The characteristics of neutrophil extracellular traps produced by all-trans retinoic acid-induced dHL-60 under PMA stimulation_Journal of Biomedical Engineering

Authors：

LIU Wang ¹ , FANG Jinhua ² , HONG Tiantian ^1,2 , HUANG Jiaqi ¹ , ZHAO Baisong ³ , FANG Ying ¹ , WU Jianhua ¹ ,  LIN Jiangguo ^2,4

1. School of Biosciences and Bioengineering, South China University of Technology, Guangzhou 510006, P. R. China;
2. Research Center of Medical Sciences, Guangdong Provincial People’s Hospital, Guangdong Academy of Medical Sciences, Guangzhou 510080, P. R. China;
3. Department of Anesthesiology, Guangzhou Women and Children’s Medical Center, Guangzhou 510623, P. R. China;
4. Department of Emergency Medicine, Guangdong Provincial People’s Hospital, Guangdong Academy of Medical Sciences, Guangzhou 510080, P. R. China;

Corresponding author：

LIN Jiangguo, Email: linjiangguo@gdph.org.cn

Keywords：

Neutrophil extracellular traps; HL-60; All-trans retinoic acid; Differentiation

DOI：

10.7507/1001-5515.202205002

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Extracellular traps released by neutrophils (neutrophil extracellular traps, NETs) are a double-edged sword, and understanding the mechanism of NET formation is of great significance for disease treatment. However, the short lifespan, the large individual differences, and the inability to perform gene editing render it difficult to decipher NET formation using neutrophils. It is necessary to find a model cell to replace neutrophils to study the mechanism of NET formation. In this study, we used different concentrations (0, 0.1, 1, and 10 μmol/L) of all-trans retinoic acid (ATRA) to differentiate HL-60 cells for different days (1, 3, 5, and 7 days). By detecting the cell viability and nuclear morphology of cells, we confirmed that HL-60 cells were differentiated to neutrophil-like cells (dHL-60) after treated with ATRA for at least 5 days. Using immunofluorescence staining to detect the formation of NETs, we demonstrated that dHL-60 cells differentiated for 5 days with 1 μmol/L ATRA could generate NETs comparable to those produced by neutrophils upon phorbol 12-myristate 13-acetate (PMA) stimulation, without histone H3 citrullination. Furthermore, the formation of NETs by dHL-60 cells were NADPH-dependent and PAD4-independent, consistent with neutrophils. Taken together, these observations suggest that dHL-60 cells differentiated with 1 μmol/L ATRA for 5 days can be used as a model cell for neutrophils to study the mechanism of NET formation.

Citation： LIU Wang, FANG Jinhua, HONG Tiantian, HUANG Jiaqi, ZHAO Baisong, FANG Ying, WU Jianhua, LIN Jiangguo. The characteristics of neutrophil extracellular traps produced by all-trans retinoic acid-induced dHL-60 under PMA stimulation. Journal of Biomedical Engineering, 2022, 39(5): 909-918. doi: 10.7507/1001-5515.202205002 Copy

