Органоспецифичная экспрессия генов воспаления в ответ на введение липополисахарида рыбам Danio rerio

Известно, что системное воспаление является ключевым компонентом прогрессирования многих инфекционных и неинфекционных заболеваний и может приводить к полиорганной недостаточности, хроническому воспалению, иммуносупрессии, катаболическому синдрому и даже смерти. Важность этого состояния обуславливает необходимость создания релевантной in vivo модели воспаления, пригодной для изучения патогенеза многих заболеваний, а также для проведения скрининга эффективности фармакологических препаратов. Рыбы Danio rerio являются одними из наиболее важных моделей исследования биологических процессов in vivo. Целью настоящего исследования было создание модели системного воспаления in vivo, индуцированного внутрибрюшинным введением липополисахарида (ЛПС) у рыб Danio rerio, с последующей идентификацией органо-специфической провоспалительной активности генов. Было проведено исследование уровня экспрессии основных провоспалительных генов у Danio rerio после инъекции ЛПС. Сравнивая 18s, eef1a1l1, gapdh и actb в качестве потенциальных генов домашнего хозяйства, мы пришли к выводу, что eef1a1l1 с эффективностью 99% является наиболее перспективным для дальнейшей нормализации в этой модели. Активность генов была наиболее выраженной в сердце, где экспрессия IL6, CXCL8a и CXCL18β была повышена до 100 раз. Кроме того, почки были наиболее вовлечены в воспалительный процесс, поскольку там было активировано наибольшее количество из проанализированных генов: уровни экспрессии CXCL18β, CXCL8a, IL1β, IL6, Mpeg1.2 и TNFa были значительно увеличены. Вероятно, это связано с активностью почек как иммунного и кроветворного органа. Самая низкая реактивность обнаружена в мышцах. Иммунные реакции демонстрируют дозозависимость, например, инфузия 20 мкг ЛПС приводила к снижению экспрессии IFNγ, Mpeg 1.2 и Mpeg 1.1 в печени и к увеличению экспрессии Mpeg 1.2 в почках по сравнению с дозировкой 10 мкг. Таким образом, Danio rerio является релевантной моделью воспаления. Наша модель продемонстрировала, что исследование изолированных органов рыб может быть полезным и информативным для исследования воспалительных процессов.

1. Al-Samadi A., Awad S.A., Tuomainen K., Zhao Y., Salem A., Parikka M., Salo T. Crosstalk between tongue carcinoma cells, extracellular vesicles, and immune cells in in vitro and in vivo models. Oncotarget, 2017, Vol. 8, no. 36, pp. 60123-60134.

2. Andersen C.L., Jensen J.L., 0rntoft T.F. Normalization of real-time quantitative reverse transcription-PCR data: A model-based variance estimation approach to identify genes suited for normalization, applied to bladder and colon cancer data sets. Cancer Res., 2004, Vol. 64, no. 15, pp. 5245-5250.

3. Arnaout R., Ferrer T., Huisken J., Spitzer K., Stainier D.Y.R., Tristani-Firouzi M., Chi N.C. Zebrafish model for human long QT syndrome. Proc. Natl Acad. Sci. USA, 2007, Vol. 104, no. 27, pp. 11316-11321.

4. Asnani A., Peterson R.T. The zebrafish as a tool to identify novel therapies for human cardiovascular disease. Dis. Model. Mech., 2014, Vol. 7, no. 7, pp. 763-767.

5. Banerjee S., Leptin M. Systemic response to ultraviolet radiation involves induction of leukocytic IL-1 в and inflammation in zebrafish. J. Immunol., 2014, Vol. 193, no. 3, pp. 1408-1415.

6. Barriuso J., Nagaraju R., Hurlstone A. Zebrafish in oncology. Aging (Albany NY), 2013, Vol. 7, no. 5, pp. 286-287.

7. Bates J.M., Akerlund J., Mittge E., Guillemin K. Intestinal alkaline phosphatase detoxifies lipopolysaccharide and prevents inflammation in zebrafish in response to the gut microbiota. Cell Host Microbe, 2007, Vol. 2, no. 6, pp. 371-382.

