Research Article

Influence of Extracellular Traps (ETs) on the Differentiation of TCD4 Cell Profiles and Macrophages in Human Autologous Culture

Guillermina Bailo
National University of Córdoba, Argentina
Fernando M. Rodríguez
National University of Córdoba, Argentina
Claudia L. Carabajal-Miotti
National University of Córdoba, Argentina
Susana G. Ruiz de Frattari
National University of Córdoba, Argentina
Adriana H. Vargas
National University of Córdoba, Argentina
Natalio E. González-Silva
National University of Córdoba, Argentina
Ivon T. C. Novak
National University of Córdoba, Argentina
* Corresponding author
DOI icon 10.24018/clinicmed.2023.4.1.240

Read Counter
369

Downloads
271

Citations

Share

Article Sidebar

Submitted 2022-11-25
Published 2023-02-23

Read counter = 369 times

Table of Contents

Article Main Content

Background: The formation of extracellular traps (ETs) as a microbicidal functional mechanism of various leukocytes has taken on importance today and they are implicated in the pathogenesis of various diseases and have even been involved in the current pandemic.

Objectives: Study ETs generated in vitro from healthy human blood leukocytes against different stimuli (LPS, fMLP) and their influence on different T cells profiles and monocyte-macrophages, in autologous cell cultures.

Methods: Heparinized human blood samples were collected with ethical consent. ETs generation was performed by stimulation with LPS and fMLP. Subsequently they were isolated.  ETs influence on cell profile differentiation was performed in samples without stimulation, with OVA addition samples, and OVA-ETs addition samples. This assay was observed through immunofluorescence (IF) labeling of molecules of T CD4 profile; Th17 and innate lymphoid cells 3 ILC3 by RORɣ; activation status of T cells by CD45RO; and M1 macrophage profile by iNOS.

Results: Significant CD4 and CD45RO positive cells percentage were observed between paired control samples and OVA addition samples (p <0.05), between paired control samples and OVA-ETs addition samples (p <0.05). In an independent experiment, significant differences were observed between OVA addition samples vs. OVA-ETs addition samples (p <0.05). At 72 h of culture, no significant differences were found between the paired samples in any case. There were no significant differences between paired control samples and OVA addition samples or OVA-ETs samples, neither 24 and 72 h of culture in RORɣ positive cells percentage or iNOS positive cells percentage.

Conclusions: Influence of ETs on T cell activation was observed and components of autologous ETs did not elicit classical activation of M1 macrophages.