1.	Brinkmann V, Reichard U, Goosmann C, et al. Neutrophil extracellular traps kill bacteria. Science, 2004, 303(5663): 1532-1535.
2.	Kaplan M J, Radic M. Neutrophil extracellular traps: double-edged swords of innate immunity. J Immunol, 2012, 189(6): 2689-2695.
3.	Cools-Lartigue J, Spicer J, Mcdonald B, et al. Neutrophil extracellular traps sequester circulating tumor cells and promote metastasis. J Clin Invest, 2013, 123(8): 3446-3458.
4.	Hakkim A, Furnrohr B G, Amann K, et al. Impairment of neutrophil extracellular trap degradation is associated with lupus nephritis. Proc Natl Acad Sci USA, 2010, 107(21): 9813-9818.
5.	Fuchs T A, Brill A, Duerschmied D, et al. Extracellular DNA traps promote thrombosis. Proc Natl Acad Sci, 2010, 107(36): 15880-15885.
6.	Wadehn H, Raluy L P, Kolman J, et al. Time- and dose-dependent inhibition of neutrophil extracellular trap formation by blocking of the interleukin-1 receptor. Cent Eur J Immunol, 2021, 46(4): 419-426.
7.	Saisorn W, Saithong S, Phuengmaung P, et al. Acute kidney injury induced lupus exacerbation through the enhanced neutrophil extracellular traps (and apoptosis) in Fcgr2b deficient lupus mice with renal ischemia reperfusion injury. Front Immunol, 2021, 12: 669162.
8.	洪天添, 刘望, 黄嘉祺, 等. LPS刺激稳定黏附于ICAM-1上的中性粒细胞形成胞外诱捕网依赖于整合素Mac-1和细胞骨架蛋白. 生物医学工程学杂志, 2021, 38(5): 903-910.
9.	Telerman A, Granot G, Leibovitch C, et al. Neutrophil extracellular traps are increased in chronic myeloid leukemia and are differentially affected by tyrosine kinase inhibitors. Cancers, 2021, 14(119): 1-10.
10.	Thiam H R, Wong S L, Qiu R, et al. NETosis proceeds by cytoskeleton and endomembrane disassembly and PAD4-mediated chromatin decondensation and nuclear envelope rupture. Proc Natl Acad Sci USA, 2020, 117(13): 7326-7337.
11.	Pilsczek F H, Salina D, Poon K, et al. A novel mechanism of rapid nuclear neutrophil extracellular trap formation in response to Staphylococcus aureus. J Immunol, 2010, 185(12): 7413-7425.
12.	Yu X, Tan J, Diamond S L. Hemodynamic force triggers rapid NETosis within sterile thrombotic occlusions. J Thromb Haemost, 2018, 16(2): 316-329.
13.	Abaricia J O, Shah A H, Olivares-Navarrete R. Substrate stiffness induces neutrophil extracellular trap (NET) formation through focal adhesion kinase activation. Biomaterials, 2021, 271: 1-24.
14.	Silva J C, Rodrigues N C, Thompson-Souza G A, et al. Mac-1 triggers neutrophil DNA extracellular trap formation to Aspergillus fumigatus independently of PAD4 histone citrullination. J Leukoc Biol, 2020, 107(1): 69-83.
15.	Etulain J, Martinod K, Wong S L, et al. P-selectin promotes neutrophil extracellular trap formation in mice. Blood, 2015, 126(2): 242-246.
16.	Shao Y, Li L, Liu L, et al. CD44/ERM/F-actin complex mediates targeted nuclear degranulation and excessive neutrophil extracellular trap formation during sepsis. J Cell Mol Med, 2022, 26(7): 2089-2103.
17.	Collins S J. The HL-60 promyelocytic leukemia cell line: proliferation, differentiation, and cellular oncogene expression. Blood, 1987, 70(5): 1233-1244.
18.	Harris P, Ralph P. Human leukemic models of myelomonocytic development: a review of the HL-60 and U937 cell lines. J Leukoc Biol, 1985, 37(4): 407-422.
19.	Hauert A B, Martinelli S, Marone C, et al. Differentiated HL-60 cells are a valid model system for the analysis of human neutrophil migration and chemotaxis. Int J Biochem Cell B, 2002, 34(7): 838-854.
20.	Bing H, Ling Y, Lin J, et al. Mechanical regulation of calcium signaling of HL-60 on P-selectin under flow. Biomed Eng Online, 2016, 15(S2): 637-646.
21.	Kawakami T, He J, Morita H, et al. Rab27a is essential for the formation of neutrophil extracellular traps (NETs) in neutrophil-like differentiated HL60 cells. PloS One, 2014, 9(1): 1-11.
22.	Wang Y, Li M, Stadler S, et al. Histone hypercitrullination mediates chromatin decondensation and neutrophil extracellular trap formation. J Cell Biol, 2009, 184(2): 205-213.