8. Benard E.L., Racz P.I., Rougeot J., Nezhinsky A.E., Verbeek F.J., Spaink H.P., Meijer A.H. Macrophage-expressed perforins Mpeg 1 and Mpeg 1.2 have an anti-bacterial function in zebrafish. J. Innate Immun., 2015, Vol. 7, no. 2, pp. 136-152.

9. Beutler B., Rietschel E.T. Innate immune sensing and its roots: the story of endotoxin. Nat. Rev. Immunol., 2003, Vol. 3, no. 2, pp. 169-176.

10. Ciura S., Lattante S., Le Ber I., Latouche M., Tostivint H., Brice A., Kabashi E. Loss of function of C9orf72 causes motor deficits in a zebrafish model of amyotrophic lateral sclerosis. Ann. Neurol., 2013, Vol. 74, no. 2, pp. 180-187.

11. Davidson A.J., Zon LI. The “definitive” (and 'primitive') guide to zebrafish hematopoiesis. Oncogene, 2004, Vol. 23, no. 43, pp. 7233-7246.

12. Dawid I.B. Developmental biology of zebrafish. Ann. N.Y. Acad. Sci., 2004, Vol. 1038, no. 4, pp. 88-93.

13. de Oliveira S., Reyes-Aldasoro C.C., Candel S., Renshaw S.A., Mulero V, Calado A. Cxc18 (IL-8) Mediates neutrophil recruitment and behavior in the zebrafish inflammatory response. J. Immunol., 2013, Vol. 190, no. 8, pp. 4349-4359.

14. Deng Q., Sarris M., Bennin D.A., Green J.M., Herbomel P., Huttenlocher A. Localized bacterial infection induces systemic activation of neutrophils through Cxcr2 signaling in zebrafish. J. Leukoc. Biol., 2013, Vol. 93, no. 5, pp. 761-769.

15. Elamm C., Fairweather D.L., Cooper L.T. Pathogenesis and diagnosis of myocarditis. Heart, 2012, Vol. 98, no. 11, pp. 835-840.

16. Forn-Cuni G., Varela M., Pereiro P, Novoa B., Figueras A. Conserved gene regulation during acute inflammation between zebrafish and mammals. Sci. Rep., 2017, Vol. 7, 41905. doi: 10.1038/srep41905.

17. Francis Stuart S.D., de Jesus N.M., Lindsey M.L., Ripplinger C.M. The crossroads of inflammation, fibrosis, and arrhythmia following myocardial infarction. J. Mol. Cell. Cardiol., 2016, Vol. 91, pp. 114-122.

18. Gupta S., Gupta S. Inflammation, atherosclerosis and coronary artery disease. Med. Updat (Vol. 17, 2007), 2007, Vol. 352, no. 16, pp. 44-44.

19. Hendriks-Balk M.C., Michel M.C., Alewijnse A.E. Pitfalls in the normalization of real-time polymerase chain reaction data. Basic Res. Cardiol., 2007, Vol. 102, no. 3, pp. 195-197.

20. Herbomel P., Thisse B., Thisse C. Ontogeny and behaviour of early macrophages in the zebrafish embryo. Development, 1999, Vol. 126, no. 17, pp. 3735-3745.

21. Howe K., Clark M.D., Torroja C.F., Torrance J., Berthelot C. The zebrafish reference genome sequence and its relationship to the human genome. Nature, 2013, Vol. 496, no. 7446, pp. 498-503.

22. Huang S., Xu M., Liu L., Yang J., Wang H., Wan C., Deng W., Tang, Q. Autophagy is involved in the protective effect of p21 on LPS-induced cardiac dysfunction. Cell Death Dis., 2020, Vol. 11, no. 7, pp. 1-15.

23. Hui S.P., Sheng D.Z., Sugimoto K., Gonzalez-Rajal A., Nakagawa S., Hesselson D., Kikuchi K. Zebrafish regulatory T cells mediate organ-specific regenerative programs. Dev. Cell, 2017, Vol. 43, no. 6, pp. 659-672.e5.

