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 process2,3,4. 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.
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All of the data supporting the findings of this study are available from the corresponding author upon reasonable request. Source data are provided with this paper.
Custom MATLAB analysis code can be found at https://github.com/niethamp/KatikaneniAndJelcic2020.
Huang, C. & Niethammer, P. Tissue damage signaling is a prerequisite for protective neutrophil recruitment to microbial infection in zebrafish. Immunity 48, 1006–1013.e6 (2018).
Niethammer, P., Grabher, C., Look, A. T. & Mitchison, T. J. A tissue-scale gradient of hydrogen peroxide mediates rapid wound detection in zebrafish. Nature 459, 996–999 (2009).
Yoo, S. K., Starnes, T. W., Deng, Q. & Huttenlocher, A. Lyn is a redox sensor that mediates leukocyte wound attraction in vivo. Nature 480, 109–112 (2011).
Enyedi, B., Kala, S., Nikolich-Zugich, T. & Niethammer, P. Tissue damage detection by osmotic surveillance. Nat. Cell Biol. 15, 1123–1130 (2013).
Enyedi, B. & Niethammer, P. Mechanisms of epithelial wound detection. Trends Cell Biol. 25, 398–407 (2015).
Klyubin, I. V., Kirpichnikova, K. M. & Gamaley, I. A. Hydrogen peroxide-induced chemotaxis of mouse peritoneal neutrophils. Eur. J. Cell Biol. 70, 347–351 (1996).
Evans, I. R., Rodrigues, F. S. L. M., Armitage, E. L. & Wood, W. Draper/CED-1 mediates an ancient damage response to control inflammatory blood cell migration in vivo. Curr. Biol. 25, 1606–1612 (2015).
Jelcic, M., Enyedi, B., Xavier, J. B. & Niethammer, P. Image-based measurement of H2O2 reaction–diffusion in wounded zebrafish larvae. Biophys. J. 112, 2011–2018 (2017).
Enyedi, B., Jelcic, M. & Niethammer, P. The cell nucleus serves as a mechanotransducer of tissue damage-induced inflammation. Cell 165, 1160–1170 (2016).
Lämmermann, T. et al. Neutrophil swarms require LTB4 and integrins at sites of cell death in vivo. Nature 498, 371–375 (2013).
Trede, N. S., Langenau, D. M., Traver, D., Look, A. T. & Zon, L. I. The use of zebrafish to understand immunity. Immunity 20, 367–379 (2004).
Hammarström, S. et al. Increased concentrations of nonesterified arachidonic acid, 12L-hydroxy-5,8,10,14-eicosatetraenoic acid, prostaglandin E2, and prostaglandin F2α in epidermis of psoriasis. Proc. Natl Acad. Sci. USA 72, 5130–5134 (1975).
Mathias, J. R. et al. Resolution of inflammation by retrograde chemotaxis of neutrophils in transgenic zebrafish. J. Leukoc. Biol. 80, 1281–1288 (2006).
Gault, W. J., Enyedi, B. & Niethammer, P. Osmotic surveillance mediates rapid wound closure through nucleotide release. J. Cell Biol. 207, 767–782 (2014).
Riendeau, D., Denis, D., Choo, L. Y. & Nathaniel, D. J. Stimulation of 5-lipoxygenase activity under conditions which promote lipid peroxidation. Biochem. J. 263, 565–572 (1989).
Rouzer, C. A. & Samuelsson, B. The importance of hydroperoxide activation for the detection and assay of mammalian 5-lipoxygenase. FEBS Lett. 204, 293–296 (1986).
Conrad, M. et al. Regulation of lipid peroxidation and ferroptosis in diverse species. Genes Dev. 32, 602–619 (2018).
Linkermann, A. et al. Synchronized renal tubular cell death involves ferroptosis. Proc. Natl Acad. Sci. USA 111, 16836–16841 (2014).
Kim, S. E. et al. Ultrasmall nanoparticles induce ferroptosis in nutrient-deprived cancer cells and suppress tumour growth. Nat. Nanotechnol. 11, 977–985 (2016).
Riegman, M. et al. Ferroptosis occurs through an osmotic mechanism and propagates independently of cell rupture. Nat. Cell Biol. (in the press).
Yang, W. S. et al. Peroxidation of polyunsaturated fatty acids by lipoxygenases drives ferroptosis. Proc. Natl Acad. Sci. USA 113, E4966–E4975 (2016).