23.	Liu C L, Tangsombatvisit S, Rosenberg J M, et al. Specific post-translational histone modifications of neutrophil extracellular traps as immunogens and potential targets of lupus autoantibodies. Arthritis Res Ther, 2012, 14(1): 1-14.
24.	Guo Y, Gao F, Wang Q, et al. Differentiation of HL-60 cells in serum-free hematopoietic cell media enhances the production of neutrophil extracellular traps. Exp Ther Med, 2021, 21(4): 1-9.
25.	Koga T, Morotomi-Yano K, Sakugawa T, et al. Nanosecond pulsed electric fields induce extracellular release of chromosomal DNA and histone citrullination in neutrophil-differentiated HL-60 cells. Sci Rep, 2019, 9(1): 1-13.
26.	Vong L, Lorentz R J, Assa A, et al. Probiotic Lactobacillus rhamnosus inhibits the formation of neutrophil extracellular traps. J Immunol, 2014, 192(4): 1870-1877.
27.	Manda-Handzlik A, Bystrzycka W, Wachowska M, et al. The influence of agents differentiating HL-60 cells toward granulocyte-like cells on their ability to release neutrophil extracellular traps. Immunol Cell Biol, 2018, 96(4): 413-425.
28.	Vakhrushev I V, Novikova S E, Tsvetkova A V, et al. Proteomic profiling of HL-60 cells during ATRA-induced differentiation. Bull Exp Biol Med, 2018, 165(4): 530-543.
29.	Lo-Coco F, Avvisati G, Vignetti M, et al. Retinoic acid and arsenic trioxide for acute promyelocytic leukemia. N Engl J Med, 2013, 369(2): 111-121.
30.	Yaseen R, Blodkamp S, Luthje P, et al. Antimicrobial activity of HL-60 cells compared to primary blood-derived neutrophils against Staphylococcus aureus. J Negat, 2017, 16(1): 1-7.
31.	Macqueen B C, Christensen R D, Yoder B A, et al. Comparing automated vs manual leukocyte differential counts for quantifying the 'left shift' in the blood of neonates. J Perinatol, 2016, 36(10): 843-848.
32.	Parker H, Dragunow M, Hampton M B, et al. Requirements for NADPH oxidase and myeloperoxidase in neutrophil extracellular trap formation differ depending on the stimulus. J Leukoc Biol, 2012, 92(4): 841-849.
33.	Walter S, Astrid O, Peter S, et al. The role of reactive oxygen species (ROS) in the formation of extracellular traps (ETs) in humans. Biomolecules, 2015, 5(2): 702-723.
34.	Li P, Li M, Lindberg M R, et al. PAD4 is essential for antibacterial innate immunity mediated by neutrophil extracellular traps. J Exp Med, 2010, 207(9): 1853-1862.
35.	Leshner M, Wang S, Lewis C, et al. PAD4 mediated histone hypercitrullination induces heterochromatin decondensation and chromatin unfolding to form neutrophil extracellular trap-like structures. Front Immunol, 2012, 3(307): 1-11.
36.	Neeli I, Khan S N, Radic M. Histone deimination as a response to inflammatory stimuli in neutrophils. J Immunol, 2008, 180(3): 1895-1902.
37.	Haijiao J, Xiaojing C, Shuoqi Z, et al. Neutrophil extracellular traps (NETs): the role of inflammation and coagulation in COVID-19. Am J Transl Res, 2021, 13(8): 8575-8588.
38.	Manda A, Pruchniak M P, Arazna M, et al. Neutrophil extracellular traps in physiology and pathology. Cent Eur J Immunol, 2014, 39(1): 116-121.
39.	Keshari R S, Verma A, Barthwal M K, et al. Reactive oxygen species-induced activation of ERK and p38 MAPK mediates PMA-induced NETs release from human neutrophils. J Cell Biochem, 2013, 114(3): 532-540.
40.	Dömer D, Walther T, Moller S, et al. Neutrophil extracellular traps activate proinflammatory functions of human neutrophils. Front Immunol, 2021, 12: 636954.
41.	Tatsiy O, Mcdonald P P. Physiological stimuli induce PAD4-dependent, ROS-independent NETosis, with early and late events controlled by discrete signaling pathways. Front Immunol, 2018, 9: 1-12.
42.	Neeli I, Radic M. Opposition between PKC isoforms regulates histone deimination and neutrophil extracellular chromatin release. Front Immunol, 2013, 4: 1-9.
43.	Kenny E F, Herzig A, Kruger R, et al. Diverse stimuli engage different neutrophil extracellular trap pathways. Elife, 2017, 6: 1-21.