Keywords: Extracellular traps ETs NETs human autologous leucocytes

References

  1. Brinkmann V, Reichard U, Goosmann C, Fauler B, Uhlemann Y, Weiss DS, et al. Neutrophil Extracellular Traps Kill Bacteria. Science (80-). 2004; 303(5663): 1532-5.
     Google Scholar
  2. Muñoz-Caro T, Rubio R MC, Silva LMR, Magdowski G, Gärtner U, McNeilly TN, et al. Leucocyte-derived extracellular trap formation significantly contributes to Haemonchus contortus larval entrapment. Parasit Vectors. 2015; 8(1): 607.
     Google Scholar
  3. Germic N, Stojkov D, Oberson K, Yousefi S, Simon H-U. Neither eosinophils nor neutrophils require ATG5-dependent autophagy for extracellular DNA trap formation. Immunology. 2017; 152(3): 517-25.
     Google Scholar
  4. Papayannopoulos V, Zychlinsky A. NETs: a new strategy for using old weapons. Trends Immunol. 2009; 30(11): 513-21.
     Google Scholar
  5. Rocha Arrieta YC, Rojas M, Vasquez G, Lopez J. The Lymphocytes Stimulation Induced DNA Release, a Phenomenon Similar to NETosis. Scand J Immunol. 2017; 86(4): 229-38.
     Google Scholar
  6. Muniz VS, Baptista-dos-Reis R, Neves JS. Functional Extracellular Eosinophil Granules: A Bomb Caught in a Trap. Int Arch Allergy Immunol. 2013; 162(4): 276-82.
     Google Scholar
  7. Ueki S, Melo RCN, Ghiran I, Spencer LA, Dvorak AM, Weller PF. Eosinophil extracellular DNA trap cell death mediates lytic release of free secretion-competent eosinophil granules in humans. Blood. 2013; 121(11): 2074-83.
     Google Scholar
  8. Hamaguchi S, Seki M, Yamamoto N, Hirose T, Matsumoto N, Irisawa T, et al. Case of invasive nontypable Haemophilus influenzae respiratory tract infection with a large quantity of neutrophil extracellular traps in sputum. Journal of Inflammation Research. New Zealand; 2012: 137-40.
     Google Scholar
  9. Urban CF, Nett JE. Neutrophil extracellular traps in fungal infection. Sem Cell Dev Biol. 2018: 1-11.
     Google Scholar
  10. Agraz-Cibrian JM, Giraldo DM, Mary F-M, Urcuqui-Inchima S. Understanding the molecular mechanisms of NETs and their role in antiviral innate immunity. Virus Res. 2017; 228: 124-33.
     Google Scholar
  11. Díaz-Godínez C, Carrero JC. The state of art of neutrophil extracellular traps in protozoan and helminthic infections. Biosci Rep. 2019; 39(1).
     Google Scholar
  12. Urban CF, Ermert D, Schmid M, Abu-Abed U, Goosmann C, Nacken W, et al. Neutrophil extracellular traps contain calprotectin, a cytosolic protein complex involved in host defense against Candida albicans. PLoS Pathog. 2009; 5(10): e1000639.
     Google Scholar
  13. Vorobjeva N V, Pinegin B V. Neutrophil extracellular traps: mechanisms of formation and role in health and disease. Biochemistry (Mosc). 2014; 79(12): 1286-96.
     Google Scholar
  14. Rodriguez FM, Novak ITC. Costimulatory Molecules CD80 and CD86 Colocalized in Neutrophil Extracellular Traps (NETs). J Immunol Infect Dis. 2016; 3(1): 1-9.
     Google Scholar
  15. Rodriguez FM, Clara Novak IT. May NETs Contain Costimulatory Molecules? J Immunobiol. 2016; 1(4): 113.
     Google Scholar
  16. Rinero R, Reyna M, Rodriguez F, Carabajal-Miotti C, Ruiz de Frattari S, Vargas A, et al. Beta Tubulin in Extracellular Traps and Mitochondrial Dynamics in Autologous Cultures of Human Leukocytes Stimulated With Lipopolysaccharide. Clin Res Immunol. 2018; 1(2): 1-5.
     Google Scholar
  17. Goldmann O, Medina E. The expanding world of extracellular traps: Not only neutrophils but much more. Front Immunol. 2012; 3: 1-11.
     Google Scholar
  18. Branzk N, Papayannopoulos V. Molecular mechanisms regulating NETosis in infection and disease. Semin Immunopathol. 2013; 35(4): 513-30.
     Google Scholar
