, Volume 49, Issue 1, pp 49–57 | Cite as

Protectin DX, a Double Lipoxygenase Product of DHA, Inhibits Both ROS Production in Human Neutrophils and Cyclooxygenase Activities

  • Miao Liu
  • Tarek Boussetta
  • Karama Makni-Maalej
  • Michèle Fay
  • Fathi Driss
  • Jamel El-Benna
  • Michel Lagarde
  • Michel Guichardant
Original Article


Neutrophils play a major role in inflammation by releasing large amounts of reactive oxygen species (ROS) produced by NADPH oxidase (NOX) and myeloperoxidase (MPO). This ROS overproduction is mediated by phosphorylation of the NOX subunits in an uncontrolled manner. Therefore, targeting neutrophil subunits would represent a promising strategy to moderate NOX activity, lower ROS, and other inflammatory agents, such as cytokines and leukotrienes, produced by neutrophils. For this purpose, we investigated the effects of protectin DX (PDX)—a docosahexaenoic acid di-hydroxylated product which inhibits blood platelet aggregation—on neutrophil activation in vitro. We found that PDX decreases ROS production, inhibits NOX activation and MPO release from neutrophils. We also confirm, that PDX is an anti-aggregatory and anti-inflammatory agent by inhibiting both cyclooxygenase-1 and -2 (COX-1 and COX-2, E.C. as well as COX-2 in lipopolysaccharides-treated human neutrophils. However, PDX has no effect on the 5-lipoxygenase pathway that produces the chemotactic agent leukotriene B4 (LTB4). Taken together, our results suggest that PDX could be a protective agent against neutrophil invasion in chronic inflammatory diseases.


Human neutrophils Inflammation Protectin DX NADPH oxidase ROS-derived oxidative stress 



Arachidonic acid




Conjugated linoleic acid


Conjugated linolenic acid




Docosahexaenoic acid


Enhanced chemiluminescence


Eicosapentaenoic acid


5-hydroxy-6E,8Z,11Z,14Z-eicosatetraenoic acid




Granulocyte macrophage-colony stimulating factor


Hank’s balanced salt solution


Hypochloric acid


Horseradish peroxidase


Inflammatory bowel disease




Linoleic acid




Leukotriene B4




NADPH oxidase


Platelet-activating factor


Polyacrylamide gel electrophoresis


Phosphate-buffered saline


Protectin DX




Phagocyte oxidase


Phorbol myristate acetate


Polymorphonuclear neutrophil


Polyunsaturated fatty acids


Reactive oxygen species


Reverse phase high performance liquid chromatography


Soybean lipoxygenase


Thin-layer chromatography


Tetramethyl benzidine


Trinitrobenzene sulfonic acid


Tumor necrosis factor-α



This study was supported by Inserm and the French Ministry of Education and Research. Miao Liu was received a grant from the China Scholarship Council.