Lebold, K. M. et al. Novel liquid chromatography–mass spectrometry method shows that vitamin E deficiency depletes arachidonic and docosahexaenoic acids in zebrafish (Danio rerio) embryos. Redox Biol. 2, 105–113 (2013).
Pap, E. H. W. et al. Ratio-fluorescence microscopy of lipid oxidation in living cells using C11-BODIPY581/591. FEBS Lett. 453, 278–282 (1999).
Brash, A. R. Arachidonic acid as a bioactive molecule. J. Clin. Invest. 107, 1339–1345 (2001).
Noguchi, N., Yoshida, Y., Kaneda, H., Yamamoto, Y. & Niki, E. Action of ebselen as an antioxidant against lipid peroxidation. Biochem. Pharmacol. 44, 39–44 (1992).
Zilka, O. et al. On the mechanism of cytoprotection by ferrostatin-1 and liproxstatin-1 and the role of lipid peroxidation in ferroptotic cell death. ACS Cent. Sci. 3, 232–243 (2017).
Adel, S., Heydeck, D., Kuhn, H. & Ufer, C. The lipoxygenase pathway in zebrafish. Expression and characterization of zebrafish ALOX5 and comparison with its human ortholog. Biochim. Biophys. Acta 1861, 1–11 (2016).
Haas, U. et al. Targeted knock-down of a structurally atypical zebrafish 12S-lipoxygenase leads to severe impairment of embryonic development. Proc. Natl Acad. Sci. USA 108, 20479–20484 (2011).
Uhlén, M. et al. A human protein atlas for normal and cancer tissues based on antibody proteomics. Mol. Cell Proteom. 4, 1920–1932 (2005).
Erlemann, K.-R. et al. Airway epithelial cells synthesize the lipid mediator 5-oxo-ETE in response to oxidative stress. Free Radic. Biol. Med. 42, 654–664 (2007).
Albadri, S. et al. Redox signaling via lipid peroxidation regulates retinal progenitor cell differentiation. Dev. Cell 50, 73–89.e6 (2019).
White, R. M. et al. Transparent adult zebrafish as a tool for in vivo transplantation analysis. Cell Stem Cell 2, 183–189 (2008).
Nüsslein-Volhard, C. & Dahm, R. Zebrafish: A Practical Approach 303 (Oxford Univ. Press, 2002).
Maeda, H. et al. Fluorescent probes for hydrogen peroxide based on a non-oxidative mechanism. Angew. Chem. Int. Ed. Engl. 43, 2389–2391 (2004).
Meijering, E., Dzyubachyk, O. & Smal, I. Methods for cell and particle tracking. Meth. Enzymol. 504, 183–200 (2012).
Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).
Rosowski, E. E. et al. Macrophages inhibit Aspergillus fumigatus germination and neutrophil-mediated fungal killing. PLoS Pathog. 14, e1007229 (2018).
Robu, M. E. et al. p53 activation by knockdown technologies. PLoS Genet. 3, e78 (2007).
Labun, K., Montague, T. G., Gagnon, J. A., Thyme, S. B. & Valen, E. CHOPCHOP v2: a web tool for the next generation of CRISPR genome engineering. Nucleic Acids Res. 44, W272–W276 (2016).
Postma, M. & Goedhart, J. PlotsOfData—a web app for visualizing data together with their summaries. PLoS Biol. 17, e3000202 (2019).
This research was supported by the NIH/NIGMS grants R01GM099970 and R01GM127356 to P.N. and the MSKCC Functional Genomics Initiative, and in part through NIH/NCI Cancer Center Support grant P30CA008748. We thank the Integrated Genomics Operation Core and Bioinformatics Core at MSKCC for assistance.