1. Brinkmann V, Reichard U, Goosmann C, et al. Neutrophil extracellular traps kill bacteria. Science, 2004, 303(5663): 1532-1535.
2. Kaplan M J, Radic M. Neutrophil extracellular traps: double-edged swords of innate immunity. J Immunol, 2012, 189(6): 2689-2695.
3. Cools-Lartigue J, Spicer J, Mcdonald B, et al. Neutrophil extracellular traps sequester circulating tumor cells and promote metastasis. J Clin Invest, 2013, 123(8): 3446-3458.
4. Hakkim A, Furnrohr B G, Amann K, et al. Impairment of neutrophil extracellular trap degradation is associated with lupus nephritis. Proc Natl Acad Sci USA, 2010, 107(21): 9813-9818.
5. Fuchs T A, Brill A, Duerschmied D, et al. Extracellular DNA traps promote thrombosis. Proc Natl Acad Sci, 2010, 107(36): 15880-15885.
6. Wadehn H, Raluy L P, Kolman J, et al. Time- and dose-dependent inhibition of neutrophil extracellular trap formation by blocking of the interleukin-1 receptor. Cent Eur J Immunol, 2021, 46(4): 419-426.
7. Saisorn W, Saithong S, Phuengmaung P, et al. Acute kidney injury induced lupus exacerbation through the enhanced neutrophil extracellular traps (and apoptosis) in Fcgr2b deficient lupus mice with renal ischemia reperfusion injury. Front Immunol, 2021, 12: 669162.
8. 洪天添, 刘望, 黄嘉祺, 等. LPS刺激稳定黏附于ICAM-1上的中性粒细胞形成胞外诱捕网依赖于整合素Mac-1和细胞骨架蛋白. 生物医学工程学杂志, 2021, 38(5): 903-910.
9. Telerman A, Granot G, Leibovitch C, et al. Neutrophil extracellular traps are increased in chronic myeloid leukemia and are differentially affected by tyrosine kinase inhibitors. Cancers, 2021, 14(119): 1-10.
10. Thiam H R, Wong S L, Qiu R, et al. NETosis proceeds by cytoskeleton and endomembrane disassembly and PAD4-mediated chromatin decondensation and nuclear envelope rupture. Proc Natl Acad Sci USA, 2020, 117(13): 7326-7337.
11. Pilsczek F H, Salina D, Poon K, et al. A novel mechanism of rapid nuclear neutrophil extracellular trap formation in response to Staphylococcus aureus. J Immunol, 2010, 185(12): 7413-7425.
12. Yu X, Tan J, Diamond S L. Hemodynamic force triggers rapid NETosis within sterile thrombotic occlusions. J Thromb Haemost, 2018, 16(2): 316-329.
13. Abaricia J O, Shah A H, Olivares-Navarrete R. Substrate stiffness induces neutrophil extracellular trap (NET) formation through focal adhesion kinase activation. Biomaterials, 2021, 271: 1-24.
14. Silva J C, Rodrigues N C, Thompson-Souza G A, et al. Mac-1 triggers neutrophil DNA extracellular trap formation to Aspergillus fumigatus independently of PAD4 histone citrullination. J Leukoc Biol, 2020, 107(1): 69-83.
15. Etulain J, Martinod K, Wong S L, et al. P-selectin promotes neutrophil extracellular trap formation in mice. Blood, 2015, 126(2): 242-246.
16. Shao Y, Li L, Liu L, et al. CD44/ERM/F-actin complex mediates targeted nuclear degranulation and excessive neutrophil extracellular trap formation during sepsis. J Cell Mol Med, 2022, 26(7): 2089-2103.
17. Collins S J. The HL-60 promyelocytic leukemia cell line: proliferation, differentiation, and cellular oncogene expression. Blood, 1987, 70(5): 1233-1244.
18. Harris P, Ralph P. Human leukemic models of myelomonocytic development: a review of the HL-60 and U937 cell lines. J Leukoc Biol, 1985, 37(4): 407-422.
19. Hauert A B, Martinelli S, Marone C, et al. Differentiated HL-60 cells are a valid model system for the analysis of human neutrophil migration and chemotaxis. Int J Biochem Cell B, 2002, 34(7): 838-854.
20. Bing H, Ling Y, Lin J, et al. Mechanical regulation of calcium signaling of HL-60 on P-selectin under flow. Biomed Eng Online, 2016, 15(S2): 637-646.
21. Kawakami T, He J, Morita H, et al. Rab27a is essential for the formation of neutrophil extracellular traps (NETs) in neutrophil-like differentiated HL60 cells. PloS One, 2014, 9(1): 1-11.
22. Wang Y, Li M, Stadler S, et al. Histone hypercitrullination mediates chromatin decondensation and neutrophil extracellular trap formation. J Cell Biol, 2009, 184(2): 205-213.