  1. 1.
    Mullenix PS, Andersen CA, Starnes BW (2005) Atherosclerosis as inflammation. Ann Vasc Surg 19:130–138PubMedCrossRefGoogle Scholar
  2. 2.
    Fiocchi C (1998) Inflammatory bowel disease: etiology and pathogenesis. Gastroenterology 115:182–205PubMedCrossRefGoogle Scholar
  3. 3.
    Podolsky DK (2002) Inflammatory bowel disease. N Engl J Med 347:417–429PubMedCrossRefGoogle Scholar
  4. 4.
    Mantovani A, Allavena P, Sica A, Balkwill F (2008) Cancer-related inflammation. Nature 454:436–444PubMedCrossRefGoogle Scholar
  5. 5.
    Pearson TA, Mensah GA, Alexander RW, Anderson JL, Cannon RO 3rd, Criqui M, Fadl YY, Fortmann SP, Hong Y, Myers GL, Rifai N, Smith SC Jr, Taubert K, Tracy RP, Vinicor F (2003) Markers of inflammation and cardiovascular disease: application to clinical and public health practice: a statement for healthcare professionals from the centers for disease control and prevention and the American Heart Association. Circulation 107:499–511PubMedCrossRefGoogle Scholar
  6. 6.
    Esposito K, Giugliano D (2004) The metabolic syndrome and inflammation: association or causation? Nutr Metab Cardiovasc Dis 14:228–232PubMedCrossRefGoogle Scholar
  7. 7.
    Choy EH, Panayi GS (2001) Cytokine pathways and joint inflammation in rheumatoid arthritis. N Engl J Med 344:907–916PubMedCrossRefGoogle Scholar
  8. 8.
    Nathan C (2002) Points of control in inflammation. Nature 420:846–852PubMedCrossRefGoogle Scholar
  9. 9.
    Santana Reyes C, García-Muñoz F, Reyes D, González G, Dominguez C, Domenech E (2003) Role of cytokines (interleukin-1beta, 6, 8, tumour necrosis factor-alpha, and soluble receptor of interleukin-2) and C-reactive protein in the diagnosis of neonatal sepsis. Acta Paediatr 92:221–227PubMedCrossRefGoogle Scholar
  10. 10.
    Bayne LJ, Beatty GL, Jhala N, Clark CE, Rhim AD, Stanger BZ, Vonderheide RH (2012) Tumor-derived granulocyte-macrophage colony-stimulating factor regulates myeloid inflammation and T cell immunity in pancreatic cancer. Cancer Cell 21:822–835PubMedCentralPubMedCrossRefGoogle Scholar
  11. 11.
    Ortega-Gómez A, Perretti M, Soehnlein O (2013) Resolution of inflammation: an integrated view. EMBO Mol Med 5:661–674PubMedCentralPubMedCrossRefGoogle Scholar
  12. 12.
    Menshchikova E, Zenkov N, Tkachev V, Potapova O, Cherdantseva L, Shkurupiy V (2013) Oxidative stress and free-radical oxidation in BCG granulomatosis development. Oxid Med Cell Longev 2013:452546PubMedCentralPubMedCrossRefGoogle Scholar
  13. 13.
    Klebanoff SJ (2005) Myeloperoxidase: friend and foe. J Leukoc Biol 77:598–625PubMedCrossRefGoogle Scholar
  14. 14.
    Roos D, van Bruggen R, Meischl C (2003) Oxidative killing of microbes by neutrophils. Microbes Infect 5:1307–1315PubMedCrossRefGoogle Scholar
  15. 15.
    Babior BM (1984) Oxidants from phagocytes: agents of defense and destruction. Blood 64:959–966PubMedGoogle Scholar
  16. 16.
    Babior BM, Lambeth JD, Nauseef W (2002) The neutrophil NADPH oxidase. Arch Biochem Biophys 397:342–344PubMedCrossRefGoogle Scholar
  17. 17.
    Sheppard FR, Kelher MR, Moore EE, McLaughlin NJ, Banerjee A, Silliman CC (2005) Structural organization of the neutrophil NADPH oxidase: phosphorylation and translocation during priming and activation. J Leukoc Biol 78:1025–1042PubMedCrossRefGoogle Scholar
  18. 18.
    Whiting CV, Bland PW, Tarlton JF (2005) Dietary n-3 polyunsaturated fatty acids reduce disease and colonic proinflammatory cytokines in a mouse model of colitis. Inflamm Bowel Dis 11:340–349PubMedCrossRefGoogle Scholar
  19. 19.
    Serhan CN (2006) Resolvins and protectins: novel lipid mediators in anti-inflammation and resolution. Scand J Food Nutr 50:68–78CrossRefGoogle Scholar
