Advertisement

Molecular Medicine

, Volume 23, Issue 1, pp 134–148 | Cite as

Development of a Zebrafish Sepsis Model for High-Throughput Drug Discovery

  • Anju M. Philip
  • Youdong Wang
  • Antonio Mauro
  • Suzan El-Rass
  • John C. Marshall
  • Warren L. Lee
  • Arthur S. Slutsky
  • Claudia C. dos Santos
  • Xiao-Yan Wen
Research Article

Abstract

Sepsis is a leading cause of death worldwide. Current treatment modalities remain largely supportive. Intervention strategies focused on inhibiting specific mediators of the inflammatory host response have been largely unsuccessful, a consequence of an inadequate understanding of the complexity and heterogeneity of the innate immune response. Moreover, the conventional drug-development pipeline is time-consuming and expensive, and the low success rates associated with cell-based screens underline the need for whole-organism screening strategies, especially for complex pathological processes. Here, we established a lipopolysaccharide (LPS)-induced zebrafish endotoxemia model, which exhibits the major hallmarks of human sepsis, including edema and tissue/organ damage, increased vascular permeability and vascular leakage accompanied by altered expression of cellular junction proteins, increased cytokine expression, immune cell activation and reactive oxygen species (ROS) production, reduced circulation and increased platelet aggregation. We tested the suitability of the model for phenotype-based drug screening using three primary readouts: mortality, vascular leakage and ROS production. Preliminary screening identified fasudil, a drug known to protect against vascular leakage in murine models, as a lead hit, thereby validating the utility of our model for sepsis drug screens. This zebrafish sepsis model has the potential to rapidly analyze sepsis-associated pathologies and cellular processes in the whole organism, as well as to screen and validate many compounds that can modify sepsis pathology in vivo.

Notes

Acknowledgments

We thank Koroboshka Brand-Arzamendi for the schematic diagrams/illustrations used in the paper, and for help with confocal imaging.

Supplementary material

10020_2017_2301134_MOESM1_ESM.pdf (903 kb)
Supplementary material, approximately 903 KB.