23. Liu C L, Tangsombatvisit S, Rosenberg J M, et al. Specific post-translational histone modifications of neutrophil extracellular traps as immunogens and potential targets of lupus autoantibodies. Arthritis Res Ther, 2012, 14(1): 1-14.
24. Guo Y, Gao F, Wang Q, et al. Differentiation of HL-60 cells in serum-free hematopoietic cell media enhances the production of neutrophil extracellular traps. Exp Ther Med, 2021, 21(4): 1-9.
25. Koga T, Morotomi-Yano K, Sakugawa T, et al. Nanosecond pulsed electric fields induce extracellular release of chromosomal DNA and histone citrullination in neutrophil-differentiated HL-60 cells. Sci Rep, 2019, 9(1): 1-13.
26. Vong L, Lorentz R J, Assa A, et al. Probiotic Lactobacillus rhamnosus inhibits the formation of neutrophil extracellular traps. J Immunol, 2014, 192(4): 1870-1877.
27. Manda-Handzlik A, Bystrzycka W, Wachowska M, et al. The influence of agents differentiating HL-60 cells toward granulocyte-like cells on their ability to release neutrophil extracellular traps. Immunol Cell Biol, 2018, 96(4): 413-425.
28. Vakhrushev I V, Novikova S E, Tsvetkova A V, et al. Proteomic profiling of HL-60 cells during ATRA-induced differentiation. Bull Exp Biol Med, 2018, 165(4): 530-543.
29. Lo-Coco F, Avvisati G, Vignetti M, et al. Retinoic acid and arsenic trioxide for acute promyelocytic leukemia. N Engl J Med, 2013, 369(2): 111-121.
30. Yaseen R, Blodkamp S, Luthje P, et al. Antimicrobial activity of HL-60 cells compared to primary blood-derived neutrophils against Staphylococcus aureus. J Negat, 2017, 16(1): 1-7.
31. Macqueen B C, Christensen R D, Yoder B A, et al. Comparing automated vs manual leukocyte differential counts for quantifying the 'left shift' in the blood of neonates. J Perinatol, 2016, 36(10): 843-848.
32. Parker H, Dragunow M, Hampton M B, et al. Requirements for NADPH oxidase and myeloperoxidase in neutrophil extracellular trap formation differ depending on the stimulus. J Leukoc Biol, 2012, 92(4): 841-849.
33. Walter S, Astrid O, Peter S, et al. The role of reactive oxygen species (ROS) in the formation of extracellular traps (ETs) in humans. Biomolecules, 2015, 5(2): 702-723.
34. Li P, Li M, Lindberg M R, et al. PAD4 is essential for antibacterial innate immunity mediated by neutrophil extracellular traps. J Exp Med, 2010, 207(9): 1853-1862.
35. Leshner M, Wang S, Lewis C, et al. PAD4 mediated histone hypercitrullination induces heterochromatin decondensation and chromatin unfolding to form neutrophil extracellular trap-like structures. Front Immunol, 2012, 3(307): 1-11.
36. Neeli I, Khan S N, Radic M. Histone deimination as a response to inflammatory stimuli in neutrophils. J Immunol, 2008, 180(3): 1895-1902.
37. Haijiao J, Xiaojing C, Shuoqi Z, et al. Neutrophil extracellular traps (NETs): the role of inflammation and coagulation in COVID-19. Am J Transl Res, 2021, 13(8): 8575-8588.
38. Manda A, Pruchniak M P, Arazna M, et al. Neutrophil extracellular traps in physiology and pathology. Cent Eur J Immunol, 2014, 39(1): 116-121.
39. Keshari R S, Verma A, Barthwal M K, et al. Reactive oxygen species-induced activation of ERK and p38 MAPK mediates PMA-induced NETs release from human neutrophils. J Cell Biochem, 2013, 114(3): 532-540.
40. Dömer D, Walther T, Moller S, et al. Neutrophil extracellular traps activate proinflammatory functions of human neutrophils. Front Immunol, 2021, 12: 636954.
41. Tatsiy O, Mcdonald P P. Physiological stimuli induce PAD4-dependent, ROS-independent NETosis, with early and late events controlled by discrete signaling pathways. Front Immunol, 2018, 9: 1-12.
42. Neeli I, Radic M. Opposition between PKC isoforms regulates histone deimination and neutrophil extracellular chromatin release. Front Immunol, 2013, 4: 1-9.
43. Kenny E F, Herzig A, Kruger R, et al. Diverse stimuli engage different neutrophil extracellular trap pathways. Elife, 2017, 6: 1-21.

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