The authors declare no competing interests.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
a, Leukocyte mobilization to different AA preparations. Circles, median. Bars, 95% confidence interval. Data aggregated from n=10 (Cayman AA), and n=10 (Sigma AA) biologically independent animal experiments. No significant difference (p=0.51) detected between groups by two-sided, heteroscedastic Student’s t-test. b, CHP stimulates H2O2. Zebrafish tail fins of wild type larvae were loaded with the H2O2 sensor dye acetyl-pentafluorobenzene sulphonyl fluorescein, and then exposed to 5 μM CHP or DMSO control (Ctr). Top panel, representative dye emission images at 0 min and 45 min after CHP exposure. Lower panel, plots (mean ± SEM) of normalized dye fluorescence over 45 min after CHP exposure. Data aggregated from n=15 (Ctr), and n=18 (CHP) biologically independent animal experiments c, Time-lapse montage of a wave-like tissue degeneration emerging from an AA (20 μM) microperfusion patch site. Pink shade highlights the affected region. Time, hh:mm:ss. Images representative of the 2 out of 241 biologically independent animal experiments with observable waves. d, Confirmation of duox knockdown by RT-PCR in 2 dpf embryos. Representative for three independent analyses. e, C11B-imaging of control larvae (Ctr) and larvae treated with 20 μM or 100 μM DPI ~15-30 min after tail fin tip amputation. Upper panel: Representative C11B ratio images. Lower panel: Wound ratio, mean C11B ratio normalized to median baseline ratio ± SEM plotted against wound distance. Inset, boxplots of baseline ratios normalized to control. Central mark, median. Box edges, 25th/75th percentiles. Whiskers, most extreme, non-outlier data points. +, outliers. Data aggregated from n=17 (Ctr, 0 μM DPI), n=9 (20 μM DPI), and n=9 (100 μM DPI) biologically independent animal experiments. Parentheses, number of different animals. Scale bar, 200 μm. Statistical source data can be found at Source data Extended Data Figure 1.
a, Schematic representation of the protein domains of zebrafish 12-lipxoygenase (Alox12). The lipoxygenase domain of Alox12 responsible for enzymatic function was targeted with a short guide RNA (sgRNA). b, Sanger sequence analysis of cDNA identified a two-base pair deletion at nucleotide position 1011-1012 within exon 10 of alox12. c, The targeted region of the lipoxygenase domain of zebrafish Alox12 is well conserved compared to human ALOX12. The frameshift mutant alox12 allele contains a premature stop codon in the coding sequence. Asterisks, fully conserved residues. Colons, conservation of strongly similar residues. Periods, conservation of weakly similar residues. d, Next-Gen Sequencing and CRISPResso analysis of the amplicon containing the sgRNA cut site was performed on genomic DNA from tail fin clips of F2 adult alox12-targeted zebrafish. Alox12-targeted F2 male and female zebrafish exhibited a 2 bp mutation positioned at the proposed sgRNA cut site in greater than 99% of sequence reads over a depth of more than 300 and 400 thousand reads, respectively. Wild type zebrafish displayed background sequence abnormalities below 1% in more than 140 thousand total reads. e, CRISPResso analysis of F2 male and female zebrafish targeted with the sgRNA against alox12 identifies a single 2 bp indel. An indel is denoted by a blue solid bar and no indel is represented by a red dashed bar. Wildtype zebrafish display no indels in most of the sequence reads.
a, Schematic representation of zebrafish Alox5a. A single guide RNA (sgRNA) was designed to target alox5a for gene disruption within the lipoxygenase domain at exon 7 (bold text). Successful target disruption is predicted to occur at a ScaI restriction enzyme recognition site (AGT^ACT) (highlighted red text) within the genomic DNA (gDNA) sequence, potentially destroying the sequence upon DNA repair. b, alox5a wild type and knockout mutant alleles. The mutant alox5a allele contains an 8-base pair (bp) deletion resulting in a frameshift and the destruction of the ScaI restriction enzyme recognition site. Red text, mutant alox5a sequence that differs from the wild type sequence. c, The targeted region of the lipoxygenase domain of zebrafish Alox5a is highly conserved compared to human ALOX5. The mutant alox5a allele contains a premature stop codon in the coding sequence, resulting in a truncated Alox5a. Asterisks, fully conserved residues. Colons, conservation of strongly similar residues. Periods, conservation of weakly similar residues. Red text, mutant Alox5a sequence that differs from the wild type sequence. d, Heterozygous alox5a larvae were crossed and gDNA from isolated progeny was PCR amplified, ScaI-digested, and analyzed by agarose gel electrophoresis for genotyping. PCR product from the wild type alox5a allele is cleaved into two smaller products upon incubation with ScaI (409 and 693 bp products). The mutant alox5a allele is not cleaved by ScaI (1094 bp product only). Heterozygotes are identified by the combined presence of a cleaved wild type allele and non-cleaved mutant allele (409, 693, and 1094 bp products). Representative of three independent analyses. e, Sudan black-stained neutrophils in the tail region of zebrafish larvae at 2.5-3 days post fertilization. Shown are three representative animals from a total of 20 different larvae stained per genotype. Scale bar, 200 μm.