References

  1. 1.
    Singer M, et al. (2016) The third international consensus definitions for sepsis and septic shock (sepsis-3). JAMA. 315:801–10.CrossRefGoogle Scholar
  2. 2.
    Marshall JC. (2014) Why have clinical trials in sepsis failed? Trends Mol. Med. 20:195–203.CrossRefGoogle Scholar
  3. 3.
    Bhatia SN, Ingber DE. (2014) Microfluidic organs-on-chips. Nature Biotech. 32:760–72.CrossRefGoogle Scholar
  4. 4.
    Roses AD. (2008) Pharmacogenetics in drug discovery and development: a translational perspective. Nat. Rev. Drug Discov. 7:807–17.CrossRefGoogle Scholar
  5. 5.
    Lieschke GJ, Currie PD. (2007) Animal models of human disease: zebrafish swim into view. Nature Rev. Genet. 8:353–67.CrossRefGoogle Scholar
  6. 6.
    Howe K, Clark M, Torroja C, et al. (2013) The zebrafish reference genome sequence and its relationship to the human genome. Nature. 496:498–503.CrossRefGoogle Scholar
  7. 7.
    Miscevic F, Rotstein O, Wen X–Y. (2012) Advances in zebrafish high content and high throughput technologies. Comb. Chem. High Throughput Screen. 15:515–21.CrossRefGoogle Scholar
  8. 8.
    Gallardo VE, et al. (2015) Phenotype-driven chemical screening in zebrafish for compounds that inhibit collective cell migration identifies multiple pathways potentially involved in metastatic invasion. Dis. Models Mech. 8:565–76.CrossRefGoogle Scholar
  9. 9.
    van der Vaart M, Spaink HP, Meijer AH. (2012) Pathogen recognition and activation of the innate immune response in zebrafish. Adv. Hematol 2012:1–19.CrossRefGoogle Scholar
  10. 10.
    Sunyer JO. (2013) Fishing for mammalian paradigms in the teleost immune system. Nat. Immunol. 14:320–26.CrossRefGoogle Scholar
  11. 11.
    Stachura DL, Svoboda O, Campbell CA, et al. (2013) The zebrafish granulocyte colonystimulating factors (Gcsfs): 2 paralogous cytokines and their roles in hematopoietic development and maintenance. Blood. 122:3918–28.CrossRefGoogle Scholar
  12. 12.
    Barros-Becker F, Romero J, Pulgar A, Feijoo CG. (2012) Persistent oxytetracycline exposure induces an inflammatory process that improves regenerative capacity in zebrafish larvae. PLoS One. 7:1–9.CrossRefGoogle Scholar
  13. 13.
    van der Vaart M, van Soest JJ, Spaink HP, Meijer AH. (2013) Functional analysis of a zebrafish myd88 mutant identifies key transcriptional components of the innate immune system. Dis. Models Mech. 6:841–54.CrossRefGoogle Scholar
  14. 14.
    Sepulcre MP, Alcaraz-Pérez F, López-Muñoz A, et al. (2009) Evolution of lipopolysaccharide (LPS) recognition and signaling: fish TLR4 does not recognize LPS and negatively regulates NF-kappaB activation. J. Immunol. 182:1836–45.CrossRefGoogle Scholar
  15. 15.
    Sullivan C, et al. (2009) The gene history of zebrafish tlr4a and tlr4b is predictive of their divergent functions. J. Immunol. 183:5896–5908.CrossRefGoogle Scholar
  16. 16.
    Philip AM, Vijayan MM. (2015) Stress-immune-growth interactions: cortisol modulates suppressors of cytokine signaling and JAK/STAT pathway in rainbow trout Liver. PLoS One. 10:e0129299.CrossRefGoogle Scholar
  17. 17.
    Remick DG. (2007) Pathophysiology of sepsis. Am. J. Pathol. 170:1435–44.CrossRefGoogle Scholar
  18. 18.
    Lee WL, Slutsky AS. (2010) Clinical implications of basic research: sepsis and endothelial permeability. N. Engl. J. Med. 363:689–91.CrossRefGoogle Scholar
  19. 19.
    Avdesh A, et al. (2012) Regular care and maintenance of a zebrafish (Danio rerio) laboratory: an introduction. J. Vis. Exp. 69:e4196.Google Scholar
  20. 20.
    Nüsslein-Volhard C, Dahm R. (2002) Zebrafish: Practical Approaches. New York: Oxford University Press.Google Scholar
  21. 21.
    Kimmel CB, Ballard WW, Kimmel SR, Ullmann B, Schilling TF. (1995) Stages of embryonic development of the zebrafish. Dev. Dynam. 203:253–310.CrossRefGoogle Scholar
  22. 22.
    Warren HS. (2009) Editorial: Mouse models to study sepsis syndrome in humans. J. Leukoc. Biol. 86:199–201.CrossRefGoogle Scholar
  23. 23.
    Rieger S, Kulkarni RP, Darcy D, Fraser SE, Köster RW. (2005) Quantum dots are powerful multipurpose vital labeling agents in zebrafish embryos. Dev. Dyn. 234:670–81.CrossRefGoogle Scholar
  24. 24.
    Li L, Yan B, Shi YQ, Zhang WQ, Wen ZL. (2012) Live imaging reveals differing roles of macrophages and neutrophils during zebrafish tail fin regeneration. J. Biol. Chem. 287:25353–60.CrossRefGoogle Scholar
  25. 25.
    Gentile LF, Moldawer LL. (2014) HMGB1 as a therapeutic target for sepsis: it’s all in the timing! Expert Opin. Ther. Targets. 18:243–45.CrossRefGoogle Scholar
  26. 26.
    Encinas P, et al. (2013) Identification of multipath genes differentially expressed in pathway-targeted microarrays in zebrafish infected and surviving spring viremia carp virus (SVCV) suggest preventive drug candidates. PLoS One. 8:1–19.CrossRefGoogle Scholar
  27. 27.
    Wallez Y, Huber P. (2008) Endothelial adherens and tight junctions in vascular homeostasis, inflammation and angiogenesis. Biochim. Biophys. Acta. 1778:794–809.CrossRefGoogle Scholar
  28. 28.
    González-Mariscal L, Betanzos A, Avila-Flores A. (2000) MAGUK proteins: structure and role in the tight junction. Semin. Cell Dev. Biol. 11:315–24.CrossRefGoogle Scholar
  29. 29.
    Li Y, Wu Y, Wang Z, Zhang XH, Wu WK. (2010) Fasudil attenuates lipopolysaccharide-induced acute lung injury in mice through the Rho/Rho kinase pathway. Med. Sci. Monit. 16:112–18.Google Scholar