  20. 20.
    Kohli P, Levy BD (2009) Resolvins and protectins: mediating solutions to inflammation. Br J Pharmacol 158:960–971PubMedCrossRefGoogle Scholar
  21. 21.
    Song HJ, Sneddon AA, Barker PA, Bestwick C, Choe SN, McClinton S, Grant I, Rotondo D, Heys SD, Wahle KW (2004) Conjugated linoleic acid inhibits proliferation and modulates protein kinase C isoforms in human prostate cancer cells. Nutr Cancer 49:100–108PubMedCrossRefGoogle Scholar
  22. 22.
    Flowers M, Thompson PA (2009) t10c12 Conjugated linoleic acid suppresses HER2 protein and enhances apoptosis in SKBr 3 breast cancer cells: possible role of COX2. PLoS One 4:e5342. doi:10.1371/journalpone0005342 PubMedCentralPubMedCrossRefGoogle Scholar
  23. 23.
    Boussetta T, Raad H, Lettéron P, Gougerot-Pocidalo MA, Marie JC, Driss F, El-Benna J (2009) Punicic acid a conjugated linolenic acid inhibits TNFα-induced neutrophil hyperactivation and protects from experimental colon inflammation in rats. PLoS One 4:e6458. doi:10.1371/journalpone0006458 PubMedCentralPubMedCrossRefGoogle Scholar
  24. 24.
    Chen P, Véricel E, Lagarde M, Guichardant M (2011) Poxytrins, a class of oxygenated products from polyunsaturated fatty acids, potently inhibit blood platelet aggregation. FASEB J 25:382–388PubMedCrossRefGoogle Scholar
  25. 25.
    Chen P, Fenet B, Michaud S, Tomczyk N, Véricel E, Lagarde M, Guichardant M (2009) Full characterization of PDX, a neuroprotectin/protectin D1 isomer, which inhibits blood platelet aggregation. FEBS Lett 583:3478–3484PubMedCrossRefGoogle Scholar
  26. 26.
    Serhan CN, Gotlinger K, Hong S, Lu Y, Siegelman J, Baer T, Yang R, Colgan SP, Petasis NA (2006) Anti-inflammatory actions of neuroprotectin D1/protectin D1 and its natural stereoisomers: assignments of dihydroxy-containing docosatrienes. J Immunol 176:1848–1859PubMedGoogle Scholar
  27. 27.
    Morita M, Kuba K, Ichikawa A, Nakayama M, Katahira J, Iwamoto R, Watanebe T, Sakabe S, Daidoji T, Nakamura S, Kadowaki A, Ohto T, Nakanishi H, Taguchi R, Nakaya T, Murakami M, Yoneda Y, Arai H, Kawaoka Y, Penninger JM, Arita M, Imai Y (2013) The lipid mediator protectin D1 inhibits influenza virus replication and improves severe influenza. Cell 153:112–125PubMedCrossRefGoogle Scholar
  28. 28.
    Dang PM, Stensballe A, Boussetta T, Raad H, Dewas C, Kroviarski Y, Hayem G, Jensen ON, Gougerot-Pocidalo MA, El-Benna J (2006) A specific p47phox -serine phosphorylated by convergent MAPKs mediates neutrophil NADPH oxidase priming at inflammatory sites. J Clin Invest 116:2033–2043PubMedCentralPubMedCrossRefGoogle Scholar
  29. 29.
    Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680–685PubMedCrossRefGoogle Scholar
  30. 30.
    Watanabe T, Narumiya S, Shimizu T, Hayaishi O (1982) Characterization of the biosynthetic pathway of prostaglandin D2 in human platelet-rich plasma. J Biol Chem 257:14847–14853PubMedGoogle Scholar
  31. 31.
    Klebanoff SJ (1999) Myeloperoxidase. Proc Assoc Am Physicians 111:383–389PubMedGoogle Scholar
  32. 32.
    Winterbourn CC, Kettle AJ (2000) Biomarkers of myeloperoxidase-derived hypochlorous acid. Free Radic Biol Med 29:403–409PubMedCrossRefGoogle Scholar
  33. 33.
    Stamp LK, Khalilova I, Tarr JM, Senthilmohan R, Turner R, Haigh RC, Winyard PG, Kettle AJ (2012) Myeloperoxidase and oxidative stress in rheumatoid arthritis. Rheumatology (Oxford) 51:1796–1803CrossRefGoogle Scholar
  34. 34.
    Papadakis KA, Targan SR (2000) Tumor necrosis factor: biology and therapeutic inhibitors. Gastroenterology 119:1148–1157PubMedCrossRefGoogle Scholar
  35. 35.