  19. Kenny EF, Herzig A, Krüger R, Muth A, Mondal S, Thompson PR, et al. Diverse stimuli engage different neutrophil extracellular trap pathways. Elife. 2017; 6: 1-21.
     Google Scholar
  20. Berthelot J-M, Le Goff B, Neel A, Maugars Y, Hamidou M. NETosis: At the crossroads of rheumatoid arthritis, lupus, and vasculitis. Jt bone Spine. 2017; 84(3): 255-62.
     Google Scholar
  21. Sollberger G, Tilley DO, Zychlinsky A. Neutrophil Extracellular Traps: The Biology of Chromatin Externalization. Dev Cell. 2018; 44(5): 542-53.
     Google Scholar
  22. Borges L, Pithon-Curi TC, Curi R, Hatanaka E. COVID-19 and Neutrophils: The Relationship between Hyperinflammation and Neutrophil Extracellular Traps. Mediators Inflamm. 2020; 2020: 8829674.
     Google Scholar
  23. Wang J, Li Q, Yin Y, Zhang Y, Cao Y, Lin X, et al. Excessive Neutrophils and Neutrophil Extracellular Traps in COVID-19. Front Immunol. 2020; 11: 2063.
     Google Scholar
  24. Janiuk K, Jabłońska E, Garley M. Significance of NETs Formation in COVID-19. Cells. 2021; 10(1).
     Google Scholar
  25. Barrientos L, Bignon A, Gueguen C, de Chaisemartin L, Gorges R, Sandré C, et al. Neutrophil extracellular traps downregulate lipopolysaccharide-induced activation of monocyte-derived dendritic cells. J Immunol. 2014; 193(11): 5689-98.
     Google Scholar
  26. Donis-Maturano L, Sánchez-Torres LE, Cerbulo-Vázquez A, Chacón-Salinas R, García-Romo GS, Orozco-Uribe MC, et al. Prolonged exposure to neutrophil extracellular traps can induce mitochondrial damage in macrophages and dendritic cells. Springerplus. 2015; 4: 161.
     Google Scholar
  27. Kahlenberg JM, Carmona-Rivera C, Smith CK, Kaplan MJ. Neutrophil extracellular trap-associated protein activation of the NLRP3 inflammasome is enhanced in lupus macrophages. J Immunol. 2013; 190(3): 1217-26.
     Google Scholar
  28. Kumar V, Sharma A. Neutrophils: Cinderella of innate immune system. Int Immunopharmacol. 2010; 10(11): 1325-34.
     Google Scholar
  29. Kolaczkowska E, Kubes P. Neutrophil recruitment and function in health and inflammation. Nat Rev Immunol. 2013; 13(3): 159-75.
     Google Scholar
  30. Rodriguez FM, Novak ITC. What about the neutrophils phenotypes? Hematol Med Oncol. 2017; 2(3): 1-6.
     Google Scholar
  31. Tillack K, Breiden P, Martin R, Sospedra M. T lymphocyte priming by neutrophil extracellular traps links innate and adaptive immune responses. J Immunol. 2012; 188(7): 3150-9.
     Google Scholar
  32. Murphy K, Weaver C. Janeway’s Immunbiology. 9th ed. Garland Science, editor. New York, USA: Taylor and Francis Group; 2017.
     Google Scholar
  33. Najmeh S, Cools-Lartigue J, Giannias B, Spicer J, Ferri LE. Simplified Human Neutrophil Extracellular Traps (NETs) Isolation and Handling. J Vis Exp. 2015; 98.
     Google Scholar
  34. Schindelin J, Arganda-Carreras I, Frise E, Kaynig V, Longair M, Pietzsch T, et al. Fiji: an open source platform for biological image analysis. Nat Methods. 2012; 9(7): 676-82.
     Google Scholar
  35. Di Rienzo JA, Casanoves F, Balzarini MG, Gonzalez L, Tablada M, Robledo CW. InfoStat versión 2020. Centro de Transferencia InfoStat, Facultad de Ciencias Agropecuarias, Universidad Nacional de Córdoba, Argentina; 2020.
     Google Scholar
  36. Tsai C-Y, Hsieh S-C, Liu C-W, Lu C-S, Wu C-H, Liao H-T, et al. Cross-Talk among Polymorphonuclear Neutrophils, Immune, and Non-Immune Cells via Released Cytokines, Granule Proteins, Microvesicles, and Neutrophil Extracellular Trap Formation: A Novel Concept of Biology and Pathobiology for Neutrophils. Int J Mol Sci. 2021; 22(6).
     Google Scholar
  37. Magna M, Pisetsky DS. The Alarmin Properties of DNA and DNA-associated Nuclear Proteins. Clin Ther. 2016; 38(5): 1029-41.
     Google Scholar
