
keywords: Drug screen; Migration; Neutrophil; Recruitment; Zebrafish