Xingang Wang1,2, Anne L. Robertson3,4, Jingyu Li5,6, Ruth Jinfen Chai1, Wang Haishan1, Pranvera Sadiku3,4, Nikolay V. Ogryzko3, Martin Everett7, Kanagasundaram Yoganathan7, Hongbo Robert Luo5, Stephen A. Renshaw3,4 and Philip W. Ingham1,3,8,*.
1 - Institute of Molecular and Cell Biology, 61 Biopolis Drive, Proteos, Singapore 138673.
2 - High-Throughput Molecular Drug Discovery Centre, Tianjin, China.
3 - MRC Centre for Developmental and Biomedical Genetics, University of Sheffield, Firth Court, Western Bank, Sheffield, S10 2TN, UK.
4 - Department of Infection and Immunity, University of Sheffield, Sheffield, S10 2JF, UK.
5 - Department of Pathology, Dana-Farber/Harvard Cancer Center, Harvard Medical School, Boston, MA 02215, USA.
6 - Department of Genetics and Cell Biology, Tianjin Key Laboratory of Protein
Science, College of Life Sciences, Nankai University, Tianjin, China.
7 - MerLion Pharmaceuticals Pte Ltd, Science Park II, Singapore 117610.
8 - Department of Biological Sciences, National University of Singapore, Singapore 117610.
* Author for correspondence (firstname.lastname@example.org)
Published online in Disease Models & Mechanisms on 28 November 2013.
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 neutrophilspecific
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
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.
Figure legend: Inhibitory effect of PF1052 and sterigmatocystin on neutrophil migration towards the wound site. Neutrophil migration assay was carried out using Tg(mpx:GFP)i114larvae according to Renshaw et al. (Renshaw etal., 2006).
(A) Neutrophils were quiescent on an uninjured 3-dpf larva.
(B) About ten neutrophils were recruited to the wound site 3 hours after the tailfin was amputated on a control larva that was treated with DMSO.
(C,D) Larvae treated by PI3K inhibitor LY294002 (50 μM) or microtubule inhibitor nocodazole (33 μM), respectively, had few neutrophils recruited to the wound.
(E) An extract (ID: XF06-5B03) from an ascomycete genus Sphaeropsidales completely blocked neutrophil migration towards the wound at 50 μg/ml.
(F) The active component identified from XF06-5B03 extract (PF1052) also completely blocked neutrophils recruitment at a very low concentration of 2 μM; the molecular structure of PF1052 is shown in J.
(G,H,K) Another extract, XF06-2A10, from a fungus, genus Aspergillus, that blocked the migration of neutrophils contains the functional component sterigmatocystin, working at 30 μM.
(I) Quantification of neutrophils recruited to the site of injury (n>15). The white line on B-H indicates the amputation site of tailfin. ***P<0.001.
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