24. Imazio M., Gaita F. Diagnosis and treatment of pericarditis. Heart, 2015, Vol. 101, no. 14, pp. 1159-1168.

25. Jiang J., Wu S., Wu C., An X., Cai L., Zhao X. Embryonic exposure to carbendazim induces the transcription of genes related to apoptosis, immunotoxicity and endocrine disruption in zebrafish (Danio rerio). Fish Shellfish Immunol., 2014, Vol. 41, no. 2, pp. 493-500.

26. Kakihana Y., Ito T., Nakahara M., Yamaguchi K., Yasuda T. Sepsis-induced myocardial dysfunction: Pathophysiology and management. J. Intensive Care, 2016, Vol. 4, no. 1, 22. doi: 10.1186/s40560-016-0148-1.

27. Kettleborough R.N.W., Busch-Nentwich E.M., Harvey S.A., Dooley C.M., de Bruijn E., van Eeden F., Sealy I., White R.J., Herd C., Nijman I.J., Fenyes F., Mehroke S., Scahill C., Gibbons R., Wali N., Carruthers S., Hall A., Yen J., Cuppen E., Stemple D.L. A systematic genome-wide analysis of zebrafish protein-coding gene function. Nature, 2013, Vol. 496, no. 7446, pp. 494-497.

28. Kondratov K., Kurapeev D., Popov M., Sidorova M., Minasian S., Galagudza M., Kostareva A., Fedorov A. Heparinase treatment of heparin-contaminated plasma from coronary artery bypass grafting patients enables reliable quantification of microRNAs. Biomol. Detect. Quantif., 2016, Vol. 8, pp. 9-14.

29. Lagos L., Tandberg J.I., Becker M.I., Winther-Larsen H.C. Immunomodulatory properties of Concholepas concholepas hemocyanin against francisellosis in a zebrafish model. Fish Shellfish Immunol., 2017, Vol. 67, pp. 571-574.

30. Libby P. Interleukin-1 beta as a target for atherosclerosis therapy: biological basis of CANTOS and beyond. J. Am. Coll. Cardiol., 2017, Vol. 70, no. 18, pp. 2278-2289.

31. Lieschke G.J., Currie P.D. Animal models of human disease: zebrafish swim into view. Nat. Rev. Genet., 2007, Vol. 8, no. 5, pp. 353-367.

32. MacRae C.A., Peterson R.T. Zebrafish as tools for drug discovery. Nat. Rev. Drug Discov., 2015, Vol. 14, no. 10, pp. 721-731.

33. Masters S.L., Simon A., Aksentijevich I., Kastner D.L. Horror autoinflammaticus: the molecular pathophysiology of autoinflammatory disease. Annu. Rev. Immunol., 2009, Vol. 27, no. 1, pp. 621-668.

34. Mazmanian S.K., Cui H.L., Tzianabos A.O., Kasper D.L. An immunomodulatory molecule of symbiotic bacteria directs maturation of the host immune system. Cell., 2005, Vol. 122, no. 1, pp. 107-118.

35. McCurley A.T., Callard G.V. Characterization of housekeeping genes in zebrafish: male-female differences and effects of tissue type, developmental stage and chemical treatment. BMC Mol. Biol., 2008, Vol. 9, no. 1, 102. doi: 10.1186/1471-2199-9-102.

36. Meeker N.D., Trede N.S. Immunology and zebrafish: spawning new models of human disease. Dev. Comp. Immunol., 2008, Vol. 32, no. 7, pp. 745-757.

37. Mukaida N. Pathophysiological roles of interleukin-8/CXCL8 in pulmonary diseases. Am. J. Physiol. Lung Cell. Mol. Physiol., 2003, Vol. 284, no. 4, pp. L566-L577.

38. Novoa B., Figueras A. Zebrafish: model for the study of inflammation and the innate immune response to infectious diseases. In: Advances in Experimental Medicine and Biology. 2012, pp. 253-275.