  38. Oberg H-H, Juricke M, Kabelitz D, Wesch D. Regulation of T cell activation by TLR ligands. Eur J Cell Biol. 2011; 90(6-7): 582-92.
     Google Scholar
  39. Lu H, Xu S, Liang X, Dai Y, Huang Z, Ren Y, et al. Advanced Glycated End Products Alter Neutrophil Effect on Regulation of CD(4)+ T Cell Differentiation Through Induction of Myeloperoxidase and Neutrophil Elastase Activities. Inflammation. 2019; 42(2): 559-71.
     Google Scholar
  40. Griffith ME, Coulthart A, Pusey CD. T cell responses to myeloperoxidase (MPO) and proteinase 3 (PR3) in patients with systemic vasculitis. Clin Exp Immunol. 1996; 103(2): 253-8.
     Google Scholar
  41. Fujiwara H, El Ouriaghli F, Grube M, Price DA, Rezvani K, Gostick E, et al. Identification and in vitro expansion of CD4+ and CD8+ T cells specific for human neutrophil elastase. Blood. 2004; 103(8): 3076-83.
     Google Scholar
  42. Tateosian NL, Reiteri RM, Amiano NO, Costa MJ, Villalonga X, Guerrieri D, et al. Neutrophil elastase treated dendritic cells promote the generation of CD4(+)FOXP3(+) regulatory T cells in vitro. Cell Immunol. 2011; 269(2): 128-34.
     Google Scholar
  43. Pulido R, Alvarez V, Mollinedo F, Sánchez-Madrid F. Biochemical and functional characterization of the leucocyte tyrosine phosphatase CD45 (CD45RO, 180 kD) from human neutrophils. In vivo upregulation of CD45RO plasma membrane expression on patients undergoing haemodialysis. Clin Exp Immunol. 1992; 87(2): 329-35.
     Google Scholar
  44. Cabral HRA, Novak ITC. Autologous rosette formation by human blood monocyte-derived macrophages and lymphocytes. Am J Hematol. 1999; 60(4): 285–8. doi: 10.1002/(SICI)1096-8652(199904)60:4<285::AID-AJH6>3.0.CO;2-A.
     Google Scholar
  45. Nikitina IY, Panteleev A V, Kosmiadi GA, Serdyuk Y V, Nenasheva TA, Nikolaev AA, et al. Th1, Th17, and Th1Th17 Lymphocytes during Tuberculosis: Th1 Lymphocytes Predominate and Appear as Low-Differentiated CXCR3(+)CCR6(+) Cells in the Blood and Highly Differentiated CXCR3(+/-)CCR6(-) Cells in the Lungs. J Immunol. 2018; 200(6): 2090-103.
     Google Scholar
  46. Petretto A, Bruschi M, Pratesi F, Croia C, Candiano G, Ghiggeri G, et al. Neutrophil extracellular traps (NET) induced by different stimuli: A comparative proteomic analysis. PLoS One. 2019; 14(7): e0218946.
     Google Scholar
  47. Lambert S, Hambro CA, Johnston A, Stuart PE, Tsoi LC, Nair RP, et al. Neutrophil Extracellular Traps Induce Human Th17 Cells: Effect of Psoriasis-Associated TRAF3IP2 Genotype. J Invest Dermatol. 2019; 139(6): 1245-53.
     Google Scholar
  48. Mariotti FR, Quatrini L, Munari E, Vacca P, Moretta L. Innate Lymphoid Cells: Expression of PD-1 and Other Checkpoints in Normal and Pathological Conditions. Front Immunol. 2019; 10: 910.
     Google Scholar
  49. Borregaard N. Neutrophils, from Marrow to Microbes. Immunity. 2010; 33(5): 657–70. doi: 10.1016/j.immuni.2010.11.011.
     Google Scholar
  50. Bogdan C. Nitric oxide synthase in innate and adaptive immunity: An update. Trends Immunol. 2015; 36(3): 161-78.
     Google Scholar
  51. Weinberg JB. Nitric oxide production and nitric oxide synthase type 2 expression by human mononuclear phagocytes: a review. Mol Med. 1998; 4(9): 557-91.
     Google Scholar
  52. Dijkstra G, Zandvoort AJH, Kobold ACM, de Jager-Krikken A, Heeringa P, van Goor H, et al. Increased expression of inducible nitric oxide synthase in circulating monocytes from patients with active inflammatory bowel disease. Scand J Gastroenterol. 2002; 37(5): 546-54.
     Google Scholar
  53. Josefs T, Barrett TJ, Brown EJ, Quezada A, Wu X, Voisin M, et al. Neutrophil extracellular traps promote macrophage inflammation and impair atherosclerosis resolution in diabetic mice. JCI insight. 2020; 5(7).
     Google Scholar