    Dewas C, Dang PM, Gougerot-Pocidalo MA, El-Benna J (2003) TNF-alpha induces phosphorylation of p47 (phox) in human neutrophils: partial phosphorylation of p47phox is a common event of priming of human neutrophils by TNF-alpha and granulocyte-macrophage colony-stimulating factor. J Immunol 171:4392–4398PubMedGoogle Scholar
  36. 36.
    El-Benna J, Dang PM, Gougerot-Pocidalo MA (2008) Priming of the neutrophil NADPH oxidase activation: role of p47phox phosphorylation and NOX2 mobilization to the plasma membrane. Semin Immunopathol 30:279–289PubMedCrossRefGoogle Scholar
  37. 37.
    Larmonier CB, Midura-Kiela MT, Ramalingam R, Laubitz D, Janikashvili N, Larmonier N, Ghishan FK, Kiela PR (2011) Modulation of neutrophil motility by curcumin: implications for inflammatory bowel disease. Inflamm Bowel Dis 17:503–515PubMedCentralPubMedCrossRefGoogle Scholar
  38. 38.
    Zock PL, Katan MB (1998) Linoleic acid intake and cancer risk: a review and meta-analysis. Am J Clin Nutr 68:142–153PubMedGoogle Scholar
  39. 39.
    Hatanaka E, Levada-Pires AC, Pithon-Curi TC, Curi R (2006) Systematic study on ROS production induced by oleic, linoleic, and gamma-linolenic acids in human and rat neutrophils. Free Radic Biol Med 41:1124–113239PubMedCrossRefGoogle Scholar
  40. 40.
    Martins de Lima-Salgado T, Coccuzzo Sampaio S, Cury-Boaventura MF, Curi R (2011) Modulatory effect of fatty acids on fungicidal activity, respiratory burst and TNF-α and IL-6 production in J774 murine macrophages. Br J Nutr 105:1173–1179PubMedCrossRefGoogle Scholar
  41. 41.
    Wanten GJ, Janssen FP, Naber AH (2002) Saturated triglycerides and fatty acids activate neutrophils depending on carbon chain-length. Eur J Clin Invest 32:285–289PubMedCrossRefGoogle Scholar
  42. 42.
    Lewis RA, Austen KF, Soberman RJ (1990) Leukotrienes and other products of the 5-lipoxygenase pathway. Biochemistry and relation to pathobiology in human diseases. N Engl J Med 323:645–655PubMedCrossRefGoogle Scholar
  43. 43.
    Sakai M, Kakutani S, Horikawa C, Tokuda H, Kawashima H, Shibata H, Okubo H, Sasaki S (2012) Arachidonic acid and cancer risk: a systematic review of observational studies. BMC Cancer 12:606PubMedCentralPubMedCrossRefGoogle Scholar
  44. 44.
    Harbige LS (2003) Fatty acids, the immune response, and autoimmunity: a question of n-6 essentiality and the balance between n-6 and n-3. Lipids 38:323–341PubMedCrossRefGoogle Scholar
  45. 45.
    Siriwardhana N, Kalupahana NS, Moustaid-Moussa N (2012) Health benefits of n-3 polyunsaturated fatty acids: eicosapentaenoic acid and docosahexaenoic acid. Adv Food Nutr Res 65:211–222PubMedGoogle Scholar
  46. 46.
    Hardy SJ, Robinson BS, Poulos A, Harvey DP, Ferrante A, Murray AW (1994) The neutrophil respiratory burst. Responses to fatty acids, N-formylmethionylleucylphenylalanine and phorbol ester suggest divergent signalling mechanisms. Eur J Biochem 198:801–806CrossRefGoogle Scholar
  47. 47.
    Paschoal VA, Vinolo MA, Crisma AR, Magdalon J, Curi R (2013) Eicosapentaenoic (EPA) and docosahexaenoic (DHA) acid differentially modulate rat neutrophil function in vitro. Lipids 48:93–103PubMedCrossRefGoogle Scholar

Copyright information

© AOCS 2013

Authors and Affiliations

  • Miao Liu
    • 1
  • Tarek Boussetta
    • 2
  • Karama Makni-Maalej
    • 2
  • Michèle Fay
    • 2
  • Fathi Driss
    • 2
  • Jamel El-Benna
    • 2
  • Michel Lagarde
    • 1
  • Michel Guichardant
    • 1
  1. 1.UMR 1060 Inserm (CarMeN), IMBL/INSA-LyonUniversité de LyonVilleurbanneFrance
  2. 2.Inserm, UMR 773, CRB3, Faculté de MédecineUniversité Paris 7 Denis DiderotParisFrance

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