  30. 30.
    Yang LL, et al. (2014) Endotoxin molecule lipopolysaccharide-induced zebrafish inflammation model: a novel screening method for anti-inflammatory drugs. Molecules. 19:2390–2409.CrossRefGoogle Scholar
  31. 31.
    Alexandraki I, Palacio C. (2010) Gram-negative versus gram-positive bacteremia: what is more alarming? Crit. Care. 14:161.CrossRefGoogle Scholar
  32. 32.
    Barber AE, Fleming BA, Mulvey MA. (2016) Similarly lethal strains of extraintestinal pathogenic Escherichia coli trigger markedly diverse host responses in a zebrafish model of sepsis. Msphere. 1:1–19.CrossRefGoogle Scholar
  33. 33.
    Goldenberg NM, Steinberg BE, Slutsky AS, Lee WL. (2011) Broken barriers: a new take on sepsis pathogenesis. Sci. Trans. Med. 3:88ps25.CrossRefGoogle Scholar
  34. 34.
    Morita K, Sasaki H, Furuse M, Tsukita S. (1999) Endothelial claudin: claudin-5/TMVCF constitutes tight junction strands in endothelial cells. J. Cell Biol. 147:185–94.CrossRefGoogle Scholar
  35. 35.
    Xie J, Farage E, Sugimoto M, Anand-Apte B. (2010) A novel transgenic zebrafish model for blood-brain and blood-retinal barrier development. BMC Dev. Biol. 10:76.CrossRefGoogle Scholar
  36. 36.
    Yang CH, et al. (2014) Simvastatin attenuates sepsis-induced blood-brain barrier integrity loss. J. Surg. Res. 194:591–98.CrossRefGoogle Scholar
  37. 37.
    Li Q, Zhang Q, Wang C, Liu X, Li N, Li J. (2009) Disruption of tight junctions during polymicrobial sepsis. J. Pathol. 218:210–22.CrossRefGoogle Scholar
  38. 38.
    Eadon MT, et al. (2012) Endotoxemia alters tight junction gene and protein expression in the kidney. Am. J. Physiol. Renal Physiol. 303: F821–30.CrossRefGoogle Scholar
  39. 39.
    Dejana E, Orsenigo F, Lampugnani MG. (2008) The role of adherens junctions and VE-cadherin in the control of vascular permeability. J. Cell Sci. 121:2115–22.CrossRefGoogle Scholar
  40. 40.
    Philip AM, Jørgensen EH, Maule AG, Vijayan MM. (2014) Tissue-specific molecular immune response to lipopolysaccharide challenge in emaciated anadromous Arctic charr. Dev. Comp. Immunol. 45:133–40.CrossRefGoogle Scholar
  41. 41.
    Herwald H, Egesten A, eds. (2011) SepsisPro-Inflammatory and Anti-Inflammatory Responses: Good, Bad or Ugly? Basel, Switzerland: Karger.Google Scholar
  42. 42.
    Stockhammer OW, Zakrzewska A, Hegedûs Z, Spaink HP, Meijer AH. (2009) Transcriptome profiling and functional analyses of the zebrafish embryonic innate immune response to Salmonella infection. J. Immunol. 182:5641–53.CrossRefGoogle Scholar
  43. 43.
    Zou J, Secombes C. (2016) The function of fish cytokines. Biology (Basel). 5:23–58.PubMedCentralGoogle Scholar
  44. 44.
    Stein C, Caccamo M, Laird G, Leptin M. (2007) Conservation and divergence of gene families encoding components of innate immune response systems in zebrafish. Genome Biol. 8: R251.CrossRefGoogle Scholar
  45. 45.
    Okusawa S, Gelfand JA, Ikejima T, Connolly RJ, Dinarello CA. (1988) Interleukin 1 induces a shock-like state in rabbits. Synergism with tumor necrosis factor and the effect of cyclooxygenase inhibition. J. Clin. Invest. 81:1162–72.CrossRefGoogle Scholar
  46. 46.
    Schulte W, Bernhagen J, Bucala R. (2013) Cytokines in sepsis: Potent immunoregulators and potential therapeutic targets. An updated view. Mediators Inflamm. 6:165974–90.Google Scholar
  47. 47.
    Cinel I, Opal SM. (2009) Molecular biology of inflammation and sepsis: a primer. Crit. Care Med. 37:291–304.CrossRefGoogle Scholar
  48. 48.
    Hinshaw LB. (1996) Sepsis/septic shock: participation of the microcirculation: an abbreviated review. Crit. Care Med. 24:1072–78.CrossRefGoogle Scholar
  49. 49.
    Secor D, et al. (2010) Impaired microvascular perfusion in sepsis requires activated coagulation and P-selectin-mediated platelet adhesion in capillaries. Intensive Care Med. 36:1928–34.CrossRefGoogle Scholar
  50. 50.
    Hotchkiss RS, Monneret G, Payen D. (2013) Immunosuppression in sepsis: a novel understanding of the disorder and a new therapeutic approach. Lancet Infect. Dis. 13:260–68.CrossRefGoogle Scholar
  51. 51.
    Makino I, Tajima H, Kitagawa H, et al. (2015) A case of severe sepsis presenting marked decrease of neutrophils and interesting findings on dynamic CT. Am. J. Case Rep. 16:322–327.CrossRefGoogle Scholar
  52. 52.
    Gregoire M, Tadie J-M, Uhel F, Gacouin A, et al. (2016) HMGB1 induces neutrophil dysfunction in experimental sepsis and in patients who survive septic shock. J. Leukoc. Biol. 101:1281–7.CrossRefGoogle Scholar
  53. 53.
    Eisa-Beygi S, Wen XY. (2015) Could pharmacological curtailment of the RhoA/Rho-kinase pathway reverse the endothelial barrier dysfunction associated with Ebola virus infection? Antiviral Res. 114:53–56.CrossRefGoogle Scholar
  54. 54.
    Winata CL, et al. (2009) Development of zebrafish swimbladder: The requirement of hedgehog signaling in specification and organization of the three tissue layers. Dev. Biol. 331:222–36.CrossRefGoogle Scholar
  55. 55.
    Hall FG. (1924) The functions of the swim bladder of fishes. Biol. Bull. 47:79–126.CrossRefGoogle Scholar
  56. 56.
    Pelster B. (1995) Metabolism of the swimbladder tissue. In: Biochemistry and Molecular Biology of Fishes. Elsevier, Amsterdam, pp. 101–18.Google Scholar