39. Oehlers S.H., Flores M.V., Okuda K.S., Hall C.J., Crosier K.E., Crosier P.S. A chemical enterocolitis model in zebrafish larvae that is dependent on microbiota and responsive to pharmacological agents. Dev. Dyn., 2011, Vol. 240, no. 1, pp. 288-298.

40. Parish R.C., Evans J.D. Inflammation in chronic heart failure. Ann. Pharmacother., 2008, Vol. 42, no. 7-8, pp. 1002-1016.

41. Renshaw S.A., Loynes C.A., Trushell D.M.I., Elworthy S., Ingham P.W., Whyte M.K.B. Atransgenic zebrafish model of neutrophilic inflammation. Blood, 2006, Vol. 108, no. 13, pp. 3976-3978.

42. Renshaw S.A., Trede N.S. A model 450 million years in the making: zebrafish and vertebrate immunity. Dis. Model. Mech., 2012, Vol. 5, no. 1, pp. 38-47.

43. Rhee C., Klompas M. New sepsis and septic shock definitions. Infect. Dis. Clin. North Am., 2017, Vol. 31, no. 3, pp. 397-413.

44. Russo R.C., Garcia C.C., Teixeira M.M., Amaral F.A. The CXCL8/IL-8 chemokine family and its receptors in inflammatory diseases. Expert Rev. Clin. Immunol., 2014, Vol. 10, no. 5, pp. 593-619.

45. Sepulcre M.P., Alcaraz-Perez F., Lopez-Munoz A., Roca F.J., Meseguer J., Cayuela M.L., Mulero V Evolution of lipopolysaccharide (LPS) recognition and signaling: fish TLR4 does not recognize LPS and negatively regulates NF-kB activation. J. Immunol., 2009, Vol. 182, no. 4, pp. 1836-1845.

46. Singer M., Deutschman C.S., Seymour C., Shankar-Hari M., Annane D., Bauer M., Bellomo R., Bernard G.R., Chiche J.D., Coopersmith C.M., Hotchkiss R.S., Levy M.M., Marshall J.C., Martin G.S., Opal S.M., Rubenfeld G.D., van der Poll T., Vincent J.L., Angus D.C. The third international consensus definitions for sepsis and septic shock (sepsis-3). JAMA, 2016, Vol. 315, no. 8, pp. 801-810.

47. Snider D.R., Clegg E.D. Alteration of phospholipids in porcine spermatozoa during in vivo uterus and oviduct incubation. J. Anim.Sci., 1975. Vol. 40, no. 2, pp. 269-274.

48. Solanas M., Moral R., Escrich E. Unsuitability of using ribosomal RNA as loading control for Northern blot analyses related to the imbalance between messenger and ribosomal RNA content in rat mammary tumors. Anal. Biochem., 2001, Vol. 288, no. 1, pp. 99-102.

49. Stockhammer O.W., Zakrzewska A., Hegedus Z., Spaink H.P, Meijer A.H. Transcriptome profiling and functional analyses of the zebrafish embryonic innate immune response to salmonella infection. J. Immunol., 2009, Vol. 182, no. 9, pp. 5641-5653.

50. Sullivan C., Charette J., Catchen J., Lage C.R., Giasson G., Postlethwait J.H., Millard P.J., Kim C.H. The gene history of zebrafish tlr4a and tlr4b is predictive of their divergent functions. J. Immunol., 2009, Vol. 183, no. 9, pp. 5896-5908.

51. Tandberg J., Oliver C., Lagos L., Gaarder M., Yanez A.J., Ropstad E., Winther-Larsen H.C. Membrane vesicles from Piscirickettsia salmonis induce protective immunity and reduce development of salmonid rickettsial septicemia in an adult zebrafish model. Fish Shellfish Immunol., 2017, Vol. 67, pp. 189-198.

52. Tanino Y., Coombe D.R., Gill S.E., Kett W.C., Kajikawa O., Proudfoot A.E.I., Wells T.N.C., Parks W.C., Wight T.N., Martin T.R., Frevert C.W. Kinetics of chemokine-glycosaminoglycan interactions control neutrophil migration into the airspaces of the lungs. J. Immunol., 2010, Vol. 184, no. 5, pp. 2677-2685.