Left panels: leukocyte speed, migration persistence, directionality, and responsiveness towards microperfused 20 μM AA tracked over 20 min after patching. Right panels: leukocyte speed, migration persistence, directionality, and responsiveness towards a tail fin wound tracked over 40 min after tail fin injury. Leukocyte speed, migration persistence, directionality was calculated as previously described (see Methods). The Responsive Fractions (red color, pie charts) denote the fractions of animals for which at least two tracks > 50 μm could be reliably measured in the caudal tail fin below the notochord line. Tracking data are only aggregated for responsive animals. Only clearly distinct positions were marked for tracking; stationary leukocytes were not tracked. The tracking analysis was performed on the time lapse data underlying Fig. 1c, 2d and 4a, b, and an additional AA/DPI pipette experiment, in which leukocyte migration to a micropipette filled with 20 μM AA + 50 μM DPI (DPI, green dots) was tracked. Gray shaded region, duox MO1 morphants were compared to duox MP animals instead of wild type animals, as both samples are also co-morphants for p53 (see Methods). Horizontal bars, mean. Data aggregated from n=46 (wt, Micropipette), n=17 (alox5a KO, Micropipette), and n=9 (alox12 KO, Micropipette), n=7 (DPI, Micropipette), n=3 (Linoleic, Micropipette), n=20 (duox MP, Micropipette), n=10 (duox MO1, Micropipette), n=56 (wt, Wound), n=42 (alox5a KO, Wound), and n=23 (alox12 KO, Wound) biologically independent animal experiments. P-values with color code, two-sided, heteroscedastic Student’s t-test for pairwise comparison of the indicated color-coded samples. P-value near brackets, two-tailed Fisher’s exact test between Responsive Fractions indicated by brackets. Parentheses, number of different animals. Statistical source data can be found at Source data Extended Data Figure 4.
Wounding activates Duox to produce H2O2 and causes hypotonic (Hypo) shock, which leads to release of PUFAs (including AA). H2O2 reacts with Fe2+ to generate HO∙ by Fenton chemistry. HO∙ reacts with PUFAs/AA to generate long-chain lipid ROS (PUFA-OO∙, PUFA-OOH) that further promote lipid peroxidation, and activate Alox5a to produce leukocyte chemoattractants. If lipid peroxidation slips out of control, it may cause cell death.
Microperfusion of a wild-type zebrafish tail fin with 20 μM AA. Note that leukocytes immediately returned to the vasculature once the pipette was removed. Time is shown in hours, minutes and seconds. Scale bar, 200 μm.
Microperfusion of control (duox MP; top) and duox knockdown (duox MO1; bottom) tail fins with 20 μM AA. The red lines show the leukocyte tracks. Time is shown in hours, minutes and seconds. Scale bar, 200 μm.
Microperfusion of a zebrafish tail fin with 20 μM AA. Note the wave emerging at 00:36:45. Time is shown in hours, minutes and seconds (after patching). Scale bar, 200 μm.
Microperfusion of zebrafish tail fins with 20 μM AA (top) or 20 μM linoleic acid (Lin; bottom). The red lines show the leukocyte tracks. Time is shown in hours, minutes and seconds. Scale bar, 200 μm.
Leukocyte recruitment to zebrafish tail fin wounds treated with DMSO carrier control (Ctr; top) or 3 μM Ebs (bottom). The red lines show the leukocyte tracks. Time is shown in hours, minutes and seconds. Scale bar, 200 μm.
Leukocyte recruitment to zebrafish tail fin wounds treated with DMSO carrier control (top), 20 μM Lpx (middle) or 100 μM α-TOC (bottom). The red lines show the leukocyte tracks. Time is shown in hours, minutes and seconds. Scale bar, 200 μm.
Microperfusion of wild-type (WT; top), alox12 KO (middle) and alox5a KO (bottom) tail fins with 20 μM AA. The red lines show the leukocyte tracks. Time is shown in hours, minutes and seconds. Scale bar, 200 μm.
Leukocyte recruitment to wounds in wild-type (top), alox12 KO (middle) or alox5a KO (bottom) tail fins. The red lines show the leukocyte tracks. Time is shown in hours, minutes and seconds. Scale bar, 200 μm.
Numerical source data for Figure 1
Numerical source data for Figure 2
Numerical source data for Figure 3
Numerical source data for Figure 4
Numerical source data for Extended Data Figure 1
Full gel scan for Extended Data Figure 3d
Numerical source data for Extended Data Figure 4
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Katikaneni, A., Jelcic, M., Gerlach, G.F. et al. Lipid peroxidation regulates long-range wound detection through 5-lipoxygenase in zebrafish. Nat Cell Biol 22, 1049–1055 (2020). https://doi.org/10.1038/s41556-020-0564-2
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