Copyright information

© The Author(s) 2017

Open Access This article is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License, which permits any non-commercial use, sharing, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, and provide a link to the Creative Commons license. You do not have permission under this license to share adapted material derived from this article or parts of it.

The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder.

To view a copy of this license, visit (https://doi.org/creativecommons.org/licenses/by-nc-nd/4.0/)

Authors and Affiliations

  • Anju M. Philip
    • 1
    • 2
    • 3
  • Youdong Wang
    • 1
    • 2
  • Antonio Mauro
    • 1
    • 2
    • 4
    • 6
  • Suzan El-Rass
    • 1
    • 2
    • 4
    • 6
  • John C. Marshall
    • 1
    • 2
    • 5
  • Warren L. Lee
    • 1
    • 2
    • 4
  • Arthur S. Slutsky
    • 1
    • 2
    • 4
    • 5
  • Claudia C. dos Santos
    • 1
    • 2
    • 4
    • 5
  • Xiao-Yan Wen
    • 1
    • 2
    • 3
    • 4
    • 6
  1. 1.Zebrafish Centre for Advanced Drug DiscoverySt. Michael’s HospitalTorontoCanada
  2. 2.Keenan Research Centre for Biomedical ScienceLi Ka Shing Knowledge Institute of St. Michael’s HospitalTorontoCanada
  3. 3.Department of PhysiologyUniversity of TorontoTorontoCanada
  4. 4.Department of Medicine and Institute of Medical ScienceUniversity of TorontoTorontoCanada
  5. 5.Interdepartmental Division of Critical CareSt. Michael’s Hospital/University of TorontoTorontoCanada
  6. 6.Collaborative Program in Cardiovascular Sciences, Faculty of MedicineUniversity of TorontoTorontoCanada

Personalised recommendations