53. Trede N.S., Langenau D.M., Traver D., Look A.T., Zon L.I.. The use of zebrafish to understand immunity. Immunity, 2004, Vol. 20, no. 4, pp. 367-379.

54. van der Vaart M., van Soest J.J., Spaink H.P., Meijer A.H. Functional analysis of a zebrafish myd88 mutant identifies key transcriptional components of the innate immune system. Dis. Model. Mech., 2013, Vol. 6, no. 3, pp. 841-854.

55. van Tassell B.W., Toldo S., Mezzaroma E., Abbate A. Targeting interleukin-1 in heart disease. Circulation, 2013, Vol. 128, no. 17, pp. 1910-1923.

56. van Tassell B.W., Raleigh J.M.V., Abbate A. Targeting Interleukin-1 in heart failure and inflammatory heart disease. Curr. Heart Fail. Rep., 2015, Vol. 12, no. 1, pp. 33-41.

57. Vandesompele J., de Preter K., Pattyn F., Poppe B., van Roy N., de Paepe A., Speleman F. Accurate normalization of real-time quantitative RT-PCR data by geometric averaging of multiple internal control genes. Genome Biol. 2002, Vol. 3, research0034.1. doi: 10.1186/gb-2002-3-7-research0034.

58. Watts S.A., Powell M., d'Abramo L.R. Fundamental approaches to the study of zebrafish nutrition. ILAR J., 2012, Vol. 53, no. 2, pp. 144-160.

59. Willett C.E., Cortes A., Zuasti A., Zapata A.G. Early hematopoiesis and developing lymphoid organs in the zebrafish. Dev. Dyn., 1999, Vol. 214, no. 4, pp. 323-336.

60. Williams M.A. Monocyte anergy in septic shock is associated with a predilection to apoptosis and is reversed by granulocyte-macrophage colony-stimulating factor ex vivo. J. Infect. Dis., 1998, Vol. 178, no. 5, pp. 1421-1433.

61. Yan В., Han P, Pan L., Lu W., Xiong J., Zhang M., Li L., Wen Z. 11-1 в and reactive oxygen species differentially regulate neutrophil directional migration and basal random motility in a zebrafish injury-induced inflammation model. J. Immunol., 2014, Vol. 192, no. 12, pp. 5998-6008.

62. Yang L.L., Wang G.Q., Yang L.M., Huang Z.B., Zhang W.Q., Yu L.Z. Endotoxin molecule lipopolysaccharide-induced zebrafish inflammation model: a novel screening method for anti-inflammatory drugs. Molecules, 2014, Vol. 19, no. 2, pp. 2390-2409.

63. Zang L., Maddison L.A., Chen W. Zebrafish as a model for obesity and diabetes. Front. Cell Dev. Biol., 2018, Vol. 6, 91. Doi: 10.3389/fcell.2018.00091.

64. Zapata A., Diez B., Cejalvo T., Gutierrez-de Frias C., Cortes A. Ontogeny of the immune system of fish. Fish Shellfish Immunol., 2006, Vol. 20, no. 2, pp. 126-136.

65. Zernecke A., Weber C. Chemokines in atherosclerosis: proceedings resumed. Arterioscler. Thromb. Vasc. Biol., 2014, Vol. 34, no. 4, pp. 742-750.

66. Zhang D., Tang J., Zhang J., Zhang D.L., Hu C.X. Responses of pro- and anti-inflammatory cytokines in zebrafish liver exposed to sublethal doses of aphanizomenon flosaquae DC-1 aphantoxins. Aquat. Toxicol., 2019, Vol. 215, 105269. doi: 10.1016/j.aquatox.2019.105269.

67. Zhang Y., Takagi N., Yuan B., Zhou Y., Si N., Wang H., Yang J., Wei X., Zhao H., Bian B. The protection of indolealkylamines from LPS-induced inflammation in zebrafish. J. Ethnopharmacol., 2019, Vol. 243, 112122. doi: 10.1016/j.jep.